Yeast population dynamics in spontaneous fermentations: Comparison between two different wine-producing areas over a period of three years

Yeast ecology, biogeography and biodiversity are important and interesting topics of research. The population dynamics of yeasts in several cellars of two Spanish wine-producing regions was analysed for three consecutive years (1996 to 1998). No yeast starter cultures had been used in these wineries which therefore provided an ideal winemaking environment to investigate the dynamics of grape-related indigenous yeast populations. Non-Saccharomyces yeast species were identified by RFLPs of their rDNA, while Saccharomyces species and strains were identified by RFLPs of their mtDNA. This study confirmed the findings of other reports that non-Saccharomyces species were limited to the early stages of fermentation whilst Saccharomyces dominated towards the end of the alcoholic fermentation. However, significant differences were found with previous studies, such as the survival of non-Saccharomyces species in stages with high alcohol content and a large variability of Saccharomyces strains (a total of 112, all of them identified as Saccharomyces cerevisiae) with no clear predominance of any strain throughout all the fermentation, probably related to the absence of killer phenotype and lack of previous inoculation with commercial strains.     

Influence of killer strains of Saccharomyces cerevisiae on wine fermentation

The effect of killer strains of Saccharomyces cerevisiae on the growth of sensitive strains during must fermentation was studied by using a new method to monitor yeast populations. The capability of killer yeast strains to eliminate sensitive strains depends on the initial proportion of killer yeasts, the susceptibility of sensitive strains, and the treatment of the must. In sterile filtered must, an initial proportion of 2-6% of killer yeasts was responsible for protracted fermentation and suppression of isogenic sensitive strains. A more variable initial proportion was needed to get the same effect with non-isogenic strains. The suspended solids that remain in the must after cold-settling decreased killer toxin effect. The addition of bentonite to the must avoided protracted fermentation and the suppression of sensitive strains; however, the addition of yeast dietary nutrients with yeast cell walls did not, although it decreased fermentation lag.     

The use of killer biotyping in an ecological survey of yeast in an old patagonian winery

An ecological study of Saccharomyces cerevisiae strains in spontaneous alcoholic fermentation has been made in the same winery on two consecutive years (1993 and 1994) with Merlot type musts, and with Malbec type must on a third year (1998). Saccharomyces cerevisiae strains associated with winery surfaces were also analysed. Differential killer sensitivity patterns related to a killer reference panel of 10 killer yeasts belonging to nine species of four genera were used as a quick and simple procedure to discriminate between indigenous S. cerevisiae isolates at the strain level. Although a great diversity of wild strains was observed, two main indigenous S. cerevisiae strains, designated as S. cerevisiae 9 and S. cerevisiae 13, took over the Merlot type fermentation in both years. These strains also appeared in Malbec must fermentation during the year 1998 and they were again found on the winery surface the next year. These results show that some few and stable indigenous S. cerevisiae strains remained in the environmental winery over the considered period of time (1993–1999) and they represent an additional evidence of the taking over of musts by local strains of S. cerevisiae.     

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.     

Role of killer effect in fermentations conducted by mixed cultures of Saccharomyces cerevisiae

Abstract The present work reports on population dynamics in musts (pH 3.2) inoculated with pairs of Saccharomyces cerevisiae wild strains. Two assays determined the growth of both killer and sensitive strains; the latter were not totally eliminated from the must and non-proliferating populations were detected. Another two were carried out with two killer or two sensitive strains, respectively; the exponential growth of the two populations was observed in both cases. The succession of Saccharomyces cerevisiae strains was seen to be common in the four assays; only one strain proved to have the ability to complete fermentation, whereas the other disappeared after 28 days of fermentation. The most important fermentation compounds were estimated at the end of fermentations.     

Synergism among lactic acid, sulfite, pH and ethanol in alcoholic fermentation of Saccharomyces cerevisiae (PE-2 and M-26)

Summary The industrial production of ethanol is affected mainly by contamination by lactic acid bacteria besides others factors that act synergistically like increased sulfite content, extremely low pH, high acidity, high alcoholic content, high temperature and osmotic pressure. In this research two strains of Saccharomyces cerevisiae PE-2 and M-26 were tested regarding the alcoholic fermentation potential in highly stressed conditions. These strains were subjected to values up to 200 mg NaHSO₃ l⁻¹, 6 g lactic acid l⁻¹, 9.5% (w/v) ethanol and pH 3.6 during fermentative processes. The low pH (3.6) was the major stressing factor on yeasts during the fermentation. The M-26 strain produced higher acidity than the other, with higher production of succinic acid, an important inhibitor of lactic bacteria. Both strains of yeasts showed similar performance during the fermentation, with no significant difference in cell viability.     

An optimized procedure for the enological selection of non-<Emphasis Type="Italic">Saccharomyces</Emphasis> starter cultures

The apiculate yeasts are the species predominating the first stage of grape must alcoholic fermentation and are important for the production of desired volatile compounds. The aim of the present investigation was to establish a protocol for the enological selection of non-Saccharomyces strains directly isolated from a natural must fermentation during the tumultuous phase. At this scope, fifty Hanseniaspora uvarum isolates were characterized at strain level by employing a new combined PCR-based approach. One isolate representative of each identified strain was used in fermentation assays to assess strain-specific enological properties. The chemical analysis indicated that all the analyzed strains were low producers of acetic acid and hydrogen sulphide, whereas they showed fructophilic character and high glycerol production. Analysis of volatile compounds indicated that one strain could positively affect, during the alcoholic fermentation process, the taste and flavour of alcoholic beverages. The statistical evaluation of obtained results indicated that the selected autochthonous H. uvarum strain possessed physiological and technological properties which satisfy the criteria indicated for non-Saccharomyces wine yeasts selection. Our data suggest that the described protocol could be advantageously applied for the selection of non-Saccharomyces strains suitable for the formulation of mixed or sequential starters together with Saccharomyces cerevisiae.     

Resistance to chlorpyriphos in the predatory mite Kampimodromus aberrans

The predatory mite Kampimodromus aberrans (Oudemans) is a key biocontrol agent in vineyards in Italy and Southern Europe. Its susceptibility to common pesticides (e.g., organophosphates) has been considered an important factor in preventing successful biocontrol of phytophagous mites. Nevertheless, populations of K. aberrans apparently resistant to organophosphates (OPs) have been reported to occur in Northern Italian vineyards. The resistance of K. aberrans to fungicides (e.g., mancozeb) has been demonstrated in the laboratory in France, but little is known about the toxicity of insecticides towards K. aberrans. Of these pesticides, the OP chlorpyriphos is extensively used in viticulture to control lepidopterans and homopterans. The present study investigated the dose–response effect of chlorpyriphos in four K. aberrans strains characterized by different levels of exposure to OP insecticides in the past: from never to frequently exposed. Resistance to chlorpyriphos is demonstrated for populations collected from vineyards and apple orchards. Resistance factors exceeded 145,000× for the three strains collected in vineyards and orchard. LC₅₀ values for resistant strains were 1.85–6.83 times higher than the recommended field dose of chlorpyriphos for vineyards and orchards (525 mg a.i./l).     

Predominance of Saccharomyces uvarum during spontaneous alcoholic fermentation, for three consecutive years, in an Alsatian winery

AIMS: The purpose of this study was to determine the origin of the yeasts involved in the spontaneous alcoholic fermentation of an Alsatian wine. METHODS AND RESULTS: During three successive years, must was collected at different stages of the winemaking process and fermented in the laboratory or in the cellar. Saccharomyces yeasts were sampled at the beginning and at the end of the fermentations. Saccharomyces cerevisiae clones were genetically characterized by inter-delta PCR. Non-S. cerevisiae clones were identified as Saccharomyces uvarum by PCR-RFLP on MET2 gene and characterized at the strain level by karyotyping. The composition of the Saccharomyces population in the vineyard, after crushing and in the vat was analyzed. This led to three main results. First, the vineyard Saccharomyces population was rather homogeneous. Second, new non-resident strains had appeared in the must during the winemaking process. Finally, the yeast population in the vat only consisted in S. uvarum strains. CONCLUSION: This 3-year study has enabled us to show the involvement of indigenous S. uvarum in the alcoholic fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: This study gives a first insight into the polymorphism of S. uvarum strains involved in a spontaneous alcoholic fermentation.     

Occurrence and Growth of Killer Yeasts during Wine Fermentation

Sixteen wine fermentations were examined for the presence of killer yeasts. Killer property and sensitivity to killer action were found in isolates of Saccharomyces cerevisiae but not in isolates of Kloeckera, Candida, Hansenula, and Torulaspora spp. Several killer and killer-sensitive strains of S. cerevisiae were differentiated by colony morphology, and this property was used to monitor their growth kinetics in mixed cultures in grape juice. Killer-sensitive strains died off within 24 to 48 h during mixed-strain fermentation. Killer action was demonstrated at pH 3.0 and pH 3.5 and over the range of 15 to 25°C but depended on the proportion of killer to killer-sensitive cells at the commencement of fermentation. The dominance of killer strains in mixed-strain fermentations was reflected in the production of ethanol, acetic acid, and glycerol.     

Changes in the intracellular concentrations of the adenosine phosphates and nicotinamide adenine dinucleotides ofSaccharomyces cerevisiae during batch fermentation

Significant changes in the intracellular concentrations of adenosine phosphates and nicotinamide adenine dinucleotides were observed during fermentation of grape must by three different strains ofSaccharomyces cerevisiae: S. cerevisiae var.cerevisiae, a typical fermentative yeast strain and two flor-veil-forming strains,S. cerevisiae var.bayanus andS. cerevisiae var.capensis. The intracellular concentration of ATP was always higher inS. cerevisiae var.cerevisiae than in the flor-veil-forming strains. NAD⁺ and NADP⁺ concentrations decreased at faster rates in the flor-veil-forming yeasts than in the other yeast but NADH concentration was the same in all yeasts for the first 10 days of fermentation. NADPH concentration was always lower inS. cerevisiae var.cerevisiae than in the other yeasts and this yeast also showed higher rates of growth and fermentation during the early stages of the fermentation and the presence of non-viable cells at the end of fermentation. In contrast, the flor-veil-forming strains maintained growth and fermentation capabilities for a relatively long time and viable cells were present throughout the entire fermentation process (31 days).The authors are with the Department of Microbiology, Faculty of Sciences, University of Cordoba, Avda. San Alberto Magno s/n, 14004-Córdoba, Spain     

Yeast species associated with spontaneous wine fermentation of Cabernet Sauvignon from Ningxia,China

The eastern base of the Helan Mountains in Ningxia is a fast developing wine production area in China. Of urgent necessity to the Ningxia wine industry is to be able to produce wines with typical regional characteristics. It is well known that autochthonous yeast species and strains play an important role in introducing local character or terroir into the winemaking practice. The aim of this study was to investigate indigenous yeast species diversity and preselect desirable S. cerevisiae strains in the Ningxia region. Four hundred wine-related yeast colonies were isolated from Cabernet Sauvignon musts in three vineyards at the beginning, middle and final stages of spontaneous fermentations. Yeast species were first classified according to colony morphologies on Wallerstein Laboratory Nutrient Agar (WL) and confirmed by sequencing of the D1/D2 domain of the 26S rRNA gene. Nine unique colony morphology types were profiled on WL agar and three new types were found. After sequence analysis of representative colonies from each WL group, nine yeast species were identified, namely H. uvarum, H. occidentalis, M. pulcherrima, C. zemplinina, H. vineae, I. orientalis, Z. bailii, P. kluyveri and S. cerevisiae. The non-Saccharomyces yeasts appeared mainly in the early stage of the fermentation while H. uvarum, I. orientalis and P. kluyveri were able to remain in the final stage. Fourty-six S. cerevisiae isolates were preselected according to physiological characteristics and twelve strains with valuable fermentation properties were obtained.     

The effect of Debina grapevine indigenous yeast strains of <Emphasis Type="Italic">Metschnikowia</Emphasis> and <Emphasis Type="Italic">Saccharomyces</Emphasis> on wine flavour

The spontaneous alcoholic fermentation of grape must is a complex microbiological process involving a large number of various yeast species, to which the flavour of every traditional wine is largely attributed. Whilst Saccharomyces cerevisiae is primarily responsible for the conversion of sugar to alcohol, the activities of various non-Saccharomyces species enhance wine flavour. In this study, indigenous yeast strains belonging to Metschnikowia pulcherrima var. zitsae as well as Saccharomyces cerevisiae were isolated and characterized from Debina must (Zitsa, Epirus, Greece). In addition, these strains were examined for their effect on the outcome of the wine fermentation process when used sequentially as starter cultures. The resulting wine, as analyzed over three consecutive years, was observed to possess a richer, more aromatic bouquet than wine from a commercial starter culture. These results emphasize the potential of employing indigenous yeast strains for the production of traditional wines with improved flavour.     

Occurrence of killer yeasts in Argentine wineries

Fifty-six wine must samples, from wineries in various regions of Argentina, were examined at different fermentation stages for the presence of killer yeast strains. The distribution of isolated killer strains was markedly different from one region to another.The authors are with the Instituto de Biotecnología, Facultad de Ingeniería, Universidad Nacional de San Juan, Av. Lib, Gral. San Martín 1109, Oeste 5400, San Juan, Argentina;     

Yeast Population Dynamics during the Fermentation and Biological Aging of Sherry Wines

Molecular and physiological analyses were used to study the evolution of the yeast population, from alcoholic fermentation to biological aging in the process of “fino” sherry wine making. The four races of “flor” Saccharomyces cerevisiae (beticus, cheresiensis, montuliensis, and rouxii) exhibited identical restriction patterns for the region spanning the internal transcribed spacers 1 and 2 (ITS-1 and ITS-2) and the 5.8S rRNA gene, but this pattern was different, from those exhibited by non-flor S. cerevisiae strains. This flor-specific pattern was detected only after wines were fortified, never during alcoholic fermentation, and all the strains isolated from the velum exhibited the typical flor yeast pattern. By restriction fragment length polymorphism of mitochondrial DNA and karyotyping, we showed that (i) the native strain is better adapted to fermentation conditions than commercial strains; (ii) two different populations of S. cerevisiae strains are involved in the process of elaboration, of fino sherry wine, one of which is responsible for must fermentation and the other, for wine aging; and (iii) one strain was dominant in the flor population integrating the velum from sherry wines produced in González Byass wineries, although other authors have described a succession of races of flor S. cerevisiae during wine aging. Analyzing all these results together, we conclude that yeast population dynamics during biological aging is a complex phenomenon and differences between yeast populations from different wineries can be observed.     

Biological acidification during grape must fermentation using mixed cultures of <Emphasis Type="Italic">Kluyveromyces thermotolerans</Emphasis> and <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Four mixed culture fermentations of grape must were carried out with Kluyveromyces thermotolerans strain TH941 and Saccharomyces cerevisiae strain SCM952. In the first culture, both yeasts were added together, whereas in the remaining three cultures S. cerevisiae was added 1, 2, and 3 days after the inoculation of K. thermotolerans. The growth and survival of the K. thermotolerans strain and the amount of the produced l-lactic acid depend on the time of inoculation of the S. cerevisiae strain and provided an effective acidification during alcoholic fermentation. The four cultures contained, respectively, at the end of fermentation 0.18, 1.80, 4.28, and 5.13 g l-lactic acid l⁻¹. The grape must with an initial pH of 3.50 was effectively acidified (70% increase in titratable acidity, 0.30 pH unit decrease) by the production of 5.13 g l-lactic acid l⁻¹.