Use of wild yeast and bacteria in wine fermentation Brewing Short Course & REG Meeting Diversity of yeast and bacteria Hanseniaspora Saccharomyces Oenococcus Pichia Candida Pediococcus Saccharomycodes Leuconostoc Dekkera Lactobacillus Debaromyces Acetobacter Metschnikowia … and many … and many more more • 11 to 20 different yeasts, every genus with sometimes more than 5 species • 5 to 7 relevant bacteria including several species • Survival factors in the vineyard are completely different from the cellar Survival factors of microorganisms In the vineyard In the cellar • High oxygen • Very little oxygen • No alcohol • Increasing ethanol level • Low sugar levels • High sugar levels • High temperatures • Low temperatures Spontanous fermentation without SO2 Candida stellata Hanseniaspora uvarum „wild yeast“ Metschnikowia pulcherrima Typical Flavor caused by wild yeast strains negative positive • band aid • Creamy character • oxidized (acetaldehyde) • Complex acidity composure • medically • Intense mouth-feel • volatile acidity and ethylacetate • Exotic fruit • hydrogensulfide • diverse fruity esters • „Mousy-taint“ impressions • Sulfur containing aroma • Lower extract and lactic flavors due compounds to flor yeast • sweaty, fleshly • Petroleum and kerosene aromas fruity, highly complex wines due to the contribution of different micro-organisms Does spontaneous fermentation lead to more interesting wines? Parallel spontaneous and inocculated fermentation of Riesling in wineries (yeast: R-HST, Lallemand) Aroma profile with fast fermentation Lemon 6 Bitterness Green apple 5 Body 4 Apple 3 • Spontaneous Fruity 2 Peach fermentation 1 considered more 0 fruit and body Acidity Quince • Positive compared to Green bean Pineapple control Honey Mango/Passionfruit Fermentation Strawberry Elder flower time ~ 30 days R-HST Spontaneous Aroma profile with slow fermentation Lemon Bitterness 6 Green apple 5 Body 4 Apple 3 • Pure culture yeast judged Fruity 2 Peach more fruity with 1 similar body 0 and bitterness Acidity Quince • Reduced effect of wild yeast flora Green bean Pineapple Fermentation Honey Mango/Passionfruit time ~ 60 days Strawberry Elder flower R-HST Spontaneous Cell count and sugar degradation 250 40 200 • The higher the cell count, 30 the faster the fermentation • The higher the temperature, 150 the faster the proliferation • The slower the proliferation, 20 the lower the final cell count 100 Sugar [g/L] cells [Mio./mL] ] [Mio./mL] cells 10 50 0 0 0 5 10 15 20 25 30 35 Sugar degradation 21°C Sugar degradation 18°C Sugar degradation 15°C Cell count 21°C Cell count 18°C Cell count 15°C Danger zone malolactic fermentation How great is the risk of unwanted bacterial activity in spontaneous fermentations? Comparison of 2 wines from the same must Mineralic Bitterness 7 Lemon 6 Mouthfeel 5 Apple 4 3 Body 2 Elder flower 1 0 Acidity Peach Buttery/Cheesy Passionfruit Green bean Honeymelon Green grass Smokey Small Scale Winery • Spontaneous malolactic fermentation can lead to buttery and Sauerkraut-like characters Effects of spontaneous MLF Mineralic 400 Bitterness Lemon 300 Mouthfeel Apple • Increase in smokey, green, 200 and buttery Body Elder flower 100 attributes 0 Acidity Peach • Increase in bitterness Buttery/Cheesy Passionfruit • Decrease in Green bean Honeymelon perceived fruity aromas Green grass Smokey Control (Siha 7) Small Scale 1 Small Scale 2 MLF during spontaneous fermentation 3.0 2.5 15°C a 2.0 15°C b 18°C a 1.5 18°C b 21°C a 1.0 Malic acid [g/L] 21°C b 0.5 0.0 0 5 10 15 20 25 30 35 40 Days • Speed of bacterial growth is depending on temperature • Only delay in MLF, no inhibition by lower temperatures Wild flora towards the end of fermentation Lactobacillus brevis Lactobacillus hilgardii Decreasing yaest activity Risk of unwanted MLF Conclusion I • With fast metabolic rates spontaneous fermentation can increase the complexity of wines • With slow metabolic rates (>60 days) spontaneous fermentations tend to mask the fruity character of wine • Cooler temperatures support growth of non-Saccharomyces yeasts and increase the risk of spoilage or stuck fermentation • Higher temperatures in the beginning increase the chance of early fermentation start and high Saccharomyces cell counts • The risk of unwanted MLF should be controlled with lysozyme; addition of SO2 would inhibit non-Saccharomyces flora and change the composition • Strategies to conduct spontaneous fermentation must be adapted to the desired style of wine! Does spontaneous fermentation enhance or mask the Terroir character of wine? • Wild yeast strains may contribute to complex aroma impressions, but also to off-flavor formation • Hypothesis: Terroir is influencing the composition of the wild yeast flora Why connecting Terroir and Yeast? Slope Sunshine Wind Terroir character Rain/Water Soil Cover crop Why connecting Terroir and Yeast? Almost the same factors have an impact on the yeast flora in the vineyard ! Sunshine Wind Rain/Water Soil Cover crop Experimental setup Normal hand- Vinification under harvesting common conditions Bottling in the wineries „sterile“ hand- harvesting Small scale „sterile“ hand- vinification under harvesting „sterile“ conditions Bottling Vinification under „sterile“ conditions up to 2 %vol. alcohol Inoculation of a pasteurized 3 different wines from one vineyard must – Spontaneous fermentation in the winery incl. Bottling Cellar-yeast (referred to as winery) – Spontaneous fermentation small scale only with yeast from the vineyard (small scale) – Inoculation of pasteurized must (inoculation) Sensory evaluation of all experiments 4 138 3 Winery wines Dried active yeast 132WGT 139 Except for a few wines, 2 134WGT grouping is possible due Small scale fermentations130 1 132A to sensory 134 properties F2 F2 %) (16,68 0 133 131 133A 132 -1 134A 131A 137A Inoculations 130A -2 133WGT 137 -3 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5 F1 (46,57 %) Biplot (Axes F1 and F2: 63.24 %) 4 Dried active yeast Winery wines 138 3 apple 132WGT 139 lemon 2 134WGT Sauerkraut peach 130 %) 1 cantaloup acid intensity 132A acidity body mouth134 feel 16.68 16.68 0 hay 133 131 smoky 132 F2 ( mineralic honey 133A -1 131A 134A Inoculations 137A bitter -2 Small scale fermentations 130A 133WGT 137 -3 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5 F1 (46.57 %) Small scale spontaneous fermentation Differences due to mineralic 7 terroir and site specific bitter lemon 6 yeasts (?) 5 body peach 4 3 2 mouthfeel honey melon 1 Sonnenschein 0 Reiterpfad Kastanienbusch acidity apple acid intensity honey Sauerkraut smoky hay • Only little differences in fruity characters Spontaneous fermentation in the wineries mineralic Differences by including 7 bitter lemon the „cellar yeast“ 6 5 body peach 4 3 2 mouthfeel honey melon 1 Sonnenschein 0 Reiterpfad Kastanienbusch acidity apple acid intensity honey Sauerkraut smoky hay • Differences between fruity characters • No differences in negative parameters Why do winery wines produce other profiles than „sterile“ small scale fermentation? • Similar sensory results in four different vintages • Hypothesis: yeast flora from the cellar dominates sensory profile. • Proof: Identification and quantification of the yeast species involved Identification of species with DGGE Extract and wash DNA Polymerase Chain Harvested yeast DNA Mix of Reaction different species Quantify by Compare with evaluating Single cultures fluorescence intensity • Differentiation on species level DGGE procedure according to • No absolute quantification; educated guess Mills et al., 2000 Yeast dynamics in spontaneous fermentation 100 9 90 8 80 7 70 6 60 5 50 4 %vol. genera [%] 40 3 30 20 2 10 1 0 0 day 2 day 4 day 6 day 8 day 10 day 13 Pichia Hanseniaspora Hansenula Saccharomyces Alcohol • At 18°C fermentation starts rapidly • Saccharomyces takes over after 6 days • Part of Hanseniaspora remains active for a longer period • Delayed onset of fermentation doubles growth time for „wild yeast“ ! Spontaneous flora without SO2 addition • Composition of yeast population changes with increasing ethanol concentration • Above 4 %vol. ethanol only Saccharomyces active ? Alkoholgehalt 4 % vol. Hansenula / Candida / Kloeckera / Saccharomyces Pichia Metschnikowia Hanseniaspora Modified from Großmann et al. 2005 Conclusions II • Spontaneous fermentation show higher batch-to-batch variation • Cooler temperatures favor growth of wild yeast and yields a different aroma profile • Composition of the wild yeast flora varies only slightly between sites • Spontaneous fermentations in the wineries had more more Saccharomyces yeasts than those fermented under sterile conditions (cellar yeast flora) • The individual cellar yeast flora seems to dominate the final sensory properties of wines from spontaneous fermentations and not the flora of individual vineyard sites Impact of yeast composition on the flavor profile of wine? • Hypothesis: Variation among volatiles in finished wines is related to yeast composition during fermentation. • Use of analytical fingerprints to display possible differences Analytical „Fingerprints“ • Comprehensive two dimensional gas chromatography yields high separation of complex mixture of volatiles • Fingerprinting method to display differences between wines Volatiles explaining the most variation Variables (Axes F1 and F2: 79.09 %) 1 74 89 19 53 0.75 • Selection of 23 compounds out of 283 0.5 160 43 based on explained 50 250 186 variability 169208 65124 0.25 185 116 %) 0 24.21 24.21 F2( 55 -0.25 -0.5 Mainly: Alcohols -0.75 Esters -1 Hydrocarbons -1 -0.75 -0.5 -0.25 0 0.25 0.5 0.75 1 F1 (54.88 %) Variation