Introduction to the Biology of Spoilage Yeasts and Brettanomyces
2017 Oregon Wine Symposium | Dr. Charles Edwards- New Insights into Preventing Brettanomyces...
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Transcript of 2017 Oregon Wine Symposium | Dr. Charles Edwards- New Insights into Preventing Brettanomyces...
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)