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The aim of the present study was to assess the antimold capacity of three Wickerhamomyces anomalus strains, both in vitro and in situ, and to identify the responsible volatile organic compounds. For that purpose, two substrates were applied; the former included brain heart infusion broth, adjusted to six initial pH values (3.5, 4.0, 4.5, 5.0, 5.5, 6.0) and supplemented with six different NaCl concentrations (0.0%, 0.5%, 1.0%, 1.5%, 2.0%, 2.5%), while the latter was a liquid dough, fortified with the six aforementioned NaCl concentrations. After a 24 h incubation at 30 °C, the maximum antimold activity was quantified for all strains at 5120 AU/mL, obtained under different combinations of initial pH value and NaCl concentration. A total of twelve volatile compounds were detected; ethanol, ethyl acetate, isoamyl alcohol and isoamyl acetate were produced by all strains. On the contrary, butanoic acid-ethyl ester, acetic acid-butyl ester, ethyl caprylate, 3-methyl-butanoic acid, 2,4-di-tert-butyl-phenol, benzaldehyde, nonanal and octanal were occasionally produced. All compounds exhibited antimold activity; the lower MIC was observed for 2,4-di-tert-butyl-phenol and benzaldehyde (0.04 and 0.06 μL/mL of headspace, respectively), while the higher MIC was observed for butanoic acid-ethyl ester and ethyl caprylate (5.14 and 6.24 μL/mL of headspace, respectively). The experimental breads made with W. anomalus strains LQC 10353, 10346 and 10360 gained an additional period of 9, 10 and 30 days of mold-free shelf life, compared to the control made by commercially available baker’s yeast. Co-culture of the W. anomalus strains with baker’s yeast did not alter the shelf-life extension, indicating the suitability of these strains as adjunct cultures.

Effect of Dough-Related Parameters on the Antimold Activity of Wickerhamomyces anomalus Strains and Mold-Free Shelf Life of Bread