Importance of Intracellular Energy Status on High-Hydrostatic-Pressure Inactivation of sake Yeast Saccharomyces cerevisiae.

The HHP inactivation behaviors of Niigata sake yeast Saccharomyces cerevisiae strain S9arg and its aerobic respiratory-deficient mutant strains were investigated after cultivating them in a YPD media containing 2% to 15% glucose, as well as in moromi mash, in a laboratory-scale sake brewing process. The piezotolerance of strain S9arg, shown after cultivation in a YPD medium containing 2% glucose, decreased to become piezosensitive with increasing glucose concentrations in YPD media. In contrast, the piezosensitivity of a mutant strain UV1, shown after cultivation in the YPD medium containing 2% glucose, decreased to become piezotolerant with increasing glucose concentrations in the YPD medium. The intracellular ATP concentrations were analyzed for an S. cerevisiae strain with intact aerobic respiratory ability, as well as for strain UV1. The higher concentration of ATP after cultivation suggested a higher energy status and may be closely related to higher piezotolerance for the yeast strains. The decreased piezotolerance of strain S9arg observed after a laboratory-scale sake brewing test may be due to a lower energy status resulting from a high glucose concentration in moromi mash during the early period of brewing, as well as a lower aeration efficiency during the brewing process, compared with cultivation in a YPD medium containing 2% glucose.

Isolation and physiological properties of methanogenic archaea that degrade tetramethylammonium hydroxide.

Tetramethylammonium hydroxide (TMAH) is a known toxic chemical used in the photolithography process of semiconductor photoelectronic processes. Significant amounts of wastewater containing TMAH are discharged from electronic industries. It is therefore attractive to apply anaerobic treatment to industrial wastewater containing TMAH. In this study, a novel TMAH-degrading methanogenic archaeon was isolated from the granular sludge of a psychrophilic upflow anaerobic sludge blanket (UASB) reactor treating synthetic wastewater containing TMAH. Although the isolate (strain NY-STAYD) was phylogenetically related to Methanomethylovorans uponensis, it was the only isolated Methanomethylovorans strain capable of TMAH degradation. Strain NY-STAYD was capable of degrading methylamine compounds, similar to the previously isolated Methanomethylovorans spp. While the strain was able to grow at temperatures ranging from 15 to 37°C, the cell yield was higher at lower temperatures. The distribution of archaeal cells affiliated with the genus Methanomethylovorans in the original granular sludge was investigated by fluorescence in situ hybridization (FISH) using specific oligonucleotide probe targeting 16S rRNA. The results demonstrated that the TMAH-degrading cells associated with the genus Methanomethylovorans were not intermingled with other microorganisms but rather isolated on the granule's surface as a lone dominant archaeon. KEY POINTS: • A TMAH-degrading methanogenic Methanomethylovorans strain was isolated • This strain was the only known Methanomethylovorans isolate that can degrade TMAH • The highest cell yield of the isolate was obtained at psychrophilic conditions.

Genomic and Metabolomic Analyses of a Piezosensitive Mutant of Saccharomyces cerevisiae and Application for Generation of Piezosensitive Niigata-Sake Yeast Strains.

A sparkling-type draft cloudy sake (Japanese rice wine), AWANAMA, was recently developed using high hydrostatic pressure (HHP) treatment as a non-thermal pasteurization method. This prototype sake has a high potential market value, since it retains the fresh taste and flavor similar to draft sake while avoiding over-fermentation. From an economic point of view, a lower pressure level for HHP pasteurization is still required. In this study, we carried out a genome analysis of a pressure-sensitive (piezosensitive) mutant strain, a924E1, which was generated by UV mutagenesis from a laboratory haploid Saccharomyces cerevisiae strain, KA31a. This mutant strain had a deletion of the COX1 gene region in the mitochondrial DNA and had deficient aerobic respiration and mitochondrial functions. A metabolomic analysis revealed restricted flux in the TCA cycle of the strain. The results enabled us to use aerobic respiration deficiency as an indicator for screening a piezosensitive mutant. Thus, we generated piezosensitive mutants from a Niigata-sake yeast strain, S9arg, which produces high levels of ethyl caproate but does not produce urea and is consequently suitable for brewing a high-quality sake. The resultant piezosensitive mutants showed brewing characteristics similar to the S9arg strain. This study provides a screening method for generating a piezosensitive yeast mutant as well as insight on a new way of applying HHP pasteurization.

Comparative analysis on inactivation kinetics of between piezotolerant and piezosensitive mutant strains of Saccharomyces cerevisiae under combinations of high hydrostatic pressure and temperature.

We previously obtained a pressure-tolerant (piezotolerant) and a pressure sensitive (piezosensitive) mutant strain, under ambient temperature, from Saccharomyces cerevisiae strain KA31a. The inactivation kinetics of these mutants were analyzed at 150 to 250MPa with 4 to 40°C. By a multiple regression analysis, the pressure and temperature dependency of the inactivation rate constants k values of both mutants, as well as the parent strain KA31a, were well approximated with high correlation coefficients (0.92 to 0.95). For both mutants, as well as strain KA31a, the lowest k value was shown at a low pressure levels with around ambient temperature. The k value approximately increased with increase in pressure level, and with increase and decrease in temperature. The piezosensitive mutant strain a924E1 showed piezosensitivity at all pressure and temperature levels, compared with the parent strain KA31a. In contrast, the piezotolerant mutant strain a2568D8 showed piezotolerance at 4 to 20°C, but did not show significant piezotolerance at 40°C. These results of the variable influence of temperature on pressure inactivation of these strains would be important for better understanding of piezosensitive and piezotolerant mechanisms, as well as the pressure inactivation mechanism of S. cerevisiae.

Effect of high pressure on the saccharification of starch in the tuberous root of sweet potato (Ipomoea batatas).

We analyzed the effect of high hydrostatic pressure (HHP) treatment on reducing sugar production in the tuberous root of sweet potato (Ipomoea batatas), based on pressure-gelatinization of starch and subsequent saccharification by internal amylases. HHP treatment at up to 600MPa at ambient temperature for 10min did not apparently affect the reducing sugar concentration in tuberous root. However, HHP treatment at 100 to 500MPa and 60°C or 70°C for 10min increased reducing sugar concentration as both the pressure and temperature increased. The reducing sugar concentration after HHP treatment at 500MPa and 70°C for 10min was roughly comparable to that of the thermal treatment control (80°C for 10min under atmospheric pressure). HHP treatment enabled the gelatinization and enzymatic saccharification of starch in the tuberous root of sweet potato, at a lower temperature than required by thermal treatment at atmospheric pressure.

Use of Pressure Activation in Food Quality Improvement.

Beside intensive studies on inactivation microorganisms by high hydrostatic pressure (HP) for food storage, pressure effects on property of food materials have also been studied based on knowledge in pressure effect on biomolecules. Pressure effects on biological membranes and mass transfer in cellular biological materials and on enzyme activity would give an idea that HP treatment can introduce two types of activations into food materials: improved mass transfer and enzyme activity. Studies focusing on these pressure activations on food materials were then reviewed. Rice flour with an exclusively fine mean particle size and small starch damage was obtained due to improved water absorption properties and/or enzyme activity by HP. HP treatment increased of free amino acids and γ-aminobutyric acid (GABA) in rice and soybeans due to improved proteolysis and amino acid metabolism. Improvement of antioxidant activity and alteration of polyphenolic-compounds composition in food materials were also demonstrated by HP treatment. The HP-induced activations on food materials could contribute towards processing technologies for food quality improvement.

Barosensitivity in Saccharomyces cerevisiae is Closely Associated with a Deletion of the COX1 Gene.

High hydrostatic pressure causes physical stress to microorganisms; therefore, this technology may be applied to food pasteurization without introducing the unfavorable effects of thermal denaturation. However, its application is limited to high-value foods because the treatment requires a robust steel vessel and expensive pressurization equipment. To reduce these costs, we studied the pasteurization of Saccharomyces cerevisiae using relatively moderate high-pressure levels. A mutant strain isolated by ultraviolet mutagenesis showed significant loss of viability under high-pressure conditions. Gene expression analysis of the mutant strain revealed that it incurred a deletion of the COX1 gene. Our results suggest that the pressure-sensitivity can readily be introduced into industrial/food microorganisms by complementing a COX1 deleted mitochondria.

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.

Dynamics of the microbial community during continuous methane fermentation in continuously stirred tank reactors.

Methane fermentation is an attractive technology for the treatment of organic wastes and wastewaters. However, the process is difficult to control, and treatment rates and digestion efficiency require further optimization. Understanding the microbiology mechanisms of methane fermentation is of fundamental importance to improving this process. In this review, we summarize the dynamics of microbial communities in methane fermentation chemostats that are operated using completely stirred tank reactors (CSTRs). Each chemostat was supplied with one substrate as the sole carbon source. The substrates include acetate, propionate, butyrate, long-chain fatty acids, glycerol, protein, glucose, and starch. These carbon sources are general substrates and intermediates of methane fermentation. The factors that affect the structure of the microbial community are discussed. The carbon source, the final product, and the operation conditions appear to be the main factors that affect methane fermentation and determine the structure of the microbial community. Understanding the structure of the microbial community during methane fermentation will guide the design and operation of practical wastewater treatments.

Diversities and similarities in pH dependency among bacterial NhaB-like Na+/H+ antiporters.

NhaB-like antiporters were the second described class of Na(+)/H(+) antiporters, identified in bacteria more than 20 years ago. While nhaB-like gene sequences have been found in a number of bacterial genomes, only a few of the NhaB-like antiporters have been functionally characterized to date. Although earlier studies have identified a few pH-sensitive and -insensitive NhaB-like antiporters, the mechanisms that determine their pH responses still remain elusive. In this study, we sought to investigate the diversities and similarities among bacterial NhaB-like antiporters, with particular emphasis on their pH responsiveness. Our phylogenetic analysis of NhaB-like antiporters, combined with pH profile analyses of activities for representative members of several phylogenetic groups, demonstrated that NhaB-like antiporters could be classified into three distinct types according to the degree of their pH dependencies. Interestingly, pH-insensitive NhaB-like antiporters were only found in a limited proportion of enterobacterial species, which constitute a subcluster that appears to have diverged relatively recently among enterobacterial NhaB-like antiporters. Furthermore, kinetic property analyses of NhaB-like antiporters at different pH values revealed that the degree of pH sensitivity of antiport activities was strongly correlated with the magnitude of pH-dependent change in apparent Km values, suggesting that the dramatic pH sensitivities observed for several NhaB-like antiporters might be mainly due to the significant increases of apparent Km at lower pH. These results strongly suggested the possibility that the loss of pH sensitivity of NhaB-like antiporters had occurred relatively recently, probably via accumulation of the mutations that impair pH-dependent change of Km in the course of molecular evolution.

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.

Polyphenol-Retaining Decaffeinated Cocoa Powder Obtained by Supercritical Carbon Dioxide Extraction and Its Antioxidant Activity.

Cocoa beans contain many functional ingredients such as theobromine and polyphenols, but also contain a relatively high amount of caffeine, which can negatively impact human health. It is therefore desirable to reduce caffeine levels in cocoa powder used to make chocolate or cocoa beverages while retaining functional ingredients. We have established conditions for supercritical carbon dioxide (SCCO2) extraction that remove 80.1% of the caffeine from cocoa powder while retaining theobromine (94.1%) and polyphenols (84.7%). The antioxidant activity of the decaffeinated cocoa powder (DCP) made with this optimized SCCO2 extraction method was 85.3% that of non-processed cocoa powder. The total procyanidin and total polyphenol concentrations of the DCPs resulting from various SCCO2 extractions showed a significant positive correlation with oxygen radical absorbance capacity (ORAC). The correlation coefficient between total polyphenols and ORAC was higher than that between total procyanidins and ORAC; thus, the concentration of total polyphenols might be a greater factor in the antioxidant activity of DCP. These results indicate that we could remove large quantities of caffeine from conventional high-cocoa products while retaining the functional benefits of high polyphenol content. This SCCO2 extraction method is expected to be applicable high-cocoa products, such as dark chocolate.

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.

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.

Detection of active, potentially acetate-oxidizing syntrophs in an anaerobic digester by flux measurement and formyltetrahydrofolate synthetase (FTHFS) expression profiling.

Syntrophic oxidation of acetate, so-called reversed reductive acetogenesis, is one of the most important degradation steps in anaerobic digesters. However, little is known about the genetic diversity of the micro-organisms involved. Here we investigated the activity and composition of potentially acetate-oxidizing syntrophs using a combinatorial approach of flux measurement and transcriptional profiling of the formyltetrahydrofolate synthetase (FTHFS) gene, an ecological biomarker for reductive acetogenesis. During the operation of a thermophilic anaerobic digester, volatile fatty acids were mostly depleted, suggesting a high turnover rate for dissolved H(2), and hydrogenotrophic methanogens were the dominant archaeal members. Batch cultivation of the digester microbiota with (13)C-labelled acetate indicated that syntrophic oxidation accounted for 13.1-21.3 % of methane production from acetate. FTHFS genes were transcribed in the absence of carbon monoxide, methoxylated compounds and inorganic electron acceptors other than CO(2), which is implicated in the activity of reversed reductive acetogenesis; however, expression itself does not distinguish whether biosynthesis or biodegradation is functioning. The mRNA- and DNA-based terminal RFLP and clone library analyses indicated that, out of nine FTHFS phylotypes detected, the FTHFS genes from the novel phylotypes I-IV in addition to the known syntroph Thermacetogenium phaeum (i.e. phylotype V) were specifically expressed. These transcripts arose from phylogenetically presumed homoacetogens. The results of this study demonstrate that hitherto unidentified phylotypes of homoacetogens are responsible for syntrophic acetate oxidation in an anaerobic digester.

Generation of free amino acids and gamma-aminobutyric acid in water-soaked soybean by high-hydrostatic pressure processing.

The effects of high-hydrostatic pressure processing (HPP) on soybean cotyledon as a cellular biological material were investigated from the viewpoints of the cell structure and enzyme reaction system. Damage to cell structure was evaluated by measuring dielectric properties using the Cole-Cole arc, the radius of which decreased as pressure level increased. Results suggested that cell structure was damaged by HPP. The distribution of free amino acids was measured after HPP (200 MPa) of soybean soaked in water or sodium glutamate (Glu) solution. HPP resulted in high accumulation of free amino acids in water-soaked soybean, due to proteolysis. HPP of soybean in Glu solution caused higher accumulation of gamma-aminobutyric acid, suggesting that both proteolysis and specific Glu metabolism were accelerated by HPP. We concluded that HPP partially degraded cell structure and accelerated biochemical reactions by allowing enzyme activities to remain. These events can be considered "high-pressure induced transformation" of soybean.

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.

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 degrees 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.

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.

Immunostimulation-mediated antitumor activity by preconditioning with rice-shochu distillation residue against implanted tumor in mice.

We investigated the ability of rice-shochu postdistillation residue (RSDR) to stimulate the activity of macrophages. RSDR significantly stimulated mouse macrophage activity and induced significant IL-12 production in vitro. In syngeneic C38 solid tumor model in mice, a diet containing 1.0% RSDR caused a significant suppression of tumor growth and prolonged the life span of the tumor-bearing mice. Further, using this model, mice fed for 21 days with RSDR showed significantly increased levels of serum IL-12 and IFN-gamma compared with controls. Moreover, the splenic NK cell activity of mice fed with RSDR was significantly elevated compared with that of mice on a normal diet and thereby suppressed C38 tumor growth. We also investigated the tumor growth suppressing effect of RSDR using a tumor model of B16-F10 melanoma cells. Dietary preconditioning with RSDR significantly suppressed B16-F10 tumor growth. Moreover, RSDR significantly increased the production of IL-12 either before or after B16-F10 tumor implantation. These results suggest that dietary RSDR suppresses tumor growth by stimulating the immune system of the host.