A composite probiotic capsule and a method for preparing the same

Abstract

The application belongs to the technical field of microcapsules, and particularly relates to a preparation method of a composite probiotic capsule, which comprises the following steps: uniformly mixing probiotic bacterial liquid, fructooligosaccharide, tea polyphenol, grape polyphenol, mannan oligosaccharide, isomaltitol and an emulsifier to obtain a core material; uniformly mixing glycerol and distilled water, heating to 60-80 DEG C, sequentially adding modified gelatin, hydroxypropyl starch and chitosan, stirring for 10-30 min, sterilizing, and cooling to room temperature to obtain a capsule material; mixing the core material and the capsule material, performing high-pressure homogenization treatment, and then performing spray drying to obtain the composite probiotic capsule; and the modified gelatin is prepared by acylation reaction of succinic acid derivatives and gelatin. The composite probiotic capsule provided by the application has excellent storage stability, antioxidant property, anti-gastric juice erosion capacity, and also has good enteric solubility and bioavailability.

Description

Technical Field

[0001] This invention belongs to the field of microcapsule technology, specifically relating to a compound probiotic capsule and its preparation method. Background Technology

[0002] The human gut is home to over 100 species and billions of different bacteria. Under normal circumstances, these bacteria coexist and regulate each other, with some harmful and others beneficial (probiotics). The main functions of probiotics include promoting digestive health, regulating immune function, alleviating lactose intolerance symptoms, and lowering serum cholesterol. With the rapid development of the probiotic industry, it is now applied in various fields such as medicine, food, and health products. However, current probiotic products generally suffer from low bacterial survival rates, unstable bacterial counts, short shelf life, and inconsistent product quality. Microencapsulation technology, by encapsulating probiotics in a larger matrix, protects them from adverse environmental factors such as pH, high temperature, and high humidity, effectively improving the activity of probiotics and the stability of product quality.

[0003] Patent application number CN201911365363.4 provides a method for preparing probiotic combined with cod liver oil capsules. First, glycerin, gelatin, and ultrapure water are mixed to prepare a gel solution. Then, cod liver oil, vitamin E, and freeze-dried probiotic powder are mixed to prepare a liquid content. Finally, the liquid content and gel solution are pressed into soft capsules, which are then shaped and dried to obtain the finished product. This invention combines the various components, balancing nutrition while not only maximizing the original effects of each component but also allowing for synergistic effects. It has excellent efficacy in improving immunity, promoting brain development, and preventing night blindness. Patent application number CN202110686051.4 provides a method for preparing slimming probiotic soft capsules, first mixing solid materials with an oil to prepare the contents. The invention involves mixing glycerin, xylitol, purified water, and gelatin to prepare a soft-shell capsule material. The contents of the soft-shell capsule are then pressed together with the soft-shell material to form soft capsules. After shaping and drying, the finished product is obtained. This invention combines various raw material components to fully utilize their effects, reducing body fat and weight without reducing water and muscle mass. All the aforementioned patents use gelatin and glycerin as capsule soft-shell materials. Gelatin, derived from collagen, is a natural biopolymer material with advantages such as biodegradability, good biocompatibility and gelling properties, and low cost. It is a widely used traditional pharmaceutical excipient in the pharmaceutical field. Gelatin is safe and effective when used as a capsule material, but as the demand for gelatin expands, its disadvantages become increasingly apparent. Due to the presence of aldehyde and imino groups, gelatin products are prone to cross-linking during storage. Furthermore, gelatin products are oxidized by air during preparation and long-term storage, further promoting cross-linking. After cross-linking, gelatin capsules form a dense cross-linked network structure, leading to problems such as difficulty in capsule disintegration, decreased solubility, and reduced bioavailability. In addition, under normal eating conditions, mixed food typically remains in the stomach for 4-6 hours before entering the small intestine, where probiotics are easily killed by stomach acid. Therefore, capsule materials should also possess excellent acid resistance to ensure that probiotics can effectively colonize the small intestine, thereby maximizing their beneficial effects and further improving the bioavailability of the capsule product. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide a compound probiotic capsule with excellent storage stability, antioxidant properties, resistance to gastric acid erosion, and solubility under neutral to slightly alkaline conditions, i.e., good enteric solubility. The present invention also provides a method for preparing the compound probiotic capsule.

[0005] The technical solution adopted by the present invention to achieve the above objectives is as follows:

[0006] A compound probiotic capsule comprises the following raw materials in parts by weight: a core material and a capsule material, wherein the mass ratio of the core material to the capsule material is 1-3:5-9; the core material comprises the following raw materials in parts by weight: 20-40 parts probiotic bacterial solution, 5-8 parts fructooligosaccharides, 2-4 parts tea polyphenols, 2-4 parts grape polyphenols, 5-8 parts mannan oligosaccharides, 3-7 parts isomaltitol, and 1-3 parts emulsifier, wherein the probiotic bacterial solution is composed of equal weights of Bifidobacterium bacterial solution and Lactobacillus acidophilus bacterial solution; the capsule material comprises the following raw materials in parts by weight: 40-60 parts modified gelatin, 50-70 parts distilled water, 5-10 parts glycerin, 20-30 parts hydroxypropyl starch, and 15-20 parts chitosan; the preparation method of the modified gelatin is as follows:

[0007] S1. Under stirring conditions, (2R,3R)-(-)-epoxysuccinic acid was dispersed in 2-methyltetrahydrofuran. After heating to 60-70℃, N-ethylmorpholine and 4-methyl-1-pentanol were added sequentially. The mixture was stirred for 5-8 hours, cooled to room temperature, and the solid and liquid were separated. The liquid was then distilled under reduced pressure, washed, and dried to obtain the intermediate.

[0008] S2. Under stirring conditions, 3,5-di-tert-butyl-4-hydroxybenzoic acid is dispersed in 2-methyltetrahydrofuran. After heating to 65-75℃, the intermediate obtained in step S1 and p-toluenesulfonic acid are added sequentially. The mixture is stirred for 4-9 hours, cooled to room temperature, and the solid and liquid are separated. The liquid is collected, and after vacuum distillation, washing, and drying, succinic acid derivatives are obtained.

[0009] S3. Under stirring conditions, gelatin is dispersed in phosphate buffer solution, heated to 50-70℃, stirred for 0.5-1.5h, then the succinic acid derivative obtained in step S2 is added, stirring is continued for 2-4h, the pH is adjusted to 6-7, and modified gelatin is obtained by dialysis and freeze drying.

[0010] The synthetic route for succinic acid derivatives is as follows:

[0011]

[0012] Gelatin is a widely used pharmaceutical excipient with good film-forming properties, gelling properties, biodegradability, and biocompatibility, but it also has some shortcomings that limit its application. Improving gelatin's properties through chemical modification allows it to acquire new properties, further broadening the application areas of this traditional excipient. This invention uses (2R,3R)-(-)-epoxysuccinic acid and 4-methyl-1-pentanol as starting materials. Under the catalysis of N-ethylmorpholine, a ring-opening reaction of the epoxide compounds occurs to obtain an intermediate. The intermediate contains a hydroxyl group, which undergoes an esterification reaction with the carboxyl group in the structure of 3,5-di-tert-butyl-4-hydroxybenzoic acid under the action of p-toluenesulfonic acid to obtain a succinic acid derivative. The carboxyl group in the succinic acid derivative further undergoes an acylation reaction with the amino group in the gelatin structure to obtain modified gelatin.

[0013] To obtain the modified gelatin, in step S1, the molar ratio of (2R,3R)-(-)-epoxysuccinic acid, N-ethylmorpholine, and 4-methyl-1-pentanol is 1:0.02-0.05:0.6-0.8, and the amount of (2R,3R)-(-)-epoxysuccinic acid added to the 2-methyltetrahydrofuran is 0.25-0.45 g / mL; in step S2, the molar ratio of 3,5-di-tert-butyl-4-hydroxybenzoic acid, the intermediate, and p-toluenesulfonic acid is 1:0.7-0.9:0.03-0.06, and the amount of 3,5-di-tert-butyl-4-hydroxybenzoic acid added to the 2-methyltetrahydrofuran is 0.2-0.4 g / mL; in step S3, the mass ratio of gelatin, phosphate buffer solution, and succinic acid derivative is 10:80-120:2-4.

[0014] Preferably, the emulsifier is one of sucrose fatty acid ester, polyglycerol fatty acid ester, Tween-80, and beeswax; the concentration of the probiotic solution is not less than 10%. 8 cfu / g.

[0015] This invention also provides a method for preparing compound probiotic capsules, comprising the following steps: mixing probiotic bacterial solution, fructooligosaccharides, tea polyphenols, grape polyphenols, mannan oligosaccharides, isomaltitol, and emulsifier evenly to obtain a core material; mixing glycerol and distilled water evenly and heating to 60-80℃, then sequentially adding modified gelatin, hydroxypropyl starch, and chitosan, stirring for 10-30 min, sterilizing, and cooling to room temperature to obtain a capsule material; mixing the core material and the capsule material, subjecting them to high-pressure homogenization, and then spray drying to obtain the final product; wherein, the high-pressure homogenization pressure is 40-60 MPa, the homogenization time is 10-20 s; the inlet air temperature of the spray drying is 120-150℃, the outlet air temperature is 70-90℃, and the feed flow rate is 10-20 mL / min.

[0016] The present invention has the following beneficial effects:

[0017] This invention utilizes a two-step method to introduce ether bonds and hindered phenolic structures into the succinic acid molecular structure to obtain succinic acid derivatives. These derivatives are then used as modifiers to modify gelatin. The reaction between the succinic acid derivatives and the amino groups in the gelatin molecular structure increases the amino group blocking rate and reduces the free amino content, while also increasing its antioxidant properties. This significantly reduces gelatin cross-linking, thereby improving the solubility, storage stability, and bioavailability of the capsule products. The introduction of hindered phenols also effectively improves the acid resistance of the modified gelatin, resulting in excellent resistance to gastric acid erosion in the capsule products. The introduction of ether bonds and hindered phenolic structures into the modified gelatin significantly improves its compatibility with raw materials such as hydroxypropyl starch, eliminating the need for additional thickeners. The components of the capsule material are evenly dispersed and more tightly bound. Furthermore, the synergistic effect of the modified gelatin with raw materials such as tea polyphenols further enhances the antioxidant properties of the capsule products. This invention constructs a compound probiotic capsule by adjusting the raw material ratio and optimizing the process conditions. The capsule product has excellent storage stability, antioxidant properties, and resistance to gastric acid erosion, while also having good enteric solubility and bioavailability. Detailed Implementation

[0018] The technical solutions in the embodiments of this application will be clearly and completely described below with reference to the embodiments of this application. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application.

[0019] Fructooligosaccharides, tea polyphenols, chitosan, and isomalt were all food-grade and purchased from Shaanxi Panier Biotechnology Co., Ltd.; grape polyphenols, grape seed extract, molecular weight 468.42, were purchased from Zhengzhou Best Food Additives Co., Ltd.; gelatin was fish skin gelatin, food-grade, purchased from Wuhan Lanabai Pharmaceutical Chemical Co., Ltd.; mannan oligosaccharides, food-grade, catalog number HH3290YMVN2F, were purchased from Shaanxi Yuanyou Biotechnology Co., Ltd.; hydroxypropyl starch (food-grade, 99% high content) and sucrose fatty acid esters (food-grade, brand Weike) were both purchased from Xi'an Musen Bioengineering Co., Ltd.; Lactobacillus acidophilus LA-G80 and Bifidobacterium longum BL-G101 were both purchased from Junyao Runying Biotechnology (Shanghai) Co., Ltd.; MRS culture medium, catalog number HB0384-5, was purchased from Qingdao Haibo Biotechnology Co., Ltd. All raw materials used in the following examples were commercially available products.

[0020] Example 1

[0021] A method for preparing modified gelatin includes the following steps:

[0022] S1. Under stirring at 160 r / min, (2R,3R)-(-)-epoxysuccinic acid was dispersed in 2-methyltetrahydrofuran. After heating to 65℃, N-ethylmorpholine and 4-methyl-1-pentanol were added sequentially. The mixture was stirred for 7 h, cooled to room temperature, and the solid and liquid were separated. The liquid was collected, and 2-methyltetrahydrofuran was removed by vacuum distillation. The liquid was washed with hot water (70℃ deionized water was added and stirred to dissolve). After cooling to room temperature, the solid and liquid were separated, and the solid was collected and dried under vacuum at 55℃ for 24 h to obtain an intermediate. The molar ratio of (2R,3R)-(-)-epoxysuccinic acid, N-ethylmorpholine, and 4-methyl-1-pentanol was 1:0.03:0.7, and the amount of (2R,3R)-(-)-epoxysuccinic acid added to 2-methyltetrahydrofuran was 0.35 g / mL.

[0023] S2. Under stirring at 160 r / min, 3,5-di-tert-butyl-4-hydroxybenzoic acid was dispersed in 2-methyltetrahydrofuran. After heating to 70℃, the intermediate obtained in step S1 and p-toluenesulfonic acid were added sequentially. The mixture was stirred for 6 h, cooled to room temperature, and the solid and liquid were separated. The liquid was collected, and 2-methyltetrahydrofuran was removed by vacuum distillation. The liquid was washed with hot water (70℃ deionized water was added and stirred to dissolve). After cooling to room temperature, the solid and liquid were separated, and the solid was collected and dried under vacuum at 55℃ for 24 h to obtain a succinic acid derivative. The molar ratio of 3,5-di-tert-butyl-4-hydroxybenzoic acid, the intermediate, and p-toluenesulfonic acid was 1:0.8:0.04, and the amount of 3,5-di-tert-butyl-4-hydroxybenzoic acid added to 2-methyltetrahydrofuran was 0.3 g / mL.

[0024] S3. Under stirring at 160 r / min, gelatin was dispersed in a phosphate buffer solution (pH 7.4), heated to 60℃, and stirred for 1 h. Then, the succinic acid derivative obtained in step S2 was added, and stirring was continued for 3 h. The pH was adjusted to 6-7 with sulfuric acid, and dialyzed for 72 h using a dialysis bag (molecular weight cutoff of 8000-14000D). The deionized water was changed every 12 hours. The solution in the dialysis bag was collected and freeze-dried to obtain modified gelatin (all indicators of the modified gelatin met the requirements of the 2020 edition of the Chinese Pharmacopoeia for capsule gelatin). The mass ratio of gelatin, phosphate buffer solution, and succinic acid derivative was 10:100:3.

[0025] The NMR results for the succinic acid derivatives are as follows: 1 H NMR (300MHz, DMSO-d6) δ14.36 (s, 2H), 7.78 (s, 2H), 7.40 (s, 1H), 6.06 (d, 1H), 5.08 (d , 1H), 3.35 (t, 2H), 1.60-1.65 (m, 1H), 1.43 (s, 20H), 1.18-1.22 (m, 2H), 0.91 (d, 6H).

[0026] A compound probiotic capsule includes a core material and a capsule material, wherein the mass ratio of the core material to the capsule material is 2:7. The core material comprises the following raw materials in parts by weight: 30 parts probiotic liquid, 6 parts fructooligosaccharides, 3 parts tea polyphenols, 3 parts grape polyphenols, 6 parts mannan oligosaccharides, 5 parts isomaltitol, and 2 parts emulsifier. The probiotic liquid is composed of equal weights of Bifidobacterium liquid and Lactobacillus acidophilus liquid. The capsule material comprises the following raw materials in parts by weight: 50 parts modified gelatin, 60 parts distilled water, 8 parts glycerin, 25 parts hydroxypropyl starch, and 17 parts chitosan. The emulsifier is sucrose fatty acid ester. The preparation method of probiotic culture is as follows: Lactobacillus acidophilus LA-G80 is inoculated into MRS medium at a 5% inoculum size and cultured at 37℃ and 100 r / min for 15 h. The bacterial cells are collected by centrifugation (centrifugation speed 8000 r / min, time 15 min), and then prepared to a concentration of 10... 9 Lactobacillus acidophilus culture (cfu / g); Bifidobacterium longum BL-G101 was inoculated into MRS medium at a 5% inoculum and cultured at 37℃ and 100 rpm for 15 h. The bacterial cells were collected by centrifugation (8000 rpm for 15 min), and then prepared to a concentration of 10... 9 Bifidobacterium bacterial suspension (cfu / g); mix equal parts by weight of Bifidobacterium bacterial suspension and Lactobacillus acidophilus bacterial suspension to obtain a concentration of 10. 9 Probiotic liquid with cfu / g.

[0027] A method for preparing a compound probiotic capsule includes the following steps:

[0028] First, fructooligosaccharides, tea polyphenols, grape polyphenols, mannan oligosaccharides, isomaltitol, and emulsifiers are sterilized at 120℃ for 10 minutes. Then, the probiotic solution is mixed evenly with the sterilized fructooligosaccharides, tea polyphenols, grape polyphenols, mannan oligosaccharides, isomaltitol, and emulsifiers to obtain the core material. Glycerin and distilled water are mixed evenly and heated to 70℃. Modified gelatin, hydroxypropyl starch, and chitosan are added sequentially and stirred for 20 minutes. The mixture is then sterilized at 120℃ for 15 minutes and cooled to room temperature to obtain the capsule material. The core material and capsule material are mixed, homogenized under high pressure, and then spray-dried to obtain the composite probiotic capsules. The encapsulation rate of probiotic cells can reach 93.6%. The homogenization pressure of the high-pressure homogenization treatment is 50 MPa, the homogenization time is 15 s, the inlet air temperature of the spray drying is 140℃, the outlet air temperature is 80℃, and the feed flow rate is 15 mL / min.

[0029] Example 2

[0030] A method for preparing modified gelatin, prepared according to the process described in Example 1, except that the mass ratio of gelatin, phosphate buffer solution and succinic acid derivative in step S3 is 10:100:2.

[0031] A compound probiotic capsule includes a core material and a capsule material, wherein the mass ratio of the core material to the capsule material is 2:7. The core material comprises the following raw materials in parts by weight: 30 parts probiotic liquid, 6 parts fructooligosaccharides, 3 parts tea polyphenols, 3 parts grape polyphenols, 6 parts mannan oligosaccharides, 5 parts isomaltitol, and 2 parts emulsifier. The probiotic liquid is composed of equal weights of Bifidobacterium liquid and Lactobacillus acidophilus liquid. The capsule material comprises the following raw materials in parts by weight: 50 parts modified gelatin, 60 parts distilled water, 8 parts glycerin, 25 parts hydroxypropyl starch, and 17 parts chitosan. The emulsifier is sucrose fatty acid ester. The preparation method of probiotic culture is as follows: Lactobacillus acidophilus LA-G80 is inoculated into MRS medium at a 5% inoculum size and cultured at 37℃ and 100 r / min for 15 h. The bacterial cells are collected by centrifugation (centrifugation speed 8000 r / min, time 15 min), and then prepared to a concentration of 10... 9 Lactobacillus acidophilus culture (cfu / g); Bifidobacterium longum BL-G101 was inoculated into MRS medium at a 5% inoculum and cultured at 37℃ and 100 rpm for 15 h. The bacterial cells were collected by centrifugation (8000 rpm for 15 min), and then prepared to a concentration of 10... 9 Bifidobacterium bacterial suspension (cfu / g); mix equal parts by weight of Bifidobacterium bacterial suspension and Lactobacillus acidophilus bacterial suspension to obtain a concentration of 10. 9 Probiotic liquid with cfu / g.

[0032] Example 3

[0033] A method for preparing modified gelatin, prepared according to the process described in Example 1, except that the mass ratio of gelatin, phosphate buffer solution and succinic acid derivative in step S3 is 10:100:4.

[0034] A compound probiotic capsule includes a core material and a capsule material, wherein the mass ratio of the core material to the capsule material is 2:7. The core material comprises the following raw materials in parts by weight: 30 parts probiotic liquid, 6 parts fructooligosaccharides, 3 parts tea polyphenols, 3 parts grape polyphenols, 6 parts mannan oligosaccharides, 5 parts isomaltitol, and 2 parts emulsifier. The probiotic liquid is composed of equal weights of Bifidobacterium liquid and Lactobacillus acidophilus liquid. The capsule material comprises the following raw materials in parts by weight: 50 parts modified gelatin, 60 parts distilled water, 8 parts glycerin, 25 parts hydroxypropyl starch, and 17 parts chitosan. The emulsifier is sucrose fatty acid ester. The preparation method of probiotic culture is as follows: Lactobacillus acidophilus LA-G80 is inoculated into MRS medium at a 5% inoculum size and cultured at 37℃ and 100 r / min for 15 h. The bacterial cells are collected by centrifugation (centrifugation speed 8000 r / min, time 15 min), and then prepared to a concentration of 10... 9 Lactobacillus acidophilus culture (cfu / g); Bifidobacterium longum BL-G101 was inoculated into MRS medium at a 5% inoculum and cultured at 37℃ and 100 rpm for 15 h. The bacterial cells were collected by centrifugation (8000 rpm for 15 min), and then prepared to a concentration of 10... 9 Bifidobacterium bacterial suspension (cfu / g); mix equal parts by weight of Bifidobacterium bacterial suspension and Lactobacillus acidophilus bacterial suspension to obtain a concentration of 10. 9 Probiotic liquid with cfu / g.

[0035] Example 4

[0036] A compound probiotic capsule includes a core material and a capsule material, wherein the mass ratio of the core material to the capsule material is 2:7. The core material comprises the following raw materials in parts by weight: 30 parts probiotic liquid, 6 parts fructooligosaccharides, 3 parts tea polyphenols, 3 parts grape polyphenols, 6 parts mannan oligosaccharides, 5 parts isomaltitol, and 2 parts emulsifier. The probiotic liquid is composed of equal weights of Bifidobacterium liquid and Lactobacillus acidophilus liquid. The capsule material comprises the following raw materials in parts by weight: 40 parts modified gelatin prepared in Example 1, 60 parts distilled water, 8 parts glycerol, 25 parts hydroxypropyl starch, and 17 parts chitosan. The emulsifier is sucrose fatty acid ester. The preparation method of probiotic culture is as follows: Lactobacillus acidophilus LA-G80 is inoculated into MRS medium at a 5% inoculum size and cultured at 37℃ and 100 r / min for 15 h. The bacterial cells are collected by centrifugation (centrifugation speed 8000 r / min, time 15 min), and then prepared to a concentration of 10... 9Lactobacillus acidophilus culture (cfu / g); Bifidobacterium longum BL-G101 was inoculated into MRS medium at a 5% inoculum and cultured at 37℃ and 100 rpm for 15 h. The bacterial cells were collected by centrifugation (8000 rpm for 15 min), and then prepared to a concentration of 10... 9 Bifidobacterium bacterial suspension (cfu / g); mix equal parts by weight of Bifidobacterium bacterial suspension and Lactobacillus acidophilus bacterial suspension to obtain a concentration of 10. 9 The probiotic liquid was prepared at cfu / g. The difference from Example 1 was that the weight percentage of modified gelatin in the capsule material was changed.

[0037] Example 5

[0038] A compound probiotic capsule includes a core material and a capsule material, wherein the mass ratio of the core material to the capsule material is 2:7. The core material comprises the following raw materials in parts by weight: 30 parts probiotic liquid, 6 parts fructooligosaccharides, 3 parts tea polyphenols, 3 parts grape polyphenols, 6 parts mannan oligosaccharides, 5 parts isomaltitol, and 2 parts emulsifier. The probiotic liquid is composed of equal weights of Bifidobacterium liquid and Lactobacillus acidophilus liquid. The capsule material comprises the following raw materials in parts by weight: 60 parts modified gelatin prepared in Example 1, 60 parts distilled water, 8 parts glycerol, 25 parts hydroxypropyl starch, and 17 parts chitosan. The emulsifier is sucrose fatty acid ester. The preparation method of probiotic culture is as follows: Lactobacillus acidophilus LA-G80 is inoculated into MRS medium at a 5% inoculum size and cultured at 37℃ and 100 r / min for 15 h. The bacterial cells are collected by centrifugation (centrifugation speed 8000 r / min, time 15 min), and then prepared to a concentration of 10... 9 Lactobacillus acidophilus culture (cfu / g); Bifidobacterium longum BL-G101 was inoculated into MRS medium at a 5% inoculum and cultured at 37℃ and 100 rpm for 15 h. The bacterial cells were collected by centrifugation (8000 rpm for 15 min), and then prepared to a concentration of 10... 9 Bifidobacterium bacterial suspension (cfu / g); mix equal parts by weight of Bifidobacterium bacterial suspension and Lactobacillus acidophilus bacterial suspension to obtain a concentration of 10. 9 The probiotic liquid was prepared at cfu / g. The difference from Example 1 was that the weight percentage of modified gelatin in the capsule material was changed.

[0039] Example 6

[0040] A compound probiotic capsule includes a core material and a capsule material, wherein the mass ratio of the core material to the capsule material is 1:5. The core material comprises the following raw materials in parts by weight: 30 parts probiotic liquid, 5 parts fructooligosaccharides, 2 parts tea polyphenols, 2 parts grape polyphenols, 8 parts mannan oligosaccharides, 7 parts isomaltitol, and 1 part emulsifier. The probiotic liquid is composed of equal weights of Bifidobacterium liquid and Lactobacillus acidophilus liquid. The capsule material comprises the following raw materials in parts by weight: 50 parts modified gelatin prepared in Example 1, 50 parts distilled water, 5 parts glycerin, 22 parts hydroxypropyl starch, and 20 parts chitosan. The emulsifier is sucrose fatty acid ester. The preparation method of probiotic culture is as follows: Lactobacillus acidophilus LA-G80 is inoculated into MRS medium at a 5% inoculum size and cultured at 37℃ and 100 r / min for 15 h. The bacterial cells are collected by centrifugation (centrifugation speed 8000 r / min, time 15 min), and then prepared to a concentration of 10... 9 Lactobacillus acidophilus culture (cfu / g); Bifidobacterium longum BL-G101 was inoculated into MRS medium at a 5% inoculum and cultured at 37℃ and 100 rpm for 15 h. The bacterial cells were collected by centrifugation (8000 rpm for 15 min), and then prepared to a concentration of 10... 9 Bifidobacterium bacterial suspension (cfu / g); mix equal parts by weight of Bifidobacterium bacterial suspension and Lactobacillus acidophilus bacterial suspension to obtain a concentration of 10. 9 Probiotic liquid with cfu / g.

[0041] Example 7

[0042] A compound probiotic capsule includes a core material and a capsule material, wherein the mass ratio of the core material to the capsule material is 3:9. The core material comprises the following raw materials in parts by weight: 30 parts probiotic liquid, 8 parts fructooligosaccharides, 4 parts tea polyphenols, 4 parts grape polyphenols, 5 parts mannan oligosaccharides, 4 parts isomaltitol, and 3 parts emulsifier. The probiotic liquid is composed of equal weights of Bifidobacterium liquid and Lactobacillus acidophilus liquid. The capsule material comprises the following raw materials in parts by weight: 50 parts modified gelatin prepared in Example 1, 70 parts distilled water, 10 parts glycerol, 30 parts hydroxypropyl starch, and 15 parts chitosan. The emulsifier is sucrose fatty acid ester. The preparation method of probiotic culture is as follows: Lactobacillus acidophilus LA-G80 is inoculated into MRS medium at a 5% inoculum size and cultured at 37℃ and 100 r / min for 15 h. The bacterial cells are collected by centrifugation (centrifugation speed 8000 r / min, time 15 min), and then prepared to a concentration of 10... 9Lactobacillus acidophilus culture (cfu / g); Bifidobacterium longum BL-G101 was inoculated into MRS medium at a 5% inoculum and cultured at 37℃ and 100 rpm for 15 h. The bacterial cells were collected by centrifugation (8000 rpm for 15 min), and then prepared to a concentration of 10... 9 Bifidobacterium bacterial suspension (cfu / g); mix equal parts by weight of Bifidobacterium bacterial suspension and Lactobacillus acidophilus bacterial suspension to obtain a concentration of 10. 9 Probiotic liquid with cfu / g.

[0043] Comparative Example 1

[0044] A compound probiotic capsule includes a core material and a capsule material, wherein the mass ratio of the core material to the capsule material is 2:7. The core material comprises the following raw materials in parts by weight: 30 parts probiotic liquid, 6 parts fructooligosaccharides, 3 parts tea polyphenols, 3 parts grape polyphenols, 6 parts mannan oligosaccharides, 5 parts isomaltitol, and 2 parts emulsifier. The probiotic liquid is composed of equal weights of Bifidobacterium liquid and Lactobacillus acidophilus liquid. The capsule material comprises the following raw materials in parts by weight: 20 parts modified gelatin prepared in Example 1, 60 parts distilled water, 8 parts glycerol, 25 parts hydroxypropyl starch, and 17 parts chitosan. The emulsifier is sucrose fatty acid ester. The preparation method of probiotic culture is as follows: Lactobacillus acidophilus LA-G80 is inoculated into MRS medium at a 5% inoculum size and cultured at 37℃ and 100 r / min for 15 h. The bacterial cells are collected by centrifugation (centrifugation speed 8000 r / min, time 15 min), and then prepared to a concentration of 10... 9 Lactobacillus acidophilus culture (cfu / g); Bifidobacterium longum BL-G101 was inoculated into MRS medium at a 5% inoculum and cultured at 37℃ and 100 rpm for 15 h. The bacterial cells were collected by centrifugation (8000 rpm for 15 min), and then prepared to a concentration of 10... 9 Bifidobacterium bacterial suspension (cfu / g); mix equal parts by weight of Bifidobacterium bacterial suspension and Lactobacillus acidophilus bacterial suspension to obtain a concentration of 10. 9 The probiotic liquid was prepared at cfu / g. The difference from Example 1 was that the weight percentage of modified gelatin in the capsule material was changed.

[0045] Comparative Example 2

[0046] A method for preparing modified gelatin includes the following steps: under stirring at 160 r / min, gelatin is dispersed in a phosphate buffer solution (pH 7.4), heated to 60℃, stirred for 1 h, then (2R,3R)-(-)-epoxysuccinic acid is added, stirring is continued for 3 h, the pH is adjusted to 6-7 with sulfuric acid, dialyzed for 72 h using a dialysis bag (molecular weight cutoff of 8000-14000D), deionized water is replaced every 12 hours, the solution in the dialysis bag is collected and freeze-dried to obtain modified gelatin, wherein the mass ratio of gelatin, phosphate buffer solution, and (2R,3R)-(-)-epoxysuccinic acid is 10:100:0.85.

[0047] A compound probiotic capsule includes a core material and a capsule material, wherein the mass ratio of the core material to the capsule material is 2:7. The core material comprises the following raw materials in parts by weight: 30 parts probiotic liquid, 6 parts fructooligosaccharides, 3 parts tea polyphenols, 3 parts grape polyphenols, 6 parts mannan oligosaccharides, 5 parts isomaltitol, and 2 parts emulsifier. The probiotic liquid is composed of equal weights of Bifidobacterium liquid and Lactobacillus acidophilus liquid. The capsule material comprises the following raw materials in parts by weight: 50 parts modified gelatin, 60 parts distilled water, 8 parts glycerin, 25 parts hydroxypropyl starch, and 17 parts chitosan. The emulsifier is sucrose fatty acid ester. The preparation method of probiotic culture is as follows: Lactobacillus acidophilus LA-G80 is inoculated into MRS medium at a 5% inoculum size and cultured at 37℃ and 100 r / min for 15 h. The bacterial cells are collected by centrifugation (centrifugation speed 8000 r / min, time 15 min), and then prepared to a concentration of 10... 9 Lactobacillus acidophilus culture (cfu / g); Bifidobacterium longum BL-G101 was inoculated into MRS medium at a 5% inoculum and cultured at 37℃ and 100 rpm for 15 h. The bacterial cells were collected by centrifugation (8000 rpm for 15 min), and then prepared to a concentration of 10... 9 Bifidobacterium bacterial suspension (cfu / g); mix equal parts by weight of Bifidobacterium bacterial suspension and Lactobacillus acidophilus bacterial suspension to obtain a concentration of 10. 9 Probiotic liquid with cfu / g.

[0048] Comparative Example 3

[0049] A compound probiotic capsule includes a core material and a capsule material, wherein the mass ratio of the core material to the capsule material is 2:7. The core material comprises the following raw materials in parts by weight: 30 parts probiotic liquid, 6 parts fructooligosaccharides, 3 parts tea polyphenols, 3 parts grape polyphenols, 6 parts mannan oligosaccharides, 5 parts isomaltitol, and 2 parts emulsifier. The probiotic liquid is composed of equal weights of Bifidobacterium liquid and Lactobacillus acidophilus liquid. The capsule material comprises the following raw materials in parts by weight: 50 parts gelatin, 60 parts distilled water, 8 parts glycerin, 25 parts hydroxypropyl starch, and 17 parts chitosan. The emulsifier is sucrose fatty acid ester. The preparation method of probiotic culture is as follows: Lactobacillus acidophilus LA-G80 is inoculated into MRS medium at a 5% inoculum size and cultured at 37℃ and 100 r / min for 15 h. The bacterial cells are collected by centrifugation (centrifugation speed 8000 r / min, time 15 min), and then prepared to a concentration of 10... 9 Lactobacillus acidophilus culture (cfu / g); Bifidobacterium longum BL-G101 was inoculated into MRS medium at a 5% inoculum and cultured at 37℃ and 100 rpm for 15 h. The bacterial cells were collected by centrifugation (8000 rpm for 15 min), and then prepared to a concentration of 10... 9 Bifidobacterium bacterial suspension (cfu / g); mix equal parts by weight of Bifidobacterium bacterial suspension and Lactobacillus acidophilus bacterial suspension to obtain a concentration of 10. 9 Probiotic liquid with cfu / g.

[0050] The compound probiotic capsules described in Examples 2-7 and Comparative Examples 1-3 were prepared according to the method described in Example 1. The resulting compound probiotic capsules had intact particles, uniform particle shape, and good uniformity. All indicators met the requirements of the 2020 edition of the Chinese Pharmacopoeia.

[0051] The following tests were conducted on the performance of the compound probiotic capsules prepared in Examples 1-7 and Comparative Examples 1-3. Storage stability test: 3g sample was placed at (25±1)℃ and (45±2)% humidity for 210 days. After removal, the viable bacteria count was detected by plate counting method, and the survival rate of probiotic cells was calculated. The calculation formula is: cell survival rate = viable bacteria count after test / viable bacteria count before test × 100%. Antioxidant performance test: The sample was placed in soybean oil, mixed evenly, and placed in the dark at (25±1)℃ and (60±2)% humidity for 60 days. The peroxide value of soybean oil was determined according to GB / T5538-2005 "Determination of Peroxide Value of Animal and Vegetable Oils". Survival rate test of probiotic cells in simulated digestive fluid: 1g sample was dispersed in 100mL of simulated gastric fluid, mixed evenly, and incubated with shaking at 37℃ and 100rpm for 6h. After centrifugation at 5000rpm for 8min, the supernatant was removed, the cells were collected, and the cells were analyzed by using a non-acidic digestive fluid. After washing with water, the bacteria were dispersed in 100 mL of simulated intestinal fluid, mixed evenly, and incubated at 37℃ and 100 rpm for 6 h with shaking. After centrifugation at 5000 rpm for 8 min, the supernatant was removed, the bacterial cells were collected, washed with sterile water, and serially diluted. The viable cell count was determined by plate counting and the bacterial survival rate was calculated using the following formulas: bacterial survival rate in simulated gastric fluid = viable cell count after incubation in gastric fluid / initial viable cell count × 100%; bacterial survival rate in simulated intestinal fluid = viable cell count after incubation in intestinal fluid / initial viable cell count × 100%. The solubility of probiotic capsules in simulated digestive fluid was tested by dispersing 1 g of sample in 100 mL of simulated gastric fluid and 100 mL of simulated intestinal fluid, respectively, and incubating at 37℃ and 100 rpm with shaking. Samples were taken at 0 h and 6 h of incubation to measure the transmittance of the samples. All tests were repeated three times, and the test results are shown in Table 1. As can be seen from the table, the storage stability, antioxidant properties, and survival rate in simulated digestive fluid of the compound probiotic capsules prepared in Examples 1-7 are significantly better than those in Comparative Examples 1-3. The solubility of the probiotic capsules in simulated digestive fluid shows that the transmittance of the compound probiotic capsules prepared in Examples 1-7 decreased by less than 1% after 6 hours of incubation in simulated gastric fluid, but decreased by more than 62% after 6 hours of incubation in simulated intestinal fluid. In contrast, the transmittance of the compound probiotic capsules prepared in Comparative Example 3 decreased by more than 13% after 6 hours of incubation in simulated gastric fluid. After being cultured in simulated intestinal fluid for 6 hours, the permeability decreased by about 64%. The above results indicate that the compound probiotic capsules prepared in Examples 1-7 have a strong ability to resist gastric acid erosion, and at the same time have good solubility in intestinal fluid, which can effectively release probiotics, give full play to the beneficial effects of probiotics, and improve the bioavailability of capsules. From the effect data of Examples 1-5 and Comparative Example 1, it can be seen that the amount of succinic acid derivative added to modified gelatin and the weight fraction of modified gelatin in capsule material have a significant impact on the overall performance of compound probiotic capsules.

[0052] Table 1 Performance test results of compound probiotic capsules

[0053]

[0054] Although embodiments of this application have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of this application, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A compound probiotic capsule, characterized in that, The product comprises a core material and a capsule material, wherein the mass ratio of the core material to the capsule material is 1-3:5-9. The core material comprises the following raw materials in parts by weight: 20-40 parts probiotic liquid, 5-8 parts fructooligosaccharides, 2-4 parts tea polyphenols, 2-4 parts grape polyphenols, 5-8 parts mannan oligosaccharides, 3-7 parts isomaltitol, and 1-3 parts emulsifier. The probiotic liquid is composed of equal weights of Bifidobacterium liquid and Lactobacillus acidophilus liquid. The capsule material comprises the following raw materials in parts by weight: 40-60 parts modified gelatin, 50-70 parts distilled water, 5-10 parts glycerin, 20-30 parts hydroxypropyl starch, and 15-20 parts chitosan. The modified gelatin is prepared as follows: S1. Under stirring conditions, (2R,3R)-(-)-epoxysuccinic acid was dispersed in 2-methyltetrahydrofuran. After heating to 60-70℃, N-ethylmorpholine and 4-methyl-1-pentanol were added sequentially. The mixture was stirred for 5-8 hours, cooled to room temperature, and the solid and liquid phases were separated. The liquid was collected, and after vacuum distillation, washing, and drying, an intermediate was obtained. The structural formula of the intermediate is: ； S2. Under stirring conditions, 3,5-di-tert-butyl-4-hydroxybenzoic acid was dispersed in 2-methyltetrahydrofuran. After heating to 65-75℃, the intermediate obtained in step S1 and p-toluenesulfonic acid were added sequentially. The mixture was stirred for 4-9 hours, cooled to room temperature, and the solid and liquid were separated. The liquid was collected, and after vacuum distillation, washing, and drying, a succinic acid derivative was obtained. The structural formula of the succinic acid derivative is: ； S3. Under stirring conditions, gelatin is dispersed in a phosphate buffer solution, heated to 50-70℃, and stirred for 0.5-1.5 hours. Then, the succinic acid derivative obtained in step S2 is added, and stirring is continued for 2-4 hours. The pH is adjusted to 6-7, and modified gelatin is obtained by dialysis and freeze-drying. The mass ratio of gelatin, phosphate buffer solution, and succinic acid derivative is 10:80-120:2-4. In step S1, the molar ratio of (2R,3R)-(-)-epoxysuccinic acid, N-ethylmorpholine, and 4-methyl-1-pentanol is 1:0.02-0.05:0.6-0.8, and the amount of (2R,3R)-(-)-epoxysuccinic acid added to the 2-methyltetrahydrofuran is 0.25-0.45 g / mL. In step S2, the molar ratio of 3,5-di-tert-butyl-4-hydroxybenzoic acid, the intermediate, and p-toluenesulfonic acid is 1:0.7-0.9:0.03-0.06, and the amount of 3,5-di-tert-butyl-4-hydroxybenzoic acid added to the 2-methyltetrahydrofuran is 0.2-0.4 g / mL.

2. The compound probiotic capsule according to claim 1, characterized in that, The emulsifier is one of sucrose fatty acid ester, polyglycerol fatty acid ester, Tween-80, and beeswax.

3. The compound probiotic capsule according to claim 1, characterized in that, The concentration of the probiotic solution is not less than 10. 8 cfu / g.

4. A method for preparing a compound probiotic capsule according to any one of claims 1-3, characterized in that, The process includes the following steps: A core material is obtained by uniformly mixing probiotic culture, fructooligosaccharides, tea polyphenols, grape polyphenols, mannan oligosaccharides, isomaltitol, and emulsifier; a capsule material is obtained by uniformly mixing glycerin and distilled water and heating the mixture to 60-80℃, then sequentially adding modified gelatin, hydroxypropyl starch, and chitosan, stirring for 10-30 minutes, sterilizing, and cooling to room temperature; and finally, a capsule material is obtained by mixing the core material and the capsule material, homogenizing under high pressure, and then spray drying.

5. The method for preparing a compound probiotic capsule according to claim 4, characterized in that, The homogenization pressure of the high-pressure homogenization process is 40-60 MPa, and the homogenization time is 10-20 s.

6. The method for preparing a compound probiotic capsule according to claim 4, characterized in that, The spray dryer has an inlet air temperature of 120-150℃, an outlet air temperature of 70-90℃, and a feed flow rate of 10-20mL / min.

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