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.     

Behaviour of ethyl caproate during the production and distillation of ethyl caproate‐rich rice shochu

To increase the popularity of rice shochu, a process was developed to produce ethyl caproate‐rich rice shochu by adding a cultured broth of a caproic acid‐producing bacterial (CAPB) consortium to the fermentation. When the CAPB consortium containing fermented mash was subjected to vacuum distillation, the distillation efficiency of ethyl caproate was up to ~300%, while the distillation efficiencies of the other flavour compounds and ethanol were <100%. The behaviour of ethyl caproate during the production and distillation of an ethyl caproate‐rich rice shochu was investigated and the results showed that ethyl caproate was synthesized by Saccharomyces cerevisiae during the shochu production process, and that some of the ethyl caproate synthesized was secreted into the medium. Ethyl caproate in the medium evaporated easily and was transferred to the distillate when distilled. The increase in ethyl caproate after distillation was mainly derived from the release of the intracellular ethyl caproate, with negligible input from the chemical esterification of caproic acid and ethanol. During vacuum distillation, although few yeast cells were disrupted, secretion of the intracellular ethyl caproate was more efficient owing to the increase in temperature, allowing the distillation efficiency of ethyl caproate to be >100%. Copyright © 2016 The Institute of Brewing & Distilling     

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.     

Development of a process for producing ethyl caproate‐ and ethyl lactate‐rich rice shochu

To increase the popularity of rice shochu, a process was developed to produce ethyl caproate‐ and ethyl lactate‐rich rice shochu. On a laboratory‐scale, there was a shochu production trial with Saccharomyces cerevisiae Y‐E. Caproic acid added in the second‐stage fermentation was esterified to ethyl caproate. Both ethyl caproate and ethyl lactate were produced by adding a caproic acid‐producing bacterial (CAPB) consortium and lactic acid bacterium (LAB) to the shochu production process. Yellow koji was more appropriate for producing a flavour‐rich shochu with the addition of a CAPB consortium and LAB than white koji. Optimal addition time for the CAPB consortium and LAB was on the first day of the second‐stage fermentation, judging from concentrations of ethyl caproate and ethyl lactate produced. Additional dosages of CAPB consortium and LAB positively affected the formation of ethyl caproate and ethyl lactate, respectively. Shochu production with the addition of 2% and 4% CAPB consortium led to ethyl caproate concentrations of 27.3 mg/L and 47.9 mg/L in genshu, respectively, and the shochu achieved the best sensory test score from the Japanese shochu brewery panellists. Copyright © 2015 The Institute of Brewing & Distilling     

酿酒酵母Y3401产己酸乙酯发酵条件的优化

以酿酒酵母Y3401为研究对象,对该菌株发酵产己酸乙酯条件进行优化。首先,通过单因素对其培养基条件(pH值和糖度)及诱导条件(温度、转速、接种量、乙醇添加量、己酸添加量、前体添加时机和诱导时间)进行优化,然后通过Plackett-Burman试验,筛选出5个显著影响酿酒酵母Y3401产己酸乙酯的因素,再通过最陡爬坡试验确定最大响应区域,最后采用Box-Behnken试验设计及响应面分析,确定其最优产己酸乙酯条件为:糖度14 Brix、初始pH 7、温度25℃、转速180 r/min、接种量3.5%、乙醇添加量8%、己酸添加量0.059%、前体添加时机30 h、诱导时间31 h。在此培养条件下己酸乙酯产量达10.1 mg/L。本研究结果有助于酿酒酵母Y3401更好地应用于白酒发酵中。     

利用己酸菌酯化液提高口子窖优质酒得率

将己酸菌酯化液应用于生产,以使口子窖基酒中己酸乙酯含量每毫升提高100mg以上,平均提高优质酒得率10%左右。     

酯化酶技术与白酒增香

利用酯化酶的催化作用,使己酸更多地合成己酯乙酯,以提高酒质。酯化酶增香技术可用于传统固态白酒,可使己酸乙酯含量成倍增加;用于新工艺白酒,使酒体具有自然发酵增香特色。（一平     

利用窖泥甲烷菌与己酸菌二元发酵技术提高泸型曲酒质量的研究

泸型曲酒生产中的一个大问题是其优质品在总出酒率中所占比例不高。而解决些问题的关键是提高酒中己酸乙酯的含量。本试验采用己酸菌与甲烷菌共栖生长,同时发酵的技术,对提高酒体中己酸乙酯含量效果很好。若以酒体中己酸乙酯含量180mg/100ml计,其优质品率可达到总出酒量的80.9％。     

仰韶酒微量组分的分析研究

甲烷菌、己酸菌二元复合菌发酵培养人工老窖技术提高了仰韶酒己酸乙酯的含量,丙酸菌的作用抑制了乳酸乙酯的生成,利用先进的毛细管气相色谱分析技术对仰韶酒微量组分进行深入分析     

红曲霉Monascus sanguineus X1处理黄水的培养基优化及酯化液制备

黄水是白酒酿造的主要副产物之一,富含多种有机质,可用于制备酯化液促进白酒增香,提高其利用率。研究了一株高产酯化酶的红曲霉菌株Monascus sanguineus X1,以酯化酶活力为指标,从碳源、碳氮比、接种量和pH值4个方面对X1菌株的培养基进行单因素实验及正交试验优化;采用酯化酶液处理黄水,制备酯化液,研究了黄水、乙醇、己酸等酯化前体物质添加量对酯化液制备的影响。结果表明,该红曲霉优化发酵培养基组分(以100 mL计):大米粉7.0 g,大豆蛋白胨2.0 g,NaNO30.2 g,KH2PO40.15 g,MgSO4·7H2O 0.1 g,培养基初始pH值5.0,接种量10%(体积分数)。在此条件下,添加体积分数为10%的黄水和体积分数为4%的乙醇,酶活力可达745.80 U/mL。向酯化酶液中加入体积分数为0.8%的己酸和体积分数为20%的乙醇继续培养1 d后,酯化液中己酸乙酯的体积分数可达0.121%;而向酯化酶液中补加体积分数为10%的黄水后,己酸乙酯和乳酸乙酯的体积分数分别可达0.055%和0.032%。该结果旨在为将黄水资源用于白酒增香提供理论参考。     

Sake: Production and flavor

Abstract

Sake, its history in Japan, the unique brewing process, and microbes concerned with the characteristics of flavor are described. The main flavor components derived mainly from the fermentation process are higher alcohols, isoamyl acetate, ethyl caproate, and phenethyl acetate. These are the compounds which give an accent to the flavor of sake. The esters are formed mainly by yeast during mash fermentation. Isoamyl acetate is produced by the reaction of acetyl CoA with isoamyl alcohol catalyzed by alcohol acetyl transferase. The enzyme, bound to the yeast cell membrane, is unstable to heat and unsaturated fatty acids. The ester formation is regulated by the amount of isoamyl alcohol produced. Acyl CoA alcohol acyl transferease catalyzes the formation of ethyl caproate from caproyl CoA and ethanol. In this reaction, the amount of caproyl CoA supplied is important. The mechanism of higher alcohol formation, including the biosynthetic pathway of amino acids and its feedback regulation, is discussed. Finally, breeding and the practical use of sake yeast with high productivity of higher concentrations of higher alcohols and esters are described.     

浓香型白酒酒醅中乳酸菌分离及其对模拟固态发酵的影响

从发酵酒醅中分离得到21株产乳酸菌株,其中16株为芽孢杆菌。利用梅里埃微生物鉴定仪鉴定,得到干燥棒杆菌（Corynebacterium xerosis）、耳葡萄球菌（Staphylococcus auricularis）、枯草芽孢杆菌（Bacillussubtilis）、巨大芽孢杆菌（Bacillus megaterium）、腊状芽孢杆菌（Bacillus cereus group）、浸麻芽孢杆菌（Paenibacillusmacerans）6种产乳酸菌。将6株产酸菌进行模拟固态发酵,结果显示所有乳酸菌都不同程度的抑制了己酸、己酸乙酯以及乳酸乙酯的产量,其中浸麻芽孢杆菌、巨大芽孢杆菌属的2株产乳酸菌严重抑制了乙醇和己酸乙酯的生成量,巨大芽孢杆菌的一株还严重抑制了己酸的产量。     

Analyses of peptides in sake mash: forming a profile of bitter-tasting peptides

Some oligopeptides and amino acids have a strong influence on the sensory qualities of sake, but the formation process of such compounds in sake mash has not been well elucidated. In this study, we investigated the formation process of bitter-tasting peptides derived from rice proteins in sake mash, because knowledge about their formation may contribute to the quality control of sake. We analyzed rice protein hydrolysates in sake mash, as well as in the enzymatic digest of steamed rice grains digested by either sake-koji or by crude enzyme extracted from sake-koji. SDS–PAGE showed that a smaller amount of polypeptides (> M.W. 10,000) accumulated in the supernatant of sake mash than in either enzymatic digest. The concentration of peptides in the supernatant of sake mash increased gradually from the early stages of fermentation. Five bitter-tasting peptides (No. 9, < QLFNPS; No. 13, < QLFNPSTNP; No. 17, < QLFNPSTNPWH; No. 18, < QLFNPSTNPWHSP; No. 20, < QLFGPNVNPWHNP), which were previously found in sake mash, were not found in significant amounts in sake-koji. On the other hand, these peptides accumulated at the early stages of both sake mash fermentation and the enzymatic digests, although the levels in sake mash were higher than those in the digests. The present study demonstrated that the 5 bitter-tasting peptides formed in high concentrations when steamed rice grains were digested under conditions of sake mash fermentation with yeast.     

保持低度浓香型白酒质量稳定技术的研究

在低度浓香型白酒勾兑过程中,通过有针对性的选择使用己酸含量较大的调味酒,适当提高己酸含量,能有效地抑制、延缓己酸乙酯的水解,达到稳定低度浓香型白酒酒质、延长其货架期的目的.     

白酒基酒典型风味物质含量的测定方法与差异性研究

建立一种新的、同时测定杜康不同级别白酒基酒中11 种典型风味物质含量的气相色谱外标法,采用AT.LZP-930型色谱柱（25 m×0.32 mm,0.1 μm）,氢火焰离子检测器检测,进样口温度220 ℃,检测器温度250 ℃,柱流速2.0 mL/min,载气为氮气（纯度99.999%）,载气流速30 mL/min,H2流速30 mL/min,空气流速300 mL/min,尾吹流速25 mL/min;采用分流进样方式,分流比10∶1,进样量1 μL。结果表明：该方法简便、准确性高;通过对不同级别白酒基酒中11 种典型风味物质含量的测定与分析,结合品酒师的品评结果可以得出：杜康不同级别中白酒基酒11 种典型风味物质中己酸乙酯、乳酸乙酯、乙酸乙酯、己酸4 种物质的含量差异显著（P＜0.05）,其中乙酸乙酯、己酸乙酯、己酸的含量随着级别的降低而显著递减,而乳酸乙酯的含量随着级别的降低而递增,其他7 种物质的含量差异不显著（P＞0.05）;己酸乙酯与乳酸乙酯、己酸乙酯与乙酸乙酯的最佳比例分别在1.2∶1以下和1.7∶1～1.4∶1,己酸含量在160 mg/100 mL时最佳。     

Effect of cellular inositol content on ethanol tolerance of Saccharomyces cerevisiae in sake brewing

The effect of cellular inositol content on the ethanol tolerance of sake yeast was investigated. In a static culture of strain K901 in a synthetic medium, when cells were grown in the presence of inositol in limited amount (L-cells), the inositol content of cells decreased by one-third that of cells grown in the presence of inositol in sufficient amount (H-cells). L-cells exhibited a higher death rate constant than H-cells in the presence of 12-20% ethanol, while no difference in specific ethanol production rate in the presence of 0-18% ethanol between the two cell types was observed. L-cells leaked more intracellular components, such as nucleotides, phosphate and potassium, in the presence of ethanol than H-cells. L-cells exhibited a lower intracellular pH value than H-cells, which represented the lowering of cell vitality by the decrease in H(+) extrusion activity. Furthermore, the plasma membrane H(+)-ATPase activity of L-cells was approximately one-half of that of H-cells. Therefore, it was considered that the decrease in viability in the presence of ethanol due to inositol limitation results from the lowering of H(+)-ATPase activity, which maintains the permeability barrier of the yeast membrane, ensuring the homeostasis of ions in the cytoplasm of yeast cells. It is assumed that the lowering of H(+)-ATPase activity due to inositol limitation is caused by the change in lipid environment of the enzyme, which is affected by inositol-containing glycerophospholipids such as phosphatidylinositol (PI), because in the PI-saturated mixed micellar assay system, the difference in H(+)-ATPase activity between L- and H-cells disappeared. In the early stage of sake mash, inositol limitation lowers the ethanol tolerance due to the decrease in H(+)-ATPase activity as in static culture. In the final stage of sake mash, the disruption of the ino1 gene responsible for inositol synthesis, resulted in a decrease in cell density. Furthermore, the ino1 disruptant, which was not capable of increasing the cellular inositol level in the final stage, exhibited a significantly higher methylene blue-staining ratio than the parental strain. It was suggested that the yeast cellular inositol level is one of the important factors which contribute to the high ethanol tolerance implied by the increased cell viability in the presence of ethanol.     

红曲霉在浓香型酒生产中的应用

研究发现，红曲霉能代谢出促进己酸与乙酸合成己酸乙酯的酯化酶。引入红曲霉菌种，制成帘曲应用于浓香型酒生产中。（1）直接混入大 发酵；（2）应用于双轮底发酵；（3）制成酯化液。红曲霉帘曲糖化力为2666u/g，用量不可过大。应用红曲霉帘曲，可提高出酒率1％－2％，提高优质品率10％。（丹妮）     

Detection of a point mutation in FAS2 gene of sake yeast strains by allele-specific PCR amplification

To identify yeast mutants with a point mutation, detection of the specific mutant alleles is necessary. For this purpose, we applied allele-specific polymerase chain reaction (PCR) to detect the FAS2-1250S dominant mutant allele that encodes an altered fatty acid synthase in Japanese brewer's yeast strains. These strains are known to produce a higher amount of ethyl caproate in Japanese sake. The mutant strains were supposed to be diploid and to contain heterozygous alleles, including wild-type FAS2 and a dominant FAS2-1250S. A set of oligonucleotide primers was designed to contain different nucleotides at their 3' termini: one type was identical to the wild type and the other to the mutant FAS2. Another set of primers was designed to have an additional mismatch at the second nucleotide from their 3' termini. By testing with control strains, we established PCR conditions for specific amplification. Using these conditions and a simple template preparation procedure with SDS, the presence of the allele was detected in commercially used sake yeast strains. The method presented here will be useful for the identification of specific yeast strains.     

试论红曲霉在传统白酒中的生产与应用

在传统白酒中,以己酸乙酯为主的酯类成分,对酒质的等级有较为明显的影响.相比于目前广泛使用的乙酸菌、红曲霉菌可以促使己酸、乙醇进行酯化反应,生成己酸乙酯,因此对白酒提质有良好效果.从斜面培养、扩大培养等方面,介绍了制作红曲霉的工艺流程,随后采用对比法,分析了红曲霉菌对于基酒中总酸、总酯、出酒率以及己酸乙酯、乙酸乙酯等各类...