Probiotic Lactiplantibacillus plantarum Tana Isolated from an International Weightlifter Enhances Exercise Performance and Promotes Antifatigue Effects in Mice

Exercise causes changes in the gut microbiota, and in turn, the composition of the gut microbiota affects exercise performance. In addition, the supplementation of probiotics is one of the most direct ways to change the gut microbiota. In recent years, the development and application of human-origin probiotics has gradually attracted attention. Therefore, we obtained intestinal Lactiplantibacillus plantarum “Tana” from a gold-medal-winning weightlifter, who has taken part in various international competitions such as the World Championships and the Olympic Games, to investigate the benefits of Tana supplementation for improving exercise performance and promoting antifatigue effects in mice. A total of 40 male Institute of Cancer Research (ICR) mice were divided into four groups (10 mice/group): (1) vehicle (0 CFU/mice/day), (2) Tana-1× (6.15 × 107 CFU/mice/day), (3) Tana-2× (1.23 × 108 CFU /mice/day), and (4) Tana-5× (3.09 × 108 CFU/mice/day). After four weeks of Tana supplementation, we found that the grip strength, endurance exercise performance, and glycogen storage in the liver and muscle were significantly improved compared to those in the vehicle group (p < 0.05). In addition, supplementation with Tana had significant effects on fatigue-related biochemical markers; lactate, ammonia, and blood urea nitrogen (BUN) levels and creatine kinase (CK) activity were significantly lowered (p < 0.05). We also found that the improved exercise performance and antifatigue benefits were significantly dose-dependent on increasing doses of Tana supplementation (p < 0.05), which increased the abundance and ratio of beneficial bacteria in the gut. Taken together, Tana supplementation for four weeks was effective in improving the gut microbiota, thereby enhancing exercise performance, and had antifatigue effects. Furthermore, supplementation did not cause any physiological or histopathological damage.


Introduction
Human gut microbiota contains trillions of microbial cells from thousands of different species, as well as a variety of archaea, eukaryotic microorganisms, and viruses, with over three million genes and enormous metabolic capacity [1]. The normal human gut microbiota includes two major phyla, Bacteroidetes and Firmicutes, as well as other smaller phyla [2]. These microorganisms inhabiting the human gut are collectively known as the gut microbiota; they form a complex community, and their coexistence, synergy, and interaction with the host are considered critical to overall health and prevention of disease [3]. Many intrinsic and extrinsic environmental factors influence the composition of the gut microbiota, resulting in a highly dynamic and individualized complex gut ecosystem [4] Forty male Institute of Cancer Research (ICR) mice (6 weeks old) from BioLASCO Taiwan (Yi-Lan Breeding Center, Yi-Lan County, Taiwan) were used. This experiment was approved by the Institutional Animal Care and Use Committee (IACUC) of National Taiwan Sport University (IACUC-10914). All mice were given a standard laboratory diet (No. 5001; PMI Nutrition International, Brentwood, MO, USA) and distilled water ad libitum, and maintained in a 12-h light/12-h dark cycle, at room temperature (22 ± 2 • C) and 60-70% humidity. After being allowed food ad libitum for 2 weeks prior to experiments, the 40 mice were randomly assigned to four groups (10 mice/group) for oral gavage treatment for 4 weeks: (1) vehicle (0 CFUs/mouse/day), (2) Tana-1× (6.15 × 10 7 CFUs/mouse/day), (3) Tana-2× (1.23 × 10 8 CFUs/mouse/day), and (4) Tana-5× (3.09 × 10 8 CFUs/mouse/day), and we recorded the body weight, water consumption, and food intake each week. The experimental flow chart is shown in Figure 1. tum, and maintained in a 12-h light/12-h dark cycle, at room temperature (22 ± 2 °C) and 60-70% humidity. After being allowed food ad libitum for 2 weeks prior to experiments, the 40 mice were randomly assigned to four groups (10 mice/group) for oral gavage treatment for 4 weeks: (1) vehicle (0 CFUs/mouse/day), (2) Tana-1× (6.15 × 10 7 CFUs/mouse/day), (3) Tana-2× (1.23 × 10 8 CFUs/mouse/day), and (4) Tana-5× (3.09 × 10 8 CFUs/mouse/day), and we recorded the body weight, water consumption, and food intake each week. The experimental flow chart is shown in Figure 1.

Forelimb Grip Strength
We used a low-force testing system (Model-RX-5, Aikoh Engineering, Nagoya, Japan) to measure the grip strength of mice undergoing vehicle or Tana supplementation. We gently held the mouse's tail to allow it to swing naturally while the two front limbs of the mouse held a tension rod (diameter 2 mm, length 7.5 cm), then we pulled gently in the opposite direction, repeating 10 times. We recorded the maximum value through the force sensor [35].

Swimming Exercise Performance Test
As previously described [36], after 4 weeks of Tana intervention on day 29 for the swimming exhaustion test, weight loading 5% of each mouse's body weight was attached to its tail, then mice were forced to swim in 27 ± 1 °C water until they lost coordinated movement or could not return to the surface within 7 s. We recorded the time from the beginning of the test until mouse exhaustion as the swimming endurance time.

Determination of Fatigue-Associated Serum Biomarkers
We evaluated the effect of Tana supplementation on biochemical markers and physiological states associated with post-exercise fatigue by swimming without weight loading; all mice were fasted for 12 h before each blood draw to reflect true physiological adaptation to exercise intervention. Day 31 after Tana intervention, serum lactate, ammonia (NH3), and glucose levels were ascertained before swimming and after 10 min of swimming and 20 min of rest by submandibular blood collection. On day 34 after Tana intervention, we also collected blood for analysis of blood urinary nitrogen (BUN) and creatine

Forelimb Grip Strength
We used a low-force testing system (Model-RX-5, Aikoh Engineering, Nagoya, Japan) to measure the grip strength of mice undergoing vehicle or Tana supplementation. We gently held the mouse's tail to allow it to swing naturally while the two front limbs of the mouse held a tension rod (diameter 2 mm, length 7.5 cm), then we pulled gently in the opposite direction, repeating 10 times. We recorded the maximum value through the force sensor [35].

Swimming Exercise Performance Test
As previously described [36], after 4 weeks of Tana intervention on day 29 for the swimming exhaustion test, weight loading 5% of each mouse's body weight was attached to its tail, then mice were forced to swim in 27 ± 1 • C water until they lost coordinated movement or could not return to the surface within 7 s. We recorded the time from the beginning of the test until mouse exhaustion as the swimming endurance time.

Determination of Fatigue-Associated Serum Biomarkers
We evaluated the effect of Tana supplementation on biochemical markers and physiological states associated with post-exercise fatigue by swimming without weight loading; all mice were fasted for 12 h before each blood draw to reflect true physiological adaptation to exercise intervention. Day 31 after Tana intervention, serum lactate, ammonia (NH 3 ), and glucose levels were ascertained before swimming and after 10 min of swimming and 20 min of rest by submandibular blood collection. On day 34 after Tana intervention, we also collected blood for analysis of blood urinary nitrogen (BUN) and creatine kinase (CK) after 90 min of swimming and 60 min of rest. Serum was collected from all blood samples by centrifugation at 1500× g for 15 min at 4 • C and was measured using an automatic analyzer (Hitachi, Tokyo, Japan, Hitachi 7060) [37].

Visceral Tissue Weight, Histology Staining, and Glycogen Determination
After the mice were euthanized, we carefully removed, excised, and weighed the liver, kidneys, heart, lungs, muscles, epididymal fat pad (EFP), and brown adipose tissue (BAT). We stored parts of the muscle and liver tissues in liquid nitrogen for glycogen content analysis, as previously described [38].

Bacterial DNA Extraction and 16S rRNA Sequencing
After the mice were euthanized, the collected cecum content samples were immediately stored at −80 • C for DNA extraction. The sample extraction, preparation, and analysis proceeded according to the methods previously used in our laboratory [39].

Statistical Analysis
All data are expressed as mean ± SD for n = 10 mice per group, and the statistical analysis software used was SAS 9.0 (SAS Inst., Cary, NC, USA); we used one-way analysis of variance (ANOVA) to measure statistical differences among groups. The Cochran-Armitage test was used for the dose-effect trend analysis. p < 0.05 was considered statistically significant.
On day 37 after 4 weeks of Tana intervention, thirty minutes after the last supplementation, all mice were euthanized by 95% CO2, and cardiac puncture blood collection was carried out immediately. After centrifugation to collect serum, the clinical biochemical variables, including aspartate aminotransferase (AST), alanine transaminase (ALT), albumin, triglycerides (TGs), blood urea nitrogen (BUN), creatinine, uric acid (UA), total protein (TP), CK, and glucose were measured using an autoanalyzer (model 717, Hitachi, Tokyo, Japan).

Visceral Tissue Weight, Histology Staining, and Glycogen Determination
After the mice were euthanized, we carefully removed, excised, and weighed the liver, kidneys, heart, lungs, muscles, epididymal fat pad (EFP), and brown adipose tissue (BAT). We stored parts of the muscle and liver tissues in liquid nitrogen for glycogen content analysis, as previously described [38].

Bacterial DNA Extraction and 16S rRNA Sequencing
After the mice were euthanized, the collected cecum content samples were immediately stored at −80 °C for DNA extraction. The sample extraction, preparation, and analysis proceeded according to the methods previously used in our laboratory [39].

Statistical Analysis
All data are expressed as mean ± SD for n = 10 mice per group, and the statistical analysis software used was SAS 9.0 (SAS Inst., Cary, NC, USA); we used one-way analysis of variance (ANOVA) to measure statistical differences among groups. The Cochran-Armitage test was used for the dose-effect trend analysis. p < 0.05 was considered statistically significant.

Effect of Tana Supplementation on Serum Lactate Levels after the 10 min Swim Test
All the mice underwent a 10 min swimming and 20 min rest test to evaluate the levels of lactate after four weeks of supplementation with Tana (Table 1). There was no significant difference in lactate levels between groups before swimming. After 10 min of swimming, the serum lactate levels of mice in the vehicle, Tana-1×, Tana-2×, and Tana-5× groups were 7.45 ± 0.65, 6.79 ± 0.55, 6.41 ± 0.51, and 5.92 ± 0.59 mmol/L, respectively. The Tana-1×, Tana-2×, and Tana-5× group lactate levels were significantly decreased by 8.86% (p = 0.0152), 13.94% (p = 0.0003), and 20.56% (p < 0.0001), respectively. The lactate production rates were determined based on the serum lactate concentration before and after 10 min of swimming; in the vehicle, Tana-1×, Tana-2×, and Tana-5× groups the lactate production rates were 1.74 ± 0.04, 1.55 ± 0.07, 1.48 ± 0.16, and 1.35 ± 0.09, respectively. The Tana-2×, and Tana-5× group rates were significantly lower than those of the vehicle group by 14.97% (p = 0.0322) and 22.17% (p = 0.0019), respectively. Lactate production rate (B/A) was the value of the lactate level after exercise (B) divided by that before exercise (A). Clearance rate (B − C)/B was defined as lactate level after swimming (B) minus that after 20 min rest (C) divided by that after swimming (B). Data are expressed as mean ± SD (n = 10 mice per group). Values in the same row with different superscript letters (a, b, c) differ significantly, p < 0.05. * Indicated significant dose dependence.

General Characteristics of Mice with Tana Supplementation for Four Weeks
After four consecutive weeks of supplementation with Tana, for the weight, food intake, and water intake of each group, there was no significant difference between the groups, and the mice showed stable growth (Table 2). However, supplementation with Tana-5× significantly improved skeletal muscle mass and reduced EFP weight compared with the vehicle group by 1.10-fold (p = 0.0326) and 17.29% (p = 0.0477), respectively. Because tissue weight is affected by body weight differences, we divided the tissue weight by the relative percentage of body weight and found that, compared with the vehicle group, supplementation with Tana-5× significantly improved relative muscle mass by 1.10-fold (p = 0.0015) and reduced relative EFP weight by 17.74% (p = 0.0168). The effects of Tana supplementation on increased muscle weight and reduced EFP weight were dosedependent (p < 0.05).

General Characteristics of Mice with Tana Supplementation for Four Weeks
After four consecutive weeks of supplementation with Tana, for the weight, food intake, and water intake of each group, there was no significant difference between the groups, and the mice showed stable growth (Table 2). However, supplementation with Tana-5× significantly improved skeletal muscle mass and reduced EFP weight compared with the vehicle group by 1.10-fold (p = 0.0326) and 17.29% (p = 0.0477), respectively. Because tissue weight is affected by body weight differences, we divided the tissue weight by the relative percentage of body weight and found that, compared with the vehicle group, supplementation with Tana-5× significantly improved relative muscle mass by 1.10-fold (p = 0.0015) and reduced relative EFP weight by 17.74% (p = 0.0168). The effects of Tana supplementation on increased muscle weight and reduced EFP weight were dosedependent (p < 0.05).

Effect of Tana Supplementation on Histopathology of Tissues and Biochemical Profiles at the End of the Study
We assessed whether four weeks of Tana supplementation had effects on health and safety and tested the biochemical parameters (Table 3). We found that all biochemical parameters were within normal ranges, and there were no significant differences between each group. In addition, we performed histological evaluation of the liver, muscle, heart, kidney, lung, EFP, and BAT of mice, and no abnormalities or pathological changes were found in any group ( Figure 5). Therefore, we believe that supplementation with Tana does not cause any harm. 3.5 ± 0.1 a 3.5 ± 0.1 a 3.5 ± 0.1 a 3.5 ± 0.2 a 0.5982 TP (g/dL) 6.0 ± 0.2 a 6.1 ± 0.1 a 6.0 ± 0.2 a 6.1 ± 0.3 a 0.1561 Data are expressed as mean ± SD (n = 10 mice per group). AST, aspartate aminotransferase; ALT, alanine transaminase; ALB, albumin; BUN, blood urea nitrogen; CREA, creatinine; UA, uric acid; TP, total protein; TG, triacylglycerol; CK, creatine kinase. The same superscript letters (a) above each bar indicate with no difference at p > 0.05.

Effect of Tana Supplementation on Gut Microbiota
At the end of the experiment, we analyzed the gut microbiota composition of mice treated with vehicle, Tana-1×, Tana-2×, and Tana-5× through 16S rRNA and observed significant changes in microbial ecology after Tana treatment. As shown in Figure 6A, principal coordinate analysis of beta diversity using an unweighted UniFrac model demonstrated that mice clustered into relatively distinct groups according to different treatments, thus indicating that Tana significantly altered gut microbial populations. At the phylum level, Firmicutes accounted for the major proportion, with the vehicle, Tana-1×, Tana-2×, and Tana-5× groups accounting for 66%, 69%, 60%, and 58%, respectively, and Bacteroidetes accounting for 31%, 27%, 36%, and 38%, respectively. The Firmicutes/Bacteroidetes (F/B) ratios in the Tana-2× (1.78) and Tana-5× (1.82) groups, respectively, were lower than those in the vehicle (2.20) and Tana-1× (2.58) groups ( Figure 6B). By observing the abundance of different bacterial genera between groups by heat map we found that, especially in the Tana-5× group, microorganisms beneficial to the gut microbiota such as Prevotellaceae, Parasutterella, Ruminococcus, and Blautia were more abundant ( Figure 6C). We also confirmed by linear discriminant analysis effect size (LEfSe) that the number of Prevotellaceae in the Tana−5× group was higher than that in the vehicle group ( Figure 6D).

Effect of Tana Supplementation on Gut Microbiota
At the end of the experiment, we analyzed the gut microbiota composition of mice treated with vehicle, Tana-1×, Tana-2×, and Tana-5× through 16S rRNA and observed tively, were lower than those in the vehicle (2.20) and Tana-1× (2.58) groups ( Figure 6B). By observing the abundance of different bacterial genera between groups by heat map we found that, especially in the Tana-5× group, microorganisms beneficial to the gut microbiota such as Prevotellaceae, Parasutterella, Ruminococcus, and Blautia were more abundant ( Figure 6C). We also confirmed by linear discriminant analysis effect size (LEfSe) that the number of Prevotellaceae in the Tana−5× group was higher than that in the vehicle group ( Figure 6D).

Discussion
Exercise and training alter gut microbial abundance and expression. Supplementation with probiotics is one way to increase the number of good bacteria in the gut [1]. In recent years, athletes have increasingly chosen to use probiotics to regulate immunity, maintain healthy gastrointestinal function, and even increase energy utilization to improve exercise performance [40]. In the current study, we obtained L. plantarum Tana from one of the world's top weightlifters and demonstrated in animal experiments that it can enhance exercise performance, improve body composition, and reduce exercise fatigue. We also observed changes in the composition of the gut microbiota.
Probiotic supplements have been shown to alter the composition and metabolic activity of the gut microbiota, thereby promoting the growth of species that increase microbial diversity and health and assist in the conversion of indigestible carbohydrates into SCFAs [41]. Previous studies have indicated that probiotic supplementation with Lactiplantibacillus strains derived from the human gut could prevent diet-induced metabolic syndrome by modulating the gut microbiota and elevated acetate. This can improve energy harvesting during exercise, providing athletes with metabolic benefits during high-intensity exercise and recovery [42]. Butyrate is primarily used by epithelial cells in the colon as a source of energy, through conversion to acetyl-CoA, and it is used in the Krebs cycle to generate ATP [43]. In addition, butyrate can maintain blood glucose homeostasis and promote glycogen metabolism through the GPR43-AKT-GSK3 signaling pathway [44]. Propionate can be used as a precursor for glucose synthesis in the liver [45]. Previous studies indicated that administration of Lactobacillus gasseri SBT2055 to normal rats for four consecutive weeks reduced the area under the curve (AUC) for blood glucose concentration and increased the molar ratio of butyrate to total SCFA in the cecum [46]. In addition, L. gasseri SBT2055 was shown to ameliorate diabetes in rats by increasing glycogen storage in the liver and quadriceps and reducing serum-free fatty acid levels [47]. This appears to validate our results showing that 4 weeks of Tana supplementation significantly increased liver and muscle glycogen storage in mice ( Figure 4A,B). Glycogen is mainly stored in the liver and muscles and is considered to be the main fuel source for long-term moderate-to-high-intensity endurance exercise [48]. When glycogen stores decrease to a certain level with increasing exercise intensity or duration, the exercise capacity of skeletal muscles is impaired, making it difficult to meet the energy demands of training and competition, resulting in increased fatigue, as well as decreased exercise capacity and endurance performance [49]. Furthermore, the modulation of SCFA production by the gut microbiota also directly affects energy metabolism during exercise and improves endurance performance through longterm maintenance of blood glucose [10]. Therefore, in this study, Tana supplementation had the effect of improving exercise endurance ( Figure 2B). Similar to past studies, in a double-blind, crossover human trial, trained male runners supplemented with a multistrain combination probiotic for 4 weeks experienced significantly longer fatigue time running in the heat compared to placebo; an increase of 16% [50]. In our past research, in addition to finding that supplementation with L. plantarum TWK10 can effectively improve the glycogen storage and endurance performance of mice, it had the effect of improving muscle strength and muscle mass. This may be related to the crosstalk pathway of the muscle-gut axis, which is affected by the composition and interaction of the microbiota and thus affects the mass, function, and energy metabolism of the muscle [51]. Muscle strength increases with muscle mass and is positively correlated [52]. Therefore, although Tana-5× supplementation alone was found to significantly improve muscle mass in this study (Table 2), 4 weeks of Tana supplementation without exercise training significantly improved grip performance (Figure 2A).
We also found that 4 weeks of Tana supplementation altered the gut microbiome, increasing the abundance and proportion of Prevotellaceae in the mouse gut, especially in the Tana-5× group ( Figure 6A). A past study investigating the guts of long-term exercise cyclists found not only an increased abundance of Akkermansia, a bacterium commonly found in the guts of athletes, but also observed that the abundance of Prevotella was associated with exercise training [53]. Prevotella is thought to promote better glucose tolerance and glycogen storage in mice [54] and might be the key flora for improving glycogen storage and endurance performance in this study. In addition, the increased abundance of Prevotella was positively associated with many amino acid and carbohydrate metabolic pathways, including the metabolic branched-chain amino acids (BCAAs) leucine, isoleucine, and valine [53]. High levels of BCAAs can reduce exercise-induced central fatigue and muscle fatigue, and promote muscle protein synthesis [55], thereby reducing muscle damage during prolonged exercise [35]. This appears to explain the benefit of reduced post-exercise CK activity in the Tana group following exercise ( Figure 3D). Additionally, lactate, NH 3 , and BUN are thought to reduce exercise performance and contribute to fatigue byproducts as exercise duration or intensity increases [56]. Among them, lactate is the product of carbohydrate glycolysis under anaerobic conditions, and during vigorous exercise, glucose is broken down into pyruvate. Under anaerobic conditions, hydrogen ions are reduced and released, resulting in lower blood and muscle tissue pH and inhibition of glycolysis, which interferes with normal cellular function [57]. In addition, when amino acid metabolism produces ATP for energy, ammonia is produced and accumulated in skeletal muscle, which may affect central fatigue [58]. At this point, ammonia is converted to BUN by the urea cycle, which is either increased in the blood or excreted in the urine [59]. However, a past study that obtained Veillonella from the guts of marathon runners and administered it to mice found that when systemic lactate produced during exercise entered the gut lumen, Veillonella, via the methylmalonyl-CoA pathway, converts the lactate to SCFAs, especially propionate, to improve exercise performance [60]. Additionally, past research has shown that probiotics can increase the hepatic clearance of ammonia and other toxins by reducing inflammation and oxidative stress in liver cells [61]. These seem to be plausible explanations, and in this study, Tana supplementation was effective in reducing post-exercise increases in blood lactate, NH 3 , and BUN ( Figure 3A-C).
In the current study, in addition to exploring the benefits of Tana supplementation on exercise performance and antifatigue effects, we further confirmed through histopathological interpretation ( Figure 5) and blood analysis ( Table 3) that Tana supplementation did not cause any adverse effects. In addition to increasing muscle mass in terms of tissue weight, Tana supplementation also exhibited a body-fat reduction benefit, especially in the Tana-5× group ( Table 2). This may be related to the F/B ratio [62]. Past research has shown that exercise training in mice increases Bacteroidetes while reducing Firmicutes, suggesting that exercise plays an important role in preventing diet-induced obesity, similar to the microbiome of lean mice [63]. This is similar to the results of this study, where higher doses of Tana supplementation alone appeared to reduce the F/B ratio in the absence of exercise training ( Figure 6B). However, there is disagreement about whether the F/B ratio should be high or low, although the balance should be optimal. Previous studies have suggested that the F/B ratio is related to exercise performance, but the supplemented L. plantarum in our study resulted in higher Prevotella levels in the gut, which requires further study to explore the possible mechanism.
In sum, in this study, we have shown that four consecutive weeks of L. plantarum Tana supplementation can effectively improve exercise performance, glycogen storage, and muscle mass in mice, as well as reducing the production of post-exercise fatigue products. In addition, it did not cause any damage to the health of the mice. However, there are still relatively few studies using human probiotics for athletic performance, even from the guts of top athletes. In this study, in addition to the efficacy evaluation, it was also necessary to explore whether supplementation caused adverse effects, so animal experiments were first conducted. In the future, it will be necessary to further explore the mechanism of action to improve exercise performance, and to verify the evaluation of human intestinal colonization ability and benefit through human trials. It will also be necessary to explore whether supplementation has an additive benefit with exercise training, and the possibility of Tana being used as a sports nutrition supplement.

Conclusions
In the present study, we found that 4 weeks of Tana supplementation significantly increased glycogen storage, forelimb grip strength, and endurance performance in mice and significantly decreased levels of fatigue markers such as lactate, BUN, ammonia, and CK. This may be related to Tana altering the gut microbiota, thereby promoting host metabolic phenotypes. Therefore, Tana has proven useful as a supplement to improve exercise performance and reduce fatigue. Further research should be conducted in the future to determine the molecular mechanism of Tana involvement in antifatigue and to conduct human clinical trials.