Safety and functional enrichment of gut microbiome in healthy subjects consuming a multi-strain fermented milk product: a randomised controlled trial

Abstract

Many clinical studies have evaluated the effect of probiotics, but only a few have assessed their dose effects on gut microbiota and host. We conducted a randomized, double-blind, controlled intervention clinical trial to assess the safety (primary endpoint) of and gut microbiota response (secondary endpoint) to the daily ingestion for 4 weeks of two doses (1 or 3 bottles/day) of a fermented milk product (Test) in 96 healthy adults. The Test product is a multi-strain fermented milk product, combining yogurt strains and probiotic candidate strains Lactobacillus paracasei subsp. paracasei CNCM I-1518 and CNCM I-3689 and Lactobacillus rhamnosus CNCM I-3690. We assessed the safety of the Test product on the following parameters: adverse events, vital signs, hematological and metabolic profile, hepatic, kidney or thyroid function, inflammatory markers, bowel habits and digestive symptoms. We explored the longitudinal gut microbiota response to product consumption and dose, by 16S rRNA gene sequencing and functional contribution by shotgun metagenomics. Safety results did not show any significant difference between the Test and Control products whatever the parameters assessed, at the two doses ingested daily over a 4-week-period. Probiotic candidate strains were detected only during consumption period, and at a significantly higher level for the three strains in subjects who consumed 3 products bottles/day. The global structure of the gut microbiota as assessed by alpha and beta-diversity, was not altered by consumption of the product for four weeks. A zero-inflated beta regression model with random effects (ZIBR) identified a few bacterial genera with differential responses to test product consumption dose compared to control. Shotgun metagenomics analysis revealed a functional contribution to the gut microbiome of probiotic candidates.

Figure 1

Clinical study design.

Figure 2

Flowchart for the study population.

Figure 3

Detection of strains in fecal samples. Quantification of three probiotic candidates by qPCR with strain-specific primers on fecal samples before (D0), during (D14 and D28) and after (D56) the period of Test product consumption. Data are expressed as Log10 gene copy number/g feces. *p < 0.05, Mann–Whitney test for the comparison between 1 daily dose (Test 1) and 3 daily doses (Test 3) of product.

Figure 4

Genera with differential abundances during the study identified by ZIBR. Effect of consumption as a function of dose, for each genus, modeled with ZIBR. The reported values are p values corrected for multiple testing (FDR).

Figure 5

Functional contribution of Test product strains to the gut microbiome and their association with resident species. (A) Ranked barplot of 50 of 798 KOs with the highest relative abundance contributions. NA corresponds to unclassified KOs. (B) Ranked barplot of the distribution of the 798 KOs within transporter and enzyme KEGG BRITE category. The colors indicate the most dominant functions. (C) Microbial co-abundance network based on the SPIEC-EASI method. Each dot represents a single MSPs. Positively and negatively co-abundant MSPs are connected by blue and red lines, respectively, the thickness of which is determined by weight in the SPIEC-EASI, model. Node colors indicate the number of shared specific functions from a list of 798 KOs contributed by the Test product probiotic candidate species (Lactobacillus rhamnosus and Lactobacillus paracasei) to the gut microbiota. Node diameter indicates the geodesic edge distance with MSPs. D. Barplot of the number of shared contributive KOs between Test product strains and dominant species as a function of geodesic distance extracted from the abundance co-variation network.