Modeling the sake brewing characteristics of rice from brown rice metabolites.

Sake, soy sauce, miso (Japanese bean paste), and beer are made from grains. The characteristics of the grain significantly affect the quality of the final product. Many studies have been performed to evaluate the sake-brewing characteristics of rice. However, current rice analysis methods are time and labor intensive and require large samples. We developed a novel method for predicting the brewing characteristics of sake rice using <1 g of sample. Brown rice extracts were analyzed by ultrahigh-performance liquid chromatography quadrupole time-of-flight mass spectrometry, and mass chromatogram data were used as explanatory variables. The objective variables were the physical and chemical properties of the rice, the enzymatic activity of the rice-koji, the fermentation properties of the sake mash, the standard analytical values of the sake, and the flavor component concentrations in the sake. Prediction models were developed using the orthogonal projections to latent structures method. The prediction performances of the models were verified, and 32 out of the 54 objective variables were used in well-performing models. In conclusion, we developed a method for predicting the rice properties and brewing characteristics from results acquired by analyzing <1 g of brown rice. The method is a powerful tool for breeding new sake rice cultivars for good brewing characteristics in early generations and will improve our understanding of fluctuations in the brewing characteristics of sake rice before each sake brewing season starts.

Effect of koji starter on metabolites in Japanese alcoholic beverage sake made from the sake rice Koshitanrei.

In sake brewing, the steamed rice is used in 2 ways, added to sake-mash and making rice-koji. Rice-koji is made from the steamed rice by using koji starter, and its quality is an important determinant of the aroma/taste of sake. The sake rice Koshitanrei (KOS) was developed in Niigata Prefecture by crossing 2 sake rice varieties, Gohyakumangoku and Yamadanishiki. Recently, we reported the characteristic components/metabolites in sake made from KOS by conducting metabolome analysis using UPLC-QTOF-MS. In this study, to investigate the effect of koji starter and sake rice cultivars on the sake metabolites, we performed small-scale sake-making tests using the above 3 rice cultivars and 3 koji starters. Finally, we demonstrated that some of the characteristic components/metabolites of sake from KOS are affected by the koji starter. Thus, in addition to rice cultivar, koji starter plays an important role for establishment/maintenance of the quality of the final product.

Analysis of metabolites in Japanese alcoholic beverage sake made from the sake rice Koshitanrei.

In sake brewing, the steamed rice is used in two ways, added to sake-mash (as kake-mai) and making koji. The rice is an important determinant for the quality of sake, as the metabolites in sake affect its taste/aroma. The sake rice Koshitanrei (KOS) was developed in Niigata Prefecture by genetically crossing two sake rice, Gohyakumangoku and Yamadanishiki. However, the metabolites in sake from KOS have not been analyzed. Here, to investigate the characteristic metabolites in sake from KOS, we performed two types of small-scale sake-fermentation tests changing only the rice used for kake-mai or total rice (both kake-mai and koji) by these three rice cultivars and examined the effect of KOS on sake metabolites by the metabolome analysis method using UPLC-QTOF-MS. We identified the peaks/metabolites, whose intensity in sake from KOS was higher/lower than those from the other cultivars. The brewing properties of KOS were partially characterized by this analysis.

Investigation of relationship between sake-making parameters and sake metabolites using a newly developed sake metabolome analysis method.

We developed a sake metabolome analysis method using liquid chromatography-quadrupole/time-of-flight mass spectrometry to investigate the metabolome of various types of sakes and other alcohol beverages. Our method identified 198 compounds by comparison with standard metabolites. Using this method, we investigated the relationship between several sake-making parameters and sake metabolites by conducting combination experiments of these parameters using small-scale fermentation. The results indicated that all parameters significantly affected sake metabolites (P < 0.005) and most peaks were affected by multiple sake-making parameters. Interestingly, the effect of the rice cultivar on sake metabolites was higher for koji rice than for kake-rice. This result suggests that the rice cultivar used has a greater effect on the characteristics of Aspergillus oryzae compared to sake yeast and affects sake metabolites. In this study, we also evaluated the combined effect of several parameters. We demonstrated the different effects of each parameter on several amino acids. The results showed a new aspect of the science of sake making. For example, the amount of α-ethylglucoside, which can affect the taste of sake, was negatively correlated with α-glucosidase activity in koji (r = -0.84). In this study, various unidentified peaks were observed; detectable peaks can be increased by analyzing additional standard reagents. Investigating these unidentified peaks and accumulating datasets for sake-making parameters will give us insight into how to improve sake taste and quality.

Suppression of ricinoleic acid toxicity by ptl2 overexpression in fission yeast Schizosaccharomyces pombe.

We previously succeeded to obtain a high content of ricinoleic acid (RA), a hydroxylated fatty acid with great values as a petrochemical replacement, in fission yeast Schizosaccharomyces pombe by introducing Claviceps purpurea oleate Δ12-hydroxylase gene (CpFAH12). Although the production was toxic to S. pombe cells, we identified plg7, encoding phospholipase A2, as a multicopy suppressor that restored the growth defect by removing RA from phospholipids and induced secretion of a part of the released free RA into culture media. In this study, we extended our analysis and examined the effect of triglyceride (TG) lipase overexpression on the tolerance to RA toxicity and RA productivity. S. pombe has three TG lipase genes, ptl1, ptl2, and ptl3, which have high protein sequence similarities to each other and to Saccharomyces cerevisiae counterparts TGL3, TGL4, and TGL5, but only ptl2 overexpression suppressed the growth defect induced by RA production, and the culture grown at 20 °C secreted free RA into media like plg7 overexpression. Suppression by ptl2 was independent of plg7, and a large amount of free RA was accumulated in the cells concomitant with the decrease in RA moieties in phospholipids. Furthermore, the suppression by ptl2 was attenuated by bromoenol lactone (BEL), a phospholipase A2 specific inhibitor, suggesting that Ptl2p may have phospholipase activity. Simultaneous overexpression of ptl2 and plg7 in the FAH12 integrant increased secretion and intracellular accumulation of RA 1.2- and 1.3-fold, respectively, compared to those with single overexpression of plg7 on day 10 at 20 °C.

Oral administration of whole dihomo-γ-linolenic acid-producing Saccharomyces cerevisiae suppresses cutaneous inflammatory responses induced by croton oil application in mice.

Polyunsaturated fatty acids have been attracting considerable interest because of their many biological activities and important roles in human health and nutrition. Dihomo-γ-linolenic acid (DGLA; C20: 3n-6) is known to have an anti-inflammatory activity, but its range of effects was not well studied because of its limited natural sources. Taking advantage of genetic tractability and increasing wealth of accessible data of Saccharomyces cerevisiae, we have previously constructed a DGLA-producing yeast strain by introducing two types of desaturase and one elongase genes to convert endogenous oleic acid (C18:1n-9) to DGLA. In this study, we investigated the efficacy of oral intake of heat-killed whole DGLA-producing yeast cells in the absence of lipid purification on cutaneous inflammation. Topical application of croton oil to mouse ears induces ear swelling in parallel with the increased production of chemokines and accumulation of infiltrating cells into the skin sites. These inflammatory reactions were significantly suppressed in a dose-dependent manner by oral intake of the DGLA-producing yeast cells for only 7 days. This suppression was not observed by the intake of the γ-linolenic acid-producing (C18:3n-6, an immediate precursor of DGLA) yeast, indicating DGLA itself suppressed the inflammation. Further analysis demonstrated that DGLA exerted an anti-inflammatory effect via prostaglandin E1 formation because naproxen, a cyclooxygenase inhibitor, attenuated the suppression. Since 25-fold of purified DGLA compared with that provided as a form of yeast was not effective, oral administration of the whole DGLA-producing yeast is considered to be a simple but efficient method to suppress inflammatory responses.

[The relationship between narcissistic personality traits and risk-taking behavior is mediated by self-monitoring].

This study investigated the hypothesis that narcissistic personality traits would affect risk-taking behaviors through self-monitoring. The Narcissistic Personality Inventory Short Version (NPI-S), the Self-monitoring Scale (SM), and the Risk-taking Behavior Scale for Undergraduates (RIBS-U) were administered to 192 university and graduate students. There were three NPI-S factors ("sense of superiority and competence", "need for attention and praise", and "self-assertion"), two SM factors ("extraversion" and "other-directedness"), and the single risk-taking factor of the RIBS-U. Covariance structure analysis was then conducted to test whether narcissistic personality traits would affect risk-taking behaviors through self-monitoring. Analysis showed that the factors of "sense of superiority and competence" and "need for attention and praise" affected risk-taking behavior through the "other-directedness" factor. However, the "self-assertion" factor was found to have a direct effect on risk-taking behavior.

Secretory production of ricinoleic acid in fission yeast Schizosaccharomyces pombe.

We have succeeded to produce a high content of ricinoleic acid (RA), a hydroxylated fatty acid with great values as a petrochemical replacement, in fission yeast Schizosaccharomyces pombe by introducing Claviceps purpurea oleate Δ12-hydroxylase gene (CpFAH12). Although the production was toxic to S. pombe cells, we solved the problem by identifying plg7, encoding phospholipase A2, as a multicopy suppressor. Characterization of the RA-tolerant strains suggested that the removal of RA moieties from phospholipids would be the suppression mechanism by plg7. In this study, we extended our analysis and report our new discovery that the overexpression of plg7 enabled cells to secrete free RA into culture media. When the FAH12 integrant in the absence of the overexpressed plg7 was grown at 20 °C for 11 days, the amount of intracellular RA reached 200.1 µg/ml of culture and only 69.3 µg/ml of RA was detected in culture media. On the other hand, the FAH12 integrant harboring the plg7 multicopy plasmid secreted RA in the media (184.5 µg/ml) without decreasing the amount in the cells, i.e., a significantly higher total secretion and a lead to making RA by its secretory production in S. pombe.

Toxicity of ricinoleic acid production in fission yeast Schizosaccharomyces pombe is suppressed by the overexpression of plg7, a phospholipase A2 of a platelet-activating factor (PAF) family homolog.

In an effort to produce ricinoleic acid (RA), an important natural raw material with great values as a petrochemical replacement, in Schizosaccharomyces pombe, we introduced Claviceps purpurea oleate Δ12-hydroxylase gene (CpFAH12) to S. pombe, putting it under the control of an inducible nmt1 promoter. However, RA was toxic to S. pombe and the cells expressing CpFAH12 grew poorly at the normal growth temperature 30 °C. To address its toxic mechanism in S. pombe, we screened for a S. pombe cDNA library and identified plg7, which encodes a phospholipase A2, as a suppressor that restored the growth defect without affecting the RA production. A lacZ fusion experiment showed that the expression of plg7 was inducible by RA. Thin layer chromatographic analysis confirmed a reduction in RA moiety in phospholipids and a concomitant increase in free RA in the plg7 overexpressed strain. Since RA is synthesized at the sn-2 position of phosphatidylcholine by Fah12p, and phospholipase A2 hydrolyzes the sn-2 acyl bond of phospholipids, we speculate that plg7 is a stress-responsive gene, and removal of RA moieties from phospholipids, major components of lipid bilayer membrane, by Plg7p would be its suppression mechanism.

Characterization of triglyceride lipase genes of fission yeast Schizosaccharomyces pombe.

Triglycerides (TG) are major storage lipids for eukaryotic cells. In this study, we characterized three genes of fission yeast Schizosaccharomyces pombe, SPCC1450.16c, SPAC1786.01c, and SPAC1A6.05c, that show high homology to Saccharomyces cerevisiae TG lipase genes, TGL3, TGL4, and TGL5. Deletion of each gene increased TG content by approximately 1.7-fold compared to the parental wild-type strain, and their triple deletion mutant further increased TG content to 2.7-fold of the wild-type strain, suggesting that all three genes encode TG lipase and are functioning in S. pombe. The triple deletion mutant showed no growth defect in rich and synthetic medium, but its growth was sensitive to cerulenin, an inhibitor of fatty acid synthesis. This growth defect by cerulenin was restored by adding oleic acid in media, suggesting that these genes were involved in the mobilization of TG in S. pombe. When ricinoleic acid was produced in the triple mutant by introducing CpFAH12 fatty acid hydroxylase gene from Claviceps purpurea, percent composition of ricinoleic acid increased by 1.1-fold compared to the wild-type strain, in addition to a 1.6-fold increase in total fatty acid content per dry cell weight (DCW). In total, the ricinoleic acid production per DCW increased by 1.8-fold in the triple deletion mutant.

Engineered high content of ricinoleic acid in fission yeast Schizosaccharomyces pombe.

In an effort to produce ricinoleic acid (12-hydroxy-octadeca-cis-9-enoic acid: C18:1-OH) as a petrochemical replacement in a variety of industrial processes, we introduced Claviceps purpurea oleate ∆12-hydroxylase gene (CpFAH12) to Schizosaccharomyces pombe, putting it under the control of inducible nmt1 promoter. Since Fah12p is able to convert oleic acid to ricinoleic acid, we thought that S. pombe, in which around 75% of total fatty acid (FA) is oleic acid, would accordingly be an ideal microorganism for high production of ricinoleic acid. Unfortunately, at the normal growth temperature of 30 °C, S. pombe cells harboring CpFAH12 grew poorly when the CpFAH12 gene expression was induced, perhaps implicating ricinoleic acid as toxic in S. pombe. However, in line with a likely thermoinstability of Fah12p, there was almost no growth inhibition at 37 °C or, by contrast with 30 °C and lower temperatures, ricinoleic acid accumulation. Accordingly, various optimization steps led to a regime with preliminary growth at 37 °C followed by a 5-day incubation at 20 °C, and the level of ricinoleic acid reached 137.4 µg/ml of culture that corresponded to 52.6% of total FA.

Efficient accumulation of oleic acid in Saccharomyces cerevisiae caused by expression of rat elongase 2 gene (rELO2) and its contribution to tolerance to alcohols.

When the cells of Saccharomyces cerevisiae are exposed to high concentration of ethanol, the content of oleic acid (C18:1n-9) increased as the initial concentration of ethanol increased. Based on this observation, we attempted to confer ethanol tolerance to S. cerevisiae by manipulating fatty acid composition of the cells. Rather than altering OLE1 expression [the desaturase making both C16:1n-7 (palmitoleic acid) and C18:1n-9], we introduced elongase genes. Introduction of rat elongase 1 gene (rELO1) into S. cerevisiae gave cis-vaccenic acid (cis-C18:1n-7) by conversion from C16:1n-7, and the increase in this C18:1 fatty acid did not confer ethanol tolerance to the cells. On the other hand, the introduction of rat elongase 2 gene (rELO2), which elongates C16:0 to C18:0, drastically increased C18:1n-9 content, and the cells acquired ethanol tolerance, emphasizing the specific role of C18:1n-9. Furthermore, the transformant of rELO2 also conferred tolerance to n-butanol, n-propanol, and 2-propanol.

Improvement of polyunsaturated fatty acids synthesis by the coexpression of CYB5 with desaturase genes in Saccharomyces cerevisiae.

Since Saccharomyces cerevisiae contains Delta9 fatty acid desaturase (OLE1) as a sole fatty acid desaturase, it produces saturated and monounsaturated fatty acids of 16- and 18-carbon compounds. We showed earlier that Kluyveromyces lactis Delta12 (KlFAD2) and omega3 (KlFAD3) fatty acid desaturase genes enabled S. cerevisiae to make also polyunsaturated fatty acids (PUFAs), linoleic (18:2n-6), and alpha-linolenic (18:3n-3) acids. Unlike Delta9 fatty acid desaturase Ole1p, the two added fatty acid desaturases (KlFAD2and KlFAD3) do not contain a cytochrome b5 domain, and we now report on effects of the overexpression of K. lactis and S. cerevisiae cytochrome b5 (CYB5) genes as well as temperature effects on PUFA synthesis. Without extra cytochrome b5, while PUFA synthesis is significant at low temperature (20 degrees C), it was marginal at 30 degrees C. Overexpression of cytochrome b5 at 20 degrees C did not affect the fatty acid synthesis so much, but it significantly enhanced the synthesis of PUFA at 30 degrees C.

Production of polyunsaturated fatty acids in yeast Saccharomyces cerevisiae and its relation to alkaline pH tolerance.

Saccharomyces cerevisiae produces saturated and monounsaturated fatty acids of 16- and 18-carbon atoms and no polyunsaturated fatty acids (PUFAs) with more than two double bonds. To study the biological significance of PUFAs in yeast, we introduced Kluyveromyces lactis Delta12 fatty acid desaturase (KlFAD2) and omega3 fatty acid desaturase (KlFAD3) genes into S. cerevisiae to produce linoleic and alpha-linolenic acids in S. cerevisiae. The strain producing linoleic and alpha-linolenic acids showed an alkaline pH-tolerant phenotype. DNA microarray analyses showed that the transcription of a set of genes whose expressions are under the repression of Rim101p were downregulated in this strain, suggesting that Rim101p, a transcriptional repressor which governs the ion tolerance, was activated. In line with this activation, the strain also showed elevated resistance to Li(+) and Na(+) ions and to zymolyase, a yeast lytic enzyme preparation containing mainly beta-1,3-glucanase, indicating that the cell wall integrity was also strengthened in this strain. Our findings demonstrate a novel influence of PUFA production on transcriptional control that is likely to play an important role in the early stage of alkaline stress response. The Accession No. for microarray data in the Center for Information Biology Gene Expression database is CBX68.

Heterologous production of dihomo-gamma-linolenic acid in Saccharomyces cerevisiae.

To make dihomo-gamma-linolenic acid (DGLA) (20:3n-6) in Saccharomyces cerevisiae, we introduced Kluyveromyces lactis Delta12 fatty acid desaturase, rat Delta6 fatty acid desaturase, and rat elongase genes. Because Fad2p is able to convert the endogenous oleic acid to linoleic acid, this allowed DGLA biosynthesis without the need to supply exogenous fatty acids on the media. Medium composition, cultivation temperature, and incubation time were examined to improve the yield of DGLA. Fatty acid content was increased by changing the medium from a standard synthetic dropout medium to a nitrogen-limited minimal medium (NSD). Production of DGLA was higher in the cells grown at 15 degrees C than in those grown at 20 degrees C, and no DGLA production was observed in the cells grown at 30 degrees C. In NSD at 15 degrees C, fatty acid content increased up until day 7 and decreased after day 10. When the cells were grown in NSD for 7 days at 15 degrees C, the yield of DGLA reached 2.19 microg/mg of cells (dry weight) and the composition of DGLA to total fatty acids was 2.74%. To our knowledge, this is the first report describing the production of polyunsaturated fatty acids in S. cerevisiae without supplying the exogenous fatty acids.

Disruption of URA7 and GAL6 improves the ethanol tolerance and fermentation capacity of Saccharomyces cerevisiae.

Screening of the homozygous diploid yeast deletion pool of 4741 non-essential genes identified two null mutants (Deltaura7 and Deltagal6) that grew faster than the wild-type strain in medium containing 8% v/v ethanol. The survival rate of the gal6 disruptant in 10% ethanol was higher than that of the wild-type strain. On the other hand, the glucose consumption rate of the ura7 disruptant was better than that of the wild-type strain in buffer containing ethanol. Both disruptants were more resistant to zymolyase, a yeast lytic enzyme containing mainly beta-1,3-glucanase, indicating that the integrity of the cell wall became more resistance to ethanol stress. The gal6 disruptant was also more resistant to Calcofluor white, but the ura7 disruptant was more sensitive to Calcofluor white than the wild-type strain. Furthermore, the mutant strains had a higher content of oleic acid (C18 : 1) in the presence of ethanol compared to the wild-type strain, suggesting that the disruptants cope with ethanol stress not only by modifying the cell wall integrity but also the membrane fluidity. When the cells were grown in medium containing 5% ethanol at 15 degrees C, the gal6 and ura7 disruptants showed 40% and 14% increases in the glucose consumption rate, respectively.

Effects of inferred social status and a beginning driver's sticker upon aggression of drivers in Japan.

The present study examined how inferred social status and a beginning driver's sticker influenced aggressive drivers' behavior on the road in Japan. A 2 x 2 x 2 factorial design was constructed to examine the effects of social status of an experimental car (high or low), with a beginning driver's sticker or no beginning driver's sticker, and a male or female driver. Analysis showed that horn-honking latency in the sticker condition was longer than that in the no-sticker condition in the low status car. In the high status car, the horn was sounded sooner in the condition with a beginning driver's sticker. A beginning driver's sticker promoted aggressive behavior oward a high status person, whereas it inhibited aggressive behavior toward low status individuals. Thus, response to a beginning driver's sticker appears to vary according to inferred social status.

[Learned helplessness, generalized self-efficacy, and immune function].

Generalized self-efficacy is considered one of important personality traits that determine psychological and physiological stress responses. The present study examined the interaction effects of generalized self-efficacy and controllability of acute stress on salivary secretory immunoglobulin A (s-IgA), task performance, and psychological stress responses in a typical learned helplessness paradigm. Twenty low and 19 high self-efficacy undergraduate women performed two response selection tasks one after another. In the first task, they were exposed to controllable or uncontrollable aversive noise. The second task was identical for all, but perceived controllability was higher for the high self-efficacy group than the low. Performance under uncontrollable condition was lower than controllable condition. The interaction of self-efficacy and controllability was observed only on the s-IgA variable; increase of secretion of s-IgA secretion under stressor uncontrollability was more prominent in the low self-efficacy group than the high. These results suggested that generalized self-efficacy was a moderator of the stressor controllability effect on secretory immunity.