Film Organisms: Acetic Acid Bacteria and Yeasts Michael S. Ramsey Teaching Laboratory Manager UCD

Outline Discussion: VA (volatile acidity) vs. acetic acid Yeast films Bacterial films Symbiosis/Synchronicities? Microbiological creation of spoilage compounds – And some chemical reaction spoilage Nutrient additions

What’s this VA thing all about anyway?

Vinegar was known early in civilization as the natural result of exposure of beer and wine to air – acetic acid-producing bacteria are present globally. The use of acetic acid in alchemy extends into the 3rd century BC, when the Greek philosopher Theophrastus described how vinegar acted on metals to produce pigments useful in art, including white lead (lead carbonate) and verdigris, a green mixture of copper salts including copper(II) acetate.

Ancient Romans boiled soured wine, reducing it to a highly sweet syrup called sapa. Sapa that was produced in lead pots was rich in lead acetate, a sweet substance also called sugar of lead or sugar of Saturn, which is believed to have contributed to lead poisoning among the Roman aristocracy. – Lead acetate was a common sweetener even into the Renaissance with notables such as Pope Clement II and Ludwig von Beethoven suspected as having died from consumption. (Carbonate of lead – white, or Venetian, lead - would be used into the 20 th Century) – Even after the substance’s use in food products was outlawed it’s use was difficult to trace until fairly modern times.

In the Renaissance, glacial acetic acid was prepared through the dry distillation of certain metal acetates (the most noticeable one being copper(II) acetate). Today, most of the acetic acid used industrially is produced chemically

VA vs. acetic acid: chemical analysis “Volatile acidity” is often wrongly assumed to be the total acetic acid content of a wine Although generally interpreted as acetic acid content, a “traditional” VA analysis includes any acid, that can be steam-distilled (or more precisely, steam-stripped), that is present in the wine – CO2 (as carbonic acid), SO2 (as sulfurous acid), sorbate, and lactic, formic, butyric, and propionic acids If acetic acid is specifically measured, as by enzymatic – spectrophotometric methods, results are strictly acetic acid

I’m Not A Cash Still! Most common apparatus – RD80 Volatile Acid Still Not really a “still”

VA vs. acetic acid: sensory analysis Acetic acid not as volatile as ethyl acetate Acetaldehyde is also often present Acetate is often called “acetic nose” – Wine concentrations range from 10 mg/L to 1200 mg/L – No legal limit

VA vs. acetic acid Some winemakers (including the late Emil Peynaud) believe the ethyl acetate component should be the legal indicator of wine spoilage – Previously, more difficult to measure analytically – Not as difficult with kits and spectrophotometry VA produced by lactic acid bacteria is often missing the ethyl acetate component (Henick- Kling, 1993)

Acetic acid Can be produced by Brett/Dekkera (but we are going with surface - film formers) Normal byproduct of Saccharomyces growth – Strains of S. cerevisiae have been shown to produce acetic acid based on increased activity of the enzyme acetyl-CoA synthetase – Fugelsang (1993) reported elevated levels when in co- culture with spoilage yeasts Can increase as a result of extended aging (1yr) in new barrels – Hydrolysis of acetyl groups in wood hemicellulose

Acetic acid Acetic acid can result from the oxidation of wine phenolics which produces hydrogen peroxide Which, in turn, oxides ethanol to acetaldehyde and then to acetic acid

Legal limits are still based on “distillable” Volatile Acidity 0.98 g/L OIV 1.4 g/L in red wine of this type of harvest (our experiment) Aroma threshold of acetic acid at around 1 g/L

Acetic acid from anaerobic bacteria Heterofermentative Lactic Acid Bacteria ferment glucose with lactic acid, ethanol/acetic acid and carbon dioxide (CO2)as by-products Important to always remember the other contributors, other than surface organisms, to acetic acid

Film Yeasts Under oxidative conditions, ethanol, glycerol, organic acids (esp. malic) can serve as growth media Can synthesize negative aroma compounds – Ethyl acetate – Acetoin (buttery cheese)

Film Yeasts Candida vini (formerly Candida mycoderma and often incorrectly identified by Kombucha makers as Saccharomyces mycoderma) Pichia species Saccharomyces cereviseae Growth may rapidly become pellicle Yeasts can initially appear as floating flowers – “Flowers of wine” – dusty

Saccharomyces cereviseae

Candida vini

Pichia kluyveri

Acetic Acid Bacteria Present on the grapes and in the winery environment Several genera and many species, most decline as ethanol is produced Acetobacter species survive through to aging, storage, and bottling – Can survive periods of anaerobic conditions – Begin again with O2 added during fining, racking, stirring, filtering, etc. – A. pasteurianus requires less O2 than A. aceti – Can survive in the bottle and regrow in days in an opened bottle

Acetobacter aceti

Acetobacter pasteurianus

A Fall Quarter Experiment Given oxidative conditions and headspace, would a late addition (post alcohol fermentation) of a commercial ML nutrient increase surface organism growth?

A Fall Quarter Experiment Barrel – aged 2011 California Malbec Controls One group received commercial ML nutrient at recommended dose Organisms added: – Flor – forming S. cereviseae – Pichia kluyveri – Candida parapsolosis – Acetobacter aceti

A note on Candida parapsilosis Most common yeast isolated from human hands and the most common cause of nail infections

Nutrients added A malolactic fermentation nutrient at recommended addition – These are generally blends of “inactive” yeasts to add amino acids, mineral cofactors, vitamins, cell wall polysaccharides, and cellulose

A Fall Quarter Experiment Visible surface film began to form within one week in both control and “plus nutrients” Added organisms could be seen under the microscope Added organisms were quickly overwhelmed by our indigenous Acetobacter pasteurianus

A Fall Experiment

By Week 5, our indigenous Acetobacter pasteurianus covered the surface of all containers No cells of any addition could be seen under the microscope

Wine data pH TA (g/L) Etoh % VA (g/L) RS (g/L) Base Wine Control Control PLUS Sacch Sacch PLUS Acetobacter aceti Acetobacter PLUS Candida Candida PLUS Pichia Pichia PLUS Although there appear to be trends…. If we exclude the Base wine, and we should, t – tests indicate there is no difference in the data sets

Drysdale and Fleet (1989) noted that the presence of Acetobacter resulted in stuck fermentations Doores(1993) found acetic acid to be inhibitory of Saccharomyces Acetobacter has been shown to be inhibitory of surface forming yeasts (Gilliland and Lacey, 1964) – Authors proposed some kind of antifungal was produced

Is there symbiosis? Remember, ethyl acetate and acetaldehyde were not measured and are not measured by Cash still or enzyme acetic

Could this spoilage have been avoided by gassing the surface?

Three Laws and the Real World Henry’s Law Law of Partial Pressures Ideal Gas Law Leaking connections and fittings Some efficacy in small containers (kegs)

Could this spoilage have been prevented strictly through sanitation?

No. We can not hope to eliminate 100% of all organisms through normal sanitation All it takes is the headspace to favor them

SO2? Largely ineffective if headspace Pichia and Candida species shown to be resistant to as much as 3 mg/L molecular SO2 – Pichia membranaefaciens is resistant to benzoate up to 1.5 grams/Liter Generally ineffective once the film is formed (Thomas and Davenport, 1985) Organisms create acetaldehyde, which binds SO2

The Best Preventative Measure Maintain topped tanks and barrels – Depriving organisms of oxygen and space to grow

Avoid moldy and damaged grapes Good sanitation helps Use of slow cellar temperatures can slow growth – Less than 60F (15C) can slow growth – Temperatures of 47F or less may inhibit growth

Conclusions Symbiosis? – Possible chemical differences early. Aerobic bacteria will win the day Did nutrient additions do anything? Number one component of creation of acetic acid was oxygen/headspace

What to do when you forgot about the headspace All methods carry some risk Blending Refermentation – Oxidatively growing yeasts can utilize acetic acid as a carbon source Reverse osmosis – Flavor and aroma modification and stripping Ion exchange – Flavor and aroma modification and stripping

Smell the glass at the side table DO NOT TASTE!

Acknowledgements FPM Group 5 – Friday afternoon – VEN 124 Wine Production class