Lactobacillus aeruginosa with muscle protection function during fat loss, its products, and applications.

Abstract

This invention discloses a strain of *Lactobacillus delbrueckii* with muscle-protecting function during fat loss, its products, and applications, belonging to the field of microbial technology. The invention discloses *Lactobacillus delbrueckii* subspecies Turb8, with accession number CGMCC No. 35627. This strain or its preparations can effectively prevent or improve muscle loss and functional decline during the use of fat-loss drugs such as GLP-1 receptor agonists (e.g., liraglutide, smegglutide), achieving the effect of "fat loss without muscle loss." While maintaining the original weight-loss effect of fat-loss drugs, the strain of this invention can significantly increase the user's lean body mass percentage, increase lower limb muscle mass, increase muscle fiber cross-sectional area, and improve muscle function indicators such as limb strength and suspension endurance. This invention also relates to bacterial agents containing this strain, various preparations, and their application in the preparation of products for improving (especially drug-induced) muscle loss.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a type of Lactobacillus deutschneidera sinensis with muscle protection function during fat loss, its products, and applications. Background Technology

[0002] Weight loss has become a widespread health concern. Drug interventions, such as those using GLP-1 receptor agonists (e.g., liraglutide, smegglutide), have achieved significant results in effective weight loss. However, while these drugs promote fat reduction, they often accompany a significant decrease in skeletal muscle mass and function—a phenomenon known as muscle loss alongside fat loss. This can lead to weakened muscle strength, decreased endurance, a lower basal metabolic rate, and even an increased risk of falls and fractures, severely impacting the quality of life and long-term health of those trying to lose weight.

[0003] However, in the clinical application and actual use of weight loss drugs, the side effects of "losing muscle while losing fat" have gradually become prominent and have become a core pain point. As the core carrier of human metabolism and motor function, the loss of skeletal muscle quality will directly lead to a series of health problems: a significant decrease in muscle strength and endurance, affecting daily activities and athletic performance; a decrease in basal metabolic rate, increasing the risk of rebound after weight loss; weakened skeletal support, increasing the incidence of falls and fractures in the elderly and those trying to lose fat; and in the long term, it may also lead to complications such as sarcopenia and metabolic syndrome, seriously undermining the long-term health value of fat loss.

[0004] Current interventions for muscle protection during fat loss have significant limitations: First, nutritional supplementation strategies, such as increasing protein intake and taking protein supplements, can only provide raw materials for muscle synthesis through exogenous nutrition and cannot prevent drug-induced muscle breakdown at the metabolic regulation level, thus having limited effectiveness for those undergoing drug intervention. Second, exercise intervention strategies, such as resistance training, can stimulate muscle synthesis, but are limited by the exercise capacity, time cost, and compliance of individuals, making it difficult to cover special fat loss groups such as the elderly, post-operative patients, and those who are sedentary. Third, existing probiotic products mostly focus on gut microbiota regulation, simple weight loss, or improvement of metabolic indicators, and have not been developed for the specific scenario of "drug-induced weight loss," lacking targeted protection functions for core indicators such as muscle mass, muscle strength, and endurance.

[0005] Relevant patent documents retrieved:

[0006] This document, published in China (CN112136897A) on December 29, 2020, discloses a high-protein nutritional meal replacement powder. Its raw materials include: milk powder, cereal powder, whey protein powder, protein synthesis promoter, dietary fiber, thickener, fish oil powder, compound vitamin powder, compound mineral powder, freeze-dried fruit and vegetable granules, flavoring, xylitol, β-carotene, and Bifidobacterium animalis subsp. lactis i797. The nutritional meal replacement powder provided by this invention is high in protein and low in fat, digests and absorbs quickly, increases muscle strength, promotes intestinal peristalsis to relieve constipation, and regulates intestinal microbiota. The product can be mixed into a thick porridge-like consistency, which not only solves swallowing difficulties for the elderly and patients but also provides a significant feeling of fullness, meeting the consumption experience and needs of consumers of different ages for meal replacement, fat loss, and muscle gain.

[0007] The prior art represented by the aforementioned documents has at least the following unresolved technical problems or defects:

[0008] Literature CN112136897A only discloses that nutritional meal replacement powder can increase muscle mass after 3 months of consumption, but it does not address the muscle loss caused by fat loss. Therefore, developing a probiotic strain that can precisely target and protect muscle quality and function while ensuring safety and effectiveness in the context of weight loss drug intervention would fill a technological gap and meet the core need of people trying to lose fat without losing muscle, which has significant clinical significance and market application prospects. Summary of the Invention

[0009] The purpose of this invention is to provide:

[0010] A type of Lactobacillus deutschneidera sinensis with muscle protection function during fat loss, its products, applications, and related technologies, in order to solve the technical problem of providing a type of Lactobacillus deutschneidera sinensis with muscle protection function during fat loss, or a combination thereof.

[0011] Terminology Explanation:

[0012] 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.

[0013] 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.

[0014] Definitions of standard chemical terms can be found in the references *Molecular Cloning: A Laboratory Manual*, Cold Spring Harbor Laboratory Science Press, 4th edition, 2017; and *Microbiology Experiments*, Higher Education Press, 4th edition, 2016.

[0015] Unless otherwise stated, conventional methods within the scope of the art, such as sequencing, strain culture, strain fermentation, strain activation, freeze drying, gavage, subcutaneous injection, etc., shall be used.

[0016] 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.

[0017] The term “CFU (colony forming unit)” used in this article refers to colony forming unit.

[0018] The term "culture medium" used in this article refers to the nutrient substrate for the growth and reproduction of microorganisms. Based on the different contents of the solidifying agent, it can be divided into three types: solid culture medium, semi-solid culture medium (semi-liquid culture medium), and liquid culture medium.

[0019] In a first aspect, the present invention provides a *Lactobacillus delbrueckii* subsp. *lactotrichum* (… Lactobacillus delbrueckii subsp.lactis Turb8, the Lactobacillus delbrueckii subsp. Turb8, has the accession number CGMCC No. 35627.

[0020] The 16S sequence of Lactobacillus delbrueckii subsp. Turb8, a technical feature, is shown in SEQ ID NO:1.

[0021] Among them, the technical characteristics of Lactobacillus delbrueckii subsp. Turb8 in MRS agar medium are that the colonies are white, opaque, raised, round, with a smooth and moist surface and neat edges.

[0022] Secondly, the present invention provides a microbial agent, wherein the microbial agent includes the aforementioned Lactobacillus delbrueckii subsp. Turb8.

[0023] The bacterial agent includes one or more of the following: Lactobacillus delbrueckii subsp. Turb8 cells, fermentation broth, fermentation broth supernatant, fermentation broth precipitate, and lyophilized powder.

[0024] The microbial agent also includes nutritionally acceptable nutrient additives.

[0025] The nutritional additives mentioned herein include, but are not limited to, any one or more of dietary fiber, prebiotics, protein, lipids, minerals, and vitamins.

[0026] Thirdly, the present invention provides a preparation of Lactobacillus delbrueckii subsp. Turb8, comprising: fermentation broth, fermentation broth precipitate, fermentation broth supernatant, live bacteria, inactivated bacteria, lyophilized powder, lysate, lysate, secondary metabolites, and exosomes.

[0027] Specifically, the fermentation broth of Lactobacillus delbrueckii subsp. Turb8 is a mixed liquid system obtained by culturing Lactobacillus delbrueckii subsp. Turb8 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.

[0028] Specifically, the precipitate of Lactobacillus delbrueckii subsp. Turb8 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.

[0029] Specifically, the supernatant of Lactobacillus delbrueckii subsp. Turb8 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.

[0030] Specifically, live Lactobacillus delbrueckii subsp. Turb8 is a bacterium with normal physiological activity, capable of carrying out life activities such as metabolism and reproduction.

[0031] Specifically, inactivated Lactobacillus delbrueckii subsp. Turb8 refers to bacteria that have lost their metabolic and reproductive activities but whose overall structure has been basically preserved after being treated by physical or chemical means.

[0032] Specifically, Lactobacillus delbrueckii subsp. lactis Turb8 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.

[0033] Specifically, Lactobacillus delbrueckii subsp. Turb8 lysate refers to a mixture of intracellular substances and cell fragments formed after live or inactivated Lactobacillus delbrueckii subsp. Turb8 cells are broken down by physical, chemical, or enzymatic methods, releasing intracellular substances.

[0034] Specifically, Lactobacillus delbrueckii subsp. Turb8 lysate refers to a mixed system containing intracellular active components and fragmented bacterial cells formed after live or inactivated Lactobacillus delbrueckii subsp. Turb8 bacteria are ruptured through physical, chemical, enzymatic, or biological lysation methods to release all intracellular substances.

[0035] Specifically, the secondary metabolites of Lactobacillus delbrueckii subsp. Turb8 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.

[0036] Specifically, Lactobacillus delbrueckii subsp. lactis Turb8 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.

[0037] Fourthly, the present invention provides a composition comprising the above-described Lactobacillus delbrueckii subsp. Turb8 or the above-described bacterial agent.

[0038] Composition: a product comprising an active ingredient and an inert component (pharmaceutically acceptable excipient) constituting a carrier, and any product obtained directly or indirectly from a combination, complexation or aggregation of two or more components, or from the decomposition of one or more components, or from other types of reactions or interactions of one or more components.

[0039] The active ingredient can be in solid and liquid dosage forms, such as capsules, tablets, lozenges, sugar lozenges, granules, and powders, and liquid dosage forms such as elixirs, syrups, emulsions, dispersions, and suspensions. Other dosage forms include ointments, creams, drops, transdermal patches, or powders; ophthalmic solutions or suspensions for use in the eyes, i.e., eye drops; and gelatin capsules containing the active ingredient and a powdered carrier, such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, etc. Similar diluents can be used to prepare compressed tablets. Both tablets and capsules can be formulated as sustained-release products for sustained release over several hours. Compressed tablets can be sugar-coated or film-coated to mask any unpleasant taste and protect the tablet from air, or they can be enteric-coated for selective disintegration in the gastrointestinal tract. Generally, water, suitable oils, saline solutions, aqueous solutions of dextrose (glucose), and related sugar solutions, as well as glycols such as propylene glycol or polyethylene glycol, are suitable carriers for parenteral solutions. The parenteral solution preferably contains a water-soluble salt of the active ingredient, a suitable stabilizer, and a buffer substance as needed. Antioxidants such as sodium bisulfite, sodium sulfite, or ascorbic acid, alone or in combination, are suitable stabilizers. Citric acid and its salts, and sodium EDTA can also be used. Furthermore, the parenteral solution may contain preservatives such as benzalkonium chloride, methylparaben, or propylparaben, and chlorobutanol. The compositions of the present invention can be conveniently delivered in aerosol form from pressurized packaging or a sprayer.

[0040] The composition described herein has a muscle protection function during the fat loss period.

[0041] Furthermore, the medications used for weight loss may include: liraglutide, smegglutide, telpoglycinide, loxenatide, orlistat, etc.

[0042] Specifically, the composition has any one or more of the following functions:

[0043] (1) It has no effect on weight loss;

[0044] (2) Improve limb strength;

[0045] (3) Improve muscle endurance;

[0046] (4) Increase lean body mass;

[0047] (5) Increase the relative mass of lower limb muscles;

[0048] (6) Maintain the mass of the gastrocnemius muscle and increase the cross-sectional area of ​​the gastrocnemius muscle fibers.

[0049] Specifically, the composition has any one or more of the following functions:

[0050] (1) Maintain the fat-reducing effect of the fat-reducing product;

[0051] (2) Improve muscle function in multiple dimensions and ensure the integrity of muscle function;

[0052] (3) Improve endurance, physical fitness and strength.

[0053] Furthermore, improving muscle function in multiple dimensions and ensuring the integrity of muscle function specifically involves: increasing lower limb muscle strength and maintaining the muscle function of the gastrocnemius muscle.

[0054] Furthermore, improving endurance, fitness, and strength specifically involves: increasing grip strength, increasing hang time, increasing lean body mass, preventing muscle loss, and increasing strength.

[0055] The composition contains at least 1 × 10⁸ Lactobacillus delbrueckii subsp. Turb8 bacteria. 8 CFU.

[0056] According to some embodiments of the present invention, the bacterial content of *Lactobacillus delbrueckii* subsp. *Turb8* may include 1 × 10⁸. 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.

[0057] Furthermore, the bacterial count of *Lactobacillus delbrueckii* subsp. Turb8 is not less than 1 × 10⁸. 9 CFU.

[0058] The composition also includes excipients;

[0059] Furthermore, the excipients are selected from any one or more of the following: diluents, excipients, fillers, disintegrants, solubilizers, osmotic pressure regulators, surfactants, pH adjusters, and antioxidants.

[0060] The composition may be in any one or more of the following forms: powder, tablet, emulsion, pill, ointment, powder, lyophilized powder for injection, gel, drops, tincture, capsule, granule or aerosol.

[0061] Fifthly, the present invention provides the use of the above-mentioned Lactobacillus delbrueckii subsp. Turb8 or the above-mentioned bacterial agent or the above-mentioned preparation in the preparation of a composition having muscle protection function during fat loss.

[0062] Specifically, the composition has any one or more of the following functions:

[0063] (1) It has no effect on weight loss;

[0064] (2) Improve limb strength;

[0065] (3) Improve muscle endurance;

[0066] (4) Increase lean body mass;

[0067] (5) Increase the relative mass of lower limb muscles;

[0068] (6) Maintain the mass of the gastrocnemius muscle and increase the cross-sectional area of ​​the gastrocnemius muscle fibers.

[0069] Specifically, the composition has any one or more of the following functions:

[0070] (1) Maintain the fat-reducing effect of the fat-reducing product;

[0071] (2) Improve muscle function in multiple dimensions and ensure the integrity of muscle function;

[0072] (3) Improve endurance, physical fitness and strength.

[0073] The composition contains at least 1 × 10⁸ Lactobacillus delbrueckii subsp. Turb8 bacteria. 8 CFU.

[0074] The composition also includes excipients;

[0075] Furthermore, the excipients are selected from any one or more of the following: diluents, excipients, fillers, disintegrants, solubilizers, osmotic pressure regulators, surfactants, pH adjusters, and antioxidants.

[0076] The composition may be in any one or more of the following forms: powder, tablet, emulsion, pill, ointment, powder, lyophilized powder for injection, gel, drops, tincture, capsule, granule or aerosol.

[0077] The present invention has at least the following beneficial effects:

[0078] (1) Lactobacillus delbrueckii subsp. Turb8 can utilize 25 kinds of carbon sources, has strong antioxidant capacity, is non-pathogenic, and has a good inhibitory effect on Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus faecalis, Shigella flexneri, and Cronobacter sakazakii. It has a high survival rate in gastric juice and intestinal juice (62.92% survival rate in gastric juice and 99.67% survival rate in intestinal juice), an adhesion index of 2.07, an adhesion rate of 72.30%, and a self-polymerization ability of 39.7%, which shows that it has a strong self-polymerization ability.

[0079] (2) Lactobacillus delbrueckii subsp. Turb8 has a muscle-protecting function during fat loss, providing a new probiotic solution for "fat loss without muscle loss." It has no impact on the fat loss effect of fat loss products, and at the same time, by targeting the muscle protection pathway, it significantly increases lean body mass, lower limb muscle mass, and muscle fiber cross-sectional area. It improves endurance, physical fitness, and strength. It has multi-dimensional muscle function improvement capabilities, not only maintaining muscle mass, but also significantly improving muscle strength (such as limb grip strength) and endurance (such as exercise suspension time) in people trying to lose fat, comprehensively ensuring the integrity of muscle function. It is suitable for people who are using GLP-1 receptor agonists or other weight loss drugs to help maintain muscle mass and function.

[0080] Considering the possibility of this invention entering other countries, this invention also provides the following technical solutions:

[0081] This invention provides a method for muscle protection during a fat loss period, comprising administering an effective amount of Lactobacillus delbrueckii subsp. Turb8 to the subject.

[0082] Among them, the bacterial content of *Lactobacillus delbrueckii* subsp. Turb8 should not be less than 1×10⁻⁶. 8 CFU.

[0083] Subjects include 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.

[0084] Preservation Instructions

[0085] Preserved strain: Lactobacillus delbrueckii subsp. Turb8;

[0086] Classification and nomenclature: Lactobacillus delbrueckii subsp. lactis Lactobacillus delbrueckii subsp.lactis ;

[0087] Accession number: CGMCC No. 35627;

[0088] Preservation period: August 15, 2025;

[0089] Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Culture Collections;

[0090] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0091] Figure 1 The colony morphology of Lactobacillus delbrueckii subsp. Turb8.

[0092] Figure 2 Gram staining microscopic morphology of Lactobacillus delbrueckii subsp. Turb8.

[0093] Figure 3 The colony characteristics of Lactobacillus delbrueckii subsp. Turb8 on Columbia blood agar plates.

[0094] Figure 4 The growth curve of Lactobacillus delbrueckii subsp. Turb8 is shown.

[0095] Figure 5 The analysis of body weight differences in mice at the end of the 20-week intervention is shown in the figure. In the figure, a represents the comparison with the high-fat diet group, and b represents the comparison with the liraglutide group. ns indicates p > 0.05. This means p < 0.0001.

[0096] Figure 6 The figure shows the grip strength of the limbs of mice at the end of the 20-week intervention. In the figure, a is the comparison with the high-fat diet group, and b is the comparison with the liraglutide group. ns indicates p > 0.05. This indicates that p < 0.01. This indicates that p < 0.001. This means p < 0.0001.

[0097] Figure 7 The suspension time of mice at the end of the 20-week intervention period is shown in the figure. Figure a represents the comparison with the high-fat diet group, and figure b represents the comparison with the liraglutide group. ns indicates p > 0.05. This indicates that p < 0.05. This means p < 0.0001.

[0098] Figure 8 The percentage of lean body mass in mice at the end of week 20 after intervention is shown in the figure. In the figure, a represents the comparison with the high-fat diet group, and b represents the comparison with the liraglutide group. ns indicates p > 0.05. This indicates that p < 0.01. This indicates that p < 0.001. This means p < 0.0001.

[0099] Figure 9 The figure shows the relative lower limb muscle mass of mice at the end of week 20 of the intervention. In the figure, a represents the comparison with the high-fat diet group, and b represents the comparison with the liraglutide group. ns indicates p > 0.05. This indicates that p < 0.05. This indicates that p < 0.01. This indicates that p < 0.001. This means p < 0.0001.

[0100] Figure 10 HE staining of cross-section of mouse gastrocnemius muscle fibers.

[0101] Figure 11 The figure shows the cross-sectional area of ​​the gastrocnemius muscle in mice at the end of the intervention. In the figure, a represents the comparison with the high-fat diet group, and b represents the comparison with the liraglutide group. ns indicates p > 0.05. This indicates that p < 0.05. This indicates that p < 0.01. This means p < 0.0001. Detailed Implementation

[0102] Unless otherwise specified, all raw materials and reagents used in this invention were purchased from commercial suppliers, and experiments were conducted in accordance with the operating instructions. Unless otherwise specified, all instruments, equipment, and apparatus used in this invention are conventional instruments, equipment, and apparatus, and experiments were conducted in accordance with the operating instructions and the accompanying reagents.

[0103] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified in the embodiments, conditions are performed under conventional conditions or conditions recommended by the manufacturer. All reagents or instruments without specified manufacturers are commercially available conventional products. Numerous specific details are provided in the following detailed embodiments to better illustrate the invention. The specific embodiments described herein are for illustrative purposes only and are not intended to constitute any limitation on the invention.

[0104] Data analysis and statistical analysis were performed using professional data processing software. One-way ANOVA was used for significance analysis, and P<0.05 was considered to indicate a significant difference.

[0105] Example 1: Isolation and Identification of Turb8 Strains

[0106] Isolated from healthy breast milk, and identified by 16S rDNA sequencing, it was identified as *Lactobacillus delbrueckii* subsp. *delbrueckii*, named *Lactobacillus delbrueckii* subsp. *delbrueckii* Turb8, 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. 35627, and classified as *Lactobacillus delbrueckii* subsp. *delbrueckii*. Lactobacillus delbrueckii subsp.lactis .

[0107] The 16S rRNA sequencing results are shown in SEQ ID NO.1:

[0108]

[0109] Example 2: Detection of the physicochemical characteristics of the strain

[0110] 1. Morphological and colony observation, hemolytic characteristics

[0111] 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.

[0112] The microbiological characteristics of Lactobacillus delbrueckii subsp. Turb8 are as follows:

[0113] (1) Colony morphology: such as Figure 1 As shown, the colonies grown in MRS agar medium are white, opaque, raised, round, with a smooth and moist surface and neat edges.

[0114] (2) Gram staining morphology: such as Figure 2 As shown, the bacteria are straight or curved rod-shaped, Gram-positive, and can be single, paired, or in chains. They do not produce spores, have no flagella, and are non-motile.

[0115] (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.

[0116] 2. Utilization of different carbohydrates

[0117] 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.

[0118] 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.

[0119] 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.

[0120] 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 after 24 and 48 hours, with 48 hours as the final result. The results are shown in Table 1. *Lactobacillus delbrueckii* subsp. *Turb8* showed an absorbance of 0.90 at OD600 and could utilize 25 carbon sources after 48 hours of culture.

[0121] Table 1. Identification results of Lactobacillus delbrueckii subsp. Turb8

[0122]

[0123] Note: "+" indicates that it can be used; "-" indicates that it cannot be used.

[0124] 3. Growth curve determination

[0125] Lactobacillus delbrueckii subsp. Turb8, preserved in glycerol, was inoculated into sterile MRS broth at a 1% inoculum and incubated statically 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 200 µl of MRS broth in a 96-well plate. Each bacterium was replicated in three wells. The plates were then shaken and incubated, with OD600 measured every hour.

[0126] The results are as follows Figure 4 The results showed that Lactobacillus delbrueckii subsp. Turb8 entered the logarithmic growth phase at 3 hours and the stationary phase at 14 hours, with an OD600 value of approximately 1.45 at the stationary phase.

[0127] 4. Antioxidant capacity

[0128] After activation, Turb8 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 2.

[0129] Table 2 Antioxidant capacity of Lactobacillus delbrueckii subsp. Turb8

[0130]

[0131] 5. Toxicity testing and safety assessment

[0132] The pathogenicity test method of food 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 delbrueckii subsp. Turb8. No abnormalities or deaths were observed in the test animals, and the body weight was not statistically significant compared with the control group (p>0.05), indicating that this strain is non-pathogenic.

[0133] 6. Detection of ability to inhibit pathogenic bacteria

[0134] 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.

[0135] 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.

[0136] 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.

[0137] 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 3.

[0138] Table 3. Diameter of the outer inhibition zone of *Lactobacillus delbrueckii* subsp. Turb8 strain against pathogens.

[0139]

[0140] The results showed that Lactobacillus delbrueckii subsp. Turb8 had a good inhibitory effect on all the above pathogenic bacteria.

[0141] 7. Gastrointestinal fluid tolerance test

[0142] 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.

[0143] 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.

[0144] 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.

[0145] 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 4 below.

[0146] Table 4. Statistical analysis of viable bacteria count of Lactobacillus delbrueckii subsp. Turb8 in simulated gastric and intestinal fluids.

[0147]

[0148] Lactobacillus delbrueckii subsp. Turb8 has an extremely high survival rate in artificial intestinal fluid and is almost unaffected.

[0149] 8. Adhesion ability test

[0150] Strain preparation: Turb8 strain was activated, cultured for three generations, centrifuged at 10000g for 5 min in 0.01M pBS (pH 7.2-7.4), washed twice, and the bacterial concentration was adjusted to approximately 1×10⁻⁶ in DMEM medium. 6 CFU / mL.

[0151] 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.

[0152] 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 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.

[0153] Adhesion index = Number of viable bacteria after adhesion / Number of cells in negative control well;

[0154] Adhesion rate (%) = Number of viable bacteria after adhesion / Number of viable bacteria before adhesion.

[0155] The results of the strain adhesion experiment are shown in Table 5.

[0156] Table 5 Adhesion ability of Lactobacillus delbrueckii subsp. Turb8 strain

[0157]

[0158] Determination of the self-polymerization ability of 9 strains

[0159] Take the fermentation broth of the experimental strain, centrifuge the broth for 10 min (4℃, 6000 r / min), collect the precipitate, wash the bacterial cells twice with sterile physiological saline, and finally resuspend the bacterial cells with physiological saline. Measure the OD600 and record it as A0. After the bacterial broth has stood for 2 h, take the supernatant and measure the OD600, record it as At. Calculate the self-polymerization ability: Self-polymerization ability (%) = (1-At / A0) 100.

[0160] Table 6. Self-aggregation ability of Lactobacillus delbrueckii subsp. Turb8 strain

[0161]

[0162] Lactobacillus delbrueckii subsp. Turb8 has a strong self-polymerization ability.

[0163] Example 3: Freeze-drying process and preparation of Lactobacillus delbrueckii subsp. Turb8 bacterial powder

[0164] Lactobacillus delbrueckii subsp. Turb8 was subcultured and activated three times at 37℃ using MRS medium. 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.

[0165] 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.

[0166] Example 4: Improvement of muscle loss during fat loss by the strain

[0167] 1. Animals and drug administration

[0168] Animal grouping and intervention: Six 8-week-old female C57 mice were divided into five groups and acclimatized for one week under constant temperature (22±2°C) and constant humidity (50±10%) conditions, while maintaining a 12-hour light / dark cycle. After 12 weeks of HFD diet feeding, the high-fat diet group showed a 20% increase in levels exceeding the normal group, indicating successful model establishment. Specific grouping and animal intervention methods are shown in Table 7. *Lactobacillus delbrueckii* subsp. *liquid* GM9-12 strain was isolated and preserved from breast milk by the inventors and is from the same batch of isolates as Turb8.

[0169] Table 7. Mouse grouping and intervention information

[0170]

[0171] 2. Mouse body weight

[0172] C57 female mice were fed different diets from week 1 to week 12. Compared with the normal group, the mean weight gain of mice in the high-fat diet group, liraglutide group, Turb8 group, and GM9-12 group was 33.63%, 34.19%, 32.51%, and 33.07%, respectively. The mean weight gain of all groups was >20%, confirming the successful modeling of obesity in mice induced by a high-fat diet. The mouse weight at the end of week 20 intervention was used as a reference. Figure 5 The results, as shown in the figure, indicate that the high-fat diet group, liraglutide group, Turb8 group, and GM9-12 group all significantly reduced mouse weight (P < 0.0001). Compared with the liraglutide group, there was no statistically significant difference in mouse weight between the Turb8 and GM9-12 groups (P > 0.05). There was no significant difference in weight loss between the Turb8 group and the liraglutide group, indicating that Turb8 itself does not interfere with the weight loss effect of liraglutide. The target of Turb8 is not fat metabolism itself, but specifically acts on the muscle protection pathway in the context of weight loss, achieving precise intervention of "fat loss without muscle loss".

[0173] 3. Grip strength of mouse limbs

[0174] Mice were weighed and their grip strength was tested using a grip strength tester (Jiangsu Saions Biotechnology Co., Ltd., model: SA415). The tester ensured that the mice's four paws fully gripped the test bar and then pulled horizontally and evenly by the tail. The test was repeated three times and the average value (gf) was calculated. The grip strength per gram of body weight of the mice was calculated using the following formula (gf / g bw).

[0175] Grip strength = average grip strength / body weight.

[0176] The results of grip strength in the limbs of mice at the end of the 20-week intervention were as follows: Figure 6 The results showed that liraglutide injection significantly reduced grip strength in all four limbs of mice, but the decrease was smaller in the Tueb8 group and was similar to that in the normal group; the GM9-12 group did not significantly improve grip strength (P>0.05). This indicates that liraglutide injection reduced grip strength in mice, and Lactobacillus delbrueckii Turb8 can improve grip strength, thus alleviating the decrease in grip strength.

[0177] 4. Mouse suspension time

[0178] Mice were suspended freely by gripping a horizontal bar with their forelimbs until their forelimbs detached from the bar, and the suspension time for each mouse was recorded. The suspension time results at the end of the 20-week intervention were referenced. Figure 7 The results, as shown in the figure, indicate that the high-fat diet group had the shortest suspension time, while the normal group had the longest. Compared to the normal group, the suspension time of mice in the high-fat diet group was significantly reduced (P < 0.0001). There was no statistically significant difference between the high-fat diet group and the liraglutide group (P > 0.05). Compared to the liraglutide group, the suspension time of mice in the Turb8 group was significantly increased (P < 0.05), while there was no difference in suspension time between the GM9-12 group and the Turb8 group (P > 0.05). This suggests that Turb8 can improve the suspension time of mice and has an endurance-enhancing effect. The GM9-12 strain did not show significant effects on grip strength and suspension time, indicating the unique function of the Turb8 strain, and that not all Lactobacillus delbrueckii strains possess this function.

[0179] 5. Lean body mass in mice

[0180] The experimental animals were weighed, euthanized by neck dissection, and the entire carcass was removed. The carcass was homogenized until it was uniform and fine. 1g of tissue was added to 15mL of a pre-prepared chloroform-methanol (V / V=2:1) ​​mixture to ensure that the chloroform-methanol fat extraction solution was thoroughly mixed with the tissue. Fat extraction was repeated three times for each sample. The extract was concentrated and dried in a fume hood and then dried at low temperature in an oven to constant weight. The fat mass of the sample was weighed and converted to mouse carcass fat mass. The lean body mass of the mice was calculated according to the following formula.

[0181] Lean body mass = Total weight - Fat mass;

[0182] Lean body mass percentage = (lean body mass / total weight) × 100%.

[0183] Lean body mass results of mice at the end of the 20-week intervention were referenced. Figure 8 The results, as shown in the figure, indicate that the lean body mass of mice treated with liraglutide increased and was similar to that of the normal group, while the lean body mass of mice treated with Turb8 was comparable to that of the normal group. Compared with the normal group, the lean body mass of mice in the high-fat diet group decreased significantly (P < 0.0001). Compared with the liraglutide group, the lean body mass of the Turb8 group increased significantly (P < 0.01), indicating that Turb8 has the effect of preventing muscle loss during fat reduction.

[0184] 6. Relative lower limb muscle mass in mice

[0185] The experimental animals were weighed, euthanized by neck dislocation, and the hind limb muscles were collected by dissection. The muscle tissue was rinsed with physiological saline, the water was absorbed with absorbent paper, and the muscle tissue was weighed. The relative hind limb muscle mass was calculated according to the following formula.

[0186] Relative lower limb muscle mass = Lower limb muscle wet weight / Mouse body weight.

[0187] The relative lower limb muscle mass of mice at the end of week 20 intervention was referenced. Figure 9 Compared to the normal group, the relative lower limb muscle mass of mice in the high-fat diet group was significantly decreased (P < 0.0001); in the high-fat diet group, the relative lower limb muscle mass of mice in the liraglutide group was significantly increased (P < 0.0001); compared to the liraglutide group, the Turb8 group showed an increase (P < 0.05), while there was no difference in the GM9-12 group (P > 0.05). This indicates that the relative lower limb muscle mass of obese model mice is significantly reduced, and Turb8 can increase the relative lower limb muscle mass of mice in this context.

[0188] Maintaining lean body mass is crucial for preventing weight rebound and maintaining a high basal metabolic rate. Turb8 further improves the lean body mass ratio and relative lower limb muscle mass based on liraglutide, indicating that Turb8 can effectively combat muscle loss.

[0189] 7. Mouse gastrocnemius muscle fibers

[0190] HE staining of cross-sections of mouse gastrocnemius muscle fibers after intervention (as reference) Figure 10 The results, as shown in the figure, indicate that compared to the normal group, the cross-sectional area of ​​the gastrocnemius muscle fibers in the high-fat diet group was significantly thinner. Subcutaneous injection of liraglutide and gavage administration of bacterial strains resulted in thicker cross-sectional areas of the gastrocnemius muscle fibers, with the Turb8 group showing similarities to the normal group. The statistical analysis of the cross-sectional area of ​​the gastrocnemius muscle fibers in mice is shown below. Figure 11The results, as shown in the figure, indicate that compared to the normal group, the cross-sectional area of ​​gastrocnemius muscle fibers in mice fed a high-fat diet was significantly decreased (P < 0.0001); compared to the high-fat diet group, the cross-sectional area of ​​gastrocnemius muscle fibers in mice fed a liraglutide diet was significantly increased (P < 0.01); compared to the liraglutide diet group, the cross-sectional area of ​​gastrocnemius muscle fibers in mice fed a Turb8 diet was increased (P < 0.05), and was close to that of the normal group, while there was no difference in the GM9-12 diet group (P > 0.05). Turb8 not only preserves muscle mass but also the structure and morphology of muscle fibers, which is fundamental to maintaining muscle function.

[0191] 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 subspecies of Lactobacillus delbrueckii ( Lactobacillus delbrueckii subsp.lactis Turb8, characterized in that, The Lactobacillus delbrueckii subsp. Turb8 has the accession number CGMCC No. 35627.

2. A microbial agent, characterized in that, The bacterial agent includes Lactobacillus delbrueckii subsp. Turb8 as described in claim 1.

3. The preparation of *Lactobacillus delbrueckii* subsp. Turb8 according to claim 1, characterized in that, include: Fermentation broth, fermentation broth sediment, live bacteria, freeze-dried powder.

4. A composition comprising Lactobacillus delbrueckii subsp. Turb8 as described in claim 1 or the bacterial agent as described in claim 2.

5. The composition according to claim 4, characterized in that, The composition described herein has a muscle protection function during fat loss.

6. The composition according to claim 5, characterized in that, The composition has any one or more of the following functions: (1) It has no effect on weight loss; (2) Improve limb strength; (3) Improve muscle endurance; (4) Increase lean body mass; (5) Increase the relative mass of lower limb muscles; (6) Maintain the mass of the gastrocnemius muscle and increase the cross-sectional area of ​​the gastrocnemius muscle fibers.

7. The composition according to claim 6, characterized in that, The composition has any one or more of the following functions: (1) Maintain the fat-reducing effect of the fat-reducing product; (2) Improve muscle function in multiple dimensions and ensure the integrity of muscle function; (3) Improve endurance, physical fitness and strength.

8. The composition according to claim 4, characterized in that, The bacterial count of *Lactobacillus delbrueckii* subsp. Turb8 should be no less than 1 × 10⁸. 8 CFU.

9. The composition according to claim 8, characterized in that, It also includes excipients, which are selected from any one or more of diluents, excipients, fillers, disintegrants, solubilizers, osmotic pressure regulators, surfactants, pH regulators, and antioxidants; the composition is in any one or more of the following forms: powder, tablet, emulsion, pill, ointment, powder, lyophilized powder for injection, gel, drops, tincture, capsule, granule, or aerosol.

10. The use of Lactobacillus delbrueckii subsp. Turb8 of claim 1, the bacterial agent of claim 2, or the preparation of claim 3 in the preparation of a composition having muscle protection function during fat loss.

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