Recipient Organization
UNIVERSITY OF CALIFORNIA, DAVIS
410 MRAK HALL
DAVIS,CA 95616-8671
Performing Department
VITICULTURE AND ENOLOGY
Non Technical Summary
Nontechnical summary Yeasts populations stressed by the addition of sulfite, a preservative, to wine fermentations will be enumerated by quantitative PCR (QPCR) and these results will be compared with those obtained by traditional enumeration methods (plating). QPCR assays will be developed for a number of yeasts important to wine fermentations such as Hanseniaspora uvarum, Zygosaccharomyces bailii and Dekkera bruxellensis. The results from the comparison between enumeration by QPCR and plating will shed light on the size and potential activity of the nonculturable yeast populations that persist in wine fermentations. In the second phase of the study experiments using the wine spoilage yeast Dekkera will be conducted. We will attempt to characterize the production of the spoilage compound 4-ethylphenol from nonculturable Dekkera populations. Also, attempts will be made to revive the nonculturable populations thereby determining if these populations, while not detected
by traditional methods, can regrow and cause spoilage of food products.
Animal Health Component
30%
Research Effort Categories
Basic
40%
Applied
30%
Developmental
30%
Goals / Objectives
1. Determine the effectiveness of Quantitative-PCR (Q-PCR) assays on enumeration of stressed (sulfite and temperature) yeasts present in early stages of the wine fermentation (Saccharomyces cerevisiae 01-158, Hanseniaspora uvarum 54-192, Pichia membranaefaciens 57-22 Metschnikowia pulcherrima 40-214, Kluyveromyces thermotolerans 55-41 and Candida sp EJ1). Compare direct counts using Q-PCR to those using the yeast Live/Dead Kit of Molecular Probes and plating on neutral or acid media. 1. Develop genera specific Q-PCR assays. 2. Propagate yeasts under stress and enumerate using Q-PCR, Live Dead yeast kit and plating. 1. Determine the effectiveness of Q-PCR assays on enumeration of yeasts present in late stages of the wine fermentation (Brettanomyces anomalus 82-27, Brettanomyces bruxellensis and Zygosaccharomyces bailii 68-113). Compare direct counts using Q-PCR to those using the yeast Live/Dead Kit of Molecular Probes, plating (at neutral or acidic pH) and production
of 4-ethylphenol and 4-ethylguaiacol by Brettanomyces sp. 1. Develop genera specific Q-PCR assays. 2. Propagate yeasts in wine with and without sulfite and enumerate using Q-PCR, Live Dead yeast kit and plating. 3. Measure 4-ethylphenol and 4-ethylguaiacol production and relate this to the results from objective 2B. 4. Establish the different Brettanomyces populations (injured, viable, and VBNC) using flow cytometry to separate cells in different physiological states. Resituate the possible VBNC Brettanomyces population using the MPN method of Kaprelyants et al. and ascertain the injured population by plating on low pH and neutral pH media. 1. Using the Q-PCR methods developed in objective 2A, survey phenolic wines received from industry for the presence of Brettanomyces. Measure 4-ethylphenol and 4-ethylguaiacol in these samples and correlate this to the level of Brettanomyces discovered in the wines.
Project Methods
Approach This proposal will investigate the detection of wine yeast under stress and the ability of spoilage yeasts to persist and affect wine, the model food system, even when not detectable by conventional methods. The project will be done in three phases. First Quantitative-PCR (Q-PCR) methods to detect yeasts present in the early stages of thewine fermentation will be developed. Saccharomyces cerevisiae 01-158, Hanseniaspora uvarum 54-192, Pichia membranaefaciens 57-22 Metschnikowia pulcherrima 40-214, Kluyveromyces thermotolerans 55-41 and Candida sp. EJ1 are yeasts that will be studied during this phase of the project. The Q-PCR assays will use primers specific to 26s rDNA and will be developed for each genera. The yeasts will be grown in grape juice and subjected to the addition of sulfite. Detection of the stressed yeasts by Q-PCR will be compared to plate counts on neutral and acidic pH media and the viable count as determined by staining. The second stage of
this project will examine the growth of spoilage yeasts, Brettanomyces anomalus 82-27, Brettanomyces bruxellensis and Zygosaccharomyces bailii 68-113, which are present at the end of the fermentation or during aging. A relationship between the Q-PCR results, staining and Brettanomyces production of 4-ethylphenol and 4-ethylguaiacol will be developed. The different populations of Brettanomyces will be separated by flow cytometry and these populations will be examined individually for their ability to regrow in wine and/or produce 4-ethylphenol. In the finial stage of this study, the relationship between Brettanomyces and 4-ethylphenol developed in stage two will be explored in wine samples obtained from industry.