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.     

Yeast interactions and wine flavour

Wine is the product of complex interactions between fungi, yeasts and bacteria that commence in the vineyard and continue throughout the fermentation process until packaging. Although grape cultivar and cultivation provide the foundations of wine flavour, microorganisms, especially yeasts, impact on the subtlety and individuality of the flavour response. Consequently, it is important to identify and understand the ecological interactions that occur between the different microbial groups, species and strains. These interactions encompass yeast-yeast, yeast-filamentous fungi and yeast-bacteria responses. The surface of healthy grapes has a predominance of Aureobasidium pullulans, Metschnikowia, Hanseniaspora (Kloeckera), Cryptococcus and Rhodotorula species depending on stage of maturity. This microflora moderates the growth of spoilage and mycotoxigenic fungi on grapes, the species and strains of yeasts that contribute to alcoholic fermentation, and the bacteria that contribute to malolactic fermentation. Damaged grapes have increased populations of lactic and acetic acid bacteria that impact on yeasts during alcoholic fermentation. Alcoholic fermentation is characterised by the successional growth of various yeast species and strains, where yeast-yeast interactions determine the ecology. Through yeast-bacterial interactions, this ecology can determine progression of the malolactic fermentation, and potential growth of spoilage bacteria in the final product. The mechanisms by which one species/strain impacts on another in grape-wine ecosystems include: production of lytic enzymes, ethanol, sulphur dioxide and killer toxin/bacteriocin like peptides; nutrient depletion including removal of oxygen, and production of carbon dioxide; and release of cell autolytic components. Cell-cell communication through quorum sensing molecules needs investigation.     

酵母种类及营养剂用量对霞多丽葡萄酒中氨基甲酸乙酯产量的影响

为研究发酵过程中酵母种类和不同酵母营养剂用量对霞多丽葡萄酒中氨基甲酸乙酯（EC）含量的影响,选择了3种酵母（X5、X16、RMS2）和3个不同质量浓度梯度酵母营养剂（THIAZOTE）（0、0.3 g/L、0.6 g/L）进行发酵试验。研究表明,土壤和葡萄汁中的氮含量与葡萄酒中的EC含量密切相关;不同酵母酿造的霞多丽葡萄酒中EC产量不同,在发酵末期阶段,与酵母X16、RMS2相比,酵母X5酿造的葡萄酒中EC产量分别增长了53.8%和17.6%;葡萄酒中的EC产量随着酵母营养剂的用量的增加而升高,在发酵末期阶段,与不加酵母营养剂的对照组相比,酵母营养剂添加量为0.3 g/L和0.6 g/L酿造的葡萄酒中EC产量分别增长了12.8%和69.2%。不同发酵处理霞多丽葡萄酒EC产量范围在2.1～3.8 μg/L,均未超过美国或者欧盟规定的EC限量标准。     

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).     

Inhibition of malolactic fermentation by Saccharomyces during alcoholic fermentation under low- and high-nitrogen conditions: a study in synthetic media

The ability of different wine yeast ( Saccharomyces cerevisiae ) to inhibit malolactic bacteria ( Oenococcus oeni ) and the influence of nitrogen were studied using a synthetic grape juice. Malolactic fermentation was induced in fermenting synthetic grape juice or synthetic wines inoculated with different commercial strains of S. cerevisiae. O. oeni was generally inhibited in wines that contained higher concentrations of total SO₂ although many yeast strains only inhibited the bacteria during fermentation under high nitrogen conditions. Yeast produced higher amounts of SO₂ during fermentation under high nitrogen conditions suggesting that nitrogen affected the malolactic fermentation by influencing yeast SO₂ production. However, the production of SO₂ by yeast did not always account for the inhibition of O. oeni , suggesting the presence of other inhibitory mechanisms.     

不同干酵母对甘蔗汁酿酒特性的影响

为研究不同酵母对甘蔗果酒的发酵影响,以新鲜的甘蔗压榨汁为原料,常温下选用葡萄酒高活性干酵母、耐高温高活性干酵母和发利干酵母三种商业活性干酵母进行液态发酵酿制甘蔗果酒。研究探讨了三种酵母菌的生长曲线、凝聚性、发酵度、发酵力、酸化力和产酒精能力等酿酒发酵特性。结果表明,三种酵母菌作用甘蔗汁的发酵过程中,酵母菌生长曲线趋势基本一致,三种酵母菌的凝聚值F<20%,均为非凝聚性酵母。三种酵母菌的发酵力基本相当的,最终产酒精的能力差距不大,其中葡萄酒高活性干酵母最终产酒精为12.2% vol,耐高温高活性干酵母和发利干酵母均为12.0% vol。从三种商用酵母菌对甘蔗汁酿酒的各发酵特性来看,葡萄酒高活性干酵母略优于耐高温高活性干酵母和发利干酵母。研究结果为甘蔗汁发酵酿制风味甘蔗果酒提供了技术参考。     

The application of malolactic fermentation process to create good-quality grape wine produced in cool-climate countries: a review

The work presents the essence of the secondary fermentation occurring in the course of grape wines production—malolactic fermentation (MLF). The selection of wine yeast and bacteria preparations, scenario of malolactic bacteria inoculation as well as control system of the process conditions are proposed to create the specific character and style of grape wines produced in cool-climate countries, in which the acidity of grape must is often significantly enhanced and the concentration of aroma compounds is considerably low. The role played by appropriately run MLF is presented, along with the advantages and disadvantages of the process, benefits, and technological drawbacks resulting from the introduction of this procedure to the vinification process. Moreover, methods to initiate and run MLF as well as interactions occurring between microorganisms used in wine production, i.e., wine yeasts from the genus Saccharomyces and wine bacteria from the genus Oenococcus, are also presented and discussed.     

Influence of different yeasts on the growth of lactic acid bacteria in wine

The influence of various yeasts on the growth of lactic acid bacteria in wine was tested by inoculating Lactobacillus hilgardii, L. brevis and two strains of Leuconostoc mesenteroides into experimental wines made with twelve different yeasts of the genus Saccharomyces. Wines made from juice which had been infected with several spoilage yeasts and then fermented with a wine yeast were also tested in this way. It was found that the yeasts differed considerably in their effects on bacterial growth. In some of the experimental wines bacterial growth was delayed or failed altogether. Generally, the unfavourable influence of any yeast on bacterial growth was much reduced if the wines were left in contact with the yeast cells for some weeks after the fermentation. The significance of these results in relation to the occurrence of malo-lactic fermentation in commercial wineries is discussed.     

红葡萄原酒发酵过程中的温度控制

论述了红葡萄酒原酒发酵过程中温度控制对酵母、酚类物质、挥发性物质的作用与影响,结合实际生产操作情况介绍了针对不同产区原料、不同葡萄品种发酵过程控制发酵温度的方法,红葡萄酒苹果酸乳酸发酵温度的控制要点。     

响应面法优化黑枸杞-葡萄复合果酒发酵工艺的研究

以黑枸杞汁和葡萄汁为原料,考察酵母添加量、发酵温度和初始糖度对复合果酒的品质的影响。在单因素试验结果的基础上,采用响应面法优化黑枸杞-葡萄复合果酒的发酵工艺参数。结果表明,黑枸杞汁与葡萄汁的最佳配比为7∶3（V/V）,最佳发酵工艺条件为发酵温度20 ℃、酵母添加量0.03%、初始糖度24%。在此条件下,发酵所得复合果酒酒精度为12.7%vol,感官评分为90分,果酒酒香浓厚,酒体澄清透明,呈酒红色。     

苹果乳酸发酵生产葡萄酒

二次发酵-苹果乳酸发酵（MLF）在葡萄酒生产过程中起非常重要的作用.酿酒需要选择合适的酿酒酵母和细菌,乳酸菌接种方案以及工艺条件控制系统.本文介绍了MLF发酵过程的优缺点、效益和技术缺陷.讨论了启动和运行MLF的方法及葡萄酒生产过程中各种微生物之间的相互作用.微生物主要是指酵母菌属中的酿酒酵母和酒类酒球菌属中的酿酒细菌.     

Engineering volatile thiol release in Saccharomyces cerevisiae for improved wine aroma

Volatile thiols, such as 4-mercapto-4-methylpentan-2-one (4MMP), 3-mercaptohexan-1-ol (3MH) and 3-mercaptohexyl acetate (3MHA), are among the most potent aroma compounds found in wine and can have a significant effect on wine quality and consumer preferences. At optimal concentrations in wine, these compounds impart flavours of passionfruit, grapefruit, gooseberry, blackcurrant, lychee, guava and box hedge. The enzymatic release of aromatic thiols from grape-derived, non-volatile cysteinylated precursors (Cys-4MMP and Cys-3MH) and the further modification thereof (conversion of 3MH into 3MHA) during fermentation, enhance the varietal characters of wines such as Sauvignon Blanc. Wine yeast strains have limited and varying capacities to produce aroma-enhancing thiols from their non-volatile counterparts in grape juice. Even under optimal fermentation conditions, the most efficient thiol-releasing Saccharomyces cerevisiae wine strain known realizes less than 5% of the thiol-related flavour potential of grape juice. The objective of this study was to develop a wine yeast able to unleash the untapped thiol aromas in grape juice during winemaking. To achieve this goal, the Escherichia coli tnaA gene, encoding a tryptophanase with strong cysteine-beta-lyase activity, was cloned and overexpressed in a commercial wine yeast strain under the control of the regulatory sequences of the yeast phosphoglycerate kinase I gene (PGK1). This modified strain expressing carbon-sulphur lyase activity released up to 25 times more 4MMP and 3MH in model ferments than the control host strain. Wines produced with the engineered strain displayed an intense passionfruit aroma. This yeast offers the potential to enhance the varietal aromas of wines to predetermined market specifications.     

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。     

Formation of higher alcohols by wine yeasts,and relationship to taste thresholds

The amounts of n-propanol, iso-butanol and iso-amyl plus active amyl alcohol produced during fermentation of grape juice were found to vary considerably according to the yeast used. The yeasts studied included 11 wine yeasts and one brewery yeast, all belonging to the genus Saccharomyces, and 4 yeasts belonging to genera which sometimes cause wine spoilage. Experiments were carried out in the laboratory and confirmed on pilot-plant scale. The average production of n-propanol in juices from 4 grape varieties varied from 13 to 106 ppm depending on the wine yeast used. The corresponding variations in the production of iso-butanol and isoamyl plus active amyl alcohol were 9 to 37 ppm and 115 to 262 ppm respectively,. Juices from different varieties' of grapes (Vitis vinifera) differed somewhat in the amounts of higher alcohols formed, irrespective of the yeast strain used. An increase in temperature of fermentation from 15° to 25° with four yeasts produced an average of 24% more iso-amyl plus active alcohol and 39% more iso-butanol, and 17% less n-propanol. A yeast/temperature interaction occurred. An increase in pH from 3.0 to 4.2 with four yeasts produced 28% more iso-amyl plus active amyl alcohol, 85% more iso-butanol, and 11% more n-propanol. A yeast /pH interaction occurred. Spoilage yeasts examined also formed higher alcohols and the amounts were not related to sugar consumed during the fermentation. Taste thresholds of the higher alcohols, with the exception of active amyl alcohol, were measured in dry white wine with 7 tasters. Iso-amyl alcohol thresholds ranged from 100 to 900 ppm (mean 300 ppm) and iso-butanol and n-propanol both exceeded 500 ppm. The average threshold of iso-amyl alcohol in distilled water was 4 ppm. For certain tasters strains of yeast can produce sufficient differences in iso-amyl alcohol to be detectable organoleptically, but differences in iso-butanol and n-propanol are not large enough to be detectable.     

Influence of different pH values and inoculation time on the growth and malolactic activity of a strain of Oenococcus oeni

The present study was undertaken to evaluate the influence of medium pH and inoculation time on the growth and malolactic activity of an Oenococcus oeni culture. Samples of a commercial white grape juice adjusted to pH 3.2, 3.4 or 3.6, and inoculated with Saccharomyces cerevisiae strain CY3079, were inoculated with a malolactic culture ( Oenococcus oeni strain 31) at the beginning, middle, and end of alcoholic fermentation. The results obtained from this single case study show that it is possible to inoculate the bacterial culture at the three different times during alcoholic fermentation without slowing down or stopping alcoholic fermentation or causing failure of MLF. However, pH and timing of bacterial inoculation were critical to how rapidly MLF starts. At pH 3.2 a lowering of bacterial viability was observed, but a more important reduction was recorded at all tested pH levels when the bacteria were inoculated halfway through alcoholic fermentation. When inoculation was carried out at the end of alcoholic fermentation, the presence of yeast seemed to favour bacterial viability and activity and bacteria performed MLF even in difficult conditions such as pH values around 3. In all wines malolactic fermentation was accompanied by total degradation of malic and citric acids and production of L-lactic acid, D-lactic and acetic acids.     

Excessive nitrogen supply and shoot trimming can impair colour development in Pinot Noir grapes and wine

We investigated the effects of nitrogen supply and shoot trimming on mature, field-grown Pinot Noir ( Vitis vinifera L.) vines. Ammonium nitrate (0, 30, 60, 90 kg N/ha) was applied at the beginning of flowering. Shoots were topped either once (at fruit set), or twice, (at fruit set and during the lag-phase of berry growth). Neither treatment affected grape berry and skin weights, yield and grape sugar, but high rates of nitrogen increased malic acid and reduced skin phenols, flavonols and anthocyanins. Malvidin-3-glucoside was the most abundant anthocyanin in skins and wine. It accounted for 75% of the total anthocyanins at the beginning of fermentation and its relative proportion increased to 95% in the finished wine.
Increases in anthocyanin concentration at the beginning of fermentation and before malolactic fermentation, were followed by declines during the later stages of alcoholic and malolactic fermentation. High nitrogen supply decreased anthocyanins in the juice and wine, increased pH and increased the percentage of malvidin-3-glucoside. Repeated shoot topping gave lower wine total phenols and anthocyanins and thus enhanced the nitrogen effect. Wine susceptibility to oxidation increased with higher pH and lower anthocyanin content. The best treatment combination for fruit and wine quality, in terms of colour and oxidative stability, was low nitrogen/single topping and the worst combination was high nitrogen/repeated topping. These results suggest that a combination of high rates of nitrogen fertiliser and repeated shoot trimming can decrease potential fruit and wine quality.     

Yeast and bacterial modulation of wine aroma and flavour

Wine is a highly complex mixture of compounds which largely define its appearance, aroma, flavour and mouth‐feel properties. The compounds responsible for those attributes have been derived in turn from three major sources, viz. grapes, microbes and, when used, wood (most commonly, oak). The grape‐derived compounds provide varietal distinction in addition to giving wine its basic structure. Thus, the floral monoterpenes largely define Muscat‐related wines and the fruity volatile thiols define Sauvignon‐related wines; the grape acids and tannins, together with alcohol, contribute the palate and mouth‐feel properties. Yeast fermentation of sugars not only produces ethanol and carbon dioxide but a range of minor but sensorially important volatile metabolites which gives wine its vinous character. These volatile metabolites, which comprise esters, higher alcohols, carbonyls, volatile fatty acids and sulfur compounds, are derived from sugar and amino acid metabolism. The malolactic fermentation, when needed, not only provides deacidification, but can enhance the flavour profile. The aroma and flavour profile of wine is the result of an almost infinite number of variations in production, whether in the vineyard or the winery. In addition to the obvious, such as the grapes selected, the winemaker employs a variety of techniques and tools to produce wines with specific flavour profiles. One of these tools is the choice of microorganism to conduct fermentation. During alcoholic fermentation, the wine yeast Saccharomyces cerevisiae brings forth the major changes between grape must and wine: modifying aroma, flavour, mouth‐feel, colour and chemical complexity. The wine bacterium Oenococcus oeni adds its contribution to wines that undergo malolactic fermentation. Thus flavour‐active yeasts and bacterial strains can produce desirable sensory results by helping to extract compounds from the solids in grape must, by modifying grape‐derived molecules and by producing flavour‐active metabolites. This article reviews some of the most important flavour compounds found in wine, and their microbiological origin.     

Effects of temperature, ammonium and glucose concentrations on yeast growth in a model wine system

In enology, alcoholic fermentation is a complex process involving several mechanisms. Slow and incomplete alcoholic fermentation is a chronic problem for the wine industry and factors leading to sluggish and stuck fermentations have been extensively studied and reviewed. The most studied cause of sluggish and stuck fermentation is the nitrogen content limitation. Nevertheless, other factors, such as temperature of fermentation and sugar concentration can affect the growth of yeasts. In this study we modelled the yeast growth‐cycle in wine model system as a function of temperature, sugar and ammonium concentrations; the individual effects and the interaction of these factors were analysed by means of a quadratic response surface methodology. Cell concentrations and weight loss were monitored in the whole wine fermentation process. The results of central composite design show that lower is the availability of nitrogen, higher is the cell growth rate; moreover, initial nitrogen concentration also influences survival time of Saccharomyces cerevisiae.     

Occurrence and significance of Bacillus thuringiensis on wine grapes

Wine grapes harvested at different stages during cultivation from several vineyards in New South Wales, Australia, harboured Bacillus thuringiensis at viable populations of 10(2)-10(6) cfu/g. Commercial preparations of B. thuringiensis had been sprayed onto the grapes as a biological insecticide. B. thuringiensis (10(1)-10(3) cfu/ml) was isolated from grape juice and fermenting grape juice in a commercial winery. Although B. thuringiensis remained viable when inoculated at 10(3)-10(4) cfu/ml into grape juice and wine (pH 3.0-6.0), it did not grow. Using in vitro agar culture assays, B. thuringiensis inhibited several grape-associated yeasts and bacteria as well as various species of fungi associated with grape spoilage and ochratoxin A production. B. thuringiensis did not inhibit Saccharomyces cerevisiae in agar culture or during alcoholic fermentation of grape juice. B. thuringiensis inhibited the malolactic bacterium, Oenococcus oeni, in agar culture but not during mixed cultures in a liquid medium.