Shigeaki Ueno

Estimation of Damage to Cells of Japanese Radish Induced by High Pressure with Drying Rate as Index

The relative drying rate of samples (RDR), which is the ratio of the drying rate of pretreated samples to that of untreated ones, might be used as a tool to investigate the damage to cells of agroproducts induced by high-pressure treatment. Damage to cells induced by high pressure was estimated by comparing the RDR after high-pressure pretreatment with the RDR after chloroform-vapor, heat, and freeze-thaw pretreatments of Japanese radish samples. The RDR after high-pressure pretreatment was similar to the RDR after chloroform-vapor pretreatment, and was lower than for heat, and for freeze-thaw pretreatment. For agroproducts, high-pressure treatment is thus comparatively moderate.

Effects of High Hydrostatic Pressure on Distribution Dynamics of Free Amino Acids in Water Soaked Brown Rice Grain

High hydrostatic pressure (HP) with approximately below 400 MPa can induce a transformation of food materials to an alternative form, where membrane systems are damaged but certain enzymes are still active. HP treatment of water soaked brown rice grain could modify the mass transfer inside and apparent activities of enzymes, resulting in HP-dependent change of distribution of free amino acids. Thus, the distribution of free amino acids in brown rice grain during preservation after HP treatment was analyzed. Just after HP treatment at 200 MPa for 10 min, the distribution of free amino acids was not apparently different from that of untreated control. In contrast, after 1 to 4 days preservation at 25°C, amino acids, such as Ala, Glu, Gly, Asp and Val, showed higher concentrations than those in control. This result suggested that HP treatment induced proteolysis to produce free amino acids. However, Gln, Thr and Cys, showed no apparent difference, suggesting that conversion of certain amino acids produced by proteolysis occurred. Moreover, the concentration of γ-aminobutyric acid (GABA) in HP-treated sample was higher than that in untreated control. These results suggested that HP treatment induced alteration of distribution of free amino acids of rice grains via proteolysis and certain amino acids metabolism pathways.

Isolation and characterization of barosensitive mutants of Saccharomyces cerevisiae obtained by UV mutagenesis.

Using UV mutagenesis, 2 high-pressure (HP) sensitive (barosensitive) mutants of Saccharomyces cerevisiae were obtained. The HP inactivation of the mutants, as well as their parent strains, followed 1st-order kinetics in the range of 175 to 250 MPa within 600 s. Both mutants showed larger 1st-order inactivation rate constant values or significant loss of viabilities, compared with their parent strains in the pressure range tested. The inactivation rate constant value of one of the mutants was comparable with that of a previously reported highly barosensitive strain, which was generated by deletion of hsp104 in a trehalose deficient strain. The activation volume values of HP inactivation reactions in the 2 mutants were apparently equivalent with those of their parent strains. This suggested that the mutation did not bring drastic volume changes of the key molecules for HP inactivation. Their auxotrophic properties, growth, and ethanol fermentation were identical in mutant and parent strains. The mutants could therefore be useful for fermentations where control by HP processing is desired.

A culture‐dependent bacterial community structure analysis based on liquid cultivation and its application to a marine environment

A method for analyzing culture-dependent bacterial community structure by liquid cultivation was established using 96-well microplates. Using 96-well microplates, this method can easily provide accurate enumeration of viable microorganisms and simultaneous separation of bacteria, which allowed us to analyze the bacterial community. Bacteria in diluted surface seawater were separated using 96-well microplates and cultivated with 1/5 ZoBell 2216E liquid medium. The 98 cultures obtained were subsequently applied to phylogenetic analysis based on 16S rRNA gene sequences. The bacterial diversity, evaluated by the Shannon-Weaver index, was relatively small but comparable to previously reported bacterial communities of several environments. The most abundant group was the family Rhodobacteraceae, which has been frequently detected in marine environments. Most bacteria were phylogenetically related to bacteria or uncultured clones detected in marine environments, but distant from published species. The analysis of bacterial community structure by liquid cultivation would be useful as an alternative culture-dependent approach.

Formation of green-blue compounds in Brassica rapa root by high pressure processing and subsequent storage.

The effect of high pressure treatment on biochemical changes during storage was investigated using Brassica rapa root. High pressure treated samples with 400 and 600 MPa formed unique green-blue color during 7-d storage at 4 °C. The mechanism of green-blue compound formation would be based on biochemical pathway for a unique green-blue pigment synthesis, containing O2-dependent steps and possibly enzymatic reactions.

Kinetic Analysis of E. coli Inactivation by High Hydrostatic Pressure with Salts

Inactivation of E. coli by high hydrostatic pressure (250 to 400 MPa) with salts was investigated based on kinetic analysis. At concentrations from 0.074 to 0.145 M and from 0.240 to 0.290 M, both the absolute activation volumes and the preexponential factors were similar in KCl, NaCl, and LiCl solutions, suggesting that pressure inactivation is not salt-specific. On the other hand, in the intermediate salt-concentration range of 0.145 to 0.240 M, inactivation kinetics in the presence of the Na(+) and K(+) differed significantly from those in the presence of Li(+) (P < 0.05). In this concentration range, effect of salt stress and osmotic stress differed significantly from those in concentrations below 0.145 M or above 0.240 M. The cellular response to pressure varies with salt type and salt concentration. These novel findings provide important clues to distinguish between salt stress and osmotic stress in the inactivation of E. coli.

High-throughput method for a kinetics analysis of the high-pressure inactivation of microorganisms using microplates

Using microplates as pressure and cultivation vessels, a high-throughput method was developed for analyzing the high-pressure inactivation kinetics of microorganisms. The loss of viability from a high-pressure treatment, measured based on the growth delay during microplate cultivation, showed reproducibility with the conventional agar plate method and was applicable for the kinetics analysis.

Comparative analyses of viable bacterial counts in foods and seawater under microplate based liquid- and conventional agar plate cultivation: increased culturability of marine bacteria under liquid cultivation.

Bacterial counts under liquid cultivation using 96-well microplates were performed. The counts under liquid and under solid cultivation were equivalent in foods, although the counts under liquid cultivation exceeded those under solid cultivation in seawater, suggesting that some bacteria in seawater were viable but did not form detectable colonies. Phylogenetic analysis of bacteria obtained under liquid cultivation was also performed.

Enzymatic production of γ-aminobutyric acid in soybeans using high hydrostatic pressure and precursor feeding.

The effects were investigated of the glutamic acid (Glu) substrate concentration on the generation and kinetics of γ-aminobutyric acid (GABA) in soybeans treated under high hydrostatic pressure (HHP; 200 MPa for 10 min at 25 °C). The conversion of Glu to GABA decreased with increasing initial Glu concentration in the soybeans. The crude glutamate decarboxylase (GAD) obtained from the HHP-treated soybeans showed substrate inhibition. The GABA production rate in the HHP-treated soybeans fitted the following substrate inhibition kinetic equation: v0=(VmaxS0)/(Km+S0+(S0)2/Ki). The Km value for the HHP-treated soybeans was significantly higher than that of the untreated soybeans. The Km values in this study show the affinity between Glu and GAD, and indicate that the HHP-treated soybeans had lower affinity between Glu and GAD than the untreated soybeans. GAD extracted from the HHP-treated soybeans showed a similar value to that in the HHP-treated soybeans. The intact biochemical system was so damaged in the HHP-treated soybeans that it showed substrate inhibition kinetics similar to that of the extracted GAD. The combination of HHP and precursor feeding proved to be a novel tool that can be used to increase the concentration of a target component.

Effects of High Hydrostatic Pressure on Water Absorption of Adzuki Beans

The effect of high hydrostatic pressure (HHP) treatment on dried soybean, adzuki bean, and kintoki kidney bean, which are low-moisture-content cellular biological materials, was investigated from the viewpoint of water absorption. The samples were vacuum-packed with distilled water and pressurized at 200 MPa and 25 °C for 10 min. After the HHP treatment, time courses of the moisture contents of the samples were measured, and the dimensionless moisture contents were estimated. Water absorption in the case of soybean could be fitted well by a simple water diffusion model. High pressures were found to have negligible effects on water absorption into the cotyledon of soybean and kintoki kidney bean. A non-linear least square method based on the Weibull equation was applied for the adzuki beans, and the effective water diffusion coefficient was found to increase significantly from 8.6 × 10−13 to 6.7 × 10−10 m2/s after HHP treatment. Approximately 30% of the testa of the adzuki bean was damaged upon HHP treatment, which was comparable to the surface area of the testa in the partially peeled adzuki bean sample. Thus, HHP was confirmed to promote mass transfer to the cotyledon of legumes with a tight testa.