Relationship between injury of living cells and cellular membrane damages arising from freeze-thawing and freeze-drying was investigated. In yeast cells, it was demonstrated that an increase of membrane permeability was brought about by freeze-thawing and freeze-drying, at the first, and then the degradation of the membrane phospholipids followed to. By the experiments using human erythrocytes, the structural change of the membranes was supposed that it can be ascribed to the dehydration from the cells. NMR study in E. coli cell membranes demonstrated that a significant structural change of phospholipids was brought about in the logarithmic phase specimen, which is sensitive to freeze-thawing and freeze-drying. In contrast, in the stationary phase specimen, which is highly resistant to the same treatments, a very slight lipid structural change was observed. Glycerol effectively endured the lipid configuration in logarithmic phase cell membranes in the same freeze-thaw treatment.

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Preservation of biological samples derived from life science experiments is essential for carrying out a variety of life science experiments on space station (Japan Experiment Module:JEM). This is because the experiments on JEM have to be carried out under a special restricted circumstance characterized by limited space and micro-gravity where analytical technique or equipment usually applied for experiments on the ground cannot be always applied. National Space Development Agency of Japan (NASDA) is surveying and developing preservation or fixation techniques of biological samples for life science experiments in space as a part of the generic experiment technology development program. The Survey and development are introduced in this paper.

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Colistin, a kind of antibiotics, is effective against gram negative bacteria. In this study, Colistin-sulfate and Colistin-citrate were manufactured as its salt, and Colistin sodiummethanesulfonate was also munufactured as its derivative. Then, they were freeze-dried. In the process of freeze-drying, rapid freezing was necessary to prepare the fusible powder. Physiological functions were maintained when they were freeze-dried at the temperature below the eutectic point. However, the temperature was raised during the process in three steps, starting from -40℃ to 55℃ finally, as it would take a long time if the temperature should be kept below the eutectic point.

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Effects of two pretreatments, that of culturing cells and tissues at 4℃ or preculture on nutrient medium containing 10% sucrose, were investigated in the cryopreservation of cells and tissues of carrot (Daucus carota) and wheat (Triticum monococcum, Aegilops squarrosa). Cryopreservation was carried out in liquid nitrogen by the methods of two-step freezing and vitrification. In carrot cells, both preculture at 4℃ for 30 to 40 days and preculture on the medium containing 10% sucrose were shown to significantly increase cell survival after cryopreservation. In the vitrification method, the survival rate of cells was very low, however, regrowth and regeneration of somatic embryos were observed in these cryopreserved cells. In cultured wheat cells, the latter pretreatment was similarly effective in survival rate increase after cryopreservation. The survival rate of the cells cryopreserved by vitrification was very low as compared with that by the two-step freezing method. The results shown here suggest that further improvement of the vitrification method is needed for the cryopreservation of these cells.

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The treatment of nonacclimated (NAC) rye protoplasts with vitrification solution (VS) consisting of 7M EG, 30%(w/v) sorbitol and 6%(w/v) BSA in buffer solution, produced the distinct plasma membrane ultrastructural changes, including, formation of aparticular domains, lamellar-tohexagonal_<II>(L-to-H_<II>) phase transitions and membrane vesiculations. Loading NAC protoplasts with 1.5M EG reduced VS-induced formation of aparticular domains and L-to-H_<II> phase transitions, but not membrane vesiculations. Cold acclimation (ACC) of rye protoplasts also reduced VS-induced formation of aparticular domains and L-to-H_<II> phase transitions, but not membrane vesiculations. The treatment of these protoplasts with hypertonic solution (13S) consisting of 60% (w/v) sorbitol brought about similar effects with VS, but did not produce membrane vesiculations. It was suggested that while formation of aparticular domains and L-to-H_<II> phase transitions were produced by dehydration due to treatment with hypertonic solutions (both VS and 13S), the membrane vesiculations were produced by biochemical toxicity due to high concentration of EG in the VS.

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The freezing and thawing behaviour of the NaCl solution was investigated by differential scanning calorimetry (DSC). Salt precipitation from the freeze-concentrated solution, which was detected by the observation of the endotherm due to eutectic melting, i.e., melting of NaCl・2 H_2O-ice, depended on the initial salt concentration. With the decrease in the initial concentration down to ca. 1 M, the amount of the salt precipitation began to decrease. Salt precipitation was inhibited in the presence of sugar molecules. The inhibition, compared on the basis of the weight percentage concentration, was more remarkable when monosaccharides were added than when polysaccharides were added. The effect of sugar addition can be explained by the consideration of the increase in the viscosity of the solution, which may inhibit nucleation and / or growth of ice and salt crystals, i. e., eutectic separation.

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An attempt has been made to preserve living water fleas (daphnia) under a frozen state at a cryogenic temperature. Although the technique of cryopreservation of living cells, tissues or organs having simple structure and with smaller sizes has been established to a certain extent, the cryopreservation of more complex living organs with larger sizes has not yet been achieved successfully. In this study water fleas with 1-3mm in stature are chosen as the subjects because they are possessed of various kinds of tissues and organs. The water fleas in an aqueous solution of glycerol are first cooled at a controlled rate down to -35℃, and then rapidly frozen in a liquid nitrogen of -196℃. If the rate of pre-cooling and the concentration of glycerol solution used as a cryoprotectant are chosen appropriately, the water fleas can be preserved for a long period of time. They can be recovered to life by thawing them at an optimal rate of warming up. A recovery ratio higher than 80% has been attained. However, there still remains a serious problem because the water fleas thus recovered would not live longer than several hours. They die because of injury to certain organs. The technique should be improved furthermore before completion.

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An autolytic enzyme of Bacillus subtilis was activated by a fast cooling, but not by a slow cooling, to a temperature below the memberane phase transition point (<5℃) and following incubation at the low temperature for more than 20 min with a modified Spizizen salt medium. The fast cooled cells were lysed during the following incubation at 37℃ with the same medium. Both of fast cooling and the following incubation at low temperature were required for the autolysis. Any suspending media for the fast cooing, water, 100mM KCl or KCl+glucose induced the lysis similarly to Spizizen medium. While, slow cooling induced the autolysis only with water or solutions without KCl, but not with solution containing more than 2.5mM KCl.

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Cells of Saccharomyces cerevisiae were subjected to heat shock treatment at 43℃ for 30-120min, followed by secondary stress treatments and then their colony forming units (CFU) were analyzed. Enhancements of more than two orders of magnitude were observed in the secondary stress tolerances against freeze-thawing, thermal (at 50℃ ), and hydrostatic pressure (at 150 MPa) treatments assessed by the CFU. NMR investigations on the cells subjected to the heat shock treatment revealed that the treatment promoted the structuralization of intracellular water and decreased the membrane fluidity. Addition of water-structuralizing salt, i.e., Na_2SO_4, induced significant decrease in the fluidity of vesicles prepared with the phospholipids which were extracted from the yeast cells. Structural breakage of the cell was observed electronmicroscopically after the physical stresses, however, the heat shock treatment protected the membranes from the breakage. Consequently, the heat shock response promoted cell water structuralization which stabilized the membrane structure or decreased its fluidity, and as the results improved the secondary stress tolerance.

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Of various changes in fish muscle during storage, these which exhibit an abnormal temperature-dependency at temperatures around the freezing point were reviewed in this paper. The growth rate of bacteria on fish skin at -4℃ was as low as 1/8 of the rate at 0℃, and the microflora after storage at -4℃ was markedly different from that at 0℃. Increase of K-value of fish muscle was effectively suppressed at temperatures below the freezing point or, in some species of fish, below the phase separation temperature of microsomal lipid. The denaturation of myofibrillar protein and the damage to muscle cell structure more or less proceeded at temperatures below the freezing points, but the degree of injury to flesh quality depended on the species of fish. On the basis of these results, the advantages and disadvantages of superchilling or partial freezing storage of fish were discussed.

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The efficacy of CO_2 anesthesia combined with low temperature in the transportation of live fish was evaluated using adult carp, Cyprinus carpio, acclimated at 23℃. All the carp became anesthetized in about 10 min with a 30 min cold treatment at 4℃ and were safely maintained in an anesthetic state for the following 9.5 hr with a cold-CO_2 treatment (14℃, Pco_2=60-90mmHg). Judging from the changes in the depth of anesthesia, the optimum anesthetic condition was Pco_2=80mmHg at 14℃. The minimum environmental and arterial Pco_2 required for anesthesia seemed to be 125mmHg and 90mmHg at 23℃, respectively. Under CO_2 anesthesia, the EEG of carp gradually became smaller in amplitude, with slowing of the wave and showed a pronounced tendency toward flatness. It can be concluded that CO_2 exerts its anesthetic action by acting on the central nervous system and probably by depressing brain activity.

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Spiked fish in a pre-rigor state called "Ikeshime Gyo" in Japanese has the same commercial value to live fish in the western Japan. Since spiked fish proceeds gradually in postmortem change to a rigor state, this type of fish has a time limitation to keep its high commercial value. The present review deals with the temperature-dependency and species-specificity of rigormortis progress of spiked fish in association with chemical changes of muscle to cope with the above limitation. Commercially valuable fishes such as yellowtail, red sea bream-and plaice are all slower in postmortem changes at 5〜15℃ than at 0℃. These results strongly suggest the acceleration of fish rigor-mortis at 0℃ is due to the increase of Ca^<2+> concentration in myofibrils during storage at this temperature, and studies along this line are now in progress.

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When fishes (horse mackerel and mackerel) were immersed in a brine at a temperature of approximately・10℃ (immediately after catching), the fish surface became semi-frozen, and then fishes assumed a state of apparent death. Immediately after such a super-rapid pre-cooling fishes were transferred into a cold brine at a temperature near the freezing point and were subjected to thermal equilibration and freshness was preserved at this temparature. On the other hand, a conventional icing procedure in which fishes were cooled directly from normal sea temperature to the temperature region near the freezing point was carried out. The results of both experiments were compared, and the following points were clarified: (1) The cooling time required to reduce the fish temperature from that at catching to the preservation temperature could be shortened significantly with the super-rapid pre-cooling. (2) Introducing a super-rapid pre-cooling technique, the number of days for the K value of fish to reach 20% during a cold storage at a temperature near the freezing point was extended to twice or more. (3) During the storage at temperatures below freezing point the appearance and organoleptic evaluation of the fish were nearly the same as those of frozen fishes, but when the temperature of fishes was risen again above the freezing point, the fish nearly resumed their states before freezing. (4) With brine used for the super-rapid pre-cooling and cold preservation, slightly higher values of salt concentration in fish boodies were detected : just under the surface skin for mackerel and in belly for horse mackerel. (5) No appreciable difference in muscular tissue due to the super-rapid pre-cooling was noticed.

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Fish lipids are labile to autoxidation due to polyunsaturated fatty acids such as eicosapentaenoic acid and docosahexaenoic acid. In the first, studies on the mechanisms of lipid oxidation were reviewed. The effects of lipid oxdation on the quality of fish flesh were discussed. Safety of the endoproducts of lipid oxidation was also studied. Another considerable deterioration of the lipids includes hydrolyses of triacylglycerol and phospholipids by lipase and phospholipases, respectively. Free fatty acids, the products of lipid hydrolysis, are accumulated in fish flesh and corelated certainy with protein denaturation during storage at low temperature. Finally, an effective technology on preventing lipid oxidation and hydrolysis was investigated.

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Several factors, such as feeding for rearing-fattening period, slaughtering process, conditioning, etc, are known to affect meat quality. In this paper, changes in pH, ATP-related compounds, hardness, glycogen and lactic acid of muscles, which may relate to conditioning, during the storage were mentioned. (Samples of various muscles were obtained immediately after slaughtering and stored at 2℃.) Additionally, conditioning and the microbiological effects for meat quality are also discussed.

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Fruits and vegetables after harvest are living tissues which are continuosly subject to physiological changes which cause lowering their quailty. Maintaining the fresh produce at low temperature close to the freezing point of individual crop will be desirable, if possible, because Q_<10> value at the range of low temperatureis higher than that at high temperature. However, there is a problem that tropical and subtropical origins are susceptible to chilling injury, depending on species and developing stages, even at the range of low temperatures suitable for the storage of common crops. Therefore, systems through transportation and storage of fresh produce at low temperature called as cold chain system must be established considering the sensitivity of the produce to low temperature. The production and action of ethylene, one of plant hormones, are closely concerned with the activity of intermediary metabolism of fresh produce and suppressed at low temperature. Hence, prompt cooling after harvest (precooling) is very effective to minimize the lowering of the quality of fruits and vegetables through transit and storage and to extend the shelf-life. Chilling injury is a disorder induced by low, but nonfreezing temperature in chilling-sensitive species of tropical and subtropical origins. The primary change of the injury has been shown to be the phase transition in cellular membranes resulted from the difference in the ratio of unsaturated fatty acid/saturated fatty acid in membrane lipids, leading to increased cellular damage. On the other hand, transferring to above critical temperature after a short chilling may reestablish normal metabolism in tissues as shown by the recovery from lowered respiratory control ratio and activities of some enzymes to normal state in cucumber mitochondria. If chilling temperature continues long, ultrastructural changes are noted as observed in mitochondria, chloroplast, or tonoplast of water convolvulus. Such structural changes may be associated with unbalanced metabolism of phenolics induced by chilling.

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An Erwinia ananas ice nucleation-active gene, named inaA, was cloned which encoded a protein with 1322 amino acid residues. When Escherichia coli was transformed by introducing this gene, it acquired an icenucleating activity which was due to production of the same protein in the inclusion body form. The possibility exists that the protein, when immobilized on the surface of a film, can be used as an ice nucleator for freeze concentration of raw and/or fresh foods under a very mild condition.

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Surface hydrophobicity of myofibrillar proteins from fishes and warm blooded animals was generally increased by freezing at -20℃ for 1-2 days. This increment was remarkable as to the sorts of proteins,in the order of myosin, actomyosin and actin, and remarkable as to species, in the order of white muscles fishes, the pink muscles fishes, and red muscles fishes or warm blooded animals. Such increase was, however, depressed by the addition of cryoprotective substances such as sucrose or sodium glutamate.

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Sardines are underutilized, although they account for the largest part of the fish catch in Japan. They are mostly processed into fish meal and oil. One problem limiting their effective utilization may be the rapid loss of protein quality during storage, since the muscle pH decreases quickly after death, easily reaching the ultimate pH of 6 or lower. During frozen storage at -20℃, myofibrillar Ca^<2+>-ATPase of sardine ordinary muscle lost half of the original activity within 2 days of storage, while the activity remained quite stable thereafter. The ordinary muscle myofibrils displayed a remaining Ca^<2+>-ATPase activity maximum at pH 7.5 and little activity below pH 6.5, when suspended in buffers of various pH values and stored at -20℃ for 2 days. Freeze denaturation of ordinary muscle myofibrils was more obviously observed in the acidic pH region, when remaining Mg^<2+>-and K^+ (EDTA)-ATPase activities were measured under the same conditions described above. Extremely low Mg^<2+>-ATPase activity was demonstrated even at pH 7.0. Compared with ATPase activities, there were much less changes in solubility in 0.5M KCl at any pH with sardine ordinary myofibrils. Sardine dark muscle, which accounts for about 30% of the total muscle, exhibited similar changes to ordinary muscle in myofibrillar ATPase activities and solubility during frozen storage at -20℃ for 2 days.

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Various methods for distinguishing fresh from frozen-thawed fish were tested. The most reliable method was the enzymatic one in which the activity of the neutral β-N-acetylglucosaminidase in blood was used as the index. The enzyme, found in fish red blood cells, was inactive in intact cells but was activated when cells were disrupted by freezing and thawing. The method was applicable for testing most common edible fish and required about 20 min using a UV-lamp.

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The denaturation of rabbit skeletal myosin B caused by freezing and thawing was measured by the change of Ca^<2+>-ATPase activity. The remarkable denaturation was observed at low protein concentrations less than 0.6 mg/ml. The denaturation caused by freezing and thawing was protected in the presence of non-ionic detergent such as Tween 20. The addition of 2% of Tween 20 was effective for the protection against of denaturation. The addition of sugars also protected the denaturation, but the effective concentration for non-ionic detergents was lower than for sugars. The sugars had protective effect for thermal denaturation but non-ionic detergents had not. The addition of glycerol showed a synergetic effect with Tween 20 for the protection of freezing-thawing denaturation.

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In the processing and distribution of domestically produced meat in Japan, priority in the distribution system is given to fresh meat, with only that amount of meat that can not be sold immediately being frozen. This is because the quality of frozen meat is reduced through "drip loss" and the palatability of the meat is adversely affected due to such changes as deterioration of juiciness. When it comes to imported meat, however, since the countries of origin are very distant from Japan, almost all of the meat comes in frozen and is distributed as such. Only a small portion of the imported beef comes in fresh. It is estimated that approximately 25-30% of the total meat sold in Japan is frozen meat. Whether or not this percentage will grow or shrink in the future depends in large part on further development of techniques. It is with this in mind that the author introduces newly developed techniques as well as those under consideration which show promise in the future. 1. Electrical Stimulation and "Hot Boning" If an electrical stimulus is applied to carcasses immediately after slaughter, the muscle tissue will rapidly contract and the ATP in the muscle tissue will be used up. This results in rigor mortis setting in very quickly. Meat from carcasses that has been so treated can be chilled, frozen and thawed without and "cold shortening"or "thaw shortening". Thus, fresh carcasses that are still warm and that are cut up and deboned before rigor mortis (called "hot boning") can be chilled and frozen very efficiently. 2. Thawing Method That do not Lead to "Thaw Shortening" Meat from carcasses that has not been treated with electrical stimulation and that is subsequently "hot boned" will usually undergo shortening when frozen and then thawed. If, however, an appropriate method of thawing is used, this will not happen. It has been known from long ago that when such frozen meat is first partially thawed and then from that half-frozen condition it is thawed very slowly at low temperature, such shortening does not occur. It is thought that this is becouse slow thawing from the half-thawing state allows the ATP in the muscle to completely decompose. However, as the authors have shown through experimenttaion, thaw shortening can be avoided even in muscle tissue that has been completely thawed and that still contains significant levels of ATP if the meat is maintained at 0℃.

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New freeze-tolerant yeasts were isolated from banana peel and undefined as Kluyveromyces thermotolerans and Saccharomyces cerevisiae. These yeasts retained their fermentative ability and bread leavening activity even after freezing fully prefermented dough. The hybrid between S.cerevisiae FRI 413 and a bakers' yeast with sexual conjugation gave good quality bread made from frozen dough containing additive sugar at low levels (0-20%). Five freeze-tolerant yeast strains suitable for frozen dough were compared with ordinary commercial bakers' yeast. These freeze-tolerant strains of S.cerevisiae showed higher surviving and trehalose-accumulating abilities than other S.cerevisiae strains, but were affected by a prolonged prefermentation period. The freeze tolerance of the yeasts was associated with the basal amount of intracellular trehalose after rapid degradation at the onset of the prefermentation period.

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