Clostridium prasrificum compositions, methods of making and uses

Abstract

This invention discloses a *Clostridium pluvialis* composition, its preparation method, and its applications, relating to the field of probiotics technology. It aims to help address obesity in the elderly. The composition comprises five active microbial flora, including *Clostridium pluvialis* and *Bifidobacterium longum*, a fructooligosaccharide-inulin prebiotic complex, and a fucoidan-glycerol encapsulation excipient. The preparation method includes four main steps: preparation of the bacterial suspension, preparation of the encapsulation medium, microencapsulation solidification, and final product mixing. Freeze-drying ensures the stability of the live bacteria. The composition can be used to prepare drugs or health products for the prevention, relief, or treatment of obesity in the elderly. This invention can help improve intestinal flora imbalance in the elderly, enhance intestinal barrier function, and improve the safety and effectiveness of obesity intervention.

Description

Technical Field

[0001] This invention relates to the field of probiotics. More specifically, this invention relates to a Clostridium plasminogen activator composition, its preparation method, and its application. Background Technology

[0002] Obesity among the elderly has become a growing global health challenge, severely impacting their quality of life and increasing the risk of complications such as diabetes and cardiovascular disease, thus exacerbating the social healthcare burden. Numerous studies have shown that gut microbiota imbalance is a key factor inducing obesity in the elderly, and probiotic intervention, due to its safety and gentleness, has become a focus of research in this field.

[0003] Certain beneficial gut bacteria (such as Clostridium plasminogen lysate) have a positive effect on regulating metabolism and improving obesity-related symptoms, but they are easily inactivated by gastric acid when used alone, resulting in poor bioavailability. Existing related products often suffer from problems such as strain incompatibility and insufficient compatibility of excipients, which limits their efficacy and fails to meet the intervention needs of elderly obesity.

[0004] Therefore, it is necessary to design a technical solution that can overcome the above-mentioned defects. Summary of the Invention

[0005] One object of the present invention is to provide a Clostridium praosporum composition, preparation method and application, which can help improve the intestinal flora imbalance in the elderly and enhance the intestinal barrier function.

[0006] To achieve these objectives and other advantages of the present invention, according to one aspect of the present invention, a Clostridium pluvialis composition is provided, comprising an active microbial community, prebiotics, and pharmaceutically acceptable excipients; wherein the active microbial community comprises Clostridium pluvialis, Bifidobacterium longum, Lactobacillus plantarum, Akkermansia myxobolus, and Bacteroides amyloliquefaciens, the prebiotics are a mixture of fructooligosaccharides and inulin, and the pharmaceutically acceptable excipients include fucoidan and glycerol.

[0007] Furthermore, the active microbial flora consists of 10-50 parts by total live bacteria; 20-100 parts by fructooligosaccharides; 10-50 parts by inulin; 5-25 parts by fucoidan; and 1-5 parts by glycerol; wherein the weight ratio of fucoidan to glycerol is 5:1.

[0008] According to another aspect of the present invention, a method for preparing a Clostridium plasminoides composition is also provided, comprising: S1: mixing an active microbial community of Clostridium plasminoides, Bifidobacterium longum, Lactobacillus plantarum, Akkermansia myxobolus, and Bacteroides amyloliquefaciens, and resuspending it in physiological saline to form a homogeneous active microbial community suspension; S2: dissolving fucoidan in water, completely dissolving it at 40-50°C with continuous stirring to form a fucoidan solution, then adding glycerol to the fucoidan solution, mixing evenly to obtain an encapsulation medium, and cooling it to 25-30°C; S3: under inert gas protection and continuous stirring conditions, the mixture from step S1... S1. The obtained active microbial flora suspension is slowly added to the embedding medium obtained in step S2. After mixing evenly, the resulting mixture is added dropwise to calcium chloride solution. During the dropwise addition, shear force is applied to form embedded microcapsules and a solidification reaction is carried out. S4. The solidified embedded microcapsules are collected, washed, and mixed with fructooligosaccharides and inulin to obtain the Clostridium praecoxibacillus composition. The active microbial flora consists of 10-50 parts by total viable bacteria; fructooligosaccharides consist of 20-100 parts; inulin consists of 10-50 parts; fucoidan consists of 5-25 parts; glycerol consists of 1-5 parts; and the weight ratio of fucoidan to glycerol is 5:1.

[0009] Further, in S1, *Clostridium plasmidon*, *Bifidobacterium longum*, *Lactobacillus plantarum*, *Ackermania mutagenes*, and *Bacteroides amyloliquefaciens* were pre-activated and expanded in culture until each strain reached the late stage of stable growth. The obtained bacterial cells of each strain were mixed with *Clostridium plasmidon*, *Bifidobacterium longum*, *Lactobacillus plantarum*, *Ackermania mutagenes*, and *Bacteroides amyloliquefaciens* in a ratio of (15-25):(10-20):(8-15):(3-8):(5-10) based on viable cell count. The mixed active microbial community was resuspended in pre-cooled, sterile physiological saline containing 0.5%-1.5% w / v reduced glutathione at 4-10℃ under inert gas protection, controlling the final total viable cell concentration of the suspension to be 5 × 10⁻⁶. 10 CFU / mL up to 2×10 11 CFU / mL, forming a uniform suspension of active microbial flora.

[0010] Further, in S2, fucoidan is uniformly dispersed in water at 40-50°C using a combination of vortex oscillation and mechanical stirring, wherein the mass of water is 15-30 times the mass of fucoidan. Under continuous stirring, 5%-15% trehalose (by mass of fucoidan) is added to the solution, and after it is completely dissolved, glycerol is added. The resulting mixed solution is homogenized at a shear rate of 2000-5000 rpm for 2-5 minutes, then allowed to stand to remove bubbles, obtaining the embedding medium, and its final viscosity is controlled to be 800-1500 cP. The embedding medium is cooled to 25-30°C under light-proof and inert gas protection.

[0011] Further, in step S3, under continuous stirring and a carbon dioxide inert gas protection, the active microbial community suspension obtained in step S1 is preheated to 25-30℃, and then added dropwise to the embedding medium obtained in step S2 at a rate of 0.5-2 mL / min, controlling the volume ratio of the active microbial community suspension to the embedding medium to be 1:(8-15). After mixing, the resulting microbial community-embedding medium mixture is added dropwise to a 1.0-2.5% w / v calcium chloride solution using a syringe pump at a rate of 10-30 drops / min, wherein the calcium chloride solution contains 0.5-1.0% pre-dissolved microorganisms. w / v trehalose; during the dropwise addition, the calcium chloride solution receiving the droplets is stirred to provide stable shear force, ensuring that the embedded microcapsules have a particle size distribution in the range of 300-600μm; after the dropwise addition is completed, the embedded microcapsules are allowed to stand and solidify in the calcium chloride solution at 4-10℃ for 20-40 minutes.

[0012] Further, in S4, the solidified embedded microcapsules are collected and washed with pre-cooled phosphate buffer solution with a pH of 6.5-7.0, containing 5%-10% (w / v) trehalose as a protective agent. The washed embedded microcapsules are then freeze-dried. The freeze-drying process is as follows: first, the temperature is lowered to below -40℃ at a rate of 0.5-1℃ / min and held for 2-4 hours; then, under a vacuum of less than 10 Pa, the main drying is carried out at a gradient temperature increase from -25℃ to 0℃ for 20-30 hours; finally, the desorption drying is carried out at 25-30℃ for 4-8 hours to obtain dried embedded microcapsule powder. The obtained dried embedded microcapsule powder is mixed with fructooligosaccharides and inulin in an inert gas protective environment with a temperature ≤25℃ and a relative humidity ≤15%.

[0013] According to another aspect of the invention, the use of the Clostridium praosporum composition is also provided for the preparation of a medicament for the prevention, relief or treatment of obesity in the elderly.

[0014] The present invention has at least the following beneficial effects: The multi-strain synergistic effect of this invention is prominent. The combination of *Clostridium pluvialis* with other beneficial bacteria can rapidly regulate the imbalance of gut microbiota in the elderly, enhance intestinal barrier function, reduce the release of inflammatory factors, and improve metabolic disorders from the source. Prebiotics and probiotics are precisely matched; fructooligosaccharides and inulin provide continuous nutrition for live bacteria, increasing intestinal colonization rate. The encapsulation system constructed from fucoidan and glycerol effectively resists gastric acid erosion, significantly improving the survival rate of live bacteria reaching the intestine. The composition is safe and gentle, suitable for the physiological characteristics of the elderly, and can both assist in weight regulation and improve lipid metabolism, reducing the risk of obesity-related complications, providing a safe and effective new solution for the prevention and relief of obesity in the elderly.

[0015] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0016] Figure 1 This is a flowchart of the method for preparing the Clostridium praosporum composition of this application. Detailed Implementation

[0017] The present invention will now be described in further detail so that those skilled in the art can implement it based on the description.

[0018] It should be understood that terms such as "having," "comprising," and "including" used in the embodiments of this application do not exclude the presence or addition of one or more other elements or combinations thereof. All directional indications (such as up, down, left, right, front, back, etc.) in the embodiments of this application are only used to explain the relative positional relationship and movement of components in a specific posture. If the specific posture changes, the directional indication will also change accordingly. When an element is referred to as "fixed to" or "set on" another element, it can be directly on the other element or may have an intervening element present. When an element is referred to as "connected to" another element, it can be directly connected to the other element or indirectly connected to the other element through an intervening element. Descriptions involving "first," "second," etc., in the embodiments of this application are for descriptive purposes only and should not be construed as indicating or implying their relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined with "first" or "second" may explicitly or implicitly include at least one of those features.

[0019] It should be noted that the technical solutions of the various embodiments of this application can be combined with each other, but only if they are based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by this application.

[0020] The embodiments of this application provide a Clostridium pluvialis composition, which comprises an active microbial community, prebiotics, and pharmaceutically acceptable excipients; wherein the active microbial community comprises Clostridium pluvialis, Bifidobacterium longum, Lactobacillus plantarum, Akkermansia myxobolus, and Bacteroides amyloliquefaciens, the prebiotics are a mixture of fructooligosaccharides and inulin, and the pharmaceutically acceptable excipients include fucoidan and glycerol.

[0021] For example, the Clostridium pluvialis composition is a compound preparation with Clostridium pluvialis as the core active ingredient. It is composed of active microbial flora, prebiotics and pharmaceutically acceptable excipients. The active microbial flora refers to a collection of various microbial strains that have physiological activity and can colonize and exert effects in the human intestine. Prebiotics are a class of indigestible carbohydrates that cannot be digested and absorbed by the human body but can selectively promote the growth and reproduction of beneficial bacteria in the intestine. Pharmaceutically acceptable excipients refer to substances that meet the national pharmacopoeia standards, have no toxic side effects on the human body, and can assist in the formation of the formulation, stabilize or enhance the efficacy of the drug. The active microbial community specifically includes *Clostridium plasminogen lysate*, *Bifidobacterium longum*, *Lactobacillus plantarum*, *Akermansia myxobolus*, and *Bacteroides amyloliquefaciens*; the prebiotic is a mixture of fructooligosaccharides and inulin. Fructooligosaccharides are oligosaccharides formed by 2-10 fructose molecules linked by β-2,1 glycosidic bonds, while inulin is a polysaccharide composed of multiple fructose molecules linked by β-2,1 glycosidic bonds with glucose as the terminal. The mixing ratio of the two can be 1:1 or 2:1; pharmaceutically acceptable excipients include fucoidan and glycerol. Fucoidan is a water-soluble polysaccharide extracted from brown algae, and glycerol is a colorless, transparent, viscous liquid with moisturizing and plasticizing effects. When preparing this composition, the active microbial flora can be in the form of freeze-dried bacterial powder or freshly cultured bacterial liquid; the prebiotics can be food-grade or pharmaceutical-grade fructooligosaccharides; the inulin can be inulin extracted from Jerusalem artichoke or inulin extracted from dandelion; the fucoidan can be fucoidan derived from kelp or fucoidan derived from wakame seaweed; and the glycerol can be pharmaceutical-grade or food-grade glycerol.

[0022] In existing technologies, microbial compositions for intestinal regulation often contain only one or two types of probiotics, combined with a single prebiotic and conventional excipients. For example, a composition containing Bifidobacterium and Lactobacillus, combined with fructooligosaccharides and starch excipients, is difficult to adapt to the complex intestinal environment. This embodiment combines five specific active microbial strains to synergistically exert intestinal regulatory effects, and uses two complementary prebiotics to provide nutrition for the probiotics. Fucoidan and glycerol are selected as excipients, which can both protect the activity of probiotics and improve the stability of the formulation. Compared with existing technologies, this composition is more compatible with the human intestinal microecological system, the synergistic effect of each component is more significant, and the selection and combination of components are based on the growth characteristics and functional needs of intestinal microorganisms.

[0023] In another embodiment, the active microbial flora consists of 10-50 parts by total live bacteria; 20-100 parts by fructooligosaccharides; 10-50 parts by inulin; 5-25 parts by fucoidan; and 1-5 parts by glycerol; wherein the weight ratio of fucoidan to glycerol is 5:1.

[0024] For example, in the above-mentioned Clostridium praosporum composition, the active microbial flora, based on the total number of viable bacteria, is 10-50 parts by weight, specifically 20 parts or 35 parts. Here, "parts by weight" refers to the relative proportions between the components, used to guide the formulation process. For instance, when the active microbial flora is selected as 20 parts, the other components are prepared according to their corresponding weight proportions. The fructooligosaccharide is 20-100 parts by weight, specifically 40 parts or 70 parts. This weight range is set to ensure sufficient nutrients are provided to the active microbial flora to promote their growth and reproduction in the intestine. The inulin is 10-50 parts by weight, specifically 25 parts or 40 parts. It works synergistically with the fructooligosaccharide to more comprehensively promote the proliferation of beneficial intestinal bacteria. The weight parts of fucoidan are 5-25 parts, specifically 10 or 20 parts, and the weight parts of glycerol are 1-5 parts, specifically 2 or 4 parts. The weight ratio of fucoidan to glycerol is strictly controlled at 5:1, that is, the weight of fucoidan is always 5 times the weight of glycerol. For example, when 2 parts of glycerol are selected, 10 parts of fucoidan are selected accordingly. This fixed ratio allows fucoidan and glycerol to form a stable encapsulation system, which better protects the active microbial community.

[0025] In existing technologies, the composition ratios of microbial compositions often lack precise design, with significant arbitrariness in the proportions of probiotics, prebiotics, and excipients, leading to low probiotic survival rates and insufficient formulation stability. This embodiment clarifies the weight range of each component and the fixed ratio of fucoidan to glycerol. The range of prebiotic dosages can adapt to the nutritional needs of different total live bacteria counts, and the fixed ratio of excipients forms a stable protective system. Compared to existing technologies, this formulation improves the storage stability of the composition and the intestinal colonization ability of probiotics. Furthermore, the formulation design is based on the functional characteristics and synergistic effects of each component, providing a reasonable technical basis.

[0026] like Figure 1As shown, embodiments of this application also provide a method for preparing a *Clostridium plasmidon* composition, comprising: S1: mixing *Clostridium plasmidon*, *Bifidobacterium longum*, *Lactobacillus plantarum*, *Akermansia myxobolus*, and *Bacteroides amyloliquefaciens*, and resuspending them in physiological saline to form a homogeneous suspension of active microbial flora; S2: dissolving fucoidan in water, completely dissolving it at 40-50°C with continuous stirring to form a fucoidan solution, then adding glycerol to the fucoidan solution, mixing evenly to obtain an encapsulation medium, and cooling it to 25-30°C; S3: under inert gas protection and continuous stirring conditions, the mixture from step S1... S1. The obtained active microbial flora suspension is slowly added to the embedding medium obtained in step S2. After mixing evenly, the resulting mixture is added dropwise to calcium chloride solution. During the dropwise addition, shear force is applied to form embedded microcapsules and a solidification reaction is carried out. S4. The solidified embedded microcapsules are collected, washed, and mixed with fructooligosaccharides and inulin to obtain the Clostridium praecoxibacillus composition. The active microbial flora consists of 10-50 parts by total viable bacteria; fructooligosaccharides consist of 20-100 parts; inulin consists of 10-50 parts; fucoidan consists of 5-25 parts; glycerol consists of 1-5 parts; and the weight ratio of fucoidan to glycerol is 5:1.

[0027] For example, the preparation method of the Clostridium praosporum composition includes four steps. First, five active microbial groups—C. praosporum, Bifidobacterium longum, Lactobacillus plantarum, Akkermansia myxobolus, and Bacteroides amyloliquefaciens—are mixed. The mixing operation can be performed in a sterile mixer to ensure uniform mixing of the microbial groups. Subsequently, the mixed microbial groups are resuspended in physiological saline, which can be a 0.9% sodium chloride aqueous solution or sterile phosphate-buffered saline. The resuspension process is completed using a magnetic stirrer, ultimately forming a homogeneous suspension of active microbial groups without obvious particle precipitation. The second step involves dissolving fucoidan in water, which can be either water for injection or sterile purified water. The dissolution process is carried out at a temperature of 40-50℃, controlled by a constant temperature water bath, to ensure complete dissolution of the fucoidan and the formation of a homogeneous fucoidan solution. Subsequently, glycerol is slowly added dropwise to the fucoidan solution using a separatory funnel or peristaltic pump. The adding device is positioned above the fucoidan solution, and the dropping rate is 1-2 ml / min. After addition, stirring is continued for 10-15 minutes until the mixture is homogeneous, resulting in the embedding medium. The embedding medium is then cooled to 25-30℃ by natural cooling in a cool, ventilated place or by cooling in a water bath. The third step involves operation under inert gas protection and continuous stirring. Nitrogen or argon can be used as the inert gas. The gas supply equipment is a high-pressure gas cylinder with a pressure reducing valve, positioned above the reaction vessel. The gas is introduced into the vessel through a conduit, ensuring the oxygen concentration inside is below 1%. Continuous stirring is achieved using a mechanical stirrer at a speed of 250-350 rpm, positioned at the center of the reaction vessel. The active microbial suspension obtained in the first step is slowly pumped into the embedding medium obtained in the second step using a metering pump, and mixed thoroughly to obtain a mixture. This mixture is then added dropwise to a 1.5% or 2.0% calcium chloride aqueous solution using a syringe pump or a dropping funnel, positioned above the calcium chloride solution. During the dropwise addition, shear force is applied using a high-speed disperser to form embedded microcapsules, which are then allowed to solidify at room temperature for 20-30 minutes. The fourth step involves collecting the solidified embedded microcapsules using a vacuum filter or centrifuge; washing them 2-3 times with sterile water; mixing the washed embedded microcapsules with fructooligosaccharides and inulin in a three-dimensional motion mixer or V-type mixer, with the mixing equipment positioned in a sterile mixing area, to finally obtain the Clostridium praosporum composition.

[0028] This embodiment reduces oxidative damage to probiotics by using inert gas protection, controls the temperature, rotation speed, and drop rate in each step, optimizes the preparation of the encapsulation medium and the microcapsule formation process, and produces microcapsules with uniform particle size and higher probiotic survival rate. Furthermore, the parameter design of the preparation process is all centered around protecting the activity of probiotics and improving the stability of the formulation.

[0029] In another embodiment, in S1, *Clostridium plasmidon*, *Bifidobacterium longum*, *Lactobacillus plantarum*, *Ackermania mutagenes*, and *Bacteroides amyloliquefaciens* are pre-activated and expanded in culture until each strain reaches the late stage of stable growth. The obtained bacterial cells of each strain are mixed with *Clostridium plasmidon*, *Bifidobacterium longum*, *Lactobacillus plantarum*, *Ackermania mutagenes*, and *Bacteroides amyloliquefaciens* in a ratio of (15-25):(10-20):(8-15):(3-8):(5-10) based on the viable count. The mixed active microbial community is resuspended in pre-cooled, sterile physiological saline containing 0.5%-1.5% w / v reduced glutathione at 4-10°C under inert gas protection, controlling the total viable bacterial concentration of the final suspension to be 5 × 10⁻⁶. 10 CFU / mL up to 2×10 11 CFU / mL, forming a uniform suspension of active microbial flora.

[0030] For example, in the step of preparing the active microbial community suspension, *Clostridium plasmidon*, *Bifidobacterium longum*, *Lactobacillus plantarum*, *Ackermania pseudomallei*, and *Bacteroides amylophilis* are first pre-activated and expanded in culture, respectively. During pre-activation, frozen bacterial powder of each strain is inoculated into the corresponding liquid culture medium. *Clostridium plasmidon* can be inoculated into BS medium or enhanced clostridium medium; *Bifidobacterium longum* can be inoculated into MRS medium or a dedicated bifidobacterium medium; *Lactobacillus plantarum* can be inoculated into MRS medium or LB medium; *Ackermania pseudomallei* can be inoculated into brain heart extract medium or modified GAM medium; and *Bacteroides amylophilis* can be inoculated into RCM medium or a dedicated *Bacteroides amylophilis* medium. The inoculation device can be a sterile inoculation loop or a pipette, assembled on a sterile operating table, with an inoculation volume of 2%-5% of the culture medium volume. Subsequently… Each strain was cultured under suitable conditions. *Clostridium plasmidon*, *Akermansia myxoma*, and *Bacteroides amyloliquefaciens* were cultured anaerobically at 37°C using an anaerobic incubator. *Bifidobacterium longum* was cultured at 37°C with 5% carbon dioxide. *Lactobacillus plantarum* was cultured aerobically at 37°C using a constant temperature incubator. For scale-up culture, 5L or 10L stainless steel fermenters were used, located in the fermentation workshop. Fermentation parameters were controlled until each strain reached the late stable growth phase. The late stable growth phase was determined by plate counting, using the same medium as the scale-up culture medium. The obtained bacterial cells were then mixed according to viable counts in a ratio of 20:15:12:5:8 or 22:18:10:6:7. The mixed active microbial flora was processed in a low-temperature environment of 4-10℃, achieved through a refrigerator or low-temperature operating table. Assembly was performed in the low-temperature operating area under nitrogen or carbon dioxide inert gas protection, with a high-pressure gas cylinder supplied next to the refrigerator. The flora was then resuspended in pre-cooled, sterile 0.9% sodium chloride saline containing 0.8% or 1.2% w / v reduced glutathione at 4℃. Resuspension was performed using a magnetic stirrer at 250-300 rpm for 8-10 minutes, controlling the final total viable bacterial concentration of the suspension to 8 × 10⁻⁶. 10 The concentration of CFU / mL was determined by plate counting, ultimately forming a homogeneous suspension of active microbial flora.

[0031] In this embodiment, each strain is pre-activated and cultured to the late stage of stable growth to ensure consistent strain activity. A specific ratio of viable bacteria is used to enable synergistic effects among the strains. Reduced glutathione is added to the resuspension for antioxidant effects, and low temperature and inert gas protection reduce activity loss. Compared with existing technologies, the suspension has higher viable bacteria stability and better synergistic effects among the strains. The technical solution is designed based on the growth characteristics and stability requirements of probiotics and has a reasonable technical basis.

[0032] In another embodiment, in S2, fucoidan is uniformly dispersed in water at 40-50°C using a combination of vortex oscillation and mechanical stirring, wherein the mass of water is 15-30 times the mass of fucoidan; under continuous stirring, 5%-15% trehalose by mass of fucoidan is added to the solution, and after it is completely dissolved, glycerol is added; the resulting mixed solution is homogenized at a shear rate of 2000-5000 rpm for 2-5 minutes, and then allowed to stand to remove bubbles to obtain the embedding medium, and its final viscosity is controlled to be 800-1500 cP; the embedding medium is cooled to 25-30°C under light-proof and inert gas protection.

[0033] For example, in the step of preparing the embedding medium, fucoidan is dispersed by a combination of vortex oscillation and mechanical stirring. The mechanical stirring equipment is an electric stirrer with a speed of 300-400 rpm and a stirring time of 8-10 minutes. Through the combination of the two stirring methods, the fucoidan is uniformly dispersed in water at 40-50°C. The water can be sterile water for injection or purified water, and the mass of the water is 20 times the mass of the fucoidan. Under continuous mechanical stirring, food-grade or pharmaceutical-grade trehalose, accounting for 8% of the mass of fucoidan, is added to the above solution. After the trehalose is completely dissolved for 5-8 minutes, pharmaceutical-grade or food-grade glycerol is added, with the amount added controlled according to a preset ratio. After adding, stirring continues for 10-15 minutes. The resulting mixed solution is homogenized at a shear rate of 3000 rpm. The homogenizing equipment can be a high-pressure homogenizer, and the homogenization time is 3 minutes. Homogenization makes the particle size in the solution more uniform. After homogenization, the solution is allowed to stand in a cool, ventilated place for 15-20 minutes to degas. After degassing, the embedded medium is obtained, and its final viscosity is measured using a rotational viscometer at 25°C, with the viscosity controlled at 1000 cP. Subsequently, the embedded medium is cooled to 25-30°C under light-proof and inert gas protection. Light protection is achieved using a black plastic or stainless steel light shield, which is mounted on the outside of the embedded medium container. The inert gas can be nitrogen or argon, supplied by a high-pressure gas cylinder, and cooled by natural cooling or a cooling water bath.

[0034] This embodiment uses a combination of vortex oscillation and mechanical stirring to disperse fucoidan, and adds trehalose to enhance the protective effect on probiotics. Homogenization and viscosity control ensure the homogeneity of the medium, and light protection and inert gas protection reduce component oxidation. The prepared encapsulation medium has high stability and good encapsulation and protection effect on probiotics.

[0035] In another embodiment, in step S3, under continuous stirring and a carbon dioxide inert gas protection, the active microbial community suspension obtained in step S1 is preheated to 25-30°C, and then added dropwise to the embedding medium obtained in step S2 at a rate of 0.5-2 mL / min, controlling the volume ratio of the active microbial community suspension to the embedding medium to be 1:(8-15). After mixing, the resulting microbial community-embedding medium mixture is added dropwise to a 1.0-2.5% w / v calcium chloride solution using a syringe pump at a rate of 10-30 drops / min, wherein the calcium chloride solution pre-dissolves 0.5-1.0% w / v of calcium chloride. w / v trehalose; during the dropwise addition, the calcium chloride solution receiving the droplets is stirred to provide stable shear force, ensuring that the embedded microcapsules have a particle size distribution in the range of 300-600μm; after the dropwise addition is completed, the embedded microcapsules are allowed to stand and solidify in the calcium chloride solution at 4-10℃ for 20-40 minutes.

[0036] For example, in the step of forming the encapsulated microcapsules, the operation is carried out under continuous stirring and inert carbon dioxide gas protection. The carbon dioxide gas can be food-grade or industrial-grade high-purity carbon dioxide. The gas supply equipment is a high-pressure carbon dioxide cylinder with a pressure reducing valve and a flow meter. The gas is introduced into the container through a conduit, and the carbon dioxide concentration in the container is maintained above 95% to isolate oxygen. The active microbial community suspension obtained in the first step is preheated to 26°C using a constant temperature water bath or heating mantle to ensure uniform temperature throughout the suspension. Then, the preheated suspension is added dropwise to the encapsulation medium obtained in the second step at a rate of 1.0 mL / min using a syringe pump or peristaltic pump. The suspension is slowly introduced through a conduit, while controlling the volume ratio of the active microbial community suspension to the encapsulation medium to be 1:10. The volume ratio is controlled by measuring with a volumetric flask or graduated cylinder. After mixing, the mixture is stirred continuously for 15-20 minutes to obtain a homogeneous microbial community-encapsulation medium mixture. The mixture was added dropwise to a 1.5% w / v calcium chloride solution using a syringe pump. The calcium chloride solution contained pre-dissolved 0.7% w / v trehalose. The calcium chloride solution was prepared beforehand using a magnetic stirrer positioned below the solution container. The dropping rate was controlled at 15 drops / min, ensuring the droplets fell vertically into the solution. During the dropping process, a mechanical stirrer was used to apply stable shear force to the calcium chloride solution at 200-300 rpm, ensuring the droplets formed uniform microcapsules in the solution with a particle size distribution within 400 μm. The particle size was measured using a laser particle size analyzer. After the dropping was complete, the embedded microcapsules were allowed to solidify in the calcium chloride solution at 6°C. The solidification environment was achieved using a refrigerator or cryogenic bath, with the assembly positioned in the solidification area. The solidification time was 25 minutes to further stabilize the microcapsule structure.

[0037] This embodiment uses carbon dioxide protection to reduce oxidative damage, precisely controls the temperature, rate, and volume ratio of each step, provides stable shear force through stirring to control particle size, and adds trehalose to the calcium chloride solution to enhance the protective effect. Compared with existing technologies, the microcapsule formation is more uniform and the survival rate of probiotics is higher.

[0038] In another embodiment, in S4, the solidified embedded microcapsules are collected and washed with pre-cooled phosphate buffer solution with a pH of 6.5-7.0, the phosphate buffer solution containing 5%-10% (w / v) trehalose as a protective agent; the washed embedded microcapsules are then freeze-dried, the freeze-drying process being as follows: first, the temperature is lowered to below -40°C at a rate of 0.5-1°C / min and held for 2-4 hours, then, under a vacuum of less than 10 Pa, the main drying is carried out at a gradient temperature increase from -25°C to 0°C for 20-30 hours, and finally, the desorption drying is carried out at 25-30°C for 4-8 hours to obtain dried embedded microcapsule powder; the obtained dried embedded microcapsule powder is mixed with fructooligosaccharides and inulin in an inert gas protected environment with a temperature ≤25°C and a relative humidity ≤15%.

[0039] For example, in the step of preparing the final composition, the solidified embedded microcapsules are first collected using a centrifuge or vacuum filtration device. The centrifugation speed is 3000-4000 rpm, and the centrifugation time is 5-8 minutes. For vacuum filtration, a Buchner funnel combined with a vacuum pump can be used, and the filtration time is 10-15 minutes. The collected embedded microcapsules are then washed with a phosphate buffer solution pre-cooled to 4°C and with a pH of 6.7. The phosphate buffer solution can be sodium dihydrogen phosphate-disodium hydrogen phosphate buffer or potassium dihydrogen phosphate-disodium hydrogen phosphate buffer, and the buffer solution contains 7% (w / v) trehalose as a protective agent. The washing process is carried out in centrifuge tubes or beakers. The microcapsules and buffer solution are mixed at a volume ratio of 1:5 and vortexed for 5-10 minutes at a frequency of 2000-3000 times / minute. This is followed by centrifugation, and the washing is repeated 2-3 times to remove residual calcium chloride from the surface. The washed embedded microcapsules are then dried using a freeze dryer. The freeze-drying process is as follows: First, the temperature is lowered to below -40℃ at a rate of 0.7℃ / min or 0.9℃ / min, and then held at that temperature for 2.5 hours or 3.5 hours. Then, under a vacuum of 5 Pa or 8 Pa, primary drying is carried out with a gradient temperature increase from -25℃ to 0℃, consisting of holding at -25℃ for 8 hours, -15℃ for 6 hours, -5℃ for 6 hours, and 0℃ for 4 hours, for a total primary drying time of 24 hours or 28 hours. Finally, desorption drying is performed at 26℃ or 29℃ for 5 hours or 7 hours, ultimately yielding dried encapsulated microcapsule powder with a moisture content of less than 5%. The obtained dried encapsulated microcapsule powder is mixed with fructooligosaccharides and inulin under specific conditions. The mixing environment is controlled at a temperature of 20℃ or 23℃ and a relative humidity of 10% or 13%, and is carried out under the protection of nitrogen or argon inert gas. The inert gas supply equipment is a high-pressure gas cylinder, assembled in the mixing area. The mixing equipment can be a three-dimensional motion mixer or a V-type mixer, with a mixing speed of 80-120 rpm and a mixing time of 15-20 minutes to ensure that all components are mixed evenly, thus obtaining the final Clostridium praosporum composition.

[0040] In this embodiment, freeze-drying is used to protect the activity of probiotics, trehalose is added to the washing buffer to enhance protection, and the temperature and humidity are controlled in the mixing environment and inert gas is introduced to avoid moisture absorption and oxidation. The dried microcapsule powder has higher stability and the composition has a longer shelf life.

[0041] Examples of this application provide the use of Clostridium pluvialis compositions for the preparation of medicaments for the prevention, relief or treatment of obesity in the elderly.

[0042] For example, the above-mentioned Clostridium pluvialis composition can be used to prepare drugs for the prevention, relief, or treatment of obesity in the elderly. Obesity in the elderly refers to metabolic diseases caused by excessive fat accumulation and a weight exceeding the standard weight by more than 20% in individuals aged 60 and above. The standard weight can be calculated according to the Chinese adult standard weight calculation formula. Prevention refers to regulating the intestinal microecology, reducing fat accumulation, and lowering the risk of obesity in elderly individuals whose weight is close to the upper limit of the standard weight and who have a tendency to become obese by taking this composition. Relief refers to improving intestinal metabolic function, slowing the weight gain trend, and alleviating obesity-related fatigue, bloating, and other discomforts in elderly individuals who have already developed obesity symptoms. Treatment refers to regulating the body's energy metabolism, reducing abnormal fat deposition in the body, and gradually restoring the weight to a reasonable range through long-term use. The drug can be formulated as capsules or tablets. Capsules can be hard capsules or soft capsules. Hard capsules can have gelatin shells or plant-based shells, while soft capsules can have gelatin-glycerin shells or polyethylene glycol shells. Tablets can be regular tablets or dispersible tablets. Tablet excipients may include microcrystalline cellulose or lactose as fillers and magnesium stearate as a lubricant. The drug is prepared in a GMP-compliant pharmaceutical facility. It is administered orally.

[0043] In existing technologies, most drugs for treating obesity in the elderly are chemical preparations that work by suppressing appetite or promoting fat metabolism. For example, one drug uses orlistat as its main ingredient, and long-term use can easily cause gastrointestinal discomfort. The drug in this embodiment is based on the intestinal microecological regulation mechanism. It improves the intestinal flora structure of the elderly through a probiotic composition, promoting fat metabolism. Compared with existing technologies, this drug is milder, has no obvious side effects, is more suitable for the physiological characteristics of the elderly, and has a simple preparation process. The components work synergistically, making it a valuable application.

[0044] The following is a description of a specific embodiment.

[0045] I. Experimental Group 1. Experimental Group 1: Active microbial flora: Total live bacteria count 30 parts (based on live bacteria ratio: Clostridium plasmidonii 20 parts, Bifidobacterium longum 15 parts, Lactobacillus plantarum 12 parts, Akkermansia myxobolus 5 parts, Bacteroides amyloliquefaciens 8 parts, all of which are freeze-dried bacterial powders with a purity ≥98%). Prebiotics: 60 parts fructooligosaccharides (food grade, Jerusalem artichoke source), 30 parts inulin (food grade, dandelion source); Pharmaceutical excipients: 15 parts fucoidan (kelp-derived, purity ≥90%), 3 parts glycerin (pharmaceutical grade), trehalose (food grade, amount is 10% of the mass of fucoidan); Other reagents: 0.9% sterile saline (containing 1.0% w / v reduced glutathione), 1.5% w / v calcium chloride solution (containing 0.8% w / v trehalose), pH 6.8 phosphate buffer (containing 8% w / v trehalose), carbon dioxide gas (purity ≥99.9%).

[0046] Preparation steps: S1: Preparation of active microbial community suspension Each strain underwent pre-activation and expansion culture separately: For pre-activation, frozen bacterial powder (purity ≥98%) of each strain was inoculated into the corresponding liquid culture medium at an inoculum volume of 5%-8% (BS medium for *Clostridium plasmidonum*, MRS medium for *Bifidobacterium longum*, MRS medium for *Lactobacillus plantarum*, modified GAM medium for *Ackermania myxobolus*, and RCM medium for *Bacteroides amyloliquefaciens*). The culture was subcultured twice at 37℃ (18 hours each time). For expansion culture, a 10L fermenter was used with a 6L liquid volume. Culture conditions were controlled according to the specific parameters for each strain (*Clostridium plasmidonum*: pH 6.0-7.0, strictly anaerobic, dissolved oxygen ≤0.5 mg / L, 24-48 hours; *Bifidobacterium longum*: pH 6.5-7.5, 5% CO2 environment, 18-24 hours; *Lactobacillus plantarum*: pH 6.2-7.2, aerobic environment, 16-20 hours; *Ackermania myxobolus*: pH 6.5-7.5, microaerophilic, dissolved oxygen 1-3 mg / L). mg / L, culture for 36-60 hours; Bacteroides amyloliquefaciens: pH 6.0-7.0, anaerobic environment, culture for 24-36 hours), until each strain reaches the late stable growth phase (judgment criteria: OD600 value difference < 0.02 between two consecutive measurements, and viable cell concentration ≥ 1×10 mg / L). 9 CFU / mL, for more than 2 hours).

[0047] Fermentation broths of each strain were collected, and the bacterial cells were collected by centrifugation at 3000 rpm for 5 minutes. The cells were then mixed according to the viable cell ratio (Clostridium plasmidoides: Bifidobacterium longum: Lactobacillus plantarum: Akkermansia myxoma: Bacteroides amyloliquefaciens = 20:15:12:5:8). Under conditions of 8°C and carbon dioxide protection (gas purity ≥99.9%, oxygen concentration in the container <1%), the mixed bacterial cell mass was resuspended in sterile physiological saline pre-cooled to 4°C and containing 1.0% w / v reduced glutathione. The cells were uniformly dispersed by magnetic stirring (300 rpm, stirring for 8 minutes), controlling the final total viable cell concentration of the suspension to 1×10⁻⁶. 11 CFU / mL, forming a homogeneous suspension of active microbial flora without obvious particle precipitation.

[0048] S2: Preparation of embedding medium Take 15 parts of fucoidan and add 25 times the mass of water for injection. Under 45°C, first vortex (2500 times / min, 4 minutes) and then mechanically stir (350 rpm, 10 minutes) to disperse and dissolve. Add 1.5 parts of trehalose and continue stirring until completely dissolved. Then add 3 parts of glycerol and continue stirring for 12 minutes. Homogenize at 4000 rpm for 4 minutes, let stand for 18 minutes to degas, and control the viscosity to 1200 cP. Cool to 28°C under light-protected and carbon dioxide-protected conditions.

[0049] S3: Microcapsule embedding preparation Under carbon dioxide protection and mechanical stirring (300 rpm), the S1 suspension was preheated to 28°C and added dropwise to the S2 embedding medium at a rate of 1.2 mL / min (volume ratio 1:11). The mixture was stirred for 20 minutes to ensure homogeneity. The mixture was then added dropwise to a 1.5% w / v calcium chloride solution (containing 0.8% w / v trehalose) at a rate of 20 drops / min using a syringe pump. The shear force was controlled at a stirring rate of 250 rpm to ensure that the microcapsule particle size was approximately 500 μm. After the addition was complete, the mixture was allowed to stand at 8°C for 30 minutes to solidify.

[0050] S4: Finished Product Preparation Microcapsules were collected by centrifugation (3500 rpm, 7 min) and washed three times with pre-cooled pH 6.8 phosphate buffer (with shaking for 7 min each time). The microcapsules were then freeze-dried (cooling rate 0.8℃ / min to below -40℃, holding for 3 hours; primary drying at -25℃ to 0℃ gradient for 26 hours under vacuum of 6 Pa; and desorption drying at 28℃ for 6 hours) to obtain microcapsule powder. The microcapsule powder was then mixed with 60 parts of fructooligosaccharide and 30 parts of inulin using a three-dimensional motion mixer (100 rpm, 18 min) at 22℃, 12% relative humidity, and under carbon dioxide protection until homogeneous. The mixture was then dispensed into capsules (each capsule containing 1×10⁻⁶ live bacteria). 11 CFU).

[0051] Experimental group 2: Compared to Experimental Group 1, the total number of viable microbial flora was modified to 25 (the ratio of viable strains was adjusted to Clostridium plasminogen lysate: Bifidobacterium longum: Lactobacillus plantarum: Akkermansia myxobolus: Bacteroides amyloliquefaciens = 22:18:13:6:9); fructooligosaccharides were modified to 80 parts, inulin to 40 parts; fucoidan to 20 parts, and glycerol to 4 parts (maintaining a 5:1 weight ratio); the remaining parameters and methods were the same as in Experimental Group 1.

[0052] Experimental group 3: Compared to Experimental Group 1, the total number of viable microbial flora was modified to 35 (the ratio of viable strains was adjusted to Clostridium plasminogen lysate: Bifidobacterium longum: Lactobacillus plantarum: Akkermansia myxobolus: Bacteroides amyloliquefaciens = 18:14:10:5:7); fructooligosaccharides were modified to 40 parts, inulin to 25 parts; fucoidan to 10 parts, and glycerol to 2 parts (maintaining a 5:1 weight ratio); the remaining parameters and methods were the same as in Experimental Group 1.

[0053] Experimental group 4: Compared to Experimental Group 1, the total number of viable microbial flora was modified to 15 parts (the ratio of viable strains was adjusted to Clostridium plasminogen lysate: Bifidobacterium longum: Lactobacillus plantarum: Akkermansia myxobolus: Bacteroides amyloliquefaciens = 15:10:8:3:5); fructooligosaccharides were modified to 100 parts, inulin to 50 parts; fucoidan to 5 parts, and glycerol to 1 part (maintaining a 5:1 weight ratio); the remaining parameters and methods were the same as in Experimental Group 1.

[0054] Experimental group 5: Compared to Experimental Group 1, the total number of viable microbial flora was modified to 50 samples (the ratio of viable strains was adjusted to Clostridium plasminogen lysate: Bifidobacterium longum: Lactobacillus plantarum: Akkermansia myxobolus: Bacteroides amyloliquefaciens = 25:20:15:8:10); fructooligosaccharides were modified to 30 samples, inulin to 15 samples; fucoidan to 25 samples, and glycerol to 5 samples (maintaining a 5:1 weight ratio); the remaining parameters and methods were the same as in Experimental Group 1.

[0055] II. Control Group Control group 1: Only *Clostridium pluvialis* was retained as a live microorganism (30 samples, live bacteria concentration 1×10⁻⁶). 11 The CFU / mL of the raw materials (60 parts of fructooligosaccharide, 30 parts of inulin, 15 parts of fucoidan, 3 parts of glycerol, etc.) and preparation steps and parameters were completely consistent with those of the experimental group (hereinafter, the experimental group refers to experimental group 1).

[0056] Control group 2: The composition and dosage of the active microbial community and the dosage of prebiotics were consistent with those of the experimental group. No encapsulation media were used (fucoidan, glycerol, trehalose and encapsulation-related reagents were removed). The preparation process was simplified as follows: the active microbial community suspension prepared in step S1 was directly freeze-dried (the process was the same as the freeze-drying parameters of experimental group S4) to obtain microbial powder, which was then mixed evenly with fructooligosaccharides and inulin according to the mixing conditions of experimental group S4, and then packaged into capsules.

[0057] Control group 3: The composition and dosage of the active microbial community, as well as the dosage of all raw materials, were consistent with the experimental group. The core adjustment was that the capsule preparation method of adding calcium chloride solution was not used in the experimental group. Instead, the microcapsules were prepared by spray drying (process parameters: inlet air temperature 110℃, outlet air temperature 50℃, feed rate 5mL / min). The remaining steps (S1 strain treatment, S4 mixing and dispensing) were consistent with the experimental group.

[0058] Control group 4: The composition and dosage of active microbial flora, the dosage of prebiotics, and the preparation process were all consistent with the experimental group; the core adjustment was that the ratio of fucoidan to glycerol in the embedding medium was changed from 5:1 to 10:1 (10 parts fucoidan and 1 part glycerol), and the amount of trehalose was still 10% (1.0 part) of the mass of fucoidan.

[0059] Control group 5: The composition and dosage of active microbial flora, the dosage of prebiotics, and the preparation process were all consistent with the experimental group; the core adjustment was that the amount of trehalose in the embedding medium was changed from 10% of the mass of fucoidan to 5% (15 parts of fucoidan, 3 parts of glycerol, and 0.75 parts of trehalose), and the ratio of fucoidan to glycerol remained at 5:1.

[0060] Control group 6: The composition and dosage of active microbial communities, the dosage of all raw materials, and the preparation method of embedded microcapsules were consistent with those of the experimental group. The core adjustment was that in step S4, vacuum drying was used instead of freeze drying for the washed embedded microcapsules (vacuum drying parameters: vacuum degree 6 Pa, temperature 35℃, drying time 12 hours). The remaining mixing and dispensing steps were consistent with those of the experimental group.

[0061] III. Performance Comparison Test Design Test 1: Storage Stability Test 1. Test Procedure Sample preparation: Take 100 capsules each from experimental group 1 and control groups 1-6, and store them in a constant temperature and humidity chamber at 25℃ and 60% relative humidity. Set up 3 parallel samples for each group.

[0062] Testing time points: Samples were taken and tested at storage time 0 days (initial), 3 months, 6 months and 12 months.

[0063] Detection methods: Viable bacterial survival rate: Three samples were ground and dissolved in sterile physiological saline, serially diluted, and then plated for counting (using specific culture media for corresponding strains). The proportion of viable bacteria to the initial viable bacteria count was calculated. Appearance of the preparation: The capsules were observed for moisture absorption, deformation, or breakage, and the abnormality rate was recorded. The results are shown in Table 1.

[0064] Table 1 Experiment 2: 1. Test Procedure Experimental animals: Seventy aged obese C57BL / 6 mice (weight ≥30g, meeting the criteria for an obese elderly model) aged 18-20 months were selected and randomly divided into 7 groups (experimental group 1 and control group 1-6), with 10 mice in each group, half male and half female, and were acclimatized for 1 week.

[0065] Administration: Each group of mice was administered 5 × 10 10 The corresponding capsule contents were administered by gavage at a dose of CFU / capsule / day for 7 consecutive days, with the patient maintaining the same diet and water intake during the administration period.

[0066] Testing time points: Samples were taken on day 1 of administration (2 hours after administration), day 7 (2 hours after the last administration), and 7 days after discontinuation of administration.

[0067] Detection method: Mice were anesthetized with ether, and 0.1g of colon contents were aseptically taken and dissolved in 0.9mL of sterile physiological saline. After serial dilution, the contents were counted on selective culture plates for each strain and the number of target strains per gram of colon contents (CFU / g) was recorded.

[0068] Table 2. Number of target bacterial strains in the intestines of mice in each group (unit: CFU / g) Experiment 3: Effects of physiological, biochemical, and intestinal metabolic indicators on aged obese mice 1. Test Procedure Experimental animals: 70 aged obese C57BL / 6 mice, the same as in Experiment 2, were grouped and fed the same adaptive diet.

[0069] Dosing regimen: Same as Experiment 2, administer the medication continuously for 4 weeks, and record feed consumption weekly.

[0070] Testing time points: baseline period (1 day before administration), mid-intervention period (2 weeks after administration), and end-intervention period (4 weeks after administration), all after fasting for 12 hours.

[0071] Sample collection and testing: Weight, fasting blood glucose, serum total cholesterol (TC), triglycerides (TG), inflammatory factors (TNF-α, IL-6) and fecal short-chain fatty acids (acetic acid, propionic acid, butyric acid) were tested. The testing methods followed the standard procedures of routine biochemical tests and ELISA and gas chromatography.

[0072] Table 3. Changes in body weight of mice in each group (unit: g) Table 4. Changes in fasting blood glucose, serum TC and TG in mice of each group (unit: mmol / L) Table 5. Serum levels of inflammatory factors (TNF-α, IL-6) in mice of each group (unit: pg / mL) Table 6. Short-chain fatty acid content in feces of mice in each group (unit: mmol / g feces) IV. Conclusion Five strains of Clostridium perfringens, Bifidobacterium longum, and others were screened and mixed in a specific ratio, which solved the problems of weak colonization ability and narrow scope of action of single strains. Experiment 2 showed that the number of target intestinal strains in the experimental group was 6.2 × 10⁻⁶ seven days after drug withdrawal. 9 CFU / g) is the same as that of the single-strain control group 1 (2.1×10). 9 The CFU / g of the experimental group was 2.9 times that of the control group. In Experiment 3, the experimental group showed significantly better weight loss, improved glucose and lipid metabolism, and anti-inflammatory effects than the control group 1, demonstrating that the synergistic effect of multiple strains can more comprehensively regulate the intestinal microecology.

[0073] An encapsulation medium constructed with fucoidan and glycerol in a 5:1 core ratio, supplemented with 10% trehalose for added protection, effectively resisted gastric acid erosion and activity loss during storage. In Experiment 1, the survival rate of live bacteria in the experimental group after 12 months of storage (78%) was 122.9% higher than that in control group 2 (35%) without encapsulation medium. In Experiment 2, the intestinal colonization of the experimental group was 4.8 times that of control group 2, fully verifying the key protective role of the encapsulation system in the activity of probiotics.

[0074] Microcapsules were prepared using a calcium chloride dropwise encapsulation method, combined with freeze-drying, which avoided the damage to bacterial activity caused by spray drying (control group 3) and vacuum drying (control group 6). Experiment 1 showed that the survival rate of live bacteria in the experimental group after 6 months of storage (85%) was 63.5% higher than that in control group 3 (52%). In Experiment 3, the experimental group showed significantly better regulatory effects on body weight, inflammatory factors, and short-chain fatty acids than control groups 3 and 6, demonstrating that process optimization is an important support for ensuring the efficacy of the composition.

[0075] After 4 weeks of intervention, the weight of elderly obese mice decreased significantly by 3.4g, fasting blood glucose, serum TC and TG all returned to normal ranges, TNF-α and IL-6 levels decreased significantly, and fecal short-chain fatty acid (especially butyrate) content increased significantly. These data indicate that the composition can improve elderly obesity by regulating gut microbiota balance, thereby improving weight control, glucose and lipid metabolism, anti-inflammation, and intestinal barrier protection, providing a safe and effective solution for the prevention and alleviation of obesity in the elderly.

[0076] In summary, the experimental group's *Clostridium plasminogen lysate* composition features five complementary beneficial strains: *Clostridium plasminogen lysate*, *Bifidobacterium longum*, *Lactobacillus plantarum*, *Akermansia myxobolus*, and *Bacteroides amyloliquefaciens*. These strains are scientifically combined with a live bacteria ratio of (15-25):(10-20):(8-15):(3-8):(5-10), effectively addressing the industry pain points of weak colonization and narrow scope of action of single strains. This results in a synergistic effect of regulating gut microbiota, strengthening the gut barrier, and improving metabolism. Based on a core ratio of 5:1 for fucoidan and glycerol, and supplemented with 5%-15% trehalose by weight of fucoidan as an auxiliary protective agent, a highly effective microcapsule structure resistant to gastric acid erosion is constructed. This significantly improves the survival rate of live bacteria during storage and digestive tract transport, resulting in nearly a 5-fold increase in intestinal colonization compared to products without an encapsulation system. In terms of optimizing the preparation process, a combination of drip-addition of calcium chloride for encapsulation and gradient freeze-drying was adopted to strictly control the microcapsule particle size within the range of 300-600μm, avoiding problems such as high-temperature damage during spray drying and loss of activity during vacuum drying. At the same time, the activity of the strain and the stability of the formulation were maximized by using inert gas protection and low-temperature operation.

[0077] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and embodiments shown and described herein.

Claims

1. A Clostridium plasminoides composition, characterized in that, The Clostridium praosporum composition consists of active microbial flora, prebiotics, and pharmaceutically acceptable excipients; The active microbial community consists of Clostridium praosporum, Bifidobacterium longum, Lactobacillus plantarum, Akkermansia myxobolus, and Bacteroides amyloliquefaciens. The prebiotics are a mixture of fructooligosaccharides and inulin. Pharmaceutically acceptable excipients include fucoidan and glycerol.

2. The Clostridium praosporum composition according to claim 1, characterized in that, Active microbial flora, calculated as total live bacteria, is 10-50 samples; The amount of fructooligosaccharides is 20-100 parts; Inulin: 10-50 parts; Fucoidan content: 5-25 parts; Glycerin is 1-5 parts; The weight ratio of fucoidan to glycerol is 5:

1.

3. A method for preparing a Clostridium plasminoides composition, characterized in that, include: S1: Mix the active microbial community of Clostridium plasminoides, Bifidobacterium longum, Lactobacillus plantarum, Akkermansia myxobolus and Bacteroides amyloliquefaciens, and resuspend them in physiological saline to form a homogeneous active microbial community suspension. S2: Dissolve fucoidan in water and stir continuously at 40-50℃ to form a fucoidan solution. Then add glycerol to the fucoidan solution and mix evenly to obtain the embedding medium. Cool to 25-30℃. S3: Under inert gas protection and continuous stirring, the active microbial community suspension obtained in step S1 is slowly added to the embedding medium obtained in step S2. After mixing evenly, the resulting mixture is added dropwise to the calcium chloride solution. During the dropwise addition, shear force is applied to form embedded microcapsules and a solidification reaction is carried out. S4: Collect the solidified embedded microcapsules, wash them, and mix them with fructooligosaccharides and inulin to obtain the Clostridium praosporum composition; The active microbial flora consists of 10-50 parts by total live bacteria; 20-100 parts by fructooligosaccharides; 10-50 parts by inulin; 5-25 parts by fucoidan; and 1-5 parts by glycerol. The weight ratio of fucoidan to glycerol is 5:

1.

4. The method for preparing the Clostridium praosporum composition according to claim 3, characterized in that, In S1, Clostridium plasminogen salina, Bifidobacterium longum, Lactobacillus plantarum, Akkermansia myxobolus and Bacteroides amyloliquefaciens were pre-activated and expanded in culture until each strain reached the late stage of stable growth. The obtained bacterial strains were mixed with Clostridium perfringens, Bifidobacterium longum, Lactobacillus plantarum, Akkermansia myxobolus, and Bacteroides amyloliquefaciens in a ratio of (15-25):(10-20):(8-15):(3-8):(5-10) according to the number of viable cells. The mixed active microbial community was resuspended in pre-cooled, sterile physiological saline containing 0.5%-1.5% w / v reduced glutathione at 4-10℃ under inert gas protection, with the total viable bacterial concentration of the final suspension controlled to be 5 × 10⁻⁶. 10 CFU / mL up to 2×10 11 CFU / mL, forming a uniform suspension of active microbial flora.

5. The method for preparing the Clostridium praosporum composition according to claim 3, characterized in that, In S2, fucoidan is uniformly dispersed in water at 40-50℃ by a combination of vortex oscillation and mechanical stirring, wherein the mass of water is 15-30 times the mass of fucoidan. Under continuous stirring, add 5%-15% trehalose (by mass of fucoidan) to the solution. After it is completely dissolved, add glycerol. The resulting mixed solution was homogenized at a shear rate of 2000-5000 rpm for 2-5 minutes, then allowed to stand to remove bubbles, and the encapsulation medium was obtained, with its final viscosity controlled at 800-1500 cP. The embedding medium was cooled to 25-30℃ under light-proof and inert gas protection.

6. The method for preparing the Clostridium praosporum composition according to claim 3, characterized in that, In step S3, under continuous stirring and carbon dioxide inert gas protection, the active microbial community suspension obtained in step S1 is preheated to 25-30℃, and then added dropwise to the embedding medium obtained in step S2 at a rate of 0.5-2 mL / min, controlling the volume ratio of the active microbial community suspension to the embedding medium to be 1: (8-15). After mixing, the microbial community-embedding medium mixture is obtained. The bacterial culture-embedding medium mixture was added dropwise to a calcium chloride solution with a concentration of 1.0-2.5% w / v at a rate of 10-30 drops / min using a syringe pump. The calcium chloride solution contained 0.5-1.0% w / v trehalose. During the droplet addition process, the calcium chloride solution receiving the droplets is stirred to provide a stable shear force, ensuring that the embedded microcapsules have a particle size distribution in the range of 300-600 μm. After the addition is complete, allow the embedded microcapsules to stand in the calcium chloride solution at 4-10℃ for 20-40 minutes to solidify.

7. The method for preparing the Clostridium praosporum composition according to claim 3, characterized in that, In S4, the solidified embedded microcapsules were collected and washed with pre-cooled phosphate buffer at pH 6.5-7.0, which contained 5%-10% (w / v) trehalose as a protective agent. The washed embedded microcapsules were freeze-dried. The freeze-drying process was as follows: first, the temperature was lowered to below -40℃ at a rate of 0.5-1℃ / min and held for 2-4 hours; then, under a vacuum of less than 10 Pa, the main drying was carried out at a gradient temperature from -25℃ to 0℃ for 20-30 hours; finally, the desorption drying was carried out at 25-30℃ for 4-8 hours to obtain the dried embedded microcapsule powder. The obtained dried encapsulated microcapsule powder was mixed with fructooligosaccharides and inulin in an inert gas protected environment with a temperature ≤25℃ and a relative humidity ≤15%.

8. The application of the Clostridium praosporum composition as described in claim 1, characterized in that, Used to prepare drugs for the prevention, relief or treatment of obesity in the elderly.

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