Bacterial Spoilage During Aging: Acetic Acid Bacteria Mike Ramsey Teaching Laboratory Manager

Bacterial Spoilage During Aging:

Acetic Acid Bacteria

Mike Ramsey

Teaching Laboratory Manager

Department of Viticulture and Enology

University of California, Davis

Acetic Acid Bacteria (AABs)

• Acetobacter

• Acidomonas

• Gluconobacter

• Gluconacetobacter

• Kozakia

• Asaia

• Big reclassification in 1998

Acetic Acid Bacteria (AABs)

• Identification on Genus level is easy; species level is difficult

• Rod –shaped obligate aerobes– metabolism is strictly respiratory– May or may not have flagella

• Very widespread– Occur mainly in sugary, acidic, or alcoholic

habitats– Peynaud in 1961 found acetic acid bacteria on

about 50% of all ripe grape clusters

Timeline

Acetobacter aceti

Impact: Acetic Acid

• Acetobacter, Acidomas, Gluconobacter, Gluconoacetobacter, and Kozakia) convert ethanol to acetic acid. – Glucose can be converted to acetic acid

or converted to gluconic acid (main acid in honey; “umami”)

• Acetobacter, Acidomonas, and Gloconacetobacter can continue to oxidize acetic acid to water and CO2 after all the ethanol has been converted.

Impact: Acetic Acid

• Acetobacter aceti is the most common of these organisms found in wine and highly ethanol tolerant, although other species are common and may dominate your winery

• The creation of VA is an impact shared by all of the organisms in this Family, except Asaia

Impact: Volatile Acidity

• The main component of VA (Volatile Aciditiy) is acetic acid– the portion of the organic acids that are

distillable away from the total


• Important to remember that acetic acid is also created by lactic acid bacteria (Lactobacillus, Pediococcus, Oenococcus) which can create acetic acid during malolactic fermentation (MLF) and can covert glucose to acetic acid– Will not produce ethyl acetate

Impact: Volatile Acidicty

• Yeast genera such as Kloeckera, Hanseniaspora, and Metchnikowia - which are found on most grapes - and winery yeast such as Brettanomyces and Dekkera produce high amounts of acetic acid

• Other acetic acid producing wine – related yeast include Zygosaccharomyces


• Saccharomyces can produce acetic acid in the range of 100 to 200 mg/L (ppm) during a normal fermentation– Amerine and Ough describe ‘normal’

fermentations as not exceeding 300 mg/L of acetic acid

• Influenced by yeast strain, fermentation temperature, and juice nutrition and happens regardless of exposure to oxygen.– However, the mechanism in yeast is poorly

understood

Why is acetic acid important?

• Legal and Sensory

• High levels of acetic acid will raise Titratable Acidity

• Both acetic acid and acetaldehyde are toxic to Saccharomyces cerevisiae and may lead to stuck fermentations.

Why is acetic acid important?

• “There are different opinions as to what level of volatile acidity is appropriate for higher quality wine.” wine faults – wikipedia (italics are mine)– some winemakers seek a low or barely

detectible level of acetic acid to add to the perceived complexity of a wine.

• Amerine and Ough found it less objectionable in older wines than in younger wines

VA Restrictions

Legal Limits: US CA OIVWhite: 1.2 g/L 1.1g/L 0.98g/LRed: 1.4 g/L 1.2g/L 0.98g/L

Threshold of Detection: 1 g/L

• Can be masked by higher alcohol and sugar content in wine

• Important to remember that the acetic acid threshold is lower than the legal limit

Acetic acid measurement

• The most accurate way to measure is by enzymatic test/spectrophotometer or HPLC/GC

• Wine lab methods– Sequential Analyzer; Segmented Flow

Analyzer• The most common method of measurement of

acidic acid is by distillation using a “Cash” still– SO2, CO2, and other organic acids

(especially lactic) interfere as well as operator error and cleanliness of equipment

Control of acetic acid production

• As the control of all the compounds created by AABs are related, we will review control at end of presentation

VA: Treatment

• Difficult to correct• Can not neutralize in wine without destroying

the wine• Refermentation is not an option

– Saccharomyces can not utilize acetic acid

• VA Filtration (Reverse osmosis coupled with ion exchange)

• Blending• Bulk/Vinegar

VA Filtration (Reverse osmosis coupled with ion exchange)

• Permeate = acetic acid, ethanol, water– Oxidative character may become more

pronounced– Winemakers report tannin and wine structure

problems– About 30% removed in one pass– The filtration companies say it doesn’t change

the wine’s character but it is detrimental to ‘wine quality’ to reduce the VA level below normal levels

Impact: Ethyl Acetate

• The most common ester in wines

• Chemical image from wikipedia (http://en.wikipedia.org/wiki/Ethyl_acetate)


• More commonly found in red wines• There is generally a relationship between acetic

acid and ethyl acetate production• Does not diminish over time, as do some

fermentation esters• Threshold values reported: 150 – 170 ppm

(Amerine and Ough, 1980)• Uncited threshold on wikipedia:120 ppm• Amerine and Ough reported values typical in US

wine: 23 – 135 ppm but it would seem the high has increased in recent years


• Can be formed in excess by indigenous yeasts, i.e Pichia spp., and Kloeckera and Hanseniaspora spp.

• Can be formed in excess by Acetobacter spp.


• Can contribute to a general “fruitiness” character in wine at about 50 ppm

• Excess amounts are considered a “fault”– Becomes more sharp and acrid as ethanol is

oxidized to acetaldehyde


• Aroma of nail polish remover– Is often used as a solvent

• No easy way to quantify– There is no set legal limit

• Can impart complexity• Is not part of Volatile Acidity, so is not measured

by Cash Still/VA Still• Can be measured by GC – FID (Gas

Chromatography – Flame Ionization Detector)

Control of Ethyl Acetate

• As the control of all the compounds created by AABs are related, we will review control at end of presentation

Treatment

• Removal with reverse osmosis and absorbent media– Can not be fully removed

• SO2 does not cover the aroma

• Can not referment out

• Blending is difficult as other reds will also have EA

• Bulk/Vinegar

Impact: Acetaldehyde

• Boiling point = 70 degrees Fahrenheit

• Called “sherry – like” character or green apple

• Sensory threshold 100 -125 ppm (mg/L)


• Can be created through the oxidation of ethanol– during “micro ox” as well as other oxygen

exposure• Increased production in the presence of acetic

acid bacteria and other wild organisms– During glycolysis– Released when conversion to ethanol is

blocked


• (Fino and Manzanillo sherries are made by micro organisms creating acetaldehyde)

• Possible carcinogen; definite mutagen

Treatment for Acetaldehyde

• No commercial filtration available

• SO2 binds – but not permanently

• Might be able to reduce by adding fresh Oenococcus after malolactic fermentation

• These compounds have much lower sensory threshold than acetic acid—both acetaldehyde and ethyl acetate are detectable at less than 200 mg/L in wine.

Impact: Mousiness

• Has been detected in wines all over the world

• Ability to perceive in tasters seems to have a genetic predisposition

• The perception is not usually in the aroma, but is a delayed perception, generally after swallowing or spitting– The compounds responsible are not volatile at

wine pH

Impact: Mousiness

• A wine that has been kept away from oxygen can have mousy taint and not exhibit it.– Aeration during filtration and/or bottling

can set it off• All three compounds can be created by

lactic acid and acetic acid bacteria.• Two of the compounds can be created by

Brettanomyces

Impact: Mousiness

• Ability to create the compounds varies within strains of the same organisms

• Generally can not be blended away and currently there is no technology to remove it

– Threshold is in parts per trillion

Impact: Mousiness

• Oxygen and iron appear to play a role

• Romano, et al. Journal of Applied Microbiology Vol 104 issue 6 2007 found if there were multigram amounts of fructose or glucose, mousiness was more likely to be caused by Brett– Can occur in the bottle

Control of AABs

• Use of undamaged, non – moldy grapes• Hand harvest in cool temperatures• Indicators of possible spoilage

– Condition of grapes on arrival– Presence of Acetobacter species– Uncontrolled malolactic fermentation

• Reject unsuitable grapes– Corison (1979) recommended rejection of grapes – White grapes: 60 mg/L ethyl acetate– Red grapes: 115 mg/L ethyl acetate

Control of AABs

• SO2 addition and monitoring– However, some AABs are very resistant to SO2– Remember your SO2 effectiveness is a function of

your wine pH

• Cold settling, clarification of infected musts• Quick fermentation with commercial strains of

yeast• Control O2 exposure

– Cap management, headspace management– Frequent topping/tight bunging

• Get rid of old cooperage

• Sanitation– Caustic cleaners– Sanitizers

Conclusion: AABs

• Can oxidize ethanol to acetic acid

• Threshold of EA and acetaldehyde much lower than acetic acid

• May cause stuck fermentations

• Sugars to organic acids

• Mousiness

• Turbidity

Bacterial Spoilage During Aging: Acetic Acid Bacteria Mike Ramsey Teaching Laboratory Manager

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