Impact of Probiotic Supplementation on Folate, Vitamin B12, and Homocysteine Levels in Humans: A Systematic Review and Meta-Analysis of Randomized Controlled Trials

Impact of Probiotic Supplementation on Folate, Vitamin B12, and Homocysteine Levels in Humans: A Systematic Review and Meta-Analysis of Randomized Controlled Trials | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Systematic Review Impact of Probiotic Supplementation on Folate, Vitamin B12, and Homocysteine Levels in Humans: A Systematic Review and Meta-Analysis of Randomized Controlled Trials MINGFANG PAN, Changming Ye, Yun Song, Qiangqiang He, Lishun Liu, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9563368/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background : Previous pre-clinical studies and human clinical trials have suggested that probiotic suppplementation may affect the homocysteine metabolism. This systematic review and meta-analysis aimed to evaluate the effects of probiotic supplementation on homocysteine level and its closely related cofactors—folate and vitamin B12—in humans. Methods : This systematic review was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. PubMed, EMBASE, Web of Science, and the Cochrane Library were searched from inception to September 2025 for randomized controlled trials investigating the effects of probiotics on homocysteine, folate, or vitamin B12 levels. Pooled effect estimates were calculated using random-effects meta-analysis. Results : Among the 13 included studies, 9 randomized controlled trials involving 395 participants were used for homocysteine analysis. Probiotic supplementation was associated with a significant reduction in homocysteine levels compared with control (mean difference [MD]: −1.61 μmol/L; 95% confidence interval [CI]: −2.61 to −0.62; P = 0.0058). Six studies (n = 300) reported folate outcomes, with no significant effect observed following probiotic intervention (MD: 0.69; 95% CI: −0.22 to 1.62; P = 0.1099). In contrast, meta-analysis of six studies including 330 participants demonstrated a significant increase in vitamin B12 levels in the probiotic group (MD: 39.13; 95% CI: 14.41 to 63.85; P = 0.0096). Conclusions : Probiotic supplementation was associated with a significant reduction in circulating homocysteine levels and an increase in vitamin B12 concentrations, but not folate levels, in humans. These findings suggest a potential role for probiotics in modulating homocysteine metabolism and micronutrients. Further well-designed trials are warranted to elucidate whether the homocysteine-lowering effect of probiotics is mediated through improvements in vitamin B12. Nutrition & Dietetics Probiotic folate homocysteine vitamin B12 meta-analysis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Folate and vitamin B12 are essential micronutrients involved in one-carbon metabolism, a fundamental biochemical pathway required for DNA synthesis, methylation reactions, and amino acid metabolism[ 1 ]. Adequate status of these B vitamins is necessary to support normal hematopoiesis, neurological function, and cardiovascular health [ 2 ]. Inadequate folate or vitamin B12 intake or absorption can lead to hyperhomocysteinemia, a metabolic disturbance that has been consistently associated with increased risks of cardiovascular disease, cognitive decline, adverse pregnancy outcomes, and all-cause mortality[ 3 , 4 ]. Consequently, strategies aimed at improving folate and vitamin B12 status and reducing circulating homocysteine levels have important clinical and public health implications. Although dietary intake remains the primary determinant of folate and vitamin B12 status, emerging evidence suggests that the gut microbiota may play a contributory role in host B-vitamin metabolism[ 5 ]. Certain commensal bacteria are capable of synthesizing folate and VB12 de novo [ 6 , 7 ], while others may influence vitamin absorption and utilization[ 7 , 8 ]. Alterations in gut microbial composition have been linked to impaired micronutrient status, particularly in vulnerable populations such as older adults and individuals with metabolic or gastrointestinal disorders[ 9 , 10 ]. These observations have stimulated interest in probiotics as a nutritional intervention to support micronutrient homeostasis through modulation of the gut microbiome [ 11 ]. Probiotics are defined as live microorganisms that, when administered in adequate amounts, confer a health benefit on the host. In addition to their established roles in gastrointestinal health and immune modulation, probiotics have been proposed to influence metabolic pathways related to B-vitamin synthesis and homocysteine regulation. Preclinical studies indicate that specific probiotic strains, including Lactobacillus and Bifidobacterium species, are capable of producing folate or vitamin B12 and may increase intestinal folate production or enhance B-vitamin bioavailability[ 12 – 14 ]. Therefore, from a clinical perspective, probiotic supplementation represents a potentially safe, accessible, and non-pharmacological approach to improve micronutrient status, particularly in populations at risk of folate or VB12 deficiency or elevated homocysteine levels. In recent years, multiple randomized controlled trials (RCTs) have evaluated the effects of probiotic supplementation on circulating folate, vitamin B12, and homocysteine levels in humans[ 15 ]. These trials have been conducted across diverse populations and clinical contexts, including healthy individuals, patients with metabolic syndrome, type 2 diabetes and pregnant women[ 16 ]. However, the results have been inconsistent, with reported effects ranging from significant improvements in folate status and reductions in homocysteine to null findings. Variability in probiotic strain composition, dosage, intervention duration, baseline nutritional status, and study design may partly explain these discrepancies. Therefore, the present study aimed to systematically review and meta-analyze data from randomized controlled trials to evaluate the effects of probiotic supplementation on circulating folate, vitamin B12, and homocysteine levels in humans. By providing a quantitative assessment of available evidence, this work seeks to clarify the clinical relevance of probiotics as an adjunct nutritional strategy for optimizing one-carbon metabolism and reducing disease-related metabolic risk. 2. Methodology 2.1. Search strategy This meta-analysis evaluated the effects of probiotic supplementation on homocysteine metabolism and related biomarkers, specifically vitamin B12 and folate, in human clinical studies providing quantitative outcome data. The study was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines[ 17 ]. The review protocol was registered in the International Prospective Register of Systematic Reviews (PROSPERO; registration number: CRD420251165113). The following databases were searched for relevant publications from inception to September 2025: PubMed, EMBASE, Web of Science, and the Cochrane Library. Publications from inception to February 2025 were included. In addition, Google Scholar was spot searched to find relevant studies that may have been published in journals that are not indexed in above databases. The primary search strategy was developed for PubMed and subsequently adapted for use in the other databases as appropriate. The search strategies are provided in Appendix A . 2.2. Eligibility criteria Only studies published in English were considered for inclusion. Case reports, book chapters, review papers, editorials and animal model or cell culture studies were excluded. Studies were selected according to the PICOS design (PICOS: patients, intervention, comparator, outcome and study design): the population include both healthy individuals and those with conditions associated with folate deficiency or hyperhomocysteinemia (e.g., cardiovascular disease, hypertension); intervention included studies that utilized probiotic supplementation with clearly defined doses, bacterial strains (such as Lactobacillus and Bifidobacterium) or substrates containing probiotics (e.g., fermented milk or yogurt); comparison included adequate control or placebo group for comparison; outcomes included studies reporting outcomes related to homocysteine metabolism and associated vitamins (VB12 and folate); study design included randomized controlled trials (RCTs). Studies were excluded if they met any of the following criteria: (1) Duplicate studies. (2) Studies where the composition of the probiotic intervention was not specified. (3) Studies with insufficient information regarding experimental details or findings. (4) Any narrative review studies, meta-analyses, or any other articles that did not meet the requirement of being an original research study were also excluded. 2.3. Data extraction Eligible studies were independently reviewed by two authors and the following data were extracted: first author's name, publication year, RCT design, sample size (intervention and control groups), participant characteristics (health status), duration of intervention, delivery format and dose of probiotic intervention, and outcome data for homocysteine, folate, and vitamin B12. Outcome data included means and standard deviations (SDs) at baseline, post-intervention, and/or change from baseline, when available. Two independent reviewers searched for additional articles by reviewing the references of articles selected from the abstract and title searches. Any discrepancies in study selection or data extraction were resolved through discussion and consensus among all authors.Extracted data were compiled and summarized in tabular form (Table 1 ). 2.4. Risk of bias We evaluated the methodological quality using the Cochrane Risk of Bias Tool (RoB 2) for the RCTs [ 18 ]. Two investigators (Changming Ye, Xinxin Zhang) independently assessed each study, with conflicts resolved by a third reviewer (Mingfang Pan). Risk of bias was categorized as low, some concerns, or high across five domains: (a) randomization process; (b) deviation from the intended interventions; (c) missing outcome data; (d) measurement of the outcome; and (e) selection of the reported results. For each criterion, bias was assessed as a judgment (expressed as “high risk of bias”, “low risk of bias”, or “some concerns”). 2.5 Sensitivity analysis To evaluate the robustness of our findings amid substantial heterogeneity, a leave-one-out sensitivity analysis was performed [ 19 ]. Each study was sequentially omitted, and the meta-analysis was re-run to assess its impact on the pooled estimate and heterogeneity. All sensitivity analyses were conducted in R statistical environment (4.4.1) using the meta package(version 8.1-0). 2.5 Meta-analysis For the studies examining probiotic effects on homocysteine/folate/VB12 levels, we conducted a meta-analysis using means and standard deviations. For studies reported data using 95% confidence interval (CI) was converted to a standard deviation (SD) using the formula SD = sqrt (n) * (CI UL–CI LL)/t, and standard error of the mean (SEM) was converted to SD using the formula SD = SEM * sqrt (n). A random-effects model was used to calculate the mean difference (MD). Effect size heterogeneity was assessed using Q-statistics for testing the null hypothesis of homogeneity. Heterogeneity was also assessed through I 2 . Restricted Maximum Likelihood (REML) method was used to estimate the between-study heterogeneity[ 20 ]. The Hartung-Knapp adjustment was applied to random-effects models to account for uncertainty in between-study variance estimations and to enhance the reliability of confidence intervals when the number of included studies was limited[ 21 ]. Results were visualized using forest plots. All analyses were conducted using meta packages (version 8.1-0) in R 4.4.1. Statistical significance was set at p < 0.05. 3. Results 3.1 Search results The study selection process is illustrated in Fig. 1 . A total of 541 records were identified through database searching, including PubMed, Web of Science, Embase, and the Cochrane Library (n = 539), with an additional 2 records identified from other sources (google scholar search). After removal of duplicates, 75 records remained for screening. During title and abstract screening, 46 records were excluded because they were case reports, reviews, book chapters, meta-analyses, editorials, not relevant to the research question, or not published in English. Full-text articles were retrieved and assessed for eligibility for 19 records, of which 7 were excluded due to insufficient data. Ultimately, 13 studies met the inclusion criteria and were included in the meta-analysis. 3.2 Study characteristics Table 1 indicates the characteristics of the pooled trials. According to our surveys, all studies were published between the years 2006–2024. Nine clinical trials had a randomized, double-blind, placebo-control design, while the remaining four trials had only randomized control design without double blinding. The population of participants in the studies included healthy individuals, obese patients undergoing OAGB/RYGB, hyperlipidemia patients, type 2 diabetes patients and patients with metabolic syndrome. The study participants were from Egypt, Turkey, Italy, France, Germany, Poland, Iran, and Brazil, with ages ranging from 11 to 85 years. Sample sizes for all 11 studies ranged from 11 to 38. The total sample size for the nine studies included in the meta-analysis for homocysteine was 395 participants. The total sample size for the six studies included in the meta-analysis for folate and VB12 was 300 and 330 participants, respectively. The duration of probiotic intervention for the trials was between 42 and ~ 180 days. There was wide variation in delivery of the probiotic, ranging from capsules, tablets, powder, yogurt or fermented milk. Probiotic composition included L. casei , L. rhamnosus , L. bulgaricus , L. acidophilus , L. salivarius , L. paracasei , L. plantarum , B. breve , B. longum , B. animalis subsp. lactis , B. bifidum , B. lactis , B. bacterium infantis and S. thermophiles . Nine studies determined the change of homocysteine, six studies measured folate values and six measured levels of VB12 (Table 1 ). Table 1 Study characteristics Study Study Design Duration Population /group Country Age (years) Intervention /probiotic strains Delivery Format Dose (daily) Hcy/FA/VB12 Mahmoud 2006[ 22 ] RCT 42 days Healthy children: Probiotic=12 Placebo = 12 Egypt 11 Probiotic: yogurt with L. acidophilus (La1), L. acidophilus (La1), 5*10 9 CFU Control: yogurt without probiotic Yogurt 2 cups Hcy↓ FA↑ VB12↑ Valentini 2015[ 23 ] RCT 56 days Healthy persons: Probiotic=31 Placebo = 31 France Germany Italy 65–85 Probiotic: probiotic blend ( B. bacterium infantis DSM 24737, B. longum DSM 24736, B. breve DSM 24732, L. acidophilus DSM 24735, L. delbrückii ssp. bulgaricus DSM 24734, L. paracasei DSM 24733, L. plantarum DSM 24730 and S. thermophilus DSM 2473),1.12*10 11 CFU Control: without addition of probiotic Capsule 2 capsules Hcy↓ FA↑ Karbaschian 2018[ 24 ] RDBPC 112 days Obese patients undergoing OAGB-MGB surgery: Placebo=23 Probiotic = 23 Iran 18–60 Probiotic: 7 probiotic bacteria ( L. casei , L. rhamnosus , S. thermophilus , B. brev e, L. acidophilus , B. longum , and L. bulgaricus and 38.5 mg fructo-oligosaccharide Control: same amount of maltodextrin Tablet 1 tablet Hcy↓ FA↑ VB12↑ Majewska 2020[ 25 ] RDBPC 84 days Obese women: Placebo=25 Probiotic = 25 Poland 45–70 Probiotic: B. bifidum W23, B. lactis W51, B. lactis W52, L. acidophilus W37, L. brevis W63, L. casei W56, L. salivarius W24, L. lactis W19 and L. lactis W58, 2.5 × 10 9 CFU/g Control: no probiotic Powder 2 sachets (4 g) Hcy↓ Okburan (a) 2024 [ 26 ] RDBPC 56 days Patients with hyperlipidemia: Placebo=16 Probiotic = 18 Turkey 30–64 Probiotic: 1*10 6 CFU L. rhamnosus GG, Control: emptied capsule Capsule 1 capsule Hcy↓ Okburan (b) 2024 [ 26 ] RDBPC 56 days Patients with hyperlipidemia: Placebo=16 Probiotic = 17 Turkey 30–64 Probiotic group: 1*10 9 CFU L. acidophilus and 1*10 9 CFU B. animalis subsp.lactis Control: emptied capsule Capsule 1 capsule Hcy↓ Bellikci-Koyu 2022[ 27 ] RCT 84 days Patients with metabolic syndrome: Probiotic=31 Placebo = 31 Turkey 18–65 Probiotic: fermented milk (Lactobacillus spp., Leuconostoc spp., and Lactococcus spp, 10 6 CFU/g) Control: non-fermented milk Fermented milk 180 ml Hcy↓ Barreto 2014[ 28 ] RCT 90 days Patients with metabolic: Probiotic=12 Placebo = 12 Brazil 58–76 Probiotic: fermented milk ( L. plantarum , 1.25*10 7 UFC/g) Control: non-fermented milk Fermented milk 80 mL Hcy↓ Alihosseini 2017[ 29 ] RDBPC 56 days Patients with type 2 diabetes: Probiotic1 = 30 Probiotic2=30 Iran 35–65 Probiotic: probiotic ( S. thermophiles , L. bulgaricus, L. casei , L.acidophilus and B. lactis ) fermented milk Control: conventional fermented milk contained S. thermophiles and L. bulgaricus Fermented milk 600 ml Hcy↓ Ramos 2021[ 30 ] RDBPC 90 days Obese patients undergoing RYGB: Placebo=33 Probiotic = 38 Brazil 26–54 Probiotic: 5*10 8 CFU L. acidophilus NCFM® Strain, 5*10 8 CFU B. lactis Bi-07® Control: manipulated tablet consisting of starch and lactose. Tablet 2 tablets FA← VB12↑ Bardosono 2018[ 31 ] RDPC ~ 180 days Pregnant women placebo=22 probiotic = 55 Indonesia 18–35 Probiotic: milk powder with B. animalis subsp. lactis DR10TM Control: milk powder Milk 200 mL FA← VB12↑ Mokhtari 2019[ 32 ] RDBPC 120 days Obese patients undergoing OAGB: Placebo=25 Probiotic = 25 Iran 18–60 Probiotic: L. casei (3.5×10 9 CFU/g), L. rhamnosus (7.5×10 8 CFU/g), S. thermophiles (1×10 8 CFU/g), B. breve (1×10 10 CFU/g), L. acidophilus (1×10 9 CFU/g), B. longum (3.5×10 9 CFU/g), and L. bulgaricus (1×10 8 CFU/g) and 38.5-mg fructooligosaccharide. Control: maltodextrin Capsule 1 capsule VB12↑ Ballini 2020[ 33 ] RDBPC 56 days Pregnant women: placebo=10 probiotic = 10 Albania Probiotic: B. infantis, L. rhamnosus, L. plantarum, L. fermentum, L. reuteri, and L. acidophilus, 5*10 8 CFU, and actinidin powder Control: actinidin powder Tablet 1 tablet FA↑ Notes: study design: RDBPC, randomized, double-blind placebo control; RCT, randomized control trial. RYGB: Roux-en-Y gastric bypass; OAGB-MGB: One Anastomosis Gastric Bypass-Mini Gastric Bypass; ↑ statistically significant increase, ↓statistically significant decrease; ← no statistically significant change. L. = Lactobacillus, B. = Bifidobacterium, S. = Streptococcus. Hcy=homocysteine 3.3 Risk of bias and publication bias According to the RoB 2.0 assessment, six of the 13 included studies were judged as having some concerns or high risk of bias in the domain bias due to deviations from intended interventions, primarily because blinding of participants and personnel was not reported (Fig. 2 ). Among them, two studies were judged as having some concerns in the domain bias arising from the randomization process due to insufficient reporting of random sequence generation. The remaining randomized controlled trials were judged as having a low overall risk of bias. Egger’s test and funnel plot analysis were conducted to evaluate potential publication bias. The funnel plot demonstrated an approximately symmetrical distribution (Supplementary Figure S1), and Egger’s test revealed no statistically significant evidence of small-study effects or publication bias (p = 0.7211) 3.4 Probiotic supplementation on homocysteine Nine studies with a total of 395 subjects were included in the meta-analysis of homocysteine concentrations. Of these subjects, 199 were in the probiotic group, and 196 were in the placebo group. All the included 9 studies reported a statistically significant reduction in homocysteine levels following probiotic supplementation (Table 1 ). Random-effects meta-analysis showed that probiotic supplementation significantly reduced the homocysteine levels (MD: -1.61, 95% confidence interval: -2.61 to -0.62, P = 0.0058), with substantial heterogeneity across studies (I² = 71.5%). To investigate potential sources of heterogeneity, a leave-one-out sensitivity analysis was performed by sequentially excluding each study. Exclusion of the study by Mahmoud et al.[ 22 ], resulted in a marked reduction in heterogeneity, with the I² statistic decreasing from 71.5% to 30.5%, indicating that this study was a major contributor to the observed heterogeneity. Importantly, the pooled effect estimate remained statistically significant (P = 0.0022) regardless of whether the Mahmoud et al. study was excluded, thereby confirming the robustness of the findings. (Fig. 4 ) 3.5 Probiotic supplementation on folate and vitamin B12 Six studies (300 subjects) evaluated serum folate levels following probiotic intervention. Of these, four reported a statistically significant increase in folate concentrations in probiotic group, whereas two studies observed no significant change (Table 1 ). Random-effects meta-analysis showed no significant difference between the two groups (MD: 0.69, 95% confidence interval: -0.22 to 1.62, P = 0.1099) (Fig. 5 A). Six studies (330 subjects) provided VB12 data, and the random meta-analysis showed a significant improvement of VB12 in probiotic group by 39.13 pg/ml (95% confidence interval: 14.41 to 63.85, P = 0.0096), accompanied by substantial between-study heterogeneity (I² = 67.5%) (Fig. 5 B). Similarly, leave-one-out sensitivity analysis showed that exclusion of the study by Mahmoud et al. reduced the I² statistic to 0%, suggesting that this study was the primary source of the observed heterogeneity (Fig. 6 ). 4. Discussion and conclusion This meta-analysis represents the first systematic evaluation of the effects of probiotic supplementation on homocysteine metabolism, with particular focus on folate and vitamin B12 (VB12) status. Overall, the pooled evidence from randomized controlled trials indicate that probiotic supplementation significantly reduces circulating homocysteine levels in humans. The observed mean reduction in homocysteine was − 1.61 µmol/L (95% confidence interval: −2.61 to − 0.62; P = 0.0058), suggesting a moderate but potentially clinically meaningful effect. This result is consistent with previous evidences from animal studies demonstrating a homocysteine-lowering effect of probiotics [ 34 , 35 ]. Given the well-established role of elevated homocysteine as a risk factor for cardiovascular and metabolic disorders, our results extend prior preclinical observations by providing quantitative clinical evidence that probiotic supplementation can modulate homocysteine metabolism in humans. Folate and VB12 play essential roles in homocysteine metabolism, and deficiencies in either vitamin are well-established contributors to elevated homocysteine levels [ 36 ]. Extensive evidence demonstrates that supplementation with folate and VB12 effectively lowers homocysteine concentrations in humans [ 37 ]. Therefore, we speculated that the homocysteine-lowering effect of probiotic supplementation observed in this meta-analysis may be mediated, at least in part, through improvements in folate and/or VB12 status. Certain probiotic strains have the capacity to synthesize B vitamins, including folate and VB12 [ 12 ]. Animal studies have shown that administration of folate-producing probiotics can enhance host folate levels [ 34 , 35 ]. Among the 13 randomized controlled trials included in this review, six reported the change of folate data after probiotic supplementation. However, no significant increases in folate levels was observed after random meta-analysis. In contrast, meta-analysis of six trials showed a significant increases in VB12 levels following probiotic innervation (MD: 39.13 39.13 pg/mL; 95% confidence interval: 14.41 to 63.85, P = 0.0096). These findings suggest that probiotic supplementation may preferentially improve VB12 status and thereby contribute to enhanced homocysteine metabolism. Beyond direct vitamin synthesis, probiotics may influence VB12 status through modulation of the gut microbiota. In silico analyses suggest that the human gut microbiome has the potential to produce a substantial proportion of the recommended daily intake of folate and VB12, thereby contributing to host B-vitamin homeostasis[ 7 , 14 ]. Gut microbiota dysbiosis resulting from antibiotic exposure, pharmacological treatments, or chronic disease has been associated with micronutrient deficiencies, including B vitamins [ 15 ]. Additionally, pilot clinical studies indicate that probiotic supplementation may enhance vitamin bioavailability, improvements in gut barrier function and intestinal absorption [ 30 ]. Despite these plausible mechanisms, further basic and translational studies are required to clarify how probiotics regulate host VB12 levels and to delineate their direct contribution to homocysteine metabolism. A relatively high degree of heterogeneity was observed in the meta-analyses of both homocysteine (I² = 71.5%) and VB12 (I² = 67.5%). Sensitivity analyses, conducted by sequential exclusion of individual trials, showed the heterogeneity appears to be largely driven by a single outlier study conducted by Mahmoud et al. [ 22 ]. Exclusion of this study reduced heterogeneity from 71.5% to 30.5% in the homocysteine analysis and from 67.5% to 0% in the VB12 analysis, while preserving statistically significant overall effects. In that trial, probiotic supplementation resulted in a markedly greater improvement of VB12 (86.2 ± 26.42 pg/ml) and a more pronounced reduction in homocysteine concentrations (− 4.8 ± 1.28 µmol/L) compared with the control group (− 0.89 ± 1.12 µmol/L). The magnitude of effect observed in this study may be explained by characteristics of the study population. The trial was conducted in Egyptian children (mean age: 11 years) who exhibited substantially higher baseline homocysteine levels (16.89 ± 1.12 µmol/L) than participants in other included studies (< 13.76 ± 8.6 µmol/L). Individuals with elevated baseline homocysteine may experience greater absolute reductions following intervention. Limitations of this meta-analysis should be acknowledged. First, because of the limitation of resources, only studies published in English language were included in this systematic review. The bias of the included studies may have affected the results of the meta-analysis. For example, four studies did not use blinding and all of the studies were lacking in justification of sample size. Moreover, the studies included in this review were widely heterogeneous in terms of participant age, probiotic species, dosage, intervention duration, and form of administration. These factors may results in high heterogeneity and have affected the overall results of the meta-analysis. More rigorously designed, randomized, blinded, and adequately powered trials using standardized probiotic formulations are needed to confirm these findings. In additon, only two clinical trials concurrently measured homocysteine alongside folate and/or VB12 levels. limiting the ability to draw firm causal inferences. Addressing these gaps will require large-scale, well-designed prospective trials that simultaneously evaluate probiotic-induced changes in B-vitamin status and homocysteine metabolism. Despite these limitations, this meta-analysis provides novel evidence that probiotic supplementation may represent a promising strategy for lowering homocysteine levels and improving VB12 status. These findings have potential clinical implications for the prevention and management of homocysteine-related conditions and warrant further investigation in rigorously designed human studies. Declarations Ethics approval and consent to participate Not applicable. Consent for publication All authors consented to the publication of the manuscript. Availability of data and material All data and material reported in this review and meta-analysis were from peer-reviewed publications. The datasets supporting the conclusions of this article are included within the article and its additional files. Competing interests No potential conflicts of interest were disclosed. Authors' contributions MFP and CMY conceived and designed the study and developed the search strategy. MFP, CMY, and XXZ independently performed the abstract and full-text screening, assessed methodological quality and risk of bias, and completed data extraction for all included studies. MFP, LSL, and QQH conducted the meta-analyses, sensitivity analyses, and publication bias assessments, and prepared the figures and tables. MFP and QQH drafted the initial manuscript and revised and finalized the manuscript. XHQ, YS, and RMW contributed to the critical interpretation of the results and provided substantial intellectual input to the manuscript. PC and MQT participated in editing and revising the final version of the manuscript. All authors read and approved the final manuscript and agreed with the authors' theoretical statements. Acknowlegements This work was supported by the Science, Technology and Innovation Committee of Shenzhen, Major Special Program (Grant No. KJZD20230923114408018): R& D of Gut Microbiota-Based Probiotic for Stroke Prevention. The funding agency had no involvement in study design, data interpretation, or manuscript preparation. References Lyon, P., et al., B Vitamins and One-Carbon Metabolism: Implications in Human Health and Disease. Nutrients, 2020. 12 (9): p. 2867. Spence, J.D., Q. Yi, and G.J. Hankey, B vitamins in stroke prevention: time to reconsider. The Lancet Neurology, 2017. 16 (9): p. 750-760. Wu, D.-F., R.-X. Yin, and J.-L. Deng, Homocysteine, hyperhomocysteinemia, and H-type hypertension. European Journal of Preventive Cardiology, 2024: p. zwae022. Guéant, J.-L., et al., Hyperhomocysteinemia in cardiovascular diseases: revisiting observational studies and clinical trials. Thrombosis and Haemostasis, 2023. 123 (03): p. 270-282. Wan, Z., et al., Intermediate role of gut microbiota in vitamin B nutrition and its influences on human health. Front Nutr, 2022. 9 : p. 1031502. LeBlanc, J.G., et al., Bacteria as vitamin suppliers to their host: a gut microbiota perspective. Current opinion in biotechnology, 2013. 24 (2): p. 160-168. Engevik, M.A., et al., Microbial metabolic capacity for intestinal folate production and modulation of host folate receptors. Frontiers in microbiology, 2019. 10 : p. 2305. Kang, W.K., et al., Vitamin B12 produced by gut bacteria modulates cholinergic signalling. Nature Cell Biology, 2024. 26 (1): p. 72-85. Hibberd, M.C., et al., The effects of micronutrient deficiencies on bacterial species from the human gut microbiota. Sci Transl Med, 2017. 9 (390). Dje Kouadio, D.K., et al., Bacteria from the gut influence the host micronutrient status. Critical Reviews in Food Science and Nutrition, 2024. 64 (29): p. 10714-10729. Voland, L., et al., Gut microbiota and vitamin status in persons with obesity: A key interplay. Obes Rev, 2022. 23 (2): p. e13377. Gu, Q. and P. Li, Biosynthesis of vitamins by probiotic bacteria. Probiotics and prebiotics in human nutrition and health, 2016: p. 135-48. LeBlanc, J.G., et al., Supplementation with engineered Lactococcus lactis improves the folate status in deficient rats. Nutrition, 2010. 26 (7-8): p. 835-41. Magnúsdóttir, S., et al., Systematic genome assessment of B-vitamin biosynthesis suggests co-operation among gut microbes. Frontiers in genetics, 2015. 6 : p. 148. Barkhidarian, B., et al., Probiotic Supplementation and Micronutrient Status in Healthy Subjects: A Systematic Review of Clinical Trials. Nutrients, 2021. 13 (9). Mahara, F.A. and D. Yusuf, A systematic review: The effect of probiotics on folate status and diseases related to its deficiency (Anemia and Homocysteinemia). Food Bioscience, 2025. 66 : p. 106130. Vrabel, M., Preferred reporting items for systematic reviews and meta-analyses. Number 5/September 2015, 2015. 42 (5): p. 552-554. Sterne, J.A.C., et al., RoB 2: a revised tool for assessing risk of bias in randomised trials. Bmj, 2019. 366 : p. l4898. Viechtbauer, W. and M.W.L. Cheung, Outlier and influence diagnostics for meta‐analysis. Research synthesis methods, 2010. 1 (2): p. 112-125. Veroniki, A.A., et al., Methods to estimate the between-study variance and its uncertainty in meta-analysis. Res Synth Methods, 2016. 7 (1): p. 55-79. IntHout, J., J.P. Ioannidis, and G.F. Borm, The Hartung-Knapp-Sidik-Jonkman method for random effects meta-analysis is straightforward and considerably outperforms the standard DerSimonian-Laird method. BMC Med Res Methodol, 2014. 14 : p. 25. Mohammad, M.A., et al., Plasma cobalamin and folate and their metabolic markers methylmalonic acid and total homocysteine among Egyptian children before and after nutritional supplementation with the probiotic bacteria Lactobacillus acidophilus in yoghurt matrix. Int J Food Sci Nutr, 2006. 57 (7-8): p. 470-80. Valentini, L., et al., Impact of personalized diet and probiotic supplementation on inflammation, nutritional parameters and intestinal microbiota - The "RISTOMED project": Randomized controlled trial in healthy older people. Clin Nutr, 2015. 34 (4): p. 593-602. Karbaschian, Z., et al., Probiotic Supplementation in Morbid Obese Patients Undergoing One Anastomosis Gastric Bypass-Mini Gastric Bypass (OAGB-MGB) Surgery: a Randomized, Double-Blind, Placebo-Controlled, Clinical Trial. Obes Surg, 2018. 28 (9): p. 2874-2885. Majewska, K., et al., The Multispecies Probiotic Effectively Reduces Homocysteine Concentration in Obese Women: A Randomized Double-Blind Placebo-Controlled Study. J Clin Med, 2020. 9 (4). Okburan, G., M. Bas, and S. Ogmen, A randomized double-blind controlled clinical trial demonstrating efficacy of different probiotic strains on serum lipids and glycemic biomarker. Nutr Hosp, 2024. 41 (4): p. 793-803. Bellikci-Koyu, E., et al., Probiotic kefir consumption improves serum apolipoprotein A1 levels in metabolic syndrome patients: a randomized controlled clinical trial. Nutr Res, 2022. 102 : p. 59-70. Barreto, F.M., et al., Beneficial effects of Lactobacillus plantarum on glycemia and homocysteine levels in postmenopausal women with metabolic syndrome. Nutrition, 2014. 30 (7-8): p. 939-42. Alihosseini, N., et al., Effect of Probiotic Fermented Milk (Kefir) on Serum Level of Insulin and Homocysteine in Type 2 Diabetes Patients. Acta Endocrinol (Buchar), 2017. 13 (4): p. 431-436. Ramos, M.R.Z., et al., Effects of Lactobacillus acidophilus NCFM and Bifidobacterium lactis Bi-07 Supplementation on Nutritional and Metabolic Parameters in the Early Postoperative Period after Roux-en-Y Gastric Bypass: a Randomized, Double-Blind, Placebo-Controlled Trial. Obes Surg, 2021. 31 (5): p. 2105-2114. Bardosono, S., et al., Plasma folate, vitamin B6 and B12 in their relationship to the presence of probiotic strain Bifidobacterium animalis subsp. Lactis HNO19 (DR10TM) among Indonesian pregnant women in their third semester. World Nutrition Journal, 2019. 2 (2): p. 56-62. Mokhtari, Z., et al., The Effects of Probiotic Supplements on Blood Markers of Endotoxin and Lipid Peroxidation in Patients Undergoing Gastric Bypass Surgery; a Randomized, Double-Blind, Placebo-Controlled, Clinical Trial with 13 Months Follow-Up. Obes Surg, 2019. 29 (4): p. 1248-1258. Ballini, A., et al., Probiotics May Improve Serum Folate Availability in Pregnant Women: A Pilot Study. Open Access Macedonian Journal of Medical Sciences, 2020. 8 (B): p. 1124-1130. Zhang, J., et al., Screening of folate-producing lactic acid bacteria and modulatory effects of folate-biofortified yogurt on gut dysbacteriosis of folate-deficient rats. Food & function, 2020. 11 (7): p. 6308-6318. Pompei, A., et al., Administration of folate-producing bifidobacteria enhances folate status in Wistar rats. The Journal of nutrition, 2007. 137 (12): p. 2742-2746. Froese, D.S., B. Fowler, and M.R. Baumgartner, Vitamin B12, folate, and the methionine remethylation cycle—biochemistry, pathways, and regulation. Journal of inherited metabolic disease, 2019. 42 (4): p. 673-685. Mohan, A., et al., Homocysteine, vitamin B12 and folate level: possible risk factors in the progression of chronic heart and kidney disorders. Current Cardiology Reviews, 2023. 19 (4): p. 66-83. Additional Declarations The authors declare no competing interests. Supplementary Files AppendixA.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9563368","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Systematic Review","associatedPublications":[],"authors":[{"id":631638088,"identity":"b36bd18f-ae86-4502-82de-2aa99433895c","order_by":0,"name":"MINGFANG PAN","email":"","orcid":"","institution":"Institute of Biopharmaceutical and Health Engineering, Shenzhen International Graduate School, Tsinghua University, Shenzhen, China","correspondingAuthor":false,"prefix":"","firstName":"MINGFANG","middleName":"","lastName":"PAN","suffix":""},{"id":631638089,"identity":"e0820463-94a8-49fe-bd10-7cc1ec1669fe","order_by":1,"name":"Changming Ye","email":"","orcid":"","institution":"Shenzhen Evergreen Medical Institute, Shenzhen, China","correspondingAuthor":false,"prefix":"","firstName":"Changming","middleName":"","lastName":"Ye","suffix":""},{"id":631638090,"identity":"8f0fa596-2f9d-450f-8eb6-7770a79a1b1c","order_by":2,"name":"Yun Song","email":"","orcid":"","institution":"Shenzhen Evergreen Medical Institute, Shenzhen, China","correspondingAuthor":false,"prefix":"","firstName":"Yun","middleName":"","lastName":"Song","suffix":""},{"id":631638091,"identity":"04e1058b-cdf4-47e7-b617-63f6955dfaf8","order_by":3,"name":"Qiangqiang He","email":"","orcid":"","institution":"Shenzhen Evergreen Medical Institute, Shenzhen, China","correspondingAuthor":false,"prefix":"","firstName":"Qiangqiang","middleName":"","lastName":"He","suffix":""},{"id":631638092,"identity":"f79967bb-dee9-4823-a344-8eecf2f34703","order_by":4,"name":"Lishun Liu","email":"","orcid":"","institution":"Shenzhen Evergreen Medical Institute, Shenzhen, China","correspondingAuthor":false,"prefix":"","firstName":"Lishun","middleName":"","lastName":"Liu","suffix":""},{"id":631638093,"identity":"06d4676c-4d04-4b58-b657-228896b75a19","order_by":5,"name":"Runming Wang","email":"","orcid":"","institution":"Institute of Biopharmaceutical and Health Engineering, Shenzhen International Graduate School, Tsinghua University, Shenzhen, China","correspondingAuthor":false,"prefix":"","firstName":"Runming","middleName":"","lastName":"Wang","suffix":""},{"id":631638094,"identity":"ad1abade-328a-4fb1-83d0-862521fd4873","order_by":6,"name":"Minqing Tian","email":"","orcid":"","institution":"Shenzhen Evergreen Medical Institute, Shenzhen, China","correspondingAuthor":false,"prefix":"","firstName":"Minqing","middleName":"","lastName":"Tian","suffix":""},{"id":631638095,"identity":"986591b9-c0b9-4f74-94f9-de80ad4c04a5","order_by":7,"name":"Xianhui Qin","email":"","orcid":"","institution":"National Clinical Research Center for Kidney Disease, State Key Laboratory for Organ Failure Research, Guangdong Provincial Key Laboratory of Renal Failure Research, Guangzhou Regenerative Medicine and Health, Guangdong Laboratory, Division of Nephrology, Nanfang Hospital, Southern Medical University, Guangzhou, China","correspondingAuthor":false,"prefix":"","firstName":"Xianhui","middleName":"","lastName":"Qin","suffix":""},{"id":631638096,"identity":"921ecb05-7e1a-411c-be1b-044bdd0d313e","order_by":8,"name":"PingChen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwklEQVRIiWNgGAWjYBAC+/b+j4///mGT4ydaiwHPAWMD3gY+Y8kGorVIJJhJ8DbIJW44QKwWc54DyQaSO8wSNx9P3sDwo2IbYS2W7Q0HHxieSTPeduZZAWPPmdtEWHPmYLNBAtsx2W03cgyYGduI0XIjmU3iANt/xs0ziNVicCONTbKxjU1xgwSxWiR7zjAbM5xhM5YA+uUgUX7hZ+9hfMxQAYzK9uSND35UEOMXBEgwOECSerAWUnWMglEwCkbBCAEAZE1AehGOxDsAAAAASUVORK5CYII=","orcid":"","institution":"Precision Nutrition Innovation \u0026 Transformation Public Service Platform, Shenzhen 518057, PR China","correspondingAuthor":true,"prefix":"","firstName":"","middleName":"","lastName":"PingChen","suffix":""}],"badges":[],"createdAt":"2026-04-29 09:15:41","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-9563368/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9563368/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108409763,"identity":"639cf461-0d41-45c2-a59f-e24aee8784cd","added_by":"auto","created_at":"2026-05-04 10:02:34","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":94671,"visible":true,"origin":"","legend":"\u003cp\u003ePRISMA flow diagram of the systematic review.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9563368/v1/1236502bd4d5791180e08d38.png"},{"id":108492604,"identity":"0e10ccf8-ce4d-4251-8d5e-3ff0a9bd578d","added_by":"auto","created_at":"2026-05-05 09:58:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":152108,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eQuality assessment for RCT studies. \u003c/strong\u003eGreen circle with plus sign = low risk of bias; yellow circle with question mark = unclear risk of bias; red circle with minus sign = high risk of bias. (a) (b) Reports two studies in one article.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9563368/v1/8307106511accac4c841f8a7.png"},{"id":108409766,"identity":"62743350-707e-46f4-89fc-5cbae98f6ed7","added_by":"auto","created_at":"2026-05-04 10:02:34","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":399619,"visible":true,"origin":"","legend":"\u003cp\u003eForest plot of randomized controlled trials investigating the effects of probiotic supplementation on homocysteine. MD: mean difference. CI: confidence interval; I-squared: variation in MD attributable to heterogeneity, I\u003csup\u003e2\u003c/sup\u003e calculated based on Q statistic; p value was associated with Q statistic; the Hartung-Knapp method was used for adjustment of the random effects model.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9563368/v1/2d1eb572c47dea0939a07e45.png"},{"id":108493354,"identity":"bb18c792-eeb5-4030-be11-8a0f88cbbf20","added_by":"auto","created_at":"2026-05-05 10:00:01","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":238884,"visible":true,"origin":"","legend":"\u003cp\u003eSensitivity analysis of the mean difference (MD) for homocysteine changes.MD: mean difference. CI: confidence interval; I-squared: variation in MD attributable to heterogeneity, I\u003csup\u003e2\u003c/sup\u003e calculated based on Q statistic; p value was associated with Q statistic; the Hartung-Knapp method was used for adjustment of the random effects model.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9563368/v1/9cfdb7dfb6f85e4dba70afec.png"},{"id":108409767,"identity":"7ce7d1d2-bd1c-4d53-9a4a-ec03caabba89","added_by":"auto","created_at":"2026-05-04 10:02:34","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":417917,"visible":true,"origin":"","legend":"\u003cp\u003eForest plot of randomized controlled trials investigating the effects of probiotic supplementation on folate(A) and VB12(B). MD: mean difference. CI: confidence interval; I-squared: variation in MD attributable to heterogeneity, I\u003csup\u003e2\u003c/sup\u003e calculated based on Q statistic; p value was associated with Q statistic; the Hartung-Knapp method was used for adjustment of the random effects model.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-9563368/v1/f267c791a14ef9c18d0d56e0.png"},{"id":108803826,"identity":"83d6a5f7-4604-41c8-803b-69074d048295","added_by":"auto","created_at":"2026-05-08 15:08:41","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":286031,"visible":true,"origin":"","legend":"\u003cp\u003eSensitivity analysis of the mean difference (MD) for VB12 changes.MD: mean difference. CI: confidence interval; I-squared: variation in MD attributable to heterogeneity, I\u003csup\u003e2\u003c/sup\u003e calculated based on Q statistic; p value was associated with Q statistic; the Hartung-Knapp method was used for adjustment of the random effects model.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-9563368/v1/5d1b5edad2b5a220e80415fe.png"},{"id":108809066,"identity":"12ddec74-6ab1-4f7a-838a-52af3b50c3ef","added_by":"auto","created_at":"2026-05-08 15:49:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1680562,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9563368/v1/e5ed2b05-e294-40ca-a9e3-a1ede5ff1d42.pdf"},{"id":108494018,"identity":"aa6e232c-e341-4852-8acc-eac61cbeea90","added_by":"auto","created_at":"2026-05-05 10:02:18","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":14799,"visible":true,"origin":"","legend":"","description":"","filename":"AppendixA.docx","url":"https://assets-eu.researchsquare.com/files/rs-9563368/v1/20162435de13c4d6d9a1f202.docx"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eImpact of Probiotic Supplementation on Folate, Vitamin B12, and Homocysteine Levels in Humans: A Systematic Review and Meta-Analysis of Randomized Controlled Trials\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eFolate and vitamin B12 are essential micronutrients involved in one-carbon metabolism, a fundamental biochemical pathway required for DNA synthesis, methylation reactions, and amino acid metabolism[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Adequate status of these B vitamins is necessary to support normal hematopoiesis, neurological function, and cardiovascular health [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Inadequate folate or vitamin B12 intake or absorption can lead to hyperhomocysteinemia, a metabolic disturbance that has been consistently associated with increased risks of cardiovascular disease, cognitive decline, adverse pregnancy outcomes, and all-cause mortality[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Consequently, strategies aimed at improving folate and vitamin B12 status and reducing circulating homocysteine levels have important clinical and public health implications.\u003c/p\u003e \u003cp\u003eAlthough dietary intake remains the primary determinant of folate and vitamin B12 status, emerging evidence suggests that the gut microbiota may play a contributory role in host B-vitamin metabolism[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Certain commensal bacteria are capable of synthesizing folate and VB12 de novo [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], while others may influence vitamin absorption and utilization[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Alterations in gut microbial composition have been linked to impaired micronutrient status, particularly in vulnerable populations such as older adults and individuals with metabolic or gastrointestinal disorders[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. These observations have stimulated interest in probiotics as a nutritional intervention to support micronutrient homeostasis through modulation of the gut microbiome [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eProbiotics are defined as live microorganisms that, when administered in adequate amounts, confer a health benefit on the host. In addition to their established roles in gastrointestinal health and immune modulation, probiotics have been proposed to influence metabolic pathways related to B-vitamin synthesis and homocysteine regulation. Preclinical studies indicate that specific probiotic strains, including Lactobacillus and Bifidobacterium species, are capable of producing folate or vitamin B12 and may increase intestinal folate production or enhance B-vitamin bioavailability[\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Therefore, from a clinical perspective, probiotic supplementation represents a potentially safe, accessible, and non-pharmacological approach to improve micronutrient status, particularly in populations at risk of folate or VB12 deficiency or elevated homocysteine levels.\u003c/p\u003e \u003cp\u003eIn recent years, multiple randomized controlled trials (RCTs) have evaluated the effects of probiotic supplementation on circulating folate, vitamin B12, and homocysteine levels in humans[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. These trials have been conducted across diverse populations and clinical contexts, including healthy individuals, patients with metabolic syndrome, type 2 diabetes and pregnant women[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. However, the results have been inconsistent, with reported effects ranging from significant improvements in folate status and reductions in homocysteine to null findings. Variability in probiotic strain composition, dosage, intervention duration, baseline nutritional status, and study design may partly explain these discrepancies.\u003c/p\u003e \u003cp\u003eTherefore, the present study aimed to systematically review and meta-analyze data from randomized controlled trials to evaluate the effects of probiotic supplementation on circulating folate, vitamin B12, and homocysteine levels in humans. By providing a quantitative assessment of available evidence, this work seeks to clarify the clinical relevance of probiotics as an adjunct nutritional strategy for optimizing one-carbon metabolism and reducing disease-related metabolic risk.\u003c/p\u003e"},{"header":"2. Methodology","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Search strategy\u003c/h2\u003e \u003cp\u003eThis meta-analysis evaluated the effects of probiotic supplementation on homocysteine metabolism and related biomarkers, specifically vitamin B12 and folate, in human clinical studies providing quantitative outcome data. The study was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The review protocol was registered in the International Prospective Register of Systematic Reviews (PROSPERO; registration number: CRD420251165113).\u003c/p\u003e \u003cp\u003eThe following databases were searched for relevant publications from inception to September 2025: PubMed, EMBASE, Web of Science, and the Cochrane Library. Publications from inception to February 2025 were included. In addition, Google Scholar was spot searched to find relevant studies that may have been published in journals that are not indexed in above databases. The primary search strategy was developed for PubMed and subsequently adapted for use in the other databases as appropriate. The search strategies are provided in \u003cspan refid=\"Sec16\" class=\"InternalRef\"\u003e\u003cb\u003eAppendix A\u003c/b\u003e\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Eligibility criteria\u003c/h2\u003e \u003cp\u003eOnly studies published in English were considered for inclusion. Case reports, book chapters, review papers, editorials and animal model or cell culture studies were excluded.\u003c/p\u003e \u003cp\u003eStudies were selected according to the PICOS design (PICOS: patients, intervention, comparator, outcome and study design): the population include both healthy individuals and those with conditions associated with folate deficiency or hyperhomocysteinemia (e.g., cardiovascular disease, hypertension); intervention included studies that utilized probiotic supplementation with clearly defined doses, bacterial strains (such as Lactobacillus and Bifidobacterium) or substrates containing probiotics (e.g., fermented milk or yogurt); comparison included adequate control or placebo group for comparison; outcomes included studies reporting outcomes related to homocysteine metabolism and associated vitamins (VB12 and folate); study design included randomized controlled trials (RCTs).\u003c/p\u003e \u003cp\u003eStudies were excluded if they met any of the following criteria: (1) Duplicate studies. (2) Studies where the composition of the probiotic intervention was not specified. (3) Studies with insufficient information regarding experimental details or findings. (4) Any narrative review studies, meta-analyses, or any other articles that did not meet the requirement of being an original research study were also excluded.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Data extraction\u003c/h2\u003e \u003cp\u003eEligible studies were independently reviewed by two authors and the following data were extracted: first author's name, publication year, RCT design, sample size (intervention and control groups), participant characteristics (health status), duration of intervention, delivery format and dose of probiotic intervention, and outcome data for homocysteine, folate, and vitamin B12. Outcome data included means and standard deviations (SDs) at baseline, post-intervention, and/or change from baseline, when available. Two independent reviewers searched for additional articles by reviewing the references of articles selected from the abstract and title searches. Any discrepancies in study selection or data extraction were resolved through discussion and consensus among all authors.Extracted data were compiled and summarized in tabular form (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Risk of bias\u003c/h2\u003e \u003cp\u003eWe evaluated the methodological quality using the Cochrane Risk of Bias Tool (RoB 2) for the RCTs [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Two investigators (Changming Ye, Xinxin Zhang) independently assessed each study, with conflicts resolved by a third reviewer (Mingfang Pan). Risk of bias was categorized as low, some concerns, or high across five domains: (a) randomization process; (b) deviation from the intended interventions; (c) missing outcome data; (d) measurement of the outcome; and (e) selection of the reported results. For each criterion, bias was assessed as a judgment (expressed as \u0026ldquo;high risk of bias\u0026rdquo;, \u0026ldquo;low risk of bias\u0026rdquo;, or \u0026ldquo;some concerns\u0026rdquo;).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Sensitivity analysis\u003c/h2\u003e \u003cp\u003eTo evaluate the robustness of our findings amid substantial heterogeneity, a leave-one-out sensitivity analysis was performed [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Each study was sequentially omitted, and the meta-analysis was re-run to assess its impact on the pooled estimate and heterogeneity. All sensitivity analyses were conducted in R statistical environment (4.4.1) using the meta package(version 8.1-0).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Meta-analysis\u003c/h2\u003e \u003cp\u003eFor the studies examining probiotic effects on homocysteine/folate/VB12 levels, we conducted a meta-analysis using means and standard deviations. For studies reported data using 95% confidence interval (CI) was converted to a standard deviation (SD) using the formula SD\u0026thinsp;=\u0026thinsp;sqrt (n) * (CI UL\u0026ndash;CI LL)/t, and standard error of the mean (SEM) was converted to SD using the formula SD\u0026thinsp;=\u0026thinsp;SEM * sqrt (n). A random-effects model was used to calculate the mean difference (MD). Effect size heterogeneity was assessed using Q-statistics for testing the null hypothesis of homogeneity. Heterogeneity was also assessed through I\u003csup\u003e2\u003c/sup\u003e. Restricted Maximum Likelihood (REML) method was used to estimate the between-study heterogeneity[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The Hartung-Knapp adjustment was applied to random-effects models to account for uncertainty in between-study variance estimations and to enhance the reliability of confidence intervals when the number of included studies was limited[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eResults were visualized using forest plots. All analyses were conducted using meta packages (version 8.1-0) in R 4.4.1. Statistical significance was set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Search results\u003c/h2\u003e \u003cp\u003eThe study selection process is illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. A total of 541 records were identified through database searching, including PubMed, Web of Science, Embase, and the Cochrane Library (n\u0026thinsp;=\u0026thinsp;539), with an additional 2 records identified from other sources (google scholar search). After removal of duplicates, 75 records remained for screening. During title and abstract screening, 46 records were excluded because they were case reports, reviews, book chapters, meta-analyses, editorials, not relevant to the research question, or not published in English. Full-text articles were retrieved and assessed for eligibility for 19 records, of which 7 were excluded due to insufficient data. Ultimately, 13 studies met the inclusion criteria and were included in the meta-analysis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Study characteristics\u003c/h2\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e indicates the characteristics of the pooled trials. According to our surveys, all studies were published between the years 2006\u0026ndash;2024. Nine clinical trials had a randomized, double-blind, placebo-control design, while the remaining four trials had only randomized control design without double blinding.\u003c/p\u003e \u003cp\u003eThe population of participants in the studies included healthy individuals, obese patients undergoing OAGB/RYGB, hyperlipidemia patients, type 2 diabetes patients and patients with metabolic syndrome. The study participants were from Egypt, Turkey, Italy, France, Germany, Poland, Iran, and Brazil, with ages ranging from 11 to 85 years. Sample sizes for all 11 studies ranged from 11 to 38. The total sample size for the nine studies included in the meta-analysis for homocysteine was 395 participants. The total sample size for the six studies included in the meta-analysis for folate and VB12 was 300 and 330 participants, respectively. The duration of probiotic intervention for the trials was between 42 and ~\u0026thinsp;180 days. There was wide variation in delivery of the probiotic, ranging from capsules, tablets, powder, yogurt or fermented milk. Probiotic composition included \u003cem\u003eL. casei\u003c/em\u003e, \u003cem\u003eL. rhamnosus\u003c/em\u003e, \u003cem\u003eL. bulgaricus\u003c/em\u003e, \u003cem\u003eL. acidophilus\u003c/em\u003e, \u003cem\u003eL. salivarius\u003c/em\u003e, \u003cem\u003eL. paracasei\u003c/em\u003e, \u003cem\u003eL. plantarum\u003c/em\u003e, \u003cem\u003eB. breve\u003c/em\u003e, \u003cem\u003eB. longum\u003c/em\u003e, \u003cem\u003eB. animalis subsp. lactis\u003c/em\u003e, \u003cem\u003eB. bifidum\u003c/em\u003e, \u003cem\u003eB. lactis\u003c/em\u003e, \u003cem\u003eB. bacterium infantis\u003c/em\u003e and \u003cem\u003eS. thermophiles\u003c/em\u003e. Nine studies determined the change of homocysteine, six studies measured folate values and six measured levels of VB12 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eStudy characteristics\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"10\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStudy\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eStudy Design\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDuration\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePopulation\u003c/p\u003e \u003cp\u003e/group\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCountry\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAge\u003c/p\u003e \u003cp\u003e(years)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eIntervention\u003c/p\u003e \u003cp\u003e/probiotic strains\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eDelivery Format\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eDose\u003c/p\u003e \u003cp\u003e(daily)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eHcy/FA/VB12\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMahmoud 2006[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRCT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e42 days\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHealthy children:\u003c/p\u003e \u003cp\u003eProbiotic=12\u003c/p\u003e \u003cp\u003ePlacebo\u0026thinsp;=\u0026thinsp;12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eEgypt\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eProbiotic: yogurt with \u003cem\u003eL. acidophilus\u003c/em\u003e (La1), \u003cem\u003eL. acidophilus\u003c/em\u003e (La1), 5*10\u003csup\u003e9\u003c/sup\u003e CFU\u003c/p\u003e \u003cp\u003eControl: yogurt without probiotic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eYogurt\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2 cups\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eHcy\u0026darr;\u003c/p\u003e \u003cp\u003eFA\u0026uarr;\u003c/p\u003e \u003cp\u003eVB12\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eValentini 2015[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRCT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e56 days\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHealthy persons:\u003c/p\u003e \u003cp\u003eProbiotic=31\u003c/p\u003e \u003cp\u003ePlacebo\u0026thinsp;=\u0026thinsp;31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eFrance\u003c/p\u003e \u003cp\u003eGermany Italy\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e65\u0026ndash;85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eProbiotic: probiotic blend (\u003cem\u003eB. bacterium infantis\u003c/em\u003e DSM 24737, \u003cem\u003eB. longum\u003c/em\u003e DSM 24736, \u003cem\u003eB. breve\u003c/em\u003e DSM 24732, \u003cem\u003eL. acidophilus\u003c/em\u003e DSM 24735, \u003cem\u003eL. delbr\u0026uuml;ckii\u003c/em\u003e ssp. bulgaricus DSM 24734, \u003cem\u003eL. paracasei\u003c/em\u003e DSM 24733, \u003cem\u003eL. plantarum\u003c/em\u003e DSM 24730 and \u003cem\u003eS. thermophilus\u003c/em\u003e DSM 2473),1.12*10\u003csup\u003e11\u003c/sup\u003eCFU\u003c/p\u003e \u003cp\u003eControl: without addition of probiotic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCapsule\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2 capsules\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eHcy\u0026darr;\u003c/p\u003e \u003cp\u003eFA\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKarbaschian 2018[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRDBPC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e112 days\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eObese patients undergoing OAGB-MGB surgery:\u003c/p\u003e \u003cp\u003ePlacebo=23\u003c/p\u003e \u003cp\u003eProbiotic\u0026thinsp;=\u0026thinsp;23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eIran\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e18\u0026ndash;60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eProbiotic: 7 probiotic bacteria (\u003cem\u003eL. casei\u003c/em\u003e, \u003cem\u003eL. rhamnosus\u003c/em\u003e, \u003cem\u003eS. thermophilus\u003c/em\u003e, \u003cem\u003eB. brev\u003c/em\u003ee, \u003cem\u003eL. acidophilus\u003c/em\u003e, \u003cem\u003eB. longum\u003c/em\u003e, and \u003cem\u003eL. bulgaricus\u003c/em\u003e and 38.5 mg fructo-oligosaccharide\u003c/p\u003e \u003cp\u003eControl: same amount of maltodextrin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eTablet\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1 tablet\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eHcy\u0026darr;\u003c/p\u003e \u003cp\u003eFA\u0026uarr;\u003c/p\u003e \u003cp\u003eVB12\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMajewska 2020[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRDBPC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e84 days\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eObese women:\u003c/p\u003e \u003cp\u003ePlacebo=25\u003c/p\u003e \u003cp\u003eProbiotic\u0026thinsp;=\u0026thinsp;25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePoland\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e45\u0026ndash;70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eProbiotic: \u003cem\u003eB. bifidum\u003c/em\u003e W23, \u003cem\u003eB. lactis\u003c/em\u003e W51, \u003cem\u003eB. lactis\u003c/em\u003e W52, \u003cem\u003eL. acidophilus\u003c/em\u003e W37, \u003cem\u003eL. brevis\u003c/em\u003e W63, \u003cem\u003eL. casei\u003c/em\u003e W56, \u003cem\u003eL. salivarius\u003c/em\u003e W24, \u003cem\u003eL. lactis\u003c/em\u003e W19 and \u003cem\u003eL. lactis\u003c/em\u003e W58, 2.5 \u0026times; 10\u003csup\u003e9\u003c/sup\u003e CFU/g\u003c/p\u003e \u003cp\u003eControl: no probiotic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003ePowder\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2 sachets (4 g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eHcy\u0026darr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOkburan (a) 2024 [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRDBPC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e56 days\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePatients with hyperlipidemia:\u003c/p\u003e \u003cp\u003ePlacebo=16\u003c/p\u003e \u003cp\u003eProbiotic\u0026thinsp;=\u0026thinsp;18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTurkey\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e30\u0026ndash;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eProbiotic: 1*10\u003csup\u003e6\u003c/sup\u003e CFU \u003cem\u003eL. rhamnosus\u003c/em\u003e GG, \u003c/p\u003e \u003cp\u003eControl: emptied capsule\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCapsule\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1 capsule\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eHcy\u0026darr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOkburan (b) 2024 [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRDBPC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e56 days\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePatients with hyperlipidemia:\u003c/p\u003e \u003cp\u003ePlacebo=16\u003c/p\u003e \u003cp\u003eProbiotic\u0026thinsp;=\u0026thinsp;17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTurkey\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e30\u0026ndash;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eProbiotic group: 1*10\u003csup\u003e9\u003c/sup\u003e CFU \u003cem\u003eL. acidophilus\u003c/em\u003e and 1*10\u003csup\u003e9\u003c/sup\u003e CFU \u003cem\u003eB. animalis\u003c/em\u003e subsp.lactis\u003c/p\u003e \u003cp\u003eControl: emptied capsule\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCapsule\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1 capsule\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eHcy\u0026darr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBellikci-Koyu 2022[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRCT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e84 days\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePatients with metabolic syndrome: \u003c/p\u003e \u003cp\u003eProbiotic=31\u003c/p\u003e \u003cp\u003ePlacebo\u0026thinsp;=\u0026thinsp;31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTurkey\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e18\u0026ndash;65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eProbiotic: fermented milk (Lactobacillus spp., Leuconostoc spp., and Lactococcus spp, 10\u003csup\u003e6\u003c/sup\u003e CFU/g)\u003c/p\u003e \u003cp\u003eControl: non-fermented milk\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eFermented milk\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e180 ml\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eHcy\u0026darr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBarreto 2014[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRCT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e90 days\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePatients with metabolic:\u003c/p\u003e \u003cp\u003eProbiotic=12\u003c/p\u003e \u003cp\u003ePlacebo\u0026thinsp;=\u0026thinsp;12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eBrazil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e58\u0026ndash;76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eProbiotic: fermented milk (\u003cem\u003eL. plantarum\u003c/em\u003e, 1.25*10\u003csup\u003e7\u003c/sup\u003e UFC/g) \u003c/p\u003e \u003cp\u003eControl: non-fermented milk\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eFermented milk\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e80 mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eHcy\u0026darr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAlihosseini 2017[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRDBPC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e56 days\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePatients with type 2 diabetes:\u003c/p\u003e \u003cp\u003eProbiotic1\u0026thinsp;=\u0026thinsp;30\u003c/p\u003e \u003cp\u003eProbiotic2=30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eIran\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e35\u0026ndash;65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eProbiotic: probiotic (\u003cem\u003eS. thermophiles\u003c/em\u003e, \u003cem\u003eL. bulgaricus, L. casei\u003c/em\u003e, \u003cem\u003eL.acidophilus\u003c/em\u003e and \u003cem\u003eB. lactis\u003c/em\u003e) fermented milk \u003c/p\u003e \u003cp\u003eControl: conventional fermented milk contained \u003cem\u003eS. thermophiles\u003c/em\u003e and \u003cem\u003eL. bulgaricus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eFermented milk\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e600 ml\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eHcy\u0026darr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRamos 2021[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRDBPC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e90 days\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eObese patients undergoing RYGB:\u003c/p\u003e \u003cp\u003ePlacebo=33\u003c/p\u003e \u003cp\u003eProbiotic\u0026thinsp;=\u0026thinsp;38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eBrazil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e26\u0026ndash;54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eProbiotic: 5*10\u003csup\u003e8\u003c/sup\u003eCFU \u003cem\u003eL. acidophilus\u003c/em\u003e NCFM\u0026reg; Strain, 5*10\u003csup\u003e8\u003c/sup\u003eCFU \u003cem\u003eB. lactis\u003c/em\u003e Bi-07\u0026reg;\u003c/p\u003e \u003cp\u003eControl: manipulated tablet consisting of starch and lactose.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eTablet\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2 tablets\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eFA\u0026larr;\u003c/p\u003e \u003cp\u003eVB12\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBardosono 2018[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRDPC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e~\u0026thinsp;180 days\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePregnant women\u003c/p\u003e \u003cp\u003eplacebo=22\u003c/p\u003e \u003cp\u003eprobiotic\u0026thinsp;=\u0026thinsp;55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eIndonesia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e18\u0026ndash;35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eProbiotic: milk powder with \u003cem\u003eB. animalis\u003c/em\u003e subsp. lactis DR10TM\u003c/p\u003e \u003cp\u003eControl: milk powder\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eMilk\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e200 mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eFA\u0026larr;\u003c/p\u003e \u003cp\u003eVB12\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMokhtari 2019[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRDBPC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e120 days\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eObese patients undergoing OAGB:\u003c/p\u003e \u003cp\u003ePlacebo=25\u003c/p\u003e \u003cp\u003eProbiotic\u0026thinsp;=\u0026thinsp;25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eIran\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e18\u0026ndash;60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eProbiotic: \u003cem\u003eL. casei\u003c/em\u003e (3.5\u0026times;10\u003csup\u003e9\u003c/sup\u003e CFU/g), \u003cem\u003eL. rhamnosus\u003c/em\u003e (7.5\u0026times;10\u003csup\u003e8\u003c/sup\u003e CFU/g), \u003cem\u003eS. thermophiles\u003c/em\u003e (1\u0026times;10\u003csup\u003e8\u003c/sup\u003e CFU/g), \u003cem\u003eB. breve\u003c/em\u003e (1\u0026times;10\u003csup\u003e10\u003c/sup\u003e CFU/g), \u003cem\u003eL. acidophilus\u003c/em\u003e (1\u0026times;10\u003csup\u003e9\u003c/sup\u003e CFU/g), \u003cem\u003eB. longum\u003c/em\u003e (3.5\u0026times;10\u003csup\u003e9\u003c/sup\u003e CFU/g), and \u003cem\u003eL. bulgaricus\u003c/em\u003e (1\u0026times;10\u003csup\u003e8\u003c/sup\u003e CFU/g) and 38.5-mg fructooligosaccharide.\u003c/p\u003e \u003cp\u003eControl: maltodextrin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCapsule\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1 capsule\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eVB12\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBallini 2020[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRDBPC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e56 days\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePregnant women: \u003c/p\u003e \u003cp\u003eplacebo=10\u003c/p\u003e \u003cp\u003eprobiotic\u0026thinsp;=\u0026thinsp;10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAlbania\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eProbiotic: B. infantis, L. rhamnosus, L. plantarum, L. fermentum, L. reuteri, and L. acidophilus, 5*10\u003csup\u003e8\u003c/sup\u003eCFU, and actinidin powder\u003c/p\u003e \u003cp\u003eControl: actinidin powder\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eTablet\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1 tablet\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eFA\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"10\"\u003eNotes: study design: RDBPC, randomized, double-blind placebo control; RCT, randomized control trial. RYGB: Roux-en-Y gastric bypass; OAGB-MGB: One Anastomosis Gastric Bypass-Mini Gastric Bypass; \u0026uarr; statistically significant increase, \u0026darr;statistically significant decrease; \u0026larr; no statistically significant change. L. = Lactobacillus, B. = Bifidobacterium, S. = Streptococcus. Hcy=homocysteine\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Risk of bias and publication bias\u003c/h2\u003e \u003cp\u003eAccording to the RoB 2.0 assessment, six of the 13 included studies were judged as having some concerns or high risk of bias in the domain bias due to deviations from intended interventions, primarily because blinding of participants and personnel was not reported (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Among them, two studies were judged as having some concerns in the domain bias arising from the randomization process due to insufficient reporting of random sequence generation. The remaining randomized controlled trials were judged as having a low overall risk of bias.\u003c/p\u003e \u003cp\u003eEgger\u0026rsquo;s test and funnel plot analysis were conducted to evaluate potential publication bias. The funnel plot demonstrated an approximately symmetrical distribution (Supplementary Figure S1), and Egger\u0026rsquo;s test revealed no statistically significant evidence of small-study effects or publication bias (p\u0026thinsp;=\u0026thinsp;0.7211)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Probiotic supplementation on homocysteine\u003c/h2\u003e \u003cp\u003eNine studies with a total of 395 subjects were included in the meta-analysis of homocysteine concentrations. Of these subjects, 199 were in the probiotic group, and 196 were in the placebo group. All the included 9 studies reported a statistically significant reduction in homocysteine levels following probiotic supplementation (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Random-effects meta-analysis showed that probiotic supplementation significantly reduced the homocysteine levels (MD: -1.61, 95% confidence interval: -2.61 to -0.62, P\u0026thinsp;=\u0026thinsp;0.0058), with substantial heterogeneity across studies (I\u0026sup2; = 71.5%).\u003c/p\u003e \u003cp\u003eTo investigate potential sources of heterogeneity, a leave-one-out sensitivity analysis was performed by sequentially excluding each study. Exclusion of the study by Mahmoud et al.[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], resulted in a marked reduction in heterogeneity, with the I\u0026sup2; statistic decreasing from 71.5% to 30.5%, indicating that this study was a major contributor to the observed heterogeneity. Importantly, the pooled effect estimate remained statistically significant (P\u0026thinsp;=\u0026thinsp;0.0022) regardless of whether the Mahmoud et al. study was excluded, thereby confirming the robustness of the findings. (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Probiotic supplementation on folate and vitamin B12\u003c/h2\u003e \u003cp\u003eSix studies (300 subjects) evaluated serum folate levels following probiotic intervention. Of these, four reported a statistically significant increase in folate concentrations in probiotic group, whereas two studies observed no significant change (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Random-effects meta-analysis showed no significant difference between the two groups (MD: 0.69, 95% confidence interval: -0.22 to 1.62, P\u0026thinsp;=\u0026thinsp;0.1099) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eSix studies (330 subjects) provided VB12 data, and the random meta-analysis showed a significant improvement of VB12 in probiotic group by 39.13 pg/ml (95% confidence interval: 14.41 to 63.85, P\u0026thinsp;=\u0026thinsp;0.0096), accompanied by substantial between-study heterogeneity (I\u0026sup2; = 67.5%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Similarly, leave-one-out sensitivity analysis showed that exclusion of the study by Mahmoud et al. reduced the I\u0026sup2; statistic to 0%, suggesting that this study was the primary source of the observed heterogeneity (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion and conclusion","content":"\u003cp\u003eThis meta-analysis represents the first systematic evaluation of the effects of probiotic supplementation on homocysteine metabolism, with particular focus on folate and vitamin B12 (VB12) status. Overall, the pooled evidence from randomized controlled trials indicate that probiotic supplementation significantly reduces circulating homocysteine levels in humans. The observed mean reduction in homocysteine was \u0026minus;\u0026thinsp;1.61 \u0026micro;mol/L (95% confidence interval: \u0026minus;2.61 to \u0026minus;\u0026thinsp;0.62; P\u0026thinsp;=\u0026thinsp;0.0058), suggesting a moderate but potentially clinically meaningful effect. This result is consistent with previous evidences from animal studies demonstrating a homocysteine-lowering effect of probiotics [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Given the well-established role of elevated homocysteine as a risk factor for cardiovascular and metabolic disorders, our results extend prior preclinical observations by providing quantitative clinical evidence that probiotic supplementation can modulate homocysteine metabolism in humans.\u003c/p\u003e \u003cp\u003eFolate and VB12 play essential roles in homocysteine metabolism, and deficiencies in either vitamin are well-established contributors to elevated homocysteine levels [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Extensive evidence demonstrates that supplementation with folate and VB12 effectively lowers homocysteine concentrations in humans [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Therefore, we speculated that the homocysteine-lowering effect of probiotic supplementation observed in this meta-analysis may be mediated, at least in part, through improvements in folate and/or VB12 status.\u003c/p\u003e \u003cp\u003eCertain probiotic strains have the capacity to synthesize B vitamins, including folate and VB12 [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Animal studies have shown that administration of folate-producing probiotics can enhance host folate levels [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Among the 13 randomized controlled trials included in this review, six reported the change of folate data after probiotic supplementation. However, no significant increases in folate levels was observed after random meta-analysis. In contrast, meta-analysis of six trials showed a significant increases in VB12 levels following probiotic innervation (MD: 39.13 39.13 pg/mL; 95% confidence interval: 14.41 to 63.85, P\u0026thinsp;=\u0026thinsp;0.0096). These findings suggest that probiotic supplementation may preferentially improve VB12 status and thereby contribute to enhanced homocysteine metabolism.\u003c/p\u003e \u003cp\u003eBeyond direct vitamin synthesis, probiotics may influence VB12 status through modulation of the gut microbiota. In silico analyses suggest that the human gut microbiome has the potential to produce a substantial proportion of the recommended daily intake of folate and VB12, thereby contributing to host B-vitamin homeostasis[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Gut microbiota dysbiosis resulting from antibiotic exposure, pharmacological treatments, or chronic disease has been associated with micronutrient deficiencies, including B vitamins [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Additionally, pilot clinical studies indicate that probiotic supplementation may enhance vitamin bioavailability, improvements in gut barrier function and intestinal absorption [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Despite these plausible mechanisms, further basic and translational studies are required to clarify how probiotics regulate host VB12 levels and to delineate their direct contribution to homocysteine metabolism.\u003c/p\u003e \u003cp\u003eA relatively high degree of heterogeneity was observed in the meta-analyses of both homocysteine (I\u0026sup2; = 71.5%) and VB12 (I\u0026sup2; = 67.5%). Sensitivity analyses, conducted by sequential exclusion of individual trials, showed the heterogeneity appears to be largely driven by a single outlier study conducted by Mahmoud et al. [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Exclusion of this study reduced heterogeneity from 71.5% to 30.5% in the homocysteine analysis and from 67.5% to 0% in the VB12 analysis, while preserving statistically significant overall effects. In that trial, probiotic supplementation resulted in a markedly greater improvement of VB12 (86.2\u0026thinsp;\u0026plusmn;\u0026thinsp;26.42 pg/ml) and a more pronounced reduction in homocysteine concentrations (\u0026minus;\u0026thinsp;4.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.28 \u0026micro;mol/L) compared with the control group (\u0026minus;\u0026thinsp;0.89\u0026thinsp;\u0026plusmn;\u0026thinsp;1.12 \u0026micro;mol/L).\u003c/p\u003e \u003cp\u003eThe magnitude of effect observed in this study may be explained by characteristics of the study population. The trial was conducted in Egyptian children (mean age: 11 years) who exhibited substantially higher baseline homocysteine levels (16.89\u0026thinsp;\u0026plusmn;\u0026thinsp;1.12 \u0026micro;mol/L) than participants in other included studies (\u0026lt;\u0026thinsp;13.76\u0026thinsp;\u0026plusmn;\u0026thinsp;8.6 \u0026micro;mol/L). Individuals with elevated baseline homocysteine may experience greater absolute reductions following intervention.\u003c/p\u003e \u003cp\u003eLimitations of this meta-analysis should be acknowledged. First, because of the limitation of resources, only studies published in English language were included in this systematic review. The bias of the included studies may have affected the results\u003c/p\u003e \u003cp\u003eof the meta-analysis. For example, four studies did not use blinding and all of the studies were lacking in justification of sample size. Moreover, the studies included in this review were widely heterogeneous in terms of participant age, probiotic species, dosage, intervention duration, and form of administration. These factors may results in high heterogeneity and have affected the overall results of the meta-analysis. More rigorously designed, randomized, blinded, and adequately powered trials using standardized probiotic formulations are needed to confirm these findings. In additon, only two clinical trials concurrently measured homocysteine alongside folate and/or VB12 levels. limiting the ability to draw firm causal inferences. Addressing these gaps will require large-scale, well-designed prospective trials that simultaneously evaluate probiotic-induced changes in B-vitamin status and homocysteine metabolism.\u003c/p\u003e \u003cp\u003eDespite these limitations, this meta-analysis provides novel evidence that probiotic supplementation may represent a promising strategy for lowering homocysteine levels and improving VB12 status. These findings have potential clinical implications for the prevention and management of homocysteine-related conditions and warrant further investigation in rigorously designed human studies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eEthics approval and consent to participate\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eConsent for publication\u003c/p\u003e\n\u003cp\u003eAll authors consented to the publication of the manuscript.\u003c/p\u003e\n\u003cp\u003eAvailability of data and material\u003c/p\u003e\n\u003cp\u003eAll data and material reported in this review and meta-analysis were from peer-reviewed publications. The datasets supporting the conclusions of this article are included within the article and its additional files.\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eNo potential conflicts of interest were disclosed.\u003c/p\u003e\n\u003cp\u003eAuthors\u0026apos; contributions\u003c/p\u003e\n\u003cp\u003eMFP and CMY conceived and designed the study and developed the search strategy. MFP, CMY, and XXZ independently performed the abstract and full-text screening, assessed methodological quality and risk of bias, and completed data extraction for all included studies. MFP, LSL, and QQH conducted the meta-analyses, sensitivity analyses, and publication bias assessments, and prepared the figures and tables. MFP and QQH drafted the initial manuscript and revised and finalized the manuscript. XHQ, YS, and RMW contributed to the critical interpretation of the results and provided substantial intellectual input to the manuscript. PC and MQT participated in editing and revising the final version of the manuscript. All authors read and approved the final manuscript and agreed with the authors\u0026apos; theoretical statements.\u003c/p\u003e\n\u003cp\u003eAcknowlegements\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Science, Technology and Innovation Committee of Shenzhen, Major Special Program (Grant No. KJZD20230923114408018): R\u0026amp; D of Gut Microbiota-Based Probiotic for Stroke Prevention. The funding agency had no involvement in study design, data interpretation, or manuscript preparation. \u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eLyon, P., et al., \u003cem\u003eB Vitamins and One-Carbon Metabolism: Implications in Human Health and Disease.\u003c/em\u003e Nutrients, 2020. \u003cstrong\u003e12\u003c/strong\u003e(9): p. 2867.\u003c/li\u003e\n \u003cli\u003eSpence, J.D., Q. Yi, and G.J. Hankey, \u003cem\u003eB vitamins in stroke prevention: time to reconsider.\u003c/em\u003e The Lancet Neurology, 2017. \u003cstrong\u003e16\u003c/strong\u003e(9): p. 750-760.\u003c/li\u003e\n \u003cli\u003eWu, D.-F., R.-X. Yin, and J.-L. Deng, \u003cem\u003eHomocysteine, hyperhomocysteinemia, and H-type hypertension.\u003c/em\u003e European Journal of Preventive Cardiology, 2024: p. zwae022.\u003c/li\u003e\n \u003cli\u003eGu\u0026eacute;ant, J.-L., et al., \u003cem\u003eHyperhomocysteinemia in cardiovascular diseases: revisiting observational studies and clinical trials.\u003c/em\u003e Thrombosis and Haemostasis, 2023. \u003cstrong\u003e123\u003c/strong\u003e(03): p. 270-282.\u003c/li\u003e\n \u003cli\u003eWan, Z., et al., \u003cem\u003eIntermediate role of gut microbiota in vitamin B nutrition and its influences on human health.\u003c/em\u003e Front Nutr, 2022. \u003cstrong\u003e9\u003c/strong\u003e: p. 1031502.\u003c/li\u003e\n \u003cli\u003eLeBlanc, J.G., et al., \u003cem\u003eBacteria as vitamin suppliers to their host: a gut microbiota perspective.\u003c/em\u003e Current opinion in biotechnology, 2013. \u003cstrong\u003e24\u003c/strong\u003e(2): p. 160-168.\u003c/li\u003e\n \u003cli\u003eEngevik, M.A., et al., \u003cem\u003eMicrobial metabolic capacity for intestinal folate production and modulation of host folate receptors.\u003c/em\u003e Frontiers in microbiology, 2019. \u003cstrong\u003e10\u003c/strong\u003e: p. 2305.\u003c/li\u003e\n \u003cli\u003eKang, W.K., et al., \u003cem\u003eVitamin B12 produced by gut bacteria modulates cholinergic signalling.\u003c/em\u003e Nature Cell Biology, 2024. \u003cstrong\u003e26\u003c/strong\u003e(1): p. 72-85.\u003c/li\u003e\n \u003cli\u003eHibberd, M.C., et al., \u003cem\u003eThe effects of micronutrient deficiencies on bacterial species from the human gut microbiota.\u003c/em\u003e Sci Transl Med, 2017. \u003cstrong\u003e9\u003c/strong\u003e(390).\u003c/li\u003e\n \u003cli\u003eDje Kouadio, D.K., et al., \u003cem\u003eBacteria from the gut influence the host micronutrient status.\u003c/em\u003e Critical Reviews in Food Science and Nutrition, 2024. \u003cstrong\u003e64\u003c/strong\u003e(29): p. 10714-10729.\u003c/li\u003e\n \u003cli\u003eVoland, L., et al., \u003cem\u003eGut microbiota and vitamin status in persons with obesity: A key interplay.\u003c/em\u003e Obes Rev, 2022. \u003cstrong\u003e23\u003c/strong\u003e(2): p. e13377.\u003c/li\u003e\n \u003cli\u003eGu, Q. and P. Li, \u003cem\u003eBiosynthesis of vitamins by probiotic bacteria.\u003c/em\u003e Probiotics and prebiotics in human nutrition and health, 2016: p. 135-48.\u003c/li\u003e\n \u003cli\u003eLeBlanc, J.G., et al., \u003cem\u003eSupplementation with engineered Lactococcus lactis improves the folate status in deficient rats.\u003c/em\u003e Nutrition, 2010. \u003cstrong\u003e26\u003c/strong\u003e(7-8): p. 835-41.\u003c/li\u003e\n \u003cli\u003eMagn\u0026uacute;sd\u0026oacute;ttir, S., et al., \u003cem\u003eSystematic genome assessment of B-vitamin biosynthesis suggests co-operation among gut microbes.\u003c/em\u003e Frontiers in genetics, 2015. \u003cstrong\u003e6\u003c/strong\u003e: p. 148.\u003c/li\u003e\n \u003cli\u003eBarkhidarian, B., et al., \u003cem\u003eProbiotic Supplementation and Micronutrient Status in Healthy Subjects: A Systematic Review of Clinical Trials.\u003c/em\u003e Nutrients, 2021. \u003cstrong\u003e13\u003c/strong\u003e(9).\u003c/li\u003e\n \u003cli\u003eMahara, F.A. and D. Yusuf, \u003cem\u003eA systematic review: The effect of probiotics on folate status and diseases related to its deficiency (Anemia and Homocysteinemia).\u003c/em\u003e Food Bioscience, 2025. \u003cstrong\u003e66\u003c/strong\u003e: p. 106130.\u003c/li\u003e\n \u003cli\u003eVrabel, M., \u003cem\u003ePreferred reporting items for systematic reviews and meta-analyses.\u003c/em\u003e Number 5/September 2015, 2015. \u003cstrong\u003e42\u003c/strong\u003e(5): p. 552-554.\u003c/li\u003e\n \u003cli\u003eSterne, J.A.C., et al., \u003cem\u003eRoB 2: a revised tool for assessing risk of bias in randomised trials.\u003c/em\u003e Bmj, 2019. \u003cstrong\u003e366\u003c/strong\u003e: p. l4898.\u003c/li\u003e\n \u003cli\u003eViechtbauer, W. and M.W.L. Cheung, \u003cem\u003eOutlier and influence diagnostics for meta‐analysis.\u003c/em\u003e Research synthesis methods, 2010. \u003cstrong\u003e1\u003c/strong\u003e(2): p. 112-125.\u003c/li\u003e\n \u003cli\u003eVeroniki, A.A., et al., \u003cem\u003eMethods to estimate the between-study variance and its uncertainty in meta-analysis.\u003c/em\u003e Res Synth Methods, 2016. \u003cstrong\u003e7\u003c/strong\u003e(1): p. 55-79.\u003c/li\u003e\n \u003cli\u003eIntHout, J., J.P. Ioannidis, and G.F. Borm, \u003cem\u003eThe Hartung-Knapp-Sidik-Jonkman method for random effects meta-analysis is straightforward and considerably outperforms the standard DerSimonian-Laird method.\u003c/em\u003e BMC Med Res Methodol, 2014. \u003cstrong\u003e14\u003c/strong\u003e: p. 25.\u003c/li\u003e\n \u003cli\u003eMohammad, M.A., et al., \u003cem\u003ePlasma cobalamin and folate and their metabolic markers methylmalonic acid and total homocysteine among Egyptian children before and after nutritional supplementation with the probiotic bacteria Lactobacillus acidophilus in yoghurt matrix.\u003c/em\u003e Int J Food Sci Nutr, 2006. \u003cstrong\u003e57\u003c/strong\u003e(7-8): p. 470-80.\u003c/li\u003e\n \u003cli\u003eValentini, L., et al., \u003cem\u003eImpact of personalized diet and probiotic supplementation on inflammation, nutritional parameters and intestinal microbiota - The \u0026quot;RISTOMED project\u0026quot;: Randomized controlled trial in healthy older people.\u003c/em\u003e Clin Nutr, 2015. \u003cstrong\u003e34\u003c/strong\u003e(4): p. 593-602.\u003c/li\u003e\n \u003cli\u003eKarbaschian, Z., et al., \u003cem\u003eProbiotic Supplementation in Morbid Obese Patients Undergoing One Anastomosis Gastric Bypass-Mini Gastric Bypass (OAGB-MGB) Surgery: a Randomized, Double-Blind, Placebo-Controlled, Clinical Trial.\u003c/em\u003e Obes Surg, 2018. \u003cstrong\u003e28\u003c/strong\u003e(9): p. 2874-2885.\u003c/li\u003e\n \u003cli\u003eMajewska, K., et al., \u003cem\u003eThe Multispecies Probiotic Effectively Reduces Homocysteine Concentration in Obese Women: A Randomized Double-Blind Placebo-Controlled Study.\u003c/em\u003e J Clin Med, 2020. \u003cstrong\u003e9\u003c/strong\u003e(4).\u003c/li\u003e\n \u003cli\u003eOkburan, G., M. Bas, and S. Ogmen, \u003cem\u003eA randomized double-blind controlled clinical trial demonstrating efficacy of different probiotic strains on serum lipids and glycemic biomarker.\u003c/em\u003e Nutr Hosp, 2024. \u003cstrong\u003e41\u003c/strong\u003e(4): p. 793-803.\u003c/li\u003e\n \u003cli\u003eBellikci-Koyu, E., et al., \u003cem\u003eProbiotic kefir consumption improves serum apolipoprotein A1 levels in metabolic syndrome patients: a randomized controlled clinical trial.\u003c/em\u003e Nutr Res, 2022. \u003cstrong\u003e102\u003c/strong\u003e: p. 59-70.\u003c/li\u003e\n \u003cli\u003eBarreto, F.M., et al., \u003cem\u003eBeneficial effects of Lactobacillus plantarum on glycemia and homocysteine levels in postmenopausal women with metabolic syndrome.\u003c/em\u003e Nutrition, 2014. \u003cstrong\u003e30\u003c/strong\u003e(7-8): p. 939-42.\u003c/li\u003e\n \u003cli\u003eAlihosseini, N., et al., \u003cem\u003eEffect of Probiotic Fermented Milk (Kefir) on Serum Level of Insulin and Homocysteine in Type 2 Diabetes Patients.\u003c/em\u003e Acta Endocrinol (Buchar), 2017. \u003cstrong\u003e13\u003c/strong\u003e(4): p. 431-436.\u003c/li\u003e\n \u003cli\u003eRamos, M.R.Z., et al., \u003cem\u003eEffects of Lactobacillus acidophilus NCFM and Bifidobacterium lactis Bi-07 Supplementation on Nutritional and Metabolic Parameters in the Early Postoperative Period after Roux-en-Y Gastric Bypass: a Randomized, Double-Blind, Placebo-Controlled Trial.\u003c/em\u003e Obes Surg, 2021. \u003cstrong\u003e31\u003c/strong\u003e(5): p. 2105-2114.\u003c/li\u003e\n \u003cli\u003eBardosono, S., et al., \u003cem\u003ePlasma folate, vitamin B6 and B12 in their relationship to the presence of probiotic strain Bifidobacterium animalis subsp. Lactis HNO19 (DR10TM) among Indonesian pregnant women in their third semester.\u003c/em\u003e World Nutrition Journal, 2019. \u003cstrong\u003e2\u003c/strong\u003e(2): p. 56-62.\u003c/li\u003e\n \u003cli\u003eMokhtari, Z., et al., \u003cem\u003eThe Effects of Probiotic Supplements on Blood Markers of Endotoxin and Lipid Peroxidation in Patients Undergoing Gastric Bypass Surgery; a Randomized, Double-Blind, Placebo-Controlled, Clinical Trial with 13 Months Follow-Up.\u003c/em\u003e Obes Surg, 2019. \u003cstrong\u003e29\u003c/strong\u003e(4): p. 1248-1258.\u003c/li\u003e\n \u003cli\u003eBallini, A., et al., \u003cem\u003eProbiotics May Improve Serum Folate Availability in Pregnant Women: A Pilot Study.\u003c/em\u003e Open Access Macedonian Journal of Medical Sciences, 2020. \u003cstrong\u003e8\u003c/strong\u003e(B): p. 1124-1130.\u003c/li\u003e\n \u003cli\u003eZhang, J., et al., \u003cem\u003eScreening of folate-producing lactic acid bacteria and modulatory effects of folate-biofortified yogurt on gut dysbacteriosis of folate-deficient rats.\u003c/em\u003e Food \u0026amp; function, 2020. \u003cstrong\u003e11\u003c/strong\u003e(7): p. 6308-6318.\u003c/li\u003e\n \u003cli\u003ePompei, A., et al., \u003cem\u003eAdministration of folate-producing bifidobacteria enhances folate status in Wistar rats.\u003c/em\u003e The Journal of nutrition, 2007. \u003cstrong\u003e137\u003c/strong\u003e(12): p. 2742-2746.\u003c/li\u003e\n \u003cli\u003eFroese, D.S., B. Fowler, and M.R. Baumgartner, \u003cem\u003eVitamin B12, folate, and the methionine remethylation cycle\u0026mdash;biochemistry, pathways, and regulation.\u003c/em\u003e Journal of inherited metabolic disease, 2019. \u003cstrong\u003e42\u003c/strong\u003e(4): p. 673-685.\u003c/li\u003e\n \u003cli\u003eMohan, A., et al., \u003cem\u003eHomocysteine, vitamin B12 and folate level: possible risk factors in the progression of chronic heart and kidney disorders.\u003c/em\u003e Current Cardiology Reviews, 2023. \u003cstrong\u003e19\u003c/strong\u003e(4): p. 66-83.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Precision Nutrition Innovation \u0026 Transformation Public Service Platform, Shenzhen 518057, PR China","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Probiotic, folate, homocysteine, vitamin B12, meta-analysis","lastPublishedDoi":"10.21203/rs.3.rs-9563368/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9563368/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e: Previous pre-clinical studies and human clinical trials have suggested that probiotic suppplementation may affect the homocysteine metabolism. This systematic review and meta-analysis aimed to evaluate the effects of probiotic supplementation on homocysteine level and its closely related cofactors—folate and vitamin B12—in humans.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e: This systematic review was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. PubMed, EMBASE, Web of Science, and the Cochrane Library were searched from inception to September 2025 for randomized controlled trials investigating the effects of probiotics on homocysteine, folate, or vitamin B12 levels. Pooled effect estimates were calculated using random-effects meta-analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: Among the 13 included studies, 9 randomized controlled trials involving 395 participants were used for homocysteine analysis. Probiotic supplementation was associated with a significant reduction in homocysteine levels compared with control (mean difference [MD]: −1.61 μmol/L; 95% confidence interval [CI]: −2.61 to −0.62; P = 0.0058). Six studies (n = 300) reported folate outcomes, with no significant effect observed following probiotic intervention (MD: 0.69; 95% CI: −0.22 to 1.62; P = 0.1099). In contrast, meta-analysis of six studies including 330 participants demonstrated a significant increase in vitamin B12 levels in the probiotic group (MD: 39.13; 95% CI: 14.41 to 63.85; P = 0.0096).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e: Probiotic supplementation was associated with a significant reduction in circulating homocysteine levels and an increase in vitamin B12 concentrations, but not folate levels, in humans. These findings suggest a potential role for probiotics in modulating homocysteine metabolism and micronutrients. Further well-designed trials are warranted to elucidate whether the homocysteine-lowering effect of probiotics is mediated through improvements in vitamin B12.\u003c/p\u003e","manuscriptTitle":"Impact of Probiotic Supplementation on Folate, Vitamin B12, and Homocysteine Levels in Humans: A Systematic Review and Meta-Analysis of Randomized Controlled Trials","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-04 10:02:29","doi":"10.21203/rs.3.rs-9563368/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3acb5b59-b721-48d9-b462-dc1bd77705ce","owner":[],"postedDate":"May 4th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":67231165,"name":"Nutrition \u0026 Dietetics"}],"tags":[],"updatedAt":"2026-05-04T10:02:30+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-04 10:02:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9563368","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9563368","identity":"rs-9563368","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}