Evidence for the existence of "survival factors" as an explanation for some peculiarities of yeast growth, especially in grape must of high sugar concentration

The retardation and arrest of fermentation, observed before the complete sugar consumption of high-sugar grape must, come from an inhibition of the yeast metabolism during its decline phase and are variable with the strain. The addition of nutritional growth factors stimulates the initial growth of the yeast but is ineffective in the decline phase. Some substances, known previously as yeast anaerobic growth factors (sterols, oleanolic acid, oxytocin), in some conditions (initially aerated grape must and aerobically cultivated yeast) act by increasing the viability of the resting cells and prolonging their fermentation activity. These substances have been named "survival factors."     

Dynamics of the yeast strain population during spontaneous alcoholic fermentation determined by CHEF gel electrophoresis

Wine yeasts were isolated from spontaneous alcoholic fermentations performed with white and red grape musts from vintages 1991 and 1992. Yeast cells were analysed by physiological tests and gel electrophoretic karyotyping. It was shown that there is a succession of different strains in the yeast population during the time course of the fermentation process. Furthermore, the composition of the yeast strain population differs from grape must to grape must and from year to year, and may therefore be considered vineyard (terrain)- and vintage-dependent.     

Utilization of KHR killer as genetic marker for purity test of starter yeast during fermentation of grape musts

An excellent wine yeast, Saccharomyces cerevisiae W3, which had KHR killer, was added as a starter yeast into grape must and behavior of the starter strain and wild yeasts was investigated during fermentation by using KHR killer as a genetic marker. The KHR killer was detected only in the strain W3 and not in other wine and wild yeast strains. Accordingly, the frequency of starter yeast W3 was monitored throughout the fermentation of grape musts by using KHR killer, W3 was discriminated efficiently from wild yeasts during fermentation by KHR killer activity and proved to lead the fermentation as a dominant yeast until their termination.     

Two-carbon metabolites, polyphenols and vitamins influence yeast chronological life span in winemaking conditions

ABSTRACT: BACKGROUND: Viability in a non dividing state is referred to as chronological life span (CLS). Most grape juice fermentation happens when Saccharomyces cerevisiae yeast cells have stopped dividing; therefore, CLS is an important factor toward winemaking success. RESULTS: We have studied both the physical and chemical determinants influencing yeast CLS. Low pH and heat shorten the maximum wine yeast life span, while hyperosmotic shock extends it. Ethanol plays an important negative role in aging under winemaking conditions, but additional metabolites produced by fermentative metabolism, such as acetaldehyde and acetate, have also a strong impact on longevity. Grape polyphenols quercetin and resveratrol have negative impacts on CLS under winemaking conditions, an unexpected behavior for these potential anti-oxidants. We observed that quercetin inhibits alcohol and aldehyde dehydrogenase activities, and that resveratrol performs a pro-oxidant role during grape juice fermentation. Vitamins nicotinic acid and nicotinamide are precursors of NAD+, and their addition reduces mean longevity during fermentation, suggesting a metabolic unbalance negative for CLS. Moreover, vitamin mix supplementation at the end of fermentation shortens CLS and enhances cell lysis, while amino acids increase life span. CONCLUSIONS: Wine S. cerevisiae strains are able to sense changes in the environmental conditions and adapt their longevity to them. Yeast death is influenced by the conditions present at the end of wine fermentation, particularly by the concentration of two-carbon metabolites produced by the fermentative metabolism, such as ethanol, acetic acid and acetaldehyde, and also by the grape juice composition, particularly its vitamin content.     

Evidence for the Existence of “Survival Factors” as an Explanation for Some Peculiarities of Yeast Growth, Especially in Grape Must of High Sugar Concentration

The retardation and arrest of fermentation, observed before the complete sugar consumption of high-sugar grape must, come from an inhibition of the yeast metabolism during its decline phase and are variable with the strain. The addition of nutritional growth factors stimulates the initial growth of the yeast but is ineffective in the decline phase. Some substances, known previously as yeast anaerobic growth factors (sterols, oleanolic acid, oxytocin), in some conditions (initially aerated grape must and aerobically cultivated yeast) act by increasing the viability of the resting cells and prolonging their fermentation activity. These substances have been named “survival factors.”     

Inhibition of Alcoholic Fermentation of Grape Must by Fatty Acids Produced by Yeasts and Their Elimination by Yeast Ghosts

In a complete nutritive medium rich in sugar, such as grape must, the inhibition of alcoholic fermentation is caused by substances produced by the yeast which, acting synergistically with ethanol, are toxic to the yeasts themselves. Among these are decanoic and octanoic acids and their corresponding ethyl esters. Their adsorption by yeast ghosts permits the prevention and treatment of fermentation stoppages.     

Assimilation of grape phytosterols by Saccharomyces cerevisiae and their impact on enological fermentations

Although yeasts are known to be able to incorporate a wide variety of exogenous sterols under strict anaerobiosis, no data are available on the assimilation of grapevine phytosterols under enological conditions and the eventual impact on fermentation kinetics. We used therefore a mixture of pure phytosterols, in a proportion representative of the different grape skins phytosterols, to supplement a synthetic fermentation medium simulating a grape must. Under anaerobiosis, normal biomass formation was achieved with 5 mg phytosterols l^–1. Similar results were obtained in comparison with the observed maximal fermentation rates. These results clearly indicated that grape phytosterols may efficiently act as a substitute for ergosterol in the yeast membrane for promoting yeast growth and initial fermentative activity. Analysis of total yeast sterols indicated that phytosterols are accumulated without further modification, mainly in their esterified form. However, all the fermentations performed with synthetic media supplemented with phytosterols led to stuck fermentations, linked to a correlative strong decrease in cell viability during the stationary phase. Therefore, grape phytosterols are easily incorporated by yeast cells under enological conditions for promoting initial growth and fermentative activity, but rapidly perturb the yeast membrane properties by being the predominant sterols.     

Comparative proteomic analysis of alcoholic fermentation employing a new environmental strain of Saccharomyces cerevisiae

The aim of this study was to investigate the biochemical pathways induced during must fermentation employing the Saccharomyces cerevisiae, strain Z622, isolated from the traditional wine area of Zitsa (Epirus, Greece). Proteomic analysis (two-dimensional gel electrophoresis followed by nano-LC ESI-MS/MS) was used to assess the protein profile response of strain Z622 cultured in Debina grape must, yeast extract–malt extract (YM) or minimal medium (MM) under fermentation conditions, at the beginning and at the end of fermentation. The proteomic results and their analysis with Gene Ontology (GO) terms revealed that the intracellular proteins induced in yeast cells grown exclusively in Debina grape must during the early stage of fermentation were mainly involved in amino acid metabolism, glycolysis and sterol formation. These three pathways reflect the adaptation of the cell to the fermentative metabolism. During the late stage, several enzymes were induced due to starvation conditions or to participation in defence mechanisms against oxidative stress and protein degradation. Several of the identified enzymes have not previously been reported to be induced during wine fermentation and thus the results presented here can contribute to a better understanding of how S. cerevisiae cells adapt to wine fermentation.     

The effect of acetic acid bacteria upon the growth and metabolism of yeasts during the fermentation of grape juice

The influence of species of Acetobacter and Gluconobacter upon growth of the wine yeasts Saccharomyces cerevisiae, Kloeckera apiculata and Candida stellata was examined during mixed culture in grape juice. Acetobacter pasteurianus, A. aceti and Gluconobacter oxydans grew in conjunction with yeasts during juice fermentation. As determined by viable counts, yeast growth was only slightly impaired by the presence of bacteria. However, as judged by the concentrations of glucose, fructose, ethanol, glycerol, acetaldehyde, ethyl acetate, iso -amyl alcohol and organic acids in the fermented juice, acetic acid bacteria significantly influenced the alcoholic fermentation by yeasts.     

Characterisation of yeast microbiota,chemical and sensory properties of organic and biodynamic Sangiovese red wines

Wine quality is closely linked to the fermentation step, which is driven by the microbial ecology of grape and the use of selected microbial strains as well. The microbial species developing during fermentation determines the type and concentration of many substances, which contribute to the sensory properties of wine and its safety. In this view, the present work aims to characterise the yeast microbiota, chemical and sensory properties of Sangiovese red wines obtained from both biodynamic and organic agriculture. The natural yeast populations of grape musts and their evolution during spontaneous were monitored and investigated. In addition, the volatile composition, physico-chemical and safety features (ethyl-carbamate) and sensory properties of wines were evaluated. The results showed that the yeast population was mostly related to the grape management, i.e. organic or biodynamic, while the wine composition was mainly affected by the winemaking process, and then by the grape management.     

Effects of grape marcs acidification treatment on the evolution of indigenous yeast populations during the production of grappa

Aims: Grappa is a typical Italian product obtained from the distillation of grape marcs, the main by‐product of grape crushing. One technological treatment frequently performed on marcs is their acidification, in order to contrast the development of unwanted spoilage bacteria during the storage period needed for alcoholic fermentation. A pilot‐scale experiment was set‐up to study the dynamics of yeast populations during a 30‐day fermentation of acidified and nonacidified Prosecco grape pomace. Methods and Results: Saccharomyces cerevisiae population, examined after 4 and 15 days of storage by mitochondrial DNA‐RFLP analysis, resulted considerably different at strain level upon acidification. In particular, although the number of different strains rescued appeared particularly high in both kind of marcs compared with what happens in must fermentation, in the acidified material such number tends to moderately decrease during storage. Conclusions: Results obtained evidence that the acidification treatment did not influence yeast population neither in terms of number of cells nor in terms of biodiversity at species level. Therefore, such treatment can be used in distillery without negatively influencing ethanol production. Significance and Impact of Study: Even though some data are available on the effects of technological treatments on the chemical composition of the distillate, no microbiological studies have been published so far on the consequence of these practices on composition, biodiversity and evolution of yeast population.     

Effect of low-temperature fermentation on yeast nitrogen metabolism

The aim of this study was to analyse the influence of low-temperature wine fermentation on nitrogen consumption and nitrogen regulation. Synthetic grape must was fermented at 25 and 13°C. Low-temperature decreased both the fermentation and the growth rates. Yeast cells growing at low-temperature consumed less nitrogen than at 25°C. Specifically, cells at 13°C consumed less ammonium and glutamine, and more tryptophan. Low-temperature seemed to relax the nitrogen catabolite repression (NCR) as deduced from the gene expression of ammonium and amino acid permeases (MEP2 and GAP1) and the uptake of some amino acids subjected to NCR (i.e. arginine and glutamine). Low-temperature influences the quantity and the quality of yeast nitrogen requirements. Nitrogen-deficient grape musts and low temperature are two of the main prevalent causes of sluggish fermentations and, therefore, the effects of both growth conditions on yeast metabolism are of considerable interest for wine making.     

Influence of specific fermentation conditions on natural microflora of pomace in “Grappa” production

As reported in the European Community regulation, grappa is a spirit beverage made in Italy from marc that has been steam distilled or distilled after the addition of water. Grape marc from red grapes has already undergone alcoholic fermentation with the must and can be distilled immediately. Grape marc from white grapes does not contain ethanol but contains sugars that are fermented by spontaneous anaerobic fermentation during a storage period. The characteristic aroma of grappa consists of a large number of volatile compounds, which arise from various sources, the most important of which is yeast. Very few studies have been undertaken to characterize the natural populations of yeast during the fermentation of grape marc. The goal of this study was to understand how different pHs, temperatures and yeast starter cultures affect the growth and dynamics of yeast species involved in pomace fermentation, which could be the basis for improving the final quality of grappa production. We found that a temperature of 15°C has the greatest effect on improving the quality of the product. Unfortunately, due to the solid state of the grape marc and the impossibility of its mixing, it appears that acidification and the addition of yeast starter cultures during the silage period are not effective.     

酵母菌在红葡萄酒酒精发酵串罐中稳定性研究

在生产条件下,对酵母菌在红葡萄酒酒精发酵串罐过程中的稳定性进行了研究。结果表明,在近1个月的时间内（相当于酵母菌细胞无性繁殖了200代）,串罐过程中的酵母菌细胞不仅能保持初始酵母菌的发酵活性和优良特性的稳定性,而且由于葡萄汁的选择作用,串罐用的酵母菌细胞的发酵活性比初始酵母菌的活性更强,因而其酒精发酵的启动和速度都更快。     

Copper Tolerance and Biosorption of Saccharomyces cerevisiae during Alcoholic Fermentation

At high levels, copper in grape mash can inhibit yeast activity and cause stuck fermentations. Wine yeast has limited tolerance of copper and can reduce copper levels in wine during fermentation. This study aimed to understand copper tolerance of wine yeast and establish the mechanism by which yeast decreases copper in the must during fermentation. Three strains of Saccharomyces cerevisiae (lab selected strain BH8 and industrial strains AWRI R2 and Freddo) and a simple model fermentation system containing 0 to 1.50 mM Cu²⁺ were used. ICP-AES determined Cu ion concentration in the must decreasing differently by strains and initial copper levels during fermentation. Fermentation performance was heavily inhibited under copper stress, paralleled a decrease in viable cell numbers. Strain BH8 showed higher copper-tolerance than strain AWRI R2 and higher adsorption than Freddo. Yeast cell surface depression and intracellular structure deformation after copper treatment were observed by scanning electron microscopy and transmission electron microscopy; electronic differential system detected higher surface Cu and no intracellular Cu on 1.50 mM copper treated yeast cells. It is most probably that surface adsorption dominated the biosorption process of Cu²⁺ for strain BH8, with saturation being accomplished in 24 h. This study demonstrated that Saccharomyces cerevisiae strain BH8 has good tolerance and adsorption of Cu, and reduces Cu²⁺ concentrations during fermentation in simple model system mainly through surface adsorption. The results indicate that the strain selected from China’s stress-tolerant wine grape is copper tolerant and can reduce copper in must when fermenting in a copper rich simple model system, and provided information for studies on mechanisms of heavy metal stress.     

Study of beta-glucosidase production by wine-related yeasts during alcoholic fermentation. A new rapid fluorimetric method to determine enzymatic activity

AIMS: The beta-glucosidase activity is involved in the hydrolysis of several important compounds for the development of varietal wine flavour. The aim of the present study was to investigate the production of beta-glucosidase in a number of wine-related yeast strains and to measure and identify this activity over the course of grape juice fermentation. METHODS AND RESULTS: beta-glucosidase activity was measured as the amount of 4-methylumbelliferone released from 4-methylumbelliferyl-beta-d-glucopyranoside substrate. Intact cells of some grape and wine-spoilage yeasts showed beta-glucosidase activity much higher than those observed in wine yeasts "sensu stricto". During fermentation, three Saccharomyces cerevisiae strains, one Hanseniaspora valbyensis strain and one Brettanomyces anomalus strain showed beta-glucosidase activity both intra- and extracellularly. CONCLUSIONS: In the studied strains, beta-glucosidase activity was at its maximum when the cells were in the active growth phase. However, a lowering of medium pH to values around 3 during fermentation led to total loss of activity. SIGNIFICANCE AND IMPACT OF THE STUDY: During the course of this study, a new, rapid and reproducible method to assay beta-glucosidase activity was developed. The fact that Saccharomyces and non-Saccharomyces yeast strains are able to express beta-glucosidase activity during the alcoholic fermentation sheds new light on the contribution of these yeasts in the aroma expression of wines.     

Wine flavor and aroma

The perception of wine flavor and aroma is the result of a multitude of interactions between a large number of chemical compounds and sensory receptors. Compounds interact and combine and show synergistic (i.e., the presence of one compound enhances the perception of another) and antagonistic (a compound suppresses the perception of another) interactions. The chemical profile of a wine is derived from the grape, the fermentation microflora (in particular the yeast Saccharomyces cerevisiae), secondary microbial fermentations that may occur, and the aging and storage conditions. Grape composition depends on the varietal and clonal genotype of the vine and on the interaction of the genotype and its phenotype with many environmental factors which, in wine terms, are usually grouped under the concept of “terroir” (macro, meso and microclimate, soil, topography). The microflora, and in particular the yeast responsible for fermentation, contributes to wine aroma by several mechanisms: firstly by utilizing grape juice constituents and biotransforming them into aroma- or flavor-impacting components, secondly by producing enzymes that transform neutral grape compounds into flavor-active compounds, and lastly by the de novo synthesis of many flavor-active primary (e.g., ethanol, glycerol, acetic acid, and acetaldehyde) and secondary metabolites (e.g., esters, higher alcohols, fatty acids). This review aims to present an overview of the formation of wine flavor and aroma-active components, including the varietal precursor molecules present in grapes and the chemical compounds produced during alcoholic fermentation by yeast, including compounds directly related to ethanol production or secondary metabolites. The contribution of malolactic fermentation, ageing, and maturation on the aroma and flavor of wine is also discussed.     

Increasing fermentation efficiency at high sugar concentrations by supplementing an additional source of nitrogen during the exponential phase of the tequila fermentation process

In the tequila industry, fermentation is traditionally achieved at sugar concentrations ranging from 50 to 100 g x L(-1). In this work, the behaviour of the Saccharomyces cerevisiae yeast (isolated from the juices of the Agave tequilana Weber blue variety) during the agave juice fermentation is compared at different sugar concentrations to determine if it is feasible for the industry to run fermentation at higher sugar concentrations. Fermentation efficiency is shown to be higher (above 90%) at a high concentration of initial sugar (170 g x L(-1)) when an additional source of nitrogen (a mixture of amino acids and ammonium sulphate, different than a grape must nitrogen composition) is added during the exponential growth phase.     

Superoxide dismutase deficiency and the toxicity of the products of autooxidation of polyunsaturated fatty acids in yeast

Mutants of Saccharomyces cerevisiae, deficient in cytosolic superoxide dismutase and catalase activities were used to study the role of various oxygen species in the process of lipid peroxidation in yeast cells. Lipid peroxidation does not occur normally in yeast, because this organism is unable to form fatty acids with more than one double bond, whereas under physiological conditions, only fatty acids with at least two double bonds undergo this process. The fatty acid content of cellular lipids was modified by growing the cells in anoxia in the presence of oleic or linolenic acid. Toxic effects of oxygen were observed almost exclusively in those cells of yeast mutants deficient in superoxide dismutase, which contain linolenic acid in cellular lipids. Hypersensitivity of the mutant cells, however, results mainly from toxic effects of the products of autooxidation of extracellular fatty acids. These facts suggest that superoxide dismutases are in some way involved in preventing toxic effects of the products of lipid peroxidation and to some extent prevent the process of lipid peroxidation.