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.

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.

Citrus medica and Cinnamomum zeylanicum Essential Oils as Potential Biopreservatives against Spoilage in Low Alcohol Wine Products.

Low alcohol wine is a new entry in the global wine market, due to the increase in consumers' concern for health, economic and modern lifestyle issues. As low alcohol products are prone to spoilage, the adoption of natural-derived products with antimicrobial activity as biopreservatives seems to be an intriguing alternative. Thus, the aim of the present study was to investigate the possible antimicrobial properties of Citrus medica and Cinnamomum zeylanicum essential oils (EOs) and assess their commercial prospective in the wine industry. The main constituents identified by GC/MS analysis were limonene (38.46%) and linalool (35.44%) in C. medica EO, whereas trans-cinnamic-aldehyde (63.58%) was the dominant compound in C. zeylanicum EO. The minimum inhibitory (MIC), non-inhibitory (NIC) and minimum lethal concentration (MLC) values against common wine spoilage microbes were initially determined. Subsequently, their efficiency was further validated in low alcohol (~6% vol) wines, either separately or in combination at 0.010% (v/v), as well as in wines deliberately inoculated with Gluconobacter cerinus, Oenococcus oeni, Pediococcus pentosaceus, Dekkera bruxellensis, Candida zemplinina, Hanseniaspora uvarum, Pichia guilliermondii or Zygosaccharomyces bailii. EO addition led to considerable spoilage and microbial growth delay during storage at room or refrigerated temperature, suggesting their potential use as wine biopreservatives.

Freeze-Dried Immobilized Kefir Culture in Low Alcohol Winemaking.

Low alcohol wines represent a rising trend in the global market. Since for ethanol removal, certain physicochemical methods that negatively affect wine quality are applied, the aim of this present study was to evaluate the efficiency of freeze-dried, immobilized kefir culture on natural supports (apple pieces, grape skins and delignified cellulosic material) in low alcohol winemaking at various temperatures (5-30 °C). Initially, genetic analysis of kefir culture was performed by Next Generation Sequencing. There was an immobilization of kefir culture on grape skins-enhanced cell survival during freeze-drying in most cases, even when no cryoprotectant was used. Simultaneous alcoholic and malolactic fermentations were performed in repeated batch fermentations for >12 months, using freeze-dried free or immobilized cells produced with no cryoprotectant, suggesting the high operational stability of the systems. Values of great industrial interest for daily ethanol productivity and malic acid conversion [up to 39.5 g/(Ld) and 67.3%, respectively] were recorded. Principal Component Analysis (PCA) showed that freeze-drying rather than the fermentation temperature affected significantly minor volatiles. All low alcohol wines produced were accepted during the preliminary sensory evaluation.

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.

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.