Long-term storage stability flora double-layer capsule and preparation method thereof

Abstract

The invention discloses a flora double-layer capsule with long-term storage stability and a preparation method thereof, and belongs to the technical field of flora double-layer capsules, and the preparation method comprises the following steps: mixing probiotic freeze-dried powder and composite lysozyme, and reacting with alpha-lactalbumin to obtain probiotic-loaded nanoparticles; mixing the nano-particles loaded with the probiotics with sodium alginate to prepare an alginic acid capsule, and carrying out surface modification on the alginic acid capsule by using oxidized starch to obtain a flora single-layer capsule; and mixing the flora single-layer capsule and the hovenia acerba methanol extract to serve as a core material, and taking a chitosan acetic acid solution as a wall material to prepare the flora double-layer capsule. The lysozyme nano-particles can be adhered to the intestinal tract through interaction with mucus, and the lysozyme nano-particles can penetrate through a mucus barrier to be absorbed by epithelial villus, so that the absorption of intestinal flora is improved; the alpha-lactalbumin is coated on the surfaces of the nano-particles, and the alpha-lactalbumin is compounded with the lysozyme nano-particles, so that intestinal mucus can be more effectively penetrated.

Description

Technical Field

[0001] The invention relates to the technical field of bacterial flora double-layer capsules, in particular to a bacterial flora double-layer capsule with long-term storage stability and a preparation method thereof. Background Art

[0002] By establishing a stable and complex microbial flora to restore the damaged intestinal microbiome, the imbalance can be corrected, and the abnormal immune status, the abnormal quantity and activity of vasoactive substances, inflammation and abnormal energy metabolism can be regulated. It has the effect of treating or preventing diseases related to intestinal flora imbalance. There are three main ways to rebuild the intestinal flora: antibiotics, probiotics and flora transplantation. The intestinal flora plays a vital role in enhancing immunity, detoxification, anti-aging, vitamin production, improving food absorption and drug metabolism. Intestinal flora disorders can lead to a rapid proliferation of intestinal pathogens, promoting the occurrence and development of colitis, obesity, diabetes and its complications, cardiovascular disease and other diseases.

[0003] Transplanting the intestinal flora from screened healthy donors to the recipient can restore the balance and diversity of the microbiome, and achieve regulation of abnormal immune status, abnormal quantity and activity of vasoactive substances, inflammation and abnormal energy metabolism, but the intestinal flora has relatively poor resistance to high temperature, acid, stress, and phage infection, and is easily inactivated. In addition, when passing through gastric juice, the intestinal flora is easily inactivated by the acid, digestive enzymes, and antibiotics in the gastric juice and cannot reach the intestine smoothly, affecting the efficacy of the intestinal flora. Microencapsulation of the intestinal flora can effectively improve the activity of the intestinal flora, but the single-layer microcapsule coating the intestinal flora has relatively weak protection ability, and the adhesion of the intestinal flora to the intestinal mucosa is weak, which is not conducive to the absorption of the intestinal flora. Summary of the invention

[0004] The invention aims to provide a long-term storage stable flora double-layer capsule and a preparation method thereof: the composite lysozyme is coated with probiotic freeze-dried powder to form lipid nanoparticles with a small size structure, the lysozyme nanoparticles can adhere to the intestinal tract by interacting with mucus, and the lysozyme nanoparticles can penetrate the mucus barrier and be absorbed by the epithelial villi, thereby improving the absorption of intestinal flora; α-lactalbumin is coated on the surface of the nanoparticles, and the α-lactalbumin and the lysozyme nanoparticles are composited to more effectively penetrate the intestinal mucus; oxidized starch is grafted on the surface of the alginate capsule, and the oxidized starch maintains the combination with the alginate capsule in the acidic gastric tract, thereby preventing the calcium ions in the alginate capsule structure from being replaced by the sodium ions or potassium ions in the gastric acid, so that the alginate microcapsules can disintegrate in the intestinal fluid; chitosan can form a flora double-layer capsule with a flora single-layer capsule, and the double-layer capsule decomposes in the intestine to release the intestinal flora and the jujube methanol extract, and the intestinal flora and the jujube methanol extract are combined, which is conducive to the production of short-chain fatty acids as metabolites of the intestinal flora, and then released into the systemic circulation to regulate the intestinal homeostasis.

[0005] The technical problems to be solved by the present invention are as follows: the intestinal flora has relatively poor resistance to high temperature, acid, stress, and phage infection, and is easily inactivated. Moreover, when passing through gastric juice, the intestinal flora is easily inactivated by the acid, digestive enzymes, and antibiotics in the gastric juice, and cannot smoothly reach the intestine, thus affecting the efficacy of the intestinal flora. Microencapsulation of the intestinal flora can effectively improve the activity of the intestinal flora, but the single-layer microcapsule is used to coat the intestinal flora, and its protective ability is relatively weak. In addition, the adhesion of the intestinal flora to the intestinal mucosa is weak, which is not conducive to the absorption of the intestinal flora.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] S1. Place the probiotics in a sterile culture medium, activate, inoculate, and centrifuge, then add a freeze-drying protective agent and freeze-dry to obtain a probiotic freeze-dried powder.

[0008] Furthermore, the probiotics are selected from one of lactic acid bacteria, bifidobacteria, and yeast;

[0009] Furthermore, the probiotics are selected from lactic acid bacteria, and the lactic acid bacteria are selected from Lactobacillus plantarum ATCC8014.

[0010] Furthermore, the freeze-drying temperature is -(15-20)°C and the freeze-drying time is 20-24h.

[0011] Furthermore, the specific operation of the freeze-dried protective agent is: trehalose, inulin, skim milk powder and betaine are mixed and stirred evenly to obtain the freeze-dried protective agent.

[0012] Furthermore, the mass ratio of trehalose, inulin, skimmed milk powder and betaine is (2-2.2):(1-1.6):(0.6-0.7):(0.62-0.66).

[0013] Furthermore, the probiotic freeze-dried powder is specifically prepared by the following steps:

[0014] The probiotics were placed in a sterile culture medium, activated for two generations at 37°C, inoculated into a new sterile culture medium at a ratio of 1%, cultured for 48 hours, the bacterial liquid was collected, and centrifuged at 8000 rpm for 5 minutes to obtain bacterial sludge. After the bacterial sludge was washed with a 5% sodium chloride solution by mass, 2.5 g of the washed bacterial sludge was added to 20 g of a freeze-drying protective agent, stirred, and freeze-dried at -18°C for 22 hours to obtain a probiotic freeze-dried powder.

[0015] Furthermore, the mass ratio of bacterial mud to freeze-drying protective agent is (2-3):(18-22).

[0016] Furthermore, the sodium chloride solution is a sterile sodium chloride solution with a mass fraction of 4-6%.

[0017] Furthermore, the sterile culture medium is prepared by the following steps: MRS broth powder is mixed with deionized water, stirred evenly, placed in a conical flask, placed in a high-temperature and high-pressure steam sterilizer, and sterilized at 120-122° C. for 18-22 minutes to obtain a sterile culture medium.

[0018] Furthermore, the dosage ratio of MRS broth powder and deionized water is (0.2-0.3) g: (8-12) mL.

[0019] S2. The probiotic freeze-dried powder and the composite lysozyme are mixed, and then reacted with α-lactalbumin to obtain nanoparticles loaded with probiotics, which are specifically prepared by the following steps:

[0020] The probiotic lyophilized powder was added to deionized water and stirred, and then added to the composite lysozyme and stirred for 30 minutes. The unincorporated probiotic lyophilized powder was removed by dialysis, and the solvent was removed by freeze drying at -20°C for 2 hours to obtain nanoparticles. The nanoparticles and α-lactalbumin were added to deionized water and stirred evenly. After incubation at room temperature for 4 hours, the nanoparticles were centrifuged at 1000 rpm for 5 minutes to obtain nanoparticles loaded with probiotics.

[0021] Among them, the probiotic freeze-dried powder is dissolved in sterile deionized water, and the amphiphilic peptides unfolded by the composite lysozyme can aggregate into a micellar structure. In the micellar process, the probiotic freeze-dried powder is coated with the composite lysozyme under the action of the amphiphilic peptides to form lipid nanoparticles. Due to its small size structure, the lysozyme nanoparticles can adhere to the intestine by interacting with mucus.

[0022] Furthermore, the surface of the lysozyme nanoparticles coated with the probiotic freeze-dried powder contains carboxyl groups produced by enzymatic hydrolysis, which can electrostatically adsorb to the amino groups in α-lactalbumin, so that the α-lactalbumin is coated on the surface of the lysozyme nanoparticles coated with the probiotic freeze-dried powder, and the α-lactalbumin and the lysozyme nanoparticles are complexed to more effectively penetrate the intestinal mucus, quickly pass through the epithelium and reach the systemic circulation.

[0023] Furthermore, the dosage ratio of probiotic freeze-dried powder, deionized water, and composite lysozyme is (0.3-0.7) g: (15-25) mL: (70-90) mL.

[0024] Furthermore, the dosage ratio of nanoparticles, α-lactalbumin, and deionized water is (0.1-0.3) g:(1.6-2) g:(45-55) mL.

[0025] Furthermore, the composite lysozyme is prepared by the following steps:

[0026] The lysozyme was added into a Tris-HCl buffer with a pH of 7.5, and stirred evenly. The protease of Bacillus licheniformis was added, and the mixture was stirred and hydrolyzed at 50° C. for 30 min. The mixture was cooled to room temperature, and 0.5 g of lysozyme nanoparticles was added, and stirred evenly to obtain a composite lysozyme.

[0027] Among them, lysozyme is dissolved in Tris-HCl buffer, and under the action of Bacillus licheniformis protease, lysozyme is enzymatically hydrolyzed to form amphiphilic peptides that can self-assemble into micelle structures. The amphiphilic peptides are incorporated into lysozyme nanoparticles, so that the amphiphilic peptides are adsorbed on the surface of the lysozyme nanoparticles, and more carboxyl groups are exposed on the surface of the lysozyme nanoparticles, which is conducive to binding with α-lactalbumin.

[0028] Furthermore, the dosage ratio of lysozyme, Tris-HCl buffer, Bacillus licheniformis protease, and lysozyme nanoparticles is (0.2-0.3) g: (15-25) mL: (0.1-0.2) mL: (0.3-0.7) g.

[0029] Furthermore, the particle size of the lysozyme nanoparticles is 20-30 nm.

[0030] S3. The nanoparticles loaded with probiotics are mixed with sodium alginate to prepare alginate capsules, and the alginate capsules are then surface-modified with oxidized starch to obtain bacterial single-layer capsules.

[0031] Furthermore, the oxidized starch is oxidized potato starch, and the degree of oxidation is 28-32%.

[0032] Furthermore, the bacterial flora single-layer capsule is specifically prepared by the following steps:

[0033] A1. Sodium alginate is added to deionized water, stirred evenly, and sterilized to obtain a sodium alginate solution. The nanoparticles loaded with probiotics are added to a sterilized peptone solution with a mass fraction of 0.1%, stirred evenly, and the sodium alginate solution is added to mix. Calcium chloride is added and stirred to allow the microcapsules to settle. The alginate capsules are filtered to obtain alginate capsules.

[0034] Among them, the nanoparticles loaded with probiotics are mixed with sodium alginate solution, and calcium chloride is used as a cross-linking agent. The calcium ions contained in the solution undergo cross-linking reaction with the carboxyl groups in alginate, embedding the nanoparticles loaded with probiotics into the cross-linked network structure to form alginate capsules.

[0035] Furthermore, the dosage ratio of the probiotic-loaded nanoparticles, sterile peptone solution, sodium alginate solution, and calcium chloride is (1.3-1.7) g: (8-12) mL: (8-12) mL: (0.1-0.3) g.

[0036] A2. Add oxidized starch and alginate capsules into deionized water, stir evenly, react at 25°C for 1 hour, and centrifuge at 1000 rpm for 5 minutes to obtain bacterial single-layer capsules.

[0037] Among them, the carboxyl groups contained on the surface of the alginate capsules can be chemically bonded to the carboxyl and aldehyde groups of the oxidized starch, so that the oxidized starch is grafted onto the surface of the alginate capsules. The oxidized starch remains combined with the alginate capsules in the acidic gastric tract, preventing the calcium ions in the alginate capsule structure from being replaced by sodium ions or potassium ions in gastric acid, causing the alginate microcapsules to disintegrate in the intestinal fluid and release the embedded probiotics.

[0038] Furthermore, the dosage ratio of oxidized starch, alginate capsule and deionized water is (1.1-1.3) g: (0.2-0.4) g: (25-35) mL.

[0039] S4. The bacterial consortium single-layer capsule and the methanol extract of jujube were mixed as the core material, and the chitosan acetic acid solution was used as the wall material to prepare the bacterial consortium double-layer capsule.

[0040] Furthermore, the chitosan acetic acid solution is prepared by mixing chitosan and acetic acid in a dosage ratio of (0.3-0.5) g:(80-120) mL.

[0041] Furthermore, the pH of the chitosan acetic acid solution is 5-7.

[0042] Furthermore, the flora double-layer capsule is specifically prepared by the following steps:

[0043] The bacterial single-layer capsules and the methanol extract of jujube were added to the chitosan acetic acid solution, and the chitosan and alginate microcapsules were reacted by magnetic stirring at 100 rpm for 10 minutes. The solution was allowed to stand, filtered, and the capsules were washed three times with sterile saline to obtain bacterial double-layer capsules. The bacterial double-layer capsules were stored in an environment of 4°C.

[0044] Among them, chitosan can undergo cross-linking reaction with the carboxyl group of oxidized starch in the single-layer microflora capsule to form a cross-linked network capsule, and the methanol extract powder of jujube is embedded in the cross-linked network capsule to form a double-layer microflora capsule, which decomposes in the intestine to release intestinal flora and jujube methanol extract. The combination of intestinal flora and jujube methanol extract contributes to the production of short-chain fatty acids, which are then released into the systemic circulation to regulate intestinal homeostasis.

[0045] Furthermore, the dosage ratio of the bacterial single-layer capsule, the methanol extract of the jujube, and the chitosan acetic acid solution is (1-1.4) g: (0.6-1) g: (8-12) mL.

[0046] Furthermore, the methanol extract of Ziziphus jujuba is prepared by the following steps:

[0047] The impurities of the jujube are washed with deionized water, dried, and then crushed in a grinder. The crushed jujube is added to a methanol aqueous solution with a mass fraction of 80%, ultrasonically treated for 25-35 minutes, centrifuged at 300 rpm for 10-15 minutes, the supernatant is collected, and vacuum concentrated to obtain the jujube methanol extract.

[0048] Furthermore, the ratio of the crushed jujube to the methanol aqueous solution is (0.5-1.5) g: (8-12) mL.

[0049] Furthermore, compared with the prior art, the present invention has the following beneficial effects:

[0050] (1) In the technical scheme of the present invention, the probiotics have the effects of reducing weight and reducing lipids, lowering blood pressure, inhibiting the growth of harmful bacteria, reducing inflammatory reactions, and anti-aging, and can improve metabolism, promote digestion and absorption of nutrients, enhance the body's immunity, improve disease resistance, and prevent and treat diarrhea caused by pathogens; the amphiphilic peptides unfolded by the composite lysozyme can aggregate into a micellar structure. In the micellar process, the probiotic freeze-dried powder is coated with the composite lysozyme under the action of the amphiphilic peptide to form lipid nanoparticles. Due to its small size structure, the lysozyme nanoparticles can adhere to the intestine by interacting with mucus, and the lysozyme nanoparticles can penetrate the mucus barrier and be absorbed by the epithelial villi, thereby improving the absorption of intestinal flora; α-lactalbumin is coated on the surface of the nanoparticles, and the α-lactalbumin is combined with the lysozyme nanoparticles to more effectively penetrate the intestinal mucus, quickly pass through the epithelium and reach the systemic circulation, and can prolong the residence time in the intestine, thereby increasing the accumulation of intestinal flora on the intestinal mucosa.

[0051] (2) In the technical solution of the present invention, nanoparticles loaded with probiotics are embedded in alginate capsules. The alginate capsules can resist the erosion of gastric acid, so that the nanoparticles loaded with probiotics can better pass through the gastric juice and reach the intestine; the carboxyl groups contained on the surface of the alginate capsules can be chemically bonded with the carboxyl groups and aldehyde groups of the oxidized starch, so that the oxidized starch is grafted on the surface of the alginate capsules. The oxidized starch remains bonded to the alginate capsules in the acidic gastric tract, avoiding the replacement of calcium ions in the alginate capsule structure by sodium ions or potassium ions in gastric acid, resulting in the alginate microcapsules being disintegrated in the intestinal juice and releasing the embedded probiotics. The oxidized starch can also resist the erosion of gastric acid and has excellent adhesion to intestinal mucus.

[0052] (3) In the technical scheme of the present invention, the phenolic acid component contained in the methanol extract of the jujube has good antioxidant activity, and contains multiple phenolic hydroxyl groups and has the ability to scavenge free radicals, which can protect the body from oxidative damage and thus regulate the intestine. The methanol extract of the jujube can reduce the number of viable Gram-negative enteric bacilli in the intestine and reduce the risk of intestinal infection in the host. In addition, the methanol extract of the jujube can promote the proliferation of intestinal probiotics and increase the content of short-chain fatty acids in intestinal flora metabolites, thereby regulating intestinal homeostasis.

[0053] (4) In the technical scheme of the present invention, chitosan can form a bacterial double-layer capsule with a bacterial single-layer capsule. The double-layer capsule decomposes in the intestine to release intestinal flora and jujube methanol extract. The intestinal flora and jujube methanol extract combine to help produce short-chain fatty acids, which are metabolites of the intestinal flora and are then released into the systemic circulation to regulate intestinal homeostasis. The outer wall material of the bacterial double-layer capsule penetrates into the intestine through gastric acid and is slowly released, which prolongs the release time of the intestinal flora and increases the duration of action with the intestinal mucosa, which is beneficial to the efficient colonization of the intestinal flora on the intestinal mucosa. The bacterial double-layer capsule can better resist the erosion of digestive juices such as gastric acid and bile, and avoid the bacterial double-layer capsule from dissolving too quickly in the gastric environment, releasing the flora, resulting in poor efficacy, thereby promoting the colonization of the intestinal flora. DETAILED DESCRIPTION

[0054] The technical solutions in the embodiments of the present invention are described clearly and completely. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0055] The raw materials used in the embodiments of the present invention are as follows:

[0056] MRS broth powder (Wuhu Ouke Biotechnology Co., Ltd., content 99%.9); lysozyme, lysozyme nanoparticles (Wuhan Huaxiang Kejie Biotechnology Co., Ltd., content 99%); α-lactalbumin (99% content, Qingdao Kester Biotechnology Co., Ltd.); sodium alginate (low viscosity, Tianjin Xiensi Biochemical Technology Co., Ltd.); sterile peptone solution (Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd.); calcium chloride (analytical grade, Tianjin Xiensi Biochemical Technology Co., Ltd.); oxidized starch (Beijing Jinming Biotechnology Co., Ltd.); jujube (purchased from Chuguiyuan Chinese medicinal materials, produced in Yulin, Guangxi).

[0057] The probiotics are selected from lactic acid bacteria, and the lactic acid bacteria are selected from Lactobacillus plantarum ATCC8014, Ruichu Biotechnology (Jiangsu) Co., Ltd.

[0058] The specific operation of the freeze-dried protective agent is as follows: 2.1 g of trehalose, 1.3 g of inulin, 0.67 g of skim milk powder, and 0.64 g of betaine are mixed and stirred evenly to obtain the freeze-dried protective agent.

[0059] The chitosan acetic acid solution is prepared by mixing chitosan and acetic acid in a dosage ratio of 0.4 g:100 mL.

[0060] Chitosan: Deacetylation degree is 95.5%, Tianjin Xiensi Biochemical Technology Co., Ltd.

[0061] Example 1

[0062] The probiotic freeze-dried powder is specifically prepared by the following steps:

[0063] A1. Mix MRS broth powder with deionized water, stir evenly, place in a conical flask, put in a high-temperature and high-pressure steam sterilizer, sterilize at 121°C for 20 minutes, and obtain a sterilized culture medium.

[0064] A2. Place the probiotics in a sterile culture medium, activate them for two generations at 37°C, inoculate them into a new sterile culture medium at a ratio of 1%, culture them for 48 hours, collect the bacterial liquid, centrifuge it at 8000 rpm for 5 minutes to obtain bacterial sludge, wash the bacterial sludge with a 5% sodium chloride solution, add 2.5 g of the washed bacterial sludge to 20 g of a freeze-drying protective agent, stir, and freeze-dry at -18°C for 22 hours to obtain probiotic freeze-dried powder.

[0065] Example 2

[0066] The probiotic-loaded nanoparticles are specifically prepared by the following steps:

[0067] B1. Add 0.25 g of lysozyme to 20 mL of Tris-HCl buffer at pH 7.5, stir evenly, add 0.15 mL of Bacillus licheniformis protease, stir and hydrolyze at 50°C for 30 min, cool to room temperature, add 0.5 g of lysozyme nanoparticles, stir evenly, and obtain composite lysozyme.

[0068] B2. Add 0.5 g of probiotic freeze-dried powder to 20 mL of deionized water, stir, add to 80 mL of composite lysozyme, stir for 30 min, remove unincorporated probiotic freeze-dried powder by dialysis, freeze-dry at -20 ° C for 2 h to remove the solvent to obtain nanoparticles, add 0.2 g of nanoparticles and 1.8 g of α-lactalbumin to 50 mL of deionized water, stir evenly, incubate at room temperature for 4 h, and centrifuge at 1000 rpm for 5 min to obtain nanoparticles loaded with probiotics.

[0069] Comparative Example 1

[0070] The difference between this comparative example and Example 2 is that no composite lysozyme is added, and the remaining steps and raw materials are the same as those of Example 2.

[0071] 0.5 g of probiotic freeze-dried powder and 1.8 g of α-lactalbumin were added to 50 mL of deionized water, stirred evenly, incubated at room temperature for 4 h, and centrifuged at 1000 rpm for 5 min to obtain a complex.

[0072] Comparative Example 2

[0073] The difference between this comparative example and Example 2 is that α-lactalbumin is not added, and the remaining steps and raw materials are the same as those of Example 2.

[0074] B1. Add 0.25 g of lysozyme to 20 mL of Tris-HCl buffer at pH 7.5, stir evenly, add 0.15 mL of Bacillus licheniformis protease, stir and hydrolyze at 50°C for 30 min, cool to room temperature, add 0.5 g of lysozyme nanoparticles, stir evenly, and obtain composite lysozyme.

[0075] B2. Add 0.5 g of probiotic freeze-dried powder to 20 mL of deionized water, stir, add to 80 mL of composite lysozyme, stir for 30 min, remove the unincorporated probiotic freeze-dried powder by dialysis, freeze-dry at -20 °C for 2 h to remove the solvent, and obtain nanoparticles loaded with probiotics.

[0076] Example 3

[0077] The bacterial flora single-layer capsule is specifically prepared by the following steps:

[0078] C1. Add 1.5 g of sodium alginate to 100 mL of deionized water, stir evenly, and sterilize to obtain a sodium alginate solution. Add 0.3 g of probiotic-loaded nanoparticles to 10 mL of a 0.1% sterilized peptone solution, stir evenly, add 10 mL of the sodium alginate solution to mix, stir, add 0.2 g of calcium chloride, stir to allow the microcapsules to settle, filter, and obtain alginate capsules.

[0079] C2. Add 1.2 g of oxidized starch and 0.3 g of alginate capsules into 30 mL of deionized water, stir evenly, react with stirring at 25°C for 1 h, and centrifuge at 1000 rpm for 5 min to obtain bacterial single-layer capsules.

[0080] Comparative Example 3

[0081] The difference between this comparative example and Example 3 is that the nanoparticles loaded with probiotics are replaced by the substance prepared in Comparative Example 1, and the remaining steps and raw materials are the same as those in Example 3.

[0082] Comparative Example 4

[0083] The difference between this comparative example and Example 3 is that the nanoparticles loaded with probiotics are replaced by the material prepared in Comparative Example 2, and the remaining steps and raw materials are the same as those in Example 3.

[0084] Comparative Example 5

[0085] The difference between this comparative example and Example 3 is that no oxidized starch is added, and the remaining steps and raw materials are the same as those of Example 3.

[0086] Add 1.5 g of sodium alginate to 100 mL of deionized water, stir evenly, and sterilize to obtain a sodium alginate solution. Add 0.3 g of nanoparticles loaded with probiotics to 10 mL of a sterilized peptone solution with a mass fraction of 0.1%, stir evenly, add 10 mL of the sodium alginate solution to mix, stir, add 0.2 g of calcium chloride, stir to allow the microcapsules to settle, filter, and obtain a bacterial single-layer capsule.

[0087] Example 4

[0088] A method for preparing a long-term storage stable flora double-layer capsule is specifically prepared by the following steps:

[0089] S1. Wash impurities from the jujube with deionized water, dry it, and grind it in a grinder. Add 0.5 g of the ground jujube into 8 mL of 80% methanol aqueous solution, ultrasonically treat it for 25-35 minutes, centrifuge it at 300 rpm for 10-15 minutes, collect the supernatant, and concentrate it in vacuo to obtain the jujube methanol extract.

[0090] S2. Add 1g of bacterial single-layer capsules and 0.6g of jujube methanol extract into 8mL of chitosan acetic acid solution, stir magnetically at 100rpm for 10min to allow chitosan to react with alginate microcapsules, let stand, filter, and wash the capsules three times with sterile saline to obtain bacterial double-layer capsules, which are stored in an environment of 4°C.

[0091] Example 5

[0092] A method for preparing a long-term storage stable flora double-layer capsule is specifically prepared by the following steps:

[0093] S1. Wash impurities from the jujube with deionized water, dry it, and grind it in a grinder. Add 1 g of the ground jujube to 10 mL of 80% methanol aqueous solution, ultrasonically treat it for 25-35 minutes, centrifuge it at 300 rpm for 10-15 minutes, collect the supernatant, and concentrate it in vacuo to obtain the jujube methanol extract.

[0094] S2. Add 1.2g of bacterial single-layer capsules and 0.8g of jujube methanol extract into 10mL of chitosan acetic acid solution, stir magnetically at 100rpm for 10min to allow chitosan to react with alginate microcapsules, let stand, filter, and wash the capsules three times with sterile saline to obtain bacterial double-layer capsules, which are stored in an environment of 4°C.

[0095] Example 6

[0096] A method for preparing a long-term storage stable flora double-layer capsule is specifically prepared by the following steps:

[0097] S1. Wash impurities from the jujube with deionized water, dry it, and grind it in a grinder. Add 1.5 g of the ground jujube into 12 mL of 80% methanol aqueous solution, ultrasonically treat it for 25-35 min, centrifuge it at 300 rpm for 10-15 min, collect the supernatant, and concentrate it in vacuo to obtain the jujube methanol extract.

[0098] S2. Add 1.4g of bacterial single-layer capsules and 1g of jujube methanol extract into 12mL of chitosan acetic acid solution, stir magnetically at 100rpm for 10min to allow chitosan to react with alginate microcapsules, let stand, filter, and wash the capsules three times with sterile saline to obtain bacterial double-layer capsules, which are stored in a 4°C environment.

[0099] Comparative Example 6

[0100] The difference between this comparative example and Example 5 is that the bacterial flora single-layer capsule is replaced by the material prepared in Comparative Example 3, and the remaining steps and raw materials are the same as those in Example 5.

[0101] Comparative Example 7

[0102] The difference between this comparative example and Example 5 is that the bacterial flora single-layer capsule is replaced by the substance prepared in Comparative Example 4, and the remaining steps and raw materials are synchronized with Example 5.

[0103] Comparative Example 8

[0104] The difference between this comparative example and Example 5 is that the bacterial flora single-layer capsule is replaced by the material prepared in Comparative Example 5, and the remaining steps and raw materials are the same as those in Example 5.

[0105] Comparative Example 9

[0106] The difference between this comparative example and Example 5 is that the methanol extract of Ziziphus jujuba is not added, and the remaining steps and raw materials are the same as those in Example 5.

[0107] The performance of the bacterial flora double-layer capsules prepared in Examples 4-6 and Comparative Examples 6-9 was tested;

[0108] The total number of live bacteria in the bacterial double-layer capsule was 1.23×10 10 cfu / mL.

[0109] Release performance test of the bacterial flora double-layer capsule in artificial intestinal fluid: take 1 mL of the bacterial flora double-layer capsule and place it in 50 mL of sterile artificial intestinal fluid with a pH of 7.4, and culture it at 37°C. Take out an appropriate amount of capsules at 8h, 16h, and 24h, break, dilute, spread on the plate, and measure the probiotic concentration. The release performance = (probiotic concentration / total number of live bacteria in the bacterial flora double-layer capsule) × 100%, and detect the colonization rate of the bacterial flora double-layer capsule in the intestine.

[0110] Determination of viability in artificial gastric juice: 1 mL of bacterial flora double-layer capsule was placed in 50 mL of sterilized simulated artificial gastric juice with a pH of 1.2, and cultured at 37°C. An appropriate amount was taken out every hour from 0 to 3 hours, and after disintegration and crushing, it was diluted and plated, and the viable bacteria concentration was measured. The survival rate = (viable bacteria concentration / total number of viable bacteria in bacterial flora double-layer capsule) × 100%;

[0111] Tolerance determination: Take 1 mL of bacterial flora double-layer capsule, put it in 50 mL of sterile bile acid (0.6% ox bile salt is added to artificial intestinal fluid, sterilized at 121°C for 15 min) at 37°C, take it out at 0h, 2h, and 4h, disintegrate and break it, dilute it, spread it on a plate, and measure its live bacteria concentration. Tolerance = (live bacteria concentration / total live bacteria in the bacterial flora double-layer capsule) × 100%.

[0112] Stability test: The bacterial double-layer capsules were stored in a refrigerator at 4°C, and taken out at 0d, 15d, and 30d, respectively, and the number of viable bacteria in the microcapsules was determined by the plate colony counting method (survival rate = number of viable bacteria after storage / number of viable bacteria in the original capsule × 100%); the results are shown in Table 1 below:

[0113] Table 1

[0114]

[0115]

[0116] It can be seen from the data in Table 1 that in Comparative Example 6, the nanoparticles loaded with probiotics prepared without adding the composite lysozyme were used to prepare the flora double-layer capsules, and their release performance and colonization performance decreased. This may be because the lysozyme nanoparticles can adhere to the intestine by interacting with the mucus, and the lysozyme nanoparticles can penetrate the mucus barrier and be absorbed by the epithelial villi, thereby improving the absorption of the intestinal flora; in Comparative Example 7, the nanoparticles loaded with probiotics were prepared without adding α-lactalbumin, and were used to prepare the flora double-layer capsules, and their release performance and colonization performance decreased. This may be because α-lactalbumin is coated on the surface of the nanoparticles, and α-lactalbumin is composited with the lysozyme nanoparticles, which more effectively penetrates the intestinal mucus, quickly passes through the epithelium and reaches the systemic circulation. It can also prolong the residence time in the intestine and increase the accumulation of intestinal flora on the intestinal mucosa; in Example 8, the double-layer flora capsules prepared without adding oxidized starch had a decreased gastric juice resistance, which may be because the oxidized starch maintained its combination with the alginate capsules in the acidic gastric tract, avoiding the replacement of calcium ions in the alginate capsule structure by sodium ions or potassium ions in gastric acid, resulting in the disintegration of alginate microcapsules in the intestinal juice; in Example 9, the double-layer flora capsules prepared without adding the methanol extract of jujube had a decreased intestinal flora balance, which may be because the phenolic acid component contained in the methanol extract of jujube has good antioxidant activity, and contains multiple phenolic hydroxyl groups and has the ability to scavenge free radicals, which can protect the body from oxidative damage and thus regulate the intestine.

[0117] The data in Table 1 show that the bacterial flora double-layer capsules prepared in Examples 4-6 meet the test performance requirements, while the bacterial flora double-layer capsules prepared in Comparative Examples 5-8 do not meet the performance requirements, indicating that the bacterial flora double-layer capsules prepared in the present invention are better able to resist the erosion of digestive juices such as gastric acid and bile.

[0118] In the description of the specification, the description with reference to the terms "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0119] The above contents are merely examples and explanations of the present invention. Those skilled in the art may make various modifications or additions to the specific embodiments described or replace them in a similar manner. As long as they do not deviate from the invention or exceed the scope defined by the claims, they shall all fall within the protection scope of the present invention.

Claims

1. A method for preparing a long-term storage stable flora double-layer capsule, characterized in that: The following steps are involved: S1. placing the probiotics in a sterile culture medium, activating, inoculating, and centrifuging, adding a freeze-drying protective agent, and freeze-drying to obtain a probiotic freeze-dried powder; S2. mixing the probiotic freeze-dried powder and the composite lysozyme, and then reacting with α-lactalbumin to obtain nanoparticles loaded with probiotics; S3. The probiotic-loaded nanoparticles are mixed with sodium alginate to prepare alginate capsules, and the alginate capsules are then surface-modified with oxidized starch to obtain bacterial monolayer capsules; S4. The bacterial consortium single-layer capsule and the methanol extract of jujube were mixed as the core material, and the chitosan acetic acid solution was used as the wall material to prepare the bacterial consortium double-layer capsule.

2. The method for preparing a long-term storage stable flora double-layer capsule according to claim 1, characterized in that: The probiotic freeze-dried powder is specifically prepared by the following steps: The probiotics were placed in a sterile culture medium, activated for two generations at 37°C, inoculated into a new sterile culture medium at a ratio of 1%, cultured for 48 hours, and the bacterial liquid was collected. The bacterial slurry was centrifuged at 8000 rpm for 5 minutes to obtain bacterial slurry. The bacterial slurry was washed with a 5% sodium chloride solution by mass, and then added to a freeze-drying protective agent, stirred, and freeze-dried at -18°C for 22 hours to obtain probiotic freeze-dried powder.

3. The method for preparing a long-term storage stable flora double-layer capsule according to claim 1, characterized in that: The sterile culture medium is prepared by the following steps: mixing MRS broth powder with deionized water, stirring evenly, placing in a conical flask, placing in a high-temperature and high-pressure steam sterilizer, sterilizing at 120-122° C. for 18-22 minutes, and obtaining the sterile culture medium.

4. The method for preparing a long-term storage stable flora double-layer capsule according to claim 1, characterized in that: The composite lysozyme is specifically prepared by the following steps: The lysozyme is added to a Tris-HCl buffer with a pH of 7-8, stirred evenly, the Bacillus licheniformis protease is added, stirred and hydrolyzed at 45-55° C. for 25-35 minutes, cooled to room temperature, the lysozyme nanoparticles are added, stirred evenly, and a composite lysozyme is obtained.

5. The method for preparing a long-term storage stable flora double-layer capsule according to claim 4, characterized in that: The dosage ratio of the lysozyme, Tris-HCl buffer, Bacillus licheniformis protease and lysozyme nanoparticles is (0.2-0.3) g: (15-25) mL: (0.1-0.2) mL: (0.3-0.7) g.

6. The method for preparing a long-term storage stable flora double-layer capsule according to claim 1, characterized in that: The oxidized starch is oxidized potato starch with an oxidation degree of 28-32%.

7. The method for preparing a long-term storage stable flora double-layer capsule according to claim 1, characterized in that: The methanol extract of Ziziphus chinensis is prepared by the following steps: The impurities of the jujube are washed with deionized water, dried, and then crushed in a grinder. The crushed jujube is added to a methanol aqueous solution with a mass fraction of 80%, ultrasonically treated for 25-35 minutes, centrifuged at 280-320 rpm for 10-15 minutes, the supernatant is collected, and vacuum concentrated to obtain the jujube methanol extract.

8. The method for preparing a long-term storage stable flora double-layer capsule according to claim 1, characterized in that: The ratio of the crushed jujube to the methanol aqueous solution is (0.5-1.5) g: (8-12) mL.

9. The method for preparing a long-term storage stable flora double-layer capsule according to claim 1, characterized in that: The chitosan acetic acid solution is prepared by mixing chitosan and acetic acid in a dosage ratio of (0.3-0.5) g:(80-120) mL; the pH value of the chitosan acetic acid solution is 5-7.

10. A bacterial flora double-layer capsule prepared according to the preparation method according to any one of claims 1 to 9.

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