Supplementary materials for Beneficial Microbes: Probiotic viability in the gastrointestinal tract in a randomised placebo controlled trial: combining molecular biology and novel cultivation techniques

Understanding the viability of ingested probiotics within the gastrointestinal tract is essential for evaluating their efficacy and deciphering their mechanisms of action. Detecting Bifidobacterium longum subspecies longum BB536 is particularly challenging owing to its indistinguishability from the naturally abundant B. longum species in the human gut. We aimed to address this challenge by developing a selective culture medium for B. longum BB536 and employing a propidium monoazide-quantitative polymerase chain reaction (PMA-qPCR) method to verify the survival of the probiotic after consumption. To achieve this, we designed a novel lactose-mupirocin-trimethoprim (LMT) medium that facilitates the cultivation of B. longum BB536 under anaerobic conditions at 42 °C. We screened 52 healthy adults and enrolled 39 who met the eligibility criteria. The participants were randomised into two groups, with 34 completing the protocol: 17 received commercial yogurt containing B. longum BB536 (9.30 log₁₀ cfu/day) and 17 received a placebo. Prior to the intervention, B. longum BB536 was undetectable in all participants. However, following supplementation, LMT culturing identified viable B. longum BB536, with average counts of 6.33 ± 0.69 log₁₀ cfu/g on day 3 and 6.16 ± 0.74 log₁₀ cfu/g on day 17. PMA–qPCR further validated these results, showing viable cell counts of 6.09 ± 0.68 log₁₀ cells/g wet faeces on day 3 and 6.44 ± 0.64 log₁₀ cells/g wet faeces on day 17. While each method detected B. longum BB536 in some participants where the other did not, no participant tested negative by both methods at any time point. This complementarity between LMT culturing and PMA–qPCR ensures a comprehensive detection strategy, confirming the presence and resilience of B. longum BB536 in the gastrointestinal tract and underscoring its potential as a beneficial probiotic strain (UMIN000052110).

Japan Conference of Clinical Research: registration number: BYG2B-01; University Hospital Medical Information Network: study protocol registration UMIN000052110.