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

The antagonistic activities of native Debaryomyces hansenii 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 D. hansenii strains, which was reflected in intra-species variation at the phenotypic level. 11 D. hansenii strains were tested for their ability to inhibit germination and growth of contaminating molds, frequently occurring at Danish dairies, i.e., Cladosporium inversicolor, Cladosporium sinuosum, Fusarium avenaceum, Mucor racemosus, and Penicillium roqueforti. Especially the germination of C. inversicolor and P. roqueforti was significantly inhibited by cell-free supernatants of all D. hansenii strains. The underlying factors behind the inhibitory effects of the D. hansenii 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 D. hansenii 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 C. inversicolor correlated strongly with three VOCs, i.e., 3-methylbutanoic acid, 2-pentanone as well as acetic acid. For P. roqueforti, 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 C. inversicolor and P. roqueforti, 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 C. inversicolor and P. roqueforti, respectively. For acetic acid, the IC50 was 1.35-2.47 × 10-3 and 1.19-2.80 × 10-3 mol/L for C. inversicolor and P. roqueforti, respectively. Finally, relative weak inhibition was observed for 2-pentanone and acetone. The current study shows that native strains of D. hansenii isolated from Danish brines have antagonistic effects against specific contaminating molds and points to the development of D. hansenii strains as bioprotective cultures, targeting cheese brines and cheese surfaces.

The Effects of NaCl and Temperature on Growth and Survival of Yeast Strains Isolated from Danish Cheese Brines.

Yeasts play an important role in cheese making, by contributing to microbial community establishment and improving flavor. This study aimed at investigating the impact of NaCl and temperature on growth and survival of 20 strains belonging to the yeast species Candida intermedia (2 strains), Debaryomyces hansenii (11), Kluyveromyces lactis (1), Papiliotrema flavescens (1), Rhodotorula glutinis (1), Sterigmatomyces halophilus (2) and Yamadazyma triangularis (2) isolated from Danish cheese brines. All yeasts could grow in Malt Yeast Glucose Peptone (MYGP) medium with low NaCl (≤ 4%, w/v) concentrations at 25 °C and 16 °C. Further, none of the strains, except for one strain of D. hansenii (KU-9), were able to grow under a condition mimicking cheese brine (MYGP with 23% (w/v) NaCl and 6.3 g/L lactate) at 25 °C, while all yeasts could grow at 16 °C, except for the two strains of C. intermedia. In the survival experiment, D. hansenii, S. halophilus and Y. triangularis survived in MYGP with 23% (w/v) NaCl throughout 13.5 days at 25 °C, with Y. triangularis and S. halophilus being the most NaCl tolerant, while the remaining yeasts survived for less than 7 days. These results enable the selection of relevant yeasts from cheese brines for potential use in the cheese industry.