Aspasia Nisiotou

Yeast Community Structures and Dynamics in Healthy and Botrytis-Affected Grape Must Fermentations

ABSTRACT Indigenous yeast population dynamics during the fermentation of healthy and Botrytis-affected grape juice samples from two regions in Greece, Attica and Arcadia, were surveyed. Species diversity was evaluated by using restriction fragment length polymorphism and sequence analyses of the 5.8S internal transcribed spacer and the D1/D2 ribosomal DNA (rDNA) regions of cultivable yeasts. Community-level profiles were also obtained by direct analysis of fermenting samples through denaturing gradient gel electrophoresis of 26S rDNA amplicons. Both approaches revealed structural divergences in yeast communities between samples of different sanitary states or geographical origins. In all cases, Botrytis infection severely perturbed the bioprocess of fermentation by dramatically altering species heterogeneity and succession during the time course. At the beginning and middle of fermentations, Botrytis-affected samples possessed higher levels of biodiversity than their healthy counterparts, being enriched with fermentative and/or spoilage species, such as Zygosaccharomyces bailii and Issatchenkia spp. or Kluyveromyces dobzhanskii and Kazachstania sp. populations that have not been reported before for wine fermentations. Importantly, Botrytis-affected samples exposed discrete final species dominance. Selection was not species specific, and two different populations, i.e., Saccharomyces cerevisiae in samples from Arcadia and Z. bailii in samples from Attica, could be recovered at the end of Botrytis-affected fermentations. The governing of wine fermentations by Z. bailii is reported for the first time and could elucidate the origins and role of this particular spoilage microbe for the wine industry. This is the first survey to compare healthy and Botrytis-affected spontaneous fermentations by using both culture-based and -independent molecular methods in an attempt to further illuminate the complex yeast ecology of grape must fermentations.

Yeast heterogeneity during spontaneous fermentation of black Conservolea olives in different brine solutions

Aims:  To assess the yeast community structure and dynamics during Greek‐style processing of natural black Conservolea olives in different brine solutions.

Yeast Populations Residing on Healthy or Botrytis-Infected Grapes from a Vineyard in Attica, Greece

ABSTRACT The yeast flora associated with healthy and Botrytis-infected grapes was assessed. Molecular identification methods assigned isolates to six genera and nine species. For the first time Hanseniaspora opuntiae was encountered as an inhabitant of the grape ecosystem. By using DraI, an informative restriction fragment length polymorphism pattern was generated to distinguish H. opuntiae from the closely related organism Hanseniaspora guilliermondii. Botrytis infection resulted in a larger population and greater diversity of yeasts enriched with fermentative or spoilage species.

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.

Bacterial species associated with sound and Botrytis-infected grapes from a Greek vineyard.

Grape bacterial microbiota plays central roles in the quality of grapes and wine, yet its diversity remains poorly described. In the present study, bacterial species associated with sound and Botrytis-infected grapes of two cultivars originating from the same vineyard were assessed. Isolates were identified by PCR-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) and sequence analysis of partial 16S rRNA gene. Comparable counts were recorded between Botrytis-infected and sound grape samples. In all cases, the majority of isolates belonged to different species of Enterobacteriaceae. The dominant species in the vineyard was Klebsiella oxytoca that was found in different combinations with Citrobacter freundii, Enterobacter spp., Erwinia sp., Pantoea dispersa, Tatumella ptyseos or other species. In fermenting musts, those populations declined while other species evolved, like Lactobacillus plantarum and Enterobacter ludwigii. Populations in botrytised samples persisted longer during spontaneous fermentations. Present study suggests that bacterial diversity on grapes may be wider than previously described.

Performance of two potential probiotic Lactobacillus strains from the olive microbiota as starters in the fermentation of heat shocked green olives

The performance of two potential probiotic Lactobacillus strains from olive microbiota, namely L. pentosus B281 and L. plantarum B282 was assessed as starter cultures in Spanish-style fermentation of heat shocked green olives cv. Halkidiki. Two different initial salt levels were studied, 10% (w/v) and 8% (w/v) NaCl, and the brines were inoculated with (a) L. pentosus B281, (b) L. plantarum B282, and (c) a mixture of both strains. A spontaneous fermentation was also taken into account as control treatment. Prior to brining, olives were heat shocked at 80 °C for 10 min to reduce the level of the indigenous microbiota on olive drupes and facilitate the dominance of the inoculated cultures. Microbiological, physicochemical and sensory analyses were conducted throughout fermentation. The composition of LAB population and the evolution of added inocula were assessed by Pulsed Field Gel Electrophoresis (PFGE). The final population of LAB was maintained above 6 log cycles in olive flesh. Both L. pentosus B281 and L. plantarum B282 were able to dominate over indigenous LAB, albeit strain B281 exhibited higher recovery percentages (100 or 94.7% for B281 and 58.8% or 55.0% for B282 in 10% or 8% NaCl, respectively). L. pentosus B281 also dominated over L. plantarum B282, when the two strains were co-inoculated in olive fermentations. The sensory assessment showed higher preference for inoculated fermentations of L. pentosus and L. plantarum separately in 8% NaCl, followed by the L. plantarum in 10% NaCl. The present study showed that probiotic strains L. pentosus B281 and L. plantarum B282, may offer a great potential for use as functional starter cultures in olive fermentation and deliver a promising probiotic food to the consumer.

Kazachstania hellenica sp. nov., a novel ascomycetous yeast from a Botrytis-affected grape must fermentation.

Four ascomycetous yeast strains (D4W13, D9W2, D9W4 and D9W17T) were isolated from Botrytis-affected fermenting grape juice originating from Attica Province, Greece. Phylogenetic analysis of rRNA gene sequences (18S, 26S and 5.8S-ITS) showed that the four strains represent a distinct species within the genus Kazachstania, closely related to Kazachstania zonata NBRC 100504T and Kazachstania gamospora NBRC 11056T. Electrophoretic karyotyping and physiological analysis support the affiliation of the four strains in a novel species for which the name Kazachstania hellenica sp. nov. is proposed, with D9W17T (=CBS 10706T=NBRC 103637T) as the type strain.

Effect of wine-based marinades on the behavior of Salmonella Typhimurium and background flora in beef fillets

The aim of this study was to evaluate the wine-based marinades to control the survival of acid-adapted and non-adapted Salmonella Typhimurium and background flora of fresh beef stored aerobically or under modified atmosphere. Beef slices were inoculated with a 3-strain cocktail of acid-adapted or non-adapted Salmonella Typhimurium strains DT 193, 4/74 and DSM 554 and marinated by immersion in wine (W) or wine supplemented with 0.3% thyme essential oil (WEO), for 12h at 4°C. Marinated slices were then stored under air or modified atmosphere conditions at 5°C. S. Typhimurium and background flora were followed for a 19-day period of storage. S. Typhimurium individual strains were monitored by pulsed field gel electrophoresis. Marination with wine significantly (P<0.05) reduced the background flora compared to the control (non-marinated). Furthermore, immersion of fillets in W or WEO marinades for 12h significantly (P<0.05) reduced the levels of S. Typhimurium compared to the non-marinated (control) samples by 1.1 and 1.4logCFU/g or 2.0 and 1.9logCFU/g for acid-adapted and non-adapted cells, respectively. Acid-adapted cells were more susceptible (P<0.05) to the addition of thyme essential oil in the wine marinade. The epidemic multi-drug resistant DT 193, the 4/74 and DSM 554 strains survived marination (for both W and WEO) and were detected at about similar proportions as revealed by PFGE results. Present results indicate that wine-based marinades are efficient, from a safety and shelf life stand point, in reducing pathogens levels as well as the background beef flora.

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)).