Though the bread making method using frozen dough after primary fermentation is common in these days. The frozen dough after secondary fermentation cannot be used for bread making method because it can cause reduced volume and poor texture of the baked bread. Addition of both raw soybean flour (RSF) and glycinebetaine (GB) resulted in formation of good dough even when the dough was frozen after secondary fermentation. Mechanical analysis by differential scanning calorimetry (DSC) indicated that addition of GB caused decrease in the amount of frozen water (free water) in the frozen dough. Results of SDS-PAGE analysis indicated that globulin proteins remained underrated in the mixed dough.

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The biological ice crystal modifiers, antifreeze proteins (AFPs), can bind onto the surface of ice crystals to alter their morphology and arrest their growth. These activities of ordinary AFPs have been evaluated using two concentration-dependent parameters, thermal hysteresis (TH) and ice recrystallization inhibition (IRI). However, it has not been subjected a parameter to correlate TH and IRI, which helps understanding of the icebinding ability of AFPs including defective species that cannot perfectly arrest the ice crystal growth. Here we examined a new parameter named “critical ice shaping concentration (CISC)”, a minimum concentration of an AFP species necessary to alter the shape of the ice crystals. We evaluated the CISC for three different types of fish AFPs including a defective AFP isoform, and examined the correlation between CISC and IRI. The obtained results showed for the first time that effective IRI concentrations of these AFPs are approximately double of their CISCs.

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To cryopreserve cells, it is important to prevent intracellular ice formation (IIF) during cooling and warming. One way to prevent IIF is to subject cells to procedures that convert cell water into a non-crystalline glass. Current belief is that to achieve this vitrification, cells must be cooled at high rates to prevent IIF during cooling and cells must be suspended in very high concentrations of cryoprotectants. We report here that these beliefs are incorrect with respect to the vitrification of 8-cell mouse embryos. We emphasize that it is important to prevent IIF (recrystallization) during warming by rapid warming. In this study, 8-cell embryos are vitrified in several dilutions of EAFS10/10 using various cooling rates and warming rates. Survival was assessed by their developmental ability to blastocysts. With a warming rate of 117,500oC/min, the percentages of embryos vitrified in 1Å~, 0.75Å~, and 0.5Å~ EAFS that developed to blastocysts were 93%, 92%, and 83%, respectively. Even when the solute concentration of the EAFS was reduced to 33% of normal, we obtained 40% functional survival of these 8-cell embryos.

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Cryopreservation is one of the essential techniques in various fields including medical and food engineering. However, it is well known that occurrence of intracellular freezing via extracellular freezing leads to the death of these cells. Cryopreservation in plants having a high water content is considered to be difficult. The authors focused on the above freezing behavior of cell that causes nearly 0% of cell’s survival rate. A new cryopreservation technique was then tested experimentally by subjecting plant tissues to an electric current during freezing. It was found that the use of electric current resulted in a finer grain size and lower growth rate of intracellular ice crystal as well as the suppression of intracellular pH (pHi)-decline as the cooling progressed. On the basis of the above results, transitions in vacuolar pH were examined at different cooling rates both with and without applying an electric current to understand the cause of this suppression in pHi-decline. It was found that as the cooling progressed, the electric current could also cause a suppression in the increase of vacuolar pH as that observed for pHi-decline.

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Microfluidic technologies have enabled cell culture and various assays for medical diagnosis, drug-screening in a small space or in an in vivo-mimicking manner. Cryopreservation of cells on such microfluidic devices that enables long-term shipping and storage is still challenging but is important in realizing stable storage and supply of the devices. We developed a versatile microfluidic channel which simplified the whole cell cryopreservation processes from freezing to restarting culture therein. The polydimethylsiloxane (PDMS)-made microchannel has two wells with smooth walls, and the flow rates can be switched using either Laplace pressure- or hydrostatic pressure-driven flow. The microchannel has two wells: one is used to collect cells to be cryopreserved; another used to wash the cryopreservation medium around thawed cells followed by restarting cultivation. We confirmed that cells introduced into the microchannel were successfully collected at the well bottom using Laplace pressure-driven flow, whereas replacement of cryopreservation medium to growth medium after thawing and transferring the cells to another well for restart culture were simultaneously done using hydrostatic pressure. We also confirmed the viability of restarted culture of COS-7 cells cryopreserved in the microfluidic channel. The cells became near confluent three days after thawing the microfluidic device.

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Compared to plants, response to cold stress in animals is regarded as less importance. However, also in animals, peripheral tissues like skin are sensitive to cold and induce shiver. In the previous study, we revealed that intracellular calcium ion uptake and mitochondrial reactive oxygen species (ROS) increase were occurred in low temperature (4°C) incubation with human keratinocyte HaCaT cells. In this study, we focused on mitochondria damage resulted from exposure to low temperature. Upon incubating HaCaT cells at 4°C, mitochondria co-localized to lysosomes. Also, a number of mitochondria seemed to be decreased, suggesting that lysosomes degraded mitochondria in autophagy process. We furthermore estimated the involvement of autophagy on HaCaT cells and murine embryonic fibroblast cells with autophagy marker protein LC3 and p62, and both markers significantly altered the protein level during low temperature incubation, presuming that autophagic process was activated during exposure to low temperature.

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The objective of this study is to establish a cryopreservation protocol for shoot apices of hybrid aspen (Populus tremula Å~ P. alba). Cryopreservation was carried out using a vitrification method on shoot apices excised from in vitro cultures. Excised shoot apices were precultured at 23oC for 2 days on hormone-free 1/2 MS medium with 0.4 M sucrose, then treated with liquid 1/2 MS medium supplemented with 0.4 M sucrose and 2.0 M glycerol (a loading solution) at 23oC for 30 min. Then shoot tips were treated with Plant Vitrification Solution 2 (PVS2) or PVS3 at 4oC for 90 min and plunged into liquid nitrogen for 1 hour. Following cryopreservation, they were rewarmed at 42oC for 2 min and washed in liquid 1/2 MS medium supplemented with 1.2 M sucrose at 23oC for 30 min. Survival rate was about 80% when shoot tips were treated with PVS2 for 90 min, however, regrowth rate after 3 weeks was about 20%.

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Group3 late embryogenesis abundant (G3LEA) proteins have the ability to protect proteins/enzymes and liposome from desiccation stress. They commonly have characteristic 11-mer repeat motif. In our previous studies, we synthesized short model peptides with two or four tandem repeats of such an 11-mer motif and demonstrated that they exhibit the protective activity similar to those of the native G3LEA proteins. However, the underlying mechanism for such protective functions remains unclear. In this study, to elucidate the mechanism at atomic level, we perform coarse-grained molecular dynamics simulations for a lysozyme-G3LEA peptide mixed system. It is shown that the G3LEA peptide molecules enter the interfacial space between two lysozyme molecules in the dry state and thus act as a barrier to avoid the direct contact between them.

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It is known that group 3 late embryogenesis abundant (G3LEA) proteins have the ability to protect membrane and proteins from desiccation stress. G3LEA proteins commonly have characteristic 11-mer repeat motif. In our previous studies using short model peptides for G3LEA proteins, we had already demonstrated that the 11-mer motif forms the core functional site of those proteins. However, the function of parts of non-repeat sequences remains unclear. Here we investigated the structure and function of the non-repeat regions using their 22-mer model peptides. The secondary structure of the peptides in solution and dry states were investigated by means of CD and FTIR spectroscopy. In addition, to test whether these peptides are able to protect liposome against drying-rehydration stress, we conducted particle size distribution measurements and fluorescence measurements. In conclusion, the non-repeat regions studied here take mainly β-sheet structure in the dry state, and have no apparent ability to protect liposome from the desiccation stress.

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The low energy vibrational dynamics of dry hen egg-white lysozyme has been investigated by the Terahertz Time-Domain Spectroscopy in the temperature range between 295 and 13 K. The low energy broad peak was clearly observed at about 0.65 THz and at 13 K in lysozyme. This broad peak is very similar to a boson peak, which has been observed in various kinds of glassy materials as the excess part of vibrational density of states.

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We developed a novel, well-controlled slow-freezing concentration-based strategy for effective protein delivery using polyampholyte NPs in the absence of a cryoprotectant. Here, we demonstrate the feasibility of this freeze concentration method, which is exceptionally favorable for cytoplasmic protein delivery, and focus on a simple slow freeze-thaw method using slow freezing program. We used confocal laser scanning microscopy to investigate the adsorption and internalization of lysozyme proteins at variable freezing temperatures. Quantification of fluorescence intensity revealed that proteins were more easily internalized by gradually decreasing the temperature. We believe that application of this concentration-based freezing technique could be extended to gene therapy and cancer immunotherapy.

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Although the commercial lactic acid bacterium is often preserved in a freezing state, the viable rate of the lactic acid bacterium is reduced largely after thawing. In this study, to understand the physical damages caused by ice crystallization during freezing for the lactic acid bacterium suspensions, the suspensions were frozen with three different freezing rate to be observed by the scanning electron microscope (SEM). Furthermore, it is considered that some freezing damages of the lactic acid bacterium would be caused by osmotic pressure alteration due to freezing concentration. So, to prove the presume, the effect of osmotic pressure alteration at room temperature for the damage of the lactic acid bacterium was observed by direct observation with the SEM. As the result, aggregated and compressed lactic acid bacterium between extracellular large ice crystals were observed in the samples which were frozen at slow and medium freezing rate. On the other hand, they could not be observed in the frozen suspension that was prepared at rapid freezing rate. Furthermore, a large step change in osmotic pressure of the medium solution for bacterium suspension, which is considered as a factor of freezing damage, caused shrinking of the configurations of the lactic acid bacterium.

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