use of wild yeast and bacteria in wine...
TRANSCRIPT
Use of wild yeast and bacteria in wine fermentation
Brewing Short Course &
REG Meeting
Diversity of yeast and bacteria
• 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
… and many more … and many more
Saccharomyces
Lactobacillus
Pediococcus
Oenococcus
Acetobacter
Leuconostoc
Candida
Metschnikowia
Pichia
Debaromyces
Dekkera
Saccharomycodes
Hanseniaspora
Survival factors of microorganisms
In the vineyard • High oxygen • No alcohol • Low sugar levels • High temperatures
In the cellar • Very little oxygen • Increasing ethanol level • High sugar levels • Low temperatures
Hanseniaspora uvarum
Metschnikowia
pulcherrima
Candida stellata
Spontanous fermentation without SO2
„wild yeast“
Typical Flavor caused by wild yeast strains
• band aid • oxidized (acetaldehyde) • medically • volatile acidity and ethylacetate • hydrogensulfide • „Mousy-taint“ impressions • Lower extract and lactic flavors due
to flor yeast • sweaty, fleshly • Petroleum and kerosene aromas
• Creamy character • Complex acidity composure • Intense mouth-feel • Exotic fruit • diverse fruity esters • Sulfur containing aroma
compounds fruity, highly complex wines due
to the contribution of different micro-organisms
negative positive
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
• Spontaneous fermentation considered more fruit and body
• Positive compared to control
Fermentation time ~ 30 days
0 1 2 3 4 5 6
Lemon Green apple
Apple
Peach
Quince
Pineapple
Mango/Passionfruit
Elder flower Strawberry
Honey
Green bean
Acidity
Fruity
Body
Bitterness
R-HST Spontaneous
Aroma profile with slow fermentation
• Pure culture yeast judged more fruity with similar body and bitterness
• Reduced effect of wild yeast flora
Fermentation time ~ 60 days
0 1 2 3 4 5 6
Lemon
Green apple
Apple
Peach
Quince
Pineapple
Mango/Passionfruit
Elder flower Strawberry
Honey
Green bean
Acidity
Fruity
Body
Bitterness
R-HST Spontaneous
0
50
100
150
200
250
0 5 10 15 20 25 30 350
10
20
30
40
cells
[Mio
./mL]
]
Sugar degradation 21°C Sugar degradation 18°C Sugar degradation 15°CCell count 21°C Cell count 18°C Cell count 15°C
Cell count and sugar degradation
• The higher the cell count, the faster the fermentation
• The higher the temperature, the faster the proliferation
• The slower the proliferation, the lower the final cell count
Suga
r [g/
L]
Danger zone malolactic fermentation
How great is the risk of unwanted bacterial
activity in spontaneous fermentations?
Comparison of 2 wines from the same must
• Spontaneous malolactic fermentation can lead to buttery and Sauerkraut-like characters
01234567
MineralicLemon
Apple
Elder flower
Peach
Passionfruit
HoneymelonSmokeyGreen grass
Green bean
Buttery/Cheesy
Acidity
Body
Mouthfeel
Bitterness
Small Scale Winery
Effects of spontaneous MLF
• Increase in smokey, green, and buttery attributes
• Increase in
bitterness • Decrease in
perceived fruity aromas
0
100
200
300
400Mineralic
Lemon
Apple
Elder flower
Peach
Passionfruit
Honeymelon
SmokeyGreen grass
Green bean
Buttery/Cheesy
Acidity
Body
Mouthfeel
Bitterness
Control (Siha 7) Small Scale 1 Small Scale 2
MLF during spontaneous fermentation
• Speed of bacterial growth is depending on temperature • Only delay in MLF, no inhibition by lower temperatures
0.0
0.5
1.0
1.5
2.0
2.5
3.0
0 5 10 15 20 25 30 35 40
Days
Mal
ic a
cid
[g/L
] 15°C a15°C b18°C a18°C b21°C a21°C b
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?
Terroir character
Soil Cover crop
Slope
Sunshine Wind
Rain/Water
Why connecting Terroir and Yeast?
Soil Cover crop
Sunshine Wind
Rain/Water
Almost the same factors have an impact on the yeast flora in the vineyard !
„sterile“ hand-harvesting
Vinification under „sterile“ conditions
up to 2 %vol. alcohol Inoculation of a
pasteurized must
Bottling
Experimental setup
3 different wines from one vineyard – Spontaneous fermentation in the winery incl.
Cellar-yeast (referred to as winery) – Spontaneous fermentation small scale only with
yeast from the vineyard (small scale) – Inoculation of pasteurized must (inoculation)
Small scale vinification under
„sterile“ conditions
„sterile“ hand-harvesting
Bottling
Vinification under common conditions
in the wineries
Normal hand-harvesting Bottling
Sensory evaluation of all experiments
Except for a few wines, grouping is possible due to sensory properties
130
130A
131
131A
132
132A
132WGT
133
133A
133WGT
134
134A
134WGT
137
137A
138
139
-3
-2
-1
0
1
2
3
4
-6 -5 -4 -3 -2 -1 0 1 2 3 4 5
F2 (1
6,68
%)
F1 (46,57 %)
Dried active yeast Winery wines
Small scale fermentations
Inoculations
130
130A
131
131A
132
132A
132WGT
133
133A
133WGT
134
134A
134WGT
137
137A
138
139 apple
bitter
hay
honey
cantaloup
body mouth feel
peach
smoky
Sauerkraut
acidity acid intensity
lemon
mineralic
-3
-2
-1
0
1
2
3
4
-6 -5 -4 -3 -2 -1 0 1 2 3 4 5
F2 (1
6.68
%)
F1 (46.57 %)
Biplot (Axes F1 and F2: 63.24 %)
Dried active yeast
Winery wines
Small scale fermentations
Inoculations
Small scale spontaneous fermentation
• Only little differences in fruity characters
0
1
2
3
4
5
6
7mineralic
lemon
peach
honey melon
apple
honey
smokyhay
Sauerkraut
acid intensity
acidity
mouthfeel
body
bitter
SonnenscheinReiterpfadKastanienbusch
Differences due to terroir and site specific yeasts (?)
0
1
2
3
4
5
6
7mineralic
lemon
peach
honey melon
apple
honey
smokyhay
Sauerkraut
acid intensity
acidity
mouthfeel
body
bitter
SonnenscheinReiterpfadKastanienbusch
Spontaneous fermentation in the wineries
• Differences between fruity characters • No differences in negative parameters
Differences by including the „cellar yeast“
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
• Differentiation on species level • No absolute quantification; educated guess
Harvested yeast
Extract and wash DNA
DNA Mix of different species
Polymerase Chain Reaction
Compare with Single cultures
Quantify by evaluating
fluorescence intensity
DGGE procedure according to Mills et al., 2000
Yeast dynamics in spontaneous fermentation
• 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“ !
0
1
2
3
4
5
6
7
8
9
0
10
20
30
40
50
60
70
80
90
100
day 2 day 4 day 6 day 8 day 10 day 13
%vo
l.
gene
ra [%
]
Pichia Hanseniaspora Hansenula Saccharomyces Alcohol
Spontaneous flora without SO2 addition
• Composition of yeast population changes with increasing ethanol concentration
• Above 4 %vol. ethanol only Saccharomyces active
Hansenula / Pichia
Candida / Metschnikowia
Kloeckera / Hanseniaspora
Saccharomyces
4 % vol. Alkoholgehalt
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
• Selection of 23 compounds out of 283 based on explained variability
Mainly: Alcohols Esters Hydrocarbons
19
43 50
53
55
65
74 89
116 124
160
169 185
186 208 250
-1
-0.75
-0.5
-0.25
0
0.25
0.5
0.75
1
-1 -0.75 -0.5 -0.25 0 0.25 0.5 0.75 1
F2 (2
4.21
%)
F1 (54.88 %)
Variables (Axes F1 and F2: 79.09 %)
Variation among wines due to volatiles
• Dried active yeast and inoculations together – Influence of wild
yeast strains less important
– Dominance of Saccharomyces
• „pure“ spontaneous fermentation batches on one side
• More variation among winery wines than among „sterile“ batches – Impact of cellar flora
increases differences between wines
130
130A
131
131A
132
132A
132WGT
133
133A
133WGT
134
134A
134WGT
137
137A
138
139
6 4 2 0 2 4 6
Winery wines
Small Scale spontaneous fermentation
Inoculations
Dried active yeast
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“
Conducted spontaneous fermentation I Yeast from successful spontaneous fermentation used to inoculate other batches
„Real“ spontaneous fermentation random population of microorganisms from vineyard and cellar
Ways to conduct spontaneous fermentation
Conducted spontaneous fermentation I (Pied de Cuve) Early harvesting of healthy grapes to establish a starter culture for further inoculation
Ways to conduct spontaneous fermentation
Conducted spontaneous fermentation I Yeast from successful spontaneous fermentation used to inoculate other batches
„Real“ spontaneous fermentation random population of microorganisms from vineyard and cellar
„optimized“ spontaneous fermentation spontaneous start followed by inoculation with dried active yeast
„Diversity due to wild yeast, safety by using Saccharomyces“
Ways to conduct spontaneous fermentation
„Real“ spontaneous fermentation random population of microorganisms from vineyard and cellar
Conducted spontaneous fermentation I Yeast from successful spontaneous fermentation used to inoculate other batches
Conducted spontaneous fermentation I (Pied de Cuve) Early harvesting of healthy grapes to establish a starter culture for further inoculation
Be on the wild or safe side of fermentation
High Low Risk of stuck fermentation
„Real“ spontaneous fermentation random population of microorganisms from vineyard and cellar
Conducted spontanous fermentation I Yeast from successful spontanous fermentation used to inocculate other batches
Conducted spontanous fermentation I (Pied de Cuve) Early harvesting of healthy grapes to establish a starter culture for further inocculation
„optimized“ spontaneous fermentation spontaneous start followed by inoculation with dried active yeast
No significant difference compared to dried active
yeast fermentation
• „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)
• 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!
Solution so far…
0
50
100
150
200
250
02468
101214
0 5 10 15 20
Citr
at [m
g/L]
Dia
cety
l [m
g/L]
Time [days]
VINIFLORA CiNe(Diacetyl)
Lalvin VP41 (Diacetyl)
VINIFLORA CiNe(Citrat)
Lalvin VP41 (Citrat)
adapted from Quintans et al., 2008
Yeast
Release during amino acid synthesis
(valine)
Uptake and reduction to 2,3-butanediol
From yeast sugar metabolism
Bacteria
Uptake towards the end of MLF
Constant release throughout growth
Release of reduced diacetyl strain depending
Citric acid degradation
OH OH
OO OH
O
OH
O
O
Experimental setup
Strong production Medium production Citrate lyase negative
VP 41 (Lallemand)
ALPHA (Lallemand)
BETA (Lallemand)
CiNe (Eaton‘s Begerow)
CLNX (FermControl)
Pinot blanc 2011 and 2012
Fermented with yeast CY 3079 (Lallemand)
Divided in simultaneous and consecutive MLF
Trials run in duplicates
Control no MLF
Influence of cell concentration
1,00E+001,00E+011,00E+021,00E+031,00E+041,00E+051,00E+061,00E+071,00E+081,00E+09
0112233445
0 5 10 15 20 25 30 35C
FU/m
L
Mal
ic a
cid
[g/L
]
vinification time [days]
Malic acid
CFU
1,00E+00
1,00E+01
1,00E+02
1,00E+03
1,00E+04
1,00E+05
1,00E+06
1,00E+07
1,00E+08
0
1
1
2
2
3
3
4
4
0 5 10 15 20 25 30 35
CFU
/mL
Mal
ic a
cid
[g/L
]
vinification time [days]
Malic acid
CFU
• no MLF with less than 106 CFU/mL
• some commercial products contain less viable cells • delayed MLF • risk of undesired flavor development
4.73
2.97
1.37
0.300
1
2
3
4
5
-1 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35
Diac
etyl
[mg/
L]
vinification [days]
Simultaneous Inoculation
ALPHA BETA CREAM control
Yeast
O. oeni
Start citrate degradation
• 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
Accumulation of diacetyl
Diacetyl concentration during vinification
1.00E+04
1.00E+05
1.00E+06
1.00E+07
1.00E+08
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0 2 4 6 8 10 12 14
CFU
/mL
Dia
cety
l [m
g/L]
vinification [days]
Diacetyl O. oeniDiacetyl controlCFU O.oeni
O. oeni and S. cerevisiae
O. oeni
• 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)
Acetolactate synthase (alsS)
Acetolactate decarboxylase (alsD)
Acetoin reductase (butA2)
Citrate lyase (citE)
Lactate dehydrogenase (ldh) House-keeping gene
adapted from Quintans et al., 2008
Citrate induced
Sugar/pyruvate induced
• first alsS over-expression induced by sugar metabolism • second alsS over-expression induced by citrate degradation
alsS
Gene expression analysis (qRT-PCR)
The central role of pyruvate
Pyruvate addition
The central role of pyruvate
Degradation von citrate also leads to an increase in pyruvate
0
10
20
30
40
50
60
0 2 4 6 8 10
Dia
cety
l [m
g/L]
vinification [days]
Diacetyl 0 mg/lDiacetyl 10 mg/LDiacetyl 20 mg/LDiacetyl 50 mg/Lcontrol
• 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
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)
• 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
What does that mean for the winemaker?
Dried active yeast (CY 3079) is able to reduce 50 mg/L diacetyl in two days Independent from the starting level, residual diacetyl of 2 mg/L always remains; reason possibly bound diacetyl
S. cerevisiae O. oeni
Correlation between biomass increase and diacetyl accumulation observed (more bacteria produce more diacetyl) Over-expression of alsS gene due to high concentrations of pyruvate as well as citrate Formation of diacetyl by citrate degradation only can be excluded
Diacetyl accumulation shows two characteristic peaks: first increase caused by yeast and bacteria, second increase due to bacteria only
Summary and conclusion