18 research outputs found

    Genetic and Technological Characterisation of Vineyard-and Winery-Associated Lactic Acid Bacteria

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    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, SO 2 , and CuSO 4 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

    New Frontiers in Wine Microbiology

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    The wine sectoris currently facing new challenges [...

    Από το σταφύλι στον οίνο: μοριακή ταυτοποίηση ζυμών και δυναμική εξέλιξη των μικροβιακών κοινοτήτων

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    The ability to assess and quantify the various populations within yeast communities is fundamental towards understanding the development and function of grape/wine ecosystems. Grape berries and juice constitute a dynamic yeast niche influenced by several geochemical constraints and certain biological attributes. Botrytis cinerea is an important grape pathogen that dramatically alters the physicochemical state of berries and consequently may affect the structure of yeast community. Nevertheless, the impact of Botrytis infection on the wine microflora is surprisingly poorly documented. In this study, the yeast flora associated with healthy or Botrytis-infected grapes cultivated in Attica and Arcadia regions and the yeast population dynamics during the alcoholic fermentation of the respective juice samples were assessed. Yeast isolates were identified at the species level by using Restriction Fragment Length Polymorphism (RFLP) and sequence analyses of the 5.8S ITS and the D1/D2 ribosomal DNA regions of cultivable yeasts. Community-level profiles were also obtained by direct analysis of fermenting samples through PCR amplification and Denaturing Gradient Gel Electrophoresis (DGGE) of 26S rDNA fragments. Structural divergences in yeast communities between samples of different sanitary state or geographical origin were observed. Botrytis infection encouraged a greater population and diversity of yeasts on grape berries, enriched with fermentative or spoilage species such as Metschnikowia pulcherrima and Issatchenkia occidentalis or Zygosaccharomyces bailii. In all cases, Botrytis infection severely perturbed the bioprocess of fermentation by dramatically altering species heterogeneity and succession during the course. At the beginning and the mid of fermentations Botrytis-affected samples possessed higher levels of biodiversity compared to their healthy counterparts, enriched with fermentative and/or spoilage species such as Zygosaccharomyces bailii and Issatchenkia spp. or Kluyveromyces dobzhanskii. Among others, strains belonging to the genus Kazachstania were isolated from Botrytis-affected fermenting grape juice. Phylogenetic analysis showed that the four strains represent a distinct species within the genus Kazachstania in close relation to Kazachstania zonata and Kazachstania gamospora. Electrophoretic karyotyping and physiological analysis supported the affiliation of the four strains to a novel species for which the name Kazachstania hellenica sp. nov. is proposed. Changes introduced by Botrytis invasion influenced final species dominance. Selection was not species-specific and two different populations could be recovered at the end of Botrytis-affected fermentations, i.e. Saccharomyces cerevisiae in samples from Arcadia and Z. bailii in samples from Attica. The governing of wine fermentations by Z. bailii is reported for the first time here and could elucidate the origins and role of this particularly spoilage microbe for the wine industry. This is the first survey to compare between healthy and Botrytis-affected grape and juice samples by using both culture-based and -independent molecular methods, in an attempt to further illuminate the complex yeast ecology of grapes and must fermentations.Οι ράγες και το γλεύκος των σταφυλιών αποτελούν ένα ιδιαίτερο οικοσύστημα, η δομή του οποίου καθορίζεται από διάφορους γεωχημικούς και βιολογικούς παράγοντες. Ο μύκητας Botrytis cinerea είναι ένα σημαντικός παθογόνος παράγοντας των σταφυλιών που επηρεάζει δραματικά τα φυσικοχημικά χαρακτηριστικά τους. Το ερώτημα που προκύπτει είναι κατά πόσο η αλλαγή στην φυσιολογική κατάσταση των βοτρυωμένων ραγών θα μπορούσε να επηρεάσει και αν ναι με ποιον τρόπο τη δομή της βιοκοινότητας των ζυμών στην επιφάνεια των σταφυλιών και ακολούθως τη δυναμική τους εξέλιξη κατά την διάρκεια της αλκοολικής ζύμωσης. Στην παρούσα μελέτη εξετάστηκαν οι ενδογενείς πληθυσμοί των ζυμών που αποικίζουν την επιφάνεια υγιών και προσβεβλημένων από Βοτρύτη σταφυλιών από αμπελώνες της Αττικής και της Αρκαδίας καθώς και η δυναμική τους εξέλιξη κατά την αλκοολική ζύμωση των αντίστοιχων γλευκών. Η ετερογένεια των ειδών προσδιορίστηκε με ανάλυση πολυμορφισμού μήκους θραυσμάτων περιορισμού (RFLP) σε συνδυασμό με ανάλυση αλληλούχησης των 5.8S ITS και D1/D2 περιοχών του ριβοσωμικού DNA των απομονωθέντων ζυμών. Επιπλέον, πρότυπα της βιοκοινότητας ανακτήθηκαν με άμεση ανάλυση των γλευκών μέσω της αλυσιδωτής αντίδρασης πολυμεράσης σε συνδυασμό με την βαθμιδωτή αποδιατακτική ηλεκτροφόρηση πηκτής (PCR-DGGE) χωρίς προηγούμενη καλλιέργεια των ζυμών σε τρυβλία. Δομικές διαφορές παρατηρήθηκαν στις κοινότητες των ζυμών μεταξύ δειγμάτων διαφορετικής φυσιολογικής κατάστασης (υγιή/βοτρυωμένα) ή γεωγραφικής προέλευσης. Η προσβολή από Βοτρύτη ευνόησε την ανάπτυξη υψηλού πληθυσμού ζυμών στην επιφάνεια των ραγών ενώ παράλληλα η ζυμοχλωρίδα εμπλουτίστηκε με νέα ζυμωτικά ή αλλοιωγόνα είδη όπως τα Metschnikowia pulcherrima και lssatchenkia occidentalis ή το Zygosaccharomyces bailii. Σε όλες τις περιπτώσεις, η προσβολή από τον Βοτρύτη διατάραξε σημαντικά και την πορεία της ζύμωσης μεταβάλλοντας δραματικά την ετερογένεια και τη διαδοχή των ζυμών. Κατά τα αρχικά και ενδιάμεσα στάδια των ζυμώσεων τα βοτρυωμένα δείγματα παρουσίαζαν υψηλότερα επίπεδα βιοποικιλότητας σε σχέση με τα αντίστοιχα υγιή. Η βιοκοινότητα των βοτρυωμένων δειγμάτων ήταν εμπλουτισμένη με ζυμωτικά και/ή αλλοιωγόνα είδη όπως τα Kluyveromyces dobzhanskii και lssatchenkia spp. ή το Zygosaccharomyces bailii. Μεταξύ άλλων, στελέχη ζυμών του γένους Kazachstania απομονώθηκαν κατά την διάρκεια αλκοολικών ζυμώσεων γλεύκους από βοτρυωμένα σταφύλια. Η φυλογενετική ανάλυση έδειξε ότι τα στελέχη αυτά αντιπροσωπεύουν ένα ξεχωριστό είδος μέσα στο γένος Kazachstania σε στενή σχέση με τα είδη Kazachstania zonata και Kazachstania gamospora. Η περαιτέρω ανάλυση καρυότυπου και οι μορφολογικοί-φυσιολογικοί χαρακτήρες υποστηρίζουν την κατάταξη των στελεχών σε ένα νέο είδος για το οποίο προτείνεται το όνομα Kazachstania hellenica sp. nov. Τα βοτρυωμένα δείγματα διαφοροποιούνταν από τα αντίστοιχα υγιή και ως προς την τελική επικράτηση είδους. Η επιλογή αυτή δεν ήταν εξειδικευμένη ως προς το είδος καθότι δυο διαφορετικοί πληθυσμοί ανακτήθηκαν από τα τελικά στάδια των ζυμώσεων. Πιο συγκεκριμένα, το Saccharomyces cerevisiae επικράτησε στα δείγματα της Αρκαδίας ενώ το Ζ. bailii στα δείγματα της Αττικής. Η κυριαρχία του Ζ. bailii σε αλκοολικές ζυμώσεις σταφυλιών αναφέρεται εδώ για πρώτη φορά. Τα αποτελέσματα που παρουσιάζονται βοηθούν στην αποσαφήνιση της προέλευσης και του ρόλου αυτού του ιδιαιτέρως αλλοιωγόνου μικροοργανισμού στην τεχνολογία παραγωγής οίνου. Η παρούσα εργασία αποτελεί την πρώτη συγκριτική μελέτη μεταξύ υγιών και βοτρυωμένων δειγμάτων με την εφαρμογή έμμεσων και άμεσων μοριακών μεθόδων ταυτοποίησης σε μια προσπάθεια να αποτυπωθεί η σύνθετη χλωρίδα του σχετιζόμενου με τον οίνο οικοσυστήματος

    Understanding Wine through Yeast Interactions

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    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

    Evaluation of Different Molecular Markers for Genotyping Non-<i>Saccharomyces</i> Wine Yeast Species

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    Wine quality is determined by the particular yeast strains prevailing at various stages of fermentation. Therefore, the ability to make an easy, fast, and unambiguous discrimination of yeasts at the strain level is of great importance. Here, the tandem repeat-tRNA (TRtRNA) method with the 5GAC or ISSR-MB primer sets and random amplified polymorphic DNA (RAPD) analysis with (GTG)3, R5, and RF2 oligonucleotides were tested on various non-Saccharomyces wine yeast species. The TRtRNA-PCR employing ISSR-MB showed the highest capacity in discriminating Lachancea thermotolerans and Metschnikowia pulcherrima isolates. RAPD with RF2 was the most efficient method in resolving Starmerella bacillaris isolates, although it produced few polymorphic bands. RAPD with R5 showed the highest capacity to discriminate among the Issatchenkia orientalis, Hanseniaspora guilliermondii, and Pichia anomala isolates. RAPD with either R5 or RF2 exhibited the highest ability to discriminate among the Torulaspora delbrueckii isolates. RAPD with (GTG)3 was the most discriminating method for the H. uvarum isolates. Here we concluded that both TRtRNA-PCR and RAPD-PCR offer rapid means for typing non-Saccharomyces species. However, each method performs better for a given species when paired with a particular primer set. The present results can be useful in wine research for the fast fingerprinting of non-Saccharomyces yeasts

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

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    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

    Grapevine Responses to Heat Stress and Global Warming

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    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

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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