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.

Cheese brines from Danish dairies reveal a complex microbiota comprising several halotolerant bacteria and yeasts.

The Danish Danbo cheese is a surface ripened semi-hard cheese, which before ripening is submerged in brine for up to 24 h. The brining is required in order to obtain the structural and organoleptic properties of the cheeses. Likewise, the content of NaCl in the cheese will influence especially the surface microbiota being of significant importance for flavour development and prevention of microbial spoilage. Even though the microbiota on cheese surfaces have been studied extensively, limited knowledge is available on the occurrence of microorganisms in cheese brine. The aim of the present study was to investigate by both culture-dependent and -independent techniques the brine microbiota in four Danish dairies producing Danbo cheese. The pH of the brines varied from 5.1 to 5.6 with a dry matter content from 20 to 27% (w/w). The content of lactate varied from 4.1 to 10.8 g/L and free amino acids from 65 to 224 mg/L. Bacteria were isolated on five different media with NaCl contents of 0.85-23.0% (w/v) NaCl. The highest count of 6.3 log CFU/mL was obtained on TSA added 4% (w/v) NaCl. For yeasts, the highest count was 3.7 log CFU/mL on MYGP added 8% (w/v) NaCl. A total of 31 bacterial and eight eukaryotic species were isolated including several halotolerant and/or halophilic species. Among bacteria, counts of ≥6.0 log CFU/mL were obtained for Tetragenococcus muriaticus and Psychrobacter celer, while counts between ≥4.5 and < 6.0 log CFU/mL were obtained for Lactococcus lactis, Staphylococcus equorum, Staphylococcus hominis, Chromohalobacter beijerinckii, Chromohalobacter japonicus and Microbacterium maritypicum. Among yeasts, counts of ≥3.5 log CFU/mL were only obtained for Debaryomyces hansenii. By amplicon-based high-throughput sequencing of 16S rRNA gene and ITS2 regions for bacteria and eukaryotes respectively, brines from the same dairy clustered together indicating the uniqueness of the dairy brine microbiota. To a great extent the results obtained by amplicon sequencing fitted with the culture-dependent technique though each of the two methodologies identified unique genera/species. Dairy brine handling procedures as e.g. microfiltration were found to influence the brine microbiota. The current study proves the occurrence of a specific dairy brine microbiota including several halotolerant and/or halophilic species most likely of sea salt origin. The importance of these species during especially the initial stages of cheese ripening and their influence on cheese quality and safety need to be investigated. Likewise, optimised brine handling procedures and microbial cultures are required to ensure an optimal brine microbiota.