THESCIENTifIC REPORTS
Of

THE Wllf\LES .RESEARCH INSIITUII ,
-

.

NO.I

THE WHALES RESEARQI INSTITUTETOKYO · JAPAN
JUNE

19(48

CONTENTS '·'f . >'Page.

1. Foreword ······························· ·················································· 1
2. Studies on Digestive Enzyme in .Whale
Shichiro Akiya · · · · · · 3
3. Studies on Tryptase from a Whale
Shichiro Akiya, Yasutaro Ishikawa,,
Setsuzo T~ima, Toyohiko Tanzawa······ 8
4. ·Studies on the Utilization of Whale meat by the use of·. Pancreatic Tryptase of Whales
Shichiro Akiya, Setsuzo Tejima, Yasutaro Ishikawa······ll
5. Test Culture of some Microorganisms with Whale-Meat Peptone.
Shichiro Akiya, Fumiharu Kobo· · · · · ·15
6. Chemical Studies on the Freshness of Whale Meat. I.
Tadashi Nakai······17
7. Chemical Studies on the Freshness of Whale Meat. II.
Tadashi Nakai··· .. ·27
8. On the Simultaneous Extraction of Vitamin A D and Vitamin
BJ Complex from the Liver of a fin Whale
Tadashi Tawara· ... · ·31
9. Studies on Whale Blood. I.
Tadashi Tawara······38
10. Sinus-hairs of the Sei Whale
Teizo Ogawa, Junnosuke Nakai, Toshitomo Shida·· .. ··41
:

i.

"

.

FOREvVORD
The establlishment of Whale Reserch Institute (Juridical Foudation)
was permitted by the Japanese Government in September 1946 but the
research itself had been carried on for a long time prior to this. The
present Institute owes its founding to the late Mr. Ikujiro Nakabe (President of the Taiyo Whaling Company) and Mr. Kenkichi Nakabe (the former Vice-President of the Taiyo Whaling Company), who found Nakabe
Research Institute in 1941 in order to carry out researches concerningwhales and for the future development of the whaling industry. However,
the outbreak of war shortly after the establishment of the Institute madeit virtually impossible to carry out researches to any extent.
The confusion of social conditions at the time of surrender made it
imperative for the· Institute to be disbanded, for the time being, at least.
Nevertheless; individual researchs were carried on, without the permissionof the Japanese Government since it would be a great loss to all concerned
if all the facilities and valuable reserchers were to be dispersed.
Japan's recovery went ahead at a far greater pace than was imagined
at first due to excellent administration of the Occupational authorities.
With regard to whefl.ing, not only that near the coast of Japan but whaling
in the Antarctic has been granted to the Japanese so that researches inwhales could once more be carried out without difficulty.
Accordingly, permission was granted by the Japanese Government to
set up a juridical foundation and the Whale Research Institute was firmly
established to carry on the work. Publication of results of research will
be made twice a year and it is hoped that exchange of date on researchin whales will be made in the near future with various colleagues all over
the world.
Tsutomu Maruyama
President
WHALE RES2ARCH INSTITUTE
Res2arch Members : Prof. S. Akiya, D. Pharm.
Y. Ishikawa, B. Agr.
T. Maruyama, D. Agr.
T. Mitsuwa, D. Sc.

2Foreeord

T. Nakai,Prof. T. Ogawa, D. Med,H. Okuda, B. Pharm.
T. Tawara,•

Studies on Digestive Enzyme m Whale
Shichiro Akiya and Setsuzo Tejima
I. Introduction

In this country where medicinal digestive enzymes are lacking due toshortages of land animals, materials for such use must be sought elsewhere.
Luckily, Japanese i~ .surrounded on all sides by the sea where resoures formarine product abounds and to which we can turn for material. It isnatural, therefore, that we should turned to one of its biggest animal, thewhales, not only for madicinal material but also for the solvation of ourfoodstuff problem.
Although biological researches on whales have been made to a large
amount in the past and many bibliographical data are available, very few
references can be found of chemical studies of whales, either in fundamental chemistry or in its application. The author, therefore, undertook tomake some chemical experiment with whales with the object of obtaining
fundamental knowledge as basis for their utilization.
The first report,herewith, present some observations obtaind regarding digestive enzymes
contained in whales.
II. Digestive enzymes of· a whale (Balaenoptera borealis Lesson)
Researchs into pepsin ·like substance.
Stomachs of whales differ in structure according to species but they
are generally divided into 4 sections. For the sake of <;onvenience, theauthor and others named then as follows : lst stomach the one directly
connected with cesophagus. 2nd, 3rd and 4th stomachs-stomachs connectedto the lst in that order.
In order to determine how pepsin is distributed in each stomach,mucous membrane of the stomach was separated from its sinews and its
dried· powder was prepared.
1) No enzyme was found in the lst stomach, very little in the 2nd, andlarge amount were found in the 3rd and 4th stomachs.
2) Contrary to the existing idea of protein decomposing enzyme ofanimals, pepsin, which acts in acid medium, the substance found inwhales work best in neutral or alkaline media.

4Shichtro Akiya

3) Certain concentrated product can be obtained from the mucuous
membrane of the stomach by a simple process.
4) The substance is activated by common salt.
EXPERIMENTAL
1) Preparation of the material
Mucous membrane from each stomach of a whale (Baleanoptera borealis Lesson) dissected at Ayukawa in Miyagi prefecture, were soaked in
acetone and sent to Tokyo. The concentration of acetone was about 50%.
Time elapsed in transportation to Tokyo was about 10 days. The membrane was then cut into small pieces with scissors, put through a mincing
machine once and soaked in two to three volumes of acetone. This wasleft for one hour with occasional shaking and then filtered by suction. This
procedure was repeated 3 to 4 times. The membrane was finally shaken
with an equal mixture of acetone and ether for dehydration and degreasing,filtered, dried in the open and finally dried in vaccuum desiccator overcalcium chloride. The dried powder here obtaind was ground in mortar
and put through a sieve.
2) Determination of suitable pH for activitya) Determination of suitable pH by the liquefaction of gelatine.
3 g gelatine was added to 10 cc each of N/:;_o buffer solutions1l of different pH values, dissolved at 50°C and placed in a thermostat of 40°C. 0.5 gof the above dried stomach powder was added to each gelatine solution
and kept for 4 .hours in a thermostet at 40°C. The gelatine solutions weretaken out, cooled by water and their solidification observed.
The protein decomposing enzyme present in stomach is called pepsin
which, a~ is well known, is active in acid m~dium of pH 1 to 2. According
to bibliography, the most suitable pH values for pepsin to work on gelatine
is at pH 2-3. However, according to the results obtained in the above
experiment, it takes 48-72 hours for the powder of 3rd and 4th stomachs
to liquefy gelatine if the pH is kept at 2 to 3. On the other hand, if the
pH is maintained at 5.0 to 6.2, powder from 3rd and 4th stomachs willliquefy gelatine completely in 4 hours. Li.:iuefaction occurs even at ph 8. 5.
1)

For buffer solution, Sorensen's hydrochloric acid sodium hydroxide, and Michaelis acetic
add-sodium acetate, were used. (Experimental Chemistry, II, 12, p. l:;.7)

Studies on Digestive Enzyme in Whale

5

Table 1
2.4

I\I,.....\

2.2
2.0

t NN

, ' ~j!

v ......, ',-': ,
A

1.8

'"·-

I

I,.-T -~
I

1.6
1.4
1.2

,~

)?'.

1.0

Q.8

r" "'"~~ .,,//

z

·z

.v---

y

.F

...v-

\/

·c, 0.2?

It"'~t 0.6~'0.4

stomack.

.......mstomacll.

1

2

3

v

4

/

'\ "-'/" I'\__ _,,,
.6

1

8

Ils

9

b) Determination of suitable pH value by titration
0.5 g Gelatine was added to 10 cc of hydrochloric acid or sodium hydroxide of various strengths, melted at 50°C and put into thermostat at 40°C.
0.1 g Dried stomach powder was added to each of these solutions, held for
4.5 hours at 40°C in the thermcstat. They were then taken out, 5 cc For- .
mol solution2 ) added and titrated with N/5 NaOH :;olution,. the end point
being coloration of the solution to red. Titration under identical conditions
were made with each of the solution of various acid or alkaline gelatine
without the addition of enzyme and the difference in the two values weretaken as the increase in acidity at each pH value. Following graph shows
the titration values at various pH values. No change in pH value wasobserved before and after the reaction. pH values were determined by thetest paper of Toyo Filter paper Co.
On the other hand, Bh.tret reaction was tested with the reaction mass
after reaction and obtained results coinciding well with the results of Formal
titration showing deep blue coloring on acidic side and light pink in neutral2)

1 cc 0.5% phenolphthalein (0.5 g phenolphthalein dissolved in 100 cc 50% alcohol) added
to 50 cc formalin (J. P.) and made into a pink solution with N/5 NaOH solution. (Biochem. Z. 7 (1907), 45 : Z. phys. C. 64 (1910), 120.

Shichlro Akiya

6

and alkaline portions.
3) Concentration of enzyme
500 cc 0.2% hydrochloric acid solution was added to 50 g dried stomach
powder and left for 5 hours at room temperature with occasional stirring.
This was then filtered and a solution of (NH1}:S01 was added the filtrate to
50% saturation and the white precipitate that salted out was filtered anddried under reduced pressure over calcium chloride. This was taken as asample and enzymic action observed.
a) Pancreatin testing (as in J. P.). Method of testing by means ofmodification in clause (6) for casein solubility.
0.1 g Casein dried over sulphuric acid was taken in 50 cc measuring
bottle, 1 cc N/10 KOH added and dissolved, and water added to make thewhole volume 50 cc of this solution was taken, 4. 75 cc water added andheld in a thermostat at 40°C. 0.1 g purified enzyme powder mentioned
above was taken in a 500 cc measuring bottle, water added to make thewhole volume 500 cc with solvation of the enzyme. 0.25 cc of this enzyme
solution. was added to the casein solution and held for 1 hour in a thermostat at 40°C. This was then taken out and 3 drops of acetic ·acid alcohol
solution (1 cc acetic acid, 9 cc water and 10 cc alcohol) was added but no
opalescence or precipitation occured. This shows that at 40°C and at pH
7, the enzyme digests 200 times its quantity of casein in 1 hour.
b) Determination of suitable pH value ·50 mg of the above mentioned salted-out enzyme was taken in a 25 cc
measuring bottle and dissolved in 50% glycerine. 1 cc of this solution was
Table 2taken as the enzyme solution.
z 2.0
-....
For a base material, 10 cc 6%tnzgelatine solution was made into
0
::c
~ 1.5
a 14 cc solutions with the addi'
tion of a suitable amount of N/5
NaOH or N/5 HCl with further
1.0
1addition of water. The enzyme
solution was added to each of the
0.5
acid or alkaline test solutions,i-~reacted for 1 hou'r at 40°C andtitreted with Formol solution. As
Ol.2345678l)l0
PH a control, a solution with 1 cc of

"

v

v

v

I \

Studieson Digetive Enzvme in Whale7

distilled water instead of enzyme solution was used and the difference intitration value was taken as the amount dissolved at each pH value.
Determination of pH was mas made as in the above (2) b). Results areshown in the following graph.
4) Activation by common salt
0.5 g Gelatine (I) was dissolved in 10 cc distilled water (II) and 10 cc
0.3% NaCl; 0.1 g dried powder of 3rd (or 4th) stomach added and reacted
for 4 hours at 40°C. It was found that the Formal titration value of (II)was 30% larger than of (I). In this case, the titration values of the basic
material a.re the same for both (I) and (II) when no addition of enzyme ismade. It is assumed that enzymic action is somewhat activated by commom salt.
(Pharmaceutical department, the Faculty of Medicine, University of Tokyoand Whales Research Institute.)

Studies on Tryptase from a Whale(Balaenoptera Borealis L.)
Shichiro Akiya, Yasutaro Ishikawa, Setsuzo Tejima
and Toyohiko Tanzawa
In order to investigate the presence of protein decomposing enzyme
in the pancreas of a whale (Balaenoptera borealis Lesson), the pancreas
was dehydrated and degreased with acetone and other, dried and preparedinto a powder. Following results were obtaind.
- 1) The powder obtained by grinding the dried powder in the mortar andputting it through No. 5 sieve can pass the test for pancreatin describedin Par. 6 of Japanese Pharmacopoeia.
2) The most suitable pH value for gelatine is in the range of pH 8-9(Fig. 1) and the most suitable temperature (for 1 hour duration of activity
is 50°C.)
3) When the amount of base material is constant and the amount of
enzymes varied, the activity curve (Fig. 2) shows that the amount of
enzyme and actiyity are proportionate.
4) The activity curve (Fig. 3) obtained by holding the amount of enzyme,
material, pH and the temperature of activity at a constant and varying thetime of activity, shows it increasing up to 4-hour period.
5) Distribution of tryptase in various parts of pancreas did not vary
greatly between those in tail, body and head parts, but the amount in the
tail part was 110% of the total of other two parts.
The purification of pancreatic tryptase is to follow the preparative
methods for chymotrypsin and trypsin crystals irom bovine pancreas asreported by Northrop. Prepartions are now being made for carrying out theexperiments. Although no definite report can yet be made, the authors areconvinced that if bovine pancreas is used, crystals of chymotrypsinogen andtrypsinogen can be prepared comparatively easily by the Northrop method.
EXPERIMENTAL

1) Preparation of enzyme powder
Pancreas obtained from a whale (Balaenoptera borealis Lesson) dissected at Ayukawa in Miyagi Prefecture was imnersed in twice its volume ofStudies on Tryptase from a Whale9acetone for 10 days, taken out, finely cut with acissors and put.' through a
mincing machine. To this was added 2 to 3 volumes of acetone, drained,
and the process repeated 3 to 4 ti:nes to dehydrate and degrease. Finally,
the residue was shaken with an equal mixture of acetone and ether, filterd, inthe open and then dried in a vacuum desiccator over calcium chloride. From
2500 g of pancreas, 300 g of dried substance were obtained. This substancewas ground in a mortar and put through a No. 5 sieve (J.P.). Yield of thepowder to the dried substance, ea 40%.
2)

Panereatin Test, Par. 6, Japan Pharmacopoeia
0.1 g Enzyme powder prepared as in the above (1) was taken in a
500 cc measuring bottle distilled water added to make the total volume
500 cc and filtered. 2 cc of the filitrate was dissolved in 1 cc N/10 KOH
and distilled water added to make the total volume 50 cc, 3 cc of this solution was added to 2 cc distilled water, reacted for 1 hour at 40°C, after which
3 drops of acetic acid alcohol solution (1 parts glacial acetic acid, 9 parts
water, 10 parts alcohol were added. Only slight opalescence could be seen.

3) Determinnation of suitable pH of adivity
3 g Enzyme powder as described in (1) was added to 30 cc 80%
glycerine and left standing over-night at room temperature. A clear upper
solution was separated centrifugaly, 9 times its volumeo of distilled water
was added and 1 cc of this solution was used
-as enzyme sample. For the base material,'.Z
0.5 g of gelatine was added to 10 cc each of ---.._•>- 1.500glycocoll-NaOH buffer solution (S:)rensen·s) 0/.l/ ~
Qlof varying pH values, and melted at 50°C. [r;· 1.25/
To this was added 1 cc enzyme sample at :r: 1.0040°C, held there for 2 hours and the amino .::i: 0.75
enacids thus formed titrated, in Willstatter's 0 .
ft0.50alcohol solution, with N/5 alcoholic KOH " 0.25
solution with thymo~-phthalein as an indicator. f>
1098
7
On the other hand, the base with 1 cc of disPH
tilled water instead of the enzyme powder
Fig. 1
was titrated and the difference between
the two values were taken as the degree of digestion at that pH value .
. Results are shown in Fig. 1.
4) Activity curve by the change in the amount of enzyme/

S. Akiya, Y•.Ishikawa:, S. Tejima · and T. Tanzawa

0.5 cc, lee, 2 cc, 3 cc, 4 cc and 5 cc of the glycerineextract of pancreas,
as described in (3), were added to solutions in which 0.5 g gelatine wasdissolved in 10 cc NaOH-glycocoll buffer solution of pH 8.1 and water added
to make the total volume 15 cc. This was reacted for 2 hours at 40°C andfinally titrated as in (3). Results are shown in Fig. 2.
4

~

3

/

2

/
v

.

/

glycerin0

extrac~ c.c.

/

/

v

/

3

Fig. 2

17
J

/

I/

2

-i~~ ~

/

,__ v

z

'CJl

/

1
2
3
Dried pancreas(g)(0.03) (Q.06) (0.()9)

I

!

I

:i>

;:;- 1

I

0

~

0

4
(0.12)

5(0.15~

i
I

1

~I
:i:

~

0

1

2

3

4

5

6

~Hours.

Fig. 35) Activity curve for varied action time
2 cc glycerine extract of pancreas as described in (3) was dissolved in
10 cc solution of NaOH-glycocoll buffer solution of pH 8.1 in which a baseof 0.5 g gelatine was dissolved. The reaction temperature was kept constant at 40°C and time varied at 15 minutes, 30 minutes, 1, 2, 3, 4, 5 and 6hours. Titration was performed as in (3). Results are shown in Fig. 3.
(Pharmaceutical department, the Facluty of Medicine, University of Tokyoand Whales Research Institute.)..

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 1
No.

Reaction time
(Hours)

12
2
6
8
:<3

12
24
48

72
120

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 effect
2)

Table 2
No.

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

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

l
2
3
4

2
3
5
7.5

1 : 3.4
l :.2.9l : 1.9
1 : 1.3

6.88
6.3$
7.46
8.00

56.0
51.5
60.7
65.0

1)

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.

12

S. 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

:

2
25
2

2
2
3

26 . 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 of
antitoxin, 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 2
2)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

13

hours 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 of
enzyme 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 filter
paper.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 4th
stomach 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 g
Water %=(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.

14

S. Akiya, S. Tejima, Y. Ishikawa

4)

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 to
medium: 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
---

11
15
Carno

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

8
7
7

6
6
6

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.

The Test Culture of some Microorganisms
with Whale Meat Peptone
Shichiro Akiya and Fumiharu Kobo
This experiment was carried out to determine the difference of valueof utilization between the samples and market·peptones. Market peptones
were used as follow ;T<: ruuchi's, Witte's, Carneau's and Chapteaut's Peptone.
Experimental
Incubation-condition;
Culture media ; Peptone·water prepared with the some kinds of peptones
as above descrived.
Test-strains ; B. coli communis, B. typhi, Pneumo coccus, B. dysenterie
Minoda, V. cholerae, B. subtilie M. B. 25, Staph. coccus aureus
Terafona, B. pyocyaneus.
Incubation ; 37°C., 24 hrs.
Result;
The amount of increased _bacillus, the formation of dyestuff and RS
are shown as follow :B. Coli cornmunis

~I

I 12

18

24

I
Sample+ I +ITeruuchiititI
Carneau
+
it
I
I
Witte
±
+
I
Chaptoteaut
it
+

it
it
it
+
it

illillillit
it

6

Order

i- - - - · - - - - -

.
I

I

!

B. typhi

3
5
4

I

I

I

P~ep_ton_~e-~ 1 _6_~1~\~I Order
SampleTeruuchiCarneau
Witte
Chaptoteaut

+
+
+
+

+

it
ill+
it
it

2
1

5

+
it
it
it
tit-

5
4

3

3
2
4
1

Pneumococcus 1

I~Hours1
2

Indol-formation
Ehrlich's Method
Kitazato's Method

it I ill- I tit I
it I ill- I tit I
it I it i it
1

it+

i

it+

2
l
4

I ittlt ! s
11

3

~I 6
.
SampleTeruuchiCarneau
Witte
Chaptoteaut

12.

i 1s

24

[it fillfil illi if!- if!- if!- if!it
it

+

tit

illif!-

illillif!-

if!illif!-

Order
4
1
5
3
2

16

Shichiro Akiya and Fumiharu Kobo
B. dysentheriae Minoda

~ 61~
p

18

24 Order+ + +SampleTeruuchiCarneau
Witte
ChaptoteautV. cholerae O.

*tft

4
1
2
5
3

--Hours'""l-6-..,.-1-2-:-1-8~1-24--:-l-o-rd_e_r,..i_In_d_o-l---.....,..1-o-rd_e_r
Peptone,I
I formation,
S a m ; l e - - l * * * tft+ +Teruuchi

1 '

* tft

4

+
+
+
+

\

5

1

4
*+ * * tft
Carnean* ·* * tft 3
± * *
Witte
+ + I* * 5
2
Chaptoteaut+ + tft 2*
1+ :1: **The experiments above described led summarily to the results as
5:l:follow:
Peptone[Order of Bac.-growth--i-- ~ -------~-~~u-P~-:h-i___ ; -~1--:

Order of Indol-formation
4

2
1
2
2
1

4

Carneau

:

2

I

3

3

3

Witte
Chaptoteaut

I

2
2

I
I

5
2

5
3

5
3

j

3
5
4

3
3
5
4
1

5
4

5
4
3

----

(i) The amount of increased Bae. Incubation; 24 hrs, Peptone-water, pH 7.0~

B. subtilis M. B. : 5 B. pyocyaneus
orr1ertft

TeruuchiWitte
SampleCarneau
Chaptoteat

1

*tft

4
2

tft
tft

3

3

tft

*tft
tft
tit

\ Staph.

01·, 1er

1
2
1
1
1

I
.

I

Jl1

_:~reus Teraji_::
Qrjer

111

1

tft
tft
tft
tft

1
1
1
1

(ii) B. typhi; H 2S-Formation tested
by Lead-acetate-paper. pH 6.8

~-

24

I

36

!'._eptone
TeruuchiWitte
SampleCame au
Chaptoteaut

tft

+

I *

tft

*
tft

(iii) Formation of dyestuff anrl JnrloJ.
B. Pyccyaneus. Incubation ; ~6hrs, pH 7/)
ReactionI
Indol-formation
I
I Dyestuff\ Ehrlich's
Kitazato's \ GrowthI
Peptonej
Method I
Method •I---1TeruuchiWitte
SampleCarneau
Chaptoteaut i

tit
tit

+

*+

*
*+

I
I

tft

tit

+
+
+
+

*ilt
tl-

tft

Authors finally deeply thank Prof. Shogo Hosoya, Prof. Michizo Asanoand Mr. Goro Urakubo of Infections Disease Institute of Tokyo University.
(Pharmaceutical department, the Faculty of Medicine, University of Tokyoand Whales Research Institute.)

Chemical Studies on the Freshness of Whale :Meat. I.
Evaluation of freshness and changes in quantity of several kindsof nitrogen in whale meat following deterioration of freshness.

Tadashi Nakai
There is generally a lapse of time on the sea between the time whena whale is harpooned and until it is dissected. There is still a longer lapseof time before this prepared whale meat gets into the hand of consumers.
It follows, therefore, ·that there should be more chance of deterioration of
freshness in whale meat than in the meat of domestic animals and fowls.
Preservation method and use of whale meat varies according to the degreeof freshness. It can be said, therefore, that the question of the freshnessof whale meat has the most important significance in the handling of whalemeat~

The author has undertaken the present studies with the object ofmaking chemical investigations on the deterioration of freshness of whale
meat and of making a contribution to the problem of maintaining freshness.
First, ·it is necessary to decide on the evaluation method of the freshness of whale meat in the author's future experiments.
An example of the evaluation method used by operators in the field inJapan is shown in Table I.1\ but it is too subjective to be adopted as themethod in the author's experiments, and even in the field it could only be
used at the time of dissection.
Table I.
Grade

' Colour andlustre of flesh.

Standard of Freshness of Whale Carcass.
Elasticity ofmuscle.

Very fresh ! Ve~y dark red,semitransparent, : Conspicuous
(gO%)
0
1
lustrous\ Offensive smell Exudation of
!
blood
1
!,

Lapse of time
between killingand dissection

1

!

none

none

Less than 6 hrs.

FreshMore reddish,i Recognizable////
Recognizable
12 //by pressing(80%) i lustrous
Satisfactory Reddish grey,
//
//
18 //
Recognizablenone(60%) I non-lustrous
Unsatisfac- I
//
//
//
24 //
Conspicuous
Recognizable
tory (40%)
11//
Flowing
Poor (Less i
Extreme!More than 25 hrs.
than 40%).___ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _~---

i

Chemical methods for determination of the quality of flesh have been
worked out by many researchers already and among them, the method based

18

Tadashi Nakaion the determination of various kinds of nitrogen has been studied mostand it seems to be more reliable than the others, as for instance, oxygen
consumption test, methylene b1ue and nitrate redution test and hydrogensulfide test.
According to Malin2), high-quality meat, fish and fowl shows 6-16 mg,
satisfactory fish and fowl, 18-:-26 mg and poor quality meat, fish and fowl,
27 or· more mg total NH3 per 100 g.
Tillmans and Otta3) reported that at the beginning of the decompositionof fish, NH3 reached 30 mg and amino acid N 100 mg per 100 g, andShimizu4 ) found ammonia N 30-40 mg and amino acid N 80 mg per 100 g
at the time.
Glassmann and Rochwarger5) reported that 0.020% and 0.025% are therespective critical quantity of NH3 indicating the beginning of putrefactionin the flesh of warm-blooded animals and of fish.
Herzner and Mann6) observed that the amount of pure albumin inmeat, which decreases with the progress of putrefaction, gave an indicationof incipient putrefaction and that a pH of over 6.2 indicated putrefaction
in meat.
Jpatov7) recommended the determination of hydrolized protein forevaluation of the freshness of meat.
For measurement of spoilage in fish, Beatty and Gibbons8) recommended the determination of trimethyl amine fraction in the filtered muscle
juice. They found that fresh muscle showed a mean value of 0.17 mg oftrimethyl amine N in each 100 cc of muscle juice; that odors appeared atabout 4.0-6.0 mg and that the trimethyl amine fraction did not increase
appreciably by autolysis.
For detecting incipient decay in meat, Brotzu9) recommended the determinations of amino acid N and pH of meat juice which transuded fromminced meat by heating, and of the bacterial count of juice obtained bypressing the minced meat. He found that 300-350 mg of amino acid N(Sorensen) per 100 cc of meat juice and pH 6.3 and above indicated incipient decomposition.
According to Yamamura10) there is a straight linear relation between
ammonia content and pH of fish. He observed that ammonia content at
the beginning of putrefaction was 30 mg per 100 g, as was found by Tillmans and Otto, and the colorimetric measurement of pH at this period

Chemical Studies on the Freshness of Whale Meat. I.

19

was 6.5.
The present paper embodies the author's observations on the changes
in the amount of some nitrogen in whale meat during the course ofdeterioration of freshness, which have been carried out with the object ofdetermining of nitrogen which would indicate the degree of freshness inwhale meat.
I. Changes in the amount of volatile basic N, non-protein N, formol
titrating N, trimethyl amine N and trimethyl amine-oxide N were determined i'n minced whale meat standing at room temperature.
The material used in the experiment was the 'oniku ', a portion offlesh between the dorsal fin and tail, of blue whale (shironagasu-kujira ;Balaenoptera musclus). Its fat content was very high, namely 29.2%.
Samples, taken from the minced material, showed a distinct acid reactionand emitted strong sour odor all through the course of experiment. The
author believes that this was due to the formation of a remarkable amountof free low fatty acids resulting from the decomposition of fat by enzyme
in muscles and that, consequently, the propagation and the kind of bacteria
was considerably restricted.
The amounts of volatile basic N and formol titrating N increased
regularly with lapse of time, and the rate of increase was great (Table III
and V). The increare of non-protein N (Table IV) and trimethyl amine N(Table V) were comparatively small. Change in the amount of trimethy.I
amine-oxide was practically unnoticed (Table V).
On account of the strong sour odor, it was difficult to detect the putrid
odor at the incipient stage of putrefaction of protein in the sample. However, assuming that the onset of putrefaction took place when ·gas generationin the interior of a sample (formation of bubbles) was detected, it was

found that the amount at this period were: volatile basic N, 30.2 and 29.7
mg per 100 g, non-protein N, 500 mg, formol titrating N, 136 mg and trimethyl amine N, 0.45 mg.
II. Comparison was then made of the percentage value of freshness
which, as shown in Table I, is determined rather subjectively by the whalefishing men in Japan for evaluating the degree of freshness at the time ofdissection, and the quantities of volatile basic nitrogen and of formol titrating nitrogen.
The material used in this experiment was flesh of sperm whale (mak-Tadashi Nakai

20

kO-kujira; Physeter macrocephalus).
The results obtained are listed in Table VI and also summarized inTable II.
Table II
Freshness%(cf. Table I)

Volatile basic N(mg/lOOg)

Formol titrating N(mg/100 g)

Number ofsamples used

61.5
70.7
81.2
90.2
95.8

I

--------·-·-

90
85
80
75
70
60
50
40

3.5
5.5
8.2
11.5
16.0
38.6
246

4
5
4
1
1
1
1

453

With the progress of deterioration from 90% to 70%, the mean valueof nitrogen for each percentage value shows gradual and yet a definite
increase, as shown in Table II. However, there are some inconsistencies
among · the individual measurement values for different percentage values,
as shown in Table VI.
During the course of deterioration under 70%, quuntitiy of volatilebasic N shows a sudden increase. Formol titrating N during this period
was not determined owing to the matter of sampling.
These results indicate that the change in the percentage values which
are determined by experts, corresponds to that in the amount of volatilebasic and formol titrating nitrogen.
From the results of these experiments, it has become clear that the
freshness of whale meat can be evaluated by determining the volatile basic
nitrogen or formol titrating nitrogen.
In author's future experiments, therefore, comparison on the degree offreshness of whale meat will be done by the comparison on the amountof volatile basic nitrogen or of formol titrating nitrogen.
Experimental
I.

Deterioration of freshness of minced whale meat ?.t room temperature.
a) Change in the quantity of volatile basic nitrogen.
Material and treatment:- The material used was the refrigerated'. oniku,' the portion of flesh between the dorsal fin and tail, of blue whale(shironagasu-kujira, Balaenoptera musclus) caught in the Antarctic Ocean.

2i

Chemical Studies on the Freshness of Whale Meat. I.

Analytical data of the material are shown in the following table.
Crude protein
MoistureJ-------54.70% -- -i----15-.5-8_%_ _Crude fatAsh

29.20%

0.64%

The minced, pounded and thoroughly mixed material was devided into
many Petri' dishes. All the dishes were put in an air-tight glass vessel
containing a small amount of water in the bottom and kept at room temprature. After standing for a definite time, a dish was taken out from thevessel for a determination, one dish for each value. The amount of a insample in one dish was about 50 g. Temperature at noon during the courseof experiments, 20"--22"C.
Method of determination:- The determination was made by theaeration method. 11 )· 12) 4 g of thoroughly mixed sample was introduced into
a test-tube, and 20 cc of distilled water, 0.5 g MgO, 0.l"g NaF, artd a few
drops of octyl alcohol to prevent foaming, were added. By means of afairly rapid flow of air current throtgh the mixture at 40"C by suction,
volatile basic substance was driven out into 10-15 cc of 0.02 N H2S04.
After 3 hrs' continued aeration, excess acid was titrated with 0.02 N NaOH
with methyl red-methylene blue indicator.
Results obtained are shown in Table III.
Table III.
Time (hrs)

Volatile basic N(mg/lOOg)

0

13.2

21

17.3

Indications

Time (hrs)165

Faint sour odor.

189

IVolatile
basic Ni I d' ti
n 1ca ons
(mg/lOO g)
I

i
!

I

35.7

I

38.4

i

i Distinct gas

!

45

20.3

69
!13

~3.6

i

27.2

117

30.2

141

33.0

------·

Strong sour odor.
Sour odur, slightly diminished
Gas generationin the interior.

21.3
262
310

I

381

II

j

I

j

evolution andputrid smell.

40.5
47.l
50.3
53.7
I

--------·--b) Change in the amount of non-protein nitrogen.
Material and treatment:- Same as in a). Temperature at noon
during the experiment, 20°-22°C.
Method of determination :- 5 g of thoroughly mixed sample wasweighed into a mortar, thoroughly pounded with 0.5 g of emery powder,transferred to a beaker, washed out with 50 cc of distilled water and 5 ccof ether and heated on a steam bath for 10 min. under occasional stirring.

Tadashi Nakai

22

25 cc each of Barnstain's CuS04 solution and NaOH solution were added
and after standing for 30 min., supernatant liquid was decanted on filterpaper, collecting the filtrate in 500 cc volumetric flask. The precipitate
was washed with 50 cc distilled water, and, after standing for 15 min., theliquid was decated as before. After the washings were repeated using six
50 cc portions of water, the combined filtrate and washings were diluted
with distilled water to the volume indicated and mixed thoroughly. 10 ccof this solution was used for the determination of nitrogen by the Kjeldahl
method.
Results obtained are listed in Table IV.
Table
Time (hrs)

0
20
44
68
92

I

Non-protein N(mg/lOOg)

460
469
478
497
500

IV.

Indications

Faint sour odor.
Distinct sour odor.

I Time (her) 'Non-protein N(mg/lOOg)

116
140
190

539

237

. Sour odor and faintputrid smell.

562
611
642

c) Changes in the amount of formol titrating N, volatile basic N, trimethyl amine N, and trimethyl amine-oxide N. Material and treatment:- Material used was the same as in a) and b), but somewhat inferior in freshness.
Treated as above. Temperature at noon during the experiment, 29°-30°C.
Method of determinaton :Formal titrating N- 100 g of throughly mixed sample was weighed
into a beaker, 300 cc of distilled water added, and heated in a water bathfor 20 min., undeJ stirring. After standing for a short time, the liquid wasdecanted into an Erlemeyer's flask, and liquid was squeezed out of the meat
residue in the beaker with a piece of gauze. The residue was extracted
three times with successive 200 cc portions of distilled water as in theforegoing. To the combined turbic liquid in the flask was added 10 cc of
20% sulfosalicylic acid, and after a few hours, the mixture was filteredthrough a moistened :filter paper, collecting the filtrate in 1 l volumetricflask. The residue on the filter was washed with 0.2% sulfosalicylic acidsolution until the combined filtrate and washings reach the marking 6n theflask. The solution thus obtained is hereafter called solution [A]. 20 cc of
the solution [A] was su~mitted to formol titration (A. 0. A. C.-method ~>)1with 0.2 N Ba(OH)2.

Chemical Studies on the Freshness of Whale Meat. I.

Volatile basic N- 20 cc of the solution [A] was neutralized with 0.2 N
Ba(OH)2, 0.5 g of MgO and a few drops of octyl alcohol . added. and the
mixture was put through the. aeration method as in a).
Trimethyl amine N- There are many worker's reports on the determination of trmethyl amine, but complicated and slow method, even if it isaccurate, cannot be used in follwing the deterioration of freshness. Determination in the present experiment was carried out according to the author's
modification of Lintzel's method14) used for humann urine. . This method isbased on the fact that ammonia combines with formaldehyd but trimethylamine does not and is driven out by aeration. Beatty and Gibbons3) reported
a determination method based on this principle for the spoilage in fish.
To 500 cc of the solution [A] was added 15% NaOH solution to render
it weakly acid to litmus paper. The solution obtained was evaporated to40-50 cc volume on a steam bath. To this were added 15 cc of 35%
formalin, 15% NaOH until slightly acid to litmus paper, and then, 2 g of
MgO and a few drops of octyl alcohol. The mixture was then immediately
submitted to aeration, continuing for 4 hrs. · at 40°C, into 5 cc of 0.02 N
RS0.1• Excess acid was titrated with 0.02 N NaOH with methyl redmethylene blue indicator.
Trimethyl amine-oxide N- The determination was based on the Hoppe·
Seyler's method, but aeration was substituted for distillation in the said
method.
200 cc of the solution [A] was heated for 1 hr. on an asbestos-wirenetting with 10 cc of hydrochloric acid, and was rendered weakly acidic
with NaOH solution. The solution obtained was evaporated on a steam
bath to 20-25 cc. 0.5 g of MgO was then added, and by means of a rapid
current of air through the mixture at 40°C for 4 hrs., volatile basic substance was driven out. The remainder was centrifuged, and the supernatant clear solution was seperated into a beaker. The precipitate waswashed 3 times by centrifuge with successive 5 cc of distillred water. To
the all combined supernatant clear solution in the beaker were addedhydrochloric acid until slightly acid to congo red paper and then 10 cc of
40% SnCl2. The mixture was heated on an asbestos-wire-netting for 20
min., and then neutralized with NaOH solution. After the addition of 0.5 g
MgO to the mixture, trimthyl amine derived from the oxide by reduction
was driven out into 0.02 N RSOJ by aeration at 40°C for 4 hrs. ExcessTadashi · Nakaiacid was titrated with 0.02 N NaOH.
From the above experiments, the following results were obtained.
Table V.
Trimethyl[ Trimethyl
I . .
I__
<!mine 1i"__ amineoxide N* ~ Ind1cabonsTime (hrs)

Volatile
basic N*

Form0ltitr;;ting N*

0

20.3

90.6

20

22.8

111

0.35

44

29.7

136

0.46

l.16

68
92
140
188
236

34.9
41.8
50.2
52.9
58.9

0.57
0.70

1.34

0.84

1.30

I

0.34

1.33

{Da~k tinge.

Famt sour odor.
[{Distinct sourodor.
:(Strong souri odor.
' Gas generation
I in the interior.

163

183
216
238
260

Putrid smell.

~~~__.__~~~~~~~~~~~~~~~~~(~'

in mg/100 g)

II. Comparison between perzentage value of fr.eshness of whale meat at
the time of dissection and the amount of volatile basic and formol
titrating nitrogen.

Material and tratment :- 18 pieces of meat taken from 9 sperm
whales which were caught off the coast of Kinka-zan in August andOcotver, 1947 were used.
The material was taken from the animal being dissected, and thepercentage value of freshness of the material ·was determined by a practised operator immediately. Without delay, the material was minced andthoroughly mixed. As soon as possible, 4 g of the minced material was
weighed into a test-tubs, and 8 cc of abso1ute alcoholic 0.01 N H2S04 wasadded as a preventive for subsequent decay. Samples thus obtained weretaken to laboratory, and the amount of nitrogen determined.
Method of determination:Volatile basic N- The sample was thoroughly pounded in a mortar,transferred to the aeration apparatus, using 15 cc of distilled water, andthe aeration performed as in a).
Formal titrating N- 40 cc of distilled water was added to the sample
in a beaker, and the mixture was heated in· a gently boilling water bathfor 15 min. under occasional stirring. The liquid was decanted throgh afilter into an Erlenmeyer's flask, and the residue in the beaker washed 3times with succesive 20 cc portions of distilled water with heating in awater bath for 5 min, each. 2 cc of 20% sulfosalicylic acid was added toChemical Studieson the -Fre91rries5' of Whale Meat. I.

the combined filtrate and washings in the flask. The mixture was allowed
to stand overnight, and filtered into 100 cc volumetric flask. The residue
on the filter was washed until the volume of combined filtrate and washings
reached the marking. Formol titration was performed using 40 cc of thesolution obtained.
Results obtained ware as follows:Table
Freshness%

Volatile
basic N*("'90
average
80

4.1
2.2
8.1
7.7
5.5

r,

6.2
7.6
8.6
. 7.9
average 8.2

Formoltitrating N*'·
61.5
69.5
62.8

VI.
Freshness% Volatilebasic N'"
75

71.1

79.5
average 70.7
87.4
79.l
79.1
72.8
87.4
average 81.2

70
60
50
40

r0.2
9.9
8.5
17.2
average 11.5
16.0
38.6
246
453

Formoltitrating N*
98.8
82.2
77.8
102.1
average 90.2

( * in mg/i.00 g)

In conclusion the author wishes to express his sincere gratitude to Dr.
Tsutomu Maruyama for his kind guidance given during the course of thework and his inspection of the manuscript. The author also wishes toexpress his gratitude to Prof. Dr. Shichiro Akiya and Mr. Hisashi Okuda
for their many kind advices during this experiment. Further, the author
wishes to thank Messrs. R. Nagaura, T. Ishimori, T. Nishihara and S.
Itani who afforded him facilities for obtaining material for the experiment.
To Mr. T. Tawara and Mr. R. Fukazawa the author is indebted for theirkind assistances during the work.
References1)

Hogei-binran (Handbook of Whaling), Vol. l, 140 (Published by Nippon Hogeigyo Suisan

2)

Malin, N. V.: C. A., 32, 8612 (1938).

3)

Tillmans, J., and Otto, R.: Z. Unters. Nahr. Genussm., 47, 25 (1924); C. A., 18, 1862.

4)

Shimizu, M.: J. Agr. Chem. Soc. Japan., 1, 730 (1925).

Kumiai (1942)).

5) . Glassmann, B., and Rochwarger, F. : Z. Unters. Lebensm., 5~, 585 (1929).

ii..

6)

Herzner, R., and Mann, 0. : Z. Unters. Lebensm., 52, 215 (1926).

7)

Ipatov, A. V.: C. A., 33, 9202 (1939).

8)

Beatty, S. A., and Gibbons, N. E.: C. A., 31, 3581 (1!137).

9)

Brotzu, G. : Z. Unters. Lebensm., 63, 503 (1932).

10)

Yamamura, Y.: BuII. Japanese Soc. Sci. Fish., 2, 118 (1933).

26

Tadashi Nakai
11)

Official and tentative methods of analysis of the Association of Official Agricultural
Chemists, Fifth Ed., p. 375.

12) Peters and Van Slyke: Quantitative clinical chemistry, Vol. II (1932), p. 547 and 577.
J.3) 11), p. 373.
14)

Lintzel;W.: Bioch· Z., 273, 243 (1~34).

(Feb. 4, 1948)

Chemical studies on the Freshness of \iVhale l\lleat. II.
On comparison between whale meat and beef
on deteioration of freshness and autolysis.

Tadashi Nakai
In the previous paper (this bulletin, p. 17) the author reported thatthe freshness of whale meat can be evaluted by determining the volatilebasic nitrogen, i. e. ammonia nitrogen, or formol titrating nitrogen, as
various workers have done on other kinds of meat. Although it cannot be
said that the freshness of whale meat and that of other kinds of meat canbe compared by the absolute quantity of these nitrogen in a definite quantityof meat, yet taking into consideration the fact that the volatile basic substances and the formol titrating sudstances are products in every stage ofdeterioration of freshness or putrefaction, the author believes that a comparison of the readiness of deteriorution of freshness or putrefaction in whaleand other meat can be made by comparing the condition of increase in theamounts of the two kinds of nitrogen with lapse of time. For this purpose,
Fig. 1.
a piece of flesh of sei-whale (I wa
PH mg,
shi-kujira; Balaenoptera borealis).9.0 900
and a piece of beef were stoodat room temperature in summer
and changes in the amount of
vofatile basic nitrogen, formol
titrating nitrogen and a change inpH value with lapse of time were
determined. Results obtained arelisted in Table I and summarized in Fig. 1. As shown in Fig.
1, the amounts of these nitrogen,especially that of volatile basic
nitrogen, makes a greater increase during the course of the•whale meatx beefvolatile basic N.
deterioration of freshness in seiformol titrating N.
whale than in beef. Formal titra-·-·--·- pH28

Tadashi Nakaiting nitrogen in sei-whale meat was far greater than beef in absolute quantity
but the rate of increase in beef was slightly above that of sei-whale meat.
Increase of pH value was more rapid in sei-whale meat than in beef.
This may be due to a large production of volatile basic substances insei-whale meat.
Fig. 2
From these results, it can be said thatmg.
the freshness of sei-whale meat is more140,,,,"""xreadily deteriorated than that of beef, pro120
vided that the kind of bacteria attacking---·--------~/-- ..
,100 _. . . -.,,...

--,,.,, . .

meat is not brought into question.
,,/
,/
x'
At the same time, autolysis which plays 80/,'
a part in the deterioration of freshness incompany with bacterial action was compared in both meat. The results obtained
are shown in Table II and summarized inFig. 2.
0
48_1:w hrs.
Increase in the amount of nitrogen• whale meatx beefresulting frorri autolysis was found to bevolatile basic N.
formol titrating N.
very small compared to that found whenin united action with bacteria which mentioned above. Increase of bothkinds of nitrogen was found to be greater in beef than in sei-whale meat
during the process of autolysis.

::(/

t::

Experimentala)

Deterioration of freshness at room temperature.
A piece of sei-whale meat preserved in an ice chamber for three days

after dissection and a piece of beef stored in a refrigerator by a butcher
were used as materials in the present experiments.
Each material was divided about equally into several Petri' dishes andlet stood at room temperature in August. Every 24 hrs. the content of adish for each material was minced and thoroughly pounded in a mortar.
4 g of each meat thus treated was weighed into a test-tube and 6 cc ofabsolute alcoholic 0.01 N H2S04 was added to prevent subsequent decay.
Samples thus obtained were put to the determination of volatile basic nitrogen and formol titrating nitrogen as mentioned in the previous report.
The determination of pH value was made as follows : 2 g of mincedChemical Studies on the Freshness of Whale Meat. II.

29material was pounded in a mortar with 10 cc of distilled water, allowed tostand for a short time, and the pH value of the supernatant liquid wasdetermined with Toyo pH test-paper (methyl red and phenol red) and its
standard table of change of colour which had been corrected with buffer.
Results obtained were as follows :Table I.

I

Formol tittating N*

Volatile basic N*
Time (hrs.) ! S . he1-w ae
1
Beef
meat
0
24
48
72

16.5
128
493
776

I

21.3
64.4
250
392

i

pH

I
I

Sei-whale
meat

I

102
211
616

52
123
345

1

i

I
II
II

I

(*

Beef

i Sei-whale
meat

5.9
6.5
7.4
7.9

I

Beef
6.0
6.2
7.1
7.1

in mg/100 g)b) Autolysis.
Materials used were the same as in a).
Each material was minced, thoroughly pounded in a mortar, and several
samples of 2 g each were placed in glass bottles.
These bottles were corked after the addition of 18 cc each of distilledwater saturated with chloroform, and allowed to stand at room temperature.
At start and after 48 and 120 hrs., 30 cc each of absolute alcoholic 0.01 N
HsS04 were added in each bottle to prevent subsequent progress of autolysis.
·Samples obtained in this manner were put to the determination of the twokinds of nitrogen.
Formol titrating nitrogen.- The sample was transferred to a beaker,
heated in a gently boiling water bath for about 15 min., and the supernatantclear liquid was filtered into an Erlenmeyer's flask. 10 cc of distilled water
was added to the meat residue in the beaker, heated in a water bath for
5 min., and filtered as before. After the washing of the precipitate wasrepeated twice more, 1 cc of 20 % sulfosalic acid was added to the combined filtrate and washings in the flask, The mixture was allowed to standovernight, and filtered into a 50 cc volumetric flask. The residue on thefilter was washed, and the combined filtrate and washings were diluted tothe marking. 20 cc of the solution thus obtained was put to the formal
titration.

Volatile basic nitrogen.- 20 cc of the solution obtained above wassubjected to the aeration method as described in the previous report.

Tadashi Nakai

30

Results obtained were as follows :Table II,
Volatile basic N*

Formol titrating N*

Time (Hrs.)
0
48

120

Sei-whale meat

Beef

Sei-whale meat

Beef

22.8
25.4
28.l

23.9
32.0
42.0

101
118
124

52.l
82.2
138

(* in mg/100 g)
In concluding this report, the author wishes to express his deepest
gratitude to Dr. Tsutomu Maruyama for his kind guidance throughout theexperiment and to Prof. Dr. Shichiro Akiya for his kind advices given
during the work. Further, the author wishes to thank Mr. T. Tawara and
Mr. R. Fukazaaw who helped him in the exi:eriment, and to Mr. R. Nag2ura and Mr. T. Nishihara for their assistance in providing the opportunities
for obtaining materials for the present experiment.

(Feb. 10, 1948)

On the simultaneous extraction of vitamin A-D andvitamin B 2 complex from the liver of a fin whale(Nagasu-kujira, Balaenoptera physals L.).

Tadashi Tawara
Since the discovery of vitamin A in 1913, by Osborne and Mender,<1>
studies of vitamin showed extraordinary development. Later, Mellanby,<2>
McCollum,<3> Sherman, Pappenheimerc4> and others distinguished between
vitamins A and D.
Then Kawakami,< 5> Kawaics> and others confirmed that vitamins A andD existed in the liver of fishes widely and in rich concentrations. Material for vitamin A and D is now almost wholly suppliep from the liver ofaquatic animals.
It was also discovered that vitamins A and D existed richly in whale
liver. Whale liver is now one of the most essential resouces of vitamin AD oil, as its volume is very large.
In 1926, vitamin B2 was distinguished from vitamin B, by Goldberger
and others,<7> and so-called " Complexity of Bz" was claimed.
Members of BJ group is now said to contain many factors including
the following: lactoflavin (riboflavin), nicotinic acid, vitamin Bs (adermin),pantothenic acid and other factors. Then, Kuhn, cs> Karrerc9> and others
discovered that liver contained a large amount of vitamin Bi complex.
B2 complex is richly contained in the liver of aquatic animal, especially,in those of tunny (Maguro; Thunnus orientalis), spearfish (Makajiki; Makaira mitsukurii), swordfish (Mekajiki; Xiphias gladius), bonito (Katsuo;
Katsuwonus vaganus), fin whale (Nagasu-kujira, Balaenoptera musculus L.),humpback (Zato-kujira ; Megaptera nodosa Bonn), etc.
There are many method of extracting liver oil, principal methods being
as follows:(A) Heating with steam-Liver is heated with steam, and then the oil separated.
(B) Resolving by standing-Liver is left standing, putrefied, resolved, and the oil separated.
(C) Vacum method-

32

Tadashi Tawara

Liver is placed in a vacuum kettle, and the oil separatEd by heatingwith low pressure steam or hot water.
(D) Refrigeration method-Liver is pressed while frozen, and the oil separated.
(E) Electrolytic methodLiver cells are destroyed by passing an electric current, and then theoil separated.
(F) Extraction with organic solvents-(i) Dry methodLiver is heated and almost dried. And the oil extracted with organic
solvents such as ether.
(ii) Resolving with caustic soda-Liver is dissolved in caustic soda, and then the oil extracted withorganic solvents, or separated by centrifuge.
At present, the last method is almost always employed because theyield of liver oil is very good, and vitamins A and D are extracted al.mostcompletely without decomposition.
The present method of preparation of liver oil is limited to the obtaining of vitamin A-D oil, and sacrificing of other effective components which
are contained in the liver is unavoidabl.
The author aimed at this point and tried to extract as completely as
possible both the vitamins A-D and B2 from one liver.
Vitamin A is quite stable to alkalis but is sensitive to acids, whilevitamin B2 (lactofiavin) is quite stable in strong mineral acids but is sensitive to alkalis. Vitamin A is sensitive to oxidation, but lactofiavin is quite
stable to agent. On account of these conflicting properties, it is difficult to
extract both A-D and B2 completely without decomposition.
Vitamins A and D are fat-soluble vitamins while B2 complex is watersoluble. The author, therefore, tried to extract first, B, complex withwater, and then A-D oil from its residue. Extraction of vitamin B2 complex
on various conditions were tested to observe how the quantity of vitamin
A changed by various conditions, and to discover conditions under which B2complex is extracted completely and decomposition of vitamih A is theleast. Vitamin B:i complex was extracted by various methods and thequantity of lactofiavin in the extract was measured. Its residue was thenresolved with caustic soda, the liver oil extracted with ether, and then theOn the simultaneous extraction of vitamin A·D andvitamin B2 complex from the liver of a fin whale

33

qunntity of vitamin A was measured.
Considerations were given to the effect of air, acids and heat on
vitamin A, and to the effect of destruction of cells, and temperature of
extraction on Bitamin H~.
Fin whale liver was used as material.
Experimental
Fin whale liver was. minced in a meat-chopper, mixed homogeneously
and 100 g of it were used for each experiment.
I. Quantitative analysis
A) Quantitative analysis of vitamin A.
Raw liver or the liver from which B2 complex had been extracted was
taken, about 100 cc of water added and warmed in a water-bath which
was later brought to a boil. After the meat had coagulated, solution of
2 g NaOH dissolved in 20 cc of water was added little by little to the
coagulated liver with agitation. In the meantime, about 200 cc of water
was also added to it, little by little.
After the whole quantity of NaOH solution and water were added, theheating was continued until the granules of liver melted completely andturned into a liquid. This liquid was then allowed to cooled, transferred
into a separating funnel, and vitamin A-Doil was extracted five times with
140 cc each of ether. The upper etherial layer was gathered, and washedwith water until the washings did not colour to phenolphthalein. Afterthe etherial solution was .dried with anhydrous Glauber's salt, ether was
distilled off, and vitamin A-D oil was obtained.
The yield of this oil was weighed, and the quantity of vitamin A in
1 g of the oil was measured with spectrophotometer by spectro-graphic
method (solvent, chloroform).
Ei~~ 328 millimicron= 1600c10) was used.
International unit (I. U.) per gram multiplied by the yield of oil gives
the total amount of vitamin A in 100 g of liver.
B) Quantitative analysis of vitamin B2 (lactofiavin)10 g of acid clay (Fuller's earth) was adrled to the liquid by which
vitamin B2 was extracted from 100 g of liver. By shaking this mixture
vigorously, lactoflavin was adsorbed completely, and then filtered. The
adsorbed earth on the filter paper was washed with a small amount ofTadashi Tawara

water,. and dried at 60°C. 50 cc of alcohol (94%) was added to the driedearth, the mixture shaken, filterd and the earth again dried at 60°C.
Lactoflavin was eluted from one-tenth quantity of this adsorbed earth
with 20 cc of .40% alcoholic 0.25 N-NaOH solution. The residual Fuller's
earth was eluted again with 20 cc of 40% alcoholic 0.1 N-NaOH solution,and the two lots of solutions were brought together. This was exactly
neutralized with HCl, concentrated at 40°C under a reduced pressure, andalcohol was then distilated off. This was further concentrcted to about
10 cc, and was made into a 0.5 N-NaOH solution by the addition of 5 NNaOH solution.
This solution was measured by Kuhn's method01 ) 02 ) with PulfrichStufenphotometer (Filter S. 47; E 10n~cc=4.75).
The value thus obtained multiplied by the gives the quantity of lacto:flavin (r) in the extract obtained from 100 g of liver.
II. Extraction of vitamin A-D oil and B2 complex under various conditions.
( 1 ) Quantative analysis of total vitamin A in 100 g of liver.
Total quantity of vitamin A extracted from 100 g of minced liver by
the abovementioned method of resolving with caustic soda, was ·measured.
Vitamin A
720,000 I. U.
( 2) Extraction of vitamin B2 compl.Ex by boiling.
100 g of liver as above was taken. 300 cc of water was added to it,. and the solution was brought to pH 5.0 with the addition of phosphoricacid. This solution was boiled in a water-bath for 2 hours, tilted withfilter-paper while hot. By the above-mentioned method, the quantity ofvitamin A in the total amount of residue, and the quantity of lactofl.avinin the filtrate were measured. In this case, owing to having boiled inacidity, the amount of vitamin A decreased greatly.
Vitamin A
550,000 I. U.
1086 rLactoflavin( 3 ) Extraction by boiling in vacuum.
Since vitamin A is oxidized easily in acidity and on heating, the boiling
was made under a reduced pressure in order to prevent decomposition.
However, the degree of the heating was to such an extent that water inthe water-bath was boiling although, it goes without saying that thetemperature of liver solution did not reach 100°C. Other conditions were

the same as above.

On the simultaneous extraction of vitamin A-D andvitamin B.i complex from the liver of a fin whale

l

r·.

:35

The quantity of vitamin A in this case, was greater, but, on accountof insufficient heating, the amount of lactoflavin decreased.
690,000 I. U.
Vitamin A
1022 rLactoflavin( 4) Extraction by digesting.
The liver was digested in order to make the the extraction of vitamin
Bi complex better by destruction of liver cells.
100 g of liver as above, was taken, 300 cc of water and 15 cc of 90%
phosphoric acid were added, and then 1 g of saccharated pepsin (J. P.)
added with stirring, and kept at 40°C for 2 hours. The digested solution
thus obtained was filtered, and the same processes as above were carriedout on the filtrate and the residue.
In this case, the extraction of B2 became somewhat better, but owing
to higher acidity the amount of vitamin A obtained was decreased.
576,000 I. u.
Vitamin A
1150 rLactoflavin( 5 ) Extraction by digesting in vacuo.
The digesting process, in this case, was carried out in vacuo. Other
conditions were the same as above.
·vitamin A
690,000 I. u.
863 r
Lactoflavin
The amount of vitamin A became somewhat better, but, on accountof insufficient heating under reduced pressure, the yield of B2 decreased
exceedingly.
( 6) Extraction by digesting and boiling.
In order to extract J.actoflavin more completely, the digested liquid wasboiled for 10 minutes, filtered while hot, and the residue and the filtrate
were treated in the same manner as above. The condition of the digestion
was the same as for (4).
456,000 I. u.
Vitamin A
1788 rLactoflavin( 7 ) Extraction by digesting und boiling in vacuo.
In the above process, vitamin A wa5 oxidized greatly because of boilingthe solution in strong acidity. Hence the same process was carried out
m vacuo.
675,000 I. u.
Vitamin A

ilf..

36

Tadashi Tawdra

1563 rLactoflavin( 8 ) Extraction by digesting, neutralizing and boiling.
In the above process satisfactory result could not be obtained on theyields of both A and B2. This must have been due to the fact that vitamin
A was boiled in acid, and also to the reduced pressure.
The liver, accordingly, was digested by the method as in (4), then thesolution exactly neutralized with NaOH solution, boiled for 10 minutes, andfiltered.
645,000 I. u.
Vitamin A
1789 rLactoflavin( 9 ) Extraction by digesting, neutalizing and boiling in vacuo.
The operations as in (8) was carried out in vacuo.
Vitamin A
703,000 I. U.
Lactoflavin1406 rIn all the methods of extractions under various conditions mentioned
above, the vacuum process for the extraction of vitamin A brought a goodresult, but it brought a bad result on B~. The inferior result of the extraction of B2 was due to the failure of the temperature to reach boilingunder reduced pressure.
Hence, it was thought that if the boiling was carried out under oridinarypressure without air, the result would be better. Carbon dioxide gas wasused as an inert gas for this purpose.
(10) Extraction by digesting, neutralizing and boiling in carbon dioxidegas.
C02 gas was used instead of a vacuum, and all the processes of digestion, neutralization and boiling were carried out in C02 gas.
711,000 I. U.
Vitamin A
Lactoflavin2110 r
The loss of vitamin A, as a result, was almost negligible, and theextraction of BJ was better.
(11) Extraction by boiling, digesting, neutralizing and boiling in CO;l gas.
The liver was coagulated by boiling so as to allow pepsin to act easily
on proteins, and cooled to 40°C by adding water, pepsin was then added.
All the process were carried out in C02 gas. Other conditions were theOn the simultaneous extraction of vitamin A-D andvitamin B2 complex from the liver of a fin whale

same as for (10).
Vitamin A
Lactoflavin!17

711,000 I. U.
2849r

Summary
Conditions under which vitamin A is decomposed least and vitamin B2
extracted completely from a liver, are as follows:
Minced liver is boiled with water in· C02 gas, cooled to 40°C by adding
water, acidified with phosphoric acid, and the liver substance is digested
with protease such as pepsin at about 40°C. After digestion is completed,the solution is exactly neutralized with NaOH, boiled again and filtered.
All the above process carried out in CO:i gas.
Vitamin B:i is obtained from this filtrate, and vitamin A-Doil from theresidue.
In conclusion, the author expresses his cordial thanks to Dr. T. Maruyama and Mr. T. Nakai for their kind advice and encouragement during
this study.
References( 1)

Osborne T. B. & Mendel L. B.: J. Biol. Chem., 16, 4'...3, (1913).

( 2)

Mellanby: Lancet, 1, 856, (1920).

(3 )McCollum : J. Biol. Chem., 45, 333, (1921).

( 4)

Sherman, pappenheimer: Proc. Exp. Biol. Med. 18, 1S3 (1921).

( 5)

Kawakami Y.: Sci. Pap. I. P. C. R., 11, 968, (1S32).

( 6)

Kawai K.: J. Pharm. Soc. Japan, 708 (1929); 249, 323, 809, &36, 951 (1930).

( 7)

Goldberger etal: U. S. Pub. Health. Rep., 41, 1025 (1926).

( 8)

Kuhn, Wagner-Jauregg: B., 67, 1770 (1S34).

(9)

Karrer Salomon, Schopp : Helv. Chim Act., 17, 419 (1934) ; 13, 1022 (1935).

(10)

Morton R. A. & Heibron I. M. : Bioch. J., 22, 987 (1928).

(11)

Kuhn, Wagner-Jauregg, Kallschmitt: B., 67, 1452 (1934).

(12)

Warburg, Christian: Biochem. Z., 254, 483, (1932); 277, 492 (1933); 2S6, 377 (1933).

STUDIES ON WHALE BLOOD. I.
On the separation of histidine from whale blood.

Tadashi Tawara
Histidine is an amino acid of the following formula :HC=C-CH2-CH-COOH

I

I

N NH

'\,/

I

NH2

c

H

This amino acid was discovered in 1896 by Kossel and is found invarious proteins, being most abundant in hemoglobin which contains about
11% histidine.
Histidine is one of the most important amino acids for nutrition and it
is contained comparatively in a large amount in fish meat. Putrefactionof meat turns histidine into a poisonous histamine. Histidine is used forgastric ulcer.
Two methods of synthesis have been worked out by Pymann.1m The
most common material now being employed for hislidine manufacture isblood. There are two principal manufacturing methods, one by precipitating
the silver salt in alkaline medium, 3)4)5) and the other using the mercuricsalt.6>7 )3>
The author used dried blood of a sperm whale (Physeter catodon L.)
as material for his experiments.
Cystin, methionine, ornithin, tryptophane and histidine are some ofamino acids that precipitate by corrosive sublimate (mercuric chloride).
Cystine almost precipitate out in neutral media, while ornitine and tryptophane are not obtained by hydrolysis with hydrochloric acid. Methionine
precipitates almost wholly in saturation of sodium chloride around pH 6.0.
The author, therefore, undertook the experiments with a special consideration for the removal of methionine.
Experimental
1 kg. Dried blood of sperm whale was hydrolysed with 4 L. 30%
hydrochloric acid for 20 hours. After cooling, pigment that precipitated

On the separation of histldine from whale blood39

out (melanine) was removed by filtration and the filtrate was neutralized topH 2.5 with concentrated sodium hydroxide solution. After standing overnight, the solution was filtered, and the precipitate was washed with a small
amount of water. The filtrate and washings were neutralized to pH 6.0
with sodium hydroxide solution and concentrated untill sodium chloride
precipitated out. The mixture was cooled and filtered. Methionine canbe obtained from this precipitate.
A hot saturated solution of 400 g mercuric chloride was added to thefiltrate with stirring and after being left overnight, was filtered. A concentrated solution of .sodum carbonate was added slowly, with stirring, until
the pH of the solution reached 7.2. After stirring overnight, the supernatant liquid was decanted and the flask filled with water to the original
volume. This procedure was repeated several times, and the mixture was
finally filtered by a Buchner funnel. From the washings, mercury can be .
recovered by passing hydrogen sulfide gas.
This precipitate (mercuric salt of histidine) was suspended in lL. ofwater and decomposed by passing hidrogen sulfid gas. This was filtered
to remove mercuric sulfide, which was then boiled with water several time
to recover histidine adsorbed in the precipitate.
•
The filtrate and washings from mercuric sulfide were concentrated toabout 150 cc, mixed with 300 cc 95% alcohol. and stood overnight ii:l an ice
chamber. Crude crytstals of histidine monohydrochloride were thus obtained.
The crude product was dissolved in five times its weight of water, andthen diluted with twice its volume of 95% alcohol. The yield of recrystallized histidine monohydrochloride was 39 g. This compound melts at 252°C
with decomposition.
In conclusion, the author wishes to express his sincere thanks to Dr.
T. Maruyama, Mr. H. Okuda and Mr. T. Nakai for their kind guid andadvice in this study.
His thnks are also due to prof. Dr. S. Akiya and prof. Dr. T. Ogawa,Tokyo University, for their valuable suggestions.
He is also indebted to Mr. R. Fukazawa for kind assistance during tl;Iework.
References
1)

Pyman: J. Chen. Soc., 99, 668, 1386 (1911).

•

Tadashi Tawara

40
2)
3)
4)

Pyman: J. Chen. Soc., 103, 159 (1941).
A. Kosse! F. Kutscher: Z. physiol. Chem., 31, 165 (1900-1).
H. B. Vickery, Leayenworth : J. Biol. Chem., 72, 403 (1927) ; 75, 115 (1927) ; 76, 707(1928) ; 79. 377 (19'.:8).

•

•

5)

H. B. Vickery, K. J. Block : J. Biol. Chem., 93, 105 (1931).

6)7)Org. Syntheses : 18, 43 (1.938).
Brig! : Z. physiol. Chem., 64, 337 (1910).

8)

M. T. Hanke, K. K. Koessler: J. Biol. Chem., 43, 521 (1920).

Sinus-hairs of the Sei-vVhale (Balaenoptera borealis)byJunnosuke Nakai and Toshitomo Shida(Director: Dr. Teizo Ogawa)
Sinus-hairs of a whale possess sensitive tactility like the whiskers of acat. The root of this hair is surrounded by blood sinus in the connectivetissue layer of the hair follicle. It is a venous sinus with numerous nerves
coming into the sinus and twining themselves around the root of the hair.
The whale-bone whales have these characteristic sinus-hairs on their
upper and lower beaks, but in dolphins they occur only in their foetusstage and degenerate after birth.
The lack of sinus-hairs in dolphins is believed, generally, to be due tothe food of them, being so large as to be easily visible that tactile hairs
become unnecessary, but, the food of the baleen whales is tiny Mysidae
and is so difficult to see that they search for the food with these hairs.
In the summer of 1946 and 1947, at Ayukawa, Miyagi Prefecture, we
had a chance to. study sinus-hairs of Sei-Whales, Balaenoptera borealis,which were caught off the coast of Kinkazan and studies were made bothat the whaling station and in the laboratory later. The distribution andnumber of sinus-hairs macroscopically and some facts on histological preparations have been studied.
D. G. Lillie (1910) merely studied thE distribution and number, while
A. Japha (1910) devoted himself to the microscopic study of histologicalpreparations.
We observed, at the whaling station, some white, weak hairs of 1 to
2 cm in length occurring on the giant body, generally symmetric~lly in
specific arrangement. The observation at the whaling station was not easy;
because of the giant body and of the speedy disposition of it. It wasalmost impossible to count and examine the hairs of both upper and lower
beaks of an individual.
The arrangement and number of these hairs are shown in Table 1 and. in Figs. 1 and 2. There are four rows of them on the dorsal surface of
the upper beak, from the anterior extremity to the blow-hole, almost 30
hairs at each distance of 20-30 cm. At the anterior end of the lower jaw,
there are two rows which occur closely together along the median line at

J, Nakai, T. Shida
upper

beak

lower

beak

I

Individual

right
left
right
left
1·
on the la- rat the an- .onthe la- 'at the an--=.
outer row mner row outer row mner row teral side terior end teral sidel terior end•-N-0-.-1--1--1-0-

5- - - - - _____ ,___9__ 1----1-,-,

No. 2

8

I

No. 3

4

11

1

14

/

14

--- -

-1---~I
I

16
14

Table 1. Number of sinus-hairs of Sei-Whale_

,,.

~

~

-

__j_._......_
,...

.....

~ ===-::::::--~--------~~-----

Fig. ?· Sinus-hairs at the anterior
end of lower beak.

Fig. I. Arrangement of sinus-hairson the head of Sei-Whale.

a distance 1 to 2 cm and contain almost 30 hairs. On each lateral side oflower jaw, there is a row of from 5 to 10 hairs. There are a total of nearly
80 hairs in all which does not greatly differ from reports of the former
investigators.
We especially made a study of the hairs at the anterior end of lower
beak. We made observations at first by naked eye or with a magnifyingglass inflicting small cuts in the sinus and its neighbouring parts. By thismeans, we were able to unearth some new facts which seemed to haveescaped notice of earlier workers.
When a large blood sinus, surrounding the hair root, was carefullySinus.hairs of the Sei·Whaleobserved, the skin above it was indented in a funnel shape and becamevery thin at the point where the hair came· out of the sinus, and the sinus
extended itself to just beneath the epidermis.
At the bottom of the funnef shaped dent, the thickness of epidermis
is only 0.3 mm, but it becomes thicker outward, being 1.3 mm thick at the
periphery of the funnel. The corium layer between the epidermis and theblood sinus is only 0.1 mm in thickness.
The sinus-hair is supported in the sinus by many threads from surrounding walls so that the hairwill move freely in all directions whenpressure is applied from outside. It is significant that this part of theepidermis where the hair comes out is very thin in order to make thismotion easily.
Observations of the cross section of the hair follicle show that thishair is situated not equally distant from inner walls ·of the sinus, but iseccentric. Furthermore, the inner walls of the sinus are different inshape, such that the side to which the hair shaft is inclined is almost
vertical, but the wall on the opposite side forms a curve, of which thecentral part swells out remarkably (Fig. 3).
The longitudinal diameter of the sinus is almost 12 mm, maximum
transverse diameter almost 2 mm. The wall of the sinus is made of denseconm:ctive tissue fibers, looking somewhat like the tendon, and is about
1 mm in thickness at the central swollen part. These fibers give off
branches radially into surrounding parts of the corium so that the swollen
part seems to be strongly pulled outwards.
In the skin of the whale, there is only a thin compact layer of connective tissue between the epidermis and the adipose tissue of the subcutaneous layer. Coarse fiber bundles run in complicated directions through theadipose tissues, but, as no remarkable connective tissue fibers are seen in
the neighbourhood of the hair follicle except the above-mentioned radial
fihers, the hair follicle lies nearly surrounded by soft adipose tissues.
We thought that there might be a rule between the eccentric locationof the hair root in the sinus, shape of the sinus walls, and the inclinationof the hair shaft. By a closer observation, it was found that the hair shaft wasalways inclined towards the side to which the hair root was located nearer.
Further observations revealed that the epidermis forming the funnel at theexit of the hair has a gentler slope on the side the hair shaft is inclined

J. Nakai, T. Shida

44

and the other side constitutes an almost perpendicular wall (Fig. 3).

sheath of the hair root
threads of connective tissue
venous sinus

covering

connective fibers coveringa nerve bundle
Fig, 3 Root of a sinus-hair and its surroundings
medial side

median line of theanterior end of thelower beakFig. 4, To show the inclination of sinus-hairs

Sinus-hairs of the Sei-Whale

45

Therefore, the direction of inclination of the hair shaft seemed to havesome intimate correlation to the structure of the sinus. Bwt since we could
not make any further generalization on a small piece cut out from the lower
jaw, we proceeded then to the examination of the entire tipe of the lower
jaw, where each direction in relation to the whole body is easily determinable. It was made clear that the hair was always inclined to the lateral
direction (Fig. 4). Therefore, the swollen part of the sinus wall is on the
medial side, namely, on the side directed to the median line.
The shaft of the sinus-hair shows no pigmentation and is 0.1-0.15 mm
in diameter, being the thinnest at the exit. Its surface is relatively smooth
only at the part, where it just comes out of epidermis and its tip has many
protrudings and looks like a slender tree trunk with many knots (Fig. 5).
While treating the hair under a microscope, it was found that dust adheres
easily to the hair shaft. The hair has no marrow; bulbus and papilla ofthe hair are divided in complex form.
Next, the nerves come into consideration. We observed both in macro
germinal layerof epidermis

4 mm

2. mm

vein
0

Fig. 5. shaft of a sinus-hairFig. 6. Lamellar corpuscles in the
superficial part of a hair-follicle

46

J. Nakai, T. Shidaand microscopical sections, a relatively thick bundle of nerve fibers ascending almost vertically and it gave off one branch entering the hair follicle
from just beneath it and the other branch ascending outside of the follicle.
According to Japha, as several hundreds of myelinated nerve fibers
are counted in one sinus-hair, there must be almost 10.000 fibers at theanterior end of the lower jaw, and he quoted in relation to this an interesting story, told by Malm (1866) that when one touched the lips of a Blue
whale washed ashore alive near Goteborg in Sweden this giant whale started
violently.
We ascertained by microscopic observations that the ascending nerve
bundles end in special terminal apparatus, in the outside of the sinus.
This apparatus resembles the Pacinian corpuscle, but since the number oflamellae is so few that, it comes nearest to the Herbsti's corpuscle.
They are nearly oval, 60-300 µ in length, 20-150 µ in transverse
diameter, and are made of concentric lamellae of connective tissues. The
axis of the corpuscle is occupied by a well developed eosinophile core,containing the termination of a nerve fiber (Fig. 6).
The corpuscles are grouped in the following wise: one group about 1
mm immediately beneath the epidermis, on the lateral side of the hair ;another group about 1.5 mm in length on the opposite side (median); thethird group on the lateral side (same side with the first group) somewhat
beneath the second, and also at the height of the hair papilla, where more
corpuscles are obviously seen on the median side than on the lateral side.
Further researches are needed as to the distribution of the terminal apparatus and differences in its size according to location. We can, however,assume that this distribution of the corpuscles has a close connection withthe functional direction of the hair, when the hair encounters pressure andresistance. Japha also observed that there were especially numerous small
terminal corpuscles (about 20 x 60 µ) in the papillae of corium immediately
beneath the epidermis around the hair.
We are endeavouring to throw more light by the method of axis
cylinder staining on the distribution of nerve fibers to the corpuscles andto study the characteristic feature of nerve fibers at the point, where thesinus continues into a vein.
The inner surface of the venous sinus, as well as nerves, blood vessels
and threads of connective tissues which are all present inside the sinus are•Sinus-hairs of the Sei-Whale47

covered by endothelial cells.
According to macroscopic studies, a relatively thick artery, being thesize of a thin needle, runs up from the under and lateral direction andenters the sinus through the lower lateral part of the hair follicle, aboutat the height of a hair papilla. The artery entering the sinus is divided
into two, runs upward and. becomes more brouched and thinner.
A large amount of blood sent into the sinus from the lower part, runs
up and seems to flow out into a vein near the epidermis. The exit issituated on the opposite side to the artery, namely on the median side.
The terminal corpuscles are crowded in this side near the exit.
It is especially worthy of notice, in the above-mentioned results, thata rule of direction exists between the sinus-hair and things accompanying
it, as a whole. For this reason, the sinus-hair seems to have a greater
significance in the life of a baleen whale than it has been assumed as atool for finding food. We suppose that by use of sinus-hairs the whales
might feel the currents of water upon their beads.
Ki.ikenthal was of the opinion that the scanty remaining hairs of whales
are merely a reduced organ, as they have no sebaceous glands. But it is
the common property of the skin of whales that it lacks not only thesebaceous glands, but also the sweat glands. We are of the opinion, thesinus-hairs of whales seem to be a very important sensory organ for thelife of them.
Literature
Japha, A. : Die Haare dtr Waltiere. 1910. Zool. Jahrb. Bd. 31-. Abt. f. An1lt. (Sonderabdruck).
K:iikenthal, W.: Untersuchungen an Walen. 1909. Jena. Z. f. Naturwiss. Bd. 45. S. 545-588.
Lillie, D. G.: Observations on the anatomy and general biology of some members of the larger
cetacea. 1910. Proc. Zool. Soc. London. p. 779-792.
Tretjakoff, D.: Die Nervenendigungen an den Sinushaaren des Rindes. 1911. z. f. wiss. Zool.
Bd. 97. S. 314-416.

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