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Pcr method for detection of tyramine producer lactic acid bacteria in wine

Tyramine is a biogenic amine (BA) frequently detected in wines and responsible of health diseases.

The early detection of tyramine-producing lactic acid bacteria (LAB) could make it possible to prevent tyramine production in wine. This is of great interest not only for public health but also from an economic point of view, because products that exceed recommended limits of BA can be rejected from the market. Several methods to detect tyramine-producing LAB have been described.

The most reliable one consists in growing LAB in an appropriated medium containing the precursor amino acids and, after the required time of growth, tyramine production is determined by HPLC. However, this method is not easy to use, requires expensive equipment and necessitates several days to be performed. Identification of the tyramine-producing pathways in Lactobacillus brevis IOEB 9809 and Carnobacterium divergens AN 508, and comparison with the known sequences of Enterococcus faecalis and Enterococcus faecium allowed us to design PCR primers, named TD2 and TD5, located in the tyrDC gene.

The TD2-TD5 primer set allowed for the amplification of a 1100-bp DNA fragment in all LAB species above-mentioned. We have screened 209 LAB strains of the IOEB collection with these primers in order to determine if they can detect tyramine-producing strains of wine. The ability of each strain to produce histamine was confirmed by HPLC.

The results obtained by PCR and HPLC were identical. A total of 37 positive strains were detected. They belong to species as diverse as L. brevis, L. hilgardii, L. casei, L. fructivorans and Pediococcus parvulus. This PCR assay allows early detection of tyramine-producing bacteria during the manufacture of wine or other foods and also it permits the selection of commercial starter strains unable to synthesise tyramine. However, this assay is limited by the impossibility to quantify the population of tyramine-producing bacteria present in a sample. To circumvent this limitation it would be interesting to develop a similar assay based on real time quantitative PCR.

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Reported by

Faculty of Enology of Bordeaux 2 University
351, Cours de la Libération
33405 Talence
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