Effects of shelf temperatures on residual activities of lactate dehydrogenase (LDH) were determined. Raffinose, trehalose and mannitol were used as excipients. The mixtures of LDH, phosphate buffer and excipients were freeze-dried. They were stored at four shelf-temperatures (-25℃, ambient temperature, 50℃ and 70℃) from 0 to 30 days. From the results, raffinose and trehalose show good ability to stabilize activities of LDH. Although mannitol is a good excipient for short-term storage, but the residual activity of LDH was decreased drastically more than two weeks storage. Comparing a trehalose to raffinose, the trehalose samples show less heat-stability. Glass transition temperature of the excipients is the most important factor for stabilizing activity of LDH and other factors seem to concerned with the results.

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To obtain the basically knowledge of the molecular mobility of low molecular carbohydrate under the glassy state, the enthalpy relaxation time (τ) of five sugars such as glucose, galactose, maltose, sucrose and trehalose, were examined through aging experiment by using DSC. The glassy samples were aged at several temperatures of (T_g-10) K to (T_g-35) K. The r at each temperature was obtained by fitting Kohlrausch-Williams-Watts (KWW) equation to the time course of enthalpy relaxation amount (ΔH). When the temperature dependence of molecular mobility was assumed as Arrhenius process, the activation energy amount (ΔE) could be estimated. Judging from the ΔE value, it can be considered that the enthalpy relaxation is mainly due to α-relaxation process governing the molecular dynamics. Furthermore, the ΔE was larger in order of trehalose>galactose>sucrose>maltose>glucose. The result would support the explanation of availability of glassy trehalose as lyoprotectant of protein or biosystem under the severe circumstance.

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Trehalose has unique properties to protect biological material and cells against severe water stresses, including desiccation, freezing, high pressure and so on. To elucidate the functional mechanism of trehalose, it is necessary to investigate the interaction of this sugar with protein, membrane and other biologically important assemblies such as gel. Here we study the effect of trehalsoe on the swelling behavior of hydrogel such as agarose and carrageenan. Cylindrically-shaped gel samples are prepared in pure water and subsequently immersed in aqueous solution with different sugar concentration. The equilibrium volumes of the gel samples are found to decrease with an increase in sugar concentration. And the degree of such volume change is most remarkable in trehalose solution than in other disaccharides (maltose and sucrose) solutions. These findings are interpreted on the basis of a thermodynamic theory of gel swelling. In conclusion, trehalose provides larger influence on the swelling property of gel due to its higher hydration ability.

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Form II is a kind of metastable crystalline form of trehalose anhydrate and is easily converted to the dihydrate crystal by absorbing water in a moist atmosphere at room temperature. It can be utilized as an edible and no poisonous desiccant and thus its efficient production from the dihydrate is significant from a viewpoint of industrial application. In this study, we attempt to extract crystal water from the dihydrate using supercritical CO_2 fluid. We examine the dependence of extraction efficiency on the extraction time, the temperature and pressure of the fluid. Consequently, we demonstrate that form II is produced from the dihydrate through supercritical CO_2 fluid extraction if appropriate temperature and pressure conditions (around 80℃ and 20 MPa) are given.

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Glass formation of emulsified aqueous solutions of MgCl_2 and AlCl_3 was investigated in relation to cryopreservation of living cells and meristems. It is pointed out that crystallization phenomenon, which is usually unavoidable in the warming-up process of a vitrified solution, is an important factor in the development for a suitable method for cryopreservation of living cells. Avoidance of the possible injuries incurred from crystallization in the warm-up process of the vitrified solution is a key factor for a successful application of the vitrification method for cryopreservation.

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The exotherm in the DSC rewarming trace observed with frozen gels made of crosslinked dextran, Sephadex G25, was confirmed to be due to crystallisation of hexagonal ice by the two-dimensional XRD (X-Ray Diffraction)-DSC simultaneous measurement. It was indicated by the measurement that small ice crystals are readily formed in the gel, and that the endothermic trend precedent to the exotherm corresponds to the melting of small ice crystals. Moreover, two-dimensional XRD images observed with various Sephadex gels in the frozen state depended on the density of crosslink.

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Ethylene can hasten an onset of endogenous rise and senescence in plants. Since ethylene exposure to plants induces a formation of abscising layer and leads to abscission of florets, water uptake to petals from flower stalk might be prevented. We indicated that exogeneous ethylene caused complex physiological changes of petals in intact flowering clones of orchid, Dendrobium phalaenopsis cv.during seven days; decrease in both ^1H-NMR relaxation times (T_1 and T_2) and water content, and increase in hue angle indicating tone of color with senescence process. Dynamic states of water are stated as several water compartments such as free water, loosely bound water and bound water which originate from vacuole, cytoplasm and apoplastic region, respectively. Long T_1 of the cellular water in the orchid petals decreased and it well corresponded to water content in the ethylene-treated clones. It suggested that vacuolar water disappeared in the petal tissues. Furthermore, long and short T_2s were not maintained in the petal tissues exposed to ethylene. From these results, change in the vacuolar water component of the petals was better monitored by T_1 while the cytosolic water which relates to their molecular mobility could be shown by short T_2 in the orchid petals. This study clearly indicated a basic information concerning petal senescing process evaluated by NMR relaxation times (T_1, T_2) of water proton in the ethylene-sensitive plant.

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Temperature dependency of Arrhenius plots of ^1H-NMR relaxation times (T_1, T_2) was investigated in fresh, stored and frozen-thawed sweet potato tubers as a chilling sensitive plant. Noteworthy converse correlation in Arrhenius plots of T_2 was observed with lowering temperature from 30 to 0℃ in the fresh tuber tissues. The tubers stored at 15℃ for one year were partly injured and the frozen-thawed tubers were dead tissues judging from the results of ion leakage. Gradient in T_2 of Arrhenius plots changed at about 14℃ in the fresh tissues. On the other hand, there were no break points in T_1 and T_2 of dead tubers. Therefore, occurrence of gradient change of Arrhenius plots in T_2 at 14℃ might respond to chilling stress for the fresh tuber tissues. In dead tuber tissues temperature dependency of Arrhenius plots was clearly observed in T_1, while no temperature dependency was observed in T_2 of that tissues. In the stored tubers, temperature dependency on T_1 and T_2 indicated similar tendency observed in the dead tuber tissues. The water contents of sweet potato tubers were about 70% while that of Vigna hypototyls, which indicate liner T_1 decrease with lowering temperature, were over 95%. Thus, inverse relaxation behavior with lowering temperature seems to be partly influenced by low-level in water content of the tubers. In sugar solutions, T_2 markedly decreased at higher concentration compared to T_1 Additionally, T_1 in the sugar solutions decreased with lowering temperature while T_2 was independent upon temperature. The difference between T_1 and T_2 observed in the solutions was similar to the dead tuber tissues. That is, T_2s of the sugar solution

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Noninvasive measurement of concentration using magnetic resonance imaging (MRI) was applied to diffusion of cryoprotectant in aqueous solution, pseudobiological tissues (agar), and biological tissues (liver tissues of chicken). The cryoprotectants were dimethyl sulfoxide and glycerol, common cryoprotectants penetrating cells. ^1H-NMR spectrum, calibration curve of MRI image intensity for cryoprotectant concentration, attenuation of MRI image intensity due to solution fraction and relaxation time in each mass transfer medium were investigated. Transient one-dimensional distribution of cryoprotectant concentration in the medium was measured to estimate apparent diffusivity of cryoprotectant within the medium as a solution in the inverse problem.

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A Micro-Sheer Image Processing System (MSIPS) has been applied to observe the ice crystal structure formed in frozen dilute solutions three-dimensionally. Several parameters were also proposed to investigate three-dimensional (3-D) morphology and distribution of ice crystals quantitatively, based on reconstructed images obtained by multi-slicing of a frozen sample with the thickness of 5 μm. The equivalent diameter of ice crystals were in the range of 85 μm up to 169 μm, and then decreased exponentially in increasing freezing rate at the freezing temperature of -20℃ to -80℃. On the other hand, no signification effects of the freezing rate were observed on two-dimensional (2-D) distribution of ice crystals at the high freezing rate over 150℃/hr. The 3-D morphology of ice crystal was found to be a bundle of continuous columns at any freezing temperature under the usual freezing conditions except supercooling.

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A simplified numerical model has been developed for predicting freezing processes of food materials. The model was comprised of the heat transfer equation and the solidification model, assuming thermal equilibrium state during freezing process as well as the existence of three layers; unfrozen, frozen and moving boundary layer. In order to investigate the applicability of the model, one-dimensional freezing processes of 10% coffee solutions have been numerically simulated based, on the thermophysical properties of 1-55% coffee solutions determined with DSC-method and the theoretical model, such as the initial freezing temperatures, phase diagrams and effective thermal conductivity. Then, the results were in good agreement with experimental freezing curves.

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Complex impedance was measured to analyze freezing-injury for food materials. High impedance and distinct Cole-Cole arc were observed for fresh plant cell tissue samples such as Japanese radish, carrot, potato, and apple. After freezing-thawing, however, impedance drastically decreased and the Cole-Cole arc disappeared reflecting the severe freezing-injury on plasma membrane. On the contrary, animal cell tissue samples like poultry and fish meat showed much lower impedance, which did not change much after freezing-thawing reflecting the stronger freezing tolerance for these samples. No distinct Cole-Cole arc was observed for these samples suggesting the different cell structure of animal tissue as compared with plant tissue. For gel-type sample like soyprotein gel, impedance rather increased after freezing-thawing.

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Normal-speed (32 frames/sec) and high-speed (up to 4,000 frames/sec) video cameras attached with a cryomicroscope were utilized to analyze the freezing process of protoplasts isolated from Arabidopsis leaves and Jerusalem artichoke tubers. With protoplasts isolated from leaves of non-acclimated Arabidopsis seedlings, intracellular freezing was observed even when protoplasts were frozen to -10℃ at rates of 0.5 to 1.0℃/min, and the frequency increased significantly at 2.5 to 10℃/min. After cold acclimation for 1 day at 2℃, the intracellular freezing did not occur at cooling rates of 1.0℃ or slower. Cold acclimation for 2 to 7 days further resulted in a shift of the temperature range over which intracellular freezing occurred to lower temperatures. High-speed video microscopy revealed that intracellular freezing was initiated, with no exceptions, at places near the plasma membrane. However, it was not possible with techniques used in the present study to determine whether ice formation initially occurs inside or outside the cell. Nevertheless, these studies demonstrate that videomicroscopy combined with cryostage provides an insight to understand the effect of cold acclimation on the freezing process of plant protoplasts.

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In vitro-grown apical shoot tips of Gentiana were successfully cryopreserved by vitrification (V) and encapsulation-vitrification (EV) protocols. We optimized and compared the V and EV protocols. Although both protocols resulted in a relatively high survival rate, the EV protocol seemed to have several distinct advantages. First, the survival rate of shoot tips cryopreserved by the EV protocol was much greater than that by the V protocol under optimized conditions. Second, the range of optimum treatment period by vitrification solution (PVS2 solution) was considerably wider with the EV protocol than with the V protocol. Third, after rewarming from liquid nitrogen temperature, the cryopreserved shoot tips showed more vigorous growth for the EV protocol than the V protocol. With the optimum EV protocol, we succeeded to cryopreserve shoot tips from 10 different lines of Gentiana and uniformly obtained high survival rate. Thus, the EV protocol appears to be promising for cryopreservation of a wide range of plant germplasm.

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When plant tissues were cryopreserved by utilizing a slow pre-freezing method, it was noticed that cotton plugs came out of the sample-containment straw during freezing only when distilled water was used as a freezing solution. This was likely due to a large ice expansion during freezing of distilled water. However, since plugs were remained on the straw when freezing with any cryoprotectant solution, it was assumed cryoprotectants might reduce ice expansion in the freezing solution. Furthermore, it seemed possible that cryoprotectants acted by reducing mechanical stress caused by extracellular ice crystals growing during the slow freezing procedure. In the present study, changes in solution volume during freezing using several types of cryoprotectant were investigated. The effect of each cryoprotectant solution on the survival of asparagus nodal segments frozen down slowly (0.5℃/min) to -40℃ was also examined. The ratio of the volume at -40℃ to the volume at +20℃ was used as an index for ice expansion, which could be calculated as a ratio of density at +20℃ to density at -40℃. Density at +20℃ was obtained by measuring both weight and volume of each solution. Density at -40℃ (r) was calculated by the following formula: r=w0^*r1/(w0-w1); rl, density of isooctane at -40℃ (known as 0.73999); w0, weight of an amount of solution in air; w1, weight of the same amount in isooctane at -40℃. Distilled water showed the largest volume change at a ratio of 1.094. The ratio gradually decreased with an increase in the molar concentration of cryoprotectant. Raffmose was the most effective in reducing ice expansion when being compared with other cryoprotectants at a same concentration. Raffinose showed it's largest cryoprotection against asparagus tissue at 0.6M where the solution became saturated. Dimethylsulfoxide (DMSO) at 1.69M had the largest effect on cryoprotecting asparagus tissue. Furthermore, DMSO was also the most effective in reducing ice expansion among plasmamembrane-permeable groups of cryoprotectants.

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During this work, We defined that vigorous growth on cryopreserved asparagus (Asparagus officinalis L.) regenerates were induced not by cryoprotectant, but by frozen-thawed treatment with different cryoprotectant 0.6M raffinose. And lateral buds of asparagus nodal segments freezing treated were dyed more deeply and extensively by 0.1% (w/v) 2, 3, 5-Triphenyl tetrazolium chloride (TTC) reduction pigment as time goes by. Histological examination showed that mitochondria were increasing after cryopreservation in the treated cell. Enhansed growth were observed by horseradish with slow freezing method, which was supposed that vigorous growth by cryopreservation might exist generally.

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Gametophytic plants of Pogonatum inflexum and Plagiomnium acutum were desiccated and stored in liquid nitrogen. The moss plants desiccated and stored in liquid nitrogen maintained about 40% (Plagiomnium acutum) or 10% (Pogonatum inflexum) of the viability according to the oxygen production test. From leaves of Pogonatum inflexum stored in liquid nitrogen, protonemata newly regenerated, when they were recultured in liquid medium. Also the protonemata of Pogonatum inflexum were desiccated, rehydrated and recultured. In the microscopic observation, survived cells and dead cells were distinguishable from the difference of morphology. These results suggest that moss plant and protonema are suitable experimental material for study the desiccation and freezing tolerance of plant cells.

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The protocorm-like body (PLB) of Oncidium hamana "elfin" was encapsulated by gelling matrix such as calcium alginate, and precultured in media with high concentrations of sucrose. The cultured PLB was desiccated on silica gel and then cooled in liquid nitrogen (LN). The effect of gelling matrices such as k-carrageenan, agar, agarose and Gelrite on the survival of PLB after desiccation and cooling in LN was investigated. In addition, the effect of ABA (abscisic acid) in preculture medium was examined on the development of the tolerance of PLB. The PLB precultured in medium with each concentration of sucrose showed a high survival rate after desiccation on silica gel. The survival rate of PLB after desiccation and cooling in LN varied depending on the kind of gelling matrices and the relatively high survival rate of PLB was obtained when agarose and calcium alginate were used as gelling matrix. Little or no effect of ABA was observed in the development of the tolerance of PLB to cooling in LN.

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Suspension-cultured cells of Arabidopsis thaliana were precultured in media with high concentrations of sucrose or sorbitol and their tolerance to desiccation and super-low temperature was examined. The tolerance of the cell to desiccation and super-low temperature substantially increased after preculturing in sucrose- or sorbitol-enriched medium. Osmotic concentration of cell-free extract increased and considerable amounts of sucrose and proline accumulated in the cell during precuturing in sucrose- or sorbitol-enriched medium. It was suggested that the accumulation of these substances play an important role in the development of the tolerance in A. thaliana cells.

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