A multivariate approach to explore the volatolomic and sensory profiles of craft Italian Grape Ale beers produced with novel Saccharomyces cerevisiae strains.

This study investigated the influence of three Saccharomyces cerevisiae strains, selected from different matrices - CHE-3 (cherry), P4 (sourdough) and TA4-10 (grape must) - on characteristics of Italian Grape Ale (IGA) beers obtained at microbrewery scale. A multidisciplinary approach, combining results from analysis of chemical, volatile and organoleptic profiles of the beers, was adopted to underline the relationships between yeast starter and the quality of final products. Detection volatile organic compounds (VOCs) by Gas-Chromatography coupled with Mass Spectrometry (GC-MS) after extraction carried out by head-space micro-extraction (HS-SPME) revealed that the beer obtained by P4 strain differed from the others for its higher concentrations of esters, alcohols, and terpenes as confirmed by PCA (principal component analysis) and Cluster heatmap. Furthermore, sensorial analysis and consumer test showed that this sample differed from others by more pronounced notes of "fruity smell and floral" and "olfactory finesse," and it was the most appreciated beer for smell, taste, and overall quality. Conversely, CHE-3 was the sample with the lowest concentrations of the identified volatiles and, together TA4-10, showed the highest scores for smoked, yeast, malt, and hop notes. As far as we know, these are the first results on the application of indigenous S. cerevisiae strains in the production of craft IGA beers analyzed through a complex multivariate approach.

Optimizing growth and biomass production of non-Saccharomyces wine yeast starters by overcoming sucrose consumption deficiency.

The use of non-Saccharomyces yeasts as starters in winemaking has increased exponentially in the last years. For instance, non-conventional yeasts have proven useful for the improvement of the organoleptic profile and biocontrol. Active dry yeast starter production has been optimized for Saccharomyces cerevisiae, which may entail problems for the propagation of non-Saccharomyces yeasts. This work shows that the poor growth of Hanseniaspora vineae and Metschnikowia pulcherrima in molasses is related to a deficient sucrose consumption, linked to their low invertase activity. In order to address this issue, simple modifications to the cultivation media based hydrolysis and the reduction of sucrose concentration were performed. We performed biomass propagation simulations at a bench-top and bioreactor scale. The results show that cultivation in a hexose-based media improved biomass production in both species, as it solves their low invertase activity. The reduction in sugar concentration promoted a metabolic shift to a respiratory metabolism, which allowed a higher biomass yield, but did not improve total biomass production, due to the lower sugar availability. To evaluate the technological performance of these adaptations, we performed mixed grape juice fermentations with biomass produced in such conditions of M. pulcherrima and S. cerevisiae. The analysis of wines produced revealed that the different treatments we have tested did not have any negative impact on wine quality, further proving their applicability at an industrial level for the improvement of biomass production.

Screening of Saccharomyces and Non-Saccharomyces Wine Yeasts for Their Decarboxylase Activity of Amino Acids.

The type and quantity of precursor amino acids present in grape must that are used by wine yeasts affect the organoleptic and health properties of wine. The aim of this work was to conduct a preliminary screening among Saccharomyces and non-Saccharomyces indigenous strains, which were previously isolated from different Italian regional grape varieties. This was performed in order to evaluate their decarboxylase activity on certain important amino acids-such as arginine, proline, serine, and tyrosine-that are present in grape must. In particular, a qualitative test on 122 wine yeasts was performed on a decarboxylase medium using arginine, proline, serine, and tyrosine as precursor amino acids. Our results showed a considerable variability among the microbial species tested for this parameter. Indeed, Saccharomyces cerevisiae strains exhibited a high decarboxylase capability of the four amino acids tested; moreover, only 10% of the total (i.e., a total of 81) did not show this trait. A high recovery of decarboxylation ability for at least one amino acid was also found for Zygosaccharomyces bailii and Hanseniaspora spp. These findings can, therefore, promote the inclusion of decarboxylase activity as an additional characteristic in a wine yeast selection program in order to choose starter cultures that possess desirable technological traits; moreover, this also can contribute to the safeguarding of consumer health.

Impact of Starmerella bacillaris and Zygosaccharomyces bailii on ethanol reduction and Saccharomyces cerevisiae metabolism during mixed wine fermentations.

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.

In Vitro Study of Probiotic, Antioxidant and Anti-Inflammatory Activities among Indigenous Saccharomyces cerevisiae Strains.

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.

Validation of a Standard Protocol to Assess the Fermentative and Chemical Properties of Saccharomyces cerevisiae Wine Strains.

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 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.

Corrigendum: Genome Sequencing and Comparative Analysis of Three Hanseniaspora uvarum Indigenous Wine Strains Reveal Remarkable Biotechnological Potential.

[This corrects the article DOI: 10.3389/fmicb.2019.03133.].

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.

Biotechnological Approach Based on Selected Saccharomyces cerevisiae Starters for Reducing the Use of Sulfur Dioxide in Wine.

Sulfites are considered the main additives in winemaking for their antimicrobial, antioxidant and anti-oxidasic activities. The current concern about the potential negative effects of sulfur dioxide (SO2) on consumer health has focused the interest on replacing or reducing SO2 use. Our work aims to develop a strategy based on the use of selected starter culture, able to perform wine fermentation without SO2 addition. Four selected Saccharomyces cerevisiae indigenous strains were tested as mixed starter cultures in laboratory scale fermentations. The starter culture, characterized by a similar percentage of dominance of both strains composing the mixed starter and able to produce a wine characterized by the best combination of chemical and aromatic characteristics, was chosen. This mixed culture was tested as a starter at pilot scale with and without SO2 addition, by using a higher inoculum level in the vinification without SO2. The selected starter confirmed higher dominance ability in vinification without SO2 addition than in SO2-added fermentation, demonstrating that sulfite addition is not a guarantee to reach an absolute dominance of starter culture on indigenous microflora. The proposed biotechnological tool allowed to produce good quality wines possessing also "functional properties", as NO-SO2 added wines were characterized by high polyphenol content and antioxidant activity.

Genome Sequencing and Comparative Analysis of Three Hanseniaspora uvarum Indigenous Wine Strains Reveal Remarkable Biotechnological Potential.

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 ß-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.

Use of Saccharomyces cerevisiae var. boulardii in co-fermentations with S. cerevisiae for the production of craft beers with potential healthy value-added.

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.

Impact of yeast starter formulations on the production of volatile compounds during wine fermentation.

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.

Indigenous yeast population from Georgian aged wines produced by traditional "Kakhetian" method.

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.

Genetic improvement of Saccharomyces cerevisiae wine strains for enhancing cell viability after desiccation stress.

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.

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.