Pichia membranifaciens as spoilage yeast in Patagonian wines: isolation, origin and volatile phenols production

SAEZ, J.; LOPES, C.A.; KIRS VERONICA; SANGORRÍN, M.P.

Córdoba. Argentina

Congreso; VI Congreso Argentino de Microbiología General,; 2009

Yeasts belonging to the species Dekkera bruxellensis have long been associated withserious economic damages in winemaking due to its ability to grow and to producephenolic compounds. In order to evaluate the potential contamination of two wines,different yeast isolation protocols were evaluated.Wine samples were filtered using 0.2 ƒÊm pore size membranes and subsequentlyseeded in two selective agar media: A (GPY+cicloheximide+ampicillin) and B (GPY+CaCO3 +ampicillin). Alicuots of the same wines were inoculated into two selective liquidmedia: C (yeast extract+glucose+NO3K+ampicillin) and D(YNB+glucose+cicloheximida+ampicillin). After incubation at 26oC, yeast cultures wereisolated in the same (A and B) solid media. Yeast colonies from each wine wereselected according with their macroscopic features and frequencies and identified byITS1-5.8S rDNA-ITS2 PCR-RFLP analysis. Only two yeast species (Saccharomycescerevisiae and Pichia membranifaciens) were detected in both wines using three out offour different media. In medium A only S. cerevisiae colonies were observed. BecauseP. membranifaciens has been related to food spoilage, we evaluated the spoilagepotential of our isolates (production of volatile phenols in wine). We also evaluated thepossible source of contamination (vineyard or cellar surfaces).Healthy and damaged grapes located in both shadowed and sunny vineyard areaswere sampled before harvest in the same familiar cellar. Samples from 13 fermentationvats and filters were obtained by streaking with sterile cotton plugs and incubationusing selective liquid medium. After growth, samples were seeded in the same solidmedia and identified. Hanseniaspora uvarum and Zygoascus hellenicus were detectedin both healthy and damaged grapes from sunny areas; the same two species as wellas iPichia guilliermondii were isolated from damaged grapes in the shadow and only H.uvarum in healthy grapes in the shadow. Thirty-eight percent of the samples from vatsurfaces did not showed yeast isolates; however, 62% evidenced the presence ofCandida boidinii and the species P. guilliermondii, Rhodotorula mucilaginosa and H.uvarum were only detected one vat each. The only species detected in filters was P.membranifaciens. The intraspecific characterization of the P. membranifaciens isolatesby mtDNA-RFLP demonstrated that a same strain was detected in both vines and filter.Finally, the capacity of producing volatile phenols by different P. membranifaciensisolates was evaluated by HPLC in laboratory scale fermentations using sterile wineadded with the precursor p-coumaric acid (100 mg/L). Average values of 0,6 mg/L of 4-etilphenol and 2,7 mg/L of 4-vinilphenol were detected, evidencing for the first time thecapacity of this yeast species to produce these compounds in wine conditions