[Abstract]

Brettanomyces species and sub-strains are a group of wine spoilage yeasts that produce metabolites associated with poor wine flavor and aroma. At high concentrations (over 10⁴ cells/ml) modified phenolics and other undesirable metabolites produced by Brett build to levels that can substantially reduce wine quality. Current methods to test for Brett include selective plating, which takes over 10 days, and skill in microbiology and DNA based testing (“PCR”) which must be sent to an outside lab and is an expensive option for routine testing and may be prone to false positives.  Recently we have developed and started to sell a rapid (3 hour) immunoassay test for Brett. The test is performed by applying a small amount of wine to a test “chip”. The chip is processed as an immunoassay with a semi-quantitative result in 3 hours. The cell number is estimated by comparing the signal produced by the wine sample to be tested with a set of internal standards. Without concentration the test is sensitive to 10⁴.  Each test costs $15.
 

A 3 hour winery and field test for Brettanomyces
¹William Edinger, ²Stewart Lebrun and ³Geoffery Anderson

                                                     1. Constellation Wines, 2. Z-Enology, 3. Unitech Scientific

[Results]

 Beta Trials: Suspect samples were obtained form 5 wineries (N=37). Samples were plated on a Brett selective agar medium and spotted on  the Z-Brett chip (as in figure 1) and assayed as outlined in the procedures section. 

10^4 cutoff for Z-Brett
TN = 21
TP = 4
FN = 0
FP =  8
Accuracy = 25/37 = 67%

SENSITIVITY = 4/(4+0) = 100%

SPECIFICITY = 21/(21+8) = 72%

10^5 cutoff  for Z-Brett
TN = 31
TP = 0
FN = 4
FP = 2
Accuracy = 84 %

SENSITIVITY = (Need Bigger “n”)

SPECIFICITY = 31/(31+2) = 94%

 <Discussion>

Results suggest that this reagent kit would have greater utility if the sensitivity were improved to 10^2 CFU/ml.  Sample concentration by filtration satisfies this sensitivity for many (easily filtered) wines.  Concentration and enrichment methods to more fully meet this goal are currently being refined and validated.  Z-Brett kits are marketed by Unitech Scientific LLC (Booth 531.)

[Introduction]

Spoilage of wines by Brettanomyces yeasts is a growing problem, due in part to currently popular winemaking practices such as extended “hang time”, post-fermentation maceration, barrel ageing, and a desire to reduce the use of SO₂.  Brettanomyces grows in finished wine, especially wine aged in barrels, but can also grow to significant numbers in fermenting wines (V. Renouf et al., 2006. Journal of Applied Microbiology 100: 1208–1219).  Brett is also involved in the formation of biogenic amines. 

Plating methods generally take a week or more to get results, and PCR methods are costly and require a high level of expertise and technique.  An antibody-based method would have the advantages of other immunological assays of specificity, speed, and low cost.  A method based on polyclonal rabbit antibodies to Brettanomyces yeasts has been developed and is being tested in Beta-Sites and winery settings.

[Procedure]
Step 1 – Apply Samples      Mix wine samples immediately before spotting. Apply 5 µL to chip using a micro-pipette (figure 1)
Dry - Gently place entire chip in covered petri dish to dry overnight (be careful moving chips after spotting to prevent spots from running or smearing.)
o         Optional Quick Drying Method – Place petri dishes in 37 -55˚C incubator or under incandescent light for rapid drying for at least 30 minutes.  

Step 2 – Destain & Block     Using measuring tube provided, pour 10 mL Destain/Blocker (A) carefully over chip. Allow to sit for 15 minutes. (figure 2)

Step 3 – Anti-Brett Antibody       Add 5 drops of Anti-Brett (B) to the petri dish and mix by hand periodically (2 or 3 times) over 30 minutes. Discard all liquid from petri dish.

Step 4 - Conjugate        Using a clean (or rinsed) measuring tube, pour 10 mL of Buffer (C)  into dish to cover chip.  Add 5 drops of Conjugate (D) and mix by hand periodically (2 or 3 times) over 30 minutes, discard liquid.

Step 5 – Wash       Using a clean (or rinsed) measuring tube, pour 10 mL of Buffer (C) into dish to cover chip.  Mix gently by hand for about 1 minutes, discard liquid. Repeat two more times for a total of 3 washes.

Step 6 – Develop Color         Using a clean (or rinsed) measuring tube, pour 10 mL of Developer (E) into dish (figure 3).