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Native Fermentations: Managing the Microbiota Linda F. Bisson Department of Viticulture and Enology University of California, Davis, CA Wine Flavor 101 July 28, 2015 Outline of Presentation Introduction to Native Fermentations – Issues in use of native microbiota – The important variables Microbial Diversity of Native Fermentations Optimizing the Microbiota Native Fermentations: Rely on microbial flora of grapes and wineries to conduct fermentation No deliberate inoculation with commercial strains May be inoculated from an existing native fermentation The Positive Impacts of Native Microbiota Complexity: Unique flavors and aromas Enhancement of varietal character – Excretion of hydrolases – Degradation of aroma precursor molecules Slower primary fermentation – Reduced temperatures – Less volatilization and entrainment in carbon dioxide stream – Stress characters produced by Saccharomyces The Negative Impacts of Native Microbiota Arrest of fermentation Spoilage character formation – – – – Ethyl acetate S-taints Volatile phenols Depends upon organisms present Loss of varietal character – Degradation of esters via esterases – Release of too high of a concentration of aroma molecules so aging of wine is impacted Native Fermentations: Many Styles 100% native; no additions of any microbes Native yeast but with ML inoculation? Native Saccharomyces only or native nonSaccharomyces? Partial natives: – At some point inoculated with a commercial strain of Saccharomyces – A blend of commercial and native fermentations – Conducted in tanks previously used for commercial yeast Manipulated microbiota natives Manipulated Microbiota Sulfur dioxide or other antimicrobial agent used Temperature of pre-incubation/fermentation pH adjustment or selection Level of aeration Nutrient addition practices Inoculation with non-Saccharomyces yeasts and bacteria Source of Native Flora Vineyard Winery Early Both are a source of microbes in season grape microbiota is most important but as microbes become established on winery surfaces, winery microbiota become more important And it all depends upon sanitation practices Successful Natives: The Most Important Variables Condition of the fruit Organisms on fruit/coming in with fruit Time of harvest Processing conditions Juice/Must amendments pH Sulfite addition Temperature Condition of the Fruit Damaged Clusters – Rot amplifies acetic acid bacteria – Mold metabolites may be present Uneven Ripening/Raisining – Leads to differences in flora on the surfaces of the fruit Presence of Material Other than Grape – Soil, leaves, bark: all contain microbes – Some of these microbes can persist early in fermentation Contribute to Winery Flora Time of Harvest During ripening the berry surface flora change – More seepage from the berry providing nutrients – Yeast population continues to increase in relative numbers postveraison Aerobic basidiomycetes are replaced by the fermentative ascomycetes – Consume available oxygen rapidly – Create localized anaerobic zones – Produce toxic waste products Aerobic bacilli and pseudomonads replaced by acetic and lactic acid bacteria – Produce toxins and toxic waste products Processing Conditions Berry Integrity – Partially damaged berries encourage growth of surface flora – Presence of juice will lead to growth and more rapid oxygen consumption Skin Contact – Allows greater extraction of surface flora – Allows leaching of berry biofilms Oxygen Exposure – Favors persistence of the aerobic microbes – Leads to production of acetic acid Temperature Juice/Must Amendments Nutrient Additions – Which population is being fed? – Partial or complete nutrients? Acidity Adjustments – Acids are nutrients for some microbes and allow adaptation to sugars Water Addition – Reduces osmolarity – High osmolarity favors Zygosaccharomyces pH Very low pH (less than 2.5) favors Brettanomyces Many bacteria require pH values above 3.5 in order to grow All berry fermentative yeasts can grow above pH 2.5 Sulfite Addition Use of antimicrobials impacts flora Sulfite impacts bacteria more than wild yeasts Level of sulfite used varies from low to high (25 ppm to 100 ppm) Effectiveness depends upon pH Temperature Of harvest, processing, fermentation In general warmer temperatures favor bacteria Cold soaks favor wild yeast populations Saccharomyces combines the tolerances of yeasts with a preference for slightly warmer temperatures than wild yeasts The combination of ethanol and higher temperature leads to dominance of fermentations by Saccharomyces MICROBIAL DIVERSITY OF NATIVE FERMENTATIONS The Native Microbiota: Bacteria Species present depends upon time of harvest Some vineyard specificity (Pediococcus) but in general the bacteria found are fairly consistent across vineyards and regions Variation in relative numbers depends upon several factors – Vineyard management practices – Insect vectors – pH of fruit – Leakiness of fruit The Desirable Bacteria Oenococcus, but this is rare Lactics that are not able to dominate the fermentation – Fastidious nutrient requirements – High sensitivity to sulfite – Low pH intolerant The Undesirable Bacteria Spoilage lactic acid bacteria – Off-characters – Negative impacts on fermentation Spoilage acetic acid bacteria – Negative impacts on fermentation May come from grapes, may come from insects associated with grapes The Native Microbiota: Yeasts Show strong impacts of maturity Show differences by climate Show seasonal effects Show differences by varietal Show site effects More strongly influenced by insect trafficking The Most Common Yeasts of the Berry Surface Yeast Kloeckera apiculata/ Hanseniaspora uvarum Candida species Issatchenkia Metschnikowia Pichia species Basidiomycetes % of Total Isolates 65-80 5-10 5-10 5-10 10-20 10-20 Saccharomyces Not often isolated from vineyards Depends upon practice of dumping yeast lees in vineyard Depends upon level of rot Is present but in very low numbers, one out of ten million yeast isolates Even at these low numbers will dominate wine fermentations Diversity of Saccharomyces Well-established variation in genome of wild and commercial strains Studies have shown the presence of recurring genotypes in specific wineries: winery-specific strains Studies in vineyards have shown great diversity of Saccharomyces but no vineyard-specific genotypes Some studies show some regionality of yeast genotypes, others do not The amount of genetic variation of wine strains is extreme with many only being able to be isolated in a single vintage Management of Native Microbiota Sulfur dioxide, sulfur dioxide, sulfur dioxide Judicious feeding of populations Need a microscope Analysis of species of microbiota present (and SO2 sensitivity) Avoid cluster rot; high insect infestation of fruit during processing Conclusions Sulfur dioxide trials of natives are key to optimizing positive impacts Some vineyards will never yield optimal native wines Microbial monitoring is advisable: organisms, both good and bad, grow exponentially
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