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