An enteric-coated lactic acid bacteria capsule and its preparation method

Abstract

The present invention specifically relates to an enteric-coated lactic acid bacteria capsule and a preparation method thereof. The present invention includes the following steps: Step S1: The lactic acid bacteria are sequentially activated and centrifuged to obtain a lactic acid bacteria sludge, which is then resuspended with sterile physiological saline to obtain a bacterial suspension; Step S2: Sodium alginate, a composite and distilled water are mixed evenly, calcium carbonate is added, and swelling is carried out for 22-24 h to obtain a composite wall material solution; Step S3: The composite wall material solution and the bacterial suspension are mixed evenly, added to soybean oil containing an emulsifier, stirred evenly to form a uniform water-in-oil emulsion, glacial acetic acid is added, stirred evenly, allowed to stand for 1-2 h to obtain a precipitate, and centrifuged to obtain the enteric-coated lactic acid bacteria capsule. The enteric-coated lactic acid bacteria capsule prepared by the present invention has high acid resistance, bile salt resistance and survival rate, and can achieve a targeted release effect in the intestine.

Description

Technical Field

[0001] The present invention specifically relates to an enteric-coated lactic acid bacteria capsule and a preparation method thereof. Background Art

[0002] With the improvement of people's living standards, probiotic products have gradually received extensive attention. As an important type of probiotic, lactic acid bacteria have physiological functions such as regulating the balance of intestinal flora, enhancing immunity, and promoting digestion and absorption.

[0003] In traditional probiotic supplementation methods, lactic acid bacteria often exist in liquid or powder form. However, these forms are prone to a large amount of inactivation in adverse environments such as storage and gastrointestinal digestion, resulting in a lack of sufficient viable bacteria to reach the human colon smoothly and exert their probiotic effects. To solve this problem, enteric-coated lactic acid bacteria capsules have emerged.

[0004] Currently, more research has been conducted on starches and plant-based proteins as capsule materials. These raw materials have certain film-forming properties, but their mechanical strength cannot meet the requirements of packaging, and there are disadvantages such as low encapsulation rate and large particle size, which limit their industrial application. Usually, chemical modification is used to improve their performance. Among them, Maillard products (MRPs) generated by the Maillard reaction are used as important encapsulants for protecting probiotics due to their activities such as film-promoting and antioxidant properties. However, the Maillard reaction is limited in application due to the generation of advanced glycation end products. To solve this problem, the present invention modifies proteins by enzymatic grafting reaction. Compared with the Maillard reaction, the by-product of the enzymatic grafting reaction is only water, without other harmful by-products, and the enzymatic reaction has specificity and high reaction efficiency, which is a safe and effective modification method.

[0005] Therefore, we propose an enteric-coated lactic acid bacteria capsule and a preparation method thereof. Summary of the Invention

[0006] The purpose of the present invention is to provide an enteric-coated lactic acid bacteria capsule and a preparation method thereof to solve the problems raised in the prior art.

[0007] To achieve the above purpose, the present invention provides the following technical solutions:

[0008] A preparation method of an enteric-coated lactic acid bacteria capsule, comprising the following steps:

[0009] Step S1: Activate and centrifuge lactic acid bacteria in sequence to obtain a lactic acid bacteria sludge, and then resuspend it with sterile physiological saline to obtain a bacterial suspension;

[0010] Step S2: Disperse rice protein in distilled water to obtain a rice protein suspension. Adjust the pH of the system to 8 - 9, keep it warm at 40 - 50 °C for 15 - 30 min, add α-chymotrypsin, react until the degree of hydrolysis reaches 4%, inactivate the enzyme and then centrifuge, collect the supernatant, and after dialysis and freeze-drying, obtain rice protein hydrolysate;

[0011] Disperse the rice protein hydrolysate in distilled water to obtain a rice protein hydrolysate solution. Adjust the pH of the system to 4 - 5, add laccase, stir for 2 - 4 h, add supported feruloyl oligosaccharide, continue to react for 22 - 24 h, inactivate the enzyme and then centrifuge, and after dialysis and freeze-drying, obtain a complex;

[0012] Mix sodium alginate, the complex and distilled water evenly, add calcium carbonate, and swell for 22 - 24 h to obtain a composite wall material solution;

[0013] Step S3: Mix the composite wall material solution and the bacterial suspension evenly, add them to soybean oil containing an emulsifier, stir evenly to form a uniform water-in-oil (W / O) emulsion, add glacial acetic acid, stir evenly, let it stand for 1 - 2 h to obtain a precipitate, centrifuge and separate to obtain enteric-coated lactic acid bacteria capsules.

[0014] Further, in the said Step S1, the specific preparation process of the lactic acid bacteria sludge is: inoculate lactic acid bacteria into MRS liquid medium, cultivate at a constant temperature of 37 °C for 24 h, after activation twice, then inoculate into MRS liquid medium with an inoculation amount of 1% (V / V), cultivate at a constant temperature of 37 °C for 24 h, and then centrifuge at 4 °C and 10000 rpm for 10 min to remove the supernatant to obtain lactic acid bacteria sludge.

[0015] Further, in the said Step S1, the viable count of the bacterial suspension is 1×10 9 -2×10 10 cfu / mL.

[0016] Further, in the said Step S2, the composite wall material solution includes raw materials with the following mass concentrations: sodium alginate 3 - 5%, the complex 5 - 10%, calcium carbonate 1 - 2%, and the balance is distilled water.

[0017] Further, the preparation method of the supported feruloyl oligosaccharide is as follows:

[0018] Step (1): Mix feruloyl oligosaccharide and distilled water evenly, stir at 70 - 80 °C for 2 - 3 h, hydrate at 3 - 5 °C for 10 - 12 h to obtain a feruloyl oligosaccharide solution, adjust the pH value to 8 - 9 with sodium hydroxide, add octenyl succinic anhydride, react at 40 - 50 °C for 1 - 2 h, adjust the pH value of the solution to 5.5 - 6.0 with hydrochloric acid, cool to room temperature, and after centrifugation, washing, dialysis and freeze-drying, obtain modified feruloyl oligosaccharide;

[0019] Step (2): Mix the modified feruloyl oligosaccharide and distilled water evenly, hydrate at 3 - 5 °C for 10 - 12 h to obtain a modified feruloyl oligosaccharide solution, add quercetin, homogenize at 40 - 50 °C for 5 - 10 min, centrifuge, collect the supernatant, and after dialysis and freeze-drying, obtain the loaded feruloyl oligosaccharide.

[0020] In the above technical solution, using feruloyl oligosaccharide rich in hydroxyl groups as the raw material, and using octenyl succinic anhydride (OSA) as a hydrophobizing agent to modify it, a kind of amphiphilic polysaccharide carrier, namely modified feruloyl oligosaccharide, is successfully prepared, which can effectively load hydrophobic quercetin; the feruloyl oligosaccharide loaded with quercetin is prepared by self-assembly to form the loaded feruloyl oligosaccharide; chymotrypsin is used to hydrolyze rice protein to expose amino acid residues such as tyrosine, and then under the catalytic action of laccase, the tyrosine residues in the rice protein hydrolysate react with ferulic acid and quercetin in the loaded feruloyl oligosaccharide to produce a complex, significantly improving the emulsifying stability and antioxidant property of the protein.

[0021] Further, in the step (1), the mass ratio of feruloyl oligosaccharide, distilled water and octenyl succinic anhydride is 1:(35 - 45):(1 - 2).

[0022] Further, in the step (1), the concentration of the sodium hydroxide solution is 3 wt%, and the concentration of the hydrochloric acid is 1 mol / L.

[0023] Further, in the step (2), the concentration of the modified feruloyl oligosaccharide solution is 0.2 - 0.4 wt%.

[0024] Further, in the step (2), the mass of quercetin is 0.2 - 0.5 times the mass of the modified feruloyl oligosaccharide.

[0025] Further, in the step S2, the concentration of the rice protein suspension is 7 - 8 wt%.

[0026] Further, in the step S2, the addition amount ratio of α-chymotrypsin to rice protein is 300 - 320 U / g.

[0027] Further, in the step S2, the concentration of the rice protein hydrolysate solution is 4 - 6 mg / mL.

[0028] Further, in the step S2, the dosage of laccase is 1.5 - 2.0 U / mL.

[0029] Further, in the step S2, the mass of the loaded feruloyl oligosaccharide is 0.5 - 1.0 times the mass of the rice protein hydrolysate.

[0030] Further, in the step S3, the mass ratio of the composite wall material solution to the bacterial suspension is 4:(1 - 3).

[0031] Further, in the step S3, the mass of soybean oil is 2 - 3 times the total mass of the composite wall material solution and the bacterial suspension.

[0032] Further, in the step S3, the emulsifier is one of Tween - 80, Span - 80, and Span - 20, and the addition amount of the emulsifier is 0.4 - 0.8% of the mass of soybean oil.

[0033] Further, in the step S3, the mass of glacial acetic acid is 0.3 - 0.6% of the total mass of the composite wall material solution and the bacterial suspension.

[0034] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0035] 1. For an enteric - coated lactic acid bacteria capsule and its preparation method of the present invention, using feruloylated oligosaccharides rich in hydroxyl groups as raw materials, and using octenyl succinic anhydride (OSA) as a hydrophobizing agent to modify it to prepare an amphiphilic carrier. Quercetin (que) is a plant polyphenol with anti - inflammatory and antioxidant properties. By self - assembly, feruloylated oligosaccharides loaded with quercetin are prepared, enabling quercetin to be effectively loaded into the structure of feruloylated oligosaccharides, improving its water solubility and bioavailability. Chymotrypsin is used to hydrolyze rice protein to expose amino acid residues such as tyrosine, and then enzymatic cross - linking catalysis is used to covalently bind the loaded feruloylated oligosaccharides to the rice protein hydrolysate to obtain a complex, significantly improving the emulsification stability and antioxidant properties of the protein. At the same time, the relatively large molecular weight and spatial structure of the loaded feruloylated oligosaccharides will produce a hindrance effect on the protein surface, reducing the binding sites of digestive enzymes on the protein surface and enhancing the steric hindrance of the protein, thereby enhancing the anti - digestion ability of the protein. Therefore, it helps to improve the stability of the encapsulated active substances during gastrointestinal digestion. Using sodium alginate, the complex, and calcium carbonate as a composite wall material, and preparing plant lactobacillus microcapsules by the endogenous emulsification gel method, which has good acid resistance and enteric solubility, can delay the release of the core material in the simulated gastric digestion environment, ensure the stability of quercetin and lactic acid bacteria in the gastric acid environment, and thus achieve precise release in the intestine. Specific Embodiments

[0036] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0037] In this example, the lactic acid bacteria: Lactobacillus plantarum, with the preservation number of CGMCC No. 16441, is preserved in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms; calcium carbonate: model ZC-1, food grade, sourced from Henan Zhongda Hengyuan Biotechnology Co., Ltd.; soybean oil: product number S817900, sourced from Shanghai Macklin Biochemical Co., Ltd.; emulsifier: Tween-80, food grade; rice protein: product number QYC03240312, sourced from Xi'an Qianye Cao Biotechnology Co., Ltd.; α-chymotrypsin: product number M10831, sourced from Hu Hui (Shanghai) Biotechnology Co., Ltd.; laccase: product number 18-2, sourced from Jinan Kangxu Biotechnology Co., Ltd.; feruloyl oligosaccharides are derived from wheat bran, and the feruloyl oligosaccharides obtained by high-temperature steaming method, and the specific preparation steps refer to the prior art (Ge Lihua, Research on the Preparation and Antioxidant Properties of Ferulic Acid Oligosaccharides [D], Northeastern University, April 1, 2007).

[0038] Example 1: A preparation method of enteric-coated lactic acid bacteria capsules, comprising the following processes:

[0039] Step S1: Inoculate the lactic acid bacteria into MRS liquid medium, cultivate at a constant temperature of 37°C for 24 h, after activation twice, then inoculate into MRS liquid medium with an inoculation amount of 1% (V / V) (the medium components are: peptone 10.0 g, beef extract 10.0 g, yeast extract 5.0 g, diammonium hydrogen citrate 2.0 g, glucose 20.0 g, Tween-80 1.0 mL, sodium acetate 5.0 g, dipotassium hydrogen phosphate 2.0 g, magnesium sulfate 0.58 g, manganese sulfate 0.25 g, distilled water 1000 mL, adjust the pH to 6.5), after cultivating at a constant temperature of 37°C for 24 h, centrifuge at 4°C and 10000 rpm for 10 min, remove the supernatant to obtain lactic acid bacteria sludge, and then resuspend with sterile normal saline to obtain a bacterial suspension;

[0040] Step S2: Disperse 10 g of rice protein in distilled water to obtain a 7 wt% rice protein suspension, adjust the system pH to 8, keep it warm at 40°C for 15 min, add 3000 U of α-chymotrypsin, react until the degree of hydrolysis is 4%, inactivate the enzyme and then centrifuge, collect the supernatant, after dialysis and freeze-drying, obtain rice protein hydrolysate;

[0041] Disperse 10 g of rice protein hydrolysate in distilled water to obtain a 4 mg / mL rice protein hydrolysate solution, adjust the system pH to 4, add 1.5 U / mL of laccase, stir for 2 h, add 5 g of supported feruloyl oligosaccharides, continue to react for 22 h, inactivate the enzyme and then centrifuge, after dialysis and freeze-drying, obtain a complex;

[0042] Mix sodium alginate, the complex, and distilled water evenly, add calcium carbonate, and swell for 22 h to obtain a composite wall material solution; the concentration of sodium alginate in the composite wall material solution is 3 wt%, the concentration of the complex is 5 wt%, and the concentration of calcium carbonate is 1 wt%.

[0043] Step S3: Mix 100 g of the composite wall material solution and 25 g of the bacterial suspension evenly, add them to 250 g of soybean oil containing 0.4 wt% emulsifier, stir evenly to form a uniform water-in-oil (W / O) emulsion, add 0.375 g of glacial acetic acid, stir evenly, let stand for 1 h to obtain a precipitate, and perform centrifugal separation to obtain enteric-coated lactic acid bacteria capsules.

[0044] The preparation method of the loaded feruloyl oligosaccharides is as follows:

[0045] Step (1): Mix 5 g of feruloyl oligosaccharides and 175 g of distilled water evenly, stir at 70 °C for 2 h, hydrate at 3 °C for 10 h to obtain a feruloyl oligosaccharide solution, adjust the pH value to 8 with 3 wt% sodium hydroxide, add 5 g of octenyl succinic anhydride, react at 40 °C for 1 h, adjust the pH value of the solution to 5.5 with 1 mol / L hydrochloric acid, cool to room temperature, and after centrifugation, washing, dialysis, and freeze-drying, obtain modified feruloyl oligosaccharides.

[0046] Step (2): Mix 5 g of the modified feruloyl oligosaccharides and distilled water evenly, hydrate at 3 °C for 10 h to obtain a 0.2 wt% modified feruloyl oligosaccharide solution, add 1 g of quercetin, homogenize at 40 °C for 5 min, centrifuge, collect the supernatant, and after dialysis and freeze-drying, obtain the loaded feruloyl oligosaccharides.

[0047] Example 2: A preparation method of enteric-coated lactic acid bacteria capsules, including the following process:

[0048] Step S1: Inoculate lactic acid bacteria into MRS liquid medium, cultivate at a constant temperature of 37 °C for 24 h, after activation twice, inoculate again into MRS liquid medium at an inoculation amount of 1% (V / V) (the medium components are: peptone 10.0 g, beef extract 10.0 g, yeast extract 5.0 g, diammonium hydrogen citrate 2.0 g, glucose 20.0 g, Tween-80 1.0 mL, sodium acetate 5.0 g, dipotassium hydrogen phosphate 2.0 g, magnesium sulfate 0.58 g, manganese sulfate 0.25 g, distilled water 1000 mL, adjust the pH to 6.5), after cultivating at a constant temperature of 37 °C for 24 h, centrifuge at 4 °C and 10000 rpm for 10 min, remove the supernatant to obtain lactic acid bacteria mud, and then resuspend it with sterile physiological saline to obtain a bacterial suspension.

[0049] Step S2: Disperse 10 g of rice protein in distilled water to obtain a 7.5 wt% rice protein suspension. Adjust the pH of the system to 8.5, keep it warm at 45 °C for 20 min, add 3100 U of α-chymotrypsin, react until the degree of hydrolysis reaches 4%, inactivate the enzyme and then centrifuge. Collect the supernatant, dialyze it, and freeze-dry it to obtain rice protein hydrolysate;

[0050] Disperse 10 g of rice protein hydrolysate in distilled water to obtain a 5 mg / mL rice protein hydrolysate solution. Adjust the pH of the system to 4.5, add 1.8 U / mL of laccase, stir for 3 h, add 8 g of supported feruloyl oligosaccharide, continue to react for 23 h, inactivate the enzyme and then centrifuge. After dialysis and freeze-drying, obtain the complex;

[0051] Mix sodium alginate, the complex, and distilled water evenly, add calcium carbonate, and swell for 23 h to obtain a composite wall material solution; the concentration of sodium alginate in the composite wall material solution is 4 wt%, the concentration of the complex is 8 wt%, and the concentration of calcium carbonate is 1.5 wt%;

[0052] Step S3: Mix 100 g of the composite wall material solution and 50 g of the bacterial suspension evenly, add them to 375 g of soybean oil containing 0.6 wt% emulsifier, stir evenly to form a uniform water-in-oil (W / O) emulsion, add 0.75 g of glacial acetic acid, stir evenly, let it stand for 1.5 h to obtain a precipitate, centrifuge and separate to obtain enteric-coated lactic acid bacteria capsules;

[0053] The preparation method of the supported feruloyl oligosaccharide is as follows:

[0054] Step (1): Mix 8 g of feruloyl oligosaccharide and 320 g of distilled water evenly, stir at 75 °C for 2.5 h, hydrate at 4 °C for 11 h to obtain a feruloyl oligosaccharide solution. Adjust the pH value to 8.5 with 3 wt% sodium hydroxide, add 12 g of octenyl succinic anhydride, react at 45 °C for 1.5 h, adjust the pH value of the solution to 5.8 with 1 mol / L hydrochloric acid, cool to room temperature, and after centrifugation, washing, dialysis, and freeze-drying, obtain modified feruloyl oligosaccharide;

[0055] Step (2): Mix 8 g of modified feruloyl oligosaccharide and distilled water evenly, hydrate at 4 °C for 11 h to obtain a 0.3 wt% modified feruloyl oligosaccharide solution, add 3.2 g of quercetin, homogenize at 45 °C for 8 min, centrifuge, collect the supernatant, and after dialysis and freeze-drying, obtain the supported feruloyl oligosaccharide.

[0056] Example 3: A preparation method of enteric-coated lactic acid bacteria capsules, including the following processes:

[0057] Step S1: Inoculate lactic acid bacteria into MRS liquid medium and incubate at 37°C for 24 h. After activation twice, inoculate it into MRS liquid medium at an inoculation amount of 1% (V / V) (the medium components are: peptone 10.0 g, beef extract 10.0 g, yeast extract 5.0 g, diammonium hydrogen citrate 2.0 g, glucose 20.0 g, Tween-80 1.0 mL, sodium acetate 5.0 g, dipotassium hydrogen phosphate 2.0 g, magnesium sulfate 0.58 g, manganese sulfate 0.25 g, distilled water 1000 mL, adjust the pH to 6.5). After incubating at 37°C for 24 h, centrifuge at 4°C and 10000 rpm for 10 min, remove the supernatant to obtain lactic acid bacteria sludge, and then resuspend it with sterile normal saline to obtain a bacterial suspension;

[0058] Step S2: Disperse 10 g of rice protein in distilled water to obtain a rice protein suspension. Adjust the pH of the system to 9, keep it warm at 50°C for 30 min, add 3200 U of α-chymotrypsin, react until the degree of hydrolysis reaches 4%, inactivate the enzyme and then centrifuge, collect the supernatant, and after dialysis and freeze-drying, obtain rice protein hydrolysate;

[0059] Disperse 10 g of rice protein hydrolysate in distilled water to obtain a 6 mg / mL rice protein hydrolysate solution. Adjust the pH of the system to 5, add 2.0 U / mL of laccase, stir for 4 h, add 10 g of supported feruloyl oligosaccharide, continue to react for 24 h, inactivate the enzyme and then centrifuge, and after dialysis and freeze-drying, obtain a complex;

[0060] Mix sodium alginate, the complex and distilled water evenly, add calcium carbonate, and swell for 24 h to obtain a composite wall material solution; the concentration of sodium alginate in the composite wall material solution is 5 wt%, the concentration of the complex is 10 wt%, and the concentration of calcium carbonate is 2 wt%;

[0061] Step S3: Mix 100 g of the composite wall material solution and 75 g of the bacterial suspension evenly, add them to 525 g of soybean oil containing 0.8 wt% emulsifier, stir evenly to form a uniform water-in-oil (W / O) emulsion, add 1.05 g of glacial acetic acid, stir evenly, let it stand for 2 h to obtain a precipitate, centrifuge and separate to obtain enteric-coated lactic acid bacteria capsules;

[0062] The preparation method of supported feruloyl oligosaccharide is as follows:

[0063] Step (1): Mix 10 g of feruloyl oligosaccharide and 450 g of distilled water evenly, stir at 80°C for 3 h, hydrate at 5°C for 12 h to obtain a feruloyl oligosaccharide solution, adjust the pH value to 9 with 3 wt% sodium hydroxide, add 20 g of octenyl succinic anhydride, react at 50°C for 2 h, adjust the pH value of the solution to 6.0 with 1 mol / L hydrochloric acid, cool to room temperature, and after centrifugation, washing, dialysis and freeze-drying, obtain modified feruloyl oligosaccharide;

[0064] Step (2): Mix 10 g of modified feruloyl oligosaccharide and distilled water evenly, hydrate at 5 °C for 12 h to obtain a 0.4 wt% modified feruloyl oligosaccharide solution, add 5 g of quercetin, homogenize at 50 °C for 10 min, centrifuge, collect the supernatant, and after dialysis and freeze-drying, obtain the loaded feruloyl oligosaccharide.

[0065] Comparative Example 1: A method for preparing an enteric-coated lactic acid bacteria capsule, including the following process:

[0066] Compared with Example 2, the composite wall material solution in Comparative Example 1 does not add the complex, and other steps are the same as those in Example 2.

[0067] Comparative Example 2: A method for preparing an enteric-coated lactic acid bacteria capsule, including the following process:

[0068] Compared with Example 2, in step S2 of Comparative Example 2, the composite wall material solution includes raw materials with the following mass concentrations: sodium alginate 4%, complex 15%, calcium carbonate 1.5%, and the balance is distilled water, and other steps are the same as those in Example 2.

[0069] Comparative Example 3: A method for preparing an enteric-coated lactic acid bacteria capsule, including the following process:

[0070] Compared with Example 2, in Comparative Example 3, the modified feruloyl oligosaccharide is replaced with the same mass of feruloyl oligosaccharide, and other steps are the same as those in Example 2.

[0071] Experiment: Take the enteric-coated lactic acid bacteria capsules obtained in Examples 1-3 and Comparative Examples 1-3, prepare specimens, and detect their properties respectively and record the detection results:

[0072] Determination of the embedding rate: Lyse the prepared enteric-coated lactic acid bacteria capsule with a sodium citrate solution. Take 1 g of the prepared enteric-coated lactic acid bacteria capsule and place it in 9 mL of 3% sodium citrate solution for lysis. The solution is placed in a 37 °C water bath constant temperature oscillator and shaken evenly at 180 r / min for 60 min. After lysis, perform viable cell counting. According to the formula, the embedding rate = N / N1×100% is calculated, where N is the number of viable bacteria encapsulated in the capsule and N1 is the number of viable bacteria added before embedding.

[0073] Determination of survival rate under continuous simulated gastrointestinal fluid conditions: The simulated gastric fluid contains 10 mg / mL pepsin, and the pH value is adjusted to 2.5 with 0.1 mol / L hydrochloric acid. Take 1 g of the prepared enteric-coated lactic acid bacteria capsules and add them to 9 mL of pre-warmed (37 °C) and filter-sterilized simulated gastric fluid. After culturing at 37 °C and 120 r / min for 2 h, perform viable plate counting to calculate the survival rate; then transfer to 9 mL of simulated intestinal fluid and continue digestion for 8 h, and then measure the survival rate; the simulated intestinal fluid contains 1 mg / mL trypsin and 0.3% bile salts, and the pH value is adjusted to 6.8 with 0.1 mol / L sodium hydroxide.

[0074] The test results are as follows:

[0075]

[0076] Based on the data in the above table, the following conclusions can be clearly obtained:

[0077] 1. Compared with Examples 1-3, the entrapment rate and survival rate of the products obtained in Comparative Example 1 and Comparative Example 2 both decreased, indicating that the addition of the complex in the present invention is beneficial to improving the entrapment rate and survival rate; as the addition amount of the complex increases, the viscosity of the mixed solution increases, resulting in a poor magnetic stirring effect, thereby affecting the emulsification process, and finally showing a significant decrease in the entrapment rate. It can be seen that the performance of the composite wall material solution prepared in the present invention is affected by its component ratio. By selecting the component ratio within the above range, a composite wall material solution with excellent performance can be prepared.

[0078] 2. Compared with Examples 1-3, the entrapment rate and survival rate of the product obtained in Comparative Example 3 both decreased, indicating that the modified feruloyl oligosaccharide prepared in the present invention has amphiphilicity and is more conducive to forming a stable complex and improving the entrapment efficiency and biocompatibility of the carrier compared with the unmodified feruloyl oligosaccharide.

[0079] For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and can be implemented in other specific forms without departing from the spirit or basic characteristics of the present invention. Therefore, from any point of view, the embodiments should be regarded as exemplary and non-limiting. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be included in the present invention.

Claims

1. A preparation method of enteric-coated lactic acid bacteria capsules, characterized in that: It includes the following steps: Step S1: Activate lactic acid bacteria successively and perform centrifugal separation to obtain lactic acid bacteria sludge, and then resuspend it with sterile physiological saline to obtain a bacterial suspension; Step S2: Disperse rice protein in distilled water to obtain a rice protein suspension, adjust the pH of the system to 8 - 9, keep it warm at 40 - 50 °C for 15 - 30 min, add α-chymotrypsin, react until the degree of hydrolysis reaches 4%, inactivate the enzyme and then centrifuge, collect the supernatant, and after dialysis and freeze-drying, obtain rice protein hydrolysate; Disperse the rice protein hydrolysate in distilled water to obtain a rice protein hydrolysate solution, adjust the pH of the system to 4 - 5, add laccase, stir for 2 - 4 h, add supported feruloyl oligosaccharides, continue to react for 22 - 24 h, inactivate the enzyme and then centrifuge, and after dialysis and freeze-drying, obtain a complex; Mix sodium alginate, the complex and distilled water evenly, add calcium carbonate, and swell for 22 - 24 h to obtain a composite wall material solution; Step S3: Mix the composite wall material solution and the bacterial suspension evenly, add them to soybean oil containing an emulsifier, stir evenly to form a uniform water-in-oil emulsion, add glacial acetic acid, stir evenly, let it stand for 1 - 2 h to obtain a precipitate, centrifuge and separate to obtain enteric-coated lactic acid bacteria capsules; The preparation method of the supported feruloyl oligosaccharides is as follows: Step (1): Mix feruloyl oligosaccharides and distilled water evenly, stir at 70 - 80 °C for 2 - 3 h, hydrate at 3 - 5 °C for 10 - 12 h to obtain a feruloyl oligosaccharide solution, adjust the pH value to 8 - 9 with sodium hydroxide, add octenyl succinic anhydride, react at 40 - 50 °C for 1 - 2 h, adjust the pH value of the solution to 5.5 - 6.0 with hydrochloric acid, cool to room temperature, and after centrifugation, washing, dialysis and freeze-drying, obtain modified feruloyl oligosaccharides; Step (2): Mix the modified feruloyl oligosaccharides and distilled water evenly, hydrate at 3 - 5 °C for 10 - 12 h to obtain a modified feruloyl oligosaccharide solution, add quercetin, homogenize at 40 - 50 °C for 5 - 10 min, centrifuge, collect the supernatant, and after dialysis and freeze-drying, obtain supported feruloyl oligosaccharides; In the step S2, the composite wall material solution includes raw materials with the following mass concentrations: 3 - 5% of sodium alginate, 5 - 10% of the complex, 1 - 2% of calcium carbonate, and the balance is distilled water.

2. The preparation method of an enteric-coated lactic acid bacteria capsule according to claim 1, characterized in that: In the step S1, the specific preparation process of the lactic acid bacteria sludge is: inoculate lactic acid bacteria in MRS liquid medium, cultivate at a constant temperature of 37 °C for 24 h, after activating twice, inoculate again in MRS liquid medium at an inoculation amount of 1% (V / V), cultivate at a constant temperature of 37 °C for 24 h, and then centrifuge at 4 °C and 10000 rpm for 10 min to remove the supernatant to obtain lactic acid bacteria sludge.

3. The preparation method of an enteric-coated lactic acid bacteria capsule according to claim 1, characterized in that: In the step (1), the mass ratio of feruloyl oligosaccharides, distilled water and octenyl succinic anhydride is 1: (35 - 45): (1 - 2).

4. The preparation method of an enteric-coated lactic acid bacteria capsule according to claim 1, characterized in that: In the step S2, the mass of the supported feruloyl oligosaccharides is 0.5 - 1.0 times the mass of the rice protein hydrolysate.

5. The preparation method of an enteric-coated lactic acid bacteria capsule according to claim 1, characterized in that: In the step S3, the mass ratio of the composite wall material solution and the bacterial suspension is 4: (1 - 3).

6. The preparation method of an enteric-coated lactic acid bacteria capsule according to claim 1, characterized in that: In the step S3, the mass of the soybean oil is 2-3 times the total mass of the composite wall material solution and the bacterial suspension.

7. The preparation method of an enteric-coated lactic acid bacteria capsule according to claim 1, characterized in that: In the step S3, the emulsifier is one of Tween-80, Span-80, and Span-20, and the addition amount of the emulsifier is 0.4-0.8% of the mass of the soybean oil.

8. An enteric-coated lactic acid bacteria capsule prepared by the preparation method according to any one of claims 1-7.

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