Glassy state is thermodynamically non-equilibrium state, and thus the various properties of glassy foods are affected more or less by thermal history such as manufacturing process and subsequent storage. Although the effect of isothermal storage on the non-equilibrium state of glassy foods has been evaluated through enthalpy relaxation measurement by using differential scanning calorimetry (DSC), there is no analytical approach for non-equilibrium state affected by its manufacturing process. To overcome this problem, the previous enthalpy relaxation measurement was extended theoretically, and an analytical approach to evaluate effect of manufacturing process on the non-equilibrium state of glassy materials was proposed. The approach involves that the value of relaxed enthalpy reflecting the thermal history of glassy materials can be evaluated from the difference between first-scanned DSC thermogram and second-scanned one. In addition, this approach was employed for freeze-dried trehalose powder, and the utility of the proposed approach was demonstrated.

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We have investigated on the additive effect of rare earth ions on the helix-coil transition of poly-L-Glutamic acid (PLGA) by Raman spectroscopy. It is found that rare earth ions seem to enhance the formation of helix structure of PLGA in the solution. We suggest that rare earth ions make a complex with PLGA in a bidentate way.

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We have investigated the low temperature-induced conformational change of aqueous tetraethylammonium chloride (Et_4NCl) having the trans-gauche.trans-gauche (tg.tg) and trans-trans.trans-trans. (tt.tt) equilibrium, chicken egg white lysozyme and bovine serum albumin (BSA) solutions using vibrational spectroscopy. A remarkable result is that conformational changes of Et_4NCl, lysozyme, and BSA are restricted by vitrification. Our results indicate that the vitrification of aqueous protein solution might restrict protein unfolding.

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Antifreeze protein (AFP) causes thermal hysteresis (TH) through its inhibitory action of the ice crystal growth, for which preservation effect is expected for a water-containing gel in a frozen state. A very weak TH activity was also reported for poly-vinyl alcohol (PVA). Here we performed TH measurements for type III AFP from Notched-fin eelpout (Zoarces elongates Kner) at various AFP concentrations in the absence and presence of PVA, and found that approximately 1.8-fold enhancement of TH is achieved by addition of 50mg/ml of PVA. We further examined structure of 0.5% agarose gel before and after freezing at -18℃ under various concentrations of AFP, PVA, and their mixture. It appeared that the gel structure was completely destroyed after freezing without addition of AFP or PVA, while it was preserved in the presence of 0.09mg/ml AFP or 50mg/ml PVA. Structure of the frozen gel was also preserved by a mixture of 0.03mg/ml AFP and 25mg/ml PVA. These results suggest that AFP provides a freeze-tolerance to water-containing gels, for which a required amount of AFP can be saved by addition of PVA.

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Antifreeze protein from Notched-fin eelpout (nfeAFP) has an ability to preserve mammalian cells in hypothermic condition. We compared the preservation ability between the naturally extracted isoform mixtures (denoted nfeAFPs) and a recombinant single isoform (denoted nfeAFP6) through observation of cultivated cells (HepG2) preserved at 4℃ for 24 hr with Euro-Collins solution in the presence of 0 - 10 mg/ml of the AFP samples. The preserved cells were examined by fluorescence microscopy after staining with calcein-AM and propidium iodide and by evaluations of extracellular lactate dehydrogenase (LDH) and intracellular ATP content. It appeared that nfeAFPs preserve the cell viability more effectively than nfeAFP6. In addition, the preservation ability increased dose-dependently and is maximized at 10 mg/ml of the protein concentration. These results suggest that addition of nfeAFP decreases hypothermic stress and the mixture of nfeAFP isoforms is more effective than the single isoform.

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The principal aim of this study was to provide a method for monitoring the product temperature during primary drying of the freeze-drying process of model pharmaceutical formulations from pressure rise based on the MTM method without insertion of thermocouples sensors in few vials, and establish a method for evaluating the ice front temperature and the mass transfer resistance to water vapor flux through the dried layer during primary drying. Effects of the formulation concentration, the shelf temperature and the chamber pressure on the ice front temperature and resistance to water vapor through dried layer were studied experimentally by MTM method, and the optimization program can be obtained. MTM method has been proposed as a more precise predictive way for determining the end of primary drying.

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The purpose of this study was to elucidate the effect of molecular interactions on the physical properties of frozen aqueous solutions and freeze-dried solids containing various carboxylic acids, amines, and alkali-metal-ions. Thermal analysis of frozen solutions containing the multivalent carboxylic acid and amines showed reduced tendency for the solute crystallization and bell-shaped profiles of the glass transition temperature of maximally freeze-concentrated amorphous solutes (T_g') depending on the number of functional groups on the solutes and their concentration ratios. Freeze-drying of the high T_g' frozen solute-mixture solutions (-40℃<T_g') resulted in cake structure amorphous solids that have the glass transition temperatures (T_g) much higher than the individual components. Network of electrostatic interactions and hydrogen-bonding between the amino and carboxyl groups, as well as anion network of strong "single hydrogen-bond" between the differently protonated carboxyl and carboxylate groups, should explain the high transition temperatures (T_g', T_g) of the frozen solutions and the freeze-dried solids.

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The glass transition of cryoprotective solutions on cooling is the key for successful cryopreservation of biological substances. We examine ethylene glycol solutions by using ultra-broadband spectroscopy. The dynamical properties from gigahertz to terahertz frequency range are investigated by using Raman scattering, Brillouin scattering and terahertz time domain spectroscopy (THz-TDS). In this frequency range the relaxation modes and vibrational modes coexist, hence it can be a powerful tool to provide important the information on dynamics of glass forming liquids. We investigate the slow and fast dynamics from the complex dielectric and Raman spectra of these solutions, and the non-coincidence effect between imaginary parts of dielectric and Raman susceptibility are discussed.

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Among the naturally occurring gluco-disaccharides, trehalose has the highest glass transition temperature (Tg) and the largest activation energy of enthalpy relaxation in the glassy state. Here, to understand why trehalose has such unique properties, we carried out FT-IR measurements and molecular dynamics simulations for trehalose and neotrehalose, and analyzed the hydrogen bond properties of both sugars in the amorphous states. By comparing the results for trehalose and neotrehalose, we reached the following interpretation: in the trehalose glassy matrix sugar molecules would be homogeneously distributed, whereas in the neotrehalose glassy matrix sugar molecules tend to be distributed separately into densely packed regions and sparsely packed ones. It is concluded that in the trehalose glassy matrix the conformational uniformity of sugar molecules brings about better (homogeneous) molecular packing, which in turn leads to the high Tg and the stability of the glassy state. Ultimately, such a unique property of trehalose originates from the presence of α, α-1, 1 glycosidic bond responsible for the conformational rigidity.

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We have studied the low-temperature properties of a hen egg white lysozyme (HEWL) crystal in cryoprotective solutions by Raman and micro-Brillouin scattering over the wide temperature range from -160℃ to 80℃. According to the observation of the characteristic Raman band of a glassy state and the continuous change of sound velocity, we have confirmed that ethylene glycol 50 mol% aqueous solution undergoes a liquid-glass transition around 150 K without crystallization even in slow cooling. We have studied a tetragonal HEWL crystal in ethylene glycol 50 mol% aqueous solution. With the decrease of temperature, the sound velocity of both solution and a crystal shows the continuous increase in all temperatures, and no abrupt jump of sound velocity due to crystallization is observed. It means that the three dimensional structure of a tetragonal HEWL crystal is preserved in low-temperatures down to -160℃.

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Fluorescence quenching is a simple method to determine the amount of molecules trapped inside phospholipid bilayer vesicles. It utilizes a fluorescent dye, calcein, the fluorescence of which can be quenched by chilate formation with cobalt (II) ions. The amount of calcein trapped inside POPC vesicles before and after freezing in presence and absence of saccharides was compared by this method. Leakage of the interior due to the damage caused by freeze-thaw process was detected. Three sacchrides were examined and sucrose exhibited the strongest protective ability while glucose exhibited the weakest. Trehalose was not very effective when it was added only in the exterior. However, the protective ability became comparable to that of sucrose when it was added to both interior and exterior of the vesicle.

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Dry weight, water content, water status in relation to kernel quality of ripening seeds of rice cultivars against thermal stresses were examined. Although a temperature of 20℃ suppressed the growth rate of developing seeds of cvs. Nikomaru and Hinohikari, the dry weight of cv. Nikomaru was entirely recovered on 21DAF. Additionally, the change in dry weight of cv. Nikomaru seeds grown at higher temperature did not differ from that of control during maturation. The water contents and T_1 values of cv. Nikomaru grown at 20℃ between 14 and 21 DAF markedly decreased in seeds indicating that the free water disappeared under low temperature stress during early ripening stage. After harvesting, thermal stresses did not severely suppress the ratio of ripened seeds and its quality of cv. Nikomaru. These results suggested that the diminish of free water in developing seeds is a reason that the quality in seeds can be maintained under low temperature stress.

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The change in seed water content has become a relatively efficient parameter for physiological maturation during seed development and maturity of of soybean (Glycine max(L.) Merr.). Fresh and dry weights of seed continuously increased from the beginning of seed filling (R5) to the end of seed filling stage (R6), while the water content gradually decreased and reached nearly 60% at R6 stage. Then the fresh weight decreased rapidly from R6 to full maturity (R8) while the decrease of the water content was accelerated. Autophagy functions in bulk degradation process and appears to have important roles in maintaining viability under various stress conditions and/or plant development, such as germination and senescence. To understand the relationship between physiological parameters and autophagy-related gene (ATG) expression, we measured seed water content, fresh/dry weight and expression profiles of soybean ATG8 homologs of seeds of soybean during pod filling stages. On the other hand, GmATG8 homologs were constitutively expressed in shell and seed during the period with high water content and the early seed setting stage(R5), and then expressions of GmATG8i and GmATG8d decreased at the end of seed setting stage (R6). These results suggested that autophagy process in seed was regulated in developmental stage and with physiological cues, possibly water content.

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Suspension cultured cells of Pogonatum inflexum survived rapid desiccation and cryopreservation, although the cells without desiccation did not survived cryopresrvation. While no protoplasts were isolated from the cells that were cryopreserved in liquid nitrogen without desiccation, many protoplasts were isolated from desiccated and desiccated-cryopreserved cells. It was likely that integrity of plasma membrane in Pogonatum remained through desiccation and cryopreservation process. Polytrichum commune and Oryza sativa cells also survived cryopreservation when they desiccated slowly. But only few protoplasts were isolated from desiccated cells of Polytrichum and Oryza. Yield of protoplasts recovered during the post-cryopreservation culture in both cells. These results suggest that plasma membrane of Polytrichum and Oryza cells was injured by desiccation stress but it was not lethal and recovered by themselves.

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Suspension cultured cells of Polytrichum commune were desiccated at various relative humidities and cryopreserved in liquid nitrogen. The water content of the desiccated cells varied from 0.06 to 0.24 (g H_2O/g dry weight). As the survival rate of the cells that were desiccated at high relative humidity was higher than those desiccated at low relative humidity, it is likely that the final water content was an important factor effecting the survival rate of the desiccated cells. However the cells that were desiccated by the two step method showed a high survival rate even if the final water content was low. Therefore, the effect of the desiccation process can be considered as important for preservation with high survival rate.

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Arabidopsis thaliana increases freezing tolerance upon exposure to low, non-freezing temperatures, which is known as cold acclimation (CA). CA results in significant changes in the lipid and protein compositions in the plasma membrane and these changes affect the freezing tolerance of plants. Recently, domains in the plasma membrane (called as microdomains), which contain membrane specific proteins associated with membrane trafficking and signal transduction, are isolated as detergent-resistant plasma membrane fractions (DRM). To investigate the role of DRM-associated proteins in CA, we characterized cold-responsive DRM-associated proteins in Arabidopsis seedlings during CA. Two-dimensional differential gel electrophoresis and subsequent mass spectrometric analyses revealed that many proteins quantitatively changed during CA, among which P-type H^+-ATPase, aquaporin and endocytosis-related proteins increased and tubulin, actin and V-type H^+-ATPase subunits decreased in DRM. Gene expression analysis indicated that the protein accumulation levels in DRM during CA were not regulated at levels of transcription. These results suggest that (1) Arabidopsis plasma membrane microdomains play a role as a platform for membrane transport, membrane trafficking and cytoskeleton interaction and (2) the changes in these proteins are not correlated with the changes in gene expression levels during CA.

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Antifreeze activity was induced in the medium of suspension cells of bromegrass (Bromus inermis) by incubation of the cultures at 4℃. We isolated a cold-inducible chitinase gene of bromegrass (BiCHT1) whose product is highly homologous to rye CHT9 known as an antifreeze protein. BiCHT1 was over-expressed in bromegrass cells using genetic transformation procedures. BiCHT1 products had chitinase activity, but the expressed proteins did not affect the growth of ice crystals in any conditions tested. The results of transgenic analyses suggest that the BiCHT1 product is a major protein with chitinase activity secreted in the medium of cold-treated cells but is unlikely to be responsible for the antifreeze activity detected in the culture medium.

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It has been shown that Marchantia polymorpha suspension-cultured cells with preculture treatment in a medium containing 0.5 M sucrose acquired a high level of desiccation tolerance. We investigated the tolerance for desiccation of protoplasts isolated from the precultured cells. Survival rate of the protoplasts (0.5 M Sucrose) after desiccation for 48 hrs was above 80%, while that of protoplasts isolated from the cultured cells without preculture (Control) was less than 30%. Furthermore, it was confirmed that the protoplasts have the ability to grow even after desiccation treatment and show almost 80% of cell division rate. We found that the protoplasts isolated from the precultured M. polymorpha cells maintained a high level of desiccation tolerance.

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The effects of cooling rates on freezing and survival numbers of the tardigrade Ramazzottius varieornatus have been examined. R. varieornatus has been cooled to subzero temperatures ranging from -2 to -20℃ at different cooling rates of 0.2, 1, or 5℃/min. The number of tardigrades frozen increases as the cooling rate increases. Survival rates are maximized at an intermediate cooling rate (1℃/min), implying that there is an optimal cooling rate for R. varieornatus to survive exposure to subzero temperatures. The freezing tolerance of R. varieornatus may be derived from the ability to survive dehydration since it is thought that common physiological mechanisms are involved in both freezing tolerance and desiccation tolerance.

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For the establishment of successful cryopreservation of Caenorhabditis elegans, freezing survival of the nematode was investigated in the presence of dimethylsulfoxide. When nematodes were cooled to -20 ℃ with the rate of 0.2 ℃/min after ice seeding treatment at -3 ℃, survival rate of the nematode increased with the time of exposure to the DMSO solution before freezing. The rate was above 80% irrespective of the cooling temperature between -20 to -75 ℃. However, the nematodes hardly survived when they were quenched by liquid nitrogen after the pre-freezing at -20 ℃, The rate of freezing survival became higher with the decrease in the pre-freezing temperature before plunging into LN_2. Heating rate also affected freezing survival. Slow heating probably prevented rapid rehydration of the nematode and increased survival rate after thawing.

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We studied the influence of dehydration conditions on the ability of dried larvae of Polypedilum vanderplanki to revive upon rehydration. High survival rates were obtained only for the larvae which had gone through the relatively slow dehydration. Final content of trehalose in the desiccated larvae varied depending on the dehydration protocol employed. The dehydrated larvae having trehalose as high as 25 wt% of their dried body mass were in glassy states up to ca. 70 ℃, whereas for the dehydrated larvae with only 15wt% trehalose their glass transition temperatures were down to ca. 30 ℃. These findings suggest that appropriate drying history is required to induce successful anhydrobiosis that enables larvae of P. vanderplanki to survive severe conditions imposed by their naturally dwelling place such as temporarily dried-up rock pool, where surface temperature can rise to 60 ℃ at midday in dry season. In addition it was made clear that larval bodies fell rapidly into glassy states at the final stage of the body water substitution for the endogenous trehalose, which supports the recent recognition that the vitrification and water substitution hypotheses are complementary to each other.

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