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JOURNAL

(FORMERLY BULLETIN)
COLLEGE OF AGRICULTURE

IMPERIAL UNIVERSITY OF TOKYO.

VOLUME I.

TOKYO,
Published by the University.

1909—1913.

——

CONTENTS

No. J, June, 1909.

Taxevucut, T.:—On the Occurrence of Urease in Higher Plants

TAKEvucHI, T. and Inour, R.:—On the Existence of an Enzyme in
the Silkworm, which produces Ammonia as a Cleavage Product
of Amino-Compounds . .... .

Suzvuxt, U. und Yosuruura, K.:—Ueber die Extraktivstoffe im Fischflei-
TONOR My -d at, AGN Sis Cul CA CaM Re eer a lin Rees ee

Suzuki, U., Yosumrura, K. und [Novye, R.:—Hydrolyse der wilden
Seiden; Antheraes Peryni Guér (Sakusan), Antheraea Yamamai
Guér (Yamamai) und Caligula Japonica Moore (Kuriwata) ...

Suzuxr, U., YosHmiura, K. und Evst, S.:—Ueber die Eiweissstoffe aus
Reissamen

YosHrmura, K.:—Ueber die chemische Zusammensetzung der Tamari-
Schoyu.

Mrrsupa, R.:—On the Carbohydrates of Shoyu

TAKAHASHI, T.:—Studies on the Microorganisms of “Tanezu” (Japanese
vinegar ferment). With 11 Figures in the Text. .

————:—A Preliminary Note on the Varieties of Aspergillus Oryzae.

Kia, Y.:—Ueber den Einfluss der héheren Temperatur beim Sterilisieren
dersViilchua cm taMemaanne fs) ss, aback osha

No. 2, June, 1909.

Aso, K. and Nisarmura, S.:—Researches on the Preseryation of Night-Soil.
With one Figure in the Text. . ....

Aso, K. and Yosnmpa, T.:—On the Manurial Value of Various Organic
Phosphoric Compounds. With Plate I.

Mrrsura, R.:—On the Availability of Phosphoric Acid in Various Forms
in Herring-guano. With Plate IL. —

Tsupa, S.:—On the Different Forms of Phosphoric Acid in Organic
Manures .

Aso, K.:—On the Influence of Different Ratios of Lime to Magnesia on
the Growth of Rice If. With Plate III .
:--On the Influence of the Ratio of Lime to Magnesia upon the
Yield in Sand Culture. With Plates 1V and V .

\-\

163

167

Yoxoyama, H.:—Is artificial Calcium Carbonate more effective than
Limestone Meal? .

SrrKer, J. N.:—On the Lime Factor for Oats. . . . ....-.

— -—~:-On the Application of Bisulphide of Carbon in Mulberry
Culture. ae:

TaKeccui, T.:—Ueber die Blatt-Ernte bei Polygonum Tinctorium bei
reichlicher Stickstoffdiingung.

Inovye, R.:—On the Application of Dicyandiamide as a Nitrogenous
Manure. With Plate VI.

TAKEUCHI, T.:—Some Improvements in Sand Culture. With Plates VI-IX.
:—Secondary Calcium Phosphate as a Manure.

———— :—On Differences of Susceptibility of Plants to Stimulation

Aso, K.:—On Manuring with Dicyandiamid. With Plate X. :

———-:—Is Dipotassium Sulphate physiologically Acid? With Plate
ie

No. 3, March, 19ll.

TAKAHASHI, T. and Sato, H.:—Some New Varieties of Willia Anomala
as Aging Yeast of Saké. With Plate XII .
:—The Quantity of Amino-acids and its Relation to the Quantity
of Saké Selb oho i eta Re al 2
Kvrono, K.:—On the Formation of Fusel Oil by Saké Yeast .
————:—On the Asparagine-splitting Enzyme in Yeast.
:—Studies on the Butyric Acid forming Bacillus of “Saké-Moromi.”
With Plates XTII and XIV.
Oxupa, Y.—On the Lactic Acid Bacillus of “Moto ”-mash
Ivo, H.:—Note on Yeasts from Quince Liquor. With Plate XV,.
Mirsupa, T.:—Note on Yeasts of “Shoyn”-mash. With Plate XVI.
YuKAWA, M.:—Zwei neue Aspergillus Arten aus ,, Katsuobushi.“ Mit
Tafeln XVII und XVIII.
Kipa, Y.:—Influence of Rice Bran upon the Manurial Value of Phosphoric

Acid contained in Oil Cakes.

No. 4, March, 1913.

Suzukt, U., SHmamura, T. und Opaxe, S.:—Ueber Oryzanin, ein Best-
andteil der Reiskleie und seine physiologische Bedeutung. Mit
Tafeln XIX-XXVI .

381

INDEX

A. ARTICLES.

Aso, K.:—On the Influence of Different Ratios of Lime to Magnesia on
the Growth of Rice IT. With Plate III .

————:—On the Influence of the Ratio of Lime to Magnesia upon the
Yield in Sand Culture. With Plates IV and V

—— :—On Manuring with Dicyandiamid. With Plate X

———— :—Is Dipotassium Sulphate physiologically Acid? With Plate XT.

Aso, K. and NisHimura, §.:—Researches on the Preservation of Night-
Soil. With one Figure in the Text.

Aso, K. and Yosuaipa, T.:—On the Manurial Value of Various Organic
Phosphoric Compounds. With Plate I

Tyouye, R.:—On the Application of Dieyandiamide as a Nitrogenous Manure.
With Plate VI. NCES. er ee erie ic Pre

Ivo, H.:—Note on Yeasts from Quinee Liquor. With Plate XV

Krpa, Y.:—Ueber den Einfluss der héheren Temperatur beim Sterilisieren
der Milch.

-———:—Influence of Rice Bran upon the Manurial Value of Phosphoric
Acid contained in Oil Cakes.

Kurono, K.—On the Formation of Fusel Oil by Saké Yeast

————:—On the Asparagine-splitting Enzyme in Yeast .

———— :—Studies on the Butyrie Acid forming Bacillus of “ Saké-Moromi.”
With Plates XTIT and XIV.

Mirsupa, R.:—On the Carbohydrates of Shoyu. oe .

Mrrsupa, T.:—Note on Yeasts of “Shoyu”-mash. With Plate XVI

Mirsupa, R.:—On the Availablity of Phosphoric Acid in Various Forms in
Herring-guano. With Plate II.

Oxupa, Y.:—On the Lactic Acid Bacillus of “ Moto ”-mash .

Srtrker, J. M.:—On the Lime Factor for Oats .

—:—On the Application of Bisulphide of Carbon in Mulberry Culture.
Suzuki, U. und Yosurmura,:—Ueber die Extraktivstoffe im Fischfleische.
Suzuxr, U., Yosmmmuras, K. und [youyr, R.:—Hydrolyse der wilden
Seiden; Antheraea Peryni Guér (Sakusan); Antheraea Yamamai
Guér (Yamamai) und Caligula Japonica Moose (Kuriwata)
Suzuxt, U., Yosurmura, K. und Fwy, §.:—Ueber die Eiweissstoffe aus

IRGISSAMen.. 4%) Iie. | epee

Suzux1, U., Scnormamura, T. und ODAKE, §.:—Ueber Oryzanin, ein Best-
andteil der Reiskleie und seine physiologische Bedeutung. Mit
Tafeln XIX-XXVI.

TAKAHASHI, T.:—Studies on the Microorganisms of “Tanezu” (Japanese
vinegar ferment). With 11 Figures in the Text.
:—A Preliminary Note on the Varieties of Aspergillus Oryzae.

TAKAHASHI, T. and Sard, H.:—Some new Varieties of Willia Anomala as
Aging Yeast of Saké. With Plate XII
:—The Quantity of Amino-acid and its Relation to the Quantity
of Sake ;

TAKEUCHI, T.:—On the Occurrence of Urease in Higher Plants .

-——— :—Ueber die Blatt-Ernte bei Polygonum Tinctorium bei reichlicher
Stickstoffdiingung . 46 Z RC aed Re Aes!
:—Some Improvements in Sand Culture. With Plate VII-IX.
:—Secondary Calcium Phosphate as a Manure. Dye
:—On Differences of Susceptibility of Plants to Stimulation .

Takeucut, T. and INovur, R.:—On the Existence of an Enzyme in the
Silkworm, which produces Ammonia as a Cleavage Product of
Amino-Compounds :

Tsuba, S.:- On the Different Forms of Phosphorie Acid in Organic Manures.

Yokoyama, H.:—Is artificial Calcium Carbonate more effective than
Limestone Meal? .

YosHrmuraA, K.:—Ueber die chemische Zusammensetzung der Tamari-
Schoyu. Pete Nex,

YuKAwWA, M.:—Zwei neue Aspergillus Arten aus ,, Katsuobushi.“ Mit Tafeln
XVII und XVIII

B. PLATES.

858

I. Manurial Value of Various Organic Phosphoric Compounds

(K. Asd and T. Yosurpa).

II. Availability of Phosphoric Acid in Various Form of Herring-

guano (R. MrrsurA),.

III. Influence of Different Ratios of Lime to Magnesia on the

Growth of Rice IT (K. Aso).

IV-Y. Influence of the Ratio of Lime to Magnesia upon the Yield in

Sund Culture (K. Aso).

VI. Application of Dicyandianide as a Nitrogenous Manure (Rh.
INOUYE).
VII-IX. Some Improvements in Sand Culture (T. TAKEUCHI).
X. Manuring with Dicyandiamid (Kk. Aso).
XL. Is Dipotassium Sulphate physiologically Acid? (K. Aso).
XII. Some New Varieties of Willia Anomala as Aging Yeast of Saké
(T. TAKAHASHI and H. Saro).
XILEXIV. Studies on the Batyric Acid forming Bacillus of “ Saké-Moromi”
(Kk. KuRoNo).
XV. Note on Yeasts from Quince Liquor (H. Ivo).
XVI. Note on Yeasts of “Shoyu”-mash (T. MirsuDA).
XVILXVII. Zwei neue Aspergillus Arten aus ,, Katsuobushi“ (M. YUKAWA).
XIX-XXVI. Ueber Oryzanin, ein Bestandteil der Reiskleie und seine physiologi-

sche Bedeutung (U. SuzuKr, T. SomuraAmura und 8. ODAKF).

C. TEXT-FIGURES.

A. No. 6. Bacterium aceti Pasteur var. Tunezu (T. TPAISAGTASHI)| clea ss) Leo
B. No. 7. Bac. wylinoides var. Tunezu (T. TAKAHASHI). . + - + + - 135
CG. No. 6. Bac. acei Brown Tunczu If (Tf. TAKAHASHI). . - . - + + 135
D. No. 6. Bac. azetv Pasteur var. Tunezu (T. TAKAWASHI). . - - = - 135
E. No. 5. Bac. acetosum Henneberg var. Tunezu 11, (YT. TAKAHASHI). . 135
F. No. 3. Bac. aceti Brown var. Tunezu I, (T. TAKAWASHI). . - + - 136
G. No. +. Bac. aceti Brown var. Tanezu ID, (T. TAKAWASHI). . . + - 136
IL No. 2. Bae. acetosum Hennberg var. Tunez I. (T. TAKAHASHI). . . 186

K. No. 7. Bae. aylinoides var. Tunezu (Gi Sco ewNScIOP oy 6 on SO ee NID

L. No. 1. Bae. astendans Henneberg var. Tunezu (T. TAKAHASHI). - - 136
M. No. 2. Bae. acetosum Henneberg var. Tunezu I. (T. TAKAHASHI) . . 136

1, Apparatus for Researches on the Preservation of Night-Soil (K. Aso

Patsee INMEEMNTTID/AN, ow wa tO) ONO) DONEC O NOs OG 5D Or TGT TO 146

On the Occurrence of Urease in Higher Plants.
BY

T. Takeuchi.

Although much has been written about urease no mention is made
of its occurrence in higher plants. Its presence has been proved only
in Uvrobacteria and certain fungi. In the course of my investigations
on a desamidizing enzyme in higher plants, I have, discovered a very
powerful urease in’ both the resting seeds and seedlings of soy-bean
(Glycine hispida). The following observations on this point may,
therefore, be not be without some value.

30 seedlings of soy-bean about 3 em. high, with

Experiment I.
their cotyledons removed were washed, crushed in a porcelain mortar,
macerated with 50 cc. water and filtered. Four such filtrates were
prepared, each of them being put in an Erlenmeyer’s flask of ca. 200 c.c.
capacity. They were made faintly alkaline with 1 ¢.c. of normal soda-
solution and coyered with enongh toluol. The special additions con-
sisted in:

(A) 0,5 g. Asparagine,

(BB) 0:>ee2 8 Urea:

After 15 hours standing at 23°C in a thermostat they were taken
out and each 5 ¢.c. of the filtrates was tested for ammonia with Nessler’s
reagent. The observations were as follows:

(A) Very weak reaction,
(B) Very strong reaction with brown precipitates,
(C) No reaction,

(D) No reaction.

T. TAKEUCHI:

The tests were repeated with a few modified additions, namely:

(a) 0.6 g Urea (weakly alkaline)
(b) 0.6 g¢. Urea after boiling (_ ,, per
(c) Control (Cs; Fee ae)
(d) 0.6 g. Asparagine ae: te eh

(e) 0.6 g. Urea (no addition of NaOH, weakly acid).
With a sheet of moistened red litmus paper hanging from a cotton
plug, they were kept at the room-temperature (9°C). After 5 minutes
ihe flask (a) already showed the paper turning blue and a little later

also the flask (e), while no change was observed in (b), (e) and (d).

Indeed, after 30 minutes the tests with Nessler’s reagent gave positive
result only for (a) and (e). Determinations’ of the ammonia liberated —

gave the followimg results:

Duration of digestion, in hours. Ammonia liberated, g. %
(a) 14 0.1622
(b) 14 0.0034
() 16 0,000 4
(e) 16 0.1603 7

lwwdrolyse urea and liberate ammonia trom it.

The writer next made similar experiments with the resting seeds
‘of soy-bean and also succeeded in proving the presence of the same
substance.

Experiment I.-—2 g. air dry seeds were finely pulverized in a_
mortar, macerated with water for one hour and filtered. 100 ec. of the —
filtarate was divided into two equal portions, one half (3) was boiled
for 15 minutes, the other half (A) not boiled. To each of them was
added 0.5 g. urea. «The flask (C) served as control. After keeping
them for some time at the*reom-temperature (9-10°C) the determination

of ammonia was made with the following results:
1. The method used was a qualified vacuum distillation method with finely powdered
magnesia usta,

ro)

ON TIIE OCCURRENCE OF UREASE IN ITIGHER PLANTS.

Intervals, hs. Ammonia, g.
(A) 15 0.1681
(B) 15 0.0026
(C) 18 9.0000

Thus the presence of a urea-splitting substance was proved beyond
doubt in the resting seeds as well as in the seedlings.

Experiment IIT.—1 g. of air dry powdered seeds of soy-bean was
kept at the room temperature with 30 ec. toluol in an Exlenmeyer’s
flask. Two such flasks were prepared, to one of which was added 2 e.e.
of 30¢ urea solution, while the second flask served as control, As the
moistened red paper of the first flask tured blue after 5 minutes, de-
terminations of the ammonia were made after 17 hours for both flasks.

The results were as follows:

(A) With urea O.1704 g. NII.
(B) Control 0.0002 ¢. NH,

This experiment shows that the urea splitting action of the substance
is not due to the living activity of the microbes, that may possibly he
present in the seeds. The digestion proceeded equally well in the
presence of chloroform. instead of toluol, so that it can not be ascribed
to putrefactive changes. The writer kas also examined the question of
bacterial agency in the process. Thus seeds were kept for 50 minutes
in a 4/,999 solution of mereurie chloride, and were then crushed weil in
a porcelain mortar, 2 ¢.c. of 304 urea solution added to it and the whole
put in 50 ee. of sterilized water. The moistened red litmus paper in
the flask began to turn blue after 15 minutes (10°C), while at the same
time the red color reaction due to ammonia formation was obtained by;
adding a few drops of phenol phthalein® as an indicator. Next 1 g. of
the powdered seeds together with 2 ce. of 30¢ urea solution was digested

with 50 ec. of 0.005¢ HgCl, solution. After 30 minutes, in this ease,

2. Phenoiphthalein has no injurious action upon the hydrolytic action. -\s the process
goes on naturally very rapidly, the colour reaction can be obfained after a few minutes. This
method of using phenolphthalein for demonstrating the presence of an enzyme may be o

some yalue.

4 T. TAKEUCHI:

the formation of ammonia in the flask was observed, although the action-
went on somewhat more slowly.

These experiments demonstrate clearly that the hydrolytie process
is due to an enzymatic action and not to the living activity of microbes.

Experiment TV.—100 healthy seedlings* of soy-bean about 3 em.
high were washed after removing their cotyledons and triturated wel!
in a mortar. To the filtrate was added a mixture of 90¢ alcohol and
ether, whereby a white amorphous precipitate was obtained. This was
sucked off after 18 hours, washed and after well pressmg with a spatula,
extracted with 30 ce. water. To the extract strong aleohol and ether
was again added until precipitation was complete. The precipitate was
collected on a Buehner’s filter, pressed, washed with aleohol and dried
on sulphuric acid. The precipitate thus obtained weighed ea. 0.43 ¢.
and was nearly pure enzyme. It was sparingly soluble in cold water
and the aqueous solution gaye very weak biuret reaction. The same
precipitate was in exactly the same way obtained from the powdered
seeds of soy-bean.

0.4 g. of the. sample thus prepared was dissolyéd in 80 ¢@c. water,
with 30 ec. of which the following experiment was made, in order to
ascertain the behavior of the enzyme.

3 flasks of ca. 100 ec. capacity were filled each with 10 cc. of the:

enzyme solution, and to them was added:

(A) 0.2 @. urea and 30 ce. water (neutral),
(B) 0.2 g urea and 30 «ec. of 1.84 NaOlIT solution,

Each flask was kept with the addition of some toluol in a thermostat
at 23°C and ammonia determination was done after a day with the-

following results:

3. The seedlings used were carefully examined, and in all cases only those which

seemed perfectly healthy and normal were employed for the purpose of the experiments.

ON THE OCCURRENCE OF UREASE IN WIGHER PLANTS.

Intervals, hs. Ammonia, ¢.
(A) 16 0.0671
(B) 17 0.00004
(C) 14 0.0010

The second experiment with 20 e.c. of the enzyme solution prepared
from 5 g. powdered seeds was made in the following manner.
Dlasis vANe eee uses eoedsone. Urea added (neutral).

3 | BE eaten se svoy ap DEPATARINE: 4, (0.194NaOH solution).

After keeping them at 20°C ammonia was determined as follows:

Intervals, hs. Ammonia, ¢.
Flask A. 18 0.1641
B. 42 0.0002

These experiments prove beyond doubt that the hydrolysis of urea
by soy-bean seeds is principally eaused by the action of the enzyme
present in them.

Dr. K. NShibata® has previously shown that the mycelium of
Aspergillus niger is able to spht urea with liberation of ammonia, but
in this case the action is far less energetic than in ours. I shall here
extract some figures from his article.

Ammonia formed, g. Fungus used, g. Urea used, ¢. Temperature (.C) Intervals

(days).
I 0.0518 0.25 0.75 37 10
I 0.0150 0.50 2.00 35 2
iil 0.0306 1.00 1.50 35 9
IV 0.0195 0.50 1.50 20 25
V 0.0174 0.50 0.50 20 28

Experiment V.—The writer considered it interesting to compare
the strength of the splitting action of the enzyme in seeds and in seedlings.

4. Both distillation and titration were repeated, but no ammonia could be demonstrated.

Ji has since been found that the enzymatic action is inhibited in such a comparatively high

concentration of NaOH as 122.

5. Hofmeister’s Beitr, zur chem, Physiol, u. Pathol. Bd. V, 1904, s. 384.

6 T. TAKEUCHT:

Unfortunately he can not give exact figures here, since the enzyme in
the seedling appears to be somewhat injured by drying. Thus much,
however, can be said that the enzymatic action is stronger in the resting
seeds than in the seedlings, as may be seen from the following.

(A) 2 ¢. of air-dried seedlings ca. 3 em. Jong, with cotyledons
removed, was erushed in a mortar with a little water, the water was
then increased to 80 ¢@e, 30 ec. of which was decanted and put in a
TaSicc One. eee (fate ;

(B) To 2 g. of air-dried powdered seeds was added 80 ec. water,
30 ce. of which was decanted and put in another flask. ...(b).

These flasks « and b were prepared in duplicates for the purpose of
control. The flasks were treated as follows:

(Ajeet .... (a) alone.
(B) ). 2:5: Seng eels.

(OC) ede to (a) 0.5 ¢. urea added.
(Dj) .. to (b) 0.5 @. urea added.

After keeping them at 27°C in the usual manner, ammonia determ-

ination was made:

Intervals, hs. Ammonia formed, g.
(A) 52 0.0010
(B) 52 0.0024
(C) 14 0.0661
(D) 14 0.2688

Experiment VI.—The action of the enzyme on urea has been thus
clarified, but its action on other urea-derivatives remained to be seen.
The writer has, therefore, undertaken to examine the following com-
pounds on this point.

(1) Binvet is only slightly attacked by the enzyme. 1 g. of powdered
seeds of soy-bean was macerated with water and filtered. The fiftrate
was divided into 4 equal portions of 20 ec. each, They were put in
flasks and treated as follows:

(\) Boiled and 0.25 ¢. biuret added,

ON THE OCCURRENCE OF UREASE IN TWIGIER PLANTS. ‘

(B) Not boiled ,, oF a ”
(C) ie a Jom ta Aion)
(D) - a » 0.5 @ biuret .
They were kept at the room+temperature (S°C) and ammonia was

determined as follows:

Intervals, hs. Ammonia formed, g.
A 15 0.0013
B 1/5) 0.0050
C 19 0.9874
D 17 0.0056

(2) Both nitrate and oxalate of urea recently prepared were tested
for the purpose. They were acted on by the enzyme as well as pure
urea, with, however, 2 slight retardation of liberation of ammonia.

(3) The following compounds were also tested, but they all gave
negative results.

Guanidine (carbonate )

Aveinine (both nitrate and methylester hydrochloride)
Benzamide,

Allantoin, —

Leucme

Alanine

Tyrosine

IXveatine

Tlistidine (hydrochloride)

Guanine ( . )

Glycocoll (ethylester hydrochloride)
Urie acid

Hippuric acid.

The results obtained thus far plainly show that there is thus one
compound—urea—which is acted on by the enzyme with an energetic
formaton of ammonia, and that biuret is acted on but slightly.

Experiment VII.—Our experiments now assumed a new trum and

8 T. TAKEUCHI:

a series of tests were made for the behavior of various kinds of plants
towards urea. 7 different kinds of seeds were used, and in all eases
50 ec. of 1% urea solution was mixed with 2 g. powdered seeds in an
Erlenmeyer’s flask. and kept at the room-temperature. The determination
of ammonia gave the following results:

Intervals, days. Ammonia. ¢

g.
Control ae so ate aS ya ews 0.0012
Glycine hispida, mawim .. .. we 0.2653
Glycine hispida, forma .. .. .. 1 0.2713
Phaseolus vulgaris, forma... .. 3 0.0165
Phaseoius radiatus .. 2. we we 0.003
Pisum sativum Ree fe ee Pcie O 0.0018
Triticum vulgare 2 S.:: .. 4 0.0018
Avena satita -eaeee ee es es 8 0.0165

Thus Glycine hispida, maxim. and Glycine hispida, forma were seen
to be analogous to soy-bean in this regard.

A second series of tests were likewise made with the resting seeds of
different plants. In these eases, however, no determination of ammonia
was made, as it was too little to be measured, The results were as follows;

+ indicating a positive and—, a negative resuli.

Control “= Maize --
Barley — Rape —
Upland rice -- Radish —
Paddy rice == Buekwheat +
Rye _ Cucumis melo oo

Thus positive results were obtained only for paddy rice, buekwheat
and cucumis melo, though the action was but slight.

Experiment VIUII.—The writer next tried to localize the enzyme in
the seed. For this purpose Glycine hispida, maxim was used and phenol
phthalein served as the indicator of ammonia formation, The tests proved
the presence of the enzyme in all parts of the seed.

Experiment [X.—The range of the enzyme action with regard to the

ON THE OCCURRENCE OF UREASIO IN HIGHER PLANTS. 9

concentration of urea solution was studied and the result was remarkable.
Indeed, with 10, 20 and 304 mea solutions the action went on vigorously,
with 40% also well, but with a little retardation.

The influence of foreign substances, such as acids, alkalis. neutral
salts, and antisepties was next observed with the enzymes of soy-bean seeds.
The color reaction with phenol phthalein previously mentioned served for
the tests which were made after neutralization of the solution. In earry-
ing out the observations the enzyme was subinitted to the action of the
reagents before beige added to the urea solution, During the tests the
room-temperature ranged from 13 to 16°C. The results are expressed in
the following table.

Foreign subst. used Concentration Appearance of red coloration.

ES Oe eOGes waittere4.5 minutes:

3 Teepe a) (OLD ey emmelis) 5
HC] Seem Mere — cre OLS a 20 5
5 mee ne (ODO Fa | ill: of
BO. tee ee een tO) <¢ = 130 uf
r. Smet (ND Lg 5 io -
MEMOMIE Ss op oe Soe eae: ye od) %
NGONGE SS 5, sah Orem Uae’ = oxi) =
(ENE SS Oo aeeione cae 0g eK -
3 eee Ae 10" 9 Peele ral(0) 53
CUSOW oc oo co oo Osby: a Oe 3
" Eee ch OL 014 55 GeO 3
INES Versace eee. OS 959 » +0 a
r- NA ov 0.204 aye lis es
OWS a5 oc oo oe) OEY: sgn ae x
” Se che ae 0.024 A 1a ;
IL.COnW ove ears OLD: a 0) i
A; ny oh Oo af uO

Lhus 14 H.SO, 1% HCl, 54 (NII,).SO,, 0.05¢ CuSO,, 0.25%
NaF and 0.05¢*HgCl, were seen to impede the action of the enzyme to

some extent.

10 T. TAKEUCHI?

Further it was observed that 1¢ NaOH, 2¢ IL,SO,, 10¢ (NI,).SO,,
0.1¢ CuSO,, and 0.1% HgCl, are strongly inhibitory, while MgO, though
present in ereat excess, has no deleterious influence on the action of the
urease.

The degree of temperature at which the enzyme is destroyed was next
studied. At 72°C more than one hour were required. Tleating fer 30
minutes to 75°C was not sufficient to destroy all the enzyme, but it injured
a great part of it. After heating for 5 minutes to 77°C a faint trace of
lidrelysing power was still observed, but no trace was noticeable after
heating for one minute to 80°C. Cooling for one hour to—5°C did not
decrease the intensity of the hydrolysing power, even at—S°C the enzymic
still retained its activity for 20 minutes.

The optimmm temperature for the working of the enzyme was found
to be 40-45°C.

Experiment X.—Trypsin, panereatin, emulsin and Taka-diastase
were tested as to their possible hydrolytic action on urea. Results obtained
by adding 0.5 g. of the enzymes to 20 e.c.of 14% urea solution, were entirely
negative, as was expected, although they had a powerful action on fibrin,
mnygdalin and starch respectively.

The necessary preliminary studies being finished the writer proceeded
to determine the value of the enzyme when applied to human urine. It
has been observed by various authors that urea, which is the chief nitro-
genous constituent of fresh urine, is not absorbed and fixed by the soil
as the ammonium compounds are, and that it is even injurious to plant
roots in a comparatively high dilution (0.5 per mille)". Fresh urine is;
therefore, subjected to a process of fermentation in order to secure the

nitrogen in an available form. The loss of ammonia during the process

has been a great disadvantage, and various plans have been devised for
saving the valuable nitrogen,
In spite of the indefatigable zeal of agricultural chemists in this
.

6. Cf. Bul. of the College of Agr., Tokyo. Vol. IV. 413.

ON THE OCCURRENCE OF UREASE IN WIGIHER PLANTS. i

direction no efficient contrivance has been found out. The application of
the new enzyme for this purpose naturally occurred to me, and led to the
desired result.

Experiment XI._—200 ¢.c. fresh urine of a healthy man was collected
and to 100 c.c. of it was added 1 gram of powdered soy-bean seeds, while
the other 109 ¢.¢. served as control. With loosely tightened cotton plugs,
they were left in the room (temperature ranged $-10°C.) for 16 days.
The mixture was then taken out of the flasks and the ammonia formed
was determined with the following results:

(A) Whithesccuseee mete, 622 62. “Uso00

a ENUIEIES
(B) Control ee ae re OS OS0no, SN EIC:

This shows that 0.322 ¢ more ammonia was produced by the addition
of the seeds. Similar experiments also showed that the alkaline fermenta-
tion of urine was favoured by the addition of the seeds, as the following
figures will show: NH. formed, g.

8—g°C. 10 days 8—g°C. 1 day.
GAY). 7 eee: OS. «60362 0.4735
(3) SU" Senet ee sec ke ~O:042 0.019

It may safely be said that the enzyme, when applied to fresh urine,
produces 4.5 g. more ammonia from 1 L, urine in a day even in colder
seasols. :

Experiment XIT.—300 ¢.e. of fresh urine collected in the morning
was mixed with several grams of powdered sov-bean seeds in an Erlen-
meyer’s flask and kept at the room-temperature (S-9°C) with a cotton
plug. Ammonia determination after several days gave the following

results. (Caleulated in terms of 100 ¢.c. urine. )

Seeds used Intervals, hs. Temp. Ammonia, g.
Control err ee erste. + Lat S-9 0.046
Od 2 2) god, See 1s 9 0.304
OlD g. eh ote Bp Cae Bea Ta S-9) 0.427
TAO ga iis UG,» SOE 7 i) 0.675
EA (GF iS) ee eb, came = v1 S-) 0.705

7. (B) is the centro], without the addition of seeds.

12 T. TAKEUCHI:

3.0 g. ; ‘eee Moll 8-9 0.726
5.0 g. Sy ¢ ca ate he ee 3 9 0.313
5.0 g. shes j 96 S-9 0.726
10.0 2. ek 140 s-) 0.687
5.0 ¢, (nourme) .. -. 140 8-9) 0.001
5.0 g. (boiled) vs) sist s-9 0.031

The percentage of total nitrogen in the urine was found after
Kjeldahl’s method to be 0.814. It is easily seen that the most favorable
ratio of seeds to urine lies between 3:500 or 1:100, and 5:300 or 1.7:
100; where 0.726 g. ammonia was produced in only + days from 100 e.e.
urine.

Tt may also be concluded that when properly managed, almost all
urea-nitrogen of urine can be changed into a form of ammonia in a few
days. Hence, it is advisable that preservative agents, e.g. sulphurie aeid
&e. be added at the proper moment to fix the ammonia produced; or if
ammonium sulphate may be manufactured on a large scale from the
hydrolysed urine.

The writer was next led to step into the biological application of the
enzyme for analytical purposes.

Experiment XITI.—10 g. of pulverized soy-bean seeds was macerated
in cold water for 30 minutes, the filtrate obtained was divided into 5 equal
parts of 10 ec. each and urea was added as follows:

(A) 0.01 g@. urea dissolved in 90 ee, water==0.01 6

(By) 0005s es = rei UA)
(CC) ‘0:0025 055; o s3) fp Ga DOORS
(D) 0:00: sf Sela? a) i SOO

Naturally at the beginning of the experiment every flask showed an
acid reaction, as proved by the addition of phenol phthalein, The room-
temperature during the test was 11°C throughout. The red colour
appeared in (A) after 20 minutes, in (B) after 30 minutes and in (C)
after 45 minutes, while in (D) it did not appear.

This shows that the enzyme is able to split urea in even '/;o900

soltition to a recognizable extent, when neutral. The enzyme can there-

ON THE OCCURRENCE OF UREASE IN HIGHER PLANTS. 13

fore be satisfactorily employed for the urea-test of various animal juices.
and further improvements will probably make quantitative determinatior

of the urea possible. This point is reserved for future study.

Conclusions

Since Musexlus! diseovered a urea-splitting enzyme in the urine
which he obtained from a patient, the behavior of urease became the
subject of carmest investigations by various authors. Lea" concluded that
its action was altogether intracellular and that it was unable to pass out
of the cell during lite. Miquel.” on the other hand, claimed that a power-
ful urease could be obtained quite free from bacterial cells. Betjerinch'
and Joll? after exhaustive series of experiments came to the conclusion
that the urease in Micrococcus ureac was by no means detached from the
eells whether the bacteria were alive or not. These conflicting statements
need to be cleared up. My present investigation has demonstrated that
urease exists not only in lower organisms, but also in ligher plants, and
that the urease in the latter in which it acts more powerfully can be
extracted with water very easily.

It is strange that the urease acts exclusively on urea and not on allied
substances. Tis natural funetion in the plant body has still to be made
out.

There is no doubt that the enzyme is important, and its urea-
splitting property can be turned to account for determining the presence
of urea even in minute quantities in various organs and juices.

Another application of the enzyme consists in its strong ammonifying

action on fresh urine, and its application for the recovery of the chiet

I. Musculus, Compt. rend. $82, 1876, 334.

2. S. Lea, Journ. of Physiol. Vol. 11, 1890.

3- P. Miquel, Compt. rend. 111, 1890, 397.

Beijerinck, Centrbl. f. Bakt. Il, Abt. VII, 1901, 33.

nan +

Moll, Beitr. z. chem, Physiol. u. Patholog., 1902, II, 344.

14 T. TAKEUCHI:

nitrogen of fresh urine is undoubtedly a step in advance in the economy

of manures. :
T have to express to Professor Dr. Us Suzulei my great indebtedness

for his advice so kindly given during the progress of the work.

On the Existence of an Enzyme in the Silkworm, which
produces Ammonia as a Cleavage Product
of Amino-Compounds.

T. Takeuchi and R. Inoue.

While engaged in an investigation of the chemical constituents of
the silkworm (Bombya mor’) at different stages of its life-cycle, especially
the mature worm, pupa and moth, at the suggestion of Professor U.
Suzuki, we found a new enzyme of which a general description is given
helow.

IT. The action of the juice of crushed silkworms on some amino-
compounds.

Ten worms were well crushed in a porcelain mortar, and divided
equally into four Erlenmeyer’s flasks, and to each was added 100 ee. of
distilled water. They were then treated as follows:

(1). No reagents.

(2). 0.5 germ. of asparagine.

(3)).. A little quantity of leucine and elyeocoll.

(4+). The same quantity of Jeueine and elycocoll as in the third

flask, and enough caustic soda to make the solution 0.174.

(The juice of the erushed worms gives a weakly alkaline reaction).

As an antiseptie enough toluole was poured into each flask to cover
the surface of the solution, the flasks were then closed with cotton stoppers,
and kept at 25°C in a thermostat. After standing for 24 hours, a
small quantity of cach solution was filtered into a test tube, and tested
for ammonia with Nessleis solution, with the following results:

The filtrate from the second flask gave a strong reaction for am-

monia, while for the others the reaction was very weak. Jn particular,

16 T. TAKEUCHI AND R. INOUE:

the fourth remained entirely without the color on the addition of Vess/er’s
solution.

This preliminary experiment appears to show the presence of an
enzyme in the juice of crushed silkworms. Similar experiment with
some pup, and moths, not only gave similar results, but the reaction
for ammonia was even stronger than in the laryre.

The next experiment was a step forward. Ten moths were well
erushed in a porcelain mortar, and transferred into two flasks of the
capacity of some 200 ec. each, and diluted with a small quantity of
water; to each was then added one half gram of asparagine, and one of
them was boiled. Toluole was used as an antiseptic, and the Hasks were
provided with cotton stoppers, as in the previous experiment. The
flasks were then kept at 25°C. for 24 hours. Then a small quantity
of the two solutions was filtered and tested for ammonia by the same
method as before. The filtrate of the wnboiled solution gave a strong
ammonia reaction, while the others gave none at all. After keeping
them for one week at the same temperature, the nitrogen of the solution
in the form of ammonia was determined with the following results:

Boiled’... "Soe. => =. OOO R mame
Unboiled: © \23° “2 eiteeeeete es -)- O20 pe Ie

The determination of the ammonia nitrogen was done by the follow-
ing method.

The solution was poured into a distiller provided with a side tube,
into which magnesia usta was dropped in small quantities by means of
a spoon, the distiller being at the same time shaken violently so as to
ensure an uniform distribution of the salt, until the solution presented a
faintly alkaline reaction. Then the distiller was connected with a
receiver, also provided with a side tube, through which the air was sucked
out. The distillation thus took place under a decreased pressure, until
all the ammonia was liberated from the solution. The ammonia was
fixed by the standard solution of sulphurie acid which had been pre-
viously put in the receiver. When the distillation was complete,

volumetric determination of the nitrogen was made.

ON THE EXISTENCE OF AN ENZYM IN THE SILKWORM. Alig

If. Isolation of the enzyme.

The existence of an enzyme in silkworm being established, our
next task was to isolate it. For this purpose, one hundred moths were
thoroughly crushed, mixed with some elean sand, in a porcelain mortar.
The mass transferred into a well washed eotton cloth, and the juice
squeezed out. The refuse was also pressed thrice in the same way. The
juice thus obtained amounted to some 100 ec. On addition to it a
mixture of absolute alcohol and a little ether enough to make 600 c.c.,
it yielded a grayish-white bulky precipitate. The solution was allowed
to stand for about 16 hours until the precipitate had settled down on the
bottom of the flask, and then filtered. The precipitate thus obtained
weighed five grams when freed from the alcohol as much as possible.
Tt is soluble im water and shows the weak biuret reaction.

In order to test ihe presence of the enzyme in the precipitate, the
following experiment was carried on.

One half of the precipitate weighing 2.5 grms., was dissolved in 25
e.c. of water, and equally divided into five flasks, and treated as follows:

(a) Diluted with water to 40 ¢¢., served as control.

(b) Also diluted with water to 40 cc. with addition of 0.4 grm.
of asparagine.
(c) Boiled, and then treated in the same way as (0b).

(d) Diluted with water to 40 ce. with addition of 0.4 grm. of
asparagine and enough caustic soda to make the strength of
0.05 per cent.

(2) Boiled, and then treated just like (d).

The flasks, with a little quantity of toluole, were closed with cotton
stoppers and kept at 28°C. for three days. Then the filtrate of the
five solutions were examined one by ene with Nessler’s reagent. The
result was as follows:—The filtrate of the flask (d) showed a
strong ammonia reaction, while the others showed none. This shows
clearly that in the case of asparagine the enzyme acts only in a faintly
alkaline solution, and that it is inactive or nearly so even when the

solution is neutral. After five days’ standing, the ammonia produced in

18 T. TAKEUCHI AND R. INOUE:

(d) and (e) was determined by the method described before, with the
following results.
(d) (e)
Nitrogen as ammonia... .. 0.0062 grm. 0.

The action of the enzyme was precisely the same after purification.

Til. The action of the enzyme on various amino-compounds,

To ascertain whether the enzyme acts on other amino-compounds
hesides asparagine, the following experiment was made.

Ten moths were well crushed in a mortar with a little water added,
transferred into a flask of the capacity of some 200 e.., and then
diluted with water to 50 ce. Several such flasks were prepared and
various amino-compounds were added in the proportion of one per cent.
They were then well shaken with enough toluole as an antiseptic and
kept at 28°C. for six days, with occasional shaking so as to well mix
the toluole and the solution to prevent putrefaction. After one week
the ammonia in each flask was determined by the previous method,

with the following results:

.\mino-compounds. Nitrogen as ammonia,
Asparasine <.7u tee | --- 5 Od OR orm:
Urea’... ee... Cee
Biuretl.;4,) 624 eee . t FOLOOSTARS
Leucine {6% teenie bk. <> ors OLOOLOD ES
Glyeocoll ot ee ics 2o DOOR SER.
Tyrosine Sa os 0 MOL OO Dames
Allantoin. <=. siamese OOO 2T eis
Guanidine carbonate... .. .. .. 0:0022 ;,
Benzamide':).) emenanee tl. :-.. SS OO0ZD ea:
Control, without any addition .. .. 0.0022 ,,

This shows that the enzyme acts chiefly on asparagine, the other
amino-compounds being affected very slightly or not at all.

IV. The cleavage products of asparagine.

We now come to the determination of the cleavage products of

asparagine. For this purpose, one-half gram of the precipitated euzyme

ON 'SHE EXISTENCE OF AN ENZYM IN TITK SILKWORM. 19:

was dissolved in 50 ec. of water, and 0.025 gram of asparagine added.
After treating the flask containing the solution as before, it was kept
at the room-temperature (about 5°C) for two weeks, then the solution
was filtered and the filtrate transferred into a separating funnel, acidified
with 5 e.e. of 20 per cent sulphuric acid. Then the solution was extracted
with ether several times until the ethereal extract presented a very faintly
acid reaction. All the extracts were combined together and_ slowly
evaporated on a water bath. White prismatic crystals were thus obtained,
which are perhaps those of succinic acid. But the sample was too
small in quantity to be used for further researches.

V. Conclusions.

The presence in the silkworm, and especially in the moth, of an
enzyme which produces ammonia by acting on asparagine has been placed
beyond doubt. Similar enzymes have been reported by many authors,
and it may uot be useless to give a brief review of their works.

K. Shibata’ has ascertained the presence of an enzyme which pro-
duces ammonia by splitting urea, biuret, acetamide, and two or three
other amino-compounds, in the filaments or mycelium of Aspergillus
niger. But this enzyme differs from ours in that it acts chiefly on urea,
and one or two other amino-compounds but very weakly on asparagine.
Moreover, as the enzyme was not isolated, the development of ammonia
in this case may have been due to the combined actions of several
enzymes instead of one. 2. Castoro* observed the production of

ammonia during the germination of one or two species of Lupinus, kept

at 25°—30°C. for two weeks with some toluole as an antiseptic, but
he has not effected the isolation of the enzyme. Again I. Jacoby® has
ascertained that ammonia is produced by the autolyse of the dog liver
but he attributes the phenomena to the action of a proteolytic enzyme

and has not determined its exact nature. .

1. Beitrage zur ch. Physiol. u. Pathol. 1904, Bd. 5, S. 384.

Z. f, physiol. Ch. 1907, Bd. 50, S. 525.
3. Z. f. physiol. Ch. 1900, Bd. 30, S, 149.

20 T. TAKEUCHI AND R. INOUE.

Lately, Effront* has discovered an enzyme which produces ar-
monia by splitting asparagine, in the heer yeast, but according to his
observations the enzyme acts most favorably in a strongly alkaline solution
(0.1—0.2¢), and not at all in neutral or weakly acid solutions, which
distinguishes it from our enzyme. Lastly, E. Weinland® has observed
that the fly Calliphora Vomitoria produces ammonia during the larval
stage, and has attributed it to the action of an enzyme. We have also
tried to repeat the experiment on the silkworm, larva and moth, but
no positive result was obtained.

In shor!, an enzyme, which produces ammonia by splitting amino-
compounds, may be fourd widely distributed in plants and animals,
but owing te want of observation in a pure state, it is not possible to
determine whether the action is due in each particular case to one or several
enzymes. We have prepared our enzyme in a comparatively pure state,
and there is no doubt that it is different from any as yet described.
What réle it plays in the metamorphosis of the silkworm is totally
unknown.

Addendum.—Since the above was communicated to the Tokyo
Chemical Society, Dr. Buthkewitsch has published “Das Ammoniak als
Umwandlungsprodukt stickstoffhaltiger Stoffe in héheren Pflanzen.”
(Biocheme Zeitschr. Bd. 16. s. 411).

4. Compt. rend., 1908, 746, 779.
5. Z. f. Biol., 1905, Bd. 47, 186.

Ueber die Extraktiv stoffe im Fischfleische.
VON

U. Suzuki und K. Yoshimura.

unter Mitwirkung von M. Yamakawa und Y. Irie.

EINLEITUNG.

Unsere Kenntnis iiber die Extraktivstoffe der tierischen Muskeln
ist in letzter Zeit durch Untersuchungen yon Gulewitsch, Krimberg’,
Kutscher?, Ackermann®, Micko* u. A. bedeutend erweitert worden. Diese
Autoren beschiaftigten sich hanptsichlich mit dem Liebig’schen Fleisch-
extrakt und haben ausser den altbekamnten Stoffen: Nanthin, Hypo-
xanthin, JXreatin, JX<reatinin, Taurin, Guanin, Adenin ete. das
Vorhandensein yon Carnosin, JXarnin, JX<arnitin, Oblitin, Neosin,
Karnomuskalin, Methylguanidin ete und auch von kleinen Mengen der
Monaminosiiuren, wie Alanin und Glutaminsiiure nachgewiesen.

So wird das physiologisch hochwichtige Problem der chemischen
Natur der Extraktivstoffe, ihre Bildung und Umwandlung, ihre Bedeutung

fiir den gesamten Kreislanf im tierischen Organisms und ferner die

1, W. 1. Gulewitsch, Krimberg u. Amiradzibi:- Zur Kenntnis der Extraktivstofte der
Muskeln 1—X. Mitteilung:—Zeitsch. f. physiol, Chem. 30 565 45 326. 47 471 48 412
49 89 50 204 36% 535. 53 514 55 466 56 417.

2. Fr. Kutscher: Zur Kenntnis des Novains:—Zeitsch. f, physiol. Chem. +9 47. 484

ch Zweite Notiz zur Kenntnis des Novains A » 50. 250,

3. D. Ackermaun u, Fr, Kutscher: Zur Konstitutions ermittelung des Novains: Zeitsch.
f. physiol, Chem. 56 220,

D, Ackermann: Ein Beitrag zur Chemie der Faulnis: Zeitsh. f. physiol. Chem. 54 1.

*r ie Ein Faulnis versuch mit Arginin ; » 50 305.

4. KX. Micko: Ueber das Vorkommen von Monaminosiuren im Fleischextrakt ,, 56 1So.

Bye U. SUZUKI UND K. YOSHIMURA:

physiologische Wirkung derselben als menschliche Nahrung immer mehr
in helleres Licht geriickt werden.

Was nun das Fischfleisch betrifft, so fehlt es dariiber an chemischer
Studien nicht; diese befassen sich aber meistens mit den Fiulnisprodukten
desselben. Auf die normalen Bestandteile dagegen hat man nur selten
die Aufmerksamkeit gerichtet.

Da der Fisch in Japan als Volks nahrungs mittel eine ebenso wichtige
Stelle cinnimmt, wie das Fleisch in Enropa, so ist es fir uns Japanern
ganz besonders wiinschenswert, die chemischen und _ physiologischen
Studien in dieser Richtung weiter vorwiirts zu bringen. Ferner sieht die
Erscheinung der Fisch vergiftung, die bei uns so oft vorkommt, mit den
Estraktivstoffen in innigstenm Zusammenhang; nur durch griindliche
Studien der Natur der letzteren kann dieselbe erkliirt werden.

Von diesen Grund gedanken ausgehend haben wir unsere Studien
begonnen and legen die bisherigen Ergebnisse der Oeffentlichkeit vor. Bis
jetzt haben wir so wohl im frischen wie im getrockneten Fleisch von
Katsuo (Bonito, Gymnosarda pelamis), Lachs (Onchorynchus keta),
Maguro (Thynnus thunnus), Lse-cbi (Hummer, Panulirus sp.), [kat
(Ommastrephes sp.) und Unagi (Siisswasser-Aal.—Anguilla fluvialitis)
ausser den bisher bekannten Stoffen, wie Kveatin, Kreatinin, Xanthin,
Hypoxanthin, Taurin ete. das Vorkommen von Carnosin, Histidin,
Arginin,? Lysin und 6-Aminovaleriansiure in ziemlich bedeutender Menge
konstatieren kénnen. Ferner konnten wir verschiedene Monaminosiiuren,
wie T'yrosin, Leucin, Alanin, Prolin ete. mit verhiiltnismiissiger Leich-
tigkeit nachweisen, Stoffe die man aus dem Liebig’schen Fleisch extrakt
nur in unbedeutender Menge zu isolieren vermochte.

Wir halten es fiir wahrscheinlich, dass man schliesslich fast alle

Spaltungsprodukte der Eiweisskérper, nebst deren Umwandlungs pro-

1. Obgleich Ise-ebi u. Ika, eigentlich nicht zu den Fischarten gehérev, so haben wir
doch der bequemlichkeit halber hier angereiht, weil sie als menschliche Nahrung ebenso wichtig
ind wie der Fisch

2. Das freie Arginin ist nur einmal in der Milz gefunden worden: Vergl. W. l,

Gulewitsch u, Jochelson:—Zeitsch, f. physiol. Chem. jo 533.

UBER DIE EXTRAKTIV STOVFE IM FISCHIPLEISCHE. 23

dukten in den tierischen Muskeln finden wird, ebenso wie dies in der
Pflanze der Fall ist. Wir sind auch zu der Annahme geneiet, dass man
diese Extraktivstoffe nicht als Ausscheidungsstoffe wie z. B. Harnstofi
betrachten darf, sondern als eine Kérpergruppe, die fiir den weiteren
Kreislauf im tierischen Organisms eine wichtige Rolle spielt. Besonders
interessant ist das reichliche Vorkommen yon Hexon basen in Fisch-(und
Hummer) muskeln, was vielleicht mit der Bildung von Sperma und Eiern
in gewissem Zusammenhang stehen diirfte, da die letzteren ausser-
ordentlich reich an basischen Stoffen sind.

Dass die giftigen Basen wie Ornithin, Putrescin, Cadaverin ete. durch
Fiulnis von Arginin und Lysin gebildet werden, ist von Ellinger, Acker-
mann u. A. nachgewiesen werden. Was aber das Histidin und Carnosin
betrifit, miissen wir noch die Ergebinsse kiimftiger Forschungen abwarten.
Eine Base, die wir im getrockneten [ka in reichlicher Menge gefunden
haben und fiir ¢-Aminovaleriansaiire halten, ist noch niemals in frischen
Muskeln geftmden worden. Es sei nur erwihnt, dass der letzt genannte
K6rper von Saikowskit aus gefaultem Pankreas isoliert und spiter vor
D. Ackermann? niher studiert worden ist. Kurzum, es bleibt in dieser
Richiung noch viel zu tun iibrig. Wir beabsichtigen daher diese Arbeit
noch weiter fortzusetzen und hoffen etwas neues in diesem Gebiete
beitragen zu kénnen.

In den folgenden Seiten teilen wir die einzelnen Ergebnisse der

bisherigen Untersuchung mit.

I KATSUO (Gymnosarda pelamis. od. Bonito),

A. Katsuobushi (Getrockneter Bonito),

Die von wos untersuchte Katsuobushi Probe hatte folgende quan-
titative Zusammensetzung:

1. E. u. H. Salkowski:—Berichte d. deutsch. chem. Gesellschaft. XVI. 1191 u,
XXXI. s, 776.
2, D. Ackermann: Ein Beitrag zur chemie der Faiilnis : Zeitsch, f. physiol. Chem. s¥ 1.

= ns » Ein Faulnisversuch mit Arginin : os o » 56 305:

24 U. SUZUKI UND K. YOSHIMURA:

In 100 Teilen Trocken substanz.

Gesamt Phosphor 1.469
In Wasser léslicher Phosphor 1.211
Gesamt Stickstoff 14.611
In Wasser léslicher Stickstoff 3.350
Darunter: { Ammoniak stickstotf 0.072
Eiweiss stickstoff 0.176

Nicht-Eiweiss stickstoff 3.102

Durch Phosphowolframsaure fillbarer

Stickstoff 1.289

(Ammoniak ausgenommen).

In Wasser loslicher Stickstoft als 100.

Ammoniak stickstotf 2.15
Eiweiss stickstoff 6,25
Nicht-Eiweiss stickstoff 92.60
Darunter: { Durch Phosphowolframsiiure fillbarer
Stickstoff 38.21
Durch Phosphowolframsiure nicht
fiillbarer Stickstoff 54.39

Unter dem durch Vhosphowolframsiiure
fillbaren Stickstoff. Derselbe als 100

( Durch Silber nitrat in neutraler

Lésung fillbarer Stickstoff 0.067 5.24
Durch Silbernitrat und Baryt

fiillbarer Stickstoff 1.008 78.75
Stickstoff in anderer Form 0,205 16.01

Tsolierung der organischen Basen.

1 Keg getrockneter und fein gepulverter Bonito wurde mit Wasser
eine Stunde gekocht und stark abgepresst. Der Riickstand wurde noch
zweimal in derselben Weise behandelt. Die vereinigten Ausziige wurden

mit Essigsiiure schwach angesiinert und mit einer wiisserigen Tannin

UEBER DIE EXTRAKTIV STOFFE IM FISCHILEISCHE. 25

lésung versetzt, wobei ein dicker Niederschlag entstand. Das Filtrat
wurde durch Bleiessig von Taunin und anderen Verunreinigungen befreit,
abfiltriert und nach dem Entfernen des Bleies durch Schwefelsiure, wurde
es mit Schwefelsiinre angesiinert bis die Fliissigkeit ungefihr 5¢ derselben
enthielt und mit emer cone. Lésung von Phosphowolframsiure gefiillt.
Yer dabei in reichlicher Menge entstandene weisse Niederschlag wurde
nach 24 Stunden abgesaugt, mit 5% Schwefelsiiure gewaschen und auf der
Tonplatte getrocknet. Der Niederschlag wurde nun in wenig Wasser
verteilt, mit Ueberschuss von Barium hydroxid verrieben. Das Gemisch
wurde 6fters umgeriihrt und bei emer Temperatur von 25-30° 24 Stunden
stehen gelassen und abgesauegt. Der Riickstand wurde nochmals in Wasser
yverteilt und mit Baryt verrieben. Diese Operation wurde dreimal
wiederholt. Die vereinigte Filtrate wurden durch Kohlensiure yon
Baryt befreit und im Vaeuum bis auf ungefaihr 200 ¢.c. eingedampft,
mit Salpetersiiure neutralisiert und mit Silbernitrat ]ésung in kleimem
Ueberschuss versetzt.

I.—Der Silbernitrat-Niederschlag (Vanthin uid Hypoxanthin). Der
Silbernitrat-Niederschlag wurde mit Ueberschuss von Ammoniak verrieben
nnd 24 Stunden stehen gelassen, um damit die Silbernitrat salze der
Basen in die Silbersalze derselben iiberzufiihren. Die Silbersalze wurden
dann mit warmer verdiinnter Salzsiiure zerlegt und heiss filtriert. Das
Filtrat wurde stark emgeengt, mit Ueberschuss von Ammoniak versetzt,
und 24 Stunden stehen gelassen. Da aber nichts dabei ansgeschieden war
(Guanin nicht vorhanden) wurde es mt Salzsiiure angesiiuert, wiederholt
zum Trocknen verdampft und zuletzt mit Alkohol verrieben. Der dabei
ungelést geblicbene Riickstand wurde in Wasser gelést und mit einer
ammoniakalischen Silberlésung versetzt. Is entstand dabei eine weisse
Pallung von Xanthin silber. Der Niederschlag von Nanthin silber wurde
mit Salzsiiure verrieben, vom ausgeschiedenen Silberchlorid abfiltriert, zum
Syrup verdampft und mit absolutem Alkohol behandelt, wm den iiberschuss
yon Salzsiiure zu entfernen. Der Riickstand wurde mit Wasser versetzt
und bei einer Temperatur von 40° 24 Stunden stehen glassen. Ein

Teil ging dabei in Lisung und hinterliess ein schwach gelblich gefirbtes

26 U. SUZUKI UND K. YOSHIMURA:

Pulver, das in kaltem Wasser schwer, in heissem Wasser etwas leichter
und in Ammoniak leicht lislich war. Es gibt die Weidels’che, so wie
die Strecker’sche Reaktion, welche eigentlich fiir Xanthin charakteristisch
sind. Leider reichte die Menge der isolierten Base zur niheren Unter-
suchung nicht aus.

Die Mutterlauge von Xanthin lieferte nach weiterem Einengen
0.74 g. Hypoxanthin. Es war in kaltem Wasser und Alkohol schwer, in
heissem Wasser aber leicht lislich. Die wiisserige Liésung reagierte
neutral. Fiir die Analyse wurde das gereinigte Priiparat im Vacuum

bei 100° getrocknet.

0.1344 ¢& Subst. gab 0.0384 ¢ Pt
Pt
(C;H,N,O. HCl), PtCl, Ber. 28.55

Gef. 28.57

11.—Durch Silbernitrat und Baryt fillbare Base (Histidin). Das
Filtrat vom Silbernitrat-Niederschlag (1) wurde nun mit Ueberschnss von
Silber nitrat und conc. Bariumhydroxid Lésung versetzt, wobei ein dunkel-
brauner Niederschlag in reichlicher Menge entstand, der abgesaugt,
mehreremal mit Wasser gewaschen, in Wasser verteilt, und mit Schwefel-
wasserstoff zerlegt wurde. Das Filtrat vom Schwefelsilber wurde im
Vacuum eingedampft; als der Schwefelwasserstoff vollstiindig von der
Fliissigkeit ausgetrieben war, wurde diese mit Schwefelsiiure angesiiuert
und mit einer cone. Lisung von Phosphowolframsiiure gefillt. Der
Phosphowolframsiiure-Niederschlag wurde wieder in oben angegebener
Weise mit Baryt zerlegt und weiter verarbeitet. Aus der so gewonnenen
stark alkalischen Fliissigkeit, die freie Base enthielt, schieden sich nach
mehreren Tage im Exikator allmihlich die farblosen Krystalle aus, die
ungefiihr 15 g. betrugen. Diese Krystalle wurden in Wasser gelést, mit
Kohlensiiure gesiittigt und mit einer wiisserigen Quecksilberchlorid
lésung gefillt. Der weisse Niederschlag wurde mit Schwefelwasserstoft
zerlegt, vom Quecksilbersulfid abfiltriert und im Vacuum verdampft, wm

Schwefelwasserstoff auszutreiben, wieder mit Schwefelsiiure angesiuert

bo
sl

UEBER DIE EXTRAKTIV STOFFE IM FISCHFLEISCHE.

und nochmals mit Phosphowolframsiiure gefallt. Die nach Zerlegung des
Phosphowolframsiiure-Niederschlags in bekennter Weise erhaltene freie
Base war nunmehr fast rein und bestand aus farblosen diinnen Plittchen,
die von der Seite betraehtet als diinne Nadel erschienen. Diese Base
war in Acther und Alkohol schwer, in Wasser aber leicht léslich. Die
wiisserige Losung reagierte ziemlich stark alkalisch. Sie gab schéne rote
Firbung mit eimer alkalischen Lésung yon Diazobenzolsulfosiiure
(Pauly’sche Reaktion), gab auch Biuretreaktion beim Erwiirmen. Im
Kapillar rohr erhitzt zersetzte sie sich bei 237-242° (ancorr.)

Fiir die Analyse wurde das sorefilltig gereimgte Priiparat im

Vacuum bei 100° getrocknet.

0.1340 ¢ Subst. 0.0367 ¢ N
0.1360 ¢ ,, 0.2300 ¢ CO, 0.0783 ¢ H,O
C iH N

CAEENeOw Bers) 4645 5.81) 27.10
Gef. 46.12 6.40 27.38

Es wurde das me#hylester salzsaure Salz der Base nach der Vorschrift
yon E. Fischer und U. Suzuki! dargestellt. Es bestand aus farblosen
Prismen. Im Kapillarrohr rasch erhitzt, schmolz es bei 195-200

(uncorr.) Fiir die Analyse wurde es im Vacuum bei 100° getrocknet.

Subst. 0.0272 ¢ N
0.0423 ¢ Cl

N Cl

C,H,N,O, (CH,). 2HCl Ber. 17.36 29.34

Gef. 17.78 28.95

0.1530
0.1412

o
t=)
oe
>

Das Pikrat:—Gelbe Prismen, leicht léslich in warmem Wasser,
enthalt ein Molbekil Krytall wasser, das bei SQ° verloren geht. Das
wasser freie Salz schmuilzt erst tiber 200°C. Fiir die Analyse wurde es

im Vacuum bei 100° getrocknet.

1. E. Fischer und U. Suzuki: Berichte d. deutsch. chem. Gesellschaft. NNXNVIIE.

Band III. 4184.

28 U. SUZUKI UND K. YOSHIMURA:

0.3111 g Subst. gab 0.1879 g Pikrinsiure
0.1052 ¢ 0.0226 ¢ N
N __ Pikrinsiure
C,HoN.O, CebisN.O7- Ber. 21.87 59.37
1.47 60.40

Nach den oben angegebenen Daten kann man kanm zweifeln, dass
die Base Histidin war.

1Ii.—Das Filtrat vom Silbcrnitrat und Baryt-Niedersehlag (11).
(Carnosin).

Das Filtrat vom Silbernitrat und Baryt-Niederschlag (11)
wurde durch Salzsiiure von Silber und durch Schwefelsiure von Baryt
befreit, mit Schwefelsiiure angesiiuert und mit Phosphowolframsiure
gefaillt, Aus dem Phosphowolframsiiure-Niederschlag wurde in_ be-
kannter Weise cine stark alkalische Fliissigkeit, die freie Base enthielt,
erhalten, Nach dem diese Fliissigkeit im Vacuum exikator langsam
eingediinstet und mit absolutem Alkohol yersetzt wurde, schieden farblose
prismatische Kvrystalle allmihlich aus, die abgesaugt, im wenig heissem
Wasser gelést und durch Zusatz von absolutem Alkohol umkrystallisiert
wurden. Die Ausbeute betrug 3.6 2.

Diese Base war in absolutem Alkohol schwer, in Wasser aber leichter
léslich. Die wiisserige Lisung reagierte stark alkalisch. Jim Kapillar
rohr erhitzt, zersetzte sie sich bei 233—235° (uneorr.) Fiir die Analyse

wurde das gereinigte Priparat im Vacuum bei 100° getrocknet.

0.1173 g Subst. gab 0.0291 ¢ N
N
CoE eas Ber. 24.78
Gef. 24.82

Ks wurde das Platin chloriddoppelsalz der Base dargestellt, indem
die freie Base zuerst mit verdiinnter Salzsiiure neutralisiert und mit
einer wiisserigen Platin chloridlésung in kleinem Ueberschuss versetzt und
Jangsam verdampft wurde. Es schieden sich dabei wnregelmiissige

Krystalle aus, die im Kapillar rohr erhitzt bei 219—221° (ameorr.) sich

UEBER DIE EXTRAKTIV STOFFE IM VISCHFLEISCHE. 29

zersetzten. Fiir die Analyse wurde das Salz im Vacuum bei 100°

vetrocknet.
0.2604 g Subst 0.02087 ¢ N
0.2262 ¢_ ,, 6.0682 & Pt
N Pt
Chr NO.) 2HCl Piel. Ber. 8.81 30.63
Gef. 8.01 30.15

Die Analysen stimmen also mit der Formel C,H,,N,O. viz, dem
Carnosin aus dem Liebig’schen Fleischextrakt. Nach den kleimen
Unterschieden in der Léslichkeit wnd den anderen Eigenschaften zn
urteilen, handelt es sich wahrscheinlich um eine Isomerie desselben.

Darauf wollen wir spiiter noch zuriickkommen,

B. Das frische Bonito fleisch.

Das Histidin, das wir im getrockneten Bonito in reichlicher Menge
gefunden haben, ist ein Spaltungs produkt der Eiweiss kérper, besonders
des Haemoglobins und bisher niemals in freiem Zustande im tierischen
Gewebe konstatiert worden. Jim Pflanzenreich scheint es jedoch ziemlich
weit verbreitet zu sein, so hat E. Schulze! in etiolierten Keimlinger
yerschiedener Pfamzenarten das WVorkommen von [Hexon-basen, viz.
Arginin, Lysin und Histidin nachgewiesen. Nach neueren Untersu-
chungen von W. L. Gulewitsch? wird das im Liebig’schen Fleisch extrakt

gefundene Carnosin durch Hydrolyse in Histidin und Alanin gespalten.

C,H, ,N,0,+H,0=C,H,N,0, + C,H,NO,

Carnosin Histidin Alanin

So kénnte man natiivlich vermuten, dass das THistidin erst wiihrend
des Trocknens aus anderen Verbindungen durch enzymatische Spaltung
gebildet wird. Um diese Frage zu entscheiden haben wir frisches Bonito-
Aeisch untersucht und bestiitigen kénnen, dass das Histidin tatstichlich

1. E. Schulze :—Hoppe-Seylers’ Zeitsch. f. physiol. Chem, 77. 507.

2. W. 1. Gulewitsch :—Hoppe-Seylers’ Zeitsch. f. physiol. Chem. 50. 535.

30 Ut. SUZUKI UND K. YOSHIMURA:

in freiem Zustande im Fleisch-gewebe vorhanden und nicht als sekundires
Produkt zu betrachten ist. Zu diesem Zwecke wurden 5 ko frisches
Bonito fleisch fein zerhacket und wiederholt mit heissem Wasser extrahiert.
Die gesamten Ausziige wurden, wie oben erwihnt, mit Tannin und
Bleiessig gefallt; das Filtrat davon wurde nach dem Entfernen des
Bleies durch Schwefelsaiire, im Vacuum zu einem Syrup verdampft, mit
Alkohol versetzt und im Exikator stehen gelassen. Die zuerst ausgeschie-
denen krystalle bestanden hauptsiichlich aus kreatin. Die Ausbeute
desselben betrug etwa 5 g.

Das Rohprodukt wurde aus heissem Wasser umkrystallisiert und

im Vaennm bei 100° getrocknet und analysiert.

0.1696 g Subst. 0.0550 ¢ N
N
C,H,N,0; Ber. 52.06
Gef. 32.43

Das Pikrat des Kreatins bestand aus kleinen gelben Nadeln, die in
kaltem Wasser nicht leicht, in heissem Wasser und Alkohol aber leicht
lislich waren. Im Kapillar rohr erhitzt zersetzt es sich bei 212—213°
(uneorr ).

Die Mutter lauge von Kreatin wurde mit Wasser verdiiunt, Kohlen-
siure bis zur Siittigung eingeleitet und mit so viel wisseriger Quecksilber
chlorid lésung versetzt, bis diese keine Fillung mehr hervorrief. Der
dabei entstandene weisse Niederschlag wurde abgesaugt, mit Wasser
vewaschen, in Wasser suspendiert und mit Schwefelwasserstoff zerlegt.
Das Filtrat vom Quecksilbersulfid lieferte nach dem Verdampfen im
Vacuum 11.6 g. salzsaures Histidin (==8.4 g. Histidin). Zur Reinigung
wurde es aus wenig Wasser umkrystallisiert und im Vacuum bei 100°

getrocknet und analysiert.

0.2120 g Subst. 0.04562 g N

O0.2012:¢ «;; 0.0374 g Cl
N Cl

C,H,N,O,. HCl Ber. 21.93 18.54

Gef. 21.52 18.61

UEBER DIE EXTRAKTIV STOFFE IM FISCHFLEISCHE. 31

Es wurde ferner das Pikrat des Histidins dargestellt, indem man
die wisserige Lésung des salzsauren Salzes mit Natrium pikrt versetzte,
kurze Zeit erwirmte und erkalten less. Das Pikrat bestand ans feinen
gelben Prismen. Es enthalt ein Molekiil Krystall wasser, welches bei
80° verloren geht. Die feimen gelben Prismen verwandeln sich dabei zu

einer dvunkel braunen Krystalimasse.

C. Der in Wasser unloesliche Rueckstand von Bonito fleisch.

Der unldsliche Riickstand von Bonito fleisch wurde getrocknet und
fein zerrieben. 100 g. davon wurden mit 700 cc. 30% iger Schwefelsaiire
30 Stunden im Riickflusskiihler gekocht. Nach dem Erkalten wurde
dieser mit Wasser verdiinnt, vom unléslichen Rickstand abfiltriert und
folgende quantitative Analyse ausgefiihrt.

In 100 ‘Teilen Trockensubstanz.

In Schwefelsaure léslich 92.20

2 unloslich 7.80

Gesamt stickstoff 14.78
In Schwefelsiure léslicher Stickstoff 14.69

a unléslicher Stickstoff 0.09
Ammoniakstickstoff im Schwefelsauren Extrakt 1.02
Durch Phosphowolframsaure fallbarer Stickstoff 4.25
Darunter : Nucleinbasen stiekstoff Spur
Histidin stickstotf 0.71

Arginin stickstoff 2.39

Lysin stickstoff 1.15

Stickstoff in anderer Form 9,42

Durch Phosphowolframsaure falibarer Stickstoff als 100.

Nuclein basen stickstott Spur
Histidin stickstoff 16.67
Arginin stickstoff 56.18
Lysin stickstoff 27.15

Isolierung der Hexon basen

32 U. SUZUKI UND K. YOSHIMURA:

{.—Histidin.

Der Schwefelsiiure-Extrakt wurde mit fiinffacher Menge Wasser
verdiinnt und mit Phosphowolframsiiure gefillt. Aus dem phosphowol-
framsauren Niederschlag wurden die Basen in bekannter Weise frei
gemacht. Die freie Basen enthaltende, stark alkalische Fliissigkeit wurde
mit Kohlensiiure gesiittigt und mit eimer Quecksilberchloridlésung
gefillt. Der dabei entstandene weisse Niederschlag wurde in Wasser
suspendiert, durch Schwefelwasscrstoff zerlegt. Aus dem Filtrate von
Quecksilbersulfid schieden sich nach dem Verdampfen farblose,
prismatische Krystalle aus. Die Ausbeute derselben betrug ungefihr
1g. Fiir die Analyse war das Salz cinmal umgelést, und im Exikator

iiber Schwefelsiiure getrocknet.

0.1242 g Subst. 0.02475 g N
0.1428 ¢ _,, 0.0235 g Cl
; N Cl
C,H,N,O,HCI.+H,O Ber. 19.93 16.94
Gef. 19.93 16.46
Das methylestersalzsanre Salz:—Farblose Prismen. Im Kapillar

rohr erhitzt schmilzt es bei 197—200° (uncorr) unter lebhaftem

Schiumen. Fiir die Analyse wurde es im Vacuum bei 100° getrocknet.

0.1213 g Subst. 0.0215 ¢ N
0.1562 ¢_s,, 0.0458 ¢ Cl
N Cl

C,H,N,O,. (CH,)2HCI Ber. 17.36 29.34
Gef. 17.74 29.29
IL.—Arginin.

Das Filtrat vom Quecksilberchlord-Niederschlag wurde durch
Schwefelwasserstoff yom Quecksilber befreit, im Vacuum verdampft, und
mit Silbernitrat versetzt, um die Salzsiiuwre zu entfernen. Zum Filtrat
von Chlorsilber wurde Silbernitrat und Baryt im Ueberschuss zugegeben,
wobei cin brauner Niedersehlag von Arginin silber in reichlicher Menge

entstand. Nach dem Zerlegen des Arginin silbers durch Selwefelwasser-

UEBER DIE EXTRAKTIV STOFFE IM FISCHILEISCHE. DO

stoff wurde das stark alkalische Filtrat im Vacuum eingedampft und
gleich zum Pikrat verwandelt. Die Ausbevte an Pikrat betrug ungefiihr
QD f£.

Das Pikrt bestand aus gelben Nadeln oder Prismen. Der Sechmelz-
punkt desselben war 207—210° (uncorr).

Fiir die Analyse war es im Vaenum bei 100° getrocknet.

0.2121 ¢ Subst. 0.05116 g N
N
Ca NgOs. C,H IN.O- Ber. 24.32
Gef. 24.12
III. Lysin.

Das Filtrat vom Argininsilber-Niederschlag wurde durch Salzsiinre
vom Silber und durch Schwefelsiiure vom Baryt befreit, mit Schwe-
felsiure angesiiuert und mit Phosphowolframsiure gefallt. Aus dem
phosphowolframsauren Niederschlag wurde 2.5 g. Lysinchlorid gewonnen.
Aus dem Chlorid wurde das methylestersalzsaure Salz dargestellt. Es
waren farblose Prismen mit dem Schmelzpunkt 20S—210° (uncorr).

Fiir die Analyse war es ii Vaeunm bei 100° getrocknet.

0.2050 & Subst. 0.02400 « N
0.2860 ¢ _,, 0.0882 ¢ Cl
N Cl

C,H, ,.N.O.2HC1 Ber. 12.02 30.47

Gef. mf 30.82

Zusammenfassung der Resultate

Ans 1 Kilo getrocknetem Bonito wurden isoliert :

Xanthin weing
Hy poxanthin 0.74
Kreatin Weing
Histidin 15.00
Carnosin 3.60

Aus 5 Kilo frischem DBonitofleisch wurder isoliert:
Histidin 8.4 &

KXreatin 5.0 ¢

34 U. SUZUKI UND K. YOSHIMURA:

Ans den Siiure spaltungs produkten von 100 g. in Wasser unlislichen

Riickstand des Bonito fleisches wurden isoliert:

Histidin 0.74 &
Arginin 10.8 ¢
Lysin 2.0 ¢

I LACHS (Onxchorynchus keta).

Das frische Lachsfleisch wurde von Haut und Knochen befreit und
fein zerhackt. 2900 . des so zubereiteten Fleisches wurden mit warmem
Wasser (40-50°) eine Stunde digeriert und stark abgepresst. Der
Riickstand wurde nochmals in derselben Weise behandelt. Diese Opera-
tion wurde dreimal wiederholt. Die vereinigten Ausziige wurden mit
Bleiessig lésung im kleinem Ueberscliuss versetzt. Das Filtrat wurde
znerst durch Schwefelsiiure von Blei befreit und mit Schwefelsiure
augesiiuert bis die Fliissigkeit ungefiihr 2¢ derselhen enthielt und so viel
Tannin lisung zugegeben, bis diese keinen Niederschlag mehr hervorrief.
Nach 24 Stunden wurde es klar abfiltriert und mit eimer cone. Lisung
von Phosphowolframsiure gefillt. Der dabei entstandene weisse flockige
Niederschlag wurde abgesaugt und mit 5¢ Schwefelsiiure gewaschen.,

A. Der Phosphowolframsiure-Niederschlag.

Der Phosphowolframsiiure-Niederschlag wurde in gewodhnlicher
Weise mit Rarvt zerlegt. Die alkalische Fliissigkeit, die Iveie Basen
enthielt, wurde im Vacuum stark eingeenet und stehen gelassen. Nach
einiger Zeit schieden sich farblose gliinzende Krystalle aus, was durch
Zusatz von Alkohol noch beschleunigt wurde. Die Ausbeute betrug 2.2 g.

Das Rohprodukt wurde aus heissem Wasser umkrystallisiert, im

Vacnum bei 100° getrocknet und analysiert.

0.1487 @ Subst. 0.2002 g CO, 0.0984 ¢ H,O
0,1482 ¢ i, 41.°° N (18° 764m)
C H N
Kreatin C,H,N,O,. Ber. 36.64 6.87 32.06
Gef. 26,67 7.42 32,15

UEBER DIE EXTRAKTIV STORFE IM FISCIEYLEISCIIE. 3)

Die Analyse stimmt also mit dem Avreatin. Aus leissem Wasser
umkrystallisiert scheidet sich das Kreatin als grosse, durehsichtige,
eliinzende Prismen oder Stibchen aus. Bei 100° getrocknet verliert es
Kvystall wasser und verwandelt sich dabei in cin weisses Pulver. Es
bildet kein Doppelsalz mit Zink chlord, gibt keine Farbune weder mit
wiasseriger Pikrinsiiure und Alkali noch mit Nitroprussidnatrium und
Alkali. Die wisserige Loésung des Kveatins reagiert neutral. Tin
Kapillarrohy erhitzt fiingt es von 240° an braun zu werden und zersetzt
sich bei héherer Temperatux.

Aus ireatin wurde leicht Kreatinin dargestellf, indem 1 @. Kreatin
in 20 ee. 10% iger Schwefelséure gelost und cine Stunde auf dem Sandbade
erhitzt wurde. Nach den Erkalten wurde die Fliissigkeit durch Baryt
yon Schwefelsiiure berreit wid stark ecingeengt, wobei dic farblosen
Krystalle sich ausschieden, die alle Eigenschaften des Kreatinins hatten.
Sie gaben das charakteristische Doppelsalz mit Zinkchlorid, auch rote
Farbong mit wisseriger Pikrinsitvre nad Alkali, eder rubimwie Firbung
durch Nitroprussid natrivm und Alkali.

Die mutterlauge von Kveatin wurde mit Wasser verdiinnt und
mit einer wiisserigen Quecksilberchloridlésung versetzt. Der weiss¢
flockige Niederschlag wurde durch Schwefelwasserstoff zerlegt,
vom Schwetelquecksilber abfillriert, im Vacuum stark cingeengt
und stehen gelassen. Nach einigen Tagen schieden sich farblose
Prismen von salzsaurem Hypoxanthin aus, die etwa 1 g. betrugen. Aus
heissem Wasser umkrystallisiert verliert es Salzsiiure und scheidet sich
das Ilypoxanthin als mikroskopisch kleine, weisse MKrystalle aus, die
abgesaugt und mit Wasser gewaschen wurden. Fiir die Analyse wurde

es bei 100° getrocknet.

0.1187 ¢ Subst. 0.1918 ¢ CO, 0.0578 g H,O
0.1015 gy, BLOOMED IST (2 ” Nataay ee)
C i N
C,H,N,0. Ber. 44.12 2.94 11.18

Gef, 44,07 3.04 AL.26

356 U. SUZUKI UND K. YOSHIMURA:

Die freie Base ]ést sich ziemlich schwer in kalten Wasser auf; in
Alkohol und Aether ist sie fast unlislich. Die wiisserige Lisung reagiert
schwach alkalisch. Der Schmelzpunkt liegt tiber 300°.

Das Hypoxanthin pikrat bestand aus gelben Tafeln, schmolz bei iiber
250°.

In der Mutter Jauge von salzsaurem Hypoxanthin war noch eine
bedeutende Menge unbekannter Basen vorhanden. Leider war es tus
nicht gelungen diese zur Krystallisation zu bringen. Wir haben nur das
Vorhandeusein von Histidin durch Pauly’sche Reaction walrscheinlich
zemacht.

Das Filtrat von Quecksilberchlorid-Niederschlag wurde dureh
Schwefelwasserstof! von Quecksilber befreit und im Vacuum eingedampft.
Nachdem der Schwetelwasserstoff ausgetrieben war, wurde die Salzsiiure
durch Silbernitrat entfernt; das Filtrat von Chlorsilber wurde nun mit
Ueberschuss yon Silbernitrat und Baryt versetzt, der dabei entstandene
braune Niederschlag wurde mit Schwefelwasserstoff zerlegt. Aus der
so erhaltenen alkalischen Fliissigkeit haben wir nichts isolieren kénnen.

Das Filtrat von Silbernitrat und Baryt-Niederschlag wurde durch
Salzsiiure von Silber und durch Schwefelsiiure yon Baryt befreit, mit
Schwefelsiiure angesiiuert und mit Phosphowoframsiure gefillt. Aus
dem phosphowolframsauren Niederschlag wurde die Base in bekannter
Weise frei gemacht. Die stark alkalische Fliissigkeit wurde im Vacuum
cingedampft. Nachdem das Wasser vollstiindig ausgetrieben war
begannen sich farblose Prismen auszuscheiden und nach einiger Zeit
verwandelte sich die ganze Masse in einen Krystallbrei. Sie wurde mit
absolutem Alkohol verrieben, abgesaugt und mit Alkohoi und Aether
vewaschen; die Ausbeute betrug 1.6 g. Zwecks Reinigung wurde die
nochmals in wenig Wasser geléste Masse klar abfiltriert und bei gelinder
Wirme langsam verdampft. Die ausgeschiedenen Krystalle wurden
abgesaugt, mit Alkohol und Aether gewaschen und fiir die Analyse im

Vacuum bei 100° getrockmet.

CEBER DIE EXLTRAKTIV STOFVE IM PISCHFLEISCHE. BT
0.1482 ¢ Subst. 0.2561 * CO, 0.0986 ¢ H,O
ONOI5i rs ,, 20.2¢¢ N (107 75am)

C iI N
Cb NGO. Ber. Mist 6.20 2h Tat
CG HwNsOs+ 200; Ber. 47.0 6.00 2363
Get. ATS 1:29 23.7

Die Analyse stimmt am besten nut der Formel Cyll,,N,O0.414C0.
dh. Carmosin tiberein; die freie Base scheimt Nohlensiiure absorbiert zu:
haben. Wir haben auch eiuen Grund anzunehmen, dass die treie Base
aus der Luft albnihlich Kollensiiure absorbiert, denn wir konuten aus
dem analysierten Praparat durch unmittelbares Erwarmen mit Pikrin-
siure kein Pikrat bekommen. Wenn man aber die Base in
Wasser l6st, mit Salzsdure schwach snsauert und kurze Zeit erwirmt,
wn die Kohlensiiure auszutreiben, dann mit ciner entsprechenden Menge
Natronlauge nentralissiert und mit Pikrinsiiure versetzt, so scheidet sich
Gas Pikrat sofort als schéne Prismen aus. Die Kohlensiiure diirfte mit
der Base ziemlich fest gebunden sein, so dass die Pikrinsiure nicht im
Stande ist, sie zu verdringen. Das freie Carnosin besteht aus langen,
farblosen, gliinzenden Prismen, leicht léslch in kaltem Wasser, schwer
aber in Alkohol. Es hat eimen siissen Geschmack, die wisserige Lisung
reagiert ziemlich stark alkalisch. In Wapillar rohr erhitzt schmulzt e:
bei 230-232° (unecorr.) unter Schiiumen.

Carnosin gibt eine weisse Fallung mit Quecksilberchlorid; diese
yerschwindet aber wenn dier detztere Reagens nicht im Ueberschuss
vorhanden ist. Ts gibt durch Kupfersulfat und Natronlange eine
schwach blau violette Fairbung, hingegen keine Farbung durch Pikrin
siure und Alkali. Pauly’sche Reaktion ist auch negativ. Durch
Ueberschnss von Silbernitrat und Baryt wird es teilweise gefallt.

Das Platinchlovid doppelsalz des Carnosins:—Das Salz wurde
dargestellt, indem die freie Base mit verdiimnter Salziiure neutralisiert, wil
kleinem Uebersehuss von Platinchlorid lésung versetzt und Jangsam

verdampft wurde. Nach einiger Zeit schieden sich die schouen Prismen

38 U. SUZUKI UND K. YOSHIMURA:

aus, was durch Zusatz yon Alkohol beschleunigt wurde. Man krystallisiert
das Salz cimmal aus Wasser um, und wisecht es mit Alkohol und Aether:

Fiir die Analyse war es im Vacuum bei 100° getrocknet.

0.0752 g Subst. Rls’ ON: (EGS) Fok)
Oia Se, 0.0584 ¢ Pt
; N Pr
GU aa ele, ELC ly: Ber. 8.81 30.63
Get. 5.81 BODS

Das Platin doppelsalz bestand aus schOnen Prisinen, Tim WKapillar
rohr erhitzt fingt es bei 200° braun zu werden an wud bei 208° (aecorr.)
zersetzt es sich unter Schiimnen.

Carnosin witral:—Zur Varstellung des Nitrats wurde das freie
Carnosin mit Salpetersiiure neutralisiert und vorsichtig eingediinstet. Es
scheidet sich als schéne farblose, meistens stern formig verwachsene
Prismen aus, die in Wasser leicht, in absolutem Alkohol schwer Jéslich
sind; die wiisserige Ldsung reagiert schwach sauer. Jin Kapillarrohr rasch
erhitzt zersetzt es sich bei 211°C. (unecorr.) unter Schiumen.

B. Das Filtrat von Phosphowolframsiure-Niederschlag (A).

Das Filtrat vom Phosphowolframsiure-Niederschlag A. wurde durch
Baryt yon Schwefelsiure und Phosphowolframsiiure befreit und der
Uberschuss von Baryt durch Sehwefelsiiure genau entfernt und im
Vacuum bis zum syrup verdampft. Es schieden sich dabei farblose
eliinzende Krystalle in reichlicher Menge aus, die abgesaugt, mit wenig
Wasser, Alkohol und Aether gewascher wurden, Die Ausbeute betrug 7 g.

Diese Krystalle bestanden fast ausschliesslich aus Arealin, Dureh
cinmaliges Umlésen war es schon vollstinding rein. Fiir die Analyse wurde

es im Vacuum bei 109° getrocknet.

0.122 ¢ Subst. 33.9c N (22° 763")
N
C,H,N,O, Ber. 32.06

Gel. d1.61

co
les)

UEBER DIE EXTRAKTIV STOFFE IM FISCHPLEISCHE,

Die aus Wasser ansgeschiedenen Krystalle sind farblose, gliinzende
Prismen, ziemlich seliver léslich in Wasser und in Alkohol und Aether
fast unloéslich; sie haben kein Drehungsvermégen, Die wiisserige
Lisung reagiert fast ucutral. Mit yerdiinmter Schwefelsiiure erhitzat
wurde sie leicht in Kreatinin iiberfiihrt, was durch Zinkehlorid doppel
salz, Pikrinsiinre und Alkali und Natriumnitroprussid und Alkali
bestiitigt wurde.

Die Mutter lange von Kreatin wurde weiter yerdampft, von aus-
geschiedenen anorganischen Salzen getrennt, mit absolutem Alkohol
versetzt und wieder eingedampft. Der wasserfreie Riickstand wurde
jetzt mit absolutem Alkohol iibergossen und trockenes Salzsiiure gas bis
zur Siittigung eingeleitet, sodann wieder eingedampfi. Diese Operation
wurde mochmals wiederholt. Der nach dem Verdampfen des Alkohols
zuriickgeblicbene dunkel braune Syrup wurde nach der E. Fischer’schen
Ester methode nach Monaminosiuren untersucht. Es wurde in der Weise
ungefiihr 0.3 @. Alanin isoliert, welches sich aus der wisserigen Lésung
als farblose Prismen ausschied, die den cigentiimlichen siissen Geschmack
besassen. In Kapillar rohr erhitzt schmolz es bei 270° (uncorr.) unter
Zersetzune. Es bildete auch schénes Kupfersalz, das in Wasser leicht
léslich war.

Nach Leuecin und Glyeoeoll wurde vergehens gesucht.

Zusammenfassung der Resultate,

Aus 1 kilo frischem Lachs fleiseh wurden isoliert:

KXreatin 3.2 ¢
KXreatinin =
Histidin Vorhanden
Hypoxanthin 0.28
Carnosin 0.55
Alanin 0.10

Il, MAGURO (7Ziyanus thunnis).

Das frische Maeuro fleisch hatte foleende Zusammensetzune.

40 U. SUZUKI UND K. YOSHIMURA:

Wasser 70.68 %

Trocken substanz 29.32 %

In 100 Teilen frischem Fleisch. In 100 Teilen Trockensubstanz

Gesamt_ stickstoff 4.36 14.86

Gesamt-Extraktivstoffe 10.85 37.00

Stickstoff im Extraktivstoffe 1593 6.58

Darunter. Eiweiss stickstoff 0.84 2.86
Durch Phosphowolframsacure

faellbarer Stickstoff 0.38 1.28

Stickstoff in anderer Form 0.71 2.44

2 Kilo frisches Maguro fleisch, welches vorher von Haut und Knochen
befreit und fein zerhackt war, wurde dreimal mit warmem Wasser (40°)
extrahiert. Die wiisserigen Ausziige wurden mit Tannin und Bleiessig
hehandelt; das durch Schwefelsiinre von tiberschussigem Blei befreite
Filtrat wurde mit Schwefeisiinre angesiiuert und mit Phosphowolfram-
siiure gefallt.

A. Der Phosphowolframsiure-Niederschlag.

Die aus dem phosphowolframsauren Niederschlag in bekannter Weise
dargestellte, stark alkalische Fliissigkeit, welche freie Basen enthielt,
wurde im Vacuum stark eingeengt. Es schieden sich dabei farblose
eliinzende Krystalle aus, die ungefiihr 2.5 @. betrugen. Aus der Mutter
lange wurde durch Zusatz yon .\lkohol noch 2.5 g. derselben Krystalle
erhalten.

Die auf beide Weisen erhaltenen Krystalle warden aus wenig Wasser

wnkrystallisiert, bei 100° getrocknet und analysiert.

0.1547 ¢& Subst. 0.2607 CO, 0.0952 ¢g H,O
0.1498 g__,, 35.1¢° N (21° 765")
C H N
CzH,N,0, Ber. 46.45 5.80 27.09
Gef. 45.95 6.84 26.86

Die Analyse stimmt also mit der Formel CyH,N,0., dem JZistidin,

iiberein, Aus heissem Wasser umeelist scheidet sich die Base als diinne

UEBER DIE EXTRAKTIV STOFFE IM FISCHFLEISCHE. 4}

perlmutter-glinzende Plittchen aus, welche mit den Leucin krystallen
grosse Aehnlichkeit haben. Sie hat fast keinen Geschmack; die wisserige
Lésung reagiert schwach alkalisch, sie ist in Wasser leicht, in Alkohol
schwer und in Aether nicht léslich. “Sie gibt schine rote Farbung mit
alkalischer Diazobenzol sulfosiiure Lésung und bildet kein Doppelsalz mit
Zinkehlorid, mit dem Millowschen Reagens gibt sie eime starke weisse
Fillung, beim Erwarmen wird sie aber nicht rot. Durch Quecksilber-
chlorid wird sie als weisser flockiger Niederschlag gefillt.

Die Base gibt auch Biuret reaktion beim Erwiirmen, was auch fiir
Histidin charakteristisch ist. Sie hat keinen Schmelzpunkt, im Kapillar-
rohr erhitzt wird sie bei 240° braun, bei 250° diinkler und zersetzt sich
bei héherer Temperatur.

Es wurde das methylestersalzsaure Salz dargestellt. Das aus methyl
alkoholischer Loésung durch Zusatz von Aethylalkohol und Aether

ausgeschiedene Salz wurde im Vacuum bei 100° getrocknet und analvsiert.

0.1253 g Subst. 19.9°° N (20° 757m)
0.1050¢ , 0,03089 g¢ Cl
N cl
C,H,,N,0,. 2HCl Ber. 17.36 29.30
Gef. 17.99 29.41

Im kapillar rohr rasch er hitzt zersetzt sich das Salz bei 197°
(uncorr.) unter lebhaftem Schiiumen.
Histidindichlorid.—Das Salz wurde in gewohnlicher Weise dargestellt

und analysiert.

0.1262 & Subst. 19.9°¢ N (20° 762™™)
0.1595 ¢ ,, 0.04945 ¢ Cl
N Cl
C,H,N,0,. 2HCl Ber. 18.42 31.11
Gef. 1>.09 31.00

Histidin dichlorid scheidet sich aus methylalkoholischer Lésyne
durch Zusatz von Aethyl alkohol und Aether als farblose Prismen aus.
999_9945

Im Kapillarrohr erhitzt, sintert es bei 232° und zersetzt sich bei 233-23

unter Schiumen.

42 ; U. SUZUKI UND K. YOSHIMURA:

Wir haben ferner bei unserem Histidin priiparate das optische
Verhalten untersucht und dasselbe mit dem aus Eiweisskérper dureh
Hydrolyse erhaltenen Praparate identisch gefunden. viz.

0.324 g freies Histidin in 19.034 g Wasser gelést, das ein

Sp. Gewicht 1.006 hatte, drehte bei 20™ Rohr das Natrium _ licht
1.29° nach links
Mithin — [a] = —38.°3

A. Kossel hat fiir sein Histidin priparat, das aus den Spaltungs
produkten des Protamins dargestellt wurde, —39.7° angegeben. Der
kleine Unterschied ist Beobachtungsfehlern zuzuschreiben.

In salzsanrer Lisung wird die Drehung verindert, wie Kossel auch

angegeben hat.

0.2788 g Subst in 21.7152 g 109% iger Salzsiiure gelést, vom Sp.
Gw. 1.041 drehte in 20™ Rohr das Natruim licht 0.46° nach rechts
Mithin [a], = + 17.94

Die Matter lange von Tstidin e1tthielt noch eine bedeutende Menge
Histidin, das von der Mutter lauge schwer zu trennen war; zu diesem
Zwecke wurde sie in 500 cc. Wasser gelést und mit einer cone, wiisserigen
Lésung von Quecksilberchlorid versetzt. Der weisse flockige Niederschlag
wurde abgesaugt, mit wenig Wasser gewaschen, in Wasser verteilt und
durch Schwefelwasserstoff zerlegt. Das Filtrat von Schwefel quecksilber
wurde im Vacuum eingedampft und in bekannter Weise das methyl ester
salzsaure Salz des Histidins dargestellt. Die Ausbeute betrug ungefihr
6.8 g. (entsprechend 4.4 g. freies Histidin).

Aus 2 kilo frischem Fleisch haben wir somit im ganzen 9.4 g. freies
Histidin isoliert.

Aus der Analyse und anderen Beobachtungen wurde das Salz mit
dem vorher von Donito gewonnenen Priiparat vollstiindig identisch
gefunden.

Das Filtrat vom Quecksilberchlorid-Niederschlag wurde dureh
Schwefel wasserstoff vom Quecksilber befreit und nach dem Entfernen

der Salzsiiure durch Silbernitrat wurde es mit Ueberschuss von Silbernitrat

UEBER DIE EXTRAKTIV STOFFE IM f£ISCHFLEISCHE. 43

und Baryt versetzt. Aus dem dabei entstandenen braunen Niederschlag
konnte man jedoch keine Base in geniigender Menge isolieren.

Das Filtrat von Silber nitrat und Baryt-Niederschlag wurde nach
dem Entfernen des Silbers durch Salzsiiure und des Baryts durch
Schwefelsiiure, mit Schwefelsiiure angesiiuert und mit Phosphowolfram-
siure gefillt. Die aus dem phosphowolframsauren Niederschlag in
bekannter Weise dargestellte alkalische Fliissigkeit wurde gleich mit
Pikrinsiiure versetzt. Das in dieser Weise dargestellte Pikrat betrug
ungefihr 8 ge.

Aus wenig heissem Wasser umeelést scheidet sich das Pikrat als
Ageregat von mikroskopisch feinen citronen gelben Nadeln aus. Im
Kapillar rohy erhitzt wird es von 200° an allmihlich brarm und zersetzt
sich bei 216° (imeorr. )

Fiir die Analvse wurde es iin Vacuum bei 100° getrocknet.

0.1418 ¢ Subst. 0.2047 » CO, 0.0605 ¢ H,O
OWA gi. 5, 2G OSE wl SO 767™™)
0.4836 ¢,, 0.2454 ¢ Pikrinsiiure

C 18f N Pikrinsiiure
C,H,,N,0,. CsH,N,O, Ber. 39.56 3.74 2154 40.8:
Gef. 39.88 4.47 21.29 50.74

Aus dem Pikrate wurde das Nitrat der Base dargestellt; zu diesem
Zweeke wurden 2 g. Pikrat in wenig heissem Wasser gelést, mit Ueberschuss
yon Salzsiiure versetzt, von der dabei ausgeschiedenen Pikrinsiiure
abfiltriert, und wiederholt mit Aether geschiittelt, um die darin vor-
handene Pikrinsiiure vollstindig zu entfernen und dann mit Phosphowol-
framsiiure gefillt. Der Phosphowolframsiiure-Niederschlag wurde in
bekannter Weise durch Baryt zerlegt. Die alkalische Fliissigkeit wurde
mit Salpetersiiure neutralisiest und bis zum Syrup eingeengt. Die ganze
Masse yverwandelte sich bald in farblose Prismen, die sich meistens
sternfémig zusammengruppierten. Diese wurden mit wenig absolutem
‘Alkohol verrieben, abgesaugt und nochmals aus wenig Wasser umgelést.

Fiir die Analyse wurden sie im Vacuum bei 100° getrocknet.

44 U. SUZUKI UND K. YOSHIMURA:

0.116 g¢ Subst. 23.6¢ N (110 764™")

0.3845 ¢ ,, 0.492 g¢ Nitron nitrat (nach Nitron methode)
N HNO,
OSE. gN OL ENG, Ber. 24,22 21.80
Gef. 24.40 21.50

Das Nitrat bestand aus farblosen Prismen; es ist in Wasser sehr
leicht, in Alkohol schwer und in Aether unléslich. Im Kapillar rohr
erhitzt schmilzt es bei 211° (uncorr.) zu einem Oel und zersetzt sich
gleich darautf.

Aus dem Pikrate wurde die freie Base dargestellt. Sie
bestand aus farblosen Prismen, reagierte zieml stark alkalisch und
zersetzte sich bei 230° (ameorr.) unter Schiumen.

Alle diese Eigenschaften stimmen mit dem Carnosin aus Lachs
iiberein.

Wir haben auch das optische Verhalten des Nitrates untersucht:

0.4881 g Carnosin nitrat in 15.014 g Wasser gelést, bei eniem
Spezifischen Gewicht von 1.008, drehte im 20° Rohr Das Natrium-
licht 0.74° nach rechts

Mithin§ a” = +11.°66

Vergleicht man nun diese Zahl mit jener des Carnosin nitrates aus
Liehig’schem Fleischextrakt a}, =+ 22.°3 so findet man, dass sie
beinahe die Hiilfte der letzteren ist. Der Schmelzpunkt der freien Base
liegt auch einige grade niedriger. Wahrscheinlich handelt es sich um
eine Isomerie, was spiiter noch eingehender wutersucht werden soll.

B. Das Filtrat von phosphowolframsaurem Niederschlag.

Aus dem Filtrate. yon phosphowolframsaurem Niederschlag wurde
6 g. reines Avreatin isoliert und in der Mutterlange von Kreatin das
Vorhandensein kleiner Mengen Monaminosiuren mittelst Ester methode
nachgewiesen; zi Analyse geniigte die Menge jedoch nicht.

Zusammenfassung der Resultate.

Aus 2 kilo frischem Fleisch wurderr isoliert.

UEBER DIE EXTRAKTIV STOFFE IM FISCHILEISCHE. 45

KXreatin 6.0
Kreatinin —
Histidin 94
Hypoxanthin =
Carnosin 40
Alanin Vorhanden

IV. HUMMER (Jse-Vebc: Panutlirus. sp.)
A. Das Hummer fleisch,

33 Sticke frische Hummem (3710 2.) wurden yon den Schalen
abgelést und das daraus gesamimelte Fieisch, das frisch gewogen 1712 g.
betrug, wurde fein zerhackt, mit warmem Wasser (60—70°) dreimal
extrahiert. Mit dem Extrakte wurde zuerst eine quantitative Analyse

anseefiilirt.

In 100 Teilen frischem Fleisch

In Wasser léslicher Stickstoff 2.78

Darunter: Kiweiss Stickstoff 0.71
Durch Phosphowolframsiiure fillbarer

Stickstoff 23,

Stickstoff in anderer Form 0.84

In Wasser loéslicher Stickstoff als 100

Eiweiss stickstoff 25.5

Durch Phosphowolfamsiiure fiillbarer
Stickstoff 44.5

Stickstoff in anderer Form 30.2

Der Rest des Extraktes wurde mit Essigsiiure angesiinert und mit
Tannin eefillt; der dabei entstandene flockige Niederschlag wurde
abgesaugt und das Filtrat davon wurde mit Bleiessig versetzt. Das
Filtrat vom Blei-Niederschlae wurde nach dem Entfernen des Bleies durch
Schwefelsiiure, mit so viel Schwefelsiimre angesiiuert, bis die Flissigkeit

dae ~ of * « . 7 a nee
ungefihr 5% derselben enthielt und mit Phosphowolframsiiure gefallt.

46 U. SUZUKI UND K. YOSHIMURA:

Nach 24 Stunden wurde der Phosphowolframsiure-Niederschlag abgesaugt
und mit 5% Schwelfelsiiure sorgfiltig gewaschen.

a). Der Phosphowolframsiure-N iederschlaq.

Der Phosphowolfrmsiure-Niederschlag wurde in bekannter Weise
durch Baryt zerlegt und der Ueberschuss von Baryt durch Schwefelsiure
entfernt; die so erhaltene alkalische Fliissigkeit wurde im Vacuum stark
eingeengt und in zwei Portionen geteilt.

1. Die eine Portion wurde gleich mit krystallinischer Pikrinsiiure
versetzt, kurze Zeit erwiimt und erkalten gelassen. Aus dieser Fliissigkeit
schieden sich eine reichliche Menge Pikrate aus, die der Lislichkeit nach
fraktioniert gesammelt wurden.

Die erste Fraktion war das Arginin pikrat; es bestand aus feinen,
citronen gelben langen Nadeln, die 6.8 g. betrugen. Nach einmaligem
Umlésen aus heissem Wasser war es vollstiindig rein. Fiir die Analyse

wurde es im Vacuum bei 100° getrocknet.

0.1519 g Subst. 0.2020 g CO, 0.0625 ¢ H,O
0146" 92 © 5 BOee Sie (ee 750.5™™ )
OA aes 0.2310 g Pikrinsiiure

Cc H N Pikrinsiiure
C3H,,N,0,.C,H;N,O, Ber. 35.73 4.22 24.32 56.82
Gef. 36.27 4.56 24.47 56.07

Die Analyse stimmt also mit dem Arginin pikrat iiberein,

Das Pikrat enthilt zwei Molekitle Krvstallwasser, das bei 80° verloren
geht; die feinen citronengelben Nadeln nehmen dabei eine orange rote
Firbung an. Im Kapillar rohr erhitzt zersetzt sich das wasserfreie Pikrat
bei 206° (uneorr.) unter Sehiiumen.

Krystallwasser bestimmnng.

1.) 0.380 ¢ Subst (vorher im Exiecator iiber Schwefelsiiure getro-
cknet) verlor im Vacuum bei 100° getrocknet 0.0312 g Wasser
2) 0.4452 & Subst. ce 1 5
- ‘5 ay 3 » OOBB2 eo)

UEBER DIE EXTRAKTIV STOFEFE IM FISCUPLEISCHE. 47

H,O

CaN, O,. CHEN. O-- 2H © Ber. 8.20

Gef. 1)) 8:19

2) -8.13
Um das Pikrat in salzsawren Methylester des Arginins zu verwandelu
wurde 3 g. Pikrat in wenig heissem Wasser geldst, 20 ce. verdiinnter
Salzsiiure (1:3) zugegeben, von der ausgeschicdenen Pikrinsiiure
getrennt und wiederholt mit Aether geschiittelt, bis die Fliissigkeit ganz
farblos wurde. Die wisserige Losung wurde dann im Vacuum verdampft
und in gewohnlicher Weise durch Methyl alkohol und Salzsiiure in das
methylester salzsaure Salz verwandelt. Zur Reinigung wurde das Salz
in wenig heissem Methyl alkohol gelést, mit Tierkohle entfiirbt und durch
Zusatz von Aethyl alkohol und Aether ausgeschieden. Fiir die Analyse

wurde es im Vacuum bei 80° getrocknet.

0.157 g Subst. 0.0424 g AgCl
(OLAS UE fem e622 Ni (129 756™™)
N Cl
C,H,,N,0,2HCl Ber. 21.47 27.15
Gef. 21.63 27.02

Das salzsaure Salz bestand aus farblosen Prismen, die sich meistens
sternférmig aneinander gruppierten, Es l6st sich in Wasser und Methyl
alkohol leicht, in Aethylalkohol etwas schwerer; in Acther, Petrolether
und Chloroform ist es fast unléslch. Im Kapillarrohr rasch erhitzt
zersetzt es sich bei 183° (uncorr.) unter lebhaftem Schiiumen,

Das optische Verhalten des isoherten Arginins soll spiter untersucht
werden.

Die zweite Fraktion war das Gemisch yon Avginin und Lysin pikrat.
Die Ausbeute derselben betrug 11.7 g.

Aus der dritten Fraktion wurde 1.5 g. Lysin pikrat in reinem
Zustande gewonnen. Ks bestand aus hellgelben langen Prismen, en-
thielt kein Krystall wasser. In Wasser und Alkohol war es ziemlich leicht,
in Aether aber schwer léslich. Im Kapillarrohr erhitzt wurde es bei 230°

braun und zersetzte sich bei héherer Temperatur.

48 U. SUZUKI UND K. YOSHIMURA:

Fiir die Analyse war es im Vaeuum bei 100° getrocknet.

0.1698 g Subst. 0.2392 ¢ CO, 0.0695 g H,O
0.1256 gs, 19:9e2N (16.59 * fares)
0.5078 g a, 0.3098 g Pikrinsiiure

C H N Pikrinsiure
C,H,,N,0,- C,H,N,O, Ber. 38.40 4.63 18:67 61,07
Gef. 38.88 455 18.44 61.01
Aus dem Pikrate wurde das methyl ester salzsaure Salz in bekannter
Weise! dargestellt. Es war farblose Prismen mit dem Schmelzpunkt

216°—218°. Fiir die Analyse war es im Vacuum bei 100° getrocknet.

0.1495 g Subst. 15.0% N (120 769mm)
0.1680 g_ 0.05025 g Cl
N cl
C,H, .N,0,2HCI Ber. 12.07 30.47
Gef. 12.05 29.91

2, Die zweite Portion wurde mit Kohlensiiure gesittigt und mit
einer wiisserigen Queckilberchlorid lésung versetzt. Aus dem Queck-
silberchlorid-Niederschlog wurde 2 g. Argininmethylester salzsauresalz
isoliert.

Das Vorhandensein von /Histidin wurde durch Diazobenzol sulfosiiure
nachgewiesen; gelang es uns jedoch nicht, diese Base in reinem Zustande
zu isolieven.

Das Filtrat vom Quecksilberchlorid Niederschlag wurde nach dem
Entfernen des Quecksilbers durch Schwefelwasserstofi und der Salzsiiure
durch Silber nitrat, mit Silber nitrat und Baryt in kleinem Ueberschuss
versetzt. Aus dem dabei enstandenen braunen Niederschlag wurde 4 g.
Arginin pikrat gewonnen.

Das Filtrat vom. Silbernitrat und Baryt Niederschlag lieferte noch
1.4 g. reines Lysin pikrat.

h). Das Filtrat von Phosphowolframsiure-N iederschlag.

Das Filtrat yon phosphowolframsiiurem Niederschlag (a) wurde

1. Vergl: E. Fischer u, U. Suzuki: Berichte d, deutsch, chem, Gesellshaft. XXXVIII.
Band III, 4180,

UEBER DIE EXTRAKTIV STOFYTE IM FISCUFLEISCHE. 49

nach dem Entfernen der Schwefelsiiure und Phosphowolframsiiure durch
Baryt und des Ueberschusses von Baryt durch Schwefelsiiure, im Vacuum
stark eingeengt. Es schieden sich dabei Tyrosin und Leucin aus; sie

wurden in 4 Fraktionen gesammelt undsgwar.

I Fraktion 0.95 ¢ bestand hauptsichlich aus Tyrosin
IL . 0.92 ¢
Til is 0.90 g Gemiseh von Tyrosin und Leucin
LV . 1.45 ¢ Leucin

1. Tyrosin:—Aus der 1 Fraktion wurde das Tyrosin rein

dargestellt, bei 100° getrocknet und analysiert.

0.1308 g Subst. 0.2831 g CO, 0.0728 ¢ H,O
0.1182 ¢ 7.9°° N (162 9751)
c H N
C,H,,NO, Ber. 59.67 6.07 7.73
Gef. 59.03 6.18 7.69

Aus heissem Wasser wnkrystallisiert, scheidet sich das Tyrosin aus;
seidenglinzende Nadeln, schwer léslich in Wasser, fast unldslich in
Alkohol und Aether. Es gibt schéne rote Farbung mit Millon’schen
Reagens, auch rote Farbung mit Diazobenzolsulfosiure in alkalischer
Loésung.

2. Leucin:—Anus der IV Fraktion; durch zweimalige Un-
krvystallisation war es gelungen das Leucin in reinem Zustaude zu isolieren.

Fir die Analyse wurde es bei 100° getrocknet.

0.1500 g Subst. 0.2921 ¢ CO, 0.1343 ¢ H,O
0.1433 ¢ _,, 13.5¢° N (150 9753)
€ H N
C,H, NO, Ber. 54.96 9,92 10.69
Gef. 53.11 9.94 10.93

Aus heissem Wasser scheidet sich das Leucin als diinne, perlmutier-
elinzende Plittchen aus; es enthalt kein Krystallwasser, hat kaum Gesch-
mack. Es list sich in Wasser, ist aber in Alkohol und Aether fast
reagiert fast neutral. Es _ bildet

unléslich. Die wisserge Losung

schénes Kupfersalz, das in Wasser schwer léslich ist,

50 U. SUZUKI UND K. YOSHIMURA:

Aus der Mutter Jauge von Tyrosin und Leucin wrude durch Easter
Methode noch 2 g. reines Leucin, ungefihr 1 g. Alanin und kleine Menge

Prolin gewonnen, Wegen Mangel an Zeit haben wir jedoch die beiden

letzteren K6rper nicht analysiert.

B. Die Hummer schale
33 Sticke Tummern (3710 g.) Heferte 1415 g. frische Schale, die
vollstiindig vom Fleisch befreit war. Diese wurde fein zerrieben, mit
heissem Wasser extrahiert und genau so bearbeitet wie das Fleisch,

In 100 Teilen frischer Schalen

In Wasser léslicher Stickstoff 1.12

Darunter: { Eiweiss stickstoff 0.32
Durch Phosphowolframsiure fillbarer ;

| Stickstoff 0.49

€ Stickstoff in anderer Form 0.31

In Wasser léslicher Stickstoff als 100

( Eiweiss stickstoff 28.6
Durch Phosphowolframsaure fillbarer

| Stickstoff 43.7
l Stickstoff in anderer Form 20.0

Aus dem phosphowolframsauren Niederschlag wurde 1.5 g. Lysin

pikrat in reinem Zustande isoliert.

0.1531 g Subst. 0.2142 g CO, 0.064 g H,O
0.1524 ¢_,, 23.8°% N (13° 768™™)
0.7083 ¢_,, 0.430 g Pikrinsiiure

C i N_ Pikrinsiiure
C,H,,N.0,. CgH,N,0,. Ber. 38.40 453 18.67 61.07
Gef. 38.16 4.65 18.65 61.14
Ausserdem haben wir noch eine Base als Pikrat isoliert, die Ausheute
desselben betrug ungefiihr 4 g. Diese Base soll spiiter nailer untersucht

werden.

UEBER DIE EXTRAKTIV STOFFE IM FISCHIFLEISCHE. D1

Wir haben ferner das Vorhandensein von Tyrosin, Leucin, Alanin,
Prolin (7%) und Histidin nachgewiesen. Zur Analyse reichte die Menge

jedoch nicht aus.

Zusammenfassing der Resultate,

Aus 1 Kilo frischem Fleisch und frischer Schale wurden isoliert.

Hummer fleisch Schale
Arginin 3.3 -
Lysin 0.66 0.42
Histidin vorhanden vorhanden
Leucin 2.3 -.
Tyrosin 1.36 7
Alanin 0.6 6
Prolin vorhanden 5

V. SURUME-IKA (Ommastrephes sp.)

Der getrocknete Surume-Ika hat foleende quantitative Zusammenset-

zug.
In 100 Teilen Luft trockensubstanz.

Wasser 23.09

Trockensubstanz 76.91

In 100 Teilen Trockensubstanz

Organische substanz 92.25
Asche 7.75
Gesamt Phosphor 2.85
In heissem Wasser loslicher Phosphor 2.01
Gesamt Stickstoff 14.96

In*heissem Wasser lislicher Stickstoff 4.90

52 U. SUZUKI UND K. YOSHIMURA:

Darunter: Ammoniak stickstoff oa ORG
Eiweiss stickstoff 2.45
Nicht-Eiweiss stickstoff 2.19
Dureh Phosphowolframsiiure fiillbarer

Stickstotf 1.50

Tn heissem Wasser léslicher Stickstoff als 100

Ammoniak stickstoff 5.48
Kiweiss stickstoff 49.98
Nicht-Eiweiss stickstoff 44.59
Durch Phosphowolframsiiure fillbarer
Stickstoff 30.58
Stickstoff in anderer Form 10.01

A). Taurin und Levcin.

500 g. Luft trocken substanz wurden mit warmem Wasser (50°C.)
wiederholt extrahiert. Die wiisserigen Ausziige wurden nach dem
Behandeln mit Tannin und Bleiessig lés~mg in bekannter Weise im
Vacuum stark eingeengt und im Exikator stehen gelassen. Nach mehreren
Tagen schieden sich die grossen monoklinischen Prismen aus, die ungef:ihr
3.7 g. betrugen. Diese Krystalle wurden aus heissem Wasser umkrystal-

lisiert, bei 100° getrocknet und analysiert.

0.5252 g¢ Subst. gab 0.0365 g N
0.1540 ¢_,, 0.2874 ¢ BaSO,
N S
C,H,NSO,. (Taurin) Ber. 11.20 25.60
Gef. 11.23 25.63

Aus der Mutter lange wurden durch Zusatz von Alkohol noch 4.3 2.
Krystalle gewornen, die unter dem Mikroskop betrachtet aus zwei
verschiedenen Kérpern bestanden. Der eine war Taurin und der andere
Lencin. Durch Fraktionieren wurde zuerst 8 g Tanrin in reinem

Zustande gewounen,
9

UEBER DIE EXTRAKTIV STOFFE IM FISCHFLEISCHE. a

0.1667 & Subst. 0.01865 ¢ N

0.1780 ¢_, 0.04611 ¢ S
N 8

C,H,NSO, Ber. 11.20 25 60

Gef. 11.19 25.79

Im ganzen haben wir somit 6.7 ¢. Taurin isoliert.
Die von Taurim getrennte Mutterlauge lieferte 0.5 g Leucin, das

unmittelbar in das Kupfersalz verwandelt und analysiert wurde.

0.1700 ¢& Subst. 0.0332 ¢ Cu
Cu
(C,H,,NO,).C€u Ber. 19.62
Gef. 19.53

B). Organische Basen.

Es wurde zuerst die quantitative Analyse ausgefiihrt.

In 100 Teilen Stickstoff der organischen Basen :

Durch Silbernitrat in neutraler Realction fiillbarer Stickstoff. 4.27

Durch Silber nitrat und Baryt fillbarer Stickstotff 31.71

Stickstoff in anderer Form 64.02

500 g. lufttrockenes Material wurde mit heissen. Wasser extrahiert.
Nach dem Proteinstoffe und andere Verunreinigungen durch Tannin
und Bleiessig losung beseitigt waren, wurde die Fliissigkeit mit Schwefel-
siure angesiiuert und mit Phosphowolframsiinre gefallt. Aus dem phospho-
wolframsauren Niederschlag wurde in bekannter Weise eine stark alkalische
Fliissigkeit der freien Base gewonnen. Diese Fliissigkeit wurde jetzt
mit Salpetersiiure neutralisiert und mit Silbernitrat versetzt. Der dabei
entstandene Niederschlag war in verhaltnismissig schr geringer Menge,
in diesem Niederschlag wurde nur das Vorhandensein von Hypoxanthin
und Xanthin identifiziert.

Das Filtrat von Silbernitrat-Niederschlag lieferte durch Zusatz

von Silber nitrat und Baryt im Ueberschuss einen braunen Niedersehlag,

54 U. SUZUKI UND K. YOSHIMURA:

aus dem ungefihr 1.5 ¢. salzsaures Salz einer unbekannten Base, deren
Natur nicht naher anfgeklirt werden konnte, dargestellt wurde.

Das Filtrat von Silbernitrat und Baryt-Niederschlag wurde nach dem
Entfernen des Silbers durch Salzsiiure und des Baryts dureh Schwefel-
siiure, mit Schwefelsaure angesauret und mit Phosphowolframsiiure
gefallt,. Aus dem phosphowolframsauren Niederschlag wurde die Base
wieder frei gemacht. Die stark alkalische Fliissigkeit lieferte nach dem
Einengen und lingeren Autbewahren im Exikator grosse farblose
monoklinische Krystalle, die ungefiihr 8 g. betrugen.

Diese Krystalle wurden aus heissem Alkohol umkrystallisiert. Fiir

die Analyse wurden sie im Vacuum bei 80° getrocknet.

1. 0.1498 ¢ Subst. 0.2825 ¢ CO, 0.1327 g H,O

2 OA5El @ 4, 0.2928 g CO, 0.1325 g H,O
3. OMA0I te 7s 15.5¢° N (130, 757™™)
4. O.1524¢ ,, 15.7¢¢ N (14.50 — 763™™)

G H N

O.Hy, NO... aber 51.28 9.40 11.97

Gef “1. 61.42 9.84 12.24

2, 51.16 9.43 }2.14

Die Base hat einen angenchmen siissen Geschmack; list sich leicht
in Wasser und in heissem Alkohol.
Sie enthilt ein Molekiil Krystall wasser, das im Vaeuum bei 100°

verloren geht.

0.4230 ¢ Subst. (im Exikator iiber Schwefelsiiure getrocknet)
verlor im Vacuum bei 100° 0.0509 ¢ Wasser

Wasser
C,H,,NO,+H,O Ber. 13.33
Gef. 12.03

Die Analyse stimmt also mit der Formel C;H,,NO., der Aminovale-
riansiure iiberein.

Das Platinchlorid doppelsalz bestand aus gold gelben monoklini-
schen Tafeln, die sich entweder aus Wasser oder aus Alkohol umkrystalli-

sieren liessen. Fiir die Analyse wurden sie bei 100° getrocknet.

Sr
or

UEBER DIE EXTRAKTIVSTOFFE IM FISCH FLEISCHE.

0.1700 g Subst (Aus Wasser umkryst.) gab 0.05120 ¢ Pt
01358 ¢ ,, (Aus Alkohol umkryst) ,, 0.0410 g¢ Pt

Pt
(C,H, NO;. Eel) Pick, Ber. 30.29
1. 30.12
Gef.
2. 30.19

Tin Kapillar rohr erhitzt zersetzt sich das Platindoppelsalz bei
246-247° (uncorr.).

Die Base bildet auch das Pikrat. Es bestand aus gelben Prismen,
die in heissem Wasser leicht, in kaltem Wasser aber schwer léslich waren.
Im Kapillarrohr erhitzt zersetzt es sich bei 235° (ameorr.).

Von den oben erwahnten Beobachtungen und besonders yor der
basichsen Kigenschaften der Base und der Fallbarkeit durch Phosphowol-
framsiure halten wir diese Base fii. d-Aminovaleriansiure, die yon
Salkowski aus gefaulten Pankreas isoliert und spiiter von Ackerman
niiher untersucht worden ist. Genauere Studien der Base hoffen wir bald

mitteilen zu k6nnen,

VI. UNAGI (Swesswasser Aal: Anguilla jiluvialits).

Das frische Fleisch yon Aal enthielt:

‘Wasser 69.24
Trockensubstanz 30 76

In 100 Teilen Trockensubstanz.

Fett 37.48
Gesamt stickstoff 9.58
Eiweiss stickstoff 8.79
Nicht-Eiweiss stickstoff 0.79
Ammoniak stickstoff Spur.
In Wasser loslicher Stickstoff 3.62

Darunter :

Liweiss stickstotf 2.83

56 U. SUZUKI UND K. YOSHIMURA:

Ammoniak stickstoff Spur.

Nicht-Eiweiss stickstotf 0.79

Durch Phosphowolframsiure fiillbarer
Stickstoif 0.30
Stickstoff in anderer Form 0.49

A. Organische Basen.

3 Kilo frisches Fleisch wurde mit warmem Wasser extrahiert und
in gewohnlicher Weise die Basen durch Phosphowolframsiiure gefillt,
Die aus dem phosphowolframsauren Niederschlag dargestellten freien
Basen wurden durch Silbernitrat und Baryt gefillt. Von diesem Nieders-
chlag wurde 8.6 g. freies Carnosin gewonnen. Dies wurde aus wenig

Wasser wmgelist, im Vacuum bei 100° getrocknet und analysiert.
.

0.1252 g Subst. 0.2180 g¢ CO, 0.0701 ¢ H,O
0.1608 g Subst. DIES aN (O°: 760™ )
C H N
C7 Ha NOs Ber. 47.70 6.20 24.77
Gef. 47.50 6.22 24.81

Das freie Carnosin bestand aus farblosen Nadeln oder Prismen; in
Wasser list es sich leicht, in Alkohol aber schwer. Die wiisserige Liésung
reagiert stark alkalisch.

Das Kupfersalz:—Dunkelblaue, sechsseitige Tafeln. Im Kapillar
rohr erhitzt zersetzt es sich bei 220° ohne zu schmelzen. Es ist in
heissem Wasser leicht, in kaltem Wasser aber schwer léslich. YF iir die

Analyse wurde es bei 100° getrocknet.

0.1936 g Subst. 0.0412 ¢ Cu
Cu
©,H;,N,0,, Cad Ber. 20.81
Gel. 21.29

Das Platinchlorid doppelsalz:—Gelbe Prismen, léslich in Wasser
und Alkohol; unlislich in Aether. Der Schmelzpunkt liegt bei 210—

222°.
Fiir die Analyse war das Salz bei 100° getrocknet.

UEBER DIE EXLPRAKTIVSTOFLE IM FISCH FLEISCHE. 57

0.1726 g¢ Subst. 0.0519 g¢ Pt
Pt
Cou 4N,O,2HCl, Ptel, Ber. 30.61
Gef. 30.06

Das Nitrat:—Farblose Prismen ; Schmelzpunkt 211°

0.1174 ¢ Subst. gab 0.02478 ¢ HNO,
HNO.,
Crue N07. HINO? Ber. 21.80
Gef. 21.11

B. Das Filtrat vom phosphowolframsauren Niederschlag.
Aus dem Filtrat yom phosphowolframsauren Niederschlag wurden
8.4 ¢. Kreatiw isoliert. Mit dem gereinigten Priparate wurde Krystall

wasser-und Stickstoff bestimmung ausgeftihrt.

0.1498 g Subst Verlor bei 100° 0.0184 ¢ Wasser
Wasser
C,H,N,0,+H,O Ber. 12.12
Gef. 12.28
0.1314 g Subst (bei 100° getrocknet) 9.0426 ¢ N
N
C,H,N,0; Ber. 32.06
Gef. 32.42

Das Kreatin bestand aus farblosen Prismen. welche bei 100° ihr
Krystallwasser verlieren und in ein undurehsichtiges, weisses Pulver sich
verwandeln. Im Kapillar rohr erhitzt wird es bei 255° schwarz braun
und zersetzt sich bei héherer Temperatur.

Zusammenfassung der Resultate.

Aus 13 Kilo frischem Fleisch wurden isoliert.

Kreatin 8.4 o
Carnosin &.6 2

Die Schleimige Substanz von Aal besteht wahrschenistich aus einem Mucinihnlichen

Korper. In ziemlich reinem Zustande enthielt sie 11.8326 N.

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Hydrolyse der wilden Seiden'; Antheraea Peryni Guér
(Sakusan), Antheraea Yamamai Guér (Yamamai)
und Caligula Japonica Moore (Kuriwata.)

VON

U. Suzuki, K. Yoshimura und R. Inouye.

Die gew6hnliche Seide yon Bombyx mori ist, wegen ihrer technischen
Wichtigkeit und ihres physiologischen JInteresses mehrfach yor
verschiedenen Seiten untersucht worden. Die als Handelsartikel minder
wertigeren, trotzdem immer noch eine wichtige Rolle spielenden wilden
Seidenarten, wie Antheraea Pernyi (Sakusan), Antheraea Yamamai
(Yamamai) und Caligula Japonica (KXuriwata) sind dagegen viel seltener
ertorscht ; besonders fehlt es an griindlichen Studien iiber die chemische
Zusammenseizung derselben.

Die gewéhnliche Seide lisst sich durch Eimwirkung von Seifer,
Alkalien oder sogar yon heissem Wasser in zwei yerschiedene Bestandteilen
trennen, nimlich das Fibroin und das Sericin. Das erstere bildet den
Hauptanteil der Seide und ist gegen die obengenannten Reagenzien viel
bestindiger als das letztere.

Die Hydrolyse des Filbroms ist schon von verschiedenen Autoren
unternommen worden. So haben Hinterberger? und Watenburger* in
den Spaltungsprodukten des Fibroins durch Salzsiiure, Tyrosin isliert;
sie wollten auch das Leucin gefunden haben. Staedlert und Cramer?
velangten auch zu denselben Resultaten; letzterer fand ausserdem noch
Glykokoll. Schiitzeuberger® und Burgeois haben das Fibroin mit Baryt-

1. Diese Abhandlung ist schon in “the Journal of the Tokyo Chemical Society.” Bd

28 heft 11. (.Vov. 1907) publiziert worden

2. Hinterberger: Jahres. f. Chem. 7853. 615
3. Waltenberger: Wien. Akad. Ber. z7 450

4. Staedler: Ann. 1859 zsz 12

5. Cramer: sour. f. prakt. Chem. 1865 96 76
6. Schutzenberger: Comptes rendus 1875. Sz 1191

60 U. SUZUKI, K. YOSHIMURA UND R. INOUYE:

wasser unter Druck erhitzt und behaupteten, neben Ammoniak, Oxalsiiure,
Kohlensiiure und Essigsinre ein Gemisch von Aminosiiuren, das aus
Tyrosin, Glykokoll, Alanin, Aminobuttersiiure und einer ungesiittigte-
siiture C,H;NO, bestehen sollte, gefunden zu haben. Ihre Angabe ist
unzuyerlissig, weil sie keine genauere Beschreibung der Methode gegeben
haben. Ferner ist der Baryt kein gecignetes Mittel fiir Hyrolyse, weil’
er bei héherer Temperatur sekiindiire Zersetzungen bewirken kann.

Weyl® hat wieder die Hydrolyse durch Siiure ausgefiihrt und neben
Tyrosin und Glykokoll, noch eine Aminopropionsiiure gefunden, die er
fiir d-Alanin hielt.. Diese drei Kérper hat er in reinem Zustande isoliert >
es gelang ihm jedoch nicht Lencin nachzuweisen.

Ueber die Diaminosiiuren liegen einige Angaben von Wetzel* vor.

Ein grosser Fortschritt anf diesem Gebiete ist aber E. Fischer’ und
seinem Mitarbeiter A. Skita zu verdanken. Diese Autoren haben dic
bekannte Ester methode, die fiir die Forschung der Spaltungsprodukten
der Eiweisskérper so grosse Erfolge gebracht hat, fiir Seiden Fibroin und’
Sericin angewendet, und ausser den bisher bekannten Stoffen: Glykokoll,
Alanin und Tyrosin, nicht nur das Vorhandensein yon Serin, Phenylalanin,
Prolin, Leucin. Arginin ete nachgewiesen sondern auch das optische
Verhalten dieser Kérper festgestellt. Unentschieden bleibt noch die
Existenz yon Lysin, und Valin im Fibroin. In Sericin ist auch nach Lysiny
und Histidin zu suchen.

Es sei noch erwahnt, dass E. Fischer® spiiter auch Spinnen seide
aus Madagascar “Nephilia madagascariens” (Soie d’araignée de Mada-
gascar) untersucht hat. Seine Angaben haben wir zum Vergleich mit
unserem Resultate, in einer Tabelle zusammengestellt.

Der Hauptzweck unserer Arbeit ist die Spaltungs produkte der wilden

6. Weyl: Ber. 1888, 27 1407 u. 1529

7. Wetzel: Zeitsch. f, Physiol. Chem. 1899 26 535

8. F. Fischer u, A. Skita: Zeitsch. f. Physiol. Chem. (1901) 33 177 1902 35 221.
(1903) 89 155

9. F. Fischer: Ueber Spinnenseide: Sitzungsberichte. Kgl. pr. Akad. Wiss, Berlin

(1907) 440-50

HAYDROLYSE DER WILDEN SEIDEN. 61

Seiden méglichst genau kennen zu lernen und mit denjenigen der gewohnli-
chen Seide zu vergleichen.
Wir geben nur zu, dass unsere Ergebnisse noch viele Liicken haben,

«lie wir spiiter noch auszufiillen hoffen.

I.—Sakusan-Seide. (Antheraea Pernyi Guér.)

Die kiiufliche Sakusan-Seide, die wir zu dieser Untersuchung an-

wendeten, hatte foleende Zusammensetzung.

Wasser 13.16 %
Trockensubstanz 86.84 9%
In 100 Teilen 'Trockensubstanz
Asche 2.92
In heisser cone. HCi léslich 92.21
5 i; unléslich 7.79
Gesamt-Stickstotf 18.87
Darunter: In heisser cone. HCl léslicher Stickstoff 16.39
a se unléslicher Stickstoff 2.48
In 100 Teilen Gesamtstickstoff
In heisser conc. HCl léslicher Stickstoff 86.87
Darunter : Ammoniak Stiekstoff ZO
Durch Phosphowolframsiiure
fallbiirer Stickstoff eB yuil
| Stickstoff in anderer Form 71.24
In heisser cone. HCl unléslicher Stickstoff 1Ssts

A, Monaminosduren in den Spaltungsprodukten,

200 g. kiiuliche Sakusan-Side wurden mit 600 e.c. Salzsiiure (1.19)
mt Riickflusskiithler zwei Tage gekoeht; nach dem Erkalten wurde die
Fliissigket mit Wasser verdiinnt, vom unléslichen Riickstand abfiltriert
und im Vaeumn stark eingeenet.

Man yersetzte nun dieselbe init 500 ¢.¢. absolutem Alkohol ynd leitete

irockenes Salzsiiure gas bis zur Siittigung ein, und dampfte wieder im

62 U. SUZUKI, K. YOSHIMURA UND R. INOUYE:

Vakuum ein. Diese Operation wurde nochmals wiederholt. Als man den
so bereiteten dicken syrup mit einigen Kyrstallen von salzsaurem-
Glvkokoll ester geimpft und in kaltem Zimmer einige Tage stehen liess,
schieden sich die Krystalle von salzsaurem Glykokoll ester in reichlicher
Menge aus, die abgesaugt, mit absolutem Alkohol und Aether gewaschen
und getrocknet wurden. Aus der Mutter lauge schieden sich noch kleine
Mengen in zweiter Ernte aus. Die Gesamt ausbeute betrug 16.4 g. Diese
Krystalle wurden aus Aethylalkohol umkrystallisiert. Sie bestanden aus
farblosen Prismen nud schmolzen bei 145° (unecorr.), Sie wurden im

Vacuum bei 100° getrocknet wnd analysiert.

0.38225 ¢ Subst 0.08136 ¢ Cl
Cl
C,H, ,0,N.Cl Ber. 25.45
Gef. 25.23

Die Mutter lange von Salzsaurem Glykokoll ester wurde im Vakuum
eingedampft und aus derselben nach der bekannten Ester Methode von
E. Fischer die freien Aminosiiureestern dargestellt, und bei niederem
Druck fraktioniert. Nach der Verseifung der enizelnen Ester fratktioner

haben wir folgende Amnésauren isoliert.

Glykokoll ieee
Alanin 8.4
Alanin + Leucin 2.0
Asparaginsiiure i
Glutaminsiiure ig
Prolin Weing

Tyrosin (aus dem undestillierbaren
Riickstand) 2.4

Analyse des Alanin praparates

0.3995 g Subst 0.06411 ¢ N
N
C,H,NO, Ber. 15.73

Gef. 16.04

HYDROLYSE DER WILDEN SEIDEN. 63

Die Menge des Leucins reichte zur Analyse nicht hin.

Asparaginsiiure wurde als Kupfersalz gereinigt. Es bestand aus
langen blanen Nadeln, ziemlich schwer léslich in Wasser und schmolz bei
218° (uncorr.) Aus dem Kupfersalz wurde freie Asparaginsiiure
dargestellt. Sie schmolz bei 228° (uncorr.)

Das bei 100° getrocknete Priparat gab 10.47¢ N. (Auf C,H,NO,
berechnet 10.534 N.).

Nach Serm und Gultaminsiiure wurde vergebens gesucht.

Zur Isolierung des Tyrosins wurde 100 ¢. Sakusan-Seide mit 300 e.c.
conc. HCI. zwei Tage gekocht, vom unléslichen Riickstand abfiltriert und
im Vacuum eingedampft, um den gréssten Teil der Salzsiiure auszutreiben.
Der Riickstand wurde mit Wasser verdiinnt, mit Natronlauge neutralisiert,
mit Tierkohle entfarbt und eimige Tage stehen gelassen. Das ausges-
chiedene Tyrosin wurde abgesaugt, mit Wasser. Alkohol und Aether ge-
waschen. Die Ausbeute betrug 1.4 g. = 1.4% der trockenen Seide.

Organische Basen.

100 g. luft trockener Substanz (==86.84 ». Trockensubstanz) wurden
mit 300 ¢c, HCl. (1.19) zwei Tage gekocht. Nach dem Erkalten wurde
die FPliissigkeit mit Wasser verdiinnt und mit Phorphowolframsiure
gefillt. Der Niederschlag wurde in bekannter Weise durch Baryt selegt.
Die so erhaltene stark alkalische Fliissigkeit wurde mit Kohlensaiure
gesittigt und mit Quecksilberchlorid versetzt. Aus dem Quecksilber-
chlorid-Niederschlag wurde das salzsaure Salz des Histidins (entsprechend
2.4 g. freies Histidin) dargestellt.

Aus dem salzsauren Salz wurde Histidin Silber bereitet, indem das
Salz in Wasser gelist, durch Silbernitrat von Salzsiiure befreit und zum
Klaren Filtrat Silber nitrat und Ammoniak zugegeben wurde, wobei ein
weisser Niederschlag von Histinsilber entstand, der mit Wasser, Alkohol

und Aether gewaschen, bei 100° getrocknet und analysiert wurde.

0.211Ig¢ Subst 0.122 ¢ Ag.
Ag
C,H,N,O,Ag. Ber. 58.53

Get. 57.82

64 U. SUZUKI, K. YOSHIMURA UND R. INOUYE:

Aus dem salzsanven Salz wurde durch Zusatz von Natrium pikrat
das Histidin pikrat als gelbe Prismen erhalten; es schmolz bei 80-90°
zu einem Oel.

Das Filtrat von Quecksilberchlorid-Niederschlag wurde dureh
Schewefelwassersto#? von Silber befreit und im Vakuvm eingedampft, um
den Schwefel wasserstoff auszutreibeu und mit Silbernitrat und Baryt
in kleinem Ueberschuss versetzt. Aus dem dabei entstandenen Nieder-
schlag wurde kein Arginin isoliert. Dagegen hat man aus dem Filtrate
von Silbernitrat und Baryt-Niederschlag in bekannter Weise 6.2 g.
Arginin pikrat (= 2.7 g. Arginin) isoliert.

Das Arginin pilkrat wurde mehreremal aus heissem Wasser um-

krystallisiert und im Vakuum bei 100° getrocknet und analysieri.
0.4725 g Subst. 0.2670 g Pikrinsaure
0.1650 gy, 0.2152 g CO, 0.0510 g H,O
O1GO0 ee” 5 36.°° N (200 75s™™)

Cc H N Pikrinsiure
C,H,,4N,O, C,H,N,O0, Ber. 35.73 4.22 24.32 56.82
Gef. 35.57 3.42 24.17 56.51
Aus dem Pikrate wurde das methylester salzsaure Salz des Arginins
dargestellt ; farblose Prismen, Schmolzen bei 175—189° (uncorr.) unter

lebhaftem Schiiumen. Fiir die Analyse war es im Vaeuum bei 100°

getrocknet.
0.1065 g Subst 0.0280 g Cl
. Cl
C,H, ,.N,O,. 2HCl Ber. 27.20
Gef. —-:26.29

Das Nitrat des Arginins:—farblose Prismen F, P. 155—165°.

II. Yamamai-Seide. (Antheraea Yamamai).

Die kaiitliche Yamamai-Seide hat foleende Zusammensetzung.
Wasser 11.29
Trockensubstanz 88.71

HYDROLYSE DER WILDEN SEIDEN. 65

In 100 Teilen Troekensubstanz

Asche 4,73
In heisser cone. HCl léslich 97.07
on 5, unléslich 2.93

Gesamt-Stickstoff 1
Daunter: Jn heisser cone. HCl léslicher Stickstof€ 17.26

Pe 5: unléslicher Stiekstoff 0.47

In 100 Teilen Gesamt-Stiekstoff

In heisser cone. HCl léslicher Stiekstoff 97.34
Darunter: { Ammoniak Stickssoff 3.85

Durch Phosphowolfamsiiare

fiillbarer Stickstoff 19.44
Stickstoff in anderer Form 74.05
In heisser cone. HCl unldéslicher Stiekstoff 2.66

Monaminosduren.
200 g. Yamamai-Seide wurden mit 600 ¢e. Salzsiiure (1.19) 30
Stunden gekocht’ und nach der Ester methode die folgenden Aminosiiuren

dargestellt.

Glycocoel ese:
Alanin HEF
Alanin + Lencin 2
Asparagin siiure_ oS
Glutaminsiiure 1.2
Riiekstand 2.6

Analyse der Aminosiiuren.
1. Glveocollester-salzsaure Salz. Aus heissem Alkohol wnkry-
stallisiert Schmolz es bei 145° (uncorr.) Fiir die Analyse

wurde es im Vakuum bei 100° eetrocknet.

1. Die Yamamai Seide ist gegen Satren und Alkalien viel bestindiger als andere

Seidenarten.

66 U. SUZUKI, K. YOSHIMURA UND R. INOUYE:

0.3110 g Subst 0.0806 ¢ Cl
Cl
C,H,,NO,Cl Ber. 25.45
Gef. 25.94
2. Alanin.
0.400 ¢ Subst 0.06262 ¢ N
CLE NO; Ber. 15.73
Gef. 15.66

3. Asparaginsiiure-Kupfersalz; bestand aus langen Nadeln oder
Prismen, der Schmelzpunkt war 218° (uncorr.)
0.232 g. Subst (iiber Schwefelsiure bei gewéhnlicher

Temperatur getrocknet) verlor bei 110° 0.0375 ¢. Wasser.

0.2485 @ Subst (Wasser frei) gab 0.0934 g¢ CuO
0.1925 ¢ ,, 3 0.01491 ¢ N
Krystall wasser
C,H,NO,Cu+2H,0 Ber. 15.45
Gef. 16.66
N Cu
C,H,NO,Cu Ber. tL 32.82
Gef. 7.50 30.06

Die Analyse stimmt nicht ganz gut, wahrscheinlich in Folge von
Verunreinigung. Zur weiteren Reinigung geniigte das Material nicht.

Das Vorhandensein von Prolin war zweifelhaft. Nach Serin und
Phenvlalanin wurden vergebens gesuchi.

In der Mutter lange von Asparaginiiure konnten wir kleine Mengen
Glutaminsiinre nachweisen; sie reagierte stark sauer und bildete schéne
prismatische Krystalle von salzsanrem Salz, das in cone. Salzsiiure schwer
loslich war; sie bildete auch schwer lisliches Kupfersalz. Zur weitere
Forschung reichte die Menge nicht aus.

Aus dem undestillierbaren Riickstand wurde durch Verseifen mit

Baryt 2.6 ¢. Tyrosin isoliert.

HYDROLYSE DER WILDEN SEIDEN. 67

Zur Gewinnunge des Tyrosins wurden 20 g@. Rohmaterial mit
Schwefelsiiure gekocht und in bekannter Weise haben wir 0.4 ¢. Tyrosin
isoliert. Das Rohprodukt wurde einmal aus Wasser umkrystallisiert und

enalysiert.

0.320 & Subst 0.22485 ¢ N
N
CANO: Ber. 7.96
Gef. 8.10

Organische Basen.

Es wurde bloss die quantitative Analyse der organischen Basen
ausgefiihrt. Zu diesem Zweeke wurden 20 ¢. Rohmaterial mit cone.
Salzsiiure drei Tage gekocht, und der salzsaure Extrakt wurde mit
Phosphowolivamsiinve gefallt. Der Niederschlag wurde in bekannter
Weise dureh Baryt zerlegt. Die stark alkalische Fliissigkeit, die freie
Baesn enthielt, wurde mit Kohlensiure gesiittigt und mit einer wiisserigen
Quecksitlberchlorid lésung versetzt. Der Quecksilber chlorid-Niederschlag
wurde unmittelbar zur Stickstoff-Bestimmung nach der Kjeldahl’schen
Methode angewendet....Zistidin stichstoff.

Das) Filtvat vom Quecksilberchlorid-Niedersehlag wurde dureh
Schwefelwasserstoff von Quecksilber befreit und im Vakuum eingedampft.
Als der Schwefelwasserstoft vollstiindig ausgetrieben war, wurde die
Fliissigkeit mit Silbernitrat versetzt, yom gebildeten Silberchlorid

adfiltviert und zum Filtrat wurde Silber nitrat und Baryt im Ueberschuss

mugegeben. Der brarne Niederschlag enthilt Arginin....Arginin
sticksloff. ..

Das Filtrat von Silbernitrat und Baryt-Niedersehlag wurde nach
dem Entfernen des Silbers und des Baryts wieder mit Schwefelsiivre
angesiiuert und mit Phosphowolframsiiure versetzt. Der Niederschlay
wurde gleich zur Stickstoff-Bestimmung nach Kjeldahl angewendet. .. .
Lysin stichstoff.

Es ist zu bemerken, dass die Trennung von <Arginin und Lysin

nach der oben angegebenan Methode nur unyollkommen war. In

68 U. SUZUKI, K. YOSHIMURA UND R. INOUYE:

der Lysin fraktion haben wir immer nicht unbedeutende Mengen Arginin

gefunden.
In 100 Teilen Trokensubstanz
Gesamt Stickstoff 17.73
Darch Phosphowolframsiiure {illbarer Stickstotf 3.45
Histidin Stickstoff 0.37
Arginin Stickstoff 1.10
Lysin Stickstoff 1.26

Als freie Base berechnet

Histidin 1.37
Arginin i 3.41
Lysin 6.57

Tin niichsten Versuche haben wir die entbastete Yamamai-Seide
analysiert. Zu diesem Zwecke wurden 20 g. Rohseide mit 5¢ Salzsiiure eine
Stunde gekocht, mit warmem Wasser gewaschen, dann wurde sie mit 10%
Marseille seife 2 Stunden gekocht, abgepresst, nnd mit warmem Wasser
gewaschen, nochmals mit 24 Natronlange eine Stunde auf 50° erwirmt

and mit Wasser gewaschen, getrocknet und analysiert. Es wurde

gefunden.
In 100 Teilen Trockensubstanz
Roh Entbastet
Verlust beim Entbasten — 27.30
In heisser cone. Salzsiiure léslich 97.07 97.23
y 5 unldéslich 2.93 2.77
Gesamt Stickstotf 17.73 18.42
In 100 Teilen Gesamt-Stickstoff
Ammoniak stickstotf 3.85 6.20
Durch Phosphowolframsiure
fiillbarer Stickstoff 19.44 15.05

Stickstoff in anderer Form 74.05 78.75

HYDROLYSE DER WILDEN SEIDEN. 69

Die Rohseide hat beim Entbasten 27¢ der Trockensubstanz verloren.
Dieser Verlust besteht hauptsiichlich aus anorganischer substanz, Leim u. a.
Der prozentische Gelialt an Gesamtstickstoff ist durch Enthbasten etwas
erhéht. Der Basen stickstoff dagegen hat etwas abgenommen, bleibt jedoch
imamcr noch hoher als 15% der Gesamtstickstoft. Vergleicht man dies mit
demjenigen des Seiden fibroins, niimlich nur 1.4% des gesamten Stichk-
stoffes, so muss man annehmen, dass die Yamamai-Seide eine stark you
Seiden fibroin abweichende Zusammensctzumg hat. Ausserdem sieht
man aus der Analyse, dass die aussere Schichte der Yamamai-Seide viel

veicher an Basenstickstoff ist als das Innere.

III._Seide. (Bombyx mori).

iL, Rohseide
Wasser 12.90
Trokensubstanz 87.10

In 100 Teilen ‘Trockensubstanz

Asche 0.63
In heisser cone. HCl léslich 99.14
of A unldéslich 0.86
Gesamt-Stickstoff 18.98
Darunter; In heisser cone. HCl lislicher Stickstoff 18.86
a », unléslicher ,, 0.12

In 100 Teilen Gesamt stickstoff

In heisser cone. HCl léslicher 99.3¢
Darunter: Ammoniak Stickstoff 4,57

Durch Phosphowolframsiiure
fiillbarer Stickstoff 1.78
Stickstoff in anderer Form 92.98
0.66

In heisser cone. HCl unléslicher Sticlkstotf
OF Entbastete Seide
Die kiiufliche Rohseide der besten Sorte wurde mit 10% Marseilleseife

2 Stunden bei 60-65° eekocht und mit Wasser gewaschen.... (A)

70 U. SUZUKI, K. YOSHIMURA UND R. INOUYE:

(A) Wurde nochmals mit 10% Marseille Seife 2 Stunden gekocht,
mit Wasser gewaschen, dann mit Wasser gekocht und mit 0.1% Natron-
lauge erwirmt und mit Wasser sorgfiiltig gewaschen. ...(B)

In 100 Teilen ‘Trockensubstanz

Rohseide — Entbastete Seide

(A) (B)

Verlust beim Entbasten — 16.0 37.0
In cone. Salzsiinre J6slich 99.14 99.33 ungeliihr 100
or 3 unloéslich 0.86 0.67 spur
Gesamt stickstoff 18.98 17.83 Ug Si

In 100 Teilen Gesamt stickstoff

Ammoniak stikstoff 457 4.71 5.85
Durch Phosphowolframsiixre

fiillbarer Stickstoff 1.78 1.38 1.47
Stickstoff’ in anderer Form 92.99 93.91 92.68

Aus dem obigen Resultaten sieht man, dass es kein merkbarer
Vnterschied zwischen Roher und entbasteter Seide gibt, bloss ist der
Gesamt stickstoff und der Basen stickstoff in der entbasteten Seide etwas
kleiner als in der Rohseide.. Doch nimmt der Basenstickstoff nie so
stark ab, dass man ihn nicht als eigentlichen Bestandteile des Fibroins
hetrachten diirfte.

Wir haben aus der Rohseide 124 Tyrosin isoliert.

IV.—Kuriwata. (Caligula japonica).
Die kiufliche Kuriwata Seide hatte fulgende Zusammensetzung
Wasser api leyial

Trockensubstanz 88.29

In 100 Yeilen Trockensubstanz

Asche | 3.85
In heisser cone. HCI léslich 88.34

a oy unléslich 11.66

THYDROLYSE DER WILDEN SEIDEN. 71

Gesamt-Stickstott 16.73
Darunter. In cone. HCl léslicher Stickstoff 15.77
a af unléslicher Stickstoff 0.96

In 100 ‘Teilen Gesamt stickstotf
In cone. HCl ldslicher Stickstoft 94.26
Darunter Ammoniok-Stickstoff 4.08

Durch Phorphowolframsaiire

fallbarer Stickstoff 15.54
Stickstoff in anderer Form 74.64
In cone. HCl unléslicher Stickstoff 5.74

200 e&. Roh-Kuriwata wurden mit kalter 5¢ iger Salzsiure 20 stunden
digeriert und mit Wasser gewaschen, dann wurde sie mit 10¢ Marseille
seife 2 Stunden eckoeht, stark abgepresst, mehreremal mit heissem dis-
tilliertem Wasser gewaschen und ectrocknet. Der Verlust an Gewicht
betrug 5.33¢ der Trockensubstanz.

200 @. der so behandelten Seide wurden mit 700 e.e. cone. Salzsiure
(1.19) S Stunden gekocht. Nach dem Erkalten wurde die Flissigkeit

mit Wasser verdtinnt und abfiltriert.

In heisser cone. HCl gelést 87.289/,

Der unlésliche Riickstand 12.72°/,

Aus dem salzsauren Extrakt wurde in bekannter Weise die freien

Aiminosiiuren dargestellt. Es wurden isoliert.

Glycocollester HCl salz 24.49 ¢ (=13.12 g Glycocoll)
Alanin 25.08
Leucin 14.05
Prolin Actives 1.06
eras 0.48
Phenylalanin 0.95
Asparaginsaiire 0.30
Tyrosin (aus 100 g) 4.87

Analyse der Aminosiiuren.

=I
bo

U. SUZUKI, K. YOSHIMURA UND R. INOUYE:

1. Glycocoil ester salzsaure Salz: Aus heissem Alkohol umkrystillisiert

und im Vacum bei 100° getrocknet ; Schmelzpuukt 144° (uncorr).

0.1584 g Subst 140°. N (12° 765™™)
0.100 ¢ 55 0.02443 ¢ Cl
N Cl
C,H,NO, HCl Ber. 10.04 25.39
Gef. 10.54 24.43

2. Alanin. Aus wiisseriger Lésung durch Zusatz yon Alkohol aus-

geschieden. Fur die Analyse wurde es im Vacuum bei 100° getrocknet.

0.1801 g Subst 2agc&) N(20o) 758m)
N
C,H ANO, Ber. 15.73
Gef. 15.75

0.3460 g Subst in 20.317 g Normal Salzsiiure gelist (sp. Gew.
1.017) drehte bei 20 © Rohr das Natrium licht 0.33° nach reclits.
mithin [a]? = 49,70
3. Leucin: Aus Wasser umkryptall.siert, im Wacuum bei 100°

getrocknet und analysiert.

0.2008 g Subst 18.9°¢ N (19° 760.5")
0.1252 ¢ _,, 0.2542 ¢ CO,
0.1169 g H,O
C H N

C,H, NO; Ber. 54.96 9.92 10.69
: Gef. 55.387 = 10.38 10.81
0.1538 g Subst in 20.252 g Normal Salzsiiure gelist (Sp. Gew.
1.014), drehte bei 20° Rohr das Natrium licht 0.22° nach rechts.
mithin [a}??= 4+14.4°
4, Tyrosin.

0.200 g Subst 13.1e% NW (89° 7640)
0.200 ¢ ,, 0.4276 « CO, 0.0906 ¢ H,O
C ist N
C,H NO. Ber. 59.67 6.08 7.74

Gef. 58.31 5.04 1.91

TIYDROLYSE DER WILDEN SEIDEN. (9

Das Vorhandensein von Prolin wurde durch seinen eigentiimlichen
Geruch, durch seine Lislichkeit in absolutem Alkohol und besonders
durch sein in Alkohol lésliches Kupfersalz sicher festgestellt, aber die
Analyse gab kein befriedigendes Resultat, weil es von anderen Amino-
siiuren Kupfersalz schwer zu trennen war und zur weiteren Reinigung
geniigte die Menge nicht.

Phenylalanin und Asparaginsiiure waren auch nicht rein dargestellt ;
wir haben bloss das Phenylalanin durch sein charakteristisches salzsaure
Salz und die Lislichkeit seimes Esters in Ather identifiziert.

Asparaginsiiure wurde analysiert und wir haben den Stickstoff gehalt
etwa um 1% héher gefunden, so dass es noch mit anderen Aminosiiuren
yerunreiniet gewesen sein muss. Von den ziemlich starken sauren
Higenschaften und dem charakteristischen Kupfersalz zu urteilen, zweifeln
wir kaum, dass es Asparaginsiiure war.

Nach Glutaminsiiure wurde mit besonderen Vorsicht gesucht; kein
positives Resultat konnten wir jedoch erzielen.

Organische Basen.

Auf die Isolierung der organischen Basen haben wir verzichtet und

nur die quantitative Analyse ausgefiihrt. Es wurde gefunden.

In 100 Teilen In 100 Teilen

Trockensubstanz. Gesamtstickstoff

Gesamt stickstoff 16.73 100.
Durch Phosphowolframsiiure fiilbarer

Stickstoff 2.62 15.54

Darunter : Histidin stickstoff 0.27 1.61

Arginin. stiekstoff 0.56 3.39

Lysin stickstoff 0.47 2.81

Als freie Base berechnet (In 100 ‘Teilen Trocken substanz)
Histidin 1.01
Arginin 1.74

Lysin WAS

74 U. SUZUKI, K. YOSHIMURA UND R, INOUYE:

CHEMISCHE ZUSAMMENSETZUNG DER VER-
SCHIEDENEN SEIDENARTEN.

Bombyx. Sakusan, Yamamai. Kuriwata.
(Roh seide) (Roh seide) (Roh seide) (Roh seide)
Wasser... we as 12.90 13.16 11.29 11.71
Trockensubstanz Sack Weer eee cee eee ee LOYELO 86.84 88.71 88.29
In 100 Teilen Trockensubstanz.
Asche ... Seats eee 0.63 2.92 4-73 3.85
In heisser conc. HCl ldslich ... ... 2... 99.14 92.21 97-07 88.34
a unléslich 0.86 7.79 2.93 11.66
Gesamte -stickstoff. | 25° Ges -eeeses eee SOS 18.87 17-73 16.73
Darunter, In conc. HCl loslicher stickstoff. 18.86 16.39 17.26 15.77
or » unldslicher ,, 0.12 2.48 0.47 0.96
In 100 Teilen Gesamt stickstoff.
In heisser conc, IICl léslicher Stickstoff. 99.34 86.87 97.34 94.26
Darunter. Ammoniak stickstoff ... ... 457 2.52 3.85 4.08
Durch Phosphowolframsaure
fallbarer Stickstoff... ... 1.78 13.11 19.44 15.54
Stickstoff in anderer Form... 92.99 71.24 74.05 74.04

In heisser canc, HCl unléslicher Stickstoff, 0,66 13.13 2,66 5-74

HYDROLYSK DER WILDEN SEIDEN. 7)

SPALTUNGS PRODUKTE DER VERSCHIEDENEN
SEIDEN ARTEN.

(Aus 100 g. Trockensubstanz.)

Spinnen
Bombyx. Sakusan. Yamamai. (Kuriwata. Seide.*
Fibroin, Sericin. (Roh) (Koh) (Entbastet) (Fischer)
(Fischer u. Skita)
Glykokoll... <.. ..- .- .. 30.0 O,[—0.2 5-7 6.3 vv 35.13
LATTE See eres sso, cass vee 21.0 5.0 4.8 7.2 15.3 234
BRIEGTIGIIO cet Ni ssh jose) Recor. ves 1.5 + 1,2 1.3 7:95 1.76
Henylalanin: 2. 6. sce 5 = — — + ae =S
ee? te eo + + Ree ea) 3.68
Glutaminsiure tne _- _ + 0.6 ? 6,16
Asparaginsaure OO Mca aide — _ 1,0 1.0 0.2
(CHELAN ama ord — = = = _
Sit ose Pee coe POON mega ons 1.6 6.6 in ? ?
Oxy prolin Soy Hone cactt, ou: — = = = _
MMUGEOSI css) wee eee oy 12.0 50 1.4 2.0 5.5 4.2
PRMISENCES ER eS aes’ brvwal pose + _ 2.7 1,6 1.01
SUTTON sey fsa) vse ess | css 1.0 4.0 31 3.8 1.74 5.242
[LSE ag SGN em ECON mDec metecien CO + + +? 7-4 2.43
Wiryptophan =. ... =. ... =f: —
Ammoniak ad eres Ce ere 1.05 1.87 0.6 0.8 0.8 1.16
WE Ree mReconmNone = cCOMecSe + 45 = =a =
Glykosamin «0... wee ee — _

1 Spinnen seide enthialt ausserdem 0.597%, Asche 0.6694 Fettsduren.

2 Diaminosiure, als Arginin berechnet.

Ueber die Hiweissstoffe aus Reissamen.!

VON

U. Suzuki, K. Yoshimura und S. Fuji.

Der geschiilte Reiskern besteht aus zwei Teilen, von denen die
diussere Schichte, die Kleie wneefihr 15% ausmacht. Die chemische
Zusammensetzung der Kleie ist von jener des entkleiten Reisinnern sehr

verschieden ; man muss natiirlich die beiden Teile fiir sich untersuchen.

In 100 Teilen Trockensubstanz

Entkleiter Reis (Hakumai) Kleie (Nuka)
Gesamt stickstoff 1.200 2.958
Eiweiss stickstotf 1.165 2.665
Nicht-Kiweiss stickstoff 0,035 0.293
Eiweiss (Kiweiss-N x 6.25) 7.282 16.655

Gesamt stickstoff als 100

Gesamt stickstoff 100.00 100.00
Eiweiss stickstoff 97.09 90,10
Nicht-Eiweiss stickstoff 2.91 9.90

Um die chemische Natur der Eiweissk6rper in der Kleie und in dem
eutkleiten Reis zu vergleichen wurden je 10 g. getrocknete Probe mit
100 ce. der folgenden Lésungsinitteln yersetzt und bei Zimimertemperatur
stehen gelassen. Nach 24 Stunden abfiltriert; mit dem Filtrate wurde

die Stickstoffbestimmung nach der IKjeldahl’schen Methode ausgefihrt.

In 100 Teilen Trockensubstanz wurde stickstotl’ gelost :

Loésungsmittel Entkleiter Reis Kleie
Destillicrtes Wasser 0.07 0.65 ¢

1. Diese Abbandlung ist schon in the Journal of the Tokyo Chemical Society Vol XXIX
No. 3. (March 1908), publiziert worden. Spiter ist eine vorliiufige Mitteilung tiber
Reisprutein yon Rosenheim. O und Kajiura S. erschienen (The Proteins of Rice :—Proe,

Physiol. Soc, 1908. Bd 54, u Journ, of Physiol. 1808, Ld 36,. No 6).

78 U. SUZUKI, K. YOSHIMURA UND §, FUJI:

609 Allkohol 0.11 0.16
10% NaCl 0.17 1.37
0.2% NaOH 0.85 1.51
0/3958 ees — 2.36
Oana: _ 2.49

Gesamt stickstoff als 100

Lésungs mittel Entkleiter Reis ]leie
Destilliertes Wasser 5.84 17.45
6096 Alkohol gol? 4.38
10% NaCl 14.17 36.87
0.2% NaOH 70.90 40.59
(O32) 3 2 _ (63.42)

(0.4% » ) — (67.02)

Wei man sielit, enthalt der entkleite Reis nur sehr wenig Eiweiss-
kérper, die in Wasser, Alkohel und 10% Natriumchlorid léslich sind.
Der grésste Teil besteht aus denjenigen Eiweisskirpern, die in verdiiunter
Natronlauge léslich sind. Die Kleie enthilt noch etwas mehr in Natrium-
chlorid lésliches Eiweiss (Globulin).

Darstellung der Biweisshorper.

Wie oben erwiihnt, ist das Eiweiss im entkleiten Reis und in der Kleie
grusstenteils in verdimuter Natrenlange léslich; so haben wir versucht,
nach Ritthausen’scher Methode das Eiweiss zu extrahieren. Zu diesem
Zwecke wurde das getrocknete, fein gepulverte Material (die Kleie vorher
entietiet) mit 0.2¢ Natronlauge mazeriert, nach 24 Stunden mit dem
Tuch koliert. Das Filtrat wurde mit verdiinnter Essigsiiure schwach
angesiiuert, wobei ein dicker, weisser Niederschlag in reichlicher Menge
entstand, der gesammelt, mit kaltem Wasser, Alkohol und zuletzt mit
Aether gewaschen wurde. Das so bereitete Rohprodukt wurde nochmals
in verdiinuter Natronlauge gelist und mit Essigsiiure gefillt,. | Weun
man mit wenig Material arbeitet, kann man aus dem entkleiten Reis etwa

70% des gesamten Eiweiss und aus der Kleie etwa 50¢ gewinnen, Wenn

UEBER DIE EIWEISSSTOFFE AUS REISSAMEN. 79

man aber mit erdsserer Menge arbeitet, so wird die Ausbeute sehlechfer,

Das in oben erwiihnter Weise dargestellte Eiweisspriiparat aus dem
enthkleiten Reis war gelblich braun gefarbt, wihrend jenes der Kleice
dunkel gefairbt und viel mareiner als der Ersteres war. Beide Eiweiss-

priparate hatten foleende Zusammensetzune.

Eiweiss aus

denientbletenikew ann irdariKleie
Asche 0.02 2.10
In heisser cone. Salzsiiure léslich 98.00 89.24
- - unl6éslich 2.00 10.76
Gesamt stickstoff 16.54 12.49
Darunter: In heisser Salzsiiure léslicher
Stickstoff 16.23 11.34
5 5 unldéslicher
Stickstoff 0.31 1.14
Ammoniack stickstoff 1.92 0.93
Durch Phosphowolframsiiure fallbarer Stickstoff
(NH,-N ausgenommen ) 4.91 4.05
Stickstoff in anderer Form 9.40 6.34
Gesamt stickstoff als 100
Tn heisser Salzsiiure léslicher Stickstoff 98.15 90.89
Darunter : Ammoniack stickstoff 11.61 7.48
t Durch Phosphowolframsiiure
fiillbarer Sticlkstoff 29.67 32.57
| Stickstoff in anderer Form 56.87 50.84
In heisser Salzsiiure unléslicher Sticlkstoff 1.85 9.11

I._Das Eiweiss aus dem entkleiten Reis.

A. Monaminosauren.

250 @. getrocknetes Eiweiss wurden mit 750 e.c. Salzsiiure (1.19)
8 Stimden eeckocht. Nach dem Erkalten wurde die Fliissigkeit vom
unlistichen Riickstand abfiltviert und im Vaenum zur Trocknuneg ein-

eedampft, mit absolutem Alkohol iibergossen und trockenes Salzsiiure gas

80 U. SUZUKI, K. YOSHIMURA UND S. FUJT:

bis zur SitHeung eingeleitet. Nach dem diese Operation nochmals
wiederholt war, wurde die Fliissigkeit stark eingedampft, md mehrere
Tage in kaltem Zimmer stehen gelassen. Da aber kein Glykokollester-
salzsaure Salz ausgeschieden war, wurde die Fliissigkeit in bekannter
Weise in die freien Aminosiinrenestern verwandelt und bei mniederem
Druck fraktioniert.

Nach der Verseifung der einzelnen Fraktionen wurden folgende

Aminosiiuren isoliert :

Alanin 9.2 ¢
Lencin 35.8
Phenylalanin 4.9
Asparaginsiiure 1.0
Actives Prolin (Xupfersalz) 7.5
Recemisches Prolin ( ,, ) 3.0
Asnalyse der Aminosiiuren
1. Alanin
0.1400 ¢ Subst 0.201 g CO, 0.0988 ¢ H,O
OHISSH oma, 220 (Sou oa)
C H N
CoHeNOs Ber. 40.45 7.86 15.73
Gef. 39.16 7.84 16.20

Die Analyse stimmt nicht ent tiberein; wahrscheinlich durch kleiner
Menge Glykokoll verunreinigt.

2. Leucin

0.1235 g¢ Subst 0.2458 « CO, 0.1075 ¢ H,O
Cc H
GiB NOs Ber 54.63 9.85
Get 4.54 9.67
3. Asparagin siiure
0.129 g Subst 11.19 W119; 7b)
N
C,H,NO, Ber. 10.52

Gef. 10,28

TEBER DIE EIWEISSSTOFFE AUS REISSAMEN. 81

4. Phenylalanin

0.1867 g Subst 0.4458 ¢ CO, 0.1135 ¢ 11,0
0.3304 ¢ ,, 23.0°°: N (6.9 765")
ec H N
C,H, ,NO, Ber. 65.42 6.66 8.40
Gef. 65.12 6.75 8.53

Das Phenylalanin bildete ein Pikrat, das aus gelben Nadeln bestand
und bei 170—173° unter lebhaftem Schiiumen schmolz. Diese Beobach-
tung stimmt mit der Beschreibung yon Mayeda? vollstindig iiberein.

Das Phenylalanin gab auch eine weisse Fillung mit Phosphowolfram
siinre in saurer Lisung. Es biledete ferner das charakteristische salz-

saure Salz.

5. Prolin

Alctives Prolin kupfer (in Aethyl alkohol léslich)

0.1622 g¢ Subst 0.0445 @ CuO
0.1900 ¢ ,, Losi (110S” '763™™)
N Cu
(C,H,NO,), Cu Ber. 9.60 21.81
Gef. 10.00 21.81

Das in Aethylalkohol unlisliche, razemische Prolin kupfer war nach
mehrmaliger Umkrystallisation immer noch unrein und hat kein befrie-
digendes Resultat gegeben.

Nach Valin und Isoleucin haben wir mit besonderer ‘Sorefalt
gesucht ohne ein positives Resultat zu bekonmen,

B. Tyrosin, Leucin und Glutaminsiiure.

100 @. trockenes Fiweiss wurden mit 609 e.c. 25¢ Schwefelséure im
Riickflusskiihler 20 Stunden gekocht. Nach dem Entfernen der Schwe-
felsiure durch Baryt wurde die Flissigkeit irs Vakuum stark eingeengt,

wobei das Tyrosin als weisse Nadeln sich ansschieden, die ans heissem

I. Mayeda. Zeitsch, f. Physiol. Chem. 57: 263

82 U. SUZUKI, K. YOSHIMURA UND §. FUJT:

Wasser wnkrystallisiert wurden. Ausbeute 0.5 g. Diese Krystalle

gaben starke Millon’sche Reaktion.

0.1908 g Subst 001499 ¢ N
N
CHO; Ber. 7.74
Gef. 7.82

Die Mutter lauge von Tyrosin lieferte nach weiterem Einengen 8.1 g.

Lencin, weleches aus heissem Wasser umkrystallisiert und analysiert

wurde.

0.2092 & Subst 0.0230 ¢ N
N
GO,B NO: Ber. 10.69
Gef. £0.99

Es wurde ferner das Kupfersalz des Leucins dargestellt und analy-

siert.
0.1868 & Subst 0.047 g CuO
Cu
(C,H,,NO,),. Cu Gef. 19.62
Gef. 20.10

Die Mutter lange von Leucin wurde weiter bis auf 100 c.c. eingeengt
wnd trockenes Salzsiinregas bis zur Siittigung eingeleitet und zwei Tage
mit Eis abgekiihlt, wobei die farblose Nadeln von salzsaurer Glutamin-
siiure in reichlicher Menge sich ausschieden. Die Ausbente betrug 18.1 ¢.
(= 14.5 & Glutaminsiinre).

Das gereinigte Salz schmolz bei 208°—204° (uncorr.)

0.4379 & Subst 0.03347 o N
OVSTGD) O° Niys 0.07512 @ Cl
N Cl
C,H,NO, HCl Ber. 7.63 19.34

Gef. 7.64 19.95

UEBER DIE EIWEISSSTOLYE AUS REISSAMEN, 85

Zur Darstellung freier Glutaminsiure wurde das salzsaure Salz
in wenig Wasser geldést, mit Natronlauge neutralisiert wid erkalten
gelassen. Es schieden sich dabei glinzende rhombische Ivystalle aus,

die bei 205—210°schmolzen. Viir die Analyse wurden sie bei 100°

getrocknet,
0.2200 & Subst 0.02161 ¢ N
C,H NOZ Ber. 9.52
Gef. 9.82
Aus 100 @, Kiweiss wurden isoliert.

Tyrosin 0.5 g

Leucin 8.1

Glutaminsiiure 14.5

C. Organische Basen.

Das Eiweiss priiparat wurde mit cone. Salzsiiure 8 Stunden gekocht
upd mit Phosphowolframsiure gefallt. Mit dem dabei entstancenen
Niederschlag wurde die Hexonbasen-Bestimmung in bekaunter Weise

nach der A. Kossel’schen Methode ausgefiihrt. Es wurden gefunden.

In 100 'Veilen Trockensubstanz

Durch Phosphowolframsiure fillbarer Stickstoff (X'3-8) 4.91

ausgen,

Darunter : Histidin stickstotf 0.57
Arginin stickstoff 224 } 4,72
Lysin stickstoff 1.91

Als freie Base berechnet

Histidin D2,
Arginin 6.95 $ 19.02
Lysin 9.95

Tsolierung der organischen Basen.
200 g. getrocknetes Eiweiss priiparat wurden mit 1200 cc, 20%

Schwefelsiiture 20 Stunden gekocht, mit Phosphowolframsiiure gefallt.

84 U. SUZUKI, K. YOSHIMURA UND Ss. FUJI!

Der Niederschlag wurde in bekannter Weise mit Baryt zerlegt. Die
alkalische Fliissigkeit, die freie Basen enthielt, wurde mit Kohlensiure
gesattigt und mit Quecksilberchlorid versetzt. Der weisse Niederschlag
wurde mit Schwefelwasserstoff zerlegt, vom Schwefelquecksilber abfil-
triert, stark eingeengt und im Exikator stehen gelassen. Nach einiger
Zeit schied sich das salzsaure Histidin als farblose Prismen oder
Platten aus, die 2.2 g. betrugen (= 1.6 g. Histidin). Es schmolz bei

228°. Fiir die Analyse wurde das gereinigte Priiparat iiber Schwefelsiiure

getrocknet.
0.1094 g¢ Subst 0.0178 g Cl
Cl
C,H,N,O,. HCl+H,O Ber. 16.94
Gef. 16.27

Das Filtrat vom Quecksilberchlorid-Niederschlag wurde durch
Sechwefelwasserstoff vom Quecksilber befreit, im Vakuum eingedampft,
wm Schwefelwasserstoff auszutreiben und die yorhandene Salzsiiure durch
Silbernitrat befreit und zum Filtrat wurde Silbernitrat und Baryt in
kleinem Ueberschuss zugegeben. Der braune Niederschlag von Arginin-
silber wurde durch Schwefelwasserstofi zerlegt, im Vacuum verdampft,
mit Salpetersiure neutralisiert, wud mit eimem Ueberschuss von Kup-
feroxydhydrat gekocht. Das dunkel blaue Filtrat wurde stark eingeengt
und im Exikator stehen gelassen. Es schieden sich dabei die dunkelblaue
Nadeln von Arginin kupfernitrat aus, die mit 50¢ Alkohol, absolutem
Alkohol und zuletzt mit Aether gewaschen wurden. Die Ausbeute betrug
5.4 g.

Das Kupfer salz verlor das Krystall wasser bei 112° und zerstzte
sich bei 230—252° (uncorr. )

0.211 g Subst verlor bei 120? 0.0194 g¢ Wasser
Krystall wasser
(C,H,,N,0,), Cu (NO;),+3H,0 Ber. 9.15
Gef. 9.21

UEBER DIE EIWEISSSTOFFE AUS REISSAMEN, 84

0.2017 g (Wasser freies Priiparat) 0.0297 g CuO
Cu

(C,H, 4N,0,),. Ca (NO,;), Ber. 11.87

Gef. Levins

Fit die Bestimmung der Salpetersiiure im Kupfer salze wurde es in
Wasser gelést, durch Schwefelwasserstoff zerlegt, vom Schwefelkupfer
abfiltriert, und stark eingeengt. Mit der so bereiteten Fliissigkeit wurde

die Bestimnuamg nach der Nitron methode ausgefiihrt. Es wurde

gefunden.
0.1857 g (Wasser freies Priiparat) 0.0432 g¢ HNO,
HNO,
(CHL NO.) Ca-(NOe): Ber. 23.52
Gel. 23.21

Das Filirat vom Arginin silber wurde durch Salzsiure und Schwetel-
siiture vom Silber und Baryt befreit, mit Schwefelsiiure angesauert und
mit Phosphowolframsiure gefallt. Aus dem Phosphowolframsaure-
Niederschlag wurde in bekannter Weise das Lysin pikrat dargestellt.
Es bestand aus gelblichbraunen Prismen. Die Ausbeute betrug 4.4 g.

(se @. Lysin).

0.3520 Subst 0.2135 ¢ Pikrinsiiure
Pikrinsiure
(Ole G pict Ot Gals ini Ber. 61.07
Gef. 60.65

Aus dem Pikrate wurde das salzsaure Salz dargestellt. Es bestand

aus farblosen Prismen.

0.1122 g Subst 0.0245 g Cl
Cl
C,H,,N,0, CHI Ber. 19.45

Gef. 20.09

$6 U. SUZUKI, K. YOSHIMURA UND §. FUJI:

Aus 100 g. trockenem Eiweiss wurden iscliert.

Histidin 0.81 ¢
Arginin 1.60
Lysin 0.86

II.—Das EBiweiss aus der Kleie.

A. Tyrosin, Leucin und Glutaminsiure.

100 g. Eiweiss aus der Kleic wurde mit 25% Schwefelsiiure hydroly-
siert und in oben erwalmter Weise Tyrosin, Leucin und Glutaminsiure

isoliert. Es wurde gefunden.

Tyrosin 0.3 ¢
Leucin 8.6 g
Glutaminsiure 47 ¢

kk 6Tyrosin gab starke Millon’sche Reaktion. Fiir die Analyse

reichte die Menge nicht aus.
2. Leucin.

0.2002 ¢ Subst 0.02195 g N

N
C,H,,NO, Ber. 10.69%
Gef. 10.96%
3. Glutaminsiiure salzsaures Salz. Schmelzpunkt 202 — 204°
(uncorr. )
0.1709 g Subst 0.01249 g N
0.2518 ¢_,, 0.04750 g Cl
N Cl
; C,H,NO,HCI. Ber. 7.63 19.34
Gef, 7.31 18.85

B. Organische Basen.

In 100 Teilen Trockensubstanz

Durch Phosphowolframesiure fillbarer Stickstoff 4.06

UEBER DIE EIWEISSSTOFEFE AUS REISSAMEN. 87

Histidin Stickstoff 0.45
Arginin Stickstoff 5 + 2.97
Lysin Stickstoff 0.97

Als freie Basen berechnet

Histidin 1.68
Areginin 4.80 $11.54
Lysin 5.06

Tsolierung der organischen Basen.

100 @. trockenes Eiweiss wurden mit 600 ec. 25% Schwefelsiinre 20
Stunden eekocht und in bekannter Weise die Hexonbasen dargestellt.

1. Salzsaures Histidin:—Ansbente 1.2 @. (= 0.88 ge. HHistidin).
Im Kapillar rohr erhitzt schmolz es bei 240—253° und zersetzt sich

eleich darauf.

0.1919 ¢ Subst 0.03849 ¢ N
0.2007 ¢ ,, 0.03415 @ Cl
N Cl
C,H,N,0,HCl+H,O Ber. 20.04 16.94
Gef. 20.05 Leo
2. Areginin nitrat:—Feine weisse Nadeln, Ausbente 2.2 @. (1.6 2.
Aveinin).
0.1664 @ Subst gab 0.0417 ¢ HNO, (Nitron methode)
HNO,
C,H,,N,0,HNO, +4$H,0 der, 25.61
Gef. 25.05
Arginin pikrat Ausbeute 4.5 @ (1.8 ¢@ Arginin)
0.3358 @ Subst 0.0265 ¢ H,O
0.2895 ¢ ,, 0.1628 @ Pikrinsiiure

H,O Pikrinsiiure
OPH IN .O,C.rHgN,O7--atl,O: Ber. ~8.20 56.82
Gef. 7.89 56.07

88 U. SUZUKI K. YOSHIMURA UND S. FUJI:

Arginin Kupfernitrat:—Dunkel griine Nadeln.
0.1839 g Subst 0.0177 g¢ H,O
0.1839 ¢ ss, 0.0243 g CuO
H,O CuO
(C,H,4N,0,), Cu (NO;),+3H,O Ber. 9.16 10.75
Gef. 9.62 10.56

3. Lysin pikrat reichte zur Analyse nicht aus.

Ans 100 @ Eiweiss wurden isoliert.

Hi-tidin 0.88
Arginin 3.40
Lysin Wenig

Gusammenfassung der Resultate.

Aus 100 @ Trocken substanz wurden isoliert.

Eiweiss aus

dem entkleiten Reis +~»«der *Kiele
(Hakumai) (Nuka)

Glykokoll ‘Vorhanden ?
Alanin 3.7
Valin ?
Leucin 14.3 8.6
Prolin 3.3
Phenylalanin 2.0
Asparaginsiiure 0.4
Glutaminsiiure 14.5 4.7
Serin =
Tyrosin 0.5 0.3
Cystin os
Lysin 0.86 --
Histidin 0.81 - 0.88
Arginin 1.60 3.40
Ammonia 2.33 1.13

Tryptophan —_ —

Ueber die chemische Zusammensetzung der
Tamari-Schoyu.
VON”

K. Yoshimura.

Die so genannte Tamari-Schoyn ist eine Art Schoyn, welche meistens
im Mittel und West Japan mit Vorliebe gebrancht wird. Anf die
Bereitune derselhen will ich hier nicht niher eingchen. Es sei nur
erwahnt, dass man als Auseangsmaterial bloss Soja-bohnen snwendet,
wihrend die sewohnliche Schoyu ans gleichen Mengen Soja nnd Weizen
hergestellt wird. Fs ist deshalb wohl begreiflich, dass es zwischen den
chemischen Zusammensetznngen heider Schoyn arten einen merkharen
Unterschied gibt.

Ueber die gewohnliche Schoyu liegt eine ausfiihrliche Abhandlung
von U. Suzuki, K. Aso und JT. Mitarai vor, welche im hiesigen Bulletin
Vol. VIT. No. 4 erschienen ist. Sie haben u. A eine Anzahl stickstoff
haltigen Verbindungen und auch organische Sinren, die wihrend des
Reifeprozesses der Maische gebildet werden, in reinem Zustande isoliert
und die chemische Natur derselben festgestellt. Was nun die Tamari-
Schoyn betrifft, so gibt es noch keine Versuche in dieser Richtrng. Es
war deshalb meine Aufgabe, zuerst die stickstoffhaltigen Koérper zu
isolieren und mit denen der Schoyn zu vergleichen. Die Tamari-Probe,
die ich fiir diese Untersuchtmg anwendete, ist von einer Brauerei aus
Mikawa bezogen. Die Maische wurde im Juli 1906 ans 10 Koku (1
Koku=1.78 Hektoliter) Soja, 580 Pfund Kochsalz und 7 Koku Wasser
hergestellt und im Marz 1908 abgepresst. Die so gewonnene Tamari
eder Pressaft betrug etwa 2.2 Koku. Diese Probe hatte foleende quantita-
tive Zusammensetzung:

Tamari. Schoyu (nach, Suzuki u.A.)
Reaktion Deutlich sauer. Sauer.
Spezifisches Gewicht (bei 15°) 1.205 al)

90 K. YOSHIMURA:

Wasser 45.68 67.15
Trockensubstanz 54.32 32.85

In 100 Teilen Trockensubstanz.

Organische substanz 58.04 49.12
Rohasche 41.96 50.88
Chlor 10.10 27.24
Als NaCl 16.64 44.94
Tamari. . Schoyu.
100 100g 100° 100¢
Gesamt stickstoff 2874 2885 1488 — 1.249
Eiweiss stickstoff 0.645 0.536 0.044 0.037
Ammoniak stickstoff 0.367 0.305 0.166 0.140
Basen stickstoff 0.457 0.379 0.361 0.330

Stickstoffin anderer Form 1.404 1.165 0.917 0.742

Gesamt stickstoff als 100.

Eiweiss stickstoff 22.47 2.96
Ammoniakstickstoff 12.78 11.16
Basen stickstoff 15.89 26.41
Stickstoff in anderer Form 48.86 59.49

Aus der Analyse sieht man, dass die Tamari viel conzentrierter und
Stickstoffreicher als gewoéhnliche Schoyu ist; der Kochsalz gehalt ist
dagegen viel geringer bei der Erstere. Die Tatsache, dass man bedeutend
mehr Eiweissstoffe in der Ersteren findet, beweist, dass die Zersetzung der
Eiweisskérper wiihrend des Reifeprozesses nicht so energiseh vor sich
geht wie in gewoéhnlicher Schoyn.

Organische Basen.

2. Liter Tamari wurden mit gleichem Volumen Wasser verdiinnt,
mit Essigsiiure schwach angesiinert und mit Tannin lisung gefallt. Das
Filtrat davon wurde mit Bleiessig lisung in kleinem Uebersehuss versetzt,
vom entstandenen Niederschlag abfiltriert und das iiberschiissige Blei
durch Schwefelsiiure entfernt, Die vom Bleisulfat abfiltrierte klare Fliis-

UEBER Dil CITEMISCILE ZUSAMMENSETZUNG DER TAMARI-SCHOYU. 91

sigkeit wurde jetzt mit Schwefelsiure angesiiuert und mit Phospho-
wolframsiiure gefillt. Der Phosphowelframsiiure-Niederschlag wurde in
bekannter Weise durch Baryt zerlegt. Mit der alkalischen Fliissigkeit,
die freie Basen enthiclt, wurde zuert cine quantitative Analyse ausgefiihrt.

Es wurde gefunden.

1 Liter Tamari. Basen. stickstoff als 100

Purin basen stickstoff 0.215 4.72
Durch Silber nitrat und Baryt

fillbarer stickstoff 2.686 58.82
Stickstoff in anderer Form — 1.665 36.46

Der Hauptanteil der Fliissigkeit diente zur Isolicrung der Basen.
Zu diesem Zsvecke wurde sie mit Salpetersiiure neutralisiert und mit
20¢ Silbernitrat in kleinem Ueberschuss versetzt. In diesem Silbernitrat-
Niederschlag sollte man die Purin basen suchen. Die menge desselben
reichte jedoch zur weiteren Forschung nicht aus.

1. Der Silbernitrat und Baryt-Niederschlag.

Das Filtrat vom Silbernitrat-Niederschlag wurde mit Silbernitrat
und Baryt in miissigem Ueberschuss versetzt. Der dabei entstandene
braune Niederschlag wurde nach sorgfiltigem Waschen in einem Moérser
gebracht und mit Salzsiiure verrieben. Man filtrierte nun vom gebildeten
Silberchlorid ab und setzte mit Phosphowolframsiiure zu. Wird der
Phosphowolframsiiure-Niederschlag in bekanuter Weise mit Baryt zerlegt,
so erhilt man die Base in freiem Zustande. Die in der Weise dargestellte
alkalische Fliissigkeit gab keine Reaktion fiir Histidin; sie wurde
mit Salzsiiure neutralisiert, bei gelinder Wirme ecingediimnstet und im
Vakuum - Exikator stchen gelassen. Nach einigen Tage schieden sich
die salzsauren Salze der Base als farblose Prismen aus, die aus heissem
Wasser umkrystallisiert wurden. Die Ausbeute betrug ungefiihr 3 ¢.

Werden diese Salze mit absolutem Methylalkohol verrieben, so geht
der Haupt anteil (etwa ?/,) in Lésung iiber wihrend ein Teil (*/;)

ungelést bleibt.

92 K. YOSHIMURA :

A. Der in Methylalkohol unlésliche Teil (Pudrescin).

Der in Methylalkohol unlésliche Teil bertug etwa 1 g. Aus heissem
Wasser umkrystailisiert schied sich das Salz als lange, farblose
Prismen aus, die in Wasser leicht, in Aethylalkohol und Aether schwer
léslich waren. Im Kapillar rohr ecrhitzt schmolz es bis 290° nicht.

Fiir die Analyse war das Praparat im Vacnem bei 100° getrecknet.

0.110 g Subst. 0.01941 ¢ N
0.220¢ ,, 0.09592 ¢ Cl
N Cl
C,H,,N.2HCI Ber. 17.39 414.09
Gef. 17.64 43.60

Dag Platin chiorid-doppel Salz:--Wird das salzsaure Salz in wenig
Wasser gelést, mit Platin chlorid lésung versetzt und langsam einge-
dampft, so scheidet sich das Platin-doppel Salz als citronen gelbe unregel-
miissige Tafeln aus. Es lést sich in kaltem Wasser ziemlich sehwer;
im Kapillar rohr erhitzt wird es gegen 230° schwarz ohne zu Schiiumen.
Das gereinigte Priiparat war im Vacuum bei 100° getrocknet und

analvsiert.

0,229 ¢ Subst. 0.0870 ¢ Pt.
Pt.
Ce nie2HCl Pe Cle Ber. 39.20
Gef. 39.17

Das Pikvat:—Zur Darstellung des Pikrats lést man das salzsaure
Salz in wenig Wasser, gibt cine entsprechenden Menge Natrium pikrat
zu. Das Gemisch wird so lange erwirmt bis klare Lisung anftritt.
Nach dem Erkalten seheidet sich das Pikrat als hellgelbe Prismen aus,
dic in kaltem Wasser schwer lislich sind. Im Kapillar rohr erhitzt wird
es gegen 240° dunkel braun und zersetzt sich bei 260° (uncorr.) Fiir

die Analyse wurde es im Vakuum bei 100° getrocknet,

UEBER DIE CHEMISCILE ZUSAMMENSETZUNG DER TAMARI-SCHOYU. 93

0.2650 ¢ Subst. 0.0412 ¢ N
N
ating in ( OnE Ons Ber. 20.52
Gef. 20.56

Die Analyse stimmt also mit der Formel C,H,.N, oder Putrescin
tiberein.

B. Der im Methylalkohol lésliche Teil (C,H,N,).

Der in Methylalkohol Jésliche Teil betrug ungefihr 2 ¢. Aus
heissem Methyl alkohol umgelést scheidet sich das Salz als farblose
Prismen aus. Im Kapillar rohr erhitzt schmilzt es scharf bei 232°

(uncorr). Fiir die Analyse war es im Vacuum bei 100° getroknet.

0.1851 g Subst. 0.08944 ¢ N
ON22510 ., 0.04471 ¢ Cl
N Cl
C,H,N,2HCl Ber. 21.43 36.23
Gef. 21.3 36.50

Ein Teil des Salzes wurde in bekannter Weise in das Pikrat ver-
wandelt. Es bestand aus citronengelben glinzenden rhombischen
Prismen oder Tafeln. Es war in heissem Wasser und <Alkohol leicht,
in kaltem Wasser schwer und in Aether unléslich. Im Kapillar rol
erhitzt schmolz es bei 230° (uncorr.) and zersetzte sich unter Schiinmen.

Fiir die Analyse war es im Vacuum bei 100° getrocknet.

0.2265 @ Subst. 004788 ¢ N
N

CO palalaisl (plat isi) Ber. 21.69

Get. 2114

Aus der Analyse und anderen Beobachtungen wurde festgestellt,
dass diese Base mit der von U. Suzuki u. A. aus Schoyu isolierte Base
Cro N. identiseh war.

Il. Das Viltrat vom Silbernitrat und Baryt-Niederschlag.

Das Filtvat vom Silbernitrat wnd Baryt-Niedersehlag wurde nach

94 K. YOSHIMURA:

dem Entfernen des Silbers durch Salzsiure und des Baryts durch Schwe-
felsiure, mit Schwefelsiiture angesiiuert und mit Phosphowolframsaure
gefallt. Die freie Base, die durch Zerlegung des Phosphowolframsiure-
Niederschlags durch Baryt in bekannter Weise erhalten war, wurde
mit Salzsiiure neutralisiert, langsam eingediinnstet und im Evikator
stehen gelassen. Die salzsauren Salze der Base schieden sich dabei als
farblose krystalle aus, die stwa 5 g. betrugen. Diese krystalle lessen
sich durch Behandeln mit absolutem Alkohol in zwei Fraktionen tremen.
A. Die in absolutem Alkohol uwnlésliche Fraktion (Ornitin).
Diese Frakktion enthielt eine erhebliche Menge anorganische Salze,
die in Methylalkohol woléslich waren. So wurde sie mit kaltem Methyl-
alkohol sorgfaltig verrieben und abfiltriert. Das Filtrat wurde langsam
bis zur Trocknen emgedampft und mit absolutem Alkohol und Aether
gewaschen. Als der Riickstand aus warmem Wasser wmngelést wurde,
schied sich das salzsaure Salz als farblose Prismen aus, die in Wasser
leicht, in absolutem Alkohol und Aether schwer léslich waren. Die
Ausbeute betrug ungefiihr 2 g. Im Kapillar rohr erhitzt schmolz das
Salz bei 203° (uncorr.) unter Schaiimen. Fiir die Analyse wurde das

gereinigte Priiparat im Vakuum bei 100° getrocknet.

0.2110 g Subst. 0.02924 ¢ N
0.1463 ¢ ,, 0.05076 g¢ Cl
N Cl

C,H,,N,0,2HCl Ber. 13.66 34.63
Gef. 14.00 ~34.69

Das Pikrat bestand aus gliinzenden gelben Tafeln oder sternférmig-
verwachsenen Prismen, die in kaltem Wasser schwer, in heissem Wasser
dagegen leicht léslich waren. Im Kapillar rohr erhitzt zersetzte es sich
bei 248—250°. Fiir die Analyse war es im Vacuum bei 100° getrocknet.

0.2120 g Subst. 0.04073 ¢ N
N
C;H,,N,0, C,H,N,O, Ber. 19.39
Get. 19.21

UEBER DIE CHEMISCHE ZUSAMMENSETZUNG DER TAMARI-SCHOYU, 95

Das Platinchlorid-doppelsalz:—Gelbe Prismen; leicht Jéslich in

Wasser, schwer in Alkohol. Im Kapillarrohr erhitzt zersetzt es sich bei

923°
0.1620 & Subst. 0.057 « Pt.
Pt.
C,H,.N,O, H,PiCl, Ber: 35.95 |
Gef. 35.19 |

B. In Methylalkohol Jésliche Fraktion.

Von dieser Fraktion haben wir nichts isolieren kénnen.

ZUSAMMENFASSUNG DER RESULTATE.

Aus 1 Liter Tamari wurden isoliert:

Putrescin 0.8 ¢
Ornitin

Base C,H,N, 0.7
NH, 4.5

Das Vorhandensein yon Arginin, Lysin und Histidin wurde nicht
constatiert. Dass das Eiweiss (Glycinin) aus Soja bohnen durch Saure
Spaltung ziemlich erhebliche Menge Hexon basen liefert haben T. B.
Osbrone und S. H. Clappt und auch der Verfasser selbst nachgewiesen.
Nach einer quantitativen Analyse wurden die folgenden Zahlen gefunden.

In 100 Teilen Glycinin.
(Osborne und Clapp?.) ( Yoshimura.)

Arginin 9.12 4.87
Lysin PATA 2.51
Histidin 1.39 1.85

Nach E. Schulze und E. Winterstein? wird das Arginin durch
Baryt wirkung im Harnstoff und Ornitin gespalten.
C,H,.N,0,+H,0=C.H,,N,0, +CH,N,O
Arginin. Ornitin. Harnustoff.

1. Amer, Jour. Physiology 1907 79 468—74.

2. E. Schulre u. E. Winterstein: Ber. deutsch. chem. Gesellsch, 1897 30 2879

96 K. YOSHIMURA:

Das Ornitin soll nach A. Ellinger’ durch Fiulnis weiter in Putresein

verwandelt werden.

CH,(NH,). CH,CH,CH(NH,)COOH

Orintin.
=CO, +CH,(NH,)CH,CH,CH, NH, ,
Putrescin.

- Es ist deshalb hochst wahrschisslich, dass das Ornitin und Putresein
in Tamari auf Kosten des Arginins gebildet wurden.

Nach U. Suzuki und K. Aso soll die Base C,H )N2, die auch in
gewohnlicher Schoyu gefunden wurde, wahrscheinlich durch Bakterien
wirkung aus Histidin gebildet werden.

Ueber das Schicksal des Lysins muss man die kiinftige Forschung

abwarten.

1. <A, Ellinger: WHoppe-seyler. Zeitsch. f. Physiol. Chem. 1902 29 334.

On the Carbohydrates of Shoyu.
BY

R. Mitsuda.

1. Isolation of sugars from shoyu.

Although the existence of sugars in shoyu is easily shown by the
reduction of Fehling solution, it is comparatively difficult to isolate them
in a pure state. At first, T have tried to isolate them as the lead com-
pounds. For this purpose 400 c.c. of shéyu were evaporated under a low
pressure. The separated sodium chloride was removed, the mother liquor
was diluted with water and precipitated with basic lead acetate. To the
filtrate of the lead precipitate was now added a moderate excess of basic
lead acetate and ammonia whereby the greater part of the carbohydrates
was thrown down. This precipitate was now suspended in water and
decomposed with hydrogen sulphide. The filtrate of lead sulphide which
contains the sugars was evaporated in vaecnum to expell the hydrogen
sulphide and the osazone was prepared in the usual manner,

Afterwards, it was found that such a long operation is unnecessary.
After removing the greater part of soldium chloride by evaporation, it
was deeolorized with animal charcoal and directly warmed with phenyl-
hydrazine solution (for 400 ¢.c. shoyu were used 25 g. phenylhydrazine
hydrochlorate and 33 ¢. sodium acetate) whereby mixture of various
osazones separated out as yellowish erystalline mass. By repeating the
reerystallisation of the erude osazones from a hot 60¢ alcoholic solution,
about 5 grs. pure glucosazone were obtained, which was dried in vacuum

at 100° and analysed.

0.1674 @ Subst. 0.3664 ¢ CO, 0.0912 ¢ H,O
0.12682 ,, 16.6" N(10° 765™™- )
(0, iB N
Glueozazone C,H, .N,O, cale, 60.14 6.14 15.64

found 59.78 6.05 15.84

98 R. MITSUDA:

Reerystallized from hot water or froma 60% alcohol, the glucosazone
separates as characteristic yellow needles which melt at 204°.

On cooling the filtrate of the glucosazone a little galactosazone was
obtained. The crude product was purified at first by washing with cold
acetone and then recrystallized from boiling 20% alcohol. In this way
T obtained about 0.6 ¢. pure galactosazone which melts at 184°. The

determination of nitrogen gave the following result:

0.1326 @ Subst. r77ee WN (49 7ee as
N
C, sHaaN,0, caleulated 15.64
found 15.75

Althongh T have recognized the presence of maltosazone under the

microscope I was not able to isolate it.

2. About furfurol.

When shéyu or tamari is exactly neutralized with dilute caustic
alkali and subjected to distillation. |The destillate produces the red

coloration with aniline acetate, which is the characteristic reaction for

furfurel. So there is no donbt that furfurol exists in shoyu or tamari .

in the free state. But the quantity being too small, it is only possible
to determine it by the colorimetrie method. For this purpose, a dilute
standard solution of furfurol was prepared by dissolving 1 g. of pure
furfurol in 5 liters of water, so that 1 ce, of the solution contained
0.0002 g. of furfurol. For the determination, 100 ¢.c. of the sample were
diluted with water to 120 e.c. exactly neutralized with alkali and subjected
to distillation until the distillate gave no more red coloration with aniline
acetate. The distillate was then dilnted up to 100 ee,

Tn two tall heakers of the equal size was put the mixture of 5 e.c.
of aniline acetate and 5 ec. strong hydrochloric acid. To one beaker
was added 10 ¢.c. of the distillate and to the other 10 ee. water and so

much standard furfurol solution from a burette, until the same intensity

of the red colouration in both heakers was obtained, Tn this way we

ON Till CARNBOUYDRATE OF SILOYU. 99

can calculate how many e.c, of the standard furfurol solution is equal to

10 ce. of the distillate. The result was as follows:

No, Trade mark. 9% of furfurol. ‘Total acidity (as butyric acid.)
1 Kikuichi 0.0001 0,28
2 Minakami 0.00015 0.65
3 Homare 0.0002 {03
4 Tamari No. 1 0.0002 0.91
5 » No2 0.0001 0.29
6 Ps No. 3 0.00005 0.26

As the aboye table shows the amount of furfurol is very little im each
sample, yet we could find out the differences clearly, because the reaction
was very delicate.

We see also in the above table that the amount of furfurol is generally
ereuter in those samples which show stronger acidity though there exists
no direct proportionality between them.

In the next experiment I found that the amount of furturol increases

with the ages of moromi. Thus:

No. Ages of moromi. Furfurol. Total acidity.
1 2 weeks Trace -—
y L year 0,0002 0,42
3 2 years 0.0004. 0.45
| 3 years 0.0006 0.58

From these facts we can probably conclude that furfurol is very
gradually produced by the action of organic acid during the ripening
process of moromi., This view is further supported by the following fact:

When shoyu or tamari is distilled without neutralization. The fur-
furol reaction in the distillate is far stronger than, when it is previously
neutralized, This fact indicates that furfurol is formed from pentose or
pentosane by the action of acids during destillation, So we can suppose
that even at ordinary temperature the formation of furburol is slowly

going on during the ripening of moromi.

100 i. WiTSUDA!

The view that furfurol is produced by microbes during the ripening
of moromi, is not probable, because we find no furfurol in the newly
prepared saké, while the preserved one contains a tolerable quantity of
it. In the case of sh6yuanoromi the analogy in also found thus; almost
no furturol was present in the moromi of two weeks.

Further investigation on this point is desirable.

3. About total pentosane.

For the determination of total pentosane 50 cc. of the sample were
mixed with 20 ¢¢. strong hydrochloric acid and subjected to distillation,
adding from time to time a 12¢ hydrochloric acid solution until the dis-
tillate reached 400 c.c. and the furfurol in the distillate was precipitated

by phloroglucin in the usual way. The result was as follows:

Mask. Drymatter. % Pentosane. 9% Pentosane in dry matter.
Kikuichi 32.80 0.41 1.26
Homare 46.43 0.50 1.08
Minakami 45.21 0.53 tsi lr)
Tamari No. | 56.74 0.91 1.18

We see from this result that the quantity of pentosan is directly
proportional to the dry matter in shoyu or tamari. We see further
that the amount of total pentosane in shoyu and tamari is comparatively
very little, while in the original material, in shdyu-koji which is pre-
pared from the same quantity of soja bean and wheat it is contained in
far greater quantity. Thus I found in one sample 8.31% of the dry matter.
This fact shows that the greater part of pentosane in koji remains in-
soluble during the ripening process of moromi.

In the next experiment I compared the amount of total pentosane in

different stages of ripening of moromi.

Ages of Moromi. % Pentosane. 96 Pentosane in dry matter,
2 weeks 2.03 6.05
1 month 1.60 4.77

5 months AES 6,42

ON THE CARBOHYDRATE OF SILOYU. 101

1 year — 1.89 5.06
2 years 1.98 5.17
3 years 1.79 4.68

The above result shows that the pentosane decreases only very
gradually, either by the assimilation of microbes or by the action of
organic acids. J found a more striking result when I determined the
total pentosane in the pressed juice of moromi at different stages of

ripening. Thus:

Age of Moromi. % Yotal Pentosane.
2 weeks 0.49
1 month 1.31
5 months 1.37
1 year 0.94
2 years 0.72
3 years 0.67

We see from the above result that the pentose and pentosane in
sh6yu-koji is gradually dissolved in salt water and reaches the maximun

at 5 months and then decreases again gradually.

;

Studies on the Microorganisms of ‘‘Tanezu’’
(Japanese vinegar ferment).

T. Takahashi,

Japanese vinegar is made from various kinds of raw materials :—
altered “saké.” altered “moromi’’-mash of “Saké,’? vinegar mash! especially
prepared for this purpose, and “saké-kasn’”? (pressed residue) of “Saké’?-
mash. “Kasuzw’ is the name generally applied to vinegar prepared from
“Saké WZasu.”

In preparing “Kasuzu’’ water and “Saké Kasu,’ which has been
allowed to ferment for about a week, are mixed in certain proportions,
and the mash is pressed in a special press. The fluid part thus obtained
is divided into two equal parts, one of which is heated to about 80O—90°C.
This heated fluid is mixed with the other part and after the addition of
some previously prepared mash? is allowed to ferment for about one month

or more.

1. This vinegar mash is prepared by mixing rice “ Koji,’ steamed rice and water, very
similar to the “ Moromi ’-mash in the manufacture of “ Sake.”
2. This mash is called “ Tanezu” in Japanese. Torazo Nishimura* analyzed « Tanezu ”

with the following results :—

Speravel(atet271 Gol). led Gn GSMEGET Mer) ass sp, 1) 1,00
In 100 c.c.

WGI EESUDStADCE MH ccm tamten mT ve) sve- sci ace QJAOZD/ EMS.
ECONO Meet ery eh er EC EE cts) cis" aes) ver QROZOP
Nolatlevacios (aspacetic acid) im ancelusesmere merit ;t. 1 «eel FeTOONNNsy
Extract Bee, Metco ee Doh AR. RSP OM CO) - OM Dey a aC A375 os
PRGtALaeNIiK OCC sts sok Were seems Ue SENy ase! Sova’ Jock tess) OLQOR

IAC" Decl arn tae Sal GAS AS. © Pee COOL. Dos 8h ee aT 0.143

* T, Nishimura: “ Kasuzu J6z0ron,”’

104 T. TAKAHASHI:

Thus the addition of a previously fermented mash (Tanezw), which

contains immense numbers of acetic bacilli, is an essential part in the
preparation of vinegar.

Tt has seemed to the writer that an investigation of the microorganisms
of “Tanezu” is very important and interesting both from the practical
and the scientific points of view.

Two samples were used for the investigation, one® from “Handa-
machi” the other! from ‘Nagoya.’ and 7 different varieties of acetic
bacillus, 2 varieties of wild yeast and 2 varieties of Aspergillus were

isolated. They are described below.

PART I. BACTERIA.
No. 1. Bacterium Ascendans Henneberq var Tanezu.

T.—Form and size: Commonly 3x1 or 44x14. — Involution
form is found in film of beereultnre (one month), but not in “Sakekasu
Sumashi’”® even after two months. Chain form was observed in the former
enlture and each eell was very short appearing almost like a cocens,
Nonmotile.

TT.—Growth: 1. Solid culture: a. Plate cultures: - “Moromi”
agar: Round, elevated, light grevish, waxy colony appears on surface.
“Saké”-agar: Somewhat bluish-white, waxy, round (almost spherical)
colony appears after 7 days at 25°C. Tts periphery is wavy when seen
with a magnification of 125. b. Surface cultures: “Saké”’-agar: Grows
as fine granular covering (40 hours at 30°C), waxy and light rose eolored

35—45°C and further 18 days at

growth of broad stream (24 honrs a

3. A vinegar factory of M. Nakano.

4. A vinegar factory of D. Sasada.

5. A mash made from “Sakekasu”: 1 Kgrms. of “Sakekasu” is mixed with 4 L. of
water; and after letting stand at the room temperature for about one week, pressed, sterillized,

puting in to a tightly corked flask.

STUDIES ON THE MICROORGANISMS OF “TANEZU.” 105

30°), or pasty yellow-grevish white covering with almost smooth® surface
(10 days at 21.2°C or 6 days at 20.5°C). “Koji’’-extract-agar: Forms
yellow-greyish white pasty coverings (6 days at 20.5). “Koji’’-extract
gelatine: Grows very feebly (5 davs at@4°C). e¢. Stab-cultures: “Saké’’-
agar: Forms lightly rose colored pasty growth along the mouth of the
stab canal (6 days at 20.5°C), or becomes lustrous at margin (10 days at
25°C). Beer wort-agar: Grows very feebly (6 days at 20.5°C). “Kojv’’-
extract-agar: Forms dirty yellow, pasty, finely granulated growth at
the mouth of the stab canal (6 days at 20.5°C). “Woji-extract-gelatine :
Forms creamy growth along the stab canal (38 days at 16.5°C).

2. Fluid culture: (6 days at 20.5°C).
Culture media T. Remarks.
Yeast water: Forms island on surface. Flnid clear. No sediment.
Yeast water glucose: Do.
Wort* (without hop): Forms ringshaped growth. Fluid clear.
Wort: Somewhat thick film’ was formed on surface. Fluid clear and

without sediment.
“Koji’-extract: Forms somewhat thick dirty yellow film. Finid clear
and almost no sediment.

Hyduck’s solution: Growth almost nil.
Bouillon: Forms ring-shaped growth.

Thus we can distinguish this bacillus from No. 2, No. 5, No. 4.
No. 5, by the formation of rather thick film on the surface of the medium.
Further, its inability to avail itself of asparagin as a nitrogen source
(Hayducek’s solution) and the cireumstance that the medium remains clear
are characteristics which distinguish it from the already known varieties

of B. aseendans.

6. Bacterium aceti. Brown. var Tanezu forms under the same conditions (10 days at
21.2°C) very lightly rose colored colony with granular surface.
7. The wort used in this investigation was prepared from malt (7022) and rice (3022).

8, Thicker than B. aceti, Brown, var Tanezu

106 T. TAKAITASHT

Culture media IT, Remarks.

“Saké Kasu’’-mash: Forms very thin (perfectly smooth) film (9 days
at 25.6°C), but sediment is comparatively conspicuous.
Fluid becomes turbid after shaking. Film becomes blue when
treated with solution of I+KT.

Beer®: Forms very thin film on the wall of the vessel. Flnid clear
(7 days 25°C), but when shaken, the film became agglutinated
into one mass. Total acidity increase by 0.9912¢ after one
month.

Wine’: No growth after 20 davs (at 30°C), but after 5 days more
a cohasive film appeared.

Pastenr’s solution'!: Forms very thin film after 6 days at 30°, fluid
remaining clear.

Beijerink’s solution'?: No growth (7 days at 25°C).

“Saké’’!?: No growth (30 days at 25°C).

Diluted “Saké” (water 20¢): No growth (30 days at 25°C).

Diluted “Saké” (water 304): Do.

Diluted “Saké” (water 50¢): Forms film on the wall of the vessel, the
medium remaining clear. Folds were formed more or less
after three months.

g. and to. Beer and wine used was “Sapporo” beer and « Kikusui shirushi” natural
wine.
9. “Sapporo” beer: It contained 4.622, of alcohol and 0.09494 of total acidity as

sucemic acid.
10. “Kikusui Shirushi” natural wine, containing 3.8694 of alcohol and 0.6794 of total

acidity.
11. Glacial acetic acid 12.5 c.c. Absolute alcohol 22.5 c.c.
Ammumonsenm SOEPUANE 3x, ny sas es kas “en, ae a
Potassium Py eT or cs OB
Calcium = asa Ivey) stv) wea’ goeebo Sees Angee EiCeinenmmen
Magnesium As pe Nees owe: |e peg Meee ne aon
Wratere: oo permet cea Bice sae sia Soe bn gn
12... Waterss ic. Secctsnte sa OO Cc. Alcohol... 5... sco! ite eases aia
Ammonium phosphate, <0. 60. ss. cen Sen ave cea’ Sen! ORRENe
Potassium a ate. Seatetant Peau, tad Cano nee 0.01 yr.
13. ‘Alcohols. di Geen 1 710% Total acidity. (as sucemic. acid)... 0.171196.
Volatile acid rye bisa gr 1024 96, Extractive matters ... 1... ... 3645796.

Sagar ss. Kess, sxe) pers O05.

STUDIES ON TUE MICROORGANISMS OF “TANEZU.” 107

Thus we see that this bacillus cen assimilate ammonium nitrogen,
and it must therefore be a varicty of B. aceti. Pasteur according to
Hoyer’s™ classification of acetic bacillus.

I11.—Behavior towards carbohydwates and alcohol. The production
of acid from carbohydrates was tested with yeast-water or bouillon!’ con-

taining a carbohydrate (at 25°C). The results are given below :—

Acid droduction.

—— ee eee —_
GB. ascendans Henn. B. ascendans
Substance. Growth. Var Tanezu Henneberg.

Glreose. A thin ring formed after 8

days. Fluid clear, not becom-

ing turbid on shaking. ah ae ae =
Fructose. Forms yery thin film after

8days. Fluid slightely turbid

and the film sinks on shaking. qe 4F =
Galactose. Forms very thin ring after 8

days. Fluid remained clear,

but beeame turbid on shaking. — =
Rhamnose. ‘Turns very slightly turbid

after 3 days. Forms white

and brittle film, which breaks

on shaking, the fluid remain-

ing clear notwithstanding. - /
Saccharose. Forms islands and ring after

7 days, fluid clear. After

8 days more Fehlings solu-

tion gave no reaction. t 2
Maltose. Forms white film along wall

of test tube, fluid clear. - ~
Arabinose. Forms trace of ring and

film afer 8 days. Film sinks

to bottom on shaking + + ~

14. Deutsche Essigindustrie 1899. Nr. 1.

15. These media were used for all the varicties,

108 f, TAKAHASHI! if

Lactose. Forms trace of ring and

slight turbidity after 7 days. trace -
Raffinose. Do. = =
Mannitol. Forms very thin and _ slight

turbidity after 3-18 days. _ =
Dextrin.1® Forms very thin film and a

little turbidity after 3 days.

Fluid became clear after 15

days more. = ~
Starch. Forms white film and a little

turbidity after 7 days. ~ =

Inulin. No growth after 3 days, but

after 15 days more a white

brittle film was formed. — /
Ethylaleohol. = fe

Thus this bacillus forms acid from arabinose, glucose, fructose,
saccharose, lactose and dextrin and hereby distinguished from the B.
ascendans Henneberg already known.

IV.—Fermentation products: Propyl; ethyl-, methylalcohol and
fuseloil were found in the distillate of koji-extract culture after + days at
25—26°C, but acetic’’ and butyric acids, acetone. and methyllactate were
not found in it.

V.—Conditions of temperature: Optimum temperature lies above
30°C. Grows very slowly below 14°C, but very energetically above 35°C
and produces a rose color. 55°C for 10 minutes in “Saké Kasu’-mash'*
does not kill the cell, which dies at SO°C,

From the above description We see that this bacillus belongs to Hoyer’s
quick vinegar bacilli or B. accti Pasteur and must be looked upon as a

variety of B. ascendans Henneberg.

16. This substance contained more or less glucose.
17. This acid was found in the culture of diluted “Saké” or “ Saké ”—agar.

18. ‘This mash was used for this purpose throughout these studies.

STUDIES ON TIE MICROORGANISMS OF “TANEZU.” L109

No. 2. Bacleritum Acetosum Henneberg var Tanezu.

J.—Form and size: Commonly 2.5 x lv or 3p x 1p and diplo-
eoecus or often long chain. Involution form is found in film of beer
euliure (one months: ..-5-. 6... .....Non-motile,

Il.—Growths: 1. Solid medium: a. Plate cultures: ‘“Moromi”-
agar: Round greyish-yellow colony with fine concentric rings and
radiations. - Central part of colony somewhat elevated but margin rather
flat with wavy'’ periphery. “Saké”-agar: Forms somewhat elevated light
yellow, waxy colony, with a special elevation in the central part. Under
the microscope the periphery is very irregular. b. Surface cultures:
“Saké”-agar: Forms dirty greyish white covering with coarse granulated
surface, and very ihick folded filn*® on surface of condensed water (24
hours at 35°—45°C and 2 days more at 30°C). After 18 days at 30°C,
the surface growth changes to milky. Forms slightly folded coyermg and
very small film on condensed water after 3 days at 22—25°C), or grows
waxy In streaks, on which watery drops form after 4 days at 23.7—24°C,
but after the lapse of 2 months the culture surface turns wet and granular.
“Koji”-extract-agar (at 23.7—24°C. 4 days): Grows in white and obscure
streaks (thick part of medium) or semi-transparent (vather thin part of
medium) and granular in middle but slant and smooth in periphery.
Surtace layer generally wet. ‘\KKoji’’-extract-gelatine: Weak growth in
streaks (4 days at 14°C), or yellowish creamy growth (38 days at 16.5°C),

In general this bacillus grows better on “Saké”-agar than on “IXoji”-
extract agar.

ce. Stab cultures: “Saké’-agar. Forms white and folded colony
at mouth of stab canal (4 days at 23.7—24°C). Wort-agar: On mouth
forms white pasty colony. “Saké’’-gelatine: Trace of growth at mouth
(4 days at 14—15°C). “Koji’-extract-gelatine: Weaker growth than
on “Sake’-gelatine (4 days at 14—15°C). Neutral “Koji’-extract-
gelatine: very weak growth.

19, The wary contour is observable from the back side of the colony.

20. At 26-29°C for the same length of time, there was no film,

110 T. TAKAHASHI:

2, Fluid medium: (4 days at 23.7—24°C).

Culture media I. Remarks.

Yeast water: Forms film. Fluid clear, with sediment.

Yeast water-glucose : Do.

Wort (without hop): Forms slimy film. Fluid clear with more or less
sedimentation.

Wort: Forms ring after 3 days at 25°C, after further 4 days appeared
film accompanying turbidity.

“Koji’-extract: Forms somewhat thick film with some deposits under
clear fluid.

Hayduck’s solution: Forms thin brittle film with some deposits under
clear fliud.

Bouillon: Forms thin film with turbid deposits.

The turbidity of the bouillon culture and the formation of a shiny
film in wort are points of similarity between this bacillus and B. aceti
Hansen. It can be distinguished from bacillus No. 1. by the thickness
of the tiln.

Culture media LI. Ntemarks.

“Saké-kasu’’mash: Forms white folded tilm?*! after 4 days (25.6°C), the
fluid becoming turbid. Growth exceedingly rapid, paralleled
only by No. 7. (B. Xylinoides). Sediment becomes rose
red in color atter very long culture.

Beer: Forms trace of deposit after 7 days (at 25°C), and thin film
oyer clear fluid after 9 days. Total acidity inereased by
1.3354¢ after one mouth,

Wine: No growth even after 30 days (at 30°C).

Pasteur’s solution: No growth at 30°C (after 26 days) and hereby dis-
tinguishable from 1, aceti, Pastorianus.

Beijerink’s solution: No growth after 7 days at 25°C.

Diluted “Saké” (water 207): No growth even after one month at 25°C,

21, This film stains yellow with I+-KI. solution.

STUDIES ON THE MICROORGANISMS OF “TD ANEDU. 111

Diluted “Saké” (water 304): Forms trace of sediment after 25 days
(25-—28'5°C),

Diluted ‘Saké”’ (water 50¢): Forms very thin film with trace of ring
over very turbid fluid after 25 days at 25—28.5°C. Film
thickens after 3 months and forms folds and a part of it
extend upwards along wall of apparatus.

I11.—Behalior towards carbohydrates and alcohol.
Acid production.
——_ Or —

B. acetosum. TB. acetosum.
Substance. Remarks. Henn. Var Tanezu Henneberg.

Arabinose. Forms film over turbid fluid
after 8 days at 25°C. Film

settles down after breaking on

shaking fe de dk =
Glucose. No growth after 5 days at

25°C. Turbidity commence on

surface part after 11 days. oP ap a +f
Fructose. Forms film with a trace of

turbidity after 8 days at 25°C. = aa
Galactose. No growth after 8 days at 25°C. / 5
Rhamnose. No growth after 3 days at

25°C, but after 15 days more

forms somewhat thick film

with production of turbidity.

Film does not breack on

shaking = =
Saccharose. Forms white film?2 after 7

days (at 25°C), it settles

down without breaking on

shaking, and therefore causes

almost no turbidity. trace =

22, Under the same condition bacillus: No. 1. (1. ascendans. Ilennebeg. var Tanezu)

forms islands and rings, but no film,

112 T, TAKAHASHI:

Maltose. Forms white and folded film,?

after 8 days at 25-26°C which

settles down without breaking

and causing almost — no

turbidity. — ~
Lactose. Forms white film?* after 7

days at 25°C, with a trace of

turbidity. Film breaks on

shaking, causing turbidity. _ ~
Raffinose. Almost no growth eyen after

17 days.25 ' + —
Mannitol. No growth after 3 days, but

after 15 days more forms very

thin film a part of which

settles on shaking. = =
Dextrin. No growth after 3 days at

25°C, but after 15 days more

became slightly turbid. tear =
Starch. Forms white2® ring with

slight turbidity after 7 days
at 25°C, and after 2 days
more forms white — easily
broken film. ss =
Inulin. No growth after 3 days at
25°C, but after 15 days
more forms film a part of
which breaks on shaking,
causing turbidity.27 - _
Ethylaleohol. eing ae ‘f
23. Bacillus No. t. does not form film under the same condition,
24. Bacillus No. 1. forms a ring under the same conditions.
25. Unlike, bacillus No. 1. which forms a ring with some turbidity after 7 days.

26. Bacillus No. 1, forms film under the same condition.

27. Bacillus No. 1. does not cause turbidity,

‘

STUDIES ON THE MICROORGANISMS OF “TANEZU.” 113

Thus. this bacillus forms acid from arabinose, saccharose, raffinose,
dextrin and hereby distinguished from the B. acetosum Henneberg already
known.

IV.—Fermentation products. Propyl, ethyl, methyl-aleohol and
fuseloil were found in the distillate of Koji-extract culture after 4 days
at 25-26°C, but acetic and butyric acids and methyl-lactate were not
found in it.

V. Conditions of temperature: Optimum temperature for growth
lies above 30°C. Grows very slightly below 14°C. Heating to 55°C
for 10 minutes in “‘saké-kasu’’ mash does not kill the eell, which dies at
80°C.

This bacillus belongs to Hoyer’s beer vinegar bacilli, Bact. rancens
Beijerink, and has many similarities to B. acetosum Hemneberg. Further,
if is distinguished from B, aceti Brown by its property of forming reducing

sugar from mannitol.

No. 3. Bacterium aceti Brown var Tanezu TI.

{—Form and size: Short bacillus commonly 24x ly, 34x 1p.
Involution form is found in film of beer culture (one month), or
“Saké-kasu’’-mash (2 months). Nonmotile.

TI.—Growth: 1. Solid medium: a. Plate culture: “Maromi’-
agar: Round elevated greyish white, moist colony appear on surface.
Round colony with smooth margin in interior of medium (at. 125/T)
“Saké’-agar: Forms round elevated greyish white, moist colony. — b,
Surface culture: “Saké’-agar Forms greyish vellow, pasty, smooth,
lustrous covering at 30°C after 40 hours, or dirty vellow, pasty, lustrous
covering with clear condensed water after 24 hours at 35-45°C and 2 days
more at 30°C. Covering becomes rose color after 18 daysyat 30°C, and
greyish white at 25°C after 18 days. Covering of old culture slimy**.

“Koji-extract”-agar (3 days at 25°C): Forms semi-tranparent

covering on surface of thin part of medium, while white pasty on thicker

28. Another variety described in this paper does not form slimy covering.

114 T. TAKAILASHT:

part. Forms film on clear condensed water. “Koji-extract’’-gelatine:

Forms dirty yellow creamy growth (38 days at 16.5°C).

C. Stab-culture: ‘‘Saké-agar’: Forms greyish white pasty growths
along mouth of stab canal (4 days at 23.7-24°C). Wort-agar: Forms
lustrous pasty growths over mouth of stab canal. (4 days at 23.7-24°O).
“Koji-extract’’-gelatine: Makes pasty pin head like growths at mouth of
stab-eanai. (4 days at 14-15°C). “Saké”-gelatine: Same as in “Koji-
extract’’-celatine.

2. Fluid media (4 day, at 23.7-24°C),

Culture media I. Remarks.

Yeast water: Forms islands on surface. Fluid clear, with some deposits.

Yeast water glucose: Forms islands on surface. Fluid clear and no
sediment.

Wort (no hop) : Forms lusterless film and ring with sedimentary growths
along wall of test tube. (after 7 days at 25°C).

Wort: Forms white ring, but no film and sediment.

“Koji’’-extract: Forms film and sediment.

Hayduek’s solution: Forms white easily broken ring.

Bouillon: No growths after 4 days, but after 6 days more there appeared
a thick ring and much sediment.

Thus, the film of this bacillus is easily to break without causing
turbidity.?°
Culture media IT, Remarks.

“Sakékasn’’-mash: Film begins to form after 5 days at 25-26°C and
after 4 days more dense turbidity comes with somewhat rose
red colored sediments. Film stains yellow with I+KI
solution. The turbidity formed in this medium was eharac-
teristic of this bacillus, and not observed in the other 6

varieties. Film became very thick after 48 days more at
16°C.

29, The culture on “Sakékasu”-mash is an exception on this point.

STUDIES ON TILE MICROORGANISMS OF “TANEZU.” 15

Beer: Forms somewhat thick film, which settles down on breaking
and forms very thin film and ring (after 6 days at 25°C).
Total acidity increased by 6.75% (of which 5.904% was acetic
acid).

Wine: No growths after 20 days at 30°C.

Pasteur’s solution: No growths atter 26 days at 30°C, hereby distinguish-
able from B. aceti Pasteur.

Beijerink’s solution: No growths after 7 days at 25°C.

“Sake”: No growth after one month at 25°C.

Diluted “Saké” (water 20¢): No growth after one month at 25°C.

i (water 304): Do.
3 (water 50%): Forms ring with turbidity and much

sediment after 25 days at 25-28.5°C. Forms thin film over
turbid fluid after 3 months more.

I11.—Behavior towards carbohydrates and alcohol:

Acid production

———_—_ =

Bact. aceti. Lact.

Brown. var aceti.
Substance. Growth, Tanezu.
Arabinose. A trace of sediment after 12 days at 25°C. + + —
Glucose. Do. 4 +
Fructose. Do. ++ =
Galactose. Almost no growths. + (2) +
Rhamnose. Forms white easily broken film after 2

days at 25°C. Film does not settle down

on shaking. =
Succharose. No growths after 7 days at 25-26°C but

after 8 days more there was formed a thin

film and ring, which caused turbidity on

shaking. : — _
Maltese. No growths after 8 days at 25-26°C, but

after further 3 days more a ring was

formed, which caused turbidity on shaking. — -- —

116 T. TAKAHASHI!

Lactose. No growths after 15 days at 25°C. — =
Raffinose. No growths after 7 days at 22.5°C, but

after 9 days more a thin and easily broken

film was formed, which caused turbidity

on shaking. = a
Mannital. Forms thin film after 2 days at 25°C.

On shaking it caused a trace of turbidity.

Fluid reduced Feliling’s solution very

districtly after 22 days culture. = -
Dextrin. Forms thin film after two days at 259°C

and a part of it formed flocculent particles

in fluid. BY, ai =
Starch. Forms a trace of turbidity after 7 days

at 22.5°C. a =
Inulin. Forms ring and flocculeat particles in

fluid after 20 days at 25°C. - -
Ethylalcohol. an ih Se =

The property of forming acid from arabinose, fructose, dextrin,
and starch is a distinguishing feature of this variety which differentiates
it from the Bacterium aceti already known. Further, its inabality to as-
similate lactose, arabinose, glucose, fructose, and galactose distinguishes
it from the other 6 varieties.

IV. Fermentation products: The growth was very poor in “Koji-
extract’? in Erlenmeyer’s flask and therefore the products were not
examined,

VY. Conditions of temporature: Optimum temperature for growth
lies near 3Q°C. erowth very slow below 14°C. Heating to 55°C for 10
minutes does not kill the cell, which dies at 80°C,

Thus, this variety belongs to Bact. rancens Beijerinck (Iloyer’s
system) and it has the character of forming reducing sugar from mannitol

in common with Baet, aceti Brown.

STUDIES ON THE MICROORGANISMS OF “TANEZU.” 117

No. 4. Bacterium aceti Brown var Tanezu II.

I.—Form and size: Short bacillus. Cells forming ring in “Saké-
kasu”’-mash culture (2 months) are irregular in size:—long ones 5 , short
ones 2-3 long, and 1.5—2y broad generally. Chain form occurs very
often, and each cell of the chain is very short appearing almost like a
coceus. Involution forms in beer or “Saké-kasu’’-nash are very irregular
(see plate) -—-l=1ip b=5.2u Non-inctile.

JI.—Growth: 1. Solid medium: a. Plate culture: ‘*Moromi’-agar
(7 days at 25°C): Forms round elevated light greyish-white lustrous
colony on surface. Marginal part has bud-like unevenness. (125/1).
“Saké”’-agar: Round elevated dity yellow, waxy colony appears on surface.
(3 days at 21.7°C). Marginal part wavy (125/1). Rose red color
developed on colonies after a very long time.

b. Surface culture: “Saké-agar’: Forms dirty white, filmy
covering (40 hours at 30°C) or lustrous dirty-white pasty growths and
smooth film on condensed water (24 hours at 35—45°C and 20 days more
at 50°C). On thicker part of medium forms mesenteric folds. (18 days
at 30°C or 18 days at 26°C). Rose red coloration of covering strongly
developed above 30°C and weakly below 26°C. Forms brown growths
after 4 days at 23.7—24°C. “Koji-extract”-agar: Forms non-lustrous
waxy covering, middle part of the track is granular, its two sides are

nearly smooth, and external sides are folded. (4 days at 23.7—24°C).
“Koji-extract’’-gelatine: No growth after 5 days at 14°C, but after 38
days at 16.5°C transparent colonies are formed along the tracks.

ce. Stab Culture: “Saké”-agar: Trace of growth. Beer-wort-agar:
Forms pasty weak growths at mouth of canal (5 days 25°C). “Koji-
extract”-gelatine: Trace of growth (5 days at 15°). “Saké’’-geletine:
Trace of growth (5 days at 15°C).

2, Ehud media: (4 days at 23.7-24°C).
Culture media I. Remarks.

Yeast water: Forms islands, with some turbidity and sediment.

138 T. TAKAHASHI?

Yeast water glucose: Film thicker*’ and turbidity greater*’ than in the
above medium.

Wort (not hoped): Forms a trace of ring (7 days at 25°C) over clear
fluid.

Wort: Forms thin film.

“Koji-extract’’: Forms a trace of island,

Hayduck’s solution: No growth, but sediment formed after 10 days more,

Bouillon: Forms thin film with more or less turbidity and sediment.

Thus the property of forming an easily broken film and the turbidity
of the nutrient fluid are characteristic of this bacillus and distinguished it
from the 3 varieties above descrived. Bact. ascendans always forms
turbidity in the nutrient fluid, but in this bacillus its formation depends
upon the kind of medium used.

Culture media LL. Remarks.

“Saké Kasuw” mash: J orms very thin film after 5 days at 25.6°C, and
after + days more it becomes somewhat rose colored and forms
many folds, but the fluid remians clear®**. Film stains yellow
with I + KI. solution.

Beer: Forms very thin®® film growing upwards along side of apparatus
atter 7 days at 25°C. Fluid below film is very turbid, as
in Bact. ascendans. Film breaks on shaking. After one month
total acidity increased by 5.056% (of which 4.926% was
acetic acid).

Wine: No growth after 20 days at 30°C,

Pasteur’s solution: No growth after 26 days at 30°C, and hereby dis-
tinguishable from Bact. aceti Pasteur.

Beijerink’s solution: No growth after 7 days at 25°C.

“Saké”: No growth after one month at 25°C,

Diluted “Saké” (water 204%): No growth after one month at 25°C..-

Diluted “Saké” (water 30¢): Do.

30. and 31. This feature was not observed in No, 1. and No. 2.

32. Same as in No, 4. and No, 2. but different from No. 3.

33- Thinner than the film of No, 1, Bacillus,

STUDIES ON TITE MICROORGANISMS oF “TANEZU.” 119

Dilated “Saké” (water 50¢): Forms non-lustrous spotted film.?! over

clear fluid after 25 days at 25-28.5°C.

I1I.—Behayior towards carbohydrate and alcohol:

Acid production.

ee ee ee
Bact. acti B. acti Bact.
Brown, var Brown. oxidans.
Tanezu, var Tanezu.
Substance. Growth. II. I.
Arabinose. No growths after 8 days at 25°C,
but after 4 days more forms very

thin film, which breaks easily on

shaking and causes turbidity. Fe gRqe gh st +
Glucose. Forms some turbidity with a trace
of ring?5 formation, after 8 days +++ + =F
at 25°C, ah Si
Fructose. Forms a trace of ring with little

turbidity and sediment. (8 days

at 25°C). +(?) = ats
Galactose. Forms a trace of ring with little

turbidity after 8 days at 25°C.

ting breaks on shaking and causes

turbidity.
Rhamnose. Forms very thin film and some

turbidity after 2 days at 25°C. -- — —-

Saccharose. Forms no film but turbidity after
7 days at 25°C. - — +
Maltose. Forms a trace of ring with little

turbidity, very similar to No. 6.
bacillus beseribed further on,
26°C. ar a5 — +

Lactose. Forms no film but trace of ring,

after 8 days at 25

with turbidity of fluid after 7

days at 25°C. After 8 days more

34. The film was thicker than in No. 3.

35- This ring does not break on shaking

120 T. TAKAHASHT:

began to form film. — Nogrowth. +
Raffinose. Forms very thin film with very

strong turbidity after 7 days at

22.5—26°C, — - -
Mannitol. Forms vyery thin film, which

easily breaks on shaking, causing

turbidity. Fluid reduces Fehling’s

solution very well. _ - _
Dextrin. Forms very thin film over clear

fluid after 2 days at 25°C. After

19 days more fluid was shaken

but fragments of film adhered to

wall without causing turbidity. a8 feet +
Starch. Forms no film but trace of

turbidity after 7 days at 28°C.

No alteration after 10 days more. + <r =
Thulin. No growth after 2 days at 25°C,

but after 19 days more the fluid

turned turbid with formation of

easily broken film. = = =

Ethylalcohol. Erte ae Me ge

The property of this bacillus to form acid from galactose, saccharose,
lactose, raffinose, mannitol and starch distinguishes it from the Bact.
oxidens already known, and that of producing reducing sugar from
mannitol assimilates it to Bact. aceti Brown.

TV. Fermentation products: Ethyl and methylaleohol and fusel
oil were found in the distillate of the eulture of “Koji-extract,”’ but
isopropyl alcohol, acetone, methyllactate, isobutyl-aleohol, acetic-acid and
butyrie acid were not found in it.

V. Conditions of temperature: Optimum temperature for growth
lies near 30°C, the erowth is very much retarded at 26°C, and almost
entirely inhibited below 14°C. Tleating to 55°C for 10 minutes will not
kill the cell which dies at 80°C,

STUDIES ON THE MICKOORGANISMS OF “TANEZU.” 121

According to Tloyer’s svstem, this bacillus belongs to Bact. rancens
Beijerink, and the property of producing reducing sugar from mannitol
assimilates it to Baet. aceti Brown, but the formation of rose red color

at high temperatures is characteristic @f this bacillus.

No. 5. Bacterium acelosim THenneberg var Tanezu.

T.—Form and size: Short bacillus, 1vx054 or 3x1 com-
monly isolated or forming chains: Inyolution form appears very fre-
quently in film of “Saké-kasnw’’anash (2 months culture), und measures
38ux1p or 504x25y. Tn film on heer, involution form appears very
seldom and many of them form chains.

1T.—Growth: 1. Selid medium: a. Plate cullure “Moromi”-agar :
Forms round colony becoming rose red in color in old eulture. “Saké-
agar: Forms round elevated light greyish creamy colony on surface, but
in deeper part it appears like lens and the margin is uneven with bud-ike
orowths. (125/17).

b. Surface culture: “Saké’-agar: Forms dirty white mesenteric
covering after 40 hours'at 30°C, or rose red sharply folded covering like
that of mycoderma veast, also folded film on condensed water. (24 hours
at 35-45 and further 2 days at 30°C), or no film on condensed water but
with intense rose covering (1S davs at 30°C).°° Still another culture
forms yellowish-white covering of which the thicker part began to beeone
rosy. “KXoji-extract’-agar: Forms semitransparent covering. (6 days at
20.6°C). “Koji-extract’’-gelatine: No growth after 5 days at 14°C, but
forms vellowish creamy growth after 38 days at 16.5°C.

c. Stab eullure: “Saké”-agar: Forms rose red pin-head-like growth
with erannlar surface at mouth of stab-eanal. (after 6 days at 20.5°C).
Wort-agar. Forms some what rose red pin-head-like erowth at month of
stab-eanal. (6 davs at 20.5°C). “Koji-extract’’-gelatine: Forms trace
yf orowih at month of stab-canal (6 days at 17-19°C). “Sak@?-eelatine :
Forms growth at mouth of stab-canal. (6 days at 17-19°C).

36. In the same culture at 26°C there was production of the rose red color but the

mass of covering was smaller than (almost half) at 30°C.

~
bo

29 T. TAKATASHT:

2. Fluid media. (6 days at 20.5°C).

Culture media I. Remarks.

Yeast water: Forms islands over clear fluid with little sediments.

Yeast water-elncose: Forms islands over clear fluid with little sediments.

Wort (not hoped): Forms islands over clear fluid.

Wort: Do.

“Koji’-extract: Forms very thin film, but thicker than in any other
culture of this bacillus.

Hayduck’s solution: No growth.

Bouillon: Forms trace of film over turbid and sediment holding fluid.

Like bacillus No. 1 (B. asceudans TIlenneberg), this bacillus can not
assimilate asparagin, and in honillon alone turbidity was observed. The
latter property was also observed bacillus No. 2. (Bact. acetosum Henne-
berg).°7 :

Culture media IT. Remarks.

“Saké-kasu”-mash: No growth after 4 days (at 25.6°C), but after 5
days growth begins and after 10 days a thick, folded,** dirty
yellow fila is formed. Film stains yellow with I+KT
solution.

Beer: Forms thin film which grows upwards along wall of apparatus,
but fluid remains clear with little sediment. (after 7 days
at 25°C). ‘After one month total acidity increased by 2.5254,

Wine: (7 days at 30°C): Forms more or less thick film, which partly
grows upwards along wall of apparatus. Film sinks down as
one mass, on shaking:—a characteristic of B. acetosum.

Pasteur’s solution: No growth after 26 days at 30°C, hereby distinguish-
able from TB. aceti Pasteur.

Beijerink’s solution: No growth afier 7 days at 25°C.

37. But Bact. acetosum Henn. forms only a film in Tlayduck’s solution, a point of
difference from this bacillus.
38. The formation of such folded film was also observed in B, aceti. Brown, var Tanezu

Tl, but in this case it was rose colored.

STUDIES ON THE MICROORGANISMS OF “YANEZUL? 125

“Saké”: No growth even after one month at 25°C.
Diluted “Saké” (water 20%): No growth even after one month at 25°C,
Diluted “Saké” (water 304): Do.
Diluted “Saké” (water 50¢): Forms*filn growing upwards along wall
of apparatus after 25 days at 25-28.5°C,
Of the 7 varieties described in the present paper, this variety alone
growths in wine.*?

I11.—Behavior towards carbohydrates and alcohol:

Acid production,

—————————- -——~“—
B. acetosum. B. acetosum. Bact.
Henn. var. Henn. var. acetosum
Substance. Growth. Tanezu. II. Tanezu. I. Henn.
Arabinose. Forms trace of film afier 4
days at 25°C, and after 3 days
more forms ring with some

turbidity. trace, +++ ~
Glucose. Begins to form film and ring
after 4 days at 250C. Ring
breaks on -shaking, causing
turbidity. ++ +++ a
Fructose. Forms thin fiim after 4 days
at 25°C, and after 3 days more
forms ring, which breaks on
shaking and causes turbidity. trace. — —
Galactose. Forms thin film and trace
of ring after 4 days at 25°C,
and after 3 days more fluid
below film beeame turbid. - —
Kthamunose. Forms very thin film, but
fluid is clear after 2 days at
39. Acetic bacilli reported lately these bulletin Vol. VI. No. 4.) behaye differently
towards wine i.e. varieties g, and j form ring, i forms thin film, B and 7 make no growth

after 20 days at30°C.

124 YT. TAKAMASIT:

25°C. After 19 days more

au slight turbidity became

apparent. - _ =
Saecharose. Forms islands over intensely

turbid fluid after 7 days at

22,5—26°C, but no invertion

of sugar took place. + trace. —
Maltose. Forms no film and turbidity,

but trace of sediment after 7

days at 25—26°C, and after

3 days more ring formed,

which breaking — easily on

shaking. - eS =
Lactose. Forms no film, but intense

turbidity after 7 days at 25°C,

and after 7 days more forms

very thin film. _ = _
Raflinose. No growth after 17 days at

22.5—26°C. ? + /
Mannital. Forms thin film oyer turbid

fluid after 2 days at 25°C,
after 9 days more appearance

remains same, but fluid reduces

Ichling’s solution. — oe =
Dextrin. Torms very thin film after 2

days at 25°C, Aes pies Je
Starch. Mo growth after 17 days at

25°C. ? =. =
Inulin, Forms very thin film after 2

days at 25°C, after 19 days
more film remains almost un-

changed ie

Sthylaleohol. ate, ahs cel") (tere om al

STUDIES ON THE MICROORGANISMS OF “TANEZU. 125

The property of this facillus of forming acid from fructose, maltose,
and starch distinguishes it from Bact. ecetosum Henneberg var. Tanezu 1,
and that of forming reducing sugar fron mannitol is common to it and
of Bact. aceti Brown. Further, the quicker growth of this bacillus as
compared with the aboye descrived 4 varieties in nutrient media contain-
ing arabinose, glucose, fructose, galactose is to be noted,

TV.—Fermentation products: races otf ethyl-alcohol, fuseloil,
acetone, methyllactate and butyrie acid were fond in the distillate of
“Koji-extract” eulture of this bacillus, but isopropyl, methyl-alcohol
isobutylalcohol and acetic-acid were not found.

V.—Conditions of temperature: Optimum temperature for growth
lies about 30-35°C; grewih retarded at 26°C and very slow at 14°C.
Heating to 55°C for 10 minutes doese not kill the cell which dies at S0°C.

By the character of the film, this bacillus would be referred to Bact.
acetosumm ILenneberg, Bact. rances Beijerink of Hoyer’s system, but its
property of forming reducing sugar from mannitol assimilates it to Bact.

aceti Brown.

No. 6. Baclerium aceli Pasteur var Tanezu.

I.—Form and size: Very short bacillus commonly 2x1 In
yolution form was not found both im “Saké-kasa’’-mash culture (2 months)
aud in beer culture (one month). Chain form appears very frequently
in beer culture. 2 cells united appear in “Saké’’-agar surface culture.
(22 days at 25°C), 2.5% long, but rarely 5 long. Nonanotile.

Tl.—Growth: 1. Solid media: a. Plate cultures: *Mavowi-agar:
Round, somewhat yellowish creamy colony appears on surface. “Saké’-
agar: Round, somewhat bluish-vellow creamy colouy appears on surtace,
Appears like 2 crossed dises in deeper part of medium (7 days at
30-21.5°C).

b. Surface culture: ‘Sake’ -agar: Forms iransparent and
Justrous covering (40 hours af 30°C), or transparent and bright covering

and film over condensed water with sediment (24 hours at 35-45°C and

126 T. TAKARASIIT

2 days more at 30°C), but after 16 days more the sediment became rose
red. Color production was almost same at 30°C (18 days) and 26°C
(18 dags), thus differing from No. 4 and No. 5. (Bact. aceti Brown.
and Bact. acetosum Henneb.).

Further, there was no difference in growth at 30°C and 22.5-25°C.
Forms semitransparent smooth growth and film on condensed water after
7 days at 25°C, “Koji-extract”-gelatine: Forms a trace of semitrans-
parent covering after 5 days at 14°C, or yellowish moist creamy growth
after 388 days at 16.5°C.

“Moromi”-agar: Forms semitrausparent filmy growth and film on
condensed water. (7 days at 25°C).

ce. Slab-cullure: “‘Saké’’-agar: Forms small colony at mouth of
canal. (7% days at 25°C).

Wort-agar: A trace of growth (7 days at 25°C). “Sake’-gelaline:
Forms semitransparent small colony at mouth of stab-canal (6 days. at
17-19°C).

2. Fluid media: (7 days at 25°C).

Culture media I. Remarks.

Yeast water: Forms uo film but a trace of turbidity.

Yeast water glucose: Forms no film but a trace of turbidity.
Wort (no hop): Forms semitransparent ring, over clear fluid.
Wort: Forms semitransparent ring, over clear fluid.
“Koji”-extract: Begins to form ring, over clear fluid,
Hayduck’s solution: No growth,

Bouillon: Forms trace of ring.

Thus, turbidity is observed only in yeast water culture, and no growth
takes place in Hayduck’s solution, as in bacillus No. 1. and No, 5., but
the semi-transparent ring is characteristic of the bacillus.

Cullure media II, Remarks.
“Saké-kasu’smash. No growth after 4 days at 25.6°C but after 6 days

more there was formed a very thin fibn, which caused no

STUDIES ON THE MICROORGANISMS oF “TANEZU.” ie,

turbility on shaking. After 48 hours more at 16°C film
altered into thick leather*®-like substance.

Beer: Ring began to be formed after 7 days at 25°C, after 2 days more,
fragment of film settled down with little turbidity of fluid,
and after 22 days more thin and easily broken film formed
again. After 34 days total acidity increased by 2.8 7924.

Wine: No growth after 20 days at 30°C.

Pasteur’s solution: Forms no film and turbidity but sediments after
21 days at 30°C, a characteristic of Bact. aceti.

Beijerink’s solution: No growth after 7 days at 35°C.

“Saké’: No erowth after one month at 25°C.

Diluted “Saké” (water 20¢): No growth after one month at 25°C.

Diluted “Saké”’ (water 304): Do.

Diluted “Saké” (water 50%): Forms ereyish easily broken film after
3 months at 25°C,

T11.—Behavior towards carbohydrates and alcohol.

Acid production.

Back acelil ManREaer
Pasteur. acetigenum.
Substance, Growth. var, Tanezu.
Arabinose. Forms trace of turbidity after 8
days, at 25°C. PSP Sr =
Glucose. Forms trace of ring, fluid remains
clear, no turbidity on shaking.
(A—8 days at 25°C). nF oF
Fructose. Trace of turbidity (8 days at 25°C). +++ a
Galactose. Forms ring which does not break
on shaking. (7 days at 25°C). ar =
Rhamnose. No growth after 2 days (25°C),

but after 19 days more there was
formed ring and white flocculent
mass, which caused turbidity on

shaking. - _

40. This occurred also with bacillus No. 3 and No, 7.

128 T. TAKAHASHI:

Saccharose. Trace of turbidity after 7 days

(at 22.5—26°C), but after 8 days

more film formed and _ turbidity

increased. Sugar was inverted. t teh =
Maltose. Begins to form ring and turbidity

after S days (25—26°C), but

after 16 days more white flocculent

mass was formed in fluid. + _
Lactose. Forms no film but trace of turbidity

after 7 days (at 25°C) but after

11 days more formed ring. + —
Raffinose. No film, but intense — turbidity,

after 8 days ring formed. trace =
Mannitol. No growth after 2 days (25°C),

but after 19 days more ring

formed and turbidity appeared. se as
Dextrin. No growth after 2 days (25°C),

but after 19 days more ring

formed, turbidity and flocculent

mass appeared. trace =
Starch. Forms no film but trace of

turbidity after 7 days (25°C) but

after 7 days more increased tur-

bidity and after 4 days more

ring commence to form ++ =
Tnulin. No growth after 2 days (25°C),

but after 19 days more ring

formed and turbidity appeared. -- =

Ehylaleohol. Bi Sis ve

Ly its property of bringing forth turbidity in all the media except
galactose yeast water, this bacillus differs from all the above deserived
varieties and Bact. acetigenum and Bact. accti Pasteur. Its property of

formng acid from sugars differentiates it clearly from Bact. acetigenum

STUDIES ON THE MICROORGANISMS oF “TANEZU.” 129

and Bact. ascendans, but the power of inverting cane sugar is also pos-
sessed by Bact. aceti. The film of the galactose veast water culture stains
reddish-brown, and that of the glucose-veast-water dark blue with the
cellulose reagent (I+H.SO,), and a similar reaction is known to ocenr
especially with the film of Bact. xvylinim.

TV.—Fermentation products: Traces of methyl-aleohol and fusel-
oil were found in the distillate of “Koji”-extract culture, but no ethyl-,
isopropyl-aleohol, methyllactate, acetone, acetic acid or butyric acid.

V.—Conditions of temperature: Optinum temperature for growth
lies near 22-30°C, minimum at 14°C. Heating to 55°C for 10 minutes
does not kill the cells.

This bacillus belongs to Bact. aceti Pasteur of Hoyer’s system, but

differs from B. acetigenum and B. aseendans in many respects.

No. 7. Bacterium xylinoides var Tanezu.

T.—Form and size: Long bacillus. Involution form was not found
in film on “Saké-kasw’-mash (2 months culture), size 54x 1p or
6ux1p. In growth of “Saké”-agar surface culture, the cell measures:
7.54x1.254 or more commonly 2.54—8x1y The cells are usually
isolated but combinations of two occur very rarely. Non-motile.

II.—Growth: 1. Solid media: a. Plate cultures: “Maromi”-agar:
Forms yellowish white creamy colony. Marginal part irregular with
bud-like outgrowths (125/T1).

“Saké’-agar: Forms brown spherical colony. (7 days at 20-21°C).

b. Surface cultures: “Saké’-agar: Forms dirty white, smooth,
non-lustrous pasty growth (40 hours at 30°C), or non-lustrous dirty
white filmy covering (1 day at 35-15°C and 2 days at 30°C.), after
16 days more growth becomes dirty brown'! and forms smooth film on
condensed water. Forms dirty white pasty growth and film on condensed
water with much sediments (8 days at 22-25°C), which becomes rose

red after 19 days more. Film, becomes vellowish-brown after 40

41. The Brown color is deeper in cultures made at 26°C than at 30°C,

130 T. TAKAIASIIT:

days. Makes better growth at 22-25°C than at 30°C. Forms dirty

yellowish brown covering with stream on margin. (7 days at 25°C),

“Maromi’-agar: Forms dirty vellow filmy covering and somewhat thick

film on condensed water. “IXoj1”’-extract-gelatine: No growth. (5 days

at 14°C).

e, Stab-cultures: “Sake”-agar: Forms filmy elevated (at central part)
growth at mouth of canal, the elevated part is colored rose red (7 days
at 25°C). “Moremi’’-agar: Form dirty yellow flat and smooth colony
at mouth of canal. (7 days at 25°C). Worl-agar: Forms small dirty
brown colony on mouth of canal. (7 davs at 25°C). “Koji’-extraet-
agar: Same as on Wort-agar.

2. Fluid media. (7 days at 25°C).

Culture media T. Remarks.

Yeast water: Forms mouldy growth remaining suspended in the medium.

Yeast water glucose. Forms mouldy growth remaining suspended in the
medium.

Wort( no hop): Forms film on clear fluid.

Wort: Forms very thin film, which does not cause turbidity on shaking.

“Koji”-extract: Same as in veast water.

Havyduck’s solution: Forms no film but dense turbidity and mouldy
suspended growth.

Zonillon: No growth after 40 days (25°C).

Thus, the mouldy growth formed by this bacillus in many nutrient
media distinguishes it from the other 6 varieties, but it has the power of
assimilating amido-mitrogen in common with Bact. xylinum.,

Culture media IT. Remarks.

“Saké-kasu”’-mash: Forms film after 4 days (25.6°C), after 6 days more,
marginal part of film alters to brown, and after 18 days more
film thickens, half leatherly half slimy, and forms thread
when treated with platinum wire. Stains very light vellow
with I+ KT solution.

3eer: Forms trace of sediment (7 days at 25°C), after one month more

total acidity increased by 0.01184,

Wine:

Pasteur’s solution:

STUDIES ON TIM MICROORGANISMS OF ““TANEZU.” 131

No growth.

Beijerink’s solution:

“Saké’:

No erowt h.

(20 days at 30°C).

(21 days at 30°C).

Do.

No growth (one month at 25°C).

Diluted ‘Saké” (water 20¢). No growth (3 months at 25°C)
Diluted “Saké” (water 50¢). Do.
Diluted “Saké” (water 50¢). Do.

[11.—Behavior towards carbohydrates and alcohol.

Acid production.

——
Lb. Xytinoides

Substance. Growth var Tanezu.
Arabinose. Forms ascending film after 8 days

at 25°C: dealt, SE
Glucose. Almost no growth. Pat
Fructose. Forms transparent film and mouldy

vrowth below, which setteles down

cause turbidity on shaking. sete
Galactose. Forms islands, which do not cause

turbidity ou shakine. =f
Kwhamnose. Forms trace of turbidity. (2 days

at 25°C). —_
Saccharose. Forms mouldy and Semitrans-

parent film, «a part of which

settles nown on skaking, aud after

8 days more film thickens. In-

vertion of sugar was distinctive. SPP Sr
Maltose. Same growth as in saccharose. ++
Lactose. Forms no growth after 7 days

(25°C) but afte 4 days more forms

mouldy growth. trace.
Rattinose. Same growth as in sacchraose. TPS i
Mannitol. Forms no growths after 2 days

(25°C), but after 18 days more

formed mouldy growth. trace.

132 YT. TAKAILASHT:!

Dextrin. Forms ring and intense turbidity

after 20 days at 25°C. ++
Starch. Forms mouldy film after 15 days

at 22°95 —26°C. trace.
Tnulin. Forms mouldy film after 20 days

(25°C). +
Etbylaleohol. Seat

The property of causing turbidity im rhanmose- and dextrin-yeast-
water culture, of the formation of mouldy growth in all the other media,
and the invertion of cane-sugar are also found in Bact. xylinum already
known.

Further, treatment with the cellulose reagent (Jodine + Potasium

iodid + concent H,SO,) of these films gave the following result :—

Media, film formed. Color produced.
Fructose yeast water. Reddih brown.
Saccharose ,, - Brown.
Maltose _,, Brown.
Raffinose _,, 5 Light yellow.

The colors produced are thus not those peculiar to cellulose, so this
bacillus must be referred to Bact. zylinoides.

iV.—Fermentation products. [sopropyl-(trace), ethyl and methyl-
alcohol, fusel-oil, methyllactate were found in the distillate of “IKXoji-
extract” culture, but not acetic acid, acetone, or butyric acid.

V.—Conditions of temperature: Optinum temperature for growth
lies near 22-25°C. At 30°C or above, the growth is rather retarded.
ILeating to 55°C for 10 minutes kills the cell.

This bacillus belongs to Bact. xylinoides of Henneberg, the tilm of
Which gives the cellulose reaction, which this variety does not, as far as

my experience goes,

PART I. YEAST AND MOULDS.

Besides bacteria, certain kinds of mycoderma and Torulaswere found.

STUDIES ON THE MICROORGANISMS OF ““LANEZU.” 35
The former grow in “IXoji’’-extract but emit no odor; the latter also
flourish in “Koji’’-extract but hardly im “Saké-kasu’-mash. Therefore,
these two varieties of yeast probably do not play an important part in
“Kasuzu” (Sake-kasu-vinegar) manufacture. Among the moulds,
Aspergillus oryzea and Asp. glaucus were isolated from Nakano’s sample,
and a variety of Aspergillus forming intensely green spores was also found

in Sasada’s.

SUMMARY.

1. The majory of the micro-organsms of “Tanezw’ which playing an
important role during the manufacture of “Kasuzu” (a kind of Japanese
vinegar), are bacteria, which may belong to.

1. Bact. rancens.

2, Bact. aceti Pasteur.

3. Bact. xylinoides.
and may be subdivided into 7 varieties.

2. The involution from of these 7 varieties was not always present
and the production of the rose red color is an interesting character of these
cultures. All varieties grow in diluted “Saké” (water 50%), except No. 7.

3. The amount of acid produced is variable according to the varie-
ties: some 5% others 14.

4, The fermentation products in alcohol free media diifer according
to the varieties:—some forms methylalcohol and fusel-oil, others form
isopropyLaleohol, ethylalcohol, methyl-aleohol and fusel-oil and one,

methyllactate or butyric acid.

Nove :—Bacillus No. 1.—No. 5. were ivlated from Nakano’s sample, Bacillus No, 6. and No,
7. from Sasadas’s. In Nakano’s sample Bacillus No. 2. predominated, and in Sasada’s

No. 6.

134 Y. TAKAMASHT:

EXPLANATION OF PLATE,

a—k. The cells of beer-agar culture (23 days).
J—m. The cells of film of beer-culture (one month).
e and g. 750/1, a—d, f, h—m 1000/1.

a. No. 6. Bact. aceti Pasteur var Tanezu.

b. No. 7. Bact. xylinoides var Tanezu.

c. No. 4. Bact. aceti Brown Tanezu II.

d. No. 6. Bact. aceti Pasteur var Tanezu.

e. No. 5. Bact. acetosum Henneberg var Tanezu II.
f. No. 3. Bact. aceti Brown var Tanezu L

g. No. 4. Bact. aceti Prown var Tanezu II.

h. No. 2. Bact. acetosuin Henneberg var Tanezu I.
k. No. 7. Bact. xylinoides var Tanezu.

l No. 1. Bact. ascendans Henneberg var Tanezu.

m. No. 2. Bact. acetosum [enneberg var Tanezu I.

STUDIES ON TITER MICROORGANISMS OF “TANEZU.”

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136

A Preliminary Note on the Varieties of
Aspergillus Oryzae.

T. Takahashi.

Since Asp. Oryzae is the fungus which plays an important réle in
the brewing of Japanese Saké, various authors have studied this funens,
but thus far on attention has been paid to the existence of its varieties.

The writer has isolated 3 varieties from 3 kinds of “Tanekoji”! from
three different sources?.

1. Variety with very long air-nyeelinm (Luft myeel) found in
Tanaka’s sample. Spores are formed very late.

2. Variety with short airmycelinm found in Ueda’s sample.

3. Variety with short airmycelium found in Tiguchi’s sample.

1. Morphology: On Koji-extract-agar the following morphological

characters were observed:

It Il. DUN

Breadth of mycelium. 2.5—3-0y) 2.5—3y, 2.5—3yy,
i d f

Length of conidiophore. 1.0 c.m. 1.5—2m.m. 1.5—2.0 m.m.
Diameter of the “ Head.” 40.— 604 7O—voyy 60—100 4,
Diameter of the “ Blase.” 25—35 55 50—60,, 30—65 ,,
Diameter of the conidiophore. 10—17.5,, 7-5—17.5 5s 75—9
Thickness of the wall of conidiophore. © —— I—I.5,, ——
Diameter of the sterigma, 2b is5 OG
Diameter of the conidia, 6— wis, oe

1, The word “tane” means “seed,” therefore Vane-koji relates to the spores of
Aspergillus Oryzae.

2. K. Tanaka at Kyoto; Yancsuke Ueda at Osaka and M, Iliguchi at Osaka.

138 T. TAKAHASHI:

The lengths of the conidiophores on koji-extract gelatine (at 10-18°C

after 30 days) were :—

I. II. III.

1.0—1,5 c.m. 3—5 mm. I—2m.m.
Further, the lengths of the same on Hayduck’s solution were :—

aE Il. Ill.

2—3c.m. 0.5—0.8 cn. 0.3—0.5 c.m.

In regard to morphological characters, the breadth of the “Blasen”
and the length of the conidiophore have to be considered as differential
characters of the varieties.

TT. Physiological differences: On koji-extract-agar plate culture at
25-22°C, there appears, after 4 months, in var. I a white niyeelium and
bluish-brown spores together with a few brown spores, in var. II dark
spores, and in var. IIT brown spores.

On koji-extract-gelatine at 10-18°C after 30 days: in var. T bright
vellow spores or bluish-vellow spores with liquefaction of gelatine; in var.
IT bluish-yellow spores and but little liquefaction: in var. TIT, bright
yellow spores with only a trace of liquefaction.

The same culture after 40 davs: All three varieties had brown
spores; after three months more, the gelatine was liquefied and crystals of
ca-oxalate® appeared in I and TIT, but none in var. II.

Tn Hayduck’s solution at 25-23°C after 16 days; in var. T very light
yellow spores appeared but the solution remained uncolored ; in IL and ITT
bluish-yellow spores and yellow solution. In glyeerint Hayduck’s solution
after 26 days at 28-32°C: in T dark brownish spores, in IT light brown
spores, in ITT brown spores and vellow solution. After 34 days more the

solution became yellow also in IT, and after 51 days® a very light yellow

3. The formation of this substance by fung has long been known, especially in Asper.
niger and Penicillium glaucum, but its formetion by Asper. Oryzae was quite recently observed
by K. Saité, who isolated this fungus from soya-ho/?.

4. Glycerin was used instead of sugar in Hayduck’s solution,

5. That is 77 days from the beginning.

A PRELIMINARY NOTE ON TILE VARIETIES OF ASPERGILLUS ORYZAE, 139

color of appeared in the solution in I. This polychrome property was
observed already by Siebermann, Welmer, and recently by Milburn and
by K. Saito.

On boiled rice at 27-28°C after T40 days: dark yellow brown spores
appear in I, and light yellow spores in II and IIT; at 31-32°C, after 4-5
days greenish-blue spores in II and III, no spores in I even after 14 days®
Thus, the first variety fructifiies well only at a rather low temperature,
but the other two varieties do so also at higher temperatures.

Enzyme production: The Extracts of the cultures of the three
Yaricties grown on boiled rice were prepared with 5¢ alcohol and to 5
e.c. of these extracts was added 3 cc. of starch paste (2.5%) and the
mixture kept at 60-G5°C. | Atter 56 minutes no starch reaction was
obtained in the case of II and II, and a moderate one with I.

Further, tests for oxidase and peroxidase showed their absence in
these extracts, while catalase was present in all the three. The extracts all
turned dark after some time in the presence of chloroform. Some more
fresh extract was precipitated by adding a large quantity of absolute
alcohol. The precipitate was collected on a filter and after washing with
ether dissolved in water. This solution was tested separately with tyrosin
and hydroquinone; a dark coloration soon appeared with hydroquinone in
the case of IT and JIT but not with I, proving that the oxydising enzyme
in question was absent in I. Tyrosinase* was absent in all the three
varieties.

On heating the three extracts or the solutions of the alcoholie precipi-
tates to 50°C for 90 seconds, the darkening power was lost, while catalase
remained active even at 52-53°C, showing that the oxidising enzyme is
different from catalose. Further, uo H.S was produced from sulphur by
these extracts. When the extracts were made after the formation of the
spores, the amount of oxidising euzvime was evidently much less.

Summary: There exist evidently three varieties of Aspergillus

6. A few spores were formed after 21 days

7- Also phloroglucin was not changed.

140 T, TAKAWASII ;

Oryzae, differing in their morphological and physiological properties; i.e.
length of the conidiophore, color production in the nutrient fluid, optimum
temperature for spore formation, speed of liquefying gelatine, and pre-

sence of an oxidising enzyme.

Ueber den Einfluss der hoheren Temperatur beim
Sterilisieren der Milch.
VON

Y. Kida.

Bekanntlich ist Soxhlet gegen Anwendung hoherer Temperatur beim
Sterilisieren der Milch und zwar deshalb, weil dadurch die Milch cite
unerwiinschte Fiarbung aununmt und die Verdaulichkeit auch dadureh
abnimuit.

Bendix! hat auch dieselbe Meinung gedussert.

Wir beobachten, dass der Geschmack und die Farbe der Mileh dureli
Sterilisation sich verindert. Das Aroma der frischen Milch geht verloren ;
sic mimmft den bekannten etwas bitteren Geschmack an und die weisse
Farbe aindert sich, wohl im Folge einer teilweisen Caramelisierung des
Milchzuckers, in cine gelbiiche bis braunliche Farbung um.

Die Gesclinacksveriinderung tritt, wie Duclaux behauptet, schon bei
70° plotzlich ein.

Da indessen Geruchs-und Geschmacks cmpfindungen beim Singlinge
und bei jungen Kindern noch ser wenig entwickelt sind, so kommen diese
Verinderungen der Milch wohl kawm in Betracht. Wichtiger ist die
Veriinderung dev Verdaulichteit und Ausuutzbarkeit der Eiweiss stoffe und
des Fettes, die die Mileh beim Sterilisieren erleidet. Wenn man die Mileh,
die zuvor tiber 100° oder auf 100° erhitzt worden ist, lingere Zeit ruhig
stehen lisst, so bildet sich eine starke Fettschicht, der Rabm, aut der
Oberiliche der Milch, der sich durch heftiges Schiitteln uur schwer
auseinander reissen liisst wid dam in grosse Kliunpen zerfillt. Dagegen
bildet sich bei frischer oder aufgekochter Milch, beim Stehen, cin locker
Rahm, der sich dureh Schiittelu wieder in feive Partikelehen in der Mileh

verteilt.

1. Vahrbuch f. Winderheilkunde m £. 38, 393

142 ¥. IenpAe

Diese mit blossem Auge wahrnchmbare Verinderung, d.h. die Um-
wandlung des Milch fettes aus dem Zustande der femen Emulsion in den
der groberen Klumpen bildung, muss natiirlich der Resorption des Fettes
nachteilig sein.

Trotzdem giebt es noch einige Forscher, die als besondere Vorziige der
sterilisterten Milch yor der rohen, neben der Keimfreiheit und Halt
barkeit, die leichte Verdaulichkeit und bessere Verwertbarkeit fiir Saug-
lingen und kinder hervorheben.

Also die Frage “Rohe oder gekochte Mileh ? bleibt immer noch un-
entschieden. Es war nun der Jauptzweck des Verfassers, die oben
erwihnten Tatsache nochmals zu priifen und experimentell festzustellen,
ob die gekochte Milch wirklich minderwertiger als die frische ist.
Dariiber hatte der Verfasser auf folgende zwei Punkte seine Aufmerk-
sankeit gerichtet. viz.

1. Die Veriinderung der Verdaulichkeit der Eiweissstoffe.

2. Die Veriinderung des Lecithin gehaltes beim Sterilisieren.

Um den Verdauungsgrad der Eiweiss stoffe zu bestimmen, wurde es in
folgender Weise ausgefiilirt.

Man bereitet zuerst cine Verdauungs fliissigkeit, idem man 1g Pepsin
in einem Erlenmeyer kolben in 500c.c. 0.2% iger Salzsiiure list; hierzu
giebt man 20g der zu untersuchenden Milch probe zu und liisst es bei einer
. Temperatur yon 87-40° 20-2 Stunden unter dfterem Umschiitteln stehen.
Nach der Versuchszeit werden die nicht yerdauten Eiweissstoffe (d.h.
durch Pepsin nicht angegriffene Eiweissstoffe ; hausptsiichlich Casein), in
der Milch in bekannter weise bestimmt. Es wurde gefunden:

Die nicht verdauten Eiweissstofle

in 100g Milch

Nicht erwarmt 0.762 ¢
30 Minuten auf 80° erwirmt * 1.153¢
» 85° 1.493
» go? » 1.4204
” Br » 1.540¢
” 100° ” 1.719¢

30 Minuten im Autoklaven (3 Atom. Druck) erhitat

VEBER DEN EINFLUSS DER NOMEREN TEMPERATUR. 143

Eiweiss gehalt in der Vollmilch 3.462
Die verdauten Eiweissstoffe in 74 der Die verdauten Eiweissstoffe in der
Gesammt-Eiweissstoffe nicht erwirmten Milch als 100
78.0 100
66.7 85.5
55.9 72.9
59.0 75.6
56.1 71.9
: 55.5 {lez
50.4 64.6

Am obigen Resultate sicht man, dass die Verdanlichkeit der Eiwissstoffe
in der erhitzten Mileh bedeutend abeenommen hat.

Es fehlt auch an Tierversuche nicht, die konstatieren, dass die
Eiweissstoffe und Fette in der frischen Milch besser ausgenutzt weren als
in der gekocliten.

2. Die Bestimmung des Lecithingehaltes der Mileh wurde in
folgender Weise ausgefiilit.

1 Liter Mileh wurde zu gewisser Temperatur erwiirmt und dann wurde
sie bei niederem Druck verdanipft. Der Riickstand wurde mit wenig
trockenem Gyps vermiseht, vorsichtig verrieben. Dieselbe wurde nun mit
Aether und dann zweimal je 2 Stunden mit kochendem Alkohol extrahiert.
Die beiden Ausziige wuden yerdampft. Der Riickstand wurde mit Soda
und Salpeter gegliiht, in verdiinnter Salpetersiiure gelést und die Phosphor-
siure bestimmune in gewohnlicher Weise nach Molybdin methode ausge-
fiihrt. Der gefundene Phosphor wurde zum Lecithin umegerechnet. Es
wurde gefunden.

KUHMILCIT (1)

30 minuten auf 95°

Nicht erwinnt erwirmt
Lecithin in 1000¢.c. 0.467 & 0.349
Abnahme O.118
2% ige Abnahme 25.27 %6

KUEMILCH (II)

30 minuten auf §0°

Nicht erwiirmt erwiirmt
Lecithin in 1000c-¢. 0.505 9.407
Abnahme 0,038

26 ige Abnahme 7.52%

2%6

144 Y. KIA?

KUHMILCH (IIT)

Nicht erwirmt 30 minuten auf 80° 30 minuten auf 75°
Lecithin 0.474 0.420 0.444
Abnahme 0.054 0,030
°% ige Abnahme 11.397 6.33%

KUNMILCH (IV)
30 minuten im Autoclayen

Nicht erwirmt 30 minuten auf 100° iiber 100°
Lecithin 0.351 0.351 ‘0.407 =
Abnahme O.1IT 0.116
% ige Abnahine 21.229 22.17%

Bei diesem Resultate sieht man die deutliche Abnahme des Lieithin-
eehaltes bei der stark erhitzten Milch.

Bordas und Sig. de Raezkowsky? nehmen an, dass die Verdaunnes
und Ernihrnngsstérung bei Neugeborenen, welche ausschliesslich mit
erhitzter Milch genihrt werden, wenigstens zum Teil auf die Verminderung
des Leeithins zuriiskzufiihren ist.

Bei der leichten Pasteurisierune bleiben die wertvollen fermente
ungestort, die Eiweisstoffe zeigen nur wenig Verindernng. Der Geschmaek
iindet sich nieht md der Lecithingehalt nimmt nicht ab. Derartige Mileh
wird nicht nur gern genommen, sondern ihre Verdaulichkeit steht kanm
hinter der rohen zuriick und ansserdem die pathogenen Keime werden un-
schiidlich gemacht.

So ist das Pasteurisicren der Mileh hei niederen Temperaturen
auszufiihren.

2. Zeitschrift. f. Nalrungs-und Genussmittel. 1094. ~B. VIT. S. o4.

(¢?

Researches on the Preservation of Night-Soil.
LY

K. Aso and §. Nishimura.

Night-soil forms the principal manure used by the farmers of Japan.
It contains all fertilizing ingredients, but of greatest importance are
nitrogenous compounds which consist chicfly of more or less decomposed
proteins. It is well known that night-soil lose a part of its nitrogen during
the storage with liberation of ammonia and free nitrogen, and various
methods for preventing, or rather of diminishing the loss of that nitrogen
have been proposed, without, however, reaching the desirable degree of
perfection. In this regard also three communications have been published
in Japan, viz. by the Agricultural College in Tokyo, by the former Sanin-
branch-station of the Imperial Agricultural Experimental Station and by
the chemical Jaboratory of the Central Experimental Station in Tokyo.
These experiments led to the conclusion that the loss of nitrogen from
night-soil may be diminished during the storage by well closing the storage-
vessels, and by the addition of superphosphate as had been also recom-
mended by others. The addition of superphosphate and gypsum had the
purpose to transform the carbonate of ammonia into unvolatile ammonium
compound, In a similar way kainit may serve to {ix ammonia on account
of its containing magnesium sulphate. According to the investigation of
the Central Imperial Experimental Station, the addition of super-
phosphate to night-soil diminished very considerably the loss of nitrogen,

especially the volatilization of ummonia as scen from the following figure

146 ik. ASO AND S. NISHIMURA:

Loss of Nitrogen. Loss of Ammonia.
— eS. ———_
Control, Superphosphate Control, Superphosphate

added. added.

After I month, 100 96.8 100 25.2
Sein SF 100 92.1 100 26.3
tS 5 100 82.0 100 19.8
= Oa as 100 71.0 100 19.1
SiS aes 100 48.6 100 18.3

It appeared to me of considerable interest to compare the effect of
the superphosphate with that of gypsuin and kainit, since the former, on
account of its acid reaction, might have merely prevented the desirable

rotting process of the night-soil.

EXPERIMENT IN THE LABORATORY.

The apparatus used for this experiment was composed of a series

of connecting bottles as seen from the following drawing:

A B Cc D E

The flask C coutained the sample of night-soil; A and E contained
strong sulphuric acid in order to retain ammonia from the air to be
passed through the apparatus. D contained a normal solution of sulphuric
acid to absorb ammonia volatilized from the sample and B contained water
to supply air saturated with moisture to the sample. A definite volume
of air was passed through this apparatus, every day, The night-soil was

passed through a sieve, then after well mixing with aa equal volume of

RESEARCHES ON TITE PRESERVATION OF NIGHT-SOIL. 147

water divided into six portions, each of 195 ¢., and mixed with various
preserving compounds.

I.—Superphosphate, 5% of the sample.

II.—Gypsum which contained SOg in an equivalent quantity to that
of the superphosphate added.1

III.—Chemically pure monocaleium phosphate in an equivalent
quantity to that of superphosphate.”

TV.—Pure ealeium sulphate and ealeium phosphate, in the same
quantities as in the second and in the third bottles.

V.—Kainit which contained SO, in an equivalent quantity to that
of gypsum in superphosphate added.

VI.—Nieht-soil alone.

The superphosphate used in this experiment contained 15.304 P.O;
soluble in water and 20.45¢ SO,, and the kainit 12.0¢ SO,. The

temperature during this experiment was:

Maximum. Minimum. Average.

EZiORG; 4.°8C. BEET:

After three weeks, the putrefied samples were analysed with the
following result:

Total nitrogen.

Original. 0:725 %
it, 0.614 %
II. 0.608 7%
Ill. 0.619 2%
IV. 0.609 %
We 0.608 2
VI. 0.513 %

From these figures the loss of nitrogen during the storage was

caleulated as follows:

1. The total content of 5O, in the superphosphate was determined although there was
certain’ difference between the total SO, and SO, in gypsum contained in the superphosphate.
2, In this case also total P,O, soluble in water was assumed to be equal to that in the

form of monocalcium phosphate.

148 K. ASO AND S. NISHIMURA:

Loss of nitrogen.

Ts O.1LI %
1 O.117 9%
III. 0,106 26
IV. 0.116 9%
Vs O17 %
VE. 0.212 %

The quantity of ammonia collected in the hottle D, was as follows:

Ammoniacal nitrogen

volatilized.
Ty 0.068 94
at. 0.088 9%
Til. 0.064 9%
IV. 0.059 9%
iv 0.069 %
VI. 0.103 %

These results show that superphosphate is very effective agent to
diminish the loss of nitrogen and to fix ammonia, and its power depends.
chiefly upon its content of monocalecium phosphate, and not to its admix-
ture of gypsum.

In regard to the loss of nitrogen in the free state further experiments

were necessary.

EXPERIMENT IN THE FIELD.

This experiment was corducted te determine the change of various
nitrogenous organie compounds in night-soil! during the time of the
usual storage in the field.

On May 17, samples well prepared according to the method above
mentioned, were placed into six porcelain jars*, each containing 3,500
grams of the sample. Each jar was put in the field soil, the upper rim
being a few inches higher than the surface of the ground, and all were
arranged in the same condition in a space enclosed with straw, only one

side of this space being opened. The reagents added and the ratios

3. Generally Japanese farmers use wooden tubs or concreted tubs for this purpose,

RESEARCHES ON TIE PRESERVATION OF NIGHT-SOIL.

149

of their quantities used were quite similar to those of the preceeding

experiment. The samples were stirred from time to time and after

eighteen days analysed with the following results:

Total Nitrogen
Ammoniacal Nitrogen
Organic basic Nitrogen...
Monoamido Nitrogen

Albuminoid Nitrogen

We
VI.

ORIGINAL SAMPLE.

Loss of weight.

705.0

700.7

364.2

545.1

598.4

700.0

TOTAL NITROGEN.
% Quantity of N.
remained, g.

1.087 30.373
0.965 27.005
1.104 34.613
1.054 31.148
0.936 27.177
0.909 25.455

Total quantity

in 3500 ¢.
34.608 g.

11.795

6.258

3.097

13.458

Remaining weight.

2795.0
2799.3
3135.8
2954-9
2901.6

2800.0

Original.
= 100

87.70
78.03
100.01
90.00
78.50

79.77

ga gl 99

From this result, it beeomes evident that the action of monoealeinm

phosphate is yery pronounced and that gypsum and kainit were not so

effective as commonly considered.

Ammoniacal {2%
Nitrogen. Total quantity
Organic basic {2
Nitrogen. Total quantity

Monoamido {7
Nitrogen. \Votal quantity

Albuminoid {2
Nitrogen. Total quantity

0.452
12.636

0,0438

1.224

vie}

0.185
5.182

0.405
g, 11.331

Il.
0.406
11.351
0.1487
4.163
0.062
1.744

0.348
9.747

Il.

0.450
14.102

0.1303
4.086

0.088
2.762

0.436
13.663

IV. NE
0.435 0:3
12.860 9.5

0.1695 0.2088
5.017 6.058

0.106 0.077
3-147 2.240
0.343 0.322

10.123 9.355

150 K. ASO AND S. NISHIMURA:

These results show that superphosphate and monocalcium phosphate*
prevent the loss of nitrogen and fix ammonia more energetically than

eypsum and kainit®, while they diminish also the decomposition of
albuminoids more than those. Gypsum and kainit have but little efficacy

in preventing loss of nitrogen.

ANALYTICAL METHODS.

To determine ammoniacal nitrogen, the sample was diluted with
water, mixed with about 2 grams of magnesia usta, and distilled at low
temperature and low pressure (40°, 40 mm.). The distillate was in-
troduced into the solution of normal titrated snlphurie acid and titrated

back with standard soda solution.

To determine organic basic nitrogen, about 20 grams of the sample
was mixed with a concentrated solution of basic lead acetate to precipitate
albuminoids, and to the filtrate obained after well washing the residue,
dilute sulphuric acid was added in a slight excess to remoye the excess
of lead acetate, filtered and washed. The filtrate therefrom was mixed
with so much concentrated sulphuric acid that the resulting fluid contained
about 5% of sulphuric acid and 10% solution of phosphotungstie acid was
added to this mixture. The precipitate was allowed to deposit for 24
hours, and after well washing with 5¢ sulphuric acid, the residue was
subjected to Kjeldahl’s method and the difference between the quantity
of nitrogen and that of ammoniacal nitrogen found before, was taken

as the nitrogen of organic bases.

The determination of albuminoid nitrogen was carried ont according
to Stutzer’s method, and that of total nitrogen, according to Kjeldahl’s

method,

4. As monocalcium phosphate was chemically pure, a proportionally larger amount was

applied than the real content of that in superphosphate.

5. It is probable that the double decomposition between ammonium carbonate and met-
allic sulphates might be not easily caused in the ordinary case, but according to the law of

mass-action this reaction might be explained.

RESEARCHES ON THE PRESERVATION OF NIGHT-SOIL. 151

The nitrogen of monoamidocompounds was calculated from the

difference.

CONCLUSION.

From the results of the above two experiments, the following con-
clusion is drawn:

1. An addition of superphosphate to night soils is very recommend-
able method to decrease the loss of nitrogen as well as the volatilization of
ammonia during the storage of excrements.

2, These effects of superphosphate are, chiefly, due to the action
of monocalcium phosphate contained in it.

3. Gypsum and kainit are not so effective in regard to fixing
amunonia.

4. The diminntion of the loss of nitrogen by the addition of super-
phosphate and monocalcinm phosphate is caused partly to the fact that
they diminish the putrefaction of albwminoids in night-soil.

5. Since superphosphate diminishes the decomposition of albu-
minoids in night-soil, its addition can not be recommended in practice,
especially not in colder climates, when the quick fermentation of night-
soil is required for successful manuring".

6. It is true that the manurial action of night-soil is very much retained by the addition

of superphosphate in the cold districts.

On the Manurial Value of Various Organic Phosphoric
Compounds.

K. Aso and T. Yoshida.

It is generally assumed that nitrogen, phosphoric acid and potash
are the three most essential ingredients of manures and that the efficacy
of manures chiefly depends upon the quantities of these three fertilising
elements present in them and upon the degree of their availability. As
for nitrogen e.g. ammoniacal, nitric and organic nitrogen, and as for
phosphorie acid, water-soluble, citrate-soluble and insoluble phosphorie
acid have been distinguished.

Tn recent times, however, various organic phosphoric compounds have
been found in plants as well as in animals. Besides nuclein and lecithin
also phytin was found widely distributed in the vegetable kingdom.
Moreover, inosic acid, jecorin, kephalin and myelin were found in the
animal body. These, as well as tricaleium phosphate, so imoprtant in
higher animals are absent in plants, while nuclei (as nucleoprotein),
lecithin and phytin play a great réle in them.

Since plants are offen used as manures, it is ef Importance to in-
vestigate the efficacy of the phosphoric acid in these different organic
forms.

From the chemical point of view, nuclein would be more diftienltly
decomposed and therefore less available than lecithin and phytin. — In
water culture, Stoklasa’ showed that lecithin jis available to oats, but its
manurial value is far below that of monocaleium phosphate, as shown

by the following figures:

I. J. Stoklasa: Die Assimilation des Lecithins durch die Pflanze: Sitz.-Ber. Wiener

Ak. ro4.

154 K. ASO AND T. YOSHIDA:

No P,O, CaH,(PO,). Lecithin.
Total weight of dried plants... see OD 29.87 g. 20.52 g.
Weight of dried grains ... ... «2 « — 7-45 g- 4.25 g.

Prof. U. Suzuki and Takaishi? in our laboratory made experiments
with barley in sand and soil cultures to observe the availability of phytin*

and the results were as follows:

SAND CULTURE.

No P,0, Ca,H,(PO,). Phytin,
Average length of plants.... ... ... 39 cm. 83 cm. 82 cm.
Total harvest (air-dried) ... ... ... 5g: 23 g. 23 g.

SOIL CULTURE.

No manure, No P,O, Ca,H,(PO,). Phytin.
Average length of plants .... 23 cm. 26 cm, 58 cm. 4t cm.
Total harvest (air-day) ... 2g. 28, 7 g. 4 &.

These results show that the manurial value of phosphoric acid in the
form of phytin is almost equal to that of dicaletumphosphate in sand
culture, but the former is far behind the latter in soil culture.

Since, in Japan, various vegetable manures such as green manures,
rice-brans, oil-cakes, straws ete are mosi widely used by practical farmers,

the practical interest of the question induced me to the following
experiments. ;

FIRST EXPERIMENT.

This experiment was carried out to compare the manurial value
of various organic and inorganie phosphoric compounds. The soil serving
for this experiment was a humus loam of a slightly acid reaction, which

was exhausted by continuous cultivation without any manures for seven

years.

Each pot containing 2.5 kilo, soil was manured as follows:

2, U. Suzuki, a. M. Takaishi: Journal of Agricultural Society, Japan. No, 323.
3. Phytin was prepared from rice-bran.

ON THE MANURIAL VALUE OF ORGANIC PHOSPHORIC COMPOUNDs. 155

Acidic manures. Basic manures.
per pot. per pot.
Ammonium sulphate... ... 0... 1 g. Sodium nitrate... ... ... 03g.
Potassium sulphate ... 2. ... £3 ¢. Potassium carbonate... I g.

Various phosphoric compounds were added as the following table

shows, the quantity of P,O; applied per pot being 0.396 grams.

Per pot.
SMUG MOS DP RAG ese weeey a) cee es, acd shee ace ose I g.
TLS acc Genco. a cece EBs OCR) TEES Rou com SERS mere mERCHuMIL HE Coy Dir-a
RHR O cel aca ace) ate) evs Cav ecco ccs Gee Set nee OSA)
INES 655 ceceot | ca «sco SESS fda tas CER eE PRRE BPX PLY ats
Alumni PHOS phate) sere ieral Gece) say ey voce ss: ez (zs) OLOSE
HE EEs PHOS PUALG es gers) tee Moench] co. Maxey | OLO42:
pnrcalcuimypnosphate: Ae ese ccecest ees esa ees ses, ce, 01865) g:

The phytin was prepared from rice-brans as follows: the rice-bran
was boiled with 95¢ aleohol twice after extracting with ether and the
dried residue was repeatedly extracted with 0.2¢ hydrochloric acid. To
the hydrochloric exiract, 95¢ alcohol was added and the white precipitate
obtained was washed with alcohol and ether, dissolved in 0.24 hydrochloric
acid, reprecipitated with alcohol and washed as before. The white
powder prepared in this way, contained 45.86% P,O;. The nuclein
used, was prepared from beer-yeast in the usual way, and contained
1.68% P.O;.

The lecithin had been prepared by Kénig & Co. and contained 7.75¢
P.O, showing that this was tolerably pure.

Sodium phosphate, ferric phosphate, aluminium phosphate and
tricalcium phosphate were chemically pure.

On Noy. 7, these compounds were mixed with the soil respectively,
and barley was sown, seven sceds in each pot. The young plants were
reduced afterwards to three of equal size in each pot. After germination,
a great difference of development was observed and on March 14, the

following measurement was made:

156 kK. ASO AND IT. YOSHIDA:

AVERAGE LENGTH OF PLANTS.

Acidic manure, Basic manure.

cm. cm.
OTT We eee asi os Feo eam ace ped) 20.4
Phytin® “sicemcestl aks eae ero ae ee 9.3 OL
Waclein. <0) Avi ass we Se oe ee OSLeStS 9.3
sodium (phosphate). ssc Gen wes lest eens ROIS 15.9
Bemicyphosphate eeuetsn ys ec eee Sos ag Oa
Aluminium phosphate ... eS 10.2
Tricalcium phosphate Yad eae 21.0
BVO aR SO Ran Macs neey dogs: tak. cess esses S1 78
Wo Amanike Soyee) messh +c @fans een ene ae 7.8 fe}

On May 13, a photograph was taken; the plants were harvested

June 16, with the following result:

Manures. Total weight. Ears. Grains.

air-dry, air-dry. air-dry.
Acidi A a 3
eye cidic 12.9 1 4.
Lecithin ... ‘+ Basic 11.7 5.7 45
ae Acidic 3-4 1.2 1.0
Phytin *\ Basic 2.5 0.7 0.5

Nuclei Acidic No test.

Nuclein ... ee ee Bai 2.3 0.65 0.45
Sodium phosphate : oe me a a3
eae Acidic A. 2.0 1.6
Verric phosphate : Basic He 13 08
Aluminium phosphate : pantie = ae oe
Tricalcium phosphate : ar a8 +7 pe
: : Acidic 0.7 ° °
No P,O, *) Basic 1.4 o_ oO
INO manure 47 Tews seee sa 0.5 ° °

From these results it is clear that lecithin is easily decomposed in
the soil and furnishes phosphoric acid available for plants. The remark-
able result with lecithin might be supposed to be partly caused by the

nitrogen contained in lecithin, but this is improbable, as there was surely

sufficient nitrogen in the general manure. Tricaleium phosphate' exerted

4. This was precipitated tricalcium phosphate.

ON THE MANURLSL VALUE OF ORGANIC PILOSPITORIC COMPOUNDS. 157

in this experiment a very favorable influence. The manurial value of
phytin was far less than that of sodium phosphate and almost eanal to
that of ferric and alwninium phosphate. Perhaps, phytin will be changed
to insoluble compounds in soils, since the iron and aluminium compounds
of phytin are insoluble. As for nuclein, the author made the experiment
only with basic manures, since nuclein is soluble in alkalies and not in
acids; nevertheless its manurial value was very small, showing that the
decomposition by bacteria in the soil is rather slow.

Among organic phosphoric compounds in plants, the manurial value
of lecithin is excelleut while those of phytin and nuelein are far less.
But, the content of lecithin in plants is generally very small, while that
of phytin much larger. This latter plays also an important part in certain

vegetable manures as the following table shows’ :

IN 100 PARTS OF DRY MATTER.

Total PAOP B.OF BEOe

POE in lecithin. in phytin. in nuclein.
soybean! Cakes.) ss 0-s0) aaa -e-s L SUE O.114 0.649 0.230
LINAS EAS ee ees retosn | cee acces mete 0.091 0.873 0.204
Red clover a cc co OHS! e.150 0.300 0.050

The inferior yalue of phosphori¢ acid in vegetable manures to that
in animal manures" is decidedly explained by these results.

However, since there exists an enzym* called phytase which splits
phytin with the production of inorganic phosphoric compounds, and which
is present in various plants, the phytin applied in the form of press cakes
will yield a somewhat better result than when applied in the pure state,
especially, when the cakes were left to putrefy before application.

SECOND ZXPHRIMENT.

This experiment was carried out to decide which part of the phosphoric

acid in rice-bran has the highest manurial value. Each pot containing
Cf. the article of Tsuda, this Journal.

5
6. Cf. Nagaoka’s result with paddy rice, these bulletins, Vol. IV.

7. U,. Suzuki and K. Yoshimura: these bulletins, Vol. VII. No. 4.

158 i, ASO AND “T. YOSHIDA:

2.5 kilo soil, was manured Noy. 8, with 5 g. ammoniuin sulphate and 2 g.
potassium carbonate. Besides, 10 g. of rice-bran treated in various ways
were added to the different pots. Seven seeds of barley were sown in
each, and the young plants reduced afterwards to three of equal size.
On March 14, the following measurement was made:

Average length

of plants.

cm.

Original rice-Hran)) Sasa yees 2.) =<.) sep aeee ee en 8.9
Wxtracted inyithy ethers eect icesc 0) acct com |) cece en 12.1
os > a and ‘aleohol.< vas. suc acute 15.1

5 ri Rs alcohol and 0:2°o6°HCI: 2c. Secemest 8.4

” ” ” ” ” fo % lich. = ache! = = gem 8.4
Sodium phosphate (equivalent to original rice-bran).. ... ... 13.2

INO (PSO OS) Brant eccMrete tree Idan) 678 ave a vce, a eee eee 7.6
Wo WManure So tee ete tewcs eee) Mavs, eeey, oct eee: ee 7-5

On June i6, the plants were harvested and weighed in air-day state

with the following result:

Total weight, Ears. Grains.
Ss: S S$:

Original wice-lran cs weceee scenic ee cee 3-3 Lt 0.65
extracted With) etners seme eee cae) u-34" Lass 4-7 1.5 1,0
a “ so) eandraleolioly) <2.) “ac. sos 4.8 1.9 1.4

a 2 s aS » and o.29¢ HCl. 0.9 — —

fs A > ‘ » and 10% HCl. 0.8 _— _
Sodium phosphate;.. 2.) jars cec sen see ven 2.3 0.6 0.35
No P,O, Boe ce sh ta cog a ET -- —
No manure Saat oon. ads 0.8 = _

A similar experiment was made with oats using rape-cake as manure
1 D ?

with the following result:

Total weight. Straw. Grains.
es 5 ae
Original rape-cake. «00 (cos ne sus see one Ze e oe
Extracted with ether 0 0.0 ccc ose soe ave oe = =
Extracted with ether and alcohol ... ... ae BA 36
. 3 Hal 1,
Vxtracted with cther, alcohol and 0.272 HCI, ee eae a

ON THE MANURIAL VALUE OF ORGANIC PHOSPHORIC compouNDs. 159

Extracted with ether, alcohol and 1074 HCl....f an Sa ot
Sodium phosphate ed a 3 oe
MapPiOish, Jiks wieder ke boa wlos ao ora
ING) MANUTe: “Peso nese ieee) ces) xem cess: se clea oe ate

These results show that riec-bran and rape cake exert a_ better
manurial effect after the fat had been extracted by ether and alkohol.
Evidently the fatty matter surrounds the particles of cake and prevents
tne root hairs to exert their absorptive power.

Since phytin is soluble in 0.2% hydrochlorie acid, the residue after
extracting with ether, alcohol and 0.2¢ hydrochloric acid has lost almost
ai value as phosphatic manure’. Tlence it must be concluded that, to

phytin is due the chief manurial value of phosphoric acid in press cakes.

THIRD EXPERIMENT.

In this experiment, various residues obtained from rice-bran as before
were analysed separately and the quantities corresponding to one gram
of sodium phosphate were used in each pot. The contents of P.O, and
the quantities of these residues were as follows:

Quantity used,

per pot.
g.
Orienalexice-brau-.. veve) = 45) ees ces pees eee 6.758 5.8
iziracted: withiether! sc. \-2<1 at--s mec deren tene 8.684 4.56
Extracted with ether and alcohol ... ... ... 8.82 4.49
Extracted with ether, alcohol and 0,293 HCl. 4-157 9.53
Extracted with ether, alcohol and 1074 HCl.... 2.525 15.68

Each pot containing 2.5 kilo soil was manured on Noy. 18, and seeds
of pea, rape, barley were sown, seven in each pot. The young plants were
reduced to two of equal size in the case of pea, to four in the ease of

rape and barley.

8. OF course, all hydrochloric acid was removed by well-washing, until no acid was per-

ceiyed in the filtrate,

160 K. ASO AND T. YOSHIDA:

On April 14, a photograph was taken, and the plants were harvested

May 30, with the following result:

PEA.
Total weight. Stems. Fruits,
Original rice-Dranisa) eects es Nerensi ee eee sno nine 1:86 0.68 118
Extracted! withinethe@nstsce acai yess) csv iicex! neo ect eeOr, 1.27 3.40
Extracted with ether and alcohol ... ... ... ... 3.61 1.59 2.02
Extracted with ether, alcohol and 0.294 HICl. ... G.gr 0.52 0.39
Extracted with ether, alcohol and 1093 HCl... 1.04 0.56 0.48
Sodium, phosphate gjss: sti eee, ese) Gate ere! 1.55 a 0.05. aims .
NOUS O PERE) 6st oy fen Aeon ce ens (7 0.47 _—
RAPE.
Total weight. Stems. Fruits.
Orxipinglixice-brans ees uere Mines see) sax, Paco ake be 127 1.06
Extratted with ether cup meses ssc) ccs wes) ne RO 1.66 1.44
Extracted with ether and alcohol ... ... ... «1. 3.50 1.95 1.55
Extracted with ether, alcohol and 0.293 HCl. ... No development.
Extracted with ether, alcohol and 109g HCl. eA
Sodium phosphates "i.e Meesnees) at) =.) ae TOLOO 3-70 2.30
BIO PO 0 ee le Al eons, se. Case, aoe No development.

The barley was harvested on June 16 and weighed in air-dry state

as follows:

Total weight. Fars. Grains. ‘
Originalyxice-brance) soo ey: ks cet ee ne ea =
Extractedwith ethers. emis er; wee se ee AO _ _
Extracted with ether and aleshol ... ... ... ... 3.6 1.3 0.9
Extracted with ether, alcohol and 0.297 IICl. ... 1.1 — _
Extracted with ether, alcohol and 107 HCl. ... 08 — —
Sodium’ phosphate? voce keer. see desl due 7 2.4 1.4
Noy EiOu oS. Oe er eee ass see) oar eae, — a2
NiosSAnUre) -5) gas Sec C EEE es oes) woe Sune OO) _ _

Although the quantity of P.O; was the same in all pots, yet the
residue obtained by extracting with ether and aleohol yielded best harvest

which coinsides with the results obtained in the second experiment. The

ON TIE MANURIAL VALUE OF ORGANIC PHOSPIIORIC cCompouNDs. 161

residue containing only nuclein as phosphoric compound, obtained by
extracting rice-bran with ether, aleohol and 10% hydrochloric acid has
no immediate manurial value as a phosphatic manure, even in large

doses, which agrees with results of the first and the second experiments.

CONCLUSION.

1. Among the organie phosphoric compounds used in these experi-
ments, the mannrial value of lecithin was highest, phytin come next and
nuclein last.

2. The manurial value of lecithin was not lower than that of
sodium phosphate, that of phytin was nearly “equivalent to that of ferric
or aluminium phosphate and that of nuclein was very low.

3. The most essential phosphoric compound in vegetable manures
is phytin.

4, As phytin is easily transformed in soils into insoluble ferric
and aluminium phosphate it is recommendable to use vegetable manures
in a putrefied state to render the phosphoric acid available.

5. In the analysis of manures, it is absolutely necessary to pay
attention to the different organic phosphoric compounds.

6. Further experiments with various organie manures along this

line and with different soils are still desirable.

Plate I. ET

Na,HPO, Ca,(PO,), Fe,(PO,), Al,(PO,), Lecithin Phytin Nuclein NoP,O, No manure

This plate shows the result of the first experiment.

PL. IL.

Extracted Extracted Original Extracted Extracted
NoP,O, with ether, withether, rice-bran. with ether. with ether Na, HPO,
alcohol and alcohol and and alcohol.

10% Hel. 0.296 Hel.

This plate shows the result of the third experiment.

On the Availability of Phosphoric Acid in Various Forms
Pie Herring-guano.

R. Mitsuta.

There exist various forms of phosphoric acid in herring-guano,*
such as calcium phosphate, lecithin, phytin, nuclein and other inorganic
phosphates. But as ‘o the availability of these different phosphoric com-
pounds, some investigation seemed desirable. | Prof. Aso compared the
manurial value of various organic phosphoric compounds derived from
rice-bran, rape-cake and yeast.

For my experiment served eight pots, each containing 2.5 Kilo soil,?
manured with 2g. potassium carbonate and 3g. sodium uitrate. These
large doses were necessary to climinate the effect of potash and nitrogen
of the herring-guano. Lesides, five grams of herring-guano treated in

various ways were added in each pot: ~

Pot. Extracted with | Removed
A, Original (not extracted). —
Bb. Ether. Oils and a part of lecithir.
ic. Ether and alcohol. Oils and leeithin.
D. Ether, alcohol and ict’ water, ae and phosphates soluble in
. * hae Oils, lecithin, phytin and phosphates
ibg Ether, alcohol and 0.2% Hel. N er amielinn alate acide,
Ether, alcohol and 107% Ilcl. Only nuclein remained.

No phosphatic manure added. —_

moo

4-3g. sodium phosphate corresponding | —
to 5g. herring-guano which contained
4.176% P05.

1, Cf, This Bulletin.

2. This soil was a humus loam of a field unmanured for cight years.

164 R. MITSUTA:

On Noy. 26, seven seeds of barley were sown in each pot, and the
young plants reduced to three afterwards. After the germination, a
great difference in development was cbserved. On May 13, a photograph

was taken and the following measurement made:

Ist plant 2nd plant 3rd plant
Pot Average Number Average Number Average Number
length of length of length of
cm. branches cm. branches cm, branches
A ORE 3 7.0 3 6.6 2
IG eke noal}@) 3 5.3 2 4.5 3
Ge he Se 3 5.1 3 5-4 3
D - 45 I 4.2 2 3-9 2
E . 1.5 ; I 1.5 I 1.2 1
an) 0.3 I 0.4 I 0.3 I
G — I “= I -- 1
ii 5-4 4 5.1 3 5-4 3

On June 26, the plants were harvested and weighed in air-dry

state:
Pot. Total weight Number of Ears. Weight of Ears,
Be g.
A re) eae 8.5 4 3
B 7 5 2.5
Cc we Yi 5 1.5
D f as 4 I
ec ety oe cee ot URC) — =
F — = =
G _ = =
be ere ec) ta EA 4 2.4

In this case, treatment with ether or ether and alcohol had not
increased the manurial value, but decreased. Jlere the cake of commerce
had been deprived of the most of its oil content* and it was chiefly lecithin,
perhaps from lecithalbumen, which had been extracted.

The great difference between D and E was caused by that part of

phosphoric acid which is soluble in dilute acids. Since the content of

3. For this experiment, Shimekasu which is a pressed residue deprived of the most part

of it's oil-content, was used.

ON TUE AVAILABILITY OF PIOSPHORIC ACID IN VARIOUS FoRMsS, 169

phytin in herring-guano is very small, the phosphoric acid soluble in
dilute acid might be present in inorganic forms, chiefly as calcium
phosphate.* Some development of plants in FE shows that there remained

still a part of tricaletumphosphate impbones onextracted, the case bemg

quite different from that of vegetable manures.” Nuclein in herring-
gnano has no immediate manuria! value as generally assumed.

Lastly, we conclude that the principal part of phosphoric acid serving
as phosphatic manure in fish-guanos is of inorganic nature, chiefly con-
sisting of calcium phosphate. Lecithin and phosphates soluble in water
exert here also a certain role. Hence a great difference regarding the
manurial value of phosphoric acid in animal and vegetable manures
‘was thus established.

4. Cf. The articles of Funatsu and Tsuda. (Thes2 Builetins).
5. Aso. This Journal.

/66~

Plate IT.

On the Different Forms of Phosphoric Acid in

Organic Manures.

S. Tsuda.

It is a well-known fact that phosphoric acid is one of the three
essential manurial elements, but there has been no systematic research
to determine the different forms under which it occurs in different organic

substances. Funatsut

determined phosphoric acid in the form of
lecithin and nuclein, and in a form soluble in dilute hydrochloric acid
in several manure cakes. Prof. U. Suzuki and Yoshimura? have found
that phytin is widely distributed in vegetable matters, forming the chief
part of the phosphoric compounds of the plant body, and the object of the
present paper is the quantitative determination of different forms of
phosphoric acid in several organic manures of vegetable as well as animal

origin.

To determine phosphoric acid in the form of lecithin, 50g. air-dry
sample was extracted for twenty hours with ether by means of Soxhlet’s
apparatus. The residue was boiled for six hours with absolute aleohol,
using a reverted cooler, and the extraction was repeated after pouring
off the alcoholic liquid. After evaporating these ether and_ alcoholic
extracts to dryness they were fused with potassium nitrate and sodium
carbonate, and the fused mass was dissolved in nitrie acid. The phosphoric
acid was then determined by molybdie method.

The residue obtained from the aleoholic extraction was dried and

I. Bul. College of Agric. Tokyo, Vol. VII. No. 3.
2. Bul, College of Agric. Tokyo. Vol. VIT. No. 4.

168 S. TSUDA:

put into a flask, and extracted with 500 cc. of 0.2¢ hydrochloric acid at
the room temperature for six hours the flask being shaken from time to
time. After filtering, the extraction was repested with the residue and
the filtrate was mixed with the first. To a part of the hydrochloric
filtrate ammonia was first added to make the reaction slightly alkaline,
then nitric acid in excess, and the phosphorie acid of this extract in the
form of inorganic salts was determined by molybdie method. To determine
the phosphoric acid in the form of phytin, another part of the
hydrochloric extract was evaporated in a large platinum basin and the
residue was well fused with potassium nitrate and sodinm carbonate.
After extracting the fused mass with nitric acid. molybdiec method was
applied. The difference between the total and the inorganic phosphoric
acid of the original hydrochloric acid is the phosphoric acid in the form
of phytin.

The residue of the 0.2% hydrochloric acid extraction was dried and
extracted with 500 ¢.. of 5¢ hydrochloric acid in the same manner as
before and its phosphoric acid, in the form of both organic and inorganic
compounds, was determined. The last residue was dried and fused with
the fusing-mixture, dissolved in dilute nitric acid, and molybdie method

was applied to determine the phosphoric in the form of nuclein. The

results obtained are shown in the following table.

IN 100 PARTS OF DRY MATTER.

Soybean Rape seeds Red clover hay

cake. cake. (before flowering)
Be) 1 ee ty ee BD 2.251 0.554

P,O, sol. in ether and alcohol (as

Lecithin) ome ete 5G em ease) ORT 0.091 0.050
PO, sol. infinorganic .., ... ... ... 0.050 9.050 Trace
0.2% YVicl.\organic (as phytin)... ... 0.640 0.873 0.300
P,O, sol. infinorganic ... ... ... ... 0.040 0.099 0.070
5% Hel. organic: sss wl an sie 0120 0.931 0.084

P.O, in the last recidue (as nuclein)... 0.236 0.204 0,050

ON THE DIFFERENT FORMS OF PITOSPITORIC ACID. 169

Pressed cake

Herring Steamed of pupa of
guano. bone dust Silk-worms Crab shells,
sO fal TP Oop ects esas coh eae 4.670 25.c60 1.350 3.23
PO, sol. in ether and alcohol
(asm ecithin) siemens 0.310 ~ 0.023 0.043 0.023
PO, sol. in inorganic... ... 1.894 5.534 1.039 0,300
0.2% HCl\organic ... ... 0.860 trace trace O.151
P,O, sol. infinorganic... ... 0,372 18.859 0.090 2.264
5% HCl. \organic ... ... 0,648 0.530 trace 0,200
P,O, in the last residue (as
nuclein) eesp, sor seas 0.583 0.112 0.169 0.302

These results show distinctly the difference between animal and
vegetable manures in the relative amount of the different forms of
phosphoric acid, the former containing it mostly in the form of inorganic
compounds and the latter mostly as organie compounds.

In vegetable manures, phosphoric acid is present principally in the
form of phytin® and the amount of nuclein is comparatively small.
Lecithin is also contained in small quantities, the phosphoric acid in this
form being always less than 10¢ of the whole. Again, as inorganic
compounds phosporie acid is present only in traces in certain cases.

On the other hand, since triealeium phosphate is the principal
ineredient of bones, most of the phosphoric acid of animal manures
containing bones is the part soluble in 5¢ hydroehloric acid; but in the
case of herring guano, the phosphoric acid soluble in 0.2¢ hydrochloric
acid is more than the former, owing to the presence of a large amount
of flesh.*

Phosphoric acid is contained in erab shells in nearly the same forms
as in hones, while in the pupa of bombyx mori silk-worms it is present in

an inorganic form easily soluble in dilute hydrochlorie acid (.2¢)°.

3. Phosphoric acid as phytin which has not been completely extracted with 0,294 hy-
drochlories acid owing to the presence of protein and other ingredients may be contained in
the organic phosphoric acid extracted with 522 hydrochloric acid.

4. Fresh and meat contain potassium phosphates and certain organic phosphoric compounds
besides nuclein and lecithin,

5. This is a very interesting fact and further study will be made in this line,

170 S. TSUDA:

The great difference in phosphoric compounds between animal and
vegetable manures proved above probably explains to some extent the
different value of animal and vegetable manures, the former being always

superior, as Prof. Nagaoka® has shown.

6. Bul. College of Agric. Tokyo, Vol. VI. No. 3.

Ce ee eR eee eee eee

On the Influence of Different Ratios of Lime to Magnesia
on the Growth of Rice II.

In my first communication on ‘his subject’ it was shown experi-
mentally that the ratio CaO: MgO=1 in the soil is the most favorable
for rice under the coudition that both these bases are about equally
available. In that experiment the manure applied was of alkaline nature;
phosphoric acid having been applied as Na,HPO, and potash as ear-
bonate. Under this condition the injurious influence of an excess of
magnesia is generally not so intense as in the presence of a manure ot
acid character. To observe the growth of rice on application of an acid
manure with varying ratios of lime fo magnesia, I selected this time
double superphosphate as the source of P,O;; potash was further apphed
as sulphate, and nitrogen half as ammonium sulphate and half as sodium
nitrate. Lime and magnesia were added in one series” as sulphates and
in a second series as carbonates,—in varying proportions,

Seven Wagner’s porcelain pots were filled with S$ kilo of air-dry
sifted soil taken from a paddy field which had not been cultivated for
several years. The quantity of available lime and magnesia in this soil
was determined by extracting the soil with cold 10¢ hydrochloric acid
for 48 hours with the following result:

In 100 parts of dry soil;

CaO 0.79
MgO 0.60

1, These Bulletins, Volt. VI, No. 2.

z. Jn my first experiment carried ont three years ago, lime an‘ magnesia were applied as

the #aruva’ carbonates in a state of very fine powder.

ihe Ik. ASO: :

The amounts of gypsum and magnesium sulphate, applied in series T,
further those of the precipitated carbonates of lime and magnesiat, in

further those of the precipitated carbonates of lime and magnesia, in

SERIES J.

Quantity of Quantity of
Pots. CaSO, + 2H,0 MgsO,+7H,O CaO: MgO.
added, g. added, g.

a 285.93 -- eh.

b 211.32 a 4:1

c 136.67 _ i

d 62.04 _ PRE

e — —_ I: 1 (nearly)
f — 100.13 ria)

g os 200,27 Ti. 2

SERIES IL.
Quantity of Quantity of basil
Pots. CaCO, added,g. Mg CO, added, g. CaO: Mg O.

A 166,24 — Ben

B 122.86 — 4:1

Cc 79.46 — St

D 36.07 ~ ru

1D _- -- 1: 1 (nearly)
Ir — 68.3 ee

G — 127.4 eS

On July 15, the following compounds were applied to each pot as

general manure:

Double superphosphate 5g.
Potassium sulphate 10 g.
Ammonium sulphate 5g
Ammonium nitrate 5 2

On July 16, three bandles of young rice plants, each bandle of 5
plants of equal size were transplanted from the seed bed into each pot.
The plants were treated as is usual for paddy plants, and eare was taken

P ‘al
7 Ca” corresponds to
0.0 MgO

nr ‘ 1,10
3. The ratio —

———-——s/aS

ON THE INFLUENCE OF DIFFERENT RATIOS OF LIME. 173

that no uimmatural conditions should occur. The accompanying photo-

graph was taken on September 7. On November 5, the plants were cut,

left to become air-dry for several weeks and weighed.

Pots. Grains, g. Straw, g. . Yotal, g.
16.5 . 61.5 78.0

De 26.5 59.0 86.5
c 40.8 58.2 99.0
d 35.5 81.5 117.0
e 60.8 80.7 141.5
f 22.0 65.0 87.0
s 13.2 41.3 54:5
A 49.5 71.0 120.5
B 42.0 70.5 112.5
Cc 47-5 76.0 123.5
D 49.0 65.0 114.0
E 62.0 80.8 142.8
F 49.8 69.2 119.0
G 51.5 73-5 125.0

This result agrees well with my former experiment carried on with
the application of lime and magnesia as natural carbonates. It shows
that any change of the ratio CaO: Mg0=1 leads to a decrease of the
harvest. The unfavorable effects of the sulphates upon the yield were
more marked than those of the carbonates, probably on account of the

slightly acid reaction in the soil*.

4- It is true that the precipitated magnesium carbonate is much more available than the
precipitated calcium carbonate, and the condition of equal availability for F and G is not
fulfilled. Tut this objection dogs rot apply to my former experiment.—Vurther the sulphates of

lime and magnesia underwent of course more or less change in the soil.

— — x

OT edie* ete ae we: 7 aw

, i
a ¥ 7%
a A a

‘
’

J
wy
: '

PL. Plate ITT. (7

a b c d e f g

Left fioeures:

This plate shows the influence of different ratios of lime to magnesia with carbonates.

instead

ioht f

)
ay

instead of a, b, ec, d read B,, By, I

igures:

On the Influence of the Ratio of Lime to Magnesia
4a i é upon the Yield in Sand Culture.

r- K. Aso.

Numerous experiments carried out here at this college as well as
at the Central Experimental Station in Nishigahara have proved ‘
that the maximum yield depends, other things being equal, also upon a
certain ratio of lime to magnesia absorbed by the plants. Some crops

are inore sensitive in this regard than others. That ratio—the lime-

Fe
factor—varies with different crops, the variations ranging mostly between 3
1:1 and 4:1. F
: But there are some agriculturists who still cling to the opinion that |
the efficient factor in this case is not the wfavorable ratio, but the
absolute excess of one base or the other. Any one who studies the ques- k
tion earefuily on the basis of experiments must become convineed of the
erroneousness of this pinion. ;
Abeve all, in regard to water cultures, the tests were earried ovt q
under the condition, that tie sum of lime and magnesia was kept constant,
while their relative amounts varied in different cases. It would be
absurd to assume that 0.2¢ lime in water culture would be in itself
injurious. Barley plants remained quite healthy for several weeks im

culture solutions with 0.5% calcium nitrate in the absence of magnesia ial
and died finally after 61 days from mere inanition’. ;

In the control case with magnesium nitrate and no lime the plants
died in 10 days. The inanition in the former case as well as the poisonous

action in the latter case was avoided by a proper ratio of lime to magnesia.

1. The discovery of Willstatter, that magnesium is essential for the formation of chlorophyll
would furnish here the simplest explanation,
’

176 K. ASO:

Further the experiments of Maki and Tanaka* have proved that
afier overliming the original fertility of the scil can be restored by the
addition of magnesia. This would be impossible if only the absolute
amount of lime and not the ratio of lime to magnesia were the cause of
the depression. On the other hand it has also been shown that the in-
jurious effect due to an excess of magnesia in the soil can be avoided by
a proper dose of lime. Tence it is of fundamental importance to bring
about a favorable ratio of lime to magnesia in the soil,—or, in case of
a very different degree of availability of the lime and magnesia com-
pounds to provide that the two bases be absorbed by the plants in
suitable ratio®. Recently two Italian authors, L. Bernardini and G.
Corso, of the Agricultural Experiment Station of Portici, have con-

tributed further experimental data in regard to this problem’.

Rye and maize were grown in culture solution, and rye, maize and
pea in pots and maize also in field enlture with varying ratios of lime to
magnesia, with the result, that the ratio 1:1 proved the best for rye, 2:1

for maize and 3:1 for pea. The authors conclude with the words:

“Queste prime ricerche confermano i risultati ottenuti dagli speri-
mentatori giapponesi e dimostrano quanta influenza abbiano sullo sviluppo
delle piante i diversi rapporti fra calee e magnesia nel terreno,”

The writer has carried out some furiher experiments on this ques-
tion in sand eulture, in which the two bases, lime and magnesia, were
offered to the plants in different quantities, but always in the same ratio.
Oats and adzunki-beans (phaseolus mungo) were selected for these tests.
Our previous experiments have indicated that for leguminous plants the
most favorable ratio is about 3:1, and that for oats it lies between 1:1

and 2:1. The ratios applied now were:

2. Bul. College of Agric. Tokyo. Vol. VII. No. 1.

3- Cf. Daikuhara’s experiments with lime as carbonate and magnesia as sulphate. (Bul.
Imp. Centr. Agric. Exp. Station Japan Vol. I. No. 1.)

4. Intorno all influenza di vari rapporti fra calce e magnesia sullo sviluppo delle piante,

Portici, 1907.

ON THE INFLUENCE OF THE RATIO OF LIME TO MAGNESIA, 17

io (2A
Sh Sp Ss
I [ I

Two series of pots, each containing 214 kilo sand, were prepared,
in one of which the absolute amounts of the two bases were five times

those of the other, namely:

1 A, AS A,
CaO Ig. 2g, 4g. 8g.
MgO 2g. 2g. 2g “28.
B, B, B, B,
CaO 5g. log. 20g. 40g.
MgO Iog. log. 10g. 10g.

Both bases were applied in the form of the natural carbonates as

very fine powders (<C0.25 mm.) in the following ratios:

A, Ish A, Teds,
Magnesite powder 4.2g. 4.2g. 4.2g. 4.22.
Calcium carbonate 1.8¢. 3.6g. 5.4g 7.26

B, B, B, B,
Magnesite powder 2tg, 21g. 2Ig. 2Ig.
Calcium carbonate og. 18g, 272. 362.

As general manure for each pot served,

K,HPO, 0.2g.
KIT,PO, 0.2¢.
K,SO, 0.2g.
NH,NO; 0.8¢.
Fe(OH), 0.5:

Oats were sown March 12, and adzuki-beans April 22, five seeds
in each pot, which were diminished afterwards to three plants in the
ease of oats and to two in the ease of adzuki-beans. Every day enongh
water was added to each pot to fill it to half its water holding capacity.
In the first stage of development, there was hardly any noticeable
difference. but it became quite marked later on, especially in the ease
of oats. The accompanying photographs and the following measnrements

date from June 18.

178 K. ASO:

LENGTH OF THE LONGEST STEM.

A, 146.6 cm. B, 149.3 cm.
A, 170.6 ,, B, 1538.6 ,,
AS 146.6 ,, By 156.0 ,,
A, 147.0 ,, BE T4260: 5

From this, it is clear that the lime-factor for oats is 1 irrespective
of the absolute quantity of lime and magnesia offered.
The plants were cut when deadripe and weighed in the air-dry state,

with the following results:

Total harvest. Roots,
(Grains and Straw.)
A, 32.0 47
A, 31.0 4.8
A, 30.0 4-7
A, 31.0 45
B, 32.0 3.4
B, 35.0 3.8
B, 33.0 3.1
B, 320 2.2

In the case of adzuki-beans, most leaves dropped off before all the
fruits had ripened. The weights of the seeds and roots in the air-dry

state were as follows:

Seeds. Roots.
A, Bi rs
A, 2.4 2.1
A, 3.6 2.1
A, 3.2 ear)
B, 2.1 0.9
B, 3.3 1.3
Be 3:5 1.7
B, giz 1.1

Altho the results obtained do not show very decisive differences’,

the lime factor for oats is shown to lie between 0.5 and 1 and that for

5. Since it is very important to give ample room to the roots, it was found too late that

three plants per pot were too many and the difference in growth was consequently less noticeable,

ON THE INFLUENCE OF THE RATIO OF LIME TO MAGNESIA. 179

adzuki-beans to be about 2 whatever be the absolute quantity of lime
and magnesia offered to the plants.

In the next experiments, paddy-rice and Italian millet were selected.
General manure and the quantities offmatural carbonates applied in each
pot were quite the same as in the former experiments.

On July 16, three young rice plants were transplanted from the
seed-bed into each pot. The accompanying photograph was taken on
September 7. On November 5 the plants were cut and weighed in the

air-dry state:

Grains. Straw. Total.
A, s a: aan
ae 14.5 15.5 30.0
cee 75 15.3 22.8
A, 5.7 14.8 20.5
B, 10.5 17.0 27.5
B, 10.7 17.5 28.2
B, 10.5 17-5 28.0
Lb, 9.5 16,0 25.5

On July 25, seven seeds of Italian millet were sown in each pot,
and the plants were reduced to three of equal size later on. On Noy. 5,

the plants were cut and weighed in the air-dry state:

Iruits. Straw. Roots. Total.
gr. gr. gr. gr.
A, 4.2 4.5 1.6 10.3
cts 2.8 4.0 1.4 8.2
A, 2. 3.9 L4 8.2
A, 2.6 3-7 0.9 7.2
B, 3.8 3:8 1,2 8.8
Bb, 4.2 4.8 1.4 10.4
BS 3-3 3-4 0.3 7:0
BE 3.2 35 0.5 7:2

From these results, it is clear that the lime-factor for rice is 1 and
that for Italian millet lies between 0.5 and 1, the absolute quantities of
lime and magnesia having no influence ou the development of the plants,

but the ratio between these bases being the chief factor concerned,

Fiat = miby hs: -
12 ~A
K. ASO:
CONCLUSION.

! 1. Certain favorable ratio of lime to magnesia for plant-growth
a exists even in sandeulture.
, 2. Absolute excess of lime or magnesia provided it be kept within”
fe certain limits, has no retarding effect on the development of the plants,
: the ratio between these bases being the chief factor for plant growth.
}
: x
)
,
;
ay
.
~ ~

Plate IV.

Bi Surksva JO oouanyus ayy Moys sajyeyd asoyy,

VISOUSeUL PUB dUIIT JO

‘AIN}[ND puvs Ul syvo uc

TH “Id

A B A, B. A, B, A, B,

This plate shows the influence of varying ratios of lime and magnesia on rice in sand culture.

Is Artificial Calcium Carbonate more Effective than
Limestone Meal ?

H. Yokoyama,

The availability of mineral nutrients for the roots of plants depeuds
to a large degree upon the fineness of the particles, and since precipitated
compounds consist generally of finer particles than pulverised minerals it
was of interest to compare the quantitative manuring effects of pre-
cipitated calcium carbonate with the finest limestone meal’, The expert-
ment was carried_out with oats in sandeulture. Each pot held 2,5 kilo

well purified quartzsand and received the following general manure:

* K,SO, ; ie
at (NH, )NO, 08 ,,
, a Na, HPO, CHS) 5
U

Fe30, 0,05 ,,

a
7

>
‘

The magnesia was applied as magnesite meal—I4g. to every pot,

while the amount of precipitated calcium carbonate was varying.

lay.
\ io
; Control.—VPot A received limestone meal=12 g,

5 received 3 g.
IG; 6 g.
D % 9 g:
BY 5 2g:

Precipitated calcium carbonate.

Hereby the following ratios were produced :

1. ‘here exists a very great difference in the manuring effects of precipitated magnesium
carbonate and magnesite meal (see these Bulletins, vol. VII p. 609) but here the physical
condition and the chemical composition also is different between the two preparations; the

former is a daséc carbonate, which is not the case with the precipitated calcium carbonate.

182 H. YOKOYAMA:

A B G D E
CaO : MgO. Bae 0,25: 1 0,50:1 0.75:1 Tek

Two parallel series of pots served for the test. Six seeds of oats
were sown, January 15, im cach pot, and the young plants after 30 days
reduced to three of equal size per pot. The plants developed well until
some time after flowering when frmgi commenced to show upon the leaves,
whereupon the plants were cut (June 19) and weighed in the air-dry state,

with the following result:

Number of Total har- Weight of
Ratio CaO : MgO. shoots. vest, g. grains, g.
(unripe)
Limestonemeal 8 37 74
Dik II 34 6,0
: 7 21 455
2 { 0,25: 1 { 9 25 st
EI : 9 26 5.4
gape { 9 24 48
ae
=e 10 32 5.4
a3 | onset 9 28 38
< ee 12 40 7:9
( : 13 29 6,1

In comparing the yield of pots A and F, it will be seen that the
artificial calcium carbonate was not essentially more effectual than fine
limestone meal. It will be further noticed that the yield commences to
sink as soon as the magnesia content exceeds the lime content in the pots,

in accordance with former observations.

On the Lime-factor for Oats.
BY

J.N. Sirker. (Caleutta.)

The lime factor i.e. the best ratio of CaO to MgO differs considerably
for different plants.
Some plants can deposit the absorbed excess of lime in the cells

as calcium oxalate. Such plants can thrive well even in sols with an excess

of lime.

Some plants, however, cannot thus dispose of the excess of lime and
the effect shows itself in the considerable decrease of the harvest. To this
group of plants belong flax’ and various cereals?. These plants yield the
best result when the ratio of lime to magnesia is 1, while the first mentioned
eroup of plants thrive much better when the ratio is greater. Generally
speaking, more lime is needed when the development of the leaf surface

is greater in a given time.

The lime factor for cereals has been determined by several authors
in soil culture. Tt was desirable therefore to repeat the experiment in
sand culture. Quartz sand was purified with dilute hydrochloric acid
and after washing well with hot water and drying, put into porcelain pots,
21% Iilos of sand to each. The following was then added to each pot as

general manure :—

K,SO, REE mn O50 1GL,
INED INO Seaieeestitesiitsssiiece)) O1S0) 2Te
Na, HPO y--.2aq) ss. a2) =. 0.50) gr.

1. Bul. College of Agric., Tokyo. VII. No, 7.
2, Cf. Bull. College of Agric, Vol. IV. No. 5, V. No, 4 and VI. No. 2.

184 J. N. SIRKER:

Further, finely powdered limestone and magnesite were added in

the following ratios :—

Limestone Magnesite C20
Pot. in grms. in grms. MgO
A Re oh cts 4.3 5:0 I,
B aeeh tewsus eee creer (Or 5.0 2
Cc 1 129 5.0 sh
D ‘ . 25.8 5.0 6.
E Ree eae eects 10.0 %.

Two pots were used for each ease. Ten seeds of oats*were sown in
each pot on the 3rd of March, 1907, and when the plants had reached
the height of about 8-10 cm. they were reduced to four plants to each
pot, care being taken that they were all of equal size as nearly as possible.

Water was added regularly to the pots, and they were occasionally
taken ont of the glass house when the weather was favourable. The

number of stems in the different pots is given below :—

Pot. A B (e D E
TE sycc) seemeeremete. $10 14 13 II 12
I Aas) Sate ner 4, 15 11 12 14
Average eee Mies ALS 14.5 12 ILS 13

The ripe crop was harvested and weighed separately in the fresh

state on May 22, 1907, with the following result :—

Pot. A B Cc D 1D}
Le etree arenes 50. Et. 58 er. 56 gr. 53 gr. 57 gr.
Lie ee nn So LT. 51 gr. 51 gr. 49 gr. 52 gr.
AVERdrE 25) ilies 515905 fr. 56.5¢0r. lee baer 51 gr. 54 gr.

From this it is obvions that the lime-factor for oats in sand eulture
is 1, thus confirming the results obtained by previous authors with other

cereals,

On the Application of Bisulphide of Carbon in
Mulberry | Culture.

J. N. Sirker. (Calcutta.)

Silk culture has occupied one of the most prominent places in the
industrial world. Most probably China was the oldest country to produce
silk. France and Italy has made a good progress in silk industry, basing
the culture of the silkworm on strictly scientific principles. But in recent
years Japan has introduced a new era in the silk industry of the world.
In Japan, silk comes close after rice in importance as an article of
domestic production, while as an article of export it has no compeer.

This important industry depends upon the rearing of the silkworm,
which again depends entirely upon the cultivation of mulberry. Recently
various authors have published their observations on the beneficial effect
of carbon-disulphide in plant culture, when applied to the soil together
with complete manures’. It seemed to me of some interest to observe
the effect of the application of carbon disulphide on the growth of mulberry,
and to compare it with that of an extra-dose of sodium nitrate applied
as top-dressing.

Three plots, of sixteen square metres each, served for this experiment.

They were deeply and loosely ploughed, and freed from roots, stones

ete. The general manure for each plot was:—

Superphosphate te ee. one eee 20 ¢.
Ammonium sulphate Ae oe LO Thee 30's,
Galciumicavbonate <5) fees acs eee) cee ves cae 20 ,,
Fotassitimlsulpliatens..n ckmeten sewn ee.) sce, aac 10 ,,
Potassinnichlonide sens rem seCMccawi sect cos «se 10

1, Although there are various opinions about the cause of the beneficial action of carbon

disulphide, there is as yet no satisfactory explanation of it.

186 J. N. SIRKER?

In the first plot, nine holes were made and 50 e.c. of carbon disulphide
was poured into each ten days before planting. The holes were filled in
immediately afterwards and water was poured on. In the second plot 40 g.
sodium nitrate was applied as top-dressing in two portions—one portion on
May 3, and the other on June 1, while the third plot received no special
compound and served as control.

Young plants of about eqnal size were planted on April 5, 1907.
Those of the first plot were planted in the same spots where the carbon
disulphide holes were made. On September 20, the harvest was gathered

with the following results:

NUMBER OF BRANCHES,

Plant.
——————— Average
Plot. I 2 3 4 5 6 7 8 9 Total. per plant.
First 5 6 18 II 4 6 12 6 15 83 9.2
pecond)..,) -.80) 5 II 5 5 7 4 7 4 54 6.0
Control ... 7 8 4 es) 5 5 7 6 52 5.8
HEIGHT OF THE PLANTS.
(in metres)
= ee Pie See Ss Average
Plot. I 2 3 4 5 6 7 8 9 Total. per plant.
First! \.5. <0) TsO) M.GQN 1.43 1,55. 9:75) oV.53ekeoe Onur 5y 14.60 1,622
Second. ;.; <.., T.90) 1:60) 1-35 1.93. 1.681050) ay 55 Toe 14.84 1.649

Control’... .. 1:67) Taz) 0.97 162° 1.46) 1-365 90.30) Xen ar.Or 12.91 1.434

NUMBER OF LEAVES.

epee Pt) Average
Plot. 1 2 3 4 5 6 ih 8 9 Total. per plant.
First ... ... %4% 233 276 199 152 203 644 319) 185 2.352 261.3
Second... ... 195 161 240 183 161 195 I4t I7 221 1,668 185,3

Control ws 250 129 143 231 157 193 135 180 215 1.633 181.4

Ten leaves were selected from each plot and weighed as follows:

First plot Fay ate ov aptinnWeat aay 74.5 £
BECOna DIORY sup) we a5 ee ie 50.5 g.
COMIOUE cede) Uecet (ecg) hexed Moga) Se 47.0 g.

ON THE APPLICATION OF DICYANDIAMIDE. 187

This result shows that the application of bisulphide of carbon to
the soil under the manuring conditions above-mentioned increased the
yield of mulberry leaves by 44¢%, while the top-dressing with an extra-
dose of nitrogen in the form of ou nitrate was of but little use in
this case*. _ ;

2. Perhaps the quantity of nitrogen applied in this case was nearly sufficient.

—

Ueber die Blatt-Ernte bei Polygonum Tinctorium bei
reichlicher Stickstoffdiingung.

VON

T. Takeuchi.

Da reichliche Stickstoffdiingung besonders dic Blattbildung beférdert,
und die litter von Polygonum tinctorium tiir Indigogewinnung in Japan
trotz der bedeutenden Einfuhr kiinstlichen Indigos noch eine grosse Rolle
spielen, war es von Interesse, zu beobachten, wie weit die Blattbildung bei
dieser Pflanze durch reichliche Stickstoffdiingung beférdert werden

kdnnte.

Ks wurden vier Parcellen zu je 12 Quadrat-metre auf eimem lehmigen
Humusboden zuniichst mit dem gleichen Grunddiinger, nimlich 2400 e,
Kompost (2000 kilo pro ha.) +300 ¢. Holzasche (== 250 kilo pro ha.)
versehen. Die Parcellen A und C dienten zur Kontrolle, wihrend B. u.
}). zweimal je 180 @. Chilisalpeter als Kopfdiigung erhielten. Die
jungen Pflanzen wurden am 21 Juni eingesetzt, die Entwicklung fand

ohne jede Stérungen statt.

Die Ernte am 13 August ergab.: Gewicht, lufttrocknen, g.

A b C D
Blatter <:. “.. 650 1160 770 1255
Eiaiime Geo a. ash 1365 1410 1550

Nun wurde nach dem Schneiden den jungen Trieben freic Entwick-
lung gelassen, um eine zweite Ernte zu erzielen. Die Parcelle D erhielt
diesmal dicselbe Dosis Nitrat wie das erste Mal, wiihrend B nur 200 eg.
Nitrat als Kopfdiineung in 2 Dosen. Die eine Koutrolparcelic © aber

erhielt dicsmal 400 c.c. Schwetelkohlenstoff, welcher in kleinen Dosen

190 T. TEKEUCHI:

in etwa if 4 Fuss tiefe Locher zwischen die Pflanzen ecingegossen wurde.

Am 9 October wurde die Pflanzen geschnitten mit folgendem Resultat:

Gewielt, luftivocknen, g.
A Lb (3 D
Blatter! <.2) <.. 630 785 760 $23
Halme ... ... 895 960 955 990

Die vermehrte Stickstoffdiingung hatte also sehr giinstig auf den
Blattertrag gewirkt, da der Mehrertrag am Bliittern (erste und zweite
Ernte zusammen) bei B=52¢ und bei D=62¢% iiber A betrug. Die
Schwefelkohlenstoffbeliandlung hat mehr auf Stengel als auf Blattbildung
gewirkt.

Auf denselben Parcellen wurde im folgenden Jahre noch ein Versuch
mit etwas abgeinderter Grunddiingung ausgefiihrt:

. z 3 kilo Kompost pro 12{_]m. (=2500 kilo pro ha.)
Grunddiingungf :
+120 g. Holzasche pro 12{_}m.(=100 kilo pro ha.)
D erhielt einige Zeit vor der Pilanzung 400 ¢.c. Schwefelkohlenstoff.

Die Kopfdiingung war:

Ao... a eee ss ~=Kontrol, keine Kopfdiingung.
B ... .. « «+ Na NO, 300g, in zwei Fraktionen.
UO ee CC : EeuyeyE a 6

Die Ernte am 24 August ergab.: Gewicht, lufttrocken, g.

g.
A B Cc D
Blatter <a 1170 1325 1665 1285
Halme 3. 4... 1680 1695 2710 1890
‘LOC MassBanse 2050 3020 4375 3175

Nach dem Schueiden wurde den Trieben freie Entwicklung gelassen.
Diese zweite Erute, am 7 October im lufttrocknen Zustande gewogen,

betrug, g.:

A B £& Dd
Bistterosom. AIS 514 572 535
Halme ...... 306 453 510 436

Total tng 719 967 1082 971

UEBER DIE BLATT-ERNTE BEI POLYGONUM TINCTORIUM, 191

Setzen wir die Ernte an Bliittern anf der

Kontrolparcelle A=100, so ist der Ertrag

bei B=116.1
” C=141.3
E D=114.3

Die Schwefelkohlenstoffbchandlung hat somit eine geringeren
Mehrertrag gebracht als reichliche Stickstoffzufuhr in Form von Kopfdiing

mit Chilisalpeter.

On the Application of Dicyandiamide as a
Nitrogenous Manure.

Since manuring with ‘Time-nitrogen’ will gradually spread according

to its importance for agriculture, it will be of some interest to investigate

the suitable method of the application of dieyandiamide as a manure.

For this purpose the followimg experiments were made.

Five pots containing 8 kilo air dry soil were manured as follows:

Pots. Superphosphate, Totasiam Ammonium Dieyandt

I. (Control) 10 g. 5 g. 5 g. fo)

I. 10 5 3:3 0.75 g.

Ill. 10 5 ahs 0.75 (as topdressing)
Ves 10 5 fo) 22

Vv. 10 5 fo) fo)

Ammonium sulphate employed contained 217 nitrogen, and dicyand'amide!l, being some-

what impure, 46.722 nitrogen.

As the above table shows, the quantities of nitrogen applied were equal
in each pots, except the pot V, which contained no nitrogenous manure at
all. Thus the pot I served as control, and contained the total nitrogen as
ammonium sulphate; the pot II, */, of the total nitrogen as ammonium
sulphate and the rest '/, of the nitrogen as dieyandiamide, while the pot
III received the manure in the same porportion as the pot IT, but
dieyandiamide being given as top-dressing later on; the pot TY, the total

nitrogen as dicyandiamide.

1. The writer prepared this compound from ‘ (ye nitrogen.’

194 R. INOUYE:

For the experimental plants served rape and barley.

On November 26, twenty seeds of each plant were sown in each pot

after selecting healthy ones by steeping them into water.

(1) Experiment with Rape.

On January 17, the young plants in each pot were reduced in
number to ten of nearly equal size. The plants in the pot I, IT, and TIT
have grown well, especially the last two very well. Eight weeks after
germination, the margins of the leaves of the plants in the pot IV
became yellowish white showing the injurious effect of dievandiamide and
their growth was very much retarded. On February 22, they were again
reduced to five. Although at the beginning of the growth the plants in
the pot II were injured, they were quite recovered after four months,
and grew so well as the control plants. On Feb. 28, 0.75 g. of dicy-
andiamide dissolved in one liter of water was topdressed to the pot ITT.
After three weeks the margin of the leaves of the rapes in this pot began

to wither, but the damage was not so severe as in the pot IY.

By this time, the plants in the pot TV have shown the same un-

favorable emmen like those in the pot V.

On March 12, almost all of the plants came to bloom, when the

accompanying photograph was taken (see the plate TI).

The crop was harvested and weighed in the fresh state on April 14.

The following results were obtained.

AN AVERAGE WEIGHT FOR ONE PLANT.

Weight of the upper

Pots part of plants oe eiclebiant
&. g.
16 57 2. 59.4
IL. 60 2.6 62.6
III. 61 3 64
aN, 7.8 0.6 8.4

Y; 5 0.4 5-4

ON THE APPIICATION OF DICYANDIAMIDE. 195

(2) Experiment with Barley.

The seeds were sown on Noyember 26. On January 17, they were
reduced to ten of equal size per pot. In the early period of the growth,
no particular phenomena were observed, but after eight weeks since they
had been sown, the leaves of the plants in the pot IV, which received 2.2 ¢g.
of dicyandiamide, began to wither from iheir tips and edges, and these
withered parts gradually developed. ‘The injurious action of the manure
was also observed on the plants in the pot II, but it was not so severe
as in the pot IV. On February 22, when the young plants had reached
to 11-15 em. high, they were again reduced to seven. On Feb. 28 ihe
pot III received a new dose of 0.75 g. dicyandiamide, dissolyed in one
liter of water. Although the plants in pot were injured to a certain
degree, they began to show the most favorable growth among all after
three weeks. In the middle of April, the beneticial effects of dicyan-
diamide became gradually more noticeable, and the plants in the pot ILL
were the best; the plants in the pot II next, and those in the control pot
third, while the growth of those in the fourth pot was not so much
retarded as that of the rapes in the fourth pot. Since some of the plants
were damaged by fungi, they were reduced to four per pot on April 30.
The accompanying photograph (the plate II.) was taken on May 14,

when they came to full growth.

The plants were harvested on June 19, and weighed in the air-dry

state with the following results.

AVERAGE WEIGHT FOR ONE PLANT.

Number Weight of dry Weight Weight of Weight
Pots. of plants (roots of ears with of
branches. taken off.) straws. grains, grains.
g. g- g. g.
I 3 8.3 4 3:5 2.7
II. 3 9 5 4 3.1
Ill. 4 9 5 4 355
IV. 2 2.5 15 1,2 I

Vv. I 1.8 I 0.8 0.7

R. INOUYE:

CONCLUSION.

Dicyandiamide may be injurious to the plants, but if the dose de

not surpass a certain limit, it does rather act beneficially to them
it is, indeed, a better manure than ammonium sulphate. . :
The pot I, I, IIT and IV received an equivalent quantity of nit (
in the different forms, but the plants in the pot IIL to which dicyandi
was topdressed, yielded the best harvest of all, on the contrary, the pl
in the pot IV, which received dicyandiamide as a only nitro
manure, were as much injured as those in the pot V which receiv

nitrogen. ‘ Bee i

From these results, we can say that one gram of nitrogen in the £ ;
of dicyandiamide for eight kilo soil is even injurious to the crops
especially to the young plants, while 0.35 g. of nitrogen in the
from yields a favorable result. It may be infered that if dieya 1
is applied as a manure, it is better to use it as topdressing. ta

Bis. LE

ria

Some Improvements in Sand Culture.
7
'
BY

T. Takeuchi.

-. Various methods of manuring sandeultures have been proposed to
overcome certain difficulties. Since sand has no absorptive power, the
reaction of the manure salts and the concentration of the ‘soil solution”
is here of much greater importance than in soil culture. The quick
evaporation of moisture from sand adds to the complication, In some
eases it was proposed to apply phosphoric acid as monopotassium phosphate,
which, however, on account of its acidity may exert a certain injurious
action owing to the rapid evaporation of the water and the consequent
concentration of the solution. The guiding principles for obtaining

— fayorable results in sand culture must be:
ss

’ (1) The diminution of the amount of soluble salts,
(2) “The insoluble ingredients of the manure must be very easily
a available for the roots,

ae (3) Care must be taken to preserve a neutral reaction as far as

possible during the time of vegetation.

2
‘hese considerations have led the writer to apply nitrogen in the

of ammonium nitrate’; further phosphoric acid in the form of a
mixture of mono-and dipotassimm phosphate to ensure neutral reaction.
Lime and magnesia were applied as finely powdered natural carbonates

a favorable ratio for the common cereals. Further, a small amount of

1 Hellriegel (Ueber das Stickstoffbedarf der Gerste) says: “Ammonium sulphate and

chilisalpetre are unfit for sandculture.” Since an acid, or alkaline reaction, will be gradually

produced by these compounds, this statement appears justified.

198 T. TAKEUCHI:

gypsum was added, not only as a source of the necessary sulphur, but also
for neutralising any alkaline reaction that might gradually result. Final-
ly a swall dose of sodium chloride was added, since the favorable action
of sodium as well as of chlorides is well established. The iron was

applied as ferric hydroxyde.

It was further desirable to institute a comparison to Hellriegel’s

sandeulture, who” used for 4 kilo sand:

11S WAZ OP MP Abs “con, cro. Cob Lele p tot
BGG cas) sce scsi) (ada) atsuemer OLA OSM

MgSO... °...) sh a eee SOLSOO IS

Further caleium as carbonate + g., while the addition of calcium
nitrate? was varied in order to observe the effect of different doses of

nitrogen.

The pots of the writer contained 4 kilo quartz sand to which was

added,
Gal (Cl & ORO roe ere Gas pane S288 In
1-12 15 @ Re Sorat cap eee 85) 4
ROE scx fast jess) oc) Seema
INET SNO sez, cea, Sats) Soccer MaRS
NaCl Treo rn. ies
CaSO 0.6 ,,

BECOR) i ie, cfs Tose ceeer Sea aoe
aia sue: een weal” MaaatlsmiOn ails

AAESEONE}’ 5 cig. conn) uecsdeasn bites

2, It seems strange that Hellriegel in two publications, namely on the “ Stickstoflbedarf
der Gerste” and on the “Assimilation of free nitrogen by Leguminose” do not mention in
what form the iron was applied in his sandcultures. In his * Grunddiingung” the addition of
iron compounds is not mentioned! The writer has, nevertheless, added iron in his pots as
ferric hydroxide.

3. In this case 1.68g was used,

4. Both these carbonates were in the form of very fine powder <0.25 m.m.

SOME IMPROVEMENTS IN SAND CULTURE. 199

To a second pot (1) 5% kaolin? was added in order to produce some
absorptive power and to increase the water retaining capacity. A third
pot (IL) was prepared exactly according to the prescription of
Hellriegel. The water was so regulated that it corresponded to 154 of
the sand, i.e. 60% of the water holding capacity of the sand. In other
words the 4 kilo of sand was so irrigated that it contained 600 @. water.
Thus the concentration of the nutrient solution would be 0.4¢. But to
avoid this somewhat high concentration,® the ammonium nitrate was
applied in two doses one half before sowing and the other half when the
young plants had reached the height of about 20 em.

The plants used were upland rice, barley, wheat and oats.

Experiment with Upland Rice.

8 seeds were sown April 21 and the young plants reduced later on
to 4 per pot, all of nearly equal size. At first, the pot (11) seemed rather
prosperous, but after the application of the secord dose of ammonium
nitrate this condition was reversed, (1) and (K) showing more rapid

progress. The plants were cut Noy. 4 with the following results (air dry).

Number of height Weight of
— ——— —_— — Ss
ears grains (average) grains straw total
44 4 288 go cm, 8.9 g. 14.6¢. 23.5 g.
it 5 207 S7ies 8.1 ,, 12.6,, 200
iH 4 107 69 ., 33h; PaXirss 10.4 ,,

5. This kaolin contained but few impurities and was of neutral reaction. 100 g. treated
with TICl of 107% yielded after evaporation of this extract only 0.63 g. residue which contained
no phosphoric acid, but only a small quantity of iron and potash. A further examination of

the kaolin yielded:

UXSOW ee Bx aki soo, Go. Pot oe
UGA) aan ace oo we ety pee ey
WTO ais MG in = Og. CEO. @ GOR MONey aay
CaO ae rete ees ces OLOK 5;

6. Tellriegel (Grundlagen des Ackerbaues, p. 275) considers 9.2 °/,,, concentration of the
solution in the sand as not injurious for the young plants, but the writer has avoided this

concentration.

200 T. TAKEUCHI:

Experiment with Oats.

The experiment was carried out just in the same manner as in
upland rice, with three pots in the glass house; but the pots contained
’ three kilo purified quertz sand instead of four kilo. The manure of (1),

therefore, consisted of

KIT PO, 2. 00) aes sae) cas) (OL225 Ry
K HPO, ... eee cee nee ee 00225
GSO nue ces 5 Sov) seen MESO Gy
NH,NO, ... 22.0 12 see oes O.900 yy
PW, had aay code pats SO
CaSO, --. =. 1. nee ewe vee 0450 5
Bel QED) = = gs asce =+s0 SEROUS
MMAPMESIIe” <2. | acs) sic. omer ee OOO
ame@stone*=.. «ss ..0 was? ees Ones

(KX) and (H) also received similar treatment.

8 seeds were sown Dec. 9 and reduced later on (Jan. 25)
to 3 per pot, all of nearly equal size. The second dose of ammonium
nitrate was applied March 7. A difference in development soon became
evident after this and on the 22 of April the plants in (1) and (K) showed
ears, almost at the same time, while those in (TT) took ten days longer.
The appended photograph was taken at this stage. The measurement on

May 10 was as follow:

Number of ears. Average height.
he (eee 9 118 cm.
A eeceitaes Vive cans 10 105 ,,
sees ee 7 86). 5

The plants were eut June 16 and weighed in the air dry state, with

the following results:

Length of Number of Weight of
stalk roots aoe ey waripened ears ripened ears straw roots
seeds seecs : seeds
K 123m. 24.6 cm, 238 114 9 9.01 g. 15.6 ¢. 434g. 9.62 g.
I 114, 22.8 5 186 135 105-4955 10.7 ,, 45-7 »» 8.14,
H 108 ,, L7G 101 86 7 3.59» 6.5 42.0,, 7:35»

SOME IMPROVEMENTS IN SAND CULTURE. 201

Experiments with Barley and Wheat.
These experiments were made exactly in the same way as in the pre-
ceding case. Sowing, reduction, topdressing and harvesting were also
carried out as in the preceding experiment. The harvests were weighed

June 16 in the air dry state with the following results:

BARLEY.

Length of Number of Weight of
— > ooo" : a
stalks roots pipcned ears mpeoed ears straw roots total
seeds seeds
Kk 87.7cm, 24.3cm. 136 II 7.89 g. 12.8 ¢. 25.3 g. 5.4 g- 43-5 g-
I 83.6 ,, 14.5 55 193 11 8.78 ,, 13.55, 17.4 5, 38,, 34-7 55
If 68.9 ,, 12.9 ,, 66 7, SHES) 6.8 ,, 14.1,, 3°35; CHGS
WHEAT.
Length of Number of Weight of
—<— —€——_—_—_ _— a ——w
stalks roots zxipencd ears teed ears straw roots total
seeds seeds
Kk 18cm. 27.2cm. 341 Io 11.35 g. 15.3 g- 28.9 g. 861g. 52.81¢.
TORI; OES 386 13 10.61 ,, TOR. 20.58 Pal San eats Sies
i 105 ,, 2InQues 217 9 9.05 ,, Less 26.0,, 5:-64,, 42.94,,

These results show that Hellriegel’s prescription for sandculture can
be improved by the modification here proposed. The addition of kaolin
(5@), however, has to be avoided in cases in which potassium compounds
come in question. When the question relates to nitrogen or phosphoric
acid, its addition can only be of benefit and not misleading, The amount
of limestone should he increased for other plants than the Graminex and

flax. The mixture of the author for Graminew in sandeculture is:

Sand ... Sar (G68) 5 Ga, cot coh Mr LoneleMommteat
K,H,PO, Ong) oy
MeteIIRO), Gas gt a ck oo @S} sp
K,SO, SP Cry aco Bes ond 0:3\5;
INDGU INTO. 8 SS O80 ocoy ako)» Boe 1.2 ,, (to be applied in 2 fractions)

af (@} Ae eerie pat bars aT eos 0.3

Magnesite ... ...

Limestone
Fe(OH),

Plate VIII.

PL. It.

Plate IX.

: in 5)
Wa Mia
oN

Secondary Caicium een as a Manure.
s

T. Takeuchi.

Repeated experiments carried out at this college have shown that the
most favorable ratio lime to magnesia for the smaller Gramineae lies
_ between 1:1 and 2:1. It remained to be tested, in how far under this
condition the harvest is influenced by yarying amounts of phosphoric acid,

when these three substances are present in the soil in about an equal

state of availability. According to the theory there ought to exist a
considerable influence, when the ratio of phosphoric acid to the limefactor
differs widely in the cells. And that there exist often varying ratios is
v- shown by the ash analyses of the same plants grown on different soils.

Further, the analyses of soils show, that the amounts of P,O, are generally
‘oe
7
_ former surpass either those of lime or of magnesia separately.

smaller than those of lime and magnesia combined, while frequently the

In my experiments lime and magnesia were applied as natural
carbonate in a very finely powdered condition (< 0.25 m.m.), while
phosphoric acid as precipitated secondary calcium phosphate, since these
compounds were suppesed to have about an equal state of availability.
Lime, magnesia and phosphoric acid were applied in sandeulture in the
following proportions ;

CaO . 1
(A) MgO: P,0,=
CaO.
MgO ~ E

ee (3 g. each)
2
5

ca0 P,0,=—: 53
28
5

ne (B)

(D) sao :P

204 T. TAKEUCHI:

From these ratios the amounts of the above compounds were as

fol lows :

CaCO,, g. MgCO,, g. CaHPO, + 2aq., g.
(A) 5.36 6.25 3.63
(B) 26.78 31.33 3.63
(C) 5.36 9.25 18.18)?
(D) 26.78 31.33 a

Four pots each holding 3 kilo well purified sand, received the follow-

ing general manure, euch:

K,SO, 05 g.
NH,.NO, 1.0) 5;
Fe (OH), 0.25 »
NaCl 0.05 5,

The ammonium nitrate was applied in 2 portions, the second half as
topdressing, when the young plants had reached 20 em in height (July 9),
the first half before sowing.

As crop served upland rice of which S$ seeds were sown in each pot
(May, 20); the young plants when 15 em. high (June 25) were reduced
to 4 per pot, all of nearly equal size. During the vegetation no disturb-
ance occurred, At the end of August the plants in A and C developed
ears, those in D 22 days later. The plants in B never showed ears at
all. The plants were harvested Oct. 2. with the following results, air
dry, g.

Average ht. Total wt. Number of ears, Number of grains, Wt. of grains

A 74 cm 7.53 4 108 2.0
B 68 ,, 22 No ear No grains °
Cc 75 » 9.78 5 151 3.3
D 70 ay 7.65 3 4 0.95

This result shows that a great excess of carbonates of lime and

magnesia can depress the absorption of phosphorie acid from secondary

1. By this increase the ratio CaO ; MgO was considerably changed.

SECONDARY CALCIUM PHOSPHATE AS \ MANURE. 205

calcium phosphate so much, that the formation of ears (with rice) becomes
impossible. ‘This depression of harvest can only partially be overcome
by raising the amount of this phosphate to the amount of calcium carbonate
present as the comparison of the hapyests in B aud D. will show. By
this increase of secondary calcium phosphate, however. also ile most
favorable ratio of lime to magnesia in the sand was altered somewhat.
In comparing harvests A and C it will be noticed that by changing the
ratio Cat): M20:P,O0, == 1:1:1 into 1:1:5 an increase of the total harvest
ot 29.9% and of grains of 65% has resulied, lence the increase of phosph-
orie acid in the form of secondary calcium phosphate has exerted such a
favorable eflect that the depression resulting by the great increase of lime
taking place by manurirg with secondary caleinm phosphate and thereby
changing the most favorable original ratio of CaO:MgO, became not
noticeabie. In comparing the harvest A and B, the detrimental effect can
be noticed, which is experienced, when soils containing phosphatic manures
in a water insoluble state are limed in a high degree.

Under certain conditions (probably when no or but little carbonates
are present in soils) the secondary calcium phosphate is an excellent
phosphatic manure, as Prianischnikow’ has shown.

It will appear to us, that although secondary calcium phosphate is
less soluble in dilute acids as calcium or magnesium carbonate so small
amounts of carbonates, as can depress the availability of tertiary calcium
phosphate, exert still but little effect on that of secondary calcium
phosphate and that this requires larger doses for the depression of its
availability.

As to superphosphate, however, probably only very large doses of
carbonates can lead to a depression of its availability. ‘This inference
may be permitted by varying a result obtained by Hamasaki*. In his
experiment disodium phosphate served as manure. In this case the change

2. Landw. Versuchs-Stationen. 56, p, 122.
3. These Bulletins, Vol. VII, p. 607.

206 T. TAKEUCHI:

of the ratio Ca0:MgO = 1:1 to ten times the amount (phosphoric acid
being constant) the harvest showed a moderate depression of 14¢.°

The question will entirely change its aspect, when lime, magnesia
and phosphoric acid are applied in water soluble forms, or when lime and
magnesia are present in soils not in the form of carbonates. Under these
conditions the varying ratios between the lime factor and phosphoric acid
will doubtless show other and very interesting results. I hope to continue

these investigations.

4. Cf. Also Westhauser and Zielsdorf, C. f. Agric. Chem., 1908.

On Differences of Susceptibility of Plants to Stimulation.

T. Takeuchi,

Since it had been observed here at this College on several occasions
that certain plant species are more susceptible of stimulation than others,
it seemed desirable to extend such observations.*

The following plants were compared under equal circumstances;
spinach, pea, barley and flax.

8 pots each holding 8 kilo of a loamy soil received the following

general manure:

8 ¢g. Double superphosphate
BN on Potassium sulphate

UW) Sodium nitrate

Sues Ammonium sulphate

Four pots served as control pots, while of four pots each received
0.2 @. crystallized managanous sulphate MnSO, + 4 aq. which was incor-

told

porated in the form of a very dilute solution (1 p. mille), while the control

pots received an equal amount of water.?

I, Jt may here be mentioned that according to Kérni%e the seeds of Vicia fade are
much more sensitive to radium rays than the seeds of Brassica.

2. A former similar experiment with 2 g. manganous sulphate per pot had shown that
this dose was too large and somewhat injurious, especially for tie young plants. These

results were: g.

Barley Pea Flax Spinach
———— ——- ++ — a
Control Mn. Control Mn. Control Mn. Control Mn,
Total Wt. 109.5 106.4 102.4 103.2 50 47 149 82
Seeds 48.1 47.7 56.2 55.1 —_ — = =
Fruits 27 26 = eS.

Mr. Muramatsu had observed further that topdressing with 1 g. MuSO, for ro kilo soil

depressed the yield also; these doses are therefore too large.

208 T. TAKEUCHI:

Spinach.
Twenty seeds were sown Noy. 10. The number of the young plants
when 3-4 em. high was reduced to 8 per pot all of nearly equal height.
The plants showed gradually a considerable difference in development:

Average height :

Control Manganese
Dec. 19 4 cm. 4.5 cm,
Jan. 29 6: os TOs.
Mar. 2 22." 9s, 20000;

The plants were eut March 22; the weight in the fresh state was:

Control plants 129 g.
Magnanese plants 182 ,,
Hence increase=41%

Pea.
Twenty seeds per pot were sown Noy. 10 and the young plants

thinned after two weeks to 8 in each pot.

Average height :

Control Manganese
Dec. 19 7.2) (cm, 9.3. cm.
Jan. 29 73:6) Des. 16:0) 1;
Mar. I 27-5. Gs 20.4) aay
April.. _ 1 56.4" - 455 626 ..

The plants were cut May 24 and left to dry; the air dry weight was:

Total Pods Seeds 100 seeds
Control 75 46.8 34.3 27.3
Mo. 85.4 48.2 26.1 29.2

Hence total harvest increase = 19.4¢ which is about one half of the

increase, observed in the previous experiment.
Barley.
Twenty seeds of Barley were sown Noy. 10 and the young plants

later on thinned to 10 of nearly equal size:

ON DIFFERENCES

Dec. 19
Jan. 29
Mar. I
Apr. I

The plants were ent May 28 and weighed in

or
s.

the followine results,

Control plants

Manganese plants

Hence increase of total harvest =5,

Twenty seeds were sown Nov. 10 and later on the young

reduced to 8 per pot, all of nearly equal size.

Average height :

OF SUSCEPTIBILITY OF PLANTS TO STIMULATION. 209

Control 7m ERR eaneee
14.2 cm. 15.1 cm.
18.5, 19.4 55
PAN Ay ZS. OMmnas
A2TA Sars; 43-9 55

Total
140.8
148.3

Flax.

Grains

51.0

50.4

the

39%

air dry state with

Straw
89.8
97.9

plants

The growth and flowering

of the control plants was behind that of the manganese plants:

Average height :

— SS

Control Manganese
Dec: 19 Bes, CM, 4.0 cm.
Jan. 29 Cape ln OMA.
Mar. I 10.1 7 12.0 oF
Apr. I 18.2 7 ZKOMa

The plants were harvested June 11 and gave the following results:

Total weight Weight of fruits Number of fruits.

Control plants S2nge I1.3 g. 169
Manganese plants 36.8 g. 13.2 g. 187
Hence increase of total harvest = 13.99%

This result shows indeed that different plant species are not stimulated
at an equal degree by manganese, under the same conditions. Former
| : A :

observations at this College seemed to prove that Leguminose and Cruei-

210 T. TAKEUCHI.

feree were more susceptible than Graminex. Also this time a Graminea,

viz. barley, was the least stimulated.* The comparison shows:

Percent of Stimulation.

Barleyeccoty «s+ jcc) fesachte-ote I
Blaze wc ee ee ee
PPM Et icis 's05 2) ise ets ae ee te
PUNCH Gis. ges # cacmeec meen

form.

On Manuring with Dicyandiamid.

Numerous experiments with calcium eyanamid showed that in many
vases this nitrogenous manure was equal or nearly so to sodium nitrate
or ammonitan sulphate.t On acid muck soils and on sandy soils, however,
less favorable results have been obtaimed.? Acid soils should at first be
neutralized with lime before lime-nitrogen is applied. Further it is
advised to avoid mixing of lime-nitrogen with superphosphate.*

Lime-nitrogen is at present manufactured with addition of calcium
chlorid, since under this condition the process goes on at a less high
temperaiure. But this product gradually attracts moisture and loses
nitrogen in form of ammonia. Hence Carlson‘ proposes to replace the
salcium chlorid by calcium fluorid. There exist however circumstances
under which lime-nitrogen would not act as favorably as expected, especial-
ly, when the soiis are not only rich in lime, but also when they contain
much more lime than magnesia, as by the application of this lime com-
pound the lime content of the soil will be unduely increased. Hence it
had been proposed some years ago to separate the lime from the eyanamid.
When the crude lime-nitrogen is treated with warm water, a separation

takes place into cyanamid and calcium hydroxid:

I. See also this bulletin. Vol. IV. No. 1. Also Tmmendorf: Fiihlings lowdw.-Ztg. 1905.
S. 794.

2. Lawdw. Presse. 16. Jan. 1907.

3. It is probable that acid liquid prevent the change of the poisonous cyanamid into the
not poisonous dicyandiamid,

4. Chem. Ze, 1906. Nr. tor,

212 I. ASO:
N NH
Caan Cat+2H,0=C— —-+Caf OH),
See Seu tha

and the cyanamid thus formed readily polymerizes to dicyandiamid:

NH 2
@: +H,N-C=N=C—NH.CN
“SNH Syn

According to Ulpiani, this dicyandiamid is not poisonous ‘while
eyanamid is poisonous. Perotti? found that higher plants are only in-
jured by solutions containing three per thousand or more dicyandiamid,
different plants having varying powers of resisting its action and lower
organisms (algae and bacteria) are more resistant than higher plants.
He concluded that calcium cyanamid is far more injurious than dieyan-
diamid and that the manurial value of calcium cyanamid probably de-
pends on its conversion into dicyandiamid. My own experiments have
convinced me that indeed dicvandiaimid is perfectly harmless, for even
at the concentration of 1%, it did not injure algae as spirogyra at least
within five days," certainly a most surprising fact. Later on the writer
observed that dievandiamid can not well be utilised by certain soil
bacteria, but cells of higher plants as a source of nitrogen at the concen-
tration of 0.1%. Ulpiani and Perotti had used mainly culture solutions,
but Wagner and Immendorf observed in poteultures with various soils
a decidedly poisonous action of dicyandiamid. This apparent contradic-
tion was cleared up by Loew,’ who observed a poisonous action of
dicyandiamid on a common soil, while in contrary a very favourable
result with sterilised soil. Hence the inference was drawn that certain

microbes in the soil produce some poisonous compound from dicyandiamid.

5. Journ, of chem. Soc, April 1907. x
Ca. >CaNH
“nu

for dicyandiamid more probable than the cyanoguanidin formula, as neither a cyanogengronp

6. This non-poisonous character renders the imido formula NIl=

nor an amidogroup would be present.

7. Chem. Ztg. 1909, Nr. 3.

ON MANURING WITIL DICYANDIAMID, 213

It was of interest, however, to make further experiments

along this

clirection.
Tn the first place, my own experiment with bacteria in pure culture
have shown that neither Bacillus myeoides nor Bacillus fluorescens liqui-

facieus nor Bacillus subtilis can utilise dicyandiamid as a favorable

source of nitrogen, the development even after three weeks consisting
only a turbidity of the liquid. The culture solution I applied had the

following composition.®

OL % dicyandiamid.
05 % mannitcl.
0.2 9% Kk, HPO,
0,022 Mg SO,

It is to be regretted that Perotti did not given a detailed characteristic

of his bacteria which could utilize dicyandiamid as a favorable source

of mitrogen.

EXPERIMENTS WITTE

On Oct, 2

developed in quartz sa

two young plants of buckwheat,

nd, were put in flasks

BUCKWHEAT.:

about

contaiing’

rarious strength and a ereat difference was observed at

centrations :

lone,
of

different con-

C@..

solutions

Dicyandiamid. Two days Thrce days Four days Seven days ‘Twe've days.
= = Th -
a5 9 The'tips of the a a lead
Wee leaves dried up. aie aie peat:
more decisive.
The tips of the| The injurious
0.2 % Normal. leaves were phenomena be-| Almost dead. —
little injured. |ci1me more clear.
O.1 % ; ” ” ” =
Injured Injurious
0.075 % 5: Normal. a phenomena Almost dead,
little. advanced.
Injured The margin of
3.05 % eA 3 Normal a leaves became a
little. little decolorized
8.

In more diluted solution of dicyandiamid, the growth of bacteria might be Juxur’ant.

214 K. ASO:

(common ” | » ” ” ”
water). |

The next experiment was carried out in culture solution. The solu-

{ion contained :

0.02 % CaH,(PO,).
0.01 % K,SO,
0.01 % Mg SO,
Trace FesO,

aud various additions of dicyandiamid. The result was as follows:

Thirty three

browni h.
developed. ,
One plant was
normal, another
withered a

Both plants Although

New leaves
withered, new as before

| appeared. little withered, {leaflets appeared
New leaves One yielded Bisa) One bud All buds
0.01 % two new leaves | “PP! F

x appeared ind other three) N° Bane flowered opened
PP ; two on other, ‘ Pa
New leaves Both plants Each plant Oae bud

Control. yielded two Normal. produced
appeare.l. new leaves, a bud, Opened.

A photograph was taken on Nov. 28, which is shown by the accom-
panying plate.

From these results, it is clear that dieyvandiamid is only injurious
at higher concentration for plants, properly diluted, however, it can serve
as a nitrogenous nutrient.

An experiment in soil enlture was also made. Each pot contained

2 kilo soil of dry land and the following general manure was applied:

ON MANURING WITH DICYANDIAMID. 215

Doub'e superphosphate ...
ROfaSsiumesSulplatesmcMlrskerrm es) Sees 9243

Nitrogenous manure per pot. (N in an equivalent quantity.)

Ammonium sulphate ... ... ... I g.
Sodium nitrate... e20nee
Lime-nitrogen Te7Olys
Dicyandiamid 0.36),

Tn this case, dieyandiamid produced a very injurious action, prevent-
ing further development of the plants in a short time. Indeed the growth

remained far behind that with lime-nitrogen.

EXPERIMENTS WITH OATS.

Similar experiments® with young plants of oats of about 10 em. long.

vielded the following results;

Dicyan- 7 ; ra ; Seventeen Twenty
Bere Two days. | Four days. | Six diys. | Ten days. dave! | Biaherdive:
The tips of
y the leaves | ‘The leaves v
a5 7 dried a dried up. donde —- =
little.
3 withered a The leaves The leaves Almost
0.2 % ; dried up 7 dead. —
little. dried up. dead.
partly.
é om am About the
OL % Normal Normal ae ee o | oe ee bal org ezch) eee
ae esha Z © 1 ke NCS SMES eahidried are hardly
bageeestae nist up. green.
Aeneaees | Dee bs of | she teaves
0.075 % * 51) <I : the leaves As before.
7 curled a ee became
little. ro brownish.
Te MPN COMla EN ae |
. = the leaves The: tips A new leaf
0.05 % ” en Normal. : became
oh curled a Wowie appeared.
little mr
| The tips Better than
0.0252 vA s is Normal. decolorized AencOttGl:
slightly.
, 1. Better than
0.01 % i » » » Normal. | the control,
a= ea = = — a — — cal <a a
Control. rs ” ” is | » Normal.

g. In these

experiments, no

nutrient was added,

216 K. ASO:

Tn the next experiment, two young plants of oats, about 10 cm. long,

were inserted in the flasks containing the following solutions;

Two days Four days. Six days, Ten days. | Seventeen days.
Ammonium
sulphate. Normal. Normal. Normal. Normal. Normal.
0.1%
Sodium Best
nitrate, Z
0.1 %

About half

Dicyandiamid.| The tips of the parts of

The tips
leaves withered 3

The leaves Almost
dried up Teves

|
|
” ” ”
development.

e H wan -
0.1% a little. withered. partly. dried up. dead.
= ee - 7 G 4 The tips decolo-
Dicyandiaraid. The tips The tips rized a little.
Normal. Normal. The development
0.05 % curle] curled. was a little infer-

ior to that in am-
morium sulphate.

These results show that dicvanciamid is not injurious for oats in

the concentration less than 0.0254.

EXPERIMENTS WITH PADDY-RICE IN POTS.

Each pot contained 8 k. paddy soil’; on July 22, the young plants
of rice were transplanted, three bundles in each pot, each bundle consisting
ot three plants. Before transplanting, the following manures were
applied''. The lime-nitrogen was applied three weeks before planting,
also the dicyandiamid.

Double Potassium

AS ul phate A |
Superphosphat sulphate Lime-nitrogen?®

A. 52. log, 8.517 g.
Ammonium sulphate

B. ne ps 5g.
Sodium nitrate
cS 3 ° 6.436 g.
Dicyandiamid'*
D. 2 ay 1.774 g.-

10, ‘The soil was humus Joam taken from a paddy field which had not been cultivated for
several years.

11. Each pot received the same amount of N, (1.062g. per pot.)
12. This double superphosphate contained 43.972P,0,.
13. This lime-nitrogen contained 12.4792 N.

14. This dicyandiamid was prepared from lime-nitrogen and contained 59,8896 N,

ON MANURING WITH DICYANDIAMID. ALT)

On Nov. 5. the plants were ent and weighed in the air dry state with

the following result:

Total Grains Straw
g. g. gs:

n i 100.00 37-2 62.8
é soe tee es TY, 99.5 40.0 59-5
: L 106.5 55-0 515
TS Ta fo 100.7 51.7 49.0
é ik 45-735 20.5 25:2
> I. 99.0 55:0 Gane
“Si Tie 98.7 55.0 43-7

This result shows that dievandiamid had produced practically the
same harvest in grains, as ammonium sulphate.
In the following experiments the influence of the time of manuring

on the paddy soil were tested.

EXPERIMENT IN POTCULTURE.

For this experiment, eighteen pots,’® each containing 8 k. soil from
a paddy field which had specially exhausted by raising crops for several
years without using manures, were used. General manures per pot applied

on June 19 were as follows:

Double superphosphate 1.265 g.
Potassium sulphate 1.029 ,,
Nitrogenous manures served for this experiment were as follows!‘
‘ ]
Ammonium sulphate 2.620 g.
Lime-nitrogen 4.704 ,,
Dicyandiamid 0,928 ,,
Time of Manuring. Time of Planting.

A. July 10 July to
B. a 3 a5

June 26
D. of oN) »
B. July 10 » [ammonium sulphate. ]

15. We have repeatedly observed and mentioned that nitrates form a very poor source
of N. for plants cultivated on swamp soil.

16. This experiment was carried out in two series.

17. The quantities of N, P,O, and KO were equal in each pot, each being 0.555 g. per

pot. The lime-nitrogen contained 11.894 and the dicyandiamid 50.8892 of N- respectively.

218 K. ‘ASO!

Three bundles of young rice-plants were planted in each pot, each
bundle consisting of three plants. In the first stage of development, some
retarding action of dicyandiamid and lime-nitrogen was observed in A, B

and C, On Noy. 25, the plants were cut and weighed in an air dry state:

Dicyandiamid. Lime-nitrogen.
—— SO a ee
grains straw. grains straw.
A, 30.5 g. 36.1 g. 28.3 g. 37-3 &
B. 33505 40.8 ,, 30.9 ,, 30.6 .,
Cc. 31,755 420 5» 29.5 55 FRCL oy
Dz 33-7 5 B78 is B32 ETA ep
E, 20:5) ics anion. 205 as 38.0!

The above result shows the average yield in two pots. This proves
that dievandiamid, like the lime-nitrogen produces a very favorable result

when it is applied to the soil at least two weeks before planting.

EXPERIMENT IN FRAME CULTURE IN OPEN FIELDS.

This experiment was carried out in wooden frames of three square
shaku (0.83 square metres), the frames being at a distance of one
metre from each other and projecting about ten centimetres above the
level of the field. The depth of these frames was about seventy centi-
metres. The same soil used in the former pot experiment, served also
in this case. As the general manures, potassium sulphate at the rate
of 100 k. potash per ha, and commen superphosphate at the rate of
100 k. phosphoric acid per ha. were applied. The quantities of
nitrogenous manures per frame were as follows, each being at the rate

of 100 k. nitrogen per ha.

Ammonium sulphate 39.24 g.
Lime-nitrogen'® 66.80 ,,
Dicyandiamid?? 17.60) 4)

18, This contaired 12.47% N.

19. ‘This contained 46.79% N,

ON MANURING WITLI DICYANDIAMID. 219

Time of Manuring. Time of Planting.
A. July 7 July 7
B. June 30 ae
G 2S oF
D. 6 a
E; UG) »
10 ree 5
G July 7 »
H. = »
kK. = »

Sixteen bundles of young rice plants were transplanted in one frame,
each bundle consisting of tweleve plants. In the first stage of development,
the plants in A were inferior to those in K, but on July 27, they com-
menced to show better growth than H. On Angust 10, there was almost
no difference observed in b, C, D, E, F and G; only in A, the plauts were
still inferior to all others. Generally the plants manured with lime-
nitrogen showed a better growth than those manured with dicyandiamid,
perhaps on account of its lime-content. On Sept. 10, the number of

branches was counted with the following average result:
t=} t=)

Number of branches per plant.

Lime-nitrogen. Dicyandiamid.

A. 17.0 14.7

B. 16.9 15.5

c 17.0 16.0

D. 18.4 18.0

I, 18.0 16.0

Ine 17.9 15.5

G,. 18.4 18.4 [ammonium sulphate. ]
H 13.5 13.5 [no Nitrogen. ]

K, 11.9 11.9 [no Manure. ]

220 K. ASO:
On Noy. 28, the plants were cut and weighed in the air dry state:

THE HARVEST PER FRAME.

Lime-nitregen. D cyandiamid.

Fullerarae eee Straw, Fullarsmnes a Bimpiserane: “Straw.
A. 197 g. 18 ¢. 399 ¢. 183 g. 12 g. 312 g.
B. 259 » 13) 5, 490 ., 208 ,, teers BEES ep
(en 260 ,, itsy ers 508 ,, 209) 4,, The 350i ks
D. 258 ,, 2155 528 ,, 238 Tomes 416 ,,
E 280 ,, D7 ay 491 ,, 244 » 14 5 394 5
F. 257 » 19 5, 46S ,, 239 » It 359
G. 266 ,, TG; AS. 266 ., TON, 482 ,,
Il. 149 ,, 10 tate 276 ,, 149 5, UKs 5 276 5;
K, Tian | Se 149 ., 75» 5» 149 »

This result again shows that dicyandiamid produced a poor result
on paddy soil when planting followed immediately after manuring; how-
ever when the planting took place three or four weeks after manuring the
result was nearly favorable as with ammonium sulphate. Tlence we may
conclude that dicvandiamid was after this time completely decomposed.

Another very interesting question was, whether dicyandiamid would
be more effective in conjunction with acidie or with alkaline manure.

For this experiment four pots, each containing 2 k. paddy soil were
used and the manures applied per pot were as follows:

Acidic Alkaline

; .
Manure. 4otassium sulphate ay Marare,

Dicyandiamid I g. Dicyandiamid I g.
Potassium carbonate 0.783 ,,

Double superphosphate 1 ,, Dicilcium phosphate 0.95 ,,

On July 25, three young rice plants in one bundle were planted;

on Noy. 5, were cut and weighed in the air dry state.

Total. Grains. Straw.
Acidic { A206, 6.38 g. 5.4 fe

A Peers 6:0) 55 6.8 ,,
Alkaline ...

{ 16.5 ¢. 8.7 g. no p
18.9 ” 9.0 ” 8.9 ”

ON MANURING WITI DICYANDIAMID. PHA |

In the following experiment with millet, no paddy soil, but dry land
soil (common field soil) served. Each pot contained 10 k. soil and

manured with the following substances:

Dicyandiamid 52.7 { Dicyandiamid 5 g
Acidic. Potassium sulphate aes Alkaline { Potassium carbonite 3.91 ,,
(Double superphosphate 4 ,, pyrene phosphate 3.8 ,,

The seeds were sewn July 23 and five plants were grown in each
pot. The plant growth was very much retarded, indecd the plants much
injured. The plants were cut at the ripening stage and the harvest

(airdry) was:

Total. Straw. Ears.
6.2 g. 2g, Rept
Acidic 2 g 33 € 29 g
6.7 ” 3:7 9 3:0) 55
Alkaline ..; of oe 4-4 & 3.6 g.
7:9 55 3-7 » Sis)

This result is exceedingly poor; but in further experiment with buek-
wheat, in which 5 g. dicyandiamid per 10 k. soil served, even poorer result
was obtained, the young plants after reaching 15-16 an. height stopping
further growth and withering.

These results show that dicvandiamid is more effective in conjunction

svith alkaline manures.

CONCLUSIONS.

In watercluture, dicyandiamid at the concentration of 0.014% proved
io be a source of nitrogen for plants. Im commou soil, however, it acted
poisonously at the rate of 5 g. dicyandiamid in 10 k. soil, but served as
a faverable nitrogenous manure with the reduced quantities. In paddy
soil the injury was less than in the soil of common dry land and when
the precaution was taken, that planting was performed three weeks after
manuring with dicyandiamid, no injury whatever was observed and the
harvest reached nearly that obtained with the equivalent quantity of

ammonium sulphate and of lime-nitrogen. This renders it very probable

292 K., ASO. { ¥

. Fa

that the bacteria in paddy soil sooner change this injurious compound
further to innocuous compound (ammonium compound?) than in common —
field soil. Furthermore dicyandiamid acts as a nitrogenous manure more
‘ favorably when it was applied in conjunction with alkaline manure. ;

- Lastly I express thanks for the assistants 8. Sanada and T. Yosh

who helped me during this investigation.

———-*

This plate shows the nutritive value of dicyandiamid in watercutture.

A 163 (e. D

Ammonium Sulphate. Sodium Nitrate. Dicyandiamid. Lime-nitrogen.

This plate shows the manurial value of dicysndiamid in pot-culture

When a compound of neutral reaction is transformed gradually into ‘
a compound of an acid reaction by the action of the roots, or undergoes a
separation into acid and base, the latter alone or chiefly heing absorbed,
it is ealled a physiologically acid manure. This is of a special interest
in the case of ammonium sulphate as Adolf Mayer has pointed out, and
eare has to be taken that sufficiently calcium carbonate is present in such
a case in the soil. Since, however, in recent times potassium sulphate is
very often applied as a manure and since the reaction of the soil has a
great influence on the yield,’ the qnestion whether this salt is a physiolo-
gically acid manure, is of practical interest. Many plants require com-
paratively little sulphur since the albuminous matters contain rarely more ;
than 1.8¢ sulphur and other organie sulphur compounds are comparatively
rare in plants being generally restricted to Cruciferse and to various kinds
of Allium. Hence soils relatively poor in sulphur may still furnish rich
crops under otherwise favorable conditions plants generally require much :
more potash than sulphur as shown by numerous analyses.

But it has not vet been decided whether in manuring with potassium
sulphate, the sulphuric acid is absorbed jn the same measure as potassa "i
or whether by preferential absorption of potassa from meutral potassium
sulphate, seme mononotassinm sulphate would be formed in the soil.
dt might also be possible that dipotassinm sulphate is absorbed as entire
molecule and that the sulphurie acid not needed in plants for protein

formation would simpiy remain in the eell-sap in form of various sulphates.

1, Compare Bull. College of Agric. Tokio, Vol. VIT. No. T. 1906.

224 K. ASO:

The questien was to be decided by sand eulture in which potassium and
sulphur were present in the form of dipotassium sulphate. A depression
of the harvest was to be experienced, when potassium sulphate was a
physiologically acid manure and no means for neutralisation was provided.

Six pots containing 2.5 k. purified quartz sand were manured with.

CaHPO, + 2aq. 0.6 g.
NH,NO, 1.0: 4,
Fe (OH), TO) sy a as
MgHPO, 0.5 5

Two pots A, and A, received each 0.62. K,SO,; two pots B, ard B,
received 0.5g. K,SO, + 0.1g. KHSO,; two pots C, and C. received 0.52.
K,SO,+0.1g. KjCO,. Six seeds of oats were sown in each pot, April 20,
ond later on tae number of plants were reduced to three, all of equa! size.
In the beginning of the development, the plants in A, and A, showed a
better growth than others, but later on the plants in C, and C, were
superior, possibly in consequence of A, and A, becoming acid. After
ripening, the plants were cut and weighed in the airdry state with the

following result.?

Total Straw Grains Roots

g. g. g- g.
A, 309.1 21.1 9.0 5.5
A, 290 20.0 9.0 4.2
B, 26.9 18.1 8.8 a5
B, 29.0 20,1 8.9 4.0
Gc 31.0 20.0 11.0 3.0
(or 30.5 20.5 10.0 3.8

The difference of harvest are not very decisive, nevertheless it will be
recognised that in case C, and C, the smallest mass of root has produced
the greatest harvest in grains. It was evidently more favorable to use
some alkaline substance along with potassium sulphate than to use some
acid substance along with it (compare C,, C, with B,, B,). Potassium
sulphate appears therefore as a weak acid manure.

2. The roots were also extracted from the sand as carefully as possible and were united

with the harvest.

bo
bo
or

IS DIPOTASSIUM SULPHATE PHYSIOLOGICALLY ACID ?

Tn the second experiment, six pots containing 2.5 k. pure quartz sand

were manured with the following compounds per pot,

CaHPO, + 2aq 0.6 g.
NH,NO, - TLOles
MgHPO, OSes
K,SO, 0.6 ,,
Fe, Cl, trace

Besides, two pots A, and A, received each 1.26g. Na, HPO, + 12aq;
B, and B., 0.67¢. NaH,PO, + 4aq.,; C, and C, 1.35g. Na,PO, + 12aq;
the quantity of P.O; being equal in each pot. Three young plants of
rice were transplanted in each pot in a bundle on July 22, and harvested

on Noy. 5 with the following yield:

Total Grains Straw
g- g. g.
A, 36.5 12.0 20.5
A, 35.0 145 20.5
B, 29.5 9.8 19.7
B, 27.5 9.0 18.5
G 32.0 12.0 20.9
c.5 22.0 6.0 16.0

This result shows a decisive depression of harvest when weak acid
manure is applied from the start and that potassium sulphate has most
manurial efficacy for rice when the other salts were applied together with
it im the neutral state (compare A, A, with B, B, and C,).

As a whole it appears that the physiological acidity of dipotassimn
sulphate is very much less pronounced than that of ammonium sulphate,

but it acts as a neutral or as weak physiologically acid manure.

3. The plants in C, were accidentally damaged and the result is not reliable

Plate XI.

/
j a
t \ |

A. Neutral,

B. Acid C. Basic.

Some New Varieties of Willia Anomala as Aging
Yeast of Saké.

T. Takahashi and H. Sato.

With Plate XTT.

Introduction.

Willia anomala, discovered by Writ! and formerly called Saccharo-
myces anomalus, forms now a large group of Saccharomyces. Tansrn?
(1891) first described this organism under the name of Sacch. anomalus.
P. Linpyer® (1892) found it on a green malt? and further he found a
new variety Willis anomala var petarcat in Belgian beer. Crrrzaszcz
(1902) reports that he has found sacch. anomalus on the surface of the
barley, and Jorcensrn® (1898) mentions a variety of saech. anomalus
which cause a turbidity in English beer (bottom fermentation system).

From a Canadian cheese factory Harrtson’ found a vaiety of sacch. ano-

1. Will: Zeit. f. d. ges. Brau. 1892. p. 75,

2. Hansen: Comp. rend. labo. Carlb. 1891.

3. Lindner: Woch. fiir B. 1892. No, 4. p. 75.
4. - : Mikrosk. Betriebskont, 1898.

5. Chrzaszez: Woch. f. Brau. 1902. p. 590.

G. Jorgensen: Mikroorganismen. 4 Auf. p. 238.
4. Lindner: Woch. f. B. 1990. Nr. 49-51.

7. Harrison: Cent, f, Bak, Bd. IX: p, 206,

228 T. TAKAHASHI AND H. SATO:

which gives a “bitter aroma” to the milk. Wzxr1’ observed an anomalus
type yeast in the wort of the cool ship, younger beer, beet-sugar and refined
cane sugar. L. Srevprer® made studies on his three varieties of Willia
anomala and one variety discovered by Witt from cherries.

Merssyer?® mentions three different types of anomala isolated from
beer, wine must and the soil from their gigantic colonies on wine must
gelatine. Xvsawskr! found a variety of anomala in plim-sauce, but his
description is short, HH. Zrkes'? found in his soil analysis a new variety
of Wirrta anomala, which forming a slimy covering on several kinds of
nutriments and thereby distinguishes itself from other varieties of Willia
anomala. Kxocxer and Scirtox1xq'* mention the presence of Willia ano-
mala in the so-called “Taka-Koji”. Koza" in his investigations on the
microbes of “Koji,” reports the occurrence of a variety of Willia anomala,
causing a feeble aleoholie fermentation and producing acetic ether in heer
wort. Ix. Sarro? isolated from Saké a kind of Willia anomala, which
produced acetic and butyric ethers in beer wort or in Koji extract. and
was capable of fermenting dextrose, levulose, saccharose, galactose and
sparingly also maltose. Quite recently S. Fukumoto" isolated five varie-
ties of Willia anomala from “Moromi-mash.’’ All of them develops well
‘

only in koji-extract containing less aleohol than 11 vol ¢, and one of

them produces amylacetate.

8. Will: Cent. f. Bak. Abt. II. Bd. VI. p. 219.

9. Steuber: Zeit. f. das g. Brau. 1900. p. 3. 17. 33.

10. Meissner: Land. Jahr. 1901.

1l. Kujawski: Zeit. f. d. g. B. Bd. XXIII. 1900. p. 111.

12. Zikes: Cent. fiir Bak. 1906. II Ab. Bd. XVII. No. 416-S. 97-111.

13. Kldcker u. Schioning: Cent. f. Bak. 1895. II Ab. Bd. I. S. 777-782.

14, Kozai: Cent. f. B, II Ab. Bd. VI, 1900. p. 385.

15. Saitd: Journal of Imp. Univ. Tokyo. College of Science. 1904. Vol. XIX.
16, Fukumoto: Jdzdkyokaizatsushi. 1910. Vol. I.

SOME NEW VARIETIES OF WILLIA ANOMALA. 229

PART I.

It is a well known fact that a new or freshly prepared saké has a very
sharp taste and is too strong as a beverage for refined people, who con-
suines almost exclusively thoroughly ripened saké. This process of after
ripening or aging occurs during the storage, which takes place after clari-
fying and continues from spring to autumn. During this storage which
continues all through the hottest part of the year, the temperature of
the saké in the vat is relatively high ‘* and the chemical changes in the
composition of the new saké must proceed at a rather quick pace and
complete the ripening. But the exact nature of these changes has not
been known. The authors isolated four varieties of Willia anomala from
a deposit ** formed on the bottom of the vat during the storage. They

are described below. Only The var. III and IV form amylacetate.

No. 1. Willia anomala Var. I.

I. Form and Size: (a) Young culture :—Koji-extract, 7 days eul-
ture at 23-25° C. The cells are round (3%3 y or 5x5 / ), elliptical (5x6.5-
7-5 # or SxS p ), club shaped (12.5x5-6 2), or oval (7.5x5 ) and many
of them contain one or two fat globules. The cells from surface cultures
on Koji-extract agar (27 days at room temp.) are small and round (2.5x
2.5 ##), small and elliptical (3x2.5 /), or somewhat elongated mostly,
but some of them are large and round (5x5 /2), oval (3x6.5 / ) or ellip-
tical (5x4.5 /# ), and contain glycogen. Old culture: The cells from
surface growths on saké-agar (10 months) are very small and round

(2.52.5 “@) or small and elliptical mostly and large cells of 4x5, 4x4,

17. In summer time the temp. of saké in the storage vat is 24-28°C,

18. The deposit of a saké from a factory of “ Nishinomiya,” « Nada.”

230 1, TAKAHASHI AND H. SATO:

or 102.5 m are very rare. Some of these cells contain 1-4 hat shaped

spores with 2.5 » ledge length and 1.5 / high.

II. Growth: In Saké-agar plate culture (10 days at 25° ©) there
was found a white flat colony’® with some folds in the centre and feathery
growth at the margin. Surface culture: A white waxy covering with
a somewhat streamy appearance margin with the presence of a promy-
celium, was formed on saké-agar, (52 days from June to July at room
tem.). A greyish white waxy and almost flat growth with pro-
mycelium on the thinner part of the medium was formed (3
months during summer). A smooth pasty covering was found
on Moromi-agar. (10 days at 20° C). The covering on the thick
part of the medium stretches out considerably with a wavy margin, while
on the thinner part it forms a white, chalky crust. A white, chalky,
thick, folded film was observed on the condensed water, which contained
the deposited cells with a sighn of fermentation. After 2 months more
the covering lost its lustre and became coarse. A white waxy covering
with concavity along the streaks, and forming folded streams
at the margin, was observed on saké-gelatine culture, (one month at room
temp.). A white chalky growth was also formed on the thinner part of
the medium. J’luid culture: In beer wort (3 days at 23° C) it forms
a thin film with somewhat massive deposit and on shaping an energetic
disengagement of gas was observed. In koji-extract (10° b, 16 days at
15-16° C) it forms a very thin film forming a foam and a deposit under
clear fluid, with the production of acetic and other esters. Some of the
cells in the film of koji-extract culture (80 days) stain red“ with methy-
leublue. r

III. Ester formation and its relation to carbon-source. As culture

19. Some of the colonies grow in three directions, as is usual in the common yeast.
19a. ‘his phenomenon will be dealt with further on. Soy-veast behaves similary

toward methylenblue,

SOME NEW VARIETIES OF WILLIA ANOMALA. 231

media solutions were used containing as carbon-source substances other

than carbohydrates.

TABLE L.

Solutions. Condition. Remarks.
No. 12° ... ...J In test tube. 2 months | Forms white film and a ring with massive de-
at room temp.: July posit, and produces fruit esters.
to August.

INTs LUE tery Do, Forms white fiim and ring with considerable
deposit. The ring does not break even on
shaking.

No. Il. --- ++ 10 days at 25-27°C. Forms no film but with a trace of acetic ester

: flavor.

No. 111.22... ...J In test tube. 2 months | Forms thin white film and ring with noticeable

at room temp. deposit.

No. IT. ... --| In sealed flask. 3 months | No film, but an energetic production of acetic

at 23-25°C. ester.

Thus, this variety forms esters from acclate and alcohel derived from
the amino-acids formed by autolyses. Further, it is also probable that
esier ts formed from the acetate and alcohol originally given in the nut-
riqgment, The function of ammonia as a nitrogen-source is also proved by

these cultures.

A further experiment was made in the same direction with different

culture media.

20. Solution No. I, contaned: KH,PO, 004g, K,5O, 0-024, MgSO, 0:0003g, C,H,0,.
(NIT,O1). 2g. Water 100c.c.

21. Solution No. II. contained: 59 of ethyl-alcohol in 100c.c. of No. I. solution.

22. Solution No. III. contained: 0.0596 of amyl-alcohol in 100c.c. of solution No. I.

22, T. TAKAHASHI AND TL. SATO:

TABLE II.

Solution. Condition. Remarks.

No. IV.23 ... | 14 days at 17-18°C.

Forms a thin film with a heavy deposit and
vigorous production of acetic and other esters.

No. V.2+ A white thin film was formed ascending to the
wall, with deposit and ester production.
No. V | In sealed flask during 2 | Forms thin film accompanying the formation of
months at 23-25°C. fruits esters.
No. VI?5 Do. Forms thin film with deposit and trace of fruit

ester. The film ts thiner than in No, 4, which
did not contain amyl-alcohol.

No. VII.2°¢... Forms relatively thick (thickest among four va-
rieties) white film ascending the wall of the
flask, with heavy deposit and a vigorous pro-

duction of acetic and fruit esters.

No. VIII.27 Forms fine folded film ascending the tube wall,

but without flavour.

| 10 days at 25-26°C.

Forms fine folded film, ascending the wall, with
trace of deposit. Deposit increased after ¥
days.

No. 1X.2° .......| 4 days at 245°C.

Seen nn nnn UEIEIEnIIIIEEIIII EEE

Thus, it is plain that the yeast assimilate carbon from butyrate,
succinate or free succinic acid and lactate, and that the formation of ester
flavour always takes place except in the case of lactate. So that, it is

highly probable that in the presence of the latter the yeast has not the

23. Solution IV. contained KH,PO, 004g, CaSO, 0:02gMgSO, 0:0003g. NH,C,11,CO,.
0.5 c.c. and alcohol (absolute)Sc.c. in 109c.c. of water.

26. Solution VII. was prepared by adling 0:05 vol9é amyl-alcvhol to svlution No. V.

24. Solution No. V- contained 0:524 of ammonium succinate instead of the ammonium
butyrate of solution No. IV.

25. Solution No. VI was prepared by adding 0:05 vol2g of amyl-alcohol to soluticn No.
Iv.

27. Solution VIII. contained 0-5c.c. of ammonium lactate instead of the ammonium buty-
rate solution No, IV.

28. It contained KH,PO, 0-04g, Asparagin 2-5g, K,SO, 0-02g, MgSO, 0-0003g, succinic
acid 0'05g, etbyl-alcohol Sc.c, and water. (to male 100c.c.)

SOME NEW VARIETIES OF WILLIA ANOMATA. 233

power of forming an alcohol of an agreeable odour by combining with
lactic acid, or of forming any acid which gives fragrant substance in com-

bining with ethyl alcohol.

Further, a remarkable fact is that the production of aroma was greater
in the enlture media containing both amyl-aleohol and snecinate than
in those containing only the latter (solution No. V). Moreover, it is
very interesting to note that fruit esters was formed in the solution No.
T, in which the only earbon source was ammonium acetate. So that it
is highly probable that aleohol, which favours the formation of esters,

must be derived from the amino acid formed by autolvses.

IV. Spore farmation. The hat shaped spores are formed on the
gypsum blocks after 38 hours at 27° C. One to four spores are found
in a cell.

V. Fermentation products. In the distillate of a 6 days old koji-
extract culture (10° B) at 22-20° C, there was found fusel oil, methyl,

alcohol, acetone and methy-lactate.

The culture contained 2.72¢ ethyl] alcohol, 0.209% total acid (as suc-

cinie acid)? and 0.638% total esters (as ethyl-suecinate )*°.

VI. Behavior towards carbohydrates*®:—Cultures at 23.5-24° C.

28. 29. In the case of other varieties described below total acid and total esters are
calculated as succinic acid and ethyl-succinate respectiveiy.

30. Such carbohydrates as arabinose, xylose, gluccse, levulosc, galactose, raffinose, lactose
maltose and a-methylglucoside and also glycerin were dissolved in the proportion of 292 in

Hayducks solutson devoid of sugar, while in the case ot cane sugar 59% was the strength used

234

T. TAKAHASHI AND H. SATO:

Substances.

Arabinose

Xylose...

Fructose...

Glucose

Galactose

Saccharose ...

Lactose

Maltose

Raffinose

Glycerin

a—Methylglucoside

Remarks.

| Forms half film after 3 days, completed after 7 days; but it is

very thin and not folded, with a trace of deposit.

-| Very thin film begins to form after 3 days and is completed after

7 days with a trace of deposit.

| Forms a very thin and almost transparent and non-folded film with

peposit and turbid fluid after 3 days. A feeble fermentation was
shown by the evolution of bubbles which increased on shaking,
Produces fruit esters which rémained even after 10 days.

Forms very thin film and rings; fluid turbid with dense foams and
white deposit after 4 days. When shaken gas bubbles were
vigorously evolved.

Forms very thin and almost transparent film, but giving no deposit
after 3 days. Davy’s test for alcohol gave a positive result after
14 days culture.

| Forms thin white film and foam with turbid fluid and a deposit

(3 days culture), which does not seatter when shaken.

Forms island-like film with no deposit and turbidity after 3 days.
A trace of deposit was formed after 14 days but Davy’s test for
alcohol gave a negative result. The degree of the growth was
almost equal to xylose-solution.

Forms white non-folded film ascending the wall after 3 days, ‘The
deposit increased after 14 days. Fruit ester was present alter 10
days but Davy’s test for alcohol gave no result.

.| Forms thin white non-folded film with white deposit after 4 days.

After 10 days gave faint reaction for alcohol.

.| Forms very thin film with trace of deposit which does not ehange

when shaken.

Forms very thin film and a trace of deposit after 3 days.

Thus, this variety assimilates xylose, arabinose, fructose, glucose,

galactose, saccharose, lactose, maltose, raftinose glycerin and a-methyl gluco-

side; especially in solutions containing fructose, glucose, saccharose the

growth is vigorous and the ferment good. But xylose and lactose are less

favourable for its growth and galactose is fermented sparingly.

SOME NEW VARIETIES OF WILLIA ANOMALA. 235

VII. Assimilation of nitrogen compounds and difference between
glycerin and cane-sugar as regards ester formation. Two series of the
solutions were prepared one containing glycerin and the others containing

saecharose, with four kinds of N-source in 4 solutions in each series,

Substances.3? Remarks.

Asparagin +glycerin _ ...] Forms white foldedless film ascending the wall (10 days at 23-24°C.).
There was deposit but not ester flovor; while in the culture of
Hayduck’s solution, a vigorous formation of fruit ester took place.

Feptone3*b +glycerin ...] Forms very thin film with ring and heavy deposit but no ester
flayor, (10 days at 23-24°C), while in peptone Hayduck’s soluton
there was an intense ester flavor.

Ammonium-phosphate**e | Forms thin film with ring shaped growth and deposit (10 days at

+glycerin 23-24°C). The film formed on ammonium phosphate Hayduck’s
solution was thinner than that of this solution, but fruit ester
formed only in the former.

Potassiumnitrate?'d ... ...| Forms thin film ascending the wall but no depesit after 3 days at
+glycerin 22-23°C. After 2 month there was formed a heavy deposit but
there was no flavor of ester, while in the culture of KNO,—

Hayduck’s solution, ester flavor was conspicuous.

Thus, this variety assimilated asparagin, peptone, ammonium and
nitrate nitrogen. Hster formation was strongly dependent upon the pre-
sence of cane sugar®** but no on glycerin. This fact explains well the
fact that this yeast when taken glycerin as a carbon source has not the
power otf forming acid, which favours the formation of ester in combining

alcohol, derivable from amino-acid by autalysis.

31. Nitrogen compound was dissolved in Hayduck’s mineral solution containing 296 ef
glycerin instead of saccharose:— peptone 194, asparagin 2.594, ammonium phosphate 19¢, K-
nitrate 1%.

31.b. Peptone Hayduck’s solution is prepared by dissolving peptone instead of asparagin
in ordinary Hayduck’s solution. So contains saccharose instead of glycerin of above solution.

31.c. Ammonium Phosphate Hayduck’s solution contains ammonium phoshpate instead of
asparagin of ordinary Hayduck’s solution.

31. d. Potassium nitrate Hayduck’s solution is prepared by dissolving KNO, instead of
the asparagin of ordinary Hayduck’s solution,

236 T. TAKAHASHI AND H. SATO:

VIII. Proteolysis. The experiment was carried on in two differ-
ent ways,°* by stab culture on the one hand and by uniformly distributing
the yeast in the medium, using neutral koji-extract gelatine (10% gelatine).

The temperature of the room was 17-20° C.

Stab culture. Uniformly distributed.
After 40 days. No liquefaction ... ...| No liquefaction.
After 56 days. Do. «+ «| About 2 c.c. of gelatin in test tube dissolved.
After 70 days. Do. ... +-| Dissolved part increased.

IX. Assimilation of amino-acids, In koji-extract culture (10° B)
after 55 days (one month at 20° C and 25 days at 10-16° C) amino-acids
were determined according to SorENsEn’s** method with the following
results.

Amino-acids in original solution ...... 0.123% as glycocoll.
Amino-acids after culture ............ 0.0052¢% as glycocoll.
Amino-acids assimilated ............. 0.116% as glycocoll.

Therefore, almost all the amino-acids contained in the original solu-

tion must have been assimilated.

X. Optimum temperature for growth: The optimum temperature**
lies about 30-31° C.

XI. Death temperature: In diluted saké (saké to water 1:1) or

beer wort heating to 56-57° C for 30 minutes kills the yeast, but not in
’

32. Other fermentable carbohydrates also contribute to the ester formation.

33. Will. Studien iiber Proteolyse durch Hefen. Cent fiir Bact. u. Paras. 2. Abt. Bd.
VII. S. 794—809.

34. Bericht d. Deut. chem. Ges. 1909.

34. To ascertain this temperatures of 20-22°C., 24-25°C., 28-32:5°C., 30-31°C., 50°C,
were tried for all the varieties described below.

SOME NEW VARIETIES OF WILLIA ANOMALA. 237

koji-extract or yeast-water. Heating to 57-58° C for 5 minutes death
will ensure in diluted saké but in koji-extracts it is retarded.

XII. Limit of alcohol contents for growth.

In koji-extract containing 10% of alcohol, it forms a foamy film with
deposit after 6 days at 27° C; while in koji-extract containing 15% of
alcohol there was no film or deposit under the same condition. The latter
culture was then kept at the room temperature (17-20° C), and after 31
days there was formed a trace of deposit which increased very slowly.

Thus, this variety decidedly belongs to the group of Willia anomala,
forming acetic—and fruit esters in cultures made in solutions containing
sugar or ethylalcohol and salt of organic acid or simply organic acid salt.
And above all, the ester formation is best in solutions containing ammo-
nium butyrate. Moreover, the assimilation of amino acid is more conspi-

cuous than in the case of sacch. sake, wine yeast or beer yeast**.

No. 2. Willia anomala Var. IT.

1. Form and size: (a) Young cultwre:—Koji-extract culture (7
days at 23-25° C). In the film smaller cells predominate which are either
round (2x2 y ), ellipsoidal (2.5x3 # ) (or 4x5 /# ), but somewhat larger
pear shaped (7.5x5 or 7.5x2.5 / ), filamental (7.5x2.5 # ), or wedge-
shaped (10x3.5 or 10x5 ~ ) ones also occur frequently. Many of the
cells contain 1-2 fat globules. (b) Old cultwre:—Surface culture on
saké-agar after 10 months. The smaller cells predominate (4x3 y ), but
large round cells (Sx8 y ), filamental cells (5x2 # or 10x5 # ) or club
shaped cells (10x2.5) occur frequently. The spore holding cells occur
very rarely and the ledge of the hat shaped spores is sometimes not evident.

Surface culture on koji-extract argar (27 days at room temperature) :

—Smaller round, elliptical, oval shapes predominate but large and long

34. b. See this Journal p. 279 (Takahashi and Yamamoto’s paper).

238 T. TAKAHASHI AND H. SATO:

elliptical, oval, sausage or club shaped cells occur rarely. The glycogen
reaction is evident in every large cell. The sizes of the cells are: 2.5x2.5 4,

5x3 p, 10x3 p, 12.5x4, 19.5%2.5

II. Growth: A faintly dirty white flat colony, with folded centre
and streamy growth on the margin, appears on saké agar plate culture (13
days at 25° C). Surface culture: A greyish white waxy covering was
found on saké-agar. The covering changed to a chalky white fine striated
growth, with conspicuous promycellium. The covering on koji-extract-
agar®* is almost same as that of variety I, but the difference consists in
that this variety does not formed a promycellium. Moreover, a more
or less dirty coloration of the covering in this medium distinguishes it
from the other three. The covering forced on Moromi-agar (10 days
at 20° C) was very like that of variety I, with the only difference that
the growth on the thinner part of the medium develops chalky white
foulds.

The growth on saké-gelatine (one month at room temp.) was almost

same as that of variety I.

Fluwd culture: In beer wort (3 days at 23° C) it forms a thin
tilm with moderate deposit. A vigorous evolution of CO, gas was obserb-
able when shaken. In koji-extract (10° B, 16 days at 15-16° C) it forms
a similar growth, but there was no foam in this variety. A faintly yellow
film is formed with vigorous fermentation on koji-extract kept at 23-25° C
for 3 days, or at 17° C for 7 days. A few of the film cells stain red

with methylenblue. (80 days culture in koji-extract.)

IIL. Ester formation and its relation to carbon-source. The solu-
tions used were the same as those mentioned connection with the foregoing

experiments.

35. ‘Ihe duration and temperature of the cultivation are exactly the same as in the case

of the variety I.

SOME NEW VARIETIES OF WILLIA ANOMALA. 239

Sclutions. Conditions. Remarks.
No. I.... ... ..| In test tube. 2 months | Same as variety I.
at room temp: July
to August.
No. II. aoe Ge Do. Forms very thin white film and fruit ester but

with no depcsit.

No, IT. .-. «4 10 days at 25-27°C. Almost same as variety I, but with formation of
free acetic flavour.

No. IT. ... «| In sealed flask 3 months | Forms no film but vigerous acetic ester flavour.
at 23-25°C.

No. III. ... ...J In test tube. 2 months | Forms thin film and ring with deposit.
at room temp: July
to August.
NOmLV.0) -- <c-|) L4udavysrat WoC, Forms no film (difference from var. I), but heavy

deposit and flavor of amyl ester.

No. V. St red Do. Forms very thin (the thinnest of the four varie-
lies) film ascending the wall. Forms deposit.

No. V. +» «4 In sealed flask for 2] Same as variety I.
months at 23-24°C.

INia), AIG" TRRacueer| Do. Forms th'n film and deposit with amyl ester.

DNOMBVIGs sc. oss Do. Forms very thin film as in the case of solution
No. V, the thinnest of the four varieties.

No. VIII. ... ..-| 10 days at 25-26°C. Same as variety I.

No: FX. ... | ...]/ 4 days et 24.5°C. Forms non-folded film with less ascending habit
as compared with variety I.

Thus, this variety assimilates butyrate, succinate or free succinic acid
and lactate as carbon source, and forms fragrant esters from alcohol and
above mentioned salts with the exception of the lactate (same as variety
I). The formation of amylester was most conspicuous when amylachol was
added to solution No. IV. (butyrate solution). Acetie ester was formed in
acetate solution (sol. No. I) devoid of ethylaleohol (same as variety I).
The production of ester flavour was greatest in butyrate solution (No.

VI. solutions).

240 T. TAKATITASHI AND H. SATO:

IV. Spore formation. The spores are formed on the gypsum block
after 38 hours at 27° C. One or two spores are found in a cell.

V. ‘Fermentation products. In the distillate of koji-extract eul-
ture**, there was found fusel oil, acetic ester, methylaleohol, acetone ( ?)
and ethylaleohol (3.42 vol. ¢ ). The quantities of acid and esters were:

0.315¢ Total ester (as ethyl-succinate).

0.0177% Total acid (as succinic acid).

VI. Behavior towards carbohydrates:

Substances. Remarks.

Arabinose .| Same as in variety I. (7 days at 23-5.-24°C).

Xylose .| Forms very thin film and a trace of ring, but without deposit. (3
days at 23.0-24°C). Same even after 7 days.

Fructose .| The growth was almost same as in variety I, but the film was
some-what thicker.

Glucose .| Same as in variety I, with the difference that the fluid remained
clear and the deposit was small. (4 days at 23.5-24°C).

Galactose .| Forms film with faint folds with ring and trace of deposit. (4 days
at 23.5-24.5°C).

Saccharose ... .| Almost same as in varicty I, except that the ring was thicker. (4
days at 23.5-24°C).

Latose .| Same as in variety I. (4 days at 23 5-24°C). Davy’s test (or alcohol)
gave a positive result after 10 days.

Maltose .| Same as in variety I. (3 days at 23.5-25°C). Davy’s test for alcohol
gave a positive result after 10 days.

Raffinose .| Almost same as in variety J. (3 days at 235-25°C), except that
the ring was thicker. Davy’s test gave a negative result after
10 days.

Glycerin | Forms a thicker ring than in variety I, and the deposit scatters

in the fluid when shaken. (3 days at 23.5-25°C).

a-methylglucoside | Almost same as in variety I, but the film is thicker.

36. The conditions of culture were the same as for variety I.

SOME NEW VARIETIES OF WILLIA ANOMALA. 241

Thus, this variety is almost exactly like var. I so far as assimilation
of carbohydrates is concerned, but the film on xylose solution, both the
film and ring on galactose, cane sugar, raffinose, glycerin, ¢-methylglucoside
solution are thicker and more prominent than in variety I. Another dis-
tinguishing point is that raffinose is fermented by variety I but now by

this, and that maltose is fermented by this variety but not by variety I.

VII. Assimilation of nitrogen compounds and difference between

glycerin and cane sugar as regards ester formation.

Substances. Remarks.

Asparagin + glycerin ...] Forms almost the same film. but some what thicker than in vriety.
I. No ester flavor was perceibable. (10 days at 23-24°C), while
in ordinary Hayduck’s solution it was copious.

Peptone + glycerin .... ...) Makes almost the same growth as vareity I, but the ring is thicker
No ester flavour was developed. (10 days at 23-24°C). But in
the culture of peptone Hayduck’s solution, there was a develop-
ment of ester flavour.

Ammoniumhposphate +] Forms same film as variety I. except that the ring is thicker (10
glycerin days at 23-24°C). A vigorous development of the ester flavour
was perceibable in the culture of the ammonium-phosphate Hay-
duck’s solution, while growth was conpicuous in this solution.??

(containing glycerin instead of cane sugar),

K-nitrate + glycerin... ...] Forms half covered film. (3 days at 22-23°C). After 2 months a
trace of butyric acid’ flavour was perceibable, with considerable
deposit, while in the culture of K-nitrate Hayduck’s solution
ester flavour was perceibable. .

Thus, variety IT assimilates asparagin, peptone, ammonium and
nitrate nitrogen. The formation of ester depends upon cane sugar, but

not on glycerin, as stated above.

37. In this property it agrees with variety I.
38. The production of butyric acid was observed by meissner’s mycoderma. (sporeless

film yeast).

242 T, TAKAHASHI AND H, SATO:

VIII. Prorrorysis.

Hanne eee eee ree rere een eee

Stab culture. Uniformly distributed
After 40 days. No liquefactior ... «++ «4 No liquefaction.
After 56 days. Do. ww. «=| About 2 c.c. of gelatine in test tube dissolved.
After 70 days. ° D>. .s ws asf Dissolvee part increased.

a nnn

TX, Assimilation of amino-acids.

Amino-acids in original koji-extract.... 0.123% (as glyeocoll)
Amino-acids after fermentation....... 0.026% (as glycocoll)
Amino-acids assimilated .........-+--- 0.097% (as glycocoll)

Thus the larger part of the amino acid contained in the original koji-

extract was assimilated.
X. Optimum temperature for growth. It lies between 30-31° C.

XI. Death temperature: In diluted saké (50¢ water) heating to
56-57° C for 30 minutes kills the cells, but not in beer wort, Koji-extract
or yeast-water. Death will ensue in diluted sake after heating to 57-58°

C for five minutes, but not in Koji-extract.

XII. Limit of aleohol content for growth.

In Koji-extract containing 10% of alcohol, it makes almost the same
growth after 6 days at 27° ©, with vigorous fermentation; but when
the alcohol was increased to 15% there was no film or deposit under the
same conditions. After this observation the culture was held at the room
temperature of 17-20° C, and after 36 days there was formed a film (the

thickest of the 4 varieties) and the turbidity was increased.

The above facts show that his yeast is also a variety of Willia
anomala, and its main distinguishing points as compared with variety

I are the form of the colonies, bad growth in solutions No, II, No. IV,

SOME NEW VARIETIES OF WILLIA ANOMALA. 243

or No. VI, inability of fermenting raffinose, and maltose fermenting

property and its greater resistance to heat and alcohol.

No. 3. Willia anomala Var. III.

I. Form and Size: (a) Young culture:—Koji-extract culture.
(7 days at 23-25° C). In the film the predominant cells are filamental
(10x2.5 ww. 7.5x2.5 yw, 12.5x2.5 m ), and round (3x3 4 ) or elliptical
(4*3.5 yw ) cells oceur very seldom. Cells, containing fat globules’ are
not found. “(b). Old culture. Tn the surface culture on saké-agar after
4 months, the majority of the cells are elliptical (3x4 y» ), bnt long ones
(12.5x2.5. 4, 6x3 pu, 5x3 pe, 13x38 yw.) also occur rarely. Spores have not
been found even in such old cultures.

In the surface culture of Koji-extract-agar (27 days at room tem-
perature) the predominant cells were small elliptical (2x3 mw), or long
elliptical ones and Jarge round (5x5 # ) or clab-shaped (152.5, 15x4 p )
cells occur very rarely. The majority of the cells gave glycogen reaction.

II. Growth. The colony in sake-agar plate culture (13 days at
25° C) was dirty white (same as variety IT). The central part of the
colony was clevated somewhat and there were no folds but the appear-
ance was granular. Both acetic ester and acetic amylester flavours were
perceived. Surface culture. A greyish white covering with more or
less mesentery like folds was found on saké-agar (52 days at room temp.).
The marginal part of the covering was composed of streamy growth but
there was no formation of the promycelium. On the surface of the con-
densed water there was formed a white folded film On Koji-extract agar
(3 months at room temp. July to September), there was formed a greyish
white waxy and almost smooth covering. A promycelial growth was ob-
served in the thinner part of the growth. The covering formed on Mo-

romi agar (10 days at 20° C) was chalky white with fine folds, On

244 T. TAKAHASHI AND H. SATO:

the surface of the condensed water, there was formed a white film with
deposit and fermentation was accompanied by turbidity. On sake-gelatin
(one month at 11-15° C) it forms a dry chalky (on the thin part of
the medium) or somewhat waxy (on thick part of the medium) covering.
The central part is elevated and surrounded by many layers of growth
and fine radiating lines of sprouting were observable at the margin.
Fluid culture: In beer wort (3 days at 23° C) it forms a thicker film
than the foregoing two varieties. A vigorous evolution of CO,-gas was
observed when shaken. In Koji-extract (10° B, 16 days at 15-16° C)
it forms a white and finely folded film which ascends the wall, with
deposit and acetic—and other ester flavour. A few of the film cells

stain red with methylenblue (80 days culture in Koji-extract).

IIT. Ester formation and its relation to carbon-source.

Salutions,

Conditions, Remarks.

No. I... ..,_ ...| In test tube. 2 months } Forms no film with a trace of deposit. So
at room temp.; July to quite differentiy from varieties I and II.
August.

ING: UN coat ena eee Do. Same as in variety II.

No. II... ... ..| 12 days at 25-27-5°C. Forms deposit but no film, Acetic ester flavour
was pronounced, which distinguishes this
yeast from var. II.

No. II... ... ...] In sealed flask. 3months | Forms no film but a trace of acetic ester

at 23-25°C. flavour.

No, II... ,..] In test tube 2 months at | Forms a white film and ring with deposit

Toom temp. (difference from var. II) and trace of fruit
ester.

No. 1V_..._...). 14 days at 17-18°C. Forms a traee of film with heavy deposit and
amylester and by the former character dis-
tinguished it from var. I or var. IT,

No, V... ... «| 14 days at 17°C, Forms a thin but spotted film (difference from
var I, II and IV), and ester flavour.

No, V..._......, In sealed flask during 2| Flavour of amylester is perceibable but on

months at 23-25°C, film is formed,

SOME NEW VARIETIES OF WILLIA ANOMALA. 245

Solutions. Conditions. Remarks

No. VI Sa Os Do. Forms trace of thin film with deposit, which
is rather copious than in the solution devoid
of amylalcohol.

Nes VIL O23) ee Do. Forms a thin®® film with white spots but no
flavour (difference from variety I).

No VIII ... ..] 10 days at 25-26°C. Forms almost the same film as var, I and II.
Fruit ester flavour is conspicuous.

No. IX aes ase 4 daiys at 27-5°C. Forms a similar film but with more conspicuous
spots than in var. I]. Deposit and turbidity
are also more conspicuous than in var. II.

Ce eee

Thus the variety III does not form a film in solution No. I, which does
not contain ethylalcohol ; and this property distinguishes from var. 1 and IT,
In other words, this variety grows very little in the solutions containing
ammonium acetate as the only carbon-source; while if a small quantity
of ethylalcohol is added the growth is good, indicating that ethylaleohol
may be a carbon source to this variety. Moreover the non-development
of ethylalcohol is added the growth is good, indicating that ethylalcohol
may be a carbon source to this variety. Moreover the non-development
this variety as carbon-source was also observed. As regards butyrate
and succinate this variety stands between var. I and var. II. The
formation of ester from lactate containing solution (No, VIII) by this
variety distinguishes it from var. I and var. IT.

IV. Spore formation. It forms on gypsum block after 38 hours
at 27° C. Four spores are contained in each cell.

V. Fermentation products. In the distillate of Koji-extraet cul-
ture, there was found fusel oil, methylaleoho]l and acetie ester but not
acetone. eside 2.02% ethyl alcohol, 0.209% total acid and 0.259% of

ester (as ethylsuccinate).

39). Thicker than in var. TI, thinner than in var, J.

946 T. TAKAHASHI AND H. SATO:

VI. Behavior towards carbolydrates.

S il stances. Remarks.

Arabinose ... ... -. --| Forms island-like growths on the surface of the solution (3 days
at 23:5-24°C). The film is not complete even after 7 days i.e.
growth is the poorest of the four varieties.

Xylose... ... ... ... -- Same as variety I.

Fructose... ... ... ...{ Forms half covered film, bat thicker than in variety II (3 days
at 23-°5-24°C),. There was no deposit or evolution of gas, indi-
cating that fermentation is vigorous less than in the two varieties
described above. Gave 2 weak Davy’s reaction for alcohol after

10 days.

Glucose... .... ... -+-| Film very similar to that of var. I and II. Fluid is turbid. with
heavy deposit and vigorous evolution of CO, eas (4 basi at
23°5-24°C).

Galactose .., ... ... ...f Same as in var. I (4 days at 25-5-24°C’. Davy's test for alcohol

gave a positive result after 10 days:—same as in var. ] and II.

Cane sugar... ... .-. -.! Same as in var. II.

Lactose se. ses sss wef Forms island-like growths on the surface of the solution as in vor.
I and JI. Gave no reaction for alcohol with Davy’s test. |

Maltose EP io. eter Makes falanust the same growth fas var. I and II, but the film is
thicker (3 days at 32:5-24°C). Davy’s reaction for alcohol after
10 days was faint as in the foregoing varieties.

Raffinose ... ... ... ---| Forms almost the same film as variety I (4 days at 23-5-24°C),
but it was not complete. Davy’s test gave a negative result
after 10 days.

Glycerin... ... ... ...J Same as in variety II-(3 days at 23-5-24°C).

amethylglucoside ... ...) The film formed was almost same as in var. IJ, but it was thicker
than in variety I (3.days at 23-5-24°C).

Thus, the assimilation of xylose and lactose by this variety was
feeble as in the ease of two previous varieties. Further, its smaller
assimilation of arabinose and its weak power of causing fermentation in
fructose distinguish it from them. Moreover, its rather “good growth in
a-methylglucoside solution and its inability of fermenting lactose distin-

guish it from variety II.

SOME NEW VARIETIES OF WILLIA ANOMALA. 247

VII. Assimilation of nitrogen compounds and difference between

glycerin and cane-sugar as regards ester formation.

Substances. Remarks.

Asparagin + glycerin...) Forms almost the same film as variety I and II, except that the
ring is thicker (10 diys at 23-24°C.. Ester flavour was absent,
while in Hayduck’s solution it was strong.

Peptone*® + glyccrin ...) Tie film was hardly perceptible (difference from the previous two
varieties), with deposit but no flavour (10 days at 23-24°C).
Ester flavour was also absent in pepton Hayduck’s solution
(after 23 days).

Ammoniumphosphate + } Forms a th'n film, but no ring growth (difference from var. I and

glycerin Il). Growth was betterin ammoniumphosphat Hayduck’s solution
(same as in var. I and JI), in which ester flavour was also
observable.

Pees neilrate +glycerin) Half-covered film was fornied after 3 days at 22-23°C. Same as
in variety I after 2 months.

Thus, the yeast behaves similarly towards nitrogen compounds, as

the previous two but differs from them in certain details.

a a Proteolysis.

Stab culture. Uniformly distributed.

After 40 days, No liquefaction takes place. | The liquefaction commences at the top.

After 56 days. About 15 cm. from surface | About 1:5 c.m. from surface of gelatine layer
of gelatine layer dissolved. liquefied.

After 70 days. The liquefied part doubled | The liquefied part increased to about five times
in comparing to the above duration. agenst to the above period,

IX. . Assimilation of amino-acids.

40). ‘The ring formed by the 4 varieties in peptone (not asparagin) Hayduck solution
was intencely yellowish brown in color while the ring formed in glycerin (not cane sugar)

Hayduck solution was very slightly yellow.

248 T. TAKAIIASHI AND H. SATO:

Amino acids in original solution ...... 0.123% (as glycocoll).
7 ue aealtermtibemonowthi ee veh 0.018% ( ys
9 ocememlbyel so. 5 554655" O.105% ( x Ne

The assimilation of amino-acids is greater than in variety II, but
almost equal to that of var. I.

X. Optimum temperature for growth. It lies between 30° and
Salo Ce

XI. Death temperature. In diluted saké (water 50%) heating to
56-57° C for 30 minutes kills the yeast, but not in koji-extract, beer wort
or yeast water; while 5 minutes heating in diluted saké to 57-58° brings
about death but not in koji-extract.

XII. Limit of alcohol content for growth.

It forms a trace of film in koji-extract containing 104% of alcohol
after 6 days at 27° ©; while in koji-extract containing 15% of alcohol,
there was no film or deposit. After this observation the culture was held
at the room temperature of 17-20° C, and after 31 days there was a trace
of deposit.

From the characters described above this yeast too belongs to the
group of Willia anomala, but it is distinguishable from the other three
varieties by its very long cell formed in the film, from var. I by its power
of fermenting maltose and inability to ferment raftinose, from var. II by
its greater assimilation of fractose, from var. I and IJ by the vigorous
growth in maltose containing solutions, from var. II by not fermenting
Yactose, and moreover from var. I and II by the non-development of
ester flavour or film in ammonium acetate solution (sol. I) devoid of
ethylaleohol. As regards proteolysis it resembles var. IV, and behaves
quite differently from var. I and II. As to the power of resistance
against aicohol this variety is the weakest of the four varieties. The
formation of fruit ester flavour by this yeast in ammonium lactate solution
(sol, VII) is an important character distinguishing it from var. I

and IT.

SOME NEW VARIETIES OF WILLIA ANOMALA. 249

No. 4. Willia anomala Var. IV.

Form and Size: (a) Young culture :—koji-extract culture at
23-25° C for 3 days. Filamental cells predominate:—curved cells
(12.5x8 pw; 15“3 p#), filamental cells (12.5x4 », 10x4 # ). Round
(55 ) or elliptical cells (2.5x4 #, 7x5 p ) are also found, but seldom.
The presence of glycogen is not certain, and fat globules are not found.
(b). Old culture: Surface culture on saké-agar during 4 months, from
January to May. In the growth elliptical cells predominate (4x3 / ),
but long (6x2.5 4, 20x2.5 # ), round (4x4 ) or elliptical ones (52.5 p )
are also found though rarely. Two hat shaped spores, are contained in
a cell and some of the spores are outside the cell. In the surface culture
of koji-extract agar at the room temperature for 27 days, the predominant
cells are elliptical (5x6, 5x8, 5x5 2) and smaller elliptical cells (2x2’5 # )
sousage shaped cells (11x38 / ), or pear shaped cells (7x4 # ) are found

rarely.

II. Growth: The colony in the saké-agar plate culture (22 days
at 25° C) is flat and almost same as in variety I, the only difference
being that the marginal part is rather coarse and not feather. Surface
culture. On koji-extract-agar (52 days at the room temperature) it forms
a greyish white (as in var. II) and almost flat covering (in var IIT it
was mesenteric). The growth on the thinner part of the medinm subse-

quently becomes chalky white and the margin contains promycellium.

On koji-extract agar (3 months at the room temp.) the growth was
similar to that cf variety JIT, with however a lusterless surface. The
covering on Moromi-agar was quite the same as in var III. On saké-

gelatine (one month at 10-15° C) the covering was intensely brown (in the

41). The ledge of the hat is fairly well developed,

250 T. TAKAHASHI AND H. SATO:

other three varieties it was white), and the central elevation was covered by
many layers of the growths with radiations in the marginal part (as in
var. III). The streamings of the marginal part were coarser than in
var ITT. .

Fluid culture: In beer wort (3 days at 23° C) it forms a thick
film as in var III, but the deposit formed is the least of the four varie-
ties, while fermentation is the most vigorous. In koji-extract (10° B,
16 days at 15-16° C) it forms a greyish white (different from var ITT),
sharply folded (mesentery like) thick film which ascends the wall.

Deposit anw acetic—and fruit ester flavour present. A few of the
film cells stain red with methylenblue.. (80 days culture in koji-extract).

III. Ester formation and its relation to carbon-source.

Salutions. Conditions. Remarks.

No. I... ... ...J Intest tube. 2 months at | Similar to var. I or IT.
room temp.

Nose eens ners Do. Similar growth as in var. I or If.
No. IT... ...° ...) 12 days at 25-27-5°C. Same as var. III.

No. H.. ......] In sealed flask. 3 months | Ester flavour has disappeared already.
at 23-25°C.

No. IIL «+» «=| In test tube. 2 months | Same as in var. III.

at room tei).

No. 1V «++... 14 days at 17-18°C. Film formed was thinner than that of var. T,
thereby distinguishable from var. II and III.
Copious deposit formed with conspicuous fruit
ester flavour.

Nos Views pes =) CARS Atl ee Film thinner than that of var. If and no spot
as in var III. Ester flavour wos perceivable.

No. V... ......J In sealed flask. 2 months | Ester flavour was notable but ro film formation.
at 23-25°C.

No. VI ars Do. Forms almost the same film as var. IT, and it
was thicker than that of solution no 1V, which
did not contain amylalcohol, while this did,

SOME NEW VARIETIES OF WILLIA ANOMATA. 251

Solutions. Conditions. Remarks.

No? VID = 228 fs Do. Forms a white but spotless film (differences from
var. III). Ester flavour was copious, dis-
tinguishing this from var. II and III.

No. VIII ... ...J 10 days at 25-26°C. The film formed was almost sime as in var. III
and the fruit ester flavour was noticeable.
Ester flavour was absent in var. I and II.

No. IX wee eel 2 dayseatiz( Doe. The folds of film distinguishes this from var.
II or JIT.

The utilization of acetate, butyrate, succinate and free suecimie acid
was also noted as carbon sources by this yeast. The mode of growth in
these solutions distinguishes this variety from the other three. The for-
mation of ester from ammonium acetate and ethylaleohol (Sol. No. IT)
or simply from ammonium acetate (solution T) took place as in var. I
or II. Moreover, the ester formation from butyrate, succinate and lactate
in presence of ethyalcohol (solution IV, V, and VIII.) was conspicuous,
especially the production of fruit ester from lactate, as we have men-

tioned in var. ITT, has a special physiological interest.

IV. Spore formation: Two or four spores are formed in a cell

on gypsum block at 27° C for 88 hours.

V. Fermentation products: Fusel oil, methyl aleohol, acetone and
fruit esters were detected in the distillate of Koji-extract culture. Quan-

titative determinations gave the following results :—

Ethylaleohol ..... is Paceen sired. such 3.42 vol.
MRO GAIN GS LOBE 5 2.c1sc5 Set erettle cove ts 0.250 (as ethyl suecinate)
MOTH ACYOS) neceacene ore a «oa + 0.0182 (as suecinie acid)

VI. Behavior towards carbohydrates, ;

252 T. TAKAHASHI AND H, SATO:

Substances. Remarks.

Arabinose ... ... ... -+-{ Makes better growth than var. III and almost the same as var. I
(3 days at 23-5-24°C.

Xylose... ... ... ... «| Wery similar to var. I and III.

Fructose ... ... ... ...{ The film was the thickest of the four varieties, but fermentation
was almost absent (3 days at 23°5-24°C). The blue coloration
obtained with Davy's test was the farntest of the four varieties.

Glucose... ... ..._...) Forms white thin folded film after 4 days at 23-5-24°C. The
folds were uot observed in the three varieties mentioned above.
The evolution of CO,-gas was vigorous.

Galactose ... ... ... ...) A very thin and almost transparent film was formed after 4 days
at 23-5-24°C. Gave a very slight blue color with Davy’s test
after 15 days.

Cane-Sugar ... ...... ...| Forms a white thia film, but the thickest of the four varieties,
after 4 days at 23°5-24°C. A trace of folds was perceptible in
the marginal part of the film, with an energetic evolution of
CO,-gas.

Lactose. ... ... ... ... Similar to var. I, II or III (4 days at 23-5-24°C). Davy’s reaction
failed after 15 days.

Maltose sts ace ee eee? As in var. III, it forms a folded film (3 days at 23-5-24°C. Fruit
ester flavour was conspicuous as in the other 3 varieties. Davy's
reaction failed after 10 days.

Raffinose ... ... ... ...J A white film was formed after 3 days at 23:5-24°C. The film was
thinner than that of var. II, 24°C. Davy’s test for alcohol
gave a negative result after 15 days.

Glycerin)... .... ... ...| Forms very thin and island like growth, after 3 days at 23.5-25°C.

a-methylglucoside .,, ...| Half covered film was formed after 3 days at 23-5-24°C. The

film was the thickest of the four varieties.

Thus, this variety also assimilates the above mentioned compounds
as earbon-souree, and is distinguished from the other three varieties by
its worst growth in glycerin containing, by the formation of a folded film

in solutions containing saccharose or maltose.

In regard to its fermenting property, this variety may be distin-

SOME NEW VARIETIES OF WILLIA ANOMALA. 253

guished from var. I or IL by not fermenting leyulose and from var. II
or III by not fermenting maltose.
VII. Assimilation of nitrogen compounds and difference between

glycerin and saccharose as regards ester formation.

Substances. Remarks.

Asparagin + glycerin ...| The film was aimost equal to that of var. III, except as regarded
the greater thickness the folded character of the film (10 days
at 23-24°C). No ester flavour was perceptible as in the culture
of Hayduck’s solution.

Peptone + glycerin... ...J The growth was almost equal except that the ring growth was
thicker than in var. III (10 days at 23-24°C). No ester flavour
was found, while in the culture of peptone-Hayduck’s solution
its formation was evident, though in traces.

Ammoniumphosphate +] Develops like var. IIL (10 days at 23:5-24°C). Ester flavour was
glycerin. not developped as in the culture of ammonium phosphate
Hayduck’s salution.

K-nitrate + glycerin ...]J Half covered film was formed (3 days at 22~23°C). After 2 months,
there was formed a deposit, but there was no ester flavour;
while in the culture of K-nitrate Hayduck’s solution ester flavour
was conpsicuous with copious deposit of a yellow color.

Thus, this variety too assimilates’ asparagin, peptone, ammonium,
and nitrate nitrogen. The non-formation of ester im glycerin-Hayduck’s
solution (changing nitrogen source as shown in above table) and its for-
mation in Hayduck’s solution or peptone; ammonium-phosphate—, patas-
siumnitrate Hayduck’s solution, clearly shows that glycerin behaves quite
differently from saccharose in regard to ester production.

VIII. Proteolysis.

Stab culture. Uniformly distributed.

After 40 days.
No liquefaction takes place. Liquefaction commences at the top.

After 46 days.
Liquefaction commences at the top of gelatine. | Liquefied part increased.

After 56 days. About 2:5 c.m. of gelatine | About 2 c.m. cf gelatine layer from the
layer from the top liquefied. top liqnefied.

After 76 days. About 4 c.m. of gelatine layer | About 3-5 c.m. of gelatine layer from the tc Pp
from the top liquefied. liquefied.

bo
or
rs

T. TAKAHASHI AND H. SATO:

IX. Assimlation of ammino-acids.

Amino-acids in original Koji-extract.. 031239, (as glycocoll)

Amino-acid after fermentation ....... 0°0104¢% (as glycocoll)

Amino acid assimilated ............ 0°1126% (as glycocoll)

X. Optimum temperature for growth: It lies between 30° and
31°C.

XI. Death temperature: In diluted saké (50% of water) or in
beer wort heating to 56-57° C for 30 minutes kills the cell, but not in
Koji-extract.

Death will ensue in diluted saké or koji-extract on heating it to
57-58° C for 5 minutes.

XII. Limit of aleohol content for growth.

In koji-extract containig 10% of alcohol it forms a film and deposit
after 7 days at 27° C, but there was no growth in koji-extract contain:
ing 15% of alcohol. The culture was subsequently held at the room tem-
perature of i7-20° C, and a white deposit formed after 31 days, to which
was added a film after 36 days.

Thus, this variety also belongs to the group of Willia anomala, and
the main properties which distinguish it from the other three are the form
of the colonies formed on saké-agar plate culture and the development
of u brown or greyish color in its growth in several media. Further, the
filamental shape of the cells distinguishes this yeast from var. I and II.

The formation of ester by this variety in a lactate solution (solution
No. VILL) devoid of carbohydrate, a property which it has in common
with var. IIT, distinguishes it from var. I and Il. The want of ferment-
ing power for maltose differentiates it from var. III. The other three
varieties are not killed by heating to 57-58° C for 5 minutes, which is
sufficient for the death of this variety.

The properties which distinguish our Willia anomala varieties from
well known varieties:

They differ from our varieties.

SOME NEW VARIETIES OF WILLIA ANOMALA, 290

Var. I. | Var. II. | Var. III. | Var. IV.

W. anomala. F :
. U B il .
V. belgica. (1) y not fermenting saccharose and absence of ester formation

By not fermenting saccharose.
Lindner’s W. anomala
from mazum. (2)

| By fermetting fructose.

By not fermenting maltose.

SENG By yellow film. By not fermenting galactose and fermenting fructose.

| By yellow film.

By the absence of forming ester and not fermenting glucose or galactose.

W. a. Steuber.
No. II. (4)

By brownish

rosy, Alla. By brownish rosy film.

By the want of ester formation and not fermenting galactose, glucose

and saccharose.
W. a. Steuber.

No. III. (5)
By yellow film.

By yellow film. |

By absence of forming ester and not fermenting galactose, glucose and
saccharose.

W. a. Steuber.

No. IV. (6) By yellow fim.

| By yellow film.

By not fermenting maltose.

By the opt. temp. 28°C, and quick liquefaction of gelatine:—14 days.
Also by death temp.:—65°C (5 m. in Koji-cxtract) and by the higher
% of alcohol produced. (59).

By greysh white
film.

Saito’s W. ano- By greysh white
mala. (7) film.
By fermenting fructose.

By fermenting

By fermenting |
fructose.

maltose.

By fermenting
maltose.

| By fermenting fructose.

Fukumoto’s ano-
mala I. (8)

By fermenting
maltose.

By less resistance agenst alcohol.

56 T. TAKAHASHI AND Il. SATO:

Var. I. Var. II. Var. III. Var. IV.

By less resistance against alcohol.

By fermenting

Fukumoto’s ano- | By fermenting
maltose.

mala II. (9) maltose.

By fermenting fructose.

By the less resistance against alcohol and much production of alcchol.
2 : (4-929).
Fukumoto'’s ano-

mala IIT. (10)

| By fermenting fructose.

By fermenting

maltose.

By fermenting
maltose.

Lindner’s anomala
from Mazum.

(11)

By fermenting raffinose.

By an energetic fermentation of fructose solution.

By fermenting raffinose.

Zeidler’s anomala. | By fermenting

(12) maltose | By fermenting fructose.

By fermenting
maltose.

By fermenting

By fermenting | :
maltose.

maltose’ By fermenting raffinose. |

from green malt. By fermenting raffinose.

Lindner’s anomala |
(13)

By fermenting fructose.

By the less production of alcohol (0992) and duration of spore forma-
tion. (17°5—19 h. at 28°C).
Hansen’s anomala,
(14) :
By fermenting raffinose.

By the active fermentation of galactose sclution.

Lindner’s anomala

from American Beer. By fermenting raffinose.

(15)
| By fermenting fructose.
Kozai’s anomala By the feeble fermention of beer wort.

(16)

SOME NEW VARIETIES OF WILLIA ANOMALA. 257

—— —————_—————————————

War. I: | Var. II. Var. III. | Var. IV.
By the higher 7% (4-069) of alcohol formed.
Meissner’s anomala.
4 (17) By chalky white By chalky white
film. film.
saecenete By the higher 9% (5:0394) of alcohol formed.
anomala 7. Sa
ss) By the SEU OwSH By yellowish-white film.
white film. ~
; By the higher 2 (5:03292) of alcohol formed.
Meissner’s "
: anomala 40. :
(19) By white fim. | By white film.

Willia Wichmanni.

(20) By the formation of slimy growth.

Summaries of Part I.

From the properties described above, it is highly probable that our
yeast are quite new varieties of Willia anomala with the exception of var I,
which behaves very similarly towards carbohydrates as the variety of
Linder’s Willia anomala isolated from an American beer (India wharf.)

However, Lindner’s variety causes an active fermention in levulose
and galactose solutions, whereas our var. I ferments the latter sugar very
sparingly and the fermentation of the former sugar is doubtful.

It is very important and interesting that these varieties behave very

(1), (2), (11), (2), (18), (15). P. Lindner: Wochenschr. Brauerei. 1900, 17. No. 49.-51.

(3), (4), (5), (6). L. Steuber. Zeit. f. gasammtes Brauw. 1900. 23. 3.

(7). Journal of the Tokyo imperi. univ. College of Science. Vol. 19, art. 18.

(8), (9), (10). J6zdkyokaizatzshi (Japanese). Y. 5. Vol. I.

(14). E. C. Hansen: Cent. Bact. Paras. Abt. II. 1904, 12. 529. Lafar. Hundb. tech.
Micol. Bd: IV. 168.

(16). Cent. Bact. Paras. Abt. II. 1900. 6. 400.

(17). (18), (19). R. Meissner: Landw. Jahrb. 1901, 30. 497-580.

(20). Zikes, cent. f. B. 1906, Abt. Bd. XVI, No. 416.-S. 97-111.

258 T. TAKAHASHI AND H. SATO:

differently as to ester formation when shavings of Crytomeria japonica are
added to the solutions; also the fact that they form esters from organic
acid salts or free acids in the presence of alcohol or simply from organic
acid salts (except var. II) in the absence of carbohydrates. Further,
the formation of ester in common media, containing carbohydrates, is well
known property of Willia anomala. So, these varieties form esters not
only from carbohydrates, but also from prexisting alcohol and organic
acids. The fact that variety III grows better in alcohol containing the
tions than in those without aleohol, in the absence of carbohydrates, shows
well that this variety also assimilates alecohol:— a property distinguishing
it from the common myccderma yeast, which is simply a destroyer of

aleohol.

A copious evolution of the fruit ester flavour in ammonium buyrate

containing solution explains the role of this salt in saké brewing*?.

As regards the difference between carbohydrates and glycerin for
ester formation, it is worth noticing that the formation of ester is always
absent in the cultures of glycerin Hayduck’s solution, whatever may be

the source of nitrogen compounds.

The great assimilability of aminoacids by our yeast, is an important
property for the aging or afler ripening of saké ; for common saké yeast
saccharomyces saké!* assimilates the acids moderately as compared with
our Willia anomala varieties, e.g. Sacch. saké. B. 21 of Oji Saké Brew-
ing Institute assimilates only 0.059% of the acids from a solution con-
taining 0.123¢ while our Willia varieties assimilate 0.116-0.097% of the

acids according to the varieties.

(42). cf. Kurono’s article of this Journal.
(43). cf. Takahashi and Yamamoto’s article. The formation and assimilation of aminoaci's

by the different varieties of yeast. P. 279 of this Journal.

SOME NEW VARIETIES OF WILLIA ANOMALA. 259

PART. Ik
Application of the Aging Yeast.

The first experiment (August 1908).

To seven flasks, each containing 700 c. ce. of newly prepared young
saké, were heated for 15 minutes to 55° C and after addition of a sterilis-
ed piece of Cryptomeria japonica were added the varieties of our yeast,
one to each flask, and kept at the room temperature. After 5 days the

result was as follows :—

Number of

Samples Quality.
1¢ A very slight change occurred,
Il. Made the best ehange in palatableness and characteristic flavor
of young saké disapeared.
ITT. Do.
IV. Almost same as no. I,
Vi Do.
VI Better than no IV. or V
VII. Do.

Thus, the ripening of saké was accelerated by the addition of the

yeast.

Seoend experiment (December 1908).

By the first experiment the role of our Willia anomala was placed
beyond doubt, therefore in this ease some of the flasks were kept at 20-
25° C, while others were kept at the room temperature below 20° C, for
the purpose of determining the influence of temperature during aging.

The flasks containing young saké were heated for 20 minutes to 60-65° C,

260 T. TAKAHASHI AND H, SATO?

and the yeast was added as in the first experiment, not omitting the addi-

tion of a piece of Cryptomeria japonica,

I. Series, where the duration of experiment was 2 weeks.

ae Cusine
Te a0 At 20-25°C. | Inferior to No XIII.
TL me, Room temp.] The worst of this series.
IV. me Room temp. Do.
V. At 20-25°C. | Stand next of No. XVIII, but better than No. V.
X. Stand next of no. V.
XIIT. Almost same as No. X.
XVIII. The best of the first series.

II. Series: The duration of aging was 3 weeks at 20-25° C.

osatol Quality.
II. The worst of this series.
Vi. Stand next to No. XI, and same as No. XIV. or XIX.
XI. The best of this series.
XIV. Some as No, XI. or No. XIX,
XIX. Some as No. VI. or XIV.

III. Series: The duration of aging was about one month at 20-

25° C.

‘

SOME NEW VARIETIES OF WILLIA ANOMALA. 261

No. of

Samples. EES

VIII. The best of this series.
ONE Stand next, in quality, to No. VIII, with a somewhat swcet taste.
XVI. Gave unpleasant flavour and was the worst of this series

Almost same as No. XII, but with a trace of bitter taste.

As the first series shows, the temperature of the fluid has a large

influence upon aging, and moreovere the duration of aging is different
according to the varieties of yeast, as shown by the other series of this
experiment.

Var I. needs the longest time and var IV. the shortest, while var
II stands in the middle.

Third experiment (January 1909).

In this experiment lactate—or butyrate of ammonium of which the
latter was the best substance for ester formation, was added to the saké
before the experiment. The temperature was 20-25° C and the duration

2 weeks.

No. of

Samples. Yeast. Organic Salt. Remarks.
1G ae — ieee, same as No, III, IV, or V. i
Il. Var. I. 000 The best of all.
III. Var. II. a Almost same as No. I.
IV. Var. III. 50 Do.
V. Var. IV. 580 Do.
VI. Var. I. 0:4 c.c, butyrate to } Inferior in quality, with flavor of butyric acid.

100 c.c, Saké,
VII. Vaz. II. Do, Do

962 T. TAKAHASHI AND H. SATO:

No. of Organic Salt. Remarks.
Samples.
VIII. 0:2 c.c. of butyrate to | Inferior in quality, with flavor of butyric acid.
100 c.e, Saké.
IX. Do. Do.
Ke Var. I. 0-2 ¢.c. of lactate to Do.
100 c.c. Sake.
XI. Var. III. Do. Do.
XU. Var, III. Do. Do.
XIII. Var. IV. Do. Do.
XIV. Var. Is 02 c.c. of both buty- Do.
rate and lactate in
100 ¢.c. Saké.
XV. Do. Do.

Do.

Tn the culture of the four varieties of our Willia in butyrate or lac-

tate containing solution, the formation of ester was conspicuous; while
in this third experiment butyric acid was formed but no ester. The cause
of this is attributable with reason to the excess of these salts, so that the
further experiments were made.

Fourth experiment (February 1909).

The duration of experiment was one month at 20° ©,

SOME NEW VARIETIES OF

WILLIA ANOMALA. 263

Seen Yeast Salts. Remarks.
ie Medium quality, showing a little change for
ripening.
II. Var. I. Better than No. I.
Ill. Var. II Do.
IV. Var, LIT Do,
V. Var. IV Better than No. II, III, or 1V.
VI. Var. I 0-Olc.c. of butyrate | Palatable,
to 100 c.c. Saké.
VII. Var. II. Do. Do.
Vill. Var, IIT. Do. Bad odor.
IX. Var. IV, Do. Do.
Xe, Var. I. 0:005 c.c. of butyrate | Medium quality as No. I.
to 100 c.c. Saké.
XI. Var. II. Do. Do.
XII Var. III. Do. Do.
XII. Var. IV. Do. De.
XIV. Q-Olc.c. of butyrate | Both the flavour and taste were not good.
to 100 ce. Sake.
XV. 0:005 c.c. of butyrate | Stands next to No. XVII.
to 100c.c. Saké.
XVI. 0-0025 c.c. of butyrate Do.
to 100 c.c. Saké,
XVII. 0°0005 c.c. of butyrate | Zhe best of all.

to 100 c.c. Saké.

264 T. TAKAHASHI AND H. SATO:

Fifth experiment (June 1909). 2 weeks at near 20° C.

mie roe ‘| Yeast. aie 10 100ce Remarks,
I The flavour of young saké still remains.
II 0-0001 c.c. | The flavour of altered saké.
III + Trace of the flavour of young saké still remains.
1V + 00001 c.c. | Do, but a little better.
Vv + Var. I ae Trace of the flavwur of young saké still remains.
VI : Var. Il Do.
Vir + Var. III i Do.
VIII + Var. 1V ai Change for ripening sufficient to make a palat-
able saké.
IX Var. I | 0-001 cc. | Almost same as no. VI and VII.
xs ae Var. II Do. Change for ripening sufficicnt to make a pala-
table saké.
XI ae Var. Ill Do. The taste was best of all samples, flavour in
less good somewhat improved.
XIL + Do. Do.
XIII + 0:0002 c.c. Do.
X1V “= Do. Do.
XV + Do. Do.
XVI + Do. Do.
XVII a 0-0001 c.c. Do. -
XVIII + Do. Do.
XIX + Do. Do.
XX + D». Change for ripening was sufficient.

This experiment proves the influence of Cryptomeria japonica for the

aging process as mentioned in the first part.
Sixth experiment (June 1909).

The fifth experiment, in which the storage was for 2 weeks, showed

(44). Cryptomeria japonica,

SOME NEW VARIETIES OF WILLIA ANOMALA. 265

that a suitable quantity of ammonium butyrate was favourable for the
aging. In this experiment the storage time was therefore prolonged to

almost one month at 20-27° C, other conditions being identical.

Bie. Biss Yeast. ate : 5 Woe Renike
I + The flavour of young saké still remained.

II + Var. II oo Change of ripening further advanced than in no. f,
III + Var, III eA The flavour and taste were not good.

IV 4. Var. IV Ses Do.
We + Var. II | 0-0001lc.c. | Almost equal no. II.

VI + Var LV Do. Palatable.

VII + Var. II | 0-001 c.c. Do.

VIII ae Var. III Do. Very Palatable.

IX + Var. IV Do. Palatable.
xX + 0:0001c.c. | Almost same as no. IX.

XI + 0-001cc. | Palatable.

Thus, the suitable quantity of butyrate for aging with yeast is seen
to be 0.00/e.r. to 100 ¢.c. of saké, and one month storage was to long espe-
cially for var III and IY. when yeast was simply added, as we observed
in the second experiment (var. IY. needs the shortest storage time for

aging). Moreover, simple addition of butyrate was effective for aging.
Seventh experiment (January 1910): 10 days at 17-22° C.
From the sixth experiment the role of our Willia anomala, by itself

or with the addition of butyrate, was positively assertained, therefore in

this case eight wooden tubs, made of Cryptomeria japonica, of about 36 L.

capacity were used instead of glass flasks.

266 T. TAKAHASHI AND H. SATO:

ee
Butyrate or

No. of tubs. Yeast. epee: Remarks.
I ae Sa Better than no. VII.
Il Var. Il 250 Very palatable.
IIL Var. III aes Do.
TV; Var. IV as Better than no. V.
Vv } Vet 00001 c.c. Better than ro. I.
VI i Do. aa Almost same in palatableness as no. I.
200 c.c. of nutri-
Vil Do. ent fluid over | The worst in quality, being thick in color.
the yeast
0:0001 c.c. of
VIII Sap butyrate to Better than no. IV.
100 c.c. of saké

The chemical composition of the saké after the experiment was as

follows :—

Origi- | Top, 1.

mal. II.

Sp Gra = 0-9930 pana 0-9923) 0-9929 0.023 0.9930) ona ones 0:9928
I

100 c.c. of saké contains in grams of.

Alcohol (vol.),.. ...J 17-60; 16-98; 17-90) 17-10} 17-00] 17-00{ 17-00) 17-10] 17-30
Extractive matters...] 3°8056| 3-9680 39568) 39792) 3-917¢) 39552) 3-9384) 3:9556] 39384
Total acid... ...), 0°1770) 0°1770) 01770) 0-1770| 0:1770] 0-1770) 01770} 0:1770) 01770
Volatile acid ... ...] 0°0360) 0:0360| 0:0300) 0-0360| 0-0360| 0:0360] 0-0360} 0-0360| 0-036)
Nonvol. acid ......} 01416) 0°1416) 0-1475) 0-1416) 0-1416] 0:1416) 1-1416} 0-1416] 0:1416
Ash... ... «2. ++] 0°0440) 0:0488) 0-0488; 0-0480) 0-0444] 0-0472) 0:0440) 0-048) 0-0464
Glycerin... ... ...] 1°1840) 11524] 11460) 11244) 1:1028} 11646) 1-:0866) 11088} 1:1440
Glucose ... ... «..{ 11984] 1-1842) 11460) 1.2048, 1:1874} 1:1608); 1:1252) 11460) 1-1660
Dextrin ... ... «| 0°5900) 0°5678) 0+5480 06678 0°5682) 05272) 05873) 05794) 05776
Crude protein... ...J 0°7158) 0°7140) 0:7140) 0:7175) 0:7105| 0:7125] 0-7105) 0:7105} 0-7095
Protein

(Stutzer’s method)} 0:0403) 0.0402! 0:0385! 0:0420) 60-0455] 0:0402) 6-0420' o-o42s] 0-04

SOME NEW VARIETIES OF WILLIA ANOMALA. 267

Fusel oil |
(after Takahashi) | 0°1500 0°1500; 0:1500 0:1500 0-1500) 0:1500 0-1500 0-1500) 0-1500

Aldehyde

(as glycocoll) ... -.- | 02126) 0-1890) =o || 0:1939) 0-2010

c.c.

Degree of) (Jod.*s 0°85, 11 10 1-0) 1:0) 1-0 11 13) 14
colorations | K. Bich. EZ, 14 1-4 10 14 1-4 1-4 1-4 Lei

Thus, there is an increase of ethylaleohol, aldelyde, and protein (in
a few examples) in the tubs, when our Willia anomala was
added. The increase of ethylaleohol is evidently the result of alco-
holic fermention due to the yeast and the addelyde must also be ascribed
to the same action; while an increase in protein contents in certain tubs
(No. IV, VII, VIIT) is explained by the presence of suspended yeast
cells in these samples. The decrease of furfurol, grycerin, and amino acids
was observable in all the tubs except in control tub. The decrease of the
last two substances is a settled matter from our Wlllia anomala, but why the
decrease of furfurol still needs explanation, Perhaps Will’s*® and Lint-
ner’s*' observation of the decrease of furfurol in wort by the growth of yeast
furnishes the only basis for the explanation of our case. Moreover, the
increase of acetic and another esters (not given in the table) and the dis-
appearance of the sharp flavor of young saké are decidedly favorable
factors for aging.

Eighth experiment (January 1910).

(45). The Todine solution contained 1-27 gr. of iodine and 1-8 grams of KI. in one litre
of water. The K-bichromate solution contained 1:94 grams of the salt in one litre of water.

(46). Zeitschift f. gessamtes Brauw. 1902. 25. 39.

(47). Lintner. (Brewer's Jonru. 1910. No. 11. §. 535). He ascribes the decrease of

furfurol to the formation of thiofurol.

268 T. TAKAHASHI AND H. SATO:

By this experiment the esters, formed by the mixed (Var. I, IT, IT)
culture in Karlsberg’s flask containing Koji-extract, were passed through

young saké twice every day:

each passage lasting 30 minutes. After
10 days the sharp flavour of younk saké disappeared and gave place to

the flavour of fruit esters.

Conclusion.

From the result of the above experiments we must conclude that
during the after ripening or the aging of saké, there must be present cer-
tain varieties of Willia anomala, which produce definite changes in the
composition of young saké, and that the artificial addition of this yeast
to young saké acceralates the ripening, producing well aged saké in a

comparatively short time.

Explanation of Figures.
Plate XII,
Colonies in saké-agar plate culture.
Fig. 1, A = Variety I.
Fig. 2, B = Variety II.
Wigaisds, GC
Fig. 4, D

Variety III.
Variety IV.

Jour. Coll. Agric. Vol. J. EAL We

The Quantity of Amino-acids and its Relation to
the Quality of Saké

T. Takahashi and H. Sato.

Tt is a fact beyond dispute that chemical composition of saké has a
certain relation to its quality, but the chemical reserches made so far arc
insufficient to determine it precisely. One? of us has a certain relation to
few exceptions, the quantity of fusel oil in saké has a certain relation to

its quality, the better the saké the less it contains fusel oil.

No other fact is known which appeared to us to have any constant
relation to the quality of saké, so we have made some reserches on the
amino acids contained in saké according to Sérensen’s* method the results

were as follows :—

(1). Takahashi. The Journal of the Tokyo chemical society Band 26, vul. VIIT.
(2). SGrensen’s method: deut. chem, Gesel 1908. Bd. I. S. 143-144.

270 T. TAKAHASIII AND H. SATO:

Name of brewers. Quality of saké. Amino acids (as glycocoll) 2.

CRORE. Gene fen ho ee ota Superior 0174
ST MOMIE@ ere ceey oer ces 5 0-178
S. Kimura... 0.0... 0 es. 3 0-210
S;elatsuuma |=) -2seeeee ee Ps 0-187
TtSanlol ccs) pecs eee Best, Ist c'ass 0163
T: Okura.sca aed ees ‘4 0-171
Si Oyagi ten 2a ude eee Py | 0-183
Mislkkawametoieesn gent stone = 0-174
j. Matsumoto ... 0... «a B 0-186
Y. Sugamoto ... ... ... =. 3 0°187
Te. Yamazaki, wee ees) eases + | 0:189
Aké Company <i. 22 ses) ae Ps | 0-195
@MKoczuka 40. 4 | 0186
B, Osabel\.cae acc cast ereee ees - 0-218
Jolanaleies7 osc esse wens 55 0-203

Eigashima Company... ... ¥. 0192
Te WaAMAM Ural. ean ee re * 0-214
(CAMUIET RE ico ie Be, oon o 0-172
Me \VaSHiwo) ae) ue ceil ceteses 0°167
ec MOjlie \ecch sagn here neers 0°155
To ADO: eee, acre tery iene .) 0-192
K. Kimura oe ee ee Pa | 0:210
S. Imanari eo HS ony 3 | 0°254
Ne Takanastiic- ce pwesten tae s 0:234
Mi Kaimural)) Gears sneer 7 0:245
J. Kutsiizawa) . Sese ess 4 0:272
Mi Hara’ oy. gve. cassie core A O-161

” Paar ec ae F 0:167
Sileite sel See : 0-230
KK. Shimois 7,0 tecsdesameets + 0°185
SislWOYA 5.5. aye cree nre eae A . 0°196

THE QUANTITIES OF AMINO-ACIDS

Name of brewers.

Quality of sake.

AND ITS RELATION,

Amino acids (as glycocoll) 7.

J. Shiwozaki
O. Takata...
I. Kato
K. Matsuoka
T. Shimo ...
K. Shimo ...
S. Kimura...
K. ihara
K. Obayashi
S. Kimura...
M. Makibayashi
Y. Fujii
N. Oto
T. Miura ...
II. Miyahara
M. Takashige ...
G. Mitsuishi
T. Murakami
T. Taketsuru
T. Morishita
»
T. Taketsuru
H. Shima ...
R. Matsui...
S. Yoshioka
S. Ishii
I. Shigeta ...
R. Horie ...
S. Nishimura
T. Hirano...
Z. Ikeda

Best

0-171
0-167
0°166
0-233
0.187
0-209
0-180
0-178
0-220
0-207
0:221
0:172
0-208

0:227

272 T. TAKAHASHI AND H. SATO:

Name of brewers. Quality of saké. Amino-acids (as glycocoll) 2.
pTshil rae esses eens Best 0-177
IS MtSUG 727k aaceee mee eas “5 0:205
MavKohiyamayr.) f--rcne mess Better, 2nd class 0.296
Dio GEECEITIES eo ees ces eos a 0-192
INS WETHER 0S og iggy Ge na Pe 0.230
HIRD KERN coy God cco cas 5 0214
Wor Giese) dav 55, eco. sccn Goo, 3rd class 0279
J. Okamura Be 0:202
R. Tada 0:263
T. Nakano Rac) pacha teanmnene = 0-291
J. Tomono i 0:272
M. Kawai... - 0-246
I. Hasegawa * 0:33
Wagkiashimotore.s e.<)iee ieee Inferior 0-263
ee Mokozekii ras snare ees P 0 250
Mee Mut rene =< orm Ms 0-286
R. Nishijo... i 0-214
R. Hirano... Ay 0200
G. Nate rf 0:273
T. Shimamura .., oF 0 243
Re Tamal 22°F. (--aont oe ” O24]
iH. Nakadyamat eee esses ” 0-349
K. Sakurai Ps 0-239
Ke Saiharay eine .caneacwan » 0218
Te, Watanabe ten vere ess ieee » 0-205
S. Hirai 4, 0-207 ;
5. Ueda ~ pene isan Sard eae ; 0:272
G, Kabachie® 20 oon ees =: 0:250
TU. Mitstiycin tte terse irre miescmats mr 0276
S, Nakamurae (220) 4.0) steeeee sy 0316
E. Takahashi... 2... soe sxe A O217

THE QUANTITIES OF AMINO-ACIDS AND ITS RELATION.

273

Name of brewers. Quality of saké. Amino-acids (as glycocoll) 7%.
eg takanoy 0S eeaee ss cee Inferior 0:277
J. Shiwoiri 7 | 0:245
T. Sakai » | 0-286
N. Ueda = 0°393
H. Takata... Fs 0°324
T. Kamaya a 0-238
T. Kitai 3 0-273
S. Sato 0-199
G. Hirata ... 3 0-324
K. Mrrata... us 0-261
H. Hirai 5 07183
S. Tikura ... 5 0-261
J. Koike . 0°337
J. Kutsusawa oF 0°255
S. Murata ... sp 0:366
S. Shiramizu “A 0-264
T. Ma'sumura .., a 0:222
K. Tanaka 7 0258
K. Ito 0-212
K. Fojishima i 0 264

In 64 samples including 4 superior and 60 first class saké we have.

Baxi ffs ere nce era cierorsieeiete a. 3's 0.272% of
Mbiavinm tim” es S<-s 0) sence nee severe erate =< 0.149% of
ANY EEN aot 3.5 Thotgo.0,5-010.0.0 2:0, COO 0.197% of
In 4 samples of the second class we haye:—

IN ER ahestriadlame oince.croe Bist: SO ACD TORING 0.296% of
iMG UIne eesti <a eee nonce Wh A OE
FAVELA DS) men ee: Sen techn emer Tepe eas as ae) OL2332) 0f

amino acids.

amino acids.
amino acids.

amino acids.

And in 46 samples including 7 of the third and 39 of the inferior

class we have :;—

S.! 7 - 7
274 T. TAKAHASHI AND H. SATO.
Maxima .2is a0 0hk- oe eee see 0.3934 of amino-acids.
Mainimim! f66525 Soe ee eee 0.183% of amino-acids.
Averace® 2 f.050. sae eee ee 0.268¢ of amino-acids.

Thus, on an average better saké contains less amino-acids than the in-

ferior ones.

- ats

The Assimilation and Formation of Amino-acids
by Saccharomyces Saké and Other

Yeast Varieties.
BY

T. Takahashi and T. Yamamoto.

As a decomposition product of the protein matters of yeast cells by
self-fermentation, leucine was found by Liebig? tyrosin by Béchamp?.
Schiitzenberger mentions still another nitrogenous compound® as a pro-
duct of self-fermentation. Recently Boullanger, Beijerink, Artari, Weh-
mer and especially Will* made laborious researches on the proteolysis of
yeast cultur. Also, Salkowski*t, Effront® and Rettger® and many others
followed with their studies on self-digestion, or autolysis and a new branch
of mycology has been established. According to Geret and Hahn the
nitrogenous matter of the pressed juice of yeast, decomposes into many
amino-acids, of which about 304 consists of di-amino-acids or bases and
70% of mono-amino-acids. Recently, Kutscher and Lohman’ analysed the
self-fermentation products of beer yeast with Kossel-Kutscher’s method
and found histidin, Iysin, arginin, leucin, tyrosin, aspartic acid, ammo-

nia and many other purin derivatives.

—

Sitzungsber. d. Kgl. bayr. Akad. d, Wiss. in Miincheu. 1868 u. 1869.
Compt. rend. de l’Ac. 1872. Bd. 74. S. 184

3. Alloxanbases and purin derivatives.

4. Zeit. f. phys. chem. 1889. Bd. 13. S, 506.

5, 6. Brewer’s Journ. 1909. P. 540.

7. Zeit. f. phys. chem, 1903. Bd. 39. S, 159 u. 313.

276 T. TAKAITASHI AND T. YAMAMOTO:

Similar decompositions of protein matters take place in the nutrient

media outside the yeast cells.

On the assimilation of amido-nitrogen by yeast, Wahl and Hantke®
found in the culture of wort, that the assimilation of amido-nitrgen is
very conspicuous as compared with that of peptone or protein nitrogen.
According to P. Petiti’s® observation the surface yeast cells consume more
than double the amido-nitrogen (asparagin) that the bottom cells do. R.
Kusserow?® too arrived at the conclusion that the decomposition products
of the protein matters are assimilated better than undecomposed proteins.
Recently, Stockhausen’! made researches on the assimilation of the de
composition products’* during the self-digestion of beer yeast, and found

that the assimilability of these decomposition products differed according
to the varieties of yeast.

About saké yeast, K. Kurono?? found that all nitrogenous substances
used in his experiment were assimilated ejually well, except tyrosin, cys-

tin, nitrate and nitrite.

It has seemed to the authors that the assimilability of amino-acids dif-
fers somewhat according to the varieties of saké yeast; for the quantities of
amino-acids of saké differs evidently in accordance with its quality as
described in another paper. The experiment was carried on with 100 c.c.

of sterilized Koji-extract (10° B) in Erlenmeyer’s flask and after the

gs. American Brewer’s Review. 1894. B. 7. S. 492.

9. Compt. Rend. T. CXXIV. 1897. P. 93.

10. Zeits. fiir Spiritind. 1897. Bd. 20. S. 97.

ll. Jahrb. der Versuch. u. Lehr. f. Brau. in Berlin. 1908. 11. S. 673.

12. The compounds used in his experiment were ;—Leucine, tyrosin, histidin, arginin,
adenin, hypoxanthin, guanidin, lysin, cholin, uracil, glutamic acid, aspartic acid, tetramethylen-
diamin, and ammonia. Further, thymin and asparagin.

13. Journal of the Scient. Agricul. Socie. No. 91. 1910 also this journal. He used as
nitrogen-source: Glycocoll, alanin, leucin, aspartic acid, (free and neutral), asparagin, tyrosin,
cystin, glutaminic acid, arginin, histidin, phosphate-and carbonate of ammonium, nitrate-nitrit

of K. The yariety of yeast used in his experiment was B. No, 25. used in our experiment.

y
‘

THE ASSIMILATION AND FORMATION OF AMINO-ACIDS. 27

addition of yeast it was kept at 25° C for 7 days. During this time the
fluid was shaken once every day. For one portion of the clear fermented
fluid the quantity of amino-acids was determined according to Sérensen’s
mehod'*. Another portion of the fluid (50 c.c.) was distilled and for it
the quantity of fusel oil was determined after T. Takahashi’s method’.

The results are given below :—

(Original koji-extract contained 0-148%4 of amino-acids).

Amino-acids (as glycocoll) 22.
Varieties of Total acid 2%.

Yeast. cee Ginmeilon LEST ON (as snccinie acid.)
emaitieds formed.
A I 0-084 0-064 Ca. 0-0125 less 0:0556
9 II 0-059 0-099 , 0:0125 more 0:0592
III 0-099 0.049 » 0:0125 less 0:0644
& IV 0-066 0 082 » 0:0125 0-0645
FC Vi 0-083 0:065 » 0:0125 0:0404
» WAL 0:074 0:074 5, 00125 less 0:1068
” VI 0-101 0-047 » OOS op 0-0488
- Vill 0-087 0-069 » 0°0125 0:0580
» IX 0-318 * 0:170 5» 0:0125 more 0:0556
” x 0:087 0-069 SO O125imr, 0:0528
* XII 0-072 0-076 » 0:0125 less 0:0716
¥ XIV 0-209 * 0-061 » 0°0125 0-0672
rs XV 0-109 0-039 » 0:0125 less 0.0891
i XVI 0:120 0-028 9 OOD 55 0:0952
a XVII 0-120 0-028 » 0:0125 ,, 0°1193
ee NeVAINT 0-068 0-080 » 00125 ,, 0:0921
- XIX 0-133 0-015 OLOLZo Iss 0:0988
x NOK 0-112 0:036 1 (OD 0:0650
4 XXI 0.224 * 0076 ey O:01250055 0°1085

14. Chem. cent. Bd. I. 1908. S. 145-144.
15. Bulletin of the College of Agric. Tokyo Imp. Univ. Vol. VI. No. 4. P. 438.

T. TAKAHASHI AND T. YAMAMOTO:

Varieties of

Amino-acids (as glycocoll) 9.

Total acid 9.

Yeast. LS Gansimnedulor et oe (as succinic acid.)
3 formed.
3 XXII 0-158 * 0:010 Ca. 0:0125 0-1428
» XXIIl 0-200 * 0-052 » 00125 0-0913
5 eh 0-178 * 0-039 » 00125 0-0654
‘ XXV 0 086 0-062 » 00126 00775
iawn Ses | 0 053 0-095 » =0°0125 0 0351
oe oe L 0-078 0-070 » 00125 0-021
kV ILI 0-118 0:030 0 1026
ee, O41 2.4 0-038 0-110 » 0°0125 less 0:0097
x XXX 0-079 0079 »» 0:0250 00454
» XXXI 0-154 * 0:005 Amylester 0:0589
» XXXII 0-004 0-043 » 00125 0 0324
0) BEEK! 0-143 0-004 » 0°0125 0:0454
» XXXVI 0-085 0-062 », 00125 less 0-0490
B. I 0-117 0-030 35 070250 0-1220
= II 0-088 0-059 » 0°0125 less 0.0887
> lL 0-058 0-089 » 00125 ,, 0-0430
m= IV 0-059 0-088 » 00250 ,, 0-0610
fr Vv 0118 0-030 » 0:0250 0-0406
3 Vi 0-041 0-106 », 0°0250 less 0:0321
rs VII 0-119 0-027 » 00125 0 0407
- VIIL 0-076 0-070 », 0°0125 less 0:0528
os IX 0-059 0:087 » 00125 ,, 00436
Fe xX 0-070 0-077 » 00125 ,, 0-0409
5 XL 0-053 0-087 » 00125 , 0:0379°
x XII 0-053 0-087 » 00500 0-0168
is XIII 0-082 0065 » 00125 0-0407
. XIV 0-59 0 087 » 0°0250 0:0321
~ XV 0.082 0-065 » 0°0250 0-0406
; XVI 6053 0 087 » —0°0250 0:0466

TITE ASSIMILATION AND FORMATION OF AMINO-ACLDS. 279

——_—_—_—_————————————————————_———————————————

Amino acids (as glycocoll) %.
Varieties of < : Total acid. %
Yeast. Fusel oil 76- (as succinic acid).

5 onsu or
Remained. Consumed

formed.
” XVII 0-065 0-083 » 00125 0 0350
Hy ease 0-041 0:106 » 00125 0-0291
P XIX 0-129 0017 » 00250 0:0032
+ XX 0-053 0-095 » 00125 0:0236
7 XXI 0 100 0:047 » 0°0250 0:0089
ny XXII 0:076 0-071 » 0°0250 0:0238
7%; XXIII 0-241 * 0:093 », 0°0250 less 00442
XXIV 0-065 0-083 » 00125 0:0292
:; XXV 0:038 0-059 » 00125 00118
7 XXVI 0-100 0:047 yy 0°0125 0:0104
5, vale 0:076 0-071 » 00125 0:0089
» XXVIII 0-059 0 089 5, 0°0250 less 00349
5 XXIX 0-094 0:053 » 0°0250 0:0239
of XXX 0029 0-118 » 00250 less 0-0108

* indicates formation.
As second series of experiments was made with beer yeast and other

well known varieties, with the following results :—

—— ———

Amino-acids'* 9.

Yeast varieties. J Fusel oil.
. Consumed or
Remained. fi
ormed.

(hampane yeasty... se.) s=s)uvees 0-112 0-064 Plenty
Tarula (red var.) EONS fo 35 0-206 +0:030 Medium
Surface beer yeast (Holland) ... 0-123 053 ”
Bottem beer yeast (Munich) .., 0.023 0-153 Least
Shiz, Sacch. Pombe ... ... ... 0-153 0-023 Medium
Wine yeast (steinberg) ... ... 0:147 0-069 Plenty
Willia anomala Hansen ... ... 0 057 0-119 Medium

16. In original koji-extract there was 0:176% of amino-acids.

280 T. TAKAHASHI AND T. YAMAMOTO:

Thus, the quantity of the amino-acids assimilated varies according
to the varieties of saké-yeast ;—from 0.064¢-0.004%. Moreover, with cer-
tain varieties there was an increase of the amino-acids, the ease of A. No.
9 being an extreme example of these varieties :—the increase being 0.170%,
or more than double the original quantity.

The increase of amino-acids corresponded with the intensity of pro-
teolysis in A. No. 9, No. 21 and B. 23 where the proteolysis was most
intense, but in A. 30, B. 8, 16, 24, where the proteolysis was also energetic,
there was no increase of amino-acids in the fluid.

In general, the culture in which the consumption of amino-acids was
large produced plenty of fusel oil, but there are exceptions: e.g. compar-

ing A. 13 and 9 we have:—

Amino-acids 2.

Fusel oil.
Consumed or

Remained. formed.

AAS YE: 0-073 —0:0760 Ca. 0:012524 less
Bs ere 03188 +0:0170 Ca. 0:012596 more

From this example, it is evident that the proteolytic enzyme acts
more efficiently when there is a suflicient quantity of amino-acids as the
nutriment of the yeast. Further, in other cases as A. 29 and 35 we

have :—

Amino-acids 2.

Fusel oil 2.

Remained. Consumed.

0-110 0:0125 less
00036 00125

The quantity of fusel oil formed is altogether inverse to the quantity
of amino-acids consumed. A similar phenomenon will be observed too in

other varieties of yeast: e.g. champagne yeast or wine yeast forms plenty

THE ASSIMILATION AND FORMATION OF AMINO-ACIDS. 281

of fusel cil, the consumption of amino-acids in this case being half that
of the bottom beer yeast, which produces the least quantity of fusel oil.
As to the cause of this inversion, we could not make it out; but perhaps it
may be due to formation of a comparatively larger quantity of some
intermediate products, such as addelyde or some derivatives of amylalco-
hol, such as esters; or to lesser assimilability of leucine as compared with
the other amino-acids, or to the assimilability of leucine as such without

the formation of fusel oil (A. 29.) At present we are not in a position

to clear up this point.

Conclusion.

I. The assimilability of amino-acids differs widely according to the
varieties of yeast, and we must select, especially in saké-brewing, such
yeast as consumes the greatest quantity of amino-acids and produces the
least quantity of fusel oil.

II. In the cultures of certain varieties of saké-yeast and Torula
(red var.), there was an in increase of amino-acids inspite of the forrna-
tion of fusel oil.

TH. The quantity of acids formed in the culture has no relation to

the other products.

——

On the Formation of Fusel oil by Saké Yeast.
BY

K. Kurono.

The formation of fusel oil during alcoholic fermentation has long
been the subject of discussion, both from the chemical and the biological
point of view but a satisfactory solution has only been given by the reent
investigation of Felix Ehrlich’. According to him, fusel oil is chiefly
formed from leucine, by the action of yeast, the chemical change being
expressed in the following general equation.

R. CHNH,COOH+H,0=R. CH,0H+CO,+NH,

Thus from d, | and inactive leucine, the corresponding amyl-alcohol
i.e. fusel oil may be formed. The ammonia produced in this process will
serve directly for the formation of proteins in the new cells. Although
Ehrlich’s view has been repeatedly confirmed by various authors and many
works have been carried out along this line, yet there remain still many
questions to be settled. Thus for instance, different kinds of yeast must
produce different amounts of fusel oil under the same condition, and more-
over there is no doubt that various factors exert great influence upon the
production of fusel oil. All these points ought to be studied thoroughly.

As Japanese saké is especially rich in fusel oil and its elimination

is one of the most important questions for the brewers, I have repeated

1. Berichte, d. Deutsch. Chem. Ges. 1906, 39, 4072, ard
» ” ” ” » 1907, 40, 1027

284 K. KURONO:

the experiments of Ehrlich with saké yeast” under different conditions
with the view of reducing the formation of fusel oil to the minimum, and
of finally applying the method on a large scale for practical purposes.

I. Experiment.

(1) 200 g. sugar were dissolved in 2 liters distilled water and put
in a capacious flask stoppered with cotton plugs. After re-
peated sterilizations, the solution was inoculated with 4 g. saké
yeast and left for fermentation at the ordinary temperature
(20-30°) for 20 days. When the fermentation was finished,

the liquid was filtered and analysed. The results was as

follows—
Alcohol ie eee oe ee 3.62%
WTS OU Me ee ee we eae te Oe he trace.

Total acids (ealeulated as succinic acid)... 0.0084

Non-volatile acids ....... ae eee 0.0024

Super ets seston." present (by Molish reaction)
Aniiniguemestr: oe ccs’. SSR en eee absent.

(2) 200 2. sugar and 4 g, leucine were dissolved in 2 liters water, .
sterilized and inoculated with 4 g. yeast. The treatment was
exactly the same as in experiment (1). After 25 days, when

the fermentation was finished the liquid was filtered and analyz-

ed.

AlepHol® Potash! tose She Ree. 5.274
Wiisel Voie. pete fs sale lore merercerear 0.125¢
Totaliaada teers ss 4. eee Recto 00844

2. The sake yeast used in this experiment was a pure culture of (A) No, 25 in k@ji-
extract. The sediment of the yeast was collected on a sterilized filter, and after washing
several times with sterilized water was pressed between the sterilized filter and immediately
added to the solutivns to be tested. ‘The fresh pressed yeast prepared in this way contained
29.9622 dry matter of which 11.9024 was nitrogen. Sugar wss also previously tested for

ammonia and nitric acid,

(4)

(5)

ON THE FORMATION OF FUSEL OIL BY SAKE YEAST. 285

Non-vOlatieracids: es ene sass Have sede OL0051%
Wolatilep acids <czaenersractsie eke varchar eae 0.00414
Susana OGIO ipo Dip con OTA RIED Btroharo ae present.
ANUANITVOTU DMs es tee) Aue nitae sears buses tes Sr oe absent.

100 @. sugar and 2.15 e. hydrochlorate of elutaminie acid were
dissolved in 2 liters water, neutralized with caustie soda,
sterilized and inoculated with 29 yeast, kept for 20 days and

analysed.

AU CONOME aem cme heater ke 5.87%
lawsail Gill gascccoc Sino ORANG Ree ee trace,
Aro pala cide meee nce or emer te tr svare.escetcuste 0.01642
INOnEVOolatilenacide eee eee Ae. ee re 0.0124¢
Wolatilesacid 2 ash. 0-0 co RA es Sanne 0.00514
MOAT Wee ene a eletee ee oss Ae absent.
UMiTOWNT, onagacdgbod ome Sb xcyO nS Oo absent.

200 @, sugar and 4 9. aspartie acid, were dissolved in 2 liters
water, neutralized with caustic soda, sterilized and inoculated

with 4 ¢ yeast, kept for 20 days and analysed.

INigolMWoll oosnceor ac eens 46, b DADA CRORE er arco 6.389%
HOSOI Only secta sy ey Sere IOC a hone: wie lo Ghia guarews 0.0614
Moi Bek sesegcex 5 Oona 6 io CHRONO TOR 0,0128%
INon=volauilem acidsieermimmmciemice « stn de eal « 0.00694
Wollehinlle, evenek) 6 uo 0506 Brees es Meas Avensietens . 0.00754
UCase ere canoe: J oerd conor fae .... absent.
JNU, caclnco oo none present in a small quantity.

100 g. sugar and 2 @. elycocoll were dissolved in 1 litre water,
inoculated with 2 @. yeast and kept for 20 days.

IKON! Gabo oe 3 dtio bon OO ao. C Renn oor 5.33%

GHISS MOU. peyse teil RRSP, at's Sask a Outs Gre
MROGHIPACIGSine FO veetaeine «cae ones MOLOUGER

WNon-volatile acids’ -. +452. eee eee 0.00624
Volatile: acids: So scar os.n seis a he ore 0.01304
SUGAR’ © o6sncse eres wets present in a small quantity.
AINMIONIA, 25.4.0 sisishe cece uber aera eee trace

(6) 100 g. sugar and 1.7 g. of the decomposition products! of rice
protein were dissolved in 1 liter water, inoculated with 2 g. yeast
and kept for 20 days.

Alcohol sc. Sere oes a eiee Pt ee 5.28%
Basel oils tasveeerenee sees sneha viele oleteeeveiete . 0.0284
Total *acvdsiecppisteie a wears cle eats ewe 0.0221%
Non-volatile acids .......... oharareheeeinre Ke . 0.0098%
Wolailemacrdsiee cies Petes aR a3 0.01574
DUPAL vac cua smi re : 0 nie cial sao eee eo ... absent.
ATPNIGUAR) cs kocenieen ela «, « si% present in a small quantity.

(7) 100 g. sugar and 0.75 g. tyrosine were dissolved in 1 liter water, |

inoculated with 2 g. yeast and kept for 20 days.

All Cobol teeters eseters) si ons= ston sete Reece at 3.46%
Mase) soil Seer. geerewesswese) «3\cio oy one eyeaeateeeae spoleys) spa URAC.
More cratels| sc, Senn EIA Atos Ecotec os 0.00734
Non-volatile acids ........... chante es ....- 0.00484
Volatile acids ..... MOIS oe reo 0.00324
Sugar. ap eeemiatess fee cater aA en ... much.
PATE WA, SRG ORO Disord 6 USK trace

(S) 50g. sugar, 1 g. leucine and 1 g.ammonium carbonate were dis-
solved in 500 ec. water, inoculated with 1 g. yeast and kept
for 18 days.

1. These decomposition products, were obtained by boiling 100g rice protein, prepared
with Ritthausen’s method, in 300 c.c. of 2594 sulphuric acid for 40 hours. After cooling, the
iquid was diluted with water and the sulphuric acid still remaining was carefully removed
with baryia, filtered and evaporated in vacuum nearly to dryness, and then dried over sulphuric
acid,

(9)

(10)

(11)

ON THE FORMATION OF FUSEL OIL BY SAKE YEAST. 287

Mkeginll scocoovcceonssoeencdcccabenube 6.444
Thtase Leola ces Meeker ee opevel cate ailo cco 1z cise te Seerey cleats 0.153%
Aol Grol yn2 5.6 oat On he DOD OLS C trace.
Woasyoleitile AEC scocanaconooegcds00006 trace.
Volatilemag@eomia accion «cou so crs acleitieters trace.
SUE ginlo ao sida. 0 On 6 Uno a kD BIO Oia absent.
INTMIAGINIE ooacosdgcngscpoosbnsonascdacc present.

50 g. sugar 1 g. leucine and 1 g. ammonium phosphate were dis-
solved in 500 c.c. water, inoculated with 1 g. yeast and kept

for 18 days.

Aeoholly -kyrenenererren: PRET svehatsiaretshsis's .. 6.444
Wine Oil cocaccavsooddsoosoqdoG GD OUOOS trace.
otal ACidsisaca chesnut reiers Grereren 3 Sepa estes 0.00484
Non-volatile acids ...... Me ayerscc ete aleve 0.0040%
Wolkinile AGG o50005000ne00000000000000 0.0011%
SiUTachos ma cipro ce Bola nrn ons Be ous s/ siekesishe absent.
AMANO 5005 c00cc0avdasa00000000050006 present.

50 @. sugar, 1 g. leucine and 1 g. ammonium citrate were dis-
solved in 500 ec. water, inoculated with 1 g. yeast and kept
for 18 days.

INO Soomas nwo tooo oan Seuameteuanerals 7.084%
Huselloilerocn ce crceurer 5 0 OOo tho pamron 0.1944
WNowl AWICK coonooaccancobcDOoDUOO DO GNO 0.0097%
INGasvoleinikey Bek! Sog0000¢ soeeao ea OUe Ode trace
Wolatilowaciclsiemyrtemeiieireir. 41: iss acasiersuoe 0.1244
SUP Ae ur slor he erent teenies: Ba aeici ep asenenc absent.
AMMO), odooocsoads0e0b SEB RHO OOUDOUde present.

50 2, sugar, 1 g. leucine and 1 g. ammonium tartarate were
dissolved in 500 e.c. water, inoculated with 1 g. yeast and left

for fermentation for 18 days.

288 K. KURONO:

Wicohol: 2.2 F oonc.c.arce ne eee ee 5.15%
Musel oilers. e caeever ee As cig wiley
Total acids: «250.6 Cass ' sense ae eee 0.00114
Nonsvolatile acids: , 5. ss0.6 28 eee ee eee 0.00624
Wolatile, acids: 2 scace, gee mies eae 0.0062¢
aren at eee ee ee vis, gaeduathen 3.802. absent.
AN gar OTL A ove cpveen aprons 2 chatet st choyalehs eaten te ae present.

For the sake of clearness the above results may be presented in a

tabular view, as follows:

alcohol | fusel oil | total acids volatile | non-volatile

No. N= b! i aci
No N-compounds % % % oa one
feo feo
1s 2 ees 3-G2 trace 0.0084 0.0076 0.0024
2 leucine 5.27 0.125 0.0084 0.0041 0.0051
3 glutaminic acid 5.87 trace 0.0164 0.0051 0.0124
4 aspartic acid 6.39 0.061 0.0128 0.0075 0 0069
5 glycocull 5.33 0.136 0.0164 0.0130 0.0062
decomposition products ca 2 = a
6 of rice protein, 5 28 0.028 0.0221 0.0157 0.0098
7 tyrosine 3.46 trace 0.0073 0 0032 0.0043
leucine + =
8 aimoniinilca bonnte 6.44 0,053 trace trace trace
leucine +
9 ammonium phosphate 6.44 trace 0.0043 0.0011 0.0004
leucine + > . - F
10 Sa aninmacioate 7-08 0.194 0.0097 0.0124 trace
11 aioe 5.15 | 0.126 | 0.0011 | 0.0062

ammonium tartarate | 0.0062

We see from the above results, that either in a pure sugar solution
(1) or in a solution containing only tyrosine (7) or glutaminie acid (3),
doubtful traces of fusel oi] were produced; while in a solution containing
leucine (2) 0.125% of it was found. This evidently proves the correctness
of Ehrlich’s view. An exception was found in the glyeocoll containing so-

lution (5), which was found to contain 0,136¢ of fusel oil with Rosr’s

ON THE FORMATION OF FUSEL OIL BY SAKE YEAST. 289

method. But this apparent exception was afterwards found to be due to a
defect of Résr’s method. The substance which behaves same as fusel oil to
chloroform was proved to be ethyl-acatate. The latter is easily distingnish-
ed from fusel oil by its giving a rose red colouration with anisaldehyde and
concentrated sulphurie acid, while fisel oil gives a violet colouration. To
make the proof more complete, the distillate of the culture of the glycocoll
solution containing ethyl-acetate was saponified by boiling with dilute caus-
tic kali in a reverted cooler for 4 hours, and was distilled after acidifying
with sulphuric acid. The distillate was neutralized with baryta and eva-
porated. The barium salts were proved to be acetates mixed with a little
butyrate. Dr. Suretx? has shown that many substances, such as, ethylace-
tate, amylacetate, aldehydes, furfurol and some higher alcohols are ab-
sorbed by chloroform and thus bring about the inaccuracy of Rosr’s me-
thod. In the glycocoll solution (5) T have tried to find out methyl-alechol,
aldehyde and furfurol, but the results were negative.

Ammonium carbonate (8) and phosphate (9), when added to the
leucine solution caused more or Jess diminution in the formation of fusel
oil, while ammonium tartarate (11) and citrate (10) had apparently no
such effect. As this deserves further consideration further exeriments

were carried out more fully.

II. Experiment.

In the following experiments, the formation of fusel oil from leucine
under different conditions was especially kept in view,

(1) 50g. sugar and 2 g. leucine were dissolved in 500 c.c. distilled

water, inoculated with 6 g@. yeast and kept for 5 days at 30° C.

(2) 50 g. sugar without addition of leucine, were dissolved in 500

e.c. water, inoculated with 6 g. yeast and kept for 5 days at

30° C.

1, Dr, E. Sell: iiber Brantwein (Berlin, 1888) S. 35.

290 K. KURONO:

The fermented fluids were analysed with the follwing results :—

Alcohol Fusel oil
6.97 % 0057 %
4.67 trace

The total quantity of fusel oil contained in solution 1) was calculated
to be 0.287 g. corresponding to 0.292 g. leucine, that is only 154 of the
leucine originally added. In the fermental liquid 0.0755 g. N was
still found to be present in the filtrate of phosphotungstie acid-precipitate,
which includes the whole protein substances together with organic bases.
If we suppose that this nitrogen belongs entirely to leucine, then it corre-
sponds to 0.706 g. We may, therefore, conclude that nearly one half of
leucine has been split up into other compounds or used up for the formation

of yeast cells.

III. Experiment.

500 ¢.c. well water, 10 ¢.c. Hayduck’s mineral solution and 50 g.
sugar, each were put into 4 Pasteur’s flasks and further to flask (1) 2 g.
Leucine, to (2) 1.007 g. ammonium phosphate (Equivalent to 2 g. leucine),
to (2) 0.733 g. ammoninm carbonate and to (4) 1.145 g. glycocoll were
added. They were sterilized and inoculated with a minute quantity of
saké yeast and kept at the ordinary temperature (20-30°) for 2 months
until the fermentation was complete and much sediment of yeast was
formed at the bottom. The liquid was then filtered and analysed, with

the following result :—

ON THE FORMATION OF FUSEL OIL BY SAKE YEAST, 291

Rk eee ee SO nan a

No. Fusel oil 94 (Rose’s method)
1 leucine 0.279
2 ammonium phosphate 0.138
3 5 carbonate 0.098
4 glycocoll 0.242

VI. Experiments to prevent the formation of fusel oil.

Since there is no doubt from the above experiments, that fusel oil
is chiefly derived from leucine during the fermentation process and further
its production is more or less diminished by the addition of ammonium
salt to the fermenting: liquid, 1 proceeded one step further to determine
the conditions, necessary to reduce it to the minimum. This is especially
important from the practical point of view, because saké usually contains
a more fusel oil than beer or wine. For example, the samples of supe-
rior qualities examined by myself contained about 0.05-0.1% and in those
of inferior qualities sometimes as much as 0.4% of it. This higher per-
centage of fusel oil in saké is most probably due to the comparative rich-
ness of the molecules of rice protein in leucine, as shown by U.t Suzuki

and others of this laboratory.

(1) 100. sugar, 1 @, leucine and 20 ¢.c. Wayduck’s mineral solution
were dissolved in 1 liter of water. The solution thus pre-

pared was equally divided and placed in 10 flasks, and to each

]. In this case the apparent increase of fusel oil in the glycocoll solution (4) was due to
the formation of ethylacetate. It was at once recognized by its characteristic agreable smell
and by the rose red colouration with anisaldehyde and concentrated sulphuric acid. So T am
sure that the decomposition products of glycocoll by sake yeast are chiefly acetic acid and
acetic ester.

2. U, Suzuki and others— this Journal; Vol. I., No. I.

bo
le)
bo

K. KURONO:

flask was added ammonium phosphate in varying quantities.
After sterilizing and inoculating with saké yeast, they were
left for fermentation at 25° for 2 weeks, when the liquid was

filtered and analysed.

No leucine added Sag Piagles Yeast Amylalcohol *

1 O01, % Q not added —
z ” 0 added Spar ae
3 r 0.05 ” crasta
4 ” 0.1 ’ ae
5 . 0.2 ” =
( , 0.3 oe +
if ” 0.4 ’ ote
8 ” 0.5 ' + 4
9 55 0.6 a +++

10 ” 0.7 5 +44

We see from this result that fusel oil gradually decreases with the
increased addition of ammonium phosphate to the solution and reaches
its minimum when the latter becomes 2-3 times of leucine, and again gra-
dually increases with the inerease of the latter.

(2) The same experiment was repeated.

1. Amyl-a’cohol was tested colourimetrically with anisaldehyde and concentrated sul-
phuric acid.

— means no reaction, <b trace, + presence of amylalcohol,

ON THE FORMATION OF FUSEL OIL BY SAKE YEAST. 293

No. L2G added amm. DEBIAN Veast Banylatca el
Ya fe |
—— — = =
1 0.1 0 no added =
2 | a 0 | added ae
3 ” 0.05 | .. fb
4 9 0.1 | ” +
5) Ay 0.2 > +
6 - 0.3 +
itm | 04 =
8 | 0.5 i
9 | - 0.7 or ih
10 » 0.9 : 4
ll | _ 15 | - we
12 5) 2,0 | 7 0
13 ; 3.0 | F 0
l4 > 0.1 | 9 —
15 ae 0.2 | : =
16 ” 0.5 as at
17 BS 1.5 | % ae

In No. 12 and 13 the growth of yeast was absolutely inhibited.

We see in this case also that fusel oil is reduced to the minimum
when 2-3 times the ammonium phosphate of leucine were added. We see
also that with ammonium phosphate alone, without leucine, a moderate

quantity of fusel oil is produced. But if this salt reaches as high as 2%

of the liquid then the growth of the yeast comes to a still-stand.

Summary.

1. Fusel oil is formed in saké chiefly from leucine, which is a decom-

position product of rice protein.

294 K. KURONO:

The formation of fusel oil is diminished in some degree by the
by the addition of ammonium carbonate or ammonium phosphate
to the fermenting liquid. The best proportion between these
ammonium salts and leucine was found to be 2-3:1. The excess
of ammonium phosphate not only increases the fusel oil but also
prevents the propagation of yeast cells... Both ammonium citrate
and tartarate are useless for this purpose.

3. Glycocoll' seems to favour the formation of acetic acid and acetic

ester in the fermenting liquid.

Tn conclusion, I wish to express my thanks to Prof. Dr. U. Suzvxr
and Prof. Dr. Taxanasut for the valuable suggestions and advices re-
ceived during the progress of my work.

This paper was already published in Japanese in the Journal of

the Tokyo Chemical Society (1910, Vol. 31, No. 2.)

1. J. Effront also reported that acetic acid is produced from glycocoll and betein by

fermentation (Zeitsch f. Spirit. indus. 1909, 32, 237).

On the Asparagine-splitting Enzyme in Yeast.
BY

K. Kurono.

The question, whether the formation of fusel oil from leucine is due
to the action of a certain enzyme, is not yet settled. Although Enrrrcn?
supposed the existence of such an enzyme, he could not prove it and
neither in the ““Pressarr’? nor in the “Acrronr Dauer hefe”*, has its
presence been shown yet. J. Errronr* has recently found an enzyme
which liberates ammonia from asparagine, and he is inclined to atribute
to this enzyme the formation of fusel oil. But as he did not isolate this
enzyme in a pure state and further, as it acts only in alkaline solution,
while the ordinary fermentation process takes place in a slightly acid fluid,
his assumption can not be accepted without further investigation. The
isolation of this enzyme from beer yeast as well as from saké yeast, and a

fuller examination of its behavior are the subjects of this paper.

Experiment I.

500 g. well washed bottom yeast were distributed in 1 liter distilled
water, and 50 ¢.c. normal caustic soda was added to it. To prevent bac-
terial growth the mixture was covered with enough toluol. After keep-

ing for 2 days in a thermostat at 87°, it was filtered, The clear filtrate

1. Berichte, d. Deutsch. Chem. Ges. 1907, Bd. 40, S. 1027.
” ” » » » 1906, Bd. 39, S. 4072.
2. E. Buchner u. J. Meissenheimer; Berichte, d. Deutsch, Chem Ges. 1906, Bd. 39, S. 3713
3. Haus Pringsheim; Berichte, d. Deutsch. Chem. Ges. 1906, Bd. 39, S. 3713.
4. Comptes Rendus, 1908, CXLVI, 779.

296 K. KURONO:

was then mixed with an equal volume of 97% alcohol, and the flocky pre-
cipitate obtained was collected on a filter and washed with dilute alcohol
and either. About 4 g. crude enzyme was thus obtained, and the following

experiments were carried out with it.

(a) 100 ec. of 2% asparagine solution was put in a flask of 200 e.c.
capacity. 1 ec. of normal caustic soda and 2 g. (erude en-
zyme) were added to it. The flask was kept in a thermostat
at 27° ©, with the addition of enough toluol to prevent putre-
faction. At the outset of the experiment a portion was taken
out for ammonia deterinination', while the remaining portions

were taken out after 28 and 72 hours respectively.

(b) For control, 2 g. ppt were dissolved in 100 ee. water without

addition of asparagine, and treated in exactly the same way

as before.
NH,—nitrogen. 9%
Time
(a) (b) ;
Asparagine solution Control solution
ACCME GUISE uc coc nce Bom eon 0.000 6.000
After 28 hours fs Salle ect dace nee P 0-056 a
After 72 hours oe ie eee 0.090 0.001

A great difference is thus seen between the control and the aspa-
ragine solution as to the production of ammonia. Nursstur’s test also re-

veals this difference at once.

Experiment II.

300 g. beer yeast were rubbed with some quartz sand in an iron mor-

1. The method used was a qualified vacuum distillation method with finely powdered

magnesia usta,

ca |

ON TITE ASPARGINE-SPLITTING ENZYME IN YEAST, 99

tar, and macerated with 1 liter distilled water. After 2 hours it was

filtered. The white precipitae obtained by the addition of 97% alcohol

was collected on a filter, and washed with absolute aleohol and ether.

About 5 g. air dry erude enzyme were thus obtained. Jt was divided
into 6 parts and the following experiments were carried out.

(A) 2g. asparagine, 0.8 2, erude enzyme and 1.5 ¢.c. normal caustic

soda were added to 150 ee. distilled water and covered with

toluol. After keeping in a thermostat at 37° C, ammona test

was made with the following results.

EL ATMG? PAGERS Tye rae erat oer sein cee: Sie ak NH.—N¢
Atethewoutcet me mctit eek ck eiotstsse mine es 0.000
PACE teatnc DOME LG mb nme ra seem RP san a oiler aa 0.112

(B) The same solution as (A) was boiled for 5 minutes. the treat-

ment being otherwise the same.

EDITNIG® rs RES OR oe ROE aston aoe nates NH.—N@
At the woulsetinces cin cee tok ate vise eevee ee es trace.
IAS GerieO QUA S8 a cust CR WEPRIM Eso co. 215 ice, ise Oi 0.011

(C) 2¢. asparagine, 0.8 g. ernde enzyme were dissolved in 150 c.e.
of water, and further as much tartarie acid was.added as was
necessary to make it 0.1% of the solution, the treatment being

otherwise the same as hefore.

Rime? Meee eee Cr eee tes mares NH.—N¢
A EtheMOULSeM ee Meio nes oie cS Ga ae 8 0.000
AH teTn (92 Mise ee een ane ecto etocsin sclale os Sidon 0.117

(D) 0.5 @ leucine, 0.8 &. ernde enzyme and 1.5 e.c. normal caustic

soda were added to 150 e.c. water and treated exactly as before.

ol Lilioa Tara eP OnE CLCKORTE: 6 p> ch oxc.lons 0. Lia Reena ea NH.—N¢Z
Nie binet OULSeU EA Ea eee. cas tical aieie 0.000
Niter OO ns. ies Apacer yee se « «all ees trace.

(FE) In this ease the caustic soda of the previous solution (D) was

replaced with tartaric acid (0.1% of the solution).

298 K. KURONO:

Timené des cartnndedbase: ee eee NH,—N@%
At, the woultset.t.ncd-eet aac re eee 0.000
After: 99 hats. ise diied eee see trace.

(F) 1 g. urea, 0.8 g. crude enzyme and 1.5 c.c. normal caustic

soda were added in 150 c.c. water.

Pima» 2c. ese eRe Ae ee ee NH,—N@
Ati-thesontseis Bite. os ht ee See ee 0.000
ATtter “OS thse oh. A. 6 er ee eee trace.

The above experiments prove beyond doubt, that an enzyme which
decomposes asparagine with the liberation of ammonia. can be easily
isolated, and that it acts not only in alkaline, but also and even more
energetically in acid solutions. Further its action is confined to aspara-
gine, but leucine and urea being never affected by it.

The writer repeated the same experiments with saké yeast and obtain-

ed the same results.

Experiment ITT.

30 gr. of saké yeast were rubbed with quartz sand in a mortar, mace-
rated with 300 e.e. of distilled water and filtered. The filtrate was dilut-
ed to 300 e.e. and divided into six equal portions, each of which was put
in a small flask of ca. 100 ec. capacity. They were subjected to the
same conditions as in the foregoing experiments.

The special additions consisted in:

(A) 0.5 gr. asparagine,

(B) 0.5 gr. asparagine after boiling.

(E) 0.2 gr. leneine, acidulated with tartaric acid,
(F) 0.5 gr. urea,

ON THE ASPARAGINE-SPILITTING ENZYME IN YEAST. 299

The determination of ammonia gave the following results:

Ammoniacal N, %

Series Nessler’s reaction
at the outset. after 48hs.
A 0.000 0.114 very strong reaction with reddish brown
precipitates,
trace * 0.059 strong reaction.
0.000 0.112 very strong reaction with reddish brown
precipitate.
0.000 0.000 no reaction
E 0.000 0.000 no reaction
0.000 0.000 no reaction

Thus the presence of an asparagine-splitting enzyme in saké yeast is

proved.

Experiment IV.

300 gr. of beer yeast were rubbed, macerated and filtered as in ex-
periment IT. From this filtrate about 5 gr. of ernde enzyme were obtain-
ed. It was again dissolved in 300 e.c. water, filtered and diluted to 500
and then divided into five equal portions, each of which was put in a small
flask of ca. 100 ¢.c. capacity. They were placed in the same conditions.
as in the foregoing experiments.

The special addition consisted in:
(A) 1 gr. asparagine,
1 gr. urea,

* This exception is perhaps due to the too short boiling, in consequence of which the

enzyme was not yet completely destroyed,

300 z K. KURONO:

(D)

1 gr. formamide, ~ &
(E) 1 gr. butylamide.

The determination of ammonia gave the following results:

Ammoniacal N, %

Series Nessler’s reaction
at the outset after 48 hs.

A 0.000 0.129 very strong reaction with reddish brown
precipitate.

B 0.000 0.000 no reaction.

(65 0.000 0.000 no reaction.

D 0.000 trace weak reaction.

E 0.000 0.000 no reaction.

Thus, it is proved that the action of this enzyme is confined to aspa-

ragine,

a Conclusion.

The presence of an enzyme which liberates ammonia from aspara-
gine is proved in saké as well as in beer yeast. This enzyme can be
extracted with water or with a dilute alkaline solution and acts equally
well both in acid and alkaline reactions.

As its action is confined to asparagine and is totally inefficient towards
leucine, urea ete., it has probably nothing to do with the formation of fusel

oil during aleoholie fermentation.

Studies on the Butyric Acid forming Bacillus of
‘‘Saké-Moromi.”

K. Kurono.

With Plates XTIT and XIV.

Although many investigations have been carried on on the microor-
ganisms of “Moromi,” the réle of the butyric acid bacillus still remains to
be made out. Moreover, there is the notable fact that the characteristic
agreeable odour of butyric ester is usually recognized in the “Takaawa’”’—
“Hochkrausen” stage. These considerations have induced me to under-
take the present study.

The samples,t which were taken from various stages of “Moromi”
fermentation, were boiled at 100°C for 15 minutes in the culture
medium,” and sealed up in Buchner’s tubes for anaérobic culture. They
were kept for three days in an incubater at 50°C.

From these cultures,* three varieties of butyric acid bacilli have

been obtained and are described below.

No. I.—Bacillus butyricus aromafaciensis moromt I.

The principal characteristic of this bacillus is that it produces

an agreeable odour of butyric ester in cultures containing alcohol. We
can distinguish two varieties:
I. Form and Size.—Facultative anaérobe; commonly slender rods

1. The samples were taken from a “ Sakura-masamune” brewery in 1908 and 1909.

2. As culture media for the isolation of bacteria was used bouillon-glucose-agar with
the addition of enough precipitat@] calcium carborate.

3. A thin greyish-white film was usually furmed in the cultures made from samples

taken before the “ Takaawa,” but not in those taken later.

302 K. KURONO:

with slightly rounded extremities; 2 to4 y» long by 0,8 to 1.2 » thick;
in old cultures (moromi-glucose agar after 5 months) long oval, 2 pz
long by 1 # thick. Involution forms have never been observed, even in

cultures over 4 months old.

If. Flagella and mobility Many flagella; actively mobile.

III. Staining reaction.—Stains with Gram’s method, but not with
methylen-blue or I+KI—solution. Granules found neither in aerobic

or anaérobie cultures (starch-bouillon-agar, glucose-bouillon-agar ete.)

IV.—Growth: 1. Solid culture:

a. Plate culture. — Bouillon-glucose-agar, bouillon-starch-agar,
koji-agar: flat, round, light greyish, waxy, slimy colonies of
2-3 m,m. diameter, appear on surface after 4 days at 30°C.
Internally the colonies present a finely granular appearance and
the periphery is perfectly even when seen under a magnification
of 120 diameters.

Frequently large colonies of even 14 inch diameter are
formed. They always present a wavy periphery and seem to
spread out along the wet surface. Such a colony obtained after
3 days at 40°C has a thin, dry, somewhat crumpled surface.

b. Surface culture: “Saké”-agar: Forms a thin, dirty greyish
white covering with dry folded surface and quickly growing out
on either side of the track with wavy margin. The condensed
water becomes turbid and covered with greyish white thick
folded film (after 3 days at 25° C). Bouillon-glucose-agar :
Growth just the same as on saké-agar, but is covered sooner
all over the surface (3 days at 25° C),

Koji-agar: Growth the same as on bouillon-glucose-agar,
but the condensed water remain clear (3 days at 25° C).

“Moromi”-agar: Growth quite the same as on bouillon-glu-
cose-agar (3 days at 25° C).

Potato-culture: Forms a dry, greyish white, thick folded

BUTYRIC ACID FORMING BACILLUS OF “SAKE-MOROMI.” 303

covering and spreads quickly all over the surface (3 days at
25° C).

e. Stab culture: Koji-agar: Forms a greyish brown wrinkled
colony spreading quickly over the surface, and forms thready
growth with twisted margin along the stab canal (3 days at 25°
C). Bouillon-starch-agar: Cultures similar in character to the
preceding.

Bouillon-glucose-gelatine: Forms a greyish white thready
growth with twisted margin, diminishing towards the bottoms
of the stab canal. First stage thin greyish white smooth pellicle
produced at the mouth of the stab canal with dish shaped de-
pression; later stage, the depressed part becomes liquefied and
shaped like a crater, the liquid being turbid; final stage, the
liquefastion proceeds downwards in the form of a cylinder‘,
with greyish brown wrinkled pellicle on the surface of the liquid
(at 15—20° C).

2. Fluid culture: (4 days at 35 C°) Neutral “koji”
extract: Forms a greyish white large wrinkled pellicle, the
fluid remaining clear; sediment formed; the pellicle is easily
scattered in the form of clouds by shaking, but the ring remains
unaltered.

Glucose-bouillon: Forms a thick, substantial folded film with some

deposits, fluid turbid; the film not seattered by shaking.

Starch-bouillon: Forms a white, somewhat shiny film with some

deposits, fluid turbid; the film seatters on shaking.

Yeast-water: Forms a white folded film. Fluid clear. No sedi-
ment.

Yeast-water glucose: Forms a white folded film. Fluid clear. No

sediment.

4. After 10 days the liquefaction proceeded half way down the thickness of the medium,

the liquefying process being most energetic of the three varieties dealt with in this paper.

304 K. KURONO:

Hayduck’s solution: Forms a thin brittle film with some deposits.
fluid turbid; in a short time the film sinks and breaks up with-
out leaving any ring.

Mayer’s solution: Just the same as above, with the only difference
that the ring remains.

Pfeffer’s solution: No growth.

Nigeli’s solution: No growth.

Milk: Is not coagulated. The growth is very difficult to observe,

as no film is formed.

V. Behavior towards carbohydrates, glycerine and calcium lactate.
The production of acid from these substances was tested with yeast water
cultures containing one of these substances’. After 10 days at 35° C,

volatile acid was determined by the steam distillation method,

Substance Volatile acid production
Glucose: ©. Eee ee. ne tHE ore we 0159
Toulin. ia: Stel Maco Seat eter 5252: bets +444
Saccharos€: c60 ps =sr ernie ames) fo6 aca +4++
Starch” “sc; .o.2y esse ee Be ees. se ee eee See nec:
Rhamnese 1.27 SeP Pi, eee esses ase ++
Maltose i plasa --0 se Rae ca) oes) |e +
Mannose Pg Cet SS loon Sener He
Mannil’ 222, ysc.inccotn een eetie ices: see (at +

Methyl glucoside 337 v-wie-mitemiece <5 oo -
Fructose Be rh se Sc) os, OE A -
Oe ES ec eee =
Melitose .— / ;*. 5 ssc tn Pees san" ane ++
Glycerine" "ic See wean eeetear s= sr Mas —
Calciumlactate)) Sao .s os enetenemeen- exe) sos =

Thus this bacillus forms quite a quantity of volatile acid from staerh
glucose end inulin, and traces of it from rhamnose, maltose and melitose.
The growth is almost the same in all these fluids i.e. a greyish folded

film is formed, accompanied by sediment and the fluid becoming turbid.

5. A 2 9 solution of each substance was used.

BUTYRIC ACID FORMING BACILLUS OF “SAKE-MOROMI.” 305

VI. Behavior towards nitrogenous compounds: This was observed
with various fluids prepared by adding to Hayduck’s solution various
nitrogenous compounds in place of the asparagin. Tests for aspartic acid,
asparagin, leucin, arginin, glutamic acid, eystin, glycocoll, tyrosin, his-
tidin, alanin, ammonium carbonate and potassium nitrate, showed that
if due attention was paid for acidity, nitrogen was assimilable in amino-
ammonia- and nitrate forms.

VII. Behavior towards acids: This bacillus can grow in “koji’-
extracts having an acidity less than 0.0894 as lactic acil, but not in 0.13%;
but, when we add pure lactic acid to the neutral “koji”-extract, it can
grow when the acidity is less than 0.054.

VIII. Behavior towards antiseptics. Only salicylic acid was
tested. 0.01% of it in “koji”-extract inhibits the growth completely, but
0.004% does not.

IX. Behavior towards alcohol. Less than 6.25 vol. of ethyl-al-
cohol in “koji”-extract does not inhibit the growth. Further, the agree-
able odour of ethyl-ester is naturally noticeable in cultures of this bacillus
when ethyl alcohol is present.

X. Although aroma-formation was tested with various alcohols",
only ethyl aleohol gaye a positive result.

XI. Fermentation products. Traces of aldehyde, ammonia, ethyl
alcohol and fusel-oil are found in the distillate of “koji’’-extract-culture
after 10 days at 25° ©, but methyl-aleohol, furfurol, acetone, acrolein
indol and hydrogen sulphide are not present. Butyrie acid is an essential
component of volatile acids, and a trace of acetic acid is found but formic

acid and propionic acid are not present.

XII. Gas formation. No formation of gas.

6. Ethyl-alcohol, methyl-alcohol, normalpropyl-alcohol, isopropyl-alcohol, normalbutyl
alcohol, isobutyl-alcohol, cinnamylic-alcohol, amyl-alcohol, heptylic-alcohol, cuminic alcohol,

and caprylic alcohol were tested by adding traces cf them to Hayduck’s solution.

306 K. KURONO:

XLII. Conditions of temperature: Optimum temperature for
grotwh lies 30—40° C. Grows very slightly below 13°C. Boiling for 2
hours in distilled water does not kill the spores of this bacillus, which,
however, are completely killed by boiling for 3 hours.

XIV. Spore formation: Long oval spores are formed after 2 days
at 40° C. medaly situated. Starch-houillon and “koji’’-agar were most
favourable for spore formation.

XV. Symbiotic culture of “saké’’-yeast and this bacillus: These
2 microorganisms were added to “koji’’-extract and kept at 30° C; after
2 days enough growth of the bacillus took place to produce the odour of
butyric acid, but the veast erew slightly; after 4 more days the yeast grew
tolerably well, but the emission of the agreeable odour of butyric ester was

replaced by the bad smell of butyric acid.

No. 2. Bacillus butyricus aromafaciensis moromi II.

This variety differs from the first in that it does not grow in saké-agar,
Tt however grows in Nigeli’s solution, while the first variety does not.

I. Form and size: Almost the same as in the first variety.

II. Flagella: Same as in the first variety.

III. Staining reaction: Same as in the first variety.

IV. Growth: 1. Solid culture:

a. Plate culture: Almost the same as in the first variety.

b. Surface culture: bouillon-glucose-agar, “koji”-agar, “Moromi”-
agar: Grows just the same as the first variety. “Saké”-agar:
No growth. Potato culture: Forms a folded covering, which
is whiter than in the first variety.

e. Stab culture: Koji-agar, bouillon-starch-agar: Almost the same
as in the first variety. Bouillon-glucose-gelatine: Liquefaction
proceeds more slowly than in the first variety (about half as first).

2. Fluid culture:..(4 days at 35° C). Neutral “koji’-

extract; Forms a greyish white wrinkled pellicle, fluid becoming

BUTYRIC ACID FORMING BACILLUS OF “SAKE-MOROMI.” 307

turbid with sediment; this pellicle is easily scattered like clouds
on shaking and no ring remains.

Neutral “Moromi”: Same as above. Bouillon-glucose, starch-
bouillon, yeast-water, and yeast-water-glucose: Grows respectively as in
the first variety.

Hayduck’s solution: Forms a thick, folded, slimy, semi-transparent
film, no deposits, medium slightly turbid; on shaking, this film does not
seatter but sinks down.

Mayer’s solution: Growth same as in the first variety.

Pfeffer’s solution: No growth.

Nageli’s solution: Forms no film; medium slightly turbid with
some white sediment; scatters like powder on shaking.

Milk: Growth same as in the first variety.

VY. Behaviour towards carbohydrates, glycerine and calcium lactate:
The production of volatile acid was determined under the same conditions

as in the first variety with the following results.

Volatile acid production

Substance
First variety Second variety

lucose Jesh the eee
Inulin Secbte de =e
Saccharose... qe araT +++
Starch +4+4+4++ ++4++
Rhamnose ++ 44
Mannose Wy, =
Maltose Prats 4
Mannit + ae
Methyl glucoside + aL
Fructose = —
Lactose = =
Melitose aye ates
Glycerine ... = at
Calcium-lactate <= =e

308 K. KURONO:

On the whole, the acid producing power of this bacillus is less than
in the first variety. Although the largest amount of acid was produced
in the cases of starch and glucose, it amounted to only 0.128 and 0.1134
respectively, as butyric acid. Moreover, the acid proluction from inulin
is far less than in the first variety.

VI. Behavior towards nitrogenous compounds. The results were
analogous to those obtained in the first variety.

VII. Behavior towards acids and alcohol; aroma-formation, gas-
formation, fermentation products, conditions of temperature, spore forma-
tion and symbiotic culture with saké-yeast gave the same results as in the

first variety.

VI. Behavior towards antiseptics: This bacillus is more resis-
tant than the first variety towards salicylic acid, 0.01% of it having no
power to inhibit the growth.

Although numerous butyric bacilli have been described, the present
form cannot be referred to any of them. The distinguishing properties of
this variety are its facultative anaérobiosis, active mobility, and absence of
granules and clostridium. Moreover, this bacillus produces butyric acid
principally from starch, glucose, ete,, but not from milk-sugar or calcium
lactate; further it has no power to ferment or coagulate milk. There is
no doubt that this bacillus is distinct from Bac. butyricus Hiippe, Bae.
Gruber ITT, and other butyrie bacilli deseribed by L. Léffler, A. Weber,
L. Adametz and O. Emmerling, although there are several characteristic

common to them and the new form.

In short, this bacillus may he considered a new species of butyric
hacillus resembling the other genuine butyric bacilli physiologically (i.e.
in producing butyric acid from carbohydrates as starch and glucose) and
standing morphologically nearest to Bac, butyrieus Hiippe, Bac. butyricus
Weber, ete. Moreover, it is characteristic for this bacillus that it produces

the agreeable odour of butyric-ester in the presence of ethyl-aleohol, On

this

BUTYRIC ACID FORMING BACILLUS OF “SAKE-MOROMI.” 309

point Bae. esterificans Massen* shows an analogous aiction, but it

produces ananas aroma in the butter without alcohol.

No. 3. Bacillus butyricus roseus moromi.

I. Form and size: Almost the same as in No. 1 and No. 2.

Il. Elagella: Just the same as in No. 1 and No. 2.

III. Staining reaction: Similar to the foregoing.

IV. Growth: 1. Solid culture:

a. Plate culture: almost the same as in No. 1 and No. 2; but the
colonies produced in the deeper parts of the medium is ball
shaped, with irregular surface and not round and flat as in No.
1 and No. 2; moreover, such colonies after 3 days at 40° C do
not present the dry, slightly crumpled surface of No. 1.

b. Surface culture: ‘“Saké”-agar: no growth as in No, 2. Mo-
romi-agar, bouillon-glucose-agar and “koji’-agar: Forms a
greyish white, wet lustrous covering. It grows quickly, spreading
out on either side of the track and ultimately covering the whole
surface, but never presenting a dry folded surface. The con-
densed water becomes turbid with some white sediment, but no
film is formed (3 days at 25° C).

Potato culture: Forms a wet, lustrous, dark red covering
spreading quickly over the whole surface (3 days at 25° C).

This is a characteristic of this bacillus differentiating it
from No. 1 and No. 2.

e. Stab culture: Koji-agar, bouillon-starch-agar: although it grows

almost like No. 1 and No. 2, it produces never a dry folded
film but a wet, smooth, lustrous one on the surface. Bouillon-glu-
cose-gelatine: growth about the same as in No, 1 and No. 2; but

the liquefaction of gelatine proceeds more slowly than in No, 2,

H. Huss; Cent. f. Bact.; IT. Ab. 569, 1907.

A similar red covering is formed also in rice and bread cultures.

310 K. KURONO:

that is after 10 days. The dish-shaped depression is found only
at the mouth of the stab canal (at 15°-20° C).

2. Fluid culture: (4 days at 35° -C) Neutral “koji”-extract:
Forms a white, semi-transparent, smooth pellicle, the fluid remaining clear.
Pellicle sinks easily but does not scatter on shaking, and a ring remains.

Neutral “moromi”: Same as in koji-extract.

Bouillon-glucose: The medium turns turbid with some cloudy sedi-
ment; the pellicle sinks gradually and a ring remains.

Starch-Bouillon: Forms a white smooth pellicle with a white sedi-
ment in the turbid fluid; the pellicle scatters like clouds on shaking.

Yeast-water: Forms almost the same growth as in bouillon-glucose
culture.

Yeast-water-glucose: Forms almost the same growth as in bouillon-
glucose-culture.

Hayduck’s solution: Forms a thin slimy film, with some deposits,
the fluid becoming slightly turbid; this film does not scatter on shaking

Mayer’s solution: Almost the same growth as in Hayduck’s solution.

But there is a trace of a ring.

Pfeffer’s solution: No growth.

Nageli’s solution: No growth,

Milk: Almost no growth.

VY. Behavior towards carbohydrates, glycerine and calcium lactate:
The phoduction of volatile acid was determinted under the same condi-
tions as in No. 1 and No. 2. The results were as follows:

Volatile acid production

Substance
Bacillus No. 1. Bacillus No. 2. Bacillus No. 3.
Glucose eta tate
Inulin re =
Saccharose estate aha sts
Starch Seeeents ee
Rhamnose es 4

BUTYRIC ACID FORMING BACILLUS OF “SAKE-MOROMI.” 311

Mannosene:.. cf: cit eses Me oa | +
Maltosem es, 8.5, wes eees ++ + ART
IVEaTIN fies seers) eeemnics + a5

Methyl glucoside ... ... == ar

irctosem:;, s/n lyse) oes = = oad
WEACEOS@) ees) cee Cena — = =
MGINEOSCg eon cae -tme=s ++ 3Rer =
Glyceriné=.. <5 seen e=- — — --
Calcium lactate ... ... aS = =

On the whole, the acid producing power of this bacillus is weaker
than in No, 1 or No. 2. Even in the eases of starch and glucose, the acid
produced amounted to only 0.094 and 0.082% respectively, as butyric acid.
It is a characteristic of this bacillus that it does not form any acid from
inulin; and in this it is more like No. 2 than No. 1.

VI. Behavior towards nitrogenous compounds: Results similar
as in No. 1 and No. 2.

VII. Behavior towards acid: This bocillus grows in ‘ koji’-ex-
tracts containing less than 0.042% acid as lactie acid, but not when the
acidity is more than 0.0847. When however pure lactic acid is added to a
neutral “koji’”-extract, it grows while the acidity is less than 0.05 but
not in 0.062.

VIII. Behavior towards antiseptics: Just the same as in No. 1.

TX. Behavior towards alcohol: Its power of resistance towards
aleohol is so weak that it cannot grow in the presence of 3¢ alcohol or
more. It does not produce butyric ester in the presence of alcohol; and
this also distinguishes it from No. 1 and No. 2.

X. The aroma formation from various alcohols and by symbiotic
culture with yeast was tested or this bacillus but with negative results.

XI. Fermentation products: Just the same as in No. 1 and No. 2.

XII. Gas formation: No gas.

XU1I.—Conditions of temperature: Optimum temperature for growth

312 K. KURONO:

is 30-40° C. Grows very slightly below 13° C. Boiling for 114 hours
in distilled water does not kill the spores of this bacillus, which die only
when boiled for 2 hours.

XIV. Spore formation: Same as in No. 1 and No, 2.

Although this bacillus closely resembles No, 1 and No. 2, it can be
distinguished by its property of not forming an ester from ethyl alcohol
and of not causing butyric fermentation in inulin as others do and by its
producing a very characteristic dark red covering in potato culture. Com-
parison with No. 1 and No. 2 leaves no doubt that this is distinct from

any of tre butyric bacilli already known.

Summary.

The three new varieties of butyric bacillus found in the ‘“Takaawa”
stage of “moromi”-fermentation are,

1. Bacillus butyricus aromafaciensis moromi I.

2. Bacillus butyricus aromafaciensis moromi ITI.

3. Bacillus butyricus roseus moromi. These 3 bacilli produce butyric
acid chiefly from starch and glucose, and 1 and 2 produce the characteristic
odour of butyric ester in the presence of ethyl-aleohol or by symbiotic eul-
ture with “saké’-yeast; moreover, these 2 bacilli grow in the presence of
as much alcohol as ca. 6%. There is no doubt that these 2 bacilli never

cause any putrefaction in ‘

‘saké” and “moromi”, because their resisting
power towards acids is comparatively very weak. It may therefore be
concluded that these 2 bacilli play an important role in producing the
characteristic aroma of “Takaawa” in “saké” brewing. They may there-
fore perhaps prove moderately useful for this purpose in future,
Bacillus No. 3 on the contrary does not appear to play such an im-
portant part, but may be considered as an agent in the production of
“akamoto’,” because it produces a red color in starch cultures.

9. During the saké fermentation, “moto”-mash sometimes changes to a dark red color,

when we call it “ akamoto.”

BUTYRIC ACID FORMING BACILLUS OF “SAKE-MOROMI.” 313

Explanation of Figures.
Plate XIII.

Fig. 1. Bacillus butyricus aromafaciensis moromi IL; after 2 days in “koji”-agar
30°C (1500/1).

Fig 2. Bacillus butyricus aromafaciensis moromi I; flagella stained, after 17 hours
at 30°C(1500/1).

Fig. 3. Bacillus butyricus aromafaciensis moromi 1; anaerobic culture; 20 hours in
“koji”’-extract at 30°C (1500/1)

Fig. 4. Bacillus butyricus aromafaciensis moromi I; spores after double staining ;
2 days in “koji”-agar at 40°C (1500/1)

Fig. 5. Bacillus butyricus aromafaciensis moromi I; 2 days in “koji”-agar at
30°C (1500/1)

Fig. 6. Bacillus butyricus aromafaciensis moromi If; flagella stained; after 17
hours at 30°C(1500/1).

Fig. 7. Bacillus butyricus aromafaciensis moromi II; anaerobic culture; after 20
hours.in “ koji”-extract at 40°C(1500/1).

Fig. 8. Bacillus butyricus aromafaciensis moromi [I; spores after double staining ;
after 2 days in “ koji”-extract at 40°C(1500/1).

Fig. 9. Bacillus butyricus roseus moromi; after 2 days in “koji”-agir at 30°C
(1500/1)

Fig. 10. Bacillus butyricus roseus moromi; flagella stained; afier 17 hours at
30°C(1500/1).

Fig. 11. Bacillus butyricus roseus moromi; anaerobic culture; after 20 hours in
“koji”-extract at 30°C(1500/1).

Fig. 12. Bacillus butyricus roseus moromi; spores after double staining; alter

2 days in “koji”-extract at 40°C(1500/1).

Plate XIV.

Fig. 13. Buicillus butyricus roseus moromi; plate culture in starch-bouillon-agar
after 2 days at 40°C; (a) are deep colonies.

Fig. 14. Bacillus butyricus roseus moromi; plate culture in glucose-Louillon-agar
after 2 days at 40°C; typical spreading out of a surface colony.

Fig. 15. Bacillus butyricus aromafaciensis moromi T; plate culture in starch-bouillon-
agar, after 2 days at 40°C.

(a) are the colonies formed in a deep position.
Fig. 16. Bacillus butyricus aromafaciensis moromi IL; plate culture in starch-

bouillon-agar, after 8 days at 40°C. (a) are colonies formed in a deep position,

ee a

AIT.

Plate

Jour. Coll. Agric. Vol. I.

Jour. Coll. Agric. Vol. I. Pl; che

On the Lactic Acid Bacillus of ‘‘Moto’’-mash.

Y. Okuda.

Although the lactie acid bacillus of saké has been studied by several
authors, there are some points which are still in need of further investi-

gation.

O. Kellner, Y. Mori, and M. Nagaoka! found that “Koji”? is liable
tp turn sour by the production of lactic acid, if it is stored in a damp
room with insufficient ventilation. T. Okumura® found that lactic
and acetic fermentations are sometimes observed in the “moto’’-mash*.
It was also pointed out by Y. Kozai® that in the earlier stage of “Moto”-
preparation lactic acid fermentation takes place, which is favorable for
yeast, T. Takahashi® has found six species of lactic acid bacilli from alter-

ed “Saké” called “Tyochi-Saké,” and one variety has been obtained by K.

J. The Bulletin of the College of Agriculture, Tokyo Imperial University. Vol. I, No. 5,
P. 28.

2. “Koji” is steamed rice upon which the mycelium of a particular species of fungus
has been developed.

3. Bull. of the Agr. College, Tokyo Imps. University. Vol. IV, P. 212.

4. “Moto”-mash or “Saké moto” is prepared by mixing steamed rice, rice “ Koji” and
Water.

5. Centralbl. f. Bakteriologie. 1900, II Abt., Bd. VI, S. 392.

6. Bull. of the Agr. College, Tokyo Imp. University. 1907, Vol. VII, 4, 531.

316 Y. OKUDA:

Eda™ from “moto”- and “Moromi’-mash. Recently S. Mori® made a
quantitative determination of lactic acid at every stage of ‘“Moto”-
preparation. iK. Eda® added some pure cultures of lactie acid
bacillus to the preparation of “Moto.” T. Shimoyama and T. Andé!°
arrived at the conclusion that a suitable application of this bacillus sim-
plifies the “Moto’’-process. The fact affirms the good effect of the applica-
tion of lactic acid in the preparation of moto-mash, made by U. Yamagata

in 1901.

Cultures of the lactie acid bacillus, Bacillus Delbriicki!! are used in
the distillery, but in the ease of “Moto”-mash the best kind of bacillus
is not known yet, therefore, it has seemed to the writer that an investi-
gation of these micro-organisms of “Moto’-mash will bring out some-
thing of importance and interest both from the practical and the scientific

point of view.

The samples used for the investigation were obtained from Nada’™
and Fushimi'® early in January, 1909, and as culture media for the
isolation of the microbes, sterilised “Koji’-extract-agar and bouillon-agar
with calcium carbonate were used, according to the method of Beyerink"*.
All the microbes isolated by the writer unfortunately produce compara-
tively small quantities of acid. The breweries which supplied the samples

the stages of “Moto”-mash at which they were taken are:—

7. Jy6zdshikenjo Hokoku (Bulletin of the Imperirl Brewing Experiment Station). 1907, 14, 5,
8. ” ” ” ” 1909, 25, 68.
9 ” ” ” » 1909, 25, 22.
10. » ” ” » 1909, 28, 45.

11. Lafar: Handbuch der technischen Mykologie 1906, V, 297.

12, Yamamura’s and Okura’s saké factories at Nada in the province of Settsu.
13. Okura’s saké factory at Fushimi in the province of Yamashiro.

14. Centralblatt fiir Bakteriologie. 1891, Abt. I, Bd, 1X, S. 781.

ON THE LACTIC ACID BACILLUS oF “MOTO”’-MASH. Sie

—— EE TEER

Bacteria. Place. Stage.
Bac. No. I. Bhs are bees Yamamura’s factory at Nada. Wakitsuki ( Ankommen”).
Coccus No. IV. Hoe kes rt » » 9 ”
BaciiNo: IT. «<=: «<0 «-- |g@kura’s af Pes +s
Goceus {No:.T., <5)... === » factory at Fushimi. Takaawa (“ Krausen”’)
(Caecus sNo NE iss. leshires rf Fy, 5) ” »

PART I. BACILLUS.
Bacillus No. I. (Bae. Aderholdi var moto).

I. Form and size: Rod shaped, 3-5 » long, 1 » broad, both ends
of the cell are round and the two sides are parallel with each other (38
days at 33-35° C in bouillon). Usually, isolated or united in pairs,
rarely in chains. In a culture on bouillon-agar somewhat elongated forms
were found after two months, but there were no remarkable involution
forms. Stains with Gram’s method. Non-motile and sporeless.

II. Growth: 1. Solid culture: a. Plate cultures:—Bouillon-
agar or 10% cane sugar-bouillon-agar: Very small, grayish white colonies
appear on the surface of the medium after 3 days at 33-35° C. The peri-
phery of the colony is smooth and the inner part is homogeneous, wher
seen with a magnification of 170. 0b. Stab-cultures: “Saké”-agar:—A
thread like, grayish white growth extends along the whole length of the
stab-canal, almost no surface growth (24 hours at 35° C., and 6 days
more at the room temperature). ouillon-gelatine: A grayish white
beaded growth along the stab canal. No liquefaction of gelatine after
9 days at the room temperature. c. Surface culture: Bouillon-agar;
“koji”-extract-agar, 10% cane sugar bouillon-agar: Forms a very feeble,
grayish white growth on the surface of the medium, and turbid condensed

water with a little sediment after 2 days at 80-33° C. The condensed

318 Y. OKUDA:

water became clear after 17 days. Bouillon-gelatine: Faint growth.
No liquefaction after 20 days at the room temperature.

2. Fluid eulture: “Koji’’-extract at 30-35° C:—This is a very
suitable medium for the growth of this bacillus. After one day diffuse
turbidity. After two days, increased turbidity, and abundant sediment.
When the tube is shaken, the sediment rises in the form of filaments and
then immediately scatters like cloud. The fluid remains clear and the
sediment appears gray in color (after 25 days). Bouillon: Fluid tur-
bid, but growth not so good as in “koji”-extract. Wort (without hop):
Intense turbidity after 3 days at 30-35° C. Yeast-water: Almost no
growth after 7 days at 30-33° C. Artificial solution Al®: No growth
even after 25 days at 30-35° C. Artificial solution B’*: Slight growth.

“Saké? : No growth even after 32 days at 28-33° C. Diluted “Sake”
(Aleohol 11.4 weight percent): Fluid turbid, some sediments, after 32
days at 28-23° C. “Moromi”-mash'*: A slight growth after 30 days
at 28-83° C. Beer!®: No growth after 32 days at 28-33° C. Milk:
No coagulation after 22 days at 30-33° C.

lie ASpAYEPiN get, ax) aioe ate 1.00 % Natrium chloride ... ... 0.50 96
Magnesium sulphate... ... 0.02 % Kalium nitrate aC ei: 0.02 96
Kalium monophosphate .., 0.10 9% Glycerit sah ces gna te 1.00 %
Ammonium Corbonate ... 0.05 %

10, Watérs: «3. 32° Vw (3000 c.c) Calcium chloride ... ... 0.2 gr.
Kalium monophosphate ... 2.0 gr. Saccharose .:. ... -. 60.0 ,
Magnesium sulphate... ... 0.2 PeptOne exc stan ext) een
Natrium carbonate ... ... RO 5,

17. Yuwao Tejima analyzed this with the following results :--

Alcohol .., ..-. ss «so 17:24 % (vol) Glycériti: ad! avvae Se 0.133 26
Extractive matters ... 2.95 ,, ASH a0) oer laecounxtie ane 0.049 ,,
Glucosé ..2° as - caesar OI ss Total acid (as lacicacid) ... 0.297 ,,
DOSS 4s, gees eg Volotile acid (as acetic acid) 0.055 ,,

18. It contained 11.5496 of alcohol and 0.409% of total acid as lactic acid.
19, It coetained 6.5 76 of alcohol and 0.187 9% of total acid as lactic acid.

ON THE LACTIC ACID BACILLUS OF “MOTO’’-MASH. 319

Il1.—Behavior towards carbohydrates, and comparison with the al-
ready described bacilli: the production of acid from carbohydrates was
tested with yeast-water containing three per cent of a carbohydrate, during

10 days at 28-33° C. The results are as follows :—

Acid production 2+
Substance. Growth?2°
a NaoH. Bac. No. I. | Bac. Aderholdi,
Pbiammose= <5. c.5 *-say--= Kk 0.0 — --
Glucose G 1.6 ++ +
Saccharose G 2.2 +++ +
Tructose ... G 16 ++ +
Galactose 3 ete Pome W 0.9 + +
INEGI) cq onda" Nesey udoon ec G 3.3 SRP ap Ar aF
HEZCLOStee cee ees, fee eae an Ww 0.8 +
Beat POSE eevee Gore cee jsn<)) (ere WwW 0.2 C+) ot
IPEREXIENWMa=cye St Gers ces) eee G 1.0 + +F
TARVIN es, ose sc) ces) aes G Tez =
SPAT CHM Scie Soult dees: 03 (+) =
IWNARIEIIE WN cer) sec) Cveyll..de-)| -og0 K 0.0 =~ —
a-methyl-glucoside ... ... Ww 0.0 — —

Growth: Saccharose, fructose, glucose and dextrin: Fluid is al-
most clear. On shaking, sediment rises, and scatters like cloud. Lae-
tose, galactose, a-methyl-glucoside: Faint growth. Mannit, rhamnose:
Now growth. Raffinose: Almost no growth. Inulin: Tums

very slightly turbid, and a sandlike sediment is produced at the bottom.

The increased amount of acid was titrated with a decinormal solu-

tion of sodicim hydroxide, the indicator being phenol-phthalein.

20. In the table, G, indicate good growth ; W, slight; K, not.

21. +, indicate production; —, not; (+), trace,

320 Y. OKUDA:

On shaking the sediment diffuses uniformly. Maltose: Fluid is very
turbid. (10 days at 28-33° C).

; Thus we see that, the growth of this bacillus in the various yeast-
waters containing carbohydrates is not so good as that of others described
in this report. Among the above mentioned carbohydrates, maltose pro-
duces, in the interval of time and at the temperature given above, the
‘greatest quantity of acid, but it is far less than in the case of “koji”-
extract.

Comparison: We can easily find that this bacillius nearly agrees
with Bacillus Aderholdi Henneberg?, in the production of acid from the
above named carbohydrates, except inulin, and also in its failure to grow
in beer, but the decisive difference between them is that milk is coagulated
by the latter and not by the former. It differs from T. Takahashi’s**
Bacillus Aderholdi var Saké by its action upon inulin and starch, and
by the thermal death point. This is also distinguishable from K. Eda’s**
bacillus by its property of production of acid from lactose, inulin, starch,
and by many other points.

IV.—Fermentation products: Lactic acid?*, acetic acid, fusel oil

?
(trace), ammonia, and methyl alcohol were found in the distillate or residue
of glucose-yeast-water culture after 21 days at 28-33° C., but formic
acid, butyric acid, methyl lactate, ethyl alcohol, aldehyde, acetone, furfu-
rol, succinic acid and indol were not found in it.

The increase in the quantity of total acid and non-volatile acid, in
100 cc. of “koji”-extract after 12 days at 35° C. was titrated with
XNaoll. Required ec. of this alkali solution was as follows:—

Total aciduas usar tee eed nc so. apsheset or cack tater eisai mee 57.0

22. Zeit. f. Spiritus industrie. 1903, 31, 343.
23. Bull. of the Agr. College, Tokyo Imy. University. 1907, VII, 4, 546.
24. Jdzdshikenjo Hokoku (Bull of the Imp. Brewing Experiment Station). 1907, 14, 13,

25. It was detected with Uffelmanu’s test and with crystals of Zinc lactate.

ON THE LACTIC ACID BACILLUS OF “MOTO”-MASH. 321

V.—Behavior under different temperatures: Optimum temperature
for growth lies near 36° C. There is no apparent growth below 17 C.,
and above 57° C. The test was done with “koji-extract, and yeast -water-
glucose. Generally the growth in “Koji’’-extract was much better than
in yeast-water-glucose.

Temperature for acid production: Acid is produced very quickly
above 30° C. in yeast-water-glucose but its production is more greater in
the long run at 26° C. than at 30° C. The minimum and maximum
temperatures for acid production are near 17° C. and 52° C. respectively.
This test was repeated with “koji’’-extract and by titration. According
to this result the optimum temperature for the production of acid for long
periods, lies near 26° C., and there was an increase of total acid by
0.8994 as lactic acid after 30 days.

Thermal death-point: Heating to 70° C. for 10 minutes exceedingly
retards the growth, but does not kill all the cells, which die at 80° C. in
10 minutes (in “koji’”-extract).

V1I.—Behavior towards alcohol, and lactie acid: In “koji’’-extract
containing 0.119¢ of total acid (as lactic acid), 3 per cent of alcohol
accelerate the growth of this bacteria. With 11% of alcohol it grows well,
but with 16% no development is observable after 15 days at 80° C. This
bacillus can not grow in yeast water containing 10% of cane sugar beside,
0.474 of lactic acid; while with 9.174 of lactic acid, it grows energetically.

VIT.—Relation to the quantity of sugar: For this purpose bouillon
containing glucose was used, the culture being continued for 15 days at
26-29° C.

Percentage of glucose 2. 5. i

Growth. + + + oe at + (4) — —
+ + a + +
- + - - ~

VIIT.—Necessity of air: Both with Buchner’s and Botkin’s me
thods proceeds energetically growth, therefore this bacillus grows well

without oxygen,

ww
bo
Lo

Y. OKUDA:

IX.—Some relation between this bacillus and yeast: Bac. No. I
was mixed with saké yeast—viz. B, No. 21 of Jézdshikenjo,—in the follow-
ing ratios, in 50 cc. of “koji’”-extract, and after 7, 10, and 27 days at
25-28° C., the number of the cells of the yeast was determined, and the

acidity of the medium was titrated with —~—NaOH solution.

Remarks. Original medium 10 c.c. corresponds to 1.4 ¢,c. =NaOH.
Number of yeast cells in 1 cc. of original eulture=7300000
=7.3 million.
Number of yeast cells in 3 drops of original culture—0.876
million

1 c.c. pipette used to Bac, No. L238 drops.

1 c.c. pipette used to saké yeast—25 drops.

In the following table, to the counting of yeast cell, one million was

adopted as the unit.

Yeast 3drops| 3drops Zero
Mixed...
Bacillus Zero 1 drop 1 drop
After 7 day Yeastcellin} i629] 160.0 0
» 27 » ” 169.5 187.0 0
sree 35 4.2 5.6
10.7

Yeast 3 drops | 3 drops
Bacillus 0.6 c.c. | 1.6 c.c.

Yeast cells in
3 Lec.

147.4 | 150.0
Acidity ‘ : : 6.6 7.2

N
2%. Cc. of To NaOH required for nentralization of 10 c.c. of the culture medium.

ON THE LACTIC ACID BACILLUS OF “MOTO”’-MASH.

The results are somewhat irregular, but we can see at least, that this
yeast grows energetically in spite of the presence of immense numbers of
this bacillus, although too many of it are not favorable for the growth of
the yeast. Further we see that this bacillus, in mixed cultures, produces

acid as much as in pure cultures.

Bacillus No. IT. (Bacillus lactis acidi Leichmanni var. moto).

T. Form and Size: Rod shaped 275-6 / long, 1-175 broad, both
ends of the cell are round, and the two sides are parallel with each other.
The cells are usually united in chains, and rarely isolated (in bouillon,
after 3 days at 35° C.). Motile. Stains with Gram’s method. Some
involution forms were found in a “koji’’-extract-agar culture after 40 days.

II. Growth: 1. Solid eulture: (a) Plate cultures: Bowillon-
or saccharose-bouillon-agar: Round or nearly round, yellowish white,
somewhat viscous colonies with more or less elevated centre. The periphery
is smooth or slightly wavy, and the internal part is homogeneous, when
seen with a magnification of 170 (2 days at 30° C.). Bouilllon-gelatine:
Liquefies in the form of hemispheres, and the liquefied part is turbid.
and in its central part exist some yellowish deposits (5 days at room tem-
perature). (b). Stab-cultures: “Saké”-agar: Forms a faint growth
along the stab canal, and a yellowish white, creamy surface growth cover-
ing the total surface of the medium (5 days at 35° C.). “‘koji”-extract-
agar: Similar as in the case of “Saké’’-agar, but the color of the surface
erowth is dirty gray (4 days at 85° C.). Bouillon-gelatine: A funnel-
shaped liquefaction cecurs, and the liquefied part is turbid (9 days at room
temperature). (ec) Surface cultures: Bouillon-agar or saccharose-
bouillon-agar: A yellowish milky growth appears on the needle track.
Condensed water is turbid and a yellowish sediment is found at the bottom
(2 days at 35° C.). After 17 days condensed water is clear. ‘“Saké-

agar: A yellowish white, flat surface growth. Exceedingly turbid con-

324 ‘ Y. OKUDA:

densed water with a yellowish white sediment (24 hours at 35° C. and
2 days more at the room temperature). “‘koji’’-extract-gelatine: Lique-

faction oceurs slowly along the needle track.

2. Fluid enlture: koji-extract: Turbid after 24 hours at 35° C.
An abundant sediment is found at the bottom. It scatters like cloud on
shaking (after 3 days). Islands appear after 5 days and the medium
remains clear after 16 days. Bouillon: turbid after 24 hours at 35° C.
Becomes clear after 6 days. Yeast-water: Fluid is slightly turbid and
sediment somewhat viscous (3 days at 35° C.). Artificial solution A:
Becomes turbid, and a small quantity of sediment is found after 10 days
at 25-27° C. “Saké”, diluted “saké”, beer: No growth after 32 hours
at 28-33° C. Milk: No change after 3 days at 30° C., after 5 days
the upper half of the milk becomes yellowish and transparent, the lower
part is being viscous. After 15 days the total medium remains complete-

ly clear.

III. Behavior towards carbohydrates and comparison with the al-

ready described bacilli: Tests made were the same as in the case of Bae.

No. I.

Acid production
Substance Growth =
Beeilles laste acidi | Bac. No: IL
veichmanni,
Rihamvose= 2... ss0, sesh oe G | — | =
Glucose Sevtylata Sccordecmeeeee G ie +
Saccharose. 3.) i... Sameera G fe age
Fructose pees See PAs Ges G ar ae
Galactose< fo. (iss'g ev et ee G + +
Maltose etn hee G + u
VSCtORO Tr... acc aed eee vee eee G + oY
Raffnose: cs) was, “ote. eee G + nage ©

Dextrin vie’ Wael vee See eee G 7 ae

ON THE LACTIC AGID BACILLUS or “ OTO’’-MASH. 325

Acid production

Thus this bacillus produces acid from many kinds of carbohydrates

but the quantity produced n each case is not much, as shown by titration.

From the above table, it will be seen that this bacillus nearly agrees
with Bac. lactis acidi in forming acid from the above named carbohydrates,
starch being an exception. But their action upon gelatine are different.

IV. Fermentation products: Acetic and lactic acids, ammonia, and
methyl-alcohol were found in the distillate and in the residue of glucose-
yeast-water culture, but formic, butyric and succinic acids, fusel oil, ethyl,-
propyl,-isoprophyl-and butyl-alcohol, aldehyde, acetone, furfurol and indol
were not found.

The ratio of the total acid to the non-volatile acid produced in glucose-
veast-water culture, after 10 days at 30° C., was 11’5: 1075.

V. Conditions of temperature: (a) Optimum temperature for
growth lies near 30° C., and no growth is observable below 17° C. and
above 52° ©. This test was done with yeast-water containing 3% of
glucose. (b) Optimum temperature for acid production lies also near
30° C. in the same medium as above. (ec) Therma] death-point:
Heating to 60° C. for 10 minutes in “Koji’’-extract retards the growth
of this bacterium very much, an exposure to 70° for 10 minutes kills
the cells.

VI. Behavior towards alcohol and lactic acid: 3% of alcohol nearly
inhibited the growth, with 7% there was no growth. No development with

0 47% of lactic acid.

326 Y. OKUDA!

VII. Influence of absence of air. This bacillus is xrobic. No

growth in anerobie culture with Buchner’s or Botokin’s methods.

PORT II. COCCUS.

Coceus No. I. (Pseudosarcina).

I. Form and Size: The diameter is about 175 4. Two, three or
eal sarcina-forms are not found. No involution form on “koji’’-extract-
eal sarcina-forms are not found. No involution form on “koji’-extract-
gelatine after 20 days. Stains with Gram’s method. Nonmotile and
sporeless.

II. Growth: 1. Solid culture: (a) Plate cultures: Bouillon-
agar, saccharose-bouillon-agar: Round somewhat elevated, milky white
colonies with fatty lustre appear on the surface of medium. The periphery
is smooth and the internal part looks homogeneous with a magnification of
170 (one day at 33° C.). The color of the colonies changed into a
light yellow tint after 2 days more. Bouillon-gelatine: Forms round,
small, yellow colonies. No liquefactoin of gelatine (15 days at the room
temperature). (b) Stab-cultures: “Saké”-agar: A needle like growth
on the surface of the medium (24 hours at 35° and 6 days more at the
room temperature. “Koji’-extract-agar: A faint growth is formed
along the needle track and creamy yellowish white colonies appear about
the mouth of the stab canal (one day at 35°). Bouillon-gelatine: Simi-
lar appearance as in the case of “koji’”-extract-agar. No liquefaction
after 10 days at the room temperature. (¢) Surface cultures: Bouil-
lon-agar: Forms a flat creamy, light-yellow growth along the streak.
Turbid condensed water has a yellowish white deposit (3 days, 35° C.).
Clear condensed water was observed after 20 days. Saccharose-bouillon-
agar: A similar growth as in the preceding dedium, but the color of
the colonies is milky white (3 days at 35°). ‘“Sake”-agar: A creamy
white growth along the needle track (24 hours at 35° O and then 2 days

ON THE LACTIC ACID BACILLUS or “t0TO”’-MASH. 32

at the room temperature). Bouillon-gelatine: A faint, grayish white
growth along the streak after 2 days at the room temperature. After 20

days no liquefaction of gelatine, the color of the colonies being yellow.

2. Fluid culture: “Koji’-extract: After 6 days at 30°C, the liquid
is turbid and sediment formed along the wall of the glass tube. Bouillon:
Fluid turbid. On shaking, the sediment rises at first as filaments and
then scatters like cloud (3 days at 85°). More or less turbid even after
11 days. Wort: Fluid turbid, and some deposits (3 days at 35°).
Yeast water: Slightly after turbid 3 days at 830° C. It becomes clear after
10 days. ‘“Saké”, diluted “saké” and beer: No growth in any of these
media, even after 32 days at 28-33° C. Milk: No coagulation after 25
days at 28-33°C. Artificial solution A: very slight growth, after 10 days

Bh 2-20 aM.

IV. Behavior towards carbohydrates, and comparison with the al-

ready described microbes.

Substance Growth Acid production
RtaMngses yl cosh Sey (ere) Stsae=sle tors Kk | —
Glucose
SACGHALOSG:s5 cfcl css fees) cect Feseece G ar arr
CLOSE Meret Face Mees) ycus. ce. ns eee G
GAICUOSE) ss: sec es) eel cee) Res cee G ae
Mialtoses: -s!))... ros a inte. Aco G ++
MEACEOISCMADS” sc ics, Susy “ees Re eee G +
Rea MOSCREAC Mt eob basi eck Vice soot Me Se G +
Dextrin 3 +
Inulin... +
Starch St a, RE WOR OSD eer! Ob --
NIECITTS © per Ptedt pa guiemer eh mran mmc rommaees G —
a-methyl-glucoside... 0.0 6. 1. a G =

328 Y. OKUDA:

Thus this coceus forms acid from many kind of carbohydrates. but

the quantity produced, in each case is not so great as in Bae. No. I.

According to Lindner?* almost every sarcina and pediococcus pro-
duces more or less lactie acid, but the descriptions for them, as regards
the production of acid from carbohydrates generally, are not sufficient. The
well known coceus, Pediococeus acidi lactic Lindner differs from our coceus
No. I, by its inability to produce acid from saccharose, raffinose, dextrin,
and inulin. Sarcina meliflava Gruber does not caurse any turbidity in
beuillon-culture, a property which distinguishes it from our Coceus No. I.

IV. Fermentation products: Lactic, succinic-(trace) and butyric
acids, fusel oil, ethyl alcohol (trace), ammonia (trace) were found in the
distillate and residue of glucose-yeast-water culture, but formic acid,
acetic acid, aldehyde, acetone, furfurol and indol were not found.

The ratio of total acid to non-volatile acid, in the culture of glucose

yeast-water, after 10 days at 30°C, was 13.0: 11.0

V. Conditions of temperature: (a) Optimum temperature for
growth lies between 26°C. and 30°C (34 glucose-yeast-water was applied
as culture medium). No visible growth below 17°C and above 52°C. (b)
Optimum temperature for acid production lies near 25°C (The same me-
dium as above). (¢) Heating to 60°C for 10 minutes does not kill all
the cells, which die only at 70°C (in bouillon).

VI. Behavior towards alcohol and lactic acid: It grows very well
in bouillon containing 7¢ of ethyl alcohol, but there was no growth with
13%. This coceus grows energetically in saccharose-yeast-waer containing
0.17% of lactie acid, but 0.47% of lactie acid inhibits its development.

VII. Relations to the quantity of sugar.

28. Lindner: Microskopische Betriebskontrolle in den Garungsgewerben, 1905, Auf. 4,

ON THE LACTIC ACID BACILLUS OF “MOTO’’-MASH. 329

Percentage of glucose in bouillon 2 5 10 1) 20 25
Growth after 15 days at 26-29C° + + a + = =
+ + + +
af ae =

VIII. Necessity of air: This coceus grows only in the presence

ot air.

Coceus No. IT. (Psendosarcina).

TI. Form and Size: The diameter of the cell is about 1 /’, similar
in appearance to Cocus No. I under the microscope. No involution forms
are found in bouillon-agar-culture after 2 months. | Non-motile and

sporless.

II. Growth: 2. Solid culture: (a) Plate cultures: Bouillon-
agar: Forms smail, round, somewhat elevated, creamy, grayish white
colonies, with smooth peripheries and homogeneous center under a magini-
fieation of 170 (24 hours at 33°C). After two days more at the same tem-
perature the color of the colonies changed into light orange-yellow. Bouil-
lon-gelatine: Forms fine, round, light orange-yellow colonies after 5 days
at the room temperature. (b) Stab-cultures: “Saké’-agar: A fila-
mentous growth appears along the stab canal, and the surface growth has
fatty looking, liget orange colonies after 24 hours at 35°C, “Koji’-extract-
agar: Good growth with an orange yellow surface (3 days at 35° C).
Bonillon-gelatine: A beaded growth appears along the stab-canal, and a
round, light orange-yellow colony about the mouth of canal. No liquefac-
tion after 12 days at the room temperature. (c) Surface cultures:
Bouillon-agar: A milky white colony grows on the streak. Condensed
water is clear and some white deposits are formed at the bottom after
20 hours at 33°C. After 20 days, both the growths on the solid medium
and at the bottom of the condensed water changed into a light orange-

yellow tint, Saccharose-bouillon-agar: Similar growth as above, but the

330 Y. OKUDA:

color of the colony remains milky white even after 20 days. “Saké’-agar:

Forms milky white ceveing after 24 hours at 35°C. “Koji’’-extract-gela-
tine: Orange-yellow colonies united together in a chain-form along the
streak. No liquefaction of gelatine after 20 days at the room temperature.

2. Fluid culture: ‘“Koji’-extract: Islands appear on the surface
of medium, and the liquid is turbid after 5 days at 35°C. Bouillon:
Turbid and some islands after 3 days at 35°C. The liquid was turbid
even after 16 days and a viscous, flocculent deposit was present. Wort:
A faint growth after 3 days at 85°C. Artificial solution A: A very slight
growth after 10 days at 30-35°C. Yeast water: Slightly turbid, and a
little sediment, which rises as filaments and scatters like cloud, when the
tube is shaken. Some islands are on the surface of the liquid. Clear,
after 10 days. “Saké”, diluted “saké’”, beer: No growth after 32 days
at 28-33°C. Milk: No coagulation after 25 days at 28-33° C.

IIT. Behavior towards carbohydrates, and comparison with the al-

ready described cocci.

Substance Growth | Acid production
Rhamnose asin eigw ih Seki seo Mn CaR ses G —
Glucose” °.6% Gesr yc, Oana coetemomiare os G +
Saecharose:.. e200 rest tees eee races G Pies
IO (ol ee ee ri a5 toot lsc recip ere G ae
Galactoss\ @.. Sut Sew” Gael Stee ee G aie
Maltose: <..0stz0 is See oe G a
Lactose) ke.8 es" cae ae pene ee G =a
Raffinose |. s- Lae ee G —
Dextrin.s 5538). 5s, aan eee Pie G GE)
Inulin Pre ee es Pie ME G =o
Starch sco) 908 <2 as #98 egy) eae _
Mangit. os5) (ssi ssa ece lee are. G —
«-methylg-lucoside ... ... 0 vs eee eee G _

ON THE LACTIC ACID BACILLUS OF ‘“‘MOTO’’-MASH. ool

Thus this microbe produces acid from comparatively few carbo-
hydrates, and the quantity produced is also small. This coccus agrees
with sarcina aurantiaca in the formation of orange-yellow color but the
coagulation of milk and the liquefaction of gelatine by the latter distin-
guishes from Coccus No. II.

TV.—Fermentation products: Butyrie-(trace) and lactie acids,
methyl-lactate, fusel oil, methyl-aleohol and ammonia (trace) were found
in a glucose-veast-water culture, but formic acid, acetie and succinic
acid, aldehyde, acetone, furfurol and indol were not found. Morver the
distillate had an odor like that of amyl-valerate, although it was not test-
ed. The ratio of total acid to non-volatile acid produced in the culture
of yeast-waterglucose, after 10 days at 30°C, was 9.5:8.0.

V. Conditions of temperature: a. Optimum temperature for
growth lies between 26° and 30° ©., and growth is not apparent below
17° or above 52° in 3% glucose-yeast-water. b. Optimum temperature
for acid production is below 30°C in the same medium as above. — e¢.
Thermal death point lies between 60° and 70°C, heating for 10 minutes in
bonillon.

VI.-—Behavior towards alcohol and lactic acid: Tt grows in bouillon
containing 7% of ethyl-aleohol, but there is no growth with 13 per cent.
of aleohol in the same medium. No growth with 0.47% of lactie acid.

VIT. Relation to the quantity of sugar:

Percentage of glucose in bouillon... 2 5 10 15 20 25
Growth after 15 days at 26-29°C, ... +5 + + 2 — —
oh 4° “5

VIII. Necessity of air: Both with Buchner’s and Botokin’s

methods this eoceus makes a slight growth in bouillon.

COCOCUS NO. III. (SARCINA).

J. Form and size: The cell is round and its diameter is about

332 Y. OKUDA:

1.2 ». Usually two or four cells are united together in a chain or a cross
form but rarely in a sareina-form in bouillon. Non-motile and sporless.

II. Growth: 1. Solid eulture: a. Plate cultures: Saccharose-
bouillon-agar: Forms a round, milky white colony on the surface of the
medium. Its periphery is smooth, and the internal parts look homogene-
ous under the microscope after 2 days at 35°C. b. — Stab-cultures:
“Saké”’-agar or “koii’’-extract-agar: A spare growth along the stab
eanal, with a flat surface growth of a creamy nature and white color (24
hours at 35°C and then 6 days at the room temperature). Bonillon-gela-
tine: Both the growths in the stab canal and on the surface of the medium
are feeble. No liquefaction of gelatine after 18 days at the room tem-
perature. ec. Surface cultures: Bouillon-agar: Round milky white
colonies form a chain along the streak, and the condensed water is turbid,
with a white deposit after 2 days at 35°C. The liquid becomes perfectly
clear after 17 days. Saccharose-bouillon-agar or “saké”’-agar: Similar
appearance is in the foregoing medium the temperature and time being

the same.

2. Fluid eultures: “Koji’’-extract: Fluid is turbid, and a white
sediment appears after 4 days at 35°. On shaking the deposit rises as
filaments and diffuses immediately like clond. Bouillon: Fluid is turbid
after 3 days at 35°C. Yeast water: Slightly turbid after 10 days at 28-
33°C. “Saké,” diluted “saké,” beer: No growth after 32 days at 28-

33°C. Milk: No coagulation after 25 days at 33°C.

IIT. Behavior towards carbohydrates, and comparison with the

already described cocci.

ON THE LACTIC ACID BACILLUS OF ‘“MOTO”-MASH. 333

Substance Growth Acid production
Rhamnose SE ee hs K —
SAIN COSE She sch he BA, Me ¢, G crete
Saccharose re ee, COU : G =f et
NICLOSG 4. Bc ge Re ieee, ce G ++
Galactose eon) 5. sMeme ee wea ae G +
NI@IOSEs fe cee Sy Bae ane K | =
ER CEOS Preis =i. ORNL OME es, ee G | —
Raffinose ... o:. . ; 006 G | (+)
EXOT seh ec, aad) wv) eoep sgt te G | +
AGC te eee m2 cena ie ec ee G +
Starch e =
IUEWRGE VTE Bes ASkeg Bry load seed actor. skies G =
a-methyl-glucoside ... 00.0... ue G =

Comparison: Sareina pulchra Henrici, and S. nivea Henrici grow
only in the presence of air, thus differing from our Coceus No. III. This
coccus agrees in many of its properties with Saito’s® Sareina Hama-
guchiae, but the differential character is that my coceus develops in bouil-
lon while 8. Hamaguchiae does not.

IV. Fermentation products: Butyrie acid (trace), lactie acid,
ethyl alcohol, ammonia, and methy alcohol (trace) were found from the
distillate and residue of glucose-yeast-water culture, but formic acid, acetic
acid, fusel oil, aldehyde, acetone, furfurol, succinic acid, and indol were
not found.

V. Conditions of temperature: a. Optimum temperature for
erowth lies between 26° and 30°C, and growth is not apparent below 17°
and above 52°C in glucose-yeast-water. b., Optimum temperature for acid

production is nearly the same as that for growth. ec. Thermal death

29. Centralblatt fiir Bakteriologie 1906, Abt, II, Bd. XVIIT, 155.

334 ¥. OKUDA:

point lies between 60° and 7 0°C, heating being done for 10 minutes in
bouillon. (

VI. Resistance against alcohol, and lactic acid: An energetic
growth takes place in bouillon containing 7% of alcohol, but with 18¢ it
ceases. It grows well in neutral-yeast-water containing 10% of saccharose
and 0.17% of lactie acid, but 0.47% of lactic acid inhibits the development
of this microbe.

VII. Necessity of air: Both with Buchner’s and Botokin’s method

it grows as well as in open air.

Summary.

1.- Two of the isolated microbes are baccilli; B. Aderholdi var moto,
B. lactis acidi Leich. var. moto, and easily distinguishable from
each other by stab-cultures. The others are cocci, and distin-
guishable irom each other by surface growths on bouillon-agar.
Bae. lactis acidi Leichm. var. moto is motile, but the others are

not.

None of the four species grows in “saké” or beer, and is there-
fore not injurious to them. They do not produce any change
in milk and do not liquefy gelatine, except Bac. No. II. (Bac.
lact. acid: Leichm. var. moto). They grow well on solid media
with the exception of Bac. Aderholdi var. moto., which does not.
The growth of all four is almost entirely inhibited by 0.47¢ of
Jactic acid in saccharose-yeast-water.

3. The fermentation products in yeast-water-glucose, especially the
production of acid from carbohydrates differs according to the
species. In all cases, lactic acid, small quantity of volatile acid,
and ammonia were found, but no aldehyde, furfurol, indol or
gases. Some slimy substances were produced by Bac. No. II.

Bae. lactis acidi Leichmanni var. moto} and Coceus No. II. All

ON THE LACTIC ACID BACILLUS oF “MoTO”-MASH. 339
four produce a relatively small quantity of acid, Bae. No. I.
(Bae. Aderholdi var. moto) producing 0.9% (as lactic acid) in
“koji”’-extract.

4. Bacillus No. IT. and Coccus No. I. grow only in aerobic cultures,
but Bac. No. I. (Bae. Aderholdi var. moto, and Coccus No. ITI.
develop well even in anaerotic culture, while Coceus No, II. Bae.

laetisacidi Leichmanni var. moto, grows slightly in anaerobic

cultures.
In conclusion my thanks are due to Prof. T. Takahashi for his kind

advices given during the progress of the work.

et nl

Note on Yeasts from Quince Liquor.

With Plate XV.

Quince liquor? was prepared from 45 litres of juice obtained by
pressing 47 k.g. of quinee. Before and during the fermentation micros-
copic examination for yeast was made on the mash. The yeasts isolated

are briefly described below :—

Saech. apiculatus. (From both fruits and mash)

I. Form and size:—Citron shaped; commonly 5x2.5 , larger ones

1. This liquor has the following composition ;—

Sih Chm (EIEN a5 05, cen sao, we 1.021

In 100 c.c.

ANGITOU QO OE )) a5 cases 6.4
BxtractnyAt. Be 02 Been ee eer eee. ee ee oe be, 8.112 grams.
BSP (1S 52°] 1 COSE)) eure eae Oe OH 2.888 - ,,
Gliycerinievssy avira ois, nace een TEE RES ces xco. vce 0.340 ,,
IAL EGUl (EGTRRA GIG) 855 an ae os oo ae 6h 0.886 ,,
Volatilevacidl(assaceticraciG)im aie seeaceaeeon &--) ee. en. 0617 ,,
Non-volatile acid (as tartaric acid ester)... ... 1... 0 2 ws 2.698
Wolatilexesteq (as acebiciesten) eeamatsmeeeen es 0.084 ,,
Non-volatile ester (as neutral tartaric diethyl Ester) lecemecrs 2600 ,,

ASD cree ee cece ct en | Seen ee vee ai waco Samer rerhe 0,286

338 H. ITO:

8.7x5 # smaller ones 3.7x1.2 #. The daughter cell separates easily from

the mother cell.

II. Contents of cells:—They are granular, clear and rich in vacu-
oles which generally contain refrective and revolving bodies or granules.

Spore was not formed.

TIT. Fluid culture :-—Culture in “k6ji’’-extract kept at 30° C for
24 huors: Fluid was turbid with sediments. When it was shaken, the
sediments assume the form of threads and diffuse in fluid. Films were
not formed on “k6ji’’-extract, veast-water and Hayduck’s solution kept

at 30° C for two months.

IV. Solid culture :—-

1. Plate culture on “k6ji’’-extract-agar kept at 25-30°C for a day.
Colony was round, slightly elevated, lustrous, waxy and gradual-
ly turned somewhat greyish white. The margin looked smooth
under the microscope.

2. Stab eculture:—(a) On “kdji”-extract-gelatine kept at 10-15°
C for 5 days. Formed feeble growth along the stab canal and
feeble, round, white, waxy, growth around the mouth of the
canal. (b) On “k6ji’’-extract-agar kept at 14-16°C for 5
days: Formed feeble growth along the stab canal and round,
waxy, dirty greyish growth around the mouth of the canal.

3. Streak culture: (a) On “kdji’-extract-gelatine kept at
10-15° © for 5 days: Feeble growth was flat, waxy, along the
track and the margin showed fine radiating lines. (b) On
“k5ji’-extract-agar kept at 14-16°C for 5 days: Growth along
th track was feeble, flat, moist, somewhat yellowish grey and
condensed water was clear, with sediments.

4. Gigantic colony: On “k6ji’-extract-gelatine kept at the room
temperature (at 10°-14° C) for one month. It was dish like,

inoist, Justrous, greyish, waxy, with wavy and radiating margin.

NOTE ON YEASTS FROM QUINCE LIQUOR. 339

V. Behavior toward different sugars:

Einhorn’s fermenting tube.

With Lindner’s method and

——sssSs.....8808089898980°08& SS....5°5°9°8°00...:.[/\—_—.__"_‘
Fermentation

Sugars

Investigated apiculatus

Sacch. apiculatus Rees.

d-Fructose
Glucose
d—-Mannose
Saccharose ..
Maltose
Galactose ...
Rhamnose ...

Dextrin

Thus this apiculatus differs from Sacch.

menting d-galactose.

apiculatus. Rees. by fer-

VI. Experiment on assimilation; according io Beijerink’s method:

2. +++ denotes comparative strong fermentation.

+4 denotes

tolerable strong fermentation.

+ denotes feeble fermentation,

— denotes

no fermentation.

340 H. ITO:

Substance as source of nitrogen. Assimilation

Peptone

Asparagine... 2... 10.0 see see one
Ammonium a 155

Sodium nitrate ...

Potassium nitrite

Substance as source of carbon.

Saccharose.:. 0 ::- csc <0 es
Maltose

Glucose

Dextrin

Galactose ...

Lactose

VIT. Degree of fermentation: In “kéji’”-extract.

Apparent fermentation index .............. Fooo bse Was

Actual fermentation index ........... i: cia toushausl steeueyoustensepeaaes

VIII. Assimilation of amino-acid: Culture in “kdji’’-extract*
kept at 30°C for 25 days. It consumed 0.0767 g. in 100 e.c. of “koji’-
extract. -

IX. Conditions of temperature: Optimum temperature was 30° C.
55° C for 30 min, was not sufficient to destroy its vitallity, but
it was killed at 60° C for 30 min.

X. Fermentation products: Ethy-, methyl-, amylaleohol and alde-
hyde were found in the distillate of “kaji”-extract culture after 7 days
at 30° © and lactie acid in the residue, but acetic-, butyric-, propionic
acid and acetone were not found. From the above description we see

that this yeast belongs to Sacch. apiculatus Rees.

3. 100 cc. of the “ kdji’’-extract contained 0,4631 g. amino acid calculated as glycocoll.

NOTE ON YEASTS FROM QUINCE LIQUOR. 341

Torula. (From both fruits and mash.).

I. Form and size: Round and somewhat elliptical; generally 6 /
in diameter, very large ones 7.5 y small ones 1 y. Generaly one or two
cells combined but three cells combined were rare.

II. Contents of cells. They were homogeneous, clear and rich in
vacuoles which sometimes contained refrective and revolving granules.
Glycogen reaction was positive.

Spore was not formed, but it formed large fatty bodies, which some-
times entirely replaced the contents.

III. Mode of growth: Generally budding, but it was noted in
droplet cultures of “k6ji’’-extract that a daughter cell sometimes hung
on to a large burst mother cell (with diameter of 7.5 / and with a cell-
membrance with 1.5 / thick.

IV. Fluid culture:

1. Culture in “koji’’-extract kept at 25° C for 4 days: Grew
well in the surface part of fluid with heavy sediment. When
it was shaken, the sediment assumes the form of threads which
easily broke into pieces, but diffused in the fluid with difficul-
ty. Formed ring after 3 days at 30° C, but no film was formed
in “k6ji’-extract culture kept at 30° C for one month more.

2. Culture in milk kept at 30° C for one day: Formed coagulum
and gradually dissolved it.

V. Solid culture:

1. Plate culture on “k6ji’-extract-agar at 25-30° C for one day:
Colony was round, elevated, waxy, slightly rose coloured, its

periphery was smooth under the microscope.

Stab culture: (a) In “kdji’-extract-gelatine kept at 10-15° C
for 4 days: Grew faintly along the stab canal and formed dry,

lightly rose coloured, gloomy, waxy, elevated growth and the

VI.

Hore s

margin was waxy. (b), In “kéji’-extract-agar kept at 14-
16° C for 3 days: It grew faintly along the stab canal and
formed moist, elevated, somewhat reddish brown growth along
the mouth of the stab canal and the margin was wavy.

Streak culture: (a) In “kdji’-extract-gelatine kept at
10-15° C for 3 days. Growth along the track was dry, gloomy,
yellowish brown, elevated, afterward it gradually changed to
brown and the margin was wavy. (b) In “koji’-extract-agar
at 14-16° C for 4 days: Along the track it formed elevated,
moist, lustrous yellowish brown growth and condensed water was
clear but contained sediments.
Gigantic colony: In “k6ji’’-extract-gelatine kept at 10-14° C
for 25 days. It was elevated, but the centre was concave and
the margin showed concentric rings and faint streams. Its peri-
phery was waxy. Root like growth* was formed under the con-
eave part of the colony. Gelatine was liquefied after 30 days
and the part was acid.

Behavior toward different sugars: With Lindner’s method

and Einhorn’s fermenting tube.

Fermentation

ace Investigated Torula et ae

d-Fructose... +44 +++
Glucose SURE Sete
d-Galactose ++ us
d-Mannose... + peers
Saccharoce ? =

Maltose - =
Rhamnose ... = —

Dextrin — —

4. Will, Central Blatt fiir Bakt. RXVIII S, 401.

NOTE ON YIASTS FROM QUINCE LIQUOR.

Thus this torula behaves almost same as torula pulcherrima.

Want,

Apparent fermentation index

Actual fermentation index

Winn,

kept at 80°C 25 days.

100 ce. of

IX. Experiment on assimilation :

Degree of fermentation:

Consumation of amino-acid:

“kojv’-extract.

In “k6ji”-extract.

see ee ww woe

6.0

343

Culture in “k6ji’-extract

Ti consumed 0.0767 g. amino-acid as glycocoll in

According to Beijerink’s method.

Sul stances as source of nitrogen

Assimilation

Peptone
Asparagi.e ..

Ammonium

Sodium nitrate ...

Patassium nitrite

Substances as source of carbon

Saccharose ...
Maltose
Glucose
extrin
d-Galactose

Lactose

+ + 4+ +

Assimilation.

+ + + + +

X. Conditions of temperature:

Optimum temperature lay 30° C.

55° C for 30 min. was not suflicient to destroy its vitallity, but it was
killed at 60° C for 30 min.

xaIe

Fermentation products:

Ethyl-, methyl-, amyl-aleohol and

aldehyde (trace) were formed in the distillate of “kdji’’-extract culture

after 7 days at 380° C and lactic acid in the residue, but acetic-, butyric-,

propionic acid, and acetone were not found.
proy )

Thus this yeast belongs to Torula pulcherrima.

344 NOTE ON YEASTS FROM QUINCE LIQUOR.

T express my great thanks to Prof. T. Takahashi for the advices

given during this work.

Explanation of Figures.

Plate XV.

Fig. 1. A, The “k6ji”-extract-gelatine culture of apiculatus.
A, The “koji’’-extract-agar culture of apiculatus.
T, The “kdji”-cxtract-gelatine culture of Torula,
T, The “koji”-extract-agar culture of 33
Fig. 2. Apiculatus: 1200/1 “ koji’’-extract culture at 30°C for 24 hours.
Fig. 3. Torula; 900/1. “ koji’’-extract culture at 30°C for 24 hours.
Fig. 4. Gigantic Colony of Torula on “ koji”-extract-gelatine for 25 days at room tem-
perature.
Fig. 5. Gigantic colony of Torula on “koji'-extract-gelatine for 50 days at recom tem

perature.

XV.

Pl.

Vol.

Agric.

Jour. Coll.

tN ner a dt i

Re A SARI a Ts EN en BN a ety

ea goed

Note on Yeasts of ‘‘Sho-yu’’-mash.
BY

T. Mitsuda.
With Plate XVI.

This short report is based on work carried on parallel with that of the
carbohydrates of “sho-yu” mash, the result of which was published a former
number of this journal’. At that time the only published report on the
microorganisms of “sho-yw” mash was that of K. Saito who described
one variety of yeast and the name of Saccharomyces Soja but recently
T. Nishimura published a long paper in the “Nogaku-Kai-Kaiho’* and
described three varieties of yeast isolated from “sho-yu’’-mash.

Three samples were used for the purpose of isolation, one from Noda,
one from Tatsuno, and the other from Handa. 8 different varieties of
yeast, 3 bacilli and 3 mould fungi were obtained from them. 3 varieties
from Noda sample, 1 variety from Tatsuno sample, 1 variety from Handa
samples, besides a red yeast and two film forming yeasts. However only

5 varieties of yeast were investigated.

Method of Isolation.

As culture medium for the isolation of microorganisms by plate cul-
ture sterllised kdji-extract-agar was used; culture temperature 28° C.;

purification by droplet culture after Lindner.

1. Vol. 1. No. 1. 1909.

2. Journal of the scient, Agricu, Society. (Japanese),

346

T. MITSUDA:

I will describe briefly the characteristics of these five :—

(2)

(b)

(ec)

(d)

The First Variety.

Form and Size: Mainly spherical, sometimes more or less
elongated, and 6-9 y in size in koji-extract. The contents

very dull, granular and rich in glycogen.

Growth: Colonies are greyish-white, very waxy and bright,
round and elevated on the surface of plate-culture; waxy, bright,
round and smooth in the inner part; periphery either smooth
or more or less zig-zag shaped (Cf. Fig. I,). Streak-
culture: a white, bright, waxy and elevated growth along the
track, with wavy surface, and streamings on margin. Stab-
culture: white and folded at the mouth of stab-canal, monili
form with gas bubbles along the line. Gigantic-colony: mealy,
white, elevated, very folded and dried appearance after 30
days at the room temperature (Cf. Fig. 6). The growth in
koji-extract, wort, yeast-water and other nutrient media is
very favorable. Yeast ring appears after 3 days at 30 C., deve-

loping into a greyish thin film over the surface of the fluid.

Spore-formation: This variety does not form spores on gyp-
sum block at 25-35 C. after 3 days.

Assimilation of Carbon and Nitrogen: This experiment was
made with Mayer’s method and was repeated three times with
the same fluid to exclude the possible influence of former
nutrition. This variety assimilates carbon from almost all
the carbohydrates and some organic salts, but not from ethyl

aleohol (Cf. Table I). Nitrogen is assimilated from peptone,

(e)

(f)

(h)

(a)

(b)

NOTE ON YEASTS oF “SHOYU”-MASII, B47

asparagine, ammonium salt and nitrate and also perhaps from
nitrite.
Behavior towards organic acids: It can grow in koji-extract

containing 2.5% snecinie or 1.5% acetic, but not in 1.75% acetic
acid.

Conditions of Temperature: Optimum temperature for growth
lies at 28-32 C. and growth is disturbed at 25 C. and vegetative
cells die at 60 C. in 4-5 minutes.

Fermentative Faculty: It was determined with Jind-
ner’s method. Jt ferments sucrose, maltose, raftinose, dextrose
ete, but not lactose ete. (Cf. Table 2). Koji-extract (12° B)
sinks to 3.5° B after fermentation, giving the apparent. fer-
mentation coefficient of 70.87.

Aleoholie production in koji-extract containing various quan-
tities of NaCl during two weeks at 28-30 C. is greatest in

5¢ NaCl, i.e. 5.25% of ethyl alcohol., 20% NaCl disturbes its

growth to a high degree (Cf. Table 3).

The Second Variety.

Cells are spherical or oval, 5-8 y in size, contents homogenous
and bright, vacnoles large, stains brownish red with KI
solution.

On plate culture: White greyish, elevated and very folded
colonies on surface; round and smooth in inner part as in
the case of the first variety (Cf. Fig. 2). Streak culture:
greyish white, elevated and dull along the track, flat and
smooth in the centre and wavy on both sides. The streak
culture on koji-extract-agar differs distinctly from that of the
first variety. Stab culture is very similar to the first variety.
Gigantic colony is white elevated and folded, with wet surface

(Cf. Fig. 7). In the above mentioned fluid media it grows

348

(c)

(d)

(a)
(b)

T. MITSUDA:

very favorably, forms a white greyish film on the surface of
media after long time, but very quickly in the media contain-
ing some NaCl.

Spore-formation was not observed on gypsum block at any
temperature as in the first variety.

Faculty of assimilation of carbon and nitrogen from various
sources comes very closely to the first; also carbon is assimilated
pretty well from ethyl-aleohol (Cf. Table 1).

This variety can grow in koji-extract containing 2,5% succinic
acid or 1.25¢ acetic, but not in 1.75% acetic acid.

The optimum temperature for growth lies at 30° C. at 34° C.
growth is much retarded.

Fermentative faculty closely resembles that of the first variety
(see Table 2). Apparent fermentation coefficient 70.8%.
Amount of aleohol produced in koji-extract containing NaCl
varies with the amount of NaCl added. 5¢ NaCl in koji-
extract gives the most favorable result i.e. 5.25% ethyl alcohol;
20% NaCl gives 3.78% aleohol (Cf. Table 3).

The Third Variety.

Spherical form: 5-8 / in diameter, bright and homogeneous.
On koji-extract-agar plate, it forms bright greyish white, round
and elevated colonies, with smooth surface and periphery (Cf.
Fig. 3). Streak culture: A white, waxy,, bright and ele-
vated growth, the folds on surface finer and less than that
of the foregoing two varieties, layers and canals at the center
of growth, periphery toothed. Stab culture: A white irre-
gular, highly elevated and smooth growth at the mouth of
stab eanal, but in inner part it is moniliform with gas bubbles.

Gigantic colony: The development of the gigantic colony

aan

eR TD

(¢)

(d)

(e)

(a)

(b)

NOTE ON YEASTS OF “‘SHOYU”-MASH. 349

is very similar that of the second (Cf. Fig. 8). It grows in
many nutrient media forms yeast ring after 5 days at 28° C..
but does not form any complete film on the surface of media
even after 15 days at 28° C. It makes most favorable growth
in koji-extraet or wort containing 10% NaCl and forms a grayish
film after a long time on the surface. In droplet culture a
single cell grows quickly and makes a large band. Spore for-
mation on gypsum block was not observed.

This variety can assimilate carbon from almost all the carbo-
hydrates and some organic salts, but not from ethyl-alcohol.
Also, nitrogen from various forms of nitrogenous compounds
except nitrite (Cf. Table I).

It can grow in 2.5% succinic acid and 1.254 acetic acid in koji-
extract, but not in 1.5% acetic. Its development is profoundly
disturbed by 10% ethyl-alcohol in koji-extract. Its apparent
fermentation coefficient in koji-extract is 73%.

Tt can develope favorably at a comparatively high temperature
i.e. its optimum temperature for growth lies at 30-32C. About
fermentative faculty and ethyl alcohol production in NaCl

solution of koji-extract of this variety, see tables 2 and 3.

The Fourth Variety.

Spherical or oval cells, mean size 5-8.5 jy, sometimes larger
cells (i.e. “Dauerzellen’” 10 ».) are found in yeast ring.
Vacuoles large, cells bright, granular, and rich in glycogen.
On plate culture are found white grayish, elevated, irregular,
and dull colonies, their surface is wavy and elevated especially
at the center, with round and smooth periphery in inner part
of the medium. (Cf. Fig. 4). Streak culture: Very

similar to that of the second variety but whiter in colour. Stab

350

(c)

(a)

(e)

(a)

(b)

T. MITSUDA:

culture: White, dried and elevated growth is found at the
mouth of the stab canal. Gigantic colony: very similar to that
of saké-yeast, white, elevated and foded in the form of a erater.
(Cf. Fig. 9). It grows in various nutritive media. No film
is found at 25° C. during 15 days, but yeast ring is formed
after 3 days 30 C: Most remarkable development was observed
10% NaCl solution in koji-extract. By droplet culture after
Lindner a single cell developes very quickly into a large band.
This variety assimilates carbon from many carbohydrates and
some organic salts, nitrogen from various form of its com-
pounds. (Cf. Table 1.)
It grows in 2.5% succinie or 1.5% acetic acid, but not in 2%
acetic acid, in koji-extract. Its growth is retarded by 10%
alcohol and no further production of alcohol takes place. Its
optimum temperature for growth lies at 30-32° C.
The fermentation faculty of sugars resembles that of the former
variety, and ihe apparent fermentation coefticient is 66.9¢
Production of ethyl-alcohol in koji-extract was more or less

disturbed when NaCl. was increased (Cf. Tables 2 and 3).

The Fifth Variety.

Cells spherical or oval, mean size 4-8 u, not bright. One or
more moving granules in the vacuoles (or “each vacuole”).

On plate culture of koji-extract-agar, round, yellowish-
white, elevated, smooth, waxy, bright colonies appear on the
surface, with smooth periphery. (Cf. Fig. 5). Streak culture
is yellowish-white, waxy, elevated and bright. Surface and
periphery smooth. Slab culture: A yellowish-white round,
elevated growth, smooth and bright at the mouth of stab canal

and many small colonies with gas bubbles along the canal.

NOTE ON YEASTS OF “SHOYU’’-MASH. 351

Gigantic colony. Yellowish white, elevated, smooth, creamy
bright colony, with smooth periphery. Growth in the fluid
media mentioned above, is favorable. The film formation was
not observed at any temperature, even in NaCl containing koji-
extract. In droplet culture a single cell developes very quickly
into a large band.

(d) Assimilation of carbon and nitrogen from various sources and
resistance against organic acides similar to those of the fore-
going varieties. The optimum temperature of growth lies at
28-30 C, but it is comparatively resistent against high tem-
perature. It ferments dextrose, saccharose, maltose and others.
but not lactose ete. It is curious that this yeast grows well
in wort, but not in koji-extract. The apparent fermentation
coefficient is 37.14¢. NaCl has a deterrent action on the pro-
duction of ethylaleohol ie. 2% NaCl in wort gives only 1.60
ethyl alcohol, while the control gives 3.57% (Cf. Table 3).

Summary.

It is highly probable that the 5 varieties described, differs from Sac-
charomyces Soja, Saito, by fermenting Sucrose and raffinose, while his
yeast does not ferment either. But the third variety somewhat resembles
to Saito’s, fermenting these two sugars very feebly. While, Sacch. Soja
forms spores only in the cells of the yeast ring, it does not form on gypsum

block, but in our yeast varieties they always absent.

352 T. MITSUDA:

TABLE I. SOURCE OF CARBONE AND NITROGENE

Variety I.

Cane sugar +++ b++ +++ +++ +44
Dextrose... +44 +++ oe fora +++
Galactose .. oF + trace hut + ++
actose! vas. eee + to trace oF ah
Fructose ... shards sParae +4+ +++ +44
Mannit Pe +44 +++ + + rhea e
Dextrin ++ +++ + ++ ttt
Arabinose a + trace — + ae
Glycerine; 55 sss: +b4e ety + + >
Inulin +5 spate te +
Na-acetate ... ... ++ + trace + ++
Na-lactate’” =. fe rm
Am-tartarate ... +++ ota: ciate +++ +44
Alcohol ... +r = + +
Am-carbonate... ... 4... Pst + SP ese + purest
Am-tartrate ... ... spies +++ Shaeat +44
Na nitrate aha hats nyse ce +4+
Nitrite - = trace =
Am-sulphate het ttt +++ +44
Asparagine... ... ar ar ar arse cearae +++
Peptone ...

NOTE ON YEASTS or “SHOYU”-MASH. 353

TABLE II. FERMENTATION FACULTY.

Variety I | li | Til VI Vv

eee

TABLE III. AMOUNT OF ALCOHOL PRODUCED IN NaCl SOLUTION OF
KOJI-EXTRACT OR WORT.

Variety I.
el
% or NaCl Sp. Gr. of Distillate Alcohol in Vol. 9%
0.0 0.9930 4.88
5.0 0.9925 5.25
10.0 0.9930 4.48
15.0 0.9540 4.18
200 0.9955 3.07

354 T. MITSUDA:

Variety IT.

Sp. Gr. of Distillate Alcohol in Vol. 9

% of NaCl

0.9925 6.25

5.0 0.9925 5.25
10.0 0.9930 4.88
15.0 0.9935 4.51

0.9945 3.78

Variety IIT.

% or NaCl Sp. Gr. of Distillate Alcohol in Vol. 9

0.9920

5.0 0.9920 5.58
10.0 0.9920 5.58
15.0 0.9930 4.88

0.9950

Variety IV.

% of NaCl Sp. Gr. of Distillate Alcohol in Vol. 9

0.0 0.9930 4.88
5.0 0.9935 4.58
10.0 0.9935 4.51
15.0 0.9940 4.18
20.0 0.9955 5.07

NOTE ON YEASTS OF “‘SHOYU”-MASI, 355

Variety V.

% of NaCl Sp. Gr. of Distillate Alcohol in Vol. 7
0.0 0.9955 3.57
2.0 0.9965 1.160
5.0 09980 1.34
10.0 0.9985 100
15.0 —- —-—
20.0 —_— =

Explanation of Figures.
Plate XVI.

Figs, 1-5, The growth of colonies of yeast on koji-extract-gelatine plate at 28C.
Figs. 6-9, The growth of gigantic colonies of yeast on koji-extract-gelatinc 30 days at
ro m temperature.
Fig. 1, Variety 1.

NED, 55 Zh

igs 13: 3:
Les se 4.
Big. 5, ; 5
Fig. 6, ]
LE Se 2

“~
~

Jour. Coll. Agric. Vol. I. PS xaVale

Zwei neue Aspergillus Arten aus ,,Katsuobushi.‘
Von

M. Yukawa.

Mit Tafeln XVII und XVIII.

In Japan ist die Verwendung des getrockneten Fisches als Volks-
nahrung schon von alters her bekannt, und vor allem wird getrockneter
Bonit (Gymnosarda pelamis) im Haushalt stark konsumiert. Zwecks
Herstellung des getrockneten Tunfisches, “Katsuobushi” genannt, wird
zunichst das in lingliche Stiicke geschnittene Fleisch des Tunfisches in
einem Kessel gekocht und, wenn es abgekiihlt ist, in ein Fass einge-
packt, worauf allmahlich Schimmelpilze auf dem Fleische wachsen.
Das mit Schimmel bedeckte Fleisch wird in der Sonne getrocknet, und
nachher die Schimmelpilzdecke abgeputzt. Diese Behandlung, d.h.
die wechselsweise Einpackung und Trocknung des Fleisches, wird so
lange wiederholt, bis die Haltbarkeit desselben vollstindig sicher ist. Im
Handel ist das getrocknete Fleisch, das mit griinlichen Schimmelpilzen
bedeckt ist, teurer als das mit gelben Pilzen.

Trotz vieler Untersuchungen zur Verbesserung des Trocknungsver-
fahrens des Tunfisches existiert bislang keine Angabe tiber die mikro-
skopische Hrforschung. und daher halte ich fiir gut. diese Studie zu
veréffentlichen. Aus verschiedenen von mir untersuchten “Katsuobushi”
Proben habe ich stets zwei Arten des Aspergillus, zuweilen ausserdem
Aspergillus albus, Verticillium glaucum, Penicillium glanecum, Mucor
racemosus und andere Pilze isoliert. Von diesen beiden Aspergillus

Pilzen wird die griine Art vorziiglich aus der teureren Ware isoliert,

358 M. YUKAWA:

wihrend die mit bernsteinartiger Farbe aus den geringeren Sorten der
Proben erhalten wird.

Es ist hoch interessant zu finden, dass diese beiden Species eine stark
peptonisierende Kraft besitzen, sie sind also unbekannte neue Arten; fiir
die bernsteinartige Sorte schlage ich den Namen Aspergillus melleus vor,

fiir die griine Art den Namen Aspergillus gymnosardae.

I. Aspergillus melleus nov. spec. (Hierzu Taf. XVII, Fig. 1-7).

Es ist bemerkenswert, dass diese Art nicht nur auf verschiedenen
Substraten reichlich Sklerotien bildet, sondern auch die sekundiren Sterig-
men oft zu langgestreckten Schliuchen auswachsen lisst, und ferner die

Eiweisstoffe sehr kriftig verfliissigt.

A. Morphologisches,

Diese Art bildet sowohl auf festen wie auf fliissigen Substraten
anfangs weisse, bald bernsteinartige Deckne, bei alteren Kulturen geht
die Farbung in weisslich-braungelb iiber. Die vegetativen Hyphen sind
farblos, hell, zart und mit Querwinden versehen; ihr Durchmesser ist
2-5 ». Der Konidientriger entwickelt sich von den Mycelien aus zu
einem Seitenzweig und erweitert sich zu einem K6pfchen. Der Stiel
nimmt in der Decke von der Blase nach dem Képfchen allmihlich zu, um
schliesslich in eine Blase iiberzugehen. Die Blase steht aufrecht oder
nickend auf dem Stiel und ist in der Regel kugelférmig (selten keulig),
aber nicht scharf gegen den Stiel abgesetzt. Die ausgewachsenen Koni-
dientriger sind ziemlich stattlich (0,7 -1mm.), und ihre Breite 7-25 yp ;
die ilteren Triger sind mit ein oder mehreren Septa versehen und zeigen
gelb-braune Fiirbung. Die sehr diinne Wand (0,5-0,7 / dick) des Stieles
ist glatt und farblos, beim Aelterwerden aber oft rauh warzig. Die Blase,
20-50 2 im Durchmesser haltend, ist allseitig mit dicht gedringten, radiar

ausstrahlenden, stets verzwveigten Sterigmen besetzt. Die primiiren

ZWEI NEUE ASPERGILLUS ARTEN AUS ,,KATSUOBUSHI“. 359

Sterigmen sind keulig und haben wieder je 3-4 langliche sekundare
Sterigmen; ihre Linge ist 10-22 » und ihre Breite 2,5-4 4. Die sekun-
diren Sterigmen sind kegelférmig, 10-16 » lang (zuweilen bis zu 30 / )
und 1-2 # breit. Missbildungen, z. B. abnormes Auswachsen der Sterig-
men, Gabelung des Stieles, unregelmiissige Verzweigung der Trigerspitze
unter Fortfall der Blalsenentwickelung u.a., sind nicht selten bei dieser
Art.

Die nicht kurzen Konidienketten sitzen an der Spitze der sekundaren
Sterigmen. Die Konidien sind klein, glatt, kugelférmig (oder selten
ellipsoidisch) und messen 2,5-4 im Durchmeseser.

Diese Art bildet reichlich sowohl auf festen wie auf fliissigen Sub-
stanzen kugelige, harte Sklerotien (Grésse 0,4-0,7 mm. Dm.)

Die Sklerotien entstehen durch Verflechten und Verwachsen gleich-
wertiger Hyphen und die Rinde derselben ist gelbbraun, aber das Mark

farblos. Askusbildung ist noch nicht bekannt.

B. Physiologisches.

Dieser Pilz wiichst iippig auf Reis, Bohnen, Kartoffeln, Brot, Koji-
gelatine und Agar, sowie in Bouillonpeptongelatine. Auch auf der Ober-
fliiche der fliissigen Nihrbéden bildet er die charakteristische Decke.

Besonders wenn die Kultur auf Pfeffer’s Lésung, Bohnen u.a., bei
23-25° C angestellt wird, erzeugt diese Art binnen 20 Tagen leicht grosse
Mengen Sklerotien auf der Myceldecke, mit Konidientragern ; aber bei
Zimmer- und héherer Temperatur wrd diese Bildung vermindert. Das
Wachstum des Pilzes zeigt keinen Unterschied in einer Losung von
Glukose, Maltose, Saccharose, Stirkekleister, ist aber spirlich in Laktose.
Bei den stickstoffhaltigen Nihrlésungen begiinstigen Ammoniumsalze,
Aminosiuren, Pepton, Eiweiss und Siiureamide das Wachstum im Grade
ihrer Reihenfolge.

Die optimale Temperatur fiir das Wachstum des Pilzes ist 23-27° C,

doch kann der Pilz auch bei 40° C noch missig gedeihen.

360 M. YUKAWA:

Das Verfliissigungsvermégen des Pilzes fiir Gelatine wurde mit paral-
lelen Kulturen von Aspergillus Oryzae, Aspergillus albus, Aspergil!us
gymnosardae verfolgt?. Der Pilz schmilzt binnen 20 Tagen bereits die
Hialfte der Gelatine, indes Asp. gymnosardae und Asp. albus ein Drittel,

Asp. Oryzae ein Sechstel vertliissigt.

Die Kraft des proteolytischen Enzyms? dieses Pilzes ist, wie oben
bezeichnet, ausserordentlich kriiftig; es verfliissigt nach kurzer Zeit
koagulierte Eiweissstoffe, Fibrin, Gelatine bei 40° C, wnd zeigt
dabei deutliche Biuretreaktion. Neutraler Zustand ist der Reaktion
am giinstigsten, im alkalischen verzégert sie sich und saurer ist ihr sehr
nachteilig; ein Teil des Enzyms list 100 Teile der 10 proz. neutralen Gela-
tine in 40 Minuten bei 40° C auf, aber unter derselben Bedingung nimmt
es eine Stunde dieselben Teile der 2 Proz. Natriumkarbonat enthaltenden
Gelatine zu verfliissigen, und ferner braucht es 114 Stunde 0,06 Proz.
Salzsiiure enthaltende Gelatine zu lésen. Die Enzymlésung verzuckert
Stirkekleister, invertiert die Saccharose, hydrolysiert auch die Maltose,
das Inulin und die Mannane. Die Guajaktinktur bei Anwesenheit von
Wassersioffsuperoxyd wird durch das Enzym blau gefirbt, auch zersetzt
es Wasserstoffperoxyd unter Sauerstoffentwicklung. — Ferner hydroly-
siert ein wisseriger Auszug dieses Pilzes Monobutyrin®. Diese Versuche
zeigen uns, dass der Pilz Amylase, Invertase, Glykase, Inulase, Semi-
nase, Pevoxydase, Katalase und Lipase ausscheiden kann.

Der Farbstaff wird durch Alkohol extrahiert.

Eine wichtige Tatsache ist, dass diese Art aus iilterer Kultur anf
Koji-gelatine Calciumoxalat-Krystalle erzeugt, und reichliche Mengen
von saurem Ammoniumoxalat auf gediimpften Bohnen.

1. Strichkultur auf Koji-gelatine (1594) bei 8-15°C.

2. Enzympraparat wurde in tiblicher Weise erzeugt.
3. Comptes rend. de l’Ac. 1897, Bd. 124 S. 370.

ZWEL NEUE ASPERGILLUS ARTEN AUS ,,KATSUOBUSHI™. 361

C. Affinitat.
Dieser Pilz steht in vielen Beziehungen Aspergillus ochraceus Wil-

helm* und Aspergillus auricomus Gueguen® nahe, jedoch stimmt nach

Wehmer® jedenfalls Asp. auricomus mit Asp. ochraceus iiberein.

Nach den Beschreibungen des Asp. ochraceus unterscheidet sich Asp.
melleus dadurch, dass der Konidientriger von Asp. ochraceus stattlich
und 2-3 mm. hoch ist (selbst 4-10 mm., aber nach Schroter* bis 1 mm.),
bei Asp. melleus ist derselbe dagegen niedrig. Auch die Konidien des
Asp. ochraceus sind 8,5 - 5 yp, aber die des Asp. melleus 2,5-4 4. Ueber-
dies ist die Farbe des Konidienrasens bei Asp. melleus im Anfang bern-
steinartig, spiiter wird er weisslich braungelb, dagegen hat Asp. ochraceus
eine Farbe von ockergelb bis braungelb. Auch bei direkten Kulturen des
Asp. ochracens Wilh. (Went) sowie Asp. ochraceus Wilh. (Tiraboschi), die
mir neulich Herr Nakazawa iibersandte, sahen wir, dass ein deutlicher
Unterschied zwischen Asp. ochraceus und Asp. melleus vorhanden ist.
Tm direkten Vergleich zeigen beide Arten night nur obenerwihnte Merk-
male, soudern Asp. ochraceus unterscheidet sich von unserer Art noch
durch foleende Tatsache: der Képfchendurchmesser des Asp. ochraceus
ist Kleiner als der des Asp. melleus, und die Konidienketten von ‘Asp.

ochraceus sind stets kiirzer als die unserer Art.

D. Diagnose.

Typhen farblos, hell, septiert, auf Fliissigkeiten zu dichten Decken

verflochten. Reife Konidienrasen bernsteinartig, spiirter weisslich braun-

4. Wilhelm, K. Beitrage zur Kenntnis der Pilzgattung Aspergillus 1877.
5. Bull. Soc. Mycol. de France T. XV. 1899 p. 171.
6. Centrabl. f. Bakt. If Abt. 1907 Bd. XVIII S.392.
Wehmer. Die Pilzgattung Aspergillus 1901 S.115,

362 M. YUKAWA:

gelb mit zahlreichen, ziemlich stattlichen Konidientriigern; Stiel farblos,
gerade oder gebogen, diimnwandig, Wand glatt oder warzig. Blase kuge-
lig, seltener keulig, nicht scharf abgesetzt und allseitig von dicht gedriingt
stehenden, verzweigten, radial ausstrahlenden Sterigmen besetzt. Primire
Sterigmen keulig, sekundiire kegelférmig, in der Regel zu 3-4. Sekun-
dire Sterigmen oft zu langer Strecke auswachsend. Konidienketten
verhiltnismiissig nicht kurz. Reife Konidien meist kugelig, selten ellip-
soidisch, stets glattwandig, kleinsporig. Sklerotien reichlich gefunden,
ohne Askusbildung. Optimale Wachstumstemperatur 23-25° C.
Vorkommen: Auf getrocknetem Tunfisch (Japan) spontan auftre-
tend. Gedeiht gut auf Bohnen, Brot, Gelatine, Fisch ete. Gebildet wer-
den Amylase, Invertase, Inulase, Glykase, Seminase, Peroxidase, Kata-

lase, Lipase, Protease.

GROESSENVERHAELTNISSE.

Elyphendorehmesser cc n---ieiece s-9)) =< lace 2-5 p

Keonidientrizer! “Sc eeeupecst fess) (cer: =) “ass 0,7—1 mm.

Stieldicke™= 2.5 Wacsae ese Mactan? cate” axes ee 7-25 wu

Sieleanddicke” Seo “sccMaecsiueee Sve nae Have 0,5—-0,7 p

Képfchendurchmesser ... 2... 0 ee eee eee wee 50—250 p

Blasendurchmesser 9 55 cc: ew cso eee ane 20—50 p

Primare Sterigmen) “sees ae eee sce oce! ees 10—22 px2,5—4 vp

Selcundare ‘Steripmenives, Wass bes facs.- cee cose 10—16 p (selten bis 30 py)x1—-2 p
Konidiendurchmesser ... ... 1... 22 see ave 25-4 pv

Sklerotiendurchmesser.,. ... 1... 0 cs+ ces 0,3—0,7 mm.

II. Aspergillus gymnosardae nov. spec. (Hierzu Taf. XVIII,
Fig. 1-7d).

Dieser Pilz ist als ,, Awokabi“* bekannt und im praktischen Gebrauch

sehr wichtig zur Fabrikation des ,,Katsuobushi“. Er tritt jedoch nicht

8. Awokabi bedeutet griiner Schimmelpilz, und gewdbnlich nennen wir Penicillium
glaucum so, aber bei Katsuobushi ist es nicht Penicillium glaucum, welches fast gar nicht in

Katsuobushi auftritt,

ZWEI NEUE ASPERGILLUS ARTEN AUS ,,KATSUOBUSHI™. 363

unter kiinstlicher Kultur auf, sondern spontan auf dem Fleische wihrend
der Einlagerung von ,,Katsuobushi“, wenn die Feuchtigkeit und die Tem-
peratur in dem Fasse miissig sind. Ueber den praktischen Wert der
Verwendung der kultivierten Sporen dieses Pilzes bei der Bereitung von
,,Katsuobushi* werde ieh mich ein andermal aussprechen. Ich weiss die
Ursache noch nirht genau, warum diese Art am besten fiir die Fabri-
kation des ,,Katsuobushi“ ist, warum die Qualitiit der mit dieser Art
bedeckten Proben fein und vorziiglich ist, wihrend die mit Asp. melleus
bedeckte Ware als gering gilt.

Nach der Farbe der Konidienrasen machen die griinen Arten das
Gros der Gattung aus, aber die Beschreibungen fiir die gréssere Reihe
dieser Arten sind unvyollstiindig. In folgendem werde ich nur diesen
Pilz mit den kenntlich beschriebenen iihnlichen Arten nach Wehmer*

vergleichen.

A. Morphologisches,

Auf den verschiedenen zur Kultur benutzten Substraten bildet dieser
Pilz einen dicken, anfangs weissen, bald gelblich griinen, seltener lanb-
griinen Konidienrasen. Bei alteren Kulturen geht die Farbung schliess-
ich von unansehnlichem Griin bis ins sehmutzig Dunkelbraune_ iiber.
Gelbliche Tone an den auf verschiedenen Substraten gezogenen Decken des
Asp. flavus treten immer tiefer auf als bei diesem Pilz. Die Konidien-
triiger des Pilzes sind stattlich, 1-2,5 mm. hoch, (beim Asp. flavus 0,5-
0,7 mm.) und meist einfach, seltener verzweigt, aber die Alteren
Traiger sind mit vielen Querwiinden versehen. Die Wand des Konidien-
trigers ist 1-2 y dick, elatt oder auch rauhwarzig. Das Ende des Triigers
quillt zu einer kugeligen oder keulenférmigen Blase (20-40 ” im Dur-

chmesser), die im der Regel nicht scharf yop dem Stiel ahgesetzt ist.

9. Wehmer. Die Pilzgattung Aspergillus 1901 S. 61.

Lafer Handbuch der Tech. Mykologie Bd. IV S. 202,

364 M. YUKAWA:

Die Sterigmen sind radial von den Seiten der Blase ausstrahlend, oder
bei kleineren Triigern mehr auf die Kuppe beschrinkt und aufwirts
gerichtet (bei Asp. pseudoflavus Saito’? nur radial ausstrahlend).
Sie sind farblos, meist einfach, doch auch verzweigt wie beim Asp. candi-
dus, Asp. Ostianus, Asp. pseudoflavus. Im Falle der Verzweigung der
Sterigmen sind die primaren Sterigmen oben breit-keulig und die sekun-
diiren zart, schlank und zu drei oder vier auftretend (bei Asp. pseudoflavus
zu zweien auftretend). Die einfachen Sterigmen haben eine Lange von
10-25 und eine Breite von 5-6 » (in den verzweigten Sterigmen: primire

Sterigmen 10-20 » x5-6 # , sekundire 10 # x 2-3 /#).

Die Linge der Sterigmen iiberschreitet den Blasenradius und ist oft
gleich dem Blasendurchmesser ; der Pilz gehért also zu der ,,Langstrahlen“ ;
diese Merkmale erméglichen eine Unterscheidung von anderen ihnlich
gefirbten Arten (in diesem Punkte unterscheidet er sich von Asp. glaucus,
Asp. Oryzae, Asp. flavus, Asp. pseudoflavus). Der Inhalt der iilteren, von
den Sterigmen befreiten Blase erscheint oft den Konidien gleich gefirbt,
wodureh wieder ein Unterschied gegen Asp. Oryzae, Asp. flavus und Asp.
psendoflavus gegeben ist. Die Konidien treten in leicht und bald zer-
fallenden langen Ketten auf; die Liinge der Konidienketten dieses Pilzes
stimmt mit Asp. flavus und Asp. Oryzae tiberein. Sie sind gross, allegemein
kugelig, seltner schwach elipsoidisch, meist warzig oder glatt und gelb-
eriinlich gefirbt. Nach der Konidiengrésse beurteilt, gehért diese Art
zu den ,,grosssporigen ; die Grisse dieser Organe ist 4-6, hierin steht
er weniger hinter Asp. Oryzae, Asp. flavus, Asp. pseudoflavus zurziick.
Misshildungen der Konidientriiger sind nicht selten.

Das Mvecel der Art besteht aus zarten, septierten, farblosen Hyphen
von 2-5 4 Durechmesser, und die iilteren TWyphen werden 6fter gelbbraun
gefiirbt wie bei Asp. glauqus und Asp. varians.

Sklerotien und Perithecien sind noch unbekannt.

10. Centralbl. f, Bak. II, Abt, XVIII S, 34.

ZWEI NEUE ASPERGILLUS ARTEN AUS ,,KATSUOBUSHI™. 365

B. Physiologisches.

Der Pilz gedeiht auf sehr verschiedenen Substanzen fliissigen wie
festen Charakters; auf Kojidekoktlésung, Bierwiirze, Reis, Brot, ist die
Art leicht zu ziehen. Doch bevorzugt der Pilz Zuckerlisung ganz
gleich ob mit Eiweiss oder mit anorganischen Stickstoffverbindungen neben
Kalimmphosphat und Magnesiumsulfat. Die Farbe der Konidienrasen
ist loslich in Alkohol, unléslich in Wasser. Auf den Kulturen in Wiirze
und Kojidekoktlésung bildet dieser Pilz Gasblasen. Auch liebt er hohe
Temperaturen, ein Wachstumsmaximum liegt 41° C, aber er kommt
mehr bei Blutwirme zur Entwickelung, bei Zimmertemperatur ist er
ein wenig schwerfiallig. Die Art bildet Amylase, Invertase, Glykase,

Peroxydase, Katalase, Lipase und Protease.

DIMENSIONEN,
iohy PAREN GUC HIN CSS calle a wei mses i Mcscimiscciisse) cite 2-5 p.
Wonidichttaceras tsumersc=-smiser ii sccdl foeel Menclitises! eke oes 1—2,5 mm.
SUAAIGHES ass ask ck Gee ceo, oes OS eo eco A oe 10—20 p.
ne Wwandd icke seme oer Can annie sears) em) ccs 0,8—1,2 p.
Maprelrendurchmesseni nage tess) (acsliescsl sees ses) se on 100—500 vu
RRR TGITC INGE 5 ces 06s oso 20—49 p
iiieedins SiG 5-5 50g beg) ca oe foo 12—25 px5—6 vu.
Brimare Slerigmen 25.5 see ec) oes Tose eee tem wee 10-20 px5—-6 v
Sekundaere(Sterigmenf) 22. s.5 cen Wen) see ar ue see 10 ux2—-3 v

ISOMIGICN esa mee mnse MERCeSIcee) Messi UeeeM iscsi yeccyo fess) isso) ese 4-6/4

Fig.

Fig.
Fig.
Fig.
Fig.
Fig.
Fig.

Fig.
Fig.
Fig.

Fig.

M. YUKAWA:

TAFELERKLARUNG:

Tafel XVIT.

1-7 Aspergillus melleus Dov. spec.

1. (Vergr. 15) Ein Stiick der Myceldecke mit den aufliegenden Sklerotien neben
Konidientrigern (halb schcmatisch).

2. (Vergr. 400) Konidientrager.

3. (Vergr. 400) Entwickelungstadien der Konidientrager.

4. (Vergr. 560) Sterigmen.

5. (Vergr. 700) Konidien,

6. (Vergr. 409) Anormale Gestalten der Konidientrager.

7. (Vergr. 125) K6pfehen mit langen Steri-zmen.

Tafel XVIII.

1-7. Aspergillus gymnosardae nov. spec.

1. (Vergr. 52) Habitusbild des Képfchens.

2-4. (Vergr. 700) Konidientrager,

2. Exemplar mit keuliger Blase und kupp-.nstindigen Sterigmen.
oD Wohlerhaltenes, gut entwickeltes Exemplar,

4. (a—c) Zwergige Konidientrager.

5 (Vergr. 700) a) einfache Sierigmen. b) verzweigte Sterigmen.

6. (Vergr. 700) Konidien.
7a—d(Vergr.700) Abnormal geformte Konidientrager.

Vil.

‘Taf. X

5665

H. Oye. Tokyo.

Lith.

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Influence of Rice Bran upon the Manurial Value
of Phosphoric Acid Contained in Oil
Cakes.

Y. Kida.

In Japan various vegetable manures are most widely used by farm-
ers, of which oi] cakes from an importana item. Oil cakes contain gen-
erally more nitrogen than phosphoric acid and they are treated rather as
nitrogenous manure. Their effect of nitrogen has been studied already by
many authors not only in our college but also in many of our prefectural
experimental stations, but on the manurial value of the phosphoric acid
contained in vegetable manures only little is known.

M. Nagaoka’ concluded from ris rice experiment that the manurial
value of vegetable phosphoric acid was inferior to animal phosphoric acid,
as the following table shows:

——_

Relative manurial va'ue

Control-Double superphosphate ... ... 1... -.. 2... 100
Shime-Kasu (Sardine)... ... 89

Shime-Kasu (Herring)... ... 82

Animal manures Arakasu (lish bone)... ... 97
Steamed bone meal... ... 87

Average Ree Nanay nies 8s

1. Bull, College of Agric, Tokyo, Vol. IV.

368 ¥. KIDA:

Rice bran! Gis. ee 42

Rape seed cake = --.) poss) yess 30

Vegetable manures Sesame Cake -c..e5 see 38
Soy beancakets 923" a 28

Average RY Pare 35

So it seems quite desirable to enhance the manurial value of phos-
phoric acid in vegetable manures; especially in oil cakes, as they are used
very largely.

In recent times, various organic phosphoric compounds have been
found in plants; besides nuclein and lecithin also phytin has been found

widely distributed in the vegetable kingdom.

According to Tsuda’s' analysis, the phosphoric acids contained in oil
cakes are chiefly in three forms: lecithin, nuclein and phytin, of which

the last contains the largest proportion of phosphoric acid, as follows:

1N 100 PARTS OF DRY MATTER

Total P.O, Pie te Bie
PO; in Lecithin in Nuclein | in Phywn
re = = a
Soy bean cake... 1.311 0.114 0236 | 0.640
Rape seed cake ... <.. 4. 2.251 0.091 0 204 | 0.873

Prof. K. Aso and T. Yosrida concluded from their experiments that
the manurial value of lecithin is much higher than that of nuclein and
phytin, and the inferior value of vegetable phosphoric acid as compared

with animal is accordingly well recognised.

This brief recapitulation shows the desirability of increasing the

1. Journal of the College of Agric. Vol. I., No. 2. P. 167.
2. The same Journal Yol. I., No. 2. P. 152.

MANURIAL VALUE OF PHOSPHORIC ACID CONTAINED IN OIL CAKES. 369

availability of phytin as a first step towards a greater utilization of vege-
table phosphoric acid for manurial purposes. Prof. U. Suzuki and K.
Yoshimura® lately found an enzym called phytase which splits phytin
with the production of a soluble inorganic phosphoric compound and

inosit, and which is widely distributed in plants.

As oil cakes are exposed to a high temperature to facilitate the separa-
tion of oil, either by streaming or by some other method, the activity of
the contained phytase seems to be greatly reduced, and it is therefore of
some interest to investigate the manuring value of the phosphoric acid of
cil cakes can be enhanced by mixing rice bran which is known to contain
much active phytin splitting enzyme. It was with this object in view that

the following experiments were made unler the direction of Prof. K. Aso.

I. Experiment: to investigate whether phytase is still active in

Rape seed cake and Soy bean cake.

A.—Rape seed cake, soy bean cake and rice bran purified of fatty
matters with petroleum ether for 24 hours, were used in this experiment.
Three Erlenmeyer’s flasks were filled with 10 grams of these fine substances
and 200 e.c. of distilled water, well stoppered with cotton and then sub-

jected to the following treatments:

Flask a.—Left at room temperature.
5, b.—2 ec. chloroform added, kept in the ineubator at 30° C.
; @—Heated for one hour in Koch’s sterilising kettle and then

left at room temperature.

Afier seven days, the contents of each flask were filtered with dried
filters and the filtrates were tested for inorganic soluble phosphorie acid

with molybdie method, with the following results:

3. Bull. College of Agric. Tokyo Vol. VII., No. 4.

370 Y. KIDA:

P,O, found

Flasks

in one flask (g) in % of total P,O,
be 0.08672 41.90
|
Rape seed cake (2.11593 P.O,)| b 0.08848 41.83
c 0.06272 29.65
|
a 0.07904 65.43
Soy bean cake (1.20896 P,O,) b 0.07856 65.03

c 0.06683 55.32
a 0.12292 39.36
Rice bran (3.12393 P,O,) b 6.19588 62.72
c 0.06276 20.09

This shows that in neither bean cake nor rape seed cake. the quantity
of phosphoric acid underwent any great change with the different treat-
ments, while in the case of rice bran the change was conspicucus.

Taking the average of b and ¢ we get:

P.O, (b— c)

in 9% of total P,O,

Rape seed cake; G2) a:-0e sme eee 0.02516 12.18
Soy ‘bean: cake nates: oes eee 0.01173 9.71
Rice (brant. ce ssierere meester ee 0.13312 42.62

Sw Shw—w—

These figures show that the activity of phytase in both rape seed and
soy bean cakes is much affected when compared with that of rice bran, as
was to be expected. As in (b) the action of microbes was excluded by
chloroform, which has no influence upon phytase, while in (¢) both these
agents were inactivated, the difference of (b—c) will represent the activity

of phytase,

MANURIAL VALUE OF PHOSPHORIC ACID CONTAINED IN OIL CAKES. 371

B.—A similar experiment as in A was repeated and a very different
result’ was obtained. The experiment was carried out with only slight

modifications in the treatment of the mixtures, as given below:

Flask a.—3 ¢.c. of chloroform added, then left at rooin tempera-
ture.
» b.—8 ee. of chloroform added, then left in the ineubator at

35°—40° C.
.. d.—Heated for one hour in Koch’s sterilising kettle, 3 cc. of
chloroform then added and left at the room temperature.
After standing for seven days, the contents of each flask was filtered
and the soluble inorganic phosphoric acid was determined with the follow-
ing results:

nnn Eryn EE nn

P,O, found

Flasks
in one flask (g) in % of total P,O,
a 0.11324 47.86
b 0.10968 46.36
Rape seed cake (2.36624 2P 0, )
c 0.12216 51.63
d 0.09104 38.48
a 0.09384 70.08
b 0.07904 59.
Soy bean cake (1.339% P,O,)
c 0.08544 63.81
d 0.06632 49.54
| ee 0.20148 47.01
| is 0.21428 | 49 99
Rice bran (4.286% P,O,)
c 0 28208 | 65.81
d 0.05816 13.57

|
SS eEEEEEEEEEEEEEEEEEEEEEEEENEEEEEEEEEEEEEE

1. Substances used in all the following experiments were of just the same quality as in

this experiment.

372 Y. KIDA:

These figures show also that under these treatments the quantity of
soluble phosphoric acids in the cakes is not conspicuously different from
that of the rice bran, the difference between (c) and (d) in particular being
very slight, but the activity of phytase was much more noticeable in rice

bran than in the pressed cakes experimented on.

The average of (c) and (d) gives the following results:

P,O, (c—d)
(g) in % of total P,O,
Rape: seedicakes <..) 25) ses) eave == 0.03112 14.27
Soy. beanveakei2- <=, con ace Meare ieee 0.01912 13.15
Ricevbranic..: 55 pen cen 0.22392 52.25

It follows that these quantities of phosprorie acid were produced from

phytin by the action of the phytase contained in these vegetable substances.

As the flasks were exposed to a high temperature, the separation
of soluble phosphoric compounds must have been more or less accelerated
by the heat, though the enzym does not exert—accordingly the amount
of P,0, in (e¢) seems in each case to be too large. The following experi-
ment was aarried out with the view of obtaining further hght on the

subject.

C.—-The experiment was done under exactly the same enoditions as
the foregoing with the only difference that the flasks of a, b and e were also
heated for one hour in Koch’s sterilising kettle (as with d) just before

being filtered after the various treatments for seven days. The results
£ ;

were as follows:

MANURIAL VALUE OF PHOSPHORIC ACID CONTAINED IN OIL CAKES. 373

—

P,O, found

Flasks ==
in one flask (g) in % of total P,O,
| a 0 11500 48 61
b 0.12140 51.31
Rape seed cake 5 ||
| c 0.12396 52.39
| d 0.09108 | 38.50
a 0.08160 60.94
b 0.07904 59.03
Soy bean cake...
c 0.08626 64.38
d 0.06555 48.96
|
| a 0.29864 69.68
|
ua 0.26040 60.76
Rice bran .. }
| c 0.39684 | 92.56
| d 0.12600 29.37

Keeping our attention to the essential factors concerned in the pro-
duction of soluble phosphoric acid we note that (a) was allow free play to
the actions of microbes, phytase and of heat, (b) to those of phytase and
heat (c) to that of phytase in its beneficial condition and of heat, at
‘Jast (d) to that of heat only, so that the action of phytase could be
clearly brought out by comparing (c), as follows:

ee —

P.O, (c—d)

(g) in % of total P,O,
eapesseedscake: | 225) "en) ace. eas, | nes 0.03288 13.89
Day WEE SR ee Sse ec! Ae) ccs 0.02065 15.42
NGS UD Sic esan act SOUR coy aoe 0.27084 63.19

374 Y. KIDA:

These figures lead me to conclude that phytase exists in these cakes
in an inactive or imperfectly active state, while in rice bran it exists
in larger quantities or in a more active state, and there remains much
phytin to be acted upon both in rape seed cake and soy bean cake. This
led me to think that it would be interesting to investigate whether the
phosphoric acid in the phytin of the cakes can be transformed into soluble
inorganic form by mixing rice bran with them, as the active phytase con-
tained in the latter seems to act well also on the phytin contained in the
pressed cakes, and further whether the manurial value of the phosphoric
acid of the pressed cakes, chiefiy produced from phytin can be enhanced

in this way.

II, Experiments to investigate the behavior of rice bran towards

the phosphoric compound contained in pressed cakes,

A.--In the following experiments the pressed cakes and rice bran
used were treated with petroleum ether and freed from fatty matter.

(i) Three Erlenmeyer’s flasks were used for this experiment. One
flask (a) was filled with 10 grams of the pressed cake and 200
c.c. of distilled water, well stoppered and heated in Koch’s
sterilising kettle for one hour, and after cooling for a short time
2 grams of rice bran were added. In (b) were put 10 grams of
the pressed cake and 200 c.e. of distilled water and heated as
in (a); in (c) 2 grams of rice bran and 200 c.c. cf disti.led water
and stoppered with cotton plug, (a) and (c) were then kept at
35°-40° C, and (b) left at the room temperature. After seven
days, filtered with dried filter, and the filtrate from each flask
analysed for soluble inorganic phosphoric acid with molybdie
method. To all the flasks were added previously 2 e.c. of chlo-
roform.

The analytical results were :—

MANURIAL VALUE OF PHOSPHORIC ACID CONTAINED IN OIL CAKES. 375

P,O, found

Flasks Remarks
in one flask (g) in % of total P,O,

10g heated bean cake = in
a 42g rice bran | ... 0.17648 83.35

b 10g heated bean cake 0.06708
0.14436 65.73

c 2g rlce bran. 0.07728
(b=) I teen cee cs 0.03212 14.62

10g heated rape seed a

a eve Oe Ege | Fe apenas 0.21016 65.20

b 10g heatedrapeseedcake}] 0.08416
0.16144 50.08

c* 2g rice bran 0.07728
a ore! ier eee 0.04872 15.12

We see that the difference a—(b+c) amounts to 20.59% of the total
phosphoric acid contained in the rape seed cake and to 23,99% of that in
soy bean cake. These quantities of P,O; must have been derived from
the phytin of the pressed cakes, and accordingly we may conclude that
the rice bran acts favorably on the phytin of the pressed cakes, as was

to be expected.

(ii) Another experiment on the same basis was carried out with the
only difference that the flasks were not exposed to heat. After
leaving for seven days at 35-40° C the filtrate from each

flask was analysed with the following results :—

* The result given is the mean of two similar flasks. This is the case for the corresp-

onding item in all the following tables.

376 Y. KIDA?

P,O, found
Flasks Remarks
in one flask (g) in 7% cf total P,OW
10g bean cake ; “
a 2g rice bean Batssiewea--10s) D088 $9.°5
b 10g bean cake 0.09488
} 0.17268 78.63
c 2g rice bran 0.07780
a=(W-6)) iI) iy a RMP ec c 0.02420 11.02
10g rape seed cake ae
2 42g rice bran veseeeeeeees 0.25656 79.60
b 10g rape seed cake 0.11704) i
0.19484 60.75
ic 2g rice bran 0.07780
a=(b-Fc), i fy meee = Sere 0.06172 18.85

The results were quite in accordance with the expectation, the soluble
phosphorie acid in (a) was increased to such an extent that it exceeded the

suin of (b) and (c); the difference a

(b+e) amounted to 18.074 of the
total phosphoric acid contained in the bean eake and to 16.06% of that in
rape seed cake, This was mainly due to the action of phytase contained

in rice bran. ,

BL. In the following experiments all the manures were used in a

finely pulverized form without freeing them from fatty matters.

(i) The sume experiment as (i) of A was repeated with the modifi-
cation that the quantity of rice bran was increased to 5 grams

in each ease. The flasks (a) and (c) were kept at 30° C, while

(b) was left at the room temperature. After seen days’ stand-

ing, their filtrate was examined for soluble phosphoric acid.

MANURIAL

VALUE OF PHOSPHORIC ACID CONTAINED IN OIL CAKES. 2

377

P,O, found

in one flask (g)

in % of total P,O,

Bete ann see 0.21268

0.05304
0.16780
0.11476

cesses ex --s 0.04488

Flasks Remarks
=i 10g heated bean cake
+5g rice bran

b 10g heated bean cake

c 5g rice bran
a—(b+c)

a 10g heated rape seed

cake +5g rice bran

b LOg heated rape seed cake

c 5g rice bran
a—\b+c)

sea apgeoone 0.20964
0.06376

0.17852
0.11476

spans os 0.03112

46.49

The difference a

(b+e) amounts to 13.154 of the total phosphorie

acid contained in rape seed cake and to 33.52% of that in soy bean eake.

From the above table it is seen that the rice bran may act on pressed

eakes in their raw condition at

value.

(ii)

30°

C so as to inerease their manurial

and the results were as follows :—

A similar experiment as (ii) of A was carried out, but in this

case 10 grams of rice bran were used with (a) and (c) each

P,O, found

Flasks Remarks
in one flask (g) in % of total P5O,

10g bean cake Rap eC
a = 410g rice bran} tt 0.26320 46.79

b 10g bean cake 0.08672
0.24840 44.16

c 10g rice bran 0.16168
a—(b+c) sreeeeeeeyy 0001480 2,63

378 Y. KIDA:

10g rape seed cake 3 ec
a ite cite brame| 0.31420 47.23
b 1 g rape seed cake 0.11860
0.28028 42.13
c 10g rice bran 0.16168
a—Gite |b oe, WR» eRe eee 0.03392 5.10

The difference a—(b+c) is small in bean cake and rape sced cake, but
compared with the total amount of phosphoric acid in each cake it is

14.33% in rape seed cake and 11.054 in bean cake,

(iii) One more experiment was made this time reducing the rice bran

to half the quantity used in the foregoing.

P,O, found
Flasks Remarks
in one flask (g) in % of total P,O,
10g beau cake Ee
a 45g rice bran} vote 0.21116 60.64
b 10g bean cake 0.08672")
0.20132 57.82
c 5g rice bran 0.11460 )
a—(D-EC)) ll IL wenteneseees 0.00984 2.82
10g rape cake 2.9995
a 45g rice bran seetasscrnes 2.29228 64.82
b 10g rape seed cake 0.11860
0.23320 51.72
c 5g rice bran 0.11460
a—(b+c) seceeeeeeees 0.05908 13.10

Rice bran brought about in this case also the decomposition of the

phosphori¢ compound contained in the pressed cakes. 22.94¢ of the total
phosphorie acid contained in the rape seed cake were changed by the action
of rice bran into a soluble form; and in soy bean cake the correspo:d-

ing figure was 7.35%,

MANURIAL VALUE OF PHOSPHORIC ACID CONTAINED IN OIL CAKES. 379

Summary or Rersvrts.

I. The existence of phytase both in rape seed cake and soy bean
cake is certain, but its action in each case is very small.

II. Rice bran acts in such a way as to transform the organic phos-
phoric compounds of the latter to simple inorganic soluble ones, when
mixed under suitable conditions; thus enhancing the manurial value of
the phosphoric acid of the pressed cake.

III. The above result can be obtained not only in the pressed cakes

freed from fatty matters, but also in the raw state.

} All the experiments were made with fresh materials.

Ueber Oryzanin, ein Bestandteil der Reiskleie und seine

physiologische Bedeutung.

U. Suzuki, T. Shimamura und §. Odake.

Mit Tafeln XIX-XXVI.

I. Hinleitung.

Im Jahre 1897 hat Erskmann* zum ersten Male beobachtet, dass
Hiihner durch ausschliessliche Fiitterung mit geschialtem, sorgfiltig yon der
Silberhaut befreitem Reis in kurzer Zeit den Appetit verlieren und unter
starker Abmagerung zugrunde gehen. Er hat ferner darauf aufmerksam
gemacht, dass diese Erscheinung mit der Beriberi-Krankheit des Men-
schen grosse Aehnlichkeit hat. Werden die Hiihner mit ungeschaltem
Reis oder mit geschiltem Reis und Kleie gefiittert, so bleiben sie nicht
nur am Leben sondern selbst die Erkvankten werden bald damit geheilt.

Seine Beobachtung ist spiiter von verschiedenen Autoren nachgepriift
und bestitigt worden. Ueber die Ursache derselben giebt es jedoch keine
befriedigende Erkliirung, so dass die Meinungen weit auseinander gehen.

Nach Ersxmann ist diese Erscheinung eine Vergiftung durch irgend

einen Giftstoff im Stiirkemehl von geschiltem Reis, oder Gifte, welche

1. EIJKMANN, Eine beriberiihnliche Krankheit der Hiihner. Virchow’s Archiv, Bd.
148, S. 523 (1897). ——, Ein Versuch zur Bekimpfung der Beriberi. Virchow's Archiv,

Bd. 149, S. 187 (1897). ——, Archiv fur Hygiene. 1906.

382 U. SUZUKI, T. SHIMAMURA UND S. ODAKE:

sich bei der Girung von Stirkemehl in den Verdaungsorganen oder durch
anomalen Stoffwechsel im KGrpe erzeugen. Nach Mavrer? ist diese
Krankheit eine Vergiftung durch Girungsprodukte von Stirkemehl in
den Verdauungsorganen, besonders des Oxalsiiure. Saxaxi" glaubte, dass
sie durch die Einwirkung der Giftstoffe, die durch Einwirkung der Bak-
terien auf geschiiltem Reis entstehen, verursacht werde. Dagegen schrieb
Marsvsuira® die Schuld dem Mangel an Eiweiss zu, Scuaumann* dem
Mangel an organischen Phosphorverbindungen.

Wir wollen hier nicht in einzelne Details eingehen. Die Geschichte
der Beobachtungen, Literatur usw. findet man in der ,,Mitteilung der
Kakke-Studienkommission des japanischen Kriegsministeriums“ (1911).

Man kann nur mit Sicherheit sagen, dass die Reiskleie irgend einen
Stoff enthilt, welcher fihig ist, die erkrankten Tiere wieder zu heilen
oder Erkrankung vorzubeugen. Was fiir ein Stoff ist das nun? Wir
haben seit vier Jahren, teils gemeinsam mit Direktor Y. Kozar und
Dr. Anpo® und teils mit Dr. Krrao® und Waranase u. a. auf diesem
Gebiete gearbeitet. Nachdem wir die Beobachtung yon ErsKMann
nachgepriift und bestitigt haben, gingen wir einen Schritt weiter, um
den wirksamen Stoff der Kleie zu isolieren und die chemische Natur des-
selben genauer kennen zu lernen.

Wir stellten zuniichst folgendes fest :—

1. Der aetherische Extrakt der Kleie hat keine Wirkung; die entfet-

tete Kleie ist ebenso wirksam wie nicht entfettete Kleie.

1. Maurer, Archiv. f. Schiffs u. Tropenhyg. Bd. 13, H. 8 u. 9, 1909.
SAKAKI, Untersuchungen iiber giftigen Reis. Tokio 1902.

3. Marsusuira, Ueber die Aetiologie der Kakke Krankheit. Zeits, f. Hygiene u.
Bakteriologie, Bd. 2, S. 437, 1906 (japanisch).

4. SCHAUMAUN, Archiv f. Schiffs- u. Tropenhyyiene. Beiheft Bd. XII, 1908, u. Bd. XIIT,
1909. Vergl. auch Nocut, Ueber den vegenwiirtigen Stand d. Beriberi-Frage. Ibidem. S. 15

5. Kozat, ANpo, SuzuKI, und SHIMAMURA, Ueber dite sog. ,, Beriberiihnliche Krankheit “
der Vozel. Special Report of the Agricultural Expertiment Station Tokio 1910, August.

6. Suzuki, Suimamura, Krrao u. A, Journal of the Tokyo Chemical Society. Vol.
32, No. 1 (1911. Januar.), No. 2 (Februar 1911), No. 3 (April 1911), No. 9 (Sept. 1911),
Vol. 33, No. 2 (Feb. 1912).

UEBER ORYZANIN, EIN BESTANDTEIL DER REISKLEIE, ETC. 383

2. Wird die entfettete Kleie mit heissem Alkohol wiederholt extra-
hiert, so geht der wirksame Stoff in die alkoholische Lésung iiber und der
Riickstand erweist sich vollstiindig wirkungslos.+ Da der geschilte Reis
sehr arm an anorganischen Bestandteilen, wie Phosphor, Eisen, Calcium,

Magnesium, Kalium ete. ist, so haben wir zuerst geglaubt, dass die Tiere

-dureh Mangel an Mineralstoffen leiden miissen, wenn sie ausschliesslich

mit geschialtem Reis gefiittert werden. Diese Annahme kann aber kaumn
richtig sein, weil der mit Aether und Alkohol extrahierte Riickstand der
KXleie immer noch reich an Eiweiss, Stiirke, Faser, Phytin, Salzen ete.
ist. Femer haben wir festgestellt, dass Kasein, Pepton, Eieralbumin,
Lecithin, Phytin, anorganische Salze ete. keine Schutz- oder Heilwirkung
gegen die Krankheit haben. Nach Kasrura® hat das in Alkohol lésliche
Eiweiss der Gerste keine spezifische Wirkung.

3. Der alkoholische Extrakt der Kleie stellt einen sauer reagierenden,
dickbraunen Syrup dar, der sehr reich an Zucker, organischen Sauren,
Lecithin, harzartigen Substanzen und anderen Verunreinigungen ist.
Wird nun dieser alkoholische Extrakt in wenig Wasser gelést, mit
Schwefelsiiure schwach angesiuert und mit Phosphorwoframsaure
yerstetzt, so entsteht ein flockiger Niederschlag der die Hauptmenge
des wirksamen Stoffes mitreisst, wihrend Zucker, organische Sauren und
andere Verunreinigungen meistens in der Mutterlauge zuriickbleiben.

Durch Zerlegung dieses Niederschlages durch Baryt erhilt man einen

- schwachsauren, hellbraunen Syrup, welcher etwa 10 mal wirksamer als
. der alkoholische Extrakt ist. Fiir dieses Praparat haben wir den Namen

_,,.Roh-Oryzanin (1) gewiahlt.*

4. Wenn man das Roh-Oryzanin (1) in wenig Wasser lést und mit

]. Verg]. H. Frasers. u. A. STAUTON, Studies from the Institute for Medical Research,
> Federated Manila States 1909; Y. TERUUCHI, Mitteilung der Kakke-Studienkommission des
Japan. Kriegsministeriums (1911); Z. Tsuzuki. Tbidem.

2. KayrurA u. O. RoseENHEIM, A Contribution to tne Etiology of Beriberi. Jour.
“Hyg. (Cambridge), 10 (1910), No. 1. pp. 45—55.

3. Diese Beobachtung ist schon in The Journal of the Tokyo Chemicul Society, Vol. 32,
No. 1 (Jan. 1911) mitgeteilt.

384 U. SUZUKI, T. SHIMAMURA UND §S. ODAKE:

Tannin versetzt, so wird cin Teil des Oryzanins mitgefillt. Nach Zerle--
gung dieses Tanninniederschlages durch Baryt und Entfernung des.
iiberschiissigen Baryts mittels Schwefelsiiure erhilt man einen hellbraunen
Syrup [Roh-Oryzanin (IT)], der nunmehr dreimal wirksamer als Roh--
Oryzanin (1) ist.*

Ein ziemlich reines Priparat kann man auch aus alkoholischem
Extrakt unmittelbar durch Tanninfillung erhalten.

Vor kurzem ist es uns gelungen aus Roh-Oryzanin (II) mittels Pik-
rinsiiure den wirksamen Stoff Oryzanin in reinem Zustande zu isolieren.? ”
Da die Ausbeute des Pikrats sehr geringfiigig ist, sind wir noch nicht im-
stande, die chemische Natur desselben aufzukliiren. Wir hoffen aber,
bald dariiber Naheres mitteilen zu kénnen.

5. Wird nun 0,005' bis 0,01 g des aus diesem Pikrate dargestellten
Oryzanins einer durch ausschliessliche Reisfiitterung erkrankten Taube -
per os gegeben oder subcutan eingespritzt, so wird das Tier in einigen
Tagen geheilt; der Appetit kommt bald zuriick und das Kérpergewicht
nimmt nach und nach zu. Man kann die Taube beliebig lange am Leben |
erhalten, wenn man dem geschiilten Reis tiglich 0,005 bis 0,01 g Oryzanin
zugiebt. Ohne dies geht das Tier in 2 bis 3 Wochen zugrunde. Da eine
Taube von ca. 300g Ké6rpergewicht tiglich 25 bis 30g Reis frisst, so
macht das Oryzanin nur 7/2599 bis 1/5000 des Futtermittels aus. Es ist
eine auffallende. Tatsache, dass eine so kleine Menge des Oryzanins cinen
so grossen Einfluss auf die Erniihrung des Tieres hat.

6. Es fragt sich nun, ob das Oryzanin bei anderen Tieren auch cine
ebenso wichtige Rolle spielt, wie bei Tauben. Bei Hiihnern, Miiuse und
Hunden haben wir beobachtet, dass das Verhalten des Oryzanins geuau
dasselbe ist wie bei Tauben. Miiuse sterben gewohnlich in 10 bis 15 Tagen
wenn sie ausschliesslich mit geschiltem Reis gefiittert werden. Sie blei-
ben aber liingere Zeit gesund und normal, wenn man den alkoholischen

Extrakt der Kleie oder das Roh-Oryzanin zugiebt.

1. Journal of the Tokyo Chemical Society, Vol. 32, Ne. 4. u. 9 (April u. Sept. 1911).
2. Ibidem. Vol. 33, Ne. 2 (Feb. 1912).

ees rer

+ ml i

em Belg Reape

SG A RE

UEBER ORYZANIN, EIN BESTANDTEIL DER REISKLEIE, ETC. 385

Wenn man Hunde mit gekochtem Reis und ausgekochtem Riickstand
des Pferdefleisches fiittert, so beobachtet man am Anfange keine Stérung.
Erst 2 bis 3 Wochen spiter geht der Appetit nach und nach zuriick und
nach 5 bis 7 Wochen gehen die Tiere unter starker Abmagerung zugrunde.
Nur 3 bis 4g alkoholischer Extakt der Kleie oder 0,3 bis 04g Roh-
Oryzanin (I) kann einen absterbenden Hund in ein paar Tagen heilen. Der
Appetit kommt bald zuriick und das Kérpergewicht nimmt sehr rasch zu.
Wird die Oryzaninzugabe eingestellt, so wird das Tier wieder krank. Mit
einem ausgewachsenen Hunde haben wir in 7 Monaten 4 mal denselben
Versuch wiederholt.

Da die Fette oder Salze keinen merkbaren Einfluss in diesen Fallen
zeigen, so nehmen wir an, dass das Oryzanin einen fiir Erhaltung des tie-
rischen Lebens unentbehrlichen Stoff bildet. Mit reinem Eiweiss, Fett,
Kohlehydraten und Salzen konnten die Tiere nicht lingere Zeit am
Leben erhalten werden. Es fehlt noch Oryzanin dazu.

Um diese Annahme weiter zu stiitzen, haben wir Tauben und Mause
mit einem Futtergemische gefiittert, welches aus einzelnen isolierten
Nahrstoffen zusammengestellt war. Zwei Tauben wurden mit Kartoffel-
stirke, Pepton, Lecithin, Phytin und Salzen gefiittert, zwei andere beka-
men noch dazu, 0,03 ¢ Roh-Oryzanin (1). Der Umnterschied zwischen bei-
den Gruppen war auffallend. Die ersten zwei Tauben gingen in 10 bis 15
Tagen unter starker Abmagerung zugrunde, wiihrend die letztere nicht
nur yollstiindig gesund blieben, sondern sogar bedeutend an Kérpergewicht
zugenommen haben. Anstatt Pepton haben wir auch Kasein, Eieralbumin
und Kleie-Eiweiss angewendet. (Das Kleie-Eiweiss wurde durch ver-

-diinntes Alkali aus der Kleie extrahiert und mit Essigsiure gefiallt.) Die
Ergebnisse waren auch genau dieselben. Ferner haben wir Tauben mit
einen eiweissfreien Futtergemisch gefiittert. Die Tiere konnten natiirlich
nicht lange leben. In kurzer Zeit gingen sie zmerunde. Trotzdem lebten
diejenigen, die Oryzanin bekamen, 3 mal linger als die, die kein Oryzanin

-erhielten. Die tigliche Abnahme des Kérpergewichts bei den ersten war

ungefihr 1/, des der letzteren.

386 CU. SUZUKI, T. SHIMAMURA UND S. ODAKE:

Welche Rolle das Oryzanin im Tierorganismus spielt, wissen wir~
gegenwirtig nicht. Es stellt nur fest, dass es zur Erhaltung des tierischen

Tebens unentbehrlich ist, wenigstens fiir Tauben, Hiihner, Mause und
Hunde.

Es sei hier erwihnt, das verschiedene Autoren schon mehrfach Ver-—
suche angestellt haben, um die Tiere langere Zeit, mit einem kiinstlichen
Futtergemische, aus einzelnen isolierten Nahrstoffen am Leben zu halten.
Die meisten Versuche erwiesen sich als erfolglos. Blos Réumann! und
Ossorne® haben in neuerer Zeit etwas bessere Resultate bekommen. Obg--
leich es keinen Zweifel giebt, dass die Konstitution des Eiweisses, die-
Mengenverhiltnisse und Verbindungsformen der Mineralbestandteile ete.
cinen grossen Einfluss auf das Gelingen der Versuche haben werden, darf
man doch bei Tierversuchen nie das Oryzanin iibersehen. Ohne Zweifel
hiitten R6umanwn und Oszorne noch bessere und befriedigendere Resultate -
gehabt, wenn sie in ihren Futtergemischen das Oryzanin zugegeben hitten

7. Da wir bis jetzt keine zuverlissige Methode ausgearbeitet haben,
um das Oryzanin in verschiedenen Futtermitteln zu bestimmen, so blieb -
uns uichts iibrvig, als durch Tierversuche die annihernde Menge desselben
zu ermitteln. Zu diesem Zwecke wurden die Tauben mit verschiedenen
Futtermitteln mit geschiltem Reis zusammen gegeben. In dieser Weise
haben wir fesgestellt, dass Weizen und Gerstenkleie, Bohnen, Hirse, Hafer, .
Gemiise ete. imstande sind, die Tiere lingere Zeit am Leben zu halten,
ohne an Kérpergewicht zu verlieren. Interessant ist dass die sorgfiltig
entkleiete Gerste, nach dem Kochen und Waschen mit Wasser, immer noch
fiihig war, die Tiere mehr als 100 Tage gesund zu halten. Auch gewohn-
liches Weizenbrot erwies sich als wirksam.

In Miso, Schoyu, Bier und Sake haben wir kein Oryzanin nach-
gewlesen.

Ob der wirksame Stoff in verschiedenen Futtermitteln immer mit
dem Oryzanin der Reiskleie identisch ist, oder ob es sich um eine Kor-
perklasse handelt, kénnen wir gegenwiirtig nicht entscheiden.

1. R6HMANN, Klin.-therap. Wochenschr. Nr. 40, 1902 und Allz. med. Zentralztg. 1908, Nr. 9
2. Osporne, Science N. S., Vol. XXXIV, No. 882, pp. 722-732 (24. Nov. 1911).

UEBER ORYZANIN, EIN BESTANDTEIL DER REISKLEIL, wre. 387

Milch, Fier, Fisch und Pferdefleisch als soleche, oder der alkoholische
Extrakt derselben haben fast keine Wirkung auf Tauben gezeigt. Bei
Hunden und Mausen war das Verhalten etwas anders. Der alkoholische
Extrakt des Pferdefleisches war fiir Hunde ebenso wirksam wie das Ory-
zanin. Fir Mause war der letztere etwas ungiinstiger, trotzdem konnten
wir in einigen Fallen die Miiuse mehr als 50 Tage vollkommen gesund
erhalten (bei Zugabe des alkoholischen Extrakis des Pferdefleisches mit
geschaltem Reis zusammen).

Der alkoholische Extrakt der Milch war auch fiahig, Miuse mehr als
50 Tage gesund zu halten, wahrend der Riickstand derselben sich vollstan-
dig als unwirksam erwies.

Es soll deshalb unsere zukiinftige Aufgabe sein, die wirksamen Stoffe
aus verschiedenen’ Futtermitteln zu isolieren und ihre chemische Natur

und physiologischen Funktionen zu studieren.

II. Darstellung des Oryzanins.

300 g entfettete Reiskleie werden in eimem 2undkolben mit 11.
Aethylalkohol (85 bis 90%) in Verbindung mit Riickflusskiihler 3 Stunden
lang gekocht. Man filtriert heiss ab, kocht den Riickstand noch 1 Stunde
mit 1% 1. Alkohol und saugt wieder ab; diese Operation wird 4 mal
wiederholt. Die gesamten alkoholischen Ausziige werden nun unter ver-
mindertem Druck so lange eingedanrpft, bis der Alkohol vollstiindig
ausgetrieben ist. Der zuriickgebliebene dickbraune Syrup wird wieder-
holt mit Aether geschiittelt, um die Fette, organischen Siiuren, Lecithinte
und andere Veruureinigungen zu entfernen, und weiter bei gelinder
Wiirme eingedampft. So erhiilt man einen ziemlich stark sauer reagieren-
den braunen Syrup, den wir der Finfachheit halber als ,,alkoholischen
Extrakt bezeichnen. Die Ausbeute desselben betragt ungefiihr 30 g, d. h.
ca. 10% des Ausgangsmaterials.*

1. Aus heissem alkoholischen Auszug scheidet sich beim Erkalten ein weisser, flockiger
Niederschlag ab, der mit wenig warmem Alkohol gewaschen, in heisem Benzol gelést und durch
Zusatz von Alkohol gereinigt wird. Es bildet ein schuppenformiges Pulver mit wachsaihnlichem
Glanz und schmilzt bei 84°C, Die Analyse gab C=80.44, H=13.339. Die cinfachste
Formel wire dann Co7H 340.

388 U. SUZUKI, T. SHIMAMURA UND S. ODAKE:

Der alkoholische Extrakt wird nun mit Wasser auf 100 cc. verdiinnt
und mit so viel Schwefelsiure angesiuert, bis sie ungefaihr 3% der Flissig-
keit ausmacht, und mit einer 30¢ igen Phosphowolframsiurelésung
versetzt. Es entsteht dabei ein brauner flockiger Niederschlag in reich-
licher Menge. Man braucht etwa 20 bis 30 c¢.c. Phosphowolframsiaure-
lésung dazu. Ein Ueberschuss des Reagens ist zu vermeiden. Nach
einigen Stunden wird der Niederschlag abgesaugt, mit 3% iger Schwefel-
siure einmal gewaschen. Man bringt den Niederschlag in einen Mérser,
gibt etwas Wasser zu, und verreibt mit iiberschiissigem Barythydrat, bis
der dicke Brei stark alkalisch reagiert (oder, man lést den Niederschlag
in acetonhaltigem Wasser und gibt so viel Barythydrat zu, bis die
Fliissigkeit stark alkalisch reagiert.) Nach einiger Zeit saugt man ab
und behandelt den Riickstand noch 3 mal in derselben Weise. Das
gesaite Filtrat wird nun durch Schwefelsiure sorgfiltig von Baryt befreit
und bei niederer Temperatur unter vermindertem Druck eingedampft.
Es bleibt dabei ein schwach sauer, hellbranner Syrup zuriick, der beim
Trocknen tiber Schwefelsiure zu einer harzartigen Masse sich verwandelt.
Wir haben fiir dieses Praparat den Namen ,,Roh-Oryzanin (1) vorgesch-
Jagen. (Friiher nannten wir es ,,Aberisiure'. Da aber das reine Ory-
zanin keine saure Natur besitzt, so haben wir den Namen fallen lassen.)
Die Ausbeute an Roh-Oryzanin (1) betriigt ca. 1,2 g aus 300 g Kleie, d. h.
0.4% des Ausgangsmaterials.

Wird nun 0,03 bis 0,04 @ Roh-Oryzanin (1) in wenig Wasser gelést,
und einer durch ausschliessliche Reisfiitterung erkrankten Taube per os
eingegeben oder subeutan eingespritzt, so wird das Tier schon am niichsten
Tage munter. Der Appetit kommt zuriick, das K6rpergewicht steigt,
und nach 3 bis 4 Tagen bemerkt man keine Zeichen der Erkrankung mehr.
Bekommt das Tier nur die halbe Dosis, so bleibt es lingere Zeit am Leben,
ohne jedoch yollstiindig geheilt zu werden, Das Kérpergewicht steigt
nicht auf. Eine gréssere Menge Oryzanin hat keine schidliche Wir-

kung. Wir haben einmal 5g alkoholischen Extrakt einer Taube gegeben,

1. U, Suzuki und T. Sutmamura, Journal of Tokyo Chemical Society, Vol. 32, No. 1
(1911).

UEBER ORYZANIN, EIN BESTANDTEIL DER REISKLEIE, ETC. 389

ohne irgend eine Stérung zu beobachten. Die Wirkung des Roh-Oryzanins
(1) ist deshalb ungefiihr 10 mal grésser als die des alkoholischen Extrakts
selbst, d. h. 0,05 g des ersteren wirkt ebenso gut wie 0.3g des letzteren.
Das Filtrat von Phosphowolframsiureniederschlag war beinahe frei
von Oryzanin. Wenn man das Filtrat mit so viel Barythydrat versetzt,
bis es schwach alkalisch reagiert, so wird die Schwefelsiure sowie die
Phosphowolframsiure vollstindig gefallt. Wird nun der dabei entstan-
dene Nieberschlag abgesangt und der Ueberschuss von Baryt durch
Schwefelsiure sorgfaltig entfernt, klar abtiltriert und bei niederer Tem-
peratur unter vermindertem Druck eingedampft, so bleibt ein hellbrauner
Syrup zuriick. Diese Praiparat hat uun kein Schutz- oder Heilwir-
:
kung mehr auf erkrankte Tauben. Es scheint also, dass der Hauptanteil
des Oryzanins durch Phosphowelframsiure gefillt, und was noch in
Lésung geblieben war, durch weitere Behandlung beinahe verloren gegan-

gen ist.

Reaktionen des Roh-Oryzanins (1).

Das in oben erwihnter Weise dargestellte Roh-Oryzanin (1) list sich
in Wasser und in.verdiinntem Alkohol sehr leicht. Die Lésung reagiert
schwach saner; gibt keine Binretreaktion; mit Miiion’schem Reagens
erwarmt farbt sich die Lésung dunkelrot; aus konzentrierter Lésung
entsteht sogar eine rotbraune Falling. Phosphowolframsiure oder Phospho-
molybdinsiure ruft in angesiuerter Lésung von Oryzanin eine flockige
Fallung hervor; Freniine’sche Lisung gibt bei gowéhnlicher Temperatar
eine schmutig-griine Firbung, beim Erwirmen entsteht ein flockiger
Niederschlag. Mit Natronkalk erhitzt, entwickelt sich Ammoniak. Wer-
den einige Tropfen Nesstrr’scher Reagens der Oryzaninlésung zugesetzt,
so wird die Fliissigkeit bei gewéhnlicher Temperatur allmahlich rotbraun
und beim Erwiirmen gibt sie einen dunkelbraunen Niederschlag. Beim
Gliihen hinterlisst das Roh-Oryzanin keine .\sche.

Eine charakterische Reaktion fiir Roh-Oryzanin (I) ist jedoch die

,,Diazo-reaktion. Wird frisch bereitete p-Diaz ybenzolsulfonsaiure in etwa

390 U. SUZUKI, T. SHIMAMURA UND S. ODAKE:

100 Teilen ganz verdiinnter Natronlauge gelést (die Lésung soll nur
schwach gelb gefarbt sein), und werden einige Tropfen Roh-Oryzanin-
lisung zugefiigt, so nimmt die Fliissigkeit sofort eine blutrote Farbung
an und zugleich merkt man eine geringe Schaumentwicklung. Nach
5 bis 10 Minuten erreicht die Maximumintensitit, die einige Tage un-
verandert bleibt.

Phosphomolybdinsiure gibt eine weisslichgriine Farbung; durch:
Zusatz von Ammoniak wird der Niederschlag gelist ; die Fliissigkeit nimmt
dabei eine tiefe indigoblaue Farbung an. Eine blaue Jod-Starke-Lésung
wird durch Zusatz von einiger Tropfen Oryzanin sofort entfiarbt.

Eine konzentrierte wasserige Lésung des Oryzanins wird dureh Blei-
essig teilweise gefallt, durch Zusatz von Ammoniak wird die Fallung
vollstiindiger, Queckilberchlorid, -acetat und -nitrat oder Gerbsiiure geberr

dabei eine unvollstindige Fillung.

Spaltungsprodukte des Roh-Oryzanins (1).

Durch verdiinnte Mineralsiuren oder Alkalien wird das Oryzanin
leicht gespalten; die eigentiimliche Wirkung geht dabei vollstiindig verlo-
ren. Emulsin wirkt auch allmihlich auf Oryzanin ein. Ohne Zweifel
ist das Oryzanin eine sehr labile Verbindung; daraus kann man die Beo-
bachtung von Erskmann, Suica! u.a., dass lingere Zeit aufbewahrte oder
verschimmelte Kleie keine Schutz- oder Heilwirkung mehr besitzt, leicht
erkliren.

Wenn mann 1g. Roh-Oryzanin (I) mit 100 ¢.. 34 iger Salz- oder
Schwefelsiiture 2 Stunden erhitzt, so wird die klare Fliissigkeit allmah-
lich triib und auf der Oberfliche der Fliissigkeit scheidet sich
eine harzartige Substanz ab. Filtriert man nun heiss ab und lisst das
Filtrat einige Stunden stehen, so scheiden sich gelbbraune Krystalle in

kleiner Menge aus; sie werden abgesaugt, mit wenig kaltem Wasser gewa-

1. SHIGA u. Kusama, Mitteilung d. Kakke-Studienkommission d. jap, Kriegsminis-
teriums (1911).

UEBER ORYZANIN, EIN BESTANDTEIL DER REISKLEIE, ETC, 39T

schen und aus heissem Alkohol wmkrystallisiert. Aus alkoholischer Lésung
erhilt man zwei verschiedene Sorten Krystalle; sie lassen sich durch un-
gleiche Léslichkeit in Alkohol leicht von einander trennen. Sie werden
vorlaufig als a-und §-Siaure bezeichnet, (Taf. XTX, IT u. IIT), die erstere
ist in Alkohol viel schwerer lislich als die letztere. Sie werden nochmal
fiir sich aus heissem Alkohol umkrystallisiert, mit wenige Alkohol und
Aether gewaschen, im Vakuum bei 100° getrocknet und analysiert. (Aus

alkoholischer Liswng scheiden sich die Krystalle durch Zusatz yon wenig
Salzsiure viel leichter aus. )

Analyse der a-Siure.

tlc 0.1129 g Subst. gaben 0.2206 g CO, 0.0426 ¢ H,O
2 0.0935¢ ,, » 0.1826 g CO, 0.0373 g H,O
3. 0.1514g =, » 0.2960 g CO, 0.0598 g H,O
4. 0.0722¢ ,, ” 4.4 c.c. N (18°, 766 mm)
bs 0.0963 ¢ ,, » 5.7 c.c. N (17°, 764 mm)
(c H N
CuEENO, Ber. 53.46 3.96 6.93
53.29 4.19 7.19
Gef. 53.26 4.43 =
53.32 4.39 6.91
Analyse der /-Siiure.
1. 0.1267 g Subst. gaben 0.2676 g CO, 0.0419 ¢ H,O
2 0.12442 |, » 0.2640 g CO, 0.0431 ¢ H,O
8 0.1005¢ ,, cf 0.2128 g CO, 0.0354 ¢ H,O
4. 0.1509 g ,, ” 9.0 c.c. N (21° 763 mm)
C H N
Cre ting Ng OG) weber. 58.25 3.88 6.80
57.60 3.68 6.82
Gef, 57.88 3.85 —
57.71 3.91 —

Die beiden Siiuren sind in kaltem Wasser schwer, in heissem Wasser

etwas leichter léslich; die wiisserige Lésung reagiert ziemlich stark sauer..

392, U. SUZUKI, T. SHIMAMURA UND S. ODAKE:

In Alkohol oder in Alkalien werden sie leicht gelést und durch Zusatz
‘von Siuren werden sie wieder ausgeschieden. Die beiden Saiuren geben
eine intensive Diezoreaktion; sie geben auch eine tief indigo-blaue Fiir-
bung durch Phosphomolybdinsaéure und Ammoniak. Sie geben auch starke
Minton’sche Reaktion. Kurzum, die charakteristischen Reaktionen des
Roh-Oryzanins (1) werden durch diese beiden Siuren hervorgerufen.
Unter den Spaltungsprodukten des Roh-Oryzanins (I) fanden wir
ausser diesen beiden Siiuren noch ziemlich viel Cholin, und Traubenzucker,
nebst einer organischen Siure, welche wir spiiter als Nikotinsiure
(m-Pyridinearbonsiure) identifiert haben. Die Menge des Cholins
und der Nikotinsiiure scheint bei verschiedenen Kleiesorten sehr verschie-
den zu sein. Wenn die Mutterlauge der und ,3-Siure mit Phosphowol-
framsiure versetzt wird, so entsteht ein weisser flockiger Niederschlag,
der nach einiger Zeit auf den Boden sich absetzt. Nach Zerlegung dieses
Niederschlages durch Baryt und Entfernung des iiberschiissigen Baryts
mittels Schwefelsiiure erhilt man eine alkalisch reagierende Fliissigkeit,
welche Cholin und Nikotinsiiure enthalt. Wird nun diese Fliissigkeit
mit Pikrinsiiure versetzt, so scheidet sich das Nikotinsiiurepikrat zuerst
aus und nach dem Einengen der Mutterlauge krystallisiert das Cholin-
pikrat als lange gelbe Prismen aus. Da das erstere in Wasser viel schwer
lislich als das letztere ist, so lassen sich die beiden Pikrate leicht von
einander trennen. Die Ausbeute an Nikotinsiurepikrat betriigt ea. 0,5 g

aus 4g Roh-Oryzanin (=1 kilo Kleie).
I. Nikotinsiurepikrat.
Aus heissem Wasser scheidet sich das Pikrat als hellgelbe kurzen
‘Stibchen ab. Es schmilzt bei 214° (unkorr.) unter Zersetzung.
Analyse des Pikrates.
1. 0.1602 g Subst, gaben 0.2376 g CO, 0.0348 g H,O

0.1291¢ » » 19.0c.c N(21.5°, 757mm)
0.1076 gy ¥ 0.0705 ¢ Pikrinsdure.

UEBER ORYZANIN, EIN BESTANDTEIL DER REISKLEIR, ETC. 398-

2) O15ll¢g =,, ” 0.2254 g CO, 0.0350 g H,O
0.1342 g ” 18.2 cc. N (12°, 763.5 mm)
(B H N Pikrinsdure.
C,H,NO,.C,H,N,0, Ber. 40.91 2.27 15.91 65.06
40.45 2.41 16.50 65.50
Gef.
40.68 2.57 16.19 —

Freie Nikotinsiure erhalt man, indem das Pikrat im Wasser gelést,
mit wenig Schwefelsiure angesaiuert, und wiederholt mit Aether geschiit-
telt wird, um die Pirkrinsaure zu entfernen. Nach Entfernung der Schwe-
felsiure durch Baryt, dampft man die wisserige Lisung bei gelinder
Warme ein, bis die freie Nikotinsiure als farblosen Nadeln sich aus-
scheidet. Nach einiger Zeit saugt man ab und wischt mit wenig Alkohol
und Aether.

Die freie Siiure reagiert ziemlich stark sauer, wird durch Phospho-
wolframsiure gefillt. Im Kapillarrohr erhitzt, schmilzt sie bei 228 bis

229° (unkorr. )

Analyse der freien Siiure.

0,1394 g Subst. gaben 00,2983 g CO. 0,0542 g HO
0,0707 g SCs, ” 7,05 c.c. N (14°, 764,5 mm)
( H N
C,H,NO, Ber. 58,54 4,07 11,38
Geb. 58,36 4,32 11,80

Ferner wurde das Kupfersalz sowie das Platinchloriddoppelsalz der
Nikotirsiure dargestellt und analysiert. Alle diese Priparate waren mit

denen aus reiner Nikotinsiiure dargestellten vollstiindig identisch.

If. Cholinpikrat.

Cholinpikrat scheidet sich aus stark konzentrierter Mutterlauge des
Nikotinsiurepikrates im grossen gelbbraunen Prismen aus. In Kapillarrohr
erhitzt, verwandelt sich die gelbe Farbe bei ca. 100° ins Orangerote und
zersetzt sich bei 240 (unkorr.) unter Schiiumen. Die Ausbeute an Cholin-
pikrat betriigt im besten Falle ca. 2 g aus 4 g Roh-Oryzanin (I) (1 kilo

Kleie).

394 U. SUZUKI, T. SHIMAMURA UND S. ODAKE? °

Die Analyse des im Vakuum bei 100° getrockneten Salzes gab fol-

-gende Zahlen :—

Analyse des Pikrates.

if 0.1493 g Subst. gaben 21,5 c.c. N (12° 756 mm)

Z 04928 ¢ , > 0.3414 g Pikrinsaure.
N Pikrinsaure.
C He NON CAHENEO: Mier. 16.87 68.97
Gef. 17.00 69.28

Aus dem Pikrate wurde das Platinchloriddoppelsalz des Cholins
-dargestellt, indem das Pikrat in wenig Wasser suspendiert, mit wenig
Salzsiiure angesiinert und wiederholt mit Aether geschiittelt wurde, um
-die Pikrinsiiure vollstiindig zu entfernen. Die farblose Lésung wurde nun
stark eingeengt und mit kleinem Ueberschiiss von Platinchloridlésung
versetzt. Es schied sich dabei das charakteristische Doppelsalz des Cholins
aus, welches bei 230 bis 232° (unkorr.) unter Verkohlung schmolz.
Das Platindoppelsalz wurde einmal aus Wasser umkrystallisiert, im

Vakuum bei 100° getrocknet und analysiert.

Analyse des Platinchloriddoppelsalzes des Cholins.

1. 0.2362 g Subst. gaben 0.0736 g Pt.
Ve

(C,H, ,.NO. Cl), Picl, Ber. 31.54
Gef. 31.54

Wir haben ferner aus reinem Cholin (Kahlbaum) das Pikrat und
«las Platinchloriddoppelsalz dargestellt und fanden, dass sie mit unserem

Priparat vollstindig identisch waren.

Til. Traubenzucker.

Aus dem Filtrat vom phosphowolframsauren Niederschlag der Nik-
tinsiure und des Cholins haben wir nach dem Entferenen der Phospho-
wolframsiure durch Baryt eine reichliche Menge Glukose als Osazon

UEBER ORYZANIN, EIN BESTANDTEIL DER REISKLEIE, ETC. 395

jsoliert. Dieses Osazon war nach einmaligem Umlésen in heissem ver-
-diimmtem Alkohol chemisch rein, hatte einen Schmelzpunkt von 202°
(unkorr.) und zeigte die charakteristischen Krystallformen.

Wir haben auch die Menge der Spaltungsprodukte anniihernd bes-
timmt. Aus 100 Teilen Roh-Oryzanin (1) wurden 10 F. a und Saure,
-30 T. Cholin und Nikotinsiiure, 23 T. Traubenzucker, ausserdem etwas
sharzartige schwarzbraune Substanz gewonnen.

1g Roh-Oryzanin gibt nach der Spaltung mit Saure

0.044 ¢ Gesamtstickstoff,

0.035 ¢ durch Phosphowolframsiure fallbaren Stickstoff,

0.009 g Stickstoff in anderer Form (hauptsichlich als a- und
-Saure) -

0.000 g Ammoniakstickstoff.

Da wir spater unmittelbar aus Roh-Oryzanin (1) ebensoviel Nikotin-
-siurepikrat isolieren konnten, wie nach dem Erhitzen des ersteren mit
verdiinnter Schwefelsiiure, so halten wir fiir wahrscheinlich, dass die letzt
.genannte Saure in der Kleie in freiem Zustande vorhanden ist.

Ob das Cholin auch als solches in der Kleie vorkommt, oder ob es
-erst nach der Erhitzen des Roh-Oryzanins mit Schwefelsaiure entsteht,
lasst sich nicht so leicht entscheiden. Es gelang uns jedenfalls nicht,
unmittelbar aus Roh-Oryzanin (I) eine nennenswerte Menge des Cholin-

~pikrates zu isolieren.
Weitere Reinigung des Oryzanins.

Zur weiteren Reinigung werden 4 g Roh-Oryzanin (I) in 100 ec. Was.
-ser gelist und mit einer 20% igen wisserigen Tanninlisung so lange ver-
-setzt, bis nur noch schwache Triibung entsteht. Man braucht dazu 15 bis 20
-eem Tanninlésung. Ein Ueberschuss von Tannin ist zu vermeiden. Die
weisslichbraune, flockige Fiallung wird abgesaugt, mit wenig 1% iger
Tanninlésung rasch gewaschen, (Ein Ueberschuss ist zu vermeiden.)
“Der Niederschlag wird nun auf Tonplatte gestrichen, getrocknet und dann
“tn einen Morser gebracht, mit wenig Wasser verrieben. Man giebt nun

-so viel Aceton zu, bis der Niederschlag gelést wird. Hierauf wird so viel

396 U. SUZUKI, T. SHIMAMURA UND S. ODAKE:

vesittigte Barytlisung zugegeben, bis die Fliissigkeit stark alkalisclr
reagiert, sorgfiltig verrieben und abgesaugt. Der Riickstand wird noch
zweimal mit Barytwasser verrieben und abgesaugt. Das verreinigte
Filtrat wird run mittels Schwefelsiiure von iiberschiissigem Baryt befreit
und im Vakuum eingedampft. Es bleibt dabei ein hellbrauner Syrup
in kleiner Menge zuriick, der nicht mehr sauer, sondern neutral oder
manchmal schwach alkalisch reagiert. Wir bezeichnen diesen Syrup mit
“Roh-Oryzanin (IT).** Die Ausbeute ist sehr gering. Aus 4 g Roh- Ory-
zanin (1) wird durchschnittlich nur 0,25 bis 0,3 g erhalten. Dieses
Priiparat war nun dreimal so wirksam als Roh-Oryzanin (I). 0,01 ¢
geniigte schon, um eine erkrankte Taube zu heilen oder von Erkrankung zu
bewahren.

Spiiter haben wir dieses Verfahren etwas modifiziert und zwar in
folgender Weise :—

Der Tanninniederschlag wird in einem Mérser mit 34 iger Schwefel-
siure sorgfiltig verreiben, abgesaugt und der -Riickstand noch mehrere
Male mit Schwefelsiure verrieben. Das Oryzanin geht dabei in Lésung
iiber. Die gesamte Fliissigkeit wird nun mit einem Ueberschuss von Baryt
versetzt, um Tannin und Schwefelsiure zu entfernen. Der dabei
entstandene Niederschlag wird abgesaugt und das Filtrat davon wird,
nach dem Entfernen des Baryts durch Schwefelsiure, bei verminderten
Druck stark eingedampft, mit Aether geschiittelt und weiter eingeengt.
Ir der Weise erhilt man einen hellbraunen Syrup, der gewéhnlich wirk-
samer als Roh-Oryzanin (IT) ist.

Wir haben auch éfters aus dem alkoholischen Extrakt der Kleie un-
mittelbar durch Tannin das Roh-Oryzanin gefillt und aus diesem Nieder-
schlaz durch weitere Behandlung mit 3¢ iger Schwefelsiiure ein ziemlich
wirksames Priiparat dargestellt. Oder man kann umgekehrt das Priparat,
das man unmittelbar durch Tannin-Verfahren erhalten hat, mit Phos-
phowolframsiure fallen.

Obgleich man in oben erwiihnter Weise schon ein ziemlich wirksames *
Priiparat erhalten konnten, konnte man es noch nicht als chemisch rein

betrachten, so lange es noch nicht krystallisieren wollte.

UEBER ORYZANIN, EIN BESTANDTEIL DER REISKLEIEL, ETC. a9T

Nach langen Bemiihungen ist es uns gegliickt, dies als Pikrinsiiures-
salz krystallinisch abzuscheiden. Wird eine konzentrierte wiisserige
Lésung des Roh-Oryzanins (Il) mit wenig Pikrinsiiure verrieben, so
scheidet sich das Oryzaninpikrat als gelbbraune, flockige Fiillung aus,
die beim Abkiihlen krystallinisch wird. Man saugt nun ab, wiischt
mit wenig kaltem Wasser und trocknet iiber Schwefelsiiure. Es bildet
ein gelbbraunes Pulver. Da das Pikrat nicht leicht krystallisieren will,
muss man nur sorgfaltig arbeiten. Gibt man zu viel Pikrinsiiure zu, so
verwandelt sich das Pikrat zu einer braunen, weichen Masse, oder, erwirmt
man die Lésung, so lést sich das Pikrat klar auf; nach dem Erkalten
krystallisiert es jedoch nichtmehr aus. Man muss auch damit rechnen,
dass das Roh-Oryzanin (IT) immer noch etwas Nikotinsiure als Verwnreini-
gung enthilt ; sie bildet auch ein Pikrat, welches sich schwer vom Oryzanin-
pikrat trennen lisst. Zu diesem Zwecke gibt man zuerst: nur ungeniigende
Menge Pikrinsiure zu und verreibt in der Kialte (nicht erkarmen!) ;
es scheidet sich das Oryzaninpikrat aus, wihrend die Nikotinsiiure in
der Lésung zuriickbleibt. Erst nach dem Erwiirmen mit viel Pikrinsiiure
scheidet sich das Nikotinsiurepikrat aus. Zu weiterer Reinigung des
Oryzaninpikrats, wird es in wenig kaltem Azeton gelést, klar abfiltriert
und iiber Schwefelsiiure langsam eingedunstet. In der Weise erhiilt man
das Pikrat als gelbbraune, mikroskopisch kleine Nadeln, welche stern-
formig sich zusammengruppieren. (Taf. XIX. I.) Es lést sich in
Aether und Petroliither nicht; in kaltem Wasser ist es ziemlich schwer,
in heissem Wasser, Alkohol und Aceton aber leichter léslich.

Die Ausbeute an Pikrat war leider sehr schlecht, so sind wir noch
nicht imstande, die genaune Beschreibung desselben wnd des freien Ory-
zanins zu geben. Ob es durch Spaltunga- und f-Siure sowie Cholin,

Traubenzucker ete. gibt, muss spiiter untersucht werden.
bo) I

Ili. Tierversuche.
Bei der Wirkung des Oryzanins auf Tiere ist vor allem zu
bemerken, dass z B. die Tauben bei ausschliesslicher Reisfiitterung

binnen zwei bis drei Wochen etwa 1/3 des urspriinglichen Kérpergewichtes

398 U. SUZUKI, T. SHIMAMURA UND 8S. ODAKE:

einbiissen und schliesslich zugrunde gehen. Eine gesunde Taube von
250 bis 300 g Kérpergewicht frisst am Anfang 20 bis 30 g Reis taglich.
Ungeschilter Reis oder geschiilter Reis mit 3 g Kleie kann das Tier lingere
Zeit vor Erkrankung hiiten, oder ein erkranktes in kurzer Zeit heilen.

Der alkoholische Extrakt, den wir in obenerwihnter Weise dargestellt
haben, hat auch dieselbe Wirkung wie Kleie selbst, wenn tiglich 0.3g
(aus 3 Kleie) per os oder mit Reis vermischt gegeben wird. Das Roh-
Oryzanin (1), das wir durch das Phosphowolframsiureverfahren aus
alkoholischem Extrakt dargestellt haben, war viel wirksamer als der al-
koholische Extrakt selbst. 0,03g geniigt um eine Taube vor Erkrankung
zu hiiten oder eine erkrankten zu heilen, d. h. 400 mal wirksamer als
Kleie.

Das durch das Tanninverfahren weiter gereinigte Priiparat ist wieder
dreimal wirksamer als Roh-Oryzanin (1) und das reine Oryzanin, das wir
als Pikrat krystallinisch erhalten haben, ist abermals doppelt so wirksam
wie das letztere. 0,005 g sind ebenso wirksam wie 3 g Kleie oder 0,3 g al-
koholischer Extrakt.

A. Tauben.

Versuch I,
Alkoholischer Kleieextrakt.

Hier wurde die Wirkung des alkoholischen Extrakts der Kleie auf
Tauben gepriift. Zwei ausgewachsene Tauben wurden erst 4 Tage mit
geschiltem Reis gefiittert, die nichsten 16 Tage, also von 5 tens bis
21 stens bekamen sie tiglich 0,3 ¢ alkoholischen Extrakt dazu; und
weitere 15 Tage wieder Reis allein. So lange sie mit alkoholischem
Extrakt versehen wurden, blieben sie gesund und munter und nahmen etwas
an Gewicht zu; aber bald nachdem der alkoholische Extrakt eingestellt
wurde, fingen sie an abzumagern, vorloren nach und nach an Gewicht und
erkrankten endlich. Hierauf bekamen sie wieder tiglich 0,3 g alkoholis-

chen Extrakt. Schon am niichsten Tage erholten sie sich erheblich. Die

UEBER ORYZANIN, EIN BESTANDTEIL DER REISKLEIE, ETC. 399

Esslust kam wieder zuriick und sie erreichten das urspriingliche Gewicht.
Gegen Ende des Versuches haben die Versuchstiere 18 Tage lang bloss
destilliertes Wasser anstatt gewohnlichen Brunnenwassers bekommen,
ohne irgend eine Stérung zu zeigen.

Aus diesem Versuchen kann man schliessen, dass der alkoholische
Extrakt fiir die Erhaltung des tierischen Lebens absolut notwendig ist,

falls die Tiere ausschliesslich mit Reis gefiittert wurden.

Versuch IT.
Geschilter Reis mit Roh-Oryzanin (I) und Salzen.

Mit 1000 g geschiltem Reis wurden 1,2 g¢ Roh-Oryzanin (1), 3,4g
Lecithin (Kahlbaum), 4,3 g Phytin, 2,6 g CaCOs;, 0.85 g CaCl, und 1,7 ¢
Na, CO, vermischt und an zwei Tauben verfiittert. Sie waren 17 Tage
yollkommen gesund und nahmen an Kérpergewicht zu. Nach 17 Tagen

avurde der Versuch unterbrochen.

Tabelle I.
Kérpergewicht
Versuchstage

(1) 2)

1 312 249
3 329 2959
i) 338 264
7 337 271
9 333 271
11 336 268
13 338 272
15 336 272

400 U. SUZUKI, T. SHIMAMURA UND S&S. ODAKE?

Versuch III. (Taf. XX.)
Roh-Oryzanin (1).

Zwei Tauben wurden zuerst mit geschiiltem Reis, Lecithin, Phytin,.
und Salzgemischen gefiittert. Nach 14 Tagen waren sie ermattet und
erkrankt. Nun wurde tiglich 0,03 g Roh-Oryzanin (I) per os gegeben.-
Schon am niichsten Tage waren sie beinahe geheilt; der Appetit kam
wieder zuriick. Nach 8 Tagen waren sie vollkommen gesund; das
Kérpergewicht nahm auch allmihlich zu. Nach 17 Tagen wurde der -

Versuch unterbrochen.

Tabelle IT.

K6rpergewicht

Versuchstage

Bemerkungen

Ohne Oryzanin
Erkrankt

Geheilt

Mit 0.03 g Reh-Oryzanin (1) taglich -

Vellstandig gesund

Versuch IV. (Taf. XX.)
Dass das Oryzanin zum gréssten Teil durch Phosphowolframsiiure-
aus dem alkoholischen Extrakt mitgerissen wird, wird dadurch bewiesen,

dass das Filtrat vom Phosphowolframsiiureniederschlag nur noch schwache

a»

UEBER ORYZANIN, EIN BESTANDTEIL DER REISKLEIE, ETC. 401

Wirkung hat. Wenn man das Filtrat vom Phosphowolframsiurenieder-
schlag durch Zusatz von Barythydrat von iiberschiissiger Phosphowolfram-

sinre und Schwefelsiiure befreit, und den Uberschuss vom Baryt wieder

- durch verdiinnte Schwefelsiiure vollstindig entfernt, klar abfiltriert und

unter vermindertem Druck verdampft, so erhilt man einen dickfliissigen
braunen Syrup, der auf erkrankte Tauben keine Wirkung hat. Die Taube
war nicht damit geheilt ; sondern wurde immer schwiicher. Nach 6 Tagen
wurden 0,02 g Roh-Oryzanin per os gegeben. Die Wirkung war iiberras-
chend. Nach zwei Tagen war die Taube schon gesund und nach 9 Tagen
hatte sie an Kérpergewicht um 38g zugenommen. Nach Einstellen der

Oryzaninzugabe magerte das Tier wieder allmihlich ab und erkrante.

Tabelle ILI.

Versuchstage KGrpergewicht Bemerkurgen
1 289 Gesund
4 277
6 280
u 278
8 261
Reis allein
9 253
10 250
ll 238
12 232
13 233 Erkrankt
14 226
15 227* *0.03 g Roh-Oryzanin (1)
(nur einmal gegeben)
Filtrat vom Phos- 16 243
phowolframsaure-
niederschlag 17 236

18 226
19 230 Erkrankt

402 U. SUZUKI, T. SHIMAMURA UND S. ODAKE:

Versuchstage Kérpergewicht Bemerkungen
20 224 |
21 235 Geheilt
22 236
23 247
24 260 Lee amg ee (1)
25 2H5
26 256
27 260
Filtrat vom Phos- 28 262 Gesund
phowolframsiiure-
niederschlag eo
29 261
30 261
31 261
32 263 Noch gesund
33 254
34 250
35 245
36 242 Allmablich schwach

Versuch V. (Taf. XX.)
Roh-Oryzanin (II).

Zu zwei Tauben, die vorher bei der Reisfiitterung erkrankt waren,
wurde 0,01 ¢ Roh-Oryzanin (11), am ersten Tagen subcutan eingespritzt
und vom zweiten Tagen an per os gegeben, Sie waren cbenso schnell
geheilt wie mit dem alkoholischen Extrakt. Nach 6 bezw. 7 Tagen be-
kamen sie wieder den Reis allein, trotzdem blieben sie noch mehrere Tage -

gesund und munter.

UEBER ORYZANIN, EIN BESTANDTEIL DER REISKLEIE, ETC. 403

Tabelle LV.

GED

Versuchstage K6érpergewicht Bemerkungen

296 Gesund

Reis allein

bo
es

229
223 Erkrankt

1 220 Geheilt
2 235
0.01g Roh-Oryzanin (II) { =
4 235
5 239

rf 6 242 Gesund
f 7 254
| 8 250
Reis allein H 9 247
| 10 245

| ll 242 Allmablich schwach

Versuchistage KGrpergewicht Bemerkungen

270 Gesund

Reis allein

223 Erkrankt

404 U. SUZUKI, T. SHIMAMURA UND 8S. ODAKE:

Versuchstage K6rpergewicht

Bemerkungen

i) te ~

0.01g Roh-Mryzanin (IT)

Reis allein 10
ll

Die Fiallung des Oryzanins durch Tannin ist nur eine unvollstindige.

< ——
=I > uw Coa

Noch nicht erkrankt

Bloss aus konzeutrieter Lisung ruft Tannin Fiallung hervor, die sowohl
durch Verdiinnung mit Wasser, als auch Zusatz von verdiinnten Sauren
und sogar durch einen Uberschuss von Tannin wieder gelést wird. Ferner,
wiihrend der Verarbeitung des Tanninniederschlages geht ein Teil Ory-
zanin verloren, so dass die Ausbeute mur eine sehr geringfiigige ist. Ein-
mal haben wir versucht, aus dem Filtrate des Tanninniederschlages Ory-
zanin zu gewinnen. Zu diesem Zwecke wurde 1g Roh-Oryzanin (1) in
100 cc, Wasser gelist und mit einer 20% igen Tanninlésung gefillt.
Das Fitrat von Tanninniederschlag wurde mit Barythydrat versetzt,
bis die Fliissigkeit stark alkalisch reagierte. Die dicke weissliche Fillung,
die eine griinlich bramne Farbe annahm, wurde abgesaugt und mit wenig
Wasser gewaschen. Das klare Filtrat wurde nun mit verdiinnter Schwefel-
siure sorgfiltig vom Baryt befreit, abfiltriert und unter vermindertem
Druck abdestilliert. Der dabei zuriickgebliebene hellbraune Syrup, der
ziemlich sauer reagierte wurde im Exsiceator tiber Schwefelsiure getrock-

net. In diesem Praparate war kein Oryzanin mehr vorhanden. Einmal

UVEBER ORYZANIN, EIN BESTANDTEIL DER REISKLEIX, ETC. 405

haben wir 0,04 g davon einer erkrankten Taube gegeben. Das Tier war
nicht damit geheilt und das Kérpergewicht ging allmablich herunter. Erst
-durch Verabreichung yon 0,01. g¢ Roh-Oryzanin (II) wurde es wieder
geheilt. Es scheint also, dass der Hauptanteil des Oryzanins in Roh-
‘Oryzanin (1) dureh Tannin gefiillt worden und was noch im Filtrat

geblieben war, durch weitere Verarbeitung verloren gegangen ist.

Zweite Versuchsreihe mit Roh-Oryzanin (II).

Hier wurde das Oryzanin durch Tannin fraktioniert gefallt. 1g Roh-
“Oryzanin (1) wurde in 100c.c. Wasser gelést und zuerst mit 10c.c. einer
20¢ igen Tanninlisung versetzt. Der Niederschlag (a) wurde abgesaugt.
Das Filtrat wurde wieder mit 10 cc. Tanninlésung versetzt. Es ent-
stand noch eine Fallung (b). Das Filtrat von (b) gab keine Fallung mehr,

Die beiden Niederschlage (a) und (b) wurden in oben angegebener
Weise mit Baryt zerlegt und daraus das freie Oryzanin dargestellt. Das
Praparat aus dem Niederschlag (a) war viel wirksamer als dasjenige
aus (b). 0,01 g des ersteren konnie eine erkrankte Taube in wenigen Tagen

heilen; wahrend das letztere nur langsam seine Wirkung entfaltete.

Tabelle V.

(i)
ee
Versuchstage Kérpergewicht Bemerkungen
283 Gesund
Reis allein | :
1 210 Erkrankt
2 212
3 226
0,01 g Roh-Oryzanin(IIy(a) Gepellt
4 218
5 27

6 220 Gesund

406 U. SUZUKI, T. SHIMAMURA UND S. ODAKE:

Versuchstage Korpergewicht Bemerkungen
7 220
s 219
9 226
10 227

IL 227 Gesund

12 223
13 229
Reis allein l4 224
15 225

16 223 Noch nicht erkrankt
17 223
18 226
19 220
20 217

21 208 Allmahlich schwach

(2)

Versuchstage Kérpergewicht Bemerkungen

Gessund

Reis allein

Erkrankt

UEBER ORYZANIN, EIN BESTANDTEIL DER REISKLEIN, ETC. 407

Versuchstage KGérpergewicht Bemerkungen
1 215
2 217
3 219
4 223 Geheilt
5 224 Gesund

6 225
ii 224
8 226
0,01 g Roh-Oryzanin 9 a5;
(11) (b) =
10 226
11 228
12 225
13 220
14 232
15 239
16 241

17 239 Kérpergewicht allmahlich

steigend

Ein ziemlich wirksames Praparat kann man auch unmittelbar aus
alkoholischem Extrakt durch Fallen mit Tannin erhalten. Zu diesem
Zwecke list man den alkoholischen Extrakt in méglichst wenig Wasser
und gibt so viel Tanninlésung zu, bis nur noch schwache Triibung entsteht.
Der braune Niederschlag wird in gewohnlicher Weise mit Baryt zerlegt
und weiter verarbeitet. Die Wirkung des so bereiteten Praparates war
nicht immer konstant. 0.01 @ desselben reichte jedoch in vielen Fallen

aus, wm eine erkrankte Taube zu heilen obgleich es nur langsam wirkte.

AQS8 U. SUZUKI, T. SHIMAMURA UND S. ODAKE:

Tabelle VI.

(ip)

Versuchstage Korpergewicht Bemerkungen

Gesund
Reis allein
Erkrant
Geheilt
0,01 g Roh-Oryzanin
Gesund

253 Gesund
Reis allein
201 Erkrankt

0,014 Roh-Oryzanin

209 Gesund

215 KGrpergewicht steigt

203
202
202
203 Nur langsam geheilt

CEBER ORYZANIN, EIN BESTANDTEIL DER REISKLEIE, ETC. 409

Versuch VI. (Taft. X XT.)

Der Tanninniederschlag selbst war auch wirksam. Da der Nieder-
schlag nicht im Wasser léslich war, wurde er in verdiinnter Natronlauge
gelést und gegeben. 9.65 @ desselben geniigte schon um eine erkrankte

Taube zu heilen.

Tabelle VIL.

Versuchstage Kérpergewicht Bemerkungen
Gesund
Reis allein
Erkrankt
1 18)
3 190
5 196 Geheilt
7 196
0,03 g Tanninniederschlag
” 200
11 202
13 204
1) 208 Gesund
17 209
19 213
21 216
Reis allein 23 211 Noch gesund
25 209
27 207
29 204 Allmahlich sehwacher

410 U. SUZUKI, T. SHIMAMURA UND S. ODAKE:

Versuch VII. (Taf. X XT.)

Reines Oryzanin.

Das reine Oryzanin, das wir aus dem Pikrate in oben erwahnter
Weise dargestellt haben, wurde nur in ganz geringer Menge gewonnen und
reichte fiir lingere Versuche nicht aus, deshalb haben wir es einmal einer

-erkrankten Taube nur 4 Tage lang und einer zweiten nur 3 Tage lang
gegeben und zwar der erstern 0.01g und der zweiten 0.005¢ tiglich. Die
Wirkung war trotzdem sehr deutlich; sie waren rasch geheilt und blieben
mehr als 10 Tage gesund und munter. Das Korpergewicht stieg auch

sehr hoch. Erst nach 10 Tage ging es wieder langsam zuriick.

Tabelle VIII.
(1)

Versuchstage KGrpergewicht Bemerkungen

Gesund

Reis allein

Allmahlich appetitlos

0,01 g

» Filtrat v. Pikraten
aus Roh-
Oryzanio (II)

0,02 g Fast keine Wirkung

Erkrankt

UEBER ORYZANIN, EIN BESTANDTEIL DER REISKLEIE, ETC. All

eee

Versuchstage KG6rperge wicht Bemerkungen
9 213 Schwach
10 207
0,02 g Pikrat=ca. 0.01 ¢ 11 217 Geheilt und munter
Orysanin 2 218
13 225 Gesund
14 233
15 245
16 238
17 243
18 245 Gesund
Reis allein 25

24 242 Noch gesund

=
to
a

Versuchstage KGrpergewicht Bemerkungen

Gesund

Reis allein

226 Erkrant

0,01 g Pikrat=0,005 g
Oryzanin

to
bo
—
Mil

3 225 Geheilt

412 U. SUZUKI, T. SHIMAMURA UND S. ODAKE:

Versuchstage Korpergewicht Bemerkungen

243
Reis allein
240
240
234 Gesund

232

225 Allm&hlich Appetit verloren

Versuch VIII. (Taf. XXII.)

Gemischtes Futter.

Vier Tauben wurden anstatt mit Reis, mit einem kiinstlich:

gemischtem Futter gefiittert. Die Mengenverhiiltnisse waren wie folgt:—-

Sticke: cc. ee Been eae 500,0 g CaCOs exe lace e eer aco are’
Pepton (3.0. Bate 25,0¢ CaGle tes. 78 ee ee he
ecithin;.<0) .cieaan wee 2,5¢g MEAG eos ee Ga ee US
Playtin: |..,aaeso ieee mans 2,5 g Nalco... ewer AN

Die Starke wurde vorher verkleistert, mit den iibrigen Stoffen
vermischt, bei niederer Temperatur getrocknet und in kleine Stiickehen:
geschnitten. Die ersten zwei (1) und (2) bekamen kein Oryzanin.
wihrend den anderen zwei (3) und (4) tiglich 0.03 g Roh-Oryzanin per:
os gegeben wurden. Der Unterschied zwischen beiden war auffallend ;-
(1) und (2) die kein Oryzanin bekamen, gingen in 10 bezw. 11 Tagen:
unter starker Abmagerung zugrunde; wihrend (3) und (4), die-
Oryzanin bekommen haben, nicht nur gesund blieben, sondern in 17 Tagen:

an Kdérpergewicht um 74 bezw. 42 @ zunahmen.
perg g£

UEBER ORYZANIN, EIN BESTANDTEIL DER REISKLEIE, ETC,

Tabelle IX.

(A) Ohne Oryzanin

K6érpergewicht u. Nr. d.
Versuchs- Versuchstieres

tage Bemerkung

413

Gesund

Erkrankt

Reis allein {
1
3
154
Starke, Pepton, Lecithin 6
Salze
(B) Mit Oryzanin

Reis allein {

Gesund

Oryzanin

Korpergewicht u. Nr. d.
Versuchs- Versuchstieres : k
tage a Sa Bemerkungen
Stirke, Pepton, |
Lecithin, Salze mit |

414 U. SUZUKI, T. SHIMAMURA UND S. ODAKE:

K6rpergewicht u. Nr, d.
Versuchstieres

Versuchs-

Bemerkungen
tage

Starke, Pepton,
Lecithin, Salze mit
Oryzanin

Gesund

317 KGrpergewicht steigt

Versuch IX. (Taf. XXII.)
Gemischtes Futter.

In diesem Versuche wurde das Pepton durch Gelatine ersetzt. Das

Futtergemisch hatte folgende Zusammensetzung :—

A. Ohne Oryzanin. B. Mit Oryzanin.

AG emer te cecum ALE A+10g alkoholischer Extrakt der
Gelatine),)~.. pase Fare Pee e Kleie.

Wecithintss --+) ca) Eee coe

Phytio ... 0 <5 sve eal gence 0 F

CaCO ee ee Oe

CaCl...) sen, Ee Oe

WaCOl Ne) ot ee Oe

KACO. an a een ee O Ee

Hier beobachtete man auch einen auffallend grossen Unterschied
gzwischen (A) und (B). Zwei Tauben, die mit (A) gefiittert wurden,
hatten von Anfang an keine Esslust. Das Kérpergewicht hat so rasch

abgenommen, dass sie nach 12 Tagen schon stark abgemagert und

UEBER ORYZANIN, EIN BESTANDTEIL DER REISKLEIE, ETC. 415

erkrankt waren. Die zwei anderen Tauben, die mit (B) gefiittert wurden,
konnten auch nicht das Gleichgewicht behalten und das Kérpergewicht
nahm auch allmahlich ab. Trotzdem waren sie nach 28 Tagen noch gesund.
Der durehschnittliche Verlust an Kd6rpergewicht betrug bei (A) 6,9
bezw. 5,2 g und bei (B) 1,4 bezw. 2,0 g. Also bei (A) war der Verlust
3 bis 4 mal egrésser als bei (B). Dieser Unterschied ist schlechthin dem
Oryzanin zuzuschreiben.

Die allgemeine Annahme, dass das Gelatin nicht das Eiweiss, wie

Kasein, Pepton ete. ersetzen kann, ist weiderum hier bestatigt.

Tabelle X.

(A) Ohne Oryzanin

Korpergewicht u. Nummer
Versuchs- des Tieres

Bemerkungen
tage

(1)

Gesund

Starke, Gelatin, etc.

Erkrankt

Taglicher Verlust an Gewicht bei A:
(1) 6,9g, (2) 5,2¢.

(B) Mit Oryzanin
EE EE EE ee EE Eee ee

K6rpergewicht u. Nummer

Versuchs- degetiexes Bemerkungen
tage —— +
(1) (2)
1 203 262 Gesund
Starke, Gelatine, etc. 3 23] id

mit Oryzanin

ur
ine]
~

416 t. SUZUKI, T. SHIMAMURA UND S. ODAKE!

KGrpergewicht u, Nummer
des Tieres

Versuchs-

tage Bemerkungenen

Starke, Gelatine usw. <t = = !
mit Oryzanin 19 226 208 :
21 221 206
23 219 202
25 220 202
27 213 205 KGrpergewicht geht

allmahlich herunter

Taglicher Verlust bei B:
Q) 14g, (2) 2,0¢

Versuch X. (Taf. XXII.)
Spaltungsprodukte des Eiweisses.

Wie oben erwiihnt, verlieren die Tauben ihr Kérpergewicht nur
lungsam, wenn sie mit eiweissfreiem aber oryzaninhaltigem Futter genahrt
werden. Darum haben wir versucht, ob das Aminosaiirengemisch, das
durch Spaltung des Eiweisses rein dargestellt wurde, den Verlust an
Képergewicht zu verhindern vermag.

Das Futtergemisch hatte folgende Zusammensetzung:

Marke .ic0 Se bee 1000 g NagCO ns. ey pas pneu Bee
Lecitittitis. 205) .o5 oe 5g Ca). [as das, <one ee lg
Phytin avd). dee». an Me eee 5g Alkoholische Extrakt d. Kleie 20¢

CACO, ss es ee «6S

UEBER ORYZANIN, EIN BESTANDTEIL DER REISKLEIE, ETC. 417

Die Starke wurden vorher verhleistert, mit iibrigen Stoffen vermischt,
getrocknet, zu kleinen Stiickchen geschnitten und zwei Tauben gegeben.

Das Aminosiiuregemisch bestand aus reinem Glykokoll, Alanin,
Leucin, Phenylalanin, Histidinhydrochlorat, Asparaginsiure, Tyrosin,
Cystin und Glutaminsiurehydrochlorat. Sie wurden ungefahr in der-
selben Mengenverhiltnisse gemischt, wie sie in gewohnlichen Eiweiss-
molekulen vorhanden sind. Leider haben wir kein reines Tryptophau
und Prolin zur Hand gehabt, und so ist dieser Versuch nicht als end-
giiltig anzusehen. Wir beabsichtigen spater nochmals diesen Versuch zu
wiederholen.,

Zwei Tauben wurden erst 33 Tage lang mit dem oben angegebenen
Futtergemisch genihrt. Sie haben dabei allmablich an Korpergewicht
verloren und waren beinahe erkrankt. Hierauf wurde vom 34. bis 49. Tage
faglich 0,3 g Aminosiuregemisch verabreicht; sie konnten das Gleich-
gewicht nicht behalten, obgleich die Abnahme des Gewichts viel lang-
sumer geworden ist. Vom 50. bis 80. Tage war die Aminosiiuremenge aut
5.0 @ vermehrt, die Abuahme wurde dabei noch langsamer und 14 Tage
lang haben sie sogar beinahe das Gleichgewicht behalten. Spater nahm
es allmablich ab und nach 95 Tagen ging das Tier schliesslich zugrunde.

Obgleich die Aminosiuren in diesem Versuch nicht imstande
waren, das Eiweiss zu ersetzen oder lingere Zeit das Gleichgewicht zu
behalten, haben die Tiere trotzdem viel liinger als ohne Aminosiiuren
gelebt, und so muss man annehmen, dass sie wenigstens den Eiweissver-
brauch im Tierkérper vermindert oder einen Teil des Eiweisses ersetzt

haben.

418 U. SUZUKI, T. SHIMAMURA UND S. ODAKE:

Tabelle XI.

Kérpergewicht u. Nr.
Versuchs- des Tieres

ee Bemerkungen
(1) (2)
254 | 276 Gesund
251 | 207
248 | 272
247 265
241 259
232 256
231 251
229 250
Starke, Lecithin,
Phytin, Salze ohne 228 246
Aminosauren
220 245
217 240
214 239
211 =
213 230
207 224
205 224 Allmahlich schwach
200 21
201 | 210
197 214
199 213 Etwas erholt
0.3 g Aiminosduren 199 205
sas 194 203
J97 201
199 203
204 201

UEBER ORYZANIN, EIN BESTANDTEIL DER REISKLEIE, ETC, 419

K6rpergewicht u. Nr.

Versuchs- des Tieres B k
tage a Sanne eae emerkungen
(1) (2
217 200
211 199
218 202
223 195
243 199
starb 198 (1) Starb) wegen mecha-
nischer Verstopfung durch
| 200 Anhaufung der Exkre-
| mente. Die angebliche
193 Zunahme des Gewichts n.
50 Tagen ist wohl d, An-
192 haufung des Exkrements
zuzurchreiben
0.5 g Aminosiuren 190
dazu
187
| 189
186
181 Allmahlich schwach
|
180
187
182
| Nits
174
174
starb

B. Hiner.
Versuch I.
Geschilter Reis.

(1) Zwei junge Hiihner (2 Monate nach dem Ausbriiten) wurden
mit geschiiltem Reis gefiittert. Nach 10 bis 13 Tagen waren die beiden

schon stark abgemagert. Am 15 ten haben sie je 1g Pepton mit kleiner

420 U. SUZUKI, T. SHIMAMURA UND S. ODAKE:

Menge Phytin, Ferratin und Salzgemisch (CaCO;, Na,CO;, KH,PO,
K.CO,, NaCl) bekommen, Sie waren aber nicht damit geheilt und

gingen bald zugrunde.

Tabelle XII.

Kérpergewicht und Nummer des Tieres

Versuchstage
(1) (2)
1 481 537
17 206 _
starb
20 — 323
starb

(2) Derselbe Versuch wurde nochmals wiederholt.

Tabelle XIII.

Kérpergewicht und Nummer des Tieres

Versuchstage
(1) | (2)
1 428 315
15 307 =
starb
18 _ 260
starh

Versuch IT,

Geschiilter Reis mit Phytin.
1. Zwei junge Hiihner (70 Tage alt) wurden mit geschiltem Reis
gefiittert; man gab tiglich 0,5 ¢ Phytin dazu. Sie waren nach 18 Tagen

stark abgemagert and erkrankten schliesslich,

UEBER ORYZANIN, EIN BESTANDTEIL DER REISKLEIE, ETC. 421

Tabelle XIV.

K6rpergewicht und Nummer des Tieres

Versuchstage
q1) (2)
t 482 418
18 356 307

2. Zwei andere Hiihner (70 Tage alt) bekamen eine kleine Menge
Salz nebst Phytin. Sie waren ebenso schnell erkrankt wie mit Reis

allein.

Versuch III.
Geschilter Reis mit Lecithin.

Zwei jungen Hiihnern wurde tiglich 0,5g Lecithin (Kahlbaum)
verabreicht. Die Abnahme des Kérpergewichts war ebenso schnell wie
mit Reis allen. Am 13 ten Tage waren sie schon schwer erkrankt.
Hieauf wurde 5g Kleie gegeben. Am niichsten Tage waren sie beinahe
geheilt und bekamen wieder Esslust. Nach 12 Tagen waren sie

vollstindig gesund und hatten an Gewicht zugenommen.

Tabelle XY.

KGrpergewicht u, Nr.

Versuchs- Ecoeplicres Bemerkungen
tage

(1) (2)

1 413 497 Gesund
3 390 492
5 372 462
Reis+ Lecithin 7 355 443
9 341 408
ll 336 3838

13 306 375 Erkrankt

422 : U. SUZUKI, T. SHIMAMURA UND 8S. ODAKE:

Kérpergewicht u. Nr.

Versuchs- des Tieres
tage Bemerkungen

(1) (2)

15 285 390

17 325 404

19 319 412 Geheilt

5g Kleie dazu

21 363 406

23 381 445

24 365 431 Gesund

Versuch LY.

Geschilter Reis mit alkoholischem Extrakt der Kleie und Salzen:

Zusammensetzung des Futtermittels:

Geschalter Reis ... ... ... 1000¢ INGO ee nace ace acon cet
Alkoholischer Extrakt... ... 20g Ki COS. Fs, Ged exw ene eey | Ae
Cantis(POl)S ostieee e) Soe NEO RRnCey cod, ath aaa cae t=
(EOE RS doy mo kD ote 3g

2 junge Hiihner (ca. 25 Tage nach dem Ausbriiten) wurden mit
obenerwahntem Futtermittel gefiittert. Anfangsgewicht war 156 bzw.
144g, Sie bheben 70 Tage lang gesund und haben allmihlich an Ge-
wicht zugenommen, bis sie ungefiihr das Doppelte ihres Gewichts erreicht
hatten. Nach 80 Tagen waren sie jedoch schwach geworden, so dass wir
den Versuch unterbrechen mussten.

Wir haben noch einige Versuche ausfiihrt, um zu sehen, ob die
jungen Hiihner ausschliesslich mit geschiiltem Reis und alkoholischem
Extrakt der Kleie lingere Zeit am Leben bleiben kénnen. In allen bis-
her untersuchten Fiillen blieben sie 60 bis 70 Tage lang gesund und
munter. Nachher ging der Appetit allmihlich zuriick. Sie starben nicht
so schnell wie mit Reis allein, sondern blieben lingere Zeit kiimmerlich.
Das Korpergewicht nimmt mach und nach ab. Da dem Reis fast alle

unentbehrlichen Mineralstoffe fehlen, so muss man genau die Mengen-

UEBER ORYZANIN, EIN BESTANDTEIL DER REISKLEIE, ETC. 423

verhiltnisse und Verbindungsformen der einzelnen Mineralstoffe kennen
lernen, um die jungen Hiihner lingere Zeit am Leben zu erhalten.

Wir wollen hier einige Boebachtungen, die Herr Dircktor Koza1}
und Dr. Anno in Nishigahara in dieser Richtung gemacht haben, er-
wahnen,

Sic haben eine Anzahl ausgewachsener Hiihner in einem beschrankten
Raume mit verschiedenen Futtermitteln gefiittert und gefunden, dass un-
geschalter Reis oder geschiilter Reis mit Kleie oder Weizenkleie nie die
Erkrankung oder Abmagerung des Tieres hervorrief. Alle Tiere, die mit
geschiltem Reis gefiittert wurden, ingen ohne Ausnahme zugrunde. Bloss
die Zeit bis zur Erkrankung war bei ihnem bedeutend langer als bei
unserem Experimente, weil sie anfangs den kiuflichen geschilten Reis
-ohne Waschen den Tieren gegeben haben, wobei natiirlich kleine Mengen
Kleie immer noch darauf haften blieben. Mit gewaschenem Reis er-
krankten die Tiere viel rascher.

Die beiden Autoren haben auch verschiedene Reissorten aus ver-
schiedenen Gegenden verglichen; sie verhielten sich manchmal sehr ver-
schiedens gegen Erkrankung, vorausgesetzt, dass der geschiilte Reis nicht
gewaschen war. Wenn er aber sorgfaltig gewaschen und von Spuren
anhaftender Kleie befreit war, so haben sie ohne eine einzige Ausnahme
Erkrankung hervorgerufen.

Die alten ausgewachsenen Hiihner waren viel widerstandsfahiger

-als die jumgen wachsenden.

Ferner muss man bemerken, dass die Hiihner, wenn sie in einem etwas
geriumigen Raume gefiittert werden, nicht selten Fliegen, Miicken u. a.
heruterschlucken, oder sie finden etwas, was sie nicht fressen sollten,

so werden sie mehr oder weniger linger vor der Erkrankung verschont.

1. Special Report of the Agricultural Experiment Station Nishigahara, Tokio.

424 U. SUZUKI, T. SHIMAMURA UND 8S. ODAKE:

C. Mause.
Versuch I. (Taf. XXIIZ.)
Geschilter Reis.

a) 4 Miuse wurden mit geschiiltem Reis und Brunnenwasser ge-
fiittert. Die ersten 4 bis 5 Tage waren sie gesund und frassen den Reis
sehr gerne. Der Appetit ging aber nach und nach zuriick, und binnen

11 bis 15 Tagen gingen sie alle zugrunde.

Tabelle XVI.

Kérpergewicht und Nummer des Tieres

Versuchstage

(2) (3) G0)

1 10,9 7,2 72

a 10,0 6,8 7,4

rf 9,9 6,4 6,6

9 9,2 6,2 6,4

ll 8,5 5,7 5,7
13 8,1 5,7 starb

15 starb starb —

b) Derselbe Versuch wurde nochmals mit zwei grésseren Miiusen
wiederholt. Die beiden Tiere starben binen 15 bis 17 Tagen unter Ab-

magerung.

Tabelle XVII.

Korpergewicht und Nummer des Tieres

Versuchstage » /
(1) (2)
l 20,5 13,1
3 20,7 13,8

5 20,5 14,2

UEBER ORYZANIN, EIN BESTANDTEIL DER REISKLEIE, ETC. 425

KGrpergewicht und Nummer des Tieres

Versuchstage

Versuch II. (Taf. XXIII.)
Ungeschilter Reis.

4 Mause wurden mit ungeschiltem Reis gefiittert; sie blieben alle
gesund. Jedes Tier frass tiiglick 2 bis 2,5 g Reis und nahm mehr oder
weniger an Koérpergewicht zu. Nach 33 Tagen wurde der Versuch unter-

brochen.

Tabelle XVIII.

Kérpergewicht und Nummer des Tieres

Versuchstage

(1) (2) (3) (4)

1 9,1 9,4 3,1 5,9
4 10,1 10,3 9,0 62
6 10,4 10,4 3,9 6,4
8 10,6 | 10,5 8,7 62
10 10,5 10,6 3,9 62
12 10,5 10,8 9,2 62
14 10,8 10,5 9,0 64
17 11,2 11,1 9,7 6,2

426 U. SUZUKI, T. SHIMAMURA UND S. ODAKE:

Kérpegewicht und Nummer des Tieres

Versuchstage

Versuch IIT. (Taf. XXIII.)
Geschilter Reis mit alkoholischem Extrakt der Kleie.

a) 500g geschiilter Reis wurden mit einer wasserigen Liésung von
7 g alkoholischen Extrakt (aus 70g Kleie) vermischt, getrocknet und
3 Miiusen gegeben. Sie blieben gesund und normal. Nach 44 Tagen.
wurde der Versuch unterbrochen. Das Kérpergewicht zeigte keine grossen.
Schwankungen.

Tabelle XTX.

Korpergewicht und Nummer des Tieres
Versuchstage

UEBER ORYZANIN, EIN BESTANDTEIL DER REISKLEIE, ETC. 427.

Kérpergewicht und Nummer des Tieres

Versuchstage
(1) (2) (3)
7,4 8,9
7,4 8,5
733 8,7
7,4 8,9
7,4 8.9
7,2 8,8
7,2 8,3
7,0 8,4
fipll 8,8
32 8,7

b) Derselbe Versuch wurde nochmals mit 2 jungen Miusen wieder-
holt. 80 Tage lang waren sie vollkommen gesund und das Kérpergewicht

nahm bedeutend zu. Nach 80 Tagen wurden der Versuch unterbrochen-

Tabelle XX.

Koérpergewicht und Nummer des Tieres

Versuchstage

(1) (2)

1 8,7 9,4

5 9.6 11,1

9 11,5 11,7

13 11,8 11,3

17 9,8 W132

21 10,3 11,2

25 10,3 11,1

29 11,6 11,3

428 U. SUZUKI, T. SHIMAMURA UND S. ODAKE:

KGrpergewicht und Nummer des Tieres

Versuchstage

c) In diesem Versuche wurden 2 Méuse mit geschaltem Reis und
alkoholischem Extrakt nebst kleinen Mengen Salzen gefiittert. Die

Mengenverhiltnisse waren wie folgt:

100g geschilter Reis,

1,5 g alkoholischer Extrakt,

0,l1g K,CO,,

0,2g Na,CO,,

01g CaCl,

0,3 g CaCo,.
Die Versuchstiere blieben lingere Zeit vollstindig gesund. Das eine

ist leider nach 57 Tagen weggelaufen. Das andere bleib 75 Tage lang

ganz normal, bis der Versuch unterbrochen wurde.

UEBER ORYZANIN, EIN BESTANDTEIL DER REISKLEIE, ETC. 429

Tabelle X XI.

KGrpergewicht und Nummer des Tieres

Versuchstage
(1) (2)

1 9,0 10,0

5 10,7 11,3

9 13,0 12,9
13 13,0 12,8
17 12,7 12,6
21 13,6 12,9
25 13,0 13,2
29 12,9 13,8
33 13,2 13,6
37 13,5 14,8
41 13,7 15,0
45 12,9 13,9
49 13,8 13,8
53 14,0 14,1
57 13,8 13,9

fortgelaufen.

430 _ . U. SUZUKI, T. SHIMAMURA UND S. ODAKE:

Versuch IV. (Taf. XXIV.)
Geschilter Reis mit Roh-Oryzanin (I).

In diesem Versuche wurden 2 Miiuse (A) mit geschiltem Reis und
Roh-Oryzanin (1) gefiittert und zwei andere (B) wurden noch dazu mit
Lecithin, Phytin und Salzen versehen. Alle blieben 35 Tage véllig ge-
sund, bis der Versuch unterbrochen wurde. Man konnte jedoch zwischen
A und B einen ziemlich grossen Unterschied merken, d. h. der Einfluss
von Lecithin, Phytin und Salzen war deutlich erkennbar. Diejenigen,
die keine Salze nebst Lecithin und Phytin bekommen haben (A), zeigten
allmiihliche Abnahme des Ké6rpergewichts, wahrend diejenigen, die sie
bekommen haben (B), etwas an Kérpergewicht zugenommen haben. Da
der geschilte Reis sehr arm an’ Mineralstoffen ist und das Roh-Oryzanin
vollkommen frei davon, so ist es wohl begreiflich, dass der Mangel an
Mineralstoffen in diesem Falle (A) fiihlbar war.

Die Futtergemische in diesem Versuche hatten folgende Zusammen-
setzung:

A. 100g Reis und 0,2 ¢ Roh-Oryzanin.

B. 100 g Reis, 0,2 g Roh-Oryzanin, 0,4 g Lecithin (Kahlbaum), 0,5¢
Phythin, 0,3 g CaCO,, 0,1 g CaCl., 0,2 g Na,CO;, 0,1 g K,CO3.

Aus oben angegebenen Beobachtungen ist also festgestellt worden,
dass die Miiuse durch ausschliessliche Reisfiitterung ohne Ausnahme
binnen 10 bis 20 Tagen unter starker Abmagerung ‘zugrunde gehen,
wihrend diejenigen, die entweder mit ungeschiiltem Reis oder mit
geschiiltem Reis nebst Kleie oder alkoholischem Extrakt gefiittert werden,
lingere Zeit gesund und normal leben kénnen.

Ganz dasselbe Resultat konnte man auch mit Roh-Oryzanin erzielen.
Nur starben einige Tiere wihrend der Versuche aus unbekannter Ursache-

Dies kann aber selbst bei gewohnlicher Fiitterung oft geschehen.

UEBER ORYZANIN, EIN BESTANDTEIL DER REISKLEIE, ETC. 431

Tabelle X XIT.

Korpergewicht und Nummer des Tieres

Versuchstage A are o B

(1) (2 (1) (2)

1 10,9 11,3 7,8 6,8
4 10,5 10,8 99 8,3
6 10.5 10,8 10,0 8,6
10 10,8 10,3 9,8 8.3
12 10,0 10,6 10,4 8,9
14 9,6 10,8 10,0 8,5
17 9,6 10,5 11,4 8,1
19 9,7 11,2 11,6 8.5
21 9,9 11.0 117 8,3
24 9,7 10,5 11,9 8.3
26 9,8 10.7 12,1 7.9
28 9,6 10.8 12,0 7,6
30 97 10,6 11,9 7,3
31 9.6 10,4 11,8 7,3
33 9,3 10,3 11,5 7,1
35 9,4 15,3 11,9 7,3

Die Zugabe von Salzen mit oder ohne Phytin, Lecithin und Phos-
phat scheint im allgemeinen einen giinstigen Einfluss gehabt zu haben.
Besonders giinstig war das Calciumphosphat. Da aber, wie gesagt, die
Individualitiit und das Alter der Tiere eine grosse Rolle dabei spielen,
so kann man noch keinen bestimmten Schluss ziehen. Wir wollen spiter

uns nochmals mit dieser Frage beschiiftigen.

432 U. SUZUKI, T. SHIMAMURA UND S. ODAKE;:

Versuch V. (Taf. XXIV.)
Hiihnereier.

500 g geschilter Reis wurden mit 250 g frischen Eiern (Schale aus-
genommen) vermischt, bei niederer Temperatur getrocknet und 3 Mausen
gegeben. Alle 3 gingen zugrunde, und zwar das erste nach 9 Tagen,
das zweite nach 23 Tagen und das dritte nach 35 Tagen. Das kleinste
lebte am kiirzesten, wohl wegen seiner Empfindlichkeit. Es scheint also,

dass Hiihnereier fast frei von Oryzanin sind.

Tabelle XXITT.

KGrpergewicht und Nummer des Tieres

Versuchstage

(2) (3)
5,9 9,8
|
3 Bye 6,0 9,6
5 5,8 6.3 9,4

5,0 5,9 8,9

starb

UEBER ORYZANIN, EIN BESTANDTEIL DER REISKLEIE, ETC. 433

Versuch VI. (Taf. XXIV.)

Geschilter Reis mit Kuhmilch.

Es wurden zwei Versuche mit Kuhmilch ausgefiihrt.

I. Die Milch wurde bei niedriger Temperatur bis zum Trocknen
eingedampft und mit Aether und dann mit heissem Alkohol dreimal ex-
trahiert. 45g des auf diese Weise gewonnenen Riickstandes wurden mit
90 @ geschiltem Reis vermischt und 4 Mausen gegeben.

II. 109g (=-800 cem frischer Milch) der hei niederer Temperatur
getrockneten Milch wurden mit 218 g Reis vermischt und 4 Miiusen ge-
geben.

Im Versuche I gingen alle vier fast in derselben Zeit zugrunde,
obgleich sie etwas linger als bei ausschliesslicher Reisfiitterung lebten.

Im Versuche II starb eine in kurzer Zeit aus unbekannter Ursache.
Die iibrigen drei blieben 34 Tage vollkommen gesund und munter; die
eine hat sogar bedeutend an Gewicht zugenommen, wihrend zwei andere
allmahlich abgenommen haben.

Man sieht also, dass die Milch sich gegen Miuse etwas anders ver-
hielt als gegen Tauben. Auf Tauben hat die Milch nimlich keine giinstige
Wirkung gezeigt. Jedenfalls enthilt die Milch irgendeinen Stoff?, der

fiir Miuse, nicht aber fiir Tauben giinstig wirkt.

Tabelle XXIV.
(1)

KGrpergewicht und Nummer des Tieres

Versuchstage - a eee. > Pe,
aly; (2) (3) | (4)
1 10,8 | 10,6 | 9,9 9,0
3 11,9 11,6 10,8 10,0
6 11,8 11,2 11,2 11,1

1. Man vergleiche die Arbeit von T. B. Osporne, The Role of different Proteins in

Nutrition and Growth, Science N. S,, Vol. 34. No. 882, p. 722—732 (24. Nov. 1911).

434 U. SUZUKI, T. SHIMAMURA UND .S. ODAKE:

KGrpergewicht und Nummer des Tieres

Versuchstage

(ly) 5 aeeren (3) (4)
11,8 | 11,4 11.4 11,4
11,1 11,5 11,0 11,0
10,1 10,4 11,4 10,4
10,0 9,9 9,6 9,5
9,2 so 8,5 9,0
8,7 | 8,5 7,9 8,5
S.4 | 8,0 75D 8.1
7,7 | 7,0 7,0 7,6
7,0 starb 63 7,0

starb | 6,0 starb

starb
(2)

Korpergewicht und Nummer des Tieres

Versuchstage

a) (3) Gy. 2h aay

1 9,2 7.5 10,4 10,1
3 10,3 3,9 11,2 11,6
6 11,0 G7 10,7 11,0
8 11,9 9,6 12,6 12
10 12,1 8,9 13,5 10,8
12 11,1 TA 12,9 10,7
14 10,6 starb 12,6 11,3
16 10,7 12,9 11,4
18 11,2 13,0 11,1
20 11,2 14,0 11,1
22 11,4 14,1 11,2
24 11,1 15,0 10,7

UEBER ORYZANIN, EIN BESTANDTEIL DER REISKLEIE, ETC.

435

KGrpergewicht und Nummer des Tieres

Versuchstage ao =
(1) (2) 8)
a 92 14,7
= 94 14,9
30 8.9 14,7
32 8.9 13,8
a 8,5 13,7

Versuch VII. (Taf. XXV.)

Geschilter Reis mit Margarine.

100g Reis wurden mit 10g Margarine vermischt und 2 Miausen

gegeben. Sie blieben nur etwas linger als bei ausschliesslicher Reistiitte-

rung am Leben.

rung.

Tabelle XXYV.

K6rpergewicht und Nummer des Tieres

Versuchstage

(1) (2)

1 17,9 | 15,9
3 18,2 16,6
5 17.9 16,8
7 17,6 16,5
9 16,7 | 16,0
ll 16,1 | 14,2
13 15,9 14.4
15 15,7 14,1
lj 14,0 13.0

Nach 22 bis 24 Tagen starben sie beide unter Abmage-

436 U. SUZUKI, T. SHIMAMURA UND 8. ODAKE:

Kérpergewicht und Nummer des Tieres

Versuchstage

(2)

21 12,3 12,0

2 12,2 11,6
starb

23 11,0

starb

Versuch VIII. (Taf. XXY.)

Geschilter Reis mit alkoholischem Extrakt von Pferdefleisch.

500 ¢ frisches Pferdefleisch wurden mit heissem 90¢ igem Alkohol
wiederholt extrahiert. Der Extrakt wurde stark eingedampft und noch-
mals mit 90¢ igem Alkohol versetzt, von ungeléstem Riickstand ab-
filtriert um Fette und andere Verunreinigungen zu entfernen, und weiter
eingeengt. Der auf diese Weise erhaltene alkoholische Extrakt wurde
mit 250 g geschiiltem Reis vermischt und 2 Miusen gegeben. Sie starben

nach 11 Tagen unter Abmagerung’.

Tabelle XXVI.

KGrpergewicht und Nummer des Tieres

Versuchstage

(1) (2)
1 215 20,4
3 21,9 20,3
5 22,1 20,2
7 22,7 19,5
9 18,5 18,7

ll 19,5 17,5
starb starb

1. Herr Dr. M,. WATANABE im hiesigen Laboratorium hat spiter diesen Versuch
wiederholt und viel bessere Resultate erzielt. Er konnte niimlich 2 Mause 80 Tage lang am
Leben erhalten. So scheint, dass der alkoholische Extrakt des Pferdefleisches auch fiir Mause
wirksam ist.

UEBER ORYZANIN, EIN BESTANDTEIL DER REISKLEIE, ETC. A:

co
oJ

Versuch IX. (Taf. XXV.)

Hier wurde ein Futtergemisch mit moglichst reiner Stiirke, Casein,
Lecithin und Salzen, mit oder ohne Oryzanin zubereitet, Die Zusammen-
setzung desselben war wie folet:

A. Ohne Oryzanin.

100 g¢ Starke, 10 ¢ Casein, 0,5 & Lecithin, 0,4 ¢ CaCO,, 0,1 ¢ CaCl,
0,2 ¢ Na,CO;, 01g K,CO,, 0,1. ¢ NaCl, 0,1 ¢ MeSO,,.

B. Mit Oryzanin.

Zu A wurde 0,2 ¢ Roh-Oryzanin (I) zugegeben. Zur Bereitung des
Futtergemisches wurde die Stirke vorher verkleistert und mit anderen
Stoffen vermischt, bei niederer Temperatur e¢trocknet und in kleine
Stiickchen geschnitten.

Simtliche Tiere in A und B starben binnen 39 Tagen. Der Unter-
schied war jedoch, dass die Tiere, die Oryzanin bekommen hatten (B),
dreimal linger lebten als die (A), die kein Oryzanin bekamen, was bloss
der Einwirkung des Oryzanins zuzuschreiben ist.

Dieser Versuch soll nochmals wiederholt werden. Die Stiirke ohne
Verkleisterung hitte vielleicht ein besseres Resultat erzielt. Auch die

anorganischen Salze hatten keine richtige Zuzammensetzung’.

1. Vor kurzem hat Herr Dr, M. WATANABE im hiesigen Laboratorium ein Salzgemisch
nach R6HMANN hergestellt, und zwar in folgendem Mengenverhiltnis :

10 g phosphors. Kalk, 40g saures phosphors. Kali, 20g Natriumchlorid,

15g Natriumcitrat, 8g Magnesiumcitrat, Sg Calciumlactat.

Er hat 20g dieses Salzgemisches mit 1000g geschiltem Reis gemischt und Mausen
gegeben. Die Tiere lebten 28 Tage, wihrend 2 Kontrolltiere ohne Salze in 14 bzw. 15 Tagen
zugrunde gingen.

Auch bei Tauben hat dieses Salzgemisch eine giinstige Wirkung gezeigt. Trotzdem konnte
er niemals die Tiere ohne Oryzaninzugabe linger als 4 Wochen am Leben erhalten. Wird
der alkoholische Extrakt der Kleie oder Roh-Oryzanin (I) nebst RGhmannschem Salzgemisch
den Tauben oder Mausen gegeben, so bleiben sie nicht nur beliebig lange am Leben, sondern
nehmen bedeutend an Gewicht zu.

Vgl. R6HMANN, Allg. med. Zentral-Ztg. 1903, Nr. 9,

438 U. SUZUKI, T. SHIMAMURA UND S. ODAKE:

Tabelle XXVIT.

A. Ohne Oryzanin.

Kérpergewicht und Nummer des Tieres

Versuchstage

(1) (2) (3)

8,7 9,2

7,1 8,2

6,4 8,0

starb

7,8

6,9

9,6
starb

B. Mit Oryzanin.

K6rperwicht und Nummer des Tieres

Versuchstage

(1) (2) (3)

1 Oo 7,3 9,5
3 10,6 8,1 11,0
5 10,3 7.4 10,1
6 10,2 7,4 9,9
8 9,6 (fil 9,8
10 9,4 6,3 9.5

11 ~ 6,0 =

3 = starb a
13 9,4 10,0
15 9,5 9,2
17 8,8 9,9
20 8,5 | 10,1
22 8,4 | 10,5
24 9,4 10,7

UEBER ORYZANIN, EIN BESTANDTEIL DER REISKLEIL, EYC. 439

KG6rpergewicht und Nummer des ‘Tieres

Versuchstage | _ i PRS
(1) (2) (3)
27 9,1 9.8
29 8,9 a
31 8,4 S31
starb
33 759
35 5,0
37 7,0
30 6,5
starb

D. Hunde.

Versuch J,

Ein ausgewachsener Hund von mittlerer Grésse (6,47 kg) wurde mit
gekochtem Reis und ausgekochtem Riickstand von Pferdefleisch nebst
einer kleinen Menge Kochsalz gefiittert. Er frass am Anfang sehr gut.
Nach 14 Tagen ging der Appetit betrichtlich zuriick. Er wollte den Reis
nicht und suchte nur nach Fleisch, und das K6érpergewicht nahm nach
und nach ab; nach 3 Wochen hatte er die Esslust vollstindig verloren.
Eine weitere Woche lebte er fast ausschliesslich von Wasser, bis er schliess-

xine bis auf

lich abgemagert und ermattet war. Das Korpergewicht ging
5,8 kg herunter. Nach 2 bis 3 Tagen wiire er zweifellos zugrunde ge-
gangen, Zu diesem Zeitpunkt, also am 29. Tage, wurden 3 g alkoholischer
Extrakt der Kleie (—30¢ Kleie) in wenig Wasser gelést und per os
gegeben. Schon am niichsten Tage kam etwas Esslust zuriick und nach
dem 3. Tage war der Appetit ganz normal. Das K6érpergewicht hat auch
allmihlich zugenommen und nach 32 Tagen (d. h. am 60. Tage vom An-
fang an) hat er 7,30 kg erreicht. Hier wurde die Zugabe des alkoholisschen
Extrakts eingestellt. Noch eine Woche hat das Kérpergewicht immer

zugenommen bis auf 7,9 kg und nachher ging es wieder allmahlich zuriiek ;

440 U. SUZUKI, 1. SHIMAMURA UND S. ODAKE!

der Appetit hatte wieder nachgelassen. Der Hund frass nur Fleisch und
schliesslich wollte er gar nichts mehr; nach 38 Tagen (d. h. am 98. Tage
vom Anfang an) kam das Kérpergewicht bis auf 6,10 kg herunter und das
Tier war sehr ermattet. So wurden am nichsten Tage 0,4 @ Roh-Oryzanin
(1) per os gegeben. Diesmal war die Wirkung noch deutlicher als beim
alkoholischen Extrakt: am 2. Tage hat er wieder normalen Appetit
bekommen wnd wurde vollstiindig gesund und munter. Nach 20 Tagen
(118 Tage vom Anfang an) stieg das Kérpergewicht bis auf 7,9 kg. Hier-
auf wurde die Oryzaninzugabe eingestellt. Die Folge dessen war genau
so wie die Erwartung. Der Hund verlor diesmal etwas schneller den
Appetit als das erste Mal und nach 20 Tagen war er schon ermattet und
das Kiérpergewicht ging bis auf 5,90kg herunter. Jetzt wurden ihm anstatt
Oryzanin ca. 3 g alkoholischer Extrakt’ des Pferdefleisches (—aus 150 g
frischem Fleisch) gegeben. Die Wirkung der letzteren war ebenso deut-
lich wie bei Oryzanin. Das Tier hat sich sehr rasch erholt und am
20. Tage erreichte es 7,5 kg. Nach Einstellung der Zugabe des alkoholis-
chen Extrakts des Pferdefleisches war er wieder sehr schnell abgemagert
und ermattet. Der Versuch wurde weiter fortgesetzt, immer mit demsel-

ben Resultate. (Vgl. Tabelle XXVIII und Taf. XXVI.)

Versuch II.

Derselbe Versuch wurde nochmals mit einem kleineren ausgewach-
senen Hunde (Kérpergewicht 4,49 kg) wiederholt. Der Verlauf war
genau derselbe wie beim ersten Hunde. Ohne Oryzaninzugabe konnte
der Hund mit gekochtem Reis und ausgekochtem Riickstand des Pferde-
fleisches nicht existieren und am 28. Tage war er vollstiindig ermattet.
Das Kérpergewicht sank bis auf 3,7 kg. Hiernach bekam er 4 g alkoholis-
chen Extrakt der Kleie. Er erholte sich ebenso rasch wie der erste Hund.

Vom 42. Tage au hat er tiglich 10 g Margarinebutter daneben bekommen.

1. 100¢ frisches Pferdefleisch lieferten 2,1 g alkoholischen Extrakt, welcher 0,2244¢
Gesamt-N, 0,0909 g Basen-N und 0,520g Asche enthielt. Eiweiss war nicht vorhanden,

UEBER ORYZANIN, EIN BESTANDTEIL DER REISKLEIE, ETC. 44]

Der Appetit wurde immer grésser. Am 60. Tage hat er das Gewicht von
4,9 ke erreicht. Hier wurde die Oryzaninzugabe eingestellt. Trotzdem
hat er noch beinahe 8 Wochen nicht an Gewicht verloren; dann ging der
Appetit allmihlich zuriick und am 98. Tage sank das Gewicht bis auf
3,8 kg herunter und das Tier ging schliesslich zugrunde. (YVgl. Tabelle

XXVIII. und Taf. X XVI.)

Versuch III.

Der dritte Versuch wurde mit einer Tiindin ausgefiihrt, die noch 2
junge Hiindchen siugte. Ohne Oryzanin war sie in 3 Wochen schon er-
mattet und gab keine Milch mehr. Hierauf wurde 0,4 ¢ Roh-Oryzanin
eingegeben. Die Wirkung desselben war ebensogut wie beim alkoholischen
Extrakt selbst. Nach 20 Tagen (am 49. Tage vom Anfang an) hat sie
wieder 6,6 kg erreicht. Nach Einstellung des Oryzanins hat sie beinahe
4 Wochen nichts an Gewicht verloren, dann ging es allmihlich herunter.

(Vgl. Tabelle X XIX und Taf. XXVI.)

Tabelle XXVIII.

Korpergewicht

Versuchs- Bemerkungen
tage
A) (B)
1] 6,47 | 4,49 (A) (B) Gesund
2 640 | 4,60
3 6,60 4,60
4 6,30 4,40
5 620 | 4,40
(A) Gein ohne 6 6,20 | 4,50
7 e =
8 6,80 4,70
9 6,80 4,70
10 6,70 4,70
ll 6,70 4,50 Allmaiblich Appetit
verloren

Versuchs-
tage

(A)

K6rpergewicht

(B)

U. SUZUKI, T. SHIMAMURA UND 8. ODAKE:

Bemerkungen

(A) und (B) ohne
Oryzanin

4,70
4,70
4,80
4,70
4,70
4,80
4,60
4,50

4,50
4,20
4,20
4,10
4,20
4,10
4,20
4,10
3,80

Frisst nur Fleisch ; (A) Er-
brechen, Faeces weich

(A) (B) Nichts gefressen

Ermattet

Schwach

(A) und (B) mit
alkoholischem
Extrakt der Kleie

6,45
6,30
¢,00
6,00

3,80
3,70
4,30
4,20
4,20
4,20
4,20
4,20
4,20
4,10
4,20
4,20

(A) (B) 3g alk. Extrakt
per os gegeben
Wieder Appetit

Viel gefressen

(A) Etwas appetitlos

UEBER ORYZANIN, EIN BESTANDTEIL DER REISKLEIE, ETC, 443

Versuchs-
tage

Kérpergewicht

(A)

(A) und (B) mit
a)koholischem
Extrakt der Kleie

(A) und (B) ohne

Oryzanin

(A) wieder Appetit bekom-
men

(B) bekommt tiglich 10¢
Margarine

(A) vollkommen gesund

(A) (B) Vollstiindig gesund

(A) (B) Alkoholextrakt
eingestellt

444 U. SUZUKI, T. SHIMAMURA UND S. ODAKE!

é icht
[eee ee Bemerkungen
(A) (B) ~

70 7,80 4,90 (A) Appetit zuriick
71 7,70 5,00
72 7,60 5,00
73 7,60 5,00
74 7,40 4,90
75 7,35 4,95
76 7,30 5,00 (B) Appetit zurtick ;
77 7,30 4,90
78 7,05 4,95
79 7,10 4,90
80 6,90 4,90
81 6,80 4,70
82 6,70 4,60 (B) Frass nur Fleisch
83 6,60 4,65

(A) und (B) ohne 84 6,80 4,75

Oryzanin ‘

85 6,70 4,55
86 6,60 4,50
87 6,60 4,40
8s 6,60 4,30 (A) Frass nur wenig Flei-
89 6,70 | 4,40 ‘ji
90 6,60 4,40
91 6,45 4,35 (B) Appetitlos
92 6,40 4,20 (A) Nichts gefressen
93 = | 4,30
94 6,35 | 4,25 (B) Schwach ; Erbrechen
95 6,30 4,20
96 6,25 4,10 (B) Sehr schwach
97 6,15 4,00

98 6,15 3,80 (A) Schwach

UEBER ORYZANIN, EIN BESVYANDTEIL DER REISKLEIE, ETC. 445.

5 KGrpergewicht ‘
tage eee Jemerkungen
(A) (B)
99 6,10 starb (A) 0,4g Roh-Oryzanin (1)
per Os
100 6,25
101 6,90 Appetit kommt wieder
102 6,95 Gesund
103 7,00
104 7,10
105 7,20
106 7,20
107 7,30
(A) 0,4 g Roh-Oryzanin 108 ue
(1) taglich 109 7,30
110 7,50
11] 7,40
112 7,50
113 7,70
114 7,60
115 7,80
116 7,80
117 7,79
118 7,90 | Vollstandig gesund
119 7,80)em|| Oryzanin eingestellt
120 7.80)mae |
121 7,80. |
122 7,70
(A) Ohne Oryzanin 123 7,60
124 7,50
125 7,40 |
126 7,20 |

446 U. SUZUKI, T. SHIMAMURA UND 8S. ODAKE:

v Korpergewicht
me a Bemerkungen
(A) | (B)
128 7,00
129 6,90
120 6,80
121 6,70
132 6,60
(A) ohne Oryzanin 133 6,45
134 6,30
135 6,30
136 6,20
137 6,05
138 5,90
139 5,90
140 6,10
141 6,30
142 6,70
143 6,80
144 6,80
145 6,90
146 7,00
Alkoholischer Extrakt 147 7,00
aus 150g Pferde-
fleisch taglich 148 6,70
149 7,00
150 7,10
151 6,90
152 6,90
153 7,00
154 7,20
155 7,40

UEBER ORYZANIN, EIN BESTANDTEIL DER REISKLEIE, ETC. 447

K6rpergewicht
Versuchs- Bemerkungen
tage —— —— g
(A) | (B®
Alkoholischer Extrakt 157 7,40
aus 150g Pferde- {
fleisch taglich 158 7,50 | Gesund und munter
159 7,50 |
160 7,40
|
161 7,20
162 7,20 |
163 7,20
164 7,20
165 7,00
166 7,00
167 7,00
168 6,90
169 6,70
170 6,60 Esslusst nachgelassen
171 6,50 |
?
» Ohne Oryzanin 172 6,40
173 6,30
174* 6,10 * Durch Phosphowolfram-
saure fallbarer Teil des
175* 6,00 alkoholischen Extrakts
} aus 250¢ Pferdefieisch.
176 6,06 Keine Wirkung.
177 5,80
178** 5,80 | ** Filtrat von Phosphor-
| wolframsaure Nieder-
179T 6,00 schlag aus 500g Pferde-
| fleisch. keine Wirkung
180 6,10 und Erbrechen ; so wurde
der Alkoholextrakt aus F)
181 6,20 200g Kleie und am
| nachsten Tage derselbe
182 6,20 aus 100g Kleie gegeben.
Das Tier wieder geheilt und
183 6,40 hat Appetit bekommen.
184 6,60

185 6,80 |

448 U. SUZUKI, T. SHIMAMURA UND S. ODAKE:

Versuchs-

tage Bemerkungen

Ohne Oryzanin 200

Etwas gefresser-

Etwas gefressen »

UEBER ORYZANIN, EIN BESTANDTEIL DER REISKLEIE, ETC,

nn ead

K6rpergewicht

Versuchs- Bemerkungen
tage — =
(A) (B)
215 6,30 Allmahlich schwach
216 6,50
217 6,40 Appetitlos
218 6,30 Nichts gefressen
Ohne Oryzanin 219 6,10
220 6,00
221 6,20
222 6,00
NY 223 5,80 Enmattet
Versuch fortgesetzt
0,5g Alkoholextrakt
Kleie taglich gegeben;
wieder geheilt und nahm
an Gewicht zu
Tabelle X XIX.
K6rpergewicht
Versuchstage | __ : Bemerkungen
(S)
1 7,00 Gesund
3 6,90
5 6,70
7 6,50
Reis + ausgekochtes a Sea
Pferdefleisch il 5,20
13 6,20 Appetit nachgelassen
15 6,10
li 5,80 Schwach

19 5,60 Ermattet

450 U. SUZUKI, T. SHIMAMURA UND S. ODAKE:

Kérpergewicht
Versuchstage Bemerkungen

: (C)
20 5,50
21 5,65
22 5,70 Geheilt ; Appetit bekommen .
23 5,75
25 . 5,90 Gesund und munter
27 5,80
29 6,20
31 6,30

0,4¢ Sepia (I) 33 6,60
35 6,70
37 6,60
39 6,60
41 6,60
43 6,60
45 6,60
47 6,60
49 6,60 Gesund
51 6,70
53 6,40
55 6,50
57 6,50
59 6,40
61* 6,50 * Herausgekommen und etwas -
Ohne Oryzanin Schweinefleisch gefressen

63 6,60
65 6,60
iy fot 6,60 ** Wieder herausgekommen
69 6,55
71 6,60

73 6,40

UEBER ORYZANIN, EIN BESTANDTEIL DER REISKLEIE, ETC. 45

Versuchstage

Ohne Oryzanin

109

KGrpergewicht

6,30

6,20
6,20
6,00
6,00
6,00
6,10
6,30
6,00
6,10

6,20

Bemerkungen

Allmihlich schwach

Appetitlos

Nichts gefressen

Ermattet ; 0,5 g Alkoholex-
trakt der Kleie

Versuch fortgesetzt

Mit 0,5g Alkoholextrakt
der Kleie wieder geheuilt
und nahm an Gewicht zu.

oS — — — — ——— ———————

452 U. SUZUKI, T. SHIMAMURA UND S. ODAKE?

Das Futtergemisch hatte folgende Zusammensetzung:
500 ¢ geschilter Reis,
120g ausgekochter Riickstand von Pferdefleisch’,
5 @ Kochsalz,
5 g Salzgemisch (KH,PO,, Ca,H, (PO,)., MgCO;, CaCO3).
Aus diesem Resultat sieht man also, dass das Leben des Hundes
vollstindig unter Kontrolle des alkoholischen Extrakts der Kleie bzw.
des Oryzanins steht. Die Wirkung des alkoholischen Extrakts aus Pfer-
defleisch war ebenso gut wie beim Oryzanin. Da der alkoholische Extrakt
aus Fleisch auf Tauben und Hiihner fast wirkungslos war, so muss man
annehmen, dass der wirksame Stoff des Fleisches mit demOryzanin nicht
identisch ist. Beim Hunde (und bei Miusen) kann der eine den anderen
vertreten, wihrend bei Tauben und Hiihnern dies nicht der Fall ist.
Wir haben schon einige Versuche angestellt, um diesen wirksamen
Stoff im Fleische ausfindig zu machen. Wenn der alkoholische Extrakt
des Fleisches in Wasser gelést und mit Bleiessig gefallt wird, so findet
man im Filtrate des Niederschlages keine Wirkung mehr. Man muss
deshalb annehmen, dass der wirksame Stoff entweder durch Bleiessig
mitgerissen wird oder wihrend der Verarbeitung zerstért wird. Wir

wollen uns mit dieser Frage weiter beschiftigen.

iV. Ueber die Verbreitung des Oryzanins in verschiedenen
Futtermitteln.
Da wir noch keine zuverliissige Bestimmungsmethode des Oryzanins

gefunden haben, so bleibt uns nights iibrig, als durch Tierversuche die

1. Das frische Fleisch wurde zweimal je eine Stunde mit Wasser gekocht und stark
abgepresst, bis keine ldslichen Stoffe mehr vorhanden waren, Der so erhaltene Riickstand

enthielt :

Wasser 46,5622,

‘Trockensubstanz 53,44%.
In 100 Teilen Trockensubstanz ;

Gesamt-N 15,0222,

Rohfette 3,51%,

Asche 0,48%.

UEBER ORYZANIN, EIN BESTANDTEIL DER REISKLEIE, ETC. 453

Verbreitung und annihernde Quantitit desselben in verschiedenen Futter-
mitteln festzustellen. Zu diesem Zwecke haben wir hauptsichlich Tauben
und Miuse benutzt. Die Tiere werden so lange mit geschiltem Reis ge-
fiittert, bis sie schliesslich krank werden, hierauf gibt man den alkoholis-
chen Extrakt der verschiedenen Futtermittel per so oder mit Reis gemischt
und beobachtet, ob sie geheilt werden oder nicht. Oder man gibt von An-
fang an den alkoholischen Extrakt der betreffenaen Futtermittel mit Reis
gemischt an gesunde Tiere, um zu sehen, ob sie schliesslich krank werden.

Diejenigen Stoffe, die die Tiere gern fressen, haben wir als solche
gegeben, z. B. wie Gerste, Weizen, Brot usw. In folgenden teilen wir

einige Versuche in dieser Richtung mit.

Versuch I.

Weizenkleie.

1. Die kiiufliche Weizenkleie wurde genau so wie Reiskleie wieder-
holt mit 85% igem Alkohol heiss extrahiert. Der alkoholische Auszug
wurde abdestilliert. Der zuriickgebliebene Sirup wurde mit wenig Wasser
verdiinnt und wiederholt mit Aether geschiittelt, der Aether verdampft und
abfiltriert. Der in der Weise aus 500 @ Weizenkleie dargestellte alkoho-
lische Extrakt wurde mit 5002 Reis gemischt, getrocknet und einer
gesunden Tauben gegeben. Die Taube blieb 16 Tage vollkommen gesund

und nahm sogar etwas an Kérpergewicht zu.

Tabelle XXX.

Versuchstage Koérpergewicht
1 300
3 308
5 314

~I
~
ty
—

454 U. SUZUKI, T. SHIMAMURA UND S, ODAKE:?:

Versuchstage KGrpergewicht

2. Wenn der alkoholische Extrakt aus 300 g Weizenkleie mit 1000 ¢
Reis vermischt wird, so geniigt dies noch nicht, um eine Taube vor der
Erkrankung zu schiitzen, und das Tier nimmt an Gewicht ab. Wenn aber
tiglich ein aus 10g Weizenkleie bereiteter alkoholischer Extrakt noch
dazugegeben wird, so wird das Tier sehr rasch geheilt und nimmt an
Gewicht zu.

3. Der alkoholische Extrakt der Weizenkleie wurde in wenig Wasser
gelést und unmittelbar mit Tannin gefallt. Der Tanninniederschlag
wurde in gewohnlicher Weise in verdiinntem Aceton gelést, mit Baryt
zerleot und genau so verarbeitet, wie bei der Reiskleie. 0,09 g¢ des so
bereiteten Roh-Oryzanins gab ein postives Resultat.

4. Das aus dem alkoholischen Extrakt der Weizenkleie durch das
Phosphorwolframsiureverfahren dargestellte Roh-Oryzanin war auch wirk-
sam. Man musste jedoch tiglich aus 60g Kleie dargestelltes Roh-Ory-

zanin verwenden, um eine Taube zu heilen.

Tabelle X XXT.

Versuchstage Korpergewicht Bemerkungen

| 310 Gesund
Reis allein 221

| 223
217 Erkrankt

UEBER ORYZANIN, EIN BESTANDTEIL DER REISKLEIE, ETC.

455

Versuchstage K6rpergewicht Bemerkungen
: 1 213
3 219
5 220) Geheilt
7 223
0,09 g Roh-Oryzanin 9 224
(Tanninverfahren)
aus Weizenkleie 11 225
13 232
15 243
17 238
19 244 Gesund
21 248
23 246
Reis allein 25 251
27 251
28 248 Noch nicht erkrankt

Tabelle XX XIT.

Versuchstage Kérpergewicht Bemerkungen
270 Gesund
Reis allein
23 | Erkrankt

te
1

wolframsdureverfahren) 3 225 Geheilt
aus 60g Kleie

1 215
: 225
Roh-Oryzanin(Phosphor-

456 U. SUZUKI, T.. SHIMAMURA UND S. ODAKE:

Versuchstage Korpergewicht Bemerkungen

Roh-Oryzanin(Phosphor-
wolframsaureverfahren)
aus 60g Kleie

Gesund

Aus dem oben angegebenen Resultate kann man schliessen, dass der

‘Gehalt an Oryzanin in der Weizenkfeie etwa '/,) des der Reiskleie ist.

Wahrend der Verarbeitung des Phosphorwolframsiureniederschlages
haben wir zeimlich viel Betain gefunden, Wenn der Niederschlag in
gewohnlicher Weise durch Baryt zerlegt und der Uebersuchuss von Baryt
mittels Schwefeliure genau entfernt wird, so erhalt man eine schwach
alkalisch reagierende Fliissigkeit, die neben Oryzanin ziemlich viel
Betain enthalt. Wird nun diese Fliissigkeit stark eingeengt und mit
Aethylalkohol und Aceton versetzt, so scheidet sich ein wenig eines an-
-organischen Salzes ab. Man filtriert davon ab, dampft das Filtrat weiter
-ein und liisst einige Tage stehen, so scheiden sich die charakteristischen
Krystalle des Betains als hygroskopische, farblose Tafeln aus; es hat einen
angenehmen, siissen Geschmack und zersetzt sich bei ca 250°, ohne zu
schmelzen.

Die Ausbeute an freiem Betain betriigt ca. 6 g aus 1 kg Weizenkleie.
Wird die wisserige Lisung des Betains mit Pikrinsiure versetzt, so erhilt
man das Betainpikrat als citronengelbe Prismen. Eimmal aus heissem
Wasser umbrystallisiert, schmilzt es bei 180° (unkorr.).

Die Analyse des Pikrats gab folgende Resultate:

1. 0,1510g Subst. gaben 0,2116g CO,, 0,0602g H,O.

2. 0,1130 ,, ” ” 0,1682 5, 5 0,0482 ,, ”
3. 0,0912 ,, ” ” 13,4 com N (21,5° 757,5 mm).
4. 0,2776,, ” » 0,1856 g_Pikrinsaure.
Cc i N Pikrinsiure
C,H, ,NO,C,H,N,O, ... ... Ber. 38,15 4,05 16,18 66,19

Gef. 38,21 4,43 16,62 66,86
38,17 4,74 _ —

_
oO
~j

UEBER ORYZANIN, EIN BESTANDTEIL DER REISKLEIE, ETC.

Weizenbrot.

Das gewoéhnliche Weizenbrot wurde zerkleinert und an zwei Tauben

verfiittert. 100 Tage lang bliebenssie gesund und normal.

Tabelle XX XITT.

Kérpegewicht und Nummer des Tieres

Versuchstage

458 U. SUZUKI, T, SHIMAMURA UND S, ODAKE;

Versuch II.

Gerstenkleie.

Wenn man den alkoholischen Extrakt aus 15 g Gerstenkleie taglich
einer erkrankten Taube gibt, so wird die Taube sehr rasch geheilt, wie
folgende Versuche zeigen.

Wie diese Tabelle zeigt, haben die beiden erkrankten Tauben nach
‘6 tigigem Zusatz vom alkoholischen Extrakt aus 15 g Gerstenkleie sich
vollstindig erholt und sind spater noch 10 Tage lang ohne alkoholischen
Extrakt vollstiindig gesund geblieben. So kann man berechnen, dass das
Oryzanin der Gerstenkleie wenigstens '/; des der Reiskleie ist. Wir
‘konnten jedoch kein wirksames Priiparat, weder durch Phosphorwolfram-

-siure noch durch das Tanninverfahren bekommen.
Tabelle XXXIV.

KGrpergewicht und

Vi hs- Nummer des Tieres
‘bee = ie ieee Bemerkungen
(G5) (2)
296 310 Gesund
Reis allein :
212 191
207 190 Erkrankt
208 188 Geheilt
222 199
Alkoholischer 229 193
Extrakt aus 15g
Gerstenkleie taglich 233 201
237 203
244 207 Gesund
247 209
Reis allein 256 210

UEBER ORYZANIN, EIN BESTANDTEIL DER REISKLEIE, ETC. 459

K6rpergewicht und

Versuchs- Nummer des Tieres 8 k
tage es =: 2 emer ungen
|

(1) | (2)

10 254 | 221

11 251 | 222

12 249 222

Reis allein

13 245 222

14 243 | 227

15 236 | 223 Noch nicht erkrankt

Versuch III.
Hafer.

Der alkoholische Extrakt aus 1ke Hafer wurde mit 1kg Reis
vermischt, getrocknet und zwei erkrankten Tauben gegeben. Die eine
war jedoch schon zu schwach, um genug Futter zu fressen und ging
schliesslich zugrunde; die andere aber erholte sich allmihlich und wurde
spiter vollstindig gesund.

Der Gebalt von Oryzanin im Hafer muss deshalb ungefihr */,9 des

der Reiskleie sein.

Tabelle XXXY.

K6rpergewicht und Nummer
des Tieres

Versuchstage Bemerkungen

1, 2 erkrankt

460 U. SUZUKI, T. SHIMAMURA UND S. ODAKE:

Kétpergewicht und Nummer
des Tieres

Versuchstage a eae a - Bemerkungen

(1) (2)

13 245 225 2 geheilt

15 239 230

17 starb 228

19 228

21 233

23 238

25 | 243

27 245 2 gesund

Versuch IV.
Hirse.

Der alkoholische Extrakt aus 50 g Hirse tiglich war geniigend, um

eine erkrankte Taube zu heilen.

Tabelle XXXVI.

Bemerkungen

Versuchstage

Korpergewicht

286 Gesund
Reis allein :
249
246 Erkankt
1 246 Geheilt
3 250
Alkoholischer Extrakt 5 257
aus 50g Hirse - 264
9 270

10 273 Gesund

UEBER ORYZANIN, EIN BESTANDTEIL DER REISKLEIE, ETC. 461

Versuchstage Koérpergewicht Bemerkungen

Reis allein
Gesund

Allmihlich schwach

Versuch V.

Brassica var. (Kyona).

400 @ der lufttrockenen Blitter und Stengel von Brassica var. wurden
mit heissem 854% igem Alkohol wiederholt extrahiert. Der alkoholische
Extrakt wurde mit 400g Reis vermischt, getrocknet und einer Taube
eingegeben. Die Taube hat 17 Tage lang nichts an Korpergewicht verlo-
ren, Nach 18 Tagen wurde der alkoholische Extrakt auf die Hilfte
reduziert. Nun nahm das Kérpergewicht allmihlich ab und schliesslich

erkrankte das Tier.
Tabelle XX XVII.

Versuchstage Korpergewicht Bemerkungen

Gesund

Alkoholischer Extrakt
aus 400¢ Brassica
var. auf 400¢
Reis

462 U. SUZUKI, T. SHIMAMURA UND S. ODAKE:

Versuchstage Kérpergewicht Bemerkungen

260

Alkoholischer Extrakt | 256
aus 400 ¢ Brassica : ae
var. auf 400¢ : 260

Reis
268 Gesund

262
258
257

250

Atkoholischer Extrakt
aus 200g Brassica 246
auf 400g Reis

239

Schwach

Erkrankt

Der Gehalt an Oryzanin in lufttrockener Brassica muss also un-
gefiihr 1/,, des der Reiskleie sein.
Weder durch Phosphorwolframsiure noch durch Tannin konnten

wir ein wirksames Priparat erhalten.

Versuch VI.
Hiihnereier.

250g frische Hiihnereier (Schale ausgenommen) wurden mit 500 g
Reis vermischt, getrocknet und an zwei Tauben verfiittert.
Nach 2 Wochen waren die beiden Tauben stark ermattet und erkrankt.
Hierauf wurde der alkoholische Extrakt der Weizenkleie per os gegeben ;
sie wurden sehr rasch geheilt und das Kérpergewicht hatte auch stark
zigenommen. Dass die Eier keine schidlichen Bestandteile fiir die
Tauben in sich enthalten, sondern dass die Erkrankung bloss durch
Mangel an Oryzanin hervorgerufen ist, geht ganz klar ans diesem Ver-

such hervor,

UEBER ORYZANIN, EIN BESTANDTEIL DER REISKLEIE, WIC. 463

Tabelle XX XVIII.

Reis+ Hiihnercier

Reis-+ Hiihnereier
+alkoholisch. Extrak:
aus Weizenkleie

Versuchs-
tage

K6rperwicht und Nummer
des Tieres

a fi dl Bemerkungen
(91 (2
242 242 Gesund
256 256
262 278
265 284
265 287
260 289
257 280
245 267
237 209
221 246
217 239
210 227
205 219
215 214 Erkavkt
203 206
210 223 Gcheilt
PN | 229
255 232
247 245
256 248
207 257

2S 260 Gesund

464 U. SUZUKI, 7. SHIMAMURA UND-S. ODAKE!

Versuch VII.
Kuhmileh.

Wenn man die Taube mit Reis und alkoholisechem Extrakt von
Kuhmilch fiittert, so lebt das Tier etwas linger als mit Reis allein,
trotzdem verliert es allmihlich an Korpergewicht und wird schliesslich
krank, Auch frische Milch wirkt kaum bessers als der alkoholische
Extrakt. Wird das Oryzanin in diesem Falle der erkrankten Taube
vegeben, so wird das Tier in einigen Tagen geheilt, oder wenn dass Ory-
zanin von Anfang an mit Milch zusammengegeben wird, so bleibt das
Tier liingere Zeit gesund und normal. Hieraus muss man schliessen,
dass der Milch nur Oryzanin fehlt. Etwas anders verhalt sich die Mileh
gegen Mause’. Das letztgenanute Tier kaun nattirlich liingere Zeit von

Reis und Milch leben, wie aut den folgenden Seiten beschrieben wird.

Versuch VIII.
Adzukibohnen.

1. Der alkoholische Extrakt aus 300g Adzukibohnen wurde mit
100g Reis vermischt und an zwei Tauben verfiittert. Sie erkrankten
ebenso schnell wie mit Reis allein. In 12 Tagen hat cine Taube von
296 bis 231 g und die andere von 287 bis 223 g abgenommen und beide
waren sehr geschwacht. Hierauf wurde der einen Taube der alkoholische
Extrakt aus 9g Adzukibohnen per os gegeben, ohne giinstige Wirkung
zu zeigen.

2. In diesem Versnche wurden zwei Tauben ausschliesslich mit. zer-
kleinerten Bohnen gefiittert. Sie frassen taglich 20 bis 25 g Bohnen und
blieben langere Zeit gesund. Das Kérpergewiecht nahm anch allmahlich

Zul.

l, P. B. Osporne, Science N. S. 34, Nr. 882, S, 722 bis 732 (24. Nov. 1911).

UEBER ORYZANIN, EIN BESTANDTEIL DER REISKLEIL, ETC. 465

Tabelle XX XIX,

Kérpergewicht und Nummer des Tieres

Versuchstage ae = =

(1) | >)

i 304 | 301
P 288 285
5 295 ain)
7 294 =
: 298 302
: ae | 305
i 300 shy
= 308 JOS
i 314 312
ia 310 308
= 306 300
= 307 305
Be 310 312
ee 315 312
a 317 ole
- 313 | 1S
f 315 315
ic 311 aus
. 315 315
* 318 320
ii 321 oie
ae 320
45 7 5
x ae 315
He ose 330
a ae 320
S54 317 21

466 t. SUZUKI, T. SUIMAMURA UND s. ODAKE!

Kérpergewicht und Nummer des Tieres

Versuchstage

Q) (2)
55 317 322
a7 299 298
59 311 324
61 313 326
63 306° 318
65 315 332
67 310 325

Demuach ist der Gehalt an Oryzanin in Adzukibohnen héchstens 1/,9

des der Reiskleie.

Versuch LX.
Sojabohnen.

1. Der alkoholische Extrakt aus 300 g Sojabohnen wurde mit 1000 g
Reis vermischt und zwei gesunden Tauben gegeben. Auch in diesem Falle
wurde keine Schutzwirkung beobachtet. Die eine hatte von 307 bis 241 g¢
und die andere yon 318 bis 258g abgenommen.

2. Wenn man aber den Tauben nicht den alkoholischen Extrakt
der Sojabohnen, sondern die Bohnen selbst gibt, so bleiben die Tiere
langere Zeit gesund.

Demnach muss man annehmen, das in Sojabohnen nicht viel Oryzanin

vorhanden ist.

UERER ORYZANIN, EIN BESTANDTEIL DER REISKLEIE, EPC.

Tabelle XL,

Kérpergewicht und Nummer des Tieres

Versuchstage
(1) (2)
1 308 303
3 310 315
5 310 323
ff 309 =
9 304 —
11 305 Ls
13 314 —
15 316 _
17 320 318
19 320 322
21 322 325
23 317 327
25 325 336
27 320 335
29 330 | 343
31 327 332
33 335 340
35 Bey 337
37 325 339
39 330 330
41 330 338
43 326 332
45 320 29
47 330 320
49 324 320
51 328 318

468 U. SUZUKI, T. SHIMAMURA UND S. ODAKE:

KGrpergewicht und Nummer des Tieres

Versuchstage

Versuch X,

Entkleiete Gerste.

1. Kinfliche entkleiete Gerste, die zum Kochen fertig ist, wurde
an 2 Tanben verfiittert, ohne dass dieselben liingere Zeit hindurch krank
wurden. Weitere 2 Tanben wurden mit in Wasser gekochter Gerste
gefiittert; sie blieben auch liingere Zeit gesund. So scheint es, dass beim
Entkleien und Kochen noch eine geniigende Menge Oryzanin zuriickbleibt,

um die Tauben vor Erkrankung zu schiitzen,

UEBER ORYZANIN, EIN BESTANDTEIL DER REISKLEIR, ETC, 469

Tabelle XLI,

Kérpergewicht und Nummer des Tieres
Versuchstage Entkleiete Gerste Dieselbe mit Wasser gekocht
(1) (2) (3) (4)

1 | 270 280 275 310

5 256 295 282 302
10 268 302 279 297
15 272 305 27 295
20 297 315 300 310
25 282 317 292 308
30 276 317 313 328
35 260 302 310 310
40 | 270 315 316 323
45 | 265 320 312 315
50 263 312 318 314
55 | 245 320 325 324
60 | 230 318 325 340
65 220 325 320 335
70 | 230 323 318 313
75 | 215 321 323 330
80 | 230 330 306 303
85 | Entflohen 333 320 | 323

Bei Gerste ist die KXleieschicht nicht so scharf yon dem inneren Teil
getrennt wie beim Reis, so dass beim Polieren immer noch etwas Sehale
zuriickbleibt. © Wahrscheinlich findet sich im Kern selbst auch etwas

Oryzanin.

470 U. SUZUKI, T, SIIIMAMURA UND S. ODAKE’

Versuch XI.

Gerstenmalz.

Das Brauermalz, das wir von Herrn Braumeister Dr. Jagi aus der
Sapporo-Brauerei bekomme haben, wurde fein zerkleinert und mit
heissem Alkohol wiederholt extrahiert. Die alkoholischen Ausziige wurden
nach dem Verdampfen des Alkohols in wenig Wasser gelést und wieder-
holt mit Aether geschiittelt, klar abfiltriert ynd weiter konzentriert. Der
auf diese Weise dargestellte alkoholische Extrakt hat sich als wirksam
erwiesen. Man musste jedoch ziemlich grosse Mengen desselben an-
wenden, Der Extrakt aus 50 @ Malz war kaum genug, um eine erkrankte
Taube zu heilen. Derselbe aus 75 @ reichte schon ans, um das Tier

lingere Zeit im Gleichgewicht zu halten,

Tabelle XLII. d

Versuchstage Korpergewicht Bemerkungen

Gesund

Re’s allein

Erkrankt
l Geheilt
2)
Alkoholischer Extrakt 3
aus 50 g Malz taglich 4
5
6 Gesund

UEBER ORYZANIN, EIN BESTANDTEIL DER REISKLEIE, ETC. 471

Versuchstage Kérpergewicht 3emerkungen

7 210

75 g Malz 12

Derselbe aus g

Gesund

Reis allein
Gesund

Erkrankt

Versuch XII.

Bier.

Das .,Miinchner“ Bier aus der Yebisu-Brauerei wurde bei gelinder
Wiirme zu einem Sirup eingedampft und mit heissem 90% igem Alkohol
extrahiert. Der alkoholische Extrakt wurde wieder eingedampft und
nochmals mit heissem Alkohol extrahiert. Der in dieser Weise aus 500
cem Bier gewonnene Extrakt wurde tiglich ciner erkrankten Taube

gegehen, hatte aber keine Schutzwirkune.
t > t

472 U. SUZUKI, T. SHIMAMURA UND S. ODAKE?

Es scheint also, dass das Oryzanin im Malz wihrend des Girprozesses

verloren gegangen ist,

Versuch XIII.

Gewohnliche Méhre: Ninjin (Daucus earota).

Der alkoholische Extrakt aus 100¢@ frischer Méhren hatte keine
Schutzwirkung. Auch konnten wir durch das Tanninyerfahren k<in wirk-
sames Priparat bekommen. Ob wirklich Oryzanin darin fehlt, muss
durch weitere Versuche noch festgestellt werden, weil Kaninchen ent-
weder mit Méhren allein oder mit Moéhren und Reis gefiittert langere

Zeit gesund geblicben sind.

Versuch XIV.
Raphanusblitter (Daikon).

Das ans 100 g Infttrockenen Raphanusblittern durch Tanninverfahren

dargestellte Roh-Oryzanin hatte Schutzwirkung, obgleich diese nicht sehr

stark war.

Versuch XY.
Miso und Schoyu.

Da Miso sehr stark salzhaltig ist, kann man sie natiirlich nicht
direkt an Tanben geben, auch dessen alkoholischer Extrakt enthilt eine
betriichtliche Mange Salze und Extraktivstoffe; so haben wir mittels des
Phosphorwolframsiureverfalirens aus alkoholischem Extrakt ein Roh-

Oryzaninpriiparat dargestellt. Die Wirkung desselben war jedoch nur
unbedeutend,

UEBER ORYZANIN, EIN BESTANDTEIL DER REISKLEIE, ETC. 473

Vielleicht kann man am Hund oder an der Katze, die gerne Miso
fressen, noch besser entscheidende Resultate bekommen, Es bleibt nur die
Frage, ob man mit grisserer Menge eim etwas wirksames Priparat be-
kommen kann. Jedenfalls ist es sicher, dass sowohl Miso wie Schoyu

keine nennenswerte Menge Oryzanin enthalten.

V. Zusammenfassung der Resultate.

1. Hiihner, Tauben, Miiuse und einige andere Tiere werden durch
ausschliessliches Fiittern mit-geschaltem Reis leicht krank und gehen
unter starker Abnahme des Kiérpergewichts zugrunde. Diese Erscheinung
ist durch Mangel an einem Stoffe im Reis, der fiir die Erhaltung des
tierischen Lebens absolut notwendig ist, bedingt.

2. Dieser unentbehrliche Stoff. is nun aus Reiskleie in reinem
Zustande isoliert worden. Wir haben fiir diesen Stoff den Namen
»Oryzanin“ vorgeschlagen.

Das Oryzanin nimmt eine ganz besondere und ebenso wichtige Stellung
im Haushalte des tierischen Lebens ein, wie Eiweiss, Fett, Kohlenhydrate
und Salze. Ohne diese kénnen die letztgenannten Stoffe keine physiolo-
gische Funktion entfalten.

3. Jedes Futtermittel, dem Oryzanin fehlt, kann das Leben des
Tieres nicht lingere Zeit erhalten.

4. Die kiinstlichen Futtergemische aus Eiweiss, Kohlenhydrat, Fett
und Salzen, ohne Oryzanin, konnten das Leben des Tieres nicht liingere
Zeit erhalten.

5. Hunde konnten nicht mit ausgekochtem Fleisch und geschaltem
Reis existieren, und nach 3 bis 4 Wochen waren sie vollstiindig abgemagert.
Wenn man aber so abgemagerten Hunden tiglich 3 g alkoholischen Extrakt
oder 0,3 ¢ Oryzanin zufiihrt, so werden sie bald geheilt.

6. Die Verbreitung des Oryzanins in verschiedenen Nahrungsmitteln
ist ziemlich gross. Da aber der geschilte Reis bei uns in Japan ein

Hauptrahrungsmittel des Volkes bildet, so kann der Mangel an Oryzanin

474 u. SUZUKI, T. SITIMAMURA UND S. ODAKE:

sehr oft eintreten, besonders bei denjenigen Leuten, die immer von
bestimmten, wenigen Nahrungsmitteln leben und keine Abwechslung
haben, wie die Leute in Werkstiitten, Liiden, Gefangnissen usw.

Was wird nun der Effekt des Mangels an Oryzanin bei Menschen
sein? Viele Mediziner behaupten, dass die Beriberikrankheit durch
geschiilten Re’s verursacht wird’. In den Philippinen ist man sogar so
weitgekommen, dass das Essen des geschilten Reises verboten wird.

Wir wollen das Studium der Beriberi, besonders der Beziehungen
zwischen dieser Krankheit und Reisnahrung, Medizinern iiberlassen und
beabsichtigen, spiiter iiber die chemische Natur des Oryzanins und seine
physiologische Funktion bei Tieren weitere Aufklirung zu geben.

1. Vergleiche die Arbeiten von BREAUDAT und DENIER: The use of rice bran in
the prevention and cure of beriberi, Annales de l’Inst. Pasteur 25, Nr. 2, S. 167 bis 189,
1911.—BrEAUDAT, Studies on the protective power of bran in a_ polished rice
diet. Bull. soc. Path. Exot. 4, Nr. 7, S. 498 bis 502, 1911—W. P. CHAMBERLAIN,
H. P. BLoomMBerGH and H. P. KiLBourng, Study of the influence of rice diet and of inanition
on the production of multiple neuritis of fowls and the bearing thereof on the etiology of
beriberi. Philippine Journ. Sci. B. Med. Sci. 6, Nr. 3, S. 177 bis 209, 1911.—G. HeEItsEr,
Practical experience with beriberi and unpolished rice in the Philippines. Philippine Journ.
Se. B. Med. Sci. 6, Nr. 3, S. 229 bis 233, 1911.—H. D. W. Greic, Rice in relation to
beriberi, in epidemic dropsy in Calcutta. Sci. Mem, Med. and Sanit. Dept. India n, ser.

1911, Nr. 45.—C. Toyama, Rice and Beriberi. Zeitschr. f. med. Mikroskopie Nr. 104, Dez.
1911.

Jour. Coll. Agric. Vol. I. Taf. XIX.

I
Oryzaninpikrat
(aus Wasser.)

“i
‘i
II
a-Saure
aus Roh-Oryzanin (I).
|
f
| Ill
B-Siiure
aus Roh-Oryzanin (1).

H, Oye. imp.

Y74
Jour. Coll. Agric. Vol. I. Tafel. XX.
Versuche. mit Sauben.
Versuch. Crab we Orysanen i) Versuch I.
350 Lecithin. Phytin fate
Geschalter Theis mit Lecithin, Cryganin. (1) fier OS.
Pitino i
300 On
Bis
250 a =
2D Seay a) aD BES io 20 30
Versuch IF
— Reis allen 5 3 mit Filtrat von Pherphowolfiam >
Bhh 2S sauce Nicdorschlag
S 03g Roh-Cryxanin
RS WV iglick 2s we
ao t
a 70 Maia apt pte 40
Versuch V1)
300). (A) ‘B)
Fheigalleim <0.0g Rohr Reis allein f
Cupganin (ID) ‘peas allein > O.0lg Foh- «—Fras alizin—
250) Bee 250|—— Cryganin HD)
wid 7 fo 20
Versuch V1), i
00 (1) 300 (2)
500 |e Aly ok Reis llein :
gone (ID Feeis allein 0.0lg Ftoh -Crysanin (Db)
a PBR eee
2.009@

Lith, H. Oye

/ 4
:
}
;
iy
i

3
Y7f

Jour. Coll. Agric. Vol. I. Tafel. XXL.
Versuche mit Taulen

Versuch V.
300 (1) 300 (2)
Reis allen 00g Rok-Crypanin
Theis altein Q0lq Reoh-Caysanin Ob2g Tech Cupyanin

200

Mess. 0.939 Fannin ~Waederchlag. Teeis hein

20 ‘. HEE ree Ree
ae I

10 20 30
Versuch VIE Peines Oryzanin
(1) +

300

Rus athin Se akaS
: 9.019 0.02g 0024 Fes
250| Flirat von Pikute Frat
aus Roi ryyanin -ea hig

()

200

150

Lith, H. Oye.

Gry”

‘fo “A WRT

0¢
= scrieeisaNtESyressrsetrs :
soesceesedgsensccceczesear Dosa
008 : Ne
= r upurthag yn (gq) ununthiag my) (Y)
unShag “CED yop unppoag mrgprh, waiyty “ayn
aga cunyprag witiye UW ig POE
Tih YP? HYG POM
06 O8 OL 09 Os Ob og 06 OL l os/

(tah) {

t TNR ee 002

VA = ia bos skso seem
2 NN
a 2S oe Es <a Ramen ff YY 1d
oN WZ
et
SS 08%
“UIYRDPOUM EL ps bo 0 WITT DOU? fe oo0 ua TROP Ulpyr~ mey ?

ape uayhnyy, UNYTTIAT IYDLL

POPLIN Faye arynprapetumnyol “Xx yng).

UaqiDy, pitt TINIE),
‘UXX ‘fol ‘IT 129A ‘omby “oD “mor

— oo — =

rN
?

Vash I. Unueschii 2 Kees

a7 manenz ace

30 40 50 60

10 20
(C) mit Phytin, tectthin a. Sabyen

B Heer tities

* (2) ausgekommen

/ 10 20 30 40 50 oo

Lith, H. Oye.

ee en,

Y7¢
Jour. Coll. Agric. Vol. I. Tafel. XXIV.
Virsche mitMauten.

Visuch IV. Geschalter Frets mit. Roh- Copyanin tH).

12 (QL) Reis-und. Teoh- Ouyzanin.

a S22 S507 6ERs SES =SeeeRee
m

9 .

2 1 16 20 30 35

ie th) Ras, Fioh-Cryzanin, Leicthin

9 =

8
1)

Tite

6 (2)
/ 10. 20 30 35
Versuch V. HKihnereser

7
Starh
6 | Tas Siaeneeeeseeaes
5)
Stark
4 Start
1 10 20 30 35

Versuch Y. Kuahmilch.
(1) mit. chhoholischem Extiakt der Kiubmilch.

i 10 20 30
15 (Z) mit pelrockniler ~ Melch

Stark 20

Lith, H. Oye.

?
E74

Jour. Coll. Agric. Vol. I. Tafel. XXV.
Versuche mit Mausen

WVerstich VW. Geschaller Reis mit Margarin.

Se Harb

HOTT 70 20 35

Versuch VI. teschalte tex Rets mit abhohotischem Fctrakt
on F ferdef leisch, 4 a

Versuch TX. Starke, huasecn, Lecithin und. Sabg.
WW (A)6fme Oryzanin. 71 (B) met Caysonin

Ytanh

Lith, H. Oye

Agric. Vol. I.

____(A)und (B) At abkoholis

: | ;
m Hi
s /
\
&
\ ;
\ \/ j
a i
ayyanan suEe 12 Chae Oryzanin J

m Extras (A) und (B)GAne Cryzanin
rérake__, A) und. (hb A

4) Ohne

150g Ferteftess

190

Tafel. XXVI.

Lith, H. Oye

.
.

Ss Tokyo Daigaku. Nogakubu
19 Journal of the College
T65 of Agriculture

Biological
& Medical
Senals

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