INVESTIGADORES
SANGORRIN marcela paula
congresos y reuniones científicas
Título:
Purification and characterization of Saccharomyces eubayanus killer toxin with potential application in wine industry.
Autor/es:
VILLALBA, L.; LOPES, C.A.; SANGORRIN, M.P.
Lugar:
RECIFE
Reunión:
Jornada; V Jornadas Sudamericanas de Biología y Biotecnología de Levadura; 2015
Resumen:
Saccharomyces eubayanus NPCC1302 produces a killer toxin (Sekt) which inhibits the growth of spoilage yeasts isolated from wine cellar environments. This novel toxin was purified and characterized in order to be used as a biocontrol agent in winemaking.The optimal toxin production conditions were determined by Statistical Experimental Design (YPD medium + triton + glycerol, pH 4.6). The crude extract of Sekt obtained using the optimal culture conditions presented fungistatic activity against sensitive yeast Candida glabrata, but not against commercial wine starters and regional Saccharomyces wine strains. Sekt was found to be active under winemaking conditions: pH range 3,5-5,temperature of 4-35°C, 50-150 ppm of SO2, 8-16% v/v ethanol and 300 g/L glucose, as well as in wine and grape must. The results obtained by plasmid curing indicate that the killer character was chromosomally codified. Sekt has glucanase and chitinase activities. The toxin reduced its killer capacity when crude extract was incubated in the presence of pustulan, curdulan, chitin and laminarin, indicating that these polymers could participate as a target in the cell wall of sensitive strains. Sekt obtained from culture supernatant (0.66 SektUnits/mg protein) was purified with a tangential ultrafiltration and Shepharose-pustulan chromatography (45.50 SektUnit/mg protein). Purified Sekt showed different percentages of its antagonistic capacity against spoilage yeasts Pichia guillermondii (60%) Pichia membranifaciens (100%), Pichia manshurica (70%) and Brettanomyces bruxellensis (40%). Sekt subcellular mode of action was analyzed. C. glabrata showed an exponential death kinetics showing 90% of dead cells after 24 h of incubation with 2 SektUnits, 15% of them showed an apoptotic phenotype. However, the incubation with 1 SektUnit caused 40% of apoptotic nuclei at the same time. The same phenomenon was observed when spoilage yeasts were used as sensitive, except for P. membranifaciens which showed no apoptosis induction. Sekt also induced chitin ultraestructural modifications of cell wall of all sensitive strains evaluated, evidenced by a decrease of fluorescence after calcofluor white staining.Based on the data presented here, Sekt present different strategiess to kill, depending on toxin concentration and according to sensitive target yeast.