Preparation method of composite probiotic preparation for removing in-vivo plasticizer

Abstract

The invention discloses a preparation method of composite probiotics for removing an in-vivo plasticizer. The method comprises the following steps: resuspending a mixed thallus of lactobacillus paracasei, lactobacillus plantarum and bifidobacterium by using a protective solution containing trehalose, mannan oligosaccharide and L-proline; mixing with a functional gel precursor composed of L-aspartic acid, carboxylated beta-cyclodextrin and porous corn starch, and carrying out cross-linking molding in a calcium ion and optional zinc ion system; then performing surface coordination treatment by using a green tea extract and ferric ions, sequentially coating a chitosan-pectin composite layer and an enteric polymer layer, and performing low-temperature drying to obtain the composite probiotic particles. The obtained preparation has high stability in a gastric acid environment, and can realize directional slow release of thalli and efficient adsorption of a plasticizer.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of probiotic agents, and in particular to a preparation method of a composite probiotic preparation for removing plasticizers in vivo. BACKGROUND

[0002] Plasticizers (such as phthalates) have lipophilic and easy migration characteristics, and are widely present in food contact materials, daily chemicals and environmental media. Human body can be exposed to them through multiple pathways such as ingestion, inhalation and percutaneous absorption. After entering the body, their metabolites can cause endocrine disruption and oxidative stress, and are closely related to dietary structure and intestinal microecological state. Therefore, the focus of research and industrial application is to reduce the burden and risk of re-exposure in the body through oral means.

[0003] There are currently three main strategies: first, supplementing small molecule substances related to antioxidant or detoxification to reduce body damage; second, using food-derived carriers (such as dietary fibers, cyclodextrins, mineral materials, etc.) with adsorption / packing effect to reduce the content of absorbable substances in the intestinal cavity; third, using probiotics or probiotics combined with prebiotics to improve intestinal barrier function and metabolic status. Although the above methods have shown certain effectiveness in antioxidant protection, reducing intestinal exposure or regulating intestinal microecology, in actual application, common limitations include: the survival rate of probiotics fluctuates greatly after passing through the gastric acid and bile salt environment; the gel or coating system is prone to burst release or breakage under strong acid environment or shear force; the selectivity and binding strength for hydrophobic plasticizers are insufficient; the intestinal segment release timing is difficult to stabilize and reproduce; and in the process of large-scale production, the batch consistency is poor due to problems in particle size distribution and layer thickness control. These problems make the overall effect of promoting excretion and preventing reabsorption unstable, with significant individual differences, making it difficult to be applied to population intervention for a long time and in a standardized manner.

[0004] Based on the above status, in the oral administration scenario, how to achieve high survival and controllable arrival of probiotics through the gastrointestinal tract while maintaining sufficient selective binding and mass transfer efficiency for hydrophobic plasticizers without increasing additional safety risks is a key problem that needs to be solved. SUMMARY

[0005] The purpose of the present application is to overcome the above-mentioned problems existing in the prior art, and to provide a preparation method of a composite probiotic preparation for removing plasticizers in vivo.

[0006] In order to achieve the above-mentioned purpose, the first aspect of the present application provides a preparation method of a composite probiotic for removing plasticizers in vivo, comprising the following steps: S1: providing a mixed bacterial body of Lactobacillus paracasei, Lactobacillus plantarum and a Bifidobacterium, to obtain a mixed bacterial body; S2: resuspend the mixed bacteria in an inner protective solution consisting of trehalose, mannose oligosaccharide and L-proline to obtain a cell suspension; S3: mix the cell suspension with a functional gel precursor consisting of L-aspartic acid, carboxylated β-cyclodextrin and porous corn starch, spray into a coagulation bath containing calcium ions and optionally zinc ions to cross-link and form, then surface coordinate with green tea extract and ferric ions to obtain bacteria-containing microparticles; S4: outer coating and low-temperature drying of the bacteria-containing microparticles, wherein the outer coating comprises: (a) a polysaccharide complex layer formed by chitosan and pectin; and (b) an enteric polymer layer, the enteric polymer being selected from one of methacrylic acid-ethyl acrylic acid copolymer aqueous dispersion, lacquer, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose succinate.

[0007] As a further improvement of the present application, the preparation method of the green tea extract is: under the condition of 20-35 ℃, using glycerol, lactic acid and water three-phase solvent as the extraction medium, microwave enhanced extraction under the condition of partially retaining polyphenol oxidase activity, and collecting the extract rich in epigallocatechin and catechin gallate through directional penetration of the permeation membrane; the total catechin content of the obtained extract is ≥80%, and the sum of epigallocatechin and catechin gallate is ≥60%.

[0008] As a further improvement of the present application, the concentration of calcium ions is 25-40 mM, the concentration of zinc ions is 2-4 mM, and the molar ratio of calcium to zinc is 9:1-12:1.

[0009] As a further improvement of the present application, the mass ratio of L-aspartic acid, carboxylated β-cyclodextrin and porous corn starch in the functional gel precursor is 100:13-75:6-31.

[0010] As a further improvement of the present application, in the preparation process of the functional gel precursor, L-aspartic acid and carboxylated β-cyclodextrin are first pre-dissolved in a low-concentration organic solvent of 10-20 wt%, then porous corn starch is added, the solution pH is controlled at 6.5±0.3, the stirring speed is controlled at 200-300 rpm, and stirring is performed for 10-15 minutes.

[0011] As a further improvement of the present application, the concentration of green tea extract in the surface coordination solution is 0.03%-0.10%(w / v), the concentration of ferric ions is 0.05-0.15 mM, and the pH is 6.3-6.7.

[0012] As a further improvement of the present invention, in the surface coordination treatment process of step S3, the pH is controlled at 6.4±0.2, the coordination time is controlled at 30-60s, and the stirring speed is controlled at 100-150rpm; and after the coordination is completed, the surface is washed with 5±0.5mM sodium citrate.

[0013] The polyphenolic compounds in green tea extract possess multiple phenolic hydroxyl sites that can form complexes with ferric ions through polydentate coordination. This allows the ferric ions to embed into the surface of the microparticles, resulting in a more compact and ordered cross-linked network. This provides additional structural stability to the microparticles, preventing dissolution, dissociation, or physical structural damage in harsh environments such as gastric acid. Furthermore, green tea extract can significantly reduce Fenton-like side effects induced by free ferric ions in the gastric environment, alleviating oxidative stress on bacterial membrane lipids and proteins, thereby improving their acid resistance.

[0014] Secondly, the complex formed by iron ions and green tea extract dissolves gradually in gastric acid, allowing the encapsulated probiotics to be released gradually in the small intestine. This enables the probiotics to maintain their activity in the gut for a longer period of time, achieving targeted release and sustained action from the stomach to the intestine.

[0015] As a further improvement of the present invention, the concentration of trehalose in the inner protective solution is 3% to 8% (w / v), the concentration of mannan oligosaccharide is 0.5% to 2.0% (w / v), the concentration of L-proline is 0.10% to 0.30% (w / v), and the pH is 6.0 to 6.8.

[0016] As a further improvement of the present invention, the particle size of the bacterial microparticles ranges from 150 to 400 μm, and the D90 is 300 ± 50 μm.

[0017] As a further improvement of the present invention, the thickness of the outer encapsulated enteric polymer layer is 0.3–0.6 μm. The present invention, by adopting the above technical solution, has the following beneficial effects: The compound probiotic preparation method provided by this invention solves the problems of poor in vivo stability, activity decline, and short-lasting detoxification effect of traditional probiotic preparations through precise process design, step optimization, and unique excipient structure design. Specifically, it is reflected in key technical aspects such as the embedded structure of L-aspartic acid and carboxylated β-cyclodextrin, metal ion crosslinking, Fe-green tea extract coordination, and multi-level coating. By rationally configuring the preparation and coating process of microparticles, stable release of probiotics is achieved, improving the stability, detoxification effect, and release control of the preparation, thus ensuring the durability of the preparation.

[0018] During the formation of microparticles, L-aspartic acid and carboxylated β-cyclodextrin form a stable embedded structure through a calcium ion cross-linking reaction. This enhances the structural stability of the microparticles, preventing rearrangement or uneven dissolution in the in vivo environment. It solves the problem of reduced activity in traditional probiotic preparations due to the influence of gastric acid, bile, and other environmental factors during use, effectively ensuring that probiotics maintain their proper biological activity after entering the intestines, thus improving the stability and reliability of probiotic preparations.

[0019] Furthermore, the L-aspartic acid molecule contains two carboxyl groups and one amino group, which allows it to react with Ca... 2+ and Zn 2+ Ions form multi-site coordination, thereby constructing a denser and more uniform ion bridge network. Moreover, the geometric symmetry of its two carboxyl groups makes the ion coordination more stable, while the amino terminus can form hydrogen bonds with the hydroxyl groups on the surface of porous corn starch and the carboxyl groups of carboxylated β-cyclodextrin, forming a network structure with flexible connecting segments and stable cross-linking points. This significantly improves the granulation strength and anti-breakage performance of the gel, and also makes the internal channels more interconnected and the specific surface area larger, which is beneficial to the dispersion of carboxylated β-cyclodextrin and the mass transfer and adsorption of plasticizer molecules.

[0020] More importantly, L-aspartic acid, as an amino acid that probiotics can metabolize, can be preferentially absorbed by bacteria after the microcapsules enter the intestinal segment and are used for cell energy restoration and membrane potential reconstruction, thereby significantly improving the bacterial recovery efficiency and subsequent metabolic activity.

[0021] A low-dose, short-time cross-linking method was employed, primarily using calcium and secondarily zinc. The entire process utilized an aqueous phase and low temperature, avoiding the reduction in bacterial survival rate caused by excessive heating or solvent stress, thus maintaining high survival rate and activity of the probiotics. Furthermore, by optimizing the process through pre-dissolving L-aspartic acid and carboxylated β-cyclodextrin in a low-concentration organic solvent, and controlling the pH of the solution after adding porous corn starch, as well as the stirring conditions, the stability of the microparticles was maintained while avoiding dissolution or rearrangement due to excessive cross-linking. This ensured that the probiotics maintained high survival rate and biological activity throughout the entire preparation process.

[0022] A stable coordination complex is formed by the binding of iron ions with the phenolic hydroxyl groups in green tea extract molecules. This coordination complex not only improves the physical structure of the particles but also provides an additional protective barrier for probiotics, thereby significantly enhancing the survival rate and activity stability of the bacteria in the complex in vivo environment.

[0023] The product employs a layered structure consisting of a composite inner layer of chitosan and pectin, a main layer of enteric polymer, and / or a porous hydrophobic thin layer. This structure enhances the resistance of probiotics in the gastric environment, enabling them to effectively pass through the stomach and achieve controlled release in the small intestine, thus ensuring maximum effectiveness in the gut. Detailed Implementation

[0024] The following provides a detailed description of specific embodiments of the present invention. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of the invention.

[0025] Unless otherwise defined, all scientific and technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art.

[0026] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0027] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0028] The present invention will now be described in detail with reference to specific embodiments, which are intended to understand rather than limit the invention.

[0029] Example 1 This embodiment discloses a method for preparing a compound probiotic preparation for removing plasticizers in vivo, including the following steps: S1: A mixed bacterial culture of *Lactobacillus paracasei*, *Lactobacillus plantarum*, and *Bifidobacterium bifidum* is provided. Specifically, the *Lactobacillus paracasei* is *Lactobacillus paracasei* DT66, the *Lactobacillus plantarum* is *Lactobacillus plantarum* DT88, and the *Bifidobacterium bifidum* is *Bifidobacterium bifidum* ATCC 29521.

[0030] All of the above strains are existing strains that can be obtained from commercially available culture centers or scientific reagent suppliers, such as strains publicly deposited by the China Center for Type Culture Collection (CCTCC), the American Center for Type Culture Collection (ATCC), or the China General Microbiological Culture Collection Center (CGMCC).

[0031] The three samples were anaerobically cultured in MRS liquid medium at 37°C for 18 hours until OD... 600 After reaching a viscosity of 1.3, centrifuge (8000 rpm, 4℃, 10 min), discard the supernatant and collect the wet bacterial cells. Wash the bacterial cells twice with sterile PBS buffer (pH=7.2), and then... Pre-freeze at 20℃ for 12 hours, then freeze-dry under vacuum. Freeze-dried bacterial powder was obtained at 45℃, vacuum degree ≤15Pa, time 18h, with a viable bacterial count of 5×10⁻⁶. 9 CFU / g.

[0032] S2: The mixed bacterial cells were resuspended in an inner layer protection solution composed of trehalose, mannooligosaccharide, and L-proline to obtain a cell suspension. The concentration of trehalose in the inner layer protection solution was 5% (w / v), the concentration of mannooligosaccharide was 1.2% (w / v), and the concentration of L-proline was 0.20% (w / v). The pH was adjusted to 6.4 using 0.01 mol / L phosphate buffer. The volume ratio of the suspension to the bacterial powder was controlled at 12:1 (mL / g). The mixture was stirred magnetically at 200 rpm for 5 min to ensure uniform dispersion of the bacterial cells. The suspension should be clear and homogeneous, with no obvious flocculation or sedimentation.

[0033] S3: The cell suspension is mixed with a functional gel precursor composed of L-aspartic acid, carboxylated β-cyclodextrin, and porous corn starch. L-aspartic acid (food grade, purity ≥99%), carboxylated β-cyclodextrin (degree of substitution 0.60), and porous corn starch (particle size 30–50 μm, specific surface area ≥8 m²) are used. 2 The mass ratio of (g / g) is 100:45:18.

[0034] First, L-aspartic acid and carboxylated β-cyclodextrin were dissolved separately in deionized water (15 wt%), and then mixed after being magnetically stirred and defoamed at 40 °C. Then, porous corn starch was slowly added and stirred for 12 min at a stirring speed of 250 rpm, while maintaining the pH at 6.5 ± 0.3, to obtain a homogeneous emulsion-like functional gel precursor.

[0035] The resulting mixture was injected via a peristaltic pump at a rate of 2 mL / min into a Ca-containing solution. 2+ and Zn 2+In a coagulation bath (CaCl2 concentration 32mM, ZnSO4 concentration 3mM, Ca:Zn molar ratio 10:1), the bath temperature was maintained at 25±2℃, and the mixture was allowed to stand for crosslinking for 15 min. The resulting initial coagulated particles were collected by filtration and washed with deionized water.

[0036] Then it was treated with a green tea extract-ferric ion surface complex solution, with a green tea extract concentration of 0.06% (w / v) and Fe... 3+ The concentration was 0.10 mM, pH was 6.5, and the mixture was stirred at 120 rpm for 45 s. After treatment, the mixture was washed with 5 ± 0.5 mM sodium citrate solution until the surface solution was colorless and transparent, thus obtaining surface-coordinated and stable microparticles containing bacteria.

[0037] The preparation method of green tea extract is as follows: (a) Take green tea powder (food-grade green tea powder produced by Nanjing Songguan Biotechnology Co., Ltd.) and mix it with citric acid and sodium citrate buffer (pH 4.0 ± 0.1), add 0.05 wt% magnesium gallate and 0.1 wt% sodium γ-polyglutamate, and let it stand at 25 °C for 15 min to balance enzyme activity.

[0038] (b) The mixture was placed in a dual-chamber extraction reactor with a PTFE nanoporous membrane diaphragm. The liquid phase was prepared using glycerol:lactic acid:water = 40:10:50 (volume fraction) as the solvent. N2 microbubbles were introduced and the mixture was continuously stirred.

[0039] (c) Start the microwave source (2.45 GHz, power 100 W) and maintain 35 °C for 20 min to allow tea polyphenols to permeate through the membrane into the collection chamber; collect the permeate by circulating it twice.

[0040] (d) The permeate was combined, concentrated by low-temperature vacuum rotary evaporation, and then freeze-dried to obtain green tea extract powder.

[0041] S4: The bacterial microparticles are double-coated and then dried and fixed at low temperature. (a) First, the polysaccharide layer was formed by alternating immersion in 1% chitosan solution (pH 5.5) and 1% pectin solution (pH 4.0) for 3 min. After standing and drying, the layer thickness was about 0.20 μm. (b) A layer of enteric polymer is then added. The enteric polymer is selected from an aqueous dispersion of methacrylic acid-ethyl acrylate copolymer, or one of shellac, hydroxypropyl methylcellulose phthalate, or hydroxypropyl methylcellulose succinate. In this embodiment, an aqueous dispersion of methacrylic acid-ethyl acrylate copolymer (Eudragit® L30D-55 dispersion) (30% solid content) is used for coating in a spray fluidized bed manner. The spray rate is 1.5 mL / min, the inlet air temperature is 35°C, and the atomizing pressure is 0.25 MPa, resulting in an enteric layer with a thickness of 0.45 μm. After coating, the layer is vacuum dried at 30°C for 8 hours, with a moisture content ≤5%.

[0042] The prepared microparticles containing bacteria were measured by a laser particle size analyzer, and the D... 50 It is 220μm, D 90 It is 300μm.

[0043] Example 2 This embodiment discloses a method for preparing a compound probiotic preparation for removing plasticizers in vivo, including the following steps: S1: Provides a mixed bacterial culture of *Lactobacillus paracasei*, *Lactobacillus plantarum*, and *Bifidobacterium bifidum*. The three bacteria were separately cultured anaerobically in MRS liquid medium at 37°C for 18 hours until OD... 600 After reaching a viscosity of 1.4, centrifuge (8000 rpm, 4°C, 10 min), discard the supernatant, and collect the wet bacterial cells. Wash the bacterial cells twice with sterile PBS buffer (pH=7.2), and then... Pre-freeze at 20℃ for 12 hours, then freeze-dry under vacuum. Freeze-dried bacterial powder was obtained at 45℃, vacuum degree ≤15Pa, time 18h, with a viable bacterial count of 8×10⁻⁶. 9 CFU / g.

[0044] S2: The mixed bacterial cells were resuspended in an inner layer protection solution composed of trehalose, mannooligosaccharide, and L-proline to obtain a cell suspension. The concentration of trehalose in the inner layer protection solution was 6% (w / v), the concentration of mannooligosaccharide was 1.5% (w / v), and the concentration of L-proline was 0.25% (w / v). The pH was adjusted to 6.6 using 0.01 mol / L phosphate buffer. The volume ratio of the suspension to the bacterial powder was controlled at 14:1 (mL / g). The mixture was stirred magnetically at 200 rpm for 5 min to ensure uniform dispersion of the bacterial cells. The suspension should be clear and homogeneous, with no obvious flocculation or sedimentation.

[0045] S3: The cell suspension is mixed with a functional gel precursor composed of L-aspartic acid, carboxylated β-cyclodextrin, and porous corn starch. L-aspartic acid (food grade, purity ≥99%), carboxylated β-cyclodextrin (degree of substitution 0.62), and porous corn starch (particle size 30–50 μm, specific surface area ≥8 m²) are used. 2 The mass ratio of (g / g) is 100:60:25.

[0046] First, L-aspartic acid and carboxylated β-cyclodextrin were dissolved separately in deionized water (18 wt%), and then mixed after being magnetically stirred and defoamed at 40 °C. Then, porous corn starch was slowly added and stirred for 14 min at a stirring speed of 280 rpm, while maintaining the pH at 6.5 ± 0.3, to obtain a homogeneous emulsion-like functional gel precursor.

[0047] The resulting mixture was injected via a peristaltic pump at a rate of 2 mL / min into a Ca-containing solution. 2+ and Zn 2+ The coagulated particles were placed in a coagulation bath (CaCl2 concentration 38mM, ZnSO4 concentration 3.5mM, Ca:Zn molar ratio 11:1) and the bath temperature was maintained at 25±2℃ for 15 min of static cross-linking. The resulting initial coagulated particles were collected by filtration and washed with deionized water.

[0048] Then it was treated in a green tea extract-ferric ion surface complex solution, with a green tea extract concentration of 0.09% (w / v) and Fe... 3+ The concentration was 0.14 mM, pH was 6.6, and the mixture was stirred at 140 rpm for 55 s. After treatment, the mixture was washed with 5 ± 0.5 mM sodium citrate solution until the surface solution was colorless and transparent, thus obtaining surface-coordinated bacterial microparticles.

[0049] S4: The bacterial microparticles are double-coated and then dried and fixed at low temperature. (a) First, the polysaccharide layer was formed by alternating immersion in 1% chitosan solution (pH 5.5) and 1% pectin solution (pH 4.0) for 3 min. After standing and drying, the layer thickness was about 0.24 μm. (b) A layer of enteric polymer is then added. The enteric polymer is selected from one of the following: an aqueous dispersion of methacrylic acid-ethyl acrylate copolymer (Eudragit® L30D-55), or shellac, hydroxypropyl methylcellulose phthalate, or hydroxypropyl methylcellulose succinate. In this embodiment, Eudragit® L30D-55 dispersion (30% solids content) is used for coating in a spray fluidized bed manner. The spray rate is 1.5 mL / min, the inlet air temperature is 35°C, and the atomizing pressure is 0.25 MPa, resulting in an enteric layer with a thickness of 0.55 μm. After coating, the layer is vacuum dried at 30°C for 8 hours, with a moisture content ≤5%.

[0050] The prepared microparticles containing bacteria were measured by a laser particle size analyzer, and the D... 50 It is 230μm, D 90 It is 320μm.

[0051] Example 3 This embodiment discloses a method for preparing a compound probiotic preparation for removing plasticizers in vivo, including the following steps: S1: Provides a mixed bacterial culture of *Lactobacillus paracasei*, *Lactobacillus plantarum*, and *Bifidobacterium bifidum*. The three bacteria were separately cultured anaerobically in MRS liquid medium at 37°C for 18 hours until OD... 600 After reaching a viscosity of 1.2, centrifuge (8000 rpm, 4℃, 10 min), discard the supernatant, and collect the wet bacterial cells. Wash the bacterial cells twice with sterile PBS buffer (pH=7.2), and then... Pre-freeze at 20℃ for 12 hours, then freeze-dry under vacuum. Freeze-dried bacterial powder was obtained at 45℃, vacuum degree ≤15Pa, time 18h, with a viable bacterial count of 3×10⁻⁶. 9 CFU / g.

[0052] S2: The mixed bacterial cells were resuspended in an inner layer protection solution composed of trehalose, mannooligosaccharide, and L-proline to obtain a cell suspension. The concentration of trehalose in the inner layer protection solution was 3% (w / v), the concentration of mannooligosaccharide was 0.5% (w / v), and the concentration of L-proline was 0.10% (w / v). The pH was adjusted to 6.0 using 0.01 mol / L phosphate buffer. The volume ratio of the suspension to the bacterial powder was controlled at 10:1 (mL / g). The mixture was stirred magnetically at 200 rpm for 5 min to ensure uniform dispersion of the bacterial cells. The suspension should be clear and homogeneous, with no obvious flocculation or sedimentation.

[0053] S3: The cell suspension is mixed with a functional gel precursor composed of L-aspartic acid, carboxylated β-cyclodextrin, and porous corn starch. L-aspartic acid (food grade, purity ≥99%), carboxylated β-cyclodextrin (degree of substitution 0.58), and porous corn starch (particle size 30–50 μm, specific surface area ≥8 m²) are used. 2 The mass ratio of (g / g) is 100:30:12.

[0054] First, L-aspartic acid and carboxylated β-cyclodextrin were dissolved separately in deionized water (12 wt%), and then mixed after being magnetically stirred and defoamed at 40 °C. Then, porous corn starch was slowly added and stirred for 11 min at a stirring speed of 220 rpm, while maintaining the pH at 6.5 ± 0.3, to obtain a homogeneous emulsion-like functional gel precursor.

[0055] The resulting mixture was injected via a peristaltic pump at a rate of 2 mL / min into a Ca-containing solution. 2+ and Zn 2+ The particles were placed in a coagulation bath (CaCl2 concentration 28 mM, ZnSO4 concentration 2.5 mM, Ca:Zn molar ratio 10:1) and the bath temperature was maintained at 25 ± 2 °C for 15 min of static crosslinking. The resulting initial coagulated particles were collected by filtration and washed with deionized water.

[0056] Then it was treated with a green tea extract-ferric ion surface complex solution, with a green tea extract concentration of 0.05% (w / v) and Fe... 3+ The concentration was 0.08 mM, pH was 6.4, and the mixture was stirred at 110 rpm for 40 s. After treatment, the microparticles were washed with 5 ± 0.5 mM sodium citrate solution until the surface was colorless and transparent, thus obtaining microparticles with stable surface coordination.

[0057] S4: The bacterial microparticles are double-coated and then dried and fixed at low temperature. (a) First, the polysaccharide layer was formed by alternating immersion in 1% chitosan solution (pH 5.5) and 1% pectin solution (pH 4.0) for 3 min. After standing and drying, the layer thickness was about 0.18 μm. (b) A layer of enteric polymer is then added. The enteric polymer is selected from one of the following: an aqueous dispersion of methacrylic acid-ethyl acrylate copolymer (Eudragit® L30D-55), or shellac, hydroxypropyl methylcellulose phthalate, or hydroxypropyl methylcellulose succinate. In this embodiment, Eudragit® L30D-55 dispersion (30% solids content) is used for coating in a spray fluidized bed manner. The spray rate is 1.5 mL / min, the inlet air temperature is 35°C, and the atomizing pressure is 0.25 MPa, resulting in an enteric layer with a thickness of 0.35 μm. After coating, the layer is vacuum dried at 30°C for 8 hours, with a moisture content ≤5%.

[0058] The prepared microparticles containing bacteria were measured by a laser particle size analyzer, and the D... 50 200μm, D 90 It is 290μm.

[0059] Example 4 The difference between this embodiment and Embodiment 1 is that step S5 was not performed. The prepared bacterial microparticles were measured using a laser particle size analyzer, and D... 50 250μm, D 90 It is 340μm.

[0060] Comparative Example 1 The difference between this comparative example and Example 1 is that L-aspartic acid is replaced with sodium alginate.

[0061] Comparative Example 2 The difference between this comparative example and Example 1 is that the green tea extract is replaced with tannins.

[0062] Comparative Example 3 This comparative example discloses a freeze-dried bacterial powder agent without any processing, including the following steps: S1: *Lactobacillus paracasei*, *Lactobacillus plantarum*, and *Bifidobacterium bifidum* were obtained, with the same bacterial strains as in Example 1. All three were inoculated into MRS liquid medium and anaerobically cultured at 37 °C for 18 h until OD (Organic Dysplasia) was reached. 600 Stop culturing when the concentration reaches 1.3. Centrifuge at 8000 rpm and 4 °C for 10 min, discard the supernatant, and collect the wet bacterial cells. Wash twice with sterile PBS buffer (pH 7.2) before use.

[0063] S2: Mix the three wet bacterial cells in a 1:1:1 ratio and resuspend them in a protective solution composed of a traditional lyophilization protectant. The protective solution consists of 10% (w / v) trehalose, 10% (w / v) skim milk powder, and 0.5% (w / v) monosodium glutamate, with the pH adjusted to 6.8. The suspension volume to bacterial cell mass ratio is 10:1 (mL / g). Mix at room temperature with magnetic stirring at 200 rpm for 5 min to obtain a homogeneous cell suspension.

[0064] S3: Transfer the above suspension directly into a lyophilization bottle, and... After pre-freezing at 20℃ for 12 hours, it is placed in a vacuum freeze dryer. Freeze-dry at 45 ℃ (vacuum degree ≤15 Pa) for 18 h to obtain traditional freeze-dried bacterial powder.

[0065] The obtained bacterial powder was used directly for subsequent testing. The samples were packaged in aluminum foil bags and stored at 4 ℃ in the dark.

[0066] Performance testing 1. Methods for detecting fungal and microbial contamination rates A certain amount of freeze-dried microparticles containing bacteria were inoculated into potato dextrose agar (PDA) and nutrient agar (NA) media, respectively, and cultured at 37°C for 7 days. Observation of colony growth in the petri dishes confirmed that no colonies were observed, and the final determination was that the fungal and microbial contamination rate was negative.

[0067] All examples and comparative examples tested negative for bacteria.

[0068] 2. Moisture content test method Take a certain amount of the microbial sample and dry it in a constant temperature drying oven at 105℃ for 6 hours until constant weight. Calculate the moisture content by weighing the difference. If the calculation result shows that the moisture content is less than 5%, the moisture content is considered to meet the requirements.

[0069] 3. Method for testing the survival rate of viable bacteria after rehydration First, a certain amount of lyophilized sample is taken and rehydrated with sterile physiological saline. The volume of the rehydrated liquid is 10 times the mass of the lyophilized sample. Then, the plate count method is used to count the colonies and obtain the viable bacterial survival rate after rehydration. If the viable bacterial survival rate after rehydration is higher than 90%, it indicates that the rehydrated microparticles can maintain high activity.

[0070] 4. Simulated gastric and intestinal fluid testing methods The simulated gastric juice was prepared using a hydrochloric acid solution with a pH of 2.0. The particulate samples were reacted with the gastric juice at 37°C for 2 hours, followed by plate counting.

[0071] The reaction was carried out at 37°C for 4 hours using simulated intestinal fluid (sodium bicarbonate and disodium hydrogen phosphate solution with a pH of 7.5), followed by plate counting.

[0072] 5. In vitro adsorption-removal kinetics test Prepare a solution of DEHP and DBP in a water-ethanol mixture (5% ethanol by volume) to a concentration of 10 mg·L⁻¹. -1 Each sample was added to an equivalent viable bacterial count of 1×10⁻⁶. 9 CFU / sample or equivalent particulate matter (0.50 g·100 mL) -1 The mixture was incubated at 37°C with gentle shaking (80 rpm) for 4 hours. The supernatant was collected by centrifugation and quantified by solid-phase microextraction-GC / MS, with blank self-adsorption subtracted.

[0073]

[0074] The test results above show that the compound probiotic preparations obtained in Examples 1-3 of this invention were negative for fungal and microbial contamination, and the moisture content remained stable at around 4%, indicating that the preparation process was hygienic, reliable, and thoroughly dried. The survival rate of live bacteria after rehydration in each example was higher than 90%, significantly higher than that of Comparative Example 3 (66.8%), which uses a traditional freeze-dried bacterial powder. This demonstrates that the present invention significantly improves the bacterial survival rate during freeze-drying and rehydration through the dual protection of the inner protective liquid and the functional gel precursor.

[0075] In tests using simulated gastric fluid (pH 2.0, 2h) and simulated intestinal fluid (pH 7.5, 4h), the cell survival rates of Examples 1-3 were between 68.5% and 71.2% and 93.6% and 95.8%, respectively, while those of Comparative Example 3 were only 18.5% and 54.2%, demonstrating that the encapsulation system of this invention exhibits excellent acid resistance and high release rate in the intestinal segment under gastric acid conditions. In particular, the microparticles containing the L-aspartic acid and carboxylated β-cyclodextrin complex effectively stabilized the gel network through their multi-point coordination structure, and also contributed to the absorption of green tea extract—Fe 3+ The surface composite layer significantly slows down acidic dissociation and enzymatic degradation.

[0076] In terms of plasticizer adsorption and removal performance, the DEHP and DBP removal rates of Example 2 reached 72.4% and 69.1%, respectively, which were higher than those of Example 1 (65.2% and 62.8%) and all comparative examples. In Comparative Example 1, after replacing L-aspartic acid with sodium alginate, the DEHP removal rate decreased to 38.7%, indicating that L-aspartic acid not only participates in ionic cross-linking in the composite network but also enhances the interfacial adsorption capacity of plasticizers through the synergistic effect of amino-carboxyl groups. In Comparative Example 2, after replacing green tea extract with tannic acid, the removal rate decreased to 59.3%, indicating that epigallocatechin and catechin gallate, which are abundant in green tea extract, play a crucial role in constructing stable Fe... 3+ The coordination layer plays a crucial role in maintaining antioxidant protection.

[0077] The above description is merely a preferred embodiment of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a composite probiotic for in vivo plasticizer removal, characterized by, The method comprises the following steps: S1: providing a mixed bacteria body of Lactobacillus paracasei, Lactobacillus plantarum and a Bifidobacterium, to obtain the mixed bacteria body; S2: resuspending the mixed bacteria body in an inner protective solution composed of trehalose, mannose oligosaccharide and L-proline to obtain a cell suspension; S3: mixing the cell suspension with a functional gel precursor composed of L-aspartic acid, carboxylated β-cyclodextrin and porous corn starch, spraying into a coagulation bath containing calcium ions and optionally zinc ions to cross-link and form, and then performing surface coordination treatment with green tea extract and ferric ions to obtain bacteria-containing microparticles; S4: performing outer coating and low-temperature drying and shaping on the bacteria-containing microparticles, wherein the outer coating comprises: (a) a polysaccharide composite layer formed by chitosan and pectin; and (b) an enteric polymer layer, the enteric polymer being selected from one of a methacrylic acid-ethyl acrylic acid copolymer aqueous dispersion, lacquer, hydroxypropyl methylcellulose phthalate and hydroxypropyl methylcellulose succinate.

2. The preparation method of the complex probiotic for in vivo plasticizer removal according to claim 1, characterized by, The green tea extract is prepared by microwave-enhanced extraction under the condition of 20-35 ℃, using a three-phase solvent of glycerol, lactic acid and water as the extraction medium, and collecting the extract liquid rich in epigallocatechin and catechin gallate through directional penetration of a penetration membrane under the condition of partially retaining polyphenol oxidase activity; the total catechin content of the obtained extract is ≥80%, and the sum of epigallocatechin and catechin gallate is ≥60%.

3. The preparation method of the complex probiotic for in vivo plasticizer removal according to claim 1, characterized by, The concentration of calcium ions is 25-40 mM, the concentration of zinc ions is 2-4 mM, and the molar ratio of calcium to zinc is 9:1-12:

1.

4. The preparation method of the complex probiotic for in vivo plasticizer removal according to claim 1, characterized by, The mass ratio of L-aspartic acid, carboxylated β-cyclodextrin and porous corn starch in the functional gel precursor is 100:13-75:6-31.

5. The method of claim 4, wherein the probiotic composition is administered to the subject in an amount of 1 x 10 L colony forming units (CFU) per kilogram of body weight of the subject. In the preparation process of the functional gel precursor, L-aspartic acid and carboxylated β-cyclodextrin are first pre-dissolved in a low-concentration solvent of 10-20 wt%, and then porous corn starch is added, the pH of the solution is controlled to be 6.5±0.3, the stirring speed is controlled to be 200-300 rpm, and stirring is performed for 10-15 minutes.

6. The method of claim 1, wherein the probiotic composition is administered to the subject in an amount of about 1 x 10 L to about 1 x 10 L. The concentration of green tea extract in the surface coordination liquid is 0.03%-0.10%(w / v), the concentration of ferric ions is 0.05-0.15 mM, and the pH is 6.3-6.

7.

7. The method of claim 7, wherein the probiotic composition is prepared by mixing the probiotic bacteria and the plasticizer-removing agent in a ratio of 1: 1 to 1:

10. In the surface coordination treatment process in the S3 step, the pH is controlled to be 6.4±0.2, the coordination time is controlled to be 30-60 s, and the stirring speed is controlled to be 100-150 rpm; and after the coordination is completed, the bacteria-containing microparticles are washed with 5±0.5 mM sodium citrate.

8. The method of claim 1, wherein the probiotic composition is prepared by mixing the probiotic bacteria and the plasticizer-removing agent in a ratio of 1: 1 to 1:

10. The concentration of trehalose in the inner protective solution is 3%-8%(w / v), the concentration of mannose oligosaccharide is 0.5%-2.0%(w / v), the concentration of L-proline is 0.10%-0.30%(w / v), and the pH is 6.0-6.

8.

9. The method of claim 1, wherein the probiotic composition is prepared by mixing the probiotic bacteria and the plasticizer-removing agent in a ratio of 1: 1 to 1: 10 (w / w). The particle size range of the bacteria-containing microparticles is 150-400 μm, and D90 is 300±50 μm.

10. The method of claim 1, wherein the probiotic composition is prepared by mixing the probiotic bacteria and the plasticizer-removing agent in a ratio of 1: 1 to 1:

10. The thickness of the enteric polymer layer of the outer coating is 0.3-0.6 μm.

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