Possible use of Biolog methodology for monitoring yeast presence in alcoholic fermentation for wine‐making

Aims: A research was undertaken to explore the possibility to use Biolog system of microbial metabolic characterization for the monitoring of yeast population evolution during alcoholic fermentation for wine production. Methods and Results: An application of Biolog system was employed for the characterization of yeasts of oenological interest, in pure cultures and mixed consortia, in various cell concentrations. The system’s capacity to discriminate among different cell concentrations of the same yeast strain was ascertained, along with the capacity to discriminate between mixed and pure populations. Conclusions: The tested application of Biolog system resulted suitable for a quick recognition (24 h) of the presence of starter cultures within mixed populations of autochthonous yeasts. Such discrimination was confirmed with the one resulting from molecular techniques. Significance and Impact of the Study: The study suggests the possibility to employ Biolog system for an early monitoring of yeast evolution in modern wine‐making fermentations, where specialized yeasts are more and more frequently used as starters and their ability to overcome autochthonous yeast populations is crucial.     

Field-flow fractionation as analytical technique for the characterization of dry yeast: correlation with wine fermentation activity

Important oenological properties of wine depend on the winemaking yeast used in the fermentation process. There is considerable controversy about the quality of yeast, and a simple and cheap analytical methodology for quality control of yeast is needed. Gravitational field flow fractionation (GFFF) was used to characterize several commercial active dry wine yeasts from Saccharomyces cerevisiae and Saccharomyces bayanus and to assess the quality of the raw material before use. Laboratory-scale fermentations were performed using two different S. cerevisiae strains as inocula, and GFFF was used to follow the behavior of yeast cells during alcoholic fermentation. The viable/nonviable cell ratio was obtained by flow cytometry (FC) using propidium iodide as fluorescent dye. In each experiment, the amount of dry wine yeast to be used was calculated in order to provide the same quantity of viable cells. Kinetic studies of the fermentation process were performed controlling the density of the must, from 1.071 to 0.989 (20/20 density), and the total residual sugars, from 170 to 3 g/L. During the wine fermentation process, differences in the peak profiles obtained by GFFF between the two types of commercial yeasts that can be related with the unlike cell growth were observed. Moreover, the strains showed different fermentation kinetic profiles that could be correlated with the corresponding fractograms monitored by GFFF. These results allow optimism that sedimentation FFF techniques could be successfully used for quality assessment of the raw material and to predict yeast behavior during yeast-based bioprocesses such as wine production.     

Oenological properties of non-Saccharomyces yeasts associated with wine-making

Several yeast cultures belonging to five non-Saccharomyces species associated with wine-making were evaluated for their oenological properties. Results showed that Candida stellata and Torulaspora delbrueckii could positively affect the taste and flavour of alcoholic beverages. Apiculate yeasts exhibited large amounts of negative byproducts, particularly ethyl acetate. Nevertheless, Kloeckera apiculata showed a significantly negative correlation between either acetic acid and ethyl acetate formation and ethanol production. Selected non-Saccharomyces yeast cultures could be applied profitably in wine-making for optimization of wine bouquet using new fermentation technologies.     

Oenological properties of non-Saccharomyces yeasts associated with wine-making

Several yeast cultures belonging to five non-Saccharomyces species associated with wine-making were evaluated for their oenological properties. Results showed that Candida stellata and Torulaspora delbrueckii could positively affect the taste and flavour of alcoholic beverages. Apiculate yeasts exhibited large amounts of negative byproducts, particularly ethyl acetate. Nevertheless, Kloeckera apiculata showed a significantly negative correlation between either acetic acid and ethyl acetate formation and ethanol production. Selected non-Saccharomyces yeast cultures could be applied profitably in wine-making for optimization of wine bouquet using new fermentation technologies.     

Molecular identification and characterization of wine yeasts isolated from Tenerife (Canary Island, Spain)

AIMS: The present study was aimed at the identification, differentiation and characterization of indigenous yeasts isolated from Tenerife vineyards (viticulture region that has never been characterized before). Microbiota were studied from 14 samples taken during fermentations carried out in the 2002 vintage, from 11 wineries belonging to five wine regions on Tenerife Island. METHODS AND RESULTS: Yeasts' strains were identified and characterized through restriction analysis of the 5.8S-internal transcribed spacer region and the mitochondrial DNA. At the beginning of alcoholic fermentation, 26 yeast species were found, where 14 species were present in significant frequencies in only one sample. Likewise, the Saccharomyces cerevisiae strains isolated are very specific, as they were only present in one wine region. CONCLUSIONS: There were isolated specific yeasts from each region on Tenerife Island. The founded yeasts may be responsible for distinctive and interesting properties of the studied wines. SIGNIFICANCE AND IMPACT OF THE STUDY: This study forms part of an extensive taxonomic survey within the ecological framework of vineyards in Tenerife. This investigation is an essential step towards the preservation and exploitation of the hidden oenological potential of the untapped wealth of yeast biodiversity in the grape growing regions of this island. The results obtained demonstrate the value of using molecular genetic methods in taxonomic and ecological surveys. The results also shed some light on the ecology and oenological potential of S. cerevisiae strains isolated from this unique environment.     

Kinetics of growth and sugar consumption in yeasts

An overview is presented of the steady- and transient state kinetics of growth and formation of metabolic byproducts in yeasts.Saccharomyces cerevisiae is strongly inclined to perform alcoholic fermentation. Even under fully aerobic conditions, ethanol is produced by this yeast when sugars are present in excess. This so-called Crabtree effect probably results from a multiplicity of factors, including the mode of sugar transport and the regulation of enzyme activities involved in respiration and alcoholic fermentation. The Crabtree effect inS. cerevisiae is not caused by an intrinsic inability to adjust its respiratory activity to high glycolytic fluxes. Under certain cultivation conditions, for example during growth in the presence of weak organic acids, very high respiration rates can be achieved by this yeast.S. cerevisiae is an exceptional yeast since, in contrast to most other species that are able to perform alcoholic fermentation, it can grow under strictly anaerobic conditions.Non-Saccharomyces yeasts require a growth-limiting supply of oxygen (i.e. oxygen-limited growth conditions) to trigger alcoholic fermentation. However, complete absence of oxygen results in cessation of growth and therefore, ultimately, of alcoholic fermentation. Since it is very difficult to reproducibly achieve the right oxygen dosage in large-scale fermentations, non-Saccharomyces yeasts are therefore not suitable for large-scale alcoholic fermentation of sugar-containing waste streams. In these yeasts, alcoholic fermentation is also dependent on the type of sugar. For example, the facultatively fermentative yeastCandida utilis does not ferment maltose, not even under oxygen-limited growth conditions, although this disaccharide supports rapid oxidative growth.     

Interactions between Brettanomyces bruxellensis and other yeast species during the initial stages of winemaking

AIMS: Wine is the product of complex interactions between yeasts and bacteria in grape must. Amongst yeast populations, two groups can be distinguished. The first, named non-Saccharomyces (NS), colonizes, with many other micro-organisms, the surface of grape berries. In the past, NS yeasts were primarily considered as spoilage micro-organisms. However, recent studies have established a positive contribution of certain NS yeasts to wine quality. Amongst the group of NS yeasts, Brettanomyces bruxellensis, which is not prevalent on wine grapes, plays an important part in the evolution of wine aroma. Some of their secondary metabolites, namely volatile phenols, are responsible for wine spoilage. The other group contributing to wine aroma, which is also the main agent of alcoholic fermentation (AF), is composed of Saccharomyces species. The fermenting must is a complex microbial ecosystem where numerous yeast strains grow and die according to their adaptation to the medium. Yeast-yeast interactions occur during winemaking right from the onset of AF. The aim of this study was to describe the interactions between B. bruxellensis, other NS and Saccharomyces cerevisiae during laboratory and practical scale winemaking. METHODS AND RESULTS: Molecular methods such as internal transcribed spacer-restriction fragment length polymorphism and polymerase chain reaction and denaturing gradient gel electrophoresis were used in laboratory scale experiments and cellar observations. The influence of different oenological practices, like the level of sulphiting at harvest time, cold maceration preceding AF, addition of commercial active dry yeasts on B. bruxellensis and other yeast interactions and their evolution during the initial stages of winemaking have been studied. Brettanomyces bruxellensis was the most adapted NS yeast at the beginning of AF, and towards the end of AF it appeared to be more resistant than S. cerevisiae to the conditions of increased alcohol and sugar limitation. CONCLUSIONS: Among all NS yeast species, B. bruxellensis is better adapted than other wild yeasts to resist in must and during AF. Moreover, B. bruxellensis appeared to be more tolerant to ethanol stress than S. cerevisiae and after AF B. bruxellensis was the main yeast species in wine. SIGNIFICANCE AND IMPACT OF THE STUDY: Brettanomyces bruxellensis interacts with other yeast species and adapts to the wine medium as the dominant yeast species at the end of AF. Contamination of B. bruxellensis might take place at the beginning of malolactic fermentation, which is a critical stage in winemaking.     

Wine yeasts for the future

International competition within the wine market, consumer demands for newer styles of wines and increasing concerns about the environmental sustainability of wine production are providing new challenges for innovation in wine fermentation. Within the total production chain, the alcoholic fermentation of grape juice by yeasts is a key process where winemakers can creatively engineer wine character and value through better yeast management and, thereby, strategically tailor wines to a changing market. This review considers the importance of yeast ecology and yeast metabolic reactions in determining wine quality, and then discusses new directions for exploiting yeasts in wine fermentation. It covers criteria for selecting and developing new commercial strains, the possibilities of using yeasts other than those in the genus of Saccharomyces, the prospects for mixed culture fermentations and explores the possibilities for high cell density, continuous fermentations.     

A low-cost procedure for production of fresh autochthonous wine yeast

A low-cost procedure was designed for easy and rapid response-on-demand production of fresh wine yeast for local wine-making. The pilot plant produced fresh yeast culture concentrate with good microbial quality and excellent oenological properties from four selected wine yeasts. The best production yields were obtained using 2% sugar beet molasses and a working culture volume of less than 60% of the fermenter capacity. The yeast yield using 2% sugar grape juice was low and had poor cell viability after freeze storage, although the resulting yeast would be directly available for use in the winery. The performance of these yeasts in commercial wineries was excellent; they dominated must fermentation and improved its kinetics, as well as improving the physicochemical parameters and the organoleptic quality of red and white wines.     

Oxygen requirements of the food spoilage yeast Zygosaccharomyces bailii in synthetic and complex media

Most yeast species can ferment sugars to ethanol, but only a few can grow in the complete absence of oxygen. Oxygen availability might, therefore, be a key parameter in spoilage of food caused by fermentative yeasts. In this study, the oxygen requirement and regulation of alcoholic fermentation were studied in batch cultures of the spoilage yeast Zygosaccharomyces bailii at a constant pH, pH 3.0. In aerobic, glucose-grown cultures, Z. bailii exhibited aerobic alcoholic fermentation similar to that of Saccharomyces cerevisiae and other Crabtree-positive yeasts. In anaerobic fermentor cultures grown on a synthetic medium supplemented with glucose, Tween 80, and ergosterol, S. cerevisiae exhibited rapid exponential growth. Growth of Z. bailii under these conditions was extremely slow and linear. These linear growth kinetics indicate that cell proliferation of Z. bailii in the anaerobic fermentors was limited by a constant, low rate of oxygen leakage into the system. Similar results were obtained with the facultatively fermentative yeast Candida utilis. When the same experimental setup was used for anaerobic cultivation, in complex YPD medium, Z. bailii exhibited exponential growth and vigorous fermentation, indicating that a nutritional requirement for anaerobic growth was met by complex-medium components. Our results demonstrate that restriction of oxygen entry into foods and beverages, which are rich in nutrients, is not a promising strategy for preventing growth and gas formation by Z. bailii. In contrast to the growth of Z. bailii, anaerobic growth of S. cerevisiae on complex YPD medium was much slower than growth in synthetic medium, which probably reflected the superior tolerance of the former yeast to organic acids at low pH.     

An inverse correlation between stress resistance and stuck fermentations in wine yeasts. A molecular study.

During alcoholic fermentations yeast cells are subjected to several stress conditions and, therefore, yeasts have developed molecular mechanisms in order to resist this adverse situation. The mechanisms involved in stress response have been studied in Saccharomyces cerevisiae laboratory strains. However a better understanding of these mechanisms in wine yeasts could open the possibility to improve the fermentation process. In this work an analysis of the stress response in three wine yeasts has been carried out by studying the expression of several representative genes under several stress conditions which occur during fermentation. We propose a simplified method to study how these stress conditions affect the viability of yeast cells. Using this approach an inverse correlation between stress-resistance and stuck fermentations has been found. We also have preliminary data about the use of the HSP12 gene as a molecular marker for stress-resistance in wine yeasts.     

Acetaldehyde addition throughout the growth phase alleviates the phenotypic effect of zinc deficiency in Saccharomyces cerevisiae

During experiments to determine the effects of exogenously added acetaldehyde on pure cultures of various yeast strains, we discovered that an early acetaldehyde perfusion during the growth phase allowed several yeasts to partially overcome the phenotypic effects of zinc depletion during alcoholic fermentation. We, therefore, performed genome-wide expression and proteomic analysis on an industrial Saccharomyces cerevisiae yeast strain (VL1) growing in zinc-replete or zinc-depleted conditions in the presence of perfused acetaldehyde to identify molecular markers of this effect. Zinc depletion severely affects ethanol production and therefore nicotinamide adenine dinucleotide (NAD) regeneration, although we observed partial compensation by the upregulation of the poorly efficient Fe-dependent Adh4p in our conditions. A coordinate metabolic response was indeed observed in response to the early acetaldehyde perfusion, and particularly of the lower part of glycolysis, leading to the cellular replenishment of NAD cofactor. These various findings suggest that acetaldehyde exchange between strains may inhibit the growth of some yeast strains while encouraging the growth of others. This phenomenon could be particularly important for understanding the ecology of colonization of complex fermentation media by S. cerevisiae after elimination of non-Saccharomyces yeasts.     

Screening and typing of Patagonian wine yeasts for glycosidase activities

AIMS: The purpose of this study was to select autochthonous glycosidase producer yeasts with potential use in industrial production of Patagonian red wines. METHODS AND RESULTS: The study was carried out in oenological autochthonous yeasts from Comahue region (Argentinean North Patagonia). A set of screenable yeast phenotypic characteristics indicative of their potential usefulness in more aromatic red wine production was defined and tested in both, Saccharomyces and non-Saccharomyces populations. Twelve isolates showing six different glycosidase phenotypes were selected and they were characterized at species and strain levels using molecular methods. A close correlation between molecular and phenotypic characteristics was observed. Five strains belonging to Candida guilliermondii, C. pulcherrima and Kloeckera apiculata with highest constitutive beta-glucosidase activity levels without anthocyanase activity were discriminated. Some of them also showed constitutive beta-xylosidase and inductive alpha-rhamnosidase activities. CONCLUSIONS: The extension of the selection of oenological yeast to non-Saccharomyces species provided strains possessing novel and interesting oenological characteristics which could have significant implications in the production of more aromatic young red wine. SIGNIFICANCE AND IMPACT OF THE STUDY: As these non-Saccharomyces are indigenous to wine, they can be used in mixed starters at the beginning or as pure cultures at the end fermentation to contribute in enhancing the wine nuance that is typical of this specific area.     

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.     

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.     

Genetic diversity in commercial wineries: effects of the farming system and vinification management on wine yeasts

Aims: Analysis of the diversity and distribution of wine yeasts isolated from organically and conventionally grown grapes, and during the subsequent fermentation with or without starter cultures in six different commercial wineries. Methods and Results: PCR‐RFLP screening of isolates revealed the involvement of ten different species. Saccharomyces cerevisiae, scarcely isolated from grapes, was the dominant species during the latter phases of fermentation, identifying 108 different genotypes by means of SSR analysis. Species and strains’ diversity and presence were strongly influenced by the farming system used to grow the grapes and the system of vinification. Conclusions: Organic farming management was more beneficial in terms of diversity and abundance than the conventional one. Induced fermentation generated a great replacement of native yeasts. Although winery‐resident yeasts resulted to be predominant in the process, some noncommercial strains originally in the vineyard were found in final stages of the fermentation, confirming that autochthonous strains of S. cerevisiae are capable to conduct the fermentation process up to its end. Significance and Impact of the Study: The study of natural yeast communities from commercial vineyards and wineries is an important step towards the preservation of native genetic resources. Our results have special relevance because it is the first time that the real situation of the yeast ecology of alcoholic fermentation in commercial wineries belonging to the relevant wine‐producing Appellation of Origin ‘Vinos de Madrid’ is shown.     

Production of a white wine and a protein-rich soy flour by yeast fermentation of soybean slurry,soybean milk and the whey from tofu production

A dry white wine with an alcoholic content of 10 to 14% v/v was produced by yeast fermentation of slurried ground soybeans, soybean milk and whey from tofu production. Wines from whey and soybean milk were judged by a 20 member taste panel to be acceptable and comparable to a commercial chablis control. Chemical analysis indicated that the high fat and protein contents of soybeans do not cause a problem in the production of wines from soybeans as the lipids and proteins are precipitated by the acid and alcohol formed during the fermentation. The less recovered following fermentation were dehydrated and ground to a flour having an enriched protein content due to the yeasts and an improved flavor resulting from the yeast fermentation.     

High-cell-density cultivation of yeasts on disaccharides in oxygen-limited batch cultures

Many facultatively fermentative yeast species exhibit a "Kluyver effect": even under oxygen-limited growth conditions, certain disaccharides that support aerobic, respiratory growth are not fermented, even though the component monosaccharides are good fermentation substrates. This article investigates the applicability of this phenomenon for high-cell-density cultivation of yeasts. In glucose-grown batch cultures of Candida utilis CBS 621, the onset of oxygen limitation led to alcoholic fermentation and, consequently, a decrease of the biomass yield on sugar. In maltose-grown cultures, alcoholic fermentation did not occur and oxygen-limited growth resulted in high biomass concentrations (90 g dry weight L(-1) from 200 g L(-1) maltose monohydrate in a simple batch fermentation). It was subsequently investigated whether this principle could also be applied to Kluyveromyces species exhibiting a Kluyver effect for lactose. In oxygen-limited, glucose-grown chemostat cultures of K. wickerhamii CBS 2745, high ethanol concentrations and low biomass yields were observed. Conversely, ethanol was absent and biomass yields on sugar were high in oxygen-limited chemostat cultures grown on lactose. Batch cultures of K. wickerhamii grown on lactose exhibited the same growth characteristics as the maltose-grown C. utilis cultures: absence of ethanol formation and high biomass yields. Within the species K. marxianus, the occurrence of a Kluyver effect for lactose is known to be strain dependent. Thus, K. marxianus CBS 7894 could be grown to high biomass densities in lactose-grown batch cultures, whereas strain CBS 5795 produced ethanol after the onset of oxygen limitation and, consequently, yielded low amounts of biomass. Because the use of yeast strains exhibiting a Kluyver effect obviates the need for controlled substrate-feeding strategies to avoid oxygen limitation, such strains should be excellently suited for the production of biomass and growth-related products from low-cost disaccharide-containing feedstocks. (c) 1996 John Wiley & Sons, Inc.     

Selection of an autochthonous Saccharomyces strain starter for alcoholic fermentation of Sherry base wines

Several indigenous Saccharomyces strains from musts were isolated in the Jerez de la Frontera region, at the end of spontaneous fermentation, in order to select the most suitable autochthonous yeast starter, during the 2007 vintage. Five strains were chosen for their oenological abilities and fermentative kinetics to elaborate a Sherry base wine. The selected autochthonous strains were characterized by molecular methods: electrophoretic karyotype and random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) and by physiological parameters: fermentative power, ethanol production, sugar consumption, acidity and volatile compound production, sensory quality, killer phenotype, desiccation, and sulphur dioxide tolerance. Laboratory- and pilot-scale fermentations were conducted with those autochthonous strains. One of them, named J4, was finally selected over all others for industrial fermentations. The J4 strain, which possesses exceptional fermentative properties and oenological qualities, prevails in industrial fermentations, and becomes the principal biological agent responsible for winemaking. Sherry base wine, industrially manufactured by means of the J4 strain, was analyzed, yielding, together with its sensory qualities, final average values of 0.9 g/l sugar content, 13.4 % (v/v) ethanol content and 0.26 g/l volatile acidity content; apart from a high acetaldehyde production, responsible for the distinctive aroma of “Fino”. This base wine was selected for “Fino” Sherry elaboration and so it was fortified; it is at present being subjected to biological aging by the so-called “flor” yeasts. The “flor” velum formed so far is very high quality. To the best of our knowledge, this is the first study covering from laboratory to industrial scale of characterization and selection of autochthonous starter intended for alcoholic fermentation in Sherry base wines. Since the 2010 vintage, the indigenous J4 strain is employed to industrially manufacture a homogeneous, exceptional Sherry base wine for “Fino” Sherry production.     

The Formation of Higher Alcohols in the Fermentation of Amino Acids by Yeast

We have found that some straight-chained α-amino acids are converted by yeast to the alcohols with correspondingly longer carbon chains in the alcoholic fermentation contrary to F. Ehrlich’s scheme, i.e., isobutyl alcohol from alanine and active amyl alcohol from α-amino-n-butyric acid or threonine.

In this report, we confirmed this fact in the alcoholic fermentation of many aliphatic amino acids by 2 yeast strains using gas chromatography. Moreover, n-propyl alcohol was proved to come from α-amino-n-butyric acid or threonine. Small quantities of n-propyl, isobutyl, active amyl and isoamyl alcohols were found in all the fermented solutions. There was some difference in the composition of higher alcohols of the alcoholic solutions fermented by different yeasts.