Reproduced with permission from the Enology-Grape Chemistry Group at Virginia Tech
By Molly Kelly, Extension Enologist, Enology Grape Chemistry Group, Virginia Tech
Wine Spoilage Yeasts
Kloeckera apiculata is the most common occurring yeast species on mature grapes. Other yeasts that may be isolated from grapes and must include species of Brettanomyces,Dekkera (the sporulating counterpart of Brettanomyces), Candida, Pichia, Hansenula andTorulopsis (Lafon Lafourcade, 1983). Diseased and damaged grapes have significantly higher populations of spoilage yeasts that can affect the outcome of the fermentation. These yeasts can metabolize the sugar in the grapes (which contain 160-240 g/L) mainly in the form of glucose and fructose. These yeasts can result in strong off-flavors and may be tolerant to sulfur dioxide.
Kloeckera apiculata has been reported to grow in the presence of up to 150 mg/L of SO2. It is also cold tolerant (10-15°C) and is able to survive until the end of fermentation. This can result in thick scum formation, ethyl acetate and amyl acetate (banana skin smell) formation. Its presence depletes nutrients needed by Saccharomyces cerevisiae for a successful fermentation. Kloeckera apiculata and its counterpart Hanseniaspora are fermentative yeasts and can grow in abundance early in native fermentations. They can represent the dominant species in unsulfited juice and must.
Using microscopy, if more than 5-10 non-Saccharomyces yeast are observed in a field under 40x magnification prefermentation, there will most likely be problems with the fermentation.Kloeckera has a distinct morphology when viewed using a microscope. It is described as lemon-shaped due to repeated budding at both poles. It will grow in 1-2 days in culture whileBrettanomyces requires 3-7 days to grow.
The most common form of yeast spoilage is due to Brettanomyces bruxellensis. This yeast produces volatile phenols and acetic acid. Examples of flaws include aromas described as “medicinal” in white wines and “leather” or “horse sweat” in red wines. Other aromas descriptors include barnyard, wet dog, tar, tobacco, creosote, plastic and band aid.
Brettanomyces can infect red wine 6-10 months after barreling and can spoil bottled wines as well. It can also be transmitted by fruit flies. It also grows on the disaccharide cellobiose, a by-product of toasting in barrel production. Control of this yeast is difficult due to its tolerance to sulfur dioxide.
Microscopically, they resemble S. cerevisiae but are usually smaller. The cells are described as ogival in shape. This shape results from repeated polar budding and looks like gothic arches. In older cultures, cells appear elongated and chains may also occur.
Prevention strategies for Brettanomyces include:
Grapes: Minimize damage to skins, pick when cool, sorting, add sulfur dioxide to picking bins, minimize transport distance and use adequate hygiene.
Winery: Winery equipment should be cleaned using regular cellar hygiene. Wines should contain adequate SO2 and air/oxygen exposure minimized. Filtration at the 0.6 micron level will eliminate Brettanomyces.
The two major groups of wine spoilage bacteria can be placed in either the acetic acid bacteria (AAB) or the lactic acid bacteria (LAB). The AAB include the genera Acetobacterand Gluconobacter. Both have aerobic metabolisms and thus their growth generally occurs on wine surfaces as a translucent film that tends to separate into a patchy appearance. In contrast, the LAB are microaerophilic to facultatively anaerobic, requiring low oxygen conditions for growth. The LAB include the genera Lactobacillus, Pediococcus andOenococcus.
During fermentation the presence of microbes may be indicated by ethyl acetate, a spontaneous or sluggish fermentation, spontaneous malolactic fermentation (MLF), volatile acidity (VA) or other off-odors.
Bacteria in this group, Acetobacter and Gluconobacter, use ethanol (and glucose) aerobically to form acetic acid. Of the two, Acetobacter is the most commonly encountered.Acetobacter can grow in barreled or bottled wines. It has the ability to grow using small amounts of oxygen absorbed during clarification and maturation. This organism is strictly aerobic (requires oxygen to grow) and appears as small rods or cocci (round) under magnification typically 0.6-0.9 microns by 1-3 microns in size. It also frequently occurs in pairs and chains.
Moldy grapes have a high population of AAB and this can lead to spoilage after crush. The most serious consequence of spoilage by AAB is the production of high levels of acetic acid (volatile acidity).
In order to control AAB, pHs should be low. Other control methods include minimizing oxygen incorporation, maintaining cool temperatures (< 50°F) and maintaining correct free SO2 levels. High VA wines (legal limit reds: 1.40 g/L, legal limit whites 1.20 g/L) can be blended with unaffected wine or treated with reverse osmosis.
Acetic acid is formed by S. cerevisiae at low levels during alcoholic fermentation. It is also produced by Oenococcus during malolactic fermentation (MLF) by the metabolism of citrate by Oenococcus. Commercial ML strains produce low levels of acetic acid but spoilage lactic acid bacteria produce more. VA can also be produced by Brettanomyces and LAB during primary fermentation.
There are two components of VA: acetic acid (smells like vinegar) and ethyl acetate (nail polish remover). Although we include ethyl acetate, this compound is an ester, not a volatile acid. Ethyl acetate is therefore not measured when using the Cash still. This only measures the level of acetic acid. Ethyl alcohol and acetic acid react to yield ethyl acetate and water. The main source for acetic acid alone is LAB. The main sources for acetic acid and ethyl acetate are Acetobacter and wild yeasts. The sensory threshold for these two compounds together is much lower than that of acetic acid alone. Post-fermentation sources of VA include headspace in barrels and oxidation of wine.
The typical spoilage times for LAB include during stuck fermentations and in finished wines with low SO2, and residual malic or sugar. These organisms appear rod-like using microscopy. The use of lysozyme will bring about destabilization of the bacterial cell wall peptidoglycan and an therefore be used to control LAB in wine.
In addition to the production of acetic acid through the metabolism of citric acid as well as glucose, LAB can result in a number of other faults. These include mousiness, geranium taint and ropiness. Mousey taint is an aftertaste. It is not volatile at wine pH but when mixed with the neutral pH of saliva, it becomes apparent. The taste is described as mouse urine and rancid nuts. This taint is usually due to LAB but can also be caused by Brettanomyces. Geranium taint is caused by the metabolism of sorbic acid by LAB. Sorbic acid is a yeast inhibitor added to prevent refermentation in the bottle. Although generally effective as a fermentative yeast inhibitor, sorbic acid shows little inhibition of LAB, AAB or film yeasts. Another fault caused by LAB, ropiness, is the result of the production of extracellular polysaccharides by LAB.
Some strains of LAB are beneficial such as Oenococcus oeni. This bacteria is involved in the decarboxylation of malic acid to lactic acid during malolactic fermentation (MLF). This reaction increases pH resulting in a “softer” mouthfeel. Diacetyl is also produced resulting in a “buttery” character. 1-4 mg/L of diacetyl is considered desirable depending on wine style, while high concentrations (>5-7 mg/L) is considered a spoilage characteristic.
In addition to the sensory implications, acetic acid and products of LAB metabolism act as inhibitors to Saccharomyces. This causes a delay in onset of fermentation or may cause a fermentation to stick. A sluggish fermentation should never be inoculated with malolactic bacteria. The bacteria can metabolize glucose and fructose to acetic acid, increasing VA by 1 g/L or more.
Other spoilage organisms that can be present during MLF include Acetobacter,Pediococcus, Brettanomyces and film yeasts. Wines should be monitored for VA increases (>0.15 g/L), surface films and off-odors/flavors.
The methods used to control/prevent LAB and AAB on grapes are the same as those for the spoilage yeasts discussed earlier. Regular cellar hygiene should be used to clean equipment. Grapes suspected to be infected should have short to no skin contact. Exclusion of air, filtration (0.45 micron) and acid additions can be used to control/prevent spoilage bacterial growth.
Some considerations when planning a winery microbiology laboratory are: space considerations, availability of trained staff to perform testing, willingness to maintain adequate record-keeping, equipment costs as well as the cost of consumables.
A microscope capable of 1000x magnification is needed to view bacteria and yeast. These can cost anywhere from $1000-$3000 but bargains can be found on used microscopes. A phase-contrast microscope requires no staining of slides. This feature also allows for rapid detection and response. The staff in the microbiology lab should have training in the proper use of a microscope as well as identification of microorganisms.
In addition to identifying spoilage organisms, a microscope can be used to monitor yeast populations. By using a simple methylene blue stain, yeast viability can be determined. Bacterial culture media is available for the growth of spoilage organisms for identification. This requires additional equipment including an incubator. This also requires further training in sterile technique and organism identification techniques. Several types of culture media exist for the detection of the organism of interest. For example, media used to plate forBrettanomyces exists that contains chloramphenicol (200 mg/L) to prevent bacterial growth while others may contain cyclohexamide to prevent Saccharomyces growth.
The membrane filter method can be used to isolate small numbers of microbes from a liquid sample. A sterile cellulose nitrate membrane (0.45 microns for bacteria, 0.65-8 microns for yeasts) is placed on a vacuum flask and filtered. Using sterile technique, the membrane is placed on the culture plate and monitored for growth. This method could be used to check bottle sterility.
The cellulose membrane can also be used to perform environmental monitoring on smooth surfaces. The sterile membrane is placed on the surface to be tested and then placed on an agar plate using sterile technique. The plate is then monitored for growth.
The swab test method is used for semi-quantitative analysis. Moist sterile cotton swabs are used to monitor dry areas (moistened with sterile saline or water). Dry swabs can be used to test moist areas. The swabs can then be used to inoculate the proper agar medium, depending on the organism of interest. Agar plates can also be used to detect airborne organisms at critical winery locations. Plates are left open for 30 minutes to 2 hours and then incubated. Airborne organisms that settle on the plate will grow and can be further identified.
It should be stressed that cellar hygiene is critical in maintaining wine integrity and quality. Poor wine quality is usually due to poor sanitation practices. Areas of spoilage organism build-up include: the vineyard, second-hand barrels, imported bulk wine and areas of the winery that are difficult to reach.