Phytobacterium plantarum with functions of improving muscle function and athletic ability as well as product and application of phytobacterium plantarum

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to plant lactobacillus with the function of improving the muscle function and the athletic ability and a product and application of the plant lactobacillus. According to the invention, the phytobacterium plantarum Vigo6 with the preservation number of CGMCC (China General Microbiological Culture Collection Center) No.35628 can promote the development of motor nerves, improve the form and density of muscle fibers, increase the muscle content and enhance the arrangement tightness of the muscle fibers; the effects of improving sarcopenia, promoting muscle growth, improving muscle strength, enhancing body coordination and balance ability, improving endurance and physical ability and improving muscle attenuation and aging equivalence are achieved. The lactobacillus plantarum Vigo6 disclosed by the invention has high intestinal tolerance, high storage stability, high safety and no pathogenicity, provides a new effective strain resource for developing related products for improving muscle health and athletic ability, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to Lactobacillus plantarum, its products, and applications, which have the effect of improving muscle function and motor ability. Background Technology

[0002] Skeletal muscle, as a core component of the human musculoskeletal system, is a key dynamic tissue for maintaining normal physiological functions. It not only undertakes basic functions such as thermoregulation and nutrient storage (especially through regulating protein balance via systemic metabolism), but also converts chemical energy into mechanical force, providing power for physical activity, social interaction, and daily physiological processes. The activity, function, and physiological state of skeletal muscle are easily regulated by various internal and external factors, including hormone levels, exercise intensity, dietary structure, and drug intervention. Lack of exercise, metabolic / chronic diseases, and natural aging are currently recognized as the three core negative factors inducing muscle atrophy.

[0003] Muscle atrophy and the resulting sarcopenia have become a public health problem affecting population health. With the accelerating aging of the population, the incidence of sarcopenia in the elderly is rising year by year, while a trend of muscle function degeneration is also emerging among younger people who are sedentary and lack exercise. The core characteristics of sarcopenia are a reduction in muscle fiber size and a significant decrease in muscle mass, accompanied by progressive decline in skeletal muscle function. Specifically, this can lead to a series of problems such as decreased muscle strength, impaired motor skills, and reduced coordination and balance. This not only weakens an individual's responsiveness to drug treatment but also significantly increases the risk of falls, fractures, and complications, even endangering life in severe cases. It greatly reduces the patient's quality of life and places a heavy medical burden and economic pressure on families and society.

[0004] Currently, the main approaches to improving muscle function and preventing sarcopenia fall into three categories: exercise intervention, nutritional supplementation, and drug therapy. While exercise intervention can slow down muscle loss to some extent, its effectiveness is limited by individual physical condition, willingness to exercise, and exercise capacity, making it difficult to cover special populations such as the elderly and those with weak constitutions. Nutritional supplementation can only provide raw materials for muscle synthesis and cannot fundamentally regulate the molecular mechanisms of muscle growth, thus its improvement effect is limited. Although drug therapy has some efficacy, it is generally accompanied by side effects, and the safety of long-term use is questionable, limiting its clinical application.

[0005] In recent years, microbial agents have received widespread attention in the health field due to their advantages such as regulating intestinal flora balance, improving metabolism, high safety, and no side effects. Patent CN120437179A utilizes a heat-treated fermentation culture complex of KM2 *Lactobacillus plantarum* as a microbiome composition for improving muscle strength. This composition increases myoprotein synthesis and shows an inhibitory effect on myoprotein degradation; it also shows effects of improving muscle strength and increasing leg muscle mass: increased grip strength, walking distance, and speed. Patent CN120899769A discloses the efficacy of *Lactobacillus plantarum* strain Lp-G18 in the treatment of skeletal muscle atrophy. *Lactobacillus plantarum* strain Lp-G18 significantly improves DEX-induced reduction in myotube diameter, decrease in fusion index, reduction in protein content, and shortening of myotube length.

[0006] Developing a strain of *Lactobacillus plantarum* that has a clear multi-pathway effect in improving muscle function and motor ability, and possesses high intestinal tolerance and high storage stability, to fill the existing technological gap, has become an urgent technical problem to be solved in this field. Summary of the Invention

[0007] To address the above shortcomings, this invention provides *Lactobacillus plantarum*, its products, and their applications, which improve muscle function and athletic ability.

[0008] Terminology Explanation: Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0009] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0010] The definition of the standard chemical term can be found in the reference "Principles and Identification Techniques of Bacterial and Archaea Systematic Taxonomy", Higher Education Press, Chief Editors Li Wenjun, Liu Lan, Jiao Jianyu, and Fang Baozhu, 2025-01.

[0011] Unless otherwise stated, conventional methods within the scope of the art shall be used, such as methods for assessing the utilization of different carbohydrates, growth curves, antioxidant capacity, toxicity experiments, ability to inhibit pathogenic bacteria, and tolerance to gastrointestinal fluids.

[0012] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0013] The terms "optional / arbitrary" or "optionally / arbitrarily" mean that the event or situation described below may or may not occur, including both the occurrence and non-occurrence of the event or situation. For example, according to the definition below: "The microbial agent comprises any one or more of *Lactobacillus plantarum* cells, fermentation broth, fermentation broth supernatant, fermentation broth precipitate, and lyophilized powder" means that the microbial agent may contain *Lactobacillus plantarum* cells, or the microbial agent may contain *Lactobacillus plantarum* fermentation broth, or the microbial agent may contain *Lactobacillus plantarum* cells, fermentation broth, fermentation broth supernatant, fermentation broth precipitate, and lyophilized powder.

[0014] In this invention, the term "Lactobacillus plantarum Vigo6" refers to a strain of Lactobacillus plantarum isolated from healthy breast milk samples and obtained through targeted domestication and screening. Lactiplantibacillus plantarum It was deposited on August 15, 2025 at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 35628.

[0015] In this invention, the term "improved muscle function and athletic ability" refers to one or more physiological effects, including promoting motor nerve development, improving sarcopenia, promoting muscle growth, increasing muscle strength, improving body coordination and balance, enhancing endurance and physical fitness, improving muscle mass that controls body balance, endurance, explosive power, and support, as well as improving muscle loss and aging.

[0016] In this invention, the term "directed acclimatization" refers to a series of acclimatization treatments on *Lactobacillus plantarum* to improve its resistance to gastric juice, intestinal juice, and low-temperature freezing, thereby enhancing the strain's intestinal tolerance and storage stability.

[0017] The technical solution of this invention is as follows: On the one hand, the present invention provides a plant lactobacillus ( Lactiplantibacillus plantarum The *Lactobacillus plantarum* mentioned is *Lactobacillus plantarum* Vigo6, with accession number CGMCC No. 35628.

[0018] In another aspect, the present invention provides a microbial agent comprising *Lactobacillus plantarum* as described in any of the preceding claims.

[0019] Specifically, the microbial agent includes any one or more of the following: fermentation broth of *Lactobacillus plantarum*, fermentation broth precipitate, fermentation broth supernatant, live bacterial cells, inactivated bacterial cells, lyophilized powder, lysate, lysate, secondary metabolites, and exosomes.

[0020] In another aspect, the present invention provides the use of any of the above-mentioned *Lactobacillus plantarum* or bacterial agents in the preparation of products that improve muscle function and athletic ability.

[0021] Specifically, the products include any one or more of the following: (1) Products that relieve physical fatigue; (2) Products that improve sarcopenia; (3) Products that promote the development of motor nerves; (4) Products that enhance athletic performance; (5) Products that promote muscle growth and enhance muscle strength; (6) Products that enhance the body's coordination and balance. (7) Products that enhance endurance and physical fitness; (8) Products that improve muscle quality, balance, endurance, explosive power, and support; (9) Products that improve muscle loss and aging.

[0022] Preferably, the *Lactobacillus plantarum* Vigo6 improves muscle function and enhances athletic ability by promoting motor nerve development, improving muscle fiber morphology and density, increasing muscle mass, and / or strengthening the compactness of muscle fiber arrangement.

[0023] In another aspect, the present invention provides a product for improving muscle function and athletic ability, the product comprising any of the above-mentioned Lactobacillus plantarum or bacterial agents.

[0024] Specifically, the product contains at least 1×10 8 CFU containing Lactobacillus plantarum Vigo6.

[0025] Preferably, the product contains 1×10 8 -1×10 12 CFU containing Lactobacillus plantarum Vigo6.

[0026] Specifically, the dosage form of the product includes solid dosage form, semi-solid dosage form, or liquid dosage form.

[0027] Specifically, the product also includes auxiliary materials.

[0028] Preferably, the excipients include any one or more of the following: diluent, excipient, filler, binder, wetting agent, disintegrant, emulsifier, cosolvent, solubilizer, osmotic pressure regulator, surfactant, coating material, colorant, pH adjuster, antioxidant, and buffer.

[0029] Considering the possibility of this invention entering other countries, this invention also provides the following technical solutions: A method for improving muscle function and athletic ability, the method comprising using the aforementioned *Lactobacillus plantarum*, bacterial agent, or product.

[0030] Specifically, the method includes administering an effective amount of *Lactobacillus plantarum*, a bacterial agent, or a product to the subject.

[0031] Among them, the technical characteristics of the subjects included mammals.

[0032] The beneficial effects of this invention are as follows: The *Lactobacillus plantarum* Vigo6, with accession number CGMCC No. 35628, can improve sarcopenia, promote muscle growth, enhance muscle strength, improve coordination and balance, increase endurance and physical fitness, and mitigate muscle loss and aging by promoting motor nerve development, improving muscle fiber morphology and density, increasing muscle mass, and enhancing muscle fiber compactness. The *Lactobacillus plantarum* Vigo6 of this invention exhibits high intestinal tolerance and high storage stability, high safety, and no pathogenicity, providing a new and effective strain resource for developing related products that improve muscle health and athletic performance, with broad application prospects.

[0033] Preservation Instructions Preserved strain: Vigo6; Classification and nomenclature: Lactobacillus plantarum Lactiplantibacillus plantarum ; Accession number: CGMCC No. 35628; Preservation period: August 15, 2025; Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Culture Collections; Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0034] Figure 1 The colony morphology of Lactobacillus plantarum Vigo6 is shown.

[0035] Figure 2 The morphology of Lactobacillus plantarum Vigo6 after Gram staining is examined under a microscope.

[0036] Figure 3 The colony characteristics of Lactobacillus plantarum Vigo 6 on Columbia blood agar plates.

[0037] Figure 4 The growth curve of Lactobacillus plantarum Vigo6 is shown.

[0038] Figure 5 The length of the peripheral motor nerves in zebrafish is shown in pixels. Compared with the model group, there was no significant difference in ns, **p<0.01, ***p<0.001.

[0039] Figure 6 This is a typical diagram showing the length of peripheral motor nerves in zebrafish; the yellow dashed box in the diagram indicates the peripheral motor nerves in the area being analyzed.

[0040] Figure 7 The total distance traveled by the zebrafish is (mm). Compared with the model group, there was no significant difference in ns in the figure, *p<0.05, **p<0.01, ***p<0.001.

[0041] Figure 8 This is a diagram showing the movement trajectory of a zebrafish.

[0042] Figure 9 Image of zebrafish muscle stained with HE.

[0043] Figure 10 The results show the grip strength of mice; compared with the control group, there was no significant difference in ns, **p<0.01.

[0044] Figure 11 The results show the results of rod time measurement in mice; compared with the control group, there was no significant difference in ns, ***p<0.001, ****p<0.0001.

[0045] Figure 12 The results show the distance measured in mice; compared with the control group, there was no significant difference in ns, *p<0.05, ***p<0.001.

[0046] Figure 13 The results show the muscle content measurement; in the figure, A represents the gastrocnemius muscle content, B represents the tibialis anterior muscle content, and C represents the soleus muscle content; there was no significant difference in ns between the figure and the normal group, *p<0.05, **p<0.01.

[0047] Figure 14 The results of HE staining of muscle are shown in the figure; A is the normal group, B is the DMF group, C is the Vigo6 group, and D is the GM918 group. There is no significant difference in ns between the normal group and the normal group, *p<0.05, ***p<0.001. Detailed Implementation

[0048] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0049] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0050] Example 1: Isolation, Identification and Domestication of Lactobacillus plantarum Vigo6 Lactobacillus plantarum was isolated from healthy breast milk samples. The samples were plated on MRS selective medium using a serial dilution method and anaerobically incubated at 37°C for 24 hours. Single colonies of yellowish, round, Gram-positive bacilli were picked and preliminarily identified as Lactobacillus plantarum. Subsequently, targeted acclimatization was carried out. Gastric juice acclimation: The strain was activated for three generations, centrifuged and the supernatant was discarded. The bacterial cells were washed once with PBS buffer and then resuspended in artificial gastric juice (NaCl 0.1g, pepsin 0.175g, water 50mL, fully dissolved, pH adjusted to 2.5 with dilute hydrochloric acid, mixed well and filtered through a 0.22μm filter under sterile conditions). After standing at 37℃ for 3h, 100μL of the bacterial suspension was spread on solid MRS medium and incubated at 37℃ for 24h. Strains with larger colonies were selected.

[0051] Screening for resistance to intestinal fluid: The obtained gastric fluid-resistant strains were further screened. After activation for three generations, the supernatant was discarded by centrifugation. The bacterial cells were washed once with PBS buffer and then resuspended in artificial intestinal fluid (0.68 g potassium dihydrogen phosphate, 1 g trypsin, 100 ml water, mixed and adjusted to pH 8, filtered through a 0.22 μm filter under sterile conditions). After standing at 37°C for 2 h, 100 μl of the bacterial suspension was spread on solid MRS medium and incubated at 37°C for 24 h. Strains with larger colonies were selected.

[0052] The results of gastric juice tolerance and intestinal juice tolerance screening are shown in Table 1: Table 1 Results of strains' acclimatization to gastrointestinal fluids

[0053] Low-temperature cryopreservation: Superior strains acclimatized to gastric and intestinal fluids were activated for three generations, then frozen at -80℃ for 2 hours, thawed at room temperature, and re-frozen at -80℃, repeated three times. The final thawed bacterial suspension was inoculated at 1% onto MRS liquid medium and cultured at 37℃ for 24 hours, constituting one acclimatization generation. After 30 generations of acclimatization, 100 μL of the bacterial suspension was spread onto solid MRS medium and cultured at 37℃ for 24 hours. Strains with larger colonies were selected. Bacterial powders were prepared from the original strain and acclimatized strains under the same conditions, stored at 37℃ for one month, and the survival rate was tested.

[0054] Table 2 Results of low-temperature acclimatization of strains

[0055] As can be seen from Table 2 above, the survival rate of the strain after 30 generations of domestication is much higher than that of the original strain.

[0056] Finally, through the above domestication and screening, a strain with both high intestinal tolerance and high freeze resistance was obtained. Identified as *Lactobacillus plantarum* by 16S rDNA sequencing, it was named *Lactobacillus plantarum* Vigo6 and deposited on August 15, 2025, at the China General Microbiological Culture Collection Center (CGMCC), address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 35628, and classified as *Lactobacillus plantarum*. Lactiplantibacillus plantarum .

[0057] The 16S rRNA sequencing results are shown in SEQ ID NO.1:

[0058] Example 2: Detection of the physicochemical characteristics of the strain 2.1 Morphological and colony observation, hemolytic characteristics Pure bacterial culture was evenly spread onto a glass slide and fixed in the outer flame of an alcohol lamp. After Gram staining, the slide was slowly rinsed with deionized water to remove excess staining solution. After the slide dried, it was observed and photographed under an oil immersion microscope. One loopful of bacterial culture was streaked onto MRS agar medium using an inoculation loop. After incubation at 37°C for 24 hours, the colony morphology was observed.

[0059] The microbiological characteristics of Lactobacillus plantarum Vigo6 are as follows: (1) Colony morphology: such as Figure 1 As shown, the colonies grown in MRS agar medium are white with a slight yellow tinge, opaque, round, with a smooth and moist surface and neat edges.

[0060] (2) Gram staining morphology: such as Figure 2 As shown, the colonies are white, round, moist, opaque, and have neat edges.

[0061] (3) Colony characteristics on Columbia blood agar plates: such as Figure 3 The white, round colonies shown are moist with regular edges, and there is no hemolysis around the colonies.

[0062] 2.2 Utilization of different carbohydrates Preparation of colony plates: The strain is streaked on MRS agar plates. After colonies grow, single colonies are picked and streaked again until single colonies grow.

[0063] Preparation of bacterial test solution: Pick the colonies on the plate into 2 mL of physiological saline, shake and mix well, take an appropriate amount of bacterial solution (v) into 5 mL of physiological saline, measure the OD value, and then take (2v) from the original bacterial solution into 10 mL of API-matched culture medium and mix well.

[0064] Incubation reaction: Add sterile deionized water to the bottom plate of the incubation box to ensure a humid environment. Take out the test strips (0-19, 20-39, 40-49) from the packaging bag, separate them, place them in the bottom plate of the incubation box, and gently tilt them forward.

[0065] Using a pipette, draw 115 µL of bacterial culture, placing the pipette tip against the edge of the beaker to add it, avoiding air bubbles. Fill only the top of the tube (beaker) completely, and seal the top with sterile liquid paraffin to maintain an anaerobic environment. Incubate the tube at 37°C. Observe the color change of the reagent strips after 24 and 48 hours, with 48 hours as the final result. The results are shown in Table 3. *Lactobacillus plantarum* Vigo6 showed an absorbance of 0.90 at OD600 and could utilize 25 carbon sources after 48 hours of culture.

[0066] Table 3. Identification results of Lactobacillus plantarum Vigo6

[0067] Note: "V" represents a variable reaction, and the results may fluctuate under different experimental conditions or batches; "+" indicates availability; "-" indicates unavailability.

[0068] 2.3 Growth Curve Glyceryl spp. Vigo6 was inoculated into sterile MRS broth at a 1% inoculum and incubated at 37°C for 16 h. 1% of the inoculum was then transferred to a sterile 96-well plate containing 200 µl of MRS broth. After two generations of incubation at 37°C, 5% of the inoculum was inoculated into a 96-well plate containing 200 µl of MRS broth. Each bacterium was replicated in three wells. The plates were then shaken and incubated. OD600 was measured every hour.

[0069] The results are as follows Figure 4 The results showed that *Lactobacillus plantarum* Vigo6 entered the logarithmic growth phase at 13 hours and the stationary phase at 15 hours, with an OD600 value of approximately 1.62 at the stationary phase.

[0070] 2.4 Antioxidant capacity After activation, *Lactobacillus plantarum* Vigo6 was cultured for three generations. Cell lysates and fermentation broth supernatants were prepared. The DPPH free radical scavenging capacity, hydroxyl free radical scavenging capacity, and total antioxidant capacity of the cell lysates and fermentation broth supernatants were tested using the Nanjing Jiancheng FRTP kit (catalog number: A015-3-1). The experimental procedures were performed in accordance with the kit instructions. The results are shown in Table 4.

[0071] Table 4 Antioxidant capacity of Lactobacillus plantarum Vigo6

[0072] 2.5 Toxicity testing and safety detection The pathogenicity test method for food-grade bacteria in Appendix A of GB 31615.2-2025 "National Food Safety Standard - Procedure for Safety Evaluation of Food-grade Microbial Strains" was used to test *Lactobacillus plantarum* Vigo6. The test animals showed no abnormalities or deaths, and their body weight was not statistically significant compared with the control group (p>0.05), indicating that this strain was non-pathogenic.

[0073] 2.6 Detection of ability to inhibit pathogenic bacteria Preparation of fermentation supernatant of the test bacteria: After three consecutive generations of activation, the third generation fermentation broth was centrifuged at 6000×g for 10 min, the supernatant was collected, filtered through a 0.22μm micromembrane, and stored at -20℃ for later use.

[0074] After activating the pathogenic bacteria (Escherichia coli ATCC25922, Staphylococcus aureus ATCC25923, Pseudomonas aeruginosa ATCC27853, Enterococcus faecalis ATCC29212, Shigella flexneri CICC 21534, and Cronobacter sakazakii CICC 21560) three times in liquid culture medium, the third-generation culture medium was adjusted to a suitable absorbance value, so that the bacterial suspension concentration was 1×10⁻⁶. 8 CFU / mL - 5 × 10 8 CFU / mL.

[0075] Preparation of test plates: Heat and dissolve the prepared NA medium, cool to 45℃-50℃, add the prepared indicator bacterial suspension to the NA medium at an addition rate of 1%, mix thoroughly, measure 20mL and pour into a sterile Petri dish, gently shake the Petri dish to spread it evenly, and let it solidify before use.

[0076] Place 4-6 Oxford cups at equal intervals on a test plate containing indicator bacteria, press gently, and slowly add 200 μL of the fermentation supernatant of the test bacteria into the Oxford cups. Repeat each treatment three times. Place the plates in a refrigerator at 4℃-6℃ for pre-diffusion for 4-10 hours. Remove the plates and place them in a constant temperature incubator at 36℃±1℃, incubating upright until the inhibition zone is clear. Measure the diameter of the inhibition zone using calipers or an inhibition zone measuring instrument. Measure each inhibition zone three times along different directions and record the average value. Results are shown in Table 5.

[0077] Table 5. Diameter of the outer inhibition zone of *Lactobacillus plantarum* Vigo6 strain against pathogens.

[0078] The results showed that Lactobacillus plantarum Vigo6 had a good inhibitory effect on all the above pathogenic bacteria.

[0079] 2.7 Gastrointestinal Fluid Tolerance Test Preparation of artificial gastric fluid: Measure 0.2 mL of hydrochloric acid, add 0.8 mL of water and mix well to obtain dilute hydrochloric acid. Weigh 0.2 g of NaCl and 0.35 g of pepsin, add 100 mL of water and dissolve completely. Adjust the pH to 2.5 with dilute hydrochloric acid, mix well, and filter under sterile conditions using a 0.22 μm filter membrane.

[0080] Preparation of artificial intestinal fluid: Dissolve 0.68g of potassium dihydrogen phosphate in 50mL of water, adjust the pH to 6.8 with 0.1mol / L sodium hydroxide solution, and dissolve 1g of pancreatic enzyme in an appropriate amount of water. Mix the two solutions, dilute with water to 100mL, mix well, and filter under sterile conditions using a 0.22μm filter membrane.

[0081] Activation of the strain: 1% of the strain cryopreservation solution was inoculated into 1.5 mL of MRS broth and incubated overnight at 37°C. The next day, 1% of the bacterial solution was inoculated into 1.5 mL of MRS broth and incubated overnight at 37°C. 1% of the activated second-generation bacterial solution was inoculated into 15 mL of MRS broth and incubated at 37°C for 20 h.

[0082] Treatment of bacterial culture with artificial gastric fluid: Take 10 mL of culture medium, centrifuge at 6000 g for 10 min, discard the supernatant, wash the bacterial cells twice with sterile physiological saline, resuspend the bacterial sludge in 3 mL of artificial gastric fluid, mix well, and then bring the volume to 10 mL with artificial gastric fluid. Take 100 μL and perform 10-fold serial dilutions, and use MRS plate counting to determine the initial viable cell count. After anaerobic static incubation at 37℃ for 3 h, take 100 μL and perform serial dilutions, and use MRS plate counting to determine the viable cell count after 3 h of gastric fluid treatment. Treatment of bacterial culture with artificial intestinal fluid: The treatment method is the same as that for bacterial culture with artificial gastric fluid, except that the artificial gastric fluid in the system is replaced with artificial intestinal fluid. The results are shown in Table 6 below.

[0083] Table 6. Statistical analysis of viable bacteria count of *Lactobacillus plantarum* Vigo6 in artificial gastric and intestinal fluids.

[0084] 2.8 Adhesion ability determination Strain preparation: Activate *Lactobacillus plantarum* Vigo6 strain, culture for three generations, centrifuge at 10000g for 5 min in 0.01M PBS (pH 7.2-7.4), wash twice, and adjust the bacterial concentration to 1×10⁻⁶ in DMEM medium. 6 CFU / mL.

[0085] Cell preparation: Resuscitate and culture Caco-2 cells to passage 3, digest the cells and plate them into 24-well cell culture plates. The Caco-2 cells should form a monolayer with a confluence of 80-90%. Wash the cells with 0.01M PBS, 1 mL / well, twice. The washing process should be gentle to avoid cell detachment.

[0086] Adhesion experiment: After cell washing, add 1 mL of solution containing 1×10⁻⁶ strains. 6 Incubate at CFU / mL in a 37℃, 5% CO2 incubator for 2 h. Use the washed bacterial suspension as a reference for co-incubation. After incubation, discard the cell culture supernatant containing unadhered probiotics. Wash cells twice with 0.01M PBS (1 mL / well). Digest cells with Trypsin-EDTA (0.25%) for 15 min (0.5 mL / well). Count the number of cells per well in a 24-well cell culture plate. Dilute the digested bacterial suspension with physiological saline and perform plate counting. Record the number of colonies formed by probiotics on MRS plates before and after adhesion, and calculate the adhesion index and adhesion rate.

[0087] Adhesion index = Number of viable bacteria after adhesion / Number of cells in negative control well; Adhesion rate (%) = Number of viable bacteria after adhesion / Number of viable bacteria before adhesion.

[0088] The results of the strain adhesion experiment are shown in Table 7.

[0089] Table 7 Adhesion ability of Lactobacillus plantarum Vigo6 strain

[0090] Example 3: Freeze-drying process and preparation of Lactobacillus plantarum Vigo6 powder Using MRS medium, *Lactobacillus plantarum* Vigo6 was subcultured and activated three times at 37℃. The bacterial culture was then inoculated into MRS liquid medium for large-scale culture (the culture volume depends on the actual needs). The culture conditions were: static culture at 37℃ for 16 hours to obtain the culture medium of the strain. Centrifuge 6000g of the cultured bacterial solution for 5 minutes, discard the supernatant, mix the bacterial sludge and skim milk at a mass ratio of 1:1, mix well, and then put it into a freeze dryer for vacuum freeze drying. After vacuum freeze drying for 48 hours, the bacterial powder is obtained.

[0091] Example 4: Improvement of sarcopenia by Lactobacillus plantarum Vigo6 4.1 Animals and Drug Administration Wild-type AB zebrafish with a 3-day pf (dpf) count were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). Water-soluble samples were administered, along with a positive control of trimetazidine hydrochloride tablets at 62.5 μg / mL. Simultaneously, a normal control group, a model control group, a Vigo6 group (administered with *Lactobacillus plantarum* Vigo6), and a GM918 group (administered with *Lactobacillus plantarum* GM918, a strain isolated from healthy breast milk samples of the same batch as *Lactobacillus plantarum* Vigo6, serving as a control strain) were set up. Each well contained 3 mL of the strain, and the sample was administered at a concentration of 1 × 10⁻⁶ μg / mL. 6 (CFU / mL). Except for the normal control group, all other experimental groups were given 2.00 μM dexamethasone in water to establish a zebrafish sarcopenia model.

[0092] 4.2 Evaluation of Promotion of Motor Neuron Development After treatment at 28℃ for 3 days, 10 zebrafish were randomly selected from each experimental group, photographed under a fluorescence microscope, and the data were analyzed and collected using NIS-ElementsD3.20 advanced image processing software to analyze the length of the motor nerves around the zebrafish.

[0093] The measurement results are shown in Table 8 and Figures 5-6As shown in the figure, compared with the model group, the length of peripheral motor nerves in the positive drug group (trimetazidine hydrochloride) and the Vigo6 group was significantly increased, and the differences were highly significant compared with the model group (p < 0.01), with no difference in effect between the two groups. Compared with the model group, the GM918 group, which is also composed of *Lactobacillus plantarum*, showed no statistically significant difference in the length of peripheral motor nerves (p > 0.05). This indicates that *Lactobacillus plantarum* Vigo6 has a role in promoting motor nerve development.

[0094] Table 8. Results of the experiment evaluating the promotion of motor nerve development (n = 10)

[0095] Note: Compared with the model group, **p<0.01, ***p<0.001.

[0096] 4.3 Improving the assessment of athletic ability After treatment at 28℃ for 4 days, 10 zebrafish were randomly selected from each group and placed in a 96-well plate (one zebrafish per well, 200 μL per well). The total movement distance of the zebrafish was measured using a behavior analyzer. The efficacy of the samples in improving sarcopenia (total movement distance) was evaluated based on the statistical analysis results of the above indicators. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS software, and p < 0.05 was considered statistically significant.

[0097] The measurement results are shown in Table 9 and Figures 7-8 As shown, compared with the model group, the Vigo6 group showed a significant increase in total movement distance, with a mean of 1.59 times that of the model group, and the difference was statistically significant (p < 0.05). However, compared with the model group, the GM918 probiotic group, which is also *Lactobacillus plantarum*, showed no statistically significant difference in total movement distance (p > 0.05). This indicates that *Lactobacillus plantarum* Vigo6 has the effect of enhancing motor ability.

[0098] Table 8 Results of the experiment on promoting motor ability assessment (n = 10)

[0099] Note: Compared with the model group, *p<0.05, **p<0.01, ***p<0.001 in the table.

[0100] 4.4 Evaluation of Muscle Development Promotion After treatment at 28℃ for 3 days, the zebrafish in each group underwent fixation, dehydration, embedding, sectioning, and H&E staining, and were then subjected to histopathological analysis, including histopathological analysis of muscle tissue.

[0101] The results are as follows Figure 9As shown, in the normal group, zebrafish skeletal muscle cells were uniformly elongated, with muscle fibers densely distributed in the body compartments. In the model group, zebrafish muscle histology exhibited severe abnormal phenotypes, with significant muscle fiber relaxation and loss, decreased muscle fiber density, and irregular interseptal boundaries or interruptions, indicating successful establishment of the sarcopenia model. In the positive drug group, zebrafish muscle fiber relaxation was improved, muscle fiber density recovered, and no irregular interseptal boundaries or interruptions appeared, suggesting that trimetazidine hydrochloride tablets have an effect on improving sarcopenia. In the GM918 group, zebrafish muscle fibers were significantly relaxed and lost, muscle fiber density decreased, and irregular interseptal boundaries or interruptions appeared, suggesting that GM918 had no significant effect on improving sarcopenia. In the Vigo6 group, the zebrafish muscle histology phenotype was restored, muscle fiber relaxation was improved, muscle fiber density recovered, and irregular interseptal boundaries or interruptions were largely improved. The results show that *Lactobacillus plantarum* Vigo6 has the effect of improving sarcopenia and promoting muscle development.

[0102] Example 5: Evaluation of the therapeutic effect of Lactobacillus plantarum Vigo6 on sarcopenia in an aging model Male C57BL / 6J mice aged 22 months were selected and acclimatized for one week under constant temperature (22±2°C) and humidity (50±10%) conditions, while maintaining a 12-hour light / dark cycle. Grip strength, stick time, and movement distance were measured in each mouse. After removing individuals with significant differences, they were randomly divided into a control group, a 5,7-dimethoxyflavone (DMF) group, a Vigo6 group (treated with *Lactobacillus plantarum* Vigo6), and a GM918 group (treated with *Lactobacillus plantarum* GM918), with 8 mice in each group. Bacterial suspension (1×10⁻⁶) was used. 8 CFU kg-1 Day 1) and DMF (25mg) kg-1 (Day 1) Mice were administered the drug orally via a gastric tube daily, while the control group received an equal volume of physiological saline via gavage daily for 8 weeks. All mice had free access to food and water. At 8 weeks, grip strength, stick time, movement distance, and muscle hemoglobin (HE) were measured in each group.

[0103] 5.1 Grip strength test To assess forelimb muscle strength in mice, grip strength tests were performed. The procedure was followed according to the manufacturer's instructions using a grip strength meter (Jiangsu Saions Biotechnology Co., Ltd., model SA415), and grip strength (gf) was the statistical indicator. When the mouse grasped the grip bar, it was pulled away from the base by grabbing its tail. Maximum grip strength was measured over 10 seconds. Results are shown in Table 9. Figure 10 As shown, compared with the control group, the forelimb grip strength of mice in the Vigo6 group was significantly improved, comparable to that in the DMF group, indicating that Lactobacillus plantarum Vigo6 has the effect of enhancing muscle strength.

[0104] Table 9 Results of forelimb gripping strength test (n = 8)

[0105] Note: Compared with the control group, **p<0.01, ***p<0.001.

[0106] 5.2 Rotating Rod Test To assess the coordination and balance of mice, they were placed on a rotating rotator (Jiangsu Saions Biotechnology Co., Ltd., model SA102). For three days prior to the formal rotator test, the mice underwent five minutes of training daily to learn how to use the device. The mice were held by their tails and placed on the rotating bar, facing the opposite direction of rotation. The time from the start of acceleration to the mouse falling was recorded. Each mouse underwent three tests, with at least a five-minute interval between each test, and the average time was calculated. The results are shown in Table 10. Figure 11 As shown, compared with the control group, the Vigo6 group mice had a 10.2% increase in rod time, and the difference was significant, indicating that Lactobacillus plantarum Vigo6 has the effect of enhancing the body's coordination and balance.

[0107] Table 10 Results of the rotating rod test (n = 8)

[0108] Note: Compared with the control group, ***p<0.001 and ****p<0.0001 were observed in the table.

[0109] 5.3 Movement Distance Test From a biomechanical perspective, poor physical performance is one of the main characteristics of sarcopenia. Before the experiment, mice underwent a 3-day adaptive treadmill training program (Jiangsu Saions Biotechnology Co., Ltd., model SA101B). Training was conducted 1-3 times daily, with 5-10 minute intervals between sessions. The total experimental duration was set at 15 minutes. The current intensity was 0.3 mA. After 3 days of treadmill adaptation, fatigue tests were performed on the experimental group mice. Mice began on the treadmill at a warm-up speed of 5 m / min uphill (20°) for 4 minutes, then the speed was increased to 14 m / min for 2 minutes, and then increased by 2 m / min until exhaustion. Exhaustion was defined as the inability to resume running within 10 seconds of direct contact with the electrical stimulation grid. Treadmill time and running distance were measured. The results are shown in Table 11 and... Figure 12 As shown, compared with the control group, the Vigo6 group had a significantly increased running distance (p<0.05), indicating that Lactobacillus plantarum Vigo6 can improve the body's endurance.

[0110] Table 11 Distance traveled (n = 8)

[0111] Note: Compared with the control group, *p<0.05, ***p<0.001.

[0112] 5.4 Muscle content and HE staining After the mice were euthanized, the four muscles of the hind limbs were carefully separated, including the gastrocnemius (GA), tibialis anterior (TA), extensor digitorum longus (EDL), and soleus (SOL), and weighed. A small piece of gastrocnemius muscle was fixed in 4% paraformaldehyde, and then subjected to dehydration, embedding, sectioning, H&E staining, and other steps for histopathological analysis of the muscle tissue.

[0113] The results showed that compared with normally aging individuals, the Vigo6 group had significantly higher levels of gastrocnemius, tibialis anterior, and soleus muscle content. Figure 13 The muscle content of the gastrocnemius and soleus muscles was comparable to that of the DMF positive control group, while the content of the tibialis anterior muscle was superior to that of the DMF group; the GM918 group showed no increase in muscle content. This indicates that *Lactobacillus plantarum* Vigo6 can increase muscle content and improve the control of balance, endurance, explosive power, and supporting muscle mass.

[0114] HE staining results showed that ( Figure 14 Compared with the normal group, the Vigo6 group significantly improved the morphology of the soleus muscle, increased the cross-sectional area of ​​muscle fibers (p<0.05), reduced the proportion of interstitial tissue, and improved the compactness of muscle fiber arrangement. The improvement effect was consistent with that of the DMF group, indicating that Lactobacillus plantarum Vigo6 can effectively improve muscle atrophy and aging.

[0115] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A plant lactobacillus ( Lactiplantibacillus plantarum ), characterized in that, The Lactobacillus plantarum mentioned is Lactobacillus plantarum Vigo6, with the accession number CGMCC No. 35628.

2. A microbial agent, characterized in that, The bacterial agent comprises *Lactobacillus plantarum* as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, The microbial agent includes any one or more of the following: fermentation broth of *Lactobacillus plantarum*, fermentation broth precipitate, fermentation broth supernatant, live bacterial cells, inactivated bacterial cells, lyophilized powder, lysate, lysate, secondary metabolites, and exosomes.

4. The use of the *Lactobacillus plantarum* as described in claim 1 or the bacterial agent as described in any one of claims 2-3 in the preparation of products that improve muscle function and athletic ability.

5. The application according to claim 1, characterized in that, The products mentioned include any one or more of the following: (1) Products that relieve physical fatigue; (2) Products that improve sarcopenia; (3) Products that promote the development of motor nerves; (4) Products that enhance athletic performance; (5) Products that promote muscle growth and enhance muscle strength; (6) Products that enhance the body's coordination and balance. (7) Products that enhance endurance and physical fitness; (8) Products that improve muscle quality, balance, endurance, explosive power, and support; (9) Products that improve muscle loss and aging.

6. The application according to claim 4, characterized in that, The aforementioned *Lactobacillus plantarum* Vigo6 improves muscle function and enhances athletic ability by promoting motor nerve development, improving muscle fiber morphology and density, increasing muscle mass, and / or strengthening the compactness of muscle fiber arrangement.

7. A product for improving muscle function and motor ability, characterized in that, The product comprises *Lactobacillus plantarum* as described in claim 1 or the bacterial agent as described in any one of claims 2-3.

8. The product according to claim 7, characterized in that, The product contains at least 1×10 8 CFU containing Lactobacillus plantarum Vigo6.

9. The product according to claim 7, characterized in that, The dosage form of the product includes solid dosage form, semi-solid dosage form, or liquid dosage form.

10. The product according to claim 7, characterized in that, The product also includes excipients, which include any one or more of the following: diluents, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH adjusters, antioxidants, and buffers.

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