Monitoring of Lactobacillus sanfranciscensis strains during wheat and rye sourdough fermentations by CRISPR locus length polymorphism PCR.

Lactobacillus (L.) sanfranciscensis is a competitive key species in sourdough fermentations. However, the principles involved in establishing the commonly observed phenomenon of strain dominance are unresolved. This has been studied little because the methods for fast and reliable differentiation of strains and their monitoring during fermentation are tedious and cannot be done with large numbers of isolates. In this contribution, we present a strain-specific, PCR-based typing method that uses length heterogeneities of the clustered regularly interspaced short palindromic repeats (CRISPR) loci as they occur in the genomes of different strains. In silico analysis of 21 genomes revealed 14 different CRISPR genotypes. We then designed a primer set to simultaneously detect different strains in a multiplex PCR assay designated CRISPR locus length polymorphism PCR (CLLP-PCR). The usefulness of this method was evaluated in lab-scale sourdough fermentations conducted with rye and wheat flours. First, the flour was mixed with water to a dough yield of 200. Then each dough was inoculated with four different L. sanfranciscensis strains (TMW 1.1150, TMW 1.392, TMW 1.2142, and TMW 1.2138) at levels of 109 cfu/g each. Sourdoughs were propagated at 28 °C for 5 days by back slopping 5% to the flour mass every 24 h. Samples were collected each day; DNA was isolated, and the presence of strains was detected qualitatively in the sourdoughs with PCR. L. sanfranciscensis TMW 1.392 became dominant as early as 2 days into the fermentation and remained the only detectable strain for the rest of the sampling period. CLLP-PCR proved to be useful in investigating the assertiveness of different strains of L. sanfranciscensis in sourdoughs. Therefore, CLLP-PCR may be used as a tool to investigate assertiveness of microorganisms in food fermentations at the strain level.