Diversity of Saccharomyces strains on grapes and winery surfaces: analysis of their contribution to fermentative flora of Malbec wine from Mendoza (Argentina) during two consecutive years

Spontaneous fermentations are still conducted by several wineries in different regions of Argentina as a common practice. Native Saccharomyces strains associated with winery equipment, grape and spontaneous fermentations of Malbec musts from "Zona Alta del Río Mendoza" region (Argentina) were investigated during 2001 and 2002 in the same cellar. Low occurrence of Saccharomyces on grapes and their limited participation during fermentation were confirmed. Strain sequential substitution during fermentation was observed. Between 30% and 60% of yeast population at the end of fermentation was coming from yeasts already present in the winery. A stable and resident Saccharomyces micro-flora in the winery was confirmed. It exhibited a dynamic behaviour during season and between years. Commercial strains were found during fermentation in different percentages, but their presence on winery equipment was low. The present work represents a first approach to winery yeast and spontaneous fermentation Saccharomyces population dynamics in an important viticultural region from Argentina that has never been characterized before. The results obtained have an important significance for the local industry, showing for the first time the real situation of the microbial ecology of alcoholic fermentation in an industrial winery from Mendoza, Argentina.     

Presence of non-Saccharomyces yeasts in cellar equipment and grape juice during harvest time

The purpose of this study was to analyze the presence of different yeasts in the facilities of four wineries from the D.O.Ca. Rioja region in Spain. The study was conducted through the identification of the yeasts via the PCR-RFLP technique of the ITS region of rDNA. The diversity of non-Saccharomyces yeasts found in wineries has previously only been studied to a limited extent, despite the fact that these yeasts take part both in the start of spontaneous fermentation and in the changes which occur in the wines during their subsequent conservation. Most earlier studies carried out on cellar ecosystems have focussed on the clonal diversity of Saccharomyces cerevisiae. The results obtained in this study indicated that the presence of non-Saccharomyces yeasts in facilities is higher than that of the S. cerevisiae, with percentages of over 60% in all the wineries analyzed. Yeasts belonging to 10 genera and 18 species were isolated, but the only genera present in all four wineries were Cryptococcus, Pichia, and Saccharomyces. The Zygosaccharomyces bailii yeast responsible for taint was detected in one cleaned winery, in both the winemaking equipment and the fermenting must. It was also noted that the quantity and type of yeasts present in the facilities are related to the product used for cleaning them. It is also necessary to point out that the cleaning of the cellars prior to the reception of the grapes does not completely eliminate the yeasts present, so that these can subsequently become part of the vinification process.     

Composition of Saccharomyces cerevisiae strains in spontaneous fermentations of Pinot Noir and Chardonnay

There is a lack of knowledge about the composition of Saccharomyces cerevisiae strains in spontaneous fermentations of Pinot Noir and Chardonnay cultivars. The objectives were to determine the relative abundance of indigenous and commercial S. cerevisiae strains in spontaneous fermentations at three wineries from the two cultivars and to compare the composition of the S. cerevisiae strains between cultivars and wineries. Three fermentation vessels were sampled at three stages of fermentation for each cultivar at each winery. Isolates were identified to the strain level using seven microsatellite loci. Commercial S. cerevisiae strains were isolated at a frequency higher than that of the indigenous strains at each winery for both cultivars. The composition of S. cerevisiae strains was different for each cultivar and at each winery. Our results illustrate the clear influence of inoculated commercial active dry yeast strains on the composition of S. cerevisiae strains in spontaneous fermentations at wineries conducting both inoculated and spontaneous fermentations.     

Quantitative and qualitative analysis of non-Saccharomyces yeasts in spontaneous alcoholic fermentations

In this study, we looked at the yeast population present in four spontaneous alcoholic fermentations in the Rioja appellation (D.O.Ca. Rioja, Spain). The study was conducted through the identification of the yeasts via the PCR–RFLP technique of the ITS region of rDNA. In a first harvest, the qualitative diversity of the species present in spontaneous alcoholic fermentation was studied, and in a second harvest, their quantitative significance. In spontaneous fermentations, 17 different yeast species were found, although only two of them, Candida stellata and Kloeckera apiculata, as well as Saccharomyces cerevisiae, appeared in major proportions during fermentation. The significance of the non-Saccharomyces yeasts during the spontaneous alcoholic fermentation was conditioned by the presence of Saccharomyces cerevisiae in the medium. Species not cited in literature in connection with winemaking and yeasts associated with wines spoilage have been detected in all the alcoholic fermentations carried out in the qualitative study.     

Selected non-Saccharomyces wine yeasts in controlled multistarter fermentations with Saccharomyces cerevisiae

Non-Saccharomyces yeasts are metabolically active during spontaneous and inoculated must fermentations, and by producing a plethora of by-products, they can contribute to the definition of the wine aroma. Thus, use of Saccharomyces and non-Saccharomyces yeasts as mixed starter cultures for inoculation of wine fermentations is of increasing interest for quality enhancement and improved complexity of wines. We initially characterized 34 non-Saccharomyces yeasts of the genera Candida, Lachancea (Kluyveromyces), Metschnikowia and Torulaspora, and evaluated their enological potential. This confirmed that non-Saccharomyces yeasts from wine-related environments represent a rich sink of unexplored biodiversity for the winemaking industry. From these, we selected four non-Saccharomyces yeasts to combine with starter cultures of Saccharomyces cerevisiae in mixed fermentation trials. The kinetics of growth and fermentation, and the analytical profiles of the wines produced indicate that these non-Saccharomyces strains can be used with S. cerevisiae starter cultures to increase polysaccharide, glycerol and volatile compound production, to reduce volatile acidity, and to increase or reduce the total acidity of the final wines, depending on yeast species and inoculum ratio used. The overall effects of the non-Saccharomyces yeasts on fermentation and wine quality were strictly dependent on the Saccharomyces/non-Saccharomyces inoculum ratio that mimicked the differences of fermentation conditions (natural or simultaneous inoculated fermentation).     

The influence of pre‐fermentative practices on the dominance of inoculated yeast starter under industrial conditions

The influence of pre‐fermentative practices on the growth dynamics of a ‘natural’ starter culture with specific phenotype (H₂S^?) concurrently with wild yeast populations was evaluated under winery conditions. Different clarification procedures and added SO₂ strongly influenced species and cell numbers isolable at the pre‐fermentation stage. Independent treatments of must with sulphite addition or vacuum‐filtering clarification caused a 30‐fold reduction in viable cells. Clarification procedures, enhanced by the selective effect of SO₂ addition, induced the appearance of Saccharomyces cerevisiae ‘wild’ yeasts. Correct application of the inoculum generally guarantees the dominance of fermentation by starter cultures. However, inoculated fermentations using unclarified white and red musts exhibited a consistent presence and persistence of non‐Saccharomyces and/or Saccharomyces ‘wild’ yeasts during fermentation. The extent and composition of the initial wild microflora at the start of fermentation may affect the presence and persistence of wild Saccharomyces and non‐Saccharomyces yeasts during guided fermentations under commercial conditions. The above findings confirm the results of previous works carried out at laboratory‐ or pilot‐scale level. Furthermore, they suggest a clear correlation between the modality of pre‐fermentative practices and the presence and persistence of ‘wild’ yeasts during fermentation. © 2002 Society of Chemical Industry     

Yeasts found in vineyards and wineries

Wine is a complex beverage, comprising thousands of metabolites that are produced through the action of a plethora of yeasts and bacteria during fermentation of grape must. These microbial communities originate in the vineyard and the winery and reflect the influence of several factors including grape variety, geographical location, climate, vineyard spraying, technological practices, processing stage and season (pre‐harvest, harvest, post‐harvest). Vineyard and winery microbial communities have the potential to participate during fermentation and influence wine flavour and aroma. Therefore, there is an enormous interest in isolating and characterising these communities, particularly non‐Saccharomyces yeast species to increase wine flavour diversity, while also exploting regional signature microbial populations to enhance regionality. In this review we describe the role and relevance of the main non‐Saccharomyces yeast species found in vineyards and wineries. This includes the latest reports covering the application of these species for winemaking; and the biotechnological characteristics and potential applications of non‐Saccharomyces species in other areas. In particular, we focus attention on the species for which molecular and genomic tools and resources are available for study. Copyright © 2016 John Wiley & Sons, Ltd.     

Effect of pure and mixed cultures of the main wine yeast species on grape must fermentations

Mixed inoculation of non-Saccharomyces yeasts and S. cerevisiae is of interest for the wine industry for technological and sensory reasons. We have analysed how mixed inocula of the main non-Saccharomyces yeasts and S. cerevisiae affect fermentation performance, nitrogen consumption and volatile compound production in a natural Macabeo grape must. Sterile must was fermented in triplicates and under the following six conditions: three pure cultures of S. cerevisiae, Hanseniaspora uvarum and Candida zemplinina and the mixtures of H. uvarum:S. cerevisiae (90:10), C. zemplinina:S. cerevisiae (90:10) and H. uvarum:C. zemplinina:S. cerevisiae (45:45:10). The presence of non-Saccharomyces yeasts slowed down the fermentations and produced higher levels of glycerol and acetic acid. Only the pure H. uvarum fermentations were unable to finish. Mixed fermentations consumed more of the available amino acids and were more complex and thus better able to synthesise volatile compounds. However, the amount of acetic acid was well above the admissible levels and compromises the immediate application of mixed cultures.     

Lachancea thermotolerans and Saccharomyces cerevisiae in simultaneous and sequential co-fermentation: A strategy to enhance acidity and improve the overall quality of wine

In the last few years there is an increasing interest on the use of mixed fermentation of Saccharomyces and non-Saccharomyces wine yeasts for inoculation of wine fermentations to enhance the quality and improve complexity of wines. In the present work Lachancea (Kluyveromyces) thermotolerans and Saccharomyces cerevisiae were evaluated in simultaneous and sequential fermentation with the aim to enhance acidity and improve the quality of wine.     

THE INCIDENCE AND SIGNIFICANCE OF WILD SACCHAROMYCES CONTAMINANTS IN THE BREWERY

The persistence of low levels of contamination by non-brewing Saccharomyces through several batch fermentations establishes the immuno-fluorescent method as a very sensitive procedure for estimating the microbiological purity of pitching yeasts. Trade return figures for draught beers show that in this brewery the principal cause for high rejection rates has, on several occasions, been contamination of pitching yeasts with “wild” Saccharomyces. The recommendation is made that pitching yeasts should be discarded when the level of infection achieves 100 cells of wild Saccharomyces per million cells of brewing yeast.     

Monitoring a mixed starter of Hanseniaspora vineae-Saccharomyces cerevisiae in natural must: impact on 2-phenylethyl acetate production

The effect of simultaneous or sequential inoculation of Hanseniaspora vineae CECT 1471 and Saccharomyces cerevisiae T73 in non-sterile must on 2-phenylethyl acetate production has been examined. In both treatments tested, no significant differences in Saccharomyces yeast growth were found, whereas non-Saccharomyces yeast growth was significantly different during all days of fermentation. Independently of the type of inoculation, S. cerevisiae was the predominant species from day 3 till the end of the fermentation. The dynamics of indigenous and inoculated yeast populations showed H. vineae to be the predominant non-Saccharomyces species at the beginning of fermentation in sequentially inoculated wines, whereas the simultaneous inoculation of S. cerevisiae did not permit any non-Saccharomyces species to become predominant. Differences found in non-Saccharomyces yeast growth in both fermentations influenced the analytical profiles of final wines and specifically 2-phenylethyl acetate concentration which was two-fold increased in sequentially inoculated wines in comparison to those co-inoculated. In conclusion we have shown that H. vineae inoculated as part of a sequential mixed starter is able to compete with native yeasts present in non-sterile must and modify the wine aroma profile.     

Indigenous lactic acid bacteria communities in alcoholic and malolactic fermentations of Tempranillo wines elaborated in ten wineries of La Rioja (Spain)

Indigenous lactic acid bacteria (LAB) communities have been analyzed for three years (2006, 2007 and 2008) during alcoholic (AF) and malolactic (MLF) fermentations of Tempranillo wines in ten wineries of La Rioja. The results showed that analytical composition of wines and physical–chemical conditions of elaboration influenced the LAB populations, the MLF duration and the percentage of each isolated species and strains. The highest diversity of LAB species was observed during AF in all the wineries. Oenococcus oeni was present in all studied stages of the fermentation process, being the predominant species at final AF stage. The study of 925 isolates of O. oeni by Pulsed Field Gel Electrophoresis (PFGE) allowed the detection of a total of 112 distinct genotypes. Most fermentation stages of both AF and MLF showed mixed O. oeni strain populations, so that there were different genotypes able to share their ecological niche or tank in spontaneous MLF. The frequency of participation of each genotype varied either from year to year or from winery to winery. Otherwise, seven genotypes were detected in the three studied years and in at least three out of the ten studied wineries, being four of them also present in the three studied subzones of this region. These results suggest the existence of an endemic microbiota in this region, the adaptation of indigenous O. oeni strains to the winery conditions every year and the interest of selecting predominant genotypes in order to preserve the biodiversity and peculiarity of these wines.     

Review: Pure non‐Saccharomyces starter cultures for beer fermentation with a focus on secondary metabolites and practical applications

Recently there has been increased interest in using non‐Saccharomyces yeasts to ferment beer. The worldwide growth of craft beer and microbreweries has revitalised the use of different yeast strains with a pronounced impact on aroma and flavour. Using non‐conventional yeast gives brewers a unique selling point to differentiate themselves. Belgian brewers have been very successful in using wild yeasts and mixed fermentations that often contain non‐Saccharomyces yeasts. Historically, ancient beers and beers produced before the domestication of commonly used Saccharomyces strains most likely included non‐Saccharomyces species. Given the renewed interest in using non‐Saccharomyces yeasts to brew traditional beers and their potential application to produce low‐alcohol or alcohol‐free beer, the fermentation and flavour characteristics of different species of non‐Saccharomyces pure culture yeast were screened for brewing potential (Brettanomyces anomalus and bruxellensis, Candida tropicalis and shehatae, Saccharomycodes ludwigii, Torulaspora delbrueckii, Pichia kluyveri, Zygosaccharomyces rouxii). Alcohol‐free beer is already industrially produced using S. ludwigii, a maltose‐negative species, which is a good example of the introduction of non‐Saccharomyces yeast to breweries. Overall, non‐Saccharomyces yeasts represent a large resource of biodiversity for the production of new beers and have the potential for wider application to other beverage and industrial applications. Almost all of the trials reviewed were conducted with varying fermentation parameters, which plays an important role in the outcome of the studies. To understand these impacts all trials were described with their major fermentation parameters. Copyright © 2016 The Institute of Brewing & Distilling     

Production of Higher Alcohols and Short Chain Fatty Acids by Different Yeasts Used in Rum Fermentations

The production of higher alcohols and short chain fatty acids by yeasts used in rum fermentations was greater by the Saccharomyces strains than by those belonging to the Schizosaccharomyces genus. There were also qualitative differences between these two genera. Among the organic acids of the raw material, citric acid and cisaconitic acid constitute a special feature of the composition of the resulting rums. Their presence entails the abundant production of biomass. Citric acid alone induced acrylic acid formation by strains of two species among the three studied.     

Outlining a future for non-Saccharomyces yeasts: selection of putative spoilage wine strains to be used in association with Saccharomyces cerevisiae for grape juice fermentation

The use of non-Saccharomyces yeasts that are generally considered as spoilage yeasts, in association with Saccharomyces cerevisiae for grape must fermentation was here evaluated. Analysis of the main oenological characteristics of pure cultures of 55 yeasts belonging to the genera Hanseniaspora, Pichia, Saccharomycodes and Zygosaccharomyces revealed wide biodiversity within each genus. Moreover, many of these non-Saccharomyces strains had interesting oenological properties in terms of fermentation purity, and ethanol and secondary metabolite production. The use of four non-Saccharomyces yeasts (one per genus) in mixed cultures with a commercial S. cerevisiae strain at different S. cerevisiae/non-Saccharomyces inoculum ratios was investigated. This revealed that most of the compounds normally produced at high concentrations by pure cultures of non-Saccharomyces, and which are considered detrimental to wine quality, do not reach threshold taste levels in these mixed fermentations. On the other hand, the analytical profiles of the wines produced by these mixed cultures indicated that depending on the yeast species and the S. cerevisiae/non-Saccharomyces inoculum ratio, these non-Saccharomyces yeasts can be used to increase production of polysaccharides and to modulate the final concentrations of acetic acid and volatile compounds, such as ethyl acetate, phenyl-ethyl acetate, 2-phenyl ethanol, and 2-methyl 1-butanol.     

Characterization of the yeast ecosystem in grape must and wine using real-time PCR

The complex microbial ecosystem of grape must and wine harbours a wide diversity of yeast species. Specific oligonucleotide primers for real-time quantitative PCR(QPCR) were designed to analyse several important non-Saccharomyces yeasts (Issatchenkia orientalis, Metschnikowia pulcherrima, Torulaspora delbrueckii, Candida zemplinina and Hanseniaspora spp.) and Saccharomyces spp. in fresh wine must, during fermentation and in the finished wine. The specificity of all primer couples for their target yeast species were validated and the QPCR methods developed were compared with a classic approach of colony identification by RFLP-ITS-PCR on cultured samples. Once the methods had been developed and validated, they were used to study these non-Saccharomyces yeasts in wine samples and to monitor their dynamics throughout the fermentation process. This study confirms the usefulness and the relevance of QPCR for studying non-Saccharomyces yeasts in the complex yeast ecosystem of grape must and wine.     

FORMULATION AND TESTING OF CUPRIC SULPHATE MEDIUM FOR WILD YEAST DETECTION

A new differential medium, cupric sulphate medium, used for the detection of wild yeasts has been formulated and tested. This medium suppressed the growth of culture yeasts and supported that of most non-Saccharomyces wild yeasts. It is not suitable for the detection of Saccharomyces wild yeasts. The contaminating wild yeasts in yeast samples and swab samples were easily detected by this medium. Since Lin's medium or modified Lin's medium is suitable for the detection of Saccharomyces wild yeasts, it is suggested that they be used in conjunction with cupric sulphate medium for detecting a more complete spectrum of wild yeasts.     

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.     

Screening for new brewing yeasts in the non‐Saccharomyces sector with Torulaspora delbrueckii as model

This study describes a screening system for future brewing yeasts focusing on non‐Saccharomyces yeasts. The aim was to find new yeast strains that can ferment beer wort into a respectable beer. Ten Torulaspora delbrueckii strains were put through the screening system, which included sugar utilization tests, hop resistance tests, ethanol resistance tests, polymerase chain reaction fingerprinting, propagation tests, amino acid catabolism and anabolism, phenolic off‐flavour tests and trial fermentations. Trial fermentations were analysed for extract reduction, pH drop, yeast concentration in bulk fluid and fermentation by‐products. All investigated strains were able to partly ferment wort sugars and showed high tolerance to hop compounds and ethanol. One of the investigated yeast strains fermented all the wort sugars and produced a respectable fruity flavour and a beer of average ethanol content with a high volatile flavour compound concentration. Two other strains could possibly be used for pre‐fermentation as a bio‐flavouring agent for beers that have been post‐fermented by Saccharomyces strains as a consequence of their low sugar utilization but good flavour‐forming properties. Copyright © 2015 John Wiley & Sons, Ltd.     

Spoilage yeasts in the wine industry

Yeasts play a central role in the spoilage of foods and beverages, mainly those with high acidity and reduced water activity (a(w)). A few species are capable of spoiling foods produced according to good manufacturing practices (GMPs). These can survive and grow under stress conditions where other microorganisms are not competitive. However, many of the aspects determining yeast spoilage have yet to be clarified. This critical review uses the wine industry as a case study where serious microbiological problems are caused by yeasts. First, the limitations of the available tools to assess the presence of spoilage yeasts in foods are discussed. Next, yeasts and factors promoting their colonisation in grapes and wines are discussed from the ecological perspective, demonstrating that a deeper knowledge of vineyard and winery ecosystems is essential to establish the origin of wine spoilage yeasts, their routes of contamination, critical points of yeast infection, and of course, their control. Further, zymological indicators are discussed as important tools to assess the microbiological quality of wines, although they are rarely used by the wine industry.The concepts of the susceptibility of wine to spoilage yeasts and wine stability are addressed based on scientific knowledge and industrial practices for monitoring yeast contamination. A discussion on acceptable levels of yeasts and microbiological criteria in the wine industry is supported by data obtained from wineries, wholesalers, and the scientific literature.Finally, future directions for applied research are proposed, involving collaboration between scientists and industry to improve the quality of wine and methods for monitoring the presence of yeast.