In the previous paper, we reported a lyophilizing method for injection preparations of antibiotic colistin salt and colistin sodium methanesulfonate which are suitable for the dissolution. In this study, we investigated the transfer process of lyophilized colistin sulfate and colistin sodium methanesulfonate by administering orally a large quantity of these antibiotics to beagles, because colistin salts are appreciated to be scarcely absorbed through the intestinal tract. The transfer of colistin sulfate to the blood was far less than that of colistin sodium methanesulfonate, and it seemed that most of colistin sulfate was discharged into faeces. We also report a method for freeze-drying a large quantity of colistin sulfate efficiently and without lowering its activity by improving the conventional dryer for the purpose of using colistin sulfate as a preservative additives for foods. We developed the colistin block lyophilization method, and the colistin raw meat ball by using freeze-dried new binding materials.

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The state of water in cells in biological systems has been studied extensively by NMR (nuclear magnetic resonance) spectroscopy. The longitudinal (T_1) or transverse (T_2) relaxation times of water protons provide invaluable information about the dynamic state of tissue water. For a decade, we have been studying changes in NMR relaxation behaviors in plants that resulted from the influence of various types of environmental stress. Our studies revealed that although Ti in plant tissues is primarily influenced by water contents, they are influenced not only by inherent factors in plants such as age, species, tissues and organs, but also by extrinsic conditions including environmental stresses. The Ti relaxation times are also influenced by the state of water balance in cells and tissues. Water balance is implied by total water contents, distribution of water in different compartments and interactions of water, macromolecules and secondary metabolites such as phenol derivatives in plants. In plant materials, however, the interpretation of the data in Tx needs special care different from the study of animal materials because plant cells contain characteristic organella such as chloroplasts, vacuoles and cell walls, and also the presence of unique water conducting systems (vascular bundles). Consequently, it is clear that the T_1 measurement in plants can be used for monitoring the primary response to various types of environmental stresses such as freezing, chilling, heat, salt, UV radiation and biotic (insect gall formation) stress and for a comparative evaluation of the stress sensitivities.

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This report describes a new shell freezing method for vials devised in our study. For aseptic pharmaceutical products, the freeze drying process should be as brief as possible not only from economical view point but also from aseptic criteria. It is well known that by use of shell freezing technique the process time must be shortened drastically. However, the conventional techniques are not suitable for large scale production because of complicated handling in which vials need to be fixed to the rotational axes one after another. In our new method, product in vials is shell frozen on the inner surface of the vials which are rolled on the cooled surface by use of the chain conveyer system. The good thermal contact between the vials and the cooled surface is obtained by a thin ethanol film laid on the cooled surface. Freezing of an aqueous solution of 20wt% lactose in volume of 1.75ml put in a vial of 18mm inner diameter has been completed within 1 minute with on effective length of one to two meter rolling. The drying time is shortened to approximately 5 hours from 20 hours required in the conventional methods. Five hour cycle will be good enough to perform continuous freeze drying of vials.

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The concentration efficiency in the progressive freeze concentration was related to the ice structure of the freezing front. A high freeze-concentration efficiency was obtained under the conditions at which a smooth solid-liquid interface was formed. An apparent distribution coefficient of a solute between the solid and liquid phases was useful to describe the concentration efficiency. The apparent distribution coefficient, reflecting the ice structure at the freezing front, was related to the operating conditions represented by the moving speed of the freezing front and the speed of stirring in the solution phase.

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Fracture stress of oil-free tofu was measured at temperatures from -20 to -196℃ by compression tests. These data were analyzed to give the fracture stress of CAS (concentrated amorphous solution) of oil-free tofu using a two-component parallel model. The calculated fracture stress of CAS of oil-free tofu was 73% larger than that of non-oil-free tofu which had oil of the amount of 50% of protein. This discrepancy was attributed to the inclusion of oil in the CAS. Hence a three-component model consisting of pure water ice, CAS (protein+water) and oil was applied to analyze the fracture stress of non-oil-free tofu, resulting in a value for calculated fracture stress of CAS of non-oil free tofu which agreed with that of oil-free tofu.

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It is well known that there is a clear relation between the thermostability of fish myofibrillar protein and the environmental temperature at which the fish species lives. The species difference in stability of the myofibrillar protein found at subzero temperatures, that is, cryostability was introduced in this review. Temperature-dependent changes in the freeze denaturation of myofibrillar protein of fish meat was studied in terms of the first-order rate constant (K_D) of inactivation of myofibrillar Ca-ATPase. The fish meat blocks were stored at different temperatures between -15〜-40℃, and the Ca-ATPase activity of myofibrillar protein from frozen-stored fish meat was assayed. It was found that K_D increased exponentially as the frozen stored temperature rose. Furthermore a certain difference in the temperature-dependence of K_D was noted among the species tested. The comparison of linear ARRHENIUS plot of K_D suggested that the lower the habitat water temperature of the fish, the lower the cryostability of myofibrillar protein.

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It is known that myosin B (MB)-Ca^<2+>-ATPase activity decreases by the salt concentration during freezing, which occurs at temperature above the eutectic point of salt solution. The salt concentration is caused by the phase transition of water. But, quantitative relationship between the phase transition of water and the decrease in MB-Ca^<2+>-ATPase activity had been investigated and clarified. To predict and control the denaturation of protein which participate in the management of fish preservation, the relationship between the cryohydration and effect of salt concentration on MB-Ca^<2+>-ATPase activity during freezing in the KCl-NaCl complex solution was examined thermodynamically. The salt concentration of KCl-NaCl with 300mM-300mM in the beginning increased to 564mM-2936mM near the eutectic point. Tilapia MB Ca^<2+>-ATPase activity decreased during reaction in high salt concentrations of prepared KCl-NaCl complex solutions at 25℃. The difference between the experimental heat quantity of solidification in the solution and the calculated one was in the range of 3%. It was considered that there was no effect of dilute MB (protein concentration, 0.37mg/ml) on the heat quantity, and MB-KCl-NaCl complex solution system was treated as the KCl-NaCl complex solution system, because the MB-KCl-NaCl complex solution had the same freezing temperature as the KCl-NaCl complex solution without MB. It was found that the changes of Gibbs free energy (ΔG) for the Ca^<2+>-ATPase reaction system was correlated to ones calculated from the heat of solidification for the KCl-NaCl complex solution.

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Two-cell embryos were vitrified after 30-sec exposure to DP solution (2.75M DMSO and 2.75M propylene glycol in PB1) supplemented with 1.0M sucrose (DPS). They exhibited significantly higher in vitro survival rate (82%) than those vitrified with DP (44%). Similar high survival rate (81%) was obtained when they were vitrified with DP plus 0.16M raffinose (DPR). In vivo survival of 2-cell embryos vitrified after 30-sec exposure to DPS or DPR was both 49%, and there was no significant difference as compared with the unvitrified control group (60%). High in vitro survival rates (80-96%) were also obtained with embryos at 1-cell and morula stages vitrified after 30-sec exposure to DPS. In vivo survival rates of embryos at the 1-cell, morula and blastocyst stages were 56, 53 and 40%, respectively, when vitrified after 15-sec exposure to DPS. The results indicate that the vitrification procedure can be used for the cryopreservation of mouse preimplantation embryos at 1-cell, 2-cell and morula stages.

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Transgenic mice have been widely utilized as a tool to evaluate the function of isolated gene in vivo. In order to develop the method of cryopreserving such genetically engineered mice, 2-cell embryos from transgenic mice expressing mouse OSF-1 (osteo-specific factor-1) cDNA were vitrified. Upon warming, 90.7% (107/118) were nomally recovered. When they were transferred to recipients, 45.8% developed to term. When the integrated pattern of transgene into the host genome and its expression level were compared between offspring derived from vitrified and those from unvitrified (control) transgenic embryos, there was neither significant alteration in the transgene integration pattern nor in the transgene expression level. These indicate that the vitrification-mediated cryopreservation does not only affect the expression level of transgene but also its integrated pattern in chromosomal DNA. By employing this technology, it will be possible to stock as many as embryos from transgenic lines without appreciable loss of transgene and its function.

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A kind of cyanobacteria in a spiral trichome shape, Spirulina platensis was fragmentated by mechanical agitation, sonication, freeze-thawing or freeze-drying. Mechanical agitation fragmentated trichomes, the size of which decreased with increasing the cutting power. Since the optimum size of trichome for growth (2-6pitch length) was obtained by mechanical agitation, destruction by this mode can be useful to increase the efficiency for the mass production of Spirulina sp. In the case of sonication, cells were destructed completely. It was indicated that can be used for biotechnological production of fine chemicals from Spirulina sp. Freeze-thawing and freeze-drying methods caused an injury to spiral trichomes and cells, and the fragmentation was easily done. We also examined the utility of these cells as competent cells for gene transfer and the possibility of the ^<31>P NMR measurment of intracellular ATP concentration, by the use of fragmentated cells.

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Screening of ice nucleating substances was carried out by using a differential scanning calorimetry (DSC). A carbon replica of ice crystals, which might have an ice structure in memory, did not show the activity of ice nucleation. It was found, in this study, that the spider's web has such activity, the degree of which seems to be almost the same as that of AgI. Moreover, the activity disappeared by the heat treatment. Although it is not certain, the result suggests the involvement of proteineous structures in the activity.

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