Physiological features of Schizosaccharomyces pombe of interest in making of white wines

This work studies the physiology of Schizosaccharomyces pombe strain 938 in the production of white wine with high malic acid levels as the sole fermentative yeast, as well as in mixed and sequential fermentations with Saccharomyces cerevisiae Cru Blanc. The induction of controlled maloalcoholic fermentation through the use of Schizosaccharomyces spp. is now being viewed with much interest. The acetic, malic and pyruvic acid concentrations, relative density and pH of the musts were measured over the entire fermentation period. In all fermentations in which Schizo. pombe 938 was involved, nearly all the malic acid was consumed and moderate acetic concentrations produced. The urea content and alcohol level of these wines were notably lower than in those made with Sacch. cerevisiae Cru Blanc alone. The pyruvic acid concentration was significantly higher in Schizo. pombe fermentations. The sensorial properties of the different final wines varied widely.     

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.     

Influence of yeast strain on binding of sulphur dioxide in wines,and on its formation during fermentation

The amounts of sulphur dioxide bound by acetaldehyde, pyruvic acid and α-ketoglutaric acid during fermentation of three grape juices by eight wine yeasts (Saccharomyces sp.) are reported. These constituents accounted for 49–83 % (mean 69) of the measured bound SO₂, depending on the yeast strain and juice. the maximum range of concentrations of the binding components for individual wines were 10–48 ppm for acetaldehyde, 9–77 ppm for pyruvic acid and 5–63 ppm for α-ketoglutaric acid, depending on yeast strain and grape juice. the validity of the calculations was verified by an experiment with SO₂ and the three binding compounds in a multicomponent model system. The acetaldehyde content was related to the total SO₂ present, which itself was determined by the strain of yeast. SOz bound in the wines after a further SO₂ addition was correlated significantly with pyruvic and α-ketoglutaric acids, but not with acetaldehyde. Certain yeasts produced SO₂ during fermentation in grape juice and in synthetic media with defined sulphur sources. More SO₂ was produced at pH 3.6 than 3.0 in the absence of added sulphate in grape juice. Sulphate was the best sulphur source for SO₂ production in synthetic media, although some yeasts were able to produce smaller amounts of SO₂ from l-cysteine and reduced glutathione.     

CONTROLLED PRODUCTION OF CIDER BY INDUCTION OF ALCOHOLIC FERMENTATION AND MALOLACTIC CONVERSION

Characterization experiments involving lactic bacteria allowed a strain of Leuconostoc oenos to be selected in terms of growth capacity at variable pH, temperature, ethanol and sulphite concentrations, malic to lactic conversion yield, acetic acid uptake and dextran production capacity. Cider was produced under controlled conditions where the effect of Kloeckera apiculata yeasts on the development of Saccharomyces cerevisiae and production of major non-volatile compounds influencing end product quality was studied. The apiculate yeast was found to produce large amounts of acetic acid and use the other organic acids; also, it hindered fermentation to a certain extent. A study of the effect of the inoculation time with L oenos as an inducer of malolactic fermentation revealed sequential inoculation of the lactic bacterium once most major sugars in the must had been depleted to be the most favourable. Using yeast cell walls boosted fermentation development, as well as degradation of malic acid and synthesis of succinic and acetic acid.     

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.     

Saccharomyces cerevisiae and Hanseniaspora uvarum mixed starter cultures: Influence of microbial/physical interactions on wine characteristics

The growing trend in the wine industry is the revaluation of the role of non-Saccharomyces yeasts, promoting the use of these yeasts in association with Saccharomyces cerevisiae. Non-Saccharomyces yeasts contribute to improve wine complexity and organoleptic composition. However, the use of mixed starters needs to better understand the effect of the interaction between these species during alcoholic fermentation. The aim of this study is to evaluate the influence of mixed starter cultures, composed by combination of different S. cerevisiae and Hanseniaspora uvarum strains, on wine characteristics and to investigate the role of cell-to-cell contact on the metabolites produced during alcoholic fermentation. In the first step, three H. uvarum and two S. cerevisiae strains, previously selected, were tested during mixed fermentations in natural red grape must in order to evaluate yeast population dynamics during inoculated fermentation and influence of mixed starter cultures on wine quality. One selected mixed starter was tested in a double-compartment fermentor in order to compare mixed inoculations of S. cerevisiae/H. uvarum with and without physical separation. Our results revealed that physical contact between S. cerevisiae and H. uvarum affected the viability of H. uvarum strain, influencing also the metabolic behaviour of the strains. Although different researches are available on the role of cell-to-cell contact-mediated interactions on cell viability of the strains included in the mixed starter, to our knowledge, very few studies have evaluated the influence of cell-to-cell contact on the chemical characteristics of wine.     

Performance of wild Saccharomyces and Non-Saccharomyces yeasts as starter cultures in dough fermentation and bread making

The use of wild Saccharomyces and non-Saccharomyces yeasts might result in bread with different and attractive sensory characteristics. This study aimed to evaluate the performance of Saccharomyces and non-Saccharomyces yeasts as starter culture in dough fermentation to bread making and the physicochemical parameters and aromatic profile of bread. All 26 wild yeasts strains isolated from Brazilian Cerrado fruit and tree bark were osmotolerant, and 19.4% were able to ferment maltose. Candida tropicalis ART101.3 and Saccharomyces cerevisiae SC5952 had the best growth capacity under high concentrations of glucose and maltose. Also, they were resistant to lyophilisation. Kinetic parameters of bioreactor cultivations showed high cell growth and lower generation time with 10 g L⁻¹ maltose. Bread produced with C. tropicalis ART101.3 and the control bread had similar physicochemical properties and acceptance of consumers. Bread with S. cerevisiae SC5951 had a lower specific volume and a different colour than control bread; however, the consumers found no significant difference. More than 70% of the consumers demonstrated purchase intention of bread produced with both wild yeasts. The present study shows the potential of native Cerrado yeasts to be used and exploited in industrial processes and contributes to the diversification of bread starter cultures.     

SCREENING FOR EXTRACELLULAR ACID PROTEASE(S) PRODUCTION BY WINE YEASTS*

Ninety-four Saccharomyces wine yeasts were screened for their ability to produce extracellular acid protease(s) under simulated vinification conditions. Seventeen strains were selected on the basis of their higher proteolytic activity on haemoglobin and casein. These strains showed maximum activity after 20 days of fermentation on haemoglobin and 25 days on casein. Wines obtained from grape juice fermented by the above 17 strains showed significative differences in the residual protein content.     

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.     

Fermentation of grape must by sequential association of yeasts: Accumulation of pyruvic and α-ketoglutaric acid

Fermentations of grape (cv. Malvar) musts from two consecutive vintages were carried out using the autochthonous microflora, a sequential association of yeasts and conventional fermentations with addition of sulfur dioxide to the must. The pyruvic and α-ketoglutaric acid content over the course of fermentation was measured and showed that for both vintages tested the maximum accumulation of the ketoacid pyruvic acid took place several days earlier in fermentations using a sequential association of yeasts than in conventional fermentations. The accumulation of pyruvic acid was higher in the must made from grapes with a higher degree of ripening and the lowest level of added SO₂. In the fermentations using either a sequential association of yeasts or the autochthonous microflora with no added SO₂, accumulation of α-ketoglutaric acid was higher in the must with the higher nitrogen content when the species making the greatest percentage contributions at the start of fermentation presented high levels of proteolytic activity.     

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.     

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     

SELECTION AND BIOCHEMICAL CHARACTERISATION OF SACCHAROMYCES CEREVISIAE AND KLOECKERA APICULATA STRAINS ISOLATED FROM SPANISH CIDER

The behaviour of different strains of Saccharomyces cerevisiae and Kloeckera apiculata in apple juice fermentation was studied. Ethanol production was higher for Saccharomyces strains while residual sugars and ethyl acetate content was higher for Kloeckera strains. Kl. apiculata fermented products showed the lowest amount of higher alcohols and the lowest content in organic acids with the exception of acetic acid, so this yeast produced an increment in volatile acidity. On the basis of ethyl acetate, hydrogen sulfide, and acetic acid production, fermentative ability, potassium metabisulfite resistance and sporulation percentage, one strain from Sacch. cerevisiae could be employed as starter for making cider.     

Adaptive response and tolerance to weak acids in Saccharomyces cerevisiae boulardii: a metabolomics approach

Weak acids inhibit the growth of probiotics, such as Saccharomyces boulardii. We explored the tolerance of S. boulardii to different weak acids. S. boulardii had better fermentation ability under lactic acid conditions compared with acetic and butyric acid conditions; however, the budding of S. boulardii was significantly stronger than that of Saccharomyces cerevisiae under acetic acid conditions. Although the surface structure of S. boulardii was destroyed, it produced more daughter cells. S. boulardii metabolites were also significantly different from S. cerevisiae under acidic stress. The growth of S. boulardii under weak acid conditions differed significantly from that of S. cerevisiae. S. boulardii-mediated fingerprints under weak acid conditions were identified as latent biomarkers, related to fructose and mannose metabolism, tricarboxylic acid cycle, and the glycolysis pathway. Identified biomarkers will aid in the genetic engineering of S. boulardii and other Saccharomyces strains for improved acid resistance and biomass yield.     

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.     

Formation of α-ketoglutaric acid by wine yeasts and its oenological significance

α-Ketoglutaric acid was measured enzymically in wines made in the laboratory from three grape varieties by pure cultures of 12 wine yeasts of the genus Saccharomyces. The results were confirmed with the same juices and 4 yeasts on pilot-plant scale in replicated 30 gallon lots. Mean values for the 12 yeasts ranged from 9 to 117 ppm (overall mean 53). In any one juice the yeasts differed by at least 10-fold in the amounts produced, and certain yeasts produced consistently high or low yields in all juices. The amounts of α-ketoglutaric acid produced depended somewhat on the grape juices used, even though these had comparable pH values, and a significant yeast-juice interaction occurred. The amount of α-ketoglutaric acid formed during fermentation at 15° was 60 per cent of that formed at 25°, and over twice as much was formed at pH 4.2 as at pH 3.0, using four yeast strains. Formation of α-ketoglutaric and pyruvic acids were not significantly correlated. The α-ketoglutaric acid content of 18 white table wines made under comparable conditions on pilot-plant scale from different grape varieties using the same yeast strain ranged from 38 to 152 ppm (mean 90). The significance of the results is discussed, particularly in relation to the binding of sulphur dioxide in wine, and recommendations are given on how to make wines which are low in α-ketoglutaric acid. Formation of α-ketoglutaric acid by three yeasts in a chemically defined medium was lower with increased amounts of nitrogen as ammonium sulphate and higher in the presence of L-glutamic acid, both being used separately as sole nitrogen sources. These findings are discussed in relation to the rǒle of α-ketoglutaric acid in nitrogen metabolism of yeasts.     

Decomposition of L-malic acid by wine yeasts

46 yeasts from 5 genera were examined for ability to decompose L -malic acid in grape juice. Saccharomyces yeasts decomposed between 3 and 45%, the amount decomposed being somewhat greater in highly acid juice. Schizosaccharomyces malidevorans brought about complete decomposition with stoicheiometric yield of ethanol. Pichia and Candida growing as oxidative films also brought about some decomposition. Mixed cultures of Saccharomyces and Schizosuccharomyces were examined quantitatively. The difference between yeast and bacterial decomposition of L-malic acid is discussed, as well as the possible selection of yeast cultures for controlled deacidification in wine-making.     

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.     

Aroma Compounds in Wine as Influenced by Apiculate Yeasts

Aroma compounds of wines resulting from fermentation of sterile grape musts from Monastrell variety inoculated with pure and mixed cultures of apiculate and Saccharomyces yeasts, were isolated and analyzed by gas chromatography with flame ionization and mass spectrometry. Samples fermented with mixed cultures produced a higher concentration of selected compounds and higher total amounts of alcohols and acids, in contrast with wines produced with pure cultures of Saccharomyces spp. Apiculate yeasts are important in the chemical composition and quality of wine.