几株啤酒酵母的筛选及发酵性能比较*

啤酒酵母是啤酒酿造的灵魂,可以直接影响啤酒品质。在啤酒酿造过程中,由于啤酒酵母被多次传代和保藏,造成优良菌种发酵性能衰退等问题,导致发酵不彻底,影响最后啤酒的风味质量。为此以8株Lager型啤酒酵母为出发菌株,通过平板分离纯化获得80株分离菌株,再经过三角瓶发酵初筛和复筛、发酵罐中试发酵实验最终获得了8株发酵性能优良的啤酒酵母。其中,6株酵母可应用于酿造双乙酰含量低于0.1 mg/L的啤酒;3株酵母发酵度高于70%,适合酿造干啤酒;1株酵母发酵度低于50%,适合酿造低醇啤酒。在风味方面:1株酵母酿造的啤酒醇酯比为3.3,啤酒酯香味较突出;另1株酵母酿造的啤酒醇酯比为4.5,啤酒高级醇含量较高。8株经过选育的啤酒酵母发酵特征明显,便于精酿啤酒厂实际应用。     

一株优良啤酒酵母菌的筛选及性能检测

以朝阳啤酒厂的啤酒酵母菌CY3为出发菌株,在分离培养基中加入0.1%土霉素溶液,以发酵液中双乙酰含量为主要测定指标,同时测定发酵度、发酵速度、死灭温度、凝聚性等指标,从而筛选出1株优良啤酒酵母菌。     

低双乙酰啤酒酵母菌株BEZ112的选育

以啤酒酿造生产菌株啤酒酵母(Saccharomyces cerevisiae)FB作为出发菌株,用甲基磺酸乙酯(EMS)诱变,经分离筛选得到一株优良的啤酒酵母菌株BEZ112。该菌株的絮凝性、发酵度、酒精度、发酵液的总酯和总高级醇的含量等特性保持了亲株的优良性状。但以12°Bx麦芽汁为培养基用500mL三角瓶在12℃下发酵,该菌株发酵至第4d,发酵液中的双乙酰含量达到峰值(0.291mg/L),比出发菌株FB发酵4d的峰值降低了30％,发酵至第8d,BEZ112发酵液中的双乙酰含量比出发菌株FB的降低了23％。以12°Bx麦芽汁为培养基用500L罐在12℃下发酵8d,BEZ112发酵液中的双乙酰含量(0.091mg/L)比出发菌株FB的(0.124mg/L)降低了27％。发酵得到的啤酒口感纯正清爽。     

优良啤酒酵母原生质体融合株GR5的构建及其发酵特性

以发酵度较高的非絮凝性的啤酒酵母菌株X6和发酵度较低、絮凝性较强的啤酒酵母菌株N1为亲本进行原生质体融合。用亚硝酸诱变原养型的菌株X6,经筛选得到一株需酪素水解物的营养缺陷型菌株X6~20。采用正交试验法分别优化菌株X6~20和N1的原生质体形成和再生的条件。用X6~20菌株的原生质体作为受体和热灭活的N1菌株原生质体作为供体进行融合。融合株经三角瓶发酵筛选,得到一株较优良的融合株GR5。该融合株的絮凝性较强(本斯值为2.7),以12°Bx麦芽汁为培养基,用500 L的发酵罐在12℃下发酵,发酵至第8 d菌株GR5的发酵度为69.2%,发酵液中的双乙酰含量为0.0498 mg/L、乙醛含量为6.34 mg/L,总高级醇含量为74.4mg/L。融合株GR5具有双亲的优点,发酵的啤酒风味较好,是一株具有工业应用前景的啤酒酿造酵母菌株。     

利用果渣产黄腐酸菌剂的筛选

以果渣发酵生产黄腐酸可实现农业有机固定废弃物的资源化利用。从腐植酸样品中分离获得4株耐高温菌,以果渣为基质发酵生产黄腐酸,通过测定黄腐酸产量和木质纤维素降解率,从中筛选出2株能够较好降解木质纤维素的黄腐酸生产菌株BFA02和BFA03,其黄腐酸产量分别为43.67、59.35 g/kg。经16S rDNA分类鉴定,初步确认BFA02为解淀粉芽孢杆菌,BFA03为地衣芽孢杆菌。选择季也蒙酵母、长枝木霉分别与BFA02、BFA03复配发酵生产黄腐酸。结果表明,复合菌剂发酵生产黄腐酸产量高于单菌发酵。其中,由BFA02、BFA03和长枝木霉组成的复合菌剂3实验组黄腐酸产量最高,达到103.19 g/kg,较BFA02、BFA03单菌发酵分别提高了136%和74%,其纤维素、半纤维素、木质素的降解率分别为46.25%、39.49%、37.5%,均高于单菌发酵。黄腐酸生产菌株与长枝木霉复配,能够有效促进木质纤维素降解率,提高黄腐酸产量,在果渣废弃物资源化利用领域有着极大的潜力。     

ILV2基因缺失对啤酒酵母生长与双乙酰代谢的影响

【目的】旨在应用分子生物学方法降低啤酒发酵液中双乙酰含量,改善啤酒感官质量。【方法】以酿酒酵母S2(Saccharomyces cerevisiae)为出发菌株,通过同源重组敲除四倍体啤酒酵母α-乙酰乳酸合成酶部分基因(ILV2),构建缺失一个和两个ILV2等位基因的突变株QI2-1和QI2-2,并进行啤酒发酵实验。【结果】ILV2基因的缺失,会导致菌株初始生长速率的降低。其中QI2-2较为明显,12 h时,突变株与出发菌株的生长速率达到一致。啤酒发酵结果表明,与出发菌株相比,突变株QI2-1双乙酰峰值与双乙酰最终含量分别降低17.50%和17.83%,而QI2-2分别降低51.67%和45.65%。其他啤酒指标如酒精度、发酵度、残糖和风味物质等略有变化,但都在优质啤酒指标范围内,符合啤酒发酵的质量要求。【结论】通过同源重组敲除部分ILV2基因和选育低产双乙酰菌株是降低啤酒双乙酰含量、提高啤酒质量的有效方法,具有一定的实际应用价值。     

啤酒生产中双乙酰形成的分子遗传学及其控制

双乙酰是啤酒中的重要风味物质,也是影响啤酒成熟和质量的关键因素之一。双乙酰是由酵母缬氨酸生物合成的中间产物α－乙酰乳酸经氧化脱羧产生的。目前,可以通过改良传统发酵工艺,添加酶制剂以及利用基因工程手段选育酵母工程菌等方法降低双乙酰在啤酒中的含量,缩短啤酒后酵期,以加速啤酒成熟。     

信息库

1.在固定化酵母生物反应器中进行啤酒快速成熟试验 在啤酒酿造中,通常α-乙酰乳酸自然氧化成双乙酰,再由酵母将双乙酰转化成乙偶姻。这两步是同时进行的,总共约需一个月时间,其中第一步是限速的关键。加温可以加速α-乙酰乳酸向双乙酰的转化,但也会影响酵母的生活力或阻断乙醇的形成。为了加速酿造过程就需要将这两个同时进行的步骤分为前后两步进行。啤酒快速酿造法的主发酵是在固定化酵母反应器中连续进行的。生产的新鲜啤酒中,α-乙酰乳酸的浓度比常规方法生产的啤酒略高。由于啤酒酵母没有α-乙酰乳酸脱羧酶,本系统的第一步是用热处理将80％α-乙酰乳酸快速转化成乙偶姻,余下的在第二步固定化酵母反应器中由酵母转化成乙偶姻。     

采用新型药物抗性选育高效黄酒酵母菌株

【目的】黄酒酵母菌种是影响大罐黄酒生产效率的关键因素之一,高发酵性能黄酒酵母的选育对于提升黄酒生产效率具有重要的实用意义。【方法】以一种药物抗性为基础设计快速初筛方法,对黄酒酿酒酵母XY进行诱变筛选得到克霉唑(CTZ)抗性的黄酒酵母突变株,进一步以酵母发酵性参数为指标筛选得到发酵速率提高的黄酒酵母新菌株XY-3。【结果】比较突变菌株与原始菌株酿造性能发现,XY-3菌株最大发酵速率较原始菌株提高5.21%。黄酒酿造小试结果显示发酵前4天XY-3菌株乙醇生成速率明显高于原始菌株,其最大乙醇生成速率从XY菌株的14.77 g/d提高到15.30 g/d。XY-3酵母菌株发酵速率的提高能够缩短黄酒发酵周期1-2天,有助于提高发酵设备的利用效率。进一步研究发现XY-3发酵速率的提升可能与XY-3菌株多药物抗性(PDR)有关。【结论】联合使用传统诱变和药物抗性筛选策略选育得到高效黄酒酵母新菌株,这种筛选策略也为发酵食品行业优良菌株的选育提供了一种新的思路。     

紫外线诱变原生质体选育核黄素高产菌株

以产核黄素生产用菌——阿舒假囊酵母RS-89为出发菌株,用对致期生长的菌丝体,经0.5%蜗牛酶+0.5%纤维素酶作用2h可获得大量原生质体,其再生率为6.1%。对经UV诱变的原生质体再生株进行初筛、发酵复筛,从中获得了菌落大、色素深、产量高于出发菌株30%的高产稳定株——UP-91。     

Enzymatic Removal of Diacetyl from Beer: II. Further Studies on the Use of Diacetyl Reductase1

Diacetyl removal from beer was studied with whole cells and crude enzyme extracts of yeasts and bacteria. Cells of Streptococcus diacetilactis 18-16 destroyed diacetyl in solutions at a rate almost equal to that achieved by the addition of whole yeast cells. Yeast cells impregnated in a diatomaceous earth filter bed removed all diacetyl from solutions percolated through the bed. Undialyzed crude enzyme extracts from yeast cells removed diacetyl very slowly from beer at its normal pH (4.1); at a pH of 5.0 or higher, rapid diacetyl removal was achieved. Dialyzed crude enzyme extracts from yeast cells were found to destroy diacetyl in a manner quite similar to that of diacetyl reductase from Aerobacter aerogenes, and both the bacterial and the yeast extracts were stimulated significantly by the addition of reduced nicotinamide adenine dinucleotide (NADH). Diacetyl reductase activity of four strains of A. aerogenes was compared; three of the strains produced enzyme with approximately twice the specific activity of the other strain (8724). Gel electrophoresis results indicated that at least three different NADH-oxidizing enzymes were present in crude extracts of diacetyl reductase. Sephadex-gel chromotography separated NADH oxidase from diacetyl reductase. It was also noted that ethyl alcohol concentrations approximately equivalent to those found in beer were quite inhibitory to diacetyl reductase.     

Identification of Sc-type ILV6 as a target to reduce diacetyl formation in lager brewers' yeast

Diacetyl causes an unwanted buttery off-flavor in lager beer. It is spontaneously generated from α-acetolactate, an intermediate of yeast's valine biosynthesis released during the main beer fermentation. Green lager beer has to undergo a maturation process lasting two to three weeks in order to reduce the diacetyl level below its taste-threshold. Therefore, a reduction of yeast's α-acetolactate/diacetyl formation without negatively affecting other brewing relevant traits has been a long-term demand of brewing industry. Previous attempts to reduce diacetyl production by either traditional approaches or rational genetic engineering had different shortcomings. Here, three lager yeast strains with marked differences in diacetyl production were studied with regard to gene copy numbers as well as mRNA abundances under conditions relevant to industrial brewing. Evaluation of data for the genes directly involved in the valine biosynthetic pathway revealed a low expression level of Sc-ILV6 as a potential molecular determinant for low diacetyl formation. This hypothesis was verified by disrupting the two copies of Sc-ILV6 in a commercially used lager brewers' yeast strain, which resulted in 65% reduction of diacetyl concentration in green beer. The Sc-ILV6 deletions did not have any perceptible impact on beer taste. To our knowledge, this has been the first study exploiting natural diversity of lager brewers' yeast strains for strain optimization.     

Enzymatic Removal of Diacetyl from Beer : III. Enzyme Protection and Regeneration of Cofactor

Use of diacetyl reductase, a reduced nicotinamide adenine dinucleotide (NADH)-requiring enzyme, to eliminate diacetyl off-flavor in beer was studied. The crude enzyme was extracted from Aerobacter aerogenes and partially purified by ammonium sulfate precipitation or Sephadex chromatography. In the semipure state, the enzyme was inactivated by lyophilization; in a crude state, the lyophilized extract remained stable for at least 4 months at - 20 C. A 50% reduction in specific activity within 5 min was observed when crude diacetyl reductase was suspended (5 mg of protein/ml) in phosphate buffer at pH 5.5 or below; a similar inactivation rate was observed when the crude enzyme was dissolved in a 5% aqueous ethyl alcohol solution. Effective crude enzyme activity in beer at a natural pH of 4.1 required protection of the enzyme in 10% gelatin. Incorporation of yeast cells with the gel-protected enzyme provided regeneration of NADH. Combinations of yeast, enzyme, and gelatin were tested to obtain data analyzed by regression analysis to determine the optimal concentration of each component of the system required to reduce the level of diacetyl in spiked (0.5 ppm) beer to less than 0.12 ppm within 48 hr at 5 C. The protected enzyme system was also effective in removing diacetyl from orange juice (pH 3.8) and some distilled liquors.     

Chromosomal Integration and Expression of Two Bacterial alpha-Acetolactate Decarboxylase Genes in Brewer's Yeast

A bacterial gene encoding alpha-acetolactate decarboxylase, isolated from Klebsiella terrigena or Enterobacter aerogenes, was expressed in brewer's yeast. The genes were expressed under either the yeast phosphoglycerokinase (PGK1) or the alcohol dehydrogenase (ADH1) promoter and were integrated by gene replacement by using cotransformation into the PGK1 or ADH1 locus, respectively, of a brewer's yeast. The expression level of the alpha-acetolactate decarboxylase gene of the PGK1 integrant strains was higher than that of the ADH1 integrants. Under pilot-scale brewing conditions, the alpha-acetolactate decarboxylase activity of the PGK1 integrant strains was sufficient to reduce the formation of diacetyl below the taste threshold value, and no lagering was needed. The brewing properties of the recombinant yeast strains were otherwise unaltered, and the quality (most importantly, the flavor) of the trial beers produced was as good as that of the control beer.     

Recombinant brewer's yeast strains suitable for accelerated brewing.

Four brewer's yeast strains carrying the alpha-ald gene of Klebsiella terrigena (ex. Aerobacter aerogenes) or of Enterobacter aerogenes on autonomously replicating plasmids were constructed. The alpha-ald genes were linked either to the ADC1 promoter or to the PGK1 promoter of yeast Saccharomyces cerevisiae. In pilot scale brewing (50 l) with three of these recombinant yeasts the formation of diacetyl in beer was so low during fermentation that lagering was not required. All other brewing properties of the strains were unaffected and the quality of finished beers was as good as that of finished beer prepared with the control strain. The total process time of beer production could therefore be reduced to 2 weeks, in contrast to about 5 weeks required in the conventional process.     

A new yeast strain for brewery: Properties and advantages

Beer is a natural product and is a multicomponent system that has both positive and negative consumer properties. Organoleptical off-flavors of beer are difficult to eliminate. Yeasts are the main active component of the system. The relationship between beer quality and yeast usage is well known. New industrial strains for brewery are continuously developed. An industrial yeast Saccharomyces cerevisiae strain was obtained and showed high technological properties, including efficient fermentation, a reduced production of sulfur hydrate, and a high diacetyl reduction rate. The advantages made it possible to develop new brands of beer and nonalcoholic products. The commercial use of the strain was patented. The strain was deposited in the Russian Collection of Industrial Microorganisms.     

Genetic engineering of brewing yeast to reduce the content of ethanol in beer

The GPD1 gene encoding the glycerol-3-phosphate dehydrogenase was overexpressed in an industrial lager brewing yeast (Saccharomyces cerevisiae ssp. carlsbergensis) to reduce the content of ethanol in beer. The amount of glycerol produced by the GPD1-overexpressing yeast in fermentation experiments simulating brewing conditions was increased 5.6 times and ethanol was decreased by 18% when compared to the wild-type. Overexpression of GPD1 does not affect the consumption of wort sugars. Only minor changes in the concentration of higher alcohols, esters and fatty acids could be observed in beer produced by the GPD1-overexpressing brewing yeast. However, the concentrations of several other by-products, particularly acetoin, diacetyl and acetaldehyde, were considerably increased.     

啤酒酵母ECM25/YJL201W基因敲除对啤酒风味稳定性的影响

以pUG6为模板, 设计含有与ECM25基因两侧序列同源的长引物, 构建了带有卡那抗性基因(kanMX)破坏盒, 转化啤酒酵母G-03, 获得一株G-03/a转化菌, 遗传稳定性良好, 测序结果证实ECM25基因敲除是成功的。有氧条件下11oC和28oC培养时转化菌G-03/a的胞外谷胱甘肽(GSH)分泌量在对数生长期分别比原菌高21.4%和14.7%。在锥形瓶中连续发酵4代后, 与原菌株相比, 转化菌G-03/a发酵液、成品酒中GSH含量分别提高32.1%和13.8%, 发酵液和成品啤酒SI系数分别提高7.7%和5.3%, 成品啤酒RSV值提高45.0%。EBC管发酵6 d后, 与原菌株相比, 转化菌G-03/a发酵液中GSH含量提高34.0%。转化菌G-03/a与G-03所酿制成品啤酒的常规指标没有显著差别。表明G-03/a是一株具有抗老化能力的优良啤酒酵母, 能够提高啤酒的风味稳定性。