3.1. Effects of LP10 on Forelimb Grip Strength
After supplementation for six weeks, the forelimb grip strengths in the vehicle, LP10-1X, and LP10-5X groups were 120 ± 5, 158 ± 3, and 168 ± 3 g, respectively (
Figure 1). Forelimb grip strengths were 1.31 and 1.40 fold higher in the LP10-1X and LP10-5X groups than in the vehicle treatment group (both
p < 0.0001). In the trend analysis, absolute forelimb grip strength dose-dependently increased with increasing LP10 dose (
p < 0.0001). In general, programmed exercise training is required to increase grip strength [
27]; however, we found that LP10 supplementation benefited grip strength even though test animals did not undergo a training intervention. Thus, long-term LP10 supplementation could benefit the muscle explosive force when no training protocol is implemented, but few studies have investigated probiotics supplementation to improve muscle strength. In previous research, supplementation of
Lactobacillus spp. reduced the expression of atrophy markers [
30] and the levels of systemic inflammatory cytokines, and restoration of these
Lactobasilli levels reduced the markers of the autophagy-lysosomal pathway, a major system of protein breakdown in gastrocnemius and tibialis muscle [
31]. Thus, supplementation of LP10 in our present data increased muscle mass to enhance forelimb grip strength. On the other hand, supplementation of prebiotics or probiotics increased gut SCFA content. SCFAs produced by the microbiota in the cecum and the colon can be found in hepatic, portal, and peripheral blood [
32,
33]. These SCFAs affect lipid, glucose, and cholesterol metabolism in various tissues and the maintenance of gut integrity [
34]. These might be possible mechanisms underlying the increase in muscle mass and strength.
3.2. Effect of LP10 on Exercise Performance in a Weight-Loaded Swimming Test
Energy metabolism during muscular activity determines the level of physiological fatigue [
35]. An important index in evaluating anti-fatigue treatment is exercise endurance. Endurance swimming times were 4.8 ± 0.9, 9.0 ± 0.6, and 23.2 ± 1.4 min with vehicle, LP10-1X, and LP10-5X treatment, respectively (
Figure 2). The exhaustive swimming time was longer, by 1.85 (
p = 0.0183) and 4.81 folds (
p < 0.0001), with LP-1X and LP-5X, respectively, than with vehicle treatment. In the trend analysis, endurance swimming time dose-dependently increased with increasing LP10 dose (
p < 0.0001). Probiotics have a wide range of benefits to promote endurance exercise performance [
36]. LP10 may improve endurance performance in the absence of training. Further investigation is required to elucidate the effects of LP10 supplementation combined with diverse gut microbiota and exercise training on endurance performance. In a recent study, certain probiotics (e.g.,
Bacillus coagulans) were shown to increase nutrient absorption, specifically protein absorption, in the form of better leucine absorption from whey protein [
37]. Improved protein utilization could increase muscle mass and thereby enhance exercise performance.
3.3. Effect of LP10 Supplementation on Serum Lactate, Ammonia, Glucose, CK and BUN Levels after Acute Exercise Challenge
Exercise-induced muscle fatigue can be evaluated by biochemical indicators such as lactate, ammonia, glucose, CK, and BUN levels [
38,
39]. Lactate accumulates in the blood and in the muscles engaged in the exercise and exceeds the aerobic metabolic capacity. When the lactic acid concentration increases, hydrogen ions accumulate, which leads to fatigue due to acidification [
40,
41]. Lactate levels in the vehicle, LP10-1X, and LP-5X groups were 6.4 ± 0.3, 4.6 ± 0.2, and 4.2 ± 0.7 mmol/L, with lower lactate levels with LP10-1X and LP10-5X supplementation (27.88%,
p = 0.0004 and 34.11%,
p = 0.0005, respectively) than with vehicle treatment (
Figure 3A). In the trend analysis, serum lactate level was dose-dependently decreased with increasing LP10 dose (
p < 0.0001). After acute exercise, relaxation is significantly affected by the blood lactate clearance rate. Approximately 75% of the total amount of lactate produced is used for oxidative production of energy in the exercising body, and it could be utilized for the
de novo synthesis of glucose in the liver [
42]. Clinical studies have found a positive correlation between probiotics supplementation and serum biochemical indicators [
43,
44]. Probiotics supplementation has rarely been used to investigate serum biochemical levels after acute exercise. LP10 supplementation may have potential for the removal and utilization of blood lactate after exercise.
Ammonia, another important metabolite produced during energy metabolism for exercise, is generated by different sources. Accumulation of ammonia in the blood and brain during exercise can negatively affect the central nervous system and cause fatigue. Although exercise-induced ammonia toxicity is transient and reversible depending on the disease state, it may affect continuing coordinated activity in critical regions of the central nervous system [
45]. The central nervous system plays a crucial role in the development of physical fatigue. In a recent study, gastrointestinal inflammation induced anxiety-like behavior and altered the biochemistry of the central nervous system [
46]. Thus, probiotics supplementation may play an important role in the central nervous system and fatigue. Serum ammonia levels were 162.1 ± 13, 102.5 ± 4.3, and 95 ± 5.5 μmol/L in the vehicle, LP10-1X, and LP10-5X groups, respectively (
Figure 3B), and levels were lower, by 36.78% (
p = 0.0004) and 41.40% (
p = 0.0001), with LP10-1X and LP10-5X, respectively, than with vehicle treatment. Thus, continuous supplementation with LP10 for six weeks could decrease ammonia levels during exercise. Trend analysis showed that the serum ammonia level dose-dependently decreased with increasing LP10 dose (
p < 0.0001).
The blood glucose level is an important index for performance maintenance during exercise [
47]. The serum glucose levels were 154.1 ± 4, 148.3 ± 6, and 139 ± 7 mg/dL in the vehicle, LP10-1X, and LP10-5X groups, respectively, with no difference among groups (
Figure 3C). Trend analysis showed that serum glucose levels dose-dependently decreased with increasing LP10 dose (
p = 0.0151). Therefore, continuous supplementation with LP10 for six weeks could increase energy utilization and improve exercise performance.
Previously, we investigated the association of intestinal bacteria and exercise performance in mice. Gut microbial status could be crucial for exercise performance. Endurance swimming time was correlated with abundant gut microbiota in mice; in fact, the greater the amount of gut microbiota, the longer the endurance swimming time and the lower the serum glucose [
48]. Therefore, gut microbiota can regulate different types of energy utilization in the host.
Serum CK level is an important clinical biomarker of muscle damage, muscular dystrophy, severe muscle breakdown, myocardial infarction, autoimmune myositides, and acute renal failure. High-intensity exercise challenge can physically or chemically cause tissue damage and muscular cell necrosis [
49]. The serum CK concentration is reduced in the normal state but increased in muscle tissue with hypoxia and the accumulation of metabolites during exercise caused by muscle cell damage, which results in decreased exercise performance [
50]. We found serum CK levels of 228.3 ± 38.4, 154.1 ± 18.8, and 147.8 ± 16.5 mg/dL in the vehicle, LP10-1X, and LP10-5X groups, respectively (
Figure 3D), with lower levels with LP-1X and LP-5X, by 32.48% (
p = 0.0165) and 35.27% (
p = 0.0142), respectively, than with vehicle treatment. Therefore, LP10 supplementation could ameliorate skeletal muscle injury induced by acute exercise challenge. Trend analysis revealed that LP10 treatment had a significant dose-dependent effect on CK level (
p = 0.0118).
BUN is an important biochemical parameter related to fatigue. The BUN level is used to measure the amount of nitrogen in blood from the waste product of urea. Urea serves an important role in the metabolism of nitrogen-containing compounds. Consequently, an increased BUN level reflects the decomposition of protein, which will adversely affect the contractive strength of muscle and lead to fatigue [
27,
28,
51]. Serum BUN level did not differ among treatment groups (
Figure 3E). Above all, LP10 may have potential as an ergogenic supplement by improving gut microbiota and regulating energy utilization.
3.4. General Characteristics of Mice with LP10 Supplementation for Six Weeks
Initial BW did not differ among the vehicle, LP10-1X, and LP10-5X groups (
Table 1). After six-week supplementation with LP10, the final BW was lower with LP10-1X and LP10-5X, by 7.47% (
p = 0.0003) and 3.46% (
p = 0.0567), respectively, than with vehicle treatment. In addition, daily intake of diet and water increased in LP10-5X fed mice. Trend analysis showed that daily intake of diet (
p < 0.0001) and water (
p < 0.0001) dose-dependently increased with LP10 supplementation, so daily diet intake was increased but BW was decreased. In addition, BW was significantly lower (
p < 0.05) with vehicle treatment at Week 3 of LP10 supplementation (
Figure 4). Thus, three-week LP10 supplementation may change the body composition and energy utilization. Probiotic supplementation could reflect the biochemistry of the conversion of carbohydrates into short-chain fatty acids (SCFAs) by the change in the bacteria composition of the gut microbial community [
52].
We measured the effect of LP10 on the muscle and epididymal fat pad (EFP) mass and relative tissue weight (different tissue weights adjusted for individual BW %). The EFP weight was lower by 34.62% (p = 0.003) and 50.30% (p < 0.0001) with LP10-1X and LP10-5X, respectively, than with vehicle treatment. Trend analysis showed that EFP weight dose-dependently decreased with LP10 supplementation (p < 0.0001). The relative weight of EFP (%) was lower by 28.93% (p = 0.0048) and 48.22% (p < 0.0001) with LP10-1X and LP10-5X, respectively, than with vehicle treatment. The relative weight (%) of muscle (gastrocnemius and soleus muscles) was greater by 1.10 (p = 0.0003) and 1.07 folds (p = 0.0098) with LP10-1X and LP10-5X, respectively, than with vehicle treatment. Trend analysis also showed a significant dose-dependent decrease and increase in relative EFP weight (%) and relative muscle weight (%), respectively, with LP10 supplementation. Thus, supplementation of LP10 for six weeks could change body composition to more fit and stronger. In addition, trend analysis showed significant increases in relative weight (%) of the kidney (p < 0.0001) and heart (p = 0.0018) with increasing LP10 dose. We found no gross abnormalities attributed to LP10 when weighing organs.
3.5. Effect of LP10 Supplementation on Biochemical Variables at the End of the Experiment
We observed beneficial effects of LP10 on grip strength and exhaustive exercise challenge, as well as other physiological effects, with six-week LP10 supplementation. We further investigated whether six-week LP10 treatment affected other biochemical markers in healthy mice. We examined tissue- and health status-related biochemical variables and major organs including skeletal muscle, heart, kidney, and lung (
Table 2).
Levels of biochemical indices, including CK, TP, creatinine, UA, TC, and glucose, did not differ among groups (
p > 0.05,
Table 2). Serum albumin levels were lower by 7.64% (
p = 0.0375) with LP10-5X than with vehicle treatment. Serum BUN levels were lower by 15.50% (
p = 0.0218) and 13.29% (
p = 0.0037) with LP10-1X and LP10-5X, respectively, than with vehicle treatment. On trend analysis, serum albumin (
p = 0.0012) and BUN (
p = 0.0017) levels were dose-dependently decreased with LP10 supplementation. Therefore, long-term daily supplementation with LP10 may have potential for tissue protection and renal benefits. In addition, serum level of TC, an index of lipid profile, was lower by 22.76% (
p = 0.0069) and 26.60% (
p = 0.0021) with LP10-1X and LP10-5X, respectively, than with vehicle treatment. Trend analysis showed significantly decreased serum TG levels (
p = 0.0005) with increasing LP10 dose. According to a previous study, the beneficial lipid profile in
Lactobacillus is attributed to the accumulation of SCFAs during fermentation [
53]. SCFAs can transport from the intestinal lumen into the blood compartment of hosts and regulate the balance in fatty acid synthesis [
54]. In addition, LP10 supplementation for six weeks had no adverse effects on major organs such as the liver, skeletal muscle, heart, kidney, lung, and EFP. Therefore, the dose of LP10 supplementation used in this study was safe (
Figure 5).