A heat-inactivated Lactobacillus rhamnosus CQFP202442 and its application in relieving exercise fatigue.

Abstract

This invention discloses a heat-inactivated Lactobacillus rhamnosus. rhamnosus This invention relates to strain CQFP202442 and its application in alleviating exercise-induced fatigue, belonging to the field of microbial technology. The preservation number of this heat-inactivated strain is CGMCC No. 29612. This invention demonstrates that this heat-inactivated strain can significantly prolong the time to exhaustion during running in mice and reduce the degree of muscle damage; it can 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 muscle tissue; simultaneously, it can upregulate the relative expression levels of related mRNAs in muscle tissue; and downregulate the levels of LDH, BUN, and CRE in mouse serum. The heat-inactivated strain of this invention can effectively alleviate exercise-induced fatigue through a dual mechanism of regulating oxidative stress and myofibrillary protection, exhibiting high safety and good stability.

Description

Technical Field

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

[0002] In today's fast-paced society, with increasing work pressure, exercise has become a key way for people to maintain health and relieve stress. However, excessive exercise or participation in high-intensity activities can easily lead to fatigue, with exercise-induced fatigue being particularly prominent. This has become a key factor affecting athletic performance and physical health. Exercise-induced fatigue is a physiological state caused by excessive exercise, mainly manifested as physical decline, 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 focal point of attention for both the academic and sports communities.

[0003] Among the many factors that induce exercise-induced fatigue, oxidative stress plays a significant role. During high-intensity exercise, the body's metabolic activity increases dramatically, producing a large amount of reactive oxygen species (ROS). These ROS are highly aggressive, capable of damaging cell membranes, proteins, and DNA structures, leading to cell damage and dysfunction, ultimately causing fatigue. Although the body has its own antioxidant defense system to eliminate excess ROS, this system may struggle to function effectively under the load of high-intensity exercise. Therefore, enhancing the body's antioxidant capacity is considered an effective strategy for alleviating exercise-induced fatigue. Oxidative stress is essentially an adverse physiological state caused by the excessive accumulation of free radicals within cells, resulting in oxidative damage. Excessive and highly reactive oxygen free radicals attack key molecular structures such as nucleic acids, proteins, and lipids within cells, causing severe damage and triggering a series of chain physiological reactions. Muscle tissue is the first and most vulnerable site.

[0004] As the basic structural unit of muscle tissue, muscle fiber damage is a key characteristic of exercise-induced fatigue. High-intensity or prolonged exercise can cause micro-tears or injuries to muscle fibers, leading to inflammation and fatigue. Muscles, as the direct executors of movement, are fully exposed to oxidative stress during exercise. Oxidative stress causes lipid peroxidation of muscle cell membranes, disrupting cell membrane integrity. Simultaneously, oxidative modification of proteins interferes with normal muscle contraction and relaxation, ultimately resulting in muscle fatigue and injury. Therefore, protecting muscle fibers from damage is crucial for alleviating exercise-induced fatigue. Currently, exploring effective methods to protect muscle fibers and reduce damage has become an important area of ​​research in sports nutrition and physiology.

[0005] In recent years, probiotics have attracted widespread research interest due to their significant health benefits, including improving gut health, enhancing immune function, and exerting antioxidant effects. Numerous studies have revealed that probiotics play an indispensable role in regulating gut microbiota balance, promoting nutrient absorption, and enhancing the body's immunity. Crucially, many studies consistently demonstrate that probiotics have a certain degree of protective effect against oxidative stress. Probiotics can alleviate muscle damage caused by oxidative stress and promote the recovery and repair of bodily functions through various complex physiological pathways, opening up new avenues and methods for alleviating exercise-induced fatigue.

[0006] Lactobacillus rhamnosus rhamnosus As a common probiotic, *Lactobacillus rhamnosus* has attracted much attention for its significant effects in regulating gut microbiota, optimizing digestive function, promoting nutrient absorption, and enhancing immunity. Importantly, existing research suggests that *Lactobacillus rhamnosus* may play a positive role in alleviating exercise-induced fatigue. However, current research on its specific mechanisms of action is insufficient and requires further in-depth exploration.

[0007] Within the field of probiotic research, heat-inactivated probiotics are gradually gaining attention. Heat inactivation involves using specific heating methods to deactivate probiotics while preserving their cell walls, metabolites, and other bioactive components. Heat-inactivated probiotics offer several advantages. First, they are safer, avoiding the potential infection risks associated with live bacteria, making them safe for use even by individuals with weakened immune systems. Second, their stability is enhanced, being less affected by environmental factors (such as temperature and humidity) during storage and transportation, facilitating product preservation and distribution. Furthermore, some components of heat-inactivated probiotics can still exert immunomodulatory and antioxidant functions, providing new insights for their application in relieving exercise-induced fatigue. However, currently, systematic research is lacking on the specific effects and mechanisms of action of heat-inactivated *Lactobacillus rhamnosus* in relieving exercise-induced fatigue, particularly its regulatory role in the antioxidant system and myofibril protection. Summary of the Invention

[0008] The purpose of this invention is to provide a heat-inactivated *Lactobacillus rhamnosus* CQFP202442 and its application in relieving exercise-induced fatigue, thereby addressing the problem of the lack of safe, stable, and effective probiotic preparations for relieving exercise-induced fatigue in the prior art. Another purpose of this invention is to provide a composition comprising the aforementioned heat-inactivated *Lactobacillus rhamnosus* CQFP202442 for relieving exercise-induced fatigue.

[0009] To achieve the above objectives, the present invention provides a heat-inactivated Lactobacillus rhamnosus. rhamnosusThe heat-inactivated Lactobacillus rhamnosus CQFP202442 has the accession number CGMCC No.29612.

[0010] The present invention also provides a composition for relieving exercise-induced fatigue, comprising an effective amount of heat-inactivated Lactobacillus rhamnosus CQFP202442 and acceptable excipients, wherein the heat-inactivated Lactobacillus rhamnosus CQFP202442 has the accession number CGMCC No. 29612.

[0011] The present invention also provides the use of the above-mentioned heat-inactivated Lactobacillus rhamnosus CQFP202442 or the above-mentioned composition in the preparation of products for relieving exercise fatigue.

[0012] Furthermore, the product alleviates exercise-induced fatigue by enhancing the body's antioxidant capacity.

[0013] Furthermore, the enhancement of the body's antioxidant capacity includes increasing the levels of total antioxidant capacity, catalase, and reduced glutathione in serum.

[0014] Furthermore, the enhancement of the body's antioxidant capacity also includes upregulating the relative mRNA expression levels of superoxide dismutase 1, superoxide dismutase 2, and catalase in muscle tissue.

[0015] Furthermore, the product alleviates exercise-induced fatigue by protecting muscle fibers.

[0016] Furthermore, the protection of muscle fibers includes reducing the degree of muscle damage and preserving the morphology and function of muscle fibers.

[0017] Furthermore, the product alleviates exercise-induced fatigue by regulating the balance of energy metabolism and metabolic products during exercise. This regulation includes increasing serum glucose levels and decreasing serum levels of lactate dehydrogenase, urea nitrogen, and creatinine.

[0018] Furthermore, the product alleviates exercise-induced fatigue by upregulating the expression of myosin heavy chain genes and energy metabolism regulatory genes in muscle tissue.

[0019] Compared with the prior art, the present invention has the following advantages and technical effects: This invention demonstrates that heat-inactivated Lactobacillus rhamnosus CQFP202442 can alleviate exercise-induced fatigue on multiple levels.

[0020] Regarding the regulation of oxidative stress, CQFP42 significantly enhanced the levels of total antioxidant capacity (T-AOC), catalase (CAT), and reduced glutathione (GSH) in the serum of exercise-fatigued mice, while simultaneously upregulating the mRNA expression levels of superoxide dismutase 1 (SOD1), superoxide dismutase 2 (SOD2), and catalase (CAT) in muscle tissue. This indicates that heat-inactivated Lactobacillus rhamnosus CQFP202442 can systematically enhance the body's antioxidant defense capabilities, effectively scavenging excess reactive oxygen species generated during high-intensity exercise, thereby reducing oxidative stress damage to muscle tissue.

[0021] In terms of energy metabolism and metabolite regulation, CQFP42 significantly prolonged the time to exhaustion in mice, effectively increased serum glucose (GLU) levels, and simultaneously decreased levels of lactate dehydrogenase (LDH), blood urea nitrogen (BUN), and creatinine (CRE). This indicates that heat-inactivated Lactobacillus rhamnosus CQFP202442 can precisely regulate the balance of energy metabolism and metabolites during exercise, improve the body's energy utilization efficiency, and thus effectively delay the onset of exercise-induced fatigue.

[0022] In terms of muscle fiber protection and functional enhancement, CQFP42- can significantly reduce skeletal muscle pathological damage caused by exercise-induced fatigue, preserving muscle fiber morphology and function. Simultaneously, CQFP42- can upregulate the expression of four myosin heavy chain genes (MyHc I, MyHc IIa, MyHc IIb, and MyHc IIx) in muscle tissue, as well as the expression of deacetylase 1 (SIRT1) and peroxisome proliferation-activating receptor gamma coactivator (PGC). This indicates that heat-inactivated *Lactobacillus rhamnosus* CQFP202442 can enhance muscle endurance and athletic performance by regulating the expression of genes related to muscle fiber composition and energy metabolism.

[0023] In summary, this embodiment systematically verified the significant effect of heat-inactivated Lactobacillus rhamnosus CQFP202442 in alleviating exercise-induced fatigue by constructing a mouse model of exercise-induced fatigue, and revealed the scientific implications of its function through a dual mechanism of antioxidant regulation and myofibril protection, providing experimental evidence and theoretical basis for the development of novel anti-fatigue food-derived antioxidants. Attached Figure Description

[0024] 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.

[0025] Figure 1 This is a graph showing the changes in mouse body weight.

[0026] Figure 2 This is a pathological analysis image of mouse muscle tissue (H&E staining).

[0027] Figure 3 The graph shows the levels of oxidation markers T-AOC, CAT, and GSH in mouse serum.

[0028] Figure 4 This is a graph showing the levels of the metabolites GLU, LDH, BUN, and CRE in mouse serum.

[0029] Figure 5 This is a graph showing the relative mRNA expression levels of oxidation-related genes SOD1, SOD2, and CAT in mouse muscle tissue.

[0030] Figure 6 This is a diagram showing the relative mRNA expression levels of the motor function-related genes MyHc I, MyHc IIa, MyHc IIb, MyHc IIx, SIRT1, and PGC in mouse muscle tissue. Detailed Implementation

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] Table 2 Instruments and Equipment 3 Experimental Methods 3.1 Preparation of experimental strains The experimental strain used in this embodiment is *Lactobacillus rhamnosus*. 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.

[0037] 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 and count the viable cells. Treat the activated bacterial solution at 121°C for 15 minutes to perform heat inactivation treatment, and obtain the heat-inactivated Lactobacillus rhamnosus CQFP42 bacterial solution for later use.

[0038] 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.

[0039] 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 heat-inactivated Lactobacillus rhamnosus CQFP42 group (CQFP42- group).

[0040] During the first week of the experiment, all mice were administered oral gavage: the normal control group and the control group were administered 0.1 mL / 10 g (based on mouse body weight) of 0.9% saline solution daily; the vitamin C group was administered 0.1 mL / 10 g (based on mouse body weight) of a 200 mg / kg vitamin C solution daily; and the CQFP42- group was administered 0.1 mL / 10 g (based on mouse body weight) of a 1.0 x 10⁻⁶ solution daily. 9CFU / mL of heat-inactivated Lactobacillus rhamnosus CQFP42 was administered. In the second week, acclimatization training was conducted on the mice. Mice were placed on a treadmill with a 0° incline and a speed controlled at 10 m / min for 6 days, 10 minutes per day, with gavage administered before each exercise session. Starting in the third week, a 2-week endurance training program was implemented. Mice trained on a 5° incline, with an initial training time of 10 minutes, an acceleration of 1 m / min, and a maximum speed of 10 m / min, 6 days of training per week with 1 day of rest. Gavage was administered before each training session. After the final gavage, the mice were fasted for 16-24 hours. All animal experiments were rigorously reviewed by the Animal Ethics Committee of the Chongqing Collaborative Innovation Center for Functional Foods, approval number 2023102701B.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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. After one week of gavage, the CQFP42- group mice showed the smallest increase in body weight; at the end of the experimental period, the CQFP42- group had the lightest body weight, while there were no significant differences among the other groups.

[0048] The weights of mouse organs and tissues are shown in Table 4. Compared with the normal group, the organ indices of the heart, liver, and muscles increased in mice that underwent exercise training, while the organ indices of the kidneys and testes decreased. Except for the heart and 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.

[0049] 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 heat-inactivated CQFP42 bacterial solution. Different letters (ac) indicate significant differences between groups in Duncan's multiple comparison test. p <0.05).

[0050] 4.2 Time to Exhaustion in Mice The time to exhaustion while running is a commonly used indicator of athletic performance, and improved athletic performance is the most powerful macroscopic manifestation of enhanced fatigue resistance. The exhaustion times of mice are shown in Table 5. The results indicate that the vitamin C group had the longest exhaustion time, followed by the CQFP42- group, and finally the control group. This result demonstrates that CQFP42- has a significant effect on improving endurance in mice, with an effect comparable to vitamin C.

[0051] 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 control group exhibited mild interstitial edema, loose connective tissue arrangement, and irregular muscle fiber arrangement and shape. Additionally, a small number of muscle fibers in the control group showed a significant reduction in area and a slight increase in spacing. Intervention in the Vc and CQFP42- groups alleviated these pathological damages. These results indicate that CQFP42- reduces skeletal muscle damage caused by exercise-induced fatigue, preserving the morphology and function of muscle fibers. This can be demonstrated by restoring the number and area of ​​healthy muscle fibers and improving muscle fiber damage.

[0052] 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). The above results indicate that CQFP42- has a good inhibitory effect on oxidative stress induced by exercise fatigue.

[0053] 4.5 Levels of the metabolites GLU, CRE, BUN, and LDH in mouse serum The results are as follows Figure 4 As shown. In the serum of control mice, GLU levels were the lowest, while LDH, BUN, and CRE levels were the highest ( p <0.05. Intake of vitamin C and CQFP42- effectively alleviated the decrease in GLU and the increase in LDH, BUN, and CRE. Among these, BUN and LDH levels in the serum of CQFP42- group mice were not significantly different from those in the vitamin C group. p >0.05). Therefore, it can be concluded that heat-inactivated Lactobacillus rhamnosus CQFP42 can effectively inhibit changes in serum indicators related to exercise function in mice induced by exercise fatigue, and enhance the endurance of mice.

[0054] 4.6 mRNA expression levels of oxidation-related genes in mouse muscle tissue The mRNA expression levels of oxidation-related genes in mouse muscle tissue showed Figure 5 In the normal group, the expression levels of SOD1, SOD2, and CAT were the highest, while they were the lowest in the control group. p <0.05). The expression levels of SOD1, SOD2, and CAT in the CQFP42 group were closest to those in the Vc group.

[0055] 4.7 mRNA expression levels of motor function-related genes in mouse muscle tissue mRNA expression levels of genes related to motor function, such as Figure 6 As shown, MyHc I, MyHc IIa, MyHc IIb, MyHc IIx, SIRT1, and PGC were expressed at the highest levels in the normal group, followed by the vitamin C group. The CQFP42- group was next in expression, while the control group showed the lowest expression levels. These results indicate that dietary supplementation with vitamin C and CQFP42- can effectively prevent oxidative damage and contribute to improved motor function in mice.

[0056] 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 heat-inactivated Lactobacillus rhamnosus rhamnosus CQFP202442, characterized in that, The heat-inactivated Lactobacillus rhamnosus CQFP202442 has the accession number CGMCC No.29612.

2. A composition for relieving exercise-induced fatigue, characterized in that, It contains an effective amount of heat-inactivated Lactobacillus rhamnosus CQFP202442 and acceptable excipients, wherein the heat-inactivated Lactobacillus rhamnosus CQFP202442 has the accession number CGMCC No.29612.

3. The use of the heat-inactivated Lactobacillus rhamnosus CQFP202442 of claim 1 or the composition of claim 2 in the preparation of a product for relieving exercise-induced fatigue.

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

5. The application according to claim 4, characterized in that, Enhancing the body's antioxidant capacity includes increasing the levels of total antioxidant capacity, catalase, and reduced glutathione in serum.

6. The application according to claim 4, characterized in that, Enhancing the body's antioxidant capacity also includes upregulating the relative mRNA expression levels of superoxide dismutase 1, superoxide dismutase 2, and catalase in muscle tissue.

7. The application according to claim 3, characterized in that, The product relieves exercise fatigue by protecting muscle fibers.

8. The application according to claim 7, characterized in that, The protection of muscle fibers includes reducing the degree of muscle damage and preserving the morphology and function of muscle fibers.

9. The application according to claim 3, characterized in that, The product alleviates exercise-induced fatigue by regulating the balance of energy metabolism and metabolic products during exercise. This regulation includes increasing serum glucose levels and decreasing serum levels of lactate dehydrogenase, urea nitrogen, and creatinine.

10. The application according to claim 3, characterized in that, The product alleviates exercise-induced fatigue by upregulating the expression of myosin heavy chain genes and energy metabolism regulatory genes in muscle tissue.

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