Trevor Phister, PhD Assistant Professor · 2012. 10. 10. · •Wine microbiology •Microbial...
Transcript of Trevor Phister, PhD Assistant Professor · 2012. 10. 10. · •Wine microbiology •Microbial...
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Microbial Faults
Trevor Phister, PhD
Assistant Professor
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Overview
• Wine microbiology
• Microbial faults
– Brettanomyces
– Lactic acid bacteria
– Cork Taint
• Controlling microbial faults
– Sanitation
– Quality programs
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Saccharomyces cerevisiae
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Saccharomyces cerevisiae
(Piskur et al 2006)
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Does Not Always Work
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Time
Sugar
ODETOH
• Metschnikowia sp.
• Pichia sp.
• Candida sp.
• Kluveromyces sp.
• Hanseniaspora sp.
• Saccharomyces
• acetic acid bacteria
• lactic acid bacteria
YeastsBacteria
Molds
• Botrytis & others
The Wine Fermentation
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Three common microbial
contaminants• Brettanomyces
• Lactobacilli
• Cork taint
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Brettanomyces
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Slow growing soft drink and wine spoilage yeast
Brettanomyces bruxellensis
Produces 4-ethylphenol or “wet dog”taint
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Am J. Enol Vitic 54:294-300
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4-ethylphenol produced in absence of platable population
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Brettanomyces bruxellensis
Isolated by
Dr. Clausen in
1904
The brewing industry just
started using yeast
Provides missing
element of traditional
beers
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Friend or Foe?
Brett
For Now
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On Your Table
4 - Ethylguaiacol (4-EG): ~175 ppb
4 - Ethylphenol (4-EP): 600 - 800 ppb
Diacetyl: 2-4 ppm
Geraniol: 0.5 – 1 ppm
Trichloroanisole (TCA): very low ppb
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Compound Character
4-ethyl phenol Band-aid, burnt plastic
4-ethyl guaiacol Smoky, Spicy, Clove
4-ethyl catechol Sweaty, Horsey
Isovaleric acid Rancid, Cheesy, Vomit
Combined Barnyard, mouldy
Typical odors
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• 4-ethylphenol 0.23 mg/L
• 4-ethylguaiacol 0.047 mg/L
• Perception is dependant on the type of wine
• Can mask varietal character
• May degrade/reduce some fruity aromas
• metallic finish
• May also produce acetic acid
Sensory threshhold
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Production of volatile phenols
Hydroxycinnamic acids Vinyl derivatives Ethyl derivatives
Cinnamate decarboxylase Vinyl phenol reductase
p-coumaric acid
p-ferluic acid
Caffeic acid
4-vinylphenol
4-vinylguaiacol
vinylcatechol
4-ethylphenol
4-ethylguaiacol
ethylcatechol
Saccharomyces
Wild yeast
Lactic acid bacteria
Brettanomyces
Pichia
Candida
Lactic acid bacteria
Plant derived
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• A low levels can enhance varietal flavors
• If you think you have it under control you
will probably not be able to keep it that
way
• Phenolic precursors from the grapes are
metabolized into ethylphenols so levels
will very with variety
Things to remember
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Where do you find Brett?
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Geographic distribution
(Conterno et al. 2006)
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Locally
• Present at low levels on grapes
– Damaged grapes may provide nutrients
• Found in fruit flies
• Present throughout the winery
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• More prevalent as a problem in reds
– More phenolic compounds
– Longer aging
• May be found in white wines
• May be in dry wines
• Likes to have oxygen but does not need
much
Growth during winemaking
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Growth during winemaking
(Renouf et al 2006)
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• Brett can even survive up to 8 millimeters into
the barrel staves
• Can consume sugars present from the wood
• Can survive in a viable nonculturable state
Growth during winemaking
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Things to remember
• Strain variation may account for differences in between wines
– We are looking for useful strains
• Present through out the winery
• Able to persist throughout fermentation
– Low numbers do not mean it is a problem
– Predominate yeast after alcoholic fermentation
– May enter viable nonculturable state
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Best to prevent Brett from
being a problem
Minimizing off flavors
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Prevention-in the vineyard
• Remove damaged grapes
• Use SO2 at harvest
• Off-flavors generally have lower sensory
thresholds in lighter wines so pay even
more attention to good winemaking
practices
Prevention-In the vineyard
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• Proper sanitation
• Long maceration
– More substrate to produce off-flavors
• Red wine more prone to Brettiness
– Higher substrate
– Longer aging
– White wines:lower pH, higher SO2
Prevention-In the winery
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• pH below 3.6
• Try to keep temperatures lower
• SO2 usage (use 0.8 ppm molecular SO2)
– Make sure to use at picking and or crushing
– Use after MLF
• Keep alcohol levels above 13%
• Keep residual sugars below 0.2 g/L
– Even dry wines can support growth
Prevention-In the winery
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• Measure must nitrogen level and add just enough nitrogen to have a strong fermentation
– Too much can leave nutrient which Brett can use
• If leaving on lees make sure to control other parameters
• Keep containers topped up
– Limits amount of oxygen avalible
Prevention-In the winery
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• Highest risk of spoilage from end of MLF
through aging
– Use an active MLF strain
• Use clean wines to top-up
– Either filter or add DMDC (velcorin)
• Beware of cheap used barrels
– Don’t use infected barrels
• Segregate infected barreles
Prevention-In the winery
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• Watch cross contamination
– Use plastic pipets to sample from barrels and change
pipets after each barrel
• Best barrel cleaning
– Cold rinse, then 70oC then steam at low pressure for
10 minutes
– Barrels cannot be sterilized even with SO2 or Ozone
• Monitor before bottling
– Check pH, SO2, alcohol, watch oxygen levels
– May need to filter or add DMDC
Prevention-In the winery
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Lactobacillus spp.
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• May cause stuck fermentations
• Higher VA-produces acetic acid and lactic acid
• “Tourne”
• Acrolein
• Geranium
• Mousiness
• Biogenic amines
• Diacetyl
• Ropiness
The problems
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• May cause stuck fermentations
• Higher VA-produces acetic acid and lactic acid
• “Tourne”
• Acrolein
• Geranium
• Mousiness
• Biogenic amines
• Diacetyl
• Ropiness
The problems
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Fermentation rates: Long lag
Brix
Time
• Usually suggest
presence of a toxin
or deficient
population of
healthy yeasts
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Fermentation rates: sluggish
throughout
Brix
Time
• Often due to nutrient deficiency– Diammonium phosphate
(DAP)
• Added at 200 mg per liter (0.03 ounces/Gal)
• Not all at once three times throughout fermentation
• Poor strain tolerance to stress
• Inhibitory compounds
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Fermentation rates: Becoming
sluggish
Brix
Time
• Toxins produced by
molds
• Mild temperature shock
• Moderate deficiency in
compounds needed to
survive at higher
ethanol concentrations
• To high an inoculum
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Fermentation rates: abrupt
arrest
Brix
Time
• Usually due to traumatic
shock
– Temperature increase or
decrease
– Higher ethanol
concentration makes it
more sensitive
• May be due to some
malolactic strains
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The other taints
• Tourne-rare breakdown of tartaric acid
– Loss acidity color becomes brown
• Acrolin-bitterness
• Mousiness- mouse cage
• Biogenic amine formation
• Ropiness
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On Your Table
4 - Ethylguaiacol (4-EG): ~175 ppb
4 - Ethylphenol (4-EP): 600 - 800 ppb
Diacetyl: 2-4 ppm
Geraniol: 0.5 – 1 ppm
Trichloroanisole (TCA): very low ppb
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Geranium taint• Most likely to occur in sweet wines
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Geranium taint• Sorbate typically used at 100 to 200 mg/L
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Geranium taint
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Geranium taint• Sorbate typically used at 100 to 200 mg/L
• Keep SO2 levels up
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Diacetyl production• Produced by lactic acid bacteria during
growth
• Use citrate
• Threshold
– 0.2 mg/L in
Chardonnay
– 2.8 mg/L in
Cabernet
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Diacetyl control• Inoculate for ML
• Don’t use citrate
• Keep pH low
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Controlling Lactic acid
bacteria• Keep pH low <3.5
• Keep SO2 levels to 50 to 75 mg/mL
• Addition of lysozyme 250 mg/mL
• Keep temp low during cold soak <59oF
• Conduct ML
• Sanitation
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Cork taint
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On Your Table
4 - Ethylguaiacol (4-EG): ~175 ppb
4 - Ethylphenol (4-EP): 600 - 800 ppb
Diacetyl: 2-4 ppm
Geraniol: 0.5 – 1 ppm
Trichloroanisole (TCA): very low ppb
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Cork taint
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Cork taint
Perception threshold
4 ng/L
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Cork taint
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Cork taint
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Cork taint
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Cork taint
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Cork taint control
• Limit use of chlorine based sanitizers
• Limit use of chlorophenol based
insecticides and fungicides
• Control humidity (high humidity
encourages mold growth)
• Have good ventilation
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Controlling microbial issues
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Winery Sanitation
• Sanitation = Disinfection
• Cleaning
• Sterilization
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Winery Sanitation
Sanitation is an attempt to
reduce the number of spoilage
microorganisms on equipment surfaces.
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Winery Sanitation
Cleaning is an attempt to physically and
chemically remove food for
microorganisms and to eliminate
hospitable environments for their growth.
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Winery Sanitation
Soil is a material in the wrong
place such as dirt dust and
organic material- tartrate
deposits
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Five Steps in a Sanitation
Program
• Rinse to remove large debris
• Apply cleaning compound- remove soil
• Rinse to remove dispersed soil
• Sanitizing- kill microorganisms
• Monitoring
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Cleaning
• Think of the soil characteristics
• Solubility– Water (salts, sugars, starches)
– Acid soluble (oxidized iron, zinc carbonates, calcium oxalates, hard water scale)
– Alkaline soluble (Fatty acids, proteins, other organic deposits)
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Methods of Sanitization
• Heat– Time
– Temperature
• Chemical– Time
– Temperature
– Concentration
– pH
– Organic matter
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Methods of Monitoring
Sanitization• Sensory
– Does the surface look clean
– Does it feel clean
– Does it smell clean
• Microbial counts
– Swab 4x4 in area for defined period of time
– Direct contact-press plate against surface
• Luciferase bioluminescence
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Methods of Monitoring
Sanitization
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Sanitation
• Develop a plan
• Have written protocols
• Place someone in charge
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Quality control
• Conduct a Hazard Analysis
• Identify the critical control points
• Establish critical limits for control measures
• Establish procedures for monitoring
• Establish corrective action to be taken when monitoring indicates there is a deviation from a critical limit
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Quality control
• Establish effective record keeping procedures that document the Quality control system
• Establish procedures for verification of the Quality control system
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Controlling microbial
issues• Use quality fruit
• Pay attention to acid levels
• Use SO2
• Watch cross contamination
• Keep barrels toped up
• Develop a sanitation plan
• Develop a quality plan
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Controlling microbial
issues• Use quality fruit
• Pay attention to acid levels
• Use SO2
• Watch cross contamination
• Keep barrels toped up
• Develop a sanitation plan
• Develop a quality plan
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How do we know Brett is in
the wine?
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Requires ~7-10 days for detection from wine
Medium contains cycloheximide
Classical Brett Enumeration
Other yeasts such as may grow on cycloheximide
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Gas Chromatography
Detects ethylphenol
Detecting metabolic
end product. If you can
measure it then it is
probably already to late
Some strain of
Brett do not
produce 4-ethylphenol
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• Need high levels of cells
– Brett does not always reach 1000 cells/mL
• Brett may be difficult to identify due to
variable morphology
• Are the cells living or dead?
– Use stain
Microscopy
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• Rapid (3-4 hours)
• Specific (sometimes to specific)
• Detect and enumerate before Brett is a
problem
• Costly
• Need lots of training
– Performed at service laboratories
PCR