Use of Wild Yeast and Bacteria in Wine Fermentation
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 among wines due to volatiles
• Dried active yeast and inoculations together 132WGT Winery wines – Influence of wild yeast strains less important – Dominance of Dried active yeast Saccharomyces 131 • „pure“ spontaneous 134WGT 139 fermentation batches on 134 132A one side • More variation among 130 winery wines than 138 among „sterile“ batches 133 131A – Impact of cellar flora 133WGT 137 increases 132 134A 130A differences between wines 133A 137A
6 4 Inoculations2 0 2 4 Small6 Scale spontaneous fermentation
How to make it work in your cellar?
What to recommend to the winemakers? Ways to conduct spontaneous fermentation
„Real“ spontaneous fermentation random population of microorganisms from vineyard and cellar
„Russian Roulette“ Ways to conduct spontaneous fermentation
„Real“ spontaneous Conducted fermentation random population of spontaneous microorganisms from vineyard and cellar fermentation I Yeast from successful spontaneous fermentation used to inoculate other batches
Ways to conduct spontaneous fermentation
„Real“ spontaneous Conducted spontaneous fermentation fermentation I Conducted spontaneous random population of Yeast from successful fermentation I microorganisms from spontaneous vineyard and cellar fermentation used to (Pied de Cuve) inoculate other batches Early harvesting of healthy grapes to establish a starter culture
for further inoculation
Ways to conduct spontaneous fermentation
„Real“ spontaneous Conducted spontaneous Conducted fermentation fermentation I spontaneous „optimized“ random population of Yeast from successful fermentation I (Pied de spontaneous microorganisms from spontaneous Cuve) vineyard and cellar fermentation used to Early harvesting of fermentation inoculate other batches healthy grapes to establish a starter spontaneous start culture for further inoculation followed by inoculation with dried active yeast „Diversity due to wild yeast, safety by using Saccharomyces“ Be on the wild or safe side of fermentation
„Real“ spontaneous Conducted spontanous Conducted spontanous „optimized“ spontaneous fermentation fermentation I fermentation I (Pied de fermentation random population of Yeast fromNo successful significant differenceCuve) spontaneous start followed by microorganisms from spontanouscompared to driedEarly harvestingactive of inoculation with dried active vineyard and cellar fermentation used to healthy grapes to yeast inocculate otheryeast batches fermentation establish a starter culture for further inocculation
High Risk of stuck fermentation Low
• „optimized“ spontaneous fermentation including established cellar yeast recommendable • => influence of cellar yeast seems to be much more important for the final flavor composition than wild yeast from the vineyards Yeast and bacteria co- inoculation
Focusing on diacetyl Diacetyl in wine
• Dicarbonyl compound with distinct buttery flavor • Sensory threshold 0.2 mg/L – 2.8 mg/L depending on wine matrix • Higher concentrations are considered as off-flavors especially in white wines • Formation and degradation by yeast and bacteria possible final concentration as a result of complex metabolic activity • Literature relates diacetyl formation to citrate degradation during MLF Use of bacteria strains that lack the responsible enzyme (citrate lyase negative, CLneg) Solution so far…
14 250 VINIFLORA CiNe 12 200 (Diacetyl) 10 Lalvin VP41 (Diacetyl) 8 150 6 100 4 VINIFLORA CiNe 50 (Citrat) 2 0 0 Citrat[mg/L] Lalvin VP41 (Citrat) Diacetyl [mg/L] Diacetyl 0 5 10 15 20 Time [days]
• CiNe (Eaton Begerow, Germany) leaves citrate untouched • VP 41 (Lallemand, Canada) is supposed to metabolize citrate without diacetyl formation • … but some of the wines contained diacetyl well above the sensory threshold! Citric acid degradation
O OH O O
HO OH OH Uptake towards the end of MLF From yeast sugar metabolism
Release Bacteria during amino acid synthesis Yeast (valine) Constant release O throughout growth
O Uptake and Release of reduced diacetyl reduction to strain depending 2,3-butanediol
adapted from Quintans et al., 2008 Experimental setup
Fermented with yeast Pinot blanc CY 3079 (Lallemand) 2011 and 2012 Divided in simultaneous and consecutive MLF Trials run in duplicates
CiNe (Eaton‘s CLNX VP 41 ALPHA BETA Begerow) (FermControl) (Lallemand) (Lallemand) (Lallemand) Control Medium Citrate lyase negative Strong production no MLF production Influence of cell concentration
4 1,00E+08 4 1,00E+07 3 1,00E+06 • no MLF with less 3 1,00E+05 6 2 1,00E+04 than 10 CFU/mL Malic acid 2 1,00E+03 1 CFU 1,00E+02 CFU/mL 1 1,00E+01 Malic acid [g/L] 0 1,00E+00 • some commercial 0 5 10 15 20 25 30 35 vinification time [days] products contain less viable cells 5 1,00E+09 4 1,00E+08 4 1,00E+07 3 1,00E+06 • delayed MLF 3 1,00E+05 2 1,00E+04 2 Malic acid 1,00E+03 1 1,00E+02 CFU/mL • risk of undesired 1 CFU 1,00E+01 Malic acid [g/L] 0 1,00E+00 flavor development 0 5 10 15 20 25 30 35 vinification time [days] Accumulation of diacetyl
Simultaneous Inoculation 5 O. oeni 4.73 4 Start citrate 3 2.97 Yeast degradation 2 1.37 1 Diacetyl [mg/L] Diacetyl 0.30 0 -1 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 vinification [days] ALPHA BETA CREAM control
• No reduction of the diacetyl formed by bacteria accumulation • Reason: low pH prior to fermentation (2.9) delays MLF and citrate degradation higher final level of diacetyl Diacetyl concentration during vinification
0.7 1.00E+08
0.6
1.00E+07 0.5 O. oeni Diacetyl O. oeni 0.4 Diacetyl control 1.00E+06 CFU O.oeni 0.3 Diacetyl [mg/L]
0.2 CFU/mL 1.00E+05
0.1 O. oeni and S. cerevisiae 0.0 1.00E+04 0 2 4 6 8 10 12 14 vinification [days] • First increase induced by both yeast and bacteria • Second increase mainly caused by O. oeni • Correlation between biomass production and diacetyl accumulation Gene expression analysis (qRT-PCR)
Citrate transporter (maeP)
Citrate lyase (citE)
Lactate dehydrogenase (ldh) House-keeping gene
Acetolactate synthase (alsS)
Acetolactate decarboxylase (alsD)
Acetoin reductase (butA2) adapted from Quintans et al., 2008 Gene expression analysis (qRT-PCR)
alsS
Sugar/pyruvate induced
Citrate induced
• first alsS over-expression induced by sugar metabolism • second alsS over-expression induced by citrate degradation The central role of pyruvate
Pyruvate addition The central role of pyruvate
Degradation von citrate also leads to an increase in pyruvate Diacetyl degradation by yeast
60
50
40
Diacetyl 0 mg/l 30 Diacetyl 10 mg/L Diacetyl 20 mg/L
Diacetyl [mg/L] 20 Diacetyl 50 mg/L 10 control
0 0 2 4 6 8 10 vinification [days]
• Diacetyl addition does not affect growth or MLF rate, even in high concentrations • The dried active yeast strain CY 3079 (Lallemand) is able to reduce 50 mg/L diacetyl in the first two days of fermentation Diacetyl degradation by yeast
Independent of the start concentration 2 mg/L diacetyl remain due to bound diacetyl (to sulfur dioxide, amino acids and others) What does that mean for the winemaker?
• Only a rapid MLF can inhibit diacetyl accumulation • At least 106 CFU/mL are required to start MLF cell count after inoculation if commercial preparation is unknown
• Initial medium must be suitable for MLF (pH at least 3.1-3.3) higher pH might favor growth of other bacteria (e.g. Pediococcus damnosus) and therefore diacetyl formation lower pH delays MLF and therefore favors diacetyl formation
• Once diacetyl is formed curative measures like fresh yeast fining may be applied
Summary and conclusion
Diacetyl accumulation shows two characteristic peaks: first increase caused by yeast and bacteria, second increase due to bacteria only
O. oeni S. cerevisiae
Correlation between biomass increase and Dried active yeast (CY 3079) is able to diacetyl accumulation observed (more reduce 50 mg/L diacetyl in two days bacteria produce more diacetyl) Independent from the starting level, Over-expression of alsS gene due to high residual diacetyl of 2 mg/L always remains; concentrations of pyruvate as well as citrate reason possibly bound diacetyl Formation of diacetyl by citrate degradation only can be excluded