Experimental approach for target selection of wild wine yeasts from spontaneous fermentation of “Inzolia” grapes

The aim of this research was the study of indigenous yeasts isolated from spontaneous fermentation of Inzolia grapes, one of the most widespread native white grapes in Sicily (Italy). The use of selective medium for the isolation and the screening for sulphur dioxide tolerance were useful for the first selection among 640 isolates. The yeasts characterized by high SO₂ tolerance were identified at species level by restriction analysis of ITS region; although the majority of isolates were identified as S. cerevisiae, some non-Saccharomyces yeasts were found. Forty-seven selected yeasts, both S. cerevisiae and non-Saccharomyces yeasts, were characterized for genetic and technological diversity. The genetic polymorphism was evaluated by RAPD-PCR analysis, whereas the technological diversity was analyzed by determining the main secondary compounds in the experimental wines obtained by inoculating these yeasts. Both the molecular and metabolic profiles of selected yeasts were able to clearly discriminate S. cerevisiae from non-Saccharomyces yeasts. This research was useful for the constitution of a collection of selected indigenous yeast strains, including S. cerevisiae and non-Saccharomyces species possessing interesting enological traits. This collection represents a source of wild yeasts, among of which it is possible to select indigenous starters able to maintain the specific organoleptic characteristics of Inzolia wine.     

Performance evaluation of Pichia kluyveri, Kluyveromyces marxianus and Saccharomyces cerevisiae in industrial tequila fermentation

Traditionally, industrial tequila production has used spontaneous fermentation or Saccharomyces cerevisiae yeast strains. Despite the potential of non-Saccharomyces strains for alcoholic fermentation, few studies have been performed at industrial level with these yeasts. Therefore, in this work, Agave tequilana juice was fermented at an industrial level using two non-Saccharomyces yeasts (Pichia kluyveri and Kluyveromyces marxianus) with fermentation efficiency higher than 85 %. Pichia kluyveri (GRO3) was more efficient for alcohol and ethyl lactate production than S. cerevisiae (AR5), while Kluyveromyces marxianus (GRO6) produced more isobutanol and ethyl-acetate than S. cerevisiae (AR5). The level of volatile compounds at the end of fermentation was compared with the tequila standard regulation. All volatile compounds were within the allowed range except for methanol, which was higher for S. cerevisiae (AR5) and K. marxianus (GRO6). The variations in methanol may have been caused by the Agave tequilana used for the tests, since this compound is not synthesized by these yeasts.     

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.     

Patagonian wines: the selection of an indigenous yeast starter

The use of selected yeasts for winemaking has clear advantages over the traditional spontaneous fermentation. The aim of this study was to select an indigenous Saccharomyces cerevisiae yeast isolate in order to develop a regional North Patagonian red wine starter culture. A two-step selection protocol developed according to physiological, technological and ecological criteria based on killer interactions was used. Following this methodology, S. cerevisiae isolate MMf9 was selected among 32 indigenous yeasts previously characterized as belonging to different strains according to molecular patterns and killer biotype. This isolate showed interesting technological and qualitative features including high fermentative power and low volatile acidity production, low foam and low sulphide production, as well as relevant ecological characteristics such as resistance to all indigenous and commercial S. cerevisiae killer strains assayed. Red wines with differential volatile profiles and interesting enological features were obtained at laboratory scale by using this selected indigenous strain.     

Enrichment of a continuous culture of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> with the yeast <Emphasis Type="Italic">Issatchenkia orientalis</Emphasis> in the production of ethanol at increasing temperatures

A fermentation system was continuously fed with sugar-cane syrup and operated with recycling of Saccharomyces cerevisiae cells at temperatures varying from 30 to 47°C. The aim of the present work was to obtain and study the colonies of isolates showing elongated cells of yeasts which were sporadically observed at the end of this continuous process. Based on a sequence of assays involving methods of classical taxonomy and RAPD-PCR, two groups of isolates showing characteristics of non-Saccharomyces yeasts were identified in the yeast population where S. cerevisiae was the dominant yeast. The largest group of non-Saccharomyces yeasts, resulting from a slow proliferation over the 2 months, reached a final level of 29.6% at the end of the process. RAPD-PCR profiles obtained for the isolates of this dominant non-Saccharomyces yeast indicated that they were isolates of Issatchenkia orientalis. Pichia membranifaciens was the only species of non-Saccharomyces yeast detected together with I. orientalis but at a very low frequency. The optimum temperature for ethanol formation shown by the isolate 195B of I. orientalis was 42°C. This strain also showed a faster ethanol formation and biomass accumulation than the thermotolerant strain of S. cerevisiae used as the starter of this fermentation process. Some isolates of I. orientalis were also able to grow better at 40°C than at 30°C on plates containing glycerol as carbon source. Yeasts able to grow and produce ethanol at high temperatures can extend the fermentation process beyond the temperature limits tolerated by S. cerevisiae.     

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.     

Tandem repeat-tRNA (TRtRNA) PCR method for the molecular typing of non-<Emphasis Type="Italic">Saccharomyces</Emphasis> subspecies

There is a worldwide trend to understand the impact of non-Saccharomyces yeast species on the process of winemaking. Although the predominant species at the end of the fermentation is Saccharomyces cerevisiae, several non-Saccharomyces species present during the first days of the process can produce and/or release aromas that improve the bouquet and complexity of the final wine. Since no genomic sequences are available for the predominant non-Saccharomyces species selected from grapes or musts (Hanseniaspora uvarum, Hanseniaspora vineae, Hanseniaspora opuntiae, Metschnikowia pulcherrima, Candida zemplinina), a reproducible PCR method was devised to discriminate strains at the subspecies level. The method combines different oligonucleotides based on tandem repeats with a second oligonucleotide based on a conserved tRNA region, specific for ascomycetes. Tandem repeats are randomly dispersed in all eukaryotic genomes and tRNA genes are conserved and present in several copies in different chromosomes. As an example, the method was applied to discriminate native M. pulcherrima strains but it could be extended to differentiate strains from other non-Saccharomyces species. The biodiversity of species and strains found in the grape ecosystem is a potential source of new enzymes, fungicides and/or novel sustainable methods for biological control of phytopathogens.     

A quick screening method to identify β-glucosidase activity in native wine yeast strains: application of Esculin Glycerol Agar (EGA) medium

One hundred and fifty-four yeast strains were isolated from grapes and musts of Uruguayan vineyards and wineries. Only thirty strains showed β-glucosidase activity in Esculin Glycerol Agar (EGA) solid medium. Twenty-one were non-Saccharomyces and nine were Saccharomyces cerevisiae strains. The objective of this study was to evaluate the suitability of Esculin Glycerol Agar (EGA) solid medium for screening β-glucosidase activity in native yeasts strains. Halo sizes measured in the EGA solid medium were correlated to the Glycosyl-Glucose (GG) indexes measured after fermentation of grape musts with each strain. The two S. cerevisiae strains with the best performance were selected for further fermentations on a Muscat Miel grape must, rich in bound monoterpenes. The levels of free linalool, hodiol I and geraniol increased significantly as compared to fermentation with a commercial wine yeast strain. These results show the suitability of this simple and economic medium to identify S. cerevisiae glucosidase producers with a potential impact on real winemaking conditions. On the other hand, great variability was found for the non-Saccharomyces strains, and this would demand further studies for each species. In conclusion, the use of EGA solid medium shows that the screening method is suitable for exploring the glucosidase activity of native strains of S. cerevisiae and shows good correlation with its real impact on free aroma compounds in the final wine.     

Metabolome segregation of four strains of Saccharomyces cerevisiae,Saccharomyces uvarum and Saccharomyces kudriavzevii conducted under low temperature oenological conditions

The monitoring of fermentation at low temperatures (12–15°C) is a current practice in the winery for retention and enhancement of the flavour volatile content of wines. Among Saccharomyces species, Saccharomyces uvarum and Saccharomyces kudriavzevii have revealed interesting industrial properties, including better adaptation at low temperatures. To gather deeper knowledge of the fermentative metabolism at a low temperature of these species together with S. cerevisiae, we performed a comparative metabolomic analysis using four representative strains. We used batch cultures to obtain an exhaustive and dynamic image of the metabolome of strains passing through the sequential stresses related to the winemaking environment. A great variety of intra- and extracellular metabolites (>500 compounds) were quantified across fermentation using distinct chromatographic methods. Besides a global decrease in the lipid composition of the four strains when they entered into the stationary phase, we reported some strain-specific high magnitude changes. Examples of these differences included divergent patterns of production of short-chain fatty acids and erythritol in the S. uvarum strain. Strains also differed in expression for aromatic amino acid biosynthesis and sulphur metabolism, including the glutathione pathway. These data will allow us to refine and obtain the most value of fermentations with this alternative Saccharomyces species.     

Yeast Diversity Isolated from Grape Musts During Spontaneous Fermentation from a Brazilian Winery

Saccharomyces and non-Saccharomyces yeast species from a winery located in Brazil were identified by ribosomal gene-sequencing analysis. A total of 130 yeast strains were isolated from grape surfaces and musts during alcoholic fermentation from Isabel, Bordeaux, and Cabernet Sauvignon varieties. Samples were submitted to PCR–RFLP analysis and genomic sequencing. Thirteen species were identified: Candida quercitrusa, Candida stellata, Cryptococcus flavescens, Cryptococcus laurentii, Hanseniaspora uvarum, Issatchenkia occidentalis, Issatchenkia orientalis, Issatchenkia terricola, Pichia kluyveri, Pichia guilliermondii, Pichia sp., Saccharomyces cerevisiae, and Sporidiobolus pararoseus. A sequential substitution of species during the different stages of fermentation, with a dominance of non-Saccharomyces yeasts at the beginning, and a successive replacement of species by S. cerevisiae strains at the final steps were observed. This is the first report about the yeast distribution present throughout the alcoholic fermentation in a Brazilian winery, providing supportive information for future studies on their contribution to wine quality.     

耐热克鲁维酵母在葡萄酒酿造中的研究进展

耐热克鲁维酵母(Lachancea thermotolerans)是一种具有优良酿造学特性的非酿酒酵母(non-Saccharomyces cevevisiae),近年来由于其对葡萄酒的发酵进程及香气、滋味等感官特性均有着重要影响而受到越来越多的关注。耐热克鲁维酵母突出的特点表现为高产乳酸、甘油、2-苯乙醇及乙酯类香气成分,低产乙醇及挥发酸类物质,并且相关研究显示不同耐热克鲁维酵母发酵对葡萄酒的影响存在明显的菌株特异性。文章围绕耐热克鲁维酵母的菌株多样性、其对葡萄酒质量的影响及在混合发酵中的应用等方面进行综述,以期为本土耐热克鲁维酵母菌株性状的筛选、产酸及产香机制的解析提供参考依据,促进我国酿酒微生物种质资源的良性发展。     

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.     

Analysis of yeasts derived from natural fermentation in a Tokaj winery

The diversity of yeast flora was investigated in a spontaneously fermenting sweet white wine in a Tokaj winery. The non-Saccharomyces yeasts dominating the first phase of fermentation were soon replaced by a heterogeneous Saccharomycespopulation, which then became dominated by Saccharomyces bayanus. Three Saccharomyces sensu stricto strains isolated from various phases of fermentation were tested for genetic stability, optimum growth temperature, tolerance to sulphur dioxide, copper and ethanol as well as for the ability to produce hydrogen sulphide and various secondary metabolites known to affect the organoleptic properties of wines. The analysis of the single-spore cultures derived from spores of dissected asci revealed high stability of electrophoretic karyotypes and various degrees of heterozygosity for mating-types, the fermentation of galactose and the production of metabolic by-products. The production levels of the by-products did not segregate in a 2:2 fashion, suggesting that the synthesis of these compounds is under polygenic control.     

Screening and evaluation of the glucoside hydrolase activity in Saccharomyces and Brettanomyces brewing yeasts

Aims: The aim of this study was to select and examine Saccharomyces and Brettanomyces brewing yeasts for hydrolase activity towards glycosidically bound volatile compounds. Methods and Results: A screening for glucoside hydrolase activity of 58 brewing yeasts belonging to the genera Saccharomyces and Brettanomyces was performed. The studied Saccharomyces brewing yeasts did not show 1,4‐β‐glucosidase activity, but a strain dependent β‐glucanase activity was observed. Some Brettanomyces species did show 1,4‐β‐glucosidase activity. The highest constitutive activity was found in Brettanomyces custersii. For the most interesting strains the substrate specificity was studied and their activity was evaluated in fermentation experiments with added hop glycosides. Fermentations with Br. custersii led to the highest release of aglycones. Conclusions: Pronounced exo‐β‐glucanase activity in Saccharomyces brewing yeasts leads to a higher release of certain aglycones. Certain Brettanomyces brewing yeasts, however, are more interesting for hydrolysis of glycosidically bound volatiles of hops. Significance and Impact of the Study: The release of flavour active compounds from hop glycosides opens perspectives for the bioflavouring and product diversification of beverages like beer. The release can be enhanced by using Saccharomyces strains with high exo‐β‐glucanase activity. Higher activities can be found in Brettanomyces species with β‐glucosidase activity.     

<Emphasis Type="Italic">Starmerella bombicola</Emphasis> influences the metabolism of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> at pyruvate decarboxylase and alcohol dehydrogenase level during mixed wine fermentation

Background  

The use of a multistarter fermentation process with Saccharomyces cerevisiae and non-Saccharomyces wine yeasts has been proposed to simulate natural must fermentation and to confer greater complexity and specificity to wine. In this context, the combined use of S. cerevisiae and immobilized Starmerella bombicola cells (formerly Candida stellata) was assayed to enhance glycerol concentration, reduce ethanol content and to improve the analytical composition of wine. In order to investigate yeast metabolic interaction during controlled mixed fermentation and to evaluate the influence of S. bombicola on S. cerevisiae, the gene expression and enzymatic activity of two key enzymes of the alcoholic fermentation pathway such as pyruvate decarboxylase (Pdc1) and alcohol dehydrogenase (Adh1) were studied.     

Redox Interactions between Saccharomyces cerevisiae and Saccharomyces uvarum in Mixed Culture under Enological Conditions

Wine yeast starters that contain a mixture of different industrial yeasts with various properties may soon be introduced to the market. The mechanisms underlying the interactions between the different strains in the starter during alcoholic fermentation have never been investigated. We identified and investigated some of these interactions in a mixed culture containing two yeast strains grown under enological conditions. The inoculum contained the same amount (each) of a strain of Saccharomyces cerevisiae and a natural hybrid strain of S. cerevisiae and Saccharomyces uvarum. We identified interactions that affected biomass, by-product formation, and fermentation kinetics, and compared the redox ratios of monocultures of each strain with that of the mixed culture. The redox status of the mixed culture differed from that of the two monocultures, showing that the interactions between the yeast strains involved the diffusion of metabolite(s) within the mixed culture. Since acetaldehyde is a potential effector of fermentation, we investigated the kinetics of acetaldehyde production by the different cultures. The S. cerevisiae-S. uvarum hybrid strain produced large amounts of acetaldehyde for which the S. cerevisiae strain acted as a receiving strain in the mixed culture. Since yeast response to acetaldehyde involves the same mechanisms that participate in the response to other forms of stress, the acetaldehyde exchange between the two strains could play an important role in inhibiting some yeast strains and allowing the growth of others. Such interactions could be of particular importance in understanding the ecology of the colonization of complex fermentation media by S. cerevisiae.     

Fermentation of Agave tequilana juice by Kloeckera africana: influence of amino-acid supplementations

This study aimed to improve the fermentation efficiency of Kloeckera africana K1, in tequila fermentations. We investigated organic and inorganic nitrogen source requirements in continuous K. africana fermentations fed with Agave tequilana juice. The addition of a mixture of 20 amino-acids greatly improved the fermentation efficiency of this yeast, increasing the consumption of reducing sugars and production of ethanol, compared with fermentations supplemented with ammonium sulfate. The preference of K. africana for each of the 20 amino-acids was further determined in batch fermentations and we found that asparagine supplementation increased K. africana biomass production, reducing sugar consumption and ethanol production (by 30, 36.7 and 45%, respectively) over fermentations supplemented with ammonium sulfate. Therefore, asparagine appears to overcome K. africana nutritional limitation in Agave juice. Surprisingly, K. africana produced a high concentration of ethanol. This contrasts to poor ethanol productivities reported for other non-Saccharomyces yeasts indicating a relatively high ethanol tolerance for the K. africana K1 strain. Kloeckera spp. strains are known to synthesize a wide variety of volatile compounds and we have shown that amino-acid supplements influenced the synthesis by K. africana of important metabolites involved in the bouquet of tequila. The findings of this study have revealed important nutritional limitations of non-Saccharomyces yeasts fermenting Agave tequilana juice, and have highlighted the potential of K. africana in tequila production processes.     

Kinetics and metabolic behavior of a composite culture of <Emphasis Type="Italic">Kloeckera apiculata</Emphasis> and <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> wine related strains

The kinetics and metabolic behavior of Kloeckera apiculata mc1 and Saccharomyces cerevisiae mc2 in composite culture was investigated. K. apiculata showed a higher viability through the fermentation; however the maximum cell density of both yeasts decreased. This behavior was not due to ethanol concentration, killer toxins production or competition for assimilable nitrogenous compounds between both yeasts. Despite the consistent production of secondary products by single culture of K. apiculata, an increase of these compounds was not observed in mixed culture. These results contribute to a better understanding of the behavior of non-Saccharomyces yeasts and their potential application in the wine industry.     

Yeast assessment during alcoholic fermentation inoculated with a natural “pied de cuve” or a commercial yeast strain

The present study has been carried out in an organic winery established in 2003 in the Denomination of Origin “Sierras de Málaga” (Southern Spain) region during the 2007 vintage. The aim of this work was to ascertain the yeast microflora present in the winery and during the vinifications and to obtain a collection of autochthonous S. cerevisiae strains from this area. Yeast populations from three vats containing fermenting musts from different grape varieties were analysed. Two of them were inoculated with a natural “pied de cuve” while the third one was sown with a rehydrated commercial yeast strain. A total of 382 yeasts were isolated and identified, initially by restriction analysis of ribosomal DNA and further by sequencing of this region. Non-Saccharomyces yeasts were found in all three musts but they practically disappeared as the fermentations progressed. Analysis of mitochondrial DNA RFLP revealed 13 different restriction patterns of Saccharomyces cerevisiae strains, five of them similar to those of commercial strains used in the winery. Commercial strains were found even in vats inoculated with a “pied de cuve” generated by spontaneous fermentation of a must sample. The analysis of samples recovered from different winery surfaces and equipments demonstrated that non-Saccharomyces and both commercial and autochthonous Saccharomyces strains were part of the resident microflora in the winery. Biodiversity of autochthonous S. cerevisiae in fermentation vats was low but two of them were able to compete with the commercial ones and they were isolated even at the end of the fermentation.     

Effect of must characteristics on the diversity of Saccharomyces strains and their prevalence in spontaneous fermentations

Aims: The aim of this study was to investigate whether grapevine variety and must characteristics influence the diversity of Saccharomyces strains and their prevalence during spontaneous fermentations. Methods and results: Musts from different grapevine varieties, all of them autochthonous from Galicia, were used to perform spontaneous fermentations. Yeasts were isolated from the must and at the beginning, in the middle and at the end of fermentations. Those yeasts identified as Saccharomyces were characterized at the strain level by analysis of mtDNA‐RFLP. The results showed a low diversity of Saccharomyces strains, which was related to must sugar content and total acidity. Moreover, from a total of 44 different Saccharomyces strains, only eleven of them appeared at frequencies higher than 20% and were able to lead fermentations. A significant correlation between yeast strains and must acidity was observed, with the predominance of certain strains at high acidity values. Conclusions: Must characteristics, such as sugar content and acidity, influence the Saccharomyces strains diversity and the leader strains during fermentation. Significance and Impact of the Study: These results showed the adaptation of certain Saccharomyces strains to must with specific characteristics; this may be considered by winemakers for yeast inocula selection. Our findings have special relevance because this is the first study carried out in Galicia dealing with the influence of must properties on yeast strains that control fermentations.