POLYMERASE CHAIN REACTION CONFIRMATORY METHOD FOR MICROBIOLOGICAL DETECTION OF BRETTANOMYCES BRUXELLENSIS IN WINES

Abstract

ABSTRACTBrettanomyces bruxellensis is one of the major causes of contamination in wine and is an important source of economic losses in this industry. In this work we developed a specific polymerase chain reaction (PCR) assay for the detection of B. bruxellensis to be used in the confirmation stage of the microbiological analysis. From a random amplification analysis using 40 primers in various B. bruxellensis strains and other yeasts that are generally present in must and wine, we designed the primers E09F and E09R that amplified a 450 bp product only in B. bruxellensis strains. We determined that the concentration of the PCR components and the annealing temperature are relevant factors in the PCR reaction, which was optimized using the response surface methodology. The protocol developed confirmed the contamination by B. bruxellensis in wines obtained from cellars, showing the capacity and speed of the technique to specifically confirm the results of the microbiological analysis.PRACTICAL APPLICATIONS Brettanomyces bruxellensis is responsible for the “Brett” character in wine that results in phenolic or medicinal aromas, leading to a decrease in its quality. The most utilized microbiological analysis for the detection of this yeast is based on the sequential use of selective media and confirmatory physiological tests that are unspecific and take up to 3 weeks to complete. However, its low cost means that this type of analysis is still widely used in the industry. On the other hand, molecular techniques are fast and specific in the amplification of DNA sequences. The polymerase chain reaction assay development in this work is specific for B. bruxellensis and allowed the confirmation of the microbiological analysis in only 2 days, compared with how long it currently takes (5–10 days), and at a lower cost than other molecular techniques.