敲除啤酒酵母 fks1 基因对啤酒酵母自溶性能的影响

以啤酒酵母G-03为模板,扩增得到铜抗性基因(cup 1)和β-葡聚糖合成酶基因(fks 1)。将fks 1连接pMD-18T Vector得到重组质粒pTK,重组质粒pTK和cup1经Bgl Ⅱ、Sal Ⅰ酶切后连接得到重组质粒pKC。Bam HI酶切重组质粒pKC得到以fks 1为整合位点包含cup1的基因片段fks 1::cup1。用此片段转化啤酒酵母工业菌株G-03,通过硫酸铜抗性筛选得到一株啤酒酵母工程菌G-03/C。工程菌连续传代10次后依然能在筛选平板上生长,遗传稳定性良好。主酵结束G-03/C和G-03的辛酸和癸酸含量基本相同。25℃诱导自溶20 d,G-03/C的辛酸、癸酸分别下降57.3%、81.8%,自溶性能减弱。G-03/C的死亡率、双乙酰、浊度及TBA均较原菌有所下降。G-03/C与G-03酿制成品啤酒的常规指标没有较大差别,品评结果表明,G-03/C风味更优。     

构建高产谷胱甘肽啤酒酵母基因工程菌提高啤酒抗老化能力的研究

根据同源重组的原理,将来源于啤酒酵母工业菌株G03的γ-谷氨酰半胱氨酸合成酶基因（GSH1）和筛选标记Kan取代质粒pRJ-5中18S rDNA内部约340bp的DNA片段,构建重组质粒pRKG。以pRKG为模版,PCR得到以18S rDNA为整合位点包含GSH1和Kan的基因片段18S rDNA::(Kan-GSH1)。用此片段转化啤酒酵母工业菌株G03,通过G418抗性筛选得到啤酒酵母工程菌。实验室小试表明,工程菌的谷胱甘肽含量比受体菌株提高16.6%,啤酒的抗老化能力得到了显著提高,而常规指标没有发生显著变化。连续传代5次后胞内GSH含量基本不变遗传稳定性良好。由于表达γ-谷氨酰半胱氨酸合成酶的基因来源于受体菌株自身,是通过自克隆技术改造工业啤酒酵母的一次有益的尝试。     

构建高产谷胱甘肽啤酒酵母基因工程菌提高啤酒抗老化能力的研究

根据同源重组的原理,将来源于啤酒酵母工业菌株G03的γ-谷氨酰半胱氨酸合成酶基因(GSH1)和筛选标记Kan取代质粒pRJ-5中18S rDNA内部约340bp的DNA片段,构建重组质粒pRKG。以pRKG为模版,PCR得到以18S rDNA为整合位点包含GSH1和Kan的基因片段18S rDNA::(Kan-GSH1)。用此片段转化啤酒酵母工业菌株G03,通过G418抗性筛选得到啤酒酵母工程菌。实验室小试表明,工程菌的谷胱甘肽含量比受体菌株提高16.6%,啤酒的抗老化能力得到了显著提高,而常规指标没有发生显著变化。连续传代5次后胞内GSH含量基本不变遗传稳定性良好。由于表达γ-谷氨酰半胱氨酸合成酶的基因来源于受体菌株自身,是通过自克隆技术改造工业啤酒酵母的一次有益的尝试。     

选育抗老化啤酒酵母提高啤酒风味稳定性的研究

对一株啤酒酵母青②进行紫外线诱变 ,在蛋氨酸浓度为 15g L的耐性平板上筛选获得一株A₂₇菌株。为使A₂₇菌株的蛋氨酸耐性特征稳定遗传 ,在 11℃、稀释率为0.035h^-1 的条件下对A₂₇菌株进行高浓度蛋氨酸 (20g/L)连续驯养 ,然后分离得到一株M^I₄菌。在锥形瓶中连续发酵5代后 ,与A₂₇菌株相比 ,M^I₄ 菌株发酵液中GSH浓度高44% ,TBA值低 24% ,主酵液RSV值高 77% ,且遗传稳定性明显提高 :连续传代 5次后 ,M^I₄ 菌株胞内GSH含量基本不变 ,而A₂₇菌则下降了22%。在相同的糖化和发酵工艺条件下 ,对优选株M^I₄和出发株啤酒酵母青②进行 1m3发酵罐中试。与出发株青②相比 ,两者的总高级醇与酯量几乎不变 ,常规指标没有显著差别 ,品尝风味也基本一致。但M^I₄菌株发酵液中GSH浓度提高了 30% ,TBA值降低了19% ,成品啤酒的RSV值则提高了92% ,表明优选株M^I₄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.     

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。     

啤酒酵母工业菌株单倍体的诱导、分离和鉴定

【目的】探索适宜的方法进行啤酒酵母工业菌株单倍体的诱导、分离和鉴定,为啤酒酵母改良和遗传学研究提供便利。【方法】首先,选择产孢效果最好的培养基进行产孢诱导,诱导产生的孢子在YPD培养基上形成菌落后,用流式细胞技术检测其DNA含量,进而判断其倍性;单倍体菌株的交配型通过MAT-PCR和杂交实验确定。【结果】啤酒酵母工业菌G-03通过产孢诱导和孢子分离、富集后得到26株菌,最终通过流式细胞技术确定了其中4株为单倍体,MATa和MATα型各2株。通过扫描电镜法观察4株单倍体菌株及出发菌G-03的细胞形态,发现单倍体菌株的形态和出发菌有较大区别,单倍体菌株长期培养没有假丝生长的现象发生。【结论】啤酒酵母工业菌单倍体育种较为困难,严格的单倍体筛选、鉴定尤其具有挑战性。     

低乙醇脱氢酶Ⅱ活性的抗老化啤酒酵母工程菌的构建

采用自克隆技术,破坏啤酒酵母工业菌株YSF31的ADH2基因,在ADH2基因位点插入来源于YSF31的编码γ-谷氨酰半胱氨酸合成酶的GSH1基因和铜抗性筛选标记CUP1基因.通过铜抗性筛选转化子,经PCR和乙醇脱氢酶Ⅱ(ADH Ⅱ)活性测定验证,获得了1株啤酒酵母工程菌.10°P麦芽汁发酵实验显示,自克隆菌株的乙醇脱氢酶Ⅱ活性是受体菌的65%,谷胱甘肽含量比受体菌YSF31的高34%.其他发酵指标并没有发生明显改变.由于DNA操作过程中没有外源基因介入,因此啤酒酵母工程菌为生物安全的自克隆菌株,具有重要的应用价值.     

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。     

全小麦啤酒酵母菌株的诱变育种及应用研究

采用10 Kev低能N~+注入啤酒酵母,经筛选获得一菌株Lz37,再用150 MPa超高压处理菌株Lz37,经双乙酰平板筛选获得一菌株Gy3,其凝聚性很强,适合于在小麦汁中发酵啤酒,其发酵度为66%～68%,双乙酰含量低于口味阈值,遗传稳定性良好。将Gy3酵母定为全小麦啤酒生产应用酵母,命名为商啤3号(Sp-03)。SP-03啤酒酵母菌株的各项生理及生产性能都较优良,特别是在全小麦芽啤酒的酿造中适用性较强,经过对发酵工艺等的调整,用其酿制的啤酒口感纯正、淡爽、柔和。     

低乙醛Lager型啤酒酵母研究进展

啤酒酵母是啤酒酿造的核心,对啤酒风味及风味稳定性具有重要影响。乙醛是影响啤酒风味和风味稳定性最重要的醛类化合物,是酒精饮料中引起人类致癌的物质之一,主要通过啤酒酵母的生物代谢产生,存在于啤酒发酵过程及成品啤酒中。因此,筛选或选育优良的低产乙醛啤酒酵母菌株将成为有效解决啤酒风味稳定性的途径之一。近年来,随着基因工程技术的发展及啤酒酵母基因组的不断阐明,人们对啤酒酵母菌种改良展开了大量的研究,以期解决啤酒酿造问题,改善啤酒质量。本文对采用传统方式及基因工程手段选育低产乙醛啤酒酵母的最新研究进展进行了综述。其中,对低乙醛啤酒酵母选育的手段及策略进行了讨论并对低乙醛啤酒酵母选育的研究热点及发展趋势进行了展望。     

Construction of amylolytic industrial brewing yeast strain with high glutathione content for manufacturing beer with improved anti-staling capability and flavor

Glutathione in beer works as the main antioxidant compounds which correlates with beer flavor stability. High residual sugars in beer contribute to major non-volatile components which correlate to high caloric content. In this work, Saccharomyces cerevisiae GSH1 gene encoding glutamylcysteine synthetase and Scharomycopsis fibuligera ALP1 gene encoding alpha-amylase were co-expressed in industrial brewing yeast strain Y31 targeting at alpha-acetolactate synthase (AHAS) gene (ILV2) and alcohol dehydrogenase gene (ADH2), and new recombinant strain TY3 was constructed. The glutathione content from the fermentation broth of TY3 increased to 43.83 mg/l compared to 33.34 mg/l from Y31. The recombinant strain showed high alpha-amylase activity and utilized more than 46% of starch after 5 days growing on starch as sole carbon source. European Brewery Convention tube fermentation tests comparing the fermentation broth of TY3 and Y31 showed that the flavor stability index increased to 1.3 fold and residual sugar concentration were reduced by 76.8%, respectively. Due to the interruption of ILV2 gene and ADH2 gene, the amounts of off-flavor compounds diacetyl and acetaldehyde were reduced by 56.93% and 31.25%, comparing with the amounts of these from Y31 fermentation broth. In addition, as no drug-resistance genes were introduced to new recombinant strain, consequently, it should be more suitable for use in beer industry because of its better flavor stability and other beneficial characteristics.     

Physiological characterization of brewer’s yeast in high-gravity beer fermentations with glucose or maltose syrups as adjuncts

High-gravity brewing, which can decrease production costs by increasing brewery yields, has become an attractive alternative to traditional brewing methods. However, as higher sugar concentration is required, the yeast is exposed to various stresses during fermentation. We evaluated the influence of high-gravity brewing on the fermentation performance of the brewer’s yeast under model brewing conditions. The lager brewer’s strain Weihenstephan 34/70 strain was characterized at three different gravities by adding either glucose or maltose syrups to the basic wort. We observed that increased gravity resulted in a lower specific growth rate, a longer lag phase before initiation of ethanol production, incomplete sugar utilization, and an increase in the concentrations of ethyl acetate and isoamyl acetate in the final beer. Increasing the gravity by adding maltose syrup as opposed to glucose syrup resulted in more balanced fermentation performance in terms of higher cell numbers, respectively, higher wort fermentability and a more favorable flavor profile of the final beer. Our study underlines the effects of the various stress factors on brewer’s yeast metabolism and the influence of the type of sugar syrups on the fermentation performance and the flavor profile of the final beer.     

Absence of fks1p in lager brewing yeast results in aberrant cell wall composition and improved beer flavor stability

The flavor stability during storage is very important to the freshness and shelf life of beer. However, beer fermented with a yeast strain which is prone to autolyze will significantly affect the flavor of product. In this study, the gene encoding β-1,3-glucan synthetase catalytic subunit (fks1) of the lager yeast was destroyed via self-clone strategy. β-1,3-glucan is the principle cell wall component, so fks1 disruption caused a decrease in β-1,3-glucan level and increase in chitin level in cell wall, resulting in the increased cell wall thickness. Comparing with wild-type strain, the mutant strain had 39.9 and 63.41 % less leakage of octanoic acid and decanoic acid which would significantly affect the flavor of beer during storage. Moreover, the results of European Brewery Convention tube fermentation test showed that the genetic manipulation to the industrial brewing yeast helped with the anti-staling ability, rather than affecting the fermentation ability. The thiobarbituric acid value reduced by 65.59 %, and the resistant staling value increased by 26.56 %. Moreover, the anti-staling index of the beer fermented with mutant strain increased by 2.64-fold than that from wild-type strain respectively. China has the most production and consumption of beer around the world, so the quality of beer has a significant impact on Chinese beer industry. The result of this study could help with the improvement of the quality of beer in China as well as around the world.     

高SO2产量啤酒酵母工业菌株的构建

二氧化硫在啤酒中具有抗氧化的重要功能,而在其形成过程中APS激酶(MET14编码)起着非常重要的作用。以二氧化硫产量较高的青岛啤酒酵母(Saccharomyces cerevisiae)YSF-5的总DNA为模板,用PCR方法克隆得到MET14基因。为使目的基因在酿酒酵母中表达,以大肠杆菌-酿酒酵母穿梭质粒YEp352为载体,以PGK1强启动子为调控元件,构建了重组表达质粒pPM,并转化酿酒酵母YS58。转化子在YNB添加亮氨酸、组氨酸和色氨酸的选择性培养基上筛选鉴定,盐酸副玫瑰苯胺法测得转化子的SO2产量是受体菌的2倍左右。在重组表达质粒pPM的基础上添加铜抗性标记基因构建了重组表达质粒pCPM,并转化青岛啤酒工业酵母菌株YSF-38,转化子在YEPD 4mmol/L CuSO4的选择性培养基上筛选鉴定,实验室条件下培养后,测得转化子YSF-38(pCPM)的SO2产量是受体菌的3.2倍。用该转化子在青岛啤酒厂进行小型发酵实验,结果表明在发酵结束时,YSF-38(pCPM)转化子的SO2产量是受体菌的1.4倍。因此,MET14基因的有效表达可以提高啤酒工业酵母的SO2产量。     

Integrated expression of the α-amylase, dextranase and glutathione gene in an industrial brewer’s yeast strain

Genetic engineering is widely used to meliorate biological characteristics of industrial brewing yeast. But how to solve multiple problems at one time has become the bottle neck in the genetic modifications of industrial yeast strains. In a newly constructed strain TYRL21, dextranase gene was expressed in addition of α-amylase to make up α-amylase’s shortcoming which can only hydrolyze α-1,4-glycosidic bond. Meanwhile, 18s rDNA repeated sequence was used as the homologous sequence for an effective and stable expression of LSD1 gene. As a result, TYRL21 consumed about twice much starch than the host strain. Moreover TYRL21 speeded up the fermentation which achieved the maximum cell number only within 3 days during EBC tube fermentation. Besides, flavor evaluation comparing TYRL21 and wild type brewing strain Y31 also confirmed TYRL21’s better performances regarding its better saccharides utilization (83% less in residual saccharides), less off-flavor compounds (57% less in diacetyl, 39% less in acetaldehyde, 67% less in pentanedione), and improved stability index (increased by 49%) which correlated with sensory evaluation of final beer product.     

高温高浓发酵工业啤酒酵母菌种的构建

高温高浓发酵技术作为一项新兴的啤酒生产技术,它为啤酒生产带来诸多利益的同时,也存在着发酵结束后酵母絮凝性下降、高级醇生成量过高等系列问题。为提高高温高浓发酵条件下酿酒酵母的絮凝性同时降低高级醇的合成能力,首先构建了以酿酒酵母BAT2基因为整合位点过表达FLO5基因的菌株,重组菌株S6-BF的絮凝性达到67.67%,比出发菌株S6提高了29%,而高级醇生成量仅降低5.9%;进一步构建以BAT2基因为整合位点再次过表达FLO5基因的菌株,与出发菌株S6相比,重组菌株S6-BF2的絮凝性提高了63%,达到85.44%,高级醇生成量下降至159.58 mg/L,降低了9.0%;通过弱化线粒体支链氨基酸转氨酶(BAT1)的表达,高级醇的生成量得到进一步的降低,达到142.13 mg/L,比原始菌株S6降低了18.4%,同时重组菌株S6-BF2B1的絮凝性没有受到影响;风味物质的测定结果表明啤酒中醇酯比例较为合理。研究结果对工业啤酒酵母发酵后的沉降分离和提高啤酒风味品质有着重要的意义。     

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.