Influence of amino acid content in seed mash on peptide uptake by yeast cells in main mash in sake brewing process

It was found that the peptide content of the main mash in the sake brewing process, seeded with kimoto, was higher than in that seeded with sokujo-moto, although the peptide content in kimoto was lower than in sokujo-moto. We investigated the underlying reasons. As a result, we found that the high concentration of free amino acids originating from kimoto decreased the peptide uptake ability of yeast cells in the main mash seeded with kimoto.     

Effect of the FAA1 gene disruption of sake yeast on the accumulation of ethyl caproate in sake mash

Fatty acid activation gene (FAA1) in sake yeast Kyokai no. 701 (K701) was disrupted to investigate the accumulation of ethyl caproate in sake mash. Ethyl caproate, recognized as an important apple-like flavor in sake, is generated by fatty acid synthesis in yeast cells. The disruptant for the FAA1 gene (K701deltafaa1) exhibited a reduced growth rate in a medium containing cerulenin and myristic acid or oleic acid compared with that of the parental strain (K701). In a sake brewing test in which the rice used was polished to 60% of its original size, the fermentation ability of K701deltafaa1 was inferior to that of K701 but the production of ethyl caproate by K701deltafaa1 was 1.6-fold higher than that by K701. These results suggest that the FAA1 gene in sake yeast plays an important role in sake brewing and the accumulation of ethyl caproate.     

Effect of the FAA1 gene disruption of sake yeast on the accumulation of ethyl caproate in sake mash

Fatty acid activation gene (FAA1) in sake yeast Kyokai no. 701 (K701) was disrupted to investigate the accumulation of ethyl caproate in sake mash. Ethyl caproate, recognized as an important apple-like flavor in sake, is generated by fatty acid synthesis in yeast cells. The disruptant for the FAA1 gene (K701Δfaa1) exhibited a reduced growth rate in a medium containing cerulenin and myristic acid or oleic acid compared with that of the parental strain (K701). In a sake brewing test in which the rice used was polished to 60% of its original size, the fermentation ability of K701Δfaa1 was inferior to that of K701 but the production of ethyl caproate by K701Δfaa1 was 1.6-fold higher than that by K701. These results suggest that the FAA1 gene in sake yeast plays an important role in sake brewing and the accumulation of ethyl caproate.     

Effect of gene disruption of succinate dehydrogenase on succinate production in a sake yeast strain

Succinate dehydrogenase (SDH) of Saccharomyces cerevisiae consists of four subunits encoded by the SDH1, SDH2, SDH3, and SDH4 genes. We determined the effect of SDH deficiency on the productivity of organic acids in a sake yeast strain Kyokai no. 9. The SDH activity of single disruptants was retained at 30-90% of that of the wild-type strain, but the activity disappeared in double disruptants of the SDH1 and SDH2 or SDH1b (the SDH1 homologue) genes. Two double disruptants showed no growth on a medium containing glycerol as the sole carbon source, while the single disruptants could utilize glycerol. These results indicate that double disruption of the SDH1 and SDH2 or SDH1b genes is required for complete loss of SDH activity and that the SDH1b gene compensates for the function of the SDH1 gene. The sdh1 sdh1b disruptant showed a marked increase in succinate productivity of up to 1.9-fold along with a decrease in malate productivity relative to the wild-type strains under shaking conditions. Under both static and sake brewing conditions, the productivity of these organic acids in the disruptants was virtually unchanged from that in the wild-type strain. Furthermore, SDH activity was undetectable in the wild-type and the disrupted strains under static conditions. These results suggest that SDH activity contributes to succinate production under shaking conditions, but not under static and sake brewing conditions.     

Effect of freeze drying and protectants on viability of the biocontrol yeast Candida sake

The effects of freezing method, freeze drying process, and the use of protective agents on the viability of the biocontrol yeast Candida sake were studied. Freezing at -20 degrees C was the best method to preserve the viability of C. sake cells after freeze drying using 10% skim milk as a protectant (28.9% survival). Liquid nitrogen freezing caused the highest level of damage to the cells with viability < 10%. Different concentrations of exogenous substances including sugars, polyols, polymers and nitrogen compounds were tested either alone or in combination with skim milk. There was little or no effect when additives were used at 1% concentration. Galactose, raffinose and sodium glutamate at 10% were the best protective agents tested alone but the viability of freeze-dried C. sake cells was always < 20%. Survival of yeast cells was increased from 0.2% to 30-40% by using appropriate protective media containing combinations of skim milk and other protectants such as 5% or 10% lactose or glucose, and 10% fructose or sucrose.     

Inhibition of mitochondrial fragmentation during sake brewing causes high malate production in sake yeast

We previously demonstrated the presence and fragmentation of mitochondria during alcohol fermentation. Here, we show that Fis1p induces mitochondrial fragmentation, and inhibition of mitochondrial fragmentation causes higher malate production during sake brewing. These findings indicate that mitochondrial morphology affects the metabolism of constituents, providing a breeding strategy for high-malate-producing yeasts.     

Isolation and characterization of sake yeast mutants deficient in gamma-aminobutyric acid utilization in sake brewing

Sake yeasts take up gamma-aminobutyric acid (GABA) derived from rice-koji in the primary stage of sake brewing. The GABA content in sake brewed with the UGA1 disruptant, which lacked GABA transaminase, was higher than that brewed with the wild-type strain K701. The UGA1 disruptant derived from sake yeast could not grow on a medium with GABA as the sole nitrogen source. We have isolated the sake yeast mutants of K701 that were unable to grow on a medium containing GABA as the sole nitrogen source. The growth defect of GAB7-1 and GAB7-2 mutants on GABA plates was complemented by UGA1, which encodes GABA transaminase, and UGA2, which encodes succinic semialdehyde dehydrogenase (SSADH), respectively. DNA sequence analysis revealed that GAB7-1 had a homozygous nonsense mutation in UGA1 and GAB7-2 had a heterozygous mutation (G247D) in UGA2. The GABA transaminase activity of GAB7-1 and the SSADH activity of GAB7-2 were markedly lower than those of K701. These GAB mutants displayed a higher intracellular GABA content. The GABA contents in sake brewed with the mutants GAB7-1 and GAB7-2 were 2.0 and 2.1 times higher, respectively, than that brewed with the wild-type strain K701. These results suggest that the reduced function of the GABA utilization pathway increases the GABA content in sake.     

Mitochondrial dynamics of yeast during sake brewing

The presence of mitochondria during alcohol fermentation has not been studied. Here, we examined the yeast mitochondrial structure during sake brewing using the green fluorescent protein. Mitochondrial structures were observed throughout brewing and they fragmented as brewing proceeded. This study is the first to show direct evidence of the presence of mitochondria during alcohol fermentation.     

Magnesium nutrition on accumulation and transport of amino acids in tea plants

BACKGROUND: Free amino acids in young tea shoots are important chemical constituents, remarkably influencing the quality of green teas. Nutrient solution, soil pot and field experiments were conducted to investigate the effect of magnesium nutritional status on accumulation and transport of free amino acids in tea plants ( Camellia sinensis (L.) O. Kuntze). RESULTS: A sufficient supply of Mg in nutrient solution increased biomass production and concentrations of free amino acids, notably theanine in young shoots and roots, without affecting total N in the leaves, absorption rates of inorganic N and glutamine synthetase activity. Amino acids in xylem saps and phloem exudates or total sugars in phloem exudates of tea plants from pot and field experiments showed their highest levels in bud breaking and shoot extension in early spring (March and early April). Application of Mg increased the amounts of amino acids and total sugars in the xylem saps and phloem exudates. CONCLUSION: Adequate supply of Mg nutrient promoted the synthesis of theanine in roots and its accumulation in the young shoots of tea plants. Magnesium nutritional status was an important factor influencing the mobility of amino acids and sugars via xylem and phloem especially when N and C reserves remobilized to support spring growth of young shoots. Copyright © 2011 Society of Chemical Industry     

QTL mapping of sake brewing characteristics of yeast

A haploid sake yeast strain derived from the commercial diploid sake yeast strain Kyokai no. 7 showed better characteristics for sake brewing compared to the haploid laboratory yeast strain X2180-1B, including higher production of ethanol and aromatic components. A hybrid of these two strains showed intermediate characteristics in most cases. After sporulation of the hybrid strain, we obtained 100 haploid segregants of the hybrid. Small-scale sake brewing tests of these segregants showed a smooth continuous distribution of the sake brewing characteristics, suggesting that these traits are determined by multiple quantitative trait loci (QTLs). To examine these sake brewing characteristics at the genomic level, we performed QTL analysis of sake brewing characteristics using 142 DNA markers that showed heterogeneity between the two parental strains. As a result, we identified 25 significant QTLs involved in the specification of sake brewing characteristics such as ethanol fermentation and the production of aromatic components.     

Competitive amino acid transport between L-tryptophan and other amino acids in Schizophyllum commune

In our study on nutritional requirement for the hyphal growth of Schizophyllum commune, we found that a Trp- mutant could not grow in the L-Trp-supplied medium in the presence of L-Ser. Further growth studies showed that not only L-Ser but also as many as 11 kinds of amino acid including L-Ala, L-Arg, L-Asn, L-His, L-Leu, L-Met, L-Phe, L-Ser, L-Thr, L-Tyr and L-Val inhibited the growth of the Trp- mutant in the L-Trp-supplied medium. However, these amino acids did not inhibit the growth of a Trp+ strain. The inhibition of growth of Trp+ strain induced by a Trp analogue of 5-fluoro-DL-tryptophan (5FT), which was usually recovered by L-Trp, was rescued by the same amino acids mentioned above. The exceptions were Gly and L-Ile, which also recovered the growth inhibition induced by 5FT. These results indicate that the permease responsible for the Trp transport in S. commune might also be active to other amino acids. However, it is considered that the permease shows high affinity to L-Trp and low affinity to other amino acids. As a result, the transport of L-Trp and 5FT may be counteracted by other amino acids.     

碱性和支链氨基酸对啤酒高浓酿造酵母发酵性能的影响

研究分别考察添加三种碱性氨基酸：精氨酸（Arg）、赖氨酸（Lys）、组氨酸（His）与支链氨基酸：缬氨酸（Val）、亮氨酸（Leu）、异亮氨酸（Ile）对24 °P高浓酿造过程中酵母发酵性能及啤酒风味的影响。结果表明,六种氨基酸的添加可促进酵母生长并提高活细胞率,显著提高麦汁发酵度及乙醇产量。其中,添加Val、Leu与Arg的高浓麦汁发酵性能较好,与对照组相比,发酵结束时总CO2失质量分别为92.4 g/L、92.7 g/L与91.2 g/L;发酵度极显著提高（P＜0.01）,分别为83.65%、82.95%和82.93%;乙醇产量极显著提高（P＜0.01）,分别为11.97%（V/V）、11.90%（V/V）和11.83%（V/V）。添加Arg可显著提高酵母总细胞数（1.64×108 cells/mL）（P＜0.05）,His、Val与Leu的添加对风味物质影响较大,可提高啤酒的醇酯比,其中,Val的添加可显著提高异丁醇含量（P＜0.05）,降低总酯含量;Leu的添加则可同时提高异戊醇及乙酸异戊酯的含量。     

清酒酿酒酵母酒精耐受机理的研究进展

清酒酿酒酵母是清酒生产过程中的关键微生物,发酵过程中逐渐积累的酒精会对酵母细胞产生毒害作用,从而抑制了细胞的生长代谢和更高浓度酒精的形成。因此,对酵母细胞酒精耐受机理的研究,为筛选、构造高产酒精和高酒精耐受性酵母菌种提供理论基础。本文从细胞壁、细胞膜、热休克蛋白、贮藏性糖类、氨基酸等方面阐述酵母细胞酒精耐受机理,并在细胞生理学的基础上分析清酒酵母具有较高的酒精产率和相对较差的酒精耐受性的原因。     

Effect of NAD+-dependent isocitrate dehydrogenase gene (IDH1, IDH2) disruption of sake yeast on organic acid composition in sake mash

The ratio of organic acids in sake mash is a very important factor affecting the taste of alcoholic beverages. To alter the organic acid composition in sake and investigate the mechanism of producing organic acids in sake mash, we examined the effect of NAD+-dependent isocitrate dehydrogenase (IDH) activity deficiency in sake yeast by disrupting the IDH1 or IDH2 gene. Two haploid strains (MATa or MATa genotype) isolated from sake yeast Kyokai no. 701 (K701) were disrupted using the aureobasidin A resistant gene (AUR1-C) as a selection marker. These disruptants were defective in the activity of IDH and failed to grow on medium containing glycerol as a sole carbon source. Sake meter, alcohol concentration, and glucose consumption in sake brewed with the disruptants were reduced in comparison with those of the parental strains. The production of citrate (including isocitrate), malate, and acetate by the disruptants was increased, but succinate production was reduced to approximately half in comparison with the parental strains. These results indicate that approximately half the amount of succinate in sake mash is produced via the oxidative pathway of the TCA cycle in sake yeast. While the diploid strain constructed by mating haploid disruptants for the IDH gene exhibited stronger fermentation ability than the haploid disruptants, almost similar profiles of components in sake were obtained for both strains.     

Effect of carbon branched-chain amino acids on gastric secretion

Experiments on dogs with Basov's gastric fistulas were made to study the effect of amino acids with branched carbon chains (L-isoleucine, L-leucine, L-valine) on gastric secretion. The mixture of amino acids in a concentration of 0.16 M exerts a powerful enough stimulant action, provoking gastric secretion amounting to 2/3 of the value obtained during sham meat feeding. The value of the secretion determined by other amino acids (L-alanine, L-proline) was the same. Unlikely, gastric secretion provoked by glycine (0.16 M) was approximately 1.5 times more intense as compared to that induced by the mixture of amino acids with branched carbon chains. The intense action of glycine is likely to be related to the fact that, apart from the properties common to other amino acids, it possesses the properties of neurotransmitters. L-glutamic acid (0.1 M) administered to the blood produces marked inhibition of gastric secretion caused by the mixture of amino acids with branched carbon chains and the same inhibition of secretion due to administration of other amino acids.