Wine Business Monthly - February 2006
New, Rapid Test for Brett Shows Promise
The new Miragene Zeta-Grip™ Brett test will require almost no special equipment, a minimum of staff training, and results can be obtained in less than three hours.
Spoilage of wines by Brettanomyces yeasts is a growing problem. This is due in part to currently popular winemaking practices such as extended "hang time," post-fermentation maceration, barrel aging, and a desire to reduce the use of SO2. Brettanomyces grows in finished wine, especially wine aged in barrels, often producing undesirable odors. The sporulating form of this yeast is known as Dekkera. Brettanomyces is a slow-growing yeast, unlike the fermentation yeast Saccharomyces cerevisiae, but it is a very tenacious yeast, being very difficult to eliminate from a winery once it becomes established.
Some strains of "Brett," as it is known in the wine industry, grow in a mold-like way. Their hair-like hyphae can penetrate into the surfaces of oak barrels. This is the reason for the caveat in the wine industry not to buy used barrels for fear they may have become infected with Brett. This yeast is rarely found in juice or fermenting wines, so its levels, if present, must be below what would be detected by peering through a microscope or plating for S. cerevisiae (i.e., less than 106 cells/mL).
After fermentation is complete, Brett can be found in high concentrations. This yeast is very alcohol-tolerant, with some strains able to use ethanol as a sole carbon source. Often, Brett is detected in barreled wine by the smell. It can produce several very aromatic substances, variously described as "Band-aid," "horsey," "sweaty" and "wet-dog-in-a-phone-booth." One of the compounds, 4-ethyl guaiacol, can have a rather pleasant spicy aroma, but it is usually overpowered by the strong-smelling 4-ethyl phenol, or may not be considered a desirable or appropriate character for the wine. Brett is also involved in the formation of biogenic amines, which can cause headaches in wine drinkers.
Monitoring wine for Brett is not an easy process for most wineries. While there are currently several methods available for detection of Brett, because it is very slow growing (one to two weeks on agar plates), spoilage can and often does occur before the winemaker is aware of contamination. Rapid methods are available but are prohibitively expensive for large-scale use and require considerable investment in equipment, time and expertise. A simple, inexpensive method that could detect Brett directly from wine in a few hours would give the winemaker an "early warning" tool in his or her campaign against Brett. For those who like a little Brett in their winemaking, for stylistic reasons, this method could serve as a monitoring device to keep the levels of Brett in check.
Rapid Immunoassays for Pathogen Detection
Immunoassay-based methods for pathogen detection are rapidly replacing microbiological methods in medicine. This trend is related to technical advances in immunochemistry that allow the development of such methods to be more rapid, specific and lower in cost than traditional methods. The application of immunochemistry to the testing for wine spoilage yeast and bacteria has, for the most part, not been attempted using the new generation of highly sensitive immunoassays that are currently available.
Another modern technique, DNA sequencing, is used in early phases of development to detect spoilage organisms, such as Brettanomyces. DNA sequencing uses "primers" for gene amplification to identify a specific species. Very little DNA is needed for such amplification to take place, and therefore, even a miniscule amount of DNA can result in a false positive. This issue confines DNA sequencing to very clean laboratory environments overseen by highly skilled technicians.
There are methods to control contamination and estimate the number of infectious cells; however these require a sterile lab environment, highly trained people and expensive equipment. These requirements result in a per-test cost that may be too high for all but the most premium wines, and may take days to send out samples and receive results. In contrast, immunoassays are very resistant to contamination and only require minimum training of staff.
The Miragene Zeta-GripTM method requires almost no special equipment, is low-cost, and results can be obtained in less than three hours. While this technique was initially developed and validated for medical applications, the researchers at California State University, Fresno and University of California, Irvine have teamed up with Miragene, Inc. to develop a rapid, low-cost test for Brett using the new Zeta-Grip technology. The new Brett test will be called Z-Wine AssayTM.
PRELIMINARY STUDIES
Assay Design The test is in an early phase of development. We envision the final test to be a white test strip in a small plastic well, approximately the size of a small matchbox. Different samples of wine are spotted, using an eyedropper, onto the strip next to identifier numbers. The numbers can be used to identify each sample from the barrel or bottle from which it originated. In some cases, the wine may be concentrated by centrifugation prior to spotting.
After spotting 10 wine samples on one test strip, several solutions (provided in kit) are added to the well, which then are shaken on a small portable shaker. The final step is the addition of a developer reagent. The developer creates a purple spot in any sample that contains Brett. The intensity of the spot is proportional to the amount of Brett in the sample. In order to determine the exact concentration of Brett, the chip is scanned using a typical flatbed computer scanner, and a computer program is used to calculate the amount of Brett contamination. Anyone with the skills involved in winemaking should be able to conduct the test easily and reliably.
Preliminary Results Preliminary tests were conducted on Brett strains Vin 1 (1) and Vin 8 (A), which are distinct morphologically and physiologically. Decreasing concentrations of Brett were used to test the sensitivity of the assay. The preliminary tests have shown promising results, with a visible reaction to Brett down to a concentration of approximately 1,000 cells/mL. The purple spots were very clear due to a strong immuno-reaction as well as the absence of background noise. From 107 cells/mL to about 10,000 cells/mL, the signal showed a linear correlation, making the prediction of Brett concentration reliable.
The method needs further development as there is still some cross-reaction with other yeasts that will be reduced by antibody purification. It was shown that there is negligible cross-reaction with bacteria and foreign contamination from the tester. With satisfactory preliminary results, the ongoing improvements in the method will be targeted towards increasing specificity and lowering the detection limit to 100 or even 10 cells/mL.
Discussion of Results Brett spoilage can occur at concentrations as low as 1,000 cells/mL. Thus, an effective assay should detect a few hundred Brett cells per mL. Such an outcome would allow a winemaker to maintain his or her winemaking style and avoid the damage that Brett can inflict on a wine. The Z-Wine Assay has demonstrated its potential to do exactly this.
Future Directions and Call for Collaboration
The Z-Wine Assay for Brett detection is an immunoassay-based test, which can be performed within a few hours without any special training or equipment, at a cost of only a few dollars per test. It is provided in kit form for use by even the smallest wineries. Plans are in the works to develop the Z-Wine Assay for other wine microorganisms (e.g., the early fermentation spoilage yeast, Hanseniaspora or Kloeckera, the bottled wine spoilage yeast, Zygosaccharomyces, and various lactic acid bacteria, such as Pediococcus and Lactobacillus). We envision being able to test for several different yeasts and bacteria on one chip. We would welcome comments from enologists and winemakers regarding other microorganisms of interest.
We are also investigating the possibility of coating 0.45µm membrane filters (47mm) with Zeta-Grip matrix. Wine would be poured through the Zeta-Grip filter in a filter funnel under vacuum in the conventional way that QC departments in the wine industry sample bottled wine to assess microbiological stability. The filter would be removed, dried and treated as described above. This new form of the Zeta-Grip technology may allow for simple and rapid detection of low levels of yeast and bacteria in wine. wbm
At California State University, Fresno: Dr. Susan Rodriguez is research fellow in the Viticulture & Enology Research Center; Dr. Roy Thornton is professor of enology in the Department of Viticulture & Enology; and Eiji Akaboshi is a graduate student in the Department of Viticulture & Enology. Dr. Stewart LeBrun is a scientist, UC Irvine and director, research and development, Miragene, Inc., Anaheim, California. Dr. William Edinger is senior research microbiologist for Constellation Wines U.S. in Madera, California. For more information, contact Susan Rodriguez at susanr@csufresno.edu or Stewart LeBrun at lebrun@miragene.com .
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