Genetic and phenotypic diversity of autochthonous cider yeasts in a cellar from Asturias

This paper analyses yeast diversity and dynamics during the production of Asturian cider. Yeasts were isolated from apple juice and at different stages of fermentation in a cellar in Villaviciosa during two Asturian cider-apple harvests. The species identified by ITS-RFLP corresponded to Hanseniaspora valbyensis, Hanseniaspora uvarum, Metschnikowia pulcherrima, Pichia guilliermondii, Candida parapsilosis, Saccharomyces cerevisiae and Saccharomyces bayanus/Saccharomyces pastorianus/Saccharomyces kudriavzevii/Saccharomyces mikatae. The species C. parapsilosis is reported here for the first time in cider. The analysis of Saccharomyces mtDNA patterns showed great diversity, sequential substitution and the presence of a small number of yeast patterns (up to 8), present in both harvests. Killer (patterns nos. 22′ and 47), sensitive (patterns nos. 12, 15, 33 and 61) and neutral phenotypes were found among the S. cerevisiae isolates. The detection of β-glucosidase activity, with arbutin as the sole carbon source, allowed two S. cerevisiae strains (patterns nos. 3′ and 19′) to be differentiated by means of this enzymatic activity. Yeast strains producing the killer toxin or with β-glucosidase activity are reported for the first time in autochthonous cider yeasts.     

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.     

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.     

Molecular characterization and oenological properties of wine yeasts isolated during spontaneous fermentation of six varieties of grape must

Fermentation by naturally occurring yeasts may produce wines of complex oenological properties that are unique to a specific region. The present work analyses the population dynamics of the yeasts during spontaneous fermentation of six varieties of grape must from the “Valle del Andarax” area (Spain). In this study we identified members of the genera Candida, Hanseniaspora, Issatchenkia, Metschnikowia, Pichia and Saccharomyces by PCR-RFLP of the ITS region. The capability of these yeasts to ferment grape must of the Macabeo variety was studied, and the volatile profile of the wine from each microvinification has been determined. Of all the yeasts isolated Candida stellata and Saccharomyces cerevisiae were able to consume virtually all the initial glucose, producing ethanol contents typical of table wines. The best profile of higher alcohols was given by Saccharomyces cerevisiae followed by Hanseniaspora uvarum, Issatchenkia orientalis and Candida stellata. Metschnikowia pulcherrima and Pichia fermentans showed the highest production of ethyl caprilate and 2-phenyl ethanol, compounds associated with pleasant aromas.     

Role of non-Saccharomyces yeasts in Korean wines produced from Campbell Early grapes: Potential use of Hanseniaspora uvarum as a starter culture

Several yeasts were isolated from Campbell Early grapes (Vitis labrusca cultivar Campbell Early), the major grape cultivar in Korea, grown in two different regions. PCR-RFLP analysis of the ITS I-5.8S-ITS II region showed that 34 isolates out of a total of 40 were in the same group. Phylogenetic analysis revealed that the major strain belonged to one species, Hanseniaspora uvarum, although they displayed some nucleotide mismatches between them. During spontaneous alcohol fermentation at 20 °C, the two grape musts containing 24 °Brix sugar exhibited similar fermentation patterns with differences in final alcohol production and yeast viable counts. PCR analysis of the yeasts randomly isolated during the fermentation using an intron splice site primer showed changes in yeast flora between 8 and 10 days of fermentation. We found that the dominant yeasts displaying various PCR patterns using the primer remained the same throughout the early stages of fermentation, as determined by molecular typing of their ITS regions using PCR-RFLP, and these yeasts were identical to those isolated from grape berries. Among the isolates, the strain designated SS6 was selected based on its potassium metabisulfite resistance, alcohol production (distillation method), and flavor (by sniffing test) of grape juice. When Campbell Early grape must was inoculated with H. uvarum SS6 cells, no differences in fermentation pattern were observed compared with that inoculated with cells of Saccharomyces cerevisiae W-3, an industrial wine yeast strain. However, SS6 wine showed higher levels of organic acid (especially lactic acid), aldehydes, and minor alcohols (except n-propyl alcohol), as well as a higher score in sensory evaluation, compared to those of W-3 wine.     

Yeast diversity during tapping and fermentation of palm wine from Cameroon

In the present study, we have investigated the occurrence of yeast flora during tapping and fermentation of palm wine from Cameroon. The yeast diversity was investigated using both traditional culture-dependent and culture-independent methods. Moreover, to characterize the isolates of the predominant yeast species (Saccharomyces cerevisiae) at the strain level, primers specific for δ sequences and minisatellites of genes encoding the cell wall were used. The results confirm the broad quantitative presence of yeast, lactic acid bacteria and acetic acid bacteria during the palm wine tapping process, and highlight a reduced diversity of yeast species using both dependent and independent methods. Together with the predominant species S. cerevisiae, during the tapping of the palm wine the other species found were Saccharomycodes ludwigii and Zygosaccharomyces bailii. In addition, denaturing gradient gel electrophoresis (DGGE) analysis detected Hanseniaspora uvarum, Candida parapsilopsis, Candida fermentati and Pichia fermentans. In contrast to the progressive simplification of yeast diversity at the species level, the molecular characterization of the S. cerevisiae isolates at the strain level showed a wide intraspecies biodiversity during the different steps of the tapping process. Indeed, 15 different biotypes were detected using a combination of three primer pairs, which were well distributed in all of the samples collected during the tapping process, indicating that a multistarter fermentation takes place in this particular natural, semi-continuous fermentation process.     

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.     

Influence of autochthonous Saccharomyces cerevisiae strains on volatile profile of Negroamaro wines

Saccharomyces cerevisiae is the yeast species predominating the alcoholic fermentation of grape must. The aim of this research was to evaluate the impact of indigenous S. cerevisiae strains biodiversity on the aroma of wines from Negroamaro grapes. Grapes collected in two different Negroamaro producing micro districts in Salento (Southern Italy), were subjected to natural fermentation and two indigenous S. cerevisiae populations were isolated. Fifteen strains for each of the two populations were selected and tested by micro fermentation assay in order to evaluate their specific contribute to the volatiles composition and sensory impact of the produced wines. The aromatic profile of wines obtained by each selected strain was characterized by different contents of acetates, ethyl esters of fatty acids, higher alcohols, thus showing to be related to the strains geographical origin. The sensorial analysis of wines produced by the six best performing strains confirmed that they are good candidates as industrial starter cultures, This study indicates that the use of a “microarea-specific” starter culture is a powerful tool to enhance the peculiarity of wines deriving from specific areas.     

A new simplified AFLP method for wine yeast strain typing

A simplified method of AFLP (Amplified Fragment Length Polymorphisms) is presented for typing yeast present during wine fermentations. The changes introduced allowed analysis by gel electrophoresis and considerably reduced the need for equipment. Another remarkable improvement was the use of non-labelled primers which reduces the cost of the analysis. This method was applied to reference strains from culture collection to test the reliability of the technique. A total of 180 colonies isolated from a spontaneous fermentation were typed into eleven different strains of Hanseniaspora uvarum, six of Hanseniaspora vineae, four of Candida zemplinina, and eleven of Saccharomyces cerevisiae. This method is suitable for typing yeast strains for routine grape and wine ecology analysis.     

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.     

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.     

Yeast population dynamics in five spontaneous fermentations of Malvasia must

The dynamics of the wine yeast strains presented in five spontaneous Malvasia wine fermentations have been studied. Samples were analysed for their microbiological characteristics and chemical substances. All 937 isolates were characterized using electrophoretic karyotyping and tested for their killer activity. The non- Saccharomyces population was identified using a combination of PCR-RFLP analysis of the rDNA spacer region and physiological testing. The total yeast population level in the must after sedimentation was 10⁵cfu ml⁻¹and included the following genera:Candida, Metschnikowia, Hanseniaspora, Rhodotorula, Issatchenkia and Debaryomyces. However, Saccharomyces sp. was not detected in fresh must samples plated on YEPD medium. Based on the chromosome length polymorphism among 649 isolates from the subsequent phases of fermentation, 46 different electrophoretic patterns of Saccharomyces cerevisiae were distinguished. The most abundant karyotypes were L₁, L₄, L₁₂, P₆. A sequential substitution of S. cerevisiae strains occurred during the different phases of fermentation. At the slow fermentation rate, karyotype L₄was most abundant in almost all fermenters. At the beginning of the tumultuous fermentation phase, the most frequent karyotype became L₁followed by karyotype L₄. Finally, during the fermentation process, pattern L₄was clearly replaced by karyotype L₁followed by pattern L₁₂. Despite the same fermentation source (grape must), differences among five spontaneous fermentations were observed. The population dynamics of S. cerevisiae yeasts, especially the dynamics of the major S. cerevisiae strains (L₁, L₄, and L₁₂) were quite similar in all five fermenters in opposite to the minor strains of S. cerevisiae.     

Comparative evaluation of some oenological properties in wine strains of Candida stellata, Candida zemplinina, Saccharomyces uvarum and Saccharomyces cerevisiae

Due to the recent changes in yeast taxonomy, a novel wine-related species Candida zemplinina as well as a “reinstated” species Saccharomyces uvarum have been accepted in addition to Candida stellata, Saccharomyces bayanus and Saccharomyces cerevisiae, and the use of the different taxon names has been inconsistent in the literature of food microbiology. The aim of this work is to make an exact comparison of genetically identified strains of these species, under oenological conditions. Dynamics and some important products of alcoholic fermentation were investigated in laboratory fermentations. The results show that C. zemplinina and C. stellata are similar in their strong fructophilic character. C. stellata produces more glycerol and fare more ethanol, which is comparable with that produced by S. uvarum. Strains of the latter species differed from S. cerevisiae mainly in low acetic acid production and lower ethanol yield. Revision of the oenological traits of these yeasts provides new data for consideration in the control of fermentation, with special regard to botrytized sweet wines, where they are frequently found in mixed population.     

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.     

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.     

Redox effect on volatile compound formation in wine during fermentation by Saccharomyces cerevisiae

Although redox state is a well-known key process parameter in microbial activity, its impact on wine volatile aroma compounds produced during fermentation has not been studied in detail. In this study we report the effect of reductive and microaerobic conditions on wine aroma compound production using different initial amounts of yeast assimilable nitrogen (YAN: 180 and 400 mg N/l) in a simil grape must defined medium and two S. cerevisiae strains commonly used in wine-making. In batch fermentation culture conditions, reductive conditions were obtained using flasks plugged with Muller valves filled with sulphuric acid; while microaerobic conditions were attained with defined cotton plugs. It was found that significant differences in redox potential were obtained using the different plugs, and with variation of over 100 mV during the main fermentation period.     

Effects of mediums on fermentation behaviour and aroma composition in pure and mixed culture of Saccharomyces cerevisiae with Torulaspora delbrueckii

The nutrient status and composition in mediums have a significant effect on yeast metabolism and phenotypic characteristics in wine fermentation. In this study, the effects of three frequently used mediums, including synthetic grape juice (SGJ), grape juice without grape pericarp and seeds (GJ) and grape must with grape pericarp and seeds (GMPS), on yeast fermentation behaviour and aroma compounds produced by pure and mixed culture of Saccharomyces cerevisiae T73 with Torulaspora delbrueckii TD20 were investigated after alcoholic fermentation. The results showed that high fermentation activities and cell population were always found in GJ medium irrespective of inoculated approach. More esters and higher alcohols were produced in GMPS medium fermented by pure S. cerevisiae, while SGJ medium had increased levels of fatty acids. Consistent with previous literatures, the mixed fermentation of T. delbrueckii and S. cerevisiae produced more acetate esters and fatty acids than the pure culture of S. cerevisiae, while this enological trait was only found in SGJ and GJ, not in GMPS. Our results highlighted that more attention should be paid to the fermentation medium when evaluating the enological and aromatic properties of selected yeasts used in industrial winemaking. In this regard, the combined use of GJ and GMPS medium might be a suitable choice.     

Selenium biotransformation by Saccharomyces cerevisiae and Saccharomyces bayanus during white wine manufacture: Laboratory-scale experiments

The main purpose of this laboratory-scale study was to evaluate the transformation of inorganic selenium, as sodium selenite, when added to white grape juice as part of the fermentation process of white wine. The participation of yeast, added in the fermentation of the must, is necessary to convert inorganic selenium into organoseleno species. Two different yeasts, Saccharomyces cerevisiae and Saccharomyces bayanus were used for fermentation. The effects of different Se concentrations on cells and their viability during fermentation were evaluated. The alcoholic fermentation that produced wine was not affected by the presence of selenium, regardless of the type of Saccharomyces used. After 21 days of fermentation, the white wine and residual yeast were separated and analysed by ICP-MS and LC–ICP-MS for determination of total selenium and speciation. Selenomethionine was found to be the main Se-species in the selenised white wine. The results obtained are preliminary but they could be considered for future studies using both pilot and full-scale vinification processes.     

Production of indole by wine-associated microorganisms under oenological conditions

A high concentration of indole has been linked to ‘plastic-like’ off-flavour in wines, predominantly in wines produced under sluggish fermentation conditions. The purpose of this study was to determine the ability of yeast and bacteria to form indole and whether tryptophan was required for indole accumulation during winemaking. Wine-associated yeast and bacteria species (Saccharomyces cerevisiae, Saccharomyces bayanus, Candida stellata, Hanseniaspora uvarum, Kluyveromyces thermoloterans, Oenococcus oeni, Lactobacillus lindneri, Pediococcus cerevisiae and Pediococcus parvulus) were screened for their potential to generate indole during alcoholic or malolactic fermentation. Tryptophan was required for the accumulation of indole in chemically defined medium, and all yeast and bacteria fermentations were able to accumulate indole. C. stellata showed the greatest potential for indole formation (1033 μg/L) and among the bacteria, the highest concentration was generated by L. lindneri (370 μg/L). Whether primary fermentation is the principle cause of indole formation remains to be determined. We hypothesise that during an efficient fermentation, indole is removed through catabolic metabolism, but, when a sluggish fermentation arises, non-Saccharomyces species might produce excess indole that is still present by end of fermentation.