Lactococcus lactis Diversity in Undefined Mixed Dairy Starter Cultures as Revealed by Comparative Genome Analyses and Targeted Amplicon Sequencing of epsD

Undefined mesophilic mixed (DL) starter cultures are used in the production of continental cheeses and contain unknown strain mixtures ofLactococcus lactis and leuconostocs. The choice of starter culture affects the taste, aroma, and quality of the final product. To gain insight into the diversity ofLactococcus lactis strains in starter cultures, we whole-genome sequenced 95 isolates from three different starter cultures. Pan-genomic analyses, which included 30 publically available complete genomes, grouped the strains into 21L. lactis subsp. lactis and 28L. lactis subsp.cremoris lineages. Only one of the 95 isolates grouped with previously sequenced strains, and the three starter cultures showed no overlap in lineage distributions. The culture diversity was assessed by targeted amplicon sequencing usingpurR , a core gene, andepsD , present in 93 of the 95 starter culture isolates but absent in most of the reference strains. This enabled an unprecedented discrimination of starter cultureLactococcus lactis and revealed substantial differences between the three starter cultures and compositional shifts during the cultivation of cultures in milk.IMPORTANCE In contemporary cheese production, standardized frozen seed stock starter cultures are used to ensure production stability, reproducibility, and quality control of the product. The dairy industry experiences significant disruptions of cheese production due to phage attacks, and one commonly used countermeasure to phage attack is to employ a starter rotation strategy, in which two or more starters with minimal overlap in phage sensitivity are used alternately. A culture-independent analysis of the lactococcal diversity in complex undefined starter cultures revealed large differences between the three starter cultures and temporal shifts in lactococcal composition during the production of bulk starters. A better understanding of the lactococcal diversity in starter cultures will enable the development of more robust starter cultures and assist in maintaining the efficiency and stability of the production process by ensuring the presence of key bacteria that are important to the characteristics of the product.