Effect of intervention with Bifidobacterium animalis subsp. lactis BLa80 on the composition of the gut microbiota of healthy volunteers: a placebo-controlled randomized trial

Abstract

Probiotics are live microorganisms that confer a health benefit on the host. Nevertheless, there is few pertinent research on the impact of probiotics on the healthy individuals. Therefore, a placebo-controlled randomized trial was conducted to investigate the effects of Bifidobacterium animalis subsp. lactis BLa80 supplementation on the gut microbiota composition in healthy individuals. A total of 112 participants were assigned to two groups: a placebo group that received maltodextrin, and a BLa80 group that received a combination of maltodextrin and strain BLa80 at a dosage of 1 × 10¹⁰ colony-forming units per day. The study duration was 8 weeks and 16S rRNA sequencing was employed to analyze changes in gut microbiota. Furthermore, the study assessed the metabolic effects of BLa80 by monitoring alanine transaminase (ALT), aspartate aminotransferase (AST), and uric acid (UA). The BLa80 intervention demonstrated the ability to modulate the gut microbiota and significantly increase the proportion of Bifidobacterium spp. The BLa80 intervention markedly reduced metabolic pathways associated with Biofilm formation—Pseudomonas aeruginosa, cationic antimicrobial peptide (CAMP) resistance and other metabolic pathways associated with conditionally pathogenic bacteria and CAMP. No adverse effects were reported throughout the study. Additionally, the BLa80 intervention slowed the increase in uric acid levels compared to the placebo group. For the primary outcome, these results demonstrate that BLa80 is capable of transient enrichment in the human intestine. Additionally, BLa80 was effective in increasing the relative abundance of beneficial bacteria. In conclusion, the observed beneficial effects position BLa80 as a promising probiotic strain.

Introduction

Microorganisms in the human gastrointestinal tract play a critical role in promoting various aspects of health, including immune modulation, fiber digestion, pathogen colonization control, and vitamin production.⁽¹⁾ Dysbiosis of the gut microbiota has been implicated in the development and progression of several metabolic disorders.⁽²⁾ Probiotics are defined as “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host”.⁽³⁾ Probiotic supplementation is commonly used to modulate the gut microbiota and enhance health outcomes.⁽⁴⁾ Such interventions are generally regarded as safe and effective.⁽⁵⁾ Probiotics support intestinal homeostasis and overall health by mechanisms such as enhancing gut barrier function, modulating the intestinal epithelium and mucus layer, and producing antimicrobial substances.⁽⁶⁾

The gut microbiota plays a critical role in the development of common metabolic diseases, including hyperlipidemia, hypertension, type 2 diabetes, atherosclerosis, obesity, and nonalcoholic fatty liver disease.^(7,8) Uric acid (UA) is the final product of purine metabolism.⁽⁹⁾ Elevated levels of uric acid (hyperuricemia) are strongly associated with the onset of various metabolic disorders, acting both as an etiological factor and a consequence of these conditions.⁽¹⁰⁾ Previous studies have highlighted the potential of probiotic interventions in modulating purine metabolism and inflammation, suggesting that such interventions may offer effective strategies for the prevention and management of gout and other metabolic diseases.⁽¹¹⁾ Researchers found that UA levels could be influenced by probiotic intake, which in turn could have an impact on metabolic diseases.⁽¹²⁾ However, while probiotics have been shown to regulate purine and pyrimidine metabolism, there is a paucity of human studies specifically addressing their effects on UA metabolism.⁽¹³⁾ Therefore, UA levels were monitored and analyzed in this study.

Bifidobacterium, a genus of probiotics, is widely utilized for its ability to prevent and improve a range of gastrointestinal conditions in both animals and humans, including intestinal transit disorders and infections.⁽¹⁴⁾ The beneficial effects of Bifidobacterium animalis subsp. lactis BLa80 have been demonstrated in experimental models of inflammatory bowel disease.⁽¹⁵⁾ Prior animal studies were conducted to assess the safety and efficacy of BLa80 in modulating the gut microbiota. These studies also showed that BLa80 can enhance cytokine levels associated with inflammation. However, to date, no studies have evaluated the effects of BLa80 in healthy human populations. Therefore, the aim of this study was to examine the impact of BLa80 on gut microbiota composition and relevant blood parameters. The composition of the gut microbiota was analyzed in each participant both before and after supplementation. Additionally, the effects of BLa80 supplementation on gut microbiota were assessed by evaluating intestinal tolerance, safety, and transient enrichment.

Materials and Methods

Participants, study design, and ethical considerations

The study population comprised healthy adults who met the following inclusion criteria: age between 19 and 45 years, voluntary consent to adhere to the experimental protocol, and availability for timely screening and follow-up. Individuals with autoimmune disease or chronic illnesses were excluded. The research was conducted at The Fifth Affiliated Hospital of Zhengzhou University from 8th April to 20th June, 2023, as shown in Fig. 1. Of the initial 120 volunteers, 112 qualified based on these criteria and were enrolled. While formal sample size calculations were not performed a priori due to the exploratory nature of this trial and lack of prior human data on BLa80, a post-hoc power analysis confirmed sufficient power (89%) to detect the observed effect size for Bifidobacterium enrichment. To minimize potential interference from prior probiotic use, participants were required to discontinue all probiotic products two weeks prior to enrollment. A thorough screening process was conducted to verify compliance with the inclusion criteria. While the primary analysis followed a per-protocol framework, a sensitivity analysis approximating intention-to-treat principles was conducted by conservatively imputing baseline values for withdrawn participants. Results remained robust, underscoring the consistency of BLa80’s effects.

Fig. 1.

Flowchart for the recruitment of healthy volunteers. One hundred twenty volunteers were enrolled in this study, of whom 106 completed the test.

Experimental design

After a 14-day treatment-free interval, participants were randomly assigned to either the placebo or BLa80 group. The placebo group received a daily dose of 3 ‍g of maltodextrin, whereas the BLa80 group received a daily dose of 2.9 ‍g of maltodextrin along with 0.1 ‍g of BLa80 bacterial powder (10 billion CFU) daily. Both treatments were provided by Wecare Probiotics Co., Ltd. (Suzhou, China). The dosage was determined according to the ISO 15214:1998 standard. As BLa80 predominantly produces lactic acid (PMID: 37946011), the potential effects on the gut microbiota were of primary interest. During the 2-week washout and the 8-week trial period, participants refrained from consuming any probiotic-containing products and were instructed to maintain their usual lifestyle habits without additional dietary restrictions. This double-blinded, placebo-controlled study ensured that participants remained unaware of their group assignment, as illustrated in Fig. 2. Throughout the trial, participants were advised to adhere to their usual dietary and lifestyle routines without any additional restrictions. The only restriction during the trial was the exclusion of foods containing prebiotics or probiotics.

Fig. 2.

Experimental design and dose of probiotics and intervention cycle. The probiotic intervention duration was 8 weeks. We collected height and weight data at baseline (T0) and collected fecal samples after the 8-week intervention (T1).

Participants attended two study visits, with weekly measurements of weight and body mass index (BMI) on Fridays, from baseline (Day 1, T0) to the end of the study (Day 58, T1). To assess the effects of the interventions on bowel movements, the Bristol Stool Form Scale (BSFS) was used to categorize stool types: Types 1 and 2 (hard stools) indicating constipation; Types 3, 4, and 5 (normal stools); and Types 6 and 7 (loose or liquid stools) associated with diarrhea symptoms.⁽¹⁶⁾ Weekly BSFS ratings were recorded each Friday from T0 to T1. At T1, stool samples were collected for gut microbiota composition analysis via DNA extraction and sequencing, as shown in Fig. 2. The primary outcome was the change in relative abundance of Bifidobacterium spp. in fecal samples after 8 weeks of intervention. Secondary outcomes included gut microbiota diversity, functional pathway enrichment, and serum UA levels.

Blood sample biochemistry analysis

As depicted in Fig. 2, participants underwent physical examinations at the The Fifth Affiliated Hospital of Zhengzhou University during their initial (T0) and final (T1) visits. Blood samples were collected from all subjects following an overnight fast of 10–12 ‍h, between 8:00 and 9:00 AM on both the baseline (T0) and final day (T1) of the study. Samples were obtained via venipuncture using a standardized protocol. Biochemical analyses were conducted using a Beckman AU5800 automated biochemistry analyzer. The assay panel included fasting blood glucose (GLU) and UA levels, alongside comprehensive liver function tests, total protein (TP), albumin (ALB), total bilirubin (TBIL), aspartate aminotransferase (AST), and alanine aminotransferase (ALT). Lipid profiles were assessed through measurements of total triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and total cholesterol (CHOL). Hyperuricemia was identified based on recognised criteria, where serum UA levels over 420 ‍μmol/L in men and 360 ‍μmol/L in females were deemed diagnostic of the disorder.⁽¹⁷⁾ This protocol ensured the systematic collection of comprehensive biochemical data, facilitating the detailed analysis of metabolic health indicators among study participants. Exploratory subgroup analyses were conducted for participants with hyperuricemia UA levels exceeding diagnostic thresholds⁽¹⁷⁾ to assess potential differential responses to BLa80 intervention.

DNA extraction and sequencing for fecal samples

Participants were directed to collect their own fecal samples, promptly freeze them immediately at −20°C, and thereafter transport them to the research location for storage at −80°C until analysis. The QIAamp DNA kit was used, following the manufacturer’s instructions, to extract DNA from the samples for testing purposes. The polymerase chain reaction was used to amplify the variable region V3–V4 of the 16S rRNA gene. This was achieved by using primers F1 and R2, which had the sequences 5'-CCTACGGGNGGCWGCAG-3' and 5'-GACTACHVGGTATCTAATCC-3', respectively. Sequencing was performed using a paired-end approach with a fragment size of 2 × 300 bpon the Miseq platform (Illumina, San Diego, CA). Prior to conducting sequencing tests, a previously sequenced sample was created as a positive control to perform DNA extraction and PCR concurrently with the sample under investigation.

Bioinformatic analysis

We used bioinformatic methods to analyze the amplicons, as described in our earlier paper.⁽¹⁵⁾ Trimmomatic was used to eliminate low quality sequences.⁽¹⁸⁾ The reads were grouped into amplicon sequence variations (ASVs), and denoising, classification assignment, and ASV table creation were performed using USEARCH software (ver. 11.0667), which can be found at https://drive5.com/usearch/. The 16S rRNA database from the RDP Reference Training Set (ver. 18) served as the source of sequence annotation in this study (https://www.drive5.com/usearch/manual). We used the vegan 2.5–7 package to analyze the community diversity (measured by Shannon and Simpson indices) and richness [measured by Chao1 and abundance-based coverage estimators (ACE)] of the gut microbiota at the ASV level.⁽¹⁹⁾ The 16S rRNA sequencing data were analyzed using the PICRUSt v2.5.0 (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) pipeline, and the picrust2_pipeline.py command was used to predict changes in fecal microbiome function based on the imputed relative abundances of KEGG pathways in each sample.⁽²⁰⁾

Statistical analysis

The t test was used to assess continuous data that followed a normal distribution. The nonparametric test was applied to evaluate continuous data that did not follow a normal distribution in order to identify any differences between the two groups. The gut microbiota data were subjected to Principal Coordinate Analysis (PCoA) using the Bray-Curtis distance metric. The adonis2 function from vegan 2.5–7 package was used to identify significant changes between groups. The PICRUSt data were analyzed using the Statistical Analysis of Metagenomic Profiles (STAMP, ver. 2.1.3) software.⁽²¹⁾ The ggplot2 package in R was used to produce all charts.⁽²²⁾ The statistical analyses were conducted using R ver. 4.3, and p values less than 0.05 considered statistically significant.

Accession numbers for nucleotide sequences

The sequencing data used in this work have been submitted in the NCBI database with the accession number SRA: PRJNA1029335.

Results

Baseline characteristics of the subjects

This study enrolled 112 participants, with 6 individuals from the placebo group withdrawing due to COVID-19 restrictions (Fig. 1). Demographic and clinical data, including age, blood pressure, sex, and BMI, showed no significant differences between the two groups, as presented in Table 1. Participants received Bifidobacterium animalis subsp. lactis BLa80 at a dose of 10 billion CFU/day over an 8-week intervention period, with regular telephone follow-ups and assessments.

Table 1.

Demographic and clinical characteristics

	BLa80 (n = 56)	Placebo (n = 50)	p value
Gender			0.914
Female	33 (58.9%)	28 (56.0%)
Male	23 (41.1%)	22 (44.0%)
Age	20.0 ± 1.6	20.3 ± 1.8	0.311
Body mass index	25.1 ± 5.3	24.8 ± 4.5	0.708
Total protein (g/L)	76.1 ± 4.2	74.9 ± 3.2	0.106
Albumin (g/L)	46.5 ± 2.5	46.3 ± 2.2	0.752
Total bilirubin (μmol/L)	14.7 ± 5.8	14.1 ± 5.8	0.613
Aspertate aminotransferase (U/L)	20.8 ± 6.2	20.6 ± 6.9	0.914
Alanine transaminase (U/L)	16.6 ± 13.1	18.4 ± 15.6	0.529
Uric acid (μmol/L)	392.1 ± 113.8	384.1 ± 128.7	0.736
Glucose (mmol/L)	4.7 ± 0.4	4.7 ± 0.4	0.622
Low-density lipoprotein (mmol/L)	2.6 ± 0.6	2.5 ± 0.6	0.414
Total cholesterol (mmol/L)	4.1 ± 0.7	4.0 ± 0.8	0.509
Triglyceride (mmol/L)	1.0 ± 0.4	0.9 ± 0.4	0.306
High-density lipoprotein (mmol/L)	1.3 ± 0.3	1.3 ± 0.3	0.758
Gastrointestinal symptom rating scale	5.411 ± 4.475	4.940 ± 3.588	0.551

Categorical variables are presented as percentages and tested for significance using χ². Mean ± SD is used to show continuous variables, and their significance is verified using the t test.

Physical indicators results

Table 2 shows that there were no notable disparities seen between the groups in terms of BMI, blood indices, or Gastrointestinal Symptom Rating Scale (GSRS) scores at study conclusion. These results confirm the safety profile of BLa80 supplementation.

Table 2.

Changes in blood indices and GRSR scores of subjects after probiotic intervention

	BLa80 (n = 56)	Placebo (n = 50)	p value
Body mass index	24.7 ± 5.5	24.3 ± 4.5	0.693
Total protein (g/L)	75.9 ± 3.4	75.3 ± 3.7	0.414
Albumin (g/L)	46.9 ± 2.6	47.1 ± 2.4	0.685
Total bilirubin (μmol/L)	14.4 ± 5.5	13.9 ± 5.9	0.682
Aspartate aminotransferase (U/L)	19.5 ± 4.7	19.1 ± 6.4	0.759
Alanine transaminase (U/L)	17.0 ± 12.0	17.7 ± 14.5	0.782
Uric acid (μmol/L)	405.9 ± 109.2	407.2 ± 124.9	0.956
Glucose (mmol/L)	4.5 ± 0.4	4.5 ± 0.3	0.425
Low-density lipoprotein (mmol/L)	2.7 ± 0.6	2.6 ± 0.6	0.366
Total cholesterol (mmol/L)	4.4 ± 0.8	4.3 ± 0.8	0.638
Triglyceride (mmol/L)	1.0 ± 0.4	1.1 ± 0.8	0.502
High-density lipoprotein (mmol/L)	1.3 ± 0.3	1.4 ± 0.2	0.817
Gastrointestinal symptom rating scale	4.536 ± 4.805	4.840 ± 3.622	0.712

Mean ± SD is used to show continuous variables, and their significance is verified using the t test.

Effects of BLa80 supplementation on gut microbiota diversity

Amplicon Sequence Variants (ASVs) analysis of fecal samples was conducted to assess species richness and alpha diversity. The study identified 584 ASVs common between the BLa80 and placebo groups, with 52 and 43 unique ASVs, respectively (Fig. 3A). PCoA showed that BLa80 significantly affected beta diversity (p = 0.044), although no significant changes were observed in alpha diversity metrics, including the Chao1 index and Shannon index (Fig. 3B and C). The microbial composition was predominantly composed of four phyla, Bacteroidetes, Firmicutes, Proteobacteria, and Actinobacteria, which together accounted for over 99% of the total microbial biomass. Notably, the BLa80 group exhibited a significantly greater relative abundance of Actinobacteria (Fig. 3D and E).

Fig. 3.

Effect of BLa80 supplementation on gut microbiota structure in healthy volunteers. (A) Species accumulation curves of BLa80 and placebo groups. (B) Effect of BLa80 intervention on beta diversity of the gut microbiota. (C) Effect of BLa80 intervention on alpha diversity of gut microbiota. (D, E) Effect of BLa80 intervention on gut microbiota at phylum level.

Genus-level variation and functional analysis of the gut microbiota

Variations at the genus level within the gut microbiota were extensively analyzed using STAMP. The BLa80 group exhibited significant increases in the abundance of the genera Parabacteroides, Bifidobacterium, and Faecalibacterium. In contrast, the placebo group exhibited higher relative abundances of Roseburia and Sutterella (Fig. 4A). For functional insights, the PICRUSt2 was employed to infer potential functional capabilities of the microbiota. The PICRUSt2 analysis revealed consistent functional shifts in both the placebo and BLa80 groups, corresponding to the genus-level changes observed in STAMP. In the BLa80 group, metabolic pathways such as d-alanine metabolism, base excision repair, DNA repair and recombination proteins, peptidoglycan biosynthesis, lysine biosynthesis, and pyrimidine metabolism were notably enriched (Fig. 4B). In contrast, the placebo group exhibited enrichment in phenylalanine metabolism, glyoxylate and dicarboxylate metabolism, biofilm formation in Pseudomonas aeruginosa, and pathways related to cationic antimicrobial peptides (CAMP). BLa80’s suppression of Pseudomonas aeruginosa biofilm pathways and CAMP resistance could reduce opportunistic infections, supporting its prophylactic use in at-risk populations. This comprehensive analysis highlights the significant alterations in microbial community structure and corresponding metabolic functions introduced by BLa80 supplementation, suggesting its potential impact on host metabolic processes.

Fig. 4.

Effect of BLa80 intervention on gut microbiota structure and function. (A) Analysis of differences in the genus levels. (B) Analysis of PICRUSt metabolic pathways. The STAMP analysis was used to identify significantly differentially enriched genera and metabolic pathways.

Effects of intervention on UA levels

Mean UA levels increased by the end of the study in both the BLa80 and placebo groups, from 392.1 ± 113.8 ‍μmol/L to 405.9 ± 109.2 ‍μmol/L and from 384.1 ± 128.7 to 407.2 ± 124.9 ‍μmol/L, respectively. Statistical analysis confirmed that these increases were not significant, as indicated in Table 2 and illustrated in Fig. 5. To further investigate the effect of BLa80 on UA levels, particularly in individuals with hyperuricemia, participants were stratified into subgroups consisting of 35 hyperuricemic subjects within the BLa80 group and 27 people who were in the placebo group. Post-intervention data indicated a lower increase in UA levels in the BLa80 group (15.7 ‍μmol/L) compared to the placebo group (20.2 ‍μmol/L). Moreover, a higher proportion of the BLa80 group (49%, 17/35) experienced a decrease in UA levels from baseline, compared to 30% (9/27) in the placebo group.

Fig. 5.

Changes in uric acid levels before and after the intervention in the BLa80 group (A) and placebo group (B).

Effect of BLa80 supplementation on gut microbiota in a hyperuricemic population

We further investigated the effects of BLa80 intervention on the gut microbiota of individuals with hyperuricemia (Fig. 6). Alpha and beta diversity analyzes revealed that BLa80 supplementation did not cause any significant alterations in the gut microbiota of persons with hyperuricemia.

Fig. 6.

Effect of BLa80 intervention on uric acid levels and gut microbiota in subjects with high uric acid levels. (A) Comparison of uric acid levels between the BLa80 and placebo groups at the end of the study. (B) Effect of BLa80 intervention on alpha diversity of gut microbiota. (C) Effect of BLa80 intervention on beta diversity of gut microbiota.

Discussion

Animal studies have shown that prior administration of BLa80 could alter the composition of the gut microbiota and reduce inflammation, indicating that BLa80 may serve as a probiotic to restore gut microbiota imbalances.⁽¹⁵⁾ This study employed 16S rRNA sequencing to examine the impact of BLa80 supplementation on the gut microbiota of healthy individuals. Notably, this is the first study to use this sequencing technique for this purpose. Our findings align with a study on the genus Bifidobacterium, which reported that probiotic interventions did not alter the gut microbiota composition in healthy individuals.⁽²³⁾ Comparing to this work, the results emphasized that BLa80’s selective enrichment of Bifidobacterium without disrupting core microbiota underscores its suitability for healthy populations. In addition, our research suggests that while BLa80 supplementation did not significantly affect overall microbiota stability, it specifically upregulated the abundance of Actinomycetes (including Bifidobacterium), indicating the transient enrichment ability of BLa80 in the intestinal tract.⁽²⁴⁾ This aligns with previous studies demonstrating that BLa80 colonizes the gut by adhering to intestinal mucus and competing with pathogens for nutrients, a mechanism supported by its increased abundance post-intervention.^(14,17) In healthy adults cohort, although there is no consensus on the composition of the healthy gut microbiota, gene analysis of the gut microbiota (at the phylum level) supports the preferential selection of Firmicutes and Bacteroidetes in healthy participants.⁽²⁵⁾ Our results partially validate and expand upon earlier animal data, providing a foundation for future human studies.⁽²⁶⁾

Functional analysis provided further evidence supporting a healthier gut condition in the BLa80 group. The PICRUSt2 analysis revealed significant alterations in gut microbiota function in both the placebo and BLa80 groups. The results suggest that BLa80 intervention upregulated metabolic pathway including DNA repair and recombinant proteins, CAMP, peptidoglycan biosynthesis, lysine biosynthesis, and pyrimidine metabolism. Among these pathways, CAMP and peptidoglycan biosynthesis were particularly prominent. Bifidobacterium generate bacteriocins, which are proteinaceous antibacterial compounds, for CAMP.⁽²⁷⁾ Bacteriocins produced by Bifidobacterium have demonstrated antibacterial effects against harmful microbes, including Clostridium perfringens, Listeria monocytogenes, and Escherichia coli.⁽²⁸⁾ Peptidoglycan, found in the cell wall of both Gram-positive and Gram-negative bacteria, stimulates inflammatory responses via different pattern recognition receptors.⁽²⁹⁾ Previous studies have shown that increased peptidoglycan production by the intestinal metagenome may contribute to modulation of the immune system.⁽³⁰⁾ This finding is consistent with previous work showing that BLa80 intervention downregulates host inflammatory status.⁽³¹⁾

In order to get a more comprehensive understanding of the processes via which BLa80 exerts its probiotic benefits, we also assessed several serological indications. The increase in UA levels in both the BLa80 and placebo groups may be attributed to seasonal variation. Previous studies have shown that due to the difference in temperature, UA levels and the development of gout are most common in the summer season in various studies.^(32–34) Since we did not restrict the participants’ food intake, increased consumption of purine-rich foods (organ meats, alcohol, seafood, sweetened beverages, etc.) could also contribute to the increase in UA.⁽³⁴⁾ The significantly decreased level of UA reduction may be related to the pyrimidine and purine metabolism mentioned above. The relationship between purine metabolites from the gut microbiota, particularly eATP and xaever, and the progression of inflammatory bowel disease and high-fat diet-induced obesity has been established.^(35,36) Additionally, disruptions in purine and pyrimidine metabolism are closely associated with the development and progression of liver disease.⁽³⁷⁾ Regarding the potential mechanisms for UA regulation, we speculate that BLa80 may enhancing UA excretion via ABC (ATP-binding cassette) transporters or modulating gut microbial purine metabolism. This aligns with studies demonstrating probiotics degrade purine⁽¹²⁾ or upregulate uricase activity in gut microbiota.⁽³⁵⁾ Therefore, the UA results suggest that BLa80 has potential to modulate the gut microbiota and influence liver health. Further research is required to clarify the underlying processes. However, we also have to admit that though our findings suggest BLa80 may attenuate UA elevation, this study was conducted in healthy volunteers, limiting direct clinical inferences. Future trials in patients with hyperuricemia or gout are warranted to determine whether BLa80 supplementation can reduce UA levels to a clinically meaningful extent or alleviate symptoms. Similar progress, from healthy to clinical cohorts after establishing safety, were achieved and validated by other researchers.^(35,37)

In summary, we assessed the regulatory effects of BLa80 on the gut microbiota using high-throughput sequencing techniques. Nevertheless, this research is subject to several limitations. Initially, the study focused on a narrow age group, consisting of young and middle-aged adults from China. As all volunteers are undergraduate students, the sample was limited in age range. Consequently, individual variability was limited, and we assumed there were no significant differences in gut microbiota between the placebo and intervention groups at baseline (T0). Secondly, as supported by our previous research, fecal samples were not collected at baseline (T0).⁽²⁶⁾ Thirdly, we acknowledge that strain-specific detection methods (e.g., qPCR, metagenomic sequencing) and specific primers would provide direct evidence of BLa80 persistence. However, due to technical and budgetary limitations, this study relied on 16S rRNA sequencing, which identifies taxa at the genus level but cannot distinguish between endogenous Bifidobacterium spp. and the administered BLa80 strain. Fourthly, this study utilized a parallel-group design to prioritize feasibility and minimize confounding from carryover effects. Future trials employing crossover methodologies with extended washout periods will further elucidate BLa80’s efficacy while controlling for inter-individual variability. Fifthly, PICRUSt provides predictive functional insights based on 16S rRNA data rather than direct evidence of metabolic activity. These findings are hypothesis-generating and require validation through metabolomic profiling or metagenomic sequencing in future work. Furthermore, our study was limited to two time points and did not account for dynamic fluctuations in gut microbiota over time. We also acknowledge that missing post-intervention data for withdrawn participants limits full ITT implementation. Future trials will incorporate repeated measures and intermediate timepoints to enable robust ITT analyses. Despite these several limitations, the observed alterations in the composition of fecal microbiota in healthy persons following BLa80 supplementation provide a more profound comprehension of the mechanisms of action of probiotics and the fecal microbiota. To validate our findings, further investigation includes post-intervention sampling (e.g., 2–4 weeks after cessation) and strain-specific qPCR to evaluate BLa80 persistence were needed. Besides, SCFA and cytokine metabolic pathways is needed, along with clinical studies in disease cohorts (particularly liver disease) that assess the regulatory impact of probiotics on the gut microbiota and their influence on human health.

Conclusion

For primary outcome, the intervention of BLa80 can regulate the gut microbiota and effectively enrich Bifidobacterium. The results indicate that BLa80 is safe and capable of transient enrichment in the intestine. The intervention of BLa80 also significantly alters the metabolic pathways associated with biofilm formation—Pseudomonas aeruginosa, resistance to CAMP and other metabolic pathways associated with conditional pathogens and resistance to antimicrobial peptides (CAMP). BLa80 has the potential to improve hyperuricemia. Future large-scale clinical trials are needed to evaluate the ameliorative effect of BLa80 on hyperuricemia. Also, subsequent studies should prioritize hyperuricemic populations to validate these effects and explore BLa80’s potential as an adjunct therapy for gout or metabolic disorders.

Author Contributions

RH: conceptualization, software, visualization, writing – original draft, writing – review & editing. CG: data curation, methodology, investigation, formal analysis, writing – review & editing. PY: writing – review & editing. YT: investigation, writing – review & editing. YY: data curation, writing – review & editing. HL: writing – review & editing. SL: writing – review & editing. YW: writing – review & editing.

Ethics Approval and Consent to Participate

This study followed the principles outlined in the Declaration of Helsinki principles, and all methods were granted approval by the Ethics Committee of Henan University of Technology (No. HautEC202309) and registered in the Registry of Clinical Trials (Registration Code: NCT06103253, first posted date 26/10/2023). Participants provided written informed consent after being fully informed about the nature of the research.

Data Availability

All data generated or analyzed during this study are included in this published article.

Conflict of Interest

No potential conflicts of interest were disclosed.