Partial characterization of the killer toxins produced by Saccharomyces cerevisiae strains Cf8 and M12

MIGUEL FERNÁNDEZ DE ULLIVARRI; LUCÍA M. MENDOZA; RAÚL R. RAYA

Mar del Plata

Congreso; VIII Congreso de Microbiología General; 2012

Sociedad Argentina de Microbiología General

Yeast strains capable of producing proteinaceous or glicoproteinaceous toxins that can kill or inhibit the growth of other microorganisms are increasingly being used as fermentation starters in winemaking to ensure the quality of fermented products. In this study, we evaluated the killer activity of two Saccharomyces cerevisiae strains (Cf8 and M12) on six wine spoilage yeasts. Cf8 and M12 yeasts were inoculated into YPD-MB broth (pH 4.6) during 96 h at 18 ºC, and their supernatants, obtained from co-cultures, sequential cultures or mixing monoculture supernatants (in a ratio 1:1), were then enriched with YPD nutrients and sterilized by filtration. The filtrates were inoculated with the spoilage strains Zigossacharomyces bailli BZb317, Pichia membranifaciens BPm481, Dekkera anomala BDa15, Schizosaccharomyces pombe BSp399, Z. bailli Ld1 and D. bruxellensis Ld2, and their killer effects were evaluated at 18 ºC after 36 h (OD600). Supernatants from monocultures were used as killer activity control, and non fermented medium as growth control. Killer activity was expressed as % reduction of absorbance measured against growth control. In order to better characterize the producer strains and their toxins, the sensitivity pattern (biotype) of Cf8 and M12 against reference killer strains (K1, K2, K3, K4, K6 and K10) by a plate test was also studied. Cf8 and M12 were seeded as a lawn in YPD-MB agar (pH 4.6). The presence of a clear zone around the spots of the reference killer strains after 96 h of incubation indicates sensitivity of the lawn yeast. Cross immunity between both strains and the effect of temperature and proteolytic enzymes on the killer activity were evaluated by OD600 in YPD-MB broth (pH 4.6). The presence of dsRNA as a genetic determinant of the killer phenotype was studied as described by Fried and Fink (1978). BDa15 and BZb317 were the most sensitive strains showing a reduction of absorbance (%) of 60.61, 69.34, 77.33 and 9.89, 41.32, 49.49 for co-culture, mix of supernatants and sequential cultures, respectively. Co-cultures were less inhibitory than the other combinations. The biotype test showed that M12 was sensitive to K1 and K2 toxins, while Cf8 was sensitive to K6 and K10 toxins. The cross immunity test indicated that both strains are sensitive to each other’s toxins. The proteinaceous nature of both toxins was confirmed by their sensitivity to chymotrypsin, trypsin and pepsin; Cf8 showed no killer activity after the treatments, while M12 exhibited a % relative activity of 14, 77 and 55%, respectively. Cf8 toxin was thermostable while M12 showed 72 % of relative activity after a 1 h treatment at 98 ºC. DsRNA plasmids were not visualized. This study evidence the differential features between the killer toxins of S. cerevisiae strains Cf8 and M12, and the cross immunity test suggest that the low killer activity in co-cultures of Cf8 and M12 strains might be caused by the inhibition between both strains.