Dehydration is one of the key steps for protein folding. While theoretical analyses suggest that the large decrease in the conformational entropy is associated with protein folding, the folding experiments show rather small decrease in the entropy, which allows us to speculate that release of hydrated water molecules from the peptide entropically contributes to the protein folding. However, no experimental data have reported on the dehydration associated with protein folding. Here, we focused on the volume changes in protein folding to discuss the dehydration and determined the volume changes associated with protein folding of reduced cytochrome c (Cyt c) from the unfolded state to the native state. The equilibrium constant between the unfolded state and native state (K_UN) was determined by a change in the absorption (420 nm) at various pressures between 0.1 and 200 MPa and at various concentrations of denaturant (guanidine hydrochloride) between 3.2 and 4.0 M. Dependence of K_UN on these factors revealed that the volume change at ambient pressure in the absence of denaturant is negative (ΔV_UN = -25(±10) cm³·mol^-1). We also followed pressure dependence of the folding rate (k) to determine the activation volume (ΔV^‡) for the process from the collapsed state (the initial intermediate for the protein folding) to the native state, by using the photo-induced protein folding reaction. The folding rate was followed by a change in the absorption (420 nm) at various pressures between 0.1 and 200 MPa and at various concentrations of the denaturant between 3.2 and 4.0 M. ΔV^‡ at ambient pressure in the absence of denaturant is also negative (-14(±8) cm³·mol^-1) and comparable to ΔV_UN. Such negative volumes can be accounted for by a decrease in volume resulting from the dehydration of hydrophobic groups, primarily the heme group, and the dehydration is mainly induced in the process from the collapsed state to the native state. The present data clearly indicates that dehydration, which increases the entropy of the protein system, compensates for a decrease in the conformational entropy accompanying the formation of the more compact and ordered transition state. We, therefore, propose that the rather small change in the entropy for the folding reaction of Cyt c is due to the dehydration of hydrophobic groups, and dehydration entropically promotes the protein folding reaction.

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Pressure shifts the equilibrium distribution of conformers of a protein through volume differences. Here, we examined the dynamics of ubiquitin and ubiquitin-like proteins by utilizing variable pressure NMR spectroscopy. Ubiquitin is a highly conserved 76 amino acid residue protein, whose sequence is extremely well conserved in all eukaryote. The same topology of folding frequently occurs in proteins, which are named ubiquitin-like proteins (ubl), despite the fact that the sequence identity among the ubl is rather poor. Further investigation of common structural motifs in a wider conformational state may provide additional information why proteins conserve similar topology. Thus by using variable pressure NMR techniques, we explored structures of ubiqitin and the two ubl, NEDD8, and SUMO-2, in a wide conformational space, namely in their energy landscape. We found similar conformational fluctuations among the ubl in the evolutionary conserved enzyme-binding region of the ubl, namely ubiquitin, NEDD8 and SUMO-2, indicating a conserved structural and thermodynamic design for their function.

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Intrinsically unfolded hen lysozyme disulfide-deficient variant spontaneously forms amyloid-like fibrils. Its early assembly reversibly dissociates under high hydrostatic pressure with a partial molar volume decrease of 100 mL per monomeric unit. The partial specific volumes of the monomeric and protofibrillar states are 0.684 and 0.724 mL g^-1, respectively, and the adiabatic compressibility coefficient of these states are -7.48 and 1.35 Mbar^-1, which indicates that the protofibrillar state is highly voluminous and compressible. Pressure accelerates the dissociation of protofibrils with a negative activation volume of -50 ml mol^-1 and a negative activation compressibility of -0.013 ml mol^-1 bar^-1, which suggests that partial hydration of existing voids takes place in the transition state of dissociation reaction.

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Deep-sea fishes, Coryphaenoides armatus and C. yaquinae, have three unique amino acid substitutions, V54 or L67P, Q137K and A155S, in comparison with non-deep sea fish, C. acrolepis. The V54A or L67P substitution plays an important role in actin polymerization under high pressures. The myosin heavy chain from deep-sea fishes has one amino acid deletion in the loop-2 region, which is one of actin-binding sites. However, the role of this deletion has remained unclear.

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Three-dimensional structures of glucose isomerase molecules in crystals prepared from supersaturated solutions in capillaries under 0.1 and 100 MPa were determined by X-ray crystallography under 0.1 MPa. With accurate temperature control, we obtained a few large crystals of good quality in the capillaries under 0.1 and 100 MPa. X-ray structures with high quality data sets were successfully obtained without removing solution around the crystals. We also determined a relation between resolution of molecular structure and size of the crystals surrounded by the solution in the capillaries. However, the r.m.s. deviation of least square fit of all atom positions of both glucose isomerase folds was about 0.05Å. This deviation was little. The distributions of water sites in both glucose isomerase structures were almost identical.

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The application of hydrostatic pressure is a well-known method for studying protein dynamics and hydration. The aims of this review are to provide a brief introduction to the thermodynamic principles of the effects of pressure on proteins and to extend such principles to the stability of proteins in solution. Finally, we refer to the significance of water for living organisms from a thermodynamic perspective.

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Studies of pressure-adapted (piezophilic) protein have lagged behind the investigation of other extremophilic proteins, however the recent characterization of proteins from deep-sea organisms has substantially accelerated the field. For example studies on piezophilic bacterium Shewanella violacea strain DSS12, which was isolated from a sediment sample collected at the Ryukyu Trench (depth: 5,110 m), has elucidated the molecular basis for gene and protein regulation at different pressure conditions. Recent experiments on dihydrofolate reductases, isopropylmalate dehydrogenases, and RNA polymerase subunits from the strain DSS12 have contributed to our understanding of protein adaptation to high pressure. These studies have also complemented previous work that had investigated the effect of pressure on the activity and stability of “normal”, unadapted proteins. Together this research has lead to the conclusion that volume changes due to the hydration effects of exposed side chains and large internal cavities drive protein unfolding under high pressure. The tight packing of internal hydrophobic core and the replacement of surface loops with β-structures have also been identified as major structural strategies that confer high-pressure adaptation to piezophilic proteins by reducing their compressibility.

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Lactate dehydrogenase (L-lactate: NAD oxidoreductase, EC 1.1.1.27; LDH) is one of the important enzymes in the glycolytic reaction cascade for its metabolic significance in catalyzing the terminating step. Cell-free extracts able to catalyze the oxidation of lactate to pyruvate were first obtained in 1932 (Banga et al., 1932). Warburg and Christian (1936) later associated the reaction with NAD , and the enzyme was first purified from bovine heart muscle (Straub, 1940). Since then, LDH has been extensively studied in vertebrates, plants and bacteria (Loewus and Stafford, 1960; Dennis and Kaplan, 1960; Biellmann and Rosenheimer, 1973). The direction of the reversible reaction depends on tissue-specific physiological conditions, such as the NAD/NADH ratio and the oxygen partial pressure. In most vertebrates, the LDH molecule forms a tetrameric structure (Markert and Moller, 1959) and two separate loci which encode A and B subunits constructing 5 tetrameric isozymes have been found (Prochazka and Wachsmuth, 1972). In cyclostomata, E. japonica has been shown to have a single subunit, while hagfishes have 2 subunits, LDH-A and LDH-B. Thus, hagfish LDH-B is the most primitive LDH-B ever examined. To examine the relationship of hagfish LDHs to lampreys and other vertebrate LDHs, we determined the cDNA sequences of LDH-A from 3 hagfishes and compared them to previously published sequences. The effect of high hydrostatic pressure on LDH activity was examined under pressures from 0.1 to 100 MPa. These results suggest that LDH-A₄ of E. okinoseanus is more adapted to high-pressure conditions than others.

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Thermodynamic quantities of phase transitions between bilayer and nonbilayer for phospholipids, phosphatidylcholines with saturated acyl chains (C18:0-PC, O-C18:0-PC) and phosphatidylethanolamines with unsaturated acyl chains (C18:1-PE(cis), C18:1-PE(trans)), were determined by means of differential scanning calorimetry under ambient pressure and light-transmittance measurements under high pressure. The thermodynamic quantities of the nonbilayer formations were much smaller than those of the transition between bilayers (gel-liquid crystal or hydrated crystal-liquid crystal transition) for the corresponding phospholipids. Although the nonbilayer formations correspond to a dynamic transformation between lamellar structure and nonlamellar structure such as interdigitated or inverted hexagonal structure, we can say that the order of the lipid molecule in both structures may not appreciably change judging from the smaller thermodynamic quantities. A notable feature of the bilayer-nonbilayer transitions is the large pressure dependence of the transition temperature as compared with that of the bilayer-bilayer transitions. This fact means that the transformation between bilayer and nonbilayer structures is remarkably influenced by pressure. Comparing the enthalpy and volume changes of the bilayer-nonbilayer transitions with those of the bilayer-bilayer transitions, we concluded that the former transitions can be regarded as volume-controlled transitions for the reconstruction of molecular packing.

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Thermotropic and barotropic phase behavior of the dinonadecanoylphosphatidylcholine (diC19-PC) bilayer membrane was investigated by use of differential scanning calorimetry and high-pressure light-transmittance technique. The constructed temperature (T)-pressure (P) phase diagram showed the polymorphism of the gel phase (i.e., lamellar gel, ripple gel and interdigitated gel phases) which is one of the common features to symmetric saturated diacyl-PC bilayer membranes. The pre- and main-transition temperatures and the critical interdigitation pressure (CIP) of the diC19-PC bilayer membrane were revealed to be 57.5 °C, 60.6 °C and 37.7 MPa, respectively, which were in good agreement with the values expected from the acyl chain-length dependence of those quantities based on the previous results for bilayer membranes of a homologous series of diacyl-PCs with shorter acyl chains. On the other hand, the enthalpy (ΔH) and volume (ΔV) changes of the main transition for the diC19-PC bilayer membrane were significantly larger than those expected from the acyl chain-length dependence. This may indicate that there are some intrinsic difference in molecular packing between bilayer membranes of diC19- and other diacyl-PCs with shorter acyl chains, and that the diC19-PC bilayer forms a more condensed gel phase than the latter bilayers.

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The bilayer phase transitions of saturated ether-linked phospholipids with different-sized polar head groups, dihexadecylphosphatidylethanolamine (DHPE), dihexadecylphos- phatidyl-N,N-dimethylethanolamine (DHMe₂PE) and dihexadecylphosphatidylcholine (DHPC), were observed by means of differential scanning calorimetry (DSC) and high-pressure light-transmittance. The temperatures of so-called main transition from the lamellar gel (L_β) or ripple gel (P_β') phase to the liquid crystalline (L_α) phase were almost linearly elevated by applying pressure. The slope of the temperature-pressure boundary (dT/dP) was in the range of 0.232 - 0.244 K MPa^-1. The main transition temperatures of these lipid bilayers decreased with increasing size of head group. On the other hand, there was no significant difference in thermodynamic quantities of the main transition among these lipid bilayers. Bilayer membranes of DHPE and DHMe₂PE underwent only a main transition from the L_β phase to the L_α phase, whereas the DHPC bilayer exhibited two phase transitions: the pretransition from the interdigitated gel (L_βI) phase to the P_β' phase and sequentially the main transition from the P_β' phase to the L_α phase. In other words, the interdigitation of DHPE and DHMe₂PE bilayers was not observed. It is well known that the interdigitation of ester-linked phosphatidylcholine bilayer, for example dipalmitoylphosphatidylcholine (DPPC) bilayer, is induced by hydrostatic pressure. Present results revealed a necessarily important condition for bilayer interdititation: lipid molecules have to include in structure a bulky head group of choline rather than the ester- or ether-linkage. A probable mechanism for pressure-induced interdigitation of bilayer is proposed.

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Bacteria living in the deep-sea have several unusual characteristic that allow them to growth in their extreme environment. The only deep-sea piezophilic bacterial species of two of these genera were named Shewanella benthica and Colwellia hadaliensis prior to the reports by the JAMSTEC group. We have identified several novel piezophilic species within the γ-subgroup of the Proteobacteria according to phylogenetic classifications based on 16S ribosomal RNA sequence information. Numerous deep-sea piezophilic bacterial strains have been isolated and characterized in an effort to understand the interaction between the deep-sea environment and its microbial inhabitants. It has been understood that the temperature and pressure influence to growth is also different depending on the strain for piezophilic bacteria.

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In this study, the inhibitory effect under compressed hydrocarbon gases on yeast growth was investigated quantitatively by microcalorimetry. The growth thermograms (heat output - incubation time curve) were obtained during incubation of yeast under various compressed hydrocarbon gases. When the gas pressure increased, the curves of thermograms shifted to the right, indicating an obvious inhibitory action on the yeast growth. After quantification of growth inhibition at various pressures, we determined the 50% inhibitory pressure (IP₅₀) and the minimum inhibitory pressure (MIP) values as indices which represent toxic potency of each gas. The lower the IP₅₀ and MIP values, the greater the growth inhibitory effects of the gas. Based on these values, the inhibitory potency of the gases increased in the order: methane < ethane < propane < i-butane < n-butane. In addition, the toxicity of some hydrocarbon gases was found to be correlated to their hydrophobicity. These results suggest that hydrocarbon gases interact with some hydrophobic region of the cell membrane, resulting in modification of membrane structure and function. However, no evidence was obtained in the yeast treated with compressed hydrocarbon gases as to the membrane damage. Therefore, we investigated the effects of hydrocarbon gases on the leakage of 260 nm absorbing material from treated cells. The release amount of 260 nm absorbing material increased with increasing pressure. This indicates that hydrocarbon gases affect the plasma membrane, particularly the nuclear membrane. Furthermore, we examined the effects of hydrocarbon gases on the morphology of the yeasts by scanning electron microscopy. Scanning electron microscopy showed that yeasts when exposed to compressed hydrocarbon gases undergo a dramatic morphological change that includes invagination of the cell surface.

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Effect of Petit-high pressure carbon dioxide treatment was investigated for microorganisms in Lyceum Barbarum Fruit Juice harvested from China. The Juice was treated with carbon dioxide gas from mild to high pressure (from 3atm to 50atm) conditions, for 1day to 14 days. Colony Favoring Unit on Petri Dishs of LB, YPD, PDF agar medium were decreased to less than 10 in 50atm-1day condition, even in 3atm-14days condition. Surprisingly, similar effect was observed between Petit-high pressure long time treatment (3atm-14days) and high-pressure short time treatment (50atm-1day). The pressure level on food distribution was restricted to be under 4atm. This data shows the possibility and reality of food distribution with mild-pressure carbon dioxide gas on the condition under 4atm.

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Microbial spoilage, including fermentation and oxidation is particularly an important factor that influences the quality of foods. In previous studies we showed that sudachi (sour citrus fruit) juice could be sterilized with oxygen gas pressurization at 10 MPa and 50°C without loss of its flavor and color. Furthermore, we reported a method to remove the dissolved oxygen in sudachi juice with nitrogen gas pressurization. However, all these results were obtained using a small-scale batch process (500 ml or less in capacity). In this research, we designed a new commercial scale food processing apparatus for the sudachi juice based on the above findings. This device consists of two parts: a sterilization part with oxygen gas pressurization and a dissolved oxygen removal part with nitrogen gas pressurization, and it has a big advantage that a large amount of sudachi juice can be treated continuously at the flow rates up to 40 L/h. Using this apparatus, the sudachi juice was sterilized under similar operation conditions (50°C and 10 MPa for 1 minute) to those of the small-scale batch system. Although the performance of dissolved oxygen removal and the quality conservation of the juice (such as flavor and color) is under investigation now, the facility and production costs are expected to be apparently less than those of high-pressure processing (liquid pressure). Because, our treatments can be completed in a short time at relatively low pressures using “oxygen-nitrogen hybrid pressurization system”. We consider further improvements of the apparatus and hope that the practical application of the method can be realized in food industry in the near future.

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Global transcriptional profiles of the yeast Saccharomyces cerevisiae were studied following changes in growth conditions to high hydrostatic pressure (25 MPa, 24 °C) and low temperature (0.1 MPa, 15 °C). These profiles were quantitatively very similar. Particularly, expression of the DAN/TIR cell wall mannoprotein genes, which are generally expressed under hypoxia, were markedly upregulated by high pressure and low temperature, suggesting the overlapping regulatory networks of transcription. In support of the role of the mannoproteins in cell wall integrity, cells acquired resistance against treatment with low concentrations of SDS and Zymolyase, and lethal level of high pressure (125 MPa) when cells were preincubated under high pressure and low temperature.

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Metabolomics is a newly emerging field of ‘omics’ research that is used for detection of small molecules or metabolites in biological fluids such as amino acids, sugars, and organic acids. Capillary electrophoresis-mass spectrometry (CE-MS) is one of the most promising approaches for comprehensive and quantitative analysis of the metabolites. The aim of the present study was to investigate the response to environmental stress using model organisms such as yeast (Saccharomyces cerevisiae) cells. CE-MS method was applied to the determination of qualitative and quantitative difference of the cationic metabolites. This paper shows that a stress response to high hydrostatic pressure (40 MPa, 16 hours) resulted in an increase in the concentration of many intracellular amino acids. Amino acid metabolism would be activated in the stress response in the yeast cells. Furthermore, it was particularly noticeable that a series of hydrophobic amino acids, such as valine, leucine, isoleucine, and phenylalanine, was increased. From the results of gene expression, the yeast cells had been damaged by the pressure treatment. Therefore, we speculated that the amino acid increases were observed caused by a decomposition product of proteins.

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The research field of high pressure processed food technology was spread from basic science to commercial basis progress. Thus, this technology is being accepted by consumer and is now on the stage of standardization. Standardization is required for the stable and safety supply of high pressure processed food to consumer. In this paper, we would like to suggest the draft version of “Technical Report” concern to indicative standard microorganisms using yeast, Saccharomyces cerevisiae. We selected the strain of S288C, medium of YPD, and cultivation time of 50-150h at 25°C. The strain was selected as the genome sequence of this strain was already analyzed and thus the most characterized strain in the yeast strains. YPD medium is widely used by basic scientist and applied scientist. The cultivation time was examined by counting CFU after and before pressure treatment and we found yeast cells during this growth phase showed the reproducible high pressure resistance. Next step is the validation studies of this “Technical Report” in different laboratories. We would like to call for participation in the validation studies to laboratories.

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Previously we showed the breakdown of mitochondrial membrane potential, cytochrome c release, caspase-3 activation, and DNA ladder in murine erythroleukemia (MEL) cells exposed to a pressure of 100 MPa. Here, to characterize pressure-induced apoptosis, the pathway of caspase-3 activation, morphological changes of the cells, and the spin-lattice relaxation time (T₁) of intracellular water were examined using 100 MPa-treated MEL cells. The results from caspase-inhibitors showed that both pathways via caspase-8 and mitochondria were involved in caspase-3 activation. Upon culture of pressure-treated cells, the plane surface of the membrane was observed using a scanning electron microscope. From the T₁ measurement, it was found that the efflux of intracellular water occurs in early stage of apoptosis, whereas the influx of water in its late stage.

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We investigated the characteristics of a decellularized porcine cornea by ultra-high hydrostatical pressurization (UHP) method. The UHP method consists of the disruption of cells by hydrostatical pressurization and the removal of components of the disrupted cells by washing process. Porcine cornea were hydrostatically pressed at 10,000 atmospheres and 10 °C for 10 min and immersed in medium for 72 hours. The turbid cornea was obtained. For H-E staining of the cornea decellularized with the UHP method, the complete removal of corneal cells and maintenance of the superstructure of collagen fibrils were confirmed. When the corneas were immersed in glycerol for 1 hour, their optical and mechanical properties were restored to those of a natural cornea. As the preliminary animal study, when the implantation of the acellular porcine cornea to rabbit cornea was carried out, the immune reaction was not occurred and the turbid cornea became clear. These results indicate that the decellularized cornea by UHP method would be useful as corneal scaffold for regeneration. These results indicate the possibility of the acellular cornea prepared by the UHP method as artificial bio-cornea.

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The digestibility of nonpressurized bovine whey protein isolate (WPI) and pressure-induced WPI gel and the effect of different pressures (200-600MPa) on digestibility were evaluated by measuring digestive rate in simulated astrointestinal digestion system and SDS-PAGE. The results showed that the digestibility of high pressure induced WPI gel significantly increased compared with that of untreated WPI after digesting by pepsin and trypsin, which were 72.59% and 61.02% respectively. When the pressure was over 400MPa, the digestibility of high pressure-induced WPI gel showed no significant change(ANOVA, p<0.05). Although the b-lactoglobulin(β-Lg) from untreated WPI sample was not easy to be digested by pepsin, β-Lg from the high pressure-induced gel (400MPa) was digested partly. Furthermore, when the pressure was up to 600MPa, β-Lg from the high pressure-induced gel was digested almost completely. In addition, the peptides with molecular masses of 3500-6500 Da were formed after pepsin digestion.

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Effects of high pressure treatment on the structure of connectin, one of the elastic protein of muscle were investigated by spectrometric analyses. Circular dichroism spectra show abundance of β-structure in the secondary structure of connectin, whereas α-helix content was less than 10%. No significant change in secondary structure was observed in the connectin pressurized up to 400 MPa, but significant decrease of β-sheet and increase of turn were observed in the connectin pressurized at 600 MPa. From the measurements of fluorescent spectra and center of spectral mass, it seems that the changes in the tertiary structure of connectin induced by relatively mild pressure of 100~200MPa were reversible, but the changes become irreversible with more higher pressure applied.

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Myosin molecules are associated into filament at physiological condition such as 0.1 M NaCl. Since myosin filaments form a gel by application of hydrostatic pressure above 200 MPa without heating, the possibility of the meat processing by the pressure treatment is suggested. Both of heat- or pressure-induced filamentous myosin gels showed similar internal structure; namely, the gels were composed of a fine-strand network, whereas the elasticities of those two gels were different. The aim of this study is to clarify the relation between morphological and rheological properties of filamentous myosin gels using atomic force microscope (AFM). The heat- or pressure-treated myosin filament was investigated by AFM in 0.1 M NaCl without chemical fixation. The thermal- and pressure-induced strands, which were formed from denatured filaments, became knobby with elevating temperature and pressure. The strands were formed by side-by-side association of several filaments above 55 °C and 300 MPa. There was also no significant morphological difference between thermal and pressure-induced strands. The elasticities of strands were also investigated using an AFM. The elasticity of heat-induced strand showed maximal value (10.24±1.16 MPa) at 55 °C. On the other hand, the elasticity of pressure-induced strand increased with elevating pressure, and the maximal value was 9.80±0.84 MPa at 500 MPa. The elasticity of the whole gel corresponded with those of the strand. Myosin molecule consists of two globular heads (S1) attached to a tail (rod). The structure and elasticities of heat- and pressure-denatured subfragments were investigated using AFM. The heat- and pressure-denatured S1 showed similar aggregated structure, and the elasticities of denatured S1 aggregates increased with elevating application of temperature and pressure. On the other hands, the elasticity of heat-denatured rod filament showed maximal value at 55 °C, while that of pressure-denatured rod filament increased with elevating pressure. From these results, we conclude that the rheological characteristics of heat-denatured rod filaments determine the elasticity of heat-induced filamentous myosin gel, whereas that of pressure-denatured head and tail affects the rheological properties of pressure-induced myosin filament gel.

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Ovomucoid (OVM) is a dominant allergen in chicken egg white. It is difficult to lower the antigenicity and allergenicity of OVM because of its stability to heat and chemical treatments. The effects of combined high-pressure/enzymatic treatment on the proteolysis, antigenicity and allergenicity of OVM were investigated. OVM (1.4 mg/ml) was pressurized to 100-600 MPa at 37°C for 30 min prior to pepsin or chymotrypsin hydrolysis, which was also conducted under high pressures (100-600 MPa). The peptide profile was analyzed by tricine sodium dodecyl sulfate polyacrylamide gel electrophoresis. The residual antigenicity was assayed by enzyme-linked immunosorbent assay, using rabbit antisera against OVM and sera from egg-allergic patients. The residual allergenicity was measured on the basis of the histamine released from KU812F human pre-basophilic cells sensitized with sera from egg-allergic patients. Chymotryptic digestion carried out under high pressures enhanced the hydrolysis of OVM and reduced its antigenicity and allergenicity. The greatest reduction of antigenicity and allergenicity was obtained at a pressure of 400 MPa. Combined high-pressure/enzymatic treatment could be useful for reducing the antigenicity of food proteins.

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The effect of high hydrostatic pressure (HHP) treatment on rooting ability of mung bean seeds was investigated. Water content of mung beans was adjusted by immersing the beans in water at 25°C for a specific time. The mung bean seeds (water content 7, 15, 25%) were treated with HHP (100-600MPa) at 25°C for 10min. Three sets of 100 seeds for each pressure condition were applied to the treatment and HHP-treated mung beans were incubated at 25°C for testing the rooting ability. The dry mung beans (water content, 7%) showed high sprouting ratio regardless of the applied pressure. As for mung beans of intermediate water content (15%), emergence of the root was delayed and the sprouting ratio was low. Furthermore, the mung bean seeds with higher water content (25%) showed very low emerged root ratio and no sprouting even at a relatively low pressure of 100 MPa. It was clarified that pressure-resistance of mung beans was significantly affected by water content. Higher water content gave lower sprouting ratio even at low pressures. For efficient microbial inactivation and spouting, the water content of mung beans should not be high.

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Water mixtures of potato and corn starches were prepared at starch contents of 10-70%w/w, and the mixtures were treated with high hydrostatic pressure (HHP) ranging from 200 to 1,200 MPa at 40°C for 1h. HHP-treated starch - water mixtures were analyzed by differential scanning calorimetry, and the enthalpy changes in gelatinization and re-gelatinization of retrograded starch were measured. Based on the enthalpy changes, the physically modified states of HHP-treated potato and corn starches were classified into five physical states: complete gelatinization, complete gelatinization with retrogradation, partial gelatinization, partial gelatinization with retrogradation, and thermodynamically no change. The classification was summarized into state diagrams for both starches.

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It has been known that bivalves can be shucked and the flesh can be detached after high hydrostatic pressure (HHP) treatment. In this regard, some patents have been issued. In Japan, one commercial scale shucking of oyster is realized by treating the bivalve at 80 MPa and 40°C. HHP shucking of oyster contributes to reducing the labor cost and the claims from consumers on the contaminated hard shell pieces. In the USA, HHP-shucked oysters receive a good reputation. However, applicability of HHP shucking to other bivalves has not been sufficiently studied and scientific publications of the relevant data have been quite limited. In this study, bivalves other than oyster were subjected to HHP treatment for shucking and flesh detaching.

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Food processing by ultra high pressure has been commercialized by leading companies in food industry. The Research Association of High Pressure Food Processing Technology in Tsukuba was established in 2004, and one of the aims of the association was to develop new equipments for high pressure food processing at low cost so that small-and-medium-sized enterprises (SMEs) can introduce the technology. Through the activity of the association, we have won a research project from the Ministry of Agriculture, Forestry, and Fisheries for developing a test model for high pressure food processing in collaboration with National Food Research Institute. As a result, we have completed a test model with a volume of 20L (250mmΦ x 420mm), a maximum pressure of 200 MPa, and a temperature range between ambient temperature and 60°C.

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