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.     

The grape must non-Saccharomyces microbial community: impact on volatile thiol release

Several studies have reported the beneficial influence of non-Saccharomyces yeasts and their potential applications in the wine industry, mainly in mixed-culture fermentation with S. cerevisiae. The potential impact of 15 non-Saccharomyces strains from 7 species on 4-methyl-4-sulfanylpentan-2-one (4MSP) and 3-sulfanylhexan-1-ol (3SH) release in model medium and Sauvignon Blanc must was evaluated after partial fermentation. Whereas the impact of non-Saccharomyces on 4MSP release in both media was low, some M. pulcherrima, T. delbrueckii and K. thermotolerans strains had a high capacity to release 3SH, despite their minimal fermentation activity. As previously demonstrated for Saccharomyces yeast, this contribution is strain dependant. Taking into account their dynamic and quantitative presence during the whole process, the real impact of non-Saccharomyces yeast on 4MSP and 3SH release was evaluated using a recreated community simulating the yeast ecosystem. Our results revealed a positive impact on 3SH release in Sauvignon Blanc wines by promoting non-Saccharomyces yeast activity and delaying the growth of S. cerevisiae. Some non-Saccharomyces yeast strains are capable of making a positive contribution to volatile thiol release in wines, essentially during the pre-fermentation stage in winemaking, when this microbiological sub-population is dominant.     

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).     

Diversity of Saccharomyces and non-Saccharomyces yeasts in three red grape varieties cultured in the Serranía de Ronda (Spain) vine-growing region

For the first time, an ecological survey of wine yeasts present in grapes growing in two vineyards located in the region of “Serranía de Ronda” (Málaga, southern Spain) has been carried out. During the 2006 and 2007 vintages, grapes from different varieties were aseptically collected and allowed to ferment spontaneously in the laboratory. From a total of 1586 colonies isolated from microvinifications, 1281 were identified according to ITS polymorphisms and their identity confirmed by sequencing of the D1/D2 region of 26S rDNA. Most of the isolates (84%) corresponded to thirteen different non-Saccharomyces species with Kluyveromyces thermotolerans, Hanseniaspora guilliermondii, Hanseniaspora uvarum and Issatchenkia orientalis accounting for 42.7% of the total. Mitochondrial DNA restriction analysis from the Saccharomyces cerevisiae isolates revealed a low diversity since only eleven different profiles were found, nine of them corresponding to local strains and two to commercial ones that had been used in different campaigns and that very likely were disseminated from the winery to the adjacent vineyard. A different distribution of strains was found in the three grape varieties studied.     

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.     

Modulation of the glycerol and ethanol syntheses in the yeast Saccharomyces kudriavzevii differs from that exhibited by Saccharomyces cerevisiae and their hybrid

In the last years there is an increasing demand to produce wines with higher glycerol levels and lower ethanol contents. The production of these compounds by yeasts is influenced by many environmental variables, and could be controlled by the choice of optimized cultivation conditions. The present work studies, in a wine model system, the effects of temperature, pH and sugar concentration on the glycerol and ethanol syntheses by yeasts Saccharomyces cerevisiae T73, the type strain of Saccharomyces kudriavzevii IFO 1802^T, and an interspecific hybrid between both species (W27), which was accomplished by the application of response surface methodology based in a central composite circumscribed design. Results show that carbon flux could be especially directed towards glycerol synthesis instead of ethanol at low pH, high sugar concentrations and low temperatures. In general, the non-wine yeast S. kudriavzevii produced higher glycerol levels and lower ethanol content than wine strains S. cerevisiae T73 and the hybrid W27, with specific and different glycerol production profiles as a function of temperature and pH. These results were congruent with the higher glycerol-3-phosphate dehydrogenase activities estimated for this species, chiefly at low temperatures (14 °C), which could explain why S. kudriavzevii is a cryotolerant yeast compared to S. cerevisiae.     

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.     

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.     

Molecular and physiological characteristics of a grape yeast strain containing atypical genetic material

The knowledge about wine yeasts remains largely dominated by the extensive studies on Saccharomyces (S.) cerevisiae. Molecular methods, allowing discrimination of both species and strains in winemaking, can profitably be applied for characterization of the microflora occurring in winemaking and for monitoring the fermentation process. Recently, some novel yeast isolates have been described as hybrid between S. cerevisiae and Saccharomyces species, leaving the Saccharomyces strains containing non-Saccharomyces hybrids essentially unexplored. In this study, we have analyzed a yeast strain isolated from “Primitivo” grape (http://www.ispa.cnr.it/index.php?page=collezioni&lang=en accession number 12998) and we found that, in addition to the S. cerevisiae genome, it has acquired genetic material from a non-Saccharomyces species. The study was focused on the analysis of chromosomal and mitochondrial gene sequences (ITS and 26S rRNA, SSU and COXII, ACTIN-1 and TEF), 2D-PAGE mitochondrial proteins, and spore viability. The results allowed us to formulate the hypothesis that in the MSH199 isolate a DNA containing an rDNA sequence from Hanseniaspora vineae, a non-Saccharomyces yeast, was incorporated through homologous recombination in the grape environment where yeast species are propagated. Moreover, physiological characterization showed that the MSH199 isolate possesses high technological quality traits (fermentation performance) and glycerol production, resistance to ethanol, SO₂ and temperature) useful for industrial application.     

Initial Saccharomyces cerevisiae concentration in single or composite cultures dictates bioprocess kinetics

The growth dynamics of three non-Saccharomyces strains in combination with Saccharomyces cerevisiae during fermentation of a sterile grape juice have been studied. The influence of the initial concentrations of S. cerevisiae on the whole yeast community was the main purpose of this research. The progression of S. cerevisiae within the first 5 days of fermentation was monitored by enumeration on selective and non-selective media. The population of each species was evaluated by morphological criteria. After 24 h, Hanseniaspora uvarum represented more than 50% of the whole yeast community, including ferments with the highest initial concentration of S. cerevisiae. As the population of S. cerevisiae increased, H. uvarum decreased. Metchnikowia pulcherrima was more inhibited by S. cerevisiae than H. uvarum, whereas the growth of Candida stellata was less inhibited. After thirty days, irrespective of the initial concentration of S. cerevisiae, only S. cerevisiae was detected in all ferments. Conversion of sugars to ethanol correlated with the initial population of S. cerevisiae. Glucose was almost completely exhausted in all cases, independent of the initial S. cerevisiae concentration used. Grape juices inoculated with composite inocula of non-S. cerevisiae and S. cerevisiae, with its initial concentration lower than 5 cfu/ml did not produce wines according to wine regulations. The concentration of ethanol in the wines did not reach the minimum amount of 9 vol%. Samples of wines fermented with a composite inoculum the concentration of S. cerevisiae represented 50 cfu/ml of grape juice, were judged to have the best sensorial properties.     

Effect of aromatic precursor addition to wine fermentations carried out with different Saccharomyces species and their hybrids

This work explores the ability of different yeast strains from different species of the genus Saccharomyces (S. cerevisiae, S. uvarum and S. kudriavzevii) and hybrids between these species to release or form varietal aroma compounds from fractions of grape odourless precursors. The de novo synthesis by the yeasts of some of the varietal aroma compounds was also evaluated. The study has shown that de novo synthesis affects some lipid derivatives, shikimic derivatives and terpenes in all species and hybrids, with some remarkable differences amongst them. The release or formation of aroma compounds from precursors was found to be strongly linked to the yeast or hybrid used, and the triple hybrid S. cerevisiae × S. bayanus × S. kudriavzevii in particular and secondarily the hybrid S. cerevisiae × S. bayanus were highly efficient in the production of most varietal aroma compounds, including γ-lactones, benzenoids, volatile phenols, vanillin derivatives and terpenols. The presence of precursors in the fermenting media caused a surprising levelling effect on the fermentative aroma composition. Altogether, these results suggest that it is possible to modulate wine aroma by employing different yeast species in order to create new wines with different aromatic notes.     

Dynamic study of yeast species and Saccharomyces cerevisiae strains during the spontaneous fermentations of Muscat blanc in Jingyang,China

The evolution of yeast species and Saccharomyces cerevisiae genotypes during spontaneous fermentations of Muscat blanc planted in 1957 in Jingyang region of China was followed in this study. Using a combination of colony morphology on Wallerstein Nutrient (WLN) medium, sequence analysis of the 26S rDNA D1/D2 domain and 5.8S-ITS-RFLP analysis, a total of 686 isolates were identified at the species level. The six species identified were S. cerevisiae, Hanseniaspora uvarum, Hanseniaspora opuntiae, Issatchenkia terricola, Pichia kudriavzevii (Issatchenkia orientalis) and Trichosporon coremiiforme. This is the first report of T. coremiiforme as an inhabitant of grape must. Three new colony morphologies on WLN medium and one new 5.8S-ITS-RFLP profile are described. Species of non-Saccharomyces, predominantly H. opuntiae, were found in early stages of fermentation. Subsequently, S. cerevisiae prevailed followed by large numbers of P. kudriavzevii that dominated at the end of fermentations. Six native genotypes of S. cerevisiae were determined by interdelta sequence analysis. Genotypes III and IV were predominant. As a first step in exploring untapped yeast resources of the region, this study is important for monitoring the yeast ecology in native fermentations and screening indigenous yeasts that will produce wines with regional characteristics.     

Monoterpene alcohols release and bioconversion by Saccharomyces species and hybrids

Terpene profile of Muscat wines fermented by Saccharomyces species and hybrid yeasts was investigated. The amount of geraniol decreased in most wines with respect to the initial must except for Saccharomyces bayanus wines. On the other hand, alpha-terpineol amount was higher in wines fermented by Saccharomyces cerevisiae and hybrid yeasts. The amount of linalool was similar in all wines and comparable to the amount in the initial must. Lower levels of beta-d-glucosidase activity were found in the hybrid yeasts with respect to S. cerevisiae. Moreover, no relationship between beta-d-glucosidase activity and terpenes profile in Muscat wines fermented with Saccharomyces species and hybrids was found. Growth of yeasts on minimum medium supplemented with geraniol showed bioconversion of geraniol into linalool and alpha-terpineol. Percentages of geraniol uptake and bioconversion were different between Saccharomyces species and hybrids. Strains within S. bayanus, Saccharomyces kudriavzevii and hybrids showed higher geraniol uptake than S. cerevisiae, whereas the percentage of produced linalool and alpha-terpineol was higher in S. cerevisiae and hybrid yeasts than in S. bayanus and S. kudriavzevii. The relationship between geraniol uptake and adaptation of Saccharomyces species to grow at low temperature is discussed.     

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.     

Acidification of grape marc for alcoholic beverage production: effects on indigenous microflora and aroma profile after distillation

Grappa is an Italian alcoholic beverage obtained from distillation of grape marc, the raw material derived from separation of must during the winemaking process. Marc is stored for a period lasting from few days to several weeks, when fermentation of residual sugars occurs mainly by yeast activity. Many distilleries have adopted different solutions to manage this critical phase in order to avoid spoilage microorganisms: marc acidification is the most widely diffused. In this work, Prosecco grape pomace was acidified with sulphuric acid (to pH 2.9) and stored, whereas non-acidified grape marc was used as control (pH 3.9). Samples for microbiological analysis were collected at the beginning of the storage period, after 15 and 43 days. At the beginning of the ensilage (time T0) the indigenous microflora was represented both by yeasts and bacteria at a concentration of about 10⁶ cfu/g. During the first 15 days, when the fermentation generally takes place, yeast population grew considerably (up to 10⁷ cfu/g) in acidified grape marc, where bacterial population was maintained at low levels. Moreover, yeast populations recovered at the three sampling times in both treated and untreated marc were genetically characterised. This analysis showed that the species succession lead to non-Saccharomyces species dominance (in particular Issatchenkia and Pichia genera) in both conditions although acidified marc showed a lower percentage of Saccharomyces at any sampling time analysed, this meaning that non-Saccharomyces species were favoured in this environment. Gas chromatographic analysis showed a remarkable change in the aromatic profile of distilled grape marcs at the end of the storage, thus evidencing that concentration of monitored volatile compounds usually produced by microflora was generally lowered by the acidification treatment.This work demonstrates for the first time the strong effect of a persistent acidification treatment both on the microbiota of grape pomace and on the aromatic profile of the distillate. Indeed, the lowering of the pH caused significant changes in yeast-bacteria populations ratio and in yeast species turnover. These microbiological changes determine an improvement of the aromatic profile of the distillate, due to the reduction of the main volatile products associated with potential off-flavours.     

Influence of indigenous yeasts on the fermentation and volatile profile of plum brandies

The aim of this study was to determine the influence of different yeasts isolated from fresh blue plum fruits (Aureobasidium sp.) and spontaneously fermenting plum musts (Kloeckera apiculata and Saccharomyces cerevisiae), as well as commercial wine and distillery strains, on the fermentation and chemical composition of plum brandies. Gas chromatography methods were used to detect major volatile components. The most rapid fermentation occurred in musts inoculated with S. cerevisiae. However, the highest concentration of ethanol was detected in samples after spontaneous fermentation (8.40% v/v). Plum brandies obtained after distillation contained from 66.3 (K. apiculata) up to 74.3% v/v ethanol (spontaneous fermentation). The samples after spontaneous fermentation were distinguished by a high content of acetoin, ethyl acetate and total esters, accompanied by a low level of methanol and fusel alcohols. Non-Saccharomyces yeasts were responsible for higher concentrations of esters and methanol, while S. cerevisiae strains resulted in increased levels of higher alcohols. It was also found that isolated indigenous strains of S. cerevisiae synthesized relatively low amounts of higher alcohols compared to commercial cultures. Samples obtained using the distillery strain of S. cerevisiae received the highest score (18.2) during sensory analysis and were characterized by a well-harmonised taste and aroma.     

Formation of vinylphenolic pyranoanthocyanins by Saccharomyces cerevisiae and Pichia guillermondii in red wines produced following different fermentation strategies

The strains of two species of yeast, Saccharomyces cerevisiae and Pichia guillermondii, both with high hydroxycinnamate decarboxylase (HCDC) activity (56% and 90% respectively), were used in the fermentation of musts enriched with grape anthocyanins, to favour the formation of highly stable vinylphenolic pyranoanthocyanin pigments. The different strains were used to ferment the must separately, simultaneously, or sequentially, the latter involving an initial period using the yeast with the greatest HCDC activity (P. guillermondii). The must was made from concentrated grape juice diluted to 220 g/l of sugar, and enriched with grape anthocyanins to 100 mg/l and with p-coumaric acid to 120 mg/l. The pH was fixed to 3.5. All 50 ml micro-fermentations were done in triplicate. The development of anthocyanin-3-O-glucoside precursors, the decarboxylation of p-coumaric acid, and the formation of 4-vinylphenol and vinylphenolic pyranoanthocyanin derivatives were studied during the fermentation. The fermentation strategy used and the yeast HCDC activity significantly influenced the formation of vinylphenolic pyranoanthocyanins. The latter molecules were separated, identified, and quantified using high performance liquid chromatograph with diode array detection and electrospray-mass spectrometry (HPLC-DAD-ESI/MS). The volatile compounds profile was screened during fermentation using gas cromatogrphy-flame ionisation detection (GC-FID), in order to detect and quantify the main molecules. The best results were reached with the sequential fermentation (3.74 mg/l of malvidin-3-O-glucoside-4-vinylphenol). This work shows that during mixed or sequential fermentations carried out with non-Saccharomyces or highly fermentative Saccharomyces strains, with high HCDC activity, the content of stable pigments can be increased without sensorial modifications.     

Effect of inoculation on strawberry fermentation and acetification processes using native strains of yeast and acetic acid bacteria

The aim of this work was to analyze the microbiota involved in the traditional vinegar elaboration of strawberry fruit during a spontaneous and inoculated process. In the spontaneous processes, low biodiversity was detected in both alcoholic fermentation (AF) and acetification. Nevertheless, a strain of Saccharomyces cerevisiae and of Acetobacter malorum were selected and tested as starter cultures in the inoculation study. The inoculated processes with these strains were compared with another spontaneous process, yielding a significant reduction in time for AF with a total imposition of the S. cerevisiae strain. The resulting strawberry wine was acetified in different containers (glass and wood) yielding an initial imposition of the A. malorum inoculated strain, although displacement by Gluconacetobacter species was observed in the wood barrels.     

Fermentative aptitude of non-Saccharomyces wine yeast for reduction in the ethanol content in wine

Over the last few decades, there has been a progressive increase in the ethanol content in wines due to global climate change and to the new wine styles that are associated with increased grape maturity. This increased ethanol content can have negative consequences on the sensory properties of the wines, human health, and economic aspects. Several microbiological approaches for decreasing the ethanol content have been suggested, such as strategies based on genetically modified yeasts, the adaptive evolution of yeasts, and the use of non-Saccharomyces yeast. In the present study, we investigated the interspecies and intraspecies variability of some non-Saccharomyces wine yeast species under anaerobic fermentation conditions. Across different grape juices and fermentation trials, Hanseniaspora uvarum, Zygosaccharomyces sapae, Zygosaccharomyces bailii, and Zygosaccharomyces bisporus promoted significant reductions in ethanol yield and fermentation efficiency in comparison with Saccharomyces cerevisiae. The diversion of alcoholic fermentation and the abundant formation of secondary compounds might explain the marked reduction in ethanol yield, as determined through the segregation of the majority of the strains according to their species attributes observed using principal component analysis. These data suggest that careful evaluation of interspecies and intraspecies biodiversity can be carried out to select yeast that produces low-ethanol yields.