Brettanomyces spoilage

Charles G. Edwards

Washington State University

Viticulture/Enology Team

School of Food Science

New insights into preventing

Impact of SO2?

Removal by filtration?

What have we learned about

controlling Brettanomyces?

Ethanol x Temperature?

Growth in oak (barrels)?

Impact of must nutrition?

Impact of SO2

A. Can SO2 cause “VBNC”?

1. “Viable-But-Not-Culturable.”

2. Yeast alive but will not grow on media.

B. How to detect VBNC?

1. Measure metabolic activity or qPCR (?).

Green = viable Orange/Red = dead

3. If valid yeast could be undetected.

2. Fluorescence microscopy.

Active cell

Dead cell

Brettanomyces

101

102

103

104

105

106

107

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

Mo

lecu

lar

SO

2 (

mg

/L)

5 10 15 20 25 30 40 0 35

108

Time (days)

<300 CFU/mL

Po

pu

lati

on

(C

FU

/mL

or

ce

lls/m

L)

0.88 mg/L molecular SO2

B. bruxellensis (B1b)

Is there a relationship between

ethanol and temperature that

Brettanomyces?

could be useful to control

Ethanol

Temperature

(low)

(high)

(low) (high)

GROWTH

Maximum ethanol

12% v/v alcohol (I1a) 108

106

104

102

<30

0 20 40 60 80 100

Cu

ltu

rab

ilit

y (

CF

U/m

L)

Time (days)

( ) 21°C ( ) 18°C ( ) 15°C ( ) 12°C

( ) 70°F ( ) 64°F ( ) 59°F ( ) 54°F

13% v/v alcohol (I1a) 108

106

104

102

<30

0 20 40 60 80 100

Cu

ltu

rab

ilit

y (

CF

U/m

L)

Time (days)

( ) 21°C ( ) 18°C ( ) 15°C ( ) 12°C

14% v/v alcohol (I1a) 108

106

104

102

<30

0 20 40 60 80 100

Cu

ltu

rab

ilit

y (

CF

U/m

L)

Time (days)

( ) 21°C ( ) 18°C ( ) 15°C ( ) 12°C

15% v/v alcohol (I1a) 108

106

104

102

<30

0 20 40 60 80 100

Cu

ltu

rab

ilit

y (

CF

U/m

L)

Time (days)

( ) 21°C ( ) 18°C ( ) 15°C ( ) 12°C

16% v/v alcohol (I1a) 108

106

104

102

<30

0 20 40 60 80 100

Cu

ltu

rab

ilit

y (

CF

U/m

L)

Time (days)

( ) 21°C ( ) 18°C ( ) 15°C ( ) 12°C

°C Ethanol (%) Strain F3 Strain I1a

Production of 4-Ethylphenol (µg/L)

21

°

12 (+) (+)

13 (+) (+)

14 (+) (+)

15 (-) (+)

16 (-) (-)

18

°

12 (+) (+)

13 (+) (+)

14 (+) (+)

15 (-) (+)

16 (-) (-)

(-) <30 µg/L; (±) 31 to 1000 µg/L; (+) >1300 µg/L

°C

Ethanol (%) Strain F3 Strain I1a

15

°

12 (+) (+)

13 (+) (+)

14 (+) (+)

15 (-) (-)

16 (-) (-)

12

°

12 (-) (±) 13 (-) (-)

14 (-) (-)

15 (-) (-)

16 (-) (-)

Production of 4-Ethylphenol (µg/L)

(-) <30 µg/L; (±) 31 to 1000 µg/L; (+) >1300 µg/L

What about filtration?

Research Focus (Filtration)

3 samples (No SO2)

3 samples (SO2)

3 samples (No SO2)

3 samples (SO2)

3 samples (No SO2)

3 samples (SO2)

Red wines (0 or 0.5 mg/L mSO2)

Replicate #1 Replicate #2 Replicate #3 Treatment Treatment Treatment

Treatment Replicate = Filter cartridge

Filter

Cartridge #1

Filter

Cartridge #2

Filter

Cartridge #3

• Multiple ferments incubated >200 days.

Filtration

107

10 20 30 40 50 60 70

106

105

104

103

<300

Cu

ltu

rab

ilit

y (

CF

U/m

L)

Time (days)

• No SO2 added

• Filtration replicates ( , , )

>212 days

1.2 µm (Strain B1b)

107

10 20 30 40 50 60 70

106

105

104

103

<300

Cu

ltu

rab

ilit

y (

CF

U/m

L)

Time (days)

• No SO2 added

• Filtration replicates ( , , )

>250 days

Filtration (Strain F3) 1.2 µm

>300 CFU/mL

107

10 20 30 40 50 60 70

106

105

104

103

<300

Cu

ltu

rab

ilit

y (

CF

U/m

L)

Time (days)

• No SO2 added

• Filtration replicates ( , , )

Filtration (Strain F3) 0.8 µm

>270 days

Growth in oak barrels?

Research Focus (Growth in Oak Barrels)

• American and French oaks (16L and 225L).

• Various toasted levels.

• D value = time for 90% reduction

(Brettanomyces 1 minute at 55°C)

Research Focus (Growth in Oak Barrels)

Cross-Sectional Layer

(transfer into sterile wine)

0 to 4 mm

5 to 9 mm

10 to 14 mm

>15 mm

French (medium-light; 0 to 4 mm)

French (medium-light; 5 to 9 mm)

American (medium-heavy; 0 to 4 mm)

American (medium-heavy; 5 to 9 mm)

Recovery of Brettanomyces

(225L barrels)

Time (days)

Cu

ltu

rab

ilit

y (

cfu

/mL

)

3125191360

106

104

102

<30

Top staves

3125191360

Bottom staves

Research Focus (Use of heat to reduce populations)

• Cut staves into 3 x 10 cm blocks/place into SS plate

• Periodically remove blocks; determine yeast recovery

• Thermocouples inserted every 4 mm

• Place plate over a steam kettle to record °C

American oak (225L)

French oak (225L)

Time (Minutes)

Te

mp

era

ture

(°C

)

2 4 6 8 10 120

20

A

40

60

80

100

20

B

40

60

80

100

0 mm depth

4.5 mm depth

9.5 mm depth

14.5 mm depth

25 mm depth

Oak barrel staves (16L)

American (light toast)

Steam treatment of staves (minutes)

108

106

104

102

<30

108

106

104

102

<30

0 to 4 mm layer

5 to 9 mm layer

0 3 6 9 12

Incubation of steamed stave layers in wine (days)

Cu

ltu

r ab

i lit

y (

CF

U/m

L)

1 7 15 30 60 1 7 15 30 60 1 7 15 30 60 1 7 15 30 60 1 7 15 30 60

American (heavy toast)

Oak barrel staves (16L)

French (light toast)

French (heavy toast)

0 to 4 mm layer

5 to 9 mm layer

Steam treatment of staves (minutes)

0 3 6 9 12

Incubation of steamed stave layers in wine (days)

1 7 15 30 60 1 7 15 30 60 1 7 15 30 60 1 7 15 30 60 1 7 15 30 60

108

106

104

102

<30

108

106

104

102

<30

Cu

l tu

r ab

i lit

y (

CF

U/m

L)

2. Does higher N in wines encourage infections?

• What amount of N is needed to support growth?

1. If more N is added to grape must

is more N present in resultant wines?

Impact of Must Nutrition (Brettanomyces)

Must treatment Wine

Yeast

strain

YAN

(mg N/L)

Sugar

(g/L)

α-Amino

nitrogen

(mg N/L)

Ammonia

nitrogen

(mg N/L)

ECA5 150 230 50b 1b

250 48bc 1b

270 45b 1b

250 230 85a 60b

250 84a 60b

270 86a 60b

Residual nitrogen in “synthetic” wines

Ethanol concentrations in wines

Sugar in musts

(g/L)

Ethanol in wines

(% v/v)

230 13.5 to 14.1 “low”

250 14.7 to 14.9 “medium”

270 15.6 to 15.9 “high”

0 10 20 30

Low YAN, Low EtOH

High YAN, Low EtOH

Low YAN, Med EtOH

High YAN, Med EtOH

Low YAN, High EtOH

High YAN, High EtOH

Brettanomyces E1 in wines C

ultura

bili

ty (

cfu

/mL)

107

106

105

104

103

102

101

Days

0 10 20 30

Uvaferm 228 ECA5

Cultura

bili

ty (

cfu

/mL)

YAN (mg N/L)

0

6

12

24

48

96

Days

0 10 20 30 40

107

106

105

0

Brettanomyces E1 in synthetic

wine (13% v/v alcohol)

Microbe Management

minimizing the risk of spoilage

OR really......

Control Spoilage By Using

Multiple “Hurdles”

Spoilage

infection

Initial

• Avoid used barrels

• Avoid importing unfiltered wine

Use of “Hurdles”

• SO2, ethanol, and low temperature

• Filtration (0.8 micron absolute)

• Cleaning / sanitizing program

Conclusions

Too few hurdles =

increased risk of spoilage.

Too many hurdles =

expense higher than benefit.

Incorporate as many hurdles as is REASONABLE for the winery

KEY Hurdle interactions exist!

Best Enology Work in 2007

References

CHARLES G. EDWARDS

www.gusmerenterprises.com

pubs.wsu.edu

C.G. Edwards and B.A. Watson

www.amazon.com

Thank you for your support!

Oregon Wine Board

Lallemand Inc.

Undergraduate/graduate students

Northwest Center for

Small Fruits Research

Washington Wine

Advisory Committee

GusmerEnterprises

Vinotech Napa

Ste Michelle Wine Estates

Washington Association

of Wine Grape Growers

Washington and

regional wineries

ETS Laboratories

Scott Laboratories

Seminar Dedication

Ken Fugelsang (CSUF)