The hexose transporters of Saccharomyces cerevisiae play different roles during enological fermentation

We investigated the role of hexose transporters in a Saccharomyces cerevisiae strain derived from an industrial wine strain by carrying out a functional analysis of HXT genes 1-7 under enological conditions. A strain in which the sugar carrier genes HXT1-HXT7 were deleted was constructed and the HXT genes were expressed individually or in combination to evaluate their role under wine alcoholic fermentation conditions. No growth or fermentation was observed in winemaking conditions for the hxt1-7 delta strain. The low-affinity carriers Hxt1 and Hxt3 were the only carriers giving complete fermentation of sugars when expressed alone, indicating that these carriers play a predominant role in wine fermentation. However, these two carriers have different functions. The Hxt3 transporter is thought to play a major role, as it was the only carrier that gave an almost normal fermentation profile when produced alone. The hxt1 carrier was much less effective during the stationary phase and its role is thought to be restricted to the beginning of fermentation. The high-affinity carriers Hxt2, Hxt6 and/or Hxt7 were also required for normal fermentation. These high-affinity transporters have different functions: hxt2 is involved in growth initiation, whereas Hxt6 and/or Hxt7 are required at the end of alcoholic fermentation. This work shows that the successful alcoholic fermentation of wine involves at least four or five hexose carriers, playing different roles at various stages in the fermentation cycle.     

Impact of oxygen addition during enological fermentation on sterol contents in yeast lees and their reactivity towards oxygen

During enological fermentations, superfluous oxygen consumption by yeast cells is observed. The superfluous oxygen consumed by the yeast cells is mainly related to the operation of non-respiratory oxygen consumption pathways resulting in an overall decrease in the total sterol fraction in yeast. On the other hand, yeast lees remaining at the end of alcoholic fermentations exhibit specific oxygen utilization rates ranging from 1 to 4 micromol O2 h- 10(-10) cells from the second to the thirteenth month of wine aging. This oxygen consumption capacity of yeast lees was independent of residual cell viability. In this study, we investigated the potential relationship between the oxygen added to commercial yeast strains during enological fermentation and the capacity of the corresponding yeast lees to interact with oxygen. Additions of low (7 mg l(-)) and excess (37 mg l(-1)) amounts of oxygen at the end of the cell growth phase were compared in terms of repercussions on the oxygen consumption activity of the corresponding yeast lees. As expected, the superfluous oxygen consumption by yeast cells during fermentation had a positive influence on the fermentation kinetics and increased cell biomass formation. Oxygen consumption rates and the total capacity of oxygen consumption by the corresponding yeast lees clearly decreased when oxygen was added during fermentation. This marked decrease in yeast lees reactivity towards oxygen was concomitantly related to an increase in ergosterol synthesis and to oxygen-dependent sterol degradation. Such degradation occurred when oxygen was added in excess. Therefore, oxygenation control during fermentation appears to be a potential way to optimize both the fermentation kinetics and control yeast lees reactivity towards oxygen. For practical applications, oxygenation control during alcoholic fermentation may be considered as a general tool for decreasing the highly reductive effect of yeast lees during wine aging.     

酿酒酵母对萝卜泡菜发酵过程的影响

以樱桃萝卜为原料,设置4个试验组,分别为乳酸菌接种发酵组、乳酸菌与酵母菌混合接种发酵组、酵母菌接种发酵组和自然发酵组。对各试验组发酵过程中pH值、总酸、亚硝酸盐、色度、硬度、感官品质、乳酸菌数量、酵母菌数量及大肠菌群数量进行研究。实验结果表明:在樱桃萝卜发酵过程中,接种酿酒酵母能够阻止发酵后期pH进一步下降,降低总酸生成量,抑制泡菜过度酸化,使泡菜保持较高的脆度,并且产生浓郁的酯香风味;酿酒酵母的加入不改变亚硝酸盐消长规律,也不影响接种乳酸菌对大肠菌群的抑制效果;但在发酵后期,加入酿酒酵母的试验组,其乳酸菌的生长受到一定程度的抑制,因此酿酒酵母的使用量还需进一步研究。     

Genetic and enological analysis of selected Saccharomyces cerevisiae strains for wine production

The non‐wine Saccharomyces cerevisiae strain of 96581 was found to be a promising candidate for the production of white wine. It produced wines with fusel alcohols that were 57% higher than those produced by the wine yeasts studied and was also more efficient in the production of 2‐phenethyl acetate and 3‐methyl‐1‐butanol acetate. This study also shows that there is a difference in the ester‐formation efficiency for acetates relative to C6, C8 and C10 fatty acid esters for all the studied yeast strains. Therefore, it supports the view that other unidentified enzymes besides those regulated by ATF1 and ATF2 genes are involved in the production of ethyl esters of C6–C10 fatty acids. DNA analysis of the 25S, 18S, 5.8S and 5S ribosomal DNA genes in these strains showed high conservation. Despite the closely related nature of these yeast strains, the chemical profiles of the wines produced were significantly different.     

Pleiotropic mutations in Saccharomyces cerevisiae affecting sterol uptake and metabolism

Sterol uptake control mutants (upc-) have been isolated via ethylmethanesulfonate mutagenesis from wild-type Saccharomyces cerevisiae. These mutants are heme and sterol competent but possess the ability to accumulate exogenous sterol(s) under aerobic conditions. Previous studies demonstrate sterol uptake only in a hem-, erg- background; however, the Upc- strains described here are Hem+ and do not require exogenous sterol for growth. We were unable to obtain viable hem+, erg-, upc+ recombinants; such combinations appear to be lethal. Isolates of Upc mutants demonstrated different levels of sterol uptake, and sterol analysis revealed a broad phenotypic range with regard to amounts and accumulation of ergosterol and non-ergosterol sterols. Assays of acyl CoA: ergosterol acyltransferase and sterol ester hydrolase showed no apparent difference in activity between Upc mutants and the wild type.     

水晶葡萄酿酒酵母菌的分离筛选及酿酒性能研究

以水晶葡萄为原料,通过杜氏管产气试验、耐受性试验分离筛选优良酿酒酵母（Saccharomyces cerevisiae）,采用分子生物学技术对其进行鉴定,并研究其酿酒性能。结果表明,筛选出两株优良酵母,分别命名为SJJM-7和SJJM-18;两株菌对糖、低pH值、乙醇和SO2的耐受性较强,均能在50%糖度、12%vol乙醇及200 mg/L SO2的培养基中生长并发酵产气;在pH值2.0的培养基中培养2 d,菌体存活率均＞20%;菌株SJJM-7和SJJM-18均被鉴定为酿酒酵母（S. cerevisiae）;利用这两株菌株发酵水晶葡萄汁后,菌株SJJM-7和SJJM-18的酿酒性能（澄清度、挥发酸含量、残糖量及酒精度）优于进口商品酵母菌株LA-PE,在生产水晶葡萄酒方面具有一定的研究意义和应用价值。     

不同时间添加氮源对酵母GJ2008果糖与葡萄糖利用的影响

甘蔗汁被完全酸水解后,添加等量果糖与葡萄糖调整总糖浓度为220g/L,对此水解液进行酒精发酵,并在发酵过程的不同时间点添加氮源。采用高效液相色谱法测定发酵过程中果糖和葡萄糖含量,通过曲线下面积法对果糖与葡萄糖代谢曲线进行分析。结果表明:酵母GJ2008始终会优先利用葡萄糖,果糖的利用滞后;在12 h前添加氮源可有效地缩小果糖与葡萄糖利用的差异性。     

Inhibition of malolactic fermentation by a peptide produced by Saccharomyces cerevisiae during alcoholic fermentation

The ability of Saccharomyces to inhibit Oenococcus oeni during the alcoholic fermentation by mechanisms other than SO(2) production was investigated. During fermentation in synthetic grape juice, S. cerevisiae strain RUBY.ferm inhibited the malolactic fermentation by O. oeni while strain EC1118 did not despite both strains producing similar amounts of SO(2). The bacterial inhibition exerted by RUBY.ferm was diminished when the wine was treated with proteases but not through the addition of nutrients. Wine fermented by RUBY.ferm was fractionated based on molecular weight and each fraction tested for the ability to inhibit the growth of O. oeni. The fraction containing compounds larger than 3 kDa was the sole inhibitory fraction. The inhibitory fraction was analyzed by SDS PAGE and showed a 5.9 kDa protein band present in wine fermented by RUBY.ferm that was not present in wine fermented by a non-antagonistic yeast, S. cerevisiae strain Saint Georges S101. The ability of the peptide to inhibit O. oeni seemed to be dependent on the presence of SO(2).     

Influence of oxygen addition during growth phase on the biosynthesis of lipids in Saccharomyces cerevisiae (M(3)30-9) in enological fermentations

The fatty acid, phospholipid and sterol composition of one strain of Saccharomyces cerevisiae under different oxygen levels during grape must fermentation was studied. Anaerobiosis partially inhibited yeast growth and resulted in a drop in the unsaturation index of the fatty acids throughout the fermentation, as well as an abnormally low ergosterol/phospholipid ratio. Aeration after 48 h of fermentation in anaerobiosis stimulated growth and increased cellular viability, as well as raised the unsaturation index of fatty acids. Although the ergosterol/phospholipid ratio remained low in the days following the aeration, by the end of the fermentation the value was that considered optimum for the maintenance of membrane functions.     

Effect of sterol alterations on conjugation in Saccharomyces cerevisiae.

Sterol auxotrophic strains of Saccharomyces cerevisiae were grown and allowed to conjugate on media supplemented with various sterols. The mating efficiency of the auxotrophs is perturbed by the replacement of the normal yeast sterol, ergosterol, with other sterols. After 4 h of mating, cells grown on ergosterol exhibited a 30-fold higher productive mating efficiency than those cells grown in stigmasterol. Aberrant budding by the conjugants was enhanced following incubation on stigmasterol and other non-ergosterol sterols. Using light and electron microscopy, we demonstrated that there is a reduced ability for stigmasterol-grown cells to undergo cytoplasmic fusion during conjugation. Many of the mated pairs remained adherent but prezygotic even after 12 h of incubation. The addition of ergosterol to cells previously grown on stigmasterol rescued the organisms, allowing for zygote formation and normal budding.     

酿酒酵母有氧发酵培养基的研究

以实验室选育的酵母菌为试验用菌株,以甜菜糖蜜为碳源发酵生产酵母菌体,通过单因素和正交试验优化实验室摇瓶发酵的培养基,优化后的培养基为总糖含量为10%的糖蜜、10%红糖、0.50%尿素、0.50%酵母粉、0.05%硫酸镁、0.05%甘氨酸,在此培养条件下,菌体干质量可达到16.83g/L。     

Effects of Saccharomyces cerevisiae culture and Saccharomyces cerevisiae live cells on in vitro mixed ruminal microorganism fermentation

The objective of this study was to examine the effects of a Saccharomyces cerevisiae live cell product and a S. cerevisiae culture product on the in vitro mixed ruminal microorganism fermentation of ground corn, soluble starch, alfalfa hay, and Coastal bermudagrass hay. In the presence of ground corn, neither concentration (0.35 or 0.73 g/L) of S. cerevisiae culture nor live cells had any effect on final pH, H2, CH4, propionate, or butyrate. The S. cerevisiae culture had no effect on acetate, but both concentrations of S. cerevisiae live cells decreased acetate and the acetate:propionate ratio. When soluble starch was the substrate, both concentrations of S. cerevisiae live cells and 0.73 g/L of S. cerevisiae culture decreased the acetate:propionate ratio. Although the treatment effects were not statistically significant, both concentrations of live cells and 0.73 g/L of the culture decreased lactate concentrations compared with the control incubations. When alfalfa hay served as the substrate, neither the S. cerevisiae culture nor the live cells had an effect on propionate, butyrate, or the acetate:propionate ratio. Both concentrations of S. cerevisiae culture decreased the final pH and in vitro dry matter disappearance, and the 0.73 g/L treatment decreased the amount of acetate. However, both treatments of S. cerevisiae live cells increased final pH and decreased acetate and in vitro dry matter disappearance. Neither yeast treatment had much effect on the Coastal bermudagrass hay fermentations. In general, both S. cerevisiae supplements seemed to have similar effects on the mixed ruminal microorganism fermentation.     

Saccharomyces cervisiae在食品发酵工业中的研究进展

从基础理论和应用技术等方面综述了国内外对酿酒酵母(Saccharomyces cervisiae)研究进展及其在食品发酵工业中的最新研究成果。     

酿酒酵母QY-1发酵过程中有机酸及游离氨基酸变化分析

该研究对酿酒酵母（Saccharomyces cerevisiae）QY-1发酵过程中的OD600 nm值、pH值、葡萄糖消耗量、乙醇生成量、有机酸及游离氨基酸种类及生成量进行监控和分析。结果表明,随着发酵时间的延长,发酵液的pH值与OD600 nm值呈负向耦合;乙醇的生成量与葡萄糖消耗量呈负向耦合,发酵36 h时,乙醇含量最高,为（26.87±2.76） g/L;有机酸含量呈现先升高后稳定的趋势,最高达（3.242±0.213） g/L,其中乙酸含量最高;游离氨基酸含量呈现先升高后降低的趋势,最高达（5.57±0.08） mg/L,其中谷氨酸含量最高。S. cerevisiae QY-1具有较强的产酸和氨基酸能力。     

优良耐酸酿酒酵母的筛选及发酵特性研究

该研究采用酸性选择培养基、氯化三苯基四氮唑（TTC）显色法、杜氏小管发酵法从实验室保藏的87株酿酒酵母（Saccharomyces cerevisiae）中筛选耐酸性强的优良菌株，并测定其最适生长条件、耐受性及发酵性能。结果表明，最终筛选出一株耐受性强及发酵性能优良的菌株SC-62，其最适生长温度为28 ℃、最适pH为4.0，可耐受乙醇体积分数14%、NaCl 100 g/L、葡萄糖500 g/L，具有发酵力[5.93 g CO2/（100 mL·24 h）]强，酒精产量（7.55%vol）高，延滞期（12 h）短、适应性强等优点。该菌株的成功筛选为后续高效生产酒精提供了良好的菌种来源。     

D-阿洛酮糖合成及偶联酿酒酵母发酵的研究

该研究利用含有组成型启动子的质粒pET-20b载体,在大肠杆菌（Escherichia coli）BL21（DE3）中对3种D-阿洛酮糖3-差向异构酶进行异源表达,其后以D-果糖为底物进行静息细胞转化。结果表明,重组菌在摇床30 ℃、200 r/min转速下发酵48 h,能够利用D-果糖为底物生产D-阿洛酮糖,转化率分别达到27.56%、23.99%和25.98%。为降低D-果糖对D-阿洛酮糖纯化过程中的影响,在产糖的过程后偶联酿酒酵母（Saccharomyces cerevisiae）好氧发酵过程,消耗掉混合糖液中的D-果糖,结果显示转化24 h后D-果糖去除率达到94.22%,该研究为下游D-阿洛酮糖的分离和纯化提供了新的思路。     

酵母菌FW-sc-08的生长及发酵特性研究

通过对影响菌体生长因素、发酵指标和香气成分的测定,研究了从自然发酵黑莓酒中筛选出的酵母菌FW-sc-08的生长和发酵特性。结果显示,酵母菌FM-sc-08对数生长期为3～9 h,对SO2最大耐受质量浓度为800 mg/L;酵母菌FM-sc-08适宜发酵的可发酵糖最大含量为30%,对糖最大耐受量为40%,可发酵酒精度最高16.3%vol。通过测定不同温度对发酵指标的影响,最终确定酵母菌FM-sc-08的最适发酵温度为25 ℃;对比20 ℃和25 ℃发酵的黑莓果酒酒样中的香气成分,可知酵母菌FM-sc-08在20 ℃发酵黑莓酒产香气成分较多（35种）。