Metagenomics-based approach for studying and selecting bioprotective strains from the bacterial community of artisanal cheeses

Abstract

A metagenome-based approach was used to assess the taxonomic affiliation and functional potential for bacteriocin production of the bacterial community in cow's milk artisanal cheeses from Northwestern Argentina. Three different samples were analyzed by high-throughput sequencing of the V4 region of the 16S rRNA gene and shotgun metagenomics. Taxonomic analysis showed that cheese A and C were quite similar whereas cheese B displayed a rather different bacterial composition. Overall, two families, Streptococceae and Enterococceae, dominated the artisanal cheese microbiota, being the former family prevalent in cheese B and the later family the most important in samples A and C. Besides the usual species associated to cheeses, a number of bacterial taxa that have not been previously found in Argentinean artisanal cheeses were reported in the present work such as Macrococcus caseolyticus and Streptococcus macedonicus Functional metagenomics analysis using the bacteriocin mining software BAGEL3, identified 2 ORFs encoding antimicrobial peptides in cheese B and 42 different peptides in sample C. The bacteriocin genes found showed good correlation with taxonomy. Based on the microbial diversity and functional features found through shotgun metagenomic sequencing, a culture-dependent approach was applied aiming to isolate bacteriocin-producing bacteria able to inhibit the growth of the foodborne pathogen Listeria monocytogenes. From 151 bacterial colonies derived from the cheese samples, 10 were associated to high anti-Listeria activity. Based on partial 16S rRNA gene sequencing and RAPD-PCR analysis, all bacteriocinogenic isolates were identified as Enterococcus faecium. Finally, we carried out a pilot experiment with L. monocytogenes-contaminated cheese using one of the enterococcal isolates as a bioprotective adjunct culture. The use of E. faecium CRL1879 during artisanal cheese manufacturing did not alter the main organoleptic properties of the cheese and ensured an efficient control of the foodborne pathogen up to 30 days. This finding supports the use of E. faecium CRL1879 as an adjunct culture in the cheese-making process with a combination of both safety and minimal processing.