Studies on the Utilization of \Nhale ·:Meat by
the use of Pancreatic Tryptase of vVhales
Shichiro Akiya, Setsuzo Tejima and Yasutaro Ishikawa•The present experiments were carried out in order to obtain enzymes
suitable for bacterial culture and production of diphtheria antitoxin fromwhale meat digested by whale pancre2s. The authors prepared 25 kindsof peptone from a definite quantity of meat at a definite pH value withvarying amounts of enzyme and at different lengths of time. With eachof the peptone obtained, the ration of total nitrigen to free amino radical-Nand the yield of peptone were measured. With each peptone obtained
under various conditions, test culture of diphtheria bacillus and productionof its arititoxin were tested. As a result of these experiments, following
observations were made :1) It was found that better yields of peptone were obtained when reaction time was prolonged. For example, when 3 g of pancreactic powder
was used, yield increased proportionately with increase in reaction time.
Table 1No.

Reaction time(Hours)

122
68:<3122448

72120

Nitrogen ofPercent of digestiveliquefied portion (g) ~ (Comment. 1)
4.42
36.0
6.63
51.5
56.5
6.93
9.51
77.3
98.0
12.06

Amino nitrogen :Total nitrogen1 : 3.0l : 2.3
l : 2.7
1 : 2.7
1: 2.2

When the reaction time was made constant, the amount of amino
nitrogen increased with the increase in the amount of enzyme but no effect2)

Table 2
No.

.Amount of usedpancreas (g)Amino nitrogen :Total nitrogen

Nitrogen of liquefied
portion (g)Percent of digestive(%)

l2
342
35
7.5

1 : 3.4
l :.2.9l : 1.91 : 1.3

6.88
6.3$
7.46
8.00

56.0
51.5
60.765.01)

As is described later, the amount of nitrogen in Frozen whale-meat is 16.4%. This value
was obtained by dividing the am~unt (in gram) of nitrosen foun:l in liquefied portion by
0.164x300x0.25 and then multiplied by 100.

12S. Akiya, S. Tejima, Y. Ishikawa

was observed in the.· yield of peptone, as can be seen from the following
graph where the reaction was limited to 24 hours.
3) Addition of duodenum powder amounting to about 1/3 the amount ofpanccreas powder results in the increase of 20% in the yield even when
the length of reaction time is the same.
Table, 3No.

Amount of used
Amount of used
_ __,_p_an_cr_e_a_s-"'(g"'-)_ 1_ _ _d_u_o_de_n_u_m__,(,,,g)'-I-~ I:

2252

2
2
326 . 1

3:~I'I

Reaction time(hours)

Yield (g)

24
24
24

35.5
43.5
44.0
50.5
44.1

1.7
0.7
1.0

___
I _ _ _:ci_!:__I ____ . _!~--·-·--·---5-5._o___As a result of experiments in diphtheria culture and production ofantitoxin, under various, it was found that some of the peptone obtainedby the authors were as good as the market products. It was also foundthat those suited to the production of antitoxin were those in which theratio of total nitrogen to amino nitrogen was in the order of 3.9.1.
4)

EXPERIMENTAL
1) Preparation of enzyme powder
Enzyme powder as described in the paragraph on 'Tryptase in a whale(baleanoptera borealis Lesson)' was used. This was prepared by drying
whale pancreas with acetone and ether, grounding in mortar and putting
through a sieve No. 5. Duodenum was also prepared in the same manner.
2) Perparation of whalemeat peptone
300 g of frozen whale-meat was chopped finely with a knife and put
through a mincing machine. 530 cc water was added to thi::; 2> and boiled~>
for 10 minutes. This was then cooled and placed in a thern-;.o'.:at at 40°C.
pH value at this juncture is 5.6 which was corrected to pH 8.6 with suitable
addition of caustic alkali. To this solution are added enzyme powder anda small amount of toluene as a preservative. This is then kept in a thermostat but for the first 6 hours, pH value must be corrected every 1 to 22)3)This amount of water was determined from the amount of water (75%) found in frozen
whale-meat as described later, Calculation: 300x0.25x10-300x0.75=530.
pH values were determined with the test papers T. B., B. T. B., and C. R. of Tokyo
Roshi Co.

Studies on the Utilization of Whale meat by the use of Pancreatic Tryptase of Whales

13hours because pH values tend to shift to ·acidic range at first. For
instance, if enzyme powder is added to a solution of pH 8.6, it will shift
to pH 7 after 90 minutes. After 6 hours from the time of the addition ofenzyme powder, pH values do not vary greatly, so that pH values a.re
determined only in 4 to 6 hours. By this correction, pH value is value ismainted at 8.6.4 ) After a lapse of definite time, the solution is taken outof the thermostat, boiled for 10 minutes to break the enzyme, pH corrected
to 4. 7 with hydrochloric acid and filtered while hot through folded filterpaper.5) The residue is washed once with 300 to 400 cc of hot water andand the washing is added to the :filtrate. The amount of filter and theresidue are weighed, 1 cc of which is taken to determine total nitrogen by
Kjeldahl method and amino nitrogen by Van Slyke method. The filtrate isneutralized with sodium hydroxide and concentrated on a steam bath asmuch as possible to a paste. This paste is tentatively called whale-meat
peptone. The time of reaction was varied at 12, 24, 48, 72 and 120 hours,
and the amount of enzyme to 1, 2, 3, 4, 5 and 7.5 g. Tests were also madein which duodenum powder to the amount of 1/3 of pancreatic powder
was added at the same time and another in which 5 g dried powder of 4thstomach was used in place of pancreatic powder.
3) Determination of the amount of water and total nitrogn in frozen
whale-meat.
5 g Frozen whale-meat forced through the mincing machine was mixed
with 5 g sand and held in a thermostat at 110°C until there was no change
in weight. The loss in weight was taken as the amount of water containedin frozen meat.
Weight at the end of 22 hours 1.2508 gWater %=(5-1.2508)5x 100=75.0% (75.0%)
The frozen whale-meat was dehydrated and degrease with acetone andether, dried and the amount of toatl nitrogen determined by Kjeldahl
method.
Sample 50 mg. N/10 H_SO~ 20 cc (F= 0.902), N/10 NaOH 13.87 cc (F= 1.000)
N% =l.4~20x0.982-13.78)x100/50=1.64%
4}
5)

pH values were determined by the test paper T. B., B. T. B., and C. R., of the Tokyo
Roshi Co.
If filtration is performed while hot, filtration is rapid enough even at this pH value.
Filtration cannot be done at alkaline state and even at pH 4.7 it becomes diffecult if
allowed to cool.

14S. Akiya, S. Tejima, Y. Ishikawa4)

Culture of diphtheria bacilli and production of antitoxin

Whale-meat peptone No. 11 and 25 were used for the culture media
for the production of diphtheria toxin. Composition of culture midium was
as follows:
Horse meat bouillon5 >............ ······· ·····250 cc
Pepton ························ ·····················2.5 g (1%)
CHzCOONa ....................................... 1.25 g (0.5%)
Mg~04 · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·0.05 g (0.02%)

NaHP04· · ·· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·0.25 g (0.1%)
CaCl2 · · ... · · · ·· · · · ··· · ·· · ·· · · .. ·· ·· · ··· · ·· · ·· ·· · · ·· · ·0.25 g (0.1%)
The pH value of the medium was corrected to 7.6 with NaOH, boiled
for 10 minutes, filtered and the pH value of the filtrated again corrected.
This solution was then poured into D-type flask of 250 cc capacity, sterilized
at ll0°C for minutes and 2.5 cc 20% rice syrup''> (amount of syrup tomedium: 0.2%) added aseptically. To_ this media was plant~d diphtheria
bacilli, strain No. 8 of park-William's, and held for 7 hours at 37°C.
Table 4
Pepton pH at inoculation---1115Carno

7.6
7.6
7.6

Growth after
7 dayspH at end time

Lf value/c.c.

Kf value

tit
tit
tit

8.8
9.0
9.0

877666

Finally, authers deeply thank Prof. Shogo Hosoya, Prof. Michizo Asanoand Mr. GOro Urakubo of Infectioins Disease Institute of Tokyo University.
(Pharmaceutical department, the Faculty of medicine, University of Tokyoand Whales Reserch Institute.)

6)

7)

8)

Preparation of horse-meat bouillon :Finely chopped horse-meat was added to 5 times its amount in weight of 5% acetic acid,kept for 30 minutes and the pH corrected to 5.8 with 20% NaOH. This was boiled for
10 minutes and filtered.
Preparation of rice syrup :Market product of rice syrup was dissolved in y.rater to obtain 20% solution, poured into
test tubes and left for 1 day. They were then disinfected at J.00°C for 15 minutes,intermittently.
Signs used to designate the amount of growth:-' Bacterial film not covering more than half of the surface after 7 days.
-r
Bacterial film covering more than half but not all of the surface after 7 days.
+ + Bacterial film covering the whole surface after 7 days.
+ + + Bacterial film covering the whole surface and constituting a layer.