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

SÁEZ J.S.; LOPES C.A.; KIRS V.; SANGORRÍN M.P.

Villa Carlos Paz, Córdoba, Argentina

Congreso; VI Congreso Nacional Sociedad Argentina de Microbiología General; 2009

Yeasts belonging to the species <i>Dekkera bruxellensis</i> have long beenassociated with serious economic damages in winemaking due to its ability to grow andto produce phenolic compounds. In order to evaluate the potential contamination of twowines, different yeast isolation protocols were evaluated.Wine samples were filtered using 0.2 μm 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(<i>Saccharomyces cerevisiae</i> and <i>Pichia membranifaciens</i>) were detectedin both wines using three out of four different media. In medium A only <i>S.cerevisiae</i> colonies were observed. Because <i>P. membranifaciens</i> has beenrelated to food spoilage, we evaluated the spoilage potential of our isolates (productionof volatile phenols in wine). We also evaluated the possible 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. <i>Hanseniaspora uvarum</i> and <i>Zygoascus hellenicus</i>were detected in both healthy and damaged grapes from sunny areas; the same twospecies as well as <i>Pichia guilliermondii</i> were isolated from damaged grapes inthe shadow and only <i>H. uvarum</i> in healthy grapes in the shadow. Thirty-eightpercent of the samples from vat surfaces did not showed yeast isolates; however, 62%evidenced the presence of <i>Candida boidinii</i> and the species <i>P.guilliermondii</i>,<i>Rhodotorula mucilaginosa</i> and <i>H. uvarum</i> were onlydetected one vat each. The only species detected in filters was <i>P.membranifaciens</i>. The intraspecific characterization of the <i>P.membranifaciens</i> isolates by mtDNA-RFLP demonstrated that a same strain wasdetected in both vines and filter.Finally, the capacity of producing volatile phenols by different <i>P.membranifaciens</i> isolates was evaluated by HPLC in laboratory scalefermentations using sterile wine added 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 weredetected, evidencing for the first time the capacity of this yeast species to producethese compounds in wine conditions.