Impact of probiotics on muscle mass, muscle strength and lean mass: a systematic review and meta‐analysis of randomized controlled trials

Abstract Probiotics have shown potential to counteract sarcopenia, although the extent to which they can influence domains of sarcopenia such as muscle mass and strength in humans is unclear. The aim of this systematic review and meta‐analysis was to explore the impact of probiotic supplementation on muscle mass, total lean mass and muscle strength in human adults. A literature search of randomized controlled trials (RCTs) was conducted through PubMed, Scopus, Web of Science and Cochrane Library from inception until June 2022. Eligible RCTs compared the effect of probiotic supplementation versus placebo on muscle and total lean mass and global muscle strength (composite score of all muscle strength outcomes) in adults (>18 years). To evaluate the differences between groups, a meta‐analysis was conducted using the random effects inverse‐variance model by utilizing standardized mean differences. Twenty‐four studies were included in the systematic review and meta‐analysis exploring the effects of probiotics on muscle mass, total lean mass and global muscle strength. Our main analysis (k = 10) revealed that muscle mass was improved following probiotics compared with placebo (SMD: 0.42, 95% CI: 0.10–0.74, I2 = 57%, P = 0.009), although no changes were revealed in relation to total lean mass (k = 12; SMD: ‐0.03, 95% CI: −0.19 – 0.13, I2 = 0%, P = 0.69). Interestingly, a significant increase in global muscle strength was also observed among six RCTs (SMD: 0.69, 95% CI: 0.33–1.06, I2 = 64%, P = 0.0002). Probiotic supplementation enhances both muscle mass and global muscle strength; however, no beneficial effects were observed in total lean mass. Investigating the physiological mechanisms underpinning different ageing groups and elucidating appropriate probiotic strains for optimal gains in muscle mass and strength are warranted.


Introduction
Sarcopenia is a progressive skeletal muscle disorder involving the accelerated loss of muscle mass and function that is associated with increased adverse outcomes including falls, functional decline, frailty and mortality in older adults 1 but can also be observed in younger populations. 2 The prognosis of sarcopenia is multifactorial and includes physical inactivity, 3 hormonal imbalances, 4 sleep disturbance 5 and malnutrition 6 that predispose loss of motor neurons and muscle fibres, 7 overproduction of reactive oxygen species and pro-inflammatory mediators, 8 immune senescence 9 and an overall state of anabolic resistance, 10 which are pathophysiologically involved in loss of muscle mass and strength.
Nutritional therapies for treating sarcopenia may include achieving a calorie intake of 24-36 kcal/kg body weight/day and a protein intake of 1.0-1.5 g/kg of body mass/day 11,12 and supplementation with antioxidants, 13 protein and essential amino acids (EAAs), 14,15 omega-3 fatty acids [eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)] 16 and creatine monohydrate. 17 Emerging evidence has also revealed a prominent role for targeting the gut microbiota to counteract sarcopenia via the gut-muscle axis. 18 Specifically, probiotics may promote anabolism via greater amino acid absorption 19,20 and digestion 21 and down-regulation of skeletal muscle catabolism by competing with pathogenic gut bacteria involved in the stimulation of pro-inflammatory pathways through systemic activation of interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNFa). 22 The use of probiotics has shown potential as a nutritional strategy to prevent and/or treat sarcopenia. 23,24 Several in vivo studies in mice have reported benefits of Lactobacillus supplementation on muscle mass preservation and physical function benefits due to a reduction in low-grade inflammation and increased mitochondrial function, [25][26][27][28] as well as inhibition of oxidative stress. 29 Additionally, an increase in muscle strength, but not mass, after gut microbial transplantation from high-functioning older adults in comparison with transplantation of microbiota from older adults with poor physical performance colonized in mice has been demonstrated. 30 Recently, a systematic review concluded that probiotic supplementation may not improve performance following resistance and aerobic exercise 31 ; however, their findings were primarily focused on markers of cardiorespiratory fitness and did not capture the overall literature around muscle mass and strength. Hence, there is a growing interest in the potential role of probiotics supplementation to improve muscle mass and strength in humans. This systematic review and meta-analysis of clinical trials aimed to investigate the effect of probiotic supplementation on muscle mass, total lean mass and muscle strength in both young and older adults across the healthspan.

Methods
This systematic review and meta-analysis was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. 32 The protocol was registered in the International Prospective Register of Systematic Reviews (PROSPERO) (CRD: 42022320115).

Search strategy
Two independent reviewers (K.P. and K.K.T.) searched PubMed, Scopus, Web of Science and Cochrane Library from inception until June 2022. The full search strategy and the search terms used are described in Table S1. A manual search of references cited in the selected articles and published reviews was also performed. As a means to minimize bias, grey literature such as published abstracts and dissertations were also screened. The searches were rerun before submission to retrieve any additional studies that met our inclusion criteria. Discrepancies in the literature search process were resolved by a third and fourth investigator (P.G. and K.S.K).

Inclusion and exclusion criteria
Studies were included based on the following criteria: (i) randomized controlled trials (RCTs); (ii) adults (i.e. >18 years old) irrespective of health status (iii) intervention group received probiotics; and (iv) comparator group receiving nothing (control) or placebo. Published articles were excluded if they (i) were reviews, letters, in vivo or in vitro experiments or commentaries; (ii) were not published as a full text; (iii) included participants younger than 18 years of age; (iv) the intervention included pharmacological agents (e.g. use of anabolic androgenic steroids); and (v) the intervention included enteral nutrition.

Data extraction and risk of bias
Two authors (K.P. and K.K.T.) extracted data independently, which included name of first author, date of publication, country of origin, number and age of participants, probiotic type (genera and species level), placebo type, dose of probiotic genera/strain, outcome measurements, method of body composition assessment and method of dietary recall assessment. Disagreements between authors were resolved by two independent reviewers (P.G. and K.S.K.). The quality of the included studies was evaluated using the Risk-of-Bias 2 (RoB2) tool 33 and performed by three independent reviewers (K.P., P.G. and K.K.T.). RoB2 is a comprehensive tool used to assess bias in RCTs based on the following domains: (i) randomization process; (ii) deviations from intended interventions; (iii) missing outcome data; (iv) measurement of the outcome; and (v) selection of the reported result. 34 According to the scoring system, study bias was defined as 'high', 'some concerns' or 'low'.

Endpoints
This meta-analysis compared changes in muscle mass, total lean mass and muscle strength in participants allocated to a probiotic supplementation or comparator group (placebo or control).

Statistical analysis
Quantitative data were treated as continuous measurements, and changes in outcomes from baseline to follow-up were compared between groups to calculate mean differences. For studies presenting multiple indices of muscle strength, pooled means and standard deviations were derived. When units of measurements were inconsistent and could not be converted to units required for inclusion in the analysis, standardized mean differences were applied. In the event that numerical data were not reported, graphical values were calculated using DigitizeIt 2.5 software. Statistical significance was assessed using the random effects model and inverse-variance method. Any missing standard deviations for changes between baseline and follow-up among outcome measurements were estimated depending on the availability of either confidence intervals, standard errors and t and P values or by calculating a correlation coefficient from a known change from baseline standard deviation derived from a similar study.
Statistical heterogeneity of outcome measurements between different studies was assessed using the overlap of their confidence interval (95% CI) and expressed as measurements of Cochran's Q (chi-square test) and I 2 . The classification of data as moderately heterogeneous was based on I 2 from 50-74.9% and highly heterogeneous from 75% and above. 35 Furthermore, sensitivity analyses were performed to evaluate the robustness of reported statistical results by discounting the effect of lifestyle advice (i.e. energy restriction and/or physical activity) on outcome measurements and according to risk of bias of the included studies. Subgroup analyses based on mean participant age, treatment duration, type of probiotic genera, participant health status, method of body composition assessment and country of origin were also performed. Publication bias was assessed using Begg's funnel plots and Egger's linear regression test 36 using R software. The meta-analysis was synthesized using Review Manager (RevMan 5.4.1) software.

Results
The initial literature search provided 2324 publications. Following the exclusion of duplicates and non-relevant abstracts, 40 full texts were identified as eligible for inclusion in the systematic review and meta-analysis. Of these 40 studies, seven studies had an incompatible intervention, five studies had missing data, two studies had an inappropriate study design, and two studies had irrelevant outcomes of interest. In total, 24 studies were included in the systematic review and meta-analysis exploring the effects of probiotics on muscle mass, total lean mass and muscle strength ( Figure 1). The characteristics of included studies are outlined in Tables 1  and 2.

Risk of bias of included studies
Seven studies were classified as having some concerns according to RoB2 [38][39][40]43,44,48,59 as displayed in Figure S18. Specifically, 10 studies had some concerns due to not providing information regarding treatment allocation. [37][38][39][43][44][45]48,51,53,59 One study had some concerns given its 1:1 allocation ratio in regard to randomization, 49 whereas in another study, there was a statistically significant difference between groups. 56 Additionally, in three studies, the score was high as investigators were aware of the intervention 40,59 and because no information about allocation treatment, randomization, and table of characteristics was provided. 52 Thirteen studies had some concerns in which no information regarding staff and participant blinding was detailed, [37][38][39][40][41][43][44][45][46]52,53,57,59 whereas one study had also some concerns due its single-blind design. 48 Furthermore, three studies had some concerns due high participant dropout rates 38,55,58 and because in two studies no information was provided in relation to participant dropout rate. 39,40 Finally, one study had some concerns as there was no prespecified plan regarding its outcome measures. 50

Publication bias
No publication bias was reported following probiotic supplementation and muscle mass (z = À0.7442, P = 0.4567) and total lean mass (z = À0.1004, P = 0.9200). Due to the limited Probiotics on musculoskeletal health: a meta-analysis number of studies, publication bias was not performed in relation to global muscle strength (k < 10). Funnel plots illustrating publication bias regarding probiotics and muscle mass and total lean mass are shown in Figure S19A and S19B, respectively.

Discussion
In the present study, we systematically reviewed RCTs investigated the effect of probiotics on muscle mass, total lean mass and muscle strength in young and older adults. Our main analyses revealed that probiotic intervention augments both muscle mass that is also supported by subgroup analyses, revealing its effectiveness across individuals of younger age (<50 years), longer duration (≥12 weeks), strain of probiotic supplementation (Bifidobacterium) and geographical location (Asia). No such positive effect of probiotics was observed on changes in total lean mass. Interestingly, we found a significant improvement on global muscle strength following probiotic supplementation that was more prominent in older adults (≥50 years), following a longer treatment duration (≥12 weeks).

Findings from subgroup analyses
Our subgroup analysis based on age revealed a prominent effect of probiotic supplementation on muscle mass in individuals below 50 years of age compared with their older counterparts. Older individuals exhibit a reduced response of muscle protein synthesis (MPS) to anabolic stimuli (i.e. exercise and amino acids) as opposed to younger adults, a phenomenon termed anabolic resistance. 61,62 This phenomenon is considered a primary factor in the development of sarcopenia. Indeed, it has been reported that older individuals (mean age 71 years) may require almost twice as much protein intake per meal to stimulate MPS to the same degree as younger counterparts (mean age 22 years). 63 Because habitual dietary protein intakes in older adults are often reported to be inadequate 64 and protein digestion kinetics (i.e. delayed absorption) also are impaired with advanced age 65 and given the increasing popularity of plant-based proteins for environmental and ethical reasons, 66 the use of probiotics to maximize the quantity of circulating amino acids in order to stimulate MPS and promote skeletal muscle mass is an area that warrants further investigation. Further explanation of our findings may pertain to the chronic elevation in levels of inflammatory biomarkers such as IL-6, C-reactive protein (CRP) and TNF-α, observed in older populations. 67 Systemic inflammation has been shown to downregulate net muscle protein balance by the suppression of anabolic pathways and upregulation of catabolic pathways [i.e. increased expression of Muscle RINGfinger protein-1 (MuRF-1) and atrogin-1], leading to reductions in muscle mass and quality. 68 When the diet of aged mice was supplemented with either Lactobacillus casei or Bifidobacterium longum, probiotics seemed to regulate levels of pro-inflammatory cytokines such as IL-6, TNF-α and monocyte chemoattractant protein-1 (MCP-1) and led to enhanced muscle strength and function. 69 Although the anabolic-induced responses that could potentially be exerted from probiotic supplementation are mediated, in part, via anti-catabolic responses suppressing systemic inflammation, it is currently unknown whether this is sufficient to suppress muscle loss in older populations. Our analysis suggests a potential role for the use of probiotic strains to enhance protein digestion and amino acid absorption that may reduce the dose of protein required to stimulate MPS; however, considering anabolic resistance during ageing, their impact on muscle mass is questionable in older populations. Further research is warranted to identify the impact of probiotic supplementation with increased protein intake in older populations and unravel the clinical significance of its anti-inflammatory properties.
Moreover, we found that longer duration above 12 weeks was superior in inducing muscle mass gains as opposed to short-term supplementation (<12 weeks), changes that may be explained by the gut microbiota. Several animal 29,70,71 and human studies 19,20 have highlighted the importance of the gut microbiome in regulating muscle mass and strength, as well as multiple mechanisms by which it may exert anabolic effects. For example, germ-free (GF) mice exhibit markedly lower levels of muscle mass/strength/locomotive capability and fatigue resistance when compared with pathogen-free (PF) mice and abundant gut microbiota. 70 More importantly, transplantation of the gut microbiota from the PF to the GF mice resulted in increases in skeletal muscle mass and improvements in muscle function of the GF mice. Furthermore, supplementation with probiotics such as Bacteroides fragilis 72 or co-housing with PF mice led to improved muscle mass and function in GF mice, further highlighting the importance of the microbiome in the regulation of muscle health. It is worth noting that, in two studies that supplemented probiotic strains in parallel with resistance exercise, the authors did not report significant changes in muscle mass. 39,59 However, both studies lasted in total for approximately 1 month, which may not be sufficient to exert significant muscle growth. 73 Future studies incorporating a long-term resistance exercise with probiotic supplementation would shed light on whether bacterial strains enhance the hypertrophic-induced responses derived by resistance exercise.
Our subgroup analysis focusing on distinct microbial species depicted a prominent role of Bifidobacterium in mediating muscle mass as opposed to supplementation with Lactobacillus strains. Both studies incorporated Bifidobacterium breve B-3 compared with placebo for 12 weeks in overweight individuals above 50 years of age. 42,43 Interestingly, we did not find a beneficial effect on muscle mass after categoriza-tion based on older age, and overweight or obese health status, implying that the positive outcomes exhibited by this bacterial strain may be attributed to study duration and any potential skeletal muscle-specific properties. However, given the small number of studies following Bifidobacterium supplementation (k = 2) along with the inability to perform subgroup analyses based on different bacterial taxa, we could not establish a causative relationship between probiotic supplementation and increased muscle mass due to B. breve B-3.
Subgroup analysis based on geographical location suggested that a link between muscle mass and probiotic supplementation may be more pronounced in people living in Asia.
Recently, a large-scale survey found a positive association of appendicular lean mass to body weight ratio and Blautia, Bifidobacterium and Eisenbergiella in Japanese adults aged 50.8 (12.8) years. 74 In our meta-analysis, both studies in which Bifidobacterium supplementation established a beneficial effect on muscle mass were conducted in Japan in adults of similar ages, 42,43 suggesting a population-specific association between muscle mass and bacterial strains. No positive findings were found in European countries following probiotic supplementation; however, these results should be treated with caution due to the low number of studies (k = 2) and the moderate heterogeneity among studies conducted in Asia (56%).
Finally, we observed a beneficial effect of probiotic supplementation on global muscle strength in individuals 50 years of age and above and following longer-term protocols (≥12 weeks). Although it is currently unknown why these differences were observed in older versus younger populations, Ni et al. found that L. casei LC122 and B. longum BL986 supplementation for 12 weeks in aged mice promoted significant increase in forelimb grip strength, suggesting a link between muscle strength and the gut microbiota. 69 Similarly, aged SAMP8 mice supplemented with Lactobacillus paracasei PS23 for 12 weeks displayed attenuated age-related decreases in grip force, exhibiting higher mitochondrial function compared with controls. 26 Interestingly, these findings are inconsistent with the aforementioned results of probiotics improving muscle mass in younger versus older populations. Whether this is based on the low number of studies related to global muscle strength or the differences among surrogate measures of muscle strength requires further investigation.

Probiotic mechanisms enhancing muscle function
A number of mechanisms have been proposed to underpin the beneficial role of the microbiome/probiotics in the regulation of muscle mass and function. From a nutritional perspective, the acute post-prandial elevation of leucine and the presence of other essential amino acids are key de-terminants of intramyocellular anabolic signalling to trigger MPS, an effect that is potentiated following acute resistance exercise. 75,76 A possible mechanism by which probiotics may stimulate increases in muscle mass or strength is by improved digestion protein and absorption of constituent amino acids into the bloodstream. Indeed, in vitro studies have revealed that the probiotic strain Bacillus coagulans GBI-30, 6086 (BC30) increases protein digestion and amino acid uptake in a model of the upper gastrointestinal tract. 19 To translate such effects to human models, Stecker et al. administered young men and women a milk protein concentrate, with or without BC30 (1 × 109 CFU) for 2 weeks in a crossover design. 19 On the final day of supplementation, blood concentrations of amino acids were measured. Findings revealed that the addition of BC30 resulted in a greater postprandial area under the curve (AUC) for arginine and isoleucine, higher peak concentrations of a number of other amino acids (arginine, serine, ornithine, methionine, glutamic acid, phenylalanine, isoleucine, tyrosine, EAAs and total amino acids) and a more rapid rise to peak concentrations for some amino acids (glutamine, citrulline, threonine and alanine). Interestingly, Jäger et al. observed equivocal effects using pea protein isolate with or without two strains of L. paracasei (5 billion CFU each). 20 The addition of the probiotics led to significantly increased maximum blood concentrations and AUC of methionine, histidine, valine, leucine, isoleucine, tyrosine, total branched chain amino acids (BCAA) and total EAAs. Furthermore, the production of short-chain fatty acids (SCFAs) is a known function of numerous bacterial species within the microbiome, and these compounds have a number of physiological effects on the host. 77 Mouse models revealed that treatment of GF mice (with reduced muscle mass and function compared with PF controls) with gut microbiota transplants or with SCFAs alone was enough to partially reverse the skeletal muscle impairments observed in the GF animals. 70 Human observational studies also demonstrate that older adult men with greater levels of lean mass and physical function exhibit higher levels of butyrateproducing bacteria as well as higher gene counts for butyrate-producing genes compared with men with lower lean mass. 71,78 Higher fibre intakes also were associated with a greater butyrate production, which is likely related to the role of dietary fibre as a prebiotic food source for probiotic bacteria. 79 In addition, creatine degradation has also been observed to be greater in the microbiomes of older compared with younger mice. 80 Creatine is a non-protein amino acid found in muscle tissue, among other body compartments where it plays a key role in energy metabolism and muscle contraction. 81 Muscle creatine levels have been observed to be lower in older populations, 82 and this decrease may contribute to the multifactorial development of sarcopenia. Taken together, these data support the notion that the gut microbiota/probiotics may play a clinically relevant and mechanistically feasible role in muscle health.

Strengths and limitations
A strength of this study is the inclusion of meta-analyses of both total lean mass and muscle mass. We propose that increasing skeletal muscle mass, rather than total lean mass, is a more impactful goal when attempting to improve muscle strength and function or reduce the prevalence of sarcopenia in older adults. Indeed, the European Working Group on Sarcopenia in Older People (EWGSOP) currently uses appendicular skeletal muscle mass as part of the diagnostic criteria for sarcopenia. 1 Due to the importance of muscle function in addition to muscle mass in ageing populations, it should also be noted that muscle mass (appendicular lean mass index) has been shown to correlate well with various measures of strength such as leg press, knee extension and knee flexion 1RM. 83 In contrast, although the umbrella term 'total lean mass' includes muscle mass, it also includes a great deal of non-muscle tissue including bone and organs, which may not decline to the extent of muscle mass that is observed in sarcopenia.
Furthermore, although a significant improvement in muscle mass was observed following probiotic supplementation, our results did not detect a notable change in total lean mass by DXA as opposed to BIA, which is thought to be a less accurate and precise tool for the measurement of body composition. This circumstance may raise some questions as to the true clinical relevance of the improvements found in muscle mass after probiotic supplementation, as DXA is considered to be a reliable clinical tool for diagnosing and monitoring the age-related loss of muscle mass. Finally, the duration of interventions was not superior to 12 weeks in all the considered studies, and as such, the long-term effects of probiotic supplementation on muscle mass and strength remain unknown. To be clinically useful, any potential treatment against sarcopenia should prove effective in the long term, as sarcopenia represents a chronic phenomenon affecting health outcomes and quality of life of older subjects beyond the horizon of just few weeks of treatment.

Conclusions
In conclusion, this is the first systematic review and meta-analysis to demonstrate that probiotic interventions augment global muscle strength and muscle mass, although no such positive effects were observed for total lean mass. Pertaining to muscle mass, the results of our meta-analysis highlight the positive effect of probiotic supplementation across populations below the mean age of 50, utilizing a longer-term (≥12 weeks) treatment duration, and specifically consuming Bifidobacterium, in Asian countries. This field warrants further research to elucidate the mechanisms of action in which probiotics may induce anabolic responses and optimize strategies related to the use of appropriate probiotic strains for the augmentation of both muscle mass and strength in a wide range of population groups.