Comparative Zymocidial Effect of Three Different Killer Toxins against Brettanomyces bruxellensis Spoilage Yeasts

Abstract

Three killer toxins that were previously investigated, one excreted by Kluyveromyces wickerhamii and two by different strains of Wickerhamomyces anomalus, were produced at the pilot scale, lyophilized and characterized, and the formulates were assessed for their zymocidial effect against Brettanomyces bruxellensis spoilage yeast. A comparative analysis allowed the evaluation of the minimum inhibitory concentration (MIC) against a sensitive strain. Fungicidal and fungistatic concentrations were used to evaluate the cytocidal effect using a cytofluorimetric approach that confirmed the lethal effect of all lyophilized formulates against B. bruxellensis spoilage yeasts. Moreover, the potential killer toxins’ cytotoxicity against human intestinal cells (Caco-2) were evaluated to exclude any possible negative effect on the consumers. Finally, the effective lethal effect of all three lyophilized killer toxins toward B. bruxellensis sensitive strain were tested. The results indicated that all of them acted without dangerous effects on the human epithelial cells, opening the way for their possible commercial application. In particular, D15 showed the lowest MIC and the highest activity, was evaluated also in wine, revealing a strong reduction of Brettamonyces yeast growth and, at the same time, a control of ethyl phenols production.

Biocontrol and Enzymatic Activity of Non-Saccharomyces Wine Yeasts: Improvements in Winemaking

“…There are a wide variety of non-Saccharomyces yeast genera that are now being used to enhance wine flavour or modulate its composition [5]. In addition, biocontrol yeasts can be applied in winemaking through two different approaches: those in which a product of the microbial metabolism is added as a biopreservative [105,106] or those in which the microorganism is added as a starter/protective culture [107][108][109]. In this section, we review the most relevant research and current knowledge on biocontrol in winemaking, highlighting the role of non-Saccharomyces wine yeasts as sources of antimicrobial metabolites or as selected strains to control wine spoilage yeasts during fermentation.…”

“…The killer character, first discovered in S. cerevisiae in 1963 [120], has been described in a variety of genera other than Saccharomyces, including Candida, Cryptococcus, Debaryomyces, Hanseniaspora, Hansenula, Kluyveromyces, Metschnikowia, Pichia, Wickerhamomyces, Williopsis, Ustilago, and Zygosaccharomyces [13,92,108,118,121,122]. The use of non-Saccharomyces KTs has been extensively studied to control the development of wine spoilage yeasts, given that these toxins typically have a broader spectrum of inhibition and, in some cases, greater stability than S. cerevisiae KTs [15,92,105,123]. Following a report of the extensive anti-Hanseniaspora killer activity produced by Kluyveromyces phaffii (reclassified as Tetrapisispora phaffii) [124], most studies have focused on killer toxins active against Dekkera/Brettanomyces, the main wine spoilage yeast present during wine fermentation and in postfermentative ageing processes.…”

“…Although the WA18 strain was demonstrated to be compatible with S. cerevisiae under coculture conditions, the WA18 mycocin was proposed as a biocontrol tool with good potential to inhibit spoilage yeasts in winemaking, especially during wine storage. Recently, three killer toxins previously investigated (named Pikt, Kwkt, and WA18), two secreted by two different W. anomalus strains and one excreted by K. wickerhamii yeast, were produced at the pilot scale, lyophilised, and characterised [105]. All preparations greatly reduced B. bruxellensis populations at different doses; in particular, the lyophilised killer toxin D15 (Kwkt) was also efficient during the industrial winemaking stage, achieving the long-term control of Brettanomyces yeasts and, at the same time, the significant reduction in 4-ethylphenol production.…”

“…Some of the killer toxins described in the literature to date are produced by yeast strains from ecological niches different from the place where they are proposed to be used, such as from olives, in soils from different environments, cheese ripening pits and, in some cases, obtained from culture collections without specifying their origin [105,115,118,122,125,129]. This is not necessarily a limitation, especially if the killer toxin is considered as a bioprotective additive in the winemaking process.…”

Biocontrol and Probiotic Function of Non-Saccharomyces Yeasts: New Insights in Agri-Food Industry

“…In a recent work, a comparative evaluation of formulates (semi-purified and lyophilized broth) of three well-characterized mycocins was assessed against B. bruxellensis . The absence of dangerous effects toward human epithelial cells opens the way for their possible commercial application [ 67 ]. Another NSY that shows antimicrobial activity is T. delbrueckii, where a mycocin exhibited glucanase and chitinase activities, it is stable in wine environmental conditions and it is active against B. bruxellensis and other potential wine spoilage yeasts.…”