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  • Combined Targeted and Non-targeted PCR Based Methods Reveal High Levels of Compliance in Probiotic Products Sold as Dietary Supplements in United States and Canada.

Combined Targeted and Non-targeted PCR Based Methods Reveal High Levels of Compliance in Probiotic Products Sold as Dietary Supplements in United States and Canada.

Frontiers in microbiology (2020-06-26)
Hanan R Shehata, Steven G Newmaster
ABSTRACT

Probiotics are defined as "live microorganisms that, when administered in adequate amounts, confer a health benefit on the host." The diverse health benefits have contributed to rapid increase in probiotic consumption and in the value of probiotic market, valued at USD 46 billion in 2019. For probiotics to be effective, the correct species/strains should be delivered viable in an adequate dose. The most commonly used methods for species/strain identification are DNA based methods including targeted and non-targeted methods (e.g., high-throughput sequencing, HTS). Using different DNA based methods, previous studies reported several cases of non-compliance in probiotic products. The objectives of this study are to evaluate levels of compliance in probiotic products (presence of all declared species/strains, absence of any contaminants or undeclared species, and meeting the declared minimum viable cell count) and to compare the performance of targeted and non-targeted methods in probiotic authentication. To the best of our knowledge, this is the largest study of its kind, testing 182 probiotic products, containing a total of 520 strains, collected from United States and Canada. Using species-specific assays, 11 species could not be detected in ten products. Missing species were Lactobacillus casei in seven products, Bifidobacterium longum and Bifidobacterium bifidum in one product, B. longum in one product while B. longum subsp. longum was mislabeled as B. longum subsp. infantis in another. Additionally, undeclared Bifidobacterium animalis subsp. lactis was detected in one product. Viable count was determined for 72 samples and was found to be lower than declared in five samples, including one product showing no viable cells. Overall, non-compliance was observed in 15 out of 182 products (8%). Additionally, undeclared species at relative abundance of ∼1-2% were found in 14 products using HTS, however, their presence could not be confirmed using species-specific assays. The results show that targeted PCR based methods enable species and strain level identification. The results also highlight the need to continue to develop strain-specific assays appropriate for use with multi-strain products. True strain-specific assays will enable strain authentication in both single-strain products and multi-strain products to ensure probiotic products meet the label claims and ensure probiotic efficacy.

MATERIALS
Product Number
Brand
Product Description

Millipore
Peptone Water, suitable for microbiology, NutriSelect® Plus
Millipore
MRS Agar, suitable for microbiology, NutriSelect® Basic
Sigma-Aldrich
L-Cysteine hydrochloride monohydrate, reagent grade, ≥98% (TLC)