Photo: Molly Kelly, Penn State
Two of the most common spoilage yeasts include Kloeckera apiculata and Brettanomyces bruxellensis. The most common form of yeast spoilage is due to Brettanomyces bruxellensis. Although mature grapes may harbor this spoilage yeast, the bigger problem can occur when winery equipment is infected due to poor sanitation practices. This yeast produces volatile phenols and acetic acid. Examples of wine 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 aids.
The two major groups of wine spoilage bacteria can be placed in either the acetic acid bacteria (AAB) group or the lactic acid bacteria (LAB) group. The AAB includes the genera Acetobacter and Gluconobacter. Both have aerobic (requiring oxygen) 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 require low oxygen conditions for growth (i.e. they are microaerophilic to facultative anaerobic micro-organisms). The LAB includes the genera Lactobacillus, Pediococcus and Oenococcus.
During fermentation the presence of such microbes may be indicated by a spontaneous or sluggish fermentation, or a spontaneous malolactic fermentation (MLF); or the presence of ethyl acetate, volatile acidity (VA) or other off-odors.
Winery Microbiology Laboratory
Because of these possible faults arising due to the presence of spoilage organisms, some wineries have incorporated sanitation monitoring and microbiological techniques into their production practices. 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.
Figure 1. (left) Brettanomyces spp. using phase contrast. Photo: David Hornack Figure 2. (right) Acetobacter spp. and yeast on WL agar. Photo: Molly Kelly, Penn State
A microscope capable of 1000x magnification is needed to view bacteria and yeast. These can cost anywhere from $1000-$3000 or more but bargains can be found on used microscopes. A phase-contrast microscope requires no staining of slides due to enhanced differences in refractive index between the microorganisms and surrounding medium. 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 for Brettanomyces contains chloramphenicol (200 mg/L) to prevent bacterial growth while others may contain cyclohexamide to prevent Saccharomyces growth. Common media used in culturing juice, wine and environmental samples include WL and WL-differential agar.
Membrane Filter Method
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.
Figures 3 and 4. Membrane filter method. Photos: Molly Kelly, Penn State
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.
Monitoring systems exist that utilize bioluminescence technology to measure adenosine triphosphate (ATP). ATP is found in all plant, animal and microbial cells and is the prime energy currency that fuels metabolic processes. It is therefore possible to detect and measure biological matter that should not be present if proper sanitation practices are followed. One system by Hygiena™ uses an enzyme found in fireflies (luciferase). In the presence of ATP, an oxidation reaction occurs that results in light formation that is directly proportional to the amount of ATP present. Results are numeric and expressed as relative light units (RLU).
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.
Figure 5. (left) Bioluminescence technology. Figure 6. Cellar hygiene. (right) Photos Molly Kelly, Penn State
There are commercial enology laboratories that provide all of the microbiological services discussed here. For more information please contact Molly Kelly.
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