Microbial Diversity and Safety in Fermented Beverages

For thousands of years, humans have exploited the natural process of fermentation of various foods to preserve them, and to enjoy the changes in the sensory characteristics that could be produced. Recently, the world of fermented beverages has gone through a rapid transformation linked to deep changes in consumer preferences, consumption habits, climate, new regulations and entry of emerging countries, accompanied by safety concerns and an important reduction in economic resources available to people. As with all food handling and preparation, we need to be sure the fermented food produced is safe. Fermentation is a complex biological process where microbial diversity takes place and the environment created inside of the fermented food provides the conditions to reduce the risk of pathogenic bacteria growth, thus providing safe food. In addition, food manufacturers fermenting food carefully control their processing and must comply with the National Food Standards Codes. Although these products have a generally good food safety record, sometimes inadequate manufacturing practices or the presence of acidophilic pathogens could compromise food safety. In fact, fermented beverages may adversely become contaminated with pathogens or microbial toxins and thereby transform into vehicles that can transmit diseases to the consumers. Moreover, many microorganisms can deteriorate the physical-chemical and sensory properties as well as the flavor of the final products. In this editorial, we present an overview of a review and six original research papers published in the Special Issue “Fermentation Process and Microbial Safety of Beverages” of the Beverages journal

Wild grape-associated yeasts as promising biocontrol agents against Vitis vinifera fungal pathogens

The increasing level of hazardous residues in the environment and food chains has led the European Union to restrict the use of chemical fungicides. Thus, exploiting new natural antagonistic microorganisms against fungal diseases could serve the agricultural production to reduce pre- and post-harvest losses, to boost safer practices for workers and to protect the consumers' health. The main aim of this work was to evaluate the antagonistic potential of epiphytic yeasts against Botrytis cinerea, Aspergillus carbonarius, and Penicillium expansum pathogen species. In particular, yeast isolation was carried out from grape berries of Vitis vinifera ssp sylvestris populations, of the Eurasian area, and V. vinifera ssp vinifera cultivars from three different farming systems (organic, biodynamic, and conventional). Strains able to inhibit or slow the growth of pathogens were selected by in vitro and in vivo experiments. The most effective antagonist yeast strains were subsequently assayed for their capability to colonize the grape berries. Finally, possible modes of action, such as nutrients and space competition, iron depletion, cell wall degrading enzymes, diffusible and volatile antimicrobial compounds, and biofilm formation, were investigated as well. Two hundred and thirty-one yeast strains belonging to 26 different species were isolated; 20 of them, ascribed to eight species, showed antagonistic action against all molds. Yeasts isolated from V. vinifera ssp sylvestris were more effective (up to 50%) against B. cinerea rather than those isolated from V. vinifera ssp vinifera. Six strains, all isolated from wild vines, belonging to four species (Meyerozyma guilliermondii, Hanseniaspora uvarum, Hanseniaspora clermontiae, and Pichia kluyveri) revealed one or more phenotypical characteristics associated to the analyzed modes of antagonistic action

Genetic Diversity and Population Structure of Saccharomyces cerevisiae Strains Isolated from Different Grape Varieties and Winemaking Regions

We herein evaluate intraspecific genetic diversity of fermentative vineyard-associated S. cerevisiae strains and evaluate relationships between grape varieties and geographical location on populational structures. From the musts obtained from 288 grape samples, collected from two wine regions (16 vineyards, nine grape varieties), 94 spontaneous fermentations were concluded and 2820 yeast isolates were obtained that belonged mainly (92%) to the species S. cerevisiae. Isolates were classified in 321 strains by the use of ten microsatellite markers. A high strain diversity (8–43 strains per fermentation) was associated with high percentage (60–100%) of fermenting samples per vineyard, whereas a lower percentage of spontaneous fermentations (0–40%) corresponded to a rather low strain diversity (1–10 strains per fermentation)

Yeast biodiversity in vineyard environments is increased by human intervention

One hundred and five grape samples were collected during two consecutive years from 33 locations on seven oceanic islands of the Azores Archipelago. Grape samples were obtained from vineyards that were either abandoned or under regular cultivation involving common viticultural interventions, to evaluate the impact of regular human intervention on grape yeast biota diversity in vineyards. A total of 3150 yeast isolates were obtained and 23 yeast species were identified. The predominant species were Hanseniaspora uvarum, Pichia terricola, Starmerella bacillaris and Issatchenkia hanoiensis. The species Barnettozyma californica, Candida azymoides and Pichia cecembensis were reported in grapes or wine-associated environments for the first time. A higher biodiversity was found in active vineyards where regular human intervention takes place (Shannon index: 1.89 and 1.53 in the first and second years, respectively) when compared to the abandoned ones (Shannon index: 0.76 and 0.31). This finding goes against the assumptions that human intervention can destroy biodiversity and lead to homogeneity in the environment. Biodiversity indices were considerably lower in the year with the heaviest rainfall. This study is the first to report on the grape yeast communities from several abandoned vineyards that have undergone no human intervention.Joao Drumonde Neves is the recipient of a fellowship of the Azorean Government (M321/006/F/2008) and PROEMPREGO. This work was supported by the strategic programme UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569) funded by national funds through the FCT I.P. and by the ERDF through the COMPETE2020 - Programa Operacional Competitividade e Internacionalizacao (POCI), and by national funds through FCT by the projects FCOMP-01-0124-008775, PTDC/AGR-ALI/103392/2008 and PTDC/AGR-ALI/121062/2010.info:eu-repo/semantics/publishedVersio

Association between Grape Yeast Communities and the Vineyard Ecosystems

The grape yeast biota from several wine-producing areas, with distinct soil types and grapevine training systems, was assessed on five islands of Azores Archipelago, and differences in yeast communities composition associated with the geographic origin of the grapes were explored. Fifty-seven grape samples belonging to the Vitis vinifera grapevine cultivars Verdelho dos Acores (Verdelho), Arinto da Terceira (Arinto) and Terrantez do Pico (Terrantez) were collected in two consecutive years and 40 spontaneous fermentations were achieved. A total of 1710 yeast isolates were obtained from freshly crushed grapes and 1200 from final stage of fermentations. Twenty-eight species were identified, Hanseniaspura uvarum, Pichia terricola and Metschnikowia pulcherrima being the three most representative species isolated. Candida carpophila was encountered for the first time as an inhabitant of grape or wine-associated environments. In both sampling years, a higher proportion of H. uvarum in fresh grapes from Verdelho cultivar was observed, in comparison with Arinto cultivar. Qualitatively significant differences were found among yeast communities from several locations on five islands of the Archipelago, particularly in locations with distinctive agro-ecological compositions. Our results are in agreement with the statement that grape-associated microbial biogeography is non-randomly associated with interactions of climate, soil, cultivar, and vine training systems in vineyard ecosystems. Our observations strongly support a possible linkage between grape yeast and wine typicality, reinforcing the statement that different viti-cultural terroirs harbor distinctive yeast biota, in particular in vineyards with very distinctive environmental conditions.Joao Drumonde Neves is the recipient of a fellowship of the Azorean Government (M321/006/F/2008) and PROEMPREGO. This work was supported by the strategic programme UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569) funded by national funds through the FCT I.P. and by the ERDF through the COMPETE2020 - Programa Operacional Competitividade e Internacionalizacao (POCI), and by national funds through FCT by the projects FCOMP-01-0124-008775, PTDC/AGR-ALI/103392/2008 and PTDC/AGR-ALI/121062/2010.info:eu-repo/semantics/publishedVersio

Non-target impact of fungicide tetraconazole on microbial communities in soils with different agricultural management

Effect of the fungicide tetraconazole on microbial community in silt loam soils from orchard with long history of triazole application and from grassland with no known history of fungicide usage was investigated. Triazole tetraconazole that had never been used on these soils before was applied at the field rate and at tenfold the FR. Response of microbial communities to tetraconazole was investigated during 28-day laboratory experiment by determination of changes in their biomass and structure (phospholipid fatty acids method—PLFA), activity (fluorescein diacetate hydrolysis—FDA) as well as changes in genetic (DGGE) and functional (Biolog) diversity. Obtained results indicated that the response of soil microorganisms to tetraconazole depended on the management of the soils. DGGE patterns revealed that both dosages of fungicide affected the structure of bacterial community and the impact on genetic diversity and richness was more prominent in orchard soil. Values of stress indices—the saturated/monounsaturated PLFAs ratio and the cyclo/monounsaturated precursors ratio, were almost twice as high and the Gram-negative/Gram-positive ratio was significantly lower in the orchard soil compared with the grassland soil. Results of principal component analysis of PLFA and Biolog profiles revealed significant impact of tetraconazole in orchard soil on day 28, whereas changes in these profiles obtained for grassland soil were insignificant or transient. Obtained results indicated that orchards soil seems to be more vulnerable to tetraconazole application compared to grassland soil. History of pesticide application and agricultural management should be taken into account in assessing of environmental impact of studied pesticides. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s10646-016-1661-7) contains supplementary material, which is available to authorized users

Prospecting of yeasts from Vitis vinifera ssp. sylvestris (Gmelin) hegi grape-berries in the Mediterranean basin and in the Caucasus Area

Vitis vinifera L. ssp. Sylvestris (Gmelin) Hegi is the only ancestral grapevine species in Europe and it is recognized as the dioecious parental generation of today\u2019s cultivars. It constitutes an important phytogenetic resource, but urbanization and industrialization of territories, climatic change and the arrival in Europe of pathogens and pests have led it to be included on the IUCN Red List of Threatened Species in 1997. Although numerous studies about the current health status of this species have been carried out, at best of our knowledge, no studies on microbial populations of grape-berry surfaces have been done. The present work has been focused on the study of yeast occurrence and diversity on grape-berries collected from wild vines (V. vinifera ssp. Sylvestris). Final outputs have allowed: i) to obtain precise information about yeast communities; ii) to provide an objective framework for the classification of the broadest range of species according to their extinction risk; iii) to select,actractive yeast strains for their biotechnological potential, offering new opportunities to winemakers. Grape-berries from wild vines were carefully harvested throughout three Mediterranean Basin countries, Spain, Italy and Georgia, in different sites for each area. In all, more than 2880 epiphytic and endophytic yeast colonies were isolated and identified as belonging to more than 20 species, including Saccharomyces cerevisiae, by 26 rDNA D1/D2 domains and ITS region sequencing. Moreover, despite of some species appeared in the three countries, yeast ecology differed in the sampled regions. This study highlights the biodiversity potential of pristine environments that still represent a fascinating avenue for the investigation of gene evolution and that offer a platform to face common problems in winemaking

Genetic diversity of yeasts isolated from Eurasian populations of Vitis vinifera ssp. Sylvestris (Gmelin) Hegi

Vitis vinifera L. ssp. Sylvestris (Gmelin) Hegi is the only ancestral grapevine species in the Euroasiatic area and it is recognized as the dioecious parental generation of today\u2019s cultivars. It constitutes an important phytogenetic resource, but urbanization and industrialization of territories, climatic change and the arrival in Europe of pathogens and pests have led it to be included on the IUCN Red List of Threatened Species in 1997. Although numerous studies about the current health status of this species have been carried out, at best of our knowledge, no studies on microbial populations of grape-berry surfaces have been done. The present work has been focused on the study of yeast occurrence and diversity on grape-berries collected from wild vines (V. vinifera ssp. Sylvestris). Final outputs have allowed: i) to obtain precise information about yeast communities; ii) to provide an objective framework for the classification of the broadest range of species according to their extinction risk; iii) to select actractive yeast strains for their biotechnological potential, offering new opportunities to winemakers. Grape-berries from wild vines were carefully harvested in Azerbaijan, Georgia, Italy, Romania and Spain, in different sites for each country. In all, 3180 epiphytic and endophytic yeast colonies were isolated and identified as belonging to 50 species, including Saccharomyces cerevisiae, by 26S rDNA D1/D2 domains and ITS region sequencing. Isolates belonging to S. cerevisiae were also analysed by SSR-PCR obtaining 163 different genotypes. Moreover, despite of some species appeared in all countries, yeast ecology differed in the sampled regions. This study highlights the biodiversity potential of pristine environments that still represent a fascinating source for the investigation of gene evolution and that offer a platform to face common problems in winemaking