Bifidobacterium animalis lactis and its use in products for enhancing athletic performance

Abstract

This invention discloses a subspecies of Bifidobacterium animalis (Lactobacillus lactis) and its application in products that enhance athletic performance, relating to the field of microbial technology. This invention provides a subspecies of Bifidobacterium animalis (Lactobacillus lactis) Bifidobacterium animalis subsp.lactis The *Bifidobacterium animalis* subsp. *lactamase* MUS+, as described in this invention, has the accession number CGMCC No. 35626. The *Bifidobacterium animalis* subsp. *lactamase* MUS+ provided by this invention can increase the length of peripheral motor nerves and promote motor nerve development; increase total motor distance and promote athletic ability and endurance; promote body length or height development; thicken muscle fibers, increase muscle fiber density, reduce interfiber spacing, and promote muscle development; and increase muscle strength. It has excellent application prospects in the preparation of products that enhance athletic ability or promote muscle growth.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a subspecies of Bifidobacterium lactis and its application in products that enhance athletic performance. Background Technology

[0002] Currently, maintaining and increasing muscle mass has become one of the core needs of public health management, encompassing not only professional fitness enthusiasts and athletes, but also middle-aged and elderly individuals experiencing sarcopenia due to aging, as well as those recovering from surgery and needing to restore muscle function. Sufficient muscle mass is directly related to physical performance, metabolic rate, immune function, and quality of life. Therefore, developing safe, effective, and long-term muscle-building products has urgent market demand and significant social value.

[0003] Currently, mainstream muscle-building methods primarily rely on a combination of "exercise stimulation + nutritional supplementation." The nutritional supplement market focuses on protein products (such as whey protein and casein), amino acids, and creatine as core ingredients. While these products can provide raw materials for muscle synthesis or improve energy supply to some extent, they generally have limitations: Firstly, some products (such as high-dose creatine) may cause gastrointestinal discomfort and other side effects, and pose a potential metabolic burden on liver and kidney function, making them unsuitable for individuals with weak liver or kidney function. Secondly, their mechanisms of action are mostly focused on raw material supply, failing to improve the muscle synthesis environment at the root level, such as the body's internal microecological balance and metabolic regulation. This makes muscle-building effects susceptible to individual differences, and their effectiveness is limited in individuals with insufficient exercise intensity or weak absorption. Furthermore, for middle-aged and elderly individuals, simple protein supplementation is insufficient to reverse the trend of muscle loss, necessitating a muscle-building program more tailored to their physiological characteristics.

[0004] Probiotics, as a core means of regulating the gut microbiota, have been widely used in digestive health, immune regulation, and other fields due to their high safety and good tolerability. However, current research on the association between probiotics and muscle growth is still in its early stages and has significant limitations. Studies have largely focused on certain strains of the Lactobacillus genus (such as Lactobacillus), with very little attention paid to the Bifidobacterium genus, especially Bifidobacterium animalis subsp. lactis. This strain, as a native human gut microbiota, has stronger intestinal colonization ability and safety, and its association with muscle metabolism has not yet been systematically explored.

[0005] Bifidobacterium animalis subsp. lactis, an important member of the Bifidobacterium genus, is a naturally occurring probiotic strain in breast milk with good human tolerance, particularly demonstrating excellent properties in regulating intestinal flora balance and promoting nutrient absorption (such as protein and calcium). Addressing the issues of significant side effects and limited mechanisms of action in existing muscle-building products, as well as the lack of application of probiotics in the field of muscle building, this study aims to screen for Bifidobacterium animalis subsp. lactis strains with definite muscle-building activity and develop their application in muscle-building products. This not only fills a technological gap but also provides the market with a safe, efficient, and widely applicable novel muscle-building solution, possessing significant scientific and market value. Summary of the Invention

[0006] The purpose of this invention is to provide a Bifidobacterium lactis subsp. animalis and its application in products that enhance athletic performance.

[0007] In this invention, the "gut-muscle axis" refers to the bidirectional signaling network established between the gut (especially gut microbiota) and skeletal muscle through multiple pathways, including neural, endocrine, immune, and metabolic pathways. It is a key regulatory system for maintaining skeletal muscle mass, function, and metabolic homeostasis. The core idea is that gut microbiota and its metabolites can act as important mediators, playing a crucial role in regulating the host's skeletal muscle synthesis and catabolism.

[0008] To achieve the above-mentioned objectives, the technical solution of the present invention is as follows:

[0009] In a first aspect, the present invention provides a subspecies of Bifidobacterium lactis (Bifidobacterium animalis) Bifidobacterium animalis subsp. lactis The Bifidobacterium animalis subsp. MUS+ has the accession number CGMCC No. 35626.

[0010] Specifically, the 16S sequence of Bifidobacterium lactis subsp. MUS+ is shown in SEQ ID NO:1.

[0011] Specifically, the Bifidobacterium animalis subsp. lactis MUS+ grows as white colonies in MRS agar medium. These colonies are opaque, round, smooth and moist, with neat edges and a raised center.

[0012] Secondly, the present invention provides a microbial agent comprising the aforementioned Bifidobacterium animalis subsp. lactis MUS+.

[0013] The bacterial agent includes one or more of the following: Bifidobacterium animalis subsp. lactis MUS+ cells, fermentation broth, fermentation broth supernatant, fermentation broth precipitate, and lyophilized powder.

[0014] The microbial agent also includes nutritionally acceptable nutrient additives.

[0015] The nutritional additives mentioned herein include, but are not limited to, any one or more of dietary fiber, prebiotics, protein, lipids, minerals, and vitamins.

[0016] Thirdly, the present invention provides a preparation of Bifidobacterium animalis subsp. lactis MUS+, comprising: fermentation broth, fermentation broth precipitate, fermentation broth supernatant, live bacteria, inactivated bacteria, lyophilized powder, lysate, lysate, secondary metabolites, and exosomes.

[0017] Specifically, the fermentation broth of Bifidobacterium animalis subsp. lactis MUS+ is a mixed liquid system obtained by culturing Bifidobacterium animalis subsp. lactis MUS+ in a culture medium under artificially controlled fermentation conditions. It contains the bacteria themselves, intracellular and extracellular metabolites, unused culture medium components, and fermentation byproducts.

[0018] Specifically, the precipitate of Bifidobacterium animalis subsp. lactis MUS+ fermentation broth is the solid phase component separated from the fermentation broth of the strain after treatment such as settling, centrifugation or filtration. It mainly includes live / dead cells of the strain, cell fragments and insoluble substances produced in the fermentation system.

[0019] Specifically, the supernatant of Bifidobacterium animalis subsp. lactis MUS+ fermentation broth is a clear liquid phase component containing extracellular metabolites of the strain, soluble culture medium residues, and soluble fermentation byproducts obtained after the fermentation broth of the strain has been allowed to stand, centrifuged, or filtered to remove solid phase precipitates such as bacterial cells.

[0020] Specifically, Bifidobacterium animalis subsp. lactis MUS+ live bacteria are bacteria with normal physiological activity and capable of carrying out life activities such as metabolism and reproduction.

[0021] Specifically, inactivated Bifidobacterium animalis subsp. lactis MUS+ refers to bacteria that have lost their metabolic and reproductive activities but whose overall structure has been basically preserved after being treated by physical, chemical or other means.

[0022] Specifically, Bifidobacterium animalis subsp. lactis MUS+ freeze-dried powder refers to a solid powder that retains the original active components, obtained by removing moisture from liquid materials such as strain fermentation broth, fermentation supernatant, and bacterial suspension through a freeze-drying process.

[0023] Specifically, Bifidobacterium animalis subsp. lactis MUS+ lysate refers to a mixture of intracellular substances and cell fragments formed after live or inactivated Bifidobacterium animalis subsp. lactis MUS+ cells are broken down by physical, chemical or enzymatic methods, releasing intracellular substances.

[0024] Specifically, Bifidobacterium animalis subsp. lactis MUS+ lysate refers to a mixed system containing intracellular active components and fragmented bacterial cells formed after live or inactivated Bifidobacterium animalis subsp. lactis MUS+ cells are ruptured through physical, chemical, enzymatic, or biological lysing methods to release all intracellular substances.

[0025] Specifically, the secondary metabolites of Bifidobacterium animalis subsp. lactis MUS+ refer to various compounds produced by microorganisms such as strains during their stable growth phase that are not essential for their own growth and reproduction and often possess specific biological activities such as anti-inflammatory and metabolic regulation.

[0026] Specifically, Bifidobacterium animalis subsp. lactis MUS+ exosomes refer to extracellular vesicles encapsulated by nanoscale lipid bilayer membranes that are actively secreted or released by Bifidobacterium animalis subsp. lactis during its growth and metabolism.

[0027] Fourthly, the present invention provides the application of the above-mentioned Bifidobacterium animalis subsp. lactis MUS+ or the above-mentioned bacterial agent or the above-mentioned preparation in the preparation of products that enhance athletic performance or build muscle.

[0028] Specifically, it includes any one or more of the following applications:

[0029] (1) Increase the length of peripheral motor nerves;

[0030] (2) Increase the total distance traveled;

[0031] (3) Increase body length;

[0032] (4) Thicken muscle fibers, increase muscle fiber density, and narrow the gaps between muscle fibers;

[0033] (5) Increase the range of increase or decrease in grip strength.

[0034] Specifically, it includes any one or more of the following applications:

[0035] (1) Promotes the development of motor nerves;

[0036] (2) Promotes athletic ability;

[0037] (3) Promotes body length development;

[0038] (4) Promotes muscle development;

[0039] (5) Improve muscle strength.

[0040] Specifically, the target population for this muscle-building regimen has the following characteristics:

[0041] (1) Poor motor nerve development;

[0042] (2) Poor athletic ability;

[0043] (3) Slow growth in height;

[0044] (4) Poor muscle development;

[0045] (5) Poor muscle strength.

[0046] Fifthly, the present invention provides a product for improving athletic performance or building muscle, wherein the product comprises the above-mentioned Bifidobacterium animalis subsp. lactis MUS+ or the above-mentioned bacterial agent or the above-mentioned preparation.

[0047] Specifically, the product comprises at least 1×10 8 CFU containing Bifidobacterium animalis subsp. MUS+.

[0048] According to some embodiments of the present invention, the product may include 1×10 8 CFU, 2×10 8 CFU, 3×10 8 CFU, 4×10 8 CFU, 5×10 8 CFU, 6×10 8 CFU, 7×10 8 CFU, 8×10 8 CFU, 9×10 8 CFU, 1×10 9 CFU, 1×10 10 CFU, 1×10 11 CFU, 1×10 12 CFU containing Bifidobacterium animalis subsp. MUS+.

[0049] Specifically, the dosage form of the product includes solid dosage form, semi-solid dosage form, or liquid dosage form.

[0050] Furthermore, the dosage forms of the product include, but are not limited to: liquid solutions, lyophilized powders, tablets, capsules, granules, sprays, oral dispersible tablets / films, sublingual tablets / lozenges, gels, creams / ointments, suppositories, in situ gels, compound preparations, or child-friendly formulations (flavored granules, oral drops).

[0051] Specifically, the products mentioned include, but are not limited to, pharmaceuticals and food.

[0052] Furthermore, the food products mentioned include, but are not limited to, dietary supplements and ordinary food products.

[0053] Furthermore, the dietary supplement uses Bifidobacterium animalis subsp. lactis MUS+ as its core functional ingredient and is added to probiotic supplements, probiotic capsules, and probiotic powders.

[0054] The common foods mentioned include, but are not limited to, beverages, dairy products, solid snacks, and fermented foods.

[0055] The beverages mentioned include, but are not limited to, at least one of the following: probiotic beverages, lactic acid bacteria beverages, and fruit juices.

[0056] The dairy products mentioned include, but are not limited to, at least one of yogurt, fermented milk, cheese, and ice cream.

[0057] The solid snacks include probiotic biscuits, probiotic oatmeal, and probiotic pastries.

[0058] The fermented foods include, but are not limited to: probiotic fermented soy products and probiotic fermented grain products.

[0059] The composite product is formed by combining Bifidobacterium animalis subsp. lactis MUS+ with prebiotics (such as fructooligosaccharides and inulin).

[0060] Specifically, the product may also include auxiliary materials.

[0061] Furthermore, the excipients 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.

[0062] Specifically, the product can be administered orally, topically, by spray, by inhalation, or a combination thereof.

[0063] In a sixth aspect, the present invention provides a method for improving athletic performance or building muscle, comprising administering to a subject an effective amount of Bifidobacterium animalis subsp. lactis MUS+.

[0064] The term "subject" includes living organisms (e.g., mammals) that can elicit an immune response. Examples of subjects include humans, primates, cattle, horses, goats, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, pigs, and their transgenic species.

[0065] The term "therapeutic effective dose" refers to a pharmaceutically considered effective dosage, that is, an amount of active drug sufficient to significantly improve the condition without causing serious side effects. Dosage depends on many factors, such as the nature and severity of the disease to be prevented or treated, the sex, age, weight, personality, and individual response of the patient or animal, the route of administration, frequency of administration, and therapeutic purpose; therefore, the dosage of this invention can vary widely.

[0066] The beneficial effects of this invention are as follows:

[0067] (1) The animal Bifidobacterium lactis subsp. MUS+ provided by the present invention can increase the length of peripheral motor nerves and promote motor nerve development; increase the total motor distance and promote motor ability; promote body length development; thicken muscle fibers, increase muscle fiber density, reduce the gap between muscle fibers and promote muscle development; and increase muscle strength.

[0068] (2) Bifidobacterium animalis subsp. lactis MUS+ can utilize 25 kinds of carbon sources, has strong antioxidant capacity, is non-pathogenic, has a good inhibitory effect on Cronobacter sakazakii, has a high survival rate in gastric juice and intestinal juice, with an adhesion index of 46.98 and an adhesion rate of 18.33%.

[0069] Preservation Instructions

[0070] Accession number: CGMCC No. 35626;

[0071] Classification and nomenclature: Bifidobacterium animalis subsp. lactis Bifidobacterium animalis subsp.lactis ;

[0072] Preservation period: August 15, 2025;

[0073] Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Culture Collections;

[0074] Abbreviation of depositary institution: CGMCC;

[0075] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0076] Figure 1 The colony morphology of Bifidobacterium animalis subsp. lactis MUS+.

[0077] Figure 2 Gram staining microscopic morphology of Bifidobacterium lactis subsp. MUS+.

[0078] Figure 3 Colony characteristics of Bifidobacterium animalis subsp. lactis MUS+ on Columbia blood agar plates.

[0079] Figure 4 The growth curve of Bifidobacterium animalis subsp. lactis MUS+.

[0080] Figure 5 The length (in pixels) of the peripheral motor nerves in zebrafish is given. Compared with the normal control group, ns indicates no significant difference. p<0.01.

[0081] Figure 6 This is a typical diagram showing the length of peripheral motor nerves in zebrafish. Note: The area within the yellow dashed box represents the peripheral motor nerves in the analysis region.

[0082] Figure 7 The total distance traveled by the zebrafish (mm) is given. Compared with the normal control group, ns indicates no significant difference. p<0.05, p<0.01.

[0083] Figure 8 This is a diagram showing the movement trajectory of a zebrafish.

[0084] Figure 9 Zebrafish body length development (pixels), compared with the normal control group, ns indicates no significant difference. p<0.01.

[0085] Figure 10 Image of zebrafish muscle (HE).

[0086] Figure 11 To intervene in the rate of change in grip strength at 8 weeks, p<0.05.

[0087] Figure 12 The change rate of grip strength in the MUS+ group at different time points is ns, p > 0.05. p<0.01, p<0.001, p<0.0001.

[0088] Figure 13 The strength increase at each muscle site was compared between the placebo group and the MUS+ group at 8 weeks of AE intervention (A. biceps brachii, B. vastus medialis, C. vastus lateralis, D. rectus abdominis, E. brachioradialis), and the strength increase at different intervention times was compared between the FJ MUS+ group (F. biceps brachii, G. vastus medialis, H. vastus lateralis, I. rectus abdominis, J. brachioradialis). Detailed Implementation

[0089] To make the technical means, creative features, and achieved objectives and effects of this invention easier to understand, the invention is further illustrated below with specific embodiments. However, the following embodiments are merely preferred embodiments of this invention and not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments described herein without creative effort are all within the protection scope of this invention. Unless otherwise specified, the operating methods and equipment used in the following embodiments are conventional operating methods, and the materials and equipment used in each embodiment are the same.

[0090] Example 1: Isolation, Identification, and Domestication of Bifidobacterium animalis subsp. Lactobacillus (MUS+)

[0091] Bifidobacterium strains were isolated from healthy breast milk samples. The samples were spread onto MRS anaerobic selective medium containing 0.5% cysteine ​​using a serial dilution method and anaerobically cultured at 37°C for 48 hours. Single colonies of Gram-positive bacilli exhibiting a forked "Y" or "V" shape were then picked and preliminarily identified as Bifidobacterium strains. Subsequently, targeted acclimatization was carried out.

[0092] For oxygen tolerance acclimatization, 100 μL of the isolated original strain was spread onto solid MRS medium and cultured at 37°C with an oxygen concentration of 0.1% for 48 h. Strains with larger colonies were selected and cultured again on MRS liquid medium at 37°C for 48 h. This process of spreading, culturing at 1% oxygen and 37°C, and selecting larger colonies was repeated approximately 30 times, with the oxygen concentration gradually increased to 0.5%, 1%, and 3%. The results of oxygen tolerance acclimatization are shown in Table 1. The final strains were then subjected to further gastric juice tolerance acclimatization.

[0093] Table 1 Results of oxygen tolerance acclimatization of strains

[0094]

[0095] Gastric juice acclimation: After centrifugation, the supernatant of the acclimated oxygen-tolerant strains was discarded. The cells were washed once with PBS buffer and then resuspended in artificial gastric juice (0.1g NaCl, 0.175g pepsin, 50mL water, fully dissolved, pH adjusted to 3 with dilute hydrochloric acid, mixed well, and filtered through a 0.22μm filter membrane under sterile conditions). After standing at 37℃ for 1.5h, 100μL of the bacterial suspension was spread onto solid MRS medium and incubated at 37℃ for 48h. Strains with larger colonies were selected.

[0096] Screening for resistance to intestinal fluid: The obtained gastric fluid-resistant strains were further screened. After activation for three generations, the strains were centrifuged and the supernatant was discarded. 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, and 100 ml water were mixed and the pH was adjusted to 8. The mixture was 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 onto solid MRS medium and incubated at 37°C for 48 h. Strains with larger colonies were selected.

[0097] The results of gastric juice tolerance and intestinal juice tolerance screening are shown in Table 2:

[0098] Table 2 Results of strains' acclimatization to gastrointestinal fluids

[0099]

[0100] Low-temperature cryopreservation: Superior strains acclimatized to oxygen, gastric juice, and intestinal juice were activated for three generations, frozen at -80℃ for 2 hours, thawed at room temperature, and then frozen again at -80℃, repeated three times. The thawed bacterial suspension was inoculated at 1% onto MRS liquid medium and cultured at 37℃ with an oxygen concentration of 3% for 24 hours, constituting one acclimatization generation. After 30 generations of acclimatization, 100 μL of bacterial suspension was spread onto solid MRS medium and cultured at 37℃ with an oxygen concentration of 3% for 48 hours. Strains with larger colonies were selected. Bacterial powder was prepared from the original strain and acclimatized strain under the same conditions and stored at 37℃ for one month to test survival rates.

[0101] Table 3 Results of low-temperature acclimatization

[0102]

[0103] As can be seen from Table 3 above, the survival rate of the strain after 30 generations of domestication is much higher than that of the original strain.

[0104] Finally, through the above domestication and screening, a strain with high oxygen tolerance, high intestinal tolerance, and high freeze resistance was obtained. It was identified as Bifidobacterium animalis subsp. lactis by 16S rDNA sequence and named Bifidobacterium animalis subsp. lactis MUS+. It was deposited at the China General Microbiological Culture Collection Center on August 15, 2025, with the accession number CGMCC No. 35626, and classified as Bifidobacterium animalis subsp. lactis.

[0105] The 16S rRNA sequencing results are shown in SEQ ID NO.1:

[0106]

[0107] Example 2: Detection of the physicochemical characteristics of the strain

[0108] 1. Morphological and colony observation, hemolytic characteristics

[0109] 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 and incubated at 37°C for 48 hours. The colony morphology was then observed.

[0110] The microbiological characteristics of Bifidobacterium animalis subsp. lactis MUS+ are as follows:

[0111] (1) Colony morphology: such as Figure 1 As shown, the colonies grown in MRS agar medium are white, opaque, round, with a smooth and moist surface, neat edges, and a raised center.

[0112] (2) Gram staining morphology: such as Figure 2 As shown, after staining, the bacteria morphology appears as short, rod-shaped rods, straight or curved, forked, arranged singly, in pairs or in a V-shape, Gram-positive, non-spore-forming, and non-motile.

[0113] (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.

[0114] 2. Utilization of different carbohydrates

[0115] 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.

[0116] 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.

[0117] 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.

[0118] Using a pipette, draw 115 µL of bacterial culture, placing the pipette tip against the edge of the cup to add it, avoiding air bubbles. Fill only the top of the tube (cup) 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 at 24 h and 48 h, with 48 h as the final result. The results are shown in Tables 4-6. The absorbance of Bifidobacterium animalis subsp. lactis MUS+ at OD600 is 0.90, and it can utilize 25 carbon sources after 48 h of culture.

[0119] Table 4. Identification results of Bifidobacterium animalis subsp. Lactobacillus MUS+

[0120]

[0121] Note: "V" represents a variable reaction, and the results may fluctuate under different experimental conditions or batches; "+" indicates availability; "-" indicates unavailability.

[0122] 3. Growth curve determination

[0123] Glyceryl animalis subsp. lactis MUS+ preserved in glycerol was inoculated into sterile MRS broth at a 1% inoculum and anaerobically cultured 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 anaerobic culture 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 purged with nitrogen, sealed, and shaken for culture. OD600 was measured every hour.

[0124] The results are as follows Figure 4 The results showed that Bifidobacterium animalis subsp. lactis MUS+ entered the logarithmic growth phase at 12 hours and the stationary phase at 25 hours, with an OD600 value of approximately 1.51 at the stationary phase.

[0125] 4. Antioxidant capacity

[0126] After MUS+ activation, the cells were cultured for three generations to prepare cell lysates and fermentation broth supernatants. 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 5.

[0127] Table 5 Antioxidant capacity of Bifidobacterium animalis subsp. lactis MUS+

[0128]

[0129] 5. Toxicity testing and safety assessment

[0130] The pathogenicity test method of 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 Bifidobacterium lactis subsp. lactis MUS+. The tested animals showed no abnormalities or deaths, and their body weight was not statistically significant compared with the control group (p>0.05), indicating that the strain was non-pathogenic.

[0131] 6. Detection of ability to inhibit pathogenic bacteria

[0132] 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 10min, the supernatant was collected, filtered through a 0.22μm micromembrane, and stored at -20℃ for later use.

[0133] After activating the pathogenic bacteria (Cronobacter sakazakii CICC 21560) three times in liquid culture medium, the third-generation culture medium was adjusted to a suitable absorbance value, resulting in a bacterial suspension concentration of 1×10⁻⁶. 8 CFU / mL ~5×10 8 CFU / mL.

[0134] 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 wait for it to solidify before use.

[0135] 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 6.

[0136] Table 6. Diameter of the outer inhibition zone of Bifidobacterium animalis subsp. lactis MUS+ strain against pathogens.

[0137]

[0138] The results showed that Bifidobacterium animalis subsp. lactis MUS+ had a good inhibitory effect on Cronobacter sakazakii.

[0139] 7. Gastrointestinal fluid tolerance test

[0140] 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.

[0141] 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.

[0142] 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.

[0143] 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 7 below.

[0144] Table 7. Statistical analysis of viable bacteria count of Bifidobacterium lactis subsp. MUS+ in artificial gastric and intestinal fluids.

[0145]

[0146] 8. Adhesion ability test

[0147] Strain preparation: Activate the MUS+ 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 cell concentration to approximately 1×10⁻⁶ in DMEM medium. 6 CFU / mL.

[0148] 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, 1mL / well, twice. The washing process should be gentle to avoid adhering cells detaching.

[0149] 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 hours. 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 cells in each well of 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.

[0150] Adhesion index = Number of viable bacteria after adhesion / Number of cells in negative control well;

[0151] Adhesion rate (%) = Number of viable bacteria after adhesion / Number of viable bacteria before adhesion.

[0152] The results of the strain adhesion experiment are shown in Table 8.

[0153] Table 8 Adhesion ability of Bifidobacterium animalis subsp. lactis MUS+ strain

[0154]

[0155] Example 3: Freeze-drying process and preparation of Bifidobacterium animalis subsp. lactis MUS+ powder

[0156] Bifidobacterium animalis subsp. lactis MUS+ was subcultured and activated three times at 37℃ using MRS medium. Then, the bacterial culture was inoculated at 5% in MRS liquid medium for expansion 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.

[0157] 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.

[0158] Example 4: Effects of bacterial strains on the locomotion ability of zebrafish

[0159] 1. Animals and drug administration

[0160] Three days post-fertilization (3 dpf) transgenic motor neuron-bearing green fluorescent zebrafish (Hb9) were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). The MUS+ and GM9-3 groups were respectively administered water-soluble samples (1 × 10⁻⁶). 8CFU / mL (total dose), positive control whey protein (31.2 μg / mL), and a normal control group were set up at the same time, with a volume of 3 mL per well.

[0161] 2. Evaluation of the promotion of motor nerve development

[0162] 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.

[0163] The results are shown in Table 9 and Figures 5-6 As shown, compared with the normal group, the MUS+ group and the whey protein group showed a significant increase in peripheral motor nerve length, and the differences were highly significant (p < 0.01). However, compared with the normal group, the GM9-3 probiotic group (GM9-3 strain isolated from healthy breast milk, animal experimental control strain of Bifidobacterium lactis) showed no statistically significant difference in peripheral motor nerve length (p > 0.05). This indicates that the MUS+ strain has a role in promoting motor nerve development.

[0164] Table 9. Results of the experiment evaluating the promotion of motor nerve development (n = 10)

[0165]

[0166] Compared with the normal control group, p<0.05, p<0.01.

[0167] 3. Promote the evaluation of athletic ability

[0168] After treatment at 28℃ for 3 days, 10 zebrafish were randomly selected from each experimental group and placed in a 96-well plate. The total movement distance of the zebrafish was measured using a behavior analyzer. The statistical analysis results of this index (total movement distance) were used to evaluate the growth-promoting effect of the samples. Statistical results are expressed as mean ± SE. Statistical analysis was performed, and p < 0.05 indicated a statistically significant difference.

[0169] The results are shown in Table 10 and Figures 7-8 As shown, compared with the normal group, the total movement distance of the MUS+ group was significantly increased, with a mean of 1.65 times that of the normal group, and the difference was highly significant (p < 0.01). Compared with the whey protein group, the mean total movement distance of the MUS+ group was higher. There was no statistically significant difference in total movement distance between the GM9-2 group of probiotics (also Bifidobacterium animalis subsp. lactis) and the normal group (p > 0.05). This indicates that the MUS+ strain has the effect of enhancing motility.

[0170] Table 10 Results of the experiment on promoting motor ability assessment (n = 10)

[0171]

[0172] Note: Compared with the normal control group, p<0.05, p<0.01.

[0173] 4. Evaluation of promoting physical length development

[0174] After treatment at 28℃ for 3 days, 10 zebrafish were randomly selected from each experimental group, photographed under a fluorescence microscope, and analyzed and collected data using NIS-ElementsD3.20 advanced image processing software to analyze the zebrafish body length.

[0175] The results are shown in Table 11 and Figure 9 As shown, compared with the normal group, the body length of the MUS+ group was significantly increased, and the difference was highly significant (p < 0.01); the body length of the whey protein group and the GM9-3 group was not statistically significant compared with the normal group (p > 0.05). This indicates that the MUS+ strain promotes body length development.

[0176] Table 11 Results of the experiment on promoting body length development (n = 10)

[0177]

[0178] Note: Compared with the normal control group, p<0.05, p<0.01.

[0179] 5. Evaluation of Muscle Development Promotion

[0180] 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.

[0181] The results are as follows Figure 10 As shown, the zebrafish skeletal muscle cells in the normal group and GM9-3 group are thin and long, fibrous, with muscle fibers distributed throughout the body compartments, but the overall structure is relaxed, with gaps between the fibers. The zebrafish skeletal muscle cells in the MUS+ group and whey protein group are thin and long, and compared with the normal group, the muscle fibers are significantly thicker, the density is increased, and the gaps between the muscle fibers are reduced, which has a significant effect on promoting muscle development.

[0182] Example 5: Effects of the strain on human muscle

[0183] Ten male and ten female volunteers aged 20-50 years with good health, no chronic diseases, and no antibiotic use within the past 15 days were randomly divided into two groups of 10 each, with a male-to-female ratio of 1:1. Participants took either MUS+ (20 billion CFU / day) or a placebo (maltodextrin) daily for 8 weeks, maintaining their daily activities and refraining from any exercise. Grip strength was tested in both hands at weeks 0, 4, and 8 using a grip strength meter, and strength-related parameters (Young's modulus) of the biceps brachii, brachioradialis, rectus femoris, vastus medialis, and vastus lateralis muscles were measured using a soft tissue biomechanical quantitative platform.

[0184] The results are as follows Figures 11-12 As shown, after 8 weeks of intervention, the strength of both hands in the MUS+ group increased to varying degrees, and the growth rate was statistically significant compared with the placebo group. Figure 11 (P < 0.05); In the MUS+ group, the left-hand strength of the subjects at week 4 was significantly different from that at week 0 (P < 0.05). Figure 12 P < 0.01, right-hand strength improved, but the difference was not statistically significant. Figure 12 (P < 0.01); In the MUS+ group, the left-hand strength of the subjects at week 4 was significantly different from that at week 0 (P < 0.01). Figure 12 P < 0.01, right-hand strength improved, but the difference was not statistically significant. Figure 12 (P < 0.01).

[0185] After 8 weeks of intervention, the increase in grip strength in both hands was significantly greater in the MUS+ group than in the placebo group. Figure 11 (Left hand: p<0.05; Right hand: p<0.05). The MUS+ group showed an overall trend of improved grip strength. MUS+ effectively promoted the enhancement of upper limb muscle strength, and the effect was relatively balanced on both sides.

[0186] Left hand grip strength: 0 weeks → 4 weeks → 8 weeks, the rate of increase in grip strength showed a gradual upward trend. Figure 12 At 4 weeks, p < 0.01; at 8 weeks, p < 0.0001, and the difference between 4 and 8 weeks was statistically significant (p < 0.0001). Right-hand grip strength: From 0 to 4 weeks, the increase in grip strength was noticeable, but not statistically significant. Figure 12 At 4 weeks, p > 0.05; however, at 8 weeks, the increase was significantly different (p < 0.001). This indicates that the promoting effect of MUS+ on left-hand grip strength strengthens with the extension of intervention time, and there is a "time-effect" relationship.

[0187] The biceps brachii, rectus femoris, vastus medialis, and vastus lateralis muscles are tested for their force exertion against constant resistance. A smaller value indicates less muscle force exertion against constant resistance, signifying greater muscle strength. The brachioradialis muscle is tested for its maximum force exertion; a larger value indicates greater force exertion, signifying greater muscle strength. From... Figure 13It can be seen that after 8 weeks of intervention, the increase in muscle strength at all points was statistically significant in both the placebo group and the MUS+ group. Figure 13 A- Figure 13 In the MUS+ group, muscle strength at all points showed positive improvement. The increase rate of brachioradialis strength was significantly different from the placebo group (p<0.0001), while the increases in biceps brachii, vastus medialis, and rectus abdominis strength were also significantly different (p<0.001), and the increase in vastus lateralis strength was significantly different (p<0.01). Analysis of intervention time showed that the significance of each muscle site in the MUS+ group gradually increased with the increase of intervention time, exhibiting a time-effect relationship. Figure 13 F- Figure 13 J in the middle.

[0188] In conclusion, the MUS+ strain can significantly improve muscle strength in different parts of the body, and the increase in muscle strength gradually increases with the duration of intervention.

[0189] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A subspecies of Bifidobacterium lactis ( Bifidobacterium animalis subsp. lactis MUS+, characterized in that, The Bifidobacterium animalis subsp. lactis MUS+ has the accession number CGMCC No. 35626.

2. A microbial agent, characterized in that, The bacterial agent includes Bifidobacterium animalis subsp. lactis MUS+ as described in claim 1.

3. The preparation of *Bifidobacterium animalis* subsp. *lactum* MUS+ according to claim 1, characterized in that, include: Fermentation broth, live bacteria, freeze-dried powder.

4. The use of the Bifidobacterium lactis subsp. MUS+ of claim 1, the bacterial agent of claim 2, or the preparation of claim 3 in the preparation of products that enhance athletic performance or build muscle.

5. The application according to claim 4, characterized in that, Including any one or more of the following applications: (1) Increase the length of peripheral motor nerves; (2) Increase the total distance traveled; (3) Increase body length; (4) Thicken muscle fibers, increase muscle fiber density, and narrow the gaps between muscle fibers; (5) Increase the range of increase or decrease in grip strength.

6. The application according to claim 5, characterized in that, Including any one or more of the following applications: (1) Promotes the development of motor nerves; (2) Promotes athletic ability; (3) Promotes body length development; (4) Promotes muscle development; (5) Improve muscle strength.

7. A product for improving athletic performance or building muscle, characterized in that, The product comprises Bifidobacterium animalis subsp. lactis+ as described in claim 1, or the bacterial agent as described in claim 2, or the preparation as described in claim 3.

8. The product according to claim 7, characterized in that, The content of Bifidobacterium animalis lactis MUS+ in the product is not less than 1 x 10 8 CFU.

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; The product 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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