Molecular and physiological comparision of Pichia kudriavzevii isolates from natural and enological origins

DEL MÓNACO S.; LOPES C.A.; RODRÍGUEZ M.E.

Congreso; VIII Congreso Nacional Sociedad Argentina de Microbiología General; 2012

SAMIGE

Pichia kudriavzevii (formerly Issatchenkia orientalis) is a biotechnologically relevant yeast species able to resist different environmental stresses. Due to its high tolerance to high ethanol and sugar concentrations, this species has been frequently isolated from wine fermentations; where its growth can influence significantly the organoleptic properties of the final product. The aim of this work was to compare genetically and physiologically 24 isolates of Pichia kudriavzevii obtained from fermented fruits of three native plants from North Patagonia (Schinus johnstonii ?Molle-, Ephedra ochreata ?Solupe-, and Lycium chilense ?Yaoyín-), 3 isolates from Patagonian wine fermentations and 10 collection strains from different origins (collection strains Y-5396T, Y-7179, Y-134, YB-236, YB-238, Y-7724, Y-7750, Y-27196, Y-420 and Y-870).Intraspecific molecular characterization was carried out by two experimental approaches: (i) RFLP of the mitochondrial DNA (mtDNA) using restriction enzymes Hinf I, Dde I and Rsa I, and (ii) RAPD patterns (using primers OPA 9, OPA 12, OPA 14 y OPA 15). The RFLP-mtDNA analysis with the endonuclease Hinf I evidenced the highest diversity among isolates (9 different patterns); while using Dde I and Rsa I only six and 4 patterns were observed respectively. Independently from the restriction enzyme used, regional isolates from both native plant fruits and wine showed the same patterns, different from those detected in foreign collection strains. A dendrogram carried out using RAPDs data obtained from all 37 isolates concurred with the results obtained after RFLP-mtDNA analysis. These results suggest for the first time that native isolates of a non-Saccharomyces yeast are involved in the fermentation of regional grape musts. The influence of these regional strains in the organoleptic properties of wines could be related to the specific terroir of North Patagonian wines.The phenotypic characterization of P. kudriavzevii isolates was based in two physiological aspects relevant for the enological industry: killer activity and sensitivity patterns, and enzymes production (protease, pectinase, xylanase, β-xylosidase, α-ramnosidase and β-glycosidase). Assays were carried out in different solid media according with bibliographic reports. Our results indicate that all the isolates showed protease activity after 16 h of culture, a feature that is not typically found in yeasts and could be relevant in winemaking, where nitrogen is a limiting substrate for yeast development. Additionally, 95 % of the isolates showed β-glycosidase activity and 100% were not able to produce killer toxins with activity against reference sensitive strains. The influence of β-glycosidase activity on wine aroma has been largely demonstrated. All these results support the hypothesis that regional Pichia Kudriavzevii strains present interesting features to be potentially applied in winemaking, enhancing the regional and particular character of wines.