High levels of copper retard the growth of Saccharomyces cerevisiae by altering cellular morphology and reducing its potential for ethanolic fermentation

Previous research has shown that high levels of Cu²⁺ inhibit the growth of yeast and induce sluggish fermentation, resulting in a decrease in alcohol production. Little data are available on the effects of copper on the cell morphology, especially the surface elasticity of Saccharomyces cerevisiae. This work investigated the effects of Cu²⁺ (0.5, 1.0 and 1.5 mm ) on the growth, surface characteristics and elasticity of two strains of S. cerevisiae in a low sugar model synthetic medium. The results indicated that high levels of Cu²⁺ retarded the growth rate of S. cerevisiae and reduced the utilisation of reducing sugars. Cells showed intercellular adhesion, became small and deformed, and the surface was uneven and rough after the adsorption of Cu²⁺. The Young's modulus also decreased with an increase in the concentration of Cu²⁺, indicating that the cells had softened. This study reveals the response mechanism of S. cerevisiae under copper stress by altering cellular morphology and mechanical properties.     

Role of non-Saccharomyces yeasts in Korean wines produced from Campbell Early grapes: Potential use of Hanseniaspora uvarum as a starter culture

Several yeasts were isolated from Campbell Early grapes (Vitis labrusca cultivar Campbell Early), the major grape cultivar in Korea, grown in two different regions. PCR-RFLP analysis of the ITS I-5.8S-ITS II region showed that 34 isolates out of a total of 40 were in the same group. Phylogenetic analysis revealed that the major strain belonged to one species, Hanseniaspora uvarum, although they displayed some nucleotide mismatches between them. During spontaneous alcohol fermentation at 20 °C, the two grape musts containing 24 °Brix sugar exhibited similar fermentation patterns with differences in final alcohol production and yeast viable counts. PCR analysis of the yeasts randomly isolated during the fermentation using an intron splice site primer showed changes in yeast flora between 8 and 10 days of fermentation. We found that the dominant yeasts displaying various PCR patterns using the primer remained the same throughout the early stages of fermentation, as determined by molecular typing of their ITS regions using PCR-RFLP, and these yeasts were identical to those isolated from grape berries. Among the isolates, the strain designated SS6 was selected based on its potassium metabisulfite resistance, alcohol production (distillation method), and flavor (by sniffing test) of grape juice. When Campbell Early grape must was inoculated with H. uvarum SS6 cells, no differences in fermentation pattern were observed compared with that inoculated with cells of Saccharomyces cerevisiae W-3, an industrial wine yeast strain. However, SS6 wine showed higher levels of organic acid (especially lactic acid), aldehydes, and minor alcohols (except n-propyl alcohol), as well as a higher score in sensory evaluation, compared to those of W-3 wine.     

Variation in content of tyramine,histamine, 2-phenylethylamine,tryptamine and their precursor amino acids in a Chardonnay wine by using different commercial active dry yeasts and nitrogen sources

Wine is a fermented beverage that could be affected by high concentrations of biogenic amines, thus altering organoleptic and health properties. In this work, the capacity of different selected yeast strains regarding to affect the content of tyramine, histamine, 2-phenylethylamine, tryptamine and their precursor amino acids (pAA) during fermentation has been described. Overall, biogenic amines (BAs) and pAA contents appeared significantly related both to the strain and to the nutrient supplementation applied. Among BAs, without and (with) nitrogen source addition, the concentrations covered a range between 0.1 and 9.5 (0.1–10.3) mg L⁻¹ for tyramine; 0.1 and 4.4 (0.1–4.6) mg L⁻¹ for histamine; 0.4 and 1.1 (0.4–1.4) mg L⁻¹ for 2-phenylethylamine; and 0.02 and 0.14 (0.01–0.12) mg L⁻¹ for tryptamine, respectively. Considering that also wine yeast species are responsible for BAs formation, the evidence from fermentation trials underlined the relevance of a careful choice of ADY strains and nitrogen sources in the management of alcoholic fermentation in wine.     

Acetic acid removal by Saccharomyces cerevisiae during fermentation in oenological conditions. Metabolic consequences

The commercial Saccharomyces cerevisiae strains used in champagne winemaking were tested for their ability to metabolise acetic acid during alcoholic fermentation. Fermentation tests were performed in conditions close to oenological ones using a Chardonnay grape juice supplemented with acetic acid. The amount of acetic acid metabolised by wine yeast increased with increasing initial acetic acid concentration and this elimination occurred during the second part of the exponential growth phase. When the initial acetic acid concentration exceeds 1 g/l, and whatever the yeast strain used, the concentration of acetic acid in the resulting wine cannot be reduced to an acceptable level according to the current legislation. Acetic acid removal modified yeast metabolism, since more acetaldehyde, less glycerol and less succinic acid were produced. Considering the reduction of the NADPH/NADP⁺ ratio following acetic acid consumption, we propose, as a new hypothesis, that acetic acid could modify yeast metabolism by reducing the activity of the NADP⁺ dependent aldehyde dehydrogenase Ald6p.     

桑椹果酒酵母筛选及发酵性能

从桑椹成熟果实、桑叶以及桑果园的土壤采样，进行桑椹果酒专用酵母的筛选，得到一株在性能上优于对照菌葡萄酒干酵母的酵母菌Y11。该菌起酵快，从第2天开始糖度大幅度下降，酒精度快速上升，发酵第4天酒精体积分数即可达到10％以上。发酵过程pH值变化趋势比较平缓，发酵结束后高级醇量符合要求。综合发酵性能及感官评定，表明该菌能代替葡萄酒干酵母用于桑椹果酒的生产。     

Isolation of a Copper‐resistant Sake Yeast Mutant with Improved Flavour Compound Production

The sake (traditional Japanese alcoholic beverage) yeast mutant A1 was previously isolated as a strain resistant to an isoprenoid analog. This strain is used for industrial sake brewing because of its increased production of isoamyl acetate. In this study, a physiological event was identified which was closely related to the elevation of alcohol acetyltransferase (AATase) activity in strain A1. This finding was applied for the isolation of another mutant with an improved capacity for flavour compound production. Strain A1 revealed an additional phenotype showing resistance to Cu²⁺, as seen from its growth and isoamyl acetate production, even in a medium with the copper ion at 6 mM. Mutant strains were successfully isolated with increased isoamyl acetate production capacity from sake yeast strain 2NF on the basis of a Cu²⁺‐resistant phenotype at a high yield. Among them, strain Cu7 was characterized by its ability to produce isoamyl acetate at the highest concentration under condition where isoamyl alcohol (its precursor) was accumulated to the lowest extent. Such a phenotype of strain Cu7 is applicable for the practical production of an alcoholic beverage of excellent quality in terms of flavour.     

Yeast viability during fermentation and sur lie ageing of a defined medium and subsequent growth of Oenococcus oeni

Interactions between the yeast strain used for primary oenological fermentation and the bacterium used to conduct subsequent malolactic fermentation were studied under model winemaking conditions. A commercial Saccharomyces cerevisiae wine yeast (strains, EC 1118, AWRI 835 and CY-3079) was grown in a defined medium whose composition approximated grape juice. Fermentations by all strains reached dryness, and retained a cell viability of greater than 90% upon completion of fermentation. Highest total viable cell number and percentage of viable cells were recorded for EC 1118. A sur lie ageing of the fermented medium over a 12 week period revealed a bi-phasic decay of culture viability for all strains. Thus 99% of cells had died within 2 weeks post-fermentation. Viabilities were then stable for the subsequent 4–6 week period before a second decline phase ensued and ended in either a minimal ( ca 100 CFU/mL, EC 1118) or no viable cells being detected at 12 weeks of ageing. The growth response of an Oenococcus oeni inoculum to yeast culture supernatants, previously aged for up to 12 weeks in the presence or absence of yeast lees, was evaluated in a bio-assay. In this way, yeast strains could be designated as being either inhibitory, neutral or stimulatory to the growth of O. oeni (strain Lc5p). Inhibition by supernatants of strain EC 1118 was evident, but found to be reduced by ageing the supernatant (with or without lees). Conversely, longer ageing on yeast lees increased the magnitude of the stimulatory response in O. oeni (strain Lc5p) to the supernatant from the wine yeast (strain CY-3079).     

产地葡萄酒发酵菌种选育及工艺优化研究

该研究从三门峡产区采集的葡萄果粒中分离到酵母株,经过耐性试验和酒精模拟发酵试验,筛选出发酵性能优良的菌株SY1和SY2,相比普通酿酒酵母发酵产酒率分别提高3.3%和2.9%;并依托原有的葡萄酒生产工艺,选取SY1作为试验菌株,分别通过改变发酵温度、酵母接种量及葡萄汁糖度的单因素试验及三因素三水平的L9(33)正交试验,最终得出以酒精度为评定指标的最佳发酵条件为主发酵温度26℃,酵母接种量6%,葡萄汁起始糖度24 Brix;以感官评价为评定指标的最佳发酵条件为主发酵温度24℃,酵母接种量10%,葡萄汁起始糖度20 Brix。     

Inhibitory effect of arginine on proline utilization in Saccharomyces cerevisiae

Proline is a predominant amino acid in grape must, but it is poorly utilized by the yeast Saccharomyces cerevisiae in wine-making processes. This sometimes leads to a nitrogen deficiency during fermentation and proline accumulation in wine. Although the presence of other nitrogen sources under fermentation conditions is likely to interfere with proline utilization, the inhibitory mechanisms of proline utilization remain unclear. In this study, we examined the effect of arginine on proline utilization in S. cerevisiae. We first constructed a proline auxotrophic yeast strain and identified an inhibitory factor by observing the growth of cells when proline was present as a sole nitrogen source. Intriguingly, we found that arginine, and not ammonium ion, clearly inhibited the growth of proline auxotrophic cells. In addition, arginine prevented the proline consumption of wild-type and proline auxotrophic cells, indicating that arginine is an inhibitory factor of proline utilization in yeast. Next, quantitative polymerase chain reaction (PCR) analysis showed that arginine partially repressed the expression of genes involved in proline degradation and uptake. We then observed that arginine induced the endocytosis of the proline transporters Put4 and Gap1, whereas ammonium induced the endocytosis of only Gap1. Hence, our results may involve an important mechanism for arginine-mediated inhibition of proline utilization in yeast. The breeding of yeast that utilizes proline efficiently could be promising for the improvement of wine quality.     

Effects of copper on the post acidification of fermented milk by St. thermophilus

Abstract: As an initial investigation of using Cu²⁺ to control post acidification, the fermentation time, pH and the viable counts of bacteria of fermented milk during fermentation and storage were studied. Streptococcus thermophilus was used as the starter culture to ferment the whole milk. Cu²⁺ was added to whole milk at a concentration ranging from 1.25 to 20 mg/kg before fermentation. It was observed that increasing the concentration of Cu²⁺, the fermentation time of yoghurt was prolonged, while the post acidification of fermented milk was decreased during storage. The results showed that Cu²⁺ at the concentration of 1.25 mg/kg could reduce the post acidification of fermented milk effectively and showed no significant effect on neither the fermentation time nor the viable counts of S. thermophilus (P > 0.05) compared with the control sample. These findings indicated that Cu²⁺ could be used as a potential additive to inhibit the post acidification of yoghurt. Practical Application: Adding copper to milk has an effect on fermented milk products such as yoghurt. Increasing the concentration of Cu2⁺ decreased the post acidification of fermented milk. The fermentation time is prolonged with the increase of Cu2⁺ concentration. Cu2⁺ at 1.25 mg/kg exhibited inhibition on post acidification and had no significant effect on fermentation time and the viable counts of fermented milk.     

Production of white wine by Saccharomyces cerevisiae immobilized on grape pomace

White wine was produced with Saccharomyces cerevisiae cells immobilized on grape pomace, by natural adsorption. The support, the main solid waste from the wine industry, consisted of the skins, seeds and stems. Immobilization was tested using different media, namely complex culture medium, raw grape must and diluted grape must. Grape pomace was revealed to be an appropriate support for yeast cell immobilization. Moreover, grape must was shown to be the most suitable medium as immobilized cells became adapted to the conditions in the subsequent alcoholic fermentation in the wine‐making process. The wines produced, either with immobilized cells or with free cells, were subjected to chemical analysis by HPLC (ethanol, glycerol, sugars and organic acids) and by gas chromatography (major and minor volatile compounds); additionally, colour (CIELab) and sensory analysis were performed. The use of immobilized systems to conduct alcoholic fermentation in white wine production proved to be a more rapid and a more efficient process, especially when large amounts of SO₂ were present in the must. Furthermore, the final wines obtained with immobilized cells demonstrated improved sensory properties related to the larger amounts of ethanol and volatile compounds produced. The more intense colour of these wines could be a drawback, which could be hindered by the reutilization of the biocatalyst in successive fermentations. Copyright © 2012 The Institute of Brewing & Distilling     

酵母菌的生存因子及其特性

麦角甾醇是酵母的“生存因子”之一,它能够延长处于衰减阶段的酵母生存时间,增强酵母菌株对酒精的抵抗能力,使酵母进一步发酵,降低葡萄酒中的残糖,但酵母细胞内麦角甾醇的富积与其生存条件、发酵环境、选择与保存条件之间有密切的关系,因而,酵母的“生存因子”对葡萄酒的酿造具有重要意义。     

The effect of nitrogen source on yeast metabolism and H2S formation

The impacts of aspartic acid and glutamine used as nitrogen supplements for alcoholic fermentations conducted by Saccharomyces were studied. Synthetic grape juice media and commercially prepared grape juices were supplemented with diammonium phosphate, aspartic acid, or glutamine to increase yeast-assimilable nitrogen concentrations to 250 mg N/L prior to fermentation. Two yeast strains, UCD522 and EC1118, were inoculated at 10⁵ CFU/mL and fermentations were monitored for soluble solids, hydrogen sulfide (H₂S), and residual amino acids. In general, unsupplemented media/juices fermented slower than supplemented ones, produced more H₂S, and contained lower concentrations of amino acids after fermentation. Among the supplemented treatments, fermentation rates, H₂S production, and amino acid utilization varied depending on the nitrogen source and yeast strain. Those fermentations supplemented with aspartic acid were generally slower and sometimes did not achieve dryness. In contrast, glutamine additions yielded fermentation rates and H₂S production equivalent or better than other supplemented treatments. Based on these results, the use of glutamine appears promising as an alternative nitrogen supplement for wine production.     

The Influence of Inoculum Level on Fermentation and Flavour Compounds of White Wines Made from cv. Emir

In this study, the influence of the addition of a commercial wine yeast (Saccharomyces cerevisiae) at inocula of 1 × 10⁴ to 1 × 10⁷ cells/ml in Emir must was investigated with a focus on yeast growth, fermentation rate, ethyl alcohol and flavour compound formation. Spontaneous fermentation without inoculation was also performed. Higher peak counts were observed with higher amounts of S. cerevisiae yeast. Addition of various amounts of yeast led to the earlier disappearance of non‐Saccharomyces yeasts. The fermentation rate was improved with higher amounts of yeast, but ethanol production was not affected. Concentrations of higher alcohols increased with increasing inoculum levels, especially inoculum sizes of 1 × 10⁶ cells/ml and 1 × 10⁷ cells/ml. The amount of ethyl acetate was reduced with increased inoculum levels.     

Removal of ochratoxin A by wine Saccharomyces cerevisiae strains

The aim of this work was to examine two wine strains of Saccharomyces cerevisiae (Syrena LOCK 0201 and Malaga LOCK 0173 strains) and thermally inactivated biomass of bakery yeast (BS strain) for their ability to remove ochratoxin A (OTA) from model YPG, white grape GM, and blackcurrant BM media. The media was initially contaminated by 1 μg/mL OTA. The influence of OTA on yeast growth parameters, kinetic of fermentation, and amount of ethanol, glycerol, and acids were determined. It was found that both yeast strains were able to decrease the toxin amount in YPG, GM, and BM media. Strain Malaga LOCK 0173 was able to remove 82.8 and 10.7 % ochratoxin A from grape and blackcurrant medium, respectively. In case of Syrena LOCK 0201 strain, the OTA reduction was higher: 85.1 % for grape and 65.2 % for blackcurrant media. From 54.1 to 64.4 % of initial ochratoxin A concentration was removed after the contaminated wine treatment by thermally inactivated baker’s yeast strain (BS) cells. The elongation of lag phase in contaminated YPG medium compared on toxin-free medium was noted. In white grape and blackcurrant medium, the differences between the final cell number, fermentation rate, moreover the ethanol, glycerol, and acids production in the medium with OTA and the control were not statistically significant. The results showed that the application of selected strains of yeasts in winemaking involving raw material contaminated with OTA might reduce the toxin contamination as well as the health risk related to human exposure to this toxin. Moreover, the application of heat-inactivated yeast’s biomass for toxin adsorption gives new possibilities in oenology.     

Emergence of [GAR+] cells in yeast from sake brewing affects the fermentation properties

In the traditional (kimoto) method of sake (Japanese rice wine) brewing, Saccharomyces cerevisiae yeast cells are exposed to lactate, which is produced by lactic acid bacteria in the seed mash. Lactate promotes the appearance of glucose-repression-resistant [GAR⁺] cells. Herein, we compared the resistance to glucose repression among kimoto, industrial, and laboratory yeast strains. We observed that the frequencies of the spontaneous emergence of [GAR⁺] cells among the kimoto strains were higher than those among the industrial and laboratory strains. The fermentation ability of a kimoto yeast (strain U44) was lower than that of an industrial strain (K701), as [GAR⁺] cells generally showed slower ethanol production. The addition of lactate decreased the fermentation abilities of the K701 strain by increasing the number of [GAR⁺] cells, but it did not affect those of the U44 strain. These results suggest that lactate controlled fermentation by promoting the appearance of [GAR⁺] cells in the industrial sake strains but not in the kimoto strains.     

Selection and modification of yeasts and lactic acid bacteria for wine fermentation

Historically, the fermentation of grape juice to wine has been carried out by indigenous yeasts found on the berry. However, in newer wine regions, e.g. the USA, inoculation with selected wine yeast strains is employed. Grape juice is high in nutritional factors and difficulties in fermentation usually arise from the inhibitory effects of the high concentration of sugar initially present and the ethanol produced. A secondary fermentation, brought about by indigenous or added lactic acid bacteria, converts malic acid to lactic acid and carbon dioxide and often occurs. This ‘malolactic’ fermentation is usually slow. For both yeast and bacterial fermentations strain selection is based more on fermentation performance than on sensory characteristics of the wine, with increased tolerance of the yeast to ethanol and of the bacteria to low pH being emphasized. Attempts to increase the malolactic fermentation rate have been made by cloning and transferring the malolactic gene from Lactobacillus to wine yeast. In early attempts to produce wines with enhanced or novel sensory characteristics a leucine-less mutant of a homothallic wine yeast has been obtained which does not produce isoamyl alcohol.     

Influence of yeast strain on binding of sulphur dioxide in wines,and on its formation during fermentation

The amounts of sulphur dioxide bound by acetaldehyde, pyruvic acid and α-ketoglutaric acid during fermentation of three grape juices by eight wine yeasts (Saccharomyces sp.) are reported. These constituents accounted for 49–83 % (mean 69) of the measured bound SO₂, depending on the yeast strain and juice. the maximum range of concentrations of the binding components for individual wines were 10–48 ppm for acetaldehyde, 9–77 ppm for pyruvic acid and 5–63 ppm for α-ketoglutaric acid, depending on yeast strain and grape juice. the validity of the calculations was verified by an experiment with SO₂ and the three binding compounds in a multicomponent model system. The acetaldehyde content was related to the total SO₂ present, which itself was determined by the strain of yeast. SOz bound in the wines after a further SO₂ addition was correlated significantly with pyruvic and α-ketoglutaric acids, but not with acetaldehyde. Certain yeasts produced SO₂ during fermentation in grape juice and in synthetic media with defined sulphur sources. More SO₂ was produced at pH 3.6 than 3.0 in the absence of added sulphate in grape juice. Sulphate was the best sulphur source for SO₂ production in synthetic media, although some yeasts were able to produce smaller amounts of SO₂ from l-cysteine and reduced glutathione.     

Standardised methodology for testing malolactic bacteria and wine yeast compatibility

Metabolic interaction of wine yeast and malolactic (ML) bacteria may stimulate or inhibit malolactic fermentation (MLF). To study this interaction in the absence of extrinsic grape-derived and processing factors, a standardised methodology using a chemically defined medium was developed to reproducibly characterise the intrinsic metabolic interaction between any wine yeast and bacteria pair. The methodology can be adapted to study the modifying effects of extrinsic factors on yeast-bacteria interaction. The methodology involves measuring the growth characteristics of the chosen ML bacterium in the wine produced by the yeast of interest under standardised conditions. The methodology was applied to four Saccharomyces cerevisiae wine yeasts which have been characterised in terms of growth response of an Oenococcus oeni strain. Three yeasts gave compatible interactions, in which the level of bacterial growth stimulation was variable; the fourth yeast inhibited bacterial growth and produced a high concentration of sulfite. To test for the possibility that the bacterial growth response was inhibited by the depletion of nutrients by the fermentation yeast, supplementation of the wines with essential nutrients gave a strain-dependent growth response. A treatment designed to remove or inactivate yeast-derived inhibitory factors was also investigated. This treatment partly alleviated bacterial inhibition caused by the sulfite-producing yeast strain. To verify that the standardised methodology relates to grape juice and wine, the synthetic grape juice medium was replaced with a Chardonnay juice of comparable composition prepared from microbiologically sound grapes; similar results were obtained, suggesting that the grape-derived extrinsic factors in this case did not appreciably modify the intrinsic yeast-bacteria metabolic interaction.     

Selenium biotransformation by Saccharomyces cerevisiae and Saccharomyces bayanus during white wine manufacture: Laboratory-scale experiments

The main purpose of this laboratory-scale study was to evaluate the transformation of inorganic selenium, as sodium selenite, when added to white grape juice as part of the fermentation process of white wine. The participation of yeast, added in the fermentation of the must, is necessary to convert inorganic selenium into organoseleno species. Two different yeasts, Saccharomyces cerevisiae and Saccharomyces bayanus were used for fermentation. The effects of different Se concentrations on cells and their viability during fermentation were evaluated. The alcoholic fermentation that produced wine was not affected by the presence of selenium, regardless of the type of Saccharomyces used. After 21 days of fermentation, the white wine and residual yeast were separated and analysed by ICP-MS and LC–ICP-MS for determination of total selenium and speciation. Selenomethionine was found to be the main Se-species in the selenised white wine. The results obtained are preliminary but they could be considered for future studies using both pilot and full-scale vinification processes.