**Background:** Probiotic supplementation is commonly used to alter gut microbiota and improve health, but evidence for effects in healthy individuals is inconsistent. Lacticaseibacillus rhamnosus LRa05 (LRa05) had shown safety and efficacy in animal models of obesity, type 2 diabetes, and immunomodulation, but had not been tested in humans. This study aimed to assess the effect of LRa05 on the gut microbiota of healthy adults.
**Methods:** This single-blind, placebo-controlled trial was conducted at Henan University of Technology from April 21 to June 31, 2022. After a 14-day washout period, 100 healthy volunteers (age 19–45, BMI 18.5–25) were enrolled; 94 completed the trial (46 placebo, 48 LRa05). The LRa05 group received 1.9 g maltodextrin plus 0.1 g LRa05 bacterial powder (1 × 10¹⁰ CFU) daily for 28 days; the placebo group received 2 g maltodextrin daily. Fecal samples were collected at baseline (T0) and after 4 weeks (T1). The V3–V4 region of the 16S rRNA gene was sequenced on the Illumina MiSeq platform. Alpha diversity (Shannon, Simpson, Chao1, ACE) and beta diversity (Bray–Curtis PCoA) were analyzed. LEfSe analysis (LDA cutoff > 2.0) identified genus-level biomarkers. PICRUSt v2.5.0 predicted functional KEGG pathways. Mantel tests assessed correlations between T0 and T1 microbiota.
**Key Results:** Baseline characteristics (age, sex, blood pressure, BMI) did not differ significantly between groups. No significant changes in BMI, body fat percentage, or Bristol Stool Form Scale scores were observed in either group. Alpha diversity analysis showed increased species richness (Chao1 and ACE) in both groups but no significant changes in community diversity (Shannon and Simpson indices). Beta diversity analysis revealed significant changes in gut microbiota composition from T0 to T1 in both groups, but no significant differences between CTL and LRa05 groups at T0 (p = 0.317) or T1 (p = 0.857). At the phylum level, both interventions led to significant reductions in Firmicutes and Actinobacteria and a significant increase in Bacteroidetes. LEfSe analysis showed that both groups had similar changes at the genus level, including increased Prevotella, Phocaeicola, Bacteroides, and Parabacteroides, and decreased Escherichia/Shigella, Gemmiger, and Bifidobacterium. However, at T1, the LRa05 group showed significant increases in Weissella, Lacticaseibacillus, Enterococcus, and Mitsuokella, and significant decreases in Dialister, Negativibacillus, and Anaeromassilibacillus compared to placebo. PICRUSt analysis showed that at T1, the LRa05 group had reduced abundance of Salmonella infection and NOD-like receptor signaling pathways and increased nitrogen metabolism pathway compared to placebo. Mantel tests showed a significant correlation between T0 and T1 microbiota in both groups (p = 0.001), but the correlation was weaker in the LRa05 group (r = 0.182) than in the CTL group (r = 0.417).
**Clinical Implications:** LRa05 supplementation was well-tolerated and safe in healthy young adults, with no adverse effects on BMI, body fat, or stool consistency. The probiotic did not disrupt overall gut microbiota stability but specifically increased Lacticaseibacillus abundance, suggesting successful gut colonization. The predicted reduction in Salmonella infection and NOD-like receptor signaling pathways hints at potential anti-pathogenic and anti-inflammatory effects. However, the majority of microbiota changes were attributable to the maltodextrin carrier, not the probiotic itself. These findings suggest that LRa05 may have subtle modulatory effects on specific bacterial taxa and functional pathways in healthy individuals, but its clinical significance requires further investigation in disease cohorts or with longer intervention periods. The study was limited by its narrow age range (Chinese college students), only two time points, and lack of blood parameter testing.