Phenotypes and brewing characteristics of sake yeast Kyokai no. 7 mutants resistant to valproate

Screening of drug‐resistant mutants of sake yeast strains has been a major method for creation of superior strains. We attempted to create a valproic acid (VPA)‐resistant mutant strain from sake yeast Kyokai No. 7 (K7). VPA is a branched‐chain fatty acid and is an inositol synthesis inhibitor in mammals and yeast. We succeeded in isolating a mutant of strain K7 that can survive long‐term in a VPA‐containing medium. This strain, K7‐VPA^LS, is significantly more resistant to not only VPA‐induced cell death but also ethanol in comparison with the parent strain. Further experiments showed that the new strain is likely to have a deficiency in inositol and/or phosphatidylinositol synthesis. The major characteristics of sake brewed by strain K7‐VPA^LS (compared with K7) were lower amino acidity, higher isoamyl acetate content without an increase in the isoamyl alcohol level and changes in constituent organic acids, particularly higher malate and succinate but lower acetate concentrations. In addition, taste sensor analysis revealed that K7‐VPA^LS‐brewed sake has milder sourness and higher saltiness or richness than K7‐brewed sake does. High isoamyl acetate production may be related to a deficiency in phosphatidylinositol because this compound directly inhibits alcohol acetyltransferase, an enzyme responsible for isoamyl acetate synthesis. Strain K7‐VPA^LS grew more rapidly than the parental strain did in a medium containing acetate as a sole carbon source, indicating that K7‐VPA^LS effectively assimilates acetate and converts it to malate and succinate through the glyoxylate cycle. Thus, strain K7‐VPA^LS shows improved characteristics for brewing of high‐quality sake. Copyright © 2017 The Institute of Brewing & Distilling     

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.     

Brewing characteristics of haploid strains isolated from sake yeast Kyokai No. 7

Sake yeast exhibit various characteristics that make them more suitable for sake brewing compared to other yeast strains. Since sake yeast strains are Saccharomyces cerevisiae heterothallic diploid strains, it is likely that they have heterozygous alleles on homologous chromosomes (heterozygosity) due to spontaneous mutations. If this is the case, segregation of phenotypic traits in haploid strains after sporulation and concomitant meiosis of sake yeast strains would be expected to occur. To examine this hypothesis, we isolated 100 haploid strains from Kyokai No. 7 (K7), a typical sake yeast strain in Japan, and compared their brewing characteristics in small‐scale sake‐brewing tests. Analyses of the resultant sake samples showed a smooth and continuous distribution of analytical values for brewing characteristics, suggesting that K7 has multiple heterozygosities that affect brewing characteristics and that these heterozygous alleles do segregate after sporulation. Correlation and principal component analyses suggested that the analytical parameters could be classified into two groups, indicating fermentation ability and sake flavour. Copyright © 2008 John Wiley & Sons, Ltd.     

Disruption of ubiquitin-related genes in laboratory yeast strains enhances ethanol production during sake brewing

Sake yeast can produce high levels of ethanol in concentrated rice mash. While both sake and laboratory yeast strains belong to the species Saccharomyces cerevisiae, the laboratory strains produce much less ethanol. This disparity in fermentation activity may be due to the strains' different responses to environmental stresses, including ethanol accumulation. To obtain more insight into the stress response of yeast cells under sake brewing conditions, we carried out small-scale sake brewing tests using laboratory yeast strains disrupted in specific stress-related genes. Surprisingly, yeast strains with disrupted ubiquitin-related genes produced more ethanol than the parental strain during sake brewing. The elevated fermentation ability conferred by disruption of the ubiquitin-coding gene UBI4 was confined to laboratory strains, and the ubi4 disruptant of a sake yeast strain did not demonstrate a comparable increase in ethanol production. These findings suggest different roles for ubiquitin in sake and laboratory yeast strains.     

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.     

Characteristics of the high malic acid production mechanism in Saccharomyces cerevisiae sake yeast strain No. 28

We characterized a high malic acid production mechanism in sake yeast strain No. 28. No considerable differences in the activity of the enzymes that were involved in malic acid synthesis were observed between strain No. 28 and its parent strain, K1001. However, compared with strain K1001, which actively took up rhodamine 123 during staining, the cells of strain No. 28 were only lightly stained, even when cultured in high glucose concentrations. In addition, malic acid production by the respiratory-deficient strain of K1001 was 2.5-fold higher than that of the wild-type K1001 and wild-type No. 28. The findings of this study demonstrated that the high malic acid production by strain No. 28 is attributed to the suppression of mitochondrial activity.     

绍兴酒酿制过程的生物学特性

阐述了绍兴酒酿制过程的生物学特性：配料的特殊性和酒种的多样性;发酵状态的多样性;低温长时间浸米;包含所有绍兴酒醪的发酵特点;微生物的多样性、复杂性;接种方式独特：菌种保存方法独特和神奇;是糖化和以多品种、高密度酵母与乳酸杆菌（细菌）协同作用的混合发酵并行的过程[即：边糖化与边酵母发酵、边乳酸杆菌（细菌）发酵同时协同进行的三边发酵];香雪酒是糖化和以多品种、高密度细菌（乳酸杆菌）发酵与少量酵母发酵协同作用的混合发酵并行的过程,酵母作用小,是真正意义上的细菌（乳酸杆菌）为主的发酵;酒液之间勾兑多;较高的灭菌温度;成品酒的贮存。     

甘蔗酒酿造酵母的筛选

选择常用商业果酒酵母菌株KD、DV10、Q23、EC1118、安琪和H7Y7作为供试酵母菌株,以川蔗17为原料发酵生产甘蔗酒,通过分析发酵液中糖含量变化情况及酒精度、澄清度,比较不同菌株发酵特性,结果发现EC1118菌株发酵彻底,产酒能力最强,发酵后甘蔗酒的高级醇含量最低;H7Y7菌株发酵的甘蔗酒中酯类物质最高,为其他菌株的3～6倍;安琪酵母发酵液最易澄清,但其发酵后的甘蔗酒中甲醇含量为其他菌株的5倍以上。感官评分结果显示,不同酵母菌株发酵的甘蔗酒感官评分依次为H7Y7EC1118DV10安琪酵母Q23KD。研究结果表明,供试酵母菌株中EC1118和H7Y7更适宜用于甘蔗酒酿造。     

糯米酒酿造酵母的优选

为了优选酵母菌种,改善液态发酵糯米酒的口感,采用酿酒酵母SJ4、扣囊复膜酵母3-1Y、粟酒裂殖酵母SP单菌种发酵及酿酒酵母/扣囊复膜酵母(1∶1)、酿酒酵母/粟酒裂殖酵母(1∶1)、酿酒酵母/扣囊复膜酵母/粟酒裂殖酵母(1∶1∶1)混合菌种发酵方式酿造糯米酒,通过曲线下面积(AUC)、主成分分析(PCA)对糯米酒风味物质及理化指标进行分析。结果表明,扣囊复膜酵母3-1Y与酿酒酵母SJ4(1∶1)混合发酵时AUC值最小,为9 371,相对贡献值最大,为3.448 7;糯米酒中总酸和氨基酸态氮含量有所提高,产品共检测了12种风味物质,总酯含量有增加,甲醇及高级醇有所降低,感官评分为7分,最适用于糯米酒的发酵。     

青岛啤酒酵母与高浓酵母筛选高浓酿造酵母融合亲株

以青岛啤酒酵母和高浓精酵母为供试菌株,筛选出生长良好的酵母,为选育具有青岛啤酒风味的高浓酵母做准备.比较了7株酵母不同糖类发酵、离子抗性、二氧化碳减重、发酵液风味品评等指标.结果表明:T1、T2和T3是传统的青岛啤酒发酵菌株,其发酵液口味符合青岛啤酒口味要求,且对Cu2+均不耐受;而G4和G6发酵减重试验和风味物质分析中的乙醛含量指标的评价均优于G5和G7菌株,且它们的发酵液的风味也接近青啤口味.因此,选择T1、T2、T3和G4、G6作100L酿造试验,进一步确定融合亲株.     

Using promoter replacement and selection for loss of heterozygosity to generate an industrially applicable sake yeast strain that homozygously overproduces isoamyl acetate

By application of the high-efficiency loss of heterozygosity (HELOH) method for disrupting genes in diploid sake yeast (Kotaka et al., Appl. Microbiol. Biotechnol., 82, 387–395 (2009)), we constructed, from a heterozygous integrant, a homozygous diploid that overexpresses the alcohol acetyltransferase gene ATF2 from the SED1 promoter, without the need for sporulation and mating. Under the conditions of sake brewing, the homozygous integrant produced 1.4 times more isoamyl acetate than the parental, heterozygous strain. Furthermore, the homozygous integrant was more genetically stable than the heterozygous recombinant. Thus, the HELOH method can produce homozygous, recombinant sake yeast that is ready to be grown on an industrial scale using the well-established procedures of sake brewing. The HELOH method, therefore, facilitates genetic modification of this rarely sporulating diploid yeast strain while maintaining those characteristics required for industrial applications.     

Constructing an amylolytic brewing yeast Saccharomyces pastorianus suitable for accelerated brewing

An amylolytic brewing yeast Saccharomyces pastorianus, free of vector sequences and drug-resistance genes, was constructed by disrupting the alpha-acetolactate synthase gene and introducing the alpha-amylase gene as a selective marker. The resulting recombinant strain was able to utilize starch as the sole carbon source and its alpha-acetolactate synthase activity was lowered by 30%. Fermentation tests confirmed that the diacetyl concentration and the residual oligosaccharide were reduced by 70% and 25%, respectively, in fermented wort by the recombinant strain, while the brewing performance of the recombinant strain was retained.     

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。     

7种酵母酿造摩尔多瓦葡萄酒的发酵特性比较

在常用的商业酵母中筛选一株酵母,应用于摩尔多瓦葡萄酒酿造,从而实现鲜食摩尔多瓦葡萄的增值。以摩尔多瓦葡萄为材料,选用VL1、F15、NS-D、P、HXD29、LE28、LD1015 7种酵母进行葡萄酒酿造试验,以自然发酵作为对照,对不同酵母发酵酒的总酸、总酚等理化性质进行检测和感官评价,比较不同酵母对摩尔多瓦葡萄酒理化性质及品质的影响。结果表明,酵母LD1015发酵的摩尔多瓦酒干浸物含量最高,为17.96 g/L;总糖含量为3.54 g/L,总酚含量为1.17 g/L,略低于酵母VL1、HXD29、LE28,感官评分最高,为91分。综合判定酵母LD1015具有较强的糖转化和干物质浸提的能力,有利于产出口感醇厚、香气馥郁的葡萄酒,是7种酵母中最适合鲜食葡萄摩尔多瓦酿酒的酵母菌种。