1. CHARACTERIZATION OF NOVEL KILLER TOXINS SECRETED BY NON-SACCHAROMYCES YEASTS
Nwabisa N. Mehlomakulu, Mathabatha E. Setati and Benoit Divol
Institute for Wine Biotechnology, Department of Viticulture and Oenology, Stellenbosch University, Stellenbosch ZA-7600, South Africa
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Introduction
Experimental layout
Brettanomyces bruxellensis is considered as a major red wine spoilage yeast, due to its ability to survive in nutrient limited, low molecular SO2 concentration and semi-aerobic conditions found at the end of fermentation and during ageing in wooden barrels (Oelofse, 2008).
B. bruxellensis produces volatile phenols in wine, causing off-flavours and odours in spoiled wine. SO2 is currently used to prevent the growth of B. bruxellensis. However, some strains of B. bruxellensis are resistant to SO2 and the antimicrobial activity of SO2 is dependent on the pH of the wine (Wedral, 2010).
The use of biological antimicrobial compounds such as killer toxins, seems to be a propitious method to control B. bruxellensis.
Killer toxins secreted by the yeasts Pichia membranifaciens, Kluyveromyces wickerhamii, Pichia anomala and the fungus Ustilago maydis respectively have been shown to display antimicrobial activity against B. bruxellensis in grape must or wine (Liu et al., 2013).
Killer toxins exert their killing action either through interaction with the cell wall, cell membrane or cell cycle of the sensitive strain leading to cell wall lysis, ion leakage or inhibition of DNA synthesis respectively (Magiali et al., 1997).
The killer toxins bind to primary receptors or secondary receptors on the cell wall or membrane of the sensitive yeast cell respectively to induce their killing action (Guo et al., 2012).
Temperature and pH activity optima and stability assays were determined on white grape juice agar plates prepared by the seeded agar method. 106 cfu/mL cells of B. bruxellensis were inoculated into 7.5 mL of white grape juice pH adjusted to 4.5 (except for the pH stability assay). 2.5 mL of 4% bacteriological agar (kept at 50°C) was mixed with the inoculated medium to a final volume of 10 mL and after brief vortexing, the medium was poured into sterile Petri dishes.
Crude extracts prepared by ultrafiltration (50kDa centrifugal filter) of the supernatant of a 24 h culture of the Candida pyralidae strains IWBT Y1140 and IWBT Y1057 was used as the killer toxin. 20 µL of the crude extracts was spotted on 7-mm wells drilled on the white grape juice agar plates. Killer activity was observed as a zone of clearance around the well after incubation at 20°C on triplicate plates.
The crude extracts were exposed to 20 mg/mL of the polysaccharides: laminarin, mannan and pullulan for 1 and 6h at 20 and 25°C for the IWBT Y1140 and IWBT Y1057 strains respectively. Residual killer activity was tested as described above in comparison to a control assayed for killer activity immediately after addition of the polysaccharide.
Results continued…
Binding affinity of killer toxins to different cell wall polysaccharides
Aims of study
Characterization of killer toxins secreted by the wine yeast C. pyralidae in conditions found in wine making
Identification of the primary cell wall receptors of the killer toxins
Results
Influence of environmental parameters on killer toxin activity
(a) CpKT1
(b) CpKT2
Figure 3: CpKT1 and CpKT2 binding to cell wall polysaccharides
(a)
(b)
Conclusion
CpKT1 and CpKT2: killer toxins secreted by C. pyralidae IWBT Y1140 and C. pyralidae IWBT Y1057 respectively
C. pyralidae IWBT Y1140 and IWBT Y1057 strains secrete 2 different killer toxins, namely CpKT1 and CpKT2 respectively.
The molecular weight of both toxins is above 50kDa.
The killer toxins exhibited killer activity and stability at temperature, pH, ethanol and sugar concentrations found during winemaking. However, differences between the two toxins were observed.
CpKT1 and CpKT2 were identified as potential biopreservatives against B. bruxellensis.
CpKT1 and CpKT2 had stronger binding affinity for laminarin and mannan, respectively than for the other polysaccharides, suggesting that the cell wall glucans and mannoproteins could act as receptors for these toxins.
(a) Temperature activity optima
(b) pH activity optima
Figure 1: Temperature and pH activity optima
Influence of environmental parameters on killer toxin stability
(a)
(b)
Acknowledgements
The authors thank the National Research Foundation, South Africa for financial support
(c)
(d)
(a) CpKT2 temperature (b) CpKT1 pH (c ) CpKT2 ethanol (d) CpKT1 sugar
Figure 2: Temperature, pH and Ethanol Stability