The Use of Hanseniaspora opuntiae to Improve 'Sideritis' Wine Quality, a Late-Ripening Greek Grape Variety.

In view of climate change and the increasingly antagonistic wine market, the exploitation of native genetic resources is revisited in relation to sustainable wine production. 'Sideritis' is a late-ripening Greek grape variety, which is quite promising for counteracting wine quality issues associated with the annual temperature rise. The aim of this study was to improve the quality and to enhance the aroma of 'Sideritis' wine through the use of native yeasts. To improve vinification, Hanseniaspora opuntiae L1 was used along with Saccharomyces cerevisiae W7 in mixed fermentations (SQ). The addition of H. οpuntiae significantly altered the chemical profile of the wine compared to the single-inoculated fermentations with W7 (IS). H. opuntiae increased all the acetate esters, except for hexyl acetate and (Z)-3-hexen-1-ol acetate. The concentration of 2-phenylethyl acetate, which imparts flowery and sweet notes, exhibited a 2.6-fold increase in SQ as compared to IS wines. SQ also showed higher levels in several ethyl esters, including ethyl butyrate, ethyl heptanoate and ethyl 7-octenoate, which are associated with fruity notes compared to IS. H. opuntiae produced citronellol, a terpene associated with rose and green notes, and increased the overall acceptance of the wine. Present results are thus quite promising for improving 'Sideritis' wine quality towards a sustainable wine production in Greece in view of global warming.

Emerging Roles of Epigenetics in Grapevine and Winegrowing.

Epigenetics refers to dynamic chemical modifications to the genome that can perpetuate gene activity without changes in the DNA sequence. Epigenetic mechanisms play important roles in growth and development. They may also drive plant adaptation to adverse environmental conditions by buffering environmental variation. Grapevine is an important perennial fruit crop cultivated worldwide, but mostly in temperate zones with hot and dry summers. The decrease in rainfall and the rise in temperature due to climate change, along with the expansion of pests and diseases, constitute serious threats to the sustainability of winegrowing. Ongoing research shows that epigenetic modifications are key regulators of important grapevine developmental processes, including berry growth and ripening. Variations in epigenetic modifications driven by genotype-environment interplay may also lead to novel phenotypes in response to environmental cues, a phenomenon called phenotypic plasticity. Here, we summarize the recent advances in the emerging field of grapevine epigenetics. We primarily highlight the impact of epigenetics to grapevine stress responses and acquisition of stress tolerance. We further discuss how epigenetics may affect winegrowing and also shape the quality of wine.

Single versus dual inoculation with indigenous Saccharomyces cerevisiae strains in winemaking.

Indigenous Saccharomyces cerevisiae strains and their combinations may be used to diversify wines and add complexity to sensory profiles. Here, two S. cerevisiae strains that represent regional genetic and phenotypic specificities for two major winegrowing areas of Greece were used in single- and mixed-culture fermentations. The kinetics and metabolic activities of the strains were analyzed to evaluate the influence of each strain individually or in combination on wine quality. The two strains differentially affected the kinetics and the outcome of fermentation. They showed significant differences in the production of important metabolites that strongly affect the organoleptic profile of wines, such as volatile acidity, acetaldehyde, certain esters, and terpenes. Furthermore, the chemical and sensory profiles of wines produced by single cultures were different from those fermented by mixed-culture inoculum. The concentration of certain metabolites was enhanced (e.g. isoamyl acetate, 1-heptanol), while others were suppressed (e.g. hexyl acetate, octyl acetate). Results highlight the potential worth of indigenous S. cerevisiae strains to differentiate local wines. The mixed-culture S. cerevisiae inoculum was shown to generate novel wine characteristics, as compared to single cultures, thus offering alternatives to further diversify wines and increase their complexity.

Torulaspora delbrueckii May Help Manage Total and Volatile Acidity of Santorini-Assyrtiko Wine in View of Global Warming.

Non-Saccharomyces (NS) yeasts are gaining popularity in modern winemaking for improving wine quality. Climate change is one of the biggest challenges winegrowing now faces in warm regions. Here, Lachancea thermotolerans LtS1 and Torulaspora delbrueckii TdS6 combined with Saccharomyces cerevisiae ScS13 isolated from Assyrtiko grapes from Santorini island were evaluated in grape must fermentation with the aim to mitigate major consequences of temperature rise. Different inoculation protocols were evaluated, including simultaneous and sequential mixed-strain inoculations, displaying significant variation in the chemical and kinetic characteristics. Both LtS1 and TdS6 could raise the titratable acidity (TA). TdS6 also reduced the volatile acidity (VA) and was thus chosen for further evaluation in microvinifications and pilot-scale fermentations. Consistent with lab-scale trials, sequential inoculation exhibited the longest persistence of TdS6 resulting in minimum VA levels. Diethyl succinate, ethyl propanoate, and ethyl isobutyrate were significantly increased in sequential inoculations, although a decline in the net total ester content was observed. On the other hand, significantly higher levels of TA, succinic acid, and 2-methylpropanoic were associated with sequential inoculation. The overall performance of TdS6 coupled with a high compatibility with S. cerevisiae suggests its use in the fermentation of Santorini-Assyrtiko or other high sugar musts for the production of structured dry or sweet wines.

The effect of vine variety and vintage on wine yeast community structure of grapes and ferments.

AIMS: The yeast community structure associated with grapes is an essential part of the wine-growing chain with a significant effect on wine quality. The aim of the present study was to evaluate the effect of the varietal factor on the yeast community assembly on grapes and during must fermentation. METHODS AND RESULTS: We analysed the wine yeast populations associated with four different grape varieties from the Greek national collection vineyard of Lykovryssi. The vintage effect was also considered by sampling the grapes for two consecutive years. Fourteen yeast species were recovered and genotyped to distinct subpopulations. A relatively stable yeast community structure was detected across vintages, with Hanseniaspora guilliermondii being the core species of the vineyard under study. The detected species subpopulations shared a relatively high genetic similarity with several genotypes persisting across vintages. CONCLUSIONS: It was shown that different grape cultivars were associated with distinct yeast communities, pointing to their possible implication on wine chemical diversity. SIGNIFICANCE AND IMPACT OF THE STUDY: Present findings show that the varietal factor is an important sharpener of the vineyard-associated wine yeast community, which may interfere with the organoleptic profile of the resulting wines.

Understanding Wine through Yeast Interactions.

Wine is a product of microbial activities and microbe-microbe interactions. Yeasts are the principal microorganisms responsible for the evolution and fulfillment of alcoholic fermentation. Several species and strains coexist and interact with their environment and with each other during the fermentation course. Yeast-yeast interactions occur even from the early stages of fermentation, determining yeast community structure and dynamics during the process. Different types of microbial interactions (e.g., mutualism and commensalism or competition and amensalism) may exert positive or negative effects, respectively, on yeast populations. Interactions are intimately linked to yeast metabolic activities that influence the wine analytical profile and shape the wine character. In this context, much attention has been given during the last years to the interactions between Saccharomyces cerevisiae (SC) and non-Saccharomyces (NS) yeast species with respect to their metabolic contribution to wine quality. Yet, there is still a significant lack of knowledge on the interaction mechanisms modulating yeast behavior during mixed culture fermentation, while much less is known about the interactions between the various NS species or between SC and Saccharomyces non-cerevisiae (SNC) yeasts. There is still much to learn about their metabolic footprints and the genetic mechanisms that alter yeast community equilibrium in favor of one species or another. Gaining deeper insights on yeast interactions in the grape-wine ecosystem sets the grounds for understanding the rules underlying the function of the wine microbial system and provides means to better control and improve oenological practices.

Biogeographical Regionalization of Wine Yeast Communities in Greece and Environmental Drivers of Species Distribution at a Local Scale.

Recent research has expanded our understanding on vineyard-associated fungal community assembly, suggesting non-random distribution and implicating regional differences in the wine terroir effect. Here, we focused on the culturable fraction of the fungal community that resides on grapes and determine wine quality, the so-called wine yeast populations. We aimed to analyze local-scale yeast community assemblages and to test whether the hypothesis of biogeographical patterns also applies to wine yeasts in particular. Surveying 34 vineyards across four main viticultural zones in Greece showed significant trends in vineyard-specific patterns. At a local scale, viticultural regions were also linked to distinct yeast community compositions. Importantly, major yeast populations directly related to wine fermentation contributed significantly to the delimitation of regions, highlighting their potential influence on the regionality of wine characteristics. In terms of the microbial terroir influence, yeast communities within an area were temporarily stable, which is critical for the regional character of the wine. Community structure could be explained only partially by environmental features. Maximum temperature, elevation, and net precipitation were the highest correlated variables with the yeast community biogeographic patterns. Finally, we also showed that certain environmental factors may drive the population size of specific yeast populations. The present results indicate that the wine yeast community has a geographical character at local scale, which is an important feature of the microbial terroir concept and thus for the wine industry.

New Frontiers in Wine Microbiology.

The wine sectoris currently facing new challenges [...].

Grapevine Responses to Heat Stress and Global Warming.

The potential effects of the forthcoming climate change include the rising of the average annual temperature and the accumulation of extreme weather events, like frequent and severe heatwaves, a phenomenon known as global warming. Temperature is an important environmental factor affecting almost all aspects of growth and development in plants. The grapevine (Vitis spp.) is quite sensitive to extreme temperatures. Over the current century, temperatures are projected to continue rising with negative impacts on viticulture. These consequences range from short-term effects on wine quality to long-term issues such as the suitability of certain varieties and the sustainability of viticulture in traditional wine regions. Many viticultural zones, particularly in Mediterranean climate regions, may not be suitable for growing winegrapes in the near future unless we develop heat-stress-adapted genotypes or identify and exploit stress-tolerant germplasm. Grapevines, like other plants, have developed strategies to maintain homeostasis and cope with high-temperature stress. These mechanisms include physiological adaptations and activation of signaling pathways and gene regulatory networks governing heat stress response and acquisition of thermotolerance. Here, we review the major impacts of global warming on grape phenology and viticulture and focus on the physiological and molecular responses of the grapevine to heat stress.

Molecular Characterization and Enological Potential of A High Lactic Acid-Producing Lachancea thermotolerans Vineyard Strain.

Lactic acid production is an important feature of the yeast Lachancea thermotolerans that has gained increasing interest in winemaking. In particular, in light of climate change, the biological acidification and ethanol reduction by the use of selected yeast strains may counteract the effect of global warming in wines. Here, the enological potential of a high lactate-producing L. thermotolerans strain (P-HO1) in mixed fermentations with S. cerevisiae was examined. Among the different inoculation schemes evaluated, the most successful implantation of L. thermotolerans was accomplished by sequential inoculation of S. cerevisiae, i.e., at 1% vol. ethanol. P-HO1produced the highest levels of lactic acid ever recorded in mixed fermentations (10.4 g/L), increasing thereby the acidity and reducing ethanol by 1.6% vol. L. thermotolerans was also associated with increases in ethyl isobutyrate (strawberry aroma), free SO2, organoleptically perceived citric nuances and aftertaste. To start uncovering the molecular mechanisms of lactate biosynthesis in L. thermotolerans, the relative expressions of the three lactate dehydrogenase (LDH) paralogous genes, which encode the key enzyme for lactate biosynthesis, along with the alcohol dehydrogenase paralogs (ADHs) were determined. Present results point to the possible implication of LDH2, but not of other LDH or ADH genes, in the high production of lactic acid in certain strains at the expense of ethanol. Taken together, the important enological features of P-HO1 highlighted here, and potentially of other L. thermotolerans strains, indicate its great importance in modern winemaking, particularly in the light of the upcoming climate change and its consequences in the grape/wine system.

Patterns of Genetic Diversity and the Invasion of Commercial Starters in Saccharomyces cerevisiae Vineyard Populations of Santorini Island.

Autochthonous Saccharomyces cerevisiae vineyard populations are important components of the grape/wine system. Besides their direct impact on winemaking, they also constitute an untapped reservoir of genotypes with special technological attributes for the wine industry. Research so far on S. cerevisiae populations has focused on spatial distribution on large scales, yet little is known about the genetic variability of populations within viticultural zones and their temporal genotypic variation. Here, S. cerevisiae populations from different vineyards in Santorini, a small Aegean island, were genotyped and their genetic diversity was assessed within and between vineyards during two consecutive years. Despite the relative geographical isolation of the island, a relatively high genetic diversity was uncovered. The vast majority of genotypes were vineyard-specific, while in one of the vintages, significant differences in the genotypic composition of vineyards were detected. Overall, higher differences were detected between vintages rather than among vineyards. Notably, only four genotypes were common for the two vintages, three of which were commercial S. cerevisiae strains, probably "escapees" from wineries. Nevertheless, the populations of the two vintages were not genetically distinct. Present results highlight the magnitude of genetic diversity in natural wine yeast populations on a small spatial scale, yet the invasion of commercial starters may constitute a potential risk for loss of local yeast biodiversity. However, present results show that industrial strains do not necessarily dominate over the natural strains or their high abundance may be temporary.

Indigenous Yeast Interactions in Dual-Starter Fermentations May Improve the Varietal Expression of Moschofilero Wine.

Multi-starter wine fermentations employing non-Saccharomyces (NS) yeasts are becoming an emerging trend in winemaking. It is therefore important to determine the impacts of different NS strains in the wine phenotype and in particular the aroma outputs in different inoculation schemes and fermentation conditions. Here, two native NS yeasts, Lachancea thermotolerans LtMM7 and Hanseniaspora uvarum HuMM19, were assessed for their ability to improve the quality of Moschofilero, a Greek aromatic white wine. The NS strains were initially examined in laboratory scale fermentations in mixed inoculations with ScMM23, a native Saccharomyces cerevisiae strain. LtMM7 was selected to be further evaluated in pilot scale fermentations. Five different inoculation schemes were considered: single inoculation of ScMM23 (IS), simultaneous inoculation of ScMM23 with HuMM19 (SMH) or LtMM7 (SML), and sequential inoculation of HuMM19 (SQH) or LtMM7 (SQL) followed by ScMM23. At laboratory scale fermentations, the chemical profiles were largely affected by both the NS species and the inoculation scheme applied. The sequential inoculation using HuMM19 produced the most divergent wine phenotype. However, HuMM19 caused significant increases in acetic acid and ethyl acetate levels that impeded its use in pilot scale trials. LtMM7 significantly affected the chemical profiles of wines produced at the winery, especially in the sequential inoculation scheme. Importantly, LtMM7 significantly increased the levels of acetate esters or ethyl esters, depending on the inoculation method applied. In particular, acetate esters like isobutyl acetate, hexyl acetate, and 2-phenylethyl acetate, which all impart fruity or floral aromas, were significantly increased in SQL. On the other hand, higher levels of total ethyl esters were associated with SML. The most striking differences were observed in the levels of fruit-impair esters like ethyl decanoate, 3-methylbutyl octanoate, and isoamyl hexanoate. This is the first study to report a significant increase in the ethyl ester fraction by L. thermotolerans. Interestingly, L. thermotolerans in SQL also increased the concentrations of damascenone and geraniol, the major teprenic compound of Moschofilero, which are associated with several typical floral and fruity aromas of the variety. Present results show that L. thermotolerans may enhance the varietal character and increase the chemical complexity of Moschofilero wines.

The use of indigenous Saccharomyces cerevisiae and Starmerella bacillaris strains as a tool to create chemical complexity in local wines.

The performance of two vineyard strains, Saccharomyces cerevisiae SacPK7 and Starmerella bacillaris StbPK9, was evaluated in laboratory and pilot scale fermentations of Cretan grape must under the following inoculation schemes: single inoculation of SacPK7 (IS), simultaneous inoculation of StbPK9 and SacPK7 (SM), and sequential inoculation of StbPK9 followed by SacPK7 (SQ). Un-inoculated (spontaneous) fermentations (SP) and fermentations inoculated with control S. cerevisiae strains (CS) were also conducted as reference. Star. bacillaris not only did not restrict but also slightly promoted the growth of S. cerevisiae when the two strains were co-inoculated at equal quantities. On the contrary, the SQ inoculation scheme conferred a competitive advantage to Star. bacillaris over S. cerevisiae, which maximum population was reduced, while increased levels of Star. bacillaris were recorded. The fermentation kinetics were also affected, accordingly. The completion of fermentation was faster in SM, IS and CS ferments than in SQ and SP. Ethanol accumulation had a predominant role in the early death of Star. bacillaris, since its growth was similarly arrested irrespective of the dominating yeast species, the magnitude of yeast population or the availability of energy sources. Interestingly, the inoculation scheme applied significantly affected the chemical profiles of the resulting wines. SQ produced the most divergent chemical profile in sterile must, with glycerol, acetic acid, acetaldehyde, residual glucose, malic acid, ethyl acetate and higher alcohols being the key compounds affected by the prolonged activity of StbPK9. In pilot scale ferments, the indigenous S. cerevisiae produced twice as high levels of esters and higher alcohols compared to the commercial starter. Star. bacillaris further increased the levels of ethyl esters in the respective ferments. The use of a mixed S. cerevisiae/Star. bacillaris starter culture instead of S. cerevisiae alone enhanced the chemical complexity of Cretan local wine. The magnitude of differentiation was even higher when the addition of Star. bacillaris preceded that of S. cerevisiae. The highest divergence in analytical profiles was recorded between wines produced by native strain combinations and commercial S. cerevisiae. Present results show that the use of indigenous yeast formulations provides significant diversification to local wines, in line with the microbial terroir concept and recent observations that indigenous yeast strains may confer regional characters to wines.

Effect of high pressure processing on the survival of Salmonella Enteritidis and shelf-life of chicken fillets.

High pressure processing (HPP) is a preservation technology alternative to heat treatment that is mild for food, but effectively inactivates the spoilage microbiota and foodborne pathogens of several foods. The purpose of the current study was to evaluate the effect of HPP on Salmonella ser. Enteritidis, indigenous microbiota and shelf-life of chicken fillets. Chicken fillets were inoculated with S. Enteritidis at three different initial inocula (3, 5, 7 log CFU/g), packed under vacuum, treated or not with HPP (500 MPa/10 min) and stored at 4 and 12 °C. Total viable counts, S. Enteritidis, pseudomonads, Brochothrix thermosphacta, lactic acid bacteria, Enterobacteriaceae and yeasts/molds populations were determined in parallel with sensory analysis of non-inoculated samples. The HPP resulted in the reduction of the pathogen population below the detection limit of the enumeration method (0.48 log CFU/g), irrespective of the inoculum. During the shelf life of the HPP samples, the pathogens population remained below or near the detection limit of the enumeration method at both temperatures, except from the high inoculum case that an increase was observed at 12 °C. At the low inoculum level, the pathogen could not be detected with the enrichment method after the first storage days (2nd day for 4 °C and 0 day for 12 °C). The survival of Salmonella strains was assessed by pulsed field gel electrophoresis and it was shown that the survival of the different strains depended on the inoculum and storage temperature. Regarding the indigenous microbiota, Br. thermosphacta was reported for the first time to be the main spoilage microorganism that survived and dominated after the HPP. From the results it was evident that, HPP may enhance the safety and increase the shelf life (6 at 4 °C and 2 days at 12 °C) of chicken meat.

Development of microsatellite markers for Lachancea thermotolerans typing and population structure of wine-associated isolates.

Lachancea (Kluyveromyces) thermotolerans is an important member of the grape/wine yeast community with great technological potential for the wine industry. Although several molecular marker techniques have been developed for typing different yeast species, no one has been designed so far for L. thermotolerans. Here we present a simple and efficient method based on a multilocus SSR analysis for molecular typing and genetic diversity assessment of L. thermotolerans isolates. Following whole genome screening, five polymorphic microsatellite markers were selected and tested on a panel of grape isolates from different vineyards of two geographically separated viticultural zones, Nemea and Peza, in Greece. The SSR method proved quite discriminatory as compared to tandem repeat-tRNA-PCR, a fingerprinting method for typing non-Saccharomyces yeasts. Genetic analysis based on SSR data revealed a clear structure between the populations of the two zones. Furthermore, significant differences were also detected in a number of phenotypic characters of enological interest. A positive correlation was observed between phenotypic and genotypic diversity. Taking together, present results support the microbial terroir concept in the case of L. thermotolerans in Greece, which is an important prerequisite for the exploitation of selected genotypes as fermentation starters with region-specific characters.

The yeast Starmerella bacillaris (synonym Candida zemplinina) shows high genetic diversity in winemaking environments.

The yeast Candida zemplinina (Starmerella bacillaris) is frequently isolated from grape and wine environments. Its enological use in mixed fermentation with Saccharomyces cerevisiae has been extensively investigated these last few years, and several interesting features including low ethanol production, fructophily, glycerol and other metabolites production, have been described. In addition, molecular tools allowing the characterization of yeast populations have been developed, both at the inter- and intraspecific levels. However, most of these fingerprinting methods are not compatible with population genetics or ecological studies. In this work, we developed 10 microsatellite markers for the C. zemplinina species that were used for the genotyping of 163 strains from nature or various enological regions (28 vineyards/wineries from seven countries). We show that the genetic diversity of C. zemplinina is shaped by geographical localization. Populations isolated from winemaking environments are quite diverse at the genetic level: neither clonal-like behaviour nor specific genetic signature were associated with the different vineyards/wineries. Altogether, these results suggest that C. zemplinina is not under selective pressure in winemaking environments.

Genetic and technological characterisation of vineyard- and winery-associated lactic acid bacteria.

Vineyard- and winery-associated lactic acid bacteria (LAB) from two major PDO regions in Greece, Peza and Nemea, were surveyed. LAB were isolated from grapes, fermenting musts, and winery tanks performing spontaneous malolactic fermentations (MLF). Higher population density and species richness were detected in Nemea than in Peza vineyards and on grapes than in fermenting musts. Pediococcus pentosaceus and Lactobacillus graminis were the most abundant LAB on grapes, while Lactobacillus plantarum dominated in fermenting musts from both regions. No particular structure of Lactobacillus plantarum populations according to the region of origin was observed, and strain distribution seems random. LAB species diversity in winery tanks differed significantly from that in vineyard samples, consisting principally of Oenococcus oeni. Different strains were analysed as per their enological characteristics and the ability to produce biogenic amines (BAs). Winery-associated species showed higher resistance to low pH, ethanol, SO2, and CuSO4 than vineyard-associated isolates. The frequency of BA-producing strains was relatively low but not negligible, considering that certain winery-associated Lactobacillus hilgardii strains were able to produce BAs. Present results show the necessity of controlling the MLF by selected starters in order to avoid BA accumulation in wine.

Weissella uvarum sp. nov., isolated from wine grapes.

Two bacterial strains (B18BM42(T) and B18NM6) were recovered during a study of bacterial diversity on wine grapes (Vitis vinifera L.) from the Nemea region in Greece. Phylogenetic analysis based on 16S rRNA gene sequences placed the two strains within the genus Weissella, and found them to be most closely related to Weissella minor NRIC 1625(T) followed by Weissella viridescens NRIC 1536(T) (99.1 and 98.9% sequence similarity, respectively). The level of DNA-DNA relatedness between strains B18NM42(T) and W. minor NRIC 1625(T) or W. viridescens NRIC 1536(T) was 31.9 and 35.0%, respectively. The two novel strains could be genetically differentiated from their closest relatives by REA-PFGE (restriction enzyme analysis-pulse field gel electrophoresis), RAPD (randomly amplified polymorphic DNA) and rep-PC R analyses (repetitive sequence-based PCR). Physiological examination showed that the novel strains can be distinguished from phylogenetically related species by their ability to grow at 42 °C and by certain carbohydrate fermentations. Based on the evidence above, the affiliation of the two strains to a novel species with the proposed name Weissella uvarum sp. nov. is suggested. The type strain is B18NM42(T) ( =DSM 28060(T) =NCCB 100484(T)).