Shotgun metagenomic and phenotypic characterization of indigenous lactic acid bacteria from raw milk artisanal cheeses: metagenomic functional insight and starter culture traits

The diversity of commercial starter cultures of lactic acid bacteria (LAB) used in fermented dairy products is limited. This has created strong demand to discover novel starter culture strains to develop unique products with appealing sensory characteristics. The current study used an integrated shotgun metagenomic and culture-based pipeline to (a) define taxonomic composition and functional potential of selected artisanal raw milk cheese microbiomes and (b) isolate and evaluate native LAB strains as potential starter cultures. Five artisanal cheeses (brie, bleu, plain gouda, mustard seed gouda, and nettle gouda) were analyzed. Shotgun metagenomics profiled the cheese microbiomes and revealed a high abundance of <i>Lactococcus cremoris</i> and <i>Lactococcus lactis</i> in gouda cheeses, whereas brie cheese contained high abundances of <i>L. lactis</i> and <i>Streptococcus thermophilus</i>. Functional profiling of metagenome-assembled genomes recovered from cheese microbiomes identified abundant pathways linked to carbon utilization, energy metabolism, and organic nitrogen metabolism. In parallel, 12 LAB isolates were recovered from all cheeses, of which five strains were classified taxonomically as <i>L. lactis</i> using whole genome sequencing. These five <i>L. lactis</i> strains displayed desirable milk and cream fermentation properties, achieving coagulation within 6 h, with final pH values of 4.5. The resulting fermented products contained 2.9%-4.2% protein content, displayed a relative increase in long-chain fatty acids, and a relative decrease in short-chain fatty acids compared to unfermented controls. The current study links cheese metagenome functional potential to dairy adaptation and identifies indigenous <i>L. lactis</i> strains as promising candidates for novel starter cultures in fermented dairy products.