乙醇对酿酒酵母Saccharomyces cerevisiae应激性的研究进展

在酿酒发酵工业中,虽然酿酒酵母Saccharomyces cerevisiae具有较高的乙醇耐受能力和乙醇产率,但也有一个乙醇的耐受能力极限。乙醇应激对酿酒酵母所产生的影响,包括乙醇应激对细胞质膜、HSP热激蛋白、抗氧化酶、海藻糖的影响以及高温高压等因素如何影响酿酒酵母的乙醇耐受力;以及当前研究中存在的问题。     

海藻糖与酿酒酵母乙醇耐受性相关性的研究进展

为了得到具有高富锌能力的酵母菌,该研究以面包来源的酵母菌DLY28为出发菌株,重复驯化后筛选获得一株优良耐锌酵母,并通过单因素试验及响应面试验对其富锌培养基进行优化。结果表明,通过驯化筛选得到一株优良耐锌酵母S7,其富锌的最优培养基组成为蔗糖含量82 g/L、胰蛋白胨含量26 g/L、锌含量404 mg/L。在此最优条件下,富锌酵母S7的锌吸附量为18.79 mg/g,生物量（OD600 nm值）为1.49。该研究为富锌酵母的生产以及食品有机锌的开发应用提供理论依据。     

酿酒酵母对木质纤维素水解液抑制物耐受性的研究进展

在木质纤维素转化为燃料乙醇的过程中,预处理产生的多种抑制物对酿酒酵母产生毒害作用,抑制细胞内相关代谢关键酶的活性,影响细胞生长和发酵过程,降低发酵过程中糖的转化率。该文介绍了木质纤维素常用的三种预处理方式及在不同预处理过程中抑制物形成的规律和作用机制,对提高酿酒酵母耐受性的方法进行了综述,并展望了未来通过基因工程和代谢工程相互耦合共同提高酿酒酵母性能的研究趋势,为提高酿酒酵母抑制物耐受性和纤维素乙醇发酵效率提供参考和借鉴。     

多耐性酒精酵母菌的选育及特性研究

以酿酒酵母spc1,Y14,S16为出发菌株,经紫外线诱变和筛选,获得了3株对温度、乙醇浓度、pH耐性有所提高的正向突变株UVs,UV14,UV16;以这些菌株为出发菌株,进行三轮原生质体融合,最终获得了能耐受48℃,19%vol乙醇浓度和耐酸(pH 2.6)的酵母菌TY33.该突变株对温度和耐酒精能力分别提高了17.1%和58.3%.该菌株在35℃下发酵72h酒精产量为101.58g/L,比出发菌株spc1在35℃发酵液中最高乙醇浓度83.82g/L提高了21.1%.     

Simultaneous accumulation of proline and trehalose in industrial baker's yeast enhances fermentation ability in frozen dough

Freeze tolerance is a necessary characteristic for industrial baker's yeast because frozen-dough baking is one of the key technologies for supplying oven-fresh bakery products to consumers. Both proline and trehalose are known to function as cryoprotectants in yeast cells. In order to enhance the freeze tolerance of yeast cells, we constructed a self-cloning diploid baker's yeast strain with simultaneous accumulation of proline, by expressing the PRO1-I150T allele, encoding the proline-feedback inhibition-less sensitive γ-glutamyl kinase, and trehalose, by disrupting the NTH1 gene, encoding neutral trehalase. The resultant strain retained higher tolerance to oxidative and freezing stresses than did the single proline- or trehalose-accumulating strain. Interestingly, our results suggest that proline and trehalose protect yeast cells from short-term and long-term freezing, respectively. Simultaneous accumulation of proline and trehalose in industrial baker's yeast also enhanced the fermentation ability in the frozen dough compared with the single accumulation of proline or trehalose. These results indicate that baker's yeast that accumulates both proline and trehalose is applicable for frozen-dough baking.     

Improving the performance of industrial ethanol‐producing yeast by expressing the aspartyl protease on the cell surface

The yeasts used in fuel ethanol manufacture are unable to metabolize soluble proteins. The PEP4 gene, encoding a vacuolar aspartyl protease in Saccharomyces cerevisiae, was either secretively or cell‐surface anchored expressed in industrial ethanol‐producing S. cerevisiae. The obtained recombinant strains APA (expressing the protease secretively) and APB (expressing the protease on the cell wall) were studied under ethanol fermentation conditions in feed barley cultures. The effects of expression of the protease on product formation, growth and cell protein content were measured. The biomass yield of the wild‐type was clearly lower than that of the recombinant strains (0.578 ± 0.12 g biomass/g glucose for APA and 0.582 ± 0.08 g biomass/g glucose for APB). In addition, nearly 98–99% of the theoretical maximum level of ethanol yield was achieved (relative to the amount of substrate consumed) for the recombinant strains, while limiting the nitrogen source resulted in dissatisfactory fermentation for the wild‐type and more than 30 g/l residual sugar was detected at the end of fermentation. In addition, higher growth rate, viability and lower yields of byproducts such as glycerol and pyruvic acid for recombinant strains were observed. Expressing acid protease can be expected to lead to a significant increase in ethanol productivity. Copyright © 2010 John Wiley & Sons, Ltd.     

发酵工业中酿酒酵母耐性机制的研究进展

由于工业生产条件和成本的限制,发酵过程中酿酒酵母会经受多种不同环境胁迫因子的影响。研究不良环境中酿酒酵母的耐性机制,旨在为提高产品质量、降低生产成本以及相应的基因改良提供理论依据。文中主要综述了酿酒酵母耐受高渗、氧化和乙醇等胁迫因素的机理以及海藻糖对酵母细胞的保护作用。      

A new yeast genetic resource for analysis and breeding

We made a library of Saccharomyces cerevisiae F₁ hybrids from all possible crosses of 16 wild‐type strains, including two common laboratory strains and two commercial winemaking varieties. Fourteen of the starting strains have been sequenced. Thus, the sequences of both genomes are known in 182 novel hybrids, and the sequence of one genome is known in 56. All tested strains sporulated. Fertilities were in the range 0–100%. Hybrids showed no more variation than parental strains for ethanol production, ethanol tolerance or growth at temperature extremes, but some F₁s appeared to display hybrid vigour (heterosis). We tested four tetrads from one hybrid for their ability to grow at low temperature or in the presence of an inhibitory concentration of ethanol. Only one F₂ was as tolerant as the most tolerant F₀ parent. A few showed intermediate tolerance, but most were less tolerant than either parent or the F₁ hybrid, consistent with uncoupling of genes contributing to an optimized quantitative trait. The diversity and structure of the library should make it useful for analysis of genetic interactions among diverse strains, quantitative inheritance and heterosis, and for breeding. Copyright © 2010 John Wiley & Sons, Ltd.     

Susceptibility and resistance to ethanol in Saccharomyces strains isolated from wild and fermentative environments

In this work, we apply statistical modelling techniques to study the influence of increasing concentrations of ethanol on the overall growth of 29 yeast strains belonging to different Saccharomyces and non‐Saccharomyces species. A modified Gompertz equation for decay was used to objectively estimate the noninhibitory concentration (NIC) and minimum inhibitory concentration (MIC) for the assayed strains to ethanol, which are related to the susceptibility and resistance of yeasts to this compound, respectively. A first ANOVA analysis, grouping strains as a function of their respective Saccharomyces species, revealed that S. cerevisiae was the yeast with the highest, and statistically significant, ethanol resistance value. Then, a second factorial ANOVA analysis, using the origin of strains (wild or fermentative) and their taxonomic classification (S. cerevisiae, S. paradoxus or S. bayanus var. uvarum) as categorical predictor variables, showed that no significant differences for the NIC and MIC parameters were found between both ecological niches within the same species, indicative that these physiological characteristics were presumably not modified throughout the adaptation to human‐manipulated fermentative environments. Finally, differences among selected strains with respect to ethanol tolerance were correlated to the initial contents of unsaturated fatty acids, mainly oleic acid. Copyright © 2010 John Wiley & Sons, Ltd.     

产香酵母菌筛选、条件优化及发酵产物在电子烟烟液中的应用

为丰富电子烟烟液香气物质来源,开发具有良好风味的电子烟产品,筛选了食源性产香酵母菌,利用气相色谱质谱联用(GC/MS)和高效液相色谱(HPLC)分析致香成分,以关键致香成分为指标对产香条件进行正交试验优化,并将最优条件下获得的发酵产物进行电子烟加香效果评价。结果表明:(1)筛选获得1株能够以杨梅汁和葡萄汁为唯一营养物的酿酒酵母菌(Saccharomyces cerevisiae)KMLY1-2,该酵母菌发酵杨梅汁和葡萄汁后可产生较高水平的苯乙醇。(2)杨梅汁和葡萄汁发酵产苯乙醇的最优条件分别为接菌量5%和10%、温度30和25℃、转速200 r/min、pH 3和9;在此条件下,杨梅汁和葡萄汁的苯乙醇产量分别为47.49和60.99 mg/L,较未优化处理分别增加11.99和27.06 mg/L,较对照(未发酵杨梅汁或未发酵葡萄汁)分别增加47.32和60.99 mg/L。(3)最优条件下得到的发酵产物的嗅香得分最高;将发酵产物按1.6%(质量分数)比例添加至电子烟烟液基础溶剂中,制备的电子烟具有浓郁玫瑰花香,且产生的气溶胶香气谐调饱满,杂气轻,余味干净,抽吸品质好。酿酒酵母KMLY1...     

Impact of dried,creamed and cake supply formats on the genetic variation and ethanol tolerance of three Saccharomyces cerevisiae distilling strains

Scotch whisky fermentations typically employ high‐gravity fermentation practices to maximize product formation and to minimize both energy and water inputs. This approach increases ethanol concentrations at the end of fermentation, creating stressful conditions for the yeast. In this work we examined the relative tolerance of four Saccharomyces cerevisiae distilling yeast strains, supplied in dried, creamed, cake or slurry format, to ethanol under CO₂‐induced anaerobic conditions. The cells were assessed for their capacity to recover and grow on inhibition spot plates and to maintain cell viability in ethanol‐dosed suspensions. Variations in ethanol tolerance were observed between strains and between the same strain supplied in different formats. The creamed yeast format typically exhibited a higher tolerance to ethanol. One possible explanation for this observation is that cells surviving the dehydration and rehydration process might incur sub‐lethal genome damage. Thus the genetic integrity of the most ethanol‐tolerant strain was assessed as a function of supply format (two dried and one creamed). The mitochondrial DNA was examined using mitochondrial restriction fragment length polymorphism and the chromosomal DNA using pulsed field gel electrophoresis and polymerase chain reaction with both ITS and delta‐specific primers. In one dried yeast sample, genetic integrity was compromised, highlighting the requirement for yeast intake quality assurance programmes. Copyright © 2012 The Institute of Brewing & Distilling     

刺葡萄园土壤酿酒酵母的筛选及耐受性分析

从铜仁市刺葡萄园采集土样,采用涂布平板法、平板划线法、酵母浸出粉胨葡萄糖（YPD）培养基分离、纯化酵母,通过WL营养琼脂培养基筛选酿酒酵母（Saccharomyces cerevisiae）,并对其乙醇、糖、酸、温度及SO2耐受性进行分析。结果表明,共获得10株酵母菌株,其中菌株TR-2、TR-3、TR-6、TR-7、TR-10为酿酒酵母（Saccharomyces cerevisiae）,且菌株TR-6、TR-10耐受性较好,均分别能在乙醇体积分数为16%、葡萄糖质量浓度为500 g/L、柠檬酸质量浓度为30 g/L、SO2质量浓度为250 mg/L的YPD液体培养基和高温45 ℃、低温5 ℃条件下生长繁殖。     

耐高浓度酒精酵母菌与酒精敏感酵母菌中海藻糖含量与耐酒精的关系

对存在葡萄糖和无葡萄糖存在情况下的耐酒精酵母菌Saccharomycescerevisiae 12 0 0菌株和酒精敏感酵母菌SaccharomycescerevisiaeK5 5A菌株的耐酒精能力进行了比较。同时分析了它们在高浓度酒精冲击过程中的海藻糖含量变化。结果发现 ,这 2株酒精酵母菌的海藻糖含量与它们的耐酒精能力、保护线粒体膜完整性、防止线粒体丢失DNA有很大的关系     

AFLP analysis of type strains and laboratory and industrial strains of Saccharomyces sensu stricto and its application to phenetic clustering

Using nine primer pairs, amplified fragment length polymorphism (AFLP) analysis was conducted to characterize industrial, laboratory and type strains of Saccharomyces sensu stricto. S. cerevisiae, S. bayanus, S. carlsbergensis and S. paradoxus had species-specific AFLP profiles, with some variations among the strains. Nineteen wine, ale, bakery, whisky and laboratory strains of S. cerevisiae were differentiated by two primer pairs, while out of 19 strains of sake yeast, two groups consisting of two and eight strains were not differentiated using nine primer pairs. A phenogram of 41 strains of S. cerevisiae, two strains of S. bayanus, the type strain of S. pastorianus, three strains of S. carlsbergensis, one hybrid strain of S. cerevisiae and S. bayanus and the type strain of S. paradoxus was obtained by the unweighted pair group method, using arithmetic averages (UPGMA) based on the percentage of shared AFLP fragments of each sample pair. This phenogram demonstrated clear separations of S. cerevisiae, S. bayanus, S. carlsbergensis and S. paradoxus. However, S. pastorianus ATCC 12752(T) showed the highest percentages of shared fragments with the strains of S. bayanus, and formed a cluster with them. Except for the type strain of S. pastorianus, the percentages of shared fragments showed a similar tendency with reported data of DNA relatedness. The cluster of S. cerevisiae separated into three subclusters: one consisting of sake and shochu strains and a whisky strain; another consisting of bakery, wine, ale and whisky strains; and a third consisting of laboratory strains.     

绿色木霉和酿酒酵母降解稻草转化为乙醇的研究

以稻草作为碳源、污泥作为氮源,利用绿色木霉（Trichoderma viride）和酿酒酵母（Saccharomyces cerevisiae）降解稻草转化为乙醇,并对该过程中影响乙醇产量的因素进行优化。结果表明,当预处理稻草与预处理污泥质量比为1∶10（g∶g）、降解培养基初始pH值为7、绿色木霉和酿酒酵母的接种量分别为4%和3%、降解温度为37 ℃和降解时间为48 h时,可得到乙醇的含量为49.25 mg/L。     

The effect of ethanol and specific growth rate on the lipid content and composition of Saccharomyces cerevisiae grown anaerobically in a chemostat

The effects of produced ethanol and specific growth rate on the lipid content and composition of Saccharomyces cerevisiae CBS 2806 were studied using anaerobic chemostat cultures. The cells adapted to increased concentrations of produced ethanol by increasing the proportion of ergosterol at the expense of lanosterol, by increasing the proportion of phosphatidylinositol at the expense of phosphatidylcholine, and by increasing the amount of C_18:0 fatty acids in total phospholipids at the expense of C_16:0 fatty acids. The produced ethanol had no effect on the phospholipid content nor on the proportion of unsaturated fatty acids in the phospholipids. The specific growth rate had no effect on the phospholipid content, the sterol composition, the phospholipid composition, the fatty acid composition of total phospholipids, or on the proportion of unsaturated fatty acids in the phospholipids of S. cerevisiae. It was not possible to separate the effects of produced ethanol and growth rate on the ergosterol content of the chemostat-grown S. cerevisiae cells.     

A new strategy for improved glutathione production from Saccharomyces cerevisiae: use of cysteine‐ and glycine‐rich chicken feather protein hydrolysate as a new cheap substrate

BACKGROUND: Glutathione (GSH) is composed of the amino acids glutamic acid, cysteine and glycine. This study investigated the usability of chicken feather protein hydrolysate (chicken feather peptone, CFP) as a substrate for GSH production from Saccharomyces cerevisiae. RESULTS: CFP was found to be rich in ash (36.7 g per 100 g), protein (61.1 g per 100 g) and minerals (S, P, K, Ca, Fe, Na and Mg). It also had high contents of cysteine and glycine. CFP augmented biomass and GSH production by 53 and 115% respectively compared with the control medium. The highest biomass (17.4 g l^?1) and GSH (271 mg L^?1) concentrations were attained in CFP medium. The second highest biomass (16.8 g l^?1) and GSH (255 mg L^?1) concentrations were obtained in fish peptone medium. It was assumed that the high mineral, cysteine and glycine contents of CFP were related to cell growth and GSH synthesis in S. cerevisiae. CONCLUSION: This is the first report on the effect of cysteine‐ and glycine‐rich protein hydrolysates on GSH production from S. cerevisiae. In this regard, CFP was tested for the first time as a GSH production substrate. As an additional contribution, a new hydrolysis process was developed for the preparation of protein hydrolysates. © 2012 Society of Chemical Industry     

酿酒酵母的乙醇耐受性分子机理与全转录工程

研究酿酒酵母对乙醇耐受性的机理,对于发展乙醇生产有重要意义.酿酒酵母乙醇耐受性涉及到基因组水平上许多基因的复杂的相互作用,已知许多影响细胞膜的完整性和通透性、细胞壁结构、蛋白质构象,以及糖和氨基酸等的吸收等基因都与乙醇耐受性有关,与乙醇诱导相关的基因往往也与其他的环境因素如渗透压、热激、化学毒性、氧化压力等诱导的基因有关或重叠.因此,从基因转录动力学研究酿酒酵母乙醇耐受性并通过全转录工程构建乙醇耐受性工程菌己成为重要的研究热点.该文对近年来酿酒酵母乙醇耐受性分子机理以及全转录工程构建工程菌的研究作一综述,旨在为了解酵母乙醇耐受性机理和培育乙醇耐受性高产酵母菌株提供参考.