Biocontrol Efficacy of the Vishniacozyma Victoriae in Semi-Commercial Assays for the Control of Postharvest Fungal Diseases of Organic Pears.

Vishniacozyma victoriae NPCC 1263 was selected for this work because of the active antagonistic effect over several fungi in postharvest organic pears. Yeast biomass production was carried out in a 15 L stirred-tank bioreactor with 12 L of working volume at 20 °C and 300 rpm and 0.64 vvm of aeration. The selected production medium was based on cheese whey powder and salts. The present study aims to evaluate the possibility of using an inexpensive growth substrate for production of added value products (yeast biomass), this innovation also requires evaluate biocontrol efficacy of yeast against fungal diseases of pears in semi-commercial assays. The yeast biomass was collected, cold stored for 60 days (Treatment 1) and 15 days (Treatment 2) and sprayed on the pears in semi-commercial level testing assays. After 180 days of postharvest conservation, significant reduction of fungal infection by Penicillium expansum, Botrytis cinerea and Cladosporium sp. was observed. The Treatment 1 reduced total diseases incidence by 71%, instead Treatment 2 reduced it by 92%. The effect of spray application on the yeast viability with different cold storage was tested. These work provides information on the bench-scale bioreactor yeast production using a new low-cost medium, viability and biocontrol efficacy of Vishniacozyma victoriae in controlling common diseases affecting pears in semi-commercial assays.

Production of a novel killer toxin from Saccharomyces eubayanus using agro-industrial waste and its application against wine spoilage yeasts.

The juicing industry generates large amounts of waste that mostly lack commercial value and, in the absence of waste treatment policies, produces environmental pollution. Also, microbiological spoilage is a major concern in the wine industry and control tools are limited. Taking these challenges into account, agro-industrial waste coming from ultrafiltrated apple and pear juice were used to grow Saccharomyces eubayanus and to produce its killer toxin (SeKT). A Plackett-Burman screening was performed in order to optimize SeKT production in ultrafiltrated apple and pear juice. The optimized medium was characterized: 75% v/v WUJ, 0.5% m/v KH2PO4, 0.5% m/v MgSO4, 0.5% m/v (NH4)SO4, 0.5% g/L urea, 10% v/v glycerol and 0.1% v/v Triton X-100. SeKT produced in WUJ optimised medium was used to perform killer assays against wine spoilage yeasts and showed antagonistic activity against Brettanomyces bruxellensis, Pichia guilliermondii, Pichia manshurica and Pichia membranifaciens. Different inhibition percentages against spoilage species in a wine environment (49-69%) were detected and preserved for at least 48 h. For the first time, this work reports the ability of S. eubayanus to produce a killer toxin with potential use as a biocontrol tool in winemaking. Producing SeKT using agro-industrial waste as an alternative medium to cultivate S. eubayanus would have industrial, economic and ecological benefits.

Purification and characterization of Saccharomyces eubayanus killer toxin: Biocontrol effectiveness against wine spoilage yeasts.

Microbiological contamination by spoilage yeasts species are frequent during winemaking, and biological control using antagonistic yeasts is considered a more beneficial alternative to conventional synthetic antimicrobials. Saccharomyces eubayanus killer toxin (SeKT) was produced and purified in a synthetic optimized medium. Purification procedure allowed the identification of SeKT as protein with an apparent molecular mass of 70 kDa and activity at physicochemical conditions suitable for winemaking process. Purified SeKT reduced the levels of volatile phenols produced by the spoilage yeasts Brettanomyces bruxellensis, Pichia membranifaciens, Meyerozyma guilliermondii and Pichia manshurica in wine-like medium. The putative mode of action of SeKT on sensitive yeast strains comprises cell wall disruption through ß-glucanase and chitinase activities as well as necrotic and apoptotic death in a toxin dose dependent manner. Thus, SeKT appears to be a promising biocontrol agent against spoilage yeasts during wine aging and storing.

Diversity and killer behaviour of indigenous yeasts isolated from the fermentation vat surfaces in four Patagonian wineries.

The diversity and killer behaviour of the yeast biota associated with surfaces of four Patagonian wineries were analyzed in the present study. These wineries were different in their technological and ecological features. Following liquid enrichment of samples from fermentation vat surfaces yeast isolates were identified by pheno- and genotyping and characterized using killer sensitivity patterns. Out of 92 isolated yeasts, 25% were Saccharomyces cerevisiae; 18% were Kloeckera apiculata and 11% were Pichia anomala; other six species representing a low percentage were also found. A particular biota composed mainly by S. cerevisiae (57%) and P. anomala (37%) was found in the winery located far from the other three wineries. As a whole, the wineries using spontaneous fermentation showed a major percentage of S. cerevisiae and a minor percentage of K. apiculata. The present study showed a pronounced heterogeneity in killer behaviour: killer, 35%, neutral, 25% and sensitive, 40%. In particular, S. cerevisiae isolates showed a higher sensitivity to killer reference yeasts than non-Saccharomyces isolates. On the other hand, most of the non-Saccharomyces yeasts isolated from fermentation vats were resistant to Saccharomyces toxins.

TdKT, a new killer toxin produced by Torulaspora delbrueckii effective against wine spoilage yeasts.

Microbiological spoilage is a major concern throughout the wine industry, and control tools are limited. This paper addresses the identification and partial characterization of a new killer toxin from Torulaspora delbrueckii with potential biocontrol activity of Brettanomyces bruxellensis, Pichia guilliermondii, Pichia manshurica and Pichia membranifaciens wine spoilage. A panel of 18 different wine strains of T. delbrueckii killer yeasts was analysed, and the strain T. delbrueckii NPCC 1033 (TdKT producer) showed a significant inhibitory effect on the growth of all different spoilage yeasts evaluated. The TdKT toxin was then subjected to a partial biochemical characterization. Its estimated molecular weight was N30 kDa and it showed glucanase and chitinase enzymatic activities. The killer activity was stable between pH 4.2 and 4.8 and inactivated at temperature above 40 °C. Pustulan and chitin ­ but not other cell wall polysaccharides ­ prevented sensitive yeast cells from being killed by TdKT, suggesting that those may be the first toxin targets in the cell wall. TdKT provoked an increase in necrosis cell death after 3 h treatment and apoptotic cell death after 24 h showing time dependence in its mechanisms of action. Killer toxin extracts were active at oenological conditions, confirming their potential use as a biocontrol tool in winemaking.