Debaryomyces hansenii Strains Isolated From Danish Cheese Brines Act as Biocontrol Agents to Inhibit Germination and Growth of Contaminating Molds

Abstract

The antagonistic activities of native <i>Debaryomyces hansenii</i> strains isolated from Danish cheese brines were evaluated against contaminating molds in the dairy industry. Determination of chromosome polymorphism by use of pulsed-field gel electrophoresis (PFGE) revealed a huge genetic heterogeneity among the <i>D. hansenii</i> strains, which was reflected in intra-species variation at the phenotypic level. 11 <i>D. hansenii</i> strains were tested for their ability to inhibit germination and growth of contaminating molds, frequently occurring at Danish dairies, i.e., <i>Cladosporium inversicolor, Cladosporium sinuosum, Fusarium avenaceum, Mucor racemosus</i>, and <i>Penicillium roqueforti.</i> Especially the germination of <i>C. inversicolor</i> and <i>P. roqueforti</i> was significantly inhibited by cell-free supernatants of all <i>D. hansenii</i> strains. The underlying factors behind the inhibitory effects of the <i>D. hansenii</i> cell-free supernatants were investigated. Based on dynamic headspace sampling followed by gas chromatography-mass spectrometry (DHS-GC-MS), 71 volatile compounds (VOCs) produced by the <i>D. hansenii</i> strains were identified, including 6 acids, 22 alcohols, 15 aldehydes, 3 benzene derivatives, 8 esters, 3 heterocyclic compounds, 12 ketones, and 2 phenols. Among the 71 identified VOCs, inhibition of germination of <i>C. inversicolor</i> correlated strongly with three VOCs, i.e., 3-methylbutanoic acid, 2-pentanone as well as acetic acid. For <i>P. roqueforti</i>, two VOCs correlated with inhibition of germination, i.e., acetone and 2-phenylethanol, of which the latter also correlated strongly with inhibition of mycelium growth. Low half-maximal inhibitory concentrations (IC₅₀) were especially observed for 3-methylbutanoic acid, i.e., 6.32-9.53 × 10^-5 and 2.00-2.67 × 10^-4 mol/L for <i>C. inversicolor</i> and <i>P. roqueforti</i>, respectively. For 2-phenylethanol, a well-known quorum sensing molecule, the IC₅₀ was 1.99-7.49 × 10^-3 and 1.73-3.45 × 10^-3 mol/L for <i>C. inversicolor</i> and <i>P. roqueforti</i>, respectively. For acetic acid, the IC₅₀ was 1.35-2.47 × 10^-3 and 1.19-2.80 × 10^-3 mol/L for <i>C. inversicolor</i> and <i>P. roqueforti</i>, respectively. Finally, relative weak inhibition was observed for 2-pentanone and acetone. The current study shows that native strains of <i>D. hansenii</i> isolated from Danish brines have antagonistic effects against specific contaminating molds and points to the development of <i>D. hansenii</i> strains as bioprotective cultures, targeting cheese brines and cheese surfaces.