Yeast communities related to honeybees: occurrence and distribution in flowers, gut mycobiota, and bee products.

Honeybee (Apis mellifera) is an important agricultural pollinator and a model for sociality. In this study, a deep knowledge on yeast community characterizing the honeybees' environmental was carried out. For this, a total of 93 samples were collected: flowers as food sources, bee gut mycobiota, and bee products (bee pollen, bee bread, propolis), and processed using culture-dependent techniques and a molecular approach for identification. The occurrence of yeast populations was quantitatively similar among flowers, bee gut mycobiota, and bee products. Overall, 27 genera and 51 species were identified. Basidiomycetes genera were predominant in the flowers while the yeast genera detected in all environments were Aureobasidium, Filobasidium, Meyerozyma, and Metschnikowia. Fermenting species belonging to the genera Debaryomyces, Saccharomyces, Starmerella, Pichia, and Lachancea occurred mainly in the gut, while most of the identified species of bee products were not found in the gut mycobiota. Five yeast species, Meyerozyma guilliermondii, Debaryomyces hansenii, Hanseniaspora uvarum, Hanseniaspora guilliermondii, and Starmerella roseus, were present in both summer and winter, thus indicating them as stable components of bee mycobiota. These findings can help understand the yeast community as a component of the bee gut microbiota and its relationship with related environments, since mycobiota characterization was still less unexplored. In addition, the gut microbiota, affecting the nutrition, endocrine signaling, immune function, and pathogen resistance of honeybees, represents a useful tool for its health evaluation and could be a possible source of functional yeasts. KEY POINTS: • The stable yeast populations are represented by M. guilliermondii, D. hansenii, H. uvarum, H. guilliermondii, and S. roseus. • A. pullulans was the most abondance yeast detective in the flowers and honeybee guts. • Aureobasidium, Meyerozyma, Pichia, and Hanseniaspora are the main genera resident in gut tract.

Agro-ecosystem of honeybees as source for native probiotic yeasts.

Probiotic microorganisms are used to improve the health and wellness of people and the research on this topic is of current relevance and interest. Fifty-five yeasts, coming from honeybee's ecosystem and belonging to Candida, Debaryomyces, Hanseniaspora, Lachancea, Metschnikowia, Meyerozyma, Starmerella and Zygosacchromyces genera and related different species, were evaluated for the probiotic traits. The resistance to gastrointestinal conditions, auto-aggregation, cell surface hydrophobicity or biofilm formation abilities as well as antimicrobial activity against common human pathogenic bacteria were evaluated. The safety analysis of strains was also carried out to exclude any possible negative effect on the consumer's health. The influence of proteinase treatment of living yeasts and their adhesion to Caco-2 cells were also evaluated. The greatest selection occurred in the first step of survival at the acidic pH and in the presence of bile salts, where more than 50% of the strains were unable to survive. Equally discriminating was the protease test which allowed the survival of only 27 strains belonging to the species Hanseniaspora guilliermondii, Hanseniaspora uvarum, Metschnikowia pulcherrima, Metschnikowia ziziphicola, Meyerozyma caribbica, Meyerozyma guilliermondii, Pichia kluyveri, Pichia kudriavzevii and Pichia terricola. An integrated analysis of the results obtained allowed the detection of seven yeast strains with probiotic aptitudes, all belonging to the Meyerozyma genus, of which three belonging to M. guillermondii and four belonging to M. caribbica species.

Comparative Zymocidial Effect of Three Different Killer Toxins against Brettanomyces bruxellensis Spoilage Yeasts.

Three killer toxins that were previously investigated, one excreted by Kluyveromyces wickerhamii and two by different strains of Wickerhamomyces anomalus, were produced at the pilot scale, lyophilized and characterized, and the formulates were assessed for their zymocidial effect against Brettanomyces bruxellensis spoilage yeast. A comparative analysis allowed the evaluation of the minimum inhibitory concentration (MIC) against a sensitive strain. Fungicidal and fungistatic concentrations were used to evaluate the cytocidal effect using a cytofluorimetric approach that confirmed the lethal effect of all lyophilized formulates against B. bruxellensis spoilage yeasts. Moreover, the potential killer toxins' cytotoxicity against human intestinal cells (Caco-2) were evaluated to exclude any possible negative effect on the consumers. Finally, the effective lethal effect of all three lyophilized killer toxins toward B. bruxellensis sensitive strain were tested. The results indicated that all of them acted without dangerous effects on the human epithelial cells, opening the way for their possible commercial application. In particular, D15 showed the lowest MIC and the highest activity, was evaluated also in wine, revealing a strong reduction of Brettamonyces yeast growth and, at the same time, a control of ethyl phenols production.

Yeast Interactions and Molecular Mechanisms in Wine Fermentation: A Comprehensive Review.

Wine can be defined as a complex microbial ecosystem, where different microorganisms interact in the function of different biotic and abiotic factors. During natural fermentation, the effect of unpredictable interactions between microorganisms and environmental factors leads to the establishment of a complex and stable microbiota that will define the kinetics of the process and the final product. Controlled multistarter fermentation represents a microbial approach to achieve the dual purpose of having a less risky process and a distinctive final product. Indeed, the interactions evolved between microbial consortium members strongly modulate the final sensorial properties of the wine. Therefore, in well-managed mixed fermentations, the knowledge of molecular mechanisms on the basis of yeast interactions, in a well-defined ecological niche, becomes fundamental to control the winemaking process, representing a tool to achieve such objectives. In the present work, the recent development on the molecular and metabolic interactions between non-Saccharomyces and Saccharomyces yeasts in wine fermentation was reviewed. A particular focus will be reserved on molecular studies regarding the role of nutrients, the production of the main byproducts and volatile compounds, ethanol reduction, and antagonistic actions for biological control in mixed fermentations.

Yeast killer toxins: from ecological significance to application.

Killer toxins are proteins that are often glycosylated and bind to specific receptors on the surface of their target microorganism, which is then killed through a target-specific mode of action. The killer phenotype is widespread among yeast and about 100 yeast killer species have been described to date. The spectrum of action of the killer toxins they produce targets spoilage and pathogenic microorganisms. Thus, they have potential as natural antimicrobials in food and for biological control of plant pathogens, as well as therapeutic agents against animal and human infections. In spite of this wide range of possible applications, their exploitation on the industrial level is still in its infancy. Here, we initially briefly report on the biodiversity of killer toxins and the ecological significance of their production. Their actual and possible applications in the agro-food industry are discussed, together with recent advances in their heterologous production and the manipulation for development of peptide-based therapeutic agents.

Volatile profile of reduced alcohol wines fermented with selected non-Saccharomyces yeasts under different aeration conditions.

Over the last decades there has been an increase in ethanol concentration in wine. High ethanol concentration may impact negatively wine flavor and can be associated with harmful effects on human health. In this study, we investigated a microbiological approach to reduce wine ethanol concentration, using three non-Saccharomyces yeast strains (Metschnikowia pulcherrima, Torulaspora delbrueckii and Zygosaccharomyces bailii) in sequential fermentations with S. cerevisiae under different aeration conditions. At the same time, we evaluated the volatile profile of the resulting reduced alcohol Chardonnay wines. Results showed that the non-Saccharomyces yeasts tested were able to reduce wine ethanol concentration when oxygen was provided. Compared to S. cerevisiae wines, ethanol reduction was 1.6% v/v, 0.9% v/v and 1.0% v/v for M. pulcherrima, T. delbrueckii and Z. bailii sequential fermentations, respectively. Under the conditions evaluated here, aeration did not affect acetic acid production for any of the non-Saccharomyces strains tested. Although aeration affected wine volatile profiles, this was depended on yeast strain. Thus, wines produced with M. pulcherrima under aeration of 0.05 volume of air per volume of culture per minute (VVM) showed excessive ethyl acetate content, while Z. bailli wines produced with 0.05 VVM aeration had increased concentrations of higher alcohols and volatile acids. Increased concentrations of these compounds over their sensory thresholds, are likely to impact negatively on wine sensory profile. Contrarily, all three non-Saccharomyces strains under 0.025 VVM aeration conditions produced wines with reduced ethanol concentration and acceptable chemical volatile profiles.

Torulaspora delbrueckii for secondary fermentation in sparkling wine production.

In the search for the desired oenological features and flavour complexity of wines, there is growing interest in the potential use of non-Saccharomyces yeast that are naturally present in the winemaking environment. Torulaspora delbrueckii is one such yeast that has seen profitable use in mixed fermentations with Saccharomyces cerevisiae and with different grape varieties. T. delbrueckii can have positive and distinctive impacts on the overall aroma of wines, and has also been used at an industrial level. Here, T. delbrueckii was successfully used in pure and mixed secondary fermentations for sparkling wine. The two selected T. delbrueckii strains used completed the secondary fermentation 'prise de mousse' in these pure and mixed fermentations. The sparkling wines obtained with T. delbrueckii showed different aromatic compositions and sensory profiles to those of S. cerevisiae. T. delbrueckii strain DiSVA 130 showed high esters production and significantly high scores for some of the aromatic descriptors that positively influence the sensory profile of sparkling wine. Thus, the use of T. delbrueckii in pure and mixed fermentations is a suitable strategy to further develop the flavour complexity during secondary fermentation of sparkling wines.

Torulaspora delbrueckii in the brewing process: A new approach to enhance bioflavour and to reduce ethanol content.

Nowadays, consumers require fermented alcoholic beverages with particular and enhanced flavour profiles while avoiding the health concerns due to high ethanol content. Here, the use of Torulaspora delbrueckii was evaluated for beer production, in both pure and in mixed cultures with a Saccharomyces cerevisiae starter strain (US-05). The yeast interactions were also evaluated. In mixed fermentations with S. cerevisiae, the main analytical characters from T. delbrueckii were comparable with those of the S. cerevisiae starter strain, but the beers were characterized by a distinctive overall analytical and aromatic profile. Indeed, there were interactions between S. cerevisiae and T. delbrueckii, with enhanced ethyl hexanoate (0.048 mg l(-1)) and ethyl octaonate (0.014 mg l(-1)) levels at the 1:20 and 1:10 inoculation ratios, respectively; while phenyl ethyl acetate increased in all mix combinations. The presence of T. delbrueckii resulted in reduced ß-phenyl ethanol and isoamyl acetate levels, which are responsible for floral and fruity aromas, respectively. Beer produced with T. delbrueckii pure cultures had a low alcohol content (2.66%; v/v), while also showing a particularly analytical and aromatic profile.

Cell-recycle batch process of Scheffersomyces stipitis and Saccharomyces cerevisiae co-culture for second generation bioethanol production.

OBJECTIVES: To achieve an optimized co-culture ratio of Scheffersomyces stipitis and Saccharomyces cerevisiae for the production of second generation bioethanol under a cell-recycle batch process. RESULTS: Three Sacc. cerevisiae strains were evaluated in co-culture with Sch. stipitis CBS 5773 at different ratios using synthetic medium containing glucose and xylose. Bioreactor trials indicated that the optimal condition for ethanol production using Sacc. cerevisiae EC1118 and Sch. stipitis co-culture was 1 % of O2 concentration. To increase ethanol production with Sacc. cerevisiae/Sch. stipitis co-culture a cell-recycle batch process was evaluated. Using this process, the maximum ethanol production (9.73 g l(-1)) and ethanol yield (0.42 g g(-1)) were achieved exhibiting a tenfold increase in ethanol productivity in comparison with batch process (2.1 g l(-1) h(-1)). In these conditions a stabilization of the cells ratio Sacc. cerevisiae/Sch. stipitis (1:5) at steady state condition was obtained. CONCLUSION: Batch cells recycling fermentation is an effective process to use Sch. stipitis/Sacc. cerevisiae co-culture for second generation ethanol production.

TpBGL2 codes for a Tetrapisispora phaffii killer toxin active against wine spoilage yeasts.

Tetrapisispora phaffii produces a killer toxin known as Kpkt that has extensive anti-Hanseniaspora/Kloeckera activity under winemaking conditions. Kpkt has a ß-glucanase activity and induces ultrastructural modifications in the cell wall of sensitive strains, with a higher specific cytocidal activity and a selective action towards target yeast cells. In this study, a two-step PCR-based approach was used to isolate the gene coding ß-glucanase of T. phaffii. Initially, a fragment of the open reading frame was isolated by degenerate PCR, with primers designed on the NH2 -terminal sequence of the protein and on conserved motifs of Bgl2p of Saccharomyces cerevisiae and Candida albicans. Subsequently, the entire sequence of the gene was obtained by inverse PCR. blast analyses of TpBGL2 highlight high identity with homologous genes in other yeast species, in which TpBGL2p shows no killer activity. However, gene disruption resulted in complete loss of the glucanase activity and the killer phenotype, thus confirming that TpBgl2p has a killer activity.

Recycled Brewer's Spent Grain (BSG) and Grape Juice: A New Tool for Non-Alcoholic (NAB) or Low-Alcoholic (LAB) Craft Beer Using Non-Conventional Yeasts.

Non-alcoholic beer (NAB) and low-alcoholic beer (LAB) are taking over the market with growing sales. Sustainable recycling and valorization of exhausted brewer's spent grain (BSG) coming from craft beer is a relevant issue in the brewing process. In this work, recycled BSG and BSG + GJ (supplemented with 10% grape juice) were used as a wort substrate to inoculate Lachancea thermotolerans, Wickeramhomyces anomalus, Torulaspora delbruecki and Pichia kluyveri non-conventional yeasts to produce NABLAB craft beer. Results showed that wort composed of only recycled BSG produced appreciated NAB beers (ethanol concentration from 0.12% to 0.54% v/v), while the addition of 10% grape juice produced LAB beers (ethanol concentration from 0.82 to 1.66% v/v). As expected, volatile compound production was highest with the addition of grape juice. L. thermotolerans showed lactic acid production, characterizing both worts with the production of ethyl butyrate and isoamyl acetate. T. delbrueckii exhibited relevant amounts of hexanol, phenyl ethyl acetate and ß-phenyl ethanol (BSG + GJ). W. anomalus and P. kluyveri showed consistent volatile production, but only in BSG + GJ where fermentation activity was exhibited. The overall results indicated that reused BSGs, non-conventional yeasts and grape juice are suitable bioprocesses for specialty NABLAB beer.

Lachancea thermotolerans and Saccharomyces cerevisiae in simultaneous and sequential co-fermentation: a strategy to enhance acidity and improve the overall quality of wine.

In the last few years there is an increasing interest on the use of mixed fermentation of Saccharomyces and non-Saccharomyces wine yeasts for inoculation of wine fermentations to enhance the quality and improve complexity of wines. In the present work Lachancea (Kluyveromyces) thermotolerans and Saccharomyces cerevisiae were evaluated in simultaneous and sequential fermentation with the aim to enhance acidity and improve the quality of wine. In this specific pairing of yeast strains in mixed fermentations (S. cerevisiae EC1118 and L. thermotolerans 101), this non-Saccharomyces yeast showed a high level of competitiveness. Nevertheless the S. cerevisiae strain dominated the fermentation over the spontaneous S. cerevisiae strains also under the industrial fermentation conditions. The different condition tested (modalities of inoculum, temperature of fermentation, different grape juice) influenced the specific interactions and the fermentation behaviour of the co-culture of S. cerevisiae and L. thermotolerans. However, some metabolic behaviours such as pH reduction and enhancement of 2-phenylethanol and glycerol, were shown here under all of the conditions tested. The specific chemical profiles of these wines were confirmed by the sensory analysis test, which expressed these results at the tasting level as significant increases in the spicy notes and in terms of total acidity increases.

Biocontrol and Probiotic Function of Non-Saccharomyces Yeasts: New Insights in Agri-Food Industry.

Fermented food matrices, including beverages, can be defined as the result of the activity of complex microbial ecosystems where different microorganisms interact according to different biotic and abiotic factors. Certainly, in industrial production, the technological processes aim to control the fermentation to place safe foods on the market. Therefore, if food safety is the essential prerogative, consumers are increasingly oriented towards a healthy and conscious diet driving the production and consequently the applied research towards natural processes. In this regard, the aim to guarantee the safety, quality and diversity of products should be reached limiting or avoiding the addition of antimicrobials or synthetic additives using the biological approach. In this paper, the recent re-evaluation of non-Saccharomyces yeasts (NSYs) has been reviewed in terms of bio-protectant and biocontrol activity with a particular focus on their antimicrobial power using different application modalities including biopackaging, probiotic features and promoting functional aspects. In this review, the authors underline the contribution of NSYs in the food production chain and their role in the technological and fermentative features for their practical and useful use as a biocontrol agent in food preparations.

Consortium of selected yeasts to produce healthy soy fermented beverage: Evaluation of microbial evolution, analytical, sensorial, and functional features.

Currently, an increasing number of intolerant and vegan consumers are driving the market towards plant-based milk alternatives. Here, selected probiotic yeasts, belonging to the Candida zeylanoides, Kluyveromyces lactis and Debaryomyces hansenii species, previously characterized for their aptitude to ferment animal milk, were tested in soy milk. Trials at different fermentation times with the developed yeast consortium (Yc) coinoculated with a lactic bacterium commercial strain were carried out. Yc showed good fermentation performance, conferring distinctive analytical and aromatic properties to the resulted soy fermented beverage, a product similar to an industrial kefir. Analytical determinations did not show significant variations between the end of fermentation and cold storage (4 weeks at 4 °C), indicating full stability. Phenol amounts and antioxidant activity were significantly increased in soy fermented beverage fermented by Yc. All yeasts remained viable until the end of storage with a final concentration of approximately 8 Log CFU/ml, a value suitable for a probiotic commercial claim. Overall, the results suggest that Yc is a promising multistarter candidate for functional soy products.

Biocontrol Using Torulaspora delbrueckii in Sequential Fermentation: New Insights into Low-Sulfite Verdicchio Wines.

Torulaspora delbrueckii has attracted renewed interest in recent years, for its biotechnological potential linked to its ability to enhance the flavor and aroma complexity of wine. Sequential fermentations with a selected native strain of T. delbrueckii (DiSVA 130) and low-sulfite native strain of Saccharomyces cerevisiae (DiSVA 709) were carried out to establish their contribution in biocontrol and the aroma profile. A first set of trials were conducted to evaluate the effect of the sulfur dioxide addition on pure and T. debrueckii/S. cerevisiae sequential fermentations. A second set of sequential fermentations without SO2 addition were conducted to evaluate the biocontrol and aromatic effectiveness of T. delbrueckii. Native T. delbrueckii showed a biocontrol action in the first two days of fermentation (wild yeasts reduced by c.a. 1 log at the second day). Finally, trials with the combination of both native and commercial T. delbrueckii/S. cerevisiae led to distinctive aromatic profiles of wines, with a significant enhancement in isoamyl acetate, phenyl ethyl acetate, supported by positive appreciations from the tasters, for ripe and tropical fruits, citrus, and balance. The whole results indicate that native T. delbrueckii could be a potential biocontrol tool against wild yeasts in the first phase of fermentation, contributing to improving the final wine aroma.

Starmerella bombicola influences the metabolism of Saccharomyces cerevisiae at pyruvate decarboxylase and alcohol dehydrogenase level during mixed wine fermentation.

BACKGROUND: The use of a multistarter fermentation process with Saccharomyces cerevisiae and non-Saccharomyces wine yeasts has been proposed to simulate natural must fermentation and to confer greater complexity and specificity to wine. In this context, the combined use of S. cerevisiae and immobilized Starmerella bombicola cells (formerly Candida stellata) was assayed to enhance glycerol concentration, reduce ethanol content and to improve the analytical composition of wine. In order to investigate yeast metabolic interaction during controlled mixed fermentation and to evaluate the influence of S. bombicola on S. cerevisiae, the gene expression and enzymatic activity of two key enzymes of the alcoholic fermentation pathway such as pyruvate decarboxylase (Pdc1) and alcohol dehydrogenase (Adh1) were studied. RESULTS: The presence of S. bombicola immobilized cells in a mixed fermentation trial confirmed an increase in fermentation rate, a combined consumption of glucose and fructose, an increase in glycerol and a reduction in the production of ethanol as well as a modification in the fermentation of by products. The alcoholic fermentation of S. cerevisiae was also influenced by S. bombicola immobilized cells. Indeed, Pdc1 activity in mixed fermentation was lower than that exhibited in pure culture while Adh1 activity showed an opposite behavior. The expression of both PDC1 and ADH1 genes was highly induced at the initial phase of fermentation. The expression level of PDC1 at the end of fermentation was much higher in pure culture while ADH1 level was similar in both pure and mixed fermentations. CONCLUSION: In mixed fermentation, S. bombicola immobilized cells greatly affected the fermentation behavior of S. cerevisiae and the analytical composition of wine. The influence of S. bombicola on S. cerevisiae was not limited to a simple additive contribution. Indeed, its presence caused metabolic modifications during S. cerevisiae fermentation causing variation in the gene expression and enzymatic activity of alcohol deydrogenase and pyruvate decarboxilase.

Lentil Fortification and Non-Conventional Yeasts as Strategy to Enhance Functionality and Aroma Profile of Craft Beer.

During the last few years, consumer demand has been increasingly oriented to fermented foods with functional properties. This work proposed to use selected non-conventional yeasts (NCY) Lachanceathermotolerans and Kazachstaniaunispora in pure and mixed fermentation to produce craft beer fortified with hydrolyzed red lentils (HRL). For this, fermentation trials using pils wort (PW) and pils wort added with HRL (PWL) were carried out. HRL in pils wort improved the fermentation kinetics both in mixed and pure fermentations without negatively affecting the main analytical characters. The addition of HRL determined a generalized increase in amino acids concentration in PW. L. thermotolerans and K. unispora affected the amino acid profile of beers (with and without adding HRL). The analysis of by-products and volatile compounds in PW trials revealed a significant increase of some higher alcohols with L. thermotolerans and ethyl butyrate with K. unispora. In PWL, the two NCY showed a different behavior: an increment of ethyl acetate (K. unispora) and ß-phenyl ethanol (L. thermotolerans). Sensory analysis showed that the presence of HRL characterized all beers, increasing the perception of the fruity aroma in both pure and mixed fermentation.

Biocontrol of Non-Saccharomyces Yeasts in Vineyard against the Gray Mold Disease Agent Botrytis cinerea.

BACKGROUND: Botrytis cinerea (B. cinerea) is responsible for grape infection and damage to the winemaking and table grape sectors. Although anti-Botrytis chemicals are available, they are considered unsustainable for resistance phenomenon and adverse effects on the environment and human health. Research is focused on developing alternative approaches, such as exploiting biological control agents (BCAs). In this context, 19 yeasts of the genera Cryptococcus, Aureobasidium, Metschnikowia, Kluyveromyces and Wickerhamomyces were tested as antimicrobial agents against B. cinerea development. METHODS: A combination of in vitro tests based on dual-culture methods, volatile organic compound production assay, laboratory tests on grape berries (punctured and sprayed with yeasts) and field experiments based on yeast treatments on grapes in vineyards allowed the selection of two potential BCAs. RESULTS: M. pulcherrima DiSVA 269 and A. pullulans DiSVA 211 exhibited the best ability to contain the development of B. cinerea, showing the severity, the decay and the McKinney index lower than a commercial biological formulation consisting of a mixture of two different A. pullulans strains, which were used as positive controls. CONCLUSIONS: The results indicated that the selected strains were effective BCA candidates to counteract B. cinerea in the field, applying them in the partial or total replacement of conventional treatments.

Epiphytic Yeasts and Bacteria as Candidate Biocontrol Agents of Green and Blue Molds of Citrus Fruits.

Overall, 180 yeasts and bacteria isolated from the peel of citrus fruits were screened for their in vitro antagonistic activity against Penicillium digitatum and P. italicum, causative agents of green and blue mold of citrus fruits, respectively. Two yeast and three bacterial isolates were selected for their inhibitory activity on mycelium growth. Based on the phylogenetic analysis of 16S rDNA and ITS rDNA sequences, the yeast isolates were identified as Candida oleophila and Debaryomyces hansenii while the bacterial isolates were identified as Bacillus amyloliquefaciens, B. pumilus and B. subtilis. All five selected isolates significantly reduced the incidence of decay incited by P. digitatum and P. italicum on 'Valencia' orange and 'Eureka' lemon fruits. Moreover, they were effective in preventing natural infections of green and blue mold of fruits stored at 4 °C. Treatments with antagonistic yeasts and bacteria did not negatively affect the quality and shelf life of fruits. The antagonistic efficacy of the five isolates depended on multiple modes of action, including the ability to form biofilms and produce antifungal lipopeptides, lytic enzymes and volatile compounds. The selected isolates are promising as biocontrol agents of postharvest green and blue molds of citrus fruits.

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.