A microbial microcapsule preparation for assisting weight loss and a preparation process thereof

Abstract

The present application relates to the technical field of microcapsule, and particularly relates to a kind of microbial microcapsule preparation for assisting weight loss and its preparation process.The present application includes the following steps: preparing fortified medium, preparing bacteria suspension, preparing composite microbial agent microcapsule and preparing microbial microcapsule preparation for assisting weight loss.The present application prepares fortified medium by compounding BHI broth with soybean peptone and other nutrients, forms the synergistic complementation of nutrition and physicochemical properties in fortified medium, protects the bacterial activity of mucinophilic Akkermansia, and lays a foundation for subsequent high-concentration active bacteria suspension.High-concentration active bacteria suspension can reduce the use amount of emulsifier, filling oil or thickening agent during the preparation of microcapsule due to high concentration of active ingredients, and does not affect the weight loss effect of microcapsule, which makes the volume of microcapsule smaller and improves the swallowing compliance of the user.

Description

Technical Field

[0001] This invention relates to the field of microcapsule technology, specifically to a microbial microcapsule preparation for weight loss and its preparation process. Background Technology

[0002] Obesity and related metabolic diseases have become a global public health problem. Currently, microbial microcapsule preparations for weight loss mainly use traditional probiotics such as lactic acid bacteria and bifidobacteria as active ingredients, and combine them with wall materials such as alginate, chitosan, and pectin to encapsulate the microbial microcapsules for weight loss. Some products also add prebiotics to enhance the colonization effect.

[0003] Akkermansia muciniphila, a next-generation probiotic that has garnered significant attention in recent years, has been extensively studied and proven to possess unique advantages in improving obesity, insulin resistance, and chronic inflammation. This bacterium colonizes the intestinal mucus layer, stimulating goblet cells to secrete mucin to repair the intestinal barrier, reducing lipopolysaccharide entry into the bloodstream to lower systemic inflammation levels, and participating in the regulation of energy metabolism pathways, thus forming a multi-target weight loss intervention mechanism.

[0004] Currently, most cultivation techniques for Akkermansia myxophilus employ complex nitrogen sources from a single source, such as brain and heart infusion medium. While these can meet basic growth requirements, they suffer from poor tolerance to oxidative stress during cultivation, resulting in low viable cell density and making it difficult to stably obtain high-concentration viable cell suspensions.

[0005] Regarding the core material design of microcapsules, existing technologies mostly focus on adding single prebiotics or protectants, lacking synergistic systems that target the growth characteristics and weight-loss mechanisms of Akkermansia muciniphila. While commonly used lyophilization protectants improve bacterial survival rates to some extent, they struggle to simultaneously address both bacterial resuscitation and colonization in the gut and functional activation. Furthermore, most existing live bacteria microcapsules employ only a single-layer wall material, offering limited protection in the acidic environment of the stomach. The lack of targeted release and functional synergistic design hinders the efficient delivery of live bacteria and their active metabolites to the intestinal target site, impacting the overall weight-loss effect of the formulation.

[0006] Therefore, the present invention provides a microbial microcapsule preparation for weight loss and its preparation process to solve the problems existing in the prior art. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the present invention aims to provide a microbial microcapsule preparation for weight loss and its preparation process.

[0008] A preparation process for a microbial microcapsule formulation for weight loss assistance specifically includes the following steps:

[0009] S1: Preparation of enhanced culture medium

[0010] BHI broth, soybean peptone, NaCl, and Na2HPO4 were dissolved in sterile water to obtain solution A; glucose and threonine were dissolved in sterile water to obtain solution B; L-cysteine ​​hydrochloride was dissolved in sterile water to obtain solution C; solutions A, B, and C were mixed and the pH was adjusted to 7.0-7.2 with NaOH solution to obtain the enhanced culture medium.

[0011] S2: Preparation of bacterial suspension

[0012] Akkermansia muciniphila was inoculated into enhanced culture medium at an inoculum rate of 1-2% to obtain activated bacterial suspension. The activated bacterial suspension was then expanded and centrifuged to separate the supernatant and the lower precipitate. The lower precipitate was resuspended in sterile physiological saline to obtain bacterial suspension.

[0013] S3: Preparation of compound microcapsules

[0014] Add arabinogalactan, bellflower polysaccharide, trehalose, and sterilized skim milk powder to the bacterial suspension and stir until dissolved to obtain a core material mixture; mix the core material mixture with sodium alginate solution to obtain a final mixture.

[0015] The mixture was added dropwise to a calcium chloride solution, solidified, filtered, and washed to obtain composite microcapsules of microbial agent.

[0016] S4: Preparation of microbial microcapsule formulations to aid weight loss

[0017] Centrifuge the supernatant obtained in step S2 to separate the precipitate, resuspend the precipitate to obtain a resuspension, filter the resuspension and dilute it to obtain vesicle fluid.

[0018] The compound microbial agent microcapsules were mixed with the vesicle fluid, and the precipitate was separated. The washed precipitate was then transferred to a pectin-chitosan mixed solution, stirred, pre-frozen, and freeze-dried to obtain a microbial microcapsule preparation for weight loss assistance.

[0019] Further, step S1 involves preparing the enhanced culture medium, including the following steps:

[0020] Dissolve 18-22 parts by weight of BHI broth, 15-18 parts by weight of soy peptone, 3-5 parts by weight of NaCl and 2-3 parts by weight of Na2HPO4 in 500 parts by weight of sterile water to obtain solution A;

[0021] Dissolve 5-7 parts by weight of glucose and 1.5-2.5 parts by weight of threonine in 200 parts by weight of sterile water to obtain solution B;

[0022] Dissolve 5-8 parts by weight of L-cysteine ​​hydrochloride in 100 parts by weight of sterile water to obtain solution C;

[0023] After mixing solutions A, B, and C, the pH value was adjusted to 7.0-7.2 with NaOH solution to obtain the enhanced culture medium.

[0024] Further, step S2, preparing the bacterial suspension, includes the following steps:

[0025] Akkermansia myxophilus was inoculated into the enhanced culture medium prepared in step S1 at an inoculation rate of 1-2%, and incubated at 35-37℃ under anaerobic conditions for 60-72 hours to obtain activated bacterial solution.

[0026] The activated bacterial solution was transferred back to the enhanced culture medium prepared in step S1 at an inoculation rate of 2-3%. The culture was carried out at 35-37℃ under anaerobic conditions for 60-72 hours. Then, the supernatant and the lower precipitate were separated at 4℃ and 9000×g for 15-20 minutes. The lower precipitate was resuspended in sterile physiological saline to obtain the bacterial suspension. The anaerobic environment was specifically a gaseous environment composed of 85% N2, 10% CO2 and 5% H2.

[0027] Further, step S3, preparing the composite microbial agent microcapsules, includes the following steps:

[0028] Add 5-8 mg / mL arabinogalactan, 15-20 mg / mL platycodon polysaccharide, 12-15 mg / mL trehalose and 8-10 mg / mL sterile skim milk powder to the bacterial suspension and stir to dissolve evenly to obtain the core material mixture.

[0029] The core material mixture and sodium alginate solution were mixed at a volume ratio of 1:(6-8) to obtain the mixture.

[0030] Add the mixture dropwise to a 2-4% calcium chloride solution, solidify for 30-35 minutes, filter and collect the microcapsules, wash twice with sterile water to obtain composite microbial agent microcapsules, and store at 4℃ for later use.

[0031] Further, step S4 involves preparing a microbial microcapsule formulation to aid weight loss, comprising the following steps:

[0032] Centrifuge the supernatant separated in step S2 at 100000×g for 10-20 min to separate the precipitate. Resuspend the precipitate with PBS buffer to obtain a resuspension. Filter the resuspension through a 0.45μm filter membrane and dilute it with PBS buffer to 5-7mg / mL to obtain vesicle fluid.

[0033] The compound microbial agent microcapsules and vesicle fluid were mixed at a material-to-liquid ratio of 1g:(4-6)mL and stirred at 100-150rpm for 2-4h at 2-4℃. The precipitate was separated and then transferred to a pectin-chitosan mixed solution and mixed at a volume ratio of 1:(3-5). The mixture was stirred for 30-35min, then pre-frozen at -60℃ for 5-6h, and then freeze-dried at -40℃ for 24-26h to obtain a microbial microcapsule preparation for weight loss assistance.

[0034] Furthermore, the anaerobic environment is specifically a gaseous environment composed of 85% N2, 10% CO2, and 5% H2.

[0035] Furthermore, the concentration of the sodium alginate solution is 2-4%.

[0036] Furthermore, the pH of the PBS buffer is 7.2-7.4.

[0037] Furthermore, the pectin-chitosan mixed solution was prepared by dissolving pectin and chitosan in sterile water, controlling the pectin content to be 1% (w / v) and the chitosan content to be 0.5% (w / v), and adjusting the pH value to 5.5-6 with HCl solution.

[0038] A microbial microcapsule preparation for aiding weight loss, which is prepared by the above-mentioned preparation process for a microbial microcapsule preparation for aiding weight loss.

[0039] The present invention has the following advantages:

[0040] 1. This invention prepares an enhanced culture medium by combining BHI broth with soybean peptone and other nutrients, achieving a synergistic complementarity in nutritional and physicochemical properties. BHI broth, prepared from a mixture of bovine brain and heart extracts, is rich in animal-derived small molecule peptides, free amino acids, and other growth factors, providing Akkermansia myxophilus with the complex nitrogen source and key coenzyme components required for its metabolism. Soybean peptone, as a plant-derived nitrogen source, supplements the specific amino acids that are low in animal-derived peptone and is rich in oligosaccharides and antioxidants. The combined use of these two substances firstly achieves complete coverage of nutrient supply, reducing the metabolic burden on the strain. Secondly, soybean peptone enhances the buffer capacity of the culture medium, effectively neutralizing pH fluctuations caused by glucose metabolism, and together with BHI broth, maintaining a stable pH environment of 7.0-7.2. Furthermore, the antioxidants in soybean peptone synergistically work with the heme provided by BHI broth to buffer the oxidative stress caused by residual dissolved oxygen in the early stages of anaerobic culture, protecting the cell activity of *Ackermania mutans* and laying the foundation for obtaining a high-concentration live bacterial suspension. The high concentration of active ingredients in the live bacterial suspension allows for a reduction in the amount of other adjuvants used in microcapsule preparation without affecting the weight-loss effect of the microcapsules. This allows for the design of smaller microcapsule volumes, improving swallowing compliance.

[0041] 2. In this invention, arabinogalactan and bellflower polysaccharide are added to the bacterial suspension. Both are high-molecular-weight polysaccharides, which can increase the viscosity of the core material mixture, so that the bacteria are uniformly suspended, avoid sedimentation, and improve the uniformity of embedding. At the same time, they act as cryoprotectants during the freeze-drying process, protecting the integrity of the bacterial cell membrane through glass formation and hydrogen bond substitution, and significantly improving the freeze-dried survival rate of Akkermansia muciniphila. The microcapsules prepared by combining these three ingredients have multiple synergistic effects in assisting weight loss. Akkermansia mucinosa colonizes the intestinal mucus layer, stimulates goblet cells to secrete mucin, repairs the intestinal barrier, reduces lipopolysaccharide entry into the blood, and reduces chronic inflammation. Platycodon grandiflorum polysaccharide promotes fat decomposition and inhibits fat synthesis by activating chemical signaling pathways, and also promotes the reproduction and culture of Akkermansia mucinosa. Arabinomelanin semi-galactosyl is fermented by intestinal flora to produce short-chain fatty acids, which increase satiety and promote fat conversion. The three ingredients form a synergistic system of probiotics-prebiotics-functional synergists. Arabinomelanin semi-galactosyl and platycodon grandiflorum polysaccharide not only act as freeze-drying protectants to ensure the stability of the bacteria during the preparation process, but also act as targeted substrates to promote the recovery and colonization of Akkermansia mucinosa in the intestine. It intervenes in obesity-related pathways through three pathways: barrier repair, flora regulation, and metabolic activation, and achieves a synergistic effect covering the entire chain of intestinal integrity, inflammation level, and energy metabolism.

[0042] 3. In step S4 of this invention, a double-layer coating of the composite bacterial agent microcapsules is achieved using a vesicle fluid and a pectin-chitosan mixed solution. The vesicle fluid is derived from the membrane vesicle components enriched by high-speed centrifugation of the supernatant of *Ackermania viride*, rich in bacterial outer membrane vesicles, which contain immunomodulatory membrane proteins, lipopolysaccharides, and metabolites. This is used as the first layer to coat the microcapsules, forming a biomimetic structure on the microcapsule surface. The adhesive properties of the membrane vesicles enhance the retention capacity of the formulation in the intestinal mucus layer. Simultaneously, the active ingredients within the membrane vesicles can directly act on intestinal epithelial cells, exerting anti-inflammatory and barrier repair functions, complementing the functions of the live bacteria within the microcapsules. The pectin-chitosan mixed solution serves as the second coating layer, utilizing the anionic properties of pectin and the cationic properties of chitosan to form a polyelectrolyte complex, constructing a pH-responsive protective layer. In the acidic gastric environment, this complex structure is tightly packed, effectively protecting the microcapsules from damage by gastric acid and digestive enzymes. Upon entering the intestine, the pH increases, causing the complex structure to loosen or dissociate, achieving targeted release. The outer pectin-chitosan layer provides a physical barrier and targeted delivery, while the inner membrane vesicle layer provides a bioactive interface and immune regulation. Together, they ensure that Akkermansia muciniphila and its active ingredients reach the intestinal target site with a high survival rate, significantly enhancing the effect of microcapsules in assisting weight loss. Attached Figure Description

[0043] Figure 1 This is a flowchart illustrating the preparation process of the microbial microcapsule formulation for weight loss assistance according to the present invention. Detailed Implementation

[0044] To enable those skilled in the art to better understand the technical solutions of this invention, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of this invention.

[0045] Example 1:

[0046] A preparation process for a microbial microcapsule formulation to aid weight loss, such as... Figure 1 As shown, it includes the following steps:

[0047] S1: Preparation of enhanced culture medium

[0048] Dissolve 18 parts by weight of BHI broth, 15 parts by weight of soy peptone, 3 parts by weight of NaCl and 2 parts by weight of Na2HPO4 in 500 parts by weight of sterile water to obtain solution A;

[0049] Dissolve 5 parts by weight of glucose and 1.5 parts by weight of threonine in 200 parts by weight of sterile water to obtain solution B;

[0050] Dissolve 5 parts by weight of L-cysteine ​​hydrochloride in 100 parts by weight of sterile water to obtain solution C;

[0051] After mixing solutions A, B, and C, the pH value was adjusted to 7.0 with NaOH solution to obtain the enhanced culture medium.

[0052] S2: Preparation of bacterial suspension

[0053] Akkermansia myxophilus was inoculated at a rate of 1% into the enhanced culture medium prepared in step S1, and the culture was allowed to stand at 35°C under anaerobic conditions for 60 hours to obtain an activated bacterial solution.

[0054] The activated bacterial solution was transferred back to the enhanced culture medium prepared in step S1 at an inoculation rate of 2%, and cultured at 35°C under anaerobic conditions for 60 h. Then, it was centrifuged at 4°C and 9000×g for 15 min to separate the supernatant and the lower precipitate. The lower precipitate was resuspended in sterile physiological saline to obtain the bacterial suspension. The anaerobic environment was specifically a gaseous environment composed of 85% N2, 10% CO2 and 5% H2.

[0055] S3: Preparation of compound microcapsules

[0056] Add 5 mg / mL arabinogalactan, 15 mg / mL platycodon polysaccharide, 12 mg / mL trehalose and 8 mg / mL sterile skim milk powder to the bacterial suspension and stir to dissolve evenly to obtain the core material mixture.

[0057] The core material mixture was mixed with a 2% sodium alginate solution at a volume ratio of 1:6 to obtain the mixture.

[0058] The mixture was added dropwise to a 2% calcium chloride solution and allowed to solidify for 30 minutes. The microcapsules were then collected by filtration and washed twice with sterile water to obtain composite microcapsules, which were stored at 4°C for later use.

[0059] S4: Preparation of microbial microcapsule formulations to aid weight loss

[0060] Centrifuge the supernatant separated in step S2 at 100000×g for 10 min to separate the precipitate. Resuspend the precipitate with PBS buffer to obtain a resuspension. Filter the resuspension through a 0.45μm filter membrane and dilute it to 5mg / mL with PBS buffer at pH 7.2 to obtain vesicle fluid.

[0061] The compound microbial agent microcapsules and vesicle fluid were mixed at a material-to-liquid ratio of 1g:4mL and stirred at 100rpm for 2h at 2℃. The precipitate was separated and then transferred to a pectin-chitosan mixed solution and mixed at a volume ratio of 1:3. The mixture was stirred for 30min, then pre-frozen at -60℃ for 5h and freeze-dried at -40℃ for 24h to obtain a microbial microcapsule preparation for weight loss assistance. The pectin-chitosan mixed solution was prepared by dissolving pectin and chitosan in sterile water, controlling the pectin content to be 1% (w / v) and the chitosan content to be 0.5% (w / v), and adjusting the pH value to 5.5 with HCl solution.

[0062] Example 2:

[0063] A preparation process for a microbial microcapsule formulation to aid weight loss, such as... Figure 1 As shown, it includes the following steps:

[0064] S1: Preparation of enhanced culture medium

[0065] Dissolve 20 parts by weight of BHI broth, 16 parts by weight of soy peptone, 4 parts by weight of NaCl and 2.5 parts by weight of Na2HPO4 in 500 parts by weight of sterile water to obtain solution A;

[0066] Dissolve 6 parts by weight of glucose and 2 parts by weight of threonine in 200 parts by weight of sterile water to obtain solution B.

[0067] Dissolve 6 parts by weight of L-cysteine ​​hydrochloride in 100 parts by weight of sterile water to obtain solution C;

[0068] After mixing solutions A, B, and C, the pH value was adjusted to 7.1 with NaOH solution to obtain the enhanced culture medium.

[0069] S2: Preparation of bacterial suspension

[0070] Akkermansia myxophilus was inoculated into the enhanced culture medium prepared in step S1 at an inoculation rate of 1.5%, and incubated at 36°C under anaerobic conditions for 66 hours to obtain activated bacterial solution.

[0071] The activated bacterial solution was transferred back to the enhanced culture medium prepared in step S1 at an inoculation rate of 2.5%. It was cultured at 36°C under anaerobic conditions for 66 hours. Then, it was centrifuged at 4°C and 9000×g for 18 minutes to separate the supernatant and the lower precipitate. The lower precipitate was resuspended in sterile physiological saline to obtain the bacterial suspension. The anaerobic environment was specifically a gaseous environment composed of 85% N2, 10% CO2 and 5% H2.

[0072] S3: Preparation of compound microcapsules

[0073] Add 7 mg / mL arabinogalactan, 17 mg / mL platycodon polysaccharide, 13 mg / mL trehalose and 9 mg / mL sterile skim milk powder to the bacterial suspension and stir to dissolve evenly to obtain the core material mixture.

[0074] The core material mixture was mixed with a 3% sodium alginate solution at a volume ratio of 1:7 to obtain the mixture.

[0075] The mixture was added dropwise to a 3% calcium chloride solution and allowed to solidify for 33 minutes. The microcapsules were then collected by filtration and washed twice with sterile water to obtain composite microcapsules, which were stored at 4°C for later use.

[0076] S4: Preparation of microbial microcapsule formulations to aid weight loss

[0077] The supernatant obtained from centrifugation in step S2 was centrifuged at 100000×g for 15 min to separate the precipitate. The precipitate was resuspended in PBS buffer to obtain a resuspension. The resuspension was filtered through a 0.45μm filter membrane and diluted to 6mg / mL with PBS buffer at pH 7.3 to obtain vesicle fluid.

[0078] The compound microbial agent microcapsules and vesicle fluid were mixed at a material-to-liquid ratio of 1g:5mL and stirred at 125rpm for 3h at 3℃. The precipitate was separated and then transferred to a pectin-chitosan mixed solution at a volume ratio of 1:4 and stirred for 33min. The mixture was then pre-frozen at -60℃ for 5.5h and freeze-dried at -40℃ for 25h to obtain a microbial microcapsule preparation for weight loss assistance. The pectin-chitosan mixed solution was prepared by dissolving pectin and chitosan in sterile water, controlling the pectin content to be 1% (w / v) and the chitosan content to be 0.5% (w / v), and adjusting the pH value to 5.7 with HCl solution.

[0079] Example 3:

[0080] A preparation process for a microbial microcapsule formulation to aid weight loss, such as... Figure 1 As shown, it includes the following steps:

[0081] S1: Preparation of enhanced culture medium

[0082] Dissolve 22 parts by weight of BHI broth, 18 parts by weight of soy peptone, 5 parts by weight of NaCl and 3 parts by weight of Na2HPO4 in 500 parts by weight of sterile water to obtain solution A;

[0083] Dissolve 7 parts by weight of glucose and 2.5 parts by weight of threonine in 200 parts by weight of sterile water to obtain solution B;

[0084] Dissolve 8 parts by weight of L-cysteine ​​hydrochloride in 100 parts by weight of sterile water to obtain solution C;

[0085] After mixing solutions A, B, and C, the pH value was adjusted to 7.2 with NaOH solution to obtain the enhanced culture medium.

[0086] S2: Preparation of bacterial suspension

[0087] Akkermansia myxophilus was inoculated at a rate of 2% into the enhanced culture medium prepared in step S1, and the culture was allowed to stand at 37°C under anaerobic conditions for 72 hours to obtain an activated bacterial solution.

[0088] The activated bacterial solution was transferred back to the enhanced culture medium prepared in step S1 at an inoculation rate of 3%, and cultured at 37°C under anaerobic conditions for 72 h. Then, it was centrifuged at 4°C and 9000×g for 20 min to separate the supernatant and the lower precipitate. The lower precipitate was resuspended in sterile physiological saline to obtain the bacterial suspension. The anaerobic environment was specifically a gaseous environment composed of 85% N2, 10% CO2 and 5% H2.

[0089] S3: Preparation of compound microcapsules

[0090] Add 8 mg / mL arabinogalactan, 20 mg / mL platycodon polysaccharide, 15 mg / mL trehalose and 10 mg / mL sterile skim milk powder to the bacterial suspension and stir to dissolve evenly to obtain the core material mixture.

[0091] The core material mixture was mixed with a 4% sodium alginate solution at a volume ratio of 1:8 to obtain the mixture.

[0092] The mixture was added dropwise to a 4% calcium chloride solution and allowed to solidify for 35 minutes. The microcapsules were then collected by filtration and washed twice with sterile water to obtain composite microcapsules, which were stored at 4°C for later use.

[0093] S4: Preparation of microbial microcapsule formulations to aid weight loss

[0094] The supernatant obtained from centrifugation in step S2 was centrifuged at 100000×g for 20 min to separate the precipitate. The precipitate was resuspended in PBS buffer to obtain a resuspension. The resuspension was filtered through a 0.45μm filter membrane and diluted to 7mg / mL with PBS buffer at pH 7.4 to obtain vesicle fluid.

[0095] The compound microbial agent microcapsules and vesicle fluid were mixed at a material-to-liquid ratio of 1g:6mL and stirred at 150rpm for 4h at 4℃. The precipitate was separated and then transferred to a pectin-chitosan mixed solution and mixed at a volume ratio of 1:5. The mixture was stirred for 35min, then pre-frozen at -60℃ for 6h and freeze-dried at -40℃ for 26h to obtain a microbial microcapsule preparation for weight loss assistance. The pectin-chitosan mixed solution was prepared by dissolving pectin and chitosan in sterile water, controlling the pectin content to be 1% (w / v) and the chitosan content to be 0.5% (w / v), and adjusting the pH value to 6 with HCl solution.

[0096] Comparative Example 1:

[0097] Compared with Example 1, the difference of Comparative Example 1 is that the BHI broth in step S1 is replaced with soy peptone, while the other steps remain unchanged. This is referred to as Comparative Example 1.

[0098] Comparative Example 2:

[0099] Compared with Example 1, Comparative Example 2 differs in that the soybean peptone in step S1 is replaced with BHI broth, while the other steps remain unchanged. This is referred to as Comparative Example 2.

[0100] Comparative Example 3:

[0101] Compared with Example 1, the difference of Comparative Example 3 is that the arabinogalactan in step S3 is replaced by Platycodon grandiflorum polysaccharide, while the other steps remain unchanged. This is referred to as Comparative Example 3.

[0102] Comparative Example 4:

[0103] Compared with Example 1, the difference of Comparative Example 4 is that the amount of Platycodon grandiflorum polysaccharide in step S3 is replaced with arabinogalactan, while the other steps remain unchanged. This is referred to as Comparative Example 4.

[0104] Comparative Example 5:

[0105] Compared with Example 1, the difference of Comparative Example 5 is that the vesicle fluid is used for two coatings in step S4, while the other steps remain the same. Specifically, the supernatant separated by centrifugation in step S2 is centrifuged at 100000×g for 10 min to separate the precipitate. The precipitate is resuspended with PBS buffer to obtain a resuspension. The resuspension is filtered through a 0.45μm filter membrane and diluted to 5mg / mL with PBS buffer at pH 7.2 to obtain the vesicle fluid.

[0106] The compound microbial agent microcapsules and vesicle fluid were mixed at a material-to-liquid ratio of 1g:4mL and stirred at 100rpm for 2h at 2℃. The precipitate was separated and then transferred to the vesicle fluid. The mixture was stirred at a volume ratio of 1:3 for 30min, then pre-frozen at -60℃ for 5h and freeze-dried at -40℃ for 24h to obtain a microbial microcapsule preparation for weight loss, which was designated as Comparative Example 5.

[0107] Comparative Example 6:

[0108] Compared with Example 1, Comparative Example 6 differs in that the pectin-chitosan mixed solution is used for two coatings in step S4, while the other steps remain unchanged. Specifically, the composite microbial agent microcapsules are mixed with the pectin-chitosan mixed solution at a material-to-liquid ratio of 1g:4mL, stirred at 100rpm for 2h at 2℃, and the precipitate is separated. The precipitate is then transferred to the pectin-chitosan mixed solution and mixed at a volume ratio of 1:3. The mixture is stirred for 30min, then pre-frozen at -60℃ for 5h, and then freeze-dried at -40℃ for 24h to obtain a microbial microcapsule preparation for weight reduction. The pectin-chitosan mixed solution is prepared by dissolving pectin and chitosan in sterile water, controlling the pectin content to be 1% (w / v) and the chitosan content to be 0.5% (w / v), and adjusting the pH value to 5.5. This is referred to as Comparative Example 6.

[0109] The bacterial suspensions prepared in step S2 of Examples 1-3 and Comparative Examples 1-2 were sampled in 1g each, and the viable bacteria count of the samples was detected by anaerobic plate counting method combined with OD600 absorbance. The results are shown in Table 1.

[0110] Table 1

[0111] Example 1 1.02 <![CDATA[2.9×10 9 ]]> Example 2 1.08 <![CDATA[3.2×10 9 ]]> Example 3 1.12 <![CDATA[3.5×10 9 ]]> Comparative Example 1 0.45 <![CDATA[5.1×10 7 ]]> Comparative Example 2 0.52 <![CDATA[6.3×10 7 ]]>

[0112] Examples 1-3 used a complete BHI broth + soy peptone complex nitrogen source, combined with growth promoters such as threonine and L-cysteine ​​hydrochloride. The cells grew well, with an OD600 exceeding 1.0 and a viable count reaching 102. 9 The concentration was in the CFU / mL range. In contrast, Comparative Example 1 replaced BHI broth with soy peptone, resulting in the absence of specific growth factors provided by bovine brain and heart extracts in the culture medium. Although the total nitrogen content was not low, the nutritional balance decreased, and bacterial growth was significantly limited, with an OD600 of only 0.45 and a viable count reduced to 10⁻⁶. 7CFU / mL level; Comparative Example 2 replaced soybean peptone with BHI broth. Although BHI broth is rich in nutrients, it lacks certain plant-derived peptides and trace elements that promote growth in soybean peptone. Compared with the original formula, the nitrogen source composition is more singular, which is also not conducive to the bacteria reaching the optimal growth density. The OD600 is 0.52, and the number of viable bacteria is slightly higher than that of Comparative Example 1, but still significantly lower than that of Example 1.

[0113] Seven groups of healthy adult male mice, weighing between 25-35g, were selected, with 10 mice in each group. Each group was fed a standard diet for one week to acclimatize to the environment. Subsequently, different feeding methods were used for different groups.

[0114] Blank control group: fed with ordinary feed for 10 weeks;

[0115] Model group: fed a high-fat diet for 10 weeks;

[0116] Example 1 group: The high-fat diet was mixed with that of Example 1 at a mass ratio of 100:1 and fed for 10 weeks;

[0117] Example 2 group: The high-fat diet was mixed with that of Example 2 at a mass ratio of 100:1 and fed for 10 weeks;

[0118] Example 3 group: The high-fat diet was mixed with that of Example 3 at a mass ratio of 100:1 and fed for 10 weeks;

[0119] Comparative Example 3: High-fat diet was mixed with Comparative Example 3 at a mass ratio of 100:1 and fed for 10 weeks;

[0120] Comparative Example 4: High-fat diet was mixed with Comparative Example 4 at a mass ratio of 100:1 and fed for 10 weeks;

[0121] Five mice from each group whose weight was close to the median were selected and fed under the same conditions for three weeks. The weight and liver weight of the mice were accurately weighed and the average value was calculated. The results are shown in Table 2.

[0122] Table 2

[0123] Blank control group 30.2±1.1 35.8±1.3 1.12±0.08 Model group 30.4±1.0 47.2±1.5 1.68±0.12 Example 1 30.1±0.9 38.9±1.2 1.28±0.09 Example 2 30.3±1.2 38.1±1.1 1.22±0.07 Example 3 30.0±1.0 38.4±1.0 1.25±0.08 Comparative Example 3 30.3±1.1 42.5±1.4 1.52±0.11 Comparative Example 4 30.3±0.9 43.6±1.3 1.59±0.10

[0124] After being fed a high-fat diet for 10 weeks, the mice in the model group gained 16.8g in weight and their liver weight was significantly higher than that of the blank control group, which is consistent with the model characteristics of obesity induced by a high-fat diet.

[0125] The weight gain of groups 1-3 in Examples 1-3 was significantly lower than that of the model group, and the liver weight was close to that of the blank control group, indicating that the microbial microcapsule preparation has the effect of assisting weight loss and reducing liver fat accumulation.

[0126] In Comparative Example 3, only Platycodon grandiflorum polysaccharide was added, without arabinogalactan. The intestinal flora could not produce short-chain fatty acids, resulting in decreased fat conversion efficiency and thus increased liver weight. Energy metabolism was also slower, and the weight loss effect in mice was weaker than in the Example group. In Comparative Example 4, only arabinogalactan was added, without Platycodon grandiflorum polysaccharide. This prevented the activation of chemical signaling pathways to promote fat breakdown and inhibit fat synthesis. Therefore, fat accumulated in the liver, leading to an increase in liver weight compared to the control group and the Example group. Furthermore, the lack of Platycodon grandiflorum polysaccharide's promoting effect on the reproduction and culture of Akkermansia obliterans resulted in insufficient Akkermansia obliterans activity, further reducing the weight loss effect.

[0127] 1g samples from Examples 1-3 and Comparative Examples 5-6 were incubated in simulated gastric fluid for 0, 0.5, 1, and 2 hours, and then transferred to simulated intestinal fluid for 0, 0.5, 1, and 2 hours. Samples were taken for viable bacterial count, as shown in Table 3.

[0128] Simulated gastric juice: Take 16.4 mL of 9.5% HCl, add 800 mL of water and 10 g of pepsin, shake well, and then dilute with water to 1000 mL.

[0129] Simulated intestinal fluid: Dissolve 6.8g of potassium dihydrogen phosphate in 500mL of water, and adjust the pH to 6.8 with 0.1mol / L sodium hydroxide solution; dissolve 10g of pancreatic enzyme in an appropriate amount of water, mix the two solutions, and dilute with water to 1000mL.

[0130] Table 3 (Unit: ×10) 8 CFU / g)

[0131] In Examples 1-3, during the gastrointestinal sequence, the viable bacterial count remained at 5.6-5.9 × 10⁻⁶ after 2 hours of gastric juice treatment. 8 After CFU / g and 2 hours of intestinal fluid treatment, the viable bacterial count was 4.3-4.5 × 10⁻⁶. 8 The CFU / g was significantly better than that of the comparative and control groups, indicating that the double-layer encapsulation provides excellent acid and bile salt resistance.

[0132] In the comparative example, five samples were encapsulated in vesicle fluid, which significantly reduced the gastric tolerance of the microbial microcapsule preparation. Furthermore, the culture of live bacteria did not yield any advantage; after 2 hours of continuous gastric fluid exposure, the number of live bacteria was only 2.1 × 10⁻⁶. 8 Around CFU / g, after 4 hours it was only 1.0×10. 8 The CFU / g level indicates that the microcapsules provide insufficient protection in the gastrointestinal tract.

[0133] Comparative Example 6 used only a pectin-chitosan mixed solution. After 4 hours, the number of viable bacteria was higher than that of Comparative Example 5, but still lower than that of Example 6. This shows that the vesicle fluid has a certain promoting effect on the activation of Akkermansia muciniphila, while the pectin-chitosan mixed solution has almost no effect. Furthermore, without the synergistic effect of the vesicle fluid and the pectin-chitosan mixed solution, the wall material is not dense enough and is difficult to resist gastric acid penetration.

[0134] It should be understood that those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims. Parts not described in detail in this specification are prior art known to those skilled in the art.

Claims

1. A preparation process for a microbial microcapsule formulation for weight loss assistance, characterized in that, Specifically, the following steps are included: S1: Preparation of enhanced culture medium BHI broth, soybean peptone, NaCl, and Na2HPO4 were dissolved in sterile water to obtain solution A; glucose and threonine were dissolved in sterile water to obtain solution B; L-cysteine ​​hydrochloride was dissolved in sterile water to obtain solution C; solutions A, B, and C were mixed and the pH was adjusted to 7.0-7.2 with NaOH solution to obtain the enhanced culture medium. S2: Preparation of bacterial suspension Akkermansia muciniphila was inoculated into enhanced culture medium at an inoculum rate of 1-2% to obtain activated bacterial suspension. The activated bacterial suspension was then expanded and centrifuged to separate the supernatant and the lower precipitate. The lower precipitate was resuspended in sterile physiological saline to obtain bacterial suspension. S3: Preparation of compound microcapsules Add arabinogalactan, bellflower polysaccharide, trehalose, and sterilized skim milk powder to the bacterial suspension and stir until dissolved to obtain a core material mixture; mix the core material mixture with sodium alginate solution to obtain a final mixture. The mixture was added dropwise to a calcium chloride solution, solidified, filtered, and washed to obtain composite microcapsules of microbial agent. S4: Preparation of microbial microcapsule formulations to aid weight loss Centrifuge the supernatant obtained in step S2 to separate the precipitate, resuspend the precipitate to obtain a resuspension, filter the resuspension and dilute it to obtain vesicle fluid. The compound microbial agent microcapsules were mixed with the vesicle fluid, and the precipitate was separated. The washed precipitate was then transferred to a pectin-chitosan mixed solution, stirred, pre-frozen, and freeze-dried to obtain a microbial microcapsule preparation for weight loss assistance.

2. The preparation process of the microbial microcapsule formulation for weight loss assistance according to claim 1, characterized in that, Step S1 involves preparing the enhanced culture medium, including the following steps: Dissolve 18-22 parts by weight of BHI broth, 15-18 parts by weight of soy peptone, 3-5 parts by weight of NaCl and 2-3 parts by weight of Na2HPO4 in 500 parts by weight of sterile water to obtain solution A; Dissolve 5-7 parts by weight of glucose and 1.5-2.5 parts by weight of threonine in 200 parts by weight of sterile water to obtain solution B; Dissolve 5-8 parts by weight of L-cysteine ​​hydrochloride in 100 parts by weight of sterile water to obtain solution C; After mixing solutions A, B, and C, the pH value was adjusted to 7.0-7.2 with NaOH solution to obtain the enhanced culture medium.

3. The preparation process of the microbial microcapsule formulation for weight loss assistance according to claim 2, characterized in that, Step S2, preparing the bacterial suspension, includes the following steps: Akkermansia myxophilus was inoculated into the enhanced culture medium prepared in step S1 at an inoculation rate of 1-2%, and incubated at 35-37℃ under anaerobic conditions for 60-72 hours to obtain activated bacterial solution. The activated bacterial solution was transferred back to the enhanced culture medium prepared in step S1 at an inoculation rate of 2-3%. The culture was carried out at 35-37℃ under anaerobic conditions for 60-72 hours. Then, the supernatant and the lower precipitate were separated at 4℃ and 9000×g for 15-20 minutes. The lower precipitate was resuspended in sterile physiological saline to obtain the bacterial suspension. The anaerobic environment was specifically a gaseous environment composed of 85% N2, 10% CO2 and 5% H2.

4. The preparation process of the microbial microcapsule formulation for weight loss assistance according to claim 3, characterized in that, Step S3 involves preparing the compound microbial agent microcapsules, including the following steps: Add 5-8 mg / mL arabinogalactan, 15-20 mg / mL platycodon polysaccharide, 12-15 mg / mL trehalose and 8-10 mg / mL sterile skim milk powder to the bacterial suspension and stir to dissolve evenly to obtain the core material mixture. The core material mixture and sodium alginate solution were mixed at a volume ratio of 1:(6-8) to obtain the mixture. Add the mixture dropwise to a 2-4% calcium chloride solution, solidify for 30-35 minutes, filter and collect the microcapsules, wash twice with sterile water to obtain composite microbial agent microcapsules, and store at 4℃ for later use.

5. The preparation process of the microbial microcapsule formulation for weight loss assistance according to claim 4, characterized in that, Step S4 involves preparing a microbial microcapsule formulation to aid weight loss, including the following steps: Centrifuge the supernatant separated in step S2 at 100000×g for 10-20 min to separate the precipitate. Resuspend the precipitate with PBS buffer to obtain a resuspension. Filter the resuspension through a 0.45μm filter membrane and dilute it with PBS buffer to 5-7mg / mL to obtain vesicle fluid. The compound microbial agent microcapsules and vesicle fluid were mixed at a material-to-liquid ratio of 1g:(4-6)mL and stirred at 100-150rpm for 2-4h at 2-4℃. The precipitate was separated and then transferred to a pectin-chitosan mixed solution and mixed at a volume ratio of 1:(3-5). The mixture was stirred for 30-35min, then pre-frozen at -60℃ for 5-6h, and then freeze-dried at -40℃ for 24-26h to obtain a microbial microcapsule preparation for weight loss assistance.

6. The preparation process of the microbial microcapsule formulation for weight loss assistance according to claim 3, characterized in that, The anaerobic environment is specifically a gaseous environment composed of 85% N2, 10% CO2 and 5% H2.

7. The preparation process of the microbial microcapsule formulation for weight loss assistance according to claim 4, characterized in that, The concentration of sodium alginate solution is 2-4%.

8. The preparation process of the microbial microcapsule formulation for weight loss assistance according to claim 5, characterized in that, The pH of the PBS buffer is 7.2-7.

4.

9. The preparation process of the microbial microcapsule formulation for weight loss assistance according to claim 5, characterized in that, The pectin-chitosan mixed solution was prepared by dissolving pectin and chitosan in sterile water, controlling the pectin content to be 1% (w / v) and the chitosan content to be 0.5% (w / v), and adjusting the pH value to 5.5-6 with HCl solution.

10. A microbial microcapsule preparation for weight loss assistance, characterized in that, It is prepared by the preparation process of the microbial microcapsule preparation for weight loss assistance as described in any one of claims 1-9.

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