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)