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.     

Comparative typing of <Emphasis Type="Italic">L. delbrueckii</Emphasis> subsp<Emphasis Type="Italic">. bulgaricus</Emphasis> strains using multilocus sequence typing and RAPD–PCR

Comparative typing analysis of 25 Lactobacillus delbrueckii subsp. bulgaricus strains, isolated from traditional yoghurts in Turkey, was performed by RAPD–PCR (randomly amplified polymorphic DNA–PCR) and MLST (multilocus sequence typing). RAPD–PCR analyses were performed using two primers; M13 and 1254. Primer 1254 produced better results than primer M13. The bands produced by primer 1254 were brighter and easier to interpret, and a higher number of bands were produced. In addition, clusters produced by primer 1254 were grouped according to the source of isolation. MLST analysis was performed using three genes, β-gal, pheS and rpoA, and isolates were successfully characterized at strain level. To our knowledge, MLST analyses were used for the first time for strain level discrimination in L. delbrueckii subsp. bulgaricus. It enabled a detailed understanding of L. delbrueckii subsp. bulgaricus strains by using allele and sequence types’ analysis. Both MLST and RAPD allowed for the typing of clusters according to the isolation source, while RAPD provided an increased differentiation. However, by increasing the number of genes analyzed, the discriminatory power of MLST could be increased.     

Selection of indigenous Saccharomyces cerevisiae strains for Nero d'Avola wine and evaluation of selected starter implantation in pilot fermentation

The present research studied Saccharomyces cerevisiae yeasts isolated from Nero d'Avola grapes, collected in different areas of the Sicily region. RAPD-PCR analysis with M13 primer was used for preliminary discrimination among 341 S. cerevisiae isolates. Inoculated fermentations with S. cerevisiae strains, exhibiting different RAPD-PCR fingerprinting, revealed the impact of selected strains on volatile compound concentration. Two selected strains were used in fermentation at cellar level and the restriction analysis of mtDNA on yeast colonies isolated during fermentation was used to control strain implantation. This study represents an important step to establish a collection of indigenous S. cerevisiae strains isolated from a unique environment, such as Nero d'Avola vineyards. Different starter implantation throughout inoculated fermentation represents an additional character, which might be considered during the selection program for wine starter cultures.     

Genetic and enological analysis of selected Saccharomyces cerevisiae strains for wine production

The non‐wine Saccharomyces cerevisiae strain of 96581 was found to be a promising candidate for the production of white wine. It produced wines with fusel alcohols that were 57% higher than those produced by the wine yeasts studied and was also more efficient in the production of 2‐phenethyl acetate and 3‐methyl‐1‐butanol acetate. This study also shows that there is a difference in the ester‐formation efficiency for acetates relative to C6, C8 and C10 fatty acid esters for all the studied yeast strains. Therefore, it supports the view that other unidentified enzymes besides those regulated by ATF1 and ATF2 genes are involved in the production of ethyl esters of C6–C10 fatty acids. DNA analysis of the 25S, 18S, 5.8S and 5S ribosomal DNA genes in these strains showed high conservation. Despite the closely related nature of these yeast strains, the chemical profiles of the wines produced were significantly different.     

Breeding of lager yeast with Saccharomyces cerevisiae improves stress resistance and fermentation performance

Lager beer brewing relies on strains collectively known as Saccharomyces carlsbergensis, which are hybrids between S. cerevisiae and S. eubayanus‐like strains. Lager yeasts are particularly adapted to low‐temperature fermentations. Selection of new yeast strains for improved traits or fermentation performance is laborious, due to the allotetraploid nature of lager yeasts. Initially, we have generated new F1 hybrids by classical genetics, using spore clones of lager yeast and S. cerevisiae and complementation of auxotrophies of the single strains upon mating. These hybrids were improved on several parameters, including growth at elevated temperature and resistance against high osmolarity or high ethanol concentrations. Due to the uncertainty of chromosomal make‐up of lager yeast spore clones, we introduced molecular markers to analyse mating‐type composition by PCR. Based on these results, new hybrids between a lager and an ale yeast strain were isolated by micromanipulation. These hybrids were not subject to genetic modification. We generated and verified 13 hybrid strains. All of these hybrid strains showed improved stress resistance as seen in the ale parent, including improved survival at the end of fermentation. Importantly, some of the strains showed improved fermentation rates using 18°Plato at 18–25°C. Uniparental mitochondrial DNA inheritance was observed mostly from the S. cerevisiae parent. Copyright © 2012 John Wiley & Sons, Ltd.     

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.     

Studies on acetate ester production by non-Saccharomyces wine yeasts

A double coupling bioreactor system was used to fast screen yeast strains for the production of acetate esters. Eleven yeast strains were used belonging to the genera Candida, Hanseniaspora, Metschnikowia, Pichia, Schizosaccharomyces and Zygosacharomyces, mainly isolated from grapes and wine, and two wine Saccharomyces cerevisiae strains. The acetate ester forming activities of yeast strains belonging to the genera Hanseniaspora (Hanseniaspora guilliermondii and H. uvarum) and Pichia (Pichia anomala) showed different substrate specificities and were able to produce ethyl acetate, geranyl acetate, isoamyl acetate and 2-phenylethyl acetate. The influence of aeration culture conditions on the formation of acetate esters by non-Saccharomyces wine yeast and S. cerevisiae was examined by growing the yeasts on synthetic microbiological medium. S. cerevisiae produced low levels of acetate esters when the cells were cultured under highly aeration conditions, while, under the same conditions, H. guilliermondii 11104 and P. anomala 10590 were found to be strong producers of 2-phenylethyl acetate and isoamyl acetate, respectively.     

Isolation of glutathione biosynthesis-deficient mutants of Saccharomyces cerevisiae and their use in baker's yeast production

 Glutathione biosynthesis-deficient mutants of Saccharomyces cerevisiae 0511 were obtained by mutation under specific conditions. A total of 3388 strains were isolated and among them were found 46 mutants sensitive to methylglyoxal. The intracellular glutathione concentration of mutant strain S. cerevisiae 3033 was 0.0172 g/g dry weight, which was a decrease of >76% compared to that of the parent. The growth of mutant strains S. cerevisiae 3033 and S. cerevisiae 1116 in the medium with glutathione present and absent was compared to that of the parent strain. The sensibility of the baker's yeast strains studied to antifoaming agents, butanol and acetic acid was also investigated. The relationship between glutathione presence in the cell and the sensitivity of strain S. cerevisiae 3033 to antifoaming agents and butanol was ascertained, while such a connection with the presence of acetic acid in the molasses medium used for baker's yeast cultivation was not observed. The higher sensitivity of strain S. cerevisiae 3033 to some chemical compounds in the molasses nutrition medium was shown. Received: 2 November 1999 / Revised version: 15 February 2000     

Higher alcohols concentration and its relation with the biological aging evolution

The production of 1-propanol, isobutanol, 2-methyl-1-butanol, 3-methyl-1-butanol and 2-phenylethanol by two flor yeast strains during accelerated biological aging of sherry wines with different aging time has been evaluated. The S. cerevisiae G1 strain has shown a higher production of higher alcohols than the S. bayanus F12 strain highlighting the 1-propanol production in the youngest wine. Differences between the two studied yeasts decreased in relation with the aging time of the initial wine used. The multiple regression analysis carried out to relate the higher alcohols concentration and the values obtained from an aging equation showed R-squared values ranging from 81.1 to 96.5%. The higher alcohols that best described such relation were 1-propanol, isobutanol and 3-methyl-1-butanol.     

Development of a multilocus variable number of tandem repeat typing method for Oenococcus oeni

Oenococcus oeni is responsible for the malolactic fermentation of wine. Genomic diversity has already been established in this species. In addition, winemakers usually report varying starter culture efficiency. The monitoring of indigenous and selected strains is essential for understanding strain survival and implantation during the winemaking process. In this study, we report the development of the first typing scheme for O. oeni using multiple-locus variable number of tandem repeat analysis (VNTR). The discriminatory power of 14 out of 44 tandem repeat loci in the genome of the PSU-1 strain was initially evaluated with a test collection of 18 genotypically distinct starter strains. Then five VNTR loci, which can be easily scored with the technology used here, were identified and used to genotype a collection of 236 strains, previously classified by restriction endonuclease analysis-pulsed-field gel electrophoresis (REA-PFGE) and multilocus sequence typing (MLST) into 136 REA-PFGE types or 110 MLST types. The discriminatory power of VNTR (as determined by Simpson's index of discrimination) was higher than that of the other two methods, with 201 VNTR types. The targeted VNTR markers were found to be stable and did not change for the clones of the same strain deposited in a collection at intervals of several years. Strains isolated from the different wine producing areas or the products were assigned to phylogenetic groups and were statistically linked with the VNTR profiles. Another interesting observation was that the loci were found in sequences homologous to regions encoding for membrane-anchored proteins.     

Evaluation of <Emphasis Type="Italic">flaA</Emphasis> Short Variable Region Sequencing,Multilocus Sequence Typing,and Fourier Transform Infrared Spectroscopy for Discrimination between <Emphasis Type="Italic">Campylobacter jejuni</Emphasis> Strains

Discriminatory and robust typing methods are needed to improve the understanding of the dynamics of food-borne Campylobacter infections and epidemiology in primary animal production. To evaluate the strain discriminatory potential of typing methods, flaA short variable region (SVR) sequencing and Fourier transform infrared (FTIR) spectroscopy were applied on a collection of 102 epidemiologically related and unrelated Campylobacter jejuni strains. Previous application of FTIR spectroscopy for subtyping of Campylobacter has been limited. A subset of isolates, initially discriminated by flaA SVR sequencing, were further subjected to multilocus sequence typing (MLST). It was found that flaA SVR sequencing had a slightly higher discriminatory power than FTIR spectroscopy, based on the Simpson diversity index. The clustering of strains indicated that FTIR spectroscopy is indeed a suitable method for discrimination of Campylobacter. The isolates were assigned to six clusters based on flaA SVR sequences and nine clusters based on the FTIR spectroscopy profiles. Furthermore, the cluster analysis of flaA SVR sequences, MLST, and FTIR spectroscopy profiles showed a high degree of congruence, assigning the isolates to similar cluster structures. In conclusion, FTIR spectroscopy can be applied for subtyping of Campylobacter, and the high discriminatory potential of both flaA SVR sequencing and FTIR spectroscopy render them suitable screening methods for large numbers of strains.     

Yeasts isolated from New Zealand vineyards and wineries

Background and Aims: The yeast flora from a range of New Zealand commercial wineries was surveyed to estimate the incidence of yeast species in grape juice. Methods and Results: Molecular analysis of the internal transcribed spacer region was performed for 1279 yeast colonies isolated from 17 different fresh grape juices sampled in eight New Zealand wineries between 2003 and 2009. The 17 juices contained at least 25 different species of yeast from nine genera. Microsatellite fingerprinting of Saccharomyces cerevisiae showed that some strains were identical to known commercial yeast varieties, but we also found evidence for local populations of S. cerevisiae common to individual wineries or regions. Five genotypes from Central Otago, New Zealand, were very closely related to a single sequenced strain derived from Chile, which in turn is related to European wine isolates. Conclusions: The yeast flora found in New Zealand grape juices is broadly similar to that found in wineries elsewhere around the world. Genotyping of S. cerevisiae suggests recent dispersal of both commercial and non-commercial yeast strains from Europe to New Zealand. Significance of the Study: These data are consistent with two human-mediated modes for the international dispersal of S. cerevisiae: one via the escape of strains traded commercially, and another via long distance dispersal of non-commercial strains.     

Multi‐gene phylogenetic analysis reveals that shochu‐fermenting Saccharomyces cerevisiae strains form a distinct sub‐clade of the Japanese sake cluster

Shochu is a traditional Japanese distilled spirit. The formation of the distinguishing flavour of shochu produced in individual distilleries is attributed to putative indigenous yeast strains. In this study, we performed the first (to our knowledge) phylogenetic classification of shochu strains based on nucleotide gene sequences. We performed phylogenetic classification of 21 putative indigenous shochu yeast strains isolated from 11 distilleries. All of these strains were shown or confirmed to be Saccharomyces cerevisiae, sharing species identification with 34 known S. cerevisiae strains (including commonly used shochu, sake, ale, whisky, bakery, bioethanol and laboratory yeast strains and clinical isolate) that were tested in parallel. Our analysis used five genes that reflect genome‐level phylogeny for the strain‐level classification. In a first step, we demonstrated that partial regions of the ZAP1, THI7, PXL1, YRR1 and GLG1 genes were sufficient to reproduce previous sub‐species classifications. In a second step, these five analysed regions from each of 25 strains (four commonly used shochu strains and the 21 putative indigenous shochu strains) were concatenated and used to generate a phylogenetic tree. Further analysis revealed that the putative indigenous shochu yeast strains form a monophyletic group that includes both the shochu yeasts and a subset of the sake group strains; this cluster is a sister group to other sake yeast strains, together comprising a sake‐shochu group. Differences among shochu strains were small, suggesting that it may be possible to correlate subtle phenotypic differences among shochu flavours with specific differences in genome sequences. Copyright © 2017 John Wiley & Sons, Ltd.     

The molecular characterization of new types of Saccharomyces cerevisiae × S. kudriavzevii hybrid yeasts unveils a high genetic diversity

New double‐ and triple‐hybrid Saccharomyces yeasts were characterized using PCR‐restriction fragment length polymorphism of 35 nuclear genes, located on different chromosome arms, and the sequencing of one nuclear and one mitochondrial gene. Most of these new hybrids were originally isolated from fermentations; however, two of them correspond to clinical and dietary supplement isolates. This is the first time that the presence of double‐hybrid S. cerevisiae × S. kudriavzevii in non‐fermentative substrates has been reported and investigated. Phylogenetic analysis of the MET6 nuclear gene confirmed the double or triple parental origin of the new hybrids. Restriction analysis of gene regions in these hybrids revealed a high diversity of genome types. From these molecular characterizations, a reduction of the S. kudriavzevii fraction of the hybrid genomes is observed in most hybrids. Mitochondrial inheritance in hybrids was deduced from the analysis of mitochondrial COX2 gene sequences, which showed that most hybrids received the mitochondrial genome from the S. kudriavzevii parent. However, two strains inherited a S. cerevisiae COX2, being the first report of S. cerevisiae × S. kudriavzevii hybrids with S. cerevisiae mitochondrial genomes. These two strains are those showing a higher S. kudriavzevii nuclear genome reduction, especially in the wine hybrid AMH. This may be due to the release of selective pressures acting on the other hybrids to maintain kudriavzevii mitochondria‐interacting genes. Copyright © 2011 John Wiley & Sons, Ltd.     

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.     

Sweet Wine Production by Two Osmotolerant Saccharomyces cerevisiae Strains

The use of Saccharomyces cerevisiae to produce sweet wine is difficult because yeast is affected by a hyperosmotic stress due to the high sugar concentrations in the fermenting must. One possible alternative could be the coimmobilization of the osmotolerant yeast strains S. cerevisiae X4 and X5 on Penicillium chrysogenum strain H3 (GRAS) for the partial fermentation of raisin musts. This immobilized has been, namely, as yeast biocapsules. Traditional sweet wine (that is, without fermentation of the must) and must partially fermented by free yeast cells were also used for comparison. Partially fermented sweet wines showed higher concentration of the volatile compounds than traditionally produced wines. The wines obtained by immobilized yeast cells reached minor concentrations of major alcohols than wines by free cells. The consumption of specific nitrogen compounds was dependent on yeast strain and the cellular immobilization. A principal component analysis shows that the compounds related to the response to osmotic stress (glycerol, acetaldehyde, acetoin, and butanediol) clearly differentiate the wines obtained with free yeasts but not the wines obtained with immobilized yeasts.     

The yeast IRC7 gene encodes a β-lyase responsible for production of the varietal thiol 4-mercapto-4-methylpentan-2-one in wine

Three varietal thiols are key aroma compounds in Sauvignon Blanc wines: 4-mercapto-4-methylpentan-2-one (4MMP), 3-mercaptohexanol (3MH) and its acetylated derivative 3-mercaptohexyl acetate (3MHA). Screening of Saccharomyces cerevisiae strains identified a clinical isolate with elevated 4MMP production after fermentation. Bulked Segregant Analysis of a cross between this isolate and the laboratory strain revealed a single major locus for 4MMP production near the telomere of chromosome 6. Deletion of the IRC7 gene from this region in YJM450 reduced 4MMP production below detectable levels, but did not affect yields of 3MH, in Sauvignon Blanc wine. Sequencing revealed that the IRC7 gene in YJM450 had been introgressed from a strain of Saccharomyces paradoxus. Most strains of S. cerevisiae, including the laboratory strain S288C, have a 38-bp deletion that inactivates IRC7. Overexpression of a full-length S. cerevisiae allele of IRC7 in a wine yeast, Zymaflore F15, increased 4MMP production in Sauvignon Blanc wine from undetectable levels (<10 ng L⁻¹) to concentrations of 1000 ng L⁻¹, and also increased 3MH and 3MHA. Biochemical analysis of soluble protein extracts showed that both the cerevisiae and paradoxus IRC7 proteins show β-lyase activity, with a substrate preference for cys-4MMP over cys-3MH.     

Effect of Different Indigenous Yeast β‐Glucosidases on the Liberation of Bound Aroma Compounds

This study investigated β‐D‐glucosidase activity in the indigenous wine yeast present on grape berries in Yantai in Shandong Province, China. Yeast population profiles from the Yantai production area in China were examined. Among the ten species identified by RFLP analysis of the 5.8S rRNA gene, four exhibited higher β‐glucosidase activity, namely, Hanseniaspora uvarum, Trichosporon asahii, Pichia fermentans and Saccharomyces cerevisiae. The β‐glucosidases from the four representative strains were chosen to hydrolyse the glycosidic precursors of Cabernet Sauvignon. After enzymatic hydrolysis, 31 compounds were identified and quantified, including terpenes, C₁₃‐noriso‐prenoid, C₆ compounds, alcohols, aldehydes and volatile phenols. Results showed that different strains exhibited different hydrolytic abilities on the bound aroma precursors. The main variables included C₆ compounds, terpenes and alcohols. The concentration of the 14 compounds showed significant differences between enzymatic treatments, with 11 treated using the β‐glucosidase of the F6 strain (T. asahii). These findings may have some applicative value for utilizing the strains or their β‐glucosidases, which are able to complement and optimize wine quality.     

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.