1. Vitamin B₁₂ content in various organs of aquatic animals was determined by H_UTNER s method and results obtained are shown in Tab. 1. The most of them, especially livers, are proved to be very rich in it.
2. Growth promoting effect of the solubles on young chicks was determined, which, Acre prepared from various parts of the following fishes, such as bonito, yellow-fin albacore, cuttlefish, oyster and baby clam. The yields and analyses of the solubles are shown in Tab. 2. By the supplement of these solubles to the all plant ration, young chicks showed considerable growth response as shown in from Tab. 3 to 5, owing to the presence of APF. However, cuttlefish meat soluble was ineffective.

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Some of seaweeds were proved to be considerably rich in vitamin B₁₂, as shown in Tab. 2. Especially, the Japanese common food, “Asakusanori”, contains much vitamin B₁₂ and shows APF activity (Tab. 1). Table 1 may suggest the presence of another active substance (s) other than vitamin B12. The active substance for Euglena in Porphyra was destroyed by alkali-treatment and moved little in paper partition chromatography with wet n-butanol just like B₁₂ (Tab. 1 and Fig. 1). The presence of vitamin B₁₂ and APF in seaweeds, a group of plant, is very interesting.

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In the present paper, the writer reports the data obtained from the experiments collecting planetonic larvae and young spats of the pearl oyster, Pinetada martensii. which were carried out, during June, July, August and September of 1951, at the Nasa Bay near Shishikui-machi, Tokushima Prefecture. The results of these experiments are summarized as follow:
(1) The natural production of the pearl oyster are closely affected by the conditions of environment during July.
(2) It is assumed that about 25 days are necessary for the D-shaped veliger to develop to the so-called full grown stage. In this year, the largest collection of the veliger larvae was occurred on July 20 th, and the largest one of the full-grown larvae on August 13th.
(3) The young spats settled on collecters of these experiments are induced to belong to the same population by the statistical measurement on the destribution of shell length.
(4) No spat settles on the fresh collecter. On the other hand, the settled spats are found abundantly, after the collecter becomes dirty to some extent. It is supposed that 16-18 days are necessary for the collecter to become dirty in a suitable degree.
(5) It is concluded, therefore, that the optimum season for setting collecter seems to be about the middle of July and the maximum settlement of the spats to be about the middle of August.

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Katsuobushi, one of the most important fishery products peculiar to Japan, is employed for giving good flavor and taste to food, being rich in some particular flavors and in nutritional value.
In manufacturing Katsuobushi and its substitutes, the moulding is one of the essential procedures. Aspergillus repens and A. ruber are well known as the excellent moulds for Katsuobushi. By inoculating them on minced meat of fishes, the authors could prove that they are responsible for the development of the characteristic flavors of Katsuobushi. (Cf Table I )
Our experiments (Tables 2-4 ) were carried out to know which factors would have effects upon the development of the Katsuobushi-like flavors.
The results are summarized as follows:
1. When fat-free mackerel meat is used as the substratum, the fatty substance from mackerel meat which is added as much as 20% of the meat in weight gives the best results as comar-ed with the fatty substances from other fishes (atka fish, herring, cod liver and flat fish), cuttl-fish liver, aid plants (soy-been and linseed). (Table 2 & 3)
2. Substrates other than fish meat, such as casein, soybean cake and tofu, give Katsuobushi-like flavor, when they are previously smoked and mixed with the fatty substance from mackerel meat which is added as much as 20% of the substratum in weight. Casein gives especially good flavor, which is quite similar to that given by fish meat. (Table 4-b)
3. Thus the development of Katsuobushi-like flavor is known to be affected by the nature and amount of the fatty substance from fish meat and by smoking, but scarcely by the nature of the proteinaceous substratum.

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The present paper deals with the biological activities of vitamin A in fin whale liver oil and the distillate distilled at 10^-3 mmHg. The results obtained are summarized as follows:
1. The biological potency (47, 800 U. S. P. units) of the whale liver oil was found to be higher than that (39, 900) assayed by spectrophotometric method of O_SER, wheras the distillate showed nearly the same potency (138, 600) biologically as spectrophotometrically.
2. Although the apparent increase of vitamin A by molecular distillation is, therefore, amounted to 123 % spectrophotometrically, that estimated by biological methed is only 85 %, which is still quite higher than those reported previously by other authors.

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It was found that the process of putrefaction, in the course of storage of shark muscles cooked at above 55°C, followed the curve general in usual fishes in place of the ammonia-developing curve characteristic of the shark. A great quantity of ammonia was however generated from the cooked muscle when it was infected with several kinds of urea-splitting bacteria separated from the shark or a salted fish. It is thus presumable that urea in the shark muscle may be decomposed only by certain limited kinds of bacteria.
We observed that these bacteria, except only one kind of species, shoud be killed or their growth considerably retarded by cooking at 55°C.

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On the basis of the results obtained from the previous experiments the author has made the assumptions as follows:
(1) Volatile acids in fish meat may consist of two kinds, one being free acids, and the other bound acids.
(2) The second distillation ratio of the acids from fresh meat of fish exceeds 100 percent because the bound acids are greater than the free acids in amount so that few acids could be obtained in the first distillation.
(3) However, the amount of the free acids increase, while the bound acids decreasing, in response of deterioration of the meat. Such fluctuations in the acids seem to account for the second distillation ratio lowering below 100 percent in case of a spoiled meat.
In order to prove the above assumptions the following experiments have been carried out.
Prepare three kinds of samples from fresh meat of fish; minced meat, its water exudation, and a protein-free solution by treating the sample with 20% sodium tungstate. The similar group of the samples is prepared from the spoiled meat of a fish. Apply steam distillation to each sample.
It was found as a result that the free acids were almost removed dy exudation, for volatile acids contained in the residue of the exudation were measured but little.
When free volatile acids were wrapped as a reagent together with gelatin, they were formed into bound acids to some extent, as the results are shown in Tables 1 to 3.

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In order to see if the ratio of steam distillation of volatile acids is feasible in determining freshness of fish meat, a series of test have been carried out using the samples taken from the bonito, Euthynnus Yaito K_ISHINOUYE. Fresh meat sampled out of the parts such as the dorsal, caudal, ventral, and the dark muscle of a carcass, was separately comminuted. The spoiled samples were prepared from the similar parts of another fish which had been left under natural condition for three days. The ratios obtained from steam distillation of the samples were compared with the values of pH and volatile basic nitrogen (abbrev. V-N) measured for each sample.
As obvious from the results shown in Tables 1 and 2 quality of the fresh samples tested with V-N or pH hardly revealed differences among the anatomical parts. However, when expressed in the ratio of second distllation, the fresh samples had different values in accordance with the parts as:100 for dorsal, 93 for caudal, 82 for ventral, and 74 for dark muscle.
Spoilage measured with V-N and pH of the deteriorated samples was fairly distinct between the parts. The second distillation ratios of the samples were 75 for dorsal, 75 for caual, 59 ventral, and 56 for dark muscle. Neglecting individual variance between the two specimens, the spoilage of the samples caused by three days' standing may be expressed as 75% for dorsal, 81% for caudal, 72% for ventral, and 75% for dark muscle. The greatest spoilage took place in the ventral muscle.
On the basis of the above experiments it may be said that freshness of two fish belonging to the same species can be examined by comparing the distillation ratios obtained from the similar parts of them, although quality of a round fish cannot be determined from the ratio of its parts.

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According to the result from the experiments on nutritive values of fish oils conducted by Yoshida (1) and Ozaki (2), highly unsaturated fatty acids in the fish oils had toxic effect upon the growth of rats. So far this has been generally believed right. And existance of highly unsaturated fatty acids in fish oils have been accounted for the reason why fish oils are inferior to other oils from nutritive point of view.
It is a well known fact that highly unsaturated acids are rapidly oxidized when they come in contact with the air. For this reason, a special attention must be paid when we carry out a feeding experiment by giving highly unsaturated acids to test animals. Otherwise, there will be a danger of supplying more or less oxidized acid to the animals and calculating nutritive value of such degenerating acids, even if the experiment has started with genuine highly unsaturated acids.
As reported in the previous paper (3), we experimented the nutritive value of ethyl ester of highly unsaturated acids with iodine value of 365 and its partially reduced products. During the experiment many hairs around the mouth and legs were falling off in the result of feeding on the ethyl ester of these acids. (see Fig. 1)
The ethyl ester used for the test was preserved in a brown bottle. Every time the bottle was opened, CO₂ gas was poured in so that the acids might be kept from oxidation. We found, however, in the later course of the experiment that it was impossible to prevent the acids from deterioration by such a treatment as that. Such being the case, the acids we used for the test were not genuine as we had taken for granted, while measuring nutritive value of slightly oxidized acids. Another experiment carried out on the same occasion revealed that the partially reduced products of highly unsaturated acid, even though iodine value being fairly high, have nutritive value almost same as oleic acids.
It is understood that oxidation of natural unsaturated acids around C₁₈ starts from the carbon which is suspended under the most unstable condition in the atoms of unsaturated acids. On the other hand, oleic acid is regarded from its stracture to be one of the stable unsaturated acids, a fact which may suggest the following. In the course of the experiment the partially reduced products of highly unsaturated acids have turned to the ones which, despite of their fairly high iodine value, are as nearly stable as oleic acid when unstable factors have been removed out of them in consequence of reduction.
On the basis of these findings we assumed that natural unsaturated acids around C₁₈, except one which has conjugate double bond, would not always become less nutritious by the increase of double bond, as it has been generally believed. In other words, the unsaturated acids with the same number of carbon, even if different in the unsaturation degree, are almost same in their nutritive values. While the long-believed difference of nutritive value among the unsaturated acids should certainly be attributable mainly to the degree of oxidation affected on each unsaturated acid. In order to prove this assumption, we chose, as test materials, some of the highly unsaturated acids which had been considered nutritively much lower than others. Because highly unsaturated acids such as contained in the fresh sardine kept unoxidized are supposed to be not only harmless, but also highly nutritious. However, it must be the oxidized ones that show a retarding action as often observed in the past experiments.
With this view in mind, we extracted highty unsaturated acids from sardine oil as genuinely as possible to carry out our experiment.

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In order to obtain in a simple method knowledge of hydration of fish muscle which plays an important role in behavior of the muscle proteins, a series of research has to be carried out on swelling of muscle. In the present study, effect of pH on swelling of comminuted muscle is examined. Degree of swelling (S) is expressed by volume percentage of sedimentary muscle in a mixture of I part muscle and 3 parts water after centrifuged.
In case of horse mackerel muscle, on either side of an isoelectric zone (about 5.0-6.0), addition of HCl up to about pH 4.5, or of NaOH up to about pH 9.5 occasions very pronounced swelling. Further addition of acid or alkali causes slight peptization and the swelled muscle shows weak fluidity (Fig. 1).
KCl, added so as to 0.5 M solution, suppresses swelling of muscle caused by HCl below an apparent isoelectric point, pH 5.5 (pH for the minimum swelling), but above pH 5.5, it exerts an exactly opposite effect, namely, very marked increase in swelling is observedin presence of salt (Fig.4).
From these facts it is deduced that swelling of muscle cannot be explained simply on basis of osmotic pressure alone, but it seems to have a close relation with hydration of muscle proteins, especially that of myosin. Because solubility of myosin'rises on the alkaline side, while decreases on acid side of isoelectric point in presence of salt. ⁽⁷⁾
According to the nature of anions or cations, different kinds of acids or bases vary in their effects upon the swelling. For instance, H₂SO₄ (Fig. 2) and Ba (OH)₂ (Fig. 3) which both interact with the proteins as a bivalent ion, do not cause so marked swelling as HCl or NaOH.
When compared the swelling of horse mackerel muscle with those of sardine, carp, and skip-jack, no appreciable differences are observed among the behaviors of their swelling. On the other hand, the muscle of squid behaves differently from the other fish muscles in the following ways: (1) its isoelectric point (pH 4.8) is lower than that of the others (pH 5.5), (2) marked swelling is observed between 5.5 and 8.5, (3) when pH stands outside of this renge, considerable peptization of the proteins occurs to form a viscous solution (Fig. 5).

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Intending to establish the criterion of putrefaction of a green shark skin, we have carried out research on volatile bases (V. B.), volatile fatty acids (V. F. A.), and trimethylamine (T. M. A.) to be formed in the course of spoilage of the green skin. Then the first two were compared with these formed in the washed skin. In addition, shrinkage temperature (Ts) that lowers in the green skin just as the spoilage proceeds has been observed for the same purpose.
The results obtained are shown in Figures 1 to 3 where fluctuations of the chemical properties, bacterial counts as well as shrinkage temperature are indicated with respective symbols, in solid lines for the test A, and in a broken lines for the test B.
As evident from Figure 2, an abrupt change in the value of each item was observed together with a sharp decrease of Ts at a certain time between 8th to 11th day of the test A, and between 5th to 6th day of the test B.
It appears for us that in the early course of the spoilage prior to the abrupt change, extractive matters (mainly urea) in the skin are decomposed by bacteria. However, the hide substance (collagen) which has remained being hardly affected at that time, is seriously degraded by bacteria in the later course.
For this reason we have reached a conclusion that in case of green shark skin, fluctuations of V. B., T. M. A., V. F. A. in amount, and of Ts in degree, cannot be used as the criterion of bacterial degradation of the hide substance. In other words, a green shark skin is not a suitable material for preservation experiments of shark skin. It is proposed that a washed skin can be conveniently used in the spoilage experiments, however.

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In the previous paper¹⁾, we suggested that a green shark skin is not a suitable material for preservation experiments, and that such experiments are practicable only for the skin washed free from urea.
The present work deals with the effects of water content upon the bacterial decompositon of the washed shark skin which has been either pressed, or dehydrated by brining. The results obtained are as follows:
(1) The pressed skin is more or less prevented from the putrefaction depending upon water content in the skin. This effect is most remarkable in accordance with the decrease of water content down to about 40 to 45 per cent, but very little when water content depletes below that extent (cf. Fig. 2).
As previously reported²⁾, water content in a shark skin ranges from 42 to 46 per cent when water present between fiber-bundles has been removed as completely as possible by pressing. Consequently it seems that propagation of bacteria in the shark skin depends on the amount of water between fiber-bundles, but hardly on the water in the spaces among individual fibers of the bundle and bound-water.
(2) The skin treated with 5 to 10 per cent brine was successfully stored as long as a week. Whereas the one treated with 10 per cent brine was found rather difficult to be kept for two weeks at 30°C. without being putrefied.
(3) When treated with brine of lower concentration, the skin could be satisfactorily preserved for one to three weeks at 30°C. The success may be due to some action or actions other than dehydration of NaCl. The actions are supposed to attain a maximum effect at the concentration of 5 per cent of NaCl.
(1) T_AKAHASHI, T., and T_ANAKA, T.: Bull. Jap. Soc. Sci. Fish., 19. 185 (1953).
(2) T_AKAHSHI, T.: Ibid., 18, 341 (1953).

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