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FISHERIES AND MARINE SERVICE
Translation Series No. 4392
Studies on nutritive value of the protein of Antarctic krill
(Report 1). Estimation of protein efficiency
ration and biological value of krill protein in rats
by M. Iwaya, Y. Kobatake
and E. Tamura
Original title: Okiami Tanpakushitsu no Eiyoka ni kansuru Kenkyu
(Dai 1 ppo) Ratto ni yoru Tanpakushitsu Koritsu, Seibutsuka
no Sokutei
From: Eiyogaku Zasshi 35: 101-107, 1977
Translated by the Translation Bureau (IHW/PS)
Multilingual Services Division
Department of the Secretary of State of Canada
Department of the Environment
Fisheries and Marine Service
Halifax Laboratory
Halifax, N. S.
1978
14
pages typescript
DEPARTMENTOFTHESECRETARYOFSTATE
••
SECRÉTARIAT D'ÉTAT
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DIVISION DES SERVICES
MULTILINGUAL SERVICES
CANADA
MULTILINGUES
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[-e'er zi312\,
TRANSLATED FROM - TRADUCTION
DE
INTO - EN
Japanese
English
AUTHOR - AUTEUR
Masako IWAYA, Yoshiki KOBATAKE, Einosuke TAMURA
TITLE IN ENGLISH - TITRE ANGLAIS
STUDIES ON NUTRITIVE VALUE OF THE PROTEIN OF ANTARCTIC KRILL (REPORT 1).
ESTIMATION
OF PROTEIN EFFICIENCY RATIO AND BIOLOGICAL VALUE OF KRILL PROTEIN IN RATS.
TITLE IN FOREIGN LANGUAGE (TRANSLITERATE FOREIGN CHARACTERS)
TITRE EN LANGUE ÉTRANGÉRE (TRANSCRIRE EN CARACTÈRES ROMAINS)
Okiami Tanpakushitsu no EiySka-ni kansuru Kenkyil (Dai 1ppi5)
Ratto ni yoru Tanpakushitsu leritsu, Seibutsuka no Sokutei.
REFERENCE IN FOREIGN LANGUAGE (NAME OF BOOK OR PUBLICATION) IN FULL. TRANSLITERATE . FOREIGN CHARACTERS.
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EiyZ)gaku Zasshi
REFERENCE IN ENGLISH - RÉFÉRENCE EN ANGLAIS
The Japanese Journal of Nutrition
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Kokumin-Eiy3-Shinkeikai, and The
Japan Dietétic Society
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Japan
DATE OF PUBLICATION
DATE DE PUBLICATION
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ANNÉE
VOLUME
1977
35
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101-107
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I
Eiya;gaku Zasshi (The Japanese Journal of Nutrition), 1977, Vol. 35,
No. 2, pp. 101-107.
STUDIES ON NUTRITIVE VALUE OF THE PROTEIN OF ANTARCTIC KRILL (REPORT 1)
ESTIMATION OF PROTEIN EFFICIENCY RATIO AND
BIOLOGICAL VALUE OF KRILL PROTEIN IN RATS
By
Masako IWAYA, Yoshiki KOBATAKE, Einosuke TAMURA
Division of Biochemistry, The National Institute of Nutrition
101
Nutritive value of the protein of antarctic krill (Eaphausia sperba) was estimated by rat assay.
Results Were as follows:
1) Weight gain, protein efficiency ratio (PER) and net protein ratio (NPR) for the protein of
precooked krill, which was . freeze-dried and defatted, were almost the same to those for casein, but
these values were obviously lower than those of whole-egg protein.
The slope ratio technique indicated that the relative nutritive values (RNV) of protein of
precooked krill and casein were 87.4 and 77.6% of the value of whole-egg protein, respectively.
3) Biological values (BV) of protein at 10% dietary level were 82.2 for precooked krill, 77.6
for raw krill, 84.9 for the extracted protein with alkaline solution, and 87.4 for eviscerated precooked
krill. The true digestibility of protein of raw krill was lower than that of precooked krill (raw
88.8%, precooked 91.7%).
2)
•
UNEDITED TRANSLATION
For information only
TRADUCTION NON REVISEE
Information se ulement
SEC 5-25 (Rev. 6/78)
•
2
Attention has been drawn to an as yet unused food resource,
the Antarctic Krill (Euphausiaswperba), as a possible source of protein.
Due to lack of experience with this species as food, it is imperative
to examine thoroughly its safety, nutritive value, and food value prior
There have been in these areas of concern a
to widespread usage.
number of research reports dealing with a determination of its chemical
composition
acid
3)9)
protein)
1)2)6)-8)
, an analysis of its fatty acid
4)5)
and amino
composition, an analysis of the nutritive value (principally
9)10)11)
, etc.
These results have demonstrated that krill
has sufficient food value and is also a promising source of protein.
In order to assess the nutritive value of the protein of
Antarctic krill more accurately, the authors have attempted to compare
its nutritive value with that of whole egg and casein, as standard
protein, employing various types of techniques generally used for
nutrition evaluation of protein.
This report deals with an examination
through animal. experimentation with rats of 1) the protein efficiency
ratio (PER), 2) biological values and digeseibility, and 3) the
relative nutritive value obtained by the slope ratio technique.
Samples
were freeze-dried raw krill, freeze-dried precooked krill, defatted
freeze-dried raw krill, and defatted freeze-dried precooked krill.
Experimental Method
1;
•
Krill Samples
The krill sample was Euphausiasuperba caught in the Antarctic
Ocean during the periods of December 1973 to January 1974, December
1974 to January 1975, and December 1975 to January 1976. It was
treated in one of two methods: freezing raw krill (without precooking)
102
3
directly after catching, or precooking raw krill in boiling sea water
(95-100°C) for 3 minutes and freezing at -40 °C for 6 hours after
cooling. Samples were kept frozen (-25 °C) for approximately 5 months
on the ship and then kept in storage (-40°C) on land for 2 to 3 months.
During the experiment, they were taken as needed from the laboratory
freezer (-20°C), in which they had been kept for 1 to 3 months.
All samples used for determination of chemical composition
and for the diet of the experimental animals were freeze-dried and
pulverized beforehand. Defatting was carried out with an etherethanol (1:2) mixture.
Soaking, filtering, and cleansing were repeated
several times, followed by cleanàing with ether in the end.
Then the
samples were air-dried at room temperature and the solvent was removed
by keeping the samples in the freeze-drier for 24 hours. Alkaline
extracted krill protein was prepared as in Figure 1 using frozen raw
aw krill
0.1N NaOH
I Homogenate I
centrifuging
IPreepitatel
I Supernatant I
•.
pH adjustment(pH 4)
0.5N NaOH
with HC1
centrifuging
centrifuging
Supernatan
N 11.5%
N 23.5%
(Krill prat.)
N 65.0'f;
Fig. Extraction of protein from raw krill
* Percentage distribution of nitrogen
4
krill, and was freeze-dried, defatted with the ether-ethanol mixture,
and again dried.
The eviscerated krill sample was prepared by removing
by hand the head (including the viscera) of individual precooked krill
and was treated in the same manner as the other samples after washing
thoroughly in water.
2. Analysis of Chemical Composition
Water content was determined by heat-drying the frozen krill in
a constant temperature drier at 105 °C for 3 hours.
For the measure-
ment of crude fat, crude protein, and ash content, frozen krill was
freeze-dried and pulverized by grinding it in a coffee mill (Shibata
Emiide).
The total nitrogen content was measured by the semi-micro-
Kjeldahl method (crude protein = N x 6.25), fat was measured in
ether-extract form prepared wiMI a &mallet
fat extractor, and ash content
was determined by ashing in an electric furnace (550 o C, 3 to 5 hours of
constant weight).
3. Experimental Animals
The experimental animals, young male rats of the Wistar strain,
weighing 30 to 40 g, were purchased from Nippon Rat Inc. Those
whose body weight changed significantly during several days with solid
diet were excluded from the experiment. During the pre-experimental
period of 5 days, a 15% casein diet (casein 15%, salt mixture 4%,
vitamin mixture 0.85%, choline chloride 0.15%, soybean oil containing
2% concentrated cod liver oil 8%, cellulose flour 2%, QC-cornstarch 70%)
was given to all the rats before they were divided into the experimental
groups.
The temperature of the room housing the animals was maintained
at 22 to 24 ° C, and the light and darkness was controlled by switching
5
on and off the fluorescent light on a 12 hour cycle (light 7:00 a.m.
to 7:00 p.m.)
4.
Analysis of the Nutritive Value of Protein
(1)
Protein Efficiency Ratio (PER)
The experimental diet was prepared by adding the respective
protein at a 9% level to the basal diet (protein-free diet), shown
in Table 1, in exchange for C‹-cornstarch.
The duration of feeding was
4 weeks during which the animals were given free access to water and
the diet, and the body weight and food intake were measured.
The
group fed with the protein-free diet, whose weight served as the basis
for calculating the net protein ratio (NPR), was treated in the saine
way
in all other respects during the same period. Casein and refined
whole egg protein were used as standard proteins.
Table 1.
Composition of basal diet
Salt mixture*
Vitamin mixture*
Choline chloride
Cellulose fl our
Soybean oil (containing 2% conc.cod liver oil)
a-Corn stach
4. 00%
0.85
0.15
2. 00
8.00
85. 00
* A. E. IIarper, J. Nutr., 68, 408 (1958)
(2)
Relative Nutritive Value (RNV)
The relative growth index was calculated by the slope ratio
technique of Hegsted
12)
.
Krill protein diet of five different levels
was prepared by adding to the basal diet 3%, 5%, 7%, 9%, and 12%
respectively of krill protein.
The standard protein diets (refined
6
whole egg protein and casein) of corresponding levels were also
prepared. Weight gain and food intake during a feeding period of 3
weeks were measured. Weight gain was plotted against nitrogen intake
and the slope of its regression line was determined.
The RNV's for
krill and casein were calculated as the percentage of the slope of the
regression line in the krill protein group and that of the casein
group, respectively, against the slope of the regression line in the
whole egg protein group, which was set at 100.
(3)
Biological Values (BV)
A diet with protein at the 10% level was prepared by adding
it to the basal diet of Table 1 in exchange for c&cornstarch.
Standard proteinsused were casein and refined whole egg.
Rats were
kept in metabolism cages throughout the feeding period of 7 days,
during which the diet was given in pellets prepared by adding an equal
amount of water at each feeding.
The diet left uneaten was dried and
pulverized in order to determine its nitrogen content. The nitrogen
content swallowed was determined by subtracting the uneaten nitrogen
content from the total nitrogen content given.
A separate group was
given a protein-free diet in order to calculate the amount of intrinsic
nitrogen per 100 g body weight.
On the basis of these data, biological
values, true digestibility, and net protein utilization (NPU: NPU =
BV x true digestibility) were calculated.
(5)
Reagents and Dietary Material
The reagents and dietary material used were as follows: Solid
diet:
Nippon Kurea Inc. AE-2;
refined whole egg protein (PEP):
Food Inc.; casein: Tanabe Seiyaku Co. Ltd.;
oi-cornstarch:
Taiyô
Nihon
7
Shokuhin Kakô Inc. Arusutà-B; pewcier filter paper : Tôyô Roshi
Inc., dietary E; salt mixture and vitamin mixture: Oriental Inc.,
Harper composition
13)
; soybean oil:
Wakô Junyaku Inc., special grade;
officinal; choline chloride:
Other reagents used were of special
grade.
Experimental Results
1.
Analysis of the Chemical Composition of the Samples
The results of the analysis of three types of krill samples
are shown in Table 2.
All figures indicate the percentage on a dry
basis. Crude protein ranged from 62% to 70%, crude fat from 8% to
11%, and ash content from 16% to 20%.
Table 2
2.
Chemical composition of krill samples (on dry basis)
Sample
no.
Catching
date
Treatment
Crude
protein
(%)
Crude
fat
(%)
Crude
ash
(%)
1
2
3
Dec. '73/Jan. '74
Dec. '74/Jan. '75
Dec. '71/Jan '75
Precooked
Precooked
Raw
62. 98
66. 50
69. 42
10. 98
8.66
8. 11
16. 50
17. 49
20. 08
Exp. 1 & 2
Exp. 3
Exp. 3
PER and NPR for the Protein of Precooked Krill (Experiment 1)
The results are shown in Table 3.
In terms of weight gain over
the 4 week period, the casein group and the krill group were almost
the same, and the whole egg protein group was clearly higher. Similar
trends are seen in the case of PER and NPR. These results indicate
that the nutritive value of the protein of precooked krill is comparable
to that of casein, but is clearly inferior to that of whole egg protein.
Table 3
Nutritive values of krill protein measured with rats
Weight gain
(g/4 weeks)
Protein source
(1)
(2)
Whole egg protein
Krill protein (precooked, freeze-dried
(3)
Milk casein
129. 5±6. 3') 4 ),,.)
102. 3±4. 3 b)
PER
PER"
(5•'• of
casein)
NPR 2)
•
3 • 6+0. 1,0
2. 8±0. 1 b)
124. 1
96 , 6
4.
0. la)
3. 7±0 1 b)
2. 9±.0. 2 b)
100
3. 8.-± 0. 1 b)
defatted)
1)
2)
98. 0..t 9. 7 b)
PER : protein efficiency ratio (protein level, 9%, period, 4 weeks)
NPR : net protein ratio (protein level, 9%, period, 4 weeks)
3)
lgean -±S.E. (n=6)
4)
Values in the same column not followed by the same letter are significantly different (p<0. 05)
3. RNV for the Protein of Precooked Krill Measured by the Slope Ratio
Technique (Experiment 2).
The RNV was calculated by Hegsted's slope ratio technique.
The
regression lines obtained were Y = 27.1X - 18.0 for the whole egg
protein, Y = 21.1X - 12.7 for casein, and Y = 23.7X - 24.9 for the
krill.
The results of the calculations based on the slopes of these
regression lines are shown in Table 4. As compared to the set value
of 100 for the whole egg protein, the value for the krill protein
is 87.4, slightly higher than the 77.6 for casein.
•
Table 4 Relative nutritive value of krill protein
Protein source
(1)
(2)
(3)
RNV*
Whole egg protein
100
Krill protein (precooked, freeze • dried defatted) 87. 4
Milk casein
77.6
* RNV : Relative nutritive value(relative growth index)
by Hegsted's slope ratio method (J. Nutrition,
85,159 (1965)).
4. Biolqgical Value, Digestibility, and Net Protein Utilization of
Precooked and Raw Krill and Extracted Krill Protein (Experiment 3)
9
The results are shown in Table 5. The biological values were
98.8 for the whole egg protein, 80.6 for casein, 82.2 for precooked
krill, 87.4 for the precooked krill whose head (including the viscera)
had been removed, 77.6 for the raw krill, and 84.9 for the extracted
krill protein. These results indicate a slightly higher biological value
for precooked krill than for casein, and a surprisingly low value for
raw krill.
Evisceration (leaving the muscles and the shells) resûlted
in a slightly higher value. The extracted protein was superior to the
raw krill, and tended to be higher than the precooked krill.
Table 5 Biological value (B V), digestibility and net protein utilization (N P U) of krill
protein
BV
(1)
(2)
(3)
(4)
(5)
(6)
Whole egg protein
Milk casein
Precooked krill (freeze-dried, defattecl)
Precooked krill (eviscerated and washed,
freeze-dried, defatted)
Raw krill (freeze-dried, defatted)
Protein extracted from raw krill (freezedried, defatted)
True digestibility
NPU
98 • 83.70. 3*)
80. 6370. 8b)
82. 2±0. 7,)
87. 4±- 1. lcn
92. 2±0. 5
95. 9±0. 3
91. 7+1. 2
91. 7+1. 2
91. 03._- 0. 7
77. 43_70. 9
75. 43_70. 9
80. 13_70. 8
77. 6±1. 2e)
84. 9±1. 4f)
88. 8±0. 9
93. 0+0. 7
68. 9±1. 3
79. 0±1. 7
* Mean_+.S.E.
Significant difference by test : p<0. 01 (a-b, a-c, a-d, a-e, a-f, b-d, c-p, c-e, d-e,
e-f)
p < 0. 05 (b-f, c-e)
All the groups except for that of raw krill (88.8%)
demonstrated a digestibility higher than 90% (91% to 96%). Since the
extracted protein (93%) did not'show a higher digestibility than the
91.7% for precooked krill, the effect of the shell could not be
demonstrated.
10
The net protein utilization is obtained as the product of the
biological value and digestibility, and indicates the ratio of the
protein in a food that is actually utilized in a body. This ratio
was 91 for the whole egg protein, 75.4 for the precooked krill, 68.9
for the raw krill, and 79.0 for the extracted krill protein. Judging
from these figures, it seems as if the nutritive value of the protein
of raw krill is somewhat lower than that of precooked krill.
Discussion
Krill contains 60% to 70% protein on a dry basis. Compared
with 68% for mysids, 78% for prawns (Metapenaeus joyneri), 68% for
spotted shrimp,
and 80% for Penaeus japonicus
is close to that of mysids
14)
,
its protein content
and spotted shrimp.
It was found that the protein of precooked krill was comparable
to casein in terms of PER measured with a 9% level of protein added
to the diet. It was also found that PER for precooked krill was
clearly inferior to that for the whole egg protein.
A similar
tendency was noted in terms of NPR measured at the same time (Table 3).
There is evidence that PER for high quality protein, such as that of
whole egg, reaches its peak when the level of protein content is
slightly lowet than 10% and that it drops rapidly when the protein
level becomes lower or higher
unaffected by the level of
15)
.
NPR, on the other hand, is relatively
protein intake, and remains fixed deperiding
on the quality of the protein
16)
.
Therefore, it is assumed that the
effect of food intake on NPR is also minimal.
Furthermore, NPR
indicates the entire effect of protein on factors involved in the
maintenance as well as increase of body weight, and differs from PER
11
in the sense that the latter indicates the effect on body weight
increase alone.
For this reason, the fact that krill protein was
comparable to casein in terms of both PER and NPR is sufficient
evidence for considering it equivalent to casein in nutritive value.
Aral et al.
9)
, incidentally, found that in comparing the PER of
dried krill, spotted shrimp, and casein at a 13% protein level,
the krill protein was comparable to casein and spotted shrimp
protein. Although the higher protein level in their study is a
difference to be noted in experimental method, their results show a
similar tendency to the present data. J.S.Sidhu et al.
1)
also noted
that krill protein possessed a nutritive value equivalent to that of
casein. Although PER is affected to a large extent by the level of
protein intake, it is often expressed in relative terms with the protein
level in the diet being at or near 10% (AOAC, 9
in 1973
18)
).
Therefore, comparison
717),
changed to 10%
of these various measurements
is considered more or less justifiable.
Applying bioassay procedures with rats to the measurement of
the amount of nitrogen (amount of protein) effective for the synthesis
of body protein, Hegsted et al.
12)
plotted weight gain against
nitrogen intake in order to obtain the ratio of the slope of the
regression line of the protein tested against that of a standard
protein. They emphasized the superiority of this ratio to other
indices such as PER and biological value.
This method is considered
to yield fairly reliable measures although it involves rather complicated
procedures. While this weight gain is plotted against nitrogen intake
in this experiment, the measurement of body protein is recommended in
order to assess more accurately the relative nutritive value
19)20)
.
As
106
12
shown in Table 4, when casein and krill protein were compared with
whole egg protein, they turned out to be close, with krill tending to
be slightly higher.
In order to examine the extent to which krill protein is
retained as body protein, and to assess its nutritive value on the
basis of this efficiency, biological values were measured.
2Table 5
contains the results. Precooked krill yielded an almost identical
value to that of casein (80--82), which was similar to the results
for PER.
Matsumoto et al, comparing the biological valueF of krill
protein with that of egg protein at a 20% protein level, obtained
84 for whole egg protein, 74 for protein extracted from raw krill,
56 for raw krill, and 66 for precooked krill.
They noted no
significant difference between the biological value
extracted from krill and that
of protein
of whole egg protein. They also reported
no significant difference between these two in terms of net protein
utilization. These results differed considerably from those of the
extracted protein obtained in the present study (biological values:
98.8 for whole egg and 84.9 for extracted krill protein). Their
biological values for raw krill and precooked krill, however, were
similar to the present results, although their figures tended to be
lower. Since the measurements of Matsumoto et al. were taken at a
20% protein level, the fact that at high protein levels biological
values of various proteirs tend to be relatively lower and the differences
among them smaller
21)
presumably has some bearing on theirresults.
In the present experiment, raw krill tended to be lower than
precooked krill in both biological value and digestibility.
This
13
requires further investigation as it is conceivable either that
components underwent certain changes during the treatment of the
samples or certain factors existed which hindered the utilization of
the protein.
The sample of eviscerated precooked krill yielded a
higher biological value than intact precooked krill.
This is probably
because nitrogen components in the viscera have inferior amino acid
composition to that of protein in the muscles.
et al.
3)
According to Suyama
, however, there is no nutritive difference between the
amino acid composition of the whole fish and of muscles alone,
although muscles contained slightly more amino acids containing
The biological value of prawns (shelled) obtained by Matsuno
sulfur.
et al.
22)
was 87, a similar value to that of eviscerated krill sample.
Summary
Same as abstract on p. 1.
At the end, the authors express
their sincere appreciation to Dr. Masaaki Yanase of Tokai Reg. Fish.
Res. Lab., and the staff of Marine Resources Development Center whol
helped obtain the krill samples.
References
1) G. S. Sidhu, W. A. Mongomery, G. L. Holloway-, A. R. Johnson and D. M. Walker : J. Sci. Fd. Agric.,
:',tMekk (W
21, 293 (1970)—Euybausia
9 Di]
2) 5
, eàil,
3) el",
rpeb,
rji
H71:Q, 30, 267 (1964)
: A(.2k, 31, 302 (1965)
4) V.;4*, hi, -gtm, 1
13, 203 (1964); 13, 477 (1961)
: U7Jii, 30, 630 (1964)
_.
"Audi :31i:es 4( t 65, 59 (1971)
77, 97 (1974)
: 7) enMell. eepeem, 83 , 1 (1978)
3p:e7e.ree, 85, 1 (1 976)
9) Me, ell,
efli*Tee, 85, 13 (1976)
10) reu, ;1e):1,, 91J,
ye*: ee e.e, 29, 307 (1976)
ee,
11) es*,
85, 159 (1965)
M. Hegstecl and YET-OY Chang : J. Nutrition,
5) 54fril,
_
12) D.
13) A. E. Harper : J. Nutrition, 68, 405 (1959)
5-Ye
, 14) 7eWeerfeWae.:a'nF 1 **MUee
35,
664
(1955)
Rev.,
Physiol.
:
15) J. B. Allison
140 (1957)
16) A. E. Bender and B. H. Doell : Brit. J. Nutr.,11,
Official Analytical Chemists, P. SOO, 7th ed.
17) Official Methods of Analysis of the Association of
_ (1970)
14
_
—
18) P. H. Derse : J. A. O. A. C., 48, 847 (1965), ibid., 56, 521 (1973)
19) D. M. Hegsted and YET-OY Chang : J. Nutrition, 87, 19 (1965)
16, 190 (1968)
20) D. M. Hegsted, R. Neff and J. Worcester : J. Agr. Food. Chem.,
21) K. M. Henry and S. K. Kon : Brit. J. Nuir., 11, 305 (1957)
22) ta, ,
,ffl :
eeegia,
29, 249 (1971)
1) G.S. Sidhu, W.A. Mongomery, G.L. Holloway, A.R. Johnson and
D.M. Walker: J. Sci. Pd. Agric., 21, 293 (1970) --
cited in
Annotated Bibliography on Euphausia by Okada et al.
2)
Hirano, Kikuchi, & Okada:
Nissuishi (Bulletin of the Japanese
Society of Scientific Fishery), 30, 267, (1964).
3) Suyama, Nakajima, & Nonaka: ibid, 31, 302 (1965)
4) Tsuyuki, Naruse, Mochizuki, &
Yukagaku (Journal of Japan
Oil Chemists' Society), 13, 203 (1964), 13, 477 (1964).
5) Nonaka & Koizumi: Nissuishi (Bulletin of the Japanese Society
of Scientific Fishery), 30, 630 (1964)
6) Yanase: T;kaisuikenhô (Bull. Tokai Reg. Fish. Res. Lab.), 65,
59 (1971)
7)
: ibid, 77, 97 (1974)
8)
: ibid, 83, 1 (1975)
9) Arai, Watanabe & Kinumaki: ibid, 85, 1 (1976)
10) Watanabe, Sugii, Wakita, & Kinumaki: ibid, 85 13 (1976)
11) Matsumoto, Masuki,Hamakura, Maekawa, & Suzuki:
Eiy-o- to Shokuryii
(J. of Japanese Soc. of Food and Nutrition), 29, 307 (1976)
14)
Science and Technology Agency.
Investigation Committee of Resources:
Standard Composition of Foods of Japan (Third Revision)
22) Matsuno, Iwaya, & Tamura:
Eiy-o-gaku zasshi (Japanese Journal of
Nutrition) 29, 249 (1971)
(Received on April 25, 1977)