Brining is an important step in the manufacture of many cheese varieties, contributing to the development of cheese structure and flavour by strongly affecting the moisture and microbial succession in cheese. Investigations of the microbial composition of cheese brine have revealed a wide diversity. However, knowledge regarding the technological properties of brine microbiota and its impact on cheese quality is lacking. Meanwhile, there is a growing interest in creating specific yeast cultures for accelerating cheese ripening and enhancing cheese flavour. This PhD project aimed to characterise the technological properties of yeast strains that were isolated from Danish cheese brines.
Firstly, we investigated the growth properties of 20 yeast strains belonging to seven different species, namely Candida intermedia, Debaryomyces hansenii, Kluyveromyces lactis, Papiliotrema flavescens, Rhodotorula glutinis, Sterigmatomyces halophilus, and Yamadazyma triangularis under different combinations of temperature and NaCl concentration. All strains could grow in media with 0% or 4% (w/v) NaCl at 16 ºC and at 25 ºC, with better growth at 25 ºC, indicating their ability to grow in conditions simulating cheese surface. Contrastingly, all strains, except for C. intermedia, grew better at 16 ºC than at 25 ºC in a medium with 23% (w/v) NaCl and 6.3 g/L lactate, a condition imitating cheese brine. Further, D. hansenii, S. halophilus, and Y. triangularis were found to be the most halotolerant species assessed by their survival days in a medium with 23% (w/v) NaCl at 25 ºC. These findings provided the basis for preliminarily selecting relevant yeasts for potential application in cheese ripening.
Then, we investigated the amino acid utilisation of D. hansenii and Y. triangularis in a cheese surface model at 16 ºC and 25 ºC. The amino acid consumption was species-specific and significantly influenced by temperatures. The consumption of most amino acids by D. hansenii was higher at 25 ºC compared to 16 ºC, whilst Y. triangularis consumed more of most amino acids at 16 ºC than at 25 ºC. Furthermore, compared to Y. triangularis, D. hansenii produced more 2- and 3-methylbutanal, 3-methyl-1-butanol and 2-phenylethanol at 25 ºC, consistent with the higher consumption of the corresponding amino acids, i.e., isoleucine, leucine and phenylalanine. In contrast, Y. triangularis produced more volatile sulphur compounds than D. hansenii at 16 °C, in line with its higher consumption of their precursor, methionine, although Y. triangularis showed a marked consumption of volatile sulphur compounds at 25 ºC. Amino acid utilisation and the consequent production of derived aroma compounds differed between species and were greatly influenced by the fermentation environment.
In order to better explore the potential of brine yeasts as adjunct cultures for cheese ripening, we investigated the aroma production and microbial interaction of the most halotolerant species, D. hansenii, S. halophilus and Y. triangularis, in a cheese-surface model at 16 ºC. As pure cultures, they showed species-specific profiles in aroma production. Compared to the other two species, D. hansenii produced the broadest range of volatile compounds, including branched-chain alcohols, aldehydes and ketones, and had an intermediate ability in amino acid consumption. Sterigmatomyces halophilus was outstanding in producing higher alcohols and sulphur volatile compounds, and showed the greatest capacity in consuming amino acids. Yamadazyma triangularis was the only species to release amino acids and was able to produce higher alcohols and ketones.
When co-cultured together, the growth of S. halophilus and Y. triangularis was significantly depressed by D. hansenii, resulting in a similar aroma pattern to D. hansenii alone, but with less abundance. Though the growth patterns of S. halophilus and Y. triangularis were not influenced by each other when grown together, the aroma production of their co-culture was similar to that of S. halophilus alone, but at a lower level. The residual amino acids demonstrated that the growth of yeasts in co-culture was at the expense of aroma production. We found that the profile of aroma production and amino acid consumption of individual yeasts were species specific and that yeast interaction had a negative effect on aroma formation, probably because more amino acids were used
to supply growth.
In conclusion, this PhD study provides a better understanding of the technological characteristics of cheese brine yeasts, most especially in the aspects of growth in conditions mimicking cheese brine and surface, as well as their amino acid utilisation, aroma production, and microbial interaction in a cheese-based medium, contributing to our understanding of their potential for cheese processing.