Bifidobacterium animalis subsp. Lactis WLP90 and application thereof in preparation of preparation for relieving obesity

Abstract

The invention belongs to the technical field of animal bifidobacterium subsp. Lactis, and particularly relates to an animal bifidobacterium subsp. Lactis WLP90 and application thereof in preparation of a preparation for relieving obesity. The bifidobacterium animalis subsp. Lactis has the performance of targeted inhibition of actinomyces chlamydoides, Enterobacter cancerogenus and Alloscedobacterium animalis, can effectively inhibit pancrelipase, stimulate intestinal cells to generate glucagon-like peptide-1, can effectively improve diseases of obese mice, and can be used for preparing a preparation for relieving obesity.

Description

Technical Field

[0001] This application belongs to the technical field of Bifidobacterium animalis subsp. lactis, specifically relating to a strain of Bifidobacterium animalis subsp. lactis WLP90 and its application in the preparation of formulations to alleviate obesity. Background Technology

[0002] Bifidobacterium animalis subsp. lactis, a star strain in the probiotic field, has become one of the most widely used probiotic strains in food, health products, and pharmaceuticals due to its excellent gastrointestinal tolerance (resistance to gastric acid and bile), intestinal colonization potential, and broad-spectrum physiological activity. Numerous studies have confirmed that this strain has irreplaceable advantages in regulating intestinal flora balance, improving metabolic disorders, and enhancing immune function.

[0003] Diabetes mellitus is a global chronic metabolic disease characterized by hyperglycemia. Its pathogenesis is primarily due to dysfunction of pancreatic islet cells, such as insufficient or abnormal insulin secretion. Irregular lifestyle, a diet high in fat and carbohydrates, and a sedentary lifestyle can all contribute to the development of diabetes. Sustained hyperglycemia can damage other organs, leading to complications such as diabetic retinopathy, diabetic nephropathy, and diabetic foot.

[0004] Studies have shown a causal relationship between gut microbiota dysbiosis and type 2 diabetes. Therefore, maintaining a healthy gut microbiota is crucial for diabetes management. Probiotics are live microorganisms ingested to supplement the body, and their core function is to maintain or restore the balance of the gut microbiota. *Bifidobacterium animalis* subsp. *lactis* is a type of Gram-positive bacillus, mainly found in breast milk and infant feces, with its abundance gradually decreasing or even disappearing with age. It plays an important probiotic role in the human body. Currently, there are relatively few commercially available *Bifidobacterium animalis* subsp. *lactis* that can safely and effectively lower blood sugar and improve diabetes. Therefore, the field needs to develop more *Bifidobacterium animalis* subsp. *lactis* with blood sugar-lowering potential. Summary of the Invention

[0005] This application provides a strain of Bifidobacterium animalis subsp. lactis WLP90, which is Bifidobacterium animalis subsp. lactis with accession number CGMCC NO.33688.

[0006] This application provides an antibacterial preparation comprising at least one or more of the following: dead cells, live cells, inactivated cells, metabiotics, and fermentation broth of Bifidobacterium lactis subsp. WLP90 as described in accession number CGMCC NO.33688.

[0007] This application provides a glucagon-like peptide-1 stimulant comprising at least one or more of the following: dead cells, live cells, inactivated cells, metabiotics, and fermentation broth of Bifidobacterium lactis subsp. WLP90 as described in accession number CGMCC NO.33688.

[0008] This application provides a probiotic preparation for assisting in reducing hepatic fat deposition, comprising at least one or more of the following: dead cells, live cells, inactivated cells, postbiotics, and fermentation broth of Bifidobacterium lactis subsp. WLP90 as described in accession number CGMCCNO.33688.

[0009] This application provides a probiotic preparation for reducing the abundance of intermittent flora in the gut, comprising at least one or more of the following: dead cells, live cells, inactivated cells, metabiotics, and fermentation broth of Bifidobacterium lactis subsp. WLP90 as described in accession number CGMCC NO.33688.

[0010] This application provides a probiotic preparation that reduces the abundance of *Alternaria* and *Dubosiella* in the gut, comprising at least one or more of the following: dead cells, live cells, inactivated cells, metabiotics, and fermentation broth of *Bifidobacterium lactis* subsp. WLP90 as described in accession number CGMCC NO.33688.

[0011] This application provides a research preparation for reducing the abundance of intermittent bacterial flora in the intestines of experimental animals, comprising at least one or more of the following: dead cells, live cells, inactivated cells, postbiotics, and fermentation broth of Bifidobacterium lactis subsp. WLP90 as described in accession number CGMCC NO.33688.

[0012] This application provides a research preparation for reducing the abundance of *Allobacterium* and *Dubosiella* in the intestines of laboratory animals, comprising at least one or more of the following: dead cells, live cells, inactivated cells, metabiotics, and fermentation broth of *Bifidobacterium lactis* subsp. WLP90 as described in accession number CGMCC NO.33688.

[0013] This application provides the application of *Bifidobacterium lactis* subsp. WLP90 described in accession number CGMCC NO.33688, including the preparation of fermentation preparations of *Bifidobacterium lactis* subsp. WLP90; the preparation of antibacterial preparations; the preparation of pancreatic lipase inhibitors; the preparation of glucagon-like peptide-1 stimulants; the preparation of probiotic preparations to help reduce hepatic fat deposition; the preparation of probiotic preparations to reduce the abundance of intermittent flora in the gut; the preparation of probiotic preparations to reduce the abundance of *Corynebacterium* and *Dubosiella* in the gut; the preparation of research preparations to reduce the abundance of intermittent flora in the gut of experimental animals; and the preparation of preparations to reduce at least one of *Corynebacterium* and *Dubosiella* in the gut of experimental animals. Attached Figure Description

[0014] Figure 1 This is a graph showing the statistical results of the body weight values ​​of mice in each group.

[0015] Figure 2 This is a graph showing the statistical results of Lee's index for each group of mice.

[0016] Figure 3 This is a statistical result graph showing the weight of white fat in the epididymis of mice in each group.

[0017] Figure 4 This is a statistical result graph showing the weight of white fat in the groin of mice in each group.

[0018] Figure 5 This is a graph showing the statistical results of perirenal fat weight in each group of mice.

[0019] Figure 6 This is a graph showing the statistical results of the brown fat index in the scapula of mice in each group.

[0020] Figure 7 This is a graph showing the statistical results of liver fat weight in each group of mice.

[0021] Figure 8 This is a graph showing the statistical results of serum TC levels in each group of mice.

[0022] Figure 9 This is a graph showing the statistical results of serum LDL-C levels in each group of mice.

[0023] Figure 10 This is a graph showing the statistical results of serum ADP levels in each group of mice.

[0024] Figure 11 These are pathological sections of liver tissue from each group of mice.

[0025] Figure 12 This is a graph showing the statistical results of the percentage of lipid droplet area in the liver tissue of mice in each group.

[0026] Figure 13These are pathological sections of white adipose tissue from the epididymis of mice in each group.

[0027] Figure 14 These are pathological sections of perirenal adipose tissue from each group of mice.

[0028] Figure 15 These are pathological sections of brown adipose tissue from the scapula of mice in each group.

[0029] Figure 16 This is a graph showing the NMDS analysis results of the gut microbiota of each group of mice.

[0030] Figure 17 This is a graph showing the analysis results of the identification of core colonies in the gut microbiota of each group of mice.

[0031] Figure 18 These are clustering diagrams of the gut microbiota of each group of mice at the phylum level and significance analysis diagrams at the individual phylum level.

[0032] Figure 19 This is a graph showing the individual significance analysis of the gut microbiota of each group of mice at the genus level.

[0033] Different letters in the figure represent statistically significant differences between groups (p < 0.05). Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application. Reagents not specifically described in detail herein are all conventional reagents and are commercially available; methods not specifically described in detail are all conventional experimental methods and can be learned from the prior art.

[0035] Current research mainly focuses on the inhibitory effect of Bifidobacterium animalis subsp. lactis WLP90 on Escherichia coli. This belongs to the classic mechanism by which probiotics inhibit the growth of pathogens by competing for living space and producing antibacterial substances (such as organic acids, hydrogen peroxide, and bacteriocins).

[0036] However, unlike its significant inhibition of Escherichia coli, Bifidobacterium animalis subsp. lactis WLP90 does not show significant inhibitory effects on other pathogenic Enterobacteriaceae. For example, there is no evidence to suggest that it has an inhibitory effect on the endogenous pathogen Alloscardovia omnicolens.

[0037] Based on this, this application obtained a strain of Bifidobacterium animalis subsp. lactis, WLP90, through screening. This strain, with accession number CGMCC NO.33688, not only exhibits significant antibacterial activity against Actinomyces ylangi, Enterobacter carcinogenicus, and Skadoviridae, but also yields multiple active ingredient forms, including live bacteria, dead cells, inactivated cells, and postbiotics. This solves the technical problems of existing strains having limited functionality, restricted formulation forms, and insufficient targeting. The specific screening and identification process of this strain is as follows:

[0038] (1) Isolation and screening of strains

[0039] The *Bifidobacterium animalis* subsp. *lactamase* WLP90 provided in this application was isolated from infant feces. The collected samples were serially diluted with 0.85% physiological saline under aseptic conditions. Appropriate gradients were selected and plated onto MRS plates containing 0.05 g / L mupirocin lithium and 0.05 g / L cysteine ​​hydrochloride, and anaerobically cultured at 37°C for 48 h. Colony morphology was observed visually. Suspected single colonies were picked for microscopic examination, followed by preliminary screening and purification. The purified strain was anaerobically cultured at 37°C for 18 h in MRS liquid medium containing 0.05 g / L cysteine ​​hydrochloride. After centrifugation to remove the supernatant, the culture was resuspended in sterile 25% glycerol aqueous solution and stored at ~80°C.

[0040] (2) Morphological characteristics and identification

[0041] The selected target strain was cultured in liquid medium, and bacterial cells were collected. Genomic DNA was extracted and amplified using universal primers to amplify its 16S rDNA fragment. The PCR amplification products were detected by agarose gel electrophoresis and sequenced. The PCR products were then sent to Wuhan Jinkairui Biotechnology Co., Ltd. for sequencing after gel electrophoresis. The identified gene sequences were compared with the NCBI database using a BLAST tool. Based on the molecular biological identification results, the Latin name of the strain was determined to be *Bifidobacterium animalis* subsp. *lactis*, confirming it as *Bifidobacterium animalis* subsp. *lactis*. This strain was named *Bifidobacterium animalis* subsp. *lactis* WLP90 and deposited for preservation. Its preservation information is as follows:

[0042] Accession number: CGMCC NO.33688

[0043] Classification and nomenclature: Bifidobacterium animalis subsp. lactis WLP90

[0044] Latin name: *Bifidobacterium animalis* subsp. *lactis*

[0045] Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee

[0046] Address of the depository: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing

[0047] Deposit date: March 3, 2025

[0048] The embodiment also provides an antibacterial preparation comprising at least one or more of the following: dead, live, and inactivated bacterial cells of Bifidobacterium animalis subsp. lactis WLP90 (accession number CGMCC NO.33688). The antibacterial effect against the target bacteria is achieved through different bacterial cell forms, adapting to different product processes and application requirements.

[0049] In some embodiments, the Bifidobacterium animalis subsp. lactis WLP90 strain according to this application may be an isolated bacterial strain or a pure culture colony.

[0050] In some of the formulations provided in the embodiments, the concentration of at least one or more of the dead bacterial cells, live bacterial cells, and inactivated bacterial cells is 10. 2 Up to 10 17 Within the range of colony-forming units per gram or per milliliter (CFU / g or CFU / mL), for example, in 10 5 ~10 17 Within the range of CFU / g or CFU / mL, for example, in the range of 10 6 ~10 17 Within the range of CFU / g or CFU / mL, for example, in the range of 10 7 ~10 17 Within the range of CFU / g or CFU / mL, for example, in the range of 10 8 ~10 17 Within the range of CFU / g or CFU / mL, for example, in the range of 10 9 ~10 17 Within the range of CFU / g or CFU / mL, for example, in the range of 10 10 ~10 17 Within the range of CFU / g or CFU / mL, for example, in the range of 10 11 ~10 16 Within the range of CFU / g or CFU / mL, for example, in the range of 10 12 ~10 16 Within the range of CFU / g or CFU / mL, for example, in the range of 10 13 ~10 16 Within the range of CFU / g or CFU / mL, for example, in the range of 10 7 ~10 16 Within the range of CFU / g or CFU / mL, for example, in the range of 10 8 ~10 15Within the range of CFU / g or CFU / mL, for example, in the range of 10 9 ~10 15 Within the range of CFU / g or CFU / mL, for example, in the range of 10 10 ~10 15 Within the range of CFU / g or CFU / mL, for example, in the range of 10 11 ~10 15 Within the range of CFU / g or CFU / mL, for example, in the range of 10 12 ~10 15 Within the range of CFU / g or CFU / mL.

[0051] In the context of this application, *Bifidobacterium animalis* subsp. *lactamase* WLP90 as defined herein may be provided in the composition according to this application in the form of at least one or more of dead cells, live cells, and inactivated cells. Live cells refer to live *Bifidobacterium animalis* subsp. *lactamase* bacteria with intact cellular structure, capable of normal metabolism and reproduction, for example, cultured in a culture medium (such as MRS medium), centrifuged, washed to retain viability, and usually preserved in lyophilized form (such as lyophilized bacterial powder). For example, live cells refer to the bacterial solid obtained by collecting the strain after fermentation in MRS medium by centrifugation and washing with physiological saline. Dead cells refer to bacterial cells that have died naturally or lost activity through physical / chemical treatment (such as high temperature, ultraviolet light), and whose cellular structure may be intact or partially destroyed. For example, dead cells are obtained by freeze-drying live cells, grinding and crushing them, and passing them through a 200-mesh sieve to obtain bacterial fragment powder (protein content ≥30%). Inactivated bacteria specifically refer to bacteria that have been killed through controlled methods (such as heat inactivation, formaldehyde treatment, or autoclaving) while retaining their cell surface structures (such as cell walls and capsules). Inactivated bacteria emphasize "structural preservation," while dead bacteria may suffer structural damage due to the treatment method. For example, inactivated bacteria are produced by sterilizing live bacteria with moist heat at 60°C for 30 minutes (or treating with 0.5% formaldehyde solution for 1 hour) to ensure no live bacteria are detected (live bacteria count <10 CFU / mL using plate counting method), thus preserving the bacterial structure and metabolic products.

[0052] In some of the formulations provided in the embodiments, Bifidobacterium animalis subsp. lactis WLP90 may be used as an active ingredient in a mixture of at least one or more of dead, live and inactivated bacterial cells, and the active ingredient accounts for 0.0001% (w / w) to 99% (w / w) of the total mass of the formulation.

[0053] In some embodiments, the preparation process of live Bifidobacterium animalis subsp. lactis WLP90 includes:

[0054] Bifidobacterium animalis subsp. lactis WLP90 strain was inoculated into seed culture medium and anaerobically cultured at 37℃ for 18 h to obtain seed culture (viable count ≥1×10⁻⁶). 9The seed culture medium contains: 10 g / L peptone, 10 g / L beef extract, 20 g / L glucose, 2 g / L sodium acetate, 5 g / L yeast extract, 2 g / L diammonium hydrogen citrate, 2 g / L dipotassium hydrogen phosphate, 0.58 g / L sulfuric acid heptahydrate, 0.25 g / L manganese sulfate, 1 mL / L Tween 80, 0.05 g / L cysteine ​​hydrochloride, and 15.0 g / L agar.

[0055] Inoculate the seed culture at a 2% inoculation rate into the fermentation medium and anaerobic ferment at 37℃ for 24 hours. The viable cell count in the fermentation broth should be ≥1×10¹. 0 CFU / mL; Fermentation medium contains: peptone 10g / L, beef extract 10g / L, glucose 20g / L, sodium acetate 2g / L, yeast extract 5g / L, diammonium hydrogen citrate 2g / L, dipotassium hydrogen phosphate 2g / L, magnesium sulfate heptahydrate 0.58g / L, manganese sulfate 0.25g / L, Tween 80 1mL / L, L-cysteine ​​hydrochloride 0.05g / L.

[0056] The fermentation broth was centrifuged at 8000 rpm for 2 minutes, and the bacterial precipitate was collected. The precipitate was washed twice with sterile physiological saline to obtain a concentrated live bacterial cell extract. This extract was then prepared with physiological saline to contain 10... 8 CFU / mL of test bacterial suspension.

[0057] In some embodiments, the preparation process of dead *Bifidobacterium animalis* subsp. *lactamase* WLP90 cells includes:

[0058] Take the above-mentioned live bacterial concentrate and sterilize it by moist heat (121℃, 0.1MPa for 20 min) or dry heat (160℃ for 2 h). If no live bacteria are detected after sterilization (plate count method), it is considered dead bacterial.

[0059] In some embodiments, the preparation process of inactivated Bifidobacterium animalis subsp. lactis WLP90 cells includes:

[0060] Take the live bacterial cell concentrate and inactivate it using low-temperature plasma inactivation (100W power, 5min) or ultraviolet inactivation (254nm, 30min). After inactivation, ensure that there are no live bacteria and retain the adhesion sites and antibacterial components on the bacterial surface.

[0061] In some embodiments, the preparation process of mixed cells of Bifidobacterium animalis subsp. lactis WLP90 includes: mixing live cells, dead cells, and inactivated cells in any mass ratio.

[0062] In some embodiments, the antibacterial preparation further includes excipients such as stabilizers, carriers, and dispersants. The stabilizers are selected from trehalose, skim milk powder, or xanthan gum; the carriers are selected from maltodextrin, lactose, or corn starch; and the dispersants are selected from polyethylene glycol or Tween 80.

[0063] In some embodiments, the antibacterial preparation is a powder, tablet, ointment, emulsion, oil, suspension, lotion, gel, paste, foam, dairy product, gel, mist, or spray.

[0064] In some embodiments, the antibacterial preparation is a powder containing live *Bifidobacterium lactis* subsp. *Lactobacillus* var. *anis* WLP90. Specifically, it contains 1×10¹¹ CFU / g of *Bifidobacterium lactis* var. *anis* WLP90 live cell concentrate, maltodextrin, and trehalose. For example, the antibacterial preparation contains 10g of 1×10¹¹ CFU / g of *Bifidobacterium lactis* var. *anis* WLP90 live cell concentrate, 80g of maltodextrin, and 10g of trehalose. The live cell concentrate of *Bifidobacterium lactis* var. *anis* WLP90 can be mixed evenly with maltodextrin and trehalose, and then aseptically packaged (1g / bag) to obtain the powdered antibacterial preparation.

[0065] In some embodiments, the antibacterial preparation is a liquid formulation containing dead *Bifidobacterium animalis* subsp. *lactospirae* WLP90 cells. Specifically, it contains 1×10¹¹ CFU / g of dead *Bifidobacterium animalis* subsp. *lactospirae* WLP90 cells, xanthan gum, and sterile physiological saline. For example, the antibacterial preparation contains 5g of 1×10¹¹ CFU / g dead *Bifidobacterium animalis* subsp. *lactospirae* WLP90 cells, 0.5g of xanthan gum, and 94.5mL of sterile physiological saline. The dead cells and xanthan gum can be added to sterile physiological saline, stirred to dissolve, and then homogenized at 10000 rpm for 10 min to obtain the liquid antibacterial preparation.

[0066] In some embodiments, the antibacterial preparation is a granule containing both live and inactivated Bifidobacterium animalis subsp. lactis 90. Specifically, it contains 5g of not less than 5 × 10⁻⁶ cells. 8 CFU / g of live Bifidobacterium animalis subsp. lactis WLP90, 5g 5×10 8 Inactivated Bifidobacterium animalis subsp. lactis WLP90 (CFU / g), 70g lactose, and 20g corn starch. 5g of this mixture should be at least 5×10⁻⁵ CFU / g. 8 CFU / g of live Bifidobacterium animalis subsp. lactis WLP90, 5g 5×10 8 Inactivated Bacterium animalis subsp. lactis WLP90 (CFU / g), 70g lactose, and 20g corn starch were mixed, and 5% povidone K30 aqueous solution was added as a binder. The mixture was granulated, dried at 60℃ for 1 hour, and then packaged to obtain granulated antibacterial preparations.

[0067] These antibacterial agents inhibit the proliferation of Actinomyces ylangi, Enterobacter carcinogens, and Skadoviridae through the antibacterial activity of live, dead, or inactivated bacteria.

[0068] Live bacteria can colonize the intestines or oral cavity, continuously proliferate, and produce antibacterial substances (such as lactic acid and bacteriocins), achieving long-lasting antibacterial effects. Dead bacteria retain their cell wall components (such as peptidoglycan) and residual antibacterial substances, exhibiting antibacterial activity without colonization, high stability, and tolerance to high temperatures, acids, and alkalis. Inactivated bacteria maintain structural integrity, possessing both the stability of dead bacteria and the ability to assist in blocking target bacterial colonization through surface adhesion sites, thus combining antibacterial and copolymerization-aiding functions. Mixed bacteria can synergistically leverage the advantages of different bacterial forms, such as the long-lasting antibacterial effect of live bacteria and the immediate antibacterial effect of dead bacteria, enhancing the overall antibacterial efficacy of the formulation.

[0069] The embodiments also provide an antibacterial preparation comprising metabiotics and / or fermentation broth of Bifidobacterium animalis subsp. lactis WLP90.

[0070] In this article, "metapiogenics" refers to the collective term for physiologically active bacterial components and metabolites produced by probiotics (such as Bifidobacterium lactis subsp. animalis in this article) after specific processing. Its core characteristic is that it can exert its functions without relying on the live state of the bacteria. Specifically, "metapiogenics" can include at least one of the following: "inactivated bacterial cells", "solutions containing inactivated bacterial cells", "fermentation broth containing inactivated bacterial cells" or inactivated fermentation solutions, or "beneficial legacy" left after the death of probiotics or their metabolites.

[0071] In some embodiments, the preparation process of Bifidobacterium animalis subsp. lactis WLP90 fermentation broth includes:

[0072] Bifidobacterium animalis subsp. lactis WLP90 strain was inoculated into seed culture medium and anaerobically cultured at 37℃ for 18 h to obtain seed culture (viable count ≥1×10⁻⁶). 9 The seed culture medium contains: 10 g / L peptone, 10 g / L beef extract, 20 g / L glucose, 2 g / L sodium acetate, 5 g / L yeast extract, 2 g / L diammonium hydrogen citrate, 2 g / L dipotassium hydrogen phosphate, 0.58 g / L magnesium sulfate heptahydrate, 0.25 g / L manganese sulfate, 1 mL / L Tween 80, 0.05 g / L cysteine ​​hydrochloride, and 15.0 g / L agar.

[0073] Inoculate the seed culture at a 2% inoculation rate into the fermentation medium and anaerobic ferment at 37℃ for 24 hours. The viable cell count in the fermentation broth should be ≥1×10¹. 0 The fermentation broth is expressed as CFU / mL. The fermentation medium contains 10 g / L peptone, 10 g / L beef extract, 20 g / L glucose, 2 g / L sodium acetate, 5 g / L yeast extract, 2 g / L diammonium hydrogen citrate, 2 g / L dipotassium hydrogen phosphate, 0.58 g / L magnesium sulfate heptahydrate, 0.25 g / L manganese sulfate, 1 mL / L Tween 80, and 0.05 g / L cysteine ​​hydrochloride.

[0074] In some embodiments, the formulation may be provided in solid, liquid, viscous, emulsion, or dry form. The formulations provided in some examples are preferably formulated as pastes, soft gelatin capsules, hard gelatin capsules, powders, talc, granules, beads, lozenges, effervescent tablets, lozenges, chewable tablets, sublingual tablets, oils, liquids, solutions, tinctures, emulsions, fruit juices, concentrates, syrups, sprays, mists, drinking ampoules, gels, gums, tablets, or coated pills.

[0075] Some of the embodiments provide formulations that are powders, tablets, ointments, emulsions, oils, suspensions, lotions, gels, pastes, foams, dairy products, gels, mists, sprays, or fermented preparations.

[0076] In some embodiments, the metabiotic is a solution obtained by passing the fermentation broth of Bifidobacterium animalis subsp. lactis WLP90 through a 70°C water bath for 30 minutes.

[0077] In some embodiments, the antibacterial preparation comprises a metabiotic of Bifidobacterium animalis subsp. lactis WLP90 and excipients. The excipients include a diluent, a preservative, and a thickener. The diluent is sterile saline or maltodextrin. The preservative is sodium benzoate or potassium sorbate. The thickener is sodium carboxymethyl cellulose or gelatin.

[0078] In some embodiments, the antibacterial agent is a liquid formulation containing Bifidobacterium animalis subsp. lactis WLP90 as a postbiotic. For example, the fermentation broth of Bifidobacterium animalis subsp. lactis is prepared to a viable count of not less than 1 × 10⁻⁶. 10 CFU / mL, incubate at 70℃ for 30 min, take 100 mL of the supernatant, add 0.1 g of potassium sorbate, stir to dissolve, sterilize, and dispense into 10 mL / bottles to obtain a liquid antibacterial preparation.

[0079] In some embodiments, the antibacterial agent is a powder containing Bifidobacterium animalis subsp. lactis WLP90 as a postbiotic. For example, the fermentation broth of Bifidobacterium animalis subsp. lactis is fermented until the viable count is not less than 1 × 10⁻⁶. 10 After being heated to CFU / mL in a water bath at 70℃ for 30 minutes, the powder was freeze-dried. 1g of the powder was mixed evenly with 99g of maltodextrin and aseptically packaged (0.5g / bag) to obtain a powdered antibacterial preparation (postbiotic content 10μg / bag).

[0080] In some embodiments, the antibacterial agent is a gel formulation containing Bifidobacterium animalis subsp. lactis WLP90 as a postbiotic. For example, the fermentation broth of Bifidobacterium animalis subsp. lactis is prepared to a viable count of not less than 1 × 10⁻⁶. 10After mixing CFU / mL with 20 mL of water at 70℃ for 30 min, homogenize the mixture with 2 g sodium carboxymethyl cellulose, 5 g glycerol, and 73 mL sterile water, sterilize at 60℃ for 30 min, and then cool to obtain a gel antibacterial preparation.

[0081] These antibacterial agents utilize active ingredients in the metabiotics (e.g., bacteriocins can disrupt the cell membrane of target bacteria, and lactic acid can lower the environmental pH and inhibit the proliferation of target bacteria) to specifically bind to receptors on the cell membrane of target bacteria, forming pores that cause the leakage of intracellular substances and death of target bacteria; they also lower the pH of the application environment, disrupting the acid-base balance of target bacteria and inhibiting their metabolism and proliferation; and they compete with target bacteria for adhesion sites, indirectly assisting in antibacterial action.

[0082] More specifically, the antibacterial agent is selected from at least one of the following: antibacterial agents of Actinomyces israelii, antibacterial agents of Enterobacter cancerogenus, and antibacterial agents of Alloscardovia omnicolens.

[0083] In some test cases, the fermentation broth of Bifidobacterium animalis subsp. lactis WLP90 was subjected to Oxford Cup tests with Actinomyces ylangiella, Enterobacter carcinogens, and Skadoviridae.

[0084] Actinomyces israelii (ATCC15909), Enterobacter cancerogenus (SHMCC(SHBCC) D72582), and Alloscardoviaomnicolens (BNCC382766) were inoculated onto BHI agar plates containing 5% defibrinated sheep blood for activation. After incubation at 37°C for 24 hours, bacterial colonies were picked and dissolved in physiological saline to prepare a bacterial suspension. The bacterial concentration was adjusted to 10-. 8 CFU / mL. Fermentation broth of Bifidobacterium animalis subsp. lactis WLP90 was prepared using the method described above.

[0085] BHI medium containing 1.5% agar was cooled to approximately 55°C and then mixed thoroughly with suspensions of *Actinomyces yewica*, *Enterobacter carcinogens*, and *Scardoviciana spectroscopy*, ensuring a viable count of 10-1 for each bacterium. 6 CFU / mL, then quickly pour into plates pre-placed with Oxford cups. After the culture medium cools and solidifies, remove the Oxford cups and inject 200 μL of viable bacteria (10⁶ CFU / mL) into each well. 8 The diameter of the inhibition zone was measured after fermentation broth of Bifidobacterium animalis subsp. lactis WLP90 at CFU / mL was incubated overnight at 37°C for 24 hours.

[0086] The results showed that the inhibition zones of the fermentation broth of Bifidobacterium animalis subsp. lactis WLP90 against Actinomyces ylangiella, Enterobacter carcinogens and Skadoviridae were 13.50±0.34 mm, 14.13±0.29 mm and 21.74±0.39 mm, respectively.

[0087] These test cases demonstrate that the *Bifidobacterium animalis* subsp. *lactamase* WLP90 provided in this application can precisely inhibit specific target bacteria, adapting to the needs of target bacteria infection control in different scenarios. *Bifidobacterium animalis* subsp. *lactamase* WLP90 avoids the impact of broad-spectrum antibacterial agents on beneficial bacteria; for example, oral preparations target only *Iscativobacterium praecoxibacterium*, without disrupting the normal oral flora. Furthermore, *Bifidobacterium animalis* subsp. *lactamase* WLP90 also solves the problem of existing antibacterial agents being "broad-spectrum but lacking specificity," improving antibacterial efficiency.

[0088] One test case revealed that the *Bifidobacterium lactis* subsp. WLP90 provided in this application exhibits 100% gastric fluid tolerance and 96% intestinal fluid tolerance. The specific testing procedure included: activating the test strain for three generations and then adjusting the bacterial concentration to 10... 8 CFU / mL. Centrifuge 1 mL of bacterial suspension to collect bacterial cells, then inoculate 1 mL of prepared artificial gastric fluid or artificial intestinal fluid and mix well. Digest at 37°C. Simultaneously, collect digestion fluid at 0 h and 3 h to detect the number of viable bacteria and calculate the survival rate. Perform the determination in triplicate. The survival rate (%) is calculated as follows: Nt / N0 × 100%, where N0 represents the number of viable bacteria at 0 h (CFU / mL) and Nt represents the number of viable bacteria at 3 h (CFU / mL). The artificial gastric fluid is prepared as follows: prepare a 0.5% sodium chloride solution, adjust the pH to 3 with 1 mol / L HCl, add 0.3% pepsin, dissolve thoroughly, and then filter sterilize using a 0.22 μm microporous membrane. The artificial intestinal fluid is prepared as follows: prepare a 0.5% sodium chloride solution, adjust the pH to 8 with 0.1 mol / L NaOH, add 0.1% trypsin, dissolve thoroughly, and then filter sterilize using a 0.22 μm microporous membrane. The results showed that the survival rate of strain WLP90 in artificial gastric fluid was 100.28±0.64%, and the survival rate in artificial intestinal fluid was 95.60±1.09%.

[0089] Based on the high tolerance of this application to gastric and intestinal fluids, embodiments of this application also provide a probiotic active formulation containing live cells of Bifidobacterium animalis subsp. lactis WLP90. In some embodiments, the content of live WLP90 cells in this probiotic active formulation is 1 trillion CFU / g. The formulation maintains a live cell survival rate of ≥85% after 12 months of storage at room temperature (25°C) and ≥70% after 3 months of storage at 40°C.

[0090] In some embodiments, based on live bacteria containing 1 trillion CFU / g of *Bifidobacterium animalis* subsp. *lactobacter* WLP90, the formulation further comprises 15-20% by weight of prebiotics, 30-35% by weight of stress protectants, 5-8% by weight of acid-base buffers, and 0.5-1% by weight of antioxidants. The prebiotics may be a mixture of fructooligosaccharides and inulin in a 1:2 weight ratio. The stress protectant may be a mixture of maltodextrin and trehalose in a 1:3 weight ratio, the acid-base buffer may be a mixture of sodium citrate and dipotassium hydrogen phosphate in a 1:1 weight ratio, and the antioxidant may be ascorbyl palmitate.

[0091] The embodiments also provide an inhibitor of pancreatic lipase, comprising at least one or more of the following: dead, live, inactivated, or metabiotic cells of Bifidobacterium animalis subsp. lactis WLP90, and fermentation broth. This agent blocks the colonization of target bacteria on mucosal surfaces (such as the intestines and oral cavity) through targeted adhesion and co-polymerization between the bacterial strain and the target bacteria, achieving dual control by inhibiting proliferation and blocking colonization through synergistic antibacterial function.

[0092] In some embodiments, the pancreatic lipase inhibitor is a powder containing a concentration of live cells of Bifidobacterium animalis subsp. lactis WLP90. For example, the pancreatic lipase inhibitor contains 10g of a concentration of 1×10¹¹ CFU / g of live cells of Bifidobacterium animalis subsp. lactis WLP90, 0.3g of chitosan, 5g of galactooligosaccharides, and 84.7g of maltodextrin. After the components are mixed evenly, they are aseptically packaged (1g / bag) with a viable count ≥1×10¹¹. 9 CFU / g.

[0093] In some embodiments, the pancreatic lipase inhibitor is a gel formulation containing inactivated Bifidobacterium animalis subsp. lactis WLP90 cells. For example, it contains 5g of inactivated Bifidobacterium animalis subsp. lactis WLP90 cells, 0.5g of sodium alginate, 8g of xylitol, 1g of sodium carboxymethyl cellulose, and 85.5mL of sterile water. After mixing and homogenizing, the mixture is sterilized at 60°C for 30min and then cooled to obtain a gel.

[0094] In some embodiments, the pancreatic lipase inhibitor is a spray formulation containing live and inactivated Bifidobacterium animalis subsp. lactis WLP90 cells. For example, it contains 6g of a bacterial mixture (live:inactivated = 3:2, total concentration 1×10¹¹ CFU / g), 0.2g of chitosan, 5g of glycerol, and 88.8mL of sterile water. After stirring to dissolve, the mixture is poured into a spray bottle, with each 0.1mL spray containing 6×10¹¹ CFU / g of the active ingredient. 8 CFU.

[0095] In some test cases, Bifidobacterium animalis subsp. lactis WLP90 was directly applied to pancreatic lipase for testing.

[0096] Fermentation broth of *Bifidobacterium animalis* subsp. *lactamase* WLP90 was prepared according to the above method. After centrifugation at 8000 r / min for 15 min, the bacterial precipitate was collected and washed twice with sterile physiological saline to obtain a concentrated live bacterial cell (live count ≥ 1 × 10⁻⁶). 8 (CFU / g), prepared with physiological saline to contain 10 CFU / g. 8 CFU / mL of test bacterial suspension. Prepare 0.1M Tris-HCl buffer (pH 8.0), and use this Tris-HCl buffer to prepare an enzyme solution containing 5 mg / mL pancreatic lipase. Prepare 10 mmol / L p-NPP as substrate solution using DMSO. Prepare 0.01 mg / mL orlistat solution as positive control solution using the above DMSO.

[0097] Mix 50 μL of sample with 50 μL of pancreatic lipase solution and incubate at 37 °C for 10 minutes. Then, immediately add 100 μL of pNPP solution and react again at 37 °C for 10 minutes. Use the diluted solution as a sample blank (blank control group). After the reaction, measure the absorbance of each reaction system at 405 nm. Calculate the absorbance using the following formula.

[0098] Pancreatic lipase inhibition rate (%) = [A_control - (A_sample - A_sample blank)] / A_control × 100%

[0099] Control group: No sample added, but pNPP solution added; Sample group: Sample added, but pNPP solution added; Sampleblank group: Sample added, but pNPP solution not added.

[0100] The results showed that the pancreatic lipase inhibition rate of Bifidobacterium animalis subsp. lactis WLP90 provided in the example was 57.00±0.01%, while the pancreatic lipase inhibition rate of the positive control group was 65.00±0.01%. This indicates that Bifidobacterium animalis subsp. lactis WLP90 possesses pancreatic lipase inhibition ability, suggesting that this strain has the ability to reduce fat accumulation in the body.

[0101] Bifidobacterium animalis subsp. lactis is a safe-grade probiotic with acid and bile salt resistance and intestinal colonization capabilities. However, existing formulations of it mostly focus on single antibacterial or broad-spectrum microbiota regulation functions, lacking effects on the stability of intestinal microbiota in obese mice.

[0102] Based on the aforementioned technical deficiencies, this embodiment also provides a glucagon-like peptide-1 stimulant, which can use at least one or more of the following as active ingredients: dead, live, inactivated, or metabiotic cells of Bifidobacterium lactis subsp. WLP90, fermentation broth, etc. Specifically, it refers to the preparation method of the active ingredients in the above-mentioned antibacterial agents or pancreatic lipase inhibitors, wherein the excipients also refer to the preparation method of the excipients in the above-mentioned antibacterial agents or pancreatic lipase inhibitors. The feasible form or formulation of this preparation also refers to the implementation method of the above-mentioned antibacterial agents or pancreatic lipase inhibitors, and will not be elaborated here.

[0103] In one test case, a mouse small intestinal endocrine cell line (STC-1 cell line) was seeded in 24-well plates and cultured until the density reached 2 × 10⁻⁶ cells / well. 5 / well, cell culture reaches 80% confluence. Use Ca-free... 2+ and Mg 2+ Wash twice with PBS buffer and incubate for 30 min in DMEM (0.5 mL) without glucose and glutamine. Prepare the fermentation broth of *Bifidobacterium animalis* subsp. *lactamase* WLP90 using the same method. Centrifuge at 8000 rpm for 15 min, collect the bacterial pellet, wash twice with sterile physiological saline to obtain a concentrated live bacterial cell extract (live count ≥ 1 × 10⁻⁶). 8 (CFU / g), prepared with physiological saline to contain 10 CFU / g. 8 CFU / mL of test bacterial suspension. Take 0.5 mL of Bifidobacterium animalis subsp. lactis WLP90 bacterial suspension (1×10⁻⁶ CFU / mL). 8 The cell suspension (CFU / mL) was added to the cell plate and incubated at 37°C for 4 h. The supernatant was collected in a 1.5 mL centrifuge tube and centrifuged at 5900×g, 4°C for 10 min to remove the cell pellet. The glucagon-like peptide-1 (GLP-1) content was measured using a mouse GLP-1 ELISA kit, with the supernatant from uninoculated STC-1 cells serving as a blank control. The results showed that the GLP-1 concentration in the blank control group was 30.84±0.49 pg / mL, while the GLP-1 concentration in the WLP90 group was 32.08±0.37 pg / mL, indicating that the WLP90 strain significantly promoted the release of GLP-1 from STC-1 cells (p<0.05).

[0104] In some test cases, the following tests were conducted:

[0105] (1) Experimental animals

[0106] Healthy male C57BL / 6j mice (5-6 weeks old; 19±1g) were purchased from the Experimental Animal Center of Huazhong Agricultural University. Animal experiment ethics number: Safety Evaluation Center Animal (Fujian) No. 202510209; Experimental Animal Use License number: SCXK (E) 2020-0019. Animal experiments were conducted at 20-22°C and 40-60% humidity, with a 12-hour light / 12-hour dark cycle. Mice had free access to food and water.

[0107] (2) Animal modeling and grouping:

[0108] Preparation of WLP90 bacterial suspension: Fermentation broth of *Bifidobacterium animalis* subsp. *lactamase* WLP90 was prepared according to the method described above. The precipitate was collected by centrifugation at 8000 r / min for 15 min, and washed twice with sterile physiological saline to obtain a concentrated live bacterial cell extract (live count ≥ 1 × 10⁻⁶). 10 (CFU / g), prepared with physiological saline to contain 10 CFU / g. 8 CFU / mL of test bacterial suspension.

[0109] After one week of acclimatization, mice were used to establish an obesity model according to the method described in (Cheng Kong, Ren Yuan Gao, Xue Bing Yan, Lin Sheng Huang, Huan Long Qin, Probiotics improve gut microbiota dysbiosis in obese mice fed a high-fat or high-sucrose diet, Nutrition, Volume 60, 2019). The obese model mice were divided into an HFD group (model group) and a WLP90 group, with healthy mice serving as the ND group. All groups underwent a 56-day intervention. Mice in the ND and HFD groups were administered sterile saline by gavage, 0.2 mL once daily for 49 days. The WLP90 group was administered WLP90 bacterial suspension by gavage, 0.2 mL once daily for 56 days.

[0110] (3) Weight and Lee's Index monitoring

[0111] During the experiment, the average weight and average Lee's index of mice in each group were measured and recorded weekly. Lee's index is a commonly used indicator for judging obesity in mice; generally, a significant difference in Lee's index between the model group and the healthy group is considered a sign of obesity.

[0112] like Figure 1As shown, from week 0 to week 8, the body weight of mice in the model group fed with a high-fat diet increased continuously; the body weight of mice after WLP90 strain intervention significantly slowed down the increasing trend, and a significant difference was reached between the model group and the WLP90 intervention group at the end of week 8 (p<0.05).

[0113] like Figure 2 As shown, compared with the model group mice, the WLP90 strain intervention reduced the Lee's index in significantly obese mice (p<0.05).

[0114] (4) Detection of organ weights in each group of mice

[0115] After the experiment, samples of epididymal white adipose tissue (eWAT), inguinal white adipose tissue (iWAT), scapular brown adipose tissue (BAT), perirenal adipose tissue, and liver tissue were taken and weighed from each group of mice. Figure 3 , Figure 4 , Figure 5 , Figure 6 and Figure 7 As shown.

[0116] The results showed that the weights of epididymal white adipose tissue, inguinal white adipose tissue, scapular brown adipose tissue, perirenal adipose tissue, and liver in the model group mice were significantly different from those in the normal group (p<0.05). Compared with the model group, the WLP90 probiotic intervention group significantly reduced the weights of epididymal white adipose tissue (p<0.05), inguinal white adipose tissue (p<0.05), scapular brown adipose tissue (p<0.05), perirenal adipose tissue (p<0.05), and liver (p<0.05) in obese model mice; indicating that WLP90 intervention can effectively reduce the weight of white adipose tissue and liver in mice, thereby effectively reducing their body weight.

[0117] (5) Monitoring of metabolic indicators in mice of each group

[0118] After the experiment, whole blood was collected from mice in each group, allowed to stand, and then centrifuged at 3000 rpm for 10 minutes. The supernatant was collected, and lipid metabolism parameters were measured in the mice according to the procedures of the biochemical and ELISA kits. These parameters included TC (total cholesterol), LDL-C (low-density lipoprotein cholesterol), and ADP (adiponectin). The results are shown below. Figure 8 , Figure 9 and Figure 10 As shown in the figure. (Different letters in the figure represent significant differences between groups, p < 0.05).

[0119] Depend on Figure 8 , Figure 9It can be seen that the serum total cholesterol and low-density lipoprotein concentrations of mice in the model group were significantly higher than those in the normal group. Compared with the model group, the WLP90 group significantly reduced the serum total cholesterol and low-density lipoprotein concentrations of mice (p<0.05) and approached those of the normal group.

[0120] Adiponectin is a key hormone secreted by adipose tissue, regulating glucose and lipid metabolism and enhancing insulin sensitivity. Figure 10 The results showed that the serum adiponectin concentration in the model group mice was significantly lower than that in the healthy group, while WLP90 intervention significantly increased serum adiponectin levels (p<0.05). This indicates that WLP90 can effectively regulate the secretion of adiponectin by adipocytes and regulate lipid metabolism.

[0121] (6) Photographs of liver and adipose tissue sections from mice in each group.

[0122] After the experiment, liver tissue, epididymal white adipose tissue, perirenal adipose tissue, and scapular brown adipose tissue were collected from mice in the ND, HFD, and WLP90 groups and fixed with 4% paraformaldehyde. Liver tissue was stained with Oil Red O and observed and photographed under a 100X microscope. The liver fat area was calculated using ImageJ software. Other adipose tissues were stained with hematoxylin and eosin (HE) (Seville Biotechnology Co., Ltd.). The tissues were observed and photographed under a 100X microscope, as shown below. Figures 11-15 As shown.

[0123] according to Figure 11 Liver tissue from the model group mice showed numerous lipid droplets in the hepatocytes, which stained red with Oil Red O. The normal group mice showed normal fat distribution in their livers.

[0124] Figure 12 To statistically analyze the percentage of lipid droplet area in the liver region of mice in each group using ImageJ software. For example... Figure 12 As shown, the proportion of liver fat area in the HFD group was significantly higher than that in the ND group, proving that the high-fat diet successfully established an obese mouse model with liver fat accumulation. The proportion of liver fat area in the WLP90 group was significantly lower than that in the HFD group, indicating that intervention with Bifidobacterium lactis subsp. animalis can effectively reduce the degree of liver fat deposition in obese mice.

[0125] This demonstrates that intervention with *Bifidobacterium animalis* subsp. *lactobacter* (WLP90) has significant health implications for obese mice. It significantly reduced hepatic fat accumulation induced by a high-fat diet and effectively improved hepatic lipid metabolism. This suggests that this strain has potential application value in preventing and alleviating obesity-related liver diseases (such as non-alcoholic fatty liver disease), providing experimental evidence for the development of probiotic preparations for regulating hepatic lipid metabolism.

[0126] Based on this, the embodiments also provide a probiotic preparation that helps reduce hepatic fat deposition. This preparation can be added as an active ingredient to foods such as yogurt, fermented milk, probiotic powder, and functional beverages, showing promise in improving the intestinal microecological balance of overweight / obese individuals, assisting in the regulation of fat metabolism, and reducing the risk of hepatic fat accumulation. In these embodiments, the probiotic preparation uses at least one or more of the following as active ingredients: dead, live, inactivated, or metabiotic cells of *Bifidobacterium animalis* subsp. *lactamase* WLP90, and fermentation broth. The formulation method of the probiotic preparation in these embodiments specifically refers to the formulation method of the active ingredient in the aforementioned antibacterial preparations or pancreatic lipase inhibitors, and the excipients also refer to the formulation method of the excipients in the aforementioned antibacterial preparations or pancreatic lipase inhibitors, which will not be elaborated here.

[0127] Enlarged fat cells are one of the important causes of obesity. Figure 13 , Figure 14 , Figure 15 The images show white adipose tissue, perirenal fat, and brown adipose tissue in the scapula, respectively. As can be seen from the figures, the model group mice exhibited severe lipid accumulation in their adipocytes, with disordered and loose cell arrangement and significantly increased adipocyte diameter. In contrast, the normal group mice had smaller, more compact, and more numerous adipocytes. Compared to the model group, the WLP90 group showed relatively smaller adipocyte diameters and more orderly and compact cell arrangement. WLP90 treatment can alleviate adipose tissue hypertrophy in obese model mice.

[0128] (7) Monitoring of gut microbiota in mice in each group

[0129] After the experiment, mice were euthanized, their abdomens were disinfected and dissected, and cecal contents from the normal group, model group, and WLP90 group were collected for microbial diversity analysis. The 16S rDNA V3-V4 hypervariable regions of all bacteria in the samples were sequenced on the Mi Seq Illumina sequencing platform to determine the characteristics of the gut microbiota.

[0130] β-diversity is used to compare the similarity of different samples in terms of species diversity. NMDS is a common analytical method that reflects the differences and distances between samples. When Stress is less than 0.2, it indicates that NMDS analysis has a certain degree of reliability; samples that are closer together on the coordinate graph are more similar.

[0131] Figure 16This is a graph showing the NMDS analysis results of the gut microbiota of each group of mice. In the graph, NMDS1 represents the major dimension of gut microbiota structural variation, and NMDS2 represents the minor dimension of gut microbiota structural variation. The stress value of 0.098 indicates that the NMDS ranking results are reliable and can well reflect the differences in microbiota structure. R = 0.83209 indicates high explanatory power for inter-group differences, and P = 0.001 indicates that the inter-group differences are statistically significant. The samples in the ND group are concentrated in the region of NMDS1 > 0 and NMDS2 ≈ -0.3 to 0.2, with moderate dispersion within the group. The samples in the HFD group are concentrated in the region of NMDS1 < 0 and NMDS2 ≈ 0 to 0.3, completely separated from the ND group, indicating that a high-fat diet significantly alters the gut microbiota structure. The samples in the WLP90 group are concentrated in the region of NMDS1 < 0 and NMDS2 ≈ -0.1 to 0, clearly separated from the HFD group and closer to the distribution trend of the ND group.

[0132] Therefore, as follows Figure 16 It was found that the model group and the healthy group differed significantly. The HFD group and the ND group were completely separated in the NMDS space, indicating that the high-fat diet successfully induced significant changes in the gut microbiota structure, forming obesity-related microbiota characteristics. The WLP90 group and the HFD group did not overlap in the NMDS space, indicating that the intervention of Bifidobacterium lactis significantly altered the gut microbiota structure of obese mice, causing it to deviate from the microbiota characteristics of the obesity model. The microbiota distribution of the WLP90 group was closer to that of the ND group, suggesting that the intervention restored the healthy characteristics of the gut microbiota to a certain extent and alleviated the dysbiosis caused by the high-fat diet. Based on this, the WLP90 strain provided in this application can significantly reverse the gut microbiota dysbiosis induced by the high-fat diet, making the microbiota structure approach the healthy state from the characteristics of the obesity model. This improvement in microbiota structure provides a microecological basis for subsequent improvement of metabolic disorders (such as hepatic fat accumulation, insulin resistance, etc.), suggesting that this strain has potential application value in the prevention and alleviation of obesity-related metabolic diseases.

[0133] Core colony identification is crucial because different environments have different microbial community compositions, partly due to the sensitivity of microorganisms to their environment. Microorganisms can be classified according to their environmental sensitivity, and further divided into transient, intermediate, and persistent species based on their detection rate (prevalence). By comparing the quantity and abundance of these species, we can study the relative environmental sensitivity of each type of microorganism in the sample. For example, a higher proportion of persistent species and a lower proportion of transient species indicate a weaker environmental sensitivity of that type of microorganism in the study sample.

[0134] Figure 17This figure shows the results of core colony identification and analysis of the gut microbiota in each group of mice. The figure displays the proportions of different stability types (transient, intermittent, and persistent) in the gut microbiota of the three groups of mice from two dimensions: abundance and number. The abundance of persistent microbiota in the HFD group (56.61%) was similar to that in the ND group (58.41%), indicating that the high-fat diet did not significantly change the abundance proportion of the core microbiota. However, after WLP90 intervention, the abundance of persistent microbiota increased significantly, further strengthening the core stability of the microbiota. The abundance of intermittent microbiota in the WLP90 group (20.27%) was much lower than that in the model group (43.22%), indicating that *Bifidobacterium animalis* subsp. *lactamase* effectively reduced the intermittent fluctuations of the microbiota and enhanced the stability of the gut microbiota.

[0135] Depend on Figure 17 It is evident that the WLP90 strain provided in this application significantly increases the abundance of persistent core flora in the gut microbiota while decreasing the abundance of intermittent flora, resulting in a more stable and resistant microbiota structure. This enhanced microbiota stability helps maintain gut microecological balance, reduces the risk of metabolic disorders, and provides microecological support for improving obesity-related pathological conditions such as hepatic steatosis. Combined with previous NMDS analysis and hepatic fat percentage results, the enhanced core stability of the microbiota is consistent with the trend of the microbiota structure approaching a healthy state and the reduction of hepatic steatosis, further supporting the mechanism by which this strain improves obesity-related metabolic diseases by optimizing the core structure of the gut microbiota.

[0136] Based on the significant effect of the WLP90 strain in reducing the abundance of intermittent gut microbiota, this application also provides a probiotic preparation for reducing the abundance of intermittent microbiota in the human gut and a research preparation for reducing the abundance of intermittent microbiota in the gut of experimental animals. In these embodiments, the probiotic preparation or research preparation uses at least one or more of the following as active ingredients: dead cells, live cells, inactivated cells, metabiotics, and fermentation broth of Bifidobacterium lactis subsp. animalis WLP90. The preparation method of the probiotic preparation in these embodiments is specifically the same as that of the active ingredient in the above-mentioned antibacterial preparation or pancreatic lipase inhibitor, and the excipients are also prepared in the same way as those in the above-mentioned antibacterial preparation or pancreatic lipase inhibitor, which will not be described in detail here.

[0137] The abundance changes of gut microbiota at the phylum and genus levels in each group of mice were analyzed. The relative abundance at different phylum levels is as follows: Figure 18As shown, the study indicates that the Firmicutes / Bacteroidetes (F / B) ratio can serve as an indicator of gut microbiota health. A higher F / B ratio may exacerbate intestinal inflammation, thereby promoting the development of obesity-related diseases. A high-fat diet led to a significant increase in Firmicutes abundance, a significant decrease in Bacteroidetes abundance, and an increase in the Firmicutes / Bacteroidetes ratio in the model group. The WLP90 group significantly improved these effects and approached those of the healthy group. It also increased the abundance of Actinobacteria in the gut microbiota.

[0138] Figure 19 The relative abundance of six key bacterial genera in the intestines of three groups of mice (ND healthy group, HFD model group, and WLP90 intervention group) is shown. Different letters (a, b, c) indicate statistically significant differences between groups (P<0.05). In the figure, the abundance of *Bifidobacterium* jumped from extremely low levels to the highest after WLP90 intervention, directly supplementing and enriching probiotics and strengthening intestinal barrier function. For *Akkermansia*, a high-fat diet in the model group led to a significant decrease in its abundance (associated with metabolic disorders), but it significantly rebounded after WLP90 intervention, approaching the levels of the healthy group, which helps improve intestinal barrier and metabolic health. For *Allobaculum* and *Dubosiella*, they were abnormally elevated in the HFD model group, associated with obesity and inflammatory responses, but both significantly decreased after WLP90 intervention, returning to the levels of the healthy group, effectively reversing dysbiosis. For Extibacter, the level was significantly elevated in the model group. Although it did not fully recover after WLP90 intervention, it was significantly reduced, demonstrating a clear inhibitory effect.

[0139] Depend on Figure 19 The results show that strain WLP90 significantly increases the abundance of Bifidobacterium, strengthening the beneficial foundation of the gut microbiota. Strain WLP90 effectively inhibits abnormal bacteria genera associated with obesity, such as *Alternaria* and *Dubosiella*, reversing dysbiosis while restoring the levels of beneficial bacteria like *Ackermania*, thus improving the gut microbiota structure. This optimized microbiota structure provides microecological support for alleviating hepatic fat accumulation and improving metabolic disorders, suggesting that this strain has important application value in the prevention and treatment of obesity-related metabolic diseases.

[0140] Combining previous NMDS analysis, core microbiota stability, and liver fat percentage results, the genus-level microbiota regulation is highly consistent with the improvement of overall microbiota structure, the enhancement of core stability, and the alleviation of pathological indicators, further confirming the mechanism by which Bifidobacterium animalis subsp. lactis improves obesity-related pathological conditions by remodeling the gut microbiota.

[0141] Based on this, this application also provides a probiotic preparation that reduces the abundance of *Codonopsis* and *Dubosiella* bacteria in the gut. This preparation uses *Bifidobacterium lactis* subsp. *animal* WLP90 as the active ingredient and has the potential to be added to common food ingredients such as fermented milk, solid beverages, and food additives. It is used to reduce the relative abundance of *Codonopsis* and *Dubosiella* bacteria in the gut of consumers and regulate the composition of the gut microbiota.

[0142] The embodiment also provides a research formulation for reducing the abundance of *Alternaria* and *Dubosiella* bacteria in the gut of experimental animals. This formulation uses *Bifidobacterium lactis* subsp. *animal* WLP90 as the active ingredient and is intended for scientific research purposes only. It is used to reduce the relative abundance of *Alternaria* and *Dubosiella* bacteria in the gut of experimental mice (or other experimental animals) and to construct a research model for the regulation of gut microbiota composition.

[0143] Based on this, embodiments of this application also provide the application of Bifidobacterium lactis subsp. WLP90 in the preparation of probiotic formulations, which are used to reduce the relative abundance of Allergenia and Dubosiella in the human gut.

[0144] Based on this, embodiments of this application also provide the application of Bifidobacterium lactis subsp. WLP90 in the preparation of experimental animal research formulations, wherein the formulations are used to reduce the relative abundance of Allostellaria and Dubosiella species in the intestines of experimental animals; the formulations are for scientific research purposes only and are not for disease treatment, food consumption or health care purposes.

[0145] The probiotic or research formulations in these embodiments all use at least one or more of the following as active ingredients: dead, live, inactivated, postbiotic, and fermentation broth of Bifidobacterium animalis subsp. lactis WLP90. The preparation methods of these probiotic formulations are specifically the same as those used in the preparation of active ingredients in the aforementioned antibacterial agents or pancreatic lipase inhibitors, and the excipients are also prepared in the same manner as those used in the aforementioned antibacterial agents or pancreatic lipase inhibitors; therefore, they will not be elaborated upon here.

[0146] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A strain of Bifidobacterium animalis subsp. lactis WLP90, which is Bifidobacterium animalis subsp. lactis with accession number CGMCC NO.33688.

2. An antibacterial preparation comprising at least one or more of the following: dead cells, live cells, inactivated cells, metabiotics, and fermentation broth of Bifidobacterium lactis subsp. WLP90 as described in claim 1.

3. The antibacterial agent according to claim 2 is selected from at least one of the following: antibacterial agents of Actinomyces israelii, antibacterial agents of Enterobacter cancerogenus, and antibacterial agents of Alloscardovia omnicolens.

4. A glucagon-like peptide-1 stimulant comprising at least one or more of the following: dead cells, live cells, inactivated cells, metabiotics, and fermentation broth of Bifidobacterium lactis subsp. WLP90 as described in claim 1.

5. A probiotic preparation for assisting in reducing liver fat deposition, comprising at least one or more of the following: dead cells, live cells, inactivated cells, postbiotics, and fermentation broth of Bifidobacterium lactis subsp. WLP90 as described in claim 1.

6. A probiotic preparation for reducing the abundance of intermittent flora in the gut, comprising at least one or more of the following: dead cells, live cells, inactivated cells, metabiotics, and fermentation broth of Bifidobacterium lactis subsp. WLP90 as described in claim 1.

7. A probiotic preparation for reducing the abundance of *Alternaria* and *Dubosiella* in the gut, comprising at least one or more of the following: dead cells, live cells, inactivated cells, metabiotics, and fermentation broth of *Bifidobacterium lactis* subsp. *Lactobacillus* WLP90 as described in claim 1.

8. A research preparation for reducing the abundance of intermittent bacterial flora in the intestines of experimental animals, comprising at least one or more of the following: dead cells, live cells, inactivated cells, metabiotics, and fermentation broth of Bifidobacterium lactis subsp. WLP90 as described in claim 1.

9. A research preparation for reducing the abundance of *Alternaria* and *Dubosiella* in the intestines of experimental animals, comprising at least one or more of the following: dead cells, live cells, inactivated cells, metabiotics, and fermentation broth of *Bifidobacterium lactis* subsp. *wLP90* as described in claim 1.

10. The application of Bifidobacterium animalis subsp. lactis WLP90 according to claim 1, wherein the application includes: Preparation of a fermentation formulation of Bifidobacterium lactis subsp. WLP90; Preparation of antibacterial agents; Preparation of inhibitors of pancreatic lipase; Preparation of stimulants for glucagon-like peptide-1; To prepare a probiotic formulation that helps reduce fat deposition in the liver; Prepare probiotic formulations that reduce the abundance of intermittent flora in the gut; Prepare a probiotic formulation that reduces the abundance of *Alternaria* and *Dubosiella* in the gut. To prepare research formulations that reduce the abundance of intermittent bacterial flora in the intestines of laboratory animals; and Prepare at least one of the following research formulations to reduce the abundance of *Alternaria* and *Dubosiella* in the intestines of laboratory animals.

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