Metagenomics Analysis of Pre-Probiotic Products Available Online in India: Bacterial Species Composition and Health Risks

Abstract

Aims: To assess the microbiological quality/species in pre-probiotic products available online in India. Study Design: gDNA extraction from pre-probiotic products, 16S metagenomic analysis, comparing the data of bacteria detected with those mentioned on the product label. Place and Duration of Study: Department of Molecular Biology, Genexplore Diagnostics and Research Centre Pvt. Ltd., Ahmedabad between December 2024 to March 2025. Methodology: Four multispecies pre-probiotics (capsule form) with an expiry of more than six months available in India were selected randomly and purchased from online platforms. gDNA was extracted. The quantity and quality of DNA was checked by Qubit 3.0 and 1.0% agarose gel electrophoresis. Library preparation was carried out using Ion Torrent 16S metagenomics kit, quantified, diluted, loaded onto Ion 550 chips by Ion Chef and sequenced by Ion S5 sequencer. Torrent Suite was used for quality control, trimming and analysis. Bioinformatics analysis was done using Metagenomics 16S w1.1, QIIME and BLAST. 16S rRNA metagenomics using Ion GeneStudio S5 Plus simple targeted sequencing system to evaluate the bacterial composition. Results: This analysis revealed that all the products failed to meet the diversity of bacterial composition mentioned on the label. Sufficient DNA could not be obtained from product P3 and was excluded from further analysis. The results were as follows: product P1 (6 out of 14) (42.85%) namely, Lactobacillus casei, L.  rhamnosus, L. paracasei, Bifidobacterium bifidum, and B. breve; P2 (0 out of 4); and P4 (3 out of 10) (30%) namely, L. rhamnosus, L. casei and Lactiplantibacillus plantarum bacterial species were detected, respectively. Other bacteria including pathogen or opportunistic pathogen such as Acinetobacter junii, Burkhoderia arboris, B. latens, Klebsiella pneumoniae, Propionibacterium acnes, Serratia marcescens, etc. were also detected. The products also contained probiotic bacteria other than those mentioned on the label. Conclusion: Metagenomic analysis revealed the non-conformity of bacterial composition in the pre-probiotics tested. Although useful molecular technique, its standardization is essential to serve as a regulatory requirement for probiotics.