A comprehensive approach, based on the use of Caenorhabditis elegans, mouse, and human models, elucidates the impact of Lactiplantibacillus plantarum TWK10 on exercise performance and longevity

Abstract

The functionality of probiotics is highly influenced by culture and processing conditions, making batch stability validation through human or mouse trials impractical. Here, we employed a comprehensive approach using Caenorhabditis elegans, mouse and human models to elucidate the beneficial effects of Lactiplantibacillus plantarum TWK10 (TWK10). In C. elegans, TWK10 administration significantly prolonged lifespan by 26.1 ± 11.9 % (p < 0.05), enhanced locomotion (p < 0.01) and muscle mass (p < 0.001), elevated glycogen storage (p < 0.05), and reduced lipid accumulation (p < 0.001), outperforming Lacticaseibacillus rhamnosus GG and L. plantarum type strain ATCC 14917^T. We also confirmed the equivalence of laboratory-prepared and mass-produced TWK10 in ergogenic efficacy using C. elegans assay. In mice, oral administration of mass-produced TWK10 significantly enhanced exercise performance and glycogen storage in muscle and liver in a dose-dependent manner. In a clinical study involving healthy male adults, significant improvements in grip strength (1.1-fold, p < 0.01) and exhaustion time (1.27-fold, p < 0.01), and significant reductions in circulating lactate and ammonia levels were observed in the TWK10 group (1 × 10¹⁰ colony-forming unit/day) compared to the control group. Both humans and mice receiving mass-produced TWK10 showed improved body composition with increased muscle mass and reduced fat mass. In conclusion, TWK10 demonstrates superior longevous and ergogenic effects in C. elegans compared to reference strains. The consistent ergogenic efficacy of mass-produced TWK10 across C. elegans, mice, and humans, highlights the utility of C. elegans as a reliable model for probiotic research and industrial application.

Keywords: Caenorhabditis elegans; Cross-species validation; Exercise performance; Humans; Lactiplantibacillus plantarum TWK10; Longevity; Mice.

Graphical abstract

Fig. 1

Lifespan and locomotion in C. elegans. (A) Lifespan assay of LAB-treated C. elegans. The survival rates of each group are presented as the mean ± SD, and the statistical differences between the four groups were analyzed using the log–rank (Mantel–Cox) test; each of the three independent experiments used 120 C. elegans (20 nematodes/well). WT, C. elegans wild-type N2 strain (B) MLS and (C) MLS extension rate of LAB-treated C. elegans from three independent experiments. Data are presented as the mean ± SD and were analyzed by the Kruskal–Wallis test with Dunn post-hoc test. Locomotion behavior analysis of LAB-treated C. elegans includes (D) crawling speed, (E) swimming speed, and (F) body bending frequency during swimming. Data are presented as the mean ± SD and were analyzed using one-way ANOVA with Tukey's post-hoc test; n ≥ 100. Different letters (a, b, c) indicate a significant difference at p < 0.05. Significant differences in the body bending frequency between Day 1 and Day 5 in each group were analyzed using the paired t-test and indicated by ∗∗p < 0.01, ∗∗∗p < 0.001. OP50, control group received only E. coli OP50; LGG, L. rhamnosus GG; ATCC 14917 ^T, L. plantarum ATCC 14917^T.

Fig. 2

Muscle mass in C. elegans. Phalloidin staining visualizes in situ filamentous actin-positive regions in LAB-treated C. elegans on (A) Day 1 and (B) Day 5. (C) Quantification of filamentous actin-positive area in single nematodes; n = 30. Data are presented as the mean ± SD and were analyzed by the Kruskal–Wallis test with Dunn's post-hoc test. Different letters (a, b, c) indicate a significant difference at p < 0.05. Scale bar = 200 μm. OP50, control group received only E. coli OP50; LGG, L. rhamnosus GG; ATCC 14917 ^T, L. plantarum ATCC 14917^T.

Fig. 3

Lipid accumulation in C. elegans. ORO staining visualizes in situ lipid droplets accumulation in LAB-treated C. elegans on (A) Day 1 and (B) Day 5. (C) Quantification of ORO staining intensity and (D) stained area ratio of single nematodes. Data are expressed as the mean ± SD and were analyzed by the one-way ANOVA with Tukey's post-hoc test for parametric comparisons and Kruskal–Wallis test with Dunn's post-hoc test for non-parametric multiple comparisons test; n = 30. Different letters (a, b, c) indicate a significant difference at p < 0.05. Scale bar = 200 μm. OP50, control group received only E. coli OP50; LGG, L. rhamnosus GG; ATCC 14917 ^T, L. plantarum ATCC 14917^T.

Fig. 4

Glycogen storage levels in C. elegans. Iodine staining visualizes in situ glycogen storage regions in LAB-treated C. elegans on (A) Day 1 and (B) Day 5. (C) Glycogen content in nematodes from three independent experiments. Data are expressed as the mean ± SD and were analyzed by the one-way ANOVA with Tukey's post-hoc test. Different letters (a, b) indicate a significant difference at p < 0.05. Scale bar = 200 μm. OP50, control group received only E. coli OP50; LGG, L. rhamnosus GG; ATCC 14917 ^T, L. plantarum ATCC 14917^T.

Fig. 5

Equivalence assessment of the ergogenic efficacy of lab-prepared TWK10 and mass-produced TWK10 bacterial powder. (A) Locomotion behavior analysis of crawling speed and (B) body bending frequency; n ≥ 100. (C) Glycogen content in nematodes from three independent experiments. (D) Quantification of (E) filamentous actin-positive area of single nematodes; n = 30. (F) ORO staining visualizes lipid accumulation in nematodes. Data are presented as the mean ± SD and were analyzed by the one-way ANOVA with Tukey's post-hoc test for parametric comparisons and Kruskal–Wallis test with Dunn's post-hoc test for non-parametric comparison. Different letters (a, b, c) indicate a significant difference at p < 0.05. Scale bar = 200 μm. OP50, control group received only E. coli OP50; BP, factory-produced bacterial powder; MRS, TWK10 bacterial cells using MRS broth.

Fig. 6

Effects of mass-produced TWK10 bacterial powder in mice on forelimb grip strength and swimming performance. (A) Total and (B) relative forelimb grip strength of mice. (C) Swimming endurance time of mice; n = 12. Data are expressed as the mean ± SD. Statistical differences among groups were analyzed by the one-way ANOVA with Tukey's post-hoc test. Non-parametric data were statistically analyzed by the Kruskal–Wallis test with Dunn's post-hoc test. Different letters (a, b, c, d) indicate a significant difference at p < 0.05.

Fig. 7

Ergogenic effects of mass-produced TWK10 bacterial powder in healthy adults. (A) Exercise performance evaluation, including grip strength of (A) right hand and (B) left hand (C) exhaustion time. (D) Serum lactate and (E) ammonia levels during exercise and rest periods after 6 weeks of administration. (F) Muscle mass and (G) body fat changes in body composition of individual subjects. Data are expressed as the mean ± SD. Statistical significance between groups was analyzed using the unpaired t-test for parametric comparison, and the Mann–Whitney U test for non-parametric comparisons. Significance is shown by: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Intragroup differences were analyzed using the paired t-test and indicated by ^###p < 0.001.