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.     

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.     

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).     

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。     

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.     

High-cell-density fermentation for ergosterol production by Saccharomyces cerevisiae

The direct feedback control of glucose using an on-line ethanol concentration monitor for ergosterol production by high-cell-density fermentation was investigated and the fermentation parameters (e.g., pH, dissolved oxygen, ethanol concentration, oxygen uptake rate, carbon dioxide evolution rate and respiratory quotient) were analyzed. Controlling glucose feeding rate in accordance with ethanol concentration and adjusting pH with ammonia during the fermentation process were effective fed-batch methods for ergosterol production. The fermentation parameters well described the variation of the whole fermentation process. Cultivation in a 5 l fermentor was carried out under the following conditions: culture temperature, 30 degrees C; pH, 5.5; agitation speed, 600 rpm; fermentation time, 60 h; controlling ethanol concentration below 1% and keeping respiratory quotient (RQ) at approximately 1.0. Under these conditions, the yeast dry weight reached 120 g/l and the ergosterol yield reached 1500 mg/l.     

Growth of non-Saccharomyces yeasts affects nutrient availability for Saccharomyces cerevisiae during wine fermentation

Yeast produces numerous secondary metabolites during fermentation that impact final wine quality. Although it is widely recognized that growth of diverse non-Saccharomyces (NS) yeast can positively affect flavor complexity during Saccharomyces cerevisiae wine fermentation, the inability to control spontaneous or co-fermentation processes by NS yeast has restricted their use in winemaking. We selected two NS yeasts from our Uruguayan native collection to study NS-S. cerevisiae interactions during wine fermentation. The selected strains of Hanseniaspora vineae and Metschnikowia pulcherrima had different yeast assimilable nitrogen consumption profiles and had different effects on S. cerevisiae fermentation and growth kinetics. Studies in which we varied inoculum size and using either simultaneous or sequential inoculation of NS yeast and S. cerevisiae suggested that competition for nutrients had a significant effect on fermentation kinetics. Sluggish fermentations were more pronounced when S. cerevisiae was inoculated 24h after the initial stage of fermentation with a NS strain compared to co-inoculation. Monitoring strain populations using differential WL nutrient agar medium and fermentation kinetics of mixed cultures allowed for a better understanding of strain interactions and nutrient addition effects. Limitation of nutrient availability for S. cerevisiae was shown to result in stuck fermentations as well as to reduce sensory desirability of the resulting wine. Addition of diammonium phosphate (DAP) and a vitamin mix to a defined medium allowed for a comparison of nutrient competition between strains. Addition of DAP and the vitamin mix was most effective in preventing stuck fermentations.     

Release of cell wall polysaccharides from Saccharomyces cerevisiae thermosensitive autolytic mutants during alcoholic fermentation

In order to increase the release of cell wall polysaccharides during alcoholic fermentation, a wine strain of Saccharomyces cerevisiae was subjected to UV mutagenesis to obtain thermosensitive autolytic mutants affected in cell wall integrity. Five mutants and the parental strain were utilized in fermentation trials conducted at 28, 32 and 34 degrees C. Results showed that at all temperatures the mutant strains released into the medium a higher polysaccharide quantity than the parental strain. In particular, at 28 degrees C there was a doubling of these macromolecules. At the end of alcoholic fermentation, all strains showed at 28 degrees C elevated and similar levels of viable cells; at 32 degrees C this parameter remained high for mutant strains ts16 and ts39 and the parental strain; at 34 degrees C all strains underwent a drop in cell viability, which was less intense in the case of strain ts16. As a relationship between cell viability and the quantity of polysaccharides released by the yeast strain was not found, it can be assumed that the mutation led to cells with a less stable wall and thus an easier release of macromolecules into the medium.     

Impact of Saccharomyces cerevisiae metabolites produced during fermentation on bread quality parameters: A review

Although bread making with the use of Baker's yeast has a long tradition in human history, little attention has been paid to the connection between yeast addition and the final bread quality. Nowadays, bakers mainly use different flour additives such as enzymes (amylases, hemicellulases, and proteases) to change and improve dough properties and/or bread quality. Another strategy is the use of modified industrial Baker's yeast. To date, there is no yeast strain used in the baking industry, which is genetically modified, despite some studies demonstrating that the application of recombinant DNA technology is a possibility for improved strains suitable for baking. However, due to the fact that the majority of consumers in Europe highly reject the use of genetically modified microorganisms in the production of food, other strategies to improve bread quality must be investigated. Such a strategy would be a reconsideration of the selection of yeast strains used for the baking process. Next to the common criteria, the requirement for adequate gas production, more attention should be paid on how yeast impacts flavor, shelf life, color, and the nutritional value of baked products, in a similar way to which yeast strains are selected in the wine and brewing industries.     

Online estimation of assimilable nitrogen by electrical conductivity measurement during alcoholic fermentation in enological conditions

The monitoring of alcoholic fermentation under enological conditions is currently poor due to the lack of sensors for online measurements. Such monitoring is currently limited to the measurement of CO(2) production or changes in density. In this study, we determined the potential value of measuring electrical conductivity. We showed that this measurement is related to the assimilation of nitrogen, which is typically the limiting nutrient, and directly correlated to ammoniacal nitrogen assimilation at any percentage of ammoniacal nitrogen in the medium. We also used electrical conductivity for the very precise monitoring of the kinetics of nitrogen assimilation after the addition of a pulse of diammonium hydrogen phosphate (DAP) during fermentation. The impact of initial conditions (e.g., must composition, grape variety, pH) remains unclear, but the robustness, precision and low price of the sensor used justify further studies of the potential value of measuring electrical conductivity on the pilot and industrial scales.     

Influence of assimilable nitrogen on volatile acidity production by Saccharomyces cerevisiae during high sugar fermentation

We analyzed the variability of volatile acidity and glycerol production by Saccharomyces cerevisiae on a large sample of high sugar musts. The production of volatile acidity was inversely correlated with the maximum cell population and the assimilable nitrogen concentration. The higher the nitrogen concentration, the less volatile acidity was produced. An approach to minimize volatile acidity production during high sugar fermentations by adjustment of assimilable nitrogen in musts was investigated in terms of both quantity and addition time. It was found that the optimal nitrogen concentration in the must is 190 mgN.l(-1). The best moment for nitrogen addition was at the beginning of fermentation. Addition at the end of the growth phase had less effect on volatile acidity reduction. We suggest that by stimulating cell growth, nitrogen addition provides NADH in the redox-equilibrating process, which in turn reduces volatile acidity formation.