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.

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

Patrizia Romano;Giuseppe Blaiotta
Membro del Collaboration Group
;
Vittorio Capozzi;
2022

Abstract

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.
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