Lactobacillus rhamnosus CQFP202442 and viable bacteria thereof are used for relieving exercise-induced fatigue

Abstract

The application discloses a lactobacillus rhamnosus (Lactobacillus rhamnosus) Lactisaceaebacillus rhamnosus ) CQFP202442 and application of live bacteria thereof in relieving exercise-induced fatigue, and belongs to the technical field of microorganisms. The application provides application of lactobacillus rhamnosus CQFP202442 with a preservation number of CGMCC No. 29612 and live bacteria thereof in preparation of products for relieving exercise-induced fatigue, and a probiotic preparation containing the strain. The application proves that the strain can improve the antioxidant capacity of mice by regulating oxidative stress, thereby reducing the damage to the body caused by exercise-induced fatigue and improving the exercise function of the body, by establishing a mouse exercise-induced fatigue model. The application effectively relieves exercise-induced fatigue through the dual mechanisms of antioxidation and muscle fiber protection, can be applied to the preparation of anti-fatigue products, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and in particular relates to the application of Lactobacillus rhamnosus CQFP202442 and its live bacteria in relieving exercise fatigue. Background Technology

[0002] With the fast pace of modern life and increasing work pressure, exercise has become an important way for people to maintain health and relieve stress. However, excessive or high-intensity exercise often leads to physical fatigue, especially exercise-induced fatigue, which is a significant factor affecting athletic performance and physical health. Exercise-induced fatigue is a physiological state caused by excessive exercise, typically manifested as decreased physical strength, lethargy, and muscle soreness. This fatigue not only reduces athletic performance but may also increase the risk of sports injuries. Therefore, finding effective ways to alleviate exercise-induced fatigue has become a hot topic of concern in academia and the sports community.

[0003] In recent years, probiotics have attracted widespread attention due to their numerous health benefits, including improving gut health, enhancing immune function, and providing antioxidant effects. Among them, *Lactobacillus rhamnosus* (Lactobacillus casei)... Lactisaceaebacillus rhamnosus As a common probiotic, *Lactobacillus rhamnosus* has attracted much attention due to its significant effects in regulating gut microbiota, improving digestive function, promoting nutrient absorption, and enhancing immunity. More importantly, research suggests that *Lactobacillus rhamnosus* may play a positive role in alleviating exercise-induced fatigue. However, current research on its specific mechanisms is insufficient and requires further exploration.

[0004] Oxidative stress is a significant factor contributing to exercise-induced fatigue. High-intensity exercise leads to the production of large amounts of reactive oxygen species (ROS) in the body. These ROS can attack cell membranes, proteins, and DNA, causing cell damage and dysfunction, thus inducing fatigue. While the body possesses an antioxidant defense system to eliminate excess ROS, this system may become overwhelmed under high-intensity exercise. Therefore, enhancing the body's antioxidant capacity is considered an effective strategy for alleviating exercise-induced fatigue.

[0005] Muscle fibers are the basic structural units of muscle tissue, and their damage is a major characteristic of exercise-induced fatigue. High-intensity or prolonged exercise can cause micro-tears or damage to muscle fibers, thereby triggering inflammatory responses and fatigue. Protecting muscle fibers from damage is crucial for alleviating exercise-induced fatigue. Therefore, exploring methods to effectively protect muscle fibers and reduce damage is an important aspect of current sports nutrition and physiology research.

[0006] Therefore, exploring active substances that can simultaneously exert antioxidant and myofibrillary protective effects is of great significance for developing novel anti-fatigue products. Probiotics, as a class of biological resources with high safety and diverse functions, have shown promising application prospects in the field of sports nutrition. However, current research on the effects and mechanisms of specific probiotic strains in alleviating exercise-induced fatigue is still relatively lacking, especially regarding whether *Lactobacillus rhamnosus* can exert anti-fatigue effects through a dual pathway of antioxidant and myofibrillary protection, which has not yet been systematically reported. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention proposes the application of *Lactobacillus rhamnosus* CQFP202442 and its live bacteria in relieving exercise-induced fatigue. One objective of this invention is to provide a probiotic preparation that can effectively relieve exercise-induced fatigue. Another objective of this invention is to reveal the mechanism of action of *Lactobacillus rhamnosus* CQFP202442 in relieving exercise-induced fatigue, providing a theoretical basis and technical support for the development of novel anti-fatigue functional foods or sports nutrition supplements.

[0008] To achieve the above objectives, the present invention provides a *Lactobacillus rhamnosus* (Lactobacillus casei) Lactisaceaebacillus rhamnosus Lactobacillus rhamnosus CQFP202442 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 29612.

[0009] The present invention also provides the application of the above-mentioned Lactobacillus rhamnosus CQFP202442 in the preparation of products for relieving exercise fatigue, wherein the Lactobacillus rhamnosus CQFP202442 is a live bacterium.

[0010] Furthermore, the product alleviates exercise-induced fatigue by enhancing the body's antioxidant capacity.

[0011] Furthermore, the product alleviates exercise-induced fatigue by upregulating the expression of antioxidant-related genes in muscle tissue, including superoxide dismutase 1 (SOD1), superoxide dismutase 2 (SOD2), and catalase (CAT).

[0012] Furthermore, the product alleviates exercise-induced fatigue by protecting muscle fiber structure and / or upregulating the expression of genes related to muscle fiber function.

[0013] Furthermore, the myofibril function-related genes include myosin heavy chain I (MyHc I), myosin heavy chain IIa (MyHc IIa), myosin heavy chain IIb (MyHc IIb), myosin heavy chain IIx (MyHc IIx), deacetylase 1 (SIRT1), and peroxisome proliferation-activating receptor γ coactivator (PGC).

[0014] The present invention also provides a probiotic preparation for relieving exercise-induced fatigue, the probiotic preparation comprising live bacteria of the above-mentioned Lactobacillus rhamnosus CQFP202442.

[0015] The present invention also provides a method for relieving exercise-induced fatigue, comprising administering an effective amount of the above-mentioned live Lactobacillus rhamnosus CQFP202442 to an individual in need.

[0016] The present invention also provides the application of the above-mentioned live bacteria of Lactobacillus rhamnosus CQFP202442 in the preparation of products for improving the body's antioxidant capacity.

[0017] The present invention also provides the use of the above-mentioned live bacteria of Lactobacillus rhamnosus CQFP202442 in the preparation of products for protecting muscle fibers or enhancing muscle endurance.

[0018] Compared with the prior art, the present invention has the following advantages and technical effects: This invention establishes a mouse model of exercise-induced fatigue and demonstrates that *Lactobacillus rhamnosus* CQFP202442 can improve the antioxidant capacity of mice by regulating oxidative stress, thereby reducing the damage caused by exercise-induced fatigue and improving the body's motor function. Experimental results show that CQFP202442 can reduce muscle damage; prolong the time to exhaustion in mice during running; increase the levels of T-AOC, CAT, GSH, and GLU in mouse serum, as well as the relative mRNA expression levels of SOD1, SOD2, and CAT in mouse liver and muscle tissue; simultaneously upregulate the relative mRNA expression levels of MyHc I, MyHc IIa, MyHc IIb, MyHc IIx, SIRT1, and PGC in muscle tissue; and downregulate the levels of LDH, BUN, and CRE in mouse serum.

[0019] This invention explores how Lactobacillus rhamnosus CQFP202442 can effectively alleviate exercise-induced fatigue by regulating oxidative stress response and myofibril expression, and elucidates its mechanism, providing a reference for future research and development of food-derived antioxidants that alleviate exercise-induced fatigue and improve bodily functions. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 This is a graph showing the changes in mouse body weight.

[0022] Figure 2 This is a pathological analysis image of mouse muscle tissue (H&E staining).

[0023] Figure 3 The graph shows the levels of oxidation markers T-AOC, CAT, and GSH in mouse serum.

[0024] Figure 4 This is a graph showing the levels of the metabolites GLU, LDH, BUN, and CRE in mouse serum.

[0025] Figure 5 This is a graph showing the relative mRNA expression levels of oxidation-related genes in mouse muscle tissue.

[0026] Figure 6 This is a graph showing the relative mRNA expression levels of genes related to motor function in mouse muscle tissue. Detailed Implementation

[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0028] All raw materials used in this invention are not particularly limited in their source; they can be purchased from the market or prepared using conventional methods known to those skilled in the art.

[0029] There are no particular restrictions on the purity of any of the raw materials used in this invention. However, this invention preferably uses raw materials of analytical grade or purity commonly used in the field of chemical synthesis.

[0030] Example 1 1. Materials and Reagents The main experimental materials and reagents used in this embodiment are shown in Table 1. All reagents were analytical grade or biological grade, with clearly identified sources, ensuring the reliability and reproducibility of the experimental results.

[0031] Table 1 Experimental Materials and Reagents 2. Instruments and Equipment The main instruments and equipment used in this embodiment are shown in Table 2. All equipment has been calibrated and is within its validity period.

[0032] Table 2 Instruments and Equipment 3 Experimental Methods 3.1 Preparation of experimental strains The experimental strain used in this embodiment was *Lactobacillus rhamnosus* (Lactobacillus casei). Lactisaceaebacillus rhamnosus CQFP202442 (hereinafter referred to as CQFP42) is a lactic acid bacteria strain isolated and purified from naturally fermented pickled vegetables in Chongqing, China. It was identified as *Lactobacillus rhamnosus* by 16S rDNA sequence analysis. This strain has been deposited at the China General Microbiological Culture Collection Center (CGMCC, Beijing, China), with accession number CGMCC No. 29612, on January 12, 2024. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0033] Take the preserved bacterial strain and inoculate it into MRS liquid medium at an inoculum rate of 2%. After culturing at 37°C for 16-24 hours, perform a second activation, collect the bacterial cells, wash and resuspend them with sterile physiological saline, and adjust the bacterial concentration to 1.0 × 10⁻⁶. 9 CFU / mL, for later use.

[0034] 3.2 Animal Models This embodiment uses 50 five-week-old male Kunming mice, weighing 20±2 g, purchased from Chongqing Enswell Biotechnology Co., Ltd. The mice were housed in an environment with a temperature of 25±2℃ and a relative humidity of 50±5%, under a 12-hour light / dark cycle. During this period, the mice were allowed unrestricted access to standard mouse feed and drinking water, and their bedding was changed every two days. All mice underwent a one-week acclimatization period before the formal experiment.

[0035] To investigate the antioxidant effect of Lactobacillus rhamnosus CQFP202442 on exercise-induced fatigue mice, mice were randomly divided into four groups of 10 mice each: normal group, control group, vitamin C group (vitamin C positive control group) and Lactobacillus rhamnosus CQFP42 group (CQFP42 group).

[0036] The experiment lasted 29 days. During the first week, all mice were administered the following treatments via gavage: Normal and control groups: 0.1 mL / 10g (mouse body weight) of 0.9% saline solution daily; Vitamin C group: 0.1 mL / 10g (mouse body weight) of a 200 mg / kg Vitamin C solution daily; CQFP42 group: 0.1 mL / 10g (mouse body weight) of a 1.0 x 10⁻⁶ solution daily.9 CFU / mL of *Lactobacillus rhamnosus* CQFP42 was administered. In the second week, mice underwent acclimatization training for 6 days on a 0° incline at a speed of 10 m / min, with 10 minutes of training per day. Gavage administration was given before each training session. In the third week, a two-week endurance training program was implemented, with 10 minutes of training on a 5° incline (acceleration of 1 m / min, maximum speed of 10 m / min). Training was conducted 6 days a week with 1 day of rest. Gavage administration was given before each training session, and mice were fasted for 16-24 hours after the last gavage. All animal experiments were reviewed by the Animal Ethics Committee of the Chongqing Collaborative Innovation Center for Functional Foods (NO. 2023102701B).

[0037] 3.3 Mouse Exhaustion Test In week 5 (day 29), an exhaustion test was conducted to analyze the exercise load of the mice, record the exhaustion rate, and document the time to exhaustion. The exhaustion test procedure was as follows: 0°, 10 m / min, 15 min; 5°, 10 m / min, 15 min; 10°, 10 m / min until exhaustion. The standard for exhaustion was that the mouse was willing to accept more than five electric shocks (1 mA current) for more than 3 seconds or remain continuously on the electric shock grid for 5 seconds. At this point, the mouse was removed from the treadmill, and the time to exhaustion was recorded. Immediately after exhaustion, blood was collected from the eyeballs of the mice, and muscle tissue was separated for later use.

[0038] 3.4 Tissue H&E staining Mouse muscle tissue was washed with physiological saline, cut in half, and fixed in 10% formalin solution. The muscle tissue was dehydrated with a gradient of ethanol, soaked in xylene and ethanol for approximately 30 minutes to clarify the tissue, embedded in paraffin, sectioned into approximately 2-3 μm sections using a microtome, and fixed onto glass slides. Hematoxylin and eosin (H&E) dyes were used to stain the cytoplasm with different shades of pink or red. Finally, morphological changes were observed under a light microscope.

[0039] 3.5 Detection of animal serum markers The obtained mouse blood was centrifuged at 4000 rpm for 10 minutes at 4°C, and then the mouse serum was separated and collected, and stored at -80°C for later use. The levels of T-AOC, CAT, GSH, GLU, BUN, and CRE in the serum were determined using appropriate biochemical kits according to the manufacturer's recommended procedures.

[0040] 3.6 Enzyme-linked immunosorbent assay (ELISA) Using preserved serum, the level of lactate dehydrogenase (LDH) in mouse serum was determined according to the ELISA instructions.

[0041] 3.7 Real-time quantitative PCR detection In this invention, messenger RNA (mRNA) expression in mouse skeletal muscle tissue was determined using the SYBR Green method. Approximately 100 mg of mouse skeletal muscle tissue was minced, and total RNA was extracted from liver and skeletal muscle tissue using Trizol reagent. RNA concentration was measured using a micro-spectrophotometer. cDNA template was obtained by reverse transcription using the Revert Aid First Strand cDNA Synthesis Kit. Amplification was then performed using a StepOnePlus real-time PCR system with 10 μL SYBR Green PCR Master Mix, 1 μL upstream and downstream primers, 1 μL cDNA template, and 7 μL DEPC. The conditions were as follows: pre-denaturation at 95°C for 3 min; followed by denaturation at 95°C for 15 s, annealing at 60°C for 30 s, and extension at 72°C for 15 s, for 40 cycles; the final melting curve was obtained at 95°C for 30 s, 60°C for 30 s, and 95°C for 15 s. Finally, the mRNA expression was determined by 2... -ΔΔCT The method calculates the relative expression level of each gene, where CT is the cycle threshold, and β-actin is used as an internal reference gene for this purpose. Table 3 shows the primer sequence information used in this invention.

[0042] Table 3 Primer sequence information 3.8 Data Analysis Serum and tissue parameters for each mouse were performed in triplicate or at least in parallel, and the average values ​​were taken. Data were statistically analyzed using IBM SPSS 22 statistical software. Results are expressed as mean ± standard deviation (SD). Differences between means were assessed using one-way ANOVA with Duncan's multiple range test. p Differences <0.05 are considered statistically significant.

[0043] 4 Results and Analysis 4.1 Mouse body weight and organ index During the experiment, the mouse's body weight changed as follows: Figure 1 As shown in the figure. The results showed that after one week of gavage, the CQFP42 group of mice had the greatest weight gain, while the weight gain slowed down after exercise training. At the end of the experimental period, there was no significant difference in weight among the four groups.

[0044] The results of organ tissue weight measurements in mice are shown in Table 4. Compared with the normal group, the organ indices of the heart, liver, and muscles were increased in mice that underwent exercise training (control group, vitamin C group, and CQFP42 group), while the organ indices of the kidneys and testes were decreased. Except for muscle tissue, there were no significant differences in the indices of all other organs between the CQFP42 group and the normal group, but significant differences were found between the control group and the normal group. This indicates that CQFP42 has almost no toxic effects on the organs of mice and can effectively increase the muscle index of mice.

[0045] Table 4. Mouse organ index Normal group: 0.1 mL / 10g 0.9% saline was administered via gavage; Control group: 0.1 mL / 10g 0.9% saline was administered via gavage; Vitamin C group: 0.1 mL / 10g 200 mg / kg Vitamin C solution was administered via gavage; CQFP42 group: 0.1 mL / 10g of a 1.0 x 10⁻⁶ solution was administered via gavage. 9 CFU / mL CQFP42 bacterial solution. Different letters (ac) indicate significant differences between groups in Duncan's multiple comparison test. p <0.05).

[0046] 4.2 Time to Exhaustion in Mice Time to exhaustion is a commonly used indicator of athletic performance, and improved athletic performance is the most powerful macroscopic manifestation of enhanced fatigue resistance. The results of the time to exhaustion measurements in mice are shown in Table 5. The results indicate that the CQFP42 group had the longest time to exhaustion, followed by the Vc group, and finally the control group. These results demonstrate that CQFP42 has a significant effect on improving endurance in mice.

[0047] Table 5. Time to Exhaustion in Mice 4.3 Pathological analysis of mouse skeletal muscle tissue Results of skeletal muscle morphological analysis as follows Figure 2 As shown in the figure, the skeletal muscle tissue of the control group mice exhibited obvious pathological changes, including mild edema of the interstitial space, loose connective tissue arrangement, irregular arrangement and shape of local muscle fibers, and a significant reduction in the area of ​​a small number of muscle fibers with slightly widened interfiber spacing. Compared with the control group, the pathological damage to the skeletal muscle tissue of the CQFP42 group mice was significantly reduced. This result indicates that CQFP42 can alleviate skeletal muscle damage caused by exercise-induced fatigue, effectively preserving the morphology and function of muscle fibers, which can be demonstrated by restoring the number and area of ​​healthy muscle fibers and improving muscle fiber damage.

[0048] 4.4 Levels of oxidative markers T-AOC, CAT, and GSH in mouse serum The levels of oxidation-related indicators in mouse serum, such as Figure 3 As shown. The levels of oxidative markers T-AOC, CAT, and GSH in the serum of mice in the control group were the lowest. Compared with the control group, the levels of T-AOC, CAT, and GSH in the serum of mice in the Vc group and CQFP42 group were significantly increased. p <0.05), among which, there was no significant difference in the levels of T-AOC and CAT in the serum of mice in the Vc group and the CQFP42 group ( p >0.05). This result indicates that CQFP42 can significantly enhance the body's antioxidant capacity and has a good inhibitory effect on oxidative stress caused by exercise fatigue.

[0049] 4.5 Levels of the metabolites GLU, LDH, BUN, and CRE in mouse serum The results of the determination of metabolites in mouse serum are as follows: Figure 4 As shown in the figure. The results showed that the control group mice had the lowest serum GLU levels, while the levels of LDH, BUN, and CRE were the highest. p <0.05%. Compared with the control group, serum GLU levels were significantly increased and LDH, BUN, and CRE levels were significantly decreased in mice in the Vc and CQFP42 groups. However, there was no significant difference in serum BUN and LDH levels between the CQFP42 group and the Vc group. p >0.05). This result indicates that CQFP42 can effectively inhibit changes in exercise-related indicators in mouse serum induced by exercise fatigue and enhance mouse endurance.

[0050] 4.6 mRNA expression levels of oxidation-related genes in mouse muscle tissue The relative mRNA expression levels of oxidation-related genes (SOD1, SOD2, CAT) in mouse skeletal muscle tissue are as follows: Figure 5 As shown in the figure. The results showed that the mRNA expression levels of SOD1, SOD2, and CAT were highest in the muscle tissue of the normal group mice, and lowest in the control group. p <0.05. The mRNA expression levels of SOD1, SOD2, and CAT in the muscle tissue of CQFP42 group mice were significantly higher than those in the control group ( p <0.05, and there was no significant difference compared with the Vc group ( p >0.05). This result indicates that CQFP42 can upregulate the expression of antioxidant-related genes in skeletal muscle tissue and enhance the antioxidant capacity of muscle tissue.

[0051] 4.7 mRNA expression levels of motor function-related genes in mouse muscle tissue The relative mRNA expression levels of motor function-related genes (MyHc I, MyHc IIa, MyHc IIb, MyHc IIx, SIRT1, PGC) in mouse skeletal muscle tissue are as follows: Figure 6 As shown in the figure. The results showed that the mRNA expression levels of the above genes were highest in the muscle tissue of the normal group mice, followed by the Vc group, then the CQFP42 group, and lowest in the control group. The differences between groups were statistically significant. p <0.05). This result indicates that CQFP42 can upregulate the expression of myofibril function-related genes, regulate myofibril composition and function, enhance the regulatory functions of SIRT1 and PGC, thereby improving the body's motor function.

[0052] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A type of Lactobacillus rhamnosus ( Lactisaceaebacillus rhamnosus CQFP202442, characterized in that, The Lactobacillus rhamnosus CQFP202442 is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 29612.

2. The use of the live bacteria of Lactobacillus rhamnosus CQFP202442 as described in claim 1 in the preparation of products for relieving exercise-induced fatigue.

3. The application according to claim 2, characterized in that, The product alleviates exercise-induced fatigue by enhancing the body's antioxidant capacity.

4. The application according to claim 2 or 3, characterized in that, The product alleviates exercise-induced fatigue by upregulating the expression of antioxidant-related genes in muscle tissue, including superoxide dismutase 1, superoxide dismutase 2, and catalase.

5. The application according to claim 2, characterized in that, The product alleviates exercise-induced fatigue by protecting muscle fiber structure and / or upregulating the expression of genes related to muscle fiber function.

6. The application according to claim 5, characterized in that, The myofibrane function-related genes include myosin heavy chain I, myosin heavy chain IIa, myosin heavy chain IIb, myosin heavy chain IIx, deacetylase 1, and peroxisome proliferation activation receptor γ coactivator.

7. A probiotic preparation for relieving exercise-induced fatigue, characterized in that, The probiotic preparation contains live bacteria of Lactobacillus rhamnosus CQFP202442 as described in claim 1.

8. A method for relieving exercise-induced fatigue, characterized in that, This includes administering an effective amount of live Lactobacillus rhamnosus CQFP202442 as described in claim 1 to individuals in need.

9. The use of the live bacteria of Lactobacillus rhamnosus CQFP202442 as described in claim 1 in the preparation of products for improving the body's antioxidant capacity.

10. The use of the live bacteria of Lactobacillus rhamnosus CQFP202442 as described in claim 1 in the preparation of articles for protecting muscle fibers or enhancing muscle endurance.

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