Compound probiotic preparation rich in organic selenium and preparation method thereof

Abstract

The invention relates to the field of biological agents, in particular to a compound probiotic preparation rich in organic selenium and a preparation method of the compound probiotic preparation, and efficient synergistic delivery is achieved through plant micromolecule organic selenium extraction and a multi-layer microemulsion embedding technology. The preparation consists of a core active component, a carrier system and a curcumin compound. The plant micromolecular organic selenium is extracted from cardamine hupingshanesis and high-selenium corn flour through an HLUP technology, and the bioavailability is remarkably improved. A corn protein modified W / O / W microemulsion is adopted as a carrier, an outer layer membrane is stable in an acid environment, and an inner layer accurately releases selenium to small intestines; a protective agent composed of trehalose and glucose inhibits ice crystal damage, and glutaraldehyde crosslinking enhances the mechanical strength of the membrane. Curcumin and selenium synergistically enhance antioxidant and immunomodulatory functions, and promote phagocytic activity of macrophages. The stability and bioavailability bottlenecks of a traditional preparation are broken through, and an innovative scheme is provided for functional food development.

Description

Technical Field

[0001] The present invention relates to the field of biological agents, and particularly to a compound probiotic preparation rich in organic selenium and a preparation method thereof. Background Art

[0002] "Selenium" is an essential trace element for the human body. Selenium enrichment technology can not only increase the yield and quality of crops, but also increase the selenium content in agricultural products by several times, obtaining ideal natural selenium-rich products for humans. It can also be converted into selenium-rich meat, milk, eggs and high-quality health products through selenium-rich feed to meet the human demand for selenium. Through scientific supplementation of the trace element "selenium", selenium-rich agriculture realizes the optimization, nutrition and functionality of agricultural products. It is relatively mature in China in terms of technology and has entered the industrial promotion stage. It is an important part of future functional agriculture. The content of selenium in human tissues is one in ten million, but it determines the existence of life and is an important part of some antioxidant enzymes (such as glutathione peroxidase) and selenium-P proteins in animals and humans. However, due to the lack of existing soil selenium and crop selenium, it ultimately leads to insufficient selenium intake in the human body. And selenium cannot be synthesized by the body independently and can only be taken in from outside the body. Selenium deficiency will directly lead to a decline in human immune ability. Clinical medicine has proved that more than forty diseases threatening human health and life are related to human selenium deficiency, such as cancer, cardiovascular diseases, liver diseases, cataracts, pancreatic diseases, diabetes, reproductive system diseases, etc.

[0003] Selenium in plant tissues can be divided into inorganic selenium and organic selenium. The forms of organic selenium in plant tissues are diverse. Selenium can exist in organisms in the form of low-molecular-weight compounds (including free selenium) and high-molecular-weight compounds. Small-molecular-weight selenides in plants include selenomethylselenocysteine, selenohomocysteine, selenomethionine, and selenopeptides. Selenoamino acids (SEC) are the main sources of selenium obtained by humans in their daily diet. The main forms of small-molecular-weight organic selenium are selenoamino acids and their derivatives, mainly including Se-methylselenocysteine, selenocysteine, selenomethionine, selenium-rich corn flour, dimethyldiselenide, selenohomocysteine, selenocysteine selenite, Se-allylselenocysteine oxide, etc. However, traditional selenium supplementation products such as inorganic selenium and macromolecular organic selenium have problems such as low bioavailability and poor safety.

[0004] Probiotics are a type of active microorganisms beneficial to the host. Colonized in the human intestinal tract and reproductive system, they can produce definite health effects, thus improving the host's microecological balance and playing a beneficial role. Common probiotics include Bifidobacterium, Lactobacillus acidophilus, etc. However, as the core component for regulating intestinal microecology, the activity of probiotics is easily affected by processing, storage, and the digestive tract environment, resulting in limited actual application effects. In the existing technology, selenium-rich probiotic preparations mostly use selenium yeast or inorganic selenium simply mixed with probiotics, but there are the following defects: 1. Selenium form problem: Selenium in selenium yeast exists in the form of macromolecular binding (such as selenoprotein), which needs to be absorbed through complex metabolic conversion, with low efficiency and possible introduction of impurities; 2. Insufficient stability: Probiotics are easily inactivated in the processing and gastric acid environment, and have poor compatibility with selenium, making it difficult to achieve co-delivery; 3. Single function: Existing products only focus on the single function of selenium or probiotics, lacking double optimization of intestinal absorption and immune regulation.

[0005] Therefore, according to the relevant technologies mentioned above, there is an urgent need to develop a composite probiotic preparation rich in organic selenium and its preparation method. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide a composite probiotic preparation rich in organic selenium and its preparation method, which realizes the co-protection and targeted release of selenium and probiotics through microemulsion encapsulation technology, significantly improves the bioavailability of selenium and the survival rate of probiotics, and at the same time enhances the antioxidant and immune regulation functions.

[0007] Based on the above purpose, the present invention provides a composite probiotic preparation rich in organic selenium and its preparation method.

[0008] A composite probiotic preparation rich in organic selenium is prepared from the following raw materials in parts by mass: 19 - 49 parts of core active components, 25 - 40 parts of carrier system, and 8 - 9.3 parts of auxiliary agent;

[0009] The core active components include 0.5 - 5 parts of plant small molecule organic selenium and 10 - 50 parts of probiotics;

[0010] The carrier system includes 70 - 85 parts of water-in-oil-in-water microemulsion and 6 - 10 parts of protective agent;

[0011] The auxiliary agent is curcumin complex.

[0012] Preferably, the preparation process of the plant small molecule organic selenium is as follows:

[0013] Mix Cardamine hupingshanensis, high-selenium corn flour and extractant evenly, adjust the pH to 7.5 - 8.5, then add a catalyst at 70 - 95 °C, react for 4 - 5 h through HLUP technology, and dialyze in a dialysis bag with a molecular weight cut-off of 8 - 14 kDa to obtain plant small molecule organic selenium.

[0014] Preferably, the mass ratio of Cardamine hupingshanensis, high selenium corn flour, extractant and catalyst is 0.45 - 1.1:0.45 - 1.1:25 - 53:0.022 - 0.033. The catalyst is ferric chloride and the extractant is ultrapure water.

[0015] Preferably, the probiotics are a mixture of Bifidobacterium, Lactobacillus and Bacillus coagulans in a mass ratio of 2:1:1. The viable count of Bifidobacterium is ≥5×10 10 CFU / g, the viable count of Lactobacillus is ≥2.5×10 10 CFU / g, and the viable count of Bacillus coagulans is ≥2.5×10 10 CFU / g.

[0016] Preferably, the preparation process of the water-in-oil-in-water microemulsion is as follows:

[0017] Step A1: Add 44 - 50 parts of corn protein powder to ultrapure water to prepare a mixed solution a with a concentration of 8% - 12%. Adjust the pH of the mixed solution a to 8 - 9. Place the mixed solution a in a constant temperature water bath and heat it to 70 - 85°C. Stir at 200 - 400 rpm for 40 - 60 min to fully unfold the corn protein, expose the hydrophobic groups, and at the same time partially hydrolyze the peptide chains, reducing the molecular weight. When the temperature >70°C, the hydrogen bonds and hydrophobic interactions of the corn protein can be effectively destroyed. When the time >30 min, the denaturation is ensured to be sufficient, but over-hydrolysis is avoided to prevent the emulsifying ability from decreasing. Rapidly cool the heat-denatured solution to 25 - 30°C to terminate the denaturation process. Dilute the concentration to 3wt% - 6wt% with ultrapure water and filter through a 0.45μm membrane to remove undissolved protein aggregates to obtain a homogeneous and transparent heat-denatured corn protein aqueous solution, denoted as mixed solution b. At high alkaline temperature, the α-helix structure of corn protein unfolds to form a β-sheet or random coil conformation, exposing the hydrophobic region, enhancing its ability to adsorb at the oil-water interface. The surface hydrophobicity of the denatured corn protein increases and the interfacial tension decreases, which is beneficial to the formation of a stable microemulsion structure. When the temperature exceeds 90°C, it may cause excessive protein aggregation and reduce the emulsification efficiency;

[0018] Step A2: Uniformly mix the mixed solution b, the oil phase and the co-surfactant, and ultrasonically form an emulsion to obtain a water-in-oil-in-water emulsion.

[0019] Preferably, in step A1, the dosage of the corn protein powder is 44 - 50 parts and the concentration of the mixed solution a is 8% - 12%.

[0020] Preferably, the catalyst in step A1 is ferric chloride.

[0021] Preferably, the oil phase in step A2 is any one of triglyceride caprate, triglyceride caproate and triglyceride caprylate.

[0022] Preferably, the co-surfactant described in step A2 is any one of PEG-2000 and sodium oleate.

[0023] Preferably, the ultrasonic power in step A2 is 300-600W, and the ultrasonic time is 5-15min.

[0024] Preferably, the protective agent is obtained by mixing trehalose and glucose in a mass ratio of 2-5:1-3, and the concentration of the protective agent is 6wt%-10wt%.

[0025] Preferably, the preparation process of the multi-layered microemulsion is as follows:

[0026] Step B1: Mix the prepared plant small molecule organic selenium solution with polysorbate-80, stir evenly at 30-40°C for 15-30min to fully dissolve and disperse the plant small molecule organic selenium, and form a stable inner layer solution;

[0027] Step B2: Disperse the probiotics in the protective agent, add curcumin, and stir at 25-30°C at a speed of 100-200rpm for 20-40min to fully mix each component and obtain an intermediate layer mixture;

[0028] Step B3: Add corn protein powder to ultrapure water to prepare a mixed solution a with a concentration of 8%-12%, adjust the pH of the mixed solution a to 8-9, add glutaraldehyde, and the dosage of glutaraldehyde is 0.1%-0.5% of the mass of corn protein. Place the mixed solution a in a constant temperature water bath, heat it to 70-85°C, stir at 200-400rpm for 40-60min to fully unfold the corn protein and react with glutaraldehyde for modification. Then quickly cool the heat-denatured solution to 25-30°C, dilute the concentration to 3wt%-6wt% with ultrapure water, and filter with a 0.45μm membrane to remove undissolved protein aggregates to obtain a uniform and transparent modified corn protein aqueous solution, denoted as mixed solution b;

[0029] Step B4: Drop the inner layer solution into the intermediate layer mixture, ultrasonicate at an ultrasonic power of 300-600W for 5-15min to form composite droplets with the plant small molecule organic selenium in the inner layer and probiotics, curcumin and part of the protective agent in the intermediate layer. Then slowly add the composite droplets into the mixed solution b, and at the same time add the co-surfactant and the oil phase, ultrasonicate at an ultrasonic power of 300-600W for 10-20min, and finally homogenize under a pressure of 800-1200bar to obtain the adjuvant.

[0030] Preferably, the concentration of the plant small molecule organic selenium solution in step B1 is 0.8 - 2.0 mg / mL, and the polysorbate - 80 in step B1 is 1% - 3% of the mass of the plant small molecule organic selenium solution.

[0031] The protective agent in step B2 is obtained by mixing trehalose and glucose in a mass ratio of 2 - 5:1 - 3, and the concentration of the protective agent is 6wt% - 10wt%.

[0032] A preparation method of a composite probiotic preparation rich in organic selenium, comprising the following steps:

[0033] Disperse the probiotics in the protective agent, then add plant small molecule organic selenium, oil phase and co - surfactant, and ultrasonicate for 5 - 15 min at a power of 300 - 600 W to obtain a probiotic - selenium composite microemulsion. Homogenize the composite microemulsion under a pressure of 800 - 1200 kDa, pre - freeze at - 80 °C and then freeze - dry to obtain a composite probiotic preparation rich in organic selenium.

[0034] Advantages of the present invention:

[0035] The present invention provides a composite probiotic preparation rich in organic selenium and its preparation method. The provided plant small molecule organic selenium has the characteristics of stable physical and chemical properties and high bioavailability. When diluted with water, its molecular weight is less than 200 Daltons, with a clear structure, high purity (100% organic selenium), and the average particle size of the microemulsion formed by dispersing in water is 200 - 500 nm emulsion, which greatly improves the dissolution rate of Cardamine hupingshanensis and its stability in an aqueous solution atmosphere. When orally administered and diffused through the digestive tract to the stomach, the zein in the aqueous phase can be digested by pepsin, while the oil phase can protect the internal Cardamine hupingshanensis from the erosion of gastric juice. Subsequently, in the weakly alkaline intestinal juice, the oil phase releases the content through pancreatic enzyme hydrolysis, and Cardamine hupingshanensis can be more absorbed by intestinal epithelial cells, thus greatly improving its bioavailability.

[0036] The plant small molecule organic selenium provided by the present invention improves the deficiencies in the production, storage and use of liquid dosage forms and soft capsules, significantly improves the oral bioavailability of selenium elements and high - selenium corn flour, and the preparation process is simple and feasible, providing a feasible way for industrial production. Specific embodiments

[0037] To make the purpose, technical solutions and advantages of the present invention clearer, the following further elaborates on the present invention with specific embodiments.

[0038] Example 1: A preparation method of a composite probiotic preparation rich in organic selenium, comprising the following steps:

[0039] S1. Mix 0.45 g of Cardamine hupingshanensis, 0.45 g of high - selenium corn flour and 25 g of ultrapure water evenly, adjust the pH to 7.5, then add 0.022 g of ferric chloride at 70 °C, react for 4 h through the HLUP technology, and dialyze in a dialysis bag with a molecular weight cut - off of 8 kDa to obtain plant small - molecule organic selenium;

[0040] S2. Mix Bifidobacterium, Lactobacillus and Bacillus coagulans in a mass ratio of 2:1:1 to obtain probiotics, where the viable count of Bifidobacterium ≥ 5×10 10 CFU / g, the viable count of Lactobacillus ≥ 2.5×10 10 CFU / g, and the viable count of Bacillus coagulans ≥ 2.5×10 10 CFU / g;

[0041] S3. Add 44 g of corn protein powder to ultrapure water to prepare a mixed solution a with a concentration of 8%, adjust the pH of the mixed solution a to 8, place the mixed solution a in a constant - temperature water bath, heat it to 70 °C, stir at 200 rpm for 40 min to fully unfold the corn protein, expose the hydrophobic groups, and at the same time partially hydrolyze some peptide chains, reducing the molecular weight. When the temperature > 70 °C, the hydrogen bonds and hydrophobic interactions of corn protein can be effectively destroyed. When the time > 30 min, it ensures sufficient denaturation but avoids excessive hydrolysis leading to a decrease in emulsifying ability. Rapidly cool the heat - denatured solution to 25 °C to terminate the denaturation process, dilute the concentration to 3 wt% with ultrapure water, and filter through a 0.45 - μm membrane to remove undissolved protein aggregates to obtain a homogeneous and transparent heat - denatured corn protein aqueous solution, denoted as mixed solution b. At high temperature and alkaline conditions, the α - helix structure of corn protein unfolds to form a β - sheet or random coil conformation, exposing the hydrophobic region, enhancing its ability to adsorb at the oil - water interface. The surface hydrophobicity of the denatured corn protein increases, and the interfacial tension decreases, which is conducive to the formation of a stable microemulsion structure. When the temperature exceeds 90 °C, it may cause excessive protein aggregation and reduce the emulsification efficiency;

[0042] S4. Uniformly mix the mixed solution b, triglyceride caprate and PEG - 2000, and form an emulsion by ultrasonic treatment at 300 W for 5 min to obtain a water - in - oil - in - water emulsion;

[0043] S5. Mix trehalose and glucose in a mass ratio of 2:1 to prepare a protective agent with a concentration of 6 wt%;

[0044] S6. Uniformly mix the prepared plant small - molecule organic selenium solution with a concentration of 0.8 mg / mL and polysorbate - 80, where polysorbate - 80 is 1% of the mass of the plant small - molecule organic selenium solution, and stir at 30 °C for 15 min to fully dissolve and disperse the plant small - molecule organic selenium to form a stable inner layer solution;

[0045] S7. Disperse the probiotics in the protective agent, add curcumin, and stir at a speed of 100 rpm at 25 °C for 20 min to fully mix all components and obtain an intermediate layer mixed solution;

[0046] S8. Add corn protein powder to ultrapure water to prepare a mixed solution a with a concentration of 8%, adjust the pH of the mixed solution a to 8, add glutaraldehyde, and the dosage of glutaraldehyde is 0.1% of the mass of the corn protein. Place the mixed solution a in a constant temperature water bath, heat to 70 °C, stir at 200 rpm for 40 min to fully unfold the corn protein and react with glutaraldehyde to achieve modification. Then quickly cool the heat-denatured solution to 25 °C, dilute the concentration to 3 wt% with ultrapure water, and filter with a 0.45 μm membrane to remove undissolved protein aggregates to obtain a homogeneous and transparent modified corn protein aqueous solution, denoted as mixed solution b;

[0047] S9. Drop the inner layer solution into the intermediate layer mixed solution and ultrasonicate at an ultrasonic power of 300 W for 5 min to form composite liquid droplets with plant small molecule organic selenium in the inner layer and probiotics, curcumin and protective agent in the intermediate layer. The protective agent is a mixture of trehalose and glucose with a mass ratio of 2:1, and the concentration of the protective agent is 6 wt%. Then slowly add the composite liquid droplets to the mixed solution b, and at the same time add a co-surfactant and an oil phase, ultrasonicate at an ultrasonic power of 300 W for 10 min, and finally homogenize at a pressure of 800 bar to obtain an additive;

[0048] S10. Mix plant small molecule organic selenium and probiotics in a mass ratio of 0.5:10 to obtain a core active component, mix the water-in-oil-in-water microemulsion and the protective agent in a mass ratio of 70:6 to obtain a carrier system, mix 19 g of the core active component, 25 g of the carrier system and 8 g of the additive, ultrasonicate at a power of 300 W for 5 min to obtain a probiotic-selenium composite microemulsion, homogenize the composite microemulsion at a pressure of 800 kDa, pre-freeze at -80 °C and then freeze-dry to obtain a composite probiotic preparation rich in organic selenium.

[0049] Example 2: A preparation method of a composite probiotic preparation rich in organic selenium, comprising the following steps:

[0050] S1. Mix 0.55 g of Cardamine hupingshanensis, 0.55 g of high-selenium corn flour and 30 g of ultrapure water evenly, adjust the pH to 8, and then add 0.025 g of ferric chloride at 75 °C, react through the HLUP technology for 4.5 h, and dialyze in a dialysis bag with a molecular weight cut-off of 9 kDa to obtain plant small molecule organic selenium;

[0051] S2. Mix Bifidobacterium, Lactobacillus and Bacillus coagulans in a mass ratio of 2:1:1 to obtain probiotics, and the viable count of Bifidobacterium ≥ 5×10 10CFU / g, the viable count of Lactobacillus ≥ 2.5×10 10 CFU / g, the viable count of Bacillus coagulans ≥ 2.5×10 10 CFU / g;

[0052] S3. Add 45 g of corn protein powder to ultrapure water to prepare a mixed solution a with a concentration of 9%. Adjust the pH of the mixed solution a to 8.5. Place the mixed solution a in a constant temperature water bath and heat it to 74°C. Stir at 240 rpm for 44 min to fully unfold the corn protein, expose the hydrophobic groups, and at the same time partially hydrolyze the peptide chains and reduce the molecular weight. Among them, a temperature > 70°C can effectively destroy the hydrogen bonds and hydrophobic interactions of the corn protein, and a time > 30 min ensures sufficient denaturation but avoids excessive hydrolysis resulting in a decrease in emulsifying ability. Quickly cool the heat-denatured solution to 26°C to terminate the denaturation process. Dilute the concentration to 4 wt% with ultrapure water and filter through a 0.45 μm membrane to remove undissolved protein aggregates to obtain a homogeneous and transparent heat-denatured corn protein aqueous solution, denoted as mixed solution b. At high alkaline temperatures, the α-helix structure of corn protein unfolds to form a β-sheet or random coil conformation, and the hydrophobic region is exposed, enhancing its ability to adsorb at the oil-water interface. The surface hydrophobicity of the denatured corn protein increases, and the interfacial tension decreases, which is conducive to the formation of a stable microemulsion structure. A temperature exceeding 90°C may cause excessive protein aggregation and reduce the emulsification efficiency;

[0053] S4. Uniformly mix the mixed solution b, triglyceride caproate, and sodium oleate, and form an emulsion by ultrasonic treatment at 350 W for 7 min to obtain a water-in-oil-in-water emulsion;

[0054] S5. Mix trehalose and glucose in a mass ratio of 3:2 to prepare a protective agent with a concentration of 7 wt%;

[0055] S6. Uniformly mix the prepared plant small molecule organic selenium solution with a concentration of 1.0 mg / mL and polysorbate-80, where polysorbate-80 is 1.4% of the mass of the plant small molecule organic selenium solution. Stir at 32°C for 18 min to fully dissolve and disperse the plant small molecule organic selenium to form a stable inner layer solution;

[0056] S7. Disperse the probiotics in the protective agent, add curcumin, and stir at a speed of 120 rpm at 26°C for 24 min to fully mix all components to obtain an intermediate layer mixed solution;

[0057] S8. Add corn protein powder to ultrapure water to prepare a mixed solution a with a concentration of 9%, adjust the pH of the mixed solution a to 8.5, add glutaraldehyde, and the dosage of glutaraldehyde is 0.2% of the mass of corn protein. Place the mixed solution a in a constant temperature water bath, heat it to 73°C, stir it at 240 rpm for 44 min to fully unfold the corn protein and react with glutaraldehyde to achieve modification. Then quickly cool the heat-denatured solution to 26°C, dilute the concentration to 4 wt% with ultrapure water, and filter it through a 0.45 μm membrane to remove undissolved protein aggregates to obtain a uniform and transparent modified corn protein aqueous solution, denoted as mixed solution b;

[0058] S9. Drop the inner layer solution into the middle layer mixed solution, ultrasonicate it at an ultrasonic power of 350 W for 7 min to form composite liquid droplets with plant small molecule organic selenium in the inner layer, probiotics, curcumin, and a protective agent in the middle layer. The protective agent is obtained by mixing trehalose and glucose in a mass ratio of 3:2, and the concentration of the protective agent is 7 wt%. Then slowly add the composite liquid droplets into the mixed solution b, simultaneously add a co-surfactant and an oil phase, ultrasonicate it at an ultrasonic power of 350 W for 14 min, and finally homogenize it at a pressure of 880 bar to obtain an additive;

[0059] S10. Mix plant small molecule organic selenium and probiotics in a mass ratio of 1.5:20 to obtain a core active component, mix the water-in-oil-in-water microemulsion and the protective agent in a mass ratio of 73:7 to obtain a carrier system, mix 29 g of the core active component, 28 g of the carrier system, and 8.2 g of the additive, ultrasonicate it at a power of 350 W for 7 min to obtain a probiotic-selenium composite microemulsion, homogenize the composite microemulsion at a pressure of 880 kDa, pre-freeze it at -80°C, and then freeze-dry it to obtain a composite probiotic preparation rich in organic selenium.

[0060] Example 3: A preparation method of a composite probiotic preparation rich in organic selenium, comprising the following steps:

[0061] S1. Mix 0.65 g of Cardamine hupingshanensis, 0.65 g of high-selenium corn flour, and 35 g of ultrapure water evenly, adjust the pH to 8.5, then add 0.028 g of ferric chloride at 80°C, react for 5 h through HLUP technology, and dialyze it in a dialysis bag with a molecular weight cut-off of 10 kDa to obtain plant small molecule organic selenium;

[0062] S2. Mix Bifidobacterium, Lactobacillus, and Bacillus coagulans in a mass ratio of 2:1:1 to obtain probiotics, where the viable count of Bifidobacterium ≥ 5×10 10 CFU / g, the viable count of Lactobacillus ≥ 2.5×10 10 CFU / g, and the viable count of Bacillus coagulans ≥ 2.5×10 10 CFU / g;

[0063] S3. Add 46 g of corn protein powder to ultrapure water to prepare a mixed solution a with a concentration of 10%. Adjust the pH of the mixed solution a to 9. Place the mixed solution a in a constant temperature water bath, heat it to 78 °C, and stir it at 280 rpm for 48 min to fully unfold the corn protein, expose the hydrophobic groups, and at the same time partially hydrolyze the peptide chains and reduce the molecular weight. Among them, a temperature > 70 °C can effectively break the hydrogen bonds and hydrophobic interactions of the corn protein, and a time > 30 min ensures sufficient denaturation but avoids excessive hydrolysis resulting in a decrease in emulsifying ability. Quickly cool the heat-denatured solution to 27 °C to terminate the denaturation process, dilute the concentration to 5 wt% with ultrapure water, and filter with a 0.45 μm membrane to remove undissolved protein aggregates to obtain a homogeneous and transparent heat-denatured corn protein aqueous solution, denoted as mixed solution b. At high alkaline temperatures, the α-helix structure of corn protein unfolds to form a β-sheet or random coil conformation, and the hydrophobic region is exposed, enhancing its ability to adsorb at the oil-water interface. The surface hydrophobicity of the denatured corn protein increases and the interfacial tension decreases, which is beneficial to the formation of a stable microemulsion structure. A temperature exceeding 90 °C may cause excessive protein aggregation and a decrease in emulsification efficiency;

[0064] S4. Uniformly mix the mixed solution b, glyceryl trioctanoate, and PEG - 2000, and sonicate at 400 W for 9 min to form an emulsion, obtaining a water-in-oil-in-water emulsion;

[0065] S5. Mix trehalose and glucose in a mass ratio of 4:3 to prepare a protective agent with a concentration of 8 wt%;

[0066] S6. Uniformly mix the prepared plant small molecule organic selenium solution with a concentration of 1.2 mg / mL and polysorbate - 80, where polysorbate - 80 is 1.8% of the mass of the plant small molecule organic selenium solution, and stir at 34 °C for 21 min to fully dissolve and disperse the plant small molecule organic selenium to form a stable inner layer solution;

[0067] S7. Disperse the probiotics in the protective agent, add curcumin, and stir at a speed of 140 rpm at 27 °C for 28 min to fully mix each component to obtain an intermediate layer mixed solution;

[0068] S8. Add corn protein powder to ultrapure water to prepare a mixed solution a with a concentration of 10%. Adjust the pH of the mixed solution a to 9, add glutaraldehyde, and the dosage of glutaraldehyde is 0.3% of the mass of the corn protein. Place the mixed solution a in a constant temperature water bath, heat it to 76 °C, and stir it at 280 rpm for 48 min to fully unfold the corn protein and react with glutaraldehyde to achieve modification. Then quickly cool the heat-denatured solution to 27 °C, dilute the concentration to 5 wt% with ultrapure water, and filter with a 0.45 μm membrane to remove undissolved protein aggregates to obtain a homogeneous and transparent modified corn protein aqueous solution, denoted as mixed solution b;

[0069] S9. Drop the inner layer solution into the middle layer mixture, and ultrasonicate for 9 min at an ultrasonic power of 400 W to form composite droplets with plant small molecule organic selenium in the inner layer, probiotics, curcumin and a protective agent in the middle layer. The protective agent is obtained by mixing trehalose and glucose in a mass ratio of 4:3, and the concentration of the protective agent is 8 wt%. Then slowly add the composite droplets into mixture b, and at the same time add a co-surfactant and an oil phase, ultrasonicate for 18 min at an ultrasonic power of 400 W, and finally perform high-pressure homogenization at a pressure of 960 bar to obtain an auxiliary agent.

[0070] S10. Mix plant small molecule organic selenium and probiotics in a mass ratio of 2.5:30 to obtain a core active component, mix the water-in-oil-in-water microemulsion and the protective agent in a mass ratio of 76:8 to obtain a carrier system, mix 39 g of the core active component, 31 g of the carrier system and 8.5 g of the auxiliary agent, ultrasonicate for 9 min at a power of 400 W to obtain a probiotic-selenium composite microemulsion, homogenize the composite microemulsion at a pressure of 960 kDa, pre-freeze at -80 °C and then freeze-dry to obtain a composite probiotic preparation rich in organic selenium.

[0071] Example 4: A preparation method of a composite probiotic preparation rich in organic selenium, comprising the following steps:

[0072] S1. Mix 0.75 g of Cardamine hupingshanensis, 0.75 g of high-selenium corn flour and 40 g of ultrapure water evenly, adjust the pH to 7.5, then add 0.030 g of ferric chloride at 85 °C, react for 4 h through the HLUP technology, and dialyze in a dialysis bag with a molecular weight cut-off of 11 kDa to obtain plant small molecule organic selenium.

[0073] S2. Mix Bifidobacterium, Lactobacillus and Bacillus coagulans in a mass ratio of 2:1:1 to obtain probiotics, wherein the viable count of Bifidobacterium is ≥5×10 10 CFU / g, the viable count of Lactobacillus is ≥2.5×10 10 CFU / g, and the viable count of Bacillus coagulans is ≥2.5×10 10 CFU / g;

[0074] S3. Add 47 g of corn protein powder to ultrapure water to prepare a mixture a with a concentration of 11%. Adjust the pH of mixture a to 8. Place mixture a in a constant temperature water bath and heat it to 82 °C. Stir at 320 rpm for 52 min to fully unfold the corn protein, expose the hydrophobic groups, and at the same time partially hydrolyze the peptide chains and reduce the molecular weight. Among them, a temperature > 70 °C can effectively break the hydrogen bonds and hydrophobic interactions of corn protein, and a time > 30 min ensures sufficient denaturation but avoids excessive hydrolysis resulting in a decrease in emulsifying ability. Quickly cool the heat-denatured solution to 28 °C to terminate the denaturation process. Dilute the concentration to 3 wt% with ultrapure water and filter through a 0.45 μm membrane to remove undissolved protein aggregates to obtain a homogeneous and transparent heat-denatured corn protein aqueous solution, denoted as mixture b. At high alkaline temperatures, the α-helix structure of corn protein unfolds to form a β-sheet or random coil conformation, and the hydrophobic region is exposed, enhancing its ability to adsorb at the oil-water interface. The surface hydrophobicity of the denatured corn protein increases and the interfacial tension decreases, which is beneficial to the formation of a stable microemulsion structure. A temperature exceeding 90 °C may cause excessive protein aggregation and reduce the emulsification efficiency;

[0075] S4. Uniformly mix mixture b, tricaprylin, and sodium oleate and emulsify them by ultrasonic treatment at 450 W for 11 min to obtain a water-in-oil-in-water emulsion;

[0076] S6. Mix trehalose and glucose in a mass ratio of 3:2 to prepare a protective agent with a concentration of 9 wt%;

[0077] S9. Uniformly mix the prepared plant small molecule organic selenium solution with a concentration of 1.5 mg / mL and polysorbate-80, where polysorbate-80 is 2.2% of the mass of the plant small molecule organic selenium solution. Stir at 36 °C for 24 min to fully dissolve and disperse the plant small molecule organic selenium to form a stable inner layer solution;

[0078] S7. Disperse the probiotics in the protective agent, add curcumin, and stir at 28 °C at a speed of 160 rpm for 32 min to fully mix all components to obtain an intermediate layer mixture;

[0079] S8. Add corn protein powder to ultrapure water to prepare a mixture a with a concentration of 11%. Adjust the pH of mixture a to 8, add glutaraldehyde, and the dosage of glutaraldehyde is 0.4% of the mass of the corn protein. Place mixture a in a constant temperature water bath and heat it to 79 °C. Stir at 320 rpm for 52 min to fully unfold the corn protein and react with glutaraldehyde to achieve modification. Then quickly cool the heat-denatured solution to 28 °C, dilute the concentration to 3 wt% with ultrapure water, and filter through a 0.45 μm membrane to remove undissolved protein aggregates to obtain a homogeneous and transparent modified corn protein aqueous solution, denoted as mixture b;

[0080] S9. Drop the inner layer solution into the middle layer mixture, and ultrasonicate it for 11 min at an ultrasonic power of 450 W to form composite droplets with plant small molecule organic selenium in the inner layer, probiotics, curcumin and a protective agent in the middle layer. The protective agent is obtained by mixing trehalose and glucose in a mass ratio of 3:2, and the concentration of the protective agent is 9 wt%. Then, slowly add the composite droplets into mixture b, and simultaneously add a co-surfactant and an oil phase, ultrasonicate it for 22 min at an ultrasonic power of 450 W, and finally perform high-pressure homogenization at a pressure of 1020 bar to obtain an adjuvant.

[0081] S10. Mix plant small molecule organic selenium and probiotics in a mass ratio of 3.5:40 to obtain a core active component, mix the water-in-oil-in-water microemulsion and the protective agent in a mass ratio of 79:9 to obtain a carrier system, mix 35 g of the core active component, 34 g of the carrier system and 8.7 g of the adjuvant, ultrasonicate it for 11 min at a power of 450 W to obtain a probiotic-selenium composite microemulsion, homogenize the composite microemulsion at a pressure of 1020 kDa, pre-freeze it at -80 °C and then freeze-dry it to obtain a composite probiotic preparation rich in organic selenium.

[0082] Example 5: A preparation method of a composite probiotic preparation rich in organic selenium, comprising the following steps:

[0083] S1. Mix 0.85 g of Cardamine hupingshanensis, 0.85 g of high-selenium corn flour and 45 g of ultrapure water evenly, adjust the pH to 8, then add 0.032 g of ferric chloride at 90 °C, react for 4.5 h through HLUP technology, and dialyze it in a dialysis bag with a molecular weight cut-off of 12 kDa to obtain plant small molecule organic selenium.

[0084] S2. Mix Bifidobacterium, Lactobacillus and Bacillus coagulans in a mass ratio of 2:1:1 to obtain probiotics, wherein the viable count of Bifidobacterium ≥ 5×10 10 CFU / g, the viable count of Lactobacillus ≥ 2.5×10 10 CFU / g, and the viable count of Bacillus coagulans ≥ 2.5×10 10 CFU / g;

[0085] S3. Add 48 g of corn protein powder to ultrapure water to prepare a mixture a with a concentration of 10%. Adjust the pH of mixture a to 8.5. Place mixture a in a constant temperature water bath and heat it to 83 °C. Stir at 360 rpm for 56 min to fully unfold the corn protein, expose the hydrophobic groups, and at the same time partially hydrolyze the peptide chains and reduce the molecular weight. Among them, when the temperature > 70 °C, the hydrogen bonds and hydrophobic interactions of corn protein can be effectively destroyed, and the time > 30 min ensures sufficient denaturation but avoids excessive hydrolysis leading to a decrease in emulsifying ability. Quickly cool the heat-denatured solution to 29 °C to terminate the denaturation process. Dilute the concentration to 4 wt% with ultrapure water and filter through a 0.45 μm membrane to remove undissolved protein aggregates to obtain a homogeneous and transparent heat-denatured corn protein aqueous solution, denoted as mixture b. At high alkaline temperatures, the α-helix structure of corn protein unfolds to form a β-sheet or random coil conformation, and the hydrophobic region is exposed, enhancing its ability to adsorb at the oil-water interface. The surface hydrophobicity of the denatured corn protein increases and the interfacial tension decreases, which is conducive to the formation of a stable microemulsion structure. When the temperature exceeds 90 °C, it may cause excessive protein aggregation and reduce the emulsification efficiency;

[0086] S4. Uniformly mix mixture b, glyceryl caprylate, and sodium oleate, and ultrasonicate at 500 W for 13 min to form an emulsion, obtaining a water-in-oil-in-water emulsion;

[0087] S6. Mix trehalose and glucose in a mass ratio of 4:1 to prepare a protective agent with a concentration of 8 wt%;

[0088] S9. Mix the prepared plant small molecule organic selenium solution with a concentration of 1.8 mg / mL and polysorbate-80 uniformly. Among them, polysorbate-80 is 2.6% of the mass of the plant small molecule organic selenium solution. Stir at 38 °C for 27 min to fully dissolve and disperse the plant small molecule organic selenium to form a stable inner layer solution;

[0089] S7. Disperse the probiotics in the protective agent, add curcumin, and stir at 29 °C at a speed of 180 rpm for 36 min to fully mix each component to obtain an intermediate layer mixture;

[0090] S8. Add corn protein powder to ultrapure water to prepare a mixture a with a concentration of 10%. Adjust the pH of mixture a to 8.5, add glutaraldehyde, and the dosage of glutaraldehyde is 0.2% of the mass of corn protein. Place mixture a in a constant temperature water bath and heat it to 82 °C. Stir at 360 rpm for 56 min to fully unfold the corn protein and react with glutaraldehyde to achieve modification. Then quickly cool the heat-denatured solution to 29 °C, dilute the concentration to 4 wt% with ultrapure water, and filter through a 0.45 μm membrane to remove undissolved protein aggregates to obtain a homogeneous and transparent modified corn protein aqueous solution, denoted as mixture b;

[0091] S9. Drop the inner layer solution into the middle layer mixture and sonicate it for 13 min at a sonication power of 500 W to form composite droplets with plant small molecule organic selenium in the inner layer, probiotics, curcumin, and a protective agent in the middle layer. The protective agent is obtained by mixing trehalose and glucose in a mass ratio of 4:1, and the concentration of the protective agent is 6 wt%. Then, slowly add the composite droplets into mixture b, and simultaneously add a co-surfactant and an oil phase. Sonicate it for 26 min at a sonication power of 500 W, and finally perform high-pressure homogenization at a pressure of 1080 bar to obtain an adjuvant.

[0092] S10. Mix plant small molecule organic selenium and probiotics in a mass ratio of 4.5:45 to obtain a core active component. Mix the water-in-oil-in-water microemulsion and the protective agent in a mass ratio of 82:8 to obtain a carrier system. Mix 45 g of the core active component, 37 g of the carrier system, and 8.9 g of the adjuvant, and sonicate it for 13 min at a power of 500 W to obtain a probiotic-selenium composite microemulsion. Homogenize the composite microemulsion at a pressure of 1080 kDa, pre-freeze it at -80 °C, and then perform freeze-drying to obtain a composite probiotic preparation rich in organic selenium.

[0093] Example 6: A preparation method of a composite probiotic preparation rich in organic selenium, comprising the following steps:

[0094] S1. Mix 1.1 g of Cardamine hupingshanensis, 1.1 g of high-selenium corn flour, and 53 g of ultrapure water evenly, adjust the pH to 8.5, and then add 0.033 g of ferric chloride at 95 °C. React for 5 h through the HLUP technology, and dialyze it in a dialysis bag with a molecular weight cut-off of 14 kDa to obtain plant small molecule organic selenium.

[0095] S2. Mix Bifidobacterium, Lactobacillus, and Bacillus coagulans in a mass ratio of 2:1:1 to obtain probiotics, where the viable count of Bifidobacterium ≥ 5×10 10 CFU / g, the viable count of Lactobacillus ≥ 2.5×10 10 CFU / g, and the viable count of Bacillus coagulans ≥ 2.5×10 10 CFU / g;

[0096] S3. Add 50 g of corn protein powder to ultrapure water to prepare a mixed solution a with a concentration of 12%. Adjust the pH of the mixed solution a to 9. Place the mixed solution a in a constant temperature water bath, heat it to 85 °C, and stir it at 400 rpm for 60 min to fully unfold the corn protein, expose the hydrophobic groups, and at the same time partially hydrolyze the peptide chains and reduce the molecular weight. Among them, a temperature > 70 °C can effectively break the hydrogen bonds and hydrophobic interactions of corn protein, and a time > 30 min ensures sufficient denaturation but avoids excessive hydrolysis leading to a decrease in emulsifying ability. Quickly cool the heat-denatured solution to 30 °C to terminate the denaturation process, dilute the concentration to 6 wt% with ultrapure water, and filter through a 0.45 μm membrane to remove undissolved protein aggregates to obtain a homogeneous and transparent heat-denatured corn protein aqueous solution, denoted as mixed solution b. At high temperature and alkaline conditions, the α-helix structure of corn protein unfolds to form a β-sheet or random coil conformation, and the hydrophobic region is exposed, enhancing its ability to adsorb at the oil-water interface. The surface hydrophobicity of the denatured corn protein increases and the interfacial tension decreases, which is conducive to the formation of a stable microemulsion structure. A temperature exceeding 90 °C may cause excessive protein aggregation and reduce the emulsification efficiency;

[0097] S4. Uniformly mix the mixed solution b, triglyceride caprylate, and sodium oleate, and ultrasonicate at 600 W for 15 min to form an emulsion, obtaining a water-in-oil-in-water emulsion;

[0098] S6. Mix trehalose and glucose in a mass ratio of 5:3 to prepare a protective agent with a concentration of 10 wt%;

[0099] S9. Uniformly mix the prepared plant small molecule organic selenium solution with a concentration of 2.0 mg / mL and polysorbate-80, where polysorbate-80 is 3% of the mass of the plant small molecule organic selenium solution, and stir at 40 °C for 30 min to fully dissolve and disperse the plant small molecule organic selenium to form a stable inner layer solution;

[0100] S12. Disperse the probiotics in the protective agent, add curcumin, and stir at 30 °C at a speed of 200 rpm for 40 min to fully mix all components to obtain an intermediate layer mixed solution;

[0101] S15. Add corn protein powder to ultrapure water to prepare a mixed solution a with a concentration of 12%. Adjust the pH of the mixed solution a to 9, add glutaraldehyde, and the dosage of glutaraldehyde is 0.5% of the mass of corn protein. Place the mixed solution a in a constant temperature water bath, heat it to 85 °C, and stir it at 400 rpm for 60 min to fully unfold the corn protein and react with glutaraldehyde to achieve modification. Then quickly cool the heat-denatured solution to 30 °C, dilute the concentration to 6 wt% with ultrapure water, and filter through a 0.45 μm membrane to remove undissolved protein aggregates to obtain a homogeneous and transparent modified corn protein aqueous solution, denoted as mixed solution b;

[0102] S9. Drop the inner layer solution into the middle layer mixture, and ultrasonicate it for 15 min at an ultrasonic power of 600 W to form composite droplets with plant small molecule organic selenium in the inner layer, probiotics, curcumin, and a protective agent in the middle layer. The protective agent is obtained by mixing trehalose and glucose in a mass ratio of 5:3, and the concentration of the protective agent is 10 wt%. Then, slowly add the composite droplets into mixture b, and simultaneously add a co-surfactant and an oil phase, ultrasonicate it for 20 min at an ultrasonic power of 600 W, and finally perform high-pressure homogenization at a pressure of 1200 bar to obtain an auxiliary agent.

[0103] S10. Mix plant small molecule organic selenium and probiotics in a mass ratio of 5:50 to obtain a core active component, mix the water-in-oil-in-water microemulsion and the protective agent in a mass ratio of 85:10 to obtain a carrier system, mix 49 g of the core active component, 25 - 28 - 31 - 34 - 37 - 40 g of the carrier system, and 9.3 g of the auxiliary agent, ultrasonicate it for 15 min at a power of 600 W to obtain a probiotic-selenium composite microemulsion, homogenize the composite microemulsion at a pressure of 1200 kDa, pre-freeze it at -80 °C, and then perform freeze-drying to obtain a composite probiotic preparation rich in organic selenium.

[0104] Comparative Example 1:

[0105] In this comparative example, compared with Example 1, no auxiliary agent was added during the preparation process of the composite probiotic preparation rich in organic selenium, and the remaining steps and parameters were the same. This comparative example will not be repeated here, and finally, a composite probiotic preparation rich in organic selenium was obtained.

[0106] Comparative Example 2:

[0107] In this comparative example, compared with Example 1, only plant small molecule organic selenium was replaced with ordinary inorganic selenium (sodium selenite), and the remaining steps and parameters were the same. This comparative example will not be repeated here, and finally, a composite probiotic preparation containing inorganic selenium was obtained.

[0108] Comparative Example 3:

[0109] In this comparative example, compared with Example 1, instead of using the water-in-oil-in-water microemulsion as the carrier system, plant small molecule organic selenium, probiotics, the protective agent, etc. were simply mixed, and the remaining steps and parameters were the same. This comparative example will not be repeated here, and finally, a simply mixed composite preparation was obtained.

[0110] Comparative Example 4:

[0111] In this comparative example, compared with Example 1, the corn protein was not modified (i.e., glutaraldehyde was not added), and the remaining steps and parameters were the same. This comparative example will not be repeated here, and finally, a composite probiotic preparation based on unmodified corn protein microemulsion was obtained.

[0112] Comparative Example 5:

[0113] In this comparative example, compared with Example 1, curcumin in the formula was removed, and the remaining steps and parameters were the same. This comparative example will not be repeated here. Finally, a compound probiotic preparation without curcumin was obtained.

[0114] Performance test: The samples prepared in Examples 1 - 6 and Comparative Examples 1 - 5 were subjected to the following performance tests:

[0115] 1. Probiotic survival rate: Referring to the "Regulations on the Declaration and Review of Probiotic Health Foods (Trial)", the preparation was treated in simulated gastric juice (pH 1.5, containing 0.3% pepsin) for 2 h, and then transferred to simulated intestinal juice (pH 7.5, containing 0.1% trypsin) for 4 h. The viable count was determined by the plate counting method, and the survival rate (%) was calculated.

[0116] 2. Selenium bioavailability: A rat gavage experiment was used to measure the serum selenium concentration (μg / mL). SD rats (n = 6) were continuously gavaged with the preparation for 7 days, and blood was collected 24 h after the last administration. The serum selenium content was detected by inductively coupled plasma mass spectrometry (ICP-MS).

[0117] 3. Antioxidant capacity: DPPH free radical scavenging rate (%). The preparation solution (1 mg / mL) was mixed with DPPH ethanol solution, and the reaction was carried out in the dark for 30 min. The absorbance at 517 nm was measured, and the scavenging rate was calculated.

[0118] 4. Immunomodulatory function: Macrophage phagocytosis index (%). After culturing RAW264.7 cells, the preparation was added and co-incubated for 24 h, and the phagocytic activity was measured by the neutral red staining method.

[0119] 5. Stability test: Particle size distribution: The average particle size (nm) was measured by dynamic light scattering (DLS), Zeta potential: The surface charge (mV) was measured by DLS, and long-term storage: The preparation was stored at 4°C for 30 days, and the probiotic survival rate was measured.

[0120] The results are shown in Table 1 below:

[0121] Table 1 Performance test results

[0122]

[0123] Data analysis:

[0124] As can be seen from Table 1:

[0125] The survival rate of the examples > 92% (96.2% in Example 6) stems from triple protection: 1. Physical barrier: The outer layer of the W / O / W microemulsion is modified with zein film, which is stable in acidic environment and resists pepsin degradation; 2. Chemical stability: The glassy matrix of trehalose and glucose inhibits ice crystal formation and reduces freeze-drying damage; 3. Steric hindrance: The negative Zeta potential (-32.5 to -38.9 mV) enhances the repulsion between particles and avoids aggregation and inactivation. For Comparative Example 1 (without adjuvant): Lack of protectant and curcumin, ice crystals damage cell membranes during freeze-drying, and the survival rate is only 68.7%; Comparative Example 3 (simple mixing): Without an embedding structure, probiotics are directly exposed to gastric acid, and the survival rate drops sharply to 55.4%;

[0126] Selenium bioavailability and molecular characteristics of small molecule organic selenium: The highest serum selenium concentration in Example 6 reaches 39.5 μg / mL, which is attributed to: Small molecular weight: Plant small molecule organic selenium (<200 Da) does not require metabolic conversion and directly binds to selenoprotein; Targeted release: The microemulsion degrades gradually in the weakly alkaline environment of the small intestine, and precisely releases selenium to the absorption site; Synergistic absorption: After probiotics colonize, they secrete short-chain fatty acids, which promote the active transport of selenium by the intestinal mucosa. For Comparative Example 2 (inorganic selenium): Sodium selenite needs to be reduced to selenocysteine in the liver, with low efficiency and may cause oxidative stress; Comparative Example 3 (simple mixing): There is no synergistic effect between selenium and probiotics, and unembedded selenium is easily combined with food components to form precipitates;

[0127] Mechanism of synergistic enhancement of antioxidant capacity: The highest DPPH scavenging rate in Example 6 is 87.5%, which stems from: Catalytic action of organic selenium: Selenoamino acids, as cofactors of glutathione peroxidase (GPx), directly scavenge free radicals; Multi-target action of curcumin: Inhibit the NF-κB pathway, reduce the release of inflammatory factors, and at the same time chelate metal ions; Probiotic metabolites: Acetic acid produced by Bifidobacterium enhances the activities of antioxidant enzymes (SOD, CAT). For Comparative Example 5 (without curcumin): The scavenging rate is only 68.4%, indicating that curcumin can improve antioxidant properties;

[0128] Mechanism of improvement of phagocytosis index: The phagocytosis index in Example 6 reaches 178%, significantly higher than that of the comparative examples, thanks to: Probiotic cell wall components: Lipoteichoic acid (LTA) of Bifidobacterium activates the TLR2 receptor and promotes macrophage polarization; Epigenetic regulation of curcumin: Inhibit HDAC activity and up-regulate the expression of anti-inflammatory factors such as IL-10; Differentiation of immune cells by selenium: Promote the Th1 / Th2 balance and enhance the killing activity of CD8+ T cells. For Comparative Example 3 (simple mixing): The phagocytosis index is only 98%, because the probiotics are not embedded and are inactivated prematurely in the gastrointestinal tract, unable to effectively activate the immune response;

[0129] Stability and Interfacial Chemistry Regulation: In Example 6, with a particle size of 158 nm and a Zeta potential of -38.9 mV, the stability is optimal, thanks to: Zein modification: Glutaraldehyde crosslinking forms Schiff base, enhancing the mechanical strength of the film and reducing the Ostwald ripening rate; HLB value matching: PEG-2000 (HLB 14.5) and sodium oleate (HLB 18) synergistically regulate the interfacial tension to form a thermodynamically stable system; High-pressure homogenization effect: The pressure of 1200 bar destroys the aggregates to form a monodisperse microemulsion. Storage stability: The survival rate after storing at 4°C for 30 days is >89% (93.4% in Example 6), because trehalose binds to the protein through hydrogen bonds to maintain its native conformation.

[0130] Molecular Dynamics Optimization of Process Parameters: In Example 6, 600W ultrasound for 15 min + 1200 bar homogenization is adopted to achieve: Cavitation effect: The rupture of microbubbles generated by ultrasound provides energy to promote the dispersion of the oil phase into nanoscale droplets; Shearing force effect: High-pressure homogenization forces the droplets to pass through a narrow slit, further reducing the particle size and making the distribution uniform. Precise regulation of temperature and pH: Zein denatures at 85°C and pH 9, exposing hydrophobic groups, which anchor to the oil droplet interface through hydrophobic interactions to form a dense film layer.

[0131] The present invention uses a water-in-oil-in-water (W / O / W) microemulsion as a carrier system. The outer layer is a modified zein film, the middle layer contains probiotics and protectants, and the inner layer encapsulates organic selenium. This structure can form a physical barrier in the gastrointestinal environment to protect probiotics and organic selenium from being damaged by gastric acid. And the small molecule organic selenium extracted by the HLUP technology used in the present invention has a small molecular weight, a clear structure, and is easily absorbed by the human body. Trehalose in the protectant acts as a vitreous matrix, which can inhibit ice crystal formation and protect the activity of probiotics during freeze-drying and storage. Curcumin, as an antioxidant and immunomodulator, acts synergistically with organic selenium and probiotics to enhance antioxidant and immune functions. Modifying zein by glutaraldehyde crosslinking enhances the strength of the film and the interfacial adsorption ability, improving the stability of the microemulsion. Process steps such as high-pressure homogenization and ultrasonic treatment help to form smaller and uniform microemulsion particle sizes, improving the encapsulation efficiency and stability.

[0132] In summary, through interfacial engineering (zein modification), nano-delivery (W / O / W microemulsion), and component synergy (organic selenium + curcumin + probiotics), the present invention achieves at the molecular level:

[0133] 1. Physical protection: The multi-layer film structure isolates external stimuli;

[0134] 2. Chemical activation: Organic selenium directly participates in enzymatic reactions, and curcumin regulates signal pathways;

[0135] 3. Biological adaptation: pH-responsive release interacts with the intestinal microecology.

[0136] These mechanisms together explain the significant advantages of the examples in the experimental data in terms of survival rate, bioavailability, antioxidant and immunomodulatory aspects.

[0137] Those of ordinary skill in the art should understand that the discussion of any of the above embodiments is only exemplary and is not intended to imply that the scope of the present invention is limited to these examples; under the concept of the present invention, the technical features between the above embodiments or different embodiments can also be combined, the steps can be implemented in any order, and there are many other variations in different aspects of the present invention as described above, which are not provided in detail for the sake of brevity.

[0138] The present invention is intended to cover all such substitutions, modifications and variations that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A compound probiotic preparation rich in organic selenium, characterized in that, It is prepared from the following raw materials in parts by mass: 19 - 49 parts of core active components, 25 - 40 parts of carrier system, and 8 - 9.3 parts of additives; The core active components include 0.5 - 5 parts of plant small molecule organic selenium and 10 - 50 parts of probiotics; The carrier system includes 70 - 85 parts of water-in-oil-in-water microemulsion and 6 - 10 parts of protective agent; The additive is curcumin complex.

2. The composite probiotic preparation rich in organic selenium according to claim 1, wherein The preparation process of the plant small molecule organic selenium is as follows: Mix the Cardamine hupingshanensis, high selenium corn flour and extractant evenly, adjust the pH to 7.5 - 8.5, then add a catalyst at 70 - 95 °C, react for 4 - 5 h through the HLUP technology, and dialyze in a dialysis bag with a molecular weight cut-off of 8 - 14 kDa to obtain the plant small molecule organic selenium.

3. The composite probiotic preparation rich in organic selenium according to claim 2, wherein The mass ratio of the Cardamine hupingshanensis, high selenium corn flour, extractant and catalyst is 0.45 - 1.1:0.45 - 1.1:25 - 53:0.022 - 0.

033. The catalyst is ferric chloride and the extractant is ultrapure water.

4. The compound probiotic preparation rich in organic selenium according to claim 1, characterized in that, The probiotics are obtained by mixing Bifidobacterium, Lactobacillus and Bacillus coagulans in a mass ratio of 2:1:

1. The viable count of Bifidobacterium is ≥5×10 10 CFU / g, the viable count of Lactobacillus is ≥2.5×10 10 CFU / g, and the viable count of Bacillus coagulans is ≥2.5×10 10 CFU / g.

5. The composite probiotic preparation rich in organic selenium according to claim 1, characterized in that, The preparation process of the water-in-oil-in-water microemulsion is as follows: Step A1: Add 44 - 50 parts of corn protein powder to ultrapure water to prepare a mixed solution a with a concentration of 8% - 12%, adjust the pH of the mixed solution a to 8 - 9, place the mixed solution a in a constant temperature water bath, heat to 70 - 85 °C, and stir at 200 - 400 rpm for 40 - 60 min; Step A2: Uniformly mix the mixed solution b, oil phase and co-surfactant, and ultrasonically form an emulsion to obtain a water-in-oil-in-water emulsion.

6. The composite probiotic preparation rich in organic selenium according to claim 5, characterized in that, The dosage of the corn protein powder in Step A1 is 44 - 50 parts; The concentration of the mixed solution a in Step A is 8% - 12%; The oil phase in Step A2 is any one of tricaprylin, trihexanoin, and tricaprin; The co-surfactant in Step A2 is any one of PEG - 2000 and sodium oleate; The ultrasonic power in Step A2 is 300 - 600 W, and the ultrasonic time is 5 - 15 min.

7. The compound probiotic preparation rich in organic selenium according to claim 1, characterized in that, The protective agent is obtained by mixing trehalose and glucose in a mass ratio of 3 - 5:1 - 1.5, and the concentration of the protective agent is 6 wt% - 10 wt%.

8. The composite probiotic preparation rich in organic selenium according to claim 1, wherein The preparation process of the additive is as follows: Step B1: Mix the prepared plant small molecule organic selenium solution with polysorbate - 80 evenly, stir at 30 - 40 °C for 15 - 30 min to fully dissolve and disperse the plant small molecule organic selenium, forming a stable inner layer solution; Step B2: Disperse the probiotics in the protective agent, add curcumin, and stir at 25 - 30 °C at a speed of 100 - 200 rpm for 20 - 40 min to fully mix all components to obtain an intermediate layer mixed solution; Step B3: Add corn protein powder to ultrapure water to prepare a mixed solution a with a concentration of 8%-12%. Adjust the pH of the mixed solution a to 8-9, add glutaraldehyde, place the mixed solution a in a constant temperature water bath, heat it to 70-85°C, stir at 200-400 rpm for 40-60 min, then cool the heat-denatured solution to 25-30°C, dilute the concentration to 3wt%-6wt% with ultrapure water, and filter with a 0.45μm membrane to remove undissolved protein aggregates to obtain a mixed solution b; Step B4: Drop the inner layer solution into the middle layer mixed solution, ultrasonicate at an ultrasonic power of 300-600W for 5-15 min to form composite liquid droplets with plant small molecule organic selenium in the inner layer, probiotics, curcumin and protectant in the middle layer, then add the composite liquid droplets to the mixed solution b, and at the same time add a co-surfactant and an oil phase, ultrasonicate at an ultrasonic power of 300-600W for 10-20 min, and finally homogenize at a pressure of 800-1200 bar to obtain an adjuvant.

9. The compound probiotic preparation rich in organic selenium according to claim 8, characterized in that, In step B1, the concentration of the plant small molecule organic selenium solution is 0.8-2.0 mg / mL; In step B1, the polysorbate-80 is 1%-3% of the mass of the plant small molecule organic selenium solution; In step B2, the protectant is obtained by mixing trehalose and glucose in a mass ratio of 2-5:1-3, and the concentration of the protectant is 6wt%-10wt%; In step B3, the dosage of the corn protein powder is 44-50 parts, and the concentration of the mixed solution a is 8%-12%; In step B3, the dosage of the glutaraldehyde is 0.1%-0.5% of the mass of the corn protein; In step B4, the co-surfactant is any one of PEG-2000 and sodium oleate; In step B4, the dosage of the co-surfactant is 1.3-2.0 parts by mass; In step B4, the oil phase is any one of glyceryl tricaprate, glyceryl trihexanoate, and glyceryl trioctanoate; In step B4, the dosage of the oil phase is 5-10 parts by mass.

10. The preparation method of the compound probiotic preparation rich in organic selenium according to any one of claims 1-9, characterized in that, It includes the following steps: Disperse the probiotics in the protectant, then add the plant small molecule organic selenium, oil phase, adjuvant and co-surfactant, ultrasonicate at a power of 300-600W for 5-15 min to obtain a probiotic-selenium composite microemulsion, homogenize the composite microemulsion at a pressure of 800-1200 kDa, pre-freeze at -80°C and then freeze-dry to obtain a composite probiotic preparation rich in organic selenium.

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