The growing application of yeasts in the food industry has spurred extensive research into their biotechnology properties. With the gradual deepening study of yeast, more biotechnology properties of yeasts have been unveiled. The potential of native yeasts isolated from food manufacturing environments for inhibiting the growth of mould contaminants in food production have attracted interest. As one of the challenging problems associated with food quality and safety worldwide, mould contamination causes economic losses and is a threat to public health. Prevention of mould growth is a matter of significant importance. The main aim of this PhD research was to characterize yeasts isolated from Danish cheese brines and to evaluate their potentials as biocontrol agents against contaminating moulds.
Brining is a vital step in the industrial production of smear-typed cheeses. Brine is used repeatedly, accumulating nutrients drained from cheese curd and becoming a natural medium for salt-tolerant microflora to grow, while it turns out to be an entry spot for salt-tolerant microorganisms to colonize on the cheese surface. This thesis aimed to investigate the impact of NaCl and temperature on the growth and survival of 20 yeast strains isolated from Danish cheese brine. Inter- and intra-species variations in both their growth and survival characteristics were observed. Notably, the condition mimicking cheese brine had less effect on the growth of brine yeasts at 16 °C compared to 25 °C. Moreover, Debaryomyces hansenii, Sterigmatomyces halophilus, and Yamadazyma triangularis survived and grew in MYGP with 23% (w/v) NaCl throughout 13.5 days, indicating salt-tolerant properties of these three yeasts. Results suggest the feasibility of selecting yeasts isolated from cheese brines as brine starter cultures for standardizing brine microbiota composition.
Debaryomyces hansenii is one of the predominate yeasts isolated from Danish cheese brines, and has been applied as a commercial culture in several dairy products. The potential use of D. hansenii as a biocontrol agent has been investigated in several types of foods, including fruits, meats, grains, and dairy products, whereas the practical application of D. hansenii isolated from food manufacturing against contaminating moulds has not been fully understood yet. This thesis found, using pulsed-field gel electrophoresis, the extensive genetic heterogeneity among the D. hansenii strains. Strain variations in chromosome profiles of D. hansenii were partially reflected in their acidification and deacidification activities. Further, the inhibitory effect of cell-free supernatants of D. hansenii strains relied on the mould species as only two mould species, Cladosporium inversicolor, and Penicillium roqueforti, were significantly inhibited, but there was little variation in inhibitory effects among D. hansenii strains per se. Strain-dependent volatile compounds (VOCs) profiles of D. hansenii strains were obtained, and five VOCs out of 71 VOCs, i.e., two acids (3-methylbutanoic acid and acetic acid), one alcohol (2‐phenylethanol), and two ketones (acetone, 2-pentanone), correlated strongly with inhibition of germination and growth of C. inversicolor and P. roqueforti by D. hansenii strains. The lowest half-maximal inhibitory concentrations (IC50) were observed for 3-methylbutanoic acid against the germination and growth of two moulds. Both 2‐phenylethanol and acetic acid had significant inhibitory effects on the growth of two moulds as well, whereas 2-pentanone and acetone had relatively weak inhibitory effects. Our results showed that native D. hansenii strains isolated from Danish cheese brines had antifungal effects against specific contaminating moulds. The inhibitory mechanisms of D. hansenii were especially noticeable due to synergistic effects of factors, i.e., five VOCs targeted in this thesis, suggesting that D. hansenii strains could be developed as a commercial brine culture or as a biocontrol agent in the future.
For 2‐phenylethanol, a well-known quorum sensing molecule, its antifungal activity was pronounced with an efficient biological concentration produced by D. hansenii strains grown in broth. Therefore, this thesis further investigated the antifungal mechanism of 2-phenylethanol in prevention of food spoilage moulds, Penicillium expansum (2 strains) and Penicillium nordicum. 2-phenylethanol significantly inhibited conidial germination of all tested moulds, and the inhibitory effect of 2-phenylethanol was in a concentration-dependent manner. Moreover, the inhibitory effect of 2-phenylethanol for preventing germination of three tested moulds was partly reversible without affecting the integrity of conidial cell membranes. Contrastingly, membrane permeability of actively growing hyphae was severely damaged when exposed to 2-phenylethanol. The results elucidate the mechanisms by which 2-phenylethanol inhibits mould growth, broadening its potentials as an optional antifungal agent for application in the food industry for controlling moulds.
In conclusion, the experiments carried out in this thesis exhibited biotechnological potentials of brine yeasts and highlighted the probability of some native D. hansenii strains isolated from Danish cheese brines as biocontrol agents. The results fill some of the gaps in our understanding of brine yeasts' growth and survival abilities and strengthen the case for using native microorganisms as biocontrol agents in the food industry.