The microbial diversity of mesophilic starter cultures used in cheese production

For ages, humankind has preserved various foods by fermentation by lactic acid bacteria (LAB), and fermentation of milk to obtain cheese can be traced back to the domestication of cattle, at least seven millennia ago. An essential ingredient in contemporary production of Dutch-type cheeses are the undefined mixed mesophilic (DL) starter cultures, which contains unknown mixtures of Lactococcus lactis strains and Leuconostoc spp.. Bacteriophages infecting Lactococcus lactis, the major contributors in the acidification of milk using mesophilic starter cultures, are recognized as the major cause of fermentation failures in dairy fermentations, disrupting the acidification process and negatively affecting the quality of the final product. The undefined mixed (DL) starter cultures are considered more robust against phage attack than the defined cultures, a characteristic gained from their large number of strains with diverse phage sensitivity. Starter cultures from different manufacturers are known to give cheeses qualitatively different characteristics, and performance differences are reported for different batches of the same starter culture, which indicates dissimilar culture compositions. Information on the microbial diversity of starter cultures is not publically available and tools to quantify the strain diversity or compare compositional differences between starter cultures does not exist. The information provided by the culture manufacturer with culture purchase does not include details beyond genus for leuconostocs, or beyond subspecies for the lactococci. In this study, the diversity of bacteria and their bacteriophages in starter cultures and dairy samples collected from three major cheese plants in Norway was investigated using molecular and DNA-sequencing based approaches. Use of a milk based-medium (GMA) in addition to the traditional M17 was instrumental in capturing a larger diversity of bacteria from starter cultures, which consequently increased the capacity to isolate bacteriophages from the dairy samples. The bacteria and bacteriophages were discriminated from each other use phage typing, revealing a large number of different bacteria as well as different bacteriophages. Interestingly, many of the strains that were only able to grow in a milkbased media, demonstrated unique phage sensitivities. A large number of phenotypically different starter bacteria with dissimilar phage sensitivities were whole-genome sequenced and characterized in pan-g