Purification and Characterization of WA18, a New Mycocin Produced by Wickerhamomyces anomalus Active in Wine Against Brettanomyces bruxellensis Spoilage Yeasts.

Francesca Comitini,Alice Agarbati,Edoardo Galli,Maurizio Ciani,Laura Canonico

doi:10.3390/microorganisms9010056

Francesca Comitini, Alice Agarbati + Show 3 more

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https://doi.org/10.3390/microorganisms9010056

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Journal: Microorganisms	Publication Date: Dec 28, 2020
Citations: 14	License type: CC BY 4.0

Affiliation: Marche Polytechnic University

Abstract

Wickerhamomyces anomalus strain 18, isolated from a natural underground cheese ripening pit, secretes a mycocin named WA18 that inhibits wine spoilage yeasts belonging to Brettanomyces bruxellensis species, with a broad-spectrum of activity. WA18 was purified, and the purified protein was digested with specific restriction enzymes (lysine K and arginine R cut sites). The LC–MS and LC–MS/MS analysis after enzymatic digestions revealed a molecular weight of 31 kDa. Bioinformatics processing and database research of digested pure killer protein showed 99% identity with a UDP-glycosyltransferase protein. Competitive inhibition assay of killer activity by cell-wall polysaccharides suggests that branched glucans represent the first receptor site of the toxin on the envelope of the sensitive target. The WA18 partially purified crude extract (PPCE) showed high stability of antimicrobial activity at the physicochemical conditions suitable for the winemaking process. Indeed, in wine WA18 was able to counteract B. bruxellensis and control the production of ethyl phenols. In addition, the strain WA18 was compatible with Saccharomyces cerevisiae in co-culture conditions with a potential application together with commercial starter cultures. These data suggest that WA18 mycocin is a promising biocontrol agent against spoilage yeasts in winemaking, particularly during wine storage.

Highlights

IntroductionThe use of killer yeasts can be considered a promising strategy in the terrestrial primary production for the control of microbial decay [6,7]
Yeast killer toxins are defined as antimicrobial proteinaceous compounds able to counteract sensitive yeast strains, they remain immune to their own toxin effect [1].Recently, killer yeasts and relative toxins have been reviewed by several authors discussing the molecular mode of action and their potential applications in view of possible use as preservatives in several fields [1,2,3,4,5].The use of killer yeasts can be considered a promising strategy in the terrestrial primary production for the control of microbial decay [6,7]
W. anomalus 18, B. bruxellensis strains coming from different sources such as grapes, winery or beer were used

Summary

Introduction

The use of killer yeasts can be considered a promising strategy in the terrestrial primary production for the control of microbial decay [6,7]. Kluyveromyces wickerhamii, Wickerhamomyces anomalus and Pichia membranifaciens represent an excellent example and have been extensively studied for their efficacy toward different sensitive spoilage yeasts in the winemaking field. W. anomalus is an ascomycetous heterothallic yeast of the family Wickerhamomycetaceae that reproduces asexually by budding and sexually by the formation of hat-shaped ascospores [8]. Strains of this species are present in several environments and have been mainly isolated from fruits and plant materials, cereal grain, maize silage, high-sugar food products, and wine [8]. W. anomalus is classified as a biosafety level 1 organism that

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