Killer yeasts as biocontrol agents of spoilage yeasts and bacteria isolated from wine

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

Mendoza

Congreso; 37th World Congress of Vine and Wine; 2014

OIV

During the winemaking process there is a predominance of fermentative yeasts as Saccharomyces cerevisiae, which are used as starter cultures. However, during fermentation other yeasts called non-Saccharomyces as well as different species of lactic acid bacteria (LAB) are also present. Yeasts of the genera Dekkera/Brettanomyces as well as Pichia guilliermondii and P. membranifaciens are generally considered wine contaminants during the fermentation stage or post-fermentation. On the other hand, among wine LAB Lactobacillus hilgardii and Pediococcus pentosaceus are also considered undesirable bacteria mainly for its ability to produce biogenic amines, compounds that affect the sanitary and sensory quality of wines. One strategy to prevent or reduce microbial contamination during the winemaking process is the use of killer yeasts. These yeasts produce proteic toxins that inhibit the growth of unwanted yeasts and fungi, but its effect on LAB has not been reported yet. The aim of this study was to evaluate the killer activity (KA) of autochthonous yeasts from Northwest region of Argentine, S. cerevisiae Cf8 and Wickerhamomyces anomalus Cf20 on different strains of spoilage yeasts and LAB of wine. The KA was evaluated using cell-free supernatants obtained from pure and mixed cultures of strains Cf8-Cf20 in YPD medium (pH 4.0, 20 ° C, 96 h). As control, the supernatant treated at 100 ° C for 1 hour to denature the toxin was used. The spoilage yeasts studied were: B. bruxellensis Ld1, D. anomala BDa15, P. membranifaciens BPm481, P. guilliermondii Cd6, Schizosaccharomyces pombe BSp399, Zygosaccharomyces bailii Ld2 and Z. bailii Bzb317. The killer supernatants were inoculated with 5x106 cfu / ml, incubated at 20 ° C for 48 h and growth inhibition was assessed by OD 600nm reading. The KA was calculated as % growth reduction in relation to the control. To study KA on LAB a screening was carried out in MRS agar medium using supernatants of each yeast 10x concentrated by ultrafiltration (Amicon 30 kDa) on a lawn of each LAB. In addition, the effect of the killer supernatants of each strain and co-culture Cf8-Cf20 on L. hilgardii growth and histamine production by RP-HPLC was evaluated. S. cerevisiae Cf8 supernatant produced a growth reduction between 10 and 35% on D. anomala BDa15, P. membranifaciens BPm481, P. guilliermondii Cd6 and Z. bailii Bzb317 while W. anomalus Cf20 exhibited KA of 20, 61 , 91 and 92% against B. bruxellensis Ld1, D. anomala BDa15, P. membranifaciens BPm481y P. guilliermondii Cd6, respectively. Killer mixed supernatants showed growth inhibition similar to strain Cf20. Screening against LAB showed that the killer toxins were able to inhibit the growth of L. hilgardii 5w, strain producing histamine. When KA of Cf8 and Cf20 supernatants was evaluated on this bacterium, it was observed a longer lag phase and lower final growth as well as a 16-31% decrease in biogenic amine production by this strain, being higher the inhibitory effect in presence of both killer toxins. These results confirm the potential of autochthonous killer yeasts as biocontrol agents in winemaking process. The mixed culture S. cerevisiae Cf8-W. anomalus Cf20 presented a wide range of KA on spoilage yeasts as well as on L. hilgardii. Therefore, the use of killer yeasts as starter cultures would permit to produce wines with controlled quality.