Temporal dynamics of probiotic Lacticaseibacillus casei and rhamnosus abundance in a fermented dairy product evaluated using a combination of cultivation-based and molecular methods

One of the central trends in functional nutrition development is fortification of products with probiotic bacteria. Establishment and optimization of methods for the quantitative determination of microbial content in food products, as well as introduction of cost-efficient methods into routine production represent great interest to the food industry. There are many methods for typing and enumerating microbial taxa in food, but the comparison of their results and applicability for analyzing fermented dairy products is yet to be undertaken. Here we assessed the levels of Lacticaseibacillus casei and rhamnosus added as probiotics in 9 different recipes of a fortified dairy product during the shelf-life using a combination of cultivation-based and -independent methods (from 10 to 270 samples depending on the method). Microbiological methods included cultivation on MRS and M-RTLV agar media. Taxon-specific qPCR tests were used to enumerate the two targeted species. Detection of viable cells was performed using a qPCR utilizing propidium monoazide (PMA). The 16S rRNA gene sequencing was used in conventional and PMA-based versions to analyze the food products, probiotics and starter cultures.All methods showed that high levels for both L. casei and rhamnosus were maintained throughout the shelf life. PMA-qPCR showed that the proportion of non-viable microbes was relatively small. According to qPCR, PMA-qPCR, cultivation analysis and sequencing, the products with three-component starter cultures (Streptococcus thermophilus, L. acidophilus and Bifidobacterium) were associated with a higher abundance of each of the probiotic strains compared to two-component starters (S. thermophilus and L. delbrueckii). The 16S rRNA sequencing results generally confirmed the expected total microbial composition, although they were not useful for estimating the dynamics of probiotic levels due to the compositionality of the data and low levels of probiotics compared to the starter culture microbes