Ice crystal structure size in frozen food is inversely proportional to the advance rate of ice front, reflecting the important role of the molecular diffusion of water in the process of ice crystal growth. In the freezing preservation of living cells, the water permeability in the plasma membrane seriously affects freezing tolerance of cells. Plant cells, in general, have much lower water permeability in the membrane compared with animal and microbial cells so that the membrane structure of plant cells is easily destroyed by intracellular ice crystal formation. By controlling the ice crystal structure size very large, the progressive freeze-concentration becomes possible. This method is very effective to make the freeze concentration system much simpler compared with the conventional method of suspension crystallization to reduce the cost of freeze concentration substantially. Thermal unfolding of proteins was analyzed in terms of water activity in aqueous solutions. The protein unfolding ratio was correlated well to the water activity of the system, which was described well by the modified Wyman-Tanford equation. From this analysis, the free energy difference (ΔΔG) for protein stability in a solution and in pure water could be obtained.

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We developed novel cryoprotective agents from carboxylated ε-poly-L-lysines (COOH-PLLs) for cell cryopreservation as alternatives to dimethyl sulfoxide (DMSO). The polymeric agents showed excellent cell viability profile for most cell types, including human mesenchymal stem cells. We investigated the molecular mechanism of cryopreservation of the polymers by using solid-state nuclear magnetic resonance (NMR). The NMR data suggested that DMSO and COOH-PLLs differently affect the states and mobility of water and solutes in ice. The polyampholytes cryoprotectants showed highly ice recrystallization inhibition properties and we are developing vitrification solution for stem cells and tissue-engineered constructs cryopreservation using the ability. Through in situ crosslinking via click chemistry, the polyampholyte cryoprotectant could become a cell scaffold hydrogel with cryoprotective properties for tissue engineering application. We also discovered novel protein delivery method by using freeze concentration mechanism by using the polyampholyte as a cryoprotectant and protein nanocarrier.

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Water confined within nanopores exhibits the melting point depression, that is described with the Gibbs- Thomson (GT) equation. Generally, the confined water follows the GT equation, but if the pore has different pore-wall or shape, the equation cannot expect the freezing behavior of the confined water. Recently, calorimetry studies have demonstrated how the confined water is affected by the pore properties such as porecrystallinity, size, and shape. Within cylindrical crystalline pores, the internal water recovers the intrinsic hydrogen-bond network, which is independent of the arrangement of the molecules in the pore-wall unlike the interfacial water, with increase in pore size. Moreover, within amorphous slit-pores, the internal water exhibits higher melting points than those within amorphous cylindrical pores. Still it remains to be seen whether the hydrogen-bond structure of the confined water depends on anisotropy of pores.

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Recrystallization of ice crystals is a cause of deterioration in frozen foods during storage and distribution. Therefore, it should be controlled and predicted for optimal storage and distribution of frozen foods. In this review, we showed that the concept of water mobility in freeze-concentrated matrix is useful for predicting recrystallization rate of ice crystals in model frozen foods; that means, the water diffusion coefficient, an index of water mobility, in a freeze-concentrated matrix would enable us to predict of recrystallization rate of ice crystals. Effects of glass transition and presence of antifreeze protein (AFP) on recrystallization behavior of ice crystal were also described.

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It is known that glass transitrion temperature (Tg) of food products is a practically important factor for their quality control and prediction. However, it is difficult to obtain a clear Tg because food products are generally complex and heterogeneous system. Thermal rheological analysis (TRA), which is almost equivalent in principal to thermal mechanical analysis, is a useful technique for the Tg determination of food products. As a typical example, effect of water content on the Tg of cookie was shown in this paper. In addition, quality control and prediction of cookie was explained based on the glass transition concept. On the other hand, Tg is useful as an indicator of viscosity at a temperature above Tg. According to the concept, the quality control of rubbery food products was proposed.

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For the cryopreservation of embryos, vitrification has many advantages, but it also has disadvantages because embryos are vitrified with considerable supercooling (i.e., in non-equilibrium state) and thus easily damaged by intracellular ice formation at over the glass transition point. We tried to develop a novel method in which embryos are vitrified in near-equilibrium state. The extent of equilibrium was assessed by examining whether vitrified embryos survive after being kept at -80oC. Mouse embryos were vitrified with ethylene glycol (EG)-based solutions, EFSc solutions, which were mixtures of EG (30-40% v/v) and an FSc solution. The FSc solution was PB1 medium containing 30%(v/v) Ficoll plus a high concentration sucrose (1.5 M). When embryos were vitrified and then kept at -80oC for 4 days, large proportions survived with EFS35c and EFS40c. When mouse embryos were vitrified with EFS35c, transported with dry ice (-79oC) from Nankoku to Tsukuba, kept at -80oC for ~ 2 days (at ~80oC for 4 days in total), and recooled with liquid nitrogen, a high proportion of the embryos developed to term after warming and transfer. In conclusion, we have developed a novel method by which embryos are vitrified in near-equilibrium, which retains the advantages of both current vitrification and equilibrium slow freezing.

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In order to know the growth shapes of ice crystals in supercooled aqueous solutions of trehalose, in-situ observations of growing ice crystals were performed varying the concentrations of trehalose and the degrees of supercooling (ΔT). The ice crystals were covered with transparent thin glassy films and the surfaces of frozen solutions were smooth enough to focus on the entire visual field of an optical microscope. The shape of each ice crystal was usually dendritic and the diameter of each branch became smaller with increasing concentrations of trehalose and ΔT. In case of 3.18 molal and ΔT=21.8 K, the shape of each ice crystal was small and spherical, and such ice crystals were separated with each other by a thin transparent glassy film.

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In this study, we evaluated the probability process of devitrification and the moisture absorption rate for the amorphous trehalose film that formed on a CaF2 glass surface. The film samples were left at longest, for 7 hours in the test chamber filled with 40 °C -relative humidity (Rh= 40-80 %) air. Using FTIR spectrometer, the IR absorption spectrum of samples was measured at 0.5 or 1 hour intervals during the storage. The phase change in the film was judged by the differential spectrum from the initial state, and the water content in a film sample was evaluated by the area of 2 bend + libration combination peak. The results are summarized as, 1) the devitrification process of amorphous trehalose film were stochastic, 2) the exposure of amorphous trehalose film at Rh= 60 % induced the crystallization of trehalose dihydrate, while the exposure at Rh= 70 % induced the crystallization and the liquefaction of sample film, 3) the water absorption coefficient and absorption flux of amorphous trehalose at 40 °C were calculated, 4) the equilibrium vapor pressure of amorphous trehalose is 3.7 kPa.

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We examined whether model peptides for group 3 late embryogenesis abundant (G3LEA) proteins that we had developed previously are capable of maintaining enzyme activities under desiccation stress. Two different peptides were tested for comparison. One is PvLEA-22, which consists of two tandem repeats of the 11-mer motif characteristic to G3LEA proteins from African sleeping chironomid. The other is PvLEA-44 made of four tandem repeat of the corresponding 11-mer motif. When lactate dehydrogenase (LDH) is dried without any of the peptides, its enzyme activity is almost lost. However, the LDH activity is preserved 70% or higher in the presence of either the above 22-mer or 44-mer peptide. In addition, the cooperative protective effect is observed when trehalose is added to LDH together with such model peptides before drying. According to these results, we conclude that the G3LEA model peptides can protect enzymes against desiccation stress.

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Callus was induced from an axillary bud of Ceratopteris thalictroides cultured on Murashige-Skoog medium containing 3% sucrose and 10 μM benzyl adenine (BA). Sporophytic plants were constantly produced from subcultured callus when transferred to BA-free medium. Callus pieces were encapsulated in arginate gel beads and stored at -23, -5, 3, 10, 20 and 30℃. Callus tissues in gel beads stored at 20 or 30℃ for 32 days successfully germinated and regenerated to sporophytic plants but callus tissues stored at -23 or -5℃ were totally dead within 4 days. Gametophytes were sometimes regenerated from callus stored for rather longer days.

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Acclimation of plants to a great variety of environmental conditions is mediated by a phytohormone abscisic acid (ABA), which induces accumulation of stress-associated transcripts such as those encoding LEA-like proteins in both vascular and non-vascular land plants. Transient assays by particle bombardment with reporter genes have been employed for studies of ABA-induced gene expression in angiosperms, but such assay system has not been established in liverworts representing basal non-vascular plants. We used gemmalings of Marchantia polymorpha, which show ABA-induced desiccation tolerance, for establishment of the transient assay system in liverworts. We introduced the β-glucuronidase (GUS) reporter gene fused with the wheat Em promoter or the endogenous MpDHN1 promoter into gemmalings of M. polymorpha by particle bombardment and showed that GUS activity was increased by exogenous ABA for the both promoter constructs. Furthermore, we observed that exogenous ABA induced accumulation of LEA-like transcripts in the gemmalings. The results suggest that the transient assay system of the gemmalings of M. polymorpha is useful for monitoring ABA and stress responses in liverworts.

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To examine the benefits of using Undaria pinnatifida residue, in this study, we added U. pinnatifida lipids to surimi gels and measured the breaking strength (BS), breaking strain (bs), and gel stiffness (Gs = BS/bs) of the gels. Extracted U. pinnatifida lipids equivalent to 0% (control) or 1% weight of frozen surimi were added, and directly heated gels and two-step heated gels were prepared. Gas chromatography revealed that U. pinnatifida lipids were composed of many ω3 polyunsaturated fatty acids. BS of the directly heated gels was not significantly different from that of control gel. On the other hand, bs was significantly decreased compared with that of control gel. BS of the two-step heated gels tended to decrease compared with that of control gel, whereas bs did not show any significant changes. The BS and Gs values indicated that U. pinnatifida lipids had little effect on gel formation. Overall, it was revealed that the addition of U. pinnatifida lipids to surimi gels suppressed the increase of BS in the setting process. Therefore, U. pinnatifida lipids might have inhibited network formation and suppressed setting of the gels.

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During non-freezing temperature, many plants have to enhance cold and freezing tolerance to survive in winter. This phenomenon is called as a cold acclimation. It has been widely accepted that calcium ion (Ca2+) may act as a second messenger in the cold acclimation process, because low temperature and/or cold shock induce the rapid increase in cytosolic Ca2+ concentration. However, its role in cold acclimation is still unknown. We established the experimental system to observe Ca2+ signals with the combination of a confocal cryomicroscope and transgenic Arabidopsis expressing FRET-based Ca2+ sensor Yellow Cameleon 3.60. Because the dispersion in Ca2+ signal was observed among samples, a moving average method was employed after averaging data to seek the peak of the Ca2+ signal. The moving average method revealed that a distinct increase in Ca2+ concentration hardly arise until the temperature decrease of more than 3°C, indicating that a certain level of temperature change may be needed to induce a specific Ca2+ signal. Ca2+ signals would be a clue to how plants sense low- temperature.

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Cowpea (Vigna unguiculata (L.) Walp) has high tolerance to drought during the pod-filling stage. However, little is known about the mechanism of drought stress tolerance during the pod-filling stage in cowpea. Therefore, it is important to investigate the response of cowpea to drought stress at this stage. We found that leaf photosynthetic rate decreased to zero by day 3 of drought stress. In leaves and hypocotyls, relative water content (RWC) and the turgid weight/dry weight (TW/DW) ratio also decreased during drought stress. In pods, RWC and TW/DW ratio tended to decrease, whereas in seeds RWC tended to decrease but TW/DW was not affected. Harvest index and germination rate were not affected by drought stress. We also investigated the effect of drought stress on the levels of tonoplast intrinsic protein (γ-TIP) and plasma membrane intrinsic protein (PIP) 1 in seeds. The expression of γ-TIP was detected under both control and drought-stress conditions, whereas PIP1 expression was upregulated longer by drought treatment. A prominent band of the PIP1 protein appeared at day 7 after the onset of drought stress. These results suggest that remobilization of photoasssimilates in leaves induced by water deficit during pod filling is coupled in cowpea, and water loss in leaves and hypocotyls may serve as a signal to increase remobilization efficiency.

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