低产乙醛啤酒酵母的定向驯化筛选

为降低啤酒中乙醛含量,采用紫外线对1株啤酒工业生产菌株MI4进行诱变,经双硫仑平板初筛、乙醛培养基驯化复筛,获得了1株低产乙醛的啤酒酵母D-A-14。与出发菌株MI4相比,采用该突变株酿制的啤酒中乙醛含量为2.86 mg/L,降低了76%;且高级醇总量降低而酯含量升高,风味更加协调。这表明筛选得到的低乙醛突变株适于啤酒工业生产。     

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     

Construction of self-cloning industrial brewing yeast with high-glutathione and low-diacetyl production

Self‐cloning strains of industrial brewing yeast were constructed, in which one allele of α‐acetohydroxyacid synthase (AHAS) gene (ILV2) was disrupted by integrating Saccharomyces cerevisiae genes, γ‐glutamylcysteine synthetase gene (GSH1) and copper resistant gene (CUP1) into the locus of ILV2. The self‐cloning strains were selected for their resistance to CuSO₄ and identified by PCR amplification. The results of AHAS and glutathione (GSH) assay from fermentation with the self‐cloning strains in 500‐mL conical flask showed that AHAS activity decreased and GSH content increased compared with that of host yeasts. The results of pilot scale brewing in 5‐L fermentation tank also indicated that GSH content in beer fermented with self‐cloning strains T5‐3 and T31‐2 was 1.3 fold and 1.5 fold of that of host QY5 and QY31, respectively; and diacetyl content decreased to 64% and 58% of their hosts, respectively. The self‐cloning strains do not contain any heterologous DNA, they may be more acceptable to the public.     

基因组改组选育低色素高产茁霉多糖生产菌株

以出芽短梗霉(Aureobasidium pullulans)高产菌P1、P2和低色素菌P3、P4为出发菌,通过5轮基因组改组,摇瓶发酵复筛选出2株产色素能力低、多糖产量较高的菌株F51、F52,其中菌株F51、F52多糖产量分别为42.38、42.60g/L,比出发菌株P1多糖产量提高41.27%、42.00%,发酵液OD值为0.378、0.363,比出发菌P3降低17.30%、20.57%。     

基因组改组技术选育苯基乳酸高产菌株

以Lactobacillus paracaseiW2为出发菌株,应用紫外诱变和亚硝基胍诱变筛选出5株突变菌株(U4、U7、U12、N3、N6),采用突变菌株进行多母本递推式原生质体融合,最终筛选出一株正向突变株F8,其发酵液中苯基乳酸含量为1.92g/L,比原始菌株提高了1.37倍。并且确定了最佳破壁条件为:溶菌酶浓度20g/L,变溶菌素浓度为15μg/mL;最佳融合条件为:常温下,聚乙二醇的浓度为50%,作用时间为10min。      

基因组改组快速提高日本小球藻脂肪产量

以日本小球藻为出发藻株,经过紫外线和甲基磺酸乙酯分别诱变处理,获得4株总脂产量有所提高的突变株。以聚乙二醇作为融合剂,对获得的突变株进行两轮递归式原生质体融合,筛选到遗传稳定的改组藻株F2C2,其总脂含量为59.01%,较原始藻株提高了101.4%。对日本小球藻的原始藻株和改组藻株F2C2的油脂含量进行分析,结果表明改组前后日本小球藻的总脂组成成分没有变化,但各组分含量有较大差别。      

基于基因组重排技术选育D-核糖高产菌株

以SFR-3A为出发菌株,构建获得一株具有正反向筛选标记的菌株SFR-3A-ZMS;并通过该菌株和菌株SFR-4进行基因组重排,实现高抗逆性D-核糖高产菌株SFRCP-100的快速高效选育。在酵母浸粉、玉米浆及硫酸铵添加量分别为7.5 g/L、3 g/L及7 g/L的优化氮源条件下,5 L发酵罐中D-核糖产量达到38.2 g/L,其转化率和整体生产效率分别达到35%和0.73 g/（L·h）。本实验成功添加正反向筛选标记,提高了基因组重排技术在枯草芽孢杆菌（Bacillus subtilis）优良菌株选育中的有效性和高效性。     

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.     

产番茄红素红酵母基因组重排实验条件的优化

目的:探索基因组重排技术应用于产番茄红素红酵母育种中关键步骤的最优条件。方法:本实验以3株产生番茄红素的红酵母cp-2-11、cp-2-6、cp-28为实验菌株,研究菌体培养时间、酶解浓度、酶解时间、灭活条件以及融合时间对基因组重排的影响。结果:当菌体培养12h,酶解浓度为2%,酶解70min时,原生质体制备与再生率最佳;15W紫外灯,照射距离为35cm,磁力搅拌50r/min的条件下照射18min,60℃下灭活35min作为双亲灭活条件;35%聚乙二醇4000（PEG-4000）作为融合剂,处理23min为最佳融合时间。结论:通过基因组重排最佳条件的确定,后续重排实验的效率得到很大的提升,最终筛选出了高产的目的菌株,产量较融合亲本提高了122%。      

基因组改组技术在微生物育种方面的应用

基因组改组技术是传统微生物菌株改良方法的一种替代方法,该项技术通过基因组水平的递归重组,高效实现整个生物体的定向进化,突破了传统微生物菌株改良方法的局限性。利用基因组改组技术可以在较短时间内得到大量性状优良的正向突变株,具有提高改良菌株产量、增强菌株对环境的耐受性、提高底物利用率等优点,被称为代谢工程和菌株选育发展过程中的一个重要里程碑。该文总结了基因组改组技术的基本概念和一般流程,重点介绍了该项技术在微生物育种方面的应用,并且对该项技术的发展前景进行了展望。     

工业化酿造过程中酵母分泌蛋白酶A的规律及影响因素的研究

对啤酒工业化规模发酵过程中酵母分泌蛋白酶A的规律进行了探讨,对酵母代数及酵母贮存条件等因素对酵母分泌蛋白酶A的影响进行了研究,并对蛋白酶A活性不同的成品纯生啤酒的泡持值、泡沫活性蛋白含量及蛋白酶A活性进行了跟踪分析。结果表明:发酵过程中,蛋白酶A的活性呈上升趋势且接种酵母的蛋白酶A活性越高,与其对应的发酵液中蛋白酶A的活性越高,成品酒的泡沫稳定性越差。另外,随着酵母代数及贮存时间的增加,酵母分泌蛋白酶A的量增加。当酵母蛋白酶A活性控制在0.015U/m L以下且成品酒的初始蛋白酶A活性在15×10-5U/m L以下时,储存4个月的成品纯生啤酒的泡沫稳定性较好。      

改良Genomeshuffling技术选育埃博霉素B高产菌株

目的：用改genomeshuffling技术选育埃博霉素高产菌株。方法：本研究对genomeshu御ing技术进行了改良,具体是在递归原生质体融合过程中对原生质体进行了紫外诱变;通过UV和DES复合诱变方法获得T4株出发菌株,并用改良genomeshuffling技术选育埃博霉素B高产菌株。结果：通过三轮基因组重组成功选育到了2株遗传稳定的高产埃博霉素B重组菌株,其中一株重组菌株S0073-45的埃博霉素B产量达到T42．5mg／L。结论：该研究证明改良后的基因组重组技术不仅能有效提高埃博霉素B的产量,而且比传统的基因组重组技术更为高效。     

Enhanced deacidification activity in Schizosaccharomyces pombe by genome shuffling

A problem frequently occurring in making some kinds of wines, particularly Vitis quinquangularis Rehd wine, is the presence of malic acid at high concentrations, which is detrimental to the quality of wines. Thus, there is a need of the ways for effectively reducing the malic acid levels in wine. This study aimed to generate shuffled fusants of Schizosaccharomyces pombe with enhanced deacidification activity for reducing the excessive malic acid content in wine. Sz. pombe CGMCC 2.1628 was used as the original strain. The starting mutant population was generated by UV treatment. The mutants with higher deacidification activity were selected and subjected to recursive protoplast fusion. The resulting fusants were screened by using the indicator of malic acid concentration of fermentation supernatants on 96‐well microtitre plates, measured with bromocresol green. After three rounds of genome shuffling, the best‐performing fusant, named GS3‐1, was obtained. Its deacidification activity (consumed 4.78 g/l malic acid within 10 days) was increased by 225.2% as compared to that of original strain. In the Vitis quinquangularis Rehd wine fermentation test, GS3‐1 consumed 4.0 g/l malic acid during the whole cycle of fermentation, providing up to 185.7% improvement in malic acid consumption compared with that of the original strain. This study shows that GS3‐1 has great potential for improving the quality of Vitis quinquangularis Rehd wine. Copyright © 2014 John Wiley & Sons, Ltd.     

Improvement of soy-sauce flavour by genome shuffling in Candida versatilis to improve salt stress resistance

Genome shuffling of Candida versatilis was applied to improve the soy-sauce flavour by further increasing salt stress resistance. A mutant S3-5, with a stronger resistance to salt, was isolated after three rounds of genome shuffling. It is found that S3-5 improved stress tolerance to higher potassium chloride and lithium chloride. S3-5 not only grew well in the YPD (peptone, yeast extract and dextrose) medium containing high concentrations of sodium chloride with various pH values, but also exhibited improvement of growth ability in soy-sauce medium. The main aroma compounds in soy sauce were distinctly improved. Notably, S3-5 produced about 2.78 times as much ethanol as the control strain. Another important aroma compound, 4-hydroxy-2 (or 5)-ethyl-5 (or 2)-methyl-3 (2H)-furanone (HEMF) was enhanced by up to 80%. Meanwhile, S3-5 accelerated flavour formation of soy sauce thus decreasing the total time required for the aroma development.     

Optimised quantification of the antiyeast activity of different barley malts towards a lager brewing yeast strain

The brewing of beer involves two major biological systems, namely malted barley (malt) and yeast. Both malt and yeast show natural variation and assessing the impact of differing malts on yeast performance is important in the optimisation of the brewing process. Currently, the brewing industry uses well-established tests to assess malt quality, but these frequently fail to predict malt-associated problem fermentations, such as incomplete fermentations, premature yeast flocculation (PYF) and gushing of the final beer product. Antimicrobial compounds, and in particular antiyeast compounds in malt, may be one of the unknown and unmeasured malt factors leading to problem fermentations. In this study, the adaptation of antimicrobial assays for the determination of antiyeast activity in malt is described. Our adapted assay was able to detect differing antiyeast activities in nine malt samples. For this sample set, malts associated with PYF during fermentation and gushing activity in beer showed high antiyeast activity. Both PYF and gushing are malt quality issues associated with fungal infection of barley in the field which may result in elevated antimicrobial activity in the barley grain. Also, two more malts that passed the normal quality control tests were also observed to have high antiyeast activity and such malts must be considered as suspect. Based on our results, this assay is a useful measure of malt quality as it quantifies the antiyeast activity in malt which may adversely impact on brewery fermentation.     

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.     

Construction of a brewing yeast expressing the glucoamylase gene STA1 by mating

Standard brewing yeast cannot utilize larger oligomers or dextrins, which represent about 25% of wort sugars. A brewing yeast strain that could ferment these additional sugars to ethanol would be useful for producing low‐carbohydrate diabetic or low‐calorie beers. In this study, a brewing yeast strain that secretes glucoamylase was constructed by mating. The resulting Saccharomyces cerevisiae 278/113371 yeast was MAT a/α diploid, but expressed the glucoamylase gene STA1 . At the early phase of the fermentation test in malt extract medium, the fermentation rate of the diploid STA1 strain was slower than those of both the parent strain S. cerevisiae MAFF113371 and the reference strain bottom‐fermenting yeast Weihenstephan 34/70. At the later phase of the fermentation test, however, the fermentation rate of the STA1 yeast strain was faster than those of the other strains. The concentration of ethanol in the culture supernatant of the STA1 yeast strain after the fermentation test was higher than those of the others. The concentration of all maltooligosaccharides in the culture supernatant of the STA1 yeast strain after the fermentation test was lower than those of the parent and reference strains, whereas the concentrations of flavour compounds in the culture supernatant were higher. These effects are due to the glucoamylase secreted by the constructed STA1 yeast strain. In summary, a glucoamylase‐secreting diploid yeast has been constructed by mating that will be useful for producing novel types of beer owing to its different fermentation pattern and concentrations of ethanol and flavour compounds. Copyright © 2017 The Institute of Brewing & Distilling     

海红果酒酵母筛选及其发酵工艺的响应面法优化

选取8种活性干酵母对海红果酒进行了发酵实验,以残糖和酒精度为考量参数,筛选出适于海红果酒酿造的最优菌株。在单因素实验的基础上,选取发酵温度、接种量、发酵时间为影响因子,以残糖为响应值,应用中心组合Box-Behnken实验设计构建二次回归方程的数学模型,进行了响应面分析。结果表明,Z2酵母是海红果酒酿造的最优菌株,优化后的海红果酒发酵工艺条件为:发酵温度为23℃;接种量为0.25g/L;发酵时间为35d,在此条件下,发酵所得的海红果酒残糖量为5g/L,且果香浓郁,酒体丰满。      

Lachancea thermotolerans as an alternative yeast for the production of beer

The ability of Lachancea thermotolerans strains to ferment brewer's wort has been investigated. Initial fermentations with three L. thermotolerans strains compared the use of maltose and maltotriose, as well as production of glycerol and lactic acid and pH evolution over the course of the fermentation. The most promising strain was subsequently tested for additional traits important for beer production, including pitching rate, generational capacity, foam stability, hop tolerance, vicinal diketone production, oxygen requirement and flocculation. These tests suggest that L. thermotolerans may be a good choice for producing sour beers in a single fermentation step without the use of lactic acid bacteria. Copyright © 2016 The Institute of Brewing & Distilling     

超高浓酿造下抗葡萄糖阻遏啤酒酵母的筛选

根据高浓发酵下(16°P)发酵度的高低,挑选下面啤酒酵母C12作为出发菌株。经过2-去氧-D-葡萄糖的定向驯养、抗性平板分离初筛以及复筛验证等步骤,筛选出一株抗葡萄糖阻遏效应的菌株CM23。将该菌株在18°P麦汁15℃条件下进行3L的EBC小型啤酒发酵实验并测定发酵指标。结果表明:与出发菌株相比,CM23的降糖速度提高了37%,达到1.8°P/d,真正发酵度达到66%,且双乙酰还原能力以及啤酒中主要风味物质含量基本不变。CM23是一株具有工业应用前景的啤酒超高浓酿造酵母菌株。