Strain specific Starmerella bacillaris and Saccharomyces cerevisiae interactions in mixed fermentations.

AIMS: Yeast interactions have a key role in the definition of the chemical profile of the wines. For this reason, winemakers are increasingly interested in mixed fermentations, employing Saccharomyces cerevisiae and non-Saccharomyces strains. However, the outcome of mixed fermentations is often contradictory because there is a great variability among strains within species. Previously, it was demonstrated that the loss of culturability of Starmerella bacillaris in mixed fermentations with S. cerevisiae was due to the physical contact between cells. Therefore, to further explore previous observations, the interaction mechanisms among different strains of Starm. bacillaris and S. cerevisiae during mixed fermentations were investigated. METHODS AND RESULTS: Fermentations were conducted under conditions that allow physical contact between cells (flasks) but also using a double-compartment fermentation system in which cells of both species were kept separate. The role of competition for nutrients and antimicrobial compounds production on yeast-yeast interaction mechanisms was also investigated. Three Starm. bacillaris and three S. cerevisiae strains were used to investigate if interaction mechanisms are modulated in a strain-specific way. Both species populations were affected by physical contact, particularly Starm. bacillaris that lost its culturability during fermentation. In addition, loss of culturability of Starm. bacillaris strains was observed earlier in flasks than in the double-compartment system. The phenomena observed occurred in a strain couple-dependent way. Starm. bacillaris disappearance seemed to be independent of nutrient depletion or the presence of inhibitory compounds (which were not measured in this study). CONCLUSION: Overall, the results of the present study reveal that cell-to-cell contact plays a role in the early death of non-Saccharomyces but the extent to which it is observed depends greatly on the Starm. bacillaris/S. cerevisiae strains tested.

Survey of the yeast ecology of dehydrated grapes and strain selection for wine fermentation.

In this study we investigated the yeast population present on partially dehydrated Nebbiolo grapes destined for 'Sforzato di Valtellina', with the aim to select indigenous starters suitable for the production of this wine. Yeasts were enumerated, isolated, and identified by molecular methods (5.8S-ITS-RFLP and D1/D2 domain sequencing). A genetic, physiological (ethanol and sulphur dioxide tolerance, potentially useful enzymatic activities, hydrogen sulphide production, adhesive properties, and killer activity) and oenological (laboratory pure micro-fermentations) characterization was also carried out. Based on relevant physiological features, seven non-Saccharomyces strains were chosen for laboratory-scale fermentations, either in pure or in mixed-culture (simultaneous and sequential inoculum) with a commercial Saccharomyces cerevisiae strain. Finally, the best couples and inoculation strategy were further tested in mixed fermentations in winery. In both laboratory and winery, microbiological and chemical analyses were conducted during fermentation. The most abundant species on grapes were Hanseniaspora uvarum (27.4 % of the isolates), followed by Metschnikowia spp. (21.0 %) and Starmerella bacillaris (12.9 %). Technological characterization highlighted several inter- and intra-species differences. The best oenological aptitude was highlighted for species Starm. bacillaris, Metschnikowia spp., Pichia kluyveri and Zygosaccharomyces bailli. The best fermentation performances in laboratory-scale fermentations were found for Starm. bacillaris and P. kluyveri, due to their ability to reduce ethanol (-0.34 % v/v) and enhance glycerol production (+0.46 g/L). This behavior was further confirmed in winery. Results of this study contribute to the knowledge of yeast communities associated with a specific environment, like those of Valtellina wine region.

Different genetic responses to oenological conditions between a flocculent wine yeast and its FLO5 deleted strain: Insights from the transcriptome.

During winemaking Saccharomyces cerevisiae strains are exposed continuously to environmental changes and this microorganism responds modifying its transcriptional profile. Yeast flocculation is considered a social trait that allows the cells to escape from hostile conditions by sedimentation. This behaviour is based on the self-interaction of flocculins, proteins encoded by FLO family genes. These are considered responsible of the facultative-helping type cooperation and were designed as green-beard genes. In order to understand the role of flocculation to stress response, the genome wide expression analysis of a wine flocculent S. cerevisiae F6789A strain and its FLO5 deleted strain (F6789A-Δflo5) were determined, using DNA microarray technology. Results highlighted that F6789A strain showed a shorter lag phase in winemaking condition. The comparison of transcriptomic profiles underlined that, while F6789A-Δflo5 strain seemed engaged in the re-organization of the cell wall and in finding different adhesion ways, F6789A strain presented an up-regulation of genes involved in stress response and higher alcohol production.

Influence of Iodine Feeding on Microbiological and Physico-Chemical Characteristics and Biogenic Amines Content in a Raw Ewes' Milk Cheese.

Iodine is an essential trace element involved in the regulation of thyroid metabolism and antioxidant status in humans and animals. The aim of this study was to evaluate the effect of ewes’ dietary iodine supplementation on biogenic amines content as well as microbiological and physico-chemical characteristics in a raw milk cheese at different ripening times (milk, curd, and 2, 7, 15, 30, 60, and 90 days). Two cheese-making trials were carried out using milk from ewes fed with unifeed (Cheese A) or with the same concentrate enriched with iodine (Cheese B). The results indicated that the counts of principal microbial groups and physico-chemical characteristics were quite similar in both cheeses at day 90. Cheese B was characterized by a higher content of biogenic amines and propionic acid. Propionic bacteria were found in both cheeses mainly in Trial B in agreement with the higher content of propionic acid detected.

Impact of Saccharomyces cerevisiae strains on traditional sparkling wines production.

Sparkling wine fermentation is a challenge for yeasts due to the hostile conditions. A phenotype sought in starters is flocculation, because it reduces riddling time. For this reason, six flocculent Saccharomyces cerevisiae wine strains with different flocculation degree and autolytic activity and two commercial strains were tested for traditional sparkling wine production in a winery. Yeast viability, free aminoacids and high molecular weight nitrogen release and physico-chemical composition of sparkling wines were evaluated. Moreover, strains were tested for their aromatic potential. Obtained data revealed that flocculent yeasts presented oenological performances (in terms of fermentation rate, maximum pressure reached, free aminoacids - AAN and high molecular weight nitrogen - HMWN release) similar to the commercial strains. All considered strains were able to complete fermentation and viable cells of all strains were detected in all sparkling wines produced even after 6 months. F6789 and F10471 strains showed slow fermentation kinetics reaching the maximum of pressure at 180 days. Regarding nitrogen compounds release, FI strain was characterized by the highest amount of AAN and HMWN released, followed by F6789. Strains showed a considerable diversification in terms of number and amount of aroma molecules produced and sparkling wines obtained with autochthonous flocculent strains presented a higher amount of alcohols and esters already after 3 months. Further studies are necessary to select starter strains to improve traditional sparkling wines production.

FLO5 gene controls flocculation phenotype and adhesive properties in a Saccharomyces cerevisiae sparkling wine strain.

Flocculation is an important feature for yeast survival in adverse conditions. The natural diversity of flocculating genes in Saccharomyces cerevisiae can also be exploited in several biotechnological applications. Flocculation is mainly regulated by the expression of genes belonging to the FLO family. These genes have a similar function, but their specific contribution to flocculation ability is still unclear. In this study, the distribution of FLO1, FLO5 and FLO8 genes in four S. cerevisiae wine strains was investigated. Subsequently, both FLO1 and FLO5 genes were separately deleted in a flocculent S. cerevisiae wine strain. After gene disruption, flocculation ability and agar adhesion were evaluated. FLO1 and FLO5 genes inheritance was also monitored. All strains presented different lengths for FLO1 and FLO5 genes. Results confirm that in S. cerevisiae strain F6789, the FLO5 gene drives flocculation and influences adhesive properties. Flocculation ability monitoring after a cross with a non-flocculent strain revealed that FLO5 is the gene responsible for flocculation development.

Aroma Profile of Montepulciano d'Abruzzo Wine Fermented by Single and Co-culture Starters of Autochthonous Saccharomyces and Non-saccharomyces Yeasts.

Montepulciano d'Abruzzo is a native grape variety of Vitis vinifera L., grown in central Italy and used for production of high quality red wines. Limited studies have been carried out to improve its enological characteristics through the use of indigenous strains of Saccharomyces cerevisiae. The main objective of the present work was to test two indigenous strains of S. cerevisiae (SRS1, RT73), a strain of Starmerella bacillaris (STS12), one of Hanseniaspora uvarum (STS45) and a co-culture of S. cerevisiae (SRS1) and S. bacillaris (STS12), in an experimental cellar to evaluate their role in the sensory characteristic of Montepulciano d'Abruzzo wine. A S. cerevisiae commercial strain was used. Fermentations were conducted under routine Montepulciano d'Abruzzo wine production, in which the main variables were the yeast strains used for fermentation. Basic winemaking parameters, some key chemical analysis and aroma compounds were considered. S. cerevisiae strain dynamics during fermentation were determined by molecular methods. The musts inoculated with the co-culture were characterized by a faster fermentation start and a higher content of glycerol after 3 days of fermentation, as well as the musts added with strains S. bacillaris (STS12) and H. uvarum (STS45). At the end of fermentation the parameters studied were quite similar in all the wines. Total biogenic amines (BA) content of all the wines was low. Ethanolamine was the predominant BA, with a concentration ranging from 21 to 24 mg/l. Wines were characterized by esters and alcohols. In particular, 2-phenylethanol, 3-methylbut-1-yl methanoate, and ethyl ethanoate were the major aroma volatile compounds in all wines. Statistical analysis highlighted the different role played by aroma compounds in the differentiation of wines, even if it was impossible to select a single class of compounds as the most important for a specific yeast. The present study represents a further step toward the use of tailored autochthonous strains to impart the specific characteristics of a given wine which are an expression of a specific terroir.

Biodiversity of autolytic ability in flocculent Saccharomyces cerevisiae strains suitable for traditional sparkling wine fermentation.

Yeasts involved in secondary fermentation of traditional sparkling wines should show specific characteristics, such as flocculation capacity and autolysis. Recently it has been postulated that autophagy may contribute to the outcome of autolysis. In this study, 28 flocculent wine Saccahromyces cerevisiae strains characterized by different flocculation degrees were studied for their autolytic and autophagic activities. Autolysis was monitored in synthetic medium through the determination of amino acid nitrogen and total proteins released. At the same time, novel primer sets were developed to determine the expression of the genes ATG1, ATG17 and ATG29. Twelve strains were selected on the basis of their autolytic rate and ATG gene expressions in synthetic medium and were inoculated in a base wine. After 30, 60 and 180 days the autolytic process and ATG gene expressions were evaluated. The obtained data showed that autolysis and ATG gene expressions differed among strains and were independent of the degree of flocculation. This biodiversity could be exploited to select new starter stains to improve sparkling wine production. Copyright © 2016 John Wiley & Sons, Ltd.

Genetic diversity of FLO1 and FLO5 genes in wine flocculent Saccharomyces cerevisiae strains.

Twenty-eight flocculent wine strains were tested for adhesion and flocculation phenotypic variability. Moreover, the expression patterns of the main genes involved in flocculation (FLO1, FLO5 and FLO8) were studied both in synthetic medium and in presence of ethanol stress. Molecular identification and typing were achieved by PCR-RFLP of the 5.8S ITS rRNA region and microsatellite PCR fingerprinting, respectively. All isolates belong to Saccharomyces cerevisiae species. The analysis of microsatellites highlighted the intraspecific genetic diversity of flocculent wine S. cerevisiae strains allowing obtaining strain-specific profiles. Moreover, strains were characterized on the basis of adhesive properties. A wide biodiversity was observed even if none of the tested strains were able to form biofilms (or 'mats'), or to adhere to polystyrene. Moreover, genetic diversity of FLO1 and FLO5 flocculating genes was determined by PCR. Genetic diversity was detected for both genes, but a relationship with the flocculation degree was not found. So, the expression patterns of FLO1, FLO5 and FLO8 genes was investigated in a synthetic medium and a relationship between the expression of FLO5 gene and the flocculation capacity was established. To study the expression of FLO1, FLO5 and FLO8 genes in floc formation and ethanol stress resistance qRT-PCR was carried out and also in this case strains with flocculent capacity showed higher levels of FLO5 gene expression. This study confirmed the diversity of flocculation phenotype and genotype in wine yeasts. Moreover, the importance of FLO5 gene in development of high flocculent characteristic of wine yeasts was highlighted. The obtained collection of S. cerevisiae flocculent wine strains could be useful to study the relationship between the genetic variation and flocculation phenotype in wine yeasts.