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

Impact: Volatile Acidity 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

Impact: Volatile Acidity 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 OIV White: 1.2 g/L 1.1g/L 0.98g/L Red: 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 (

Impact: 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

Impact: Ethyl Acetate 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.

Impact: Ethyl Acetate 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

Impact: Ethyl Acetate 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 ppm (mg/L)

Impact: Acetaldehyde 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

Impact: Acetaldehyde (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 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