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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.

Nowadays, the interest toward products containing probiotics is growing due to their potential health benefits to the host and the research is focusing on search of new probiotic microorganisms. The present work was focused on the characterization of indigenous

The bulk of grape juice fermentation is carried out by the yeast Saccharomyces cerevisiae, but non-Saccharomyces yeasts can modulate many sensorial aspects of the final products in ways not well understood. In this study, some of such non-conventional yeasts were screened as mixed starter cultures in a defined growth medium in both simultaneous and sequential inoculations. One strain of Starmerella bacillaris and another of Zygosaccharomyces bailii were chosen by their distinct phenotypic footprint and their ability to reduce ethanol levels at the end of fermentation. S. bacillaris losses viability strongly at the end of mixed fermentations, while Z. bailii remains viable. S. cerevisiae viability was unchanged by the presence of the other yeasts. Physiological characterization of both strains indicates that S. bacillaris behavior is overall more different from S. cerevisiae than Z. bailii. In addition, S. cerevisiae transcriptome changes to a bigger degree in the presence of S. bacillaris in comparison to mixed fermentation with Z. bailii. S. bacillaris induces the translation machinery and repress vesicular transport. Both non-Saccharomyces yeasts induce S. cerevisiae glycolytic genes, and that may be related to ethanol lowering, but some aspects of carbon-related mechanisms are specific for each strain. Z. bailii presence increases the stress-related polysaccharides trehalose and glycogen, while S. bacillaris induces gluconeogenesis genes.

This paper reports on a common experiment performed by 17 Research Units of the Italian Group of Microbiology of Vine and Wine (GMVV), which belongs to the Scientific Society SIMTREA, with the aim to validate a protocol for the characterization of wine strains of Saccharomyces cerevisiae. For this purpose, two commercial S. cerevisiae strains (EC 1118 and AWRI796) were used to carry out inter-laboratory- scale comparative fermentations using both synthetic medium and grape musts and applying the same protocol to obtain reproducible, replicable, and statistically valid results. Ethanol yield, production of acetic acid, glycerol, higher alcohols, and other volatile compounds were assessed. Moreover, the Fourier transform infrared spectroscopy was also applied to define the metabolomic fingerprint of yeast cells from each experimental trial. Data were standardized as unit of compounds or yield per gram of sugar (glucose and fructose) consumed throughout fermentation, and analyzed through parametric and non-parametric tests, and multivariate approaches (cluster Frontiers in Microbiology | www.frontiersin.org 1 February 2022 | Volume 13 | Article 830277 Romano et al. Wine S. cerevisiae Protocol Validation INTRODUCTION Saccharomyces cerevisiae is the predominant yeast species in winemaking. Due to its adaptability to the stressful conditions imposed by grape must fermentation, it easily competes with other yeasts and bacteria, and being the main actor of the transformation of grape must into wine, it is universally known as the “wine yeast.” In the last decades, a wide number of molecular and physiological studies demonstrated the high genotypic and phenotypic diversity of S. cerevisiae wine strains (Romano et al., 2008; Csoma et al., 2010; Mercado et al., 2011; Capece et al., 2013; Tristezza et al., 2013; Legras et al., 2018; Peter et al., 2018). This biodiversity is strictly associated with a significant high technological variability (Pretorius, 2000) and is of great importance for a successful strain selection and the development of new starters able to modulate the organoleptic quality of wine (Romano et al., 2003). Wild strains of S. cerevisiae are genetically and phenotypically distinguished from the selected commercial starter strains that are the result of selection programs (Peter et al., 2018; Pontes et al., 2020). In general, the commercial strains are characterized by high ethanol and low-pH tolerance, and they exhibit scarce production of aromatic compounds and low sporulation activity and biodiversity level (Duan et al., 2018; Kang et al., 2019). On the contrary, the wild strains, possessing high genotypic and phenotypic diversity, produce relatively high amounts of different secondary metabolites, thus offering considerable potential for utilization in industrial applications (Kang et al., 2019). Therefore, wild isolates from flowers and sugar-rich sources can lead to an aromatic profile characterized by specific volatile compounds capable of characterizing wine (Pontes et al., 2020; Alfonzo et al., 2021). As an example, wine fermentations using native wild strains obtained from oaks produce earthy and sulfurous organoleptic characteristics but intense of citrus and floral attributes (Hyma et al., 2011). Thus, although industrial yeast strains represent a fundamental tool for reproducing the final quality of table wines, their massive use is not recommended for traditional wines in which peculiar traits are desired (Spano et al., 2010; Capozzi and Spano, 2011). For these reasons, indigenous yeast starters, which are supposedly well adapted to a specific grape must and reflect the biodiversity of a particular “terroir” are more and more requested by winemakers (Bokulich et al., 2014; Gilbert et al., 2014; Feghali et al., 2020). Indeed, it is hypothesized that in different vitivinicultural regions, specific yeast strains are naturally selected and that they are able to exalt the sensorial and aromatic profile of wine produced in that area. In fact, Knight and Goddard (2015) showed that genetically differentiated population of S. cerevisiae in New Zealand had a different impact on wine quality due to the production of a different complex mix of chemicals. Setati et al. (2012), while studying the spatial distribution of fungal microbial communities within and between vineyards from the same “terroir” found higher yeast heterogeneity on grape samples collected at different points inside individual vineyards than between vineyards with very contrasting farming strategies. Thus, the myriad of microclimates occurring within each vineyard due to differential shading of grapes by leaves, and the aspect of each grape cluster, greatly affects the qualitative/quantitative composition of the vineyard-associated yeast microbiota. Bokulich et al. (2014) used a high-throughput short-amplicon sequencing approach to demonstrate that specific regional and grape-variety factors shape the biodiversity of fungal and bacterial consortia inhabiting wine-grape surfaces. Indeed, the microbial assemblages correlate with specific climatic features, and this suggests a link between vineyard environmental conditions and microbial residence patterns. Taken together, these findings reveal the importance of microbial populations for the regional identity of wine (Bokulich et al., 2016) and underline that the utilization of S. cerevisiae indigenous strain with selected traits is fundamental to modulate the final characteristics of the wine. The first step toward the attainment of indigenous S. cerevisiae wine starters is the clonal selection of the yeast strains associated with the wine-producing area of interest. Clonal selection is based on the evaluation of a number of phenotypic characteristics that are requested to guarantee the production of wines with peculiar sensorial properties. Traditionally, these are distinguished in technological and qualitative characteristics. Technological characteristics, such as fermentation power (ethanol production), analysis, two-way joining, and principal component analysis). The results of experiments carried out by using synthetic must showed that it was possible to gain comparable results from three different laboratories by using the same strains. Then, the use of the standardized protocol on different grape musts allowed pointing out the goodness and the reproducibility of the method; it showed the main traits of the two yeast strains and allowed reducing variability amongst independent batches (biological replicates) to acceptable levels. In conclusion, the findings of this collaborative study contributed to the validation of a protocol in a specific synthetic medium and in grape must and showed how data should be treated to gain reproducible and robust results, which could allow direct comparison of the experimental data obtained during the characterization of wine yeasts carried out by different research laboratories.

This paper reports on a common experiment performed by 17 Research Units of the Italian Group of Microbiology of Vine and Wine (GMVV), which belongs to the Scientific Society SIMTREA, with the aim to validate a protocol for the characterization of wine strains of

The olive oil microbiota mainly consists of yeasts, which may positively or negatively affect the physicochemical and sensory features of product. In this study, 17 yeast strains belonging to Candida boidinii, Lachancea fermentati, Nakazawaea molendinolei, N. wickerhamii and Schwanniomyces polymorphus species were collected during olive oil production, identified and tested for the ability to ferment sugars, to grow at low temperatures, for the occurrence of different enzymatic activities, for the tolerance and degradation of phenolic compounds, radical scavenging activities, biofilm formation, survival to simulated gastro-intestinal (GIT) tract. Yeasts were also inoculated in extra virgin olive oils (EVOO; from Leccino and Coratina cultivar) to evaluate their survival and their effect on EVOO quality (changes in analytical indices) during 6-months of storage. Most of strains were able to grow at 15°C, while the ability to ferment different sugars was strain-specific. All strains had β-glucosidase activity, while none exhibited lipolytic activity; peroxidase was widespread among the strains, while protease activity was strain-dependent. Esterase and the ability to hydrolyse oleuropein and form hydroxytyrosol was present only in N. wickerhamii strains. All strains were able to survive in olive mill wastewater, used as a model of phenolic compounds-rich matrix. A potential biofilm formation was observed only in N. wickerhamii, while the ability to scavenge radical and to cope with GIT-associated stresses were strain-dependent. High levels of survival were observed for almost strains (except S. polymorphus), in both Leccino and Coratina samples. Yeasts limited the acidity rise in olive oils, but overtime they contributed to increase the parameters related to oxidative phenomena (i.e. peroxides, K

The bulk of grape juice fermentation is carried out by the yeast Saccharomyces cerevisiae, but non-Saccharomyces yeasts can modulate many sensorial aspects of the final products in ways not well understood. In this study, some of such non-conventional yeasts were screened as mixed starter cultures in a fermentation defined medium in both simultaneous and sequential inoculations. One strain of Starmerella bacillaris and another of Zygosaccharomyces bailii were chosen by their distinct phenotypic footprint and their ability to reduce ethanol levels at the end of fermentation, particularly during simultaneous vinification. S. bacillaris losses viability strongly at the end of mixed fermentation, while Z. bailii remains viable until the end of vinification. Interestingly, for most non-Saccharomyces yeasts, simultaneous inoculation helps for survival at the end of fermentation compared to sequential inoculation. S. cerevisiae viability was unchanged by the presence of the either yeast. Characterization of both strains indicates that S. bacillaris behavior is overall more different from S. cerevisiae than Z. bailii. S. bacillaris has a less strict glucose repression mechanism and molecular markers like catabolite repression kinase Snf1 is quite different in size. Besides, S. cerevisiae transcriptome changes to a bigger degree in the presence of S. bacillaris than when inoculated with Z. bailii. S. bacillaris induces the translation machinery and repress vesicular transport. Both non-Saccharomyces yeast induce S. cerevisiae glycolytic genes, and that may be related to ethanol lowering, but there are specific aspects of carbon-related mechanisms between strains: Z. bailii presence increases the stress-related polysaccharides trehalose and glycogen while S. bacillaris induces gluconeogenesis genes.

Preservation technologies for winemaking have relied mainly on the addition of sulfur dioxide (SO2), in consequence of the large spectrum of action of this compound, linked to the control of undesirable microorganisms and the prevention of oxidative phenomena. However, its potential negative effects on consumer health have addressed the interest of the international research on alternative treatments to substitute or minimize the SO2 content in grape must and wine. This review is aimed at analyzing chemical methods, both traditional and innovative, useful for the microbiological stabilization of wine. After a preliminary description of the antimicrobial and technological properties of SO2, the additive traditionally used during wine production, the effects of the addition (in must and wine) of other compounds officially permitted in winemaking, such as sorbic acid, dimethyl dicarbonate (DMDC), lysozyme and chitosan, are discussed and evaluated. Furthermore, other substances showing antimicrobial properties, for which the use for wine microbiological stabilization is not yet permitted in EU, are investigated. Even if these treatments exhibit a good efficacy, a single compound able to completely replace SO2 is not currently available, but a combination of different procedures might be useful to reduce the sulfite content in wine. Among the strategies proposed, particular interest is directed towards the use of insect-based chitosan as a reliable alternative to SO2, mainly due to its low environmental impact. The production of wines containing low sulfite levels by using pro-environmental practices can meet both the consumers’ expectations, who are even more interested in the healthy traits of foods, and wine-producers’ needs, who are interested in the use of sustainable practices to promote the profile of their brand.

A current trend in winemaking has highlighted the beneficial contribution of non-Saccharomyces yeasts to wine quality. Hanseniaspora uvarum is one of the more represented non-Saccharomyces species onto grape berries and plays a critical role in influencing the wine sensory profile, in terms of complexity and organoleptic richness. In this work, we analyzed a group of H. uvarum indigenous wine strains as for genetic as for technological traits, such as resistance to SO2 and β-glucosidase activity. Three strains were selected for genome sequencing, assembly and comparative genomic analyses at species and genus level. Hanseniaspora genomes appeared compact and contained a moderate number of genes, while rarefaction analyses suggested an open accessory genome, reflecting a rather incomplete representation of the Hanseniaspora gene pool in the currently available genomes. The analyses of patterns of functional annotation in the three indigenous H. uvarum strains showed distinct enrichment for several PFAM protein domains. In particular, for certain traits, such as flocculation related protein domains, the genetic prediction correlated well with relative flocculation phenotypes at lab-scale. This feature, together with the enrichment for oligo-peptide transport and lipid and amino acid metabolism domains, reveals a promising potential of these indigenous strains to be applied in fermentation processes and modulation of wine flavor and aroma. This study also contributes to increasing the catalog of publicly available genomes from H. uvarum strains isolated from natural grape samples and provides a good roadmap for unraveling the biodiversity and the biotechnological potential of these non-Saccharomyces yeasts.

Nowadays, the interest toward products containing probiotics is growing due to their potential health benefits to the host and the research is focusing on search of new probiotic microorganisms. The present work was focused on the characterization of indigenous Saccharomyces cerevisiae strains, isolated from different food matrixes, with the goal to select strains with probiotic or health-beneficial potential. A preliminary screening performed on fifty S. cerevisiae indigenous strains, in comparison to a commercial probiotic strain, allowed to individuate the most suitable ones for potential probiotic aptitude. Fourteen selected strains were tested for survival ability in the gastrointestinal tract and finally, the strains characterized for the most important probiotic features were analyzed for health-beneficial traits, such as the content of glucan, antioxidant and potential anti-inflammatory activities. Three strains, 4LBI-3, LL-1, TA4-10, showing better attributes compared to the commercial probiotic S.cerevisiae var. boulardii strain, were characterized by interesting health-beneficial traits, such as high content of glucan, high antioxidant and potential anti-inflammatory activities. Our results suggest that some of the tested S. cerevisiae strains have potential as probiotics and candidate for different applications, such as dietary supplements, and starter for the production of functional foods or as probiotic to be used therapeutically.

In recent years, the awareness of consumers about the impact of food on health is constantly increasing. A high amount of dietary antioxidant intake can be supplied by beverages widely consumed, such as wine, coffee, beer. Recently, an increase in the consumer interest was observed for beer, in consequence of the high phenolic antioxidants and low ethanol content present in this beverage. Among all beer types, in recent years, consumption of craft beers has gained popularity. Being an unpasteurized and unfiltered, craft beer is potentially a new vehicle for delivering health effects. While health benefits of lactic acid bacteria as probiotics are well known, few data are available on probiotic yeasts in fermented food. Therefore, this study was aimed to analyse the effect of integrating the well-known probiotic yeast strain of S. cerevisiae var. boulardii (S.b) in mixed cultures with S. cerevisiae strains for production of beers with increased healthy benefits. The probiotic strain of S.b was tested in mixed cultures with selected S. cerevisiae strains, during wort fermentation. As the viability during processing operations is one of the criteria for selecting suitable strains of probiotic microorganisms, the survival of probiotic yeast during the fermentation and the presence of highly viable cells at the end of fermentations were evaluated. In almost all the mixed fermentations, at the end of the process the probiotic yeast was predominant on S. cerevisiae strain, and the experimental beers contained a high number of viable cells of S.b strain (ranging between 8 × 106 and 7.0 × 107/mL). The analysis of experimental beers for the content of main volatile compounds showed that the inclusion of S.b strain in mixed starter did not affect negatively beer aroma. Moreover, the inclusion of S.b strain in mixed starters determined an increase in the antioxidant activity and polyphenols content, in comparison to beers from single starter fermentations, indicating the influence of S.b strain on these parameters. Some mixed starter cultures tested in this study resulted a very promising tool to increase the healthy quality of the product, such as the improve the antioxidant activity and polyphenols content of beer.

During active dry yeast (ADY) production process, cells are exposed to multiple stresses, such as thermal, oxidative and hyperosmotic shock. Previously, by analysing cells in exponential growth phase, we selected an indigenous Saccharomyces cerevisiae wine strain, namely CD-6Sc, for its higher tolerance to desiccation and higher expression of specific desiccation stress-related genes in comparison to other yeast strains. In this study, we performed a desiccation treatment on stationary phase cells by comparing the efficacy of two different methods: a 'laboratory dry test' on a small scale (mild stress) and a treatment by spray-drying (severe stress), one of the most appropriate preservation method for yeasts and other micro-organisms. The expression of selected desiccation-related genes has been also assessed in order to validate predictive markers for desiccation tolerance. Our data demonstrate that the 'mild' and the 'severe' desiccation treatments give similar results in terms of cell recovery, but the choice of marker genes strictly depends on the growth phase in which cells undergo desiccation. The indigenous CD-6Sc was ultimately identified as a high dehydration stress-tolerant indigenous strain suitable for ADY production. This study highlights the exploitation of natural yeast biodiversity as a source of hidden technological features and as an alternative approach to strain improvement by genetic modifications. Copyright © 2017 John Wiley & Sons, Ltd.

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.

Yeast characteristics aimed to satisfy the new tendencies in wine sector will be explored. The use of indigenous starters, selected among yeast biota of specific environments, is proposed as a tool for production of premium quality wines possessing characteristics correlated to the specific production environment. Wine aromatic complexity can be improved also by the use of mixed starter cultures, composed by Saccharomyces cerevisiae and various non-Saccharomyces species, which can allow contemporary the control of undesirable microflora and/or reduction of ethanol content in wine. Moreover, the increasing request of organic wines determines changes in yeast microbiota and fermentation conditions, requiring starter strains with peculiar features, such as low production of H 2 S, SO 2 , acetaldehyde, and ability to reduce copper content in wine.

The aim of this work was to evaluate the possibility of using capillary electrophoresis for rapid and sensitive detection of microorganisms, such as yeasts, in order to propose it as an alternative to traditional microbiology methods both in manufacturing and research areas. After optimization of condition for the electrophoretic separation, three Saccharomyces cerevisiae strains, cultured in YPD medium, were analyzed by capillary electrophoresis at different growth times (3, 7, 24 and 48 h). The analysis was carried out on undamaged and non-pretreated cells. For all the strains, the electrophoretic profile was characterised by two distinct peaks, at each time of growth. Nevertheless some differences in the electropherograms of each strain were evident, probably in consequence of changing in the surface charge of yeasts at various stage of growth. The results demonstrated the high efficacy of capillary electrophoresis for intact cells separation of yeast and support the application of this technique for the evaluation of the viability of cultured cells.

Organoleptic characteristics of wine, aroma and flavour, are the most important characteristics that define the differences among the vast array of products throughout the world. Yeasts have a prominent role in determining the chemical composition of wine by several mechanisms: by producing enzymes that transform neutral grape compounds into flavour active compounds (pre-fermentative aroma), secondly by producing many hundreds of flavour active, secondary metabolites (fermentative and post-fermentative aroma) and lastly by extracting flavour components from grape solids and by autolytic degradation of dead yeast cells. These reactions vary with the yeast species and strains contributing to the fermentation. This review aims to present an overview on major achievements of yeast role in the formation of wine flavour. Firstly we illustrate the yeast metabolic activities involved in wine aroma production in function of wine styles as well as the main factors affecting flavour quality of wine. Furthermore, the influence of starter cultures (single or mixed) on wine flavour is discussed. Finally, novel methodologies to select wine yeasts in function of their influence on wine aroma are also summarized.

During active dry yeast (ADY) production process, cells are exposed to multiple stresses, such as thermal, oxidative and hyperosmotic shock. Previously, by analysing cells in exponential growth phase, we selected an indigenous Saccharomyces cerevisiae wine strain, namely CD-6Sc, for its higher tolerance to desiccation and higher expression of specific desiccation stress-related genes in comparison to other yeast strains. In this study, we performed a desiccation treatment on stationary phase cells by comparing the efficacy of two different methods: a 'laboratory dry test' on a small scale (mild stress) and a treatment by spray-drying (severe stress), one of the most appropriate preservation method for yeasts and other micro-organisms. The expression of selected desiccation-related genes has been also assessed in order to validate predictive markers for desiccation tolerance. Our data demonstrate that the 'mild' and the 'severe' desiccation treatments give similar results in terms of cell recovery, but the choice of marker genes strictly depends on the growth phase in which cells undergo desiccation. The indigenous CD-6Sc was ultimately identified as a high dehydration stress-tolerant indigenous strain suitable for ADY production. This study highlights the exploitation of natural yeast biodiversity as a source of hidden technological features and as an alternative approach to strain improvement by genetic modifications. Copyright © 2017 John WileySons, Ltd.

Wine production by inoculated fermentation with commercial Saccharomyces cerevisiae strains is an ordinary practice in modern winemaking in order to assure the final quality of wine, although this procedure results in the production of highly homogeneous wines. The use of indigenous selected starters represents a useful tool to control alcoholic grape must fermentation, safeguarding the typical sensory characteristics of wine produced from specific regions. In this study, we selected three indigenous S. cerevisiae strains among 16 indigenous strains previously isolated from the spontaneous fermentation of Primitivo grapes, which were collected from the vineyards of three different cellars. The three selected starters (one for each cellar) were tested during fermentations at pilot scale by performing in each cellar two trials: one with an indigenous starter (specific for the winery), and one with the commercial starter AWRI796 (common to all the cellars). Starter dominance ability and influence on aromatic quality of the wine were used as criteria to test the suitability of these indigenous starters to be used at the cellar scale. The results obtained in this study showed that the indigenous strains were characterized by very high dominance ability, and the aromatic quality of wine was strongly influenced both by the inoculated strain and the interaction strain/grape must.

The yeast microbiota present in wines produced by the ancient "Kakhetian" method in Georgia (EU) was studied. This technique involves the use of terracotta vessels (amphoras), during spontaneous fermentation, maceration phase and wine ageing. The analysed yeasts were collected from wines after maturation for one year in ten amphoras from a Georgian winery. The 260 isolates were all identified as Saccharomyces cerevisiae, and the majority were classified as flor yeasts by restriction analysis of ITS region. A first technological and molecular screening was used to select 70 strains for further characterization. Both genetic and metabolic characterization discriminated flor from non-flor strains. The combined results obtained by analysis of interdelta region and mtDNA-RFLP yielded 23 different biotypes; no biotype was common to flor and non-flor strains. The wines produced by flor yeasts showed a high content in acetaldehyde, acetic acid, acetoin, whereas the level of other compounds was similar to wines obtained by non-flor strains. This study represents the first report on the composition of yeast microbiota involved in the maturation of this traditional wine. These flor strains represent an interesting yeast population, in possession of peculiar characteristics allowing them to survive during wine ageing, becoming the dominant flora in the final wine.

In this study we analyzed Saccharomyces cerevisiae isolates obtained from spontaneous fermentations of grapes collected in one vineyard (same rows), during four harvest seasons (2006–2010). A total of 160 isolates were characterized by PCR amplification of inter-δ region. Six strains, representative of the different inter-δ profiles obtained, were tested in single and mixed fermentations at laboratory scale. In all the mixed fermentations, the strain possessing profile A (the only biotype found for two consecutive years) was tested in co-cultures with each of strains showing the other profiles. The strain “A” dominated in almost all the mixed fermentations. The experimental wines obtained from single and co-culture fermentations were analyzed for the content of main secondary compounds. The wines produced by mono-cultures were very different from the wines obtained by co-fermentations, except the wine by strain “A”, which grouped together with mixed-culture wines. Furthermore, the comparison between volatile components determined in mixed culture and blended wines, obtained by mixing monoculture wines, can suggest the existence of yeast metabolic interactions during mixed fermentations. The strain exhibiting the profile A resulted a good “competitor” not only among natural yeast population, but it was also capable to dominate mixed fermentations at lab scale.

In the last few decades spontaneous grape must fermentations have been replaced by inoculated fermentation with Saccharomyces cerevisiae strains as active dry yeast (ADY). Among the essential genes previously characterized to overcome the cell-drying/rehydration process, six belong to the group of very hydrophilic proteins known as hydrophilins. Among them, only SIP18 has shown early transcriptional response during dehydration stress. In fact, the overexpression in S. cerevisiae of gene SIP18 increases cell viability after the dehydration process. The purpose of this study was to characterize dehydration stress tolerance of three wild and one commercial S. cerevisiae strains of wine origin. The four strains were submitted to transformation by insertion of the gene SIP18. Selected transformants were submitted to the cell-drying–rehydration process and yeast viability was evaluated by both viable cell count and flow cytometry. The antioxidant capacity of SIP18p was illustrated by ROS accumulation reduction after H2O2 attack. Growth data as cellular duplication times and lag times were calculated to estimate cell vitality after the cell rehydration process. The overexpressing SIP18 strains showed significantly longer time of lag phase despite less time needed to stop the leakage of intracellular compounds during the rehydration process. Subsequently, the transformants were tested in inoculated grape must fermentation at laboratory scale in comparison to untransformed strains. Chemical analyses of the resultant wines indicated that no significant change for the content of secondary compounds was detected. The obtained data showed that the transformation enhances the viability of ADY without affecting fermentation efficiency and metabolic behaviour. Copyright © 2013 John Wiley & Sons, Ltd.

In this study, wild Saccharomyces cerevisiae strains, isolated from spontaneously fermenting grapes of different varieties and origins, were submitted to genetic analysis using different molecular techniques, such as amplification of genes coding for cell wall proteins and containing minisatellite-like sequences, karyotyping, mtDNA-RFLP, and analysis of the δ region. The lowest discriminative power was obtained by minisatellites analysis, in particular the amplification of AGA1 genes. Karyotyping and mtDNA-RFLP analysis yielded the same differentiation among the strains, whereas the PCR amplification of δ sequences resulted the best method as it was fast and it showed a very high discriminative power. In any case, it has to be underlined that some strains, showing the same delta profiles, exhibited a different mtDNA restriction profile and electrophoretic karyotype, suggesting that more than one molecular marker is required for reliable strain discrimination. Although the techniques used revealed a different resolution power, they all revealed a genetic relationship among strains isolated from spontaneous fermentation of grapes of different origins. In fact, none of the typing methods was able to discriminate some strains isolated from different areas.

Autochthonous strains of Saccharomyces cerevisiae from traditional starters used for the production of rice-based ethnic fermented beverage in North East India were examined for their genetic polymorphism using mitochondrial DNA-RFLP and electrophoretic karyotyping. Mitochondrial DNA-RFLP analysis of S. cerevisiae strains with similar technological origins from hamei starter of Manipur and marcha starter of Sikkim revealed widely separated clusters based on their geographical origin. Electrophoretic karyotyping showed high polymorphism amongst the hamei strains within similar mitochondrial DNA-RFLP cluster and one unique karyotype of marcha strain was widely distributed in the Sikkim-Himalayan region. We conceptualized the possibility of separate domestication events for hamei strains in Manipur (located in the Indo-Burma biodiversity hotspot) and marcha strains in Sikkim (located in Himalayan biodiversity hotspot), as a consequence of less homogeneity in the genomic structure between these two groups, their clear separation being based on geographical origin, but not on technological origin and low strain level diversity within each group. The molecular markers developed based on HinfI-mtDNA-RFLP profile and the chromosomal doublets in chromosome VIII position of Sikkim-Himalayan strains could be effectively used as geographical markers for authenticating the above starter strains and differentiating them from other commercial strains.

The aim of this research was the study of indigenous yeasts isolated from spontaneous fermentation of Inzolia grapes, one of the most widespread native white grapes in Sicily (Italy). The use of selective medium for the isolation and the screening for sulphur dioxide tolerance were useful for the first selection among 640 isolates. The yeasts characterized by high SO2 tolerance were identified at species level by restriction analysis of ITS region; although the majority of isolates were identified as S. cerevisiae, some non-Saccharomyces yeasts were found. Forty-seven selected yeasts, both S. cerevisiae and non-Saccharomyces yeasts, were characterized for genetic and technological diversity. The genetic polymorphism was evaluated by RAPD-PCR analysis, whereas the technological diversity was analyzed by determining the main secondary compounds in the experimental wines obtained by inoculating these yeasts. Both the molecular and metabolic profiles of selected yeasts were able to clearly discriminate S. cerevisiae from non-Saccharomyces yeasts. This research was useful for the constitution of a collection of selected indigenous yeast strains, including S. cerevisiae and non-Saccharomyces species possessing interesting enological traits. This collection represents a source of wild yeasts, among of which it is possible to select indigenous starters able to maintain the specific organoleptic characteristics of Inzolia wine.

The main purpose of this work was to test the effective dominance of the inoculated strain during the fermentation process. During this research activity, two Saccharomyces cerevisiae selected strains, isolated from Aglianico del Vulture grapes, were tested during inoculated fermentations at pilot scale in three wine cellars producing Aglianico del Vulture wine and characterized by different typologies. Yeast colonies sampled during the processes of fermentation were identified by restriction analysis of the internal transcribed spacer (ITS) region, and S. cerevisiae strains were differentiated by restriction fragment length polymorphism of mitochondrial DNA (RFLP-mtDNA). Analysis of the yeast population during the inoculated fermentations evidenced a significant presence of non-Saccharomyces, which varied with the cellar. The presence of non-Saccharomyces yeasts at different levels affected the aromatic composition of the experimental wines, as determined by gas-chromatographic analysis. The RFLP-mtDNA of S. cerevisiae isolates revealed the differing dominance of inoculated strains as a function of the wine cellar. In two cellars, all the isolates showed the same restriction profile, which was identical to that of the starter pattern. In contrast, in the third cellar, a significant percentage of S. cerevisiae isolates exhibited mtDNA-RFLP patterns different from the yeast starter profile, showing that, in this case, the starter exhibited low dominance in the fermentation. Our results demonstrate that, although the inoculated strains were found with high frequency, other yeasts (S. cerevisiae and non-Saccharomyces) developed, contributing to the fermentative process and to organoleptic quality of the final wine.

Olive mill wastewater (OMW) samples from two traditional varieties (Peranzana and Ogliarola Garganica) of Apulian region (southern Italy) and produced through continuous and traditional methods were microbiologically and chemically examined; thus, 104 yeasts were isolated and selected for further analyses. The strains were identified as Candida boidinii, Pichia holstii, Pichia membranifaciens, and Saccharomyces cerevisiae and analyzed to assess their suitability to metabolize phenols. Based on phenol metabolism, 27 strains were selected and inoculated into OMW aliquots to determine their ability to reduce phenols in vivo; then, five strains (identified with the codes 682-C. boidinii and 625, 642, 647, and 941-P. holstii) were used as a cocktail in wastewaters for a final validation step. In this last experiment, the effects of the temperature (10-30 degrees C) and (NH(4))(2)SO(4) (0.0-6.0 g l(-1)) were studied through a central composite design approach, and the results highlighted that the cocktail was able to reduce phenols by 40% at 10 degrees C with 6.0 g l(-1) of (NH(4))(2)SO(4) added.

article i nfo Combination of molecular genetic analysis (karyotyping, PCR-RFLP of MET2, the ITS1-ITS2 region and the NTS region) and physiological examination (melibiose and mannitol utilization, sugar-, ethanol- and copper tolerance, killer activity, fermentation vigor and production of metabolites) of yeasts isolated from spontaneously fermenting wines in four wine regions revealed very high diversity in the Saccharomyces cerevisiae populations. Practically each S. cerevisiae isolate showed a unique pattern of properties. Although the strains originating from the same wine were quite similar in certain traits, they showed diversity in other properties. These results indicate that alcoholic fermentation in grape wines is performed by highly diverse yeast consortia rather than by one or two dominating strains. The less frequent Saccharomyces uvarum strains were less diverse, showed lower karyotype variability, were Mel + , Man + , more sensitive to 60% sugar, and ethanol or copper in the medium. They produced less acetic acid and fermented better at 14 °C than most of the S. cerevisiae isolates, but certain S. cerevisiae strains showed comparably high fermentation rates at this temperature, indicating that it is not a general rule that S. uvarum ferments better than S. cerevisiae at low temperatures. The segregation of certain traits (melibiose utilization, mannitol utilization and copper resistance) in both species indicates that the genomes can easily change during vegetative propagation. The higher diversity among the S. cerevisiae isolates suggests that the S. cerevisiae genome may be more flexible than the S. uvarum genome and may allow more efficient adaptation to the continuously changing environment in the fermenting wine. © 2010 Published by Elsevier B.V.

The composition of yeast microflora in artisanal "Pecorino di Filiano" cheese, a typical product of the Basilicata region of Southern Italy, was studied during ripening. The isolates were identified by restriction analysis of the 18S rDNA amplified region with the combined use of Hinf I and Cfo I enzymes. The majority of the isolates were identified as Debaryomyces hansenii, whereas two yeasts were identified as Kluyveromyces lactis and one as Dekkera anomala. To evaluate natural biodiversity, D. hansenii "Pecorino di Filiano" isolates were submitted to genetic and technological characterization. RAPD-PCR analysis with P80 (5CGCGTGCCCA3) primer revealed significant polymorphism among D. hansenii isolates. About 30% of the isolates showed single molecular profiles, whereas the other D. hansenii yeasts were separated into three main patterns, differing for both the ripening time and the isolation source. Furthermore, the yeasts showed significant variability in their, "proteolytic activity". This work demonstrated the high predominance of D. hansenii among the yeast population of "Pecorino di Filiano" cheese, probably in consequence of the traditional salting process, which was selected for this salt tolerant species. This preliminary study allowed us to isolate autochthonous D. hansenii yeasts potentially useful as starters for the production of this artisanal cheese.

The principal agent in winemaking is the yeast Saccharomyces cerevisiae, which is characterized by a significant strain biodiversity. Here we report the characterization of 80 wild S. cerevisiae strains, isolated from grapes of different varieties in southern Italy, for genetic and technological variability. By PCR amplification with M13 primer a significant polymorphism was recorded and 12 different biotypes were identified among the strains. The specific strain-pattern could be used to follow the dynamics of different biotypes during the fermentation process. The analysis of experimental wines obtained by inoculated fermentations with the 80 strains showed significant differences among the wines. The level of each compound was a function of the strain performing the fermentative process. The main variables for the strain differentiation were the production of acetaldehyde and acetic acid, which ranged from 53 to 282 mg/l and from 0.20 to 1.88 g/l, respectively. Selected strains were tested in fermentation with two different grape musts, yielding experimental wines differing in the levels of secondary compounds and polyphenol content, in function of the interaction “grape must composition/yeast strain”. This finding has an applicative value for the potentiality of utilizing the resource of strain variability as a tool to individuate suitable starter cultures, which are able to complement and optimize grape quality.

The most diffused formulation of starter for winemaking is active dry yeast (ADY). ADYs production process is essentially characterized by air-drying stress, a combination of several stresses, including thermal, hyperosmotic and oxidative and cell capacity to counteract such multiple stresses will determine its survival. The molecular mechanisms underlying cell stress response to desiccation have been mostly studied in laboratory and commercial yeast strains, but a growing interest is currently developing for indigenous yeast strains which represent a valuable and alternative source of genetic and molecular biodiversity to be exploited. In this work, a comparative study of different Saccharomyces cerevisiae indigenous wine strains, previously selected for their technological traits, has been carried out to identify potentially relevant genes involved in desiccation stress tolerance. Cell viability was evaluated along desiccation treatment and gene expression was analyzed by real-time PCR before and during the stress. Our data show that the observed differences in individual strain sensitivity to desiccation stress could be associated to specific gene expression over time. In particular, either the basal or the stress-induced mRNA levels of certain genes, such as HSP12, SSA3, TPS1, TPS2, CTT1 and SOD1 , result tightly correlated to the strain survival advantage. This study provides a reliable and sensitive method to predict desiccation stress tolerance of indigenous wine yeast strains which could be preliminary to biotechnological applications.

Aims: The characterization by molecular and physiological methods of wild apiculate strains, isolated from ‘Aglianico del Vulture’ grape must. Methods and Results: The restriction analysis of 18S rDNA allowed the identification of strains at the species level, which were predominantly Hanseniaspora uvarum. The RAPD analysis and the evaluation of technological traits, such as the metabolic and enzymatic activities, were useful to evaluate the polymorphism of this species. Conclusions: The RAPD analysis clustered the wild H. uvarum strains in four main genetic groups and a very high phenotypic variability confirmed this genetic polymorphism. The technological variables, which determined the strain biodiversity differed significantly, demonstrating that these technological traits are strain dependent. A certain correlation was found between the strain behaviour and its isolation zone, indicating the influence of the environment on the genetic patrimony of the population. Significance and Impact of the Study: The genetic and technological biodiversity recorded among H. uvarum wild strains represents the basis for organizing a collection of apiculate strains exhibiting oenological characteristics at different levels, such as high/low production of secondary compounds, and, therefore, potentially useful for a selection programme.

In grapevine (Vitis vinifera L.,), cultivar identification problems have frequently been solved using ampelographic and chemical analysis. However, these methods result in several ambiguities, particularly when different clones of the same cultivar have to be identified. The availability of reliable and reproducible tools to identify genetic differences at clonal level would facilitate the classification of clones and cultivars. At the same time, molecular tools are also well developed in order to classify the autochthonous yeast strains (Saccharomyces cerevisiae) isolated in the area of Aglianico del Vulture cultivation. In this work, six vineyards of the ancient cultivar Aglianico del Vulture and 60 Saccharomyces sensu stricto strains were characterised. Molecular tools such as RAPD-polymerase chain reaction analysis, microsatellites, amplified ribosomal DNA restriction analysis and AFLP analysis were applied in order to study the genetic variability among the vineyards of Aglianico del Vulture...

The diversity and the composition of the yeast micropopulation significantly contribute to the sensory characteristics of wine. The growth of each wine yeast species is characterized by a specific metabolic activity, which determines concentrations of flavour compounds in the final wine. However, it must be underlined that, within each species, significant strain variability has been recorded. The wide use of starter cultures, mainly applied to reduce the risk of spoilage and unpredictable changes of wine flavour, can ensure a balanced wine flavour, but it may also cause a loss of characteristic aroma and flavour determinants. Thus, the beneficial contribution from the yeast increases when starter cultures for winemaking are selected on the basis of scientifically verified characteristics and are able to complement and optimise grape quality and individual characteristics. Here we report the characterization of a large number of strains of different wine yeast species, isolated from spontaneous wine fermentations and included in the culture collection of the Basilicata University.

A total of 32 yeast strains belonging to four non-Saccharomyces species associated with winemaking was characterized by different molecular techniques. The PCR amplification of 18S rRNA-coding DNA and nontranscribed spacer, followed by restriction analysis with the endonucleases HaeIII and MspI, and PCR fingerprinting with microsatellite primers (GAC)5 and (GTG)5 were used. The methods used provided species-specific profiles and proved to be fast and reliable for monitoring the evolution of the four non-Saccharomyces yeast populations throughout wine fermentation. D 2002 Elsevier Science B.V. All rights reserved.

One hundred and fifteen Saccharomyces cerevisiae strains from Aglianico del Vulture, a red wine produced in Southern Italy, were characterized for the production of some secondary compounds involved in the aroma and taste of alcoholic beverages. The strains exhibited a uniform behaviour in the production levels of n-propanol, active amyl alcohol and ethyl acetate, whereas isobutanol, isoamyl alcohol and acetaldehyde were formed with a wide variability. Only five strains produced wines close to the reference Aglianico del Vulture wine for the traits considered. Of these, two strains were selected, underwent to tetrad analysis and the single spore cultures were tested in grape must fermentation. The progeny of one strain showed a significant metabolic variability, confirming the necessity to test starter cultures for the segregation of traits of technological interest. Our findings suggest the selection of specific strains for specific fermentations as a function of the vine variety characteristics in order to take the major advantage from the combination grape must/S. cerevisiae strain.

Aims: Kloeckera apiculata and Saccharomyces cerevisiae yeast species are dominant, respectively, at the early and at the following stages of wine fermentation. In the present study, PCR fingerprinting and NTS region amplification and restriction were applied as techniques for monitoring yeast population performing Aglianico of Vulture grape must fermentation. Methods and Results: Thirty S. cerevisiae and 30 K. apiculata strains were typed by PCR fingerprinting with (GAC)5 and (GTG)5 primers and by complete NTS region amplification followed by restriction with HaeIII and MspI enzymes. S. cerevisiae strains generated two patterns with (GAC)5 primer, while (GTG)5 primer yielded a higher genetic polymorphism. Conversely, in K. apiculata Aglianico wine strains (GAC)5 and (GTG)5 primers generated the same profile for all strains. Restriction analysis of the amplified NTS region gave the same profile for all strains within the same species, except for one strain of S. cerevisiae. Conclusions: The PCR fingerprinting technique was useful in discriminating at strain level S. cerevisiae, particularly with the primer (GTG)5. RFLP patterns generated from the NTS region of the two species can be more easily compared than the patterns resulting from PCR fingerprinting, thus RFLP is more suitable for the rapid monitoring of the species involved in different stages of fermentation. Significance and Impact of the Study: The molecular techniques used allow discrimination of S. cerevisiae at strain level and monitoring of the ratio of S. cerevisiae/K. apiculata during the fermentation process. Thus, their application can assure technological adjustments in a suitable time.

Two wine strains of Saccharomyces cerevisiae, characterized by a different degree of copper resistance, were tested in grape must fermentation in the presence of different copper concentrations. The sensitive strain SN9 was strongly affected by copper concentration (32 ppm, (32 mg/l)), whereas the resistant strain SN41 exhibited a good growth activity in presence of 32 ppm of copper and only a reduced activity in presence of 320 ppm. The different strain fermentation performance in response to the copper addition corresponded to a different capability to accumulate copper inside the cells. Both strains exhibited the capacity to reduce the copper content in the final product, eventhough a significantly greater reducing activity was exerted by the resistant strain SN41, which was able to reduce by 90% the copper concentration in the final product and to accumulate the metal in great concentrations in the cells. As high concentrations of copper can be responsible for wine alterations, the selection of S. cerevisiae strains possessing high copper resistance and the ability to reduce the copper content of wine has a great technological interest, in particular for the fermentation of biological products. From the results obtained, the technique proposed is not only suitable for the assay of copper residues in must, wine and yeast cells, but it also offers the advantage of easy sample preparation and low detection limit in the ppb (μg/l) range.

The most diffused starter formulation in winemaking is actually represented by active dry yeast (ADY). Spray-drying has been reported as an appropriate preservation method for yeast and other micro-organisms. Despite the numerous advantages of this method, the high air temperatures used can negatively affect cell viability and the fermentative performance of dried cells. In the present study, 11 wine S. cerevisiae strains (both indigenous and commercial) were submitted to spray-drying; different process conditions were tested in order to select the conditions allowing the highest strain survival. The strains exhibited high variability for tolerance to spray-drying treatment. Selected strains were tested in fermentation at laboratory scale in different formulations (free fresh cells, free dried cells, immobilized fresh cells and immobilized dried cells), in order to assess the influence of starter formulation on fermentative fitness of strains and aromatic quality of wine. The analysis of volatile fraction in the experimental wines produced by selected strains in different formulations allowed identification of > 50 aromatic compounds (alcohols, esters, ketones, aldehydes and terpenes). The results obtained showed that the starter formulation significantly influenced the content of volatile compounds. In particular, the wines obtained by strains in dried forms (as both free and immobilized cells) contained higher numbers of volatile compounds than wines obtained from fresh cells.

The most diffused starter formulation in winemaking is actually represented by active dry yeast (ADY). Spray-drying has been reported as an appropriate preservation method for yeast and other micro-organisms. Despite the numerous advantages of this method, the high air temperatures used can negatively affect cell viability and the fermentative performance of dried cells. In the present study, 11 wine S. cerevisiae strains (both indigenous and commercial) were submitted to spray-drying; different process conditions were tested in order to select the conditions allowing the highest strain survival. The strains exhibited high variability for tolerance to spray-drying treatment. Selected strains were tested in fermentation at laboratory scale in different formulations (free fresh cells, free dried cells, immobilized fresh cells and immobilized dried cells), in order to assess the influence of starter formulation on fermentative fitness of strains and aromatic quality of wine. The analysis of volatile fraction in the experimental wines produced by selected strains in different formulations allowed identification of 50 aromatic compounds (alcohols, esters, ketones, aldehydes and terpenes). The results obtained showed that the starter formulation significantly influenced the content of volatile compounds. In particular, the wines obtained by strains in dried forms (as both free and immobilized cells) contained higher numbers of volatile compounds than wines obtained from fresh cells.

In the last few decades spontaneous grape must fermentations have been replaced by inoculated fermentation with Saccharomyces cerevisiae strains as active dry yeast (ADY). Among the essential genes previously characterized to overcome the cell-drying/rehydration process, six belong to the group of very hydrophilic proteins known as hydrophilins. Among them, only SIP18 has shown early transcriptional response during dehydration stress. In fact, the overexpression in S. cerevisiae of gene SIP18 increases cell viability after the dehydration process. The purpose of this study was to characterize dehydration stress tolerance of three wild and one commercial S. cerevisiae strains of wine origin. The four strains were submitted to transformation by insertion of the gene SIP18. Selected transformants were submitted to the cell-drying-rehydration process and yeast viability was evaluated by both viable cell count and flow cytometry. The antioxidant capacity of SIP18p was illustrated by ROS accumulation reduction after H2 O2 attack. Growth data as cellular duplication times and lag times were calculated to estimate cell vitality after the cell rehydration process. The overexpressing SIP18 strains showed significantly longer time of lag phase despite less time needed to stop the leakage of intracellular compounds during the rehydration process. Subsequently, the transformants were tested in inoculated grape must fermentation at laboratory scale in comparison to untransformed strains. Chemical analyses of the resultant wines indicated that no significant change for the content of secondary compounds was detected. The obtained data showed that the transformation enhances the viability of ADY without affecting fermentation efficiency and metabolic behaviour.

Bioethanol can be produced from several biomasses including lignocellulosic materials. Besides 6-carbon sugars that represent the prevalent carbohydrates, some of these feedstocks contain significant amounts of 5-carbon sugars. One common limit of the major part of the xylose-fermenting yeasts is the diauxic shift between the uptake of glucose and xylose during the fermentation of mixed syrups. Thus, optimized fermentation strategies are required.In this paper the ability of Scheffersomyces stipitis strain NRRLY-11544 to ferment mixed syrups with a total sugar concentration in the range 40-80. g/L was investigated by using mono cultures, co-cultures with Saccharomyces cerevisiae strain Bakers Yeast Type II and single cultures immobilized in silica-hydrogel films. The experimental design for the fermentations with immobilized cells included the process analysis in function of two parameters: the fraction of the gel in the broth and the concentration of the cells loaded in the gel. Furthermore, for each total sugars level, the fermentative course of S. stipitis was analyzed at several glucose-to xylose ratios.The results indicated that the use of S. stipitis and S. cerevisiae in free co-cultures ensured faster processes than single cultures of S. stipitis either free or immobilized. However, the rapid production of ethanol by S. cerevisiae inhibited S. stipitis and caused a stuck of the process.Immobilization of S. stipitis in silica-hydrogel increased the relative consumption rate of xylose-to-glucose by 2-6 times depending on the composition of the fermentation medium. Furthermore the films performances appeared stable over three weeks of continuous operations. However, on the whole, the final process yields obtained with the immobilized cells were not meaningfully different from that of the free cells. This was probably due to concurrent fermentations operated by the cells released in the broth. Optimization of the carrier characteristics could improve the performances of the process with immobilized cells. © 2013 Elsevier B.V.

The main aim of this work was to analyse the diversity of wild Saccharomyces cerevisiae isolated from spontaneous fermentations of grapes collected from heroic vine-growing area. A first screening based on several technological traits was used to select 39 strains among 132 isolates. By using three molecular typing techniques (evaluation of cell wall gene polymorphisms, mtDNA restriction analysis, inter-delta amplification analysis) a significant genetic variability was found. The analysis of principal aromatic compounds produced during inoculated fermentation of two grape musts demonstrated the strain impact on wine flavour and a significant influence of grape must on strain metabolic behavior. One selected strain was used in fermentation at cellar level and the analysis of inter-delta region on yeast colonies isolated during the process revealed the high-implantation ability of this strain. The obtained results demonstrate the usefulness of different molecular and technological markers for the evaluation of natural biodiversity among S. cerevisiae strains. This study represents an essential step towards the exploitation and the preservation of biodiversity of strains isolated from heroic vine-growing area. Selected S. cerevisiae strains could represent starter cultures available for winemakers addressed to production of quality premium wines maintaining differential properties of their own area.

Wine is the result of the performance of different yeast strains throughout the fermentation in both spontaneous and inoculated processes. 22 Saccharomyces cerevisiae strains were characterized by microsatellite fingerprinting, selecting 6 of them to formulate S. cerevisiae mixed cultures. The aim of this study was to ascertain a potential benefit to use mixed cultures to improve wine quality. For this purpose yeasts behavior was studied during co-inoculated fermentations. Aromatic composition of the wines obtained was analyzed, and despite the fact that only one strain dominated at the end of the process, co-cultures released different concentrations of major volatile compounds than single strains, especially higher alcohols and acetaldehydes. Nevertheless, no significant differences were found in the type and quantity of the amino acids assimilated. This study demonstrates that the final wine composition may be modulated and enhanced by using suitable combinations of yeast strains.