Studies on the wine spoilage yeast Brettanomyces bruxellensis: 4-ethylphenol pr...

• Author(s): Fernandes, Miguel Tiago Nunes
• Date: 2013
• Persistent ID: http://hdl.handle.net/10362/10986
• Origin: Repositório Institucional da UNL
• Subject(s): Brettanomyces bruxellensis; Wine spoilage; Viable but nonculturable state; 4-ethylphenol; Selective growth media; Real-time PCR
• Document Type: Master Thesis
• Language: English
• Advisor(s): Ferreira, Manuel

Description

Dissertação para obtenção do Grau de Mestre em Biotecnologia Brettanomyces bruxellensis is a major threat to wine industry due to its spoilage ability characterized by high production of volatile phenols, mainly 4-ethylphenol. The horse sweat odor, characteristic of this phenol, causes large economic losses to wineries. A better understanding of the behavior of this yeast and better detection methods may lead to a decrease in 4-ethylphenol incidence in red wines worldwide. In the present work, we studied: (i) the ability of B. bruxellensis to enter the viable but nonculturable state by using both vital staining and plate counts to distinguish between viable and culturable cells; (ii) the production of 4-ethylphenol at different growth phases; (iii) the improvement of selective culture media; (iv) the application of a Real-Time PCR protocol for the rapid detection of B. bruxellensis. The existence of a viable but nonculturable state was evidenced during growth in synthetic medium ranging from 2% in strain ISA 2211 to 71% in strain ISA 1791 of the viable cells. The production rate of 4-ethylphenol was maximum when the precursor p-coumaric acid was added during exponential growth and decreased in stationary phase with incubation time. The developed selective medium presented recovery rates higher than the general purpose medium GYP and selectivity similar to DBDM. Response time lasted from 3 to 5 days while DBDM colonies appeared only after 12 days or more of incubation. Real-Time PCR showed to be an easy and faster method for a highly selective detection, taking 3 hours to obtain a positive response. The detection threshold was 700 cells/mL which may be decreased using sample concentration by centrifugation. However, results were 3.7 times higher than the viable counts, probably due to the DNA of dead or lysed cells. Collectively, this work represented a step forward in understanding the spoiling behavior of this yeast species and enabled the development of better detection methods for B. bruxellensis.