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.

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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.

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.

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.

Vineyards in Nemea, the most important viticultural zone in Greece, were surveyed for indigenous non-Saccharomyces (NS) yeasts of enological potential. NS populations were isolated from the final stage of alcoholic fermentation and identified by a range of molecular methods. The enological profiles of Hanseniaspora guilliermondii, H. osmophila, Lachancea thermotolerans, Starmerella bacillaris and Torulaspora delbrueckii strains were evaluated. Significant interspecies variation was observed in fermentation kinetics. H. osmophila and T. delbrueckii showed the highest capacity for prompt initiation of fermentation, while S. bacillaris achieved a higher fermentation rate in the second half of the process. Significant differences were also observed in the chemical parameters of NS strains. S. bacillaris SbS42 and T. delbrueckii TdS45 were further evaluated in mixed-culture fermentations with Saccharomyces cerevisiae. NS strains achieved lower population densities than S. cerevisiae. SbS42 exhibited a higher death rate than TdS45. The chemical profiles of different ferments were separated by principal component analysis (PCA). Both NS strains were associated with lower levels of ethanol, when compared to single S. cerevisiae inoculation. TdS45 increased the ethyl acetate levels, while SbS42 caused a different production pattern of higher alcohols. This is the first report to explore the enological potential of NS wine yeast populations from Nemea. Based on prominent enological traits identified, the selected S. bacillaris and T. delbrueckii strains may be further exploited as co-culture starters for improving the quality and enhancing the regional character of local wines.Although NS yeasts initiate fermentation, most of them are not detectable at the end of the course, either because they are ethanol-intolerant or incapable of withstanding microbial antagonism [8,9]. However, in various later studies it has been shown that several NS species, such as I. occidentalis, L. thermotolerans, M. pulcherrima, Starmerella bacillaris (synonym C. zemplinina) and Z. bailii, were able to maintain high viable populations (up to 6-7 log CFU/mL) for longer periods than previously thought and were even isolated from the final stages of the alcoholic fermentation [4,7,10,11]. These species may play a crucial role in winemaking, due to the production of important metabolites that shape the flavor and contribute to the style of wines [1,12]. These metabolites include both "volatile" compounds, like aldehydes and esters, and "non-volatile" compounds, like glycerol, acetic acid and lactic acid.In this context, there is a growing interest in the use of well-selected NS yeasts such as Torulaspora, Candida, Metschnikowia and Lachancea/Kluyveromyces species, which when combined with S. cerevisiae in mixed-culture starters can improve wine fermentation and final product quality [13]. Several NS strains have been produced commercially as active dry yeasts and novel starter cultures are continuously developing to address specific challenges in m...

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.

Low alcohol wines (≤10.5% vol) are novel products that have gradually been gaining the consumers’ and market’s interest over the last decade. Taking into account the technological properties of immobilized cell systems alongside with the commercial need for dry cultures, the aim of the present study was to assess the suitability of thermally-dried immobilized kefir cells on DCM, apples pieces, and grape skins in low alcohol wine production. Storage of thermally-dried kefir culture in various temperatures (−18, 5, and 20 °C) resulted in high viability rates for immobilized cells (up to 93% for yeasts/molds immobilized on grape skins and stored at −18 °C for 6 months). Fermentation activity was maintained after storage in all cases, while high operational stability was confirmed in repeated batch fermentations for a period of 6 months. Principal Component Analysis (PCA) revealed that the fermentation temperature rather than the state of kefir culture affected significantly volatiles detected by Head Space Solid-Phase Microextraction Gas Chromatography–Mass Spectrometry analysis. Notably, all new products were of high quality and approved by the sensory panel.