The use of atmospheric and room temperature plasma mutagenesis to create a brewing yeast with reduced acetaldehyde production

High acetaldehyde levels in beer from yeast metabolism is a major concern for brewers in China. To obtain a strain with lower acetaldehyde production, this work reports a novel approach based on atmospheric and room temperature plasma mutagenesis and high‐throughput screening using 4‐methylpyrazole + disulphiram plating. A mutant LAL‐8a with lower acetaldehyde‐producing capability was obtained. The alcohol dehydrogenase activity decreased by 54% compared with the wild‐type M14 and the aldehyde dehydrogenase activity increased by 64% of the wild‐type strain. Through domestication and fermentation in EBC tubes, the mutant LAL‐8a was shown to produce 2.2 mg/L acetaldehyde, 88.2% less than the wild‐type strain M14. In addition, the ratio of higher esters to alcohols in beer fermented by the mutant LAL‐8a (0.28) was higher than M14 (0.16). The fermentation performance of LAL‐8a was similar to that of the wild‐type M14. This work suggests strain LAL‐8a a promising option for the brewing industry. © 2018 The Institute of Brewing & Distilling     

低产乙酸啤酒酵母菌株A12的选育及中试

以啤酒酿造生产菌株啤酒酵母JM-36为出发菌株,用甲基磺酸乙酯(EMS)诱变,用含浅蓝菌素的麦芽汁琼脂平板分离抗浅蓝菌素的突变株,在低温下发酵,以发酵液的乙酸、双乙酰、乙醛、高级醇、发酵度和凝聚性为筛选指标,得到1株发酵特性优良的菌株A12。以13°BX麦芽汁为培养基,用100L发酵罐在10℃下发酵14d,菌株A12发酵液的发酵度为68.2%,乙酸、双乙酰、乙醛和高级醇的含量分别为62.3mg/L、0.081mg/L、5.321mg/L和76.43mg/L。菌株A12的主要发酵特性优良且稳定,啤酒口感良好。     

降低上面发酵小麦啤酒头痛感的酿造条件优化

啤酒上头的原因主要是较高的醇酯比和乙醛含量。为降低上面发酵小麦啤酒头痛感,对其发酵条件进行优化,并通过气相色谱（GC）检测啤酒中高级醇、酯类和乙醛等风味物质含量。结果表明,最优发酵条件为发酵温度18 ℃（醇酯比2.02、乙醛含量3.51 mg/L）、发酵压力0.12 MPa（醇酯比2.01、乙醛含量2.75 mg/L）和零代酵母（醇酯比2.20、乙醛含量2.76 mg/L）。在此条件下发酵的小麦啤酒醇酯比为2.0～2.5,乙醛含量＜4 mg/L,不易引起上头。     

Reduced acetaldehyde production by genome shuffling of an industrial brewing yeast strain

High levels of acetaldehyde produced by yeast during fermentation can be of concern to product quality. A novel approach, based on genome shuffling, was applied to reduce the production of acetaldehyde by industrial brewing strain YS86. Four isolates with different impacts of acetaldehyde concentration were obtained from populations generated by ultraviolet irradiation and nitrosoguanidine mutagenesis. These yeast strains were then subjected to recursive pool‐wise protoplast fusion. A strain library that was likely to yield positive colonies was created by fusing the lethal protoplasts obtained from both UV irradiation and heat treatments. After two rounds of genome shuffling, a recombinant YSF2–9 strain produced less acetaldehyde than wild‐type strain YS86, by 64.5 and 66.2% in laboratory and pilot plant fermentations, respectively. The shuffled yeast strain YSF2–9 was genetically stable and may have a potential application in brewing industry for managing acetaldehyde in beer. Copyright © 2017 The Institute of Brewing & Distilling     

Construction of recombinant industrial brewer’s yeast with lower diacetyl production and proteinase A activity

The characteristic buttery taste of diacetyl has long been a major problem in the brewing industry, and the foam stability of unpasteurized beer is often influenced by proteinase A (PrA), which is encoded by PEP4 and released from yeast cells into beer during brewing. A recombinant industrial brewer’s yeast strain that reduces the diacetyl content of beer and improves foam stability was constructed. We constructed a PGK1p-ILV5-PGK1t expression cassette, which was introduced into one of the PEP4 alleles via PCR-mediated homologous recombination. Then, the second PEP4 allele was disrupted using the Cre-loxP recombination system, and the recombinant strain was designated as S-CSIK12. The results show that the diacetyl production of S-CSIK12 is always lower than that of the host strain at all stages of beer fermentation. In addition, brewing with S-CSIK12 reduced the PrA activity of the final beer by 44 % compared with that using the wild-type strain. The head retention of the beer brewed with S-CSIK12 (260 ± 2 s) was better than that of the host strain S-6 (212 ± 3 s). Considering that more PrA is released from yeast cells during the final stage of main fermentation and that the timing of yeast cropping is determined by diacetyl reduction, brewing with strains that have low diacetyl production also reduced the PrA activity of the beer and improved its head retention. The present study provides reference for the brewing industry as well as research on the diacetyl reduction and foam stability of beer.     

Fed‐batch fermentation with glucose syrup as an adjunct for high‐ethanol beer brewing

To produce a beer with a high ethanol content, preliminary research on fed‐batch fermentation profiles with glucose syrup as an adjunct during the primary fermentation period was conducted. The ethanol concentration of the beer was elevated by feeding a glucose syrup into the fermentors at a later stage of primary fermentation. Fermentation trials were carried out using a typical lager strain, SC‐9, with a pitching rate at 7.0 × 10⁶ cells/mL. An all‐malt wort (12.5°P) was employed and the primary fermentation temperature was 14 °C. Glucose syrup was supplemented when the concentration of residual reducing sugars was decreased to ~10 g/L. Results showed that the supplemented glucose was consumed rapidly and that the ethanol concentration in the final beer was raised to 67.9 g/L. Additional growth of yeast was observed after feeding accompanied by a low yield of ethanol (~0.46 g/g). Formation of diacetyl was enhanced by yeast growth and two additional peaks were obtained after feeding. The peak value of the diacetyl concentration was 1.90 mg/L. The fed‐batch fermentation resulted in a beer with an overproduction of higher alcohols and esters, indicating that brewing under these experimental conditions led to an unbalanced flavour profile. Results of optimization demonstrated that the optimal conditions were found to be 15°P for initial wort extract, 10 °C for fermentation temperature and 20 × 10⁶ cells/mL for yeast pitching rate, leading to total higher alcohols of 173.8 mg/L, total esters of 22.8 mg/L and an acetaldehyde concentration of 40.5 mg/L. A 12 day maturation and fermentation temperature of 8 °C was needed to reduce the acetaldehyde to 14.3 mg/L. Copyright © 2014 The Institute of Brewing & Distilling     

啤酒酵母高级醇的代谢与调控研究进展

高级醇是啤酒酵母在啤酒酿造过程中代谢产生的,是啤酒风味物质的重要组成部分。适量的高级醇能赋予啤酒独特的香味,但其含量过高或者过低都会影响啤酒的质量。该文重点综述啤酒酵母中高级醇的生成途径、关键基因、代谢调控机理及选育低产高级醇优良菌株的主要方法,为适当降低啤酒中高级醇含量,进而推动啤酒行业的健康发展提供理论基础。     

抗老化啤酒酵母的选育

以无锡轻工大学生物工程学院保藏的一株啤酒酵母为出发菌株，经紫外线诱变及蛋氨酸连续驯养后，选育得到一株抗老化性能较为优良的菌株Ｍ４．在１ｍ３发酵罐中的中试结果表明，与出发菌株相比，其羰基化合物（ＴＢＡ）含量降低１９．１％，谷胱甘肽（ＧＳＨ）含量增加２９．６％．在不改变其它生产条件的情况下，啤酒风味稳定性提高９２％，工业应用前景良好。     

The Impact of Xanthohumol on a Brewing Yeast's Viability,Vitality and Metabolite Formation

In order to understand how xanthohumol affects a brewing yeast's metabolism, yeast viability and vitality were studied during the production of a xanthohumol enriched beer (10 mg/L xanthohumol) on a 50 L pilot plant scale. The results showed that yeast viability was not significantly affected, but yeast vitality in the xanthohumol enriched brewing trials was slightly better. The content of higher alcohols, esters and organic acids was similar to the control in all the xanthohumol enriched brewing trials, however the content of sulphur dioxide, acetaldehyde and saturated fatty acids was lower in the xanthohumol enriched brewing trials. To the authors' knowledge, this is the first time brewing trials have been carried out showing that xanthohumol has a positive effect on a yeast's physiological condition.     

不同大小酵母细胞对啤酒发酵的影响

采用4株不同大小的酵母菌进行啤酒发酵,在比较不同大小酵母菌发酵性能的基础上,考察了相应酿造参数和理化指标的变化;另外,还对啤酒发酵过程中酵母菌大小对啤酒风味物质形成的影响进行了相应的研究。结果发现,不同大小的酵母菌对啤酒发酵的影响存在一定的差异,细胞较小的酵母发酵性能较好,生产的啤酒高级醇含量相对较高、物理性能好;而细胞偏大的酵母产醋酸酯较多。     

啤酒酵母融合株GR8发酵特性的初步研究

研究了啤酒酵母原生质体融合株GR8的主要发酵特性。以12°Bx麦芽汁为三角瓶发酵培养基,在12℃下发酵8d,融合株GR8的发酵度为66.5%,凝絮性(本斯值)为3.0mL;发酵液中的双乙酰、乙醛、高级醇和乙酸乙酯等风味物质的含量分别为0.0583mg/L、9.66mg/L、91.20mg/L和22.8mg/L。从主要发酵特性的指标和口感品评以及遗传稳定性的结果表明,融合株GR8是一株具有工业应用前景的啤酒酿造酵母菌株。     

Mutagenizing brewing yeast strain for improving fermentation property of beer

A brewing yeast mutant with perfect sugar fermentation capacity was isolated by mutagenizing the Saccharomyces pastorianus transformant, which carries an integrated glucoamylase gene and has one copy of non-functional alpha-acetolactate synthase gene. The mutant was able to utilize maltotriose efficiently, and the maltotriose fermentability in YNB-2% maltotriose medium increased from 32.4% to 72.0% after 5 d in shaking culture. The wort fermentation test confirmed that the sugar fermentation property of the mutant was greatly improved, while its brewing performances were analogous to that of the wild-type strain and the characteristic trait of shortened beer maturation period was retained. Therefore, we believe that the brewing yeast mutant would benefit the beer industry and would be useful for low caloric beer production.     

改进酿酒酵母谷物发酵性能的诱变研究

酿酒酵母经诱变获得有效利用麦汁糖的优良性能。突变菌株经5d摇床培养使YNB基础培养基的麦芽三糖发酵度从32.4%提高到81.8%。薄层层析证明突变株麦芽三糖转运能力得到改善。模拟工业发酵实验证明突变菌株的麦汁糖发酵性能大大改善,而原有发酵优良性状未受到影响。说明该突变株益于工业啤酒生产和低热啤酒的生产。     

压力对啤酒发酵的影响

啤酒发酵过程中,随着压力的升高,发酵速度、耗糖率、乙醇生成量、双乙酰的生成和还原速率有所下降;风味物质中的醇类、脂类下降,而乙醛含量却上升。压力还使酵母细胞形态产生了变化。     

比利时/德国小麦啤酒风味物质差异研究

以大麦芽、小麦芽和未发芽的小麦为原料,添加酒花、橘皮和芫荽籽,使用上面发酵酵母No.303,酿造比利时风格和德国风格小麦啤酒。该研究介绍了两种风格小麦啤酒的酿造工艺,对两种风格的成品小麦啤酒进行风味物质检测分析以及感官品评,探讨了比利时风格小麦啤酒和德国风格小麦啤酒风味物质的差异。结果表明,比利时风格小麦啤酒乙醛含量更为适宜（约为2.6 mg/L）,高级醇和乙酸含量较高（分别为113 mg/L和160 mg/L）,酯类物质含量偏低（约为50 mg/L）,成品啤酒橘香味突出,但酯香味不够充足;德国风格小麦啤酒乙醛和酯类物质含量略高（分别为3 mg/L和63 mg/L）,高级醇含量稍低（约为104 mg/L）,乙酸含量适宜（约为135 mg/L）。     

啤酒酵母乙醛代谢关键酶相关性研究

乙醛是啤酒中的主要风味物质,其代谢主要来自酵母细胞。酵母中乙醇脱氢酶及乙醛脱氢酶是乙醛代谢的关键酶,对乙醛变化起着重要作用。跟踪啤酒酵母发酵过程中相对酶活力及乙醛变化,发现两种乙醇脱氢酶和乙醛脱氢酶的相对酶活力与发酵过程乙醛含量变化具有一定相关性。同时对低产乙醛啤酒酿酒酵母kb2-4与出发菌株啤酒酵母kb进行发酵试验,跟踪检测相对酶活力及乙醛含量,其乙醇脱氢酶Ⅰ和乙醇脱氢酶Ⅱ及乙醛脱氢酶相对酶活力均高于出发菌株,平均增幅分别为15.5%,11.6%和5%。3种酶活性的变化协同作用可以使乙醛含量降幅最大为33.8%。     

Using Faster Gas Chromatography Analyses in Brewing Analytics

Dominant trends in analytical chemistry include miniaturization in sample preparation techniques and faster run times to provide high‐throughput screening, fast process monitoring and fast method development. This study focused on the application of narrow bore gas chromatographic capillary columns, 0.18 mm internal diameter, for brewing analyses. On these capillary columns, faster analyses could be performed compared to conventional GC capillary columns using 0.32 to 0.53 mm internal diameters. The robustness of the state‐of‐the‐art faster capillary gas chromatography, without compromising resolution, has been demonstrated with the analyses of beer flavour compounds such as lower and higher alcohols, esters and other volatile compounds such as acetaldehyde and dimethyl sulphide in beer. These methods were able to reduce sample run times by 60%.     

ATF1过表达和BAT2敲除酿酒酵母发酵性能的研究

以啤酒酵母工业菌株S5为对照,对过表达醇乙酰基转移酶编码基因ATF1同时敲除氨基酸转氨酶编码基因BAT2酿酒酵母工程菌株S5-1进行发酵性能的研究。结果表明,与出发菌株相比,突变株生长状况、发酵速度、酒精度等基本发酵性能没有明显变化,而突变株发酵后的乙酸酯总量有较大程度的提升,为85.44mg/L,提高了6.96倍,高级醇总量有较大程度的下降,为79.25mg/L,降低了27.28%。研究结果为啤酒风味的改善奠定了良好的基础。     

啤酒挥发性风味成分研究进展

气味作为啤酒重要的风味特征,由啤酒的挥发性成分决定。高级醇、有机酸和酯类是其中最重要的三类化合物,主要与酿造使用的酵母菌有关。在啤酒花提供的几百种挥发性化合物中,萜烯类化合物所占比例最高,它赋予啤酒花香、松木香等气味。在啤酒的生产和贮藏过程中发生的美拉德反应是引起啤酒老化的原因之一。老化过程中产生的反-2-壬烯醛、Strecker醛、双乙酰等物质会导致啤酒产生异味。该文综述近年来关于啤酒挥发性成分和气味的研究进展,并对改善啤酒的品质提出建议。     

Application of Plackett–Burman experimental design for investigating the effect of wort amino acids on flavour‐active compounds production during lager yeast fermentation

Aroma‐active higher alcohols and esters are produced intracellularly in the cytosol by fermenting lager yeast cells, which are of major industrial interest because they determine aroma and taste characteristics of the fermented beer. Wort amino acid composition and their utilization by yeast during brewer's wort fermentation influence both the yeast fermentation performance and the flavour profile of the finished product. To better understand the relationship between the yeast cell and wort amino acid composition, Plackett–Burman screening design was applied to measure the changes in nitrogen composition associated with yeast amino acids uptake and flavour formation during fermentation. Here, using an industrial lager brewing strain of Saccharomyces pastorianus , we investigated the effect of amino acid composition on the accumulation of higher alcohols and volatile esters. The objective of this study was to identify the significant amino acids involved in the flavour production during beer fermentation. Our results showed that even though different flavour substances were produced with different amino acid composition in the fermentation experiments, the discrepancies were not related to the total amount of amino acids in the synthetic medium. The most significant effect on higher alcohol production was exercised by the content of glutamic acid, aromatic amino acids and branch chain amino acids. Leucine, valine, glutamic acid, phenylalanine, serine and lysine were identified as important determinants for the formation of esters. The future applications of this information could drastically improve the current regime of selecting malt and adjunct or their formula with desired amino acids in wort. Copyright © 2017 The Institute of Brewing & Distilling