Enteric lactobacillus capsule and preparation method thereof

Abstract

The invention relates to the technical field of food biology, in particular to an enteric lactobacillus capsule and a preparation method thereof. The method comprises the following steps: S1, sequentially activating and centrifugally separating lactic acid bacteria to obtain lactic acid bacteria sludge, and resuspending with sterile normal saline to obtain a bacterial suspension; s2, uniformly mixing sodium alginate, the compound and distilled water, adding calcium carbonate, and swelling for 22-24 hours to obtain a composite wall material solution; and S3, uniformly mixing the composite wall material solution and the bacterial suspension, adding the mixture into soybean oil containing an emulsifier, uniformly stirring to form a uniform water-in-oil emulsion, adding glacial acetic acid, uniformly stirring, standing for 1-2 hours to obtain a precipitate, and centrifugally separating to obtain the enteric lactobacillus capsule. The prepared enteric lactic acid bacteria capsule has high acid resistance, cholate resistance and survival rate, and can achieve a directional release effect in intestinal tracts.

Description

Technical Field

[0001] The invention relates to the field of food biotechnology, in particular to an enteric lactic acid bacteria capsule and a preparation method thereof. Background Art

[0002] As people's living standards improve and their health awareness increases, probiotic products are gradually gaining widespread attention. As an important type of probiotic, lactic acid bacteria are widely used in food, health products, and medicines because of their ability to regulate the balance of intestinal flora, enhance immunity, and promote digestion and absorption.

[0003] In traditional probiotic supplementation, lactic acid bacteria are often in liquid or powder form, but these forms are easily inactivated in large quantities in adverse environments such as food processing, storage and gastrointestinal digestion, resulting in a lack of sufficient live bacteria to successfully reach the human colon and exert their probiotic effects. In order to solve this problem, enteric-coated lactic acid bacteria capsules came into being.

[0004] At present, starch and plant proteins are widely studied as capsule materials. These raw materials have certain film-forming properties, but their mechanical strength cannot meet the needs of food packaging, and they have disadvantages such as low embedding rate and large particle size, which limit their industrial application. Chemical modification is usually used to improve their performance. Among them, Maillard products (MRPs) generated by the Maillard reaction are used as important encapsulation agents to protect probiotics due to their film-promoting and antioxidant activities. However, the application of the Maillard reaction is limited because it produces late terminal glycosylation products. In order to solve this problem, the present invention uses an enzymatic grafting reaction to modify the protein. Compared with the Maillard reaction, the byproduct of the enzymatic grafting reaction is only water, without other harmful byproducts, and the enzyme 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 object of the present invention is to provide an enteric-coated lactic acid bacteria capsule and a preparation method thereof, so as to solve the problems raised in the prior art.

[0007] To achieve the above object, the present invention provides the following technical solutions: A method for preparing enteric-coated lactic acid bacteria capsules comprises the following steps: Step S1: activating and centrifuging the lactic acid bacteria in sequence to obtain lactic acid bacteria mud, and then resuspending the lactic acid bacteria in sterile physiological saline to obtain a bacterial suspension; Step S2: dispersing rice protein in distilled water to obtain a rice protein suspension, adjusting the pH of the system to 8-9, keeping warm at 40-50° C. for 15-30 min, adding α-chymotrypsin, reacting until the degree of hydrolysis is 4%, inactivating the enzyme, centrifuging, collecting the supernatant, dialyzing, and freeze-drying to obtain a rice protein hydrolyzate; The rice protein hydrolysate is dispersed in distilled water to obtain a rice protein hydrolysate solution, the pH of the system is adjusted to 4-5, laccase is added, stirred for 2-4 hours, the loaded feruloyl oligosaccharide is added, the reaction is continued for 22-24 hours, the enzyme is inactivated and then centrifuged, and a complex is obtained after dialyzing and freeze-drying; Mix the sodium alginate, the composite and distilled water evenly, add calcium carbonate, and swell for 22-24 hours to obtain a composite wall material solution; Step S3: Evenly mix the composite wall material solution and the bacterial suspension, add them into soybean oil containing an emulsifier, stir evenly to form a uniform water-in-oil (W / O) emulsion, add glacial acetic acid, stir evenly, let stand for 1-2 hours, obtain a precipitate, centrifuge and separate, and obtain enteric lactic acid bacteria capsules.

[0008] Furthermore, in step S1, the specific preparation process of the lactic acid bacteria sludge is as follows: inoculate the lactic acid bacteria in MRS liquid culture medium, culture at a constant temperature of 37°C for 24 hours, activate twice, and then inoculate in MRS liquid culture medium with a 1% inoculation amount, culture at a constant temperature of 37°C for 24 hours, centrifuge at 4°C and 10000rpm for 10 minutes, remove the supernatant, and obtain lactic acid bacteria sludge.

[0009] Furthermore, in step S1, the number of viable bacteria in the bacterial suspension is 1×10 9 -2×10 10 cfu / mL.

[0010] Furthermore, in step S2, the composite wall material solution includes the following raw materials in mass concentrations: 3-5% sodium alginate, 5-10% composite material, 1-2% calcium carbonate, and the remainder is distilled water.

[0011] Furthermore, the preparation method of the supported feruloyl oligosaccharide is as follows: Step (1): mixing feruloyl oligosaccharide and distilled water uniformly, stirring at 70-80° C. for 2-3 hours, hydrating at 3-5° C. for 10-12 hours to obtain a feruloyl oligosaccharide solution, adjusting the pH value to 8-9 with sodium hydroxide, adding octenyl succinic anhydride, reacting at 40-50° C. for 1-2 hours, adjusting the pH value of the solution to 5.5-6.0 with hydrochloric acid, cooling to room temperature, centrifuging, washing, dialyzing, and freeze-drying to obtain modified feruloyl oligosaccharide; Step (2): uniformly mix the modified feruloyl oligosaccharide and distilled water, hydrate at 3-5°C for 10-12 hours to obtain a modified feruloyl oligosaccharide solution, add quercetin, homogenize at 40-50°C for 5-10 minutes, centrifuge, collect the supernatant, dialyze, and freeze-dry to obtain the loaded feruloyl oligosaccharide.

[0012] In the above technical scheme, hydroxyl-rich feruloyl oligosaccharides are used as raw materials, and octenyl succinic anhydride (OSA) is used as a hydrophobic agent to modify them, and an amphiphilic polysaccharide carrier, namely modified feruloyl oligosaccharides, is successfully prepared, which can effectively load hydrophobic quercetin; feruloyl oligosaccharides loaded with quercetin are prepared by self-assembly to form loaded feruloyl oligosaccharides; chymosin is used to hydrolyze rice protein to expose amino acid residues such as tyrosine, and then under the catalysis of laccase, the tyrosine residues in the rice protein hydrolysate undergo cross-linking reaction with ferulic acid and quercetin in the loaded feruloyl oligosaccharides to obtain a complex, which significantly improves the emulsification stability and antioxidant properties of the protein.

[0013] Furthermore, in the step (1), the mass ratio of feruloyl oligosaccharide, distilled water and octenyl succinic anhydride is 1:(35-45):(1-2).

[0014] Furthermore, in step (1), the concentration of the sodium hydroxide solution is 3 wt %, and the concentration of the hydrochloric acid is 1 mol / L.

[0015] Furthermore, in the step (2), the concentration of the modified feruloyl oligosaccharide solution is 0.2-0.4 wt %.

[0016] Furthermore, in step (2), the mass of quercetin is 0.2-0.5 times the mass of the modified feruloyl oligosaccharide.

[0017] Further, in step S2, the concentration of the rice protein suspension is 7-8wt%.

[0018] Furthermore, in step S2, the ratio of the added amount of α-chymotrypsin to the rice protein is 300-320 U / g.

[0019] Furthermore, in step S2, the concentration of the rice protein hydrolysate solution is 4-6 mg / mL.

[0020] Furthermore, in step S2, the dosage of laccase is 1.5-2.0 U / mL.

[0021] Furthermore, in step S2, the mass of the loaded feruloyl oligosaccharide is 0.5-1.0 times the mass of the rice protein hydrolysate.

[0022] Furthermore, in step S3, the mass ratio of the composite wall material solution to the bacterial suspension is 4:(1-3).

[0023] Furthermore, in step S3, the mass of soybean oil is 2-3 times the total mass of the composite wall material solution and the bacterial suspension.

[0024] Furthermore, in step S3, the emulsifier is one of Tween-80, Span-80 and Span-20, and the amount of the emulsifier added is 0.4-0.8% of the mass of the soybean oil.

[0025] Furthermore, in 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.

[0026] Compared with the prior art, the present invention has the following beneficial effects: 1. An enteric-coated lactic acid bacteria capsule and a preparation method thereof of the present invention comprises the following steps: using hydroxyl-rich feruloyl oligosaccharides as raw materials, modifying them with octenyl succinic anhydride (OSA) as a hydrophobic agent, and preparing an amphiphilic carrier; quercetin (que) is a plant polyphenol with anti-inflammatory and antioxidant properties; and preparing quercetin-loaded feruloyl oligosaccharides by self-assembly, so that quercetin can be effectively loaded in the structure of feruloyl oligosaccharides, thereby improving its water solubility and bioavailability; using chymosin to hydrolyze rice protein to expose amino acid residues such as tyrosine, and then covalently bonding the loaded feruloyl oligosaccharides with rice protein hydrolysates by enzymatic cross-linking catalysis to obtain a complex, thereby significantly improving the protein At the same time, the larger molecular weight and spatial structure of the loaded feruloyl oligosaccharide will have an inhibitory effect on the protein surface, reducing the available binding sites for digestive enzymes on the protein surface, enhancing the spatial hindrance of the protein, and thus enhancing the protein's anti-digestion ability. Therefore, it is helpful to the stability of the encapsulated active substances in the gastrointestinal digestion process; sodium alginate, complex and calcium carbonate are used as composite wall materials; the endogenous emulsification gel method is used to prepare plant lactobacillus microcapsules, which have good acid resistance and enteric solubility, can delay the release of the core material in a simulated gastric digestion environment, and ensure the stability of quercetin and lactic acid bacteria in the gastric acid environment, thereby achieving precise release in the intestine. DETAILED DESCRIPTION

[0027] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. 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.

[0028] In the present embodiment, the lactic acid bacteria: Lactobacillus plantarum, with a deposit number of CGMCC No. 16441, is deposited in the General Microbiology Center of the China National Committee for the Preservation and Administration of Microbiological Cultures; calcium carbonate: model ZC-1, food grade, sourced from Henan Zhongda Hengyuan Biotechnology Co., Ltd.; soybean oil: item number S817900, sourced from Shanghai MacLean Biochemical Technology Co., Ltd.; emulsifier: Tween-80, food grade; rice protein: item number QYC03240312, sourced from Xi'an Qianyecao Biotechnology Co., Ltd.; α-chymotrypsin: item number M10831, sourced from Huhui (Shanghai) Biotechnology Co., Ltd.; laccase: item number 18-2, sourced from Jinan Kangxu Biotechnology Co., Ltd.; feruloyl oligosaccharide is derived from wheat bran, and the feruloyl oligosaccharide obtained by high temperature cooking method, and its specific preparation steps refer to the prior art (Ge Lihua, Preparation of feruloyl oligosaccharide and study on its antioxidant properties [D], Northeastern University, 2007-04-01).

[0029] Embodiment 1: A method for preparing enteric lactic acid bacteria capsules, comprising the following process: Step S1: inoculate the lactic acid bacteria in MRS liquid culture medium, culture at 37°C for 24 hours, activate twice, and then inoculate in MRS liquid culture medium at 1% (V / V) inoculation amount (the culture medium components are: 10.0 g of peptone, 10.0 g of beef extract, 5.0 g of yeast extract, 2.0 g of diammonium hydrogen citrate, 20.0 g of glucose, 1.0 mL of Tween-80, 5.0 g of sodium acetate, 2.0 g of dipotassium hydrogen phosphate, 0.58 g of magnesium sulfate, 0.25 g of manganese sulfate, 1000 mL of distilled water, and adjust the pH to 6.5), culture at 37°C for 24 hours, centrifuge at 4°C and 10000 rpm for 10 minutes, remove the supernatant, obtain lactic acid bacteria mud, and then resuspend with sterile physiological saline to obtain a bacterial suspension; Step S2: dispersing 10g of rice protein in distilled water to obtain a 7wt% rice protein suspension, adjusting the system pH to 8, keeping warm at 40°C for 15min, adding 3000U of α-chymotrypsin, reacting until the degree of hydrolysis is 4%, inactivating the enzyme and centrifuging, collecting the supernatant, dialyzing, and freeze-drying to obtain a rice protein hydrolysate; 10 g of rice protein hydrolysate was dispersed in distilled water to obtain a 4 mg / mL rice protein hydrolysate solution, the pH of the system was adjusted to 4, 1.5 U / mL laccase was added, and the mixture was stirred for 2 h. 5 g of supported feruloyl oligosaccharide was added, and the reaction was continued for 22 h. After the enzyme was inactivated, the mixture was centrifuged, and a complex was obtained after dialysis and freeze-drying. The sodium alginate, the composite and distilled water were mixed evenly, and calcium carbonate was added and swelled for 22 hours to obtain a composite wall material solution; the concentration of sodium alginate in the composite wall material solution was 3wt%, the concentration of the composite was 5wt%, and the concentration of calcium carbonate was 1wt%; Step S3: 100 g of the composite wall material solution and 25 g of the bacterial suspension are mixed evenly, added to 250 g of soybean oil containing 0.4 wt% of an emulsifier, stirred evenly to form a uniform water-in-oil (W / O) emulsion, 0.375 g of glacial acetic acid is added, stirred evenly, and allowed to stand for 1 hour to obtain a precipitate, which is centrifuged to obtain an enteric-coated lactic acid bacteria capsule; The preparation method of the loaded feruloyl oligosaccharide is as follows: Step (1): 5 g of feruloyl oligosaccharide and 175 g of distilled water are mixed uniformly, stirred at 70° C. for 2 h, and hydrated at 3° C. for 10 h to obtain a feruloyl oligosaccharide solution, the pH value of which is adjusted to 8 with 3 wt % sodium hydroxide, 5 g of octenyl succinic anhydride is added, reacted at 40° C. for 1 h, the pH value of the solution is adjusted to 5.5 with 1 mol / L hydrochloric acid, cooled to room temperature, centrifuged, washed, dialyzed, and freeze-dried to obtain modified feruloyl oligosaccharide; Step (2): 5 g of modified feruloyl oligosaccharide and distilled water were mixed evenly, and hydrated at 3°C ​​for 10 h to obtain a 0.2 wt% modified feruloyl oligosaccharide solution, 1 g of quercetin was added, and the mixture was homogenized at 40°C for 5 min. The mixture was centrifuged, and the supernatant was collected. After dialyzation and freeze-drying, the loaded feruloyl oligosaccharide was obtained.

[0030] Embodiment 2: A method for preparing enteric lactic acid bacteria capsules, comprising the following process: Step S1: inoculate the lactic acid bacteria in MRS liquid culture medium, culture at 37°C for 24 hours, activate twice, and then inoculate in MRS liquid culture medium at 1% (V / V) inoculation amount (the culture medium components are: 10.0 g of peptone, 10.0 g of beef extract, 5.0 g of yeast extract, 2.0 g of diammonium hydrogen citrate, 20.0 g of glucose, 1.0 mL of Tween-80, 5.0 g of sodium acetate, 2.0 g of dipotassium hydrogen phosphate, 0.58 g of magnesium sulfate, 0.25 g of manganese sulfate, 1000 mL of distilled water, and adjust the pH to 6.5), culture at 37°C for 24 hours, centrifuge at 4°C and 10000 rpm for 10 minutes, remove the supernatant, obtain lactic acid bacteria mud, and then resuspend with sterile physiological saline to obtain a bacterial suspension; Step S2: dispersing 10g of rice protein in distilled water to obtain a 7.5wt% rice protein suspension, adjusting the pH of the system to 8.5, keeping warm at 45°C for 20min, adding 3100U of α-chymotrypsin, reacting until the degree of hydrolysis is 4%, inactivating the enzyme and centrifuging, collecting the supernatant, dialyzing, and freeze-drying to obtain a rice protein hydrolysate; 10 g of rice protein hydrolysate was dispersed in distilled water to obtain a 5 mg / mL rice protein hydrolysate solution, the pH of the system was adjusted to 4.5, 1.8 U / mL laccase was added, and the mixture was stirred for 3 h. 8 g of supported feruloyl oligosaccharide was added, and the reaction was continued for 23 h. After the enzyme was inactivated, the mixture was centrifuged, and a complex was obtained after dialysis and freeze-drying; The sodium alginate, the composite and distilled water were mixed evenly, and calcium carbonate was added and swelled for 23 hours to obtain a composite wall material solution; the concentration of sodium alginate in the composite wall material solution was 4wt%, the concentration of the composite was 8wt%, and the concentration of calcium carbonate was 1.5wt%; Step S3: 100 g of the composite wall material solution and 50 g of the bacterial suspension were mixed evenly, added to 375 g of soybean oil containing 0.6 wt % of an emulsifier, stirred evenly to form a uniform water-in-oil (W / O) emulsion, 0.75 g of glacial acetic acid was added, stirred evenly, and allowed to stand for 1.5 h to obtain a precipitate, which was centrifuged to obtain an enteric-coated lactic acid bacteria capsule; The preparation method of the loaded feruloyl oligosaccharide is as follows: Step (1): 8 g of feruloyl oligosaccharide and 320 g of distilled water are mixed uniformly, stirred at 75° C. for 2.5 h, and hydrated at 4° C. for 11 h to obtain a feruloyl oligosaccharide solution, the pH value of which is adjusted to 8.5 with 3 wt % sodium hydroxide, 12 g of octenyl succinic anhydride is added, the mixture is reacted at 45° C. for 1.5 h, the pH value of the solution is adjusted to 5.8 with 1 mol / L hydrochloric acid, the mixture is cooled to room temperature, and the mixture is centrifuged, washed, dialyzed, and freeze-dried to obtain modified feruloyl oligosaccharide; Step (2): 8 g of modified feruloyl oligosaccharide and distilled water were mixed evenly, and hydrated at 4°C for 11 h to obtain a 0.3 wt% modified feruloyl oligosaccharide solution, 3.2 g of quercetin was added, and the mixture was homogenized at 45°C for 8 min. The mixture was centrifuged, and the supernatant was collected. After dialyzation and freeze-drying, the loaded feruloyl oligosaccharide was obtained.

[0031] Embodiment 3: A method for preparing enteric lactic acid bacteria capsules, comprising the following process: Step S1: inoculate the lactic acid bacteria in MRS liquid culture medium, culture at 37°C for 24 hours, activate twice, and then inoculate in MRS liquid culture medium at a 1% inoculum amount (the culture medium components are: 10.0 g of peptone, 10.0 g of beef extract, 5.0 g of yeast extract, 2.0 g of diammonium hydrogen citrate, 20.0 g of glucose, 1.0 mL of Tween-80, 5.0 g of sodium acetate, 2.0 g of dipotassium hydrogen phosphate, 0.58 g of magnesium sulfate, 0.25 g of manganese sulfate, 1000 mL of distilled water, and adjust the pH to 6.5), culture at 37°C for 24 hours, centrifuge at 4°C and 10000 rpm for 10 minutes, remove the supernatant, obtain lactic acid bacteria mud, and then resuspend with sterile physiological saline to obtain a bacterial suspension; Step S2: dispersing 10 g of rice protein in distilled water to obtain a rice protein suspension, adjusting the pH of the system to 9, keeping warm at 50° C. for 30 min, adding 3200 U of α-chymotrypsin, reacting until the degree of hydrolysis is 4%, inactivating the enzyme, centrifuging, collecting the supernatant, dialyzing, and freeze-drying to obtain a rice protein hydrolyzate; 10 g of rice protein hydrolysate was dispersed in distilled water to obtain a 6 mg / mL rice protein hydrolysate solution, the pH of the system was adjusted to 5, 2.0 U / mL laccase was added, and the mixture was stirred for 4 h. 10 g of supported feruloyl oligosaccharide was added, and the reaction was continued for 24 h. After the enzyme was inactivated, the mixture was centrifuged, and a complex was obtained after dialysis and freeze-drying. The sodium alginate, the composite and distilled water are mixed evenly, and calcium carbonate is added and swelled for 24 hours to obtain a composite wall material solution; the concentration of sodium alginate in the composite wall material solution is 5wt%, the concentration of the composite is 10wt%, and the concentration of calcium carbonate is 2wt%; Step S3: 100 g of the composite wall material solution and 75 g of the bacterial suspension were mixed evenly, added to 525 g of soybean oil containing 0.8 wt % of an emulsifier, stirred evenly to form a uniform water-in-oil (W / O) emulsion, 1.05 g of glacial acetic acid was added, stirred evenly, and allowed to stand for 2 h to obtain a precipitate, which was centrifuged to obtain an enteric-coated lactic acid bacteria capsule; The preparation method of the loaded feruloyl oligosaccharide is as follows: Step (1): 10 g of feruloyl oligosaccharide and 450 g of distilled water are mixed uniformly, stirred at 80° C. for 3 h, and hydrated at 5° C. for 12 h to obtain a feruloyl oligosaccharide solution, the pH value of which is adjusted to 9 with 3 wt % sodium hydroxide, 20 g of octenyl succinic anhydride is added, and the mixture is reacted at 50° C. for 2 h, the pH value of the solution is adjusted to 6.0 with 1 mol / L hydrochloric acid, and the mixture is cooled to room temperature, and after centrifugation, washing, dialysis, and freeze-drying, a modified feruloyl oligosaccharide is obtained; Step (2): 10 g of modified feruloyl oligosaccharide and distilled water were mixed evenly, and hydrated at 5° C. for 12 h to obtain a 0.4 wt% modified feruloyl oligosaccharide solution, 5 g of quercetin was added, and the mixture was homogenized at 50° C. for 10 min. The mixture was centrifuged, and the supernatant was collected. After dialyzation and freeze-drying, the loaded feruloyl oligosaccharide was obtained.

[0032] Comparative Example 1: A method for preparing enteric lactic acid bacteria capsules, comprising the following processes: Compared with Example 2, no composite material is added to the composite wall material solution of Comparative Example 1, and other steps are the same as those of Example 2.

[0033] Comparative Example 2: A method for preparing enteric lactic acid bacteria capsules, comprising the following processes: Compared with Example 2, in step S2 of Comparative Example 1, the composite wall material solution includes the following raw materials in mass concentrations: 4% sodium alginate, 15% composite, 1.5% calcium carbonate, and the balance is distilled water. The other steps are the same as those in Example 2.

[0034] Comparative Example 3: A method for preparing enteric lactic acid bacteria capsules, comprising the following processes: Compared with Example 2, in Comparative Example 3, the modified feruloyl oligosaccharide is replaced with feruloyl oligosaccharide of the same mass, and the other steps are the same as those in Example 2.

[0035] Experiment: Enteric-coated lactic acid bacteria capsules obtained in Examples 1-3 and Comparative Examples 1-3 were taken to prepare samples, and their performance was tested respectively and the test results were recorded: Determination of embedding rate: The prepared enteric-coated lactic acid bacteria capsules were lysed with sodium citrate solution. 1 g of the prepared enteric-coated lactic acid bacteria capsules were placed in 9 mL of 3% sodium citrate solution for lysis. The solution was placed in a 37°C water bath constant temperature oscillator and shaken at 180 r / min for 60 min. After lysis, the live bacteria were counted. The embedding rate was calculated according to the formula: N / N1×100%, where N is the number of live bacteria embedded in the capsules and N1 is the number of live bacteria added before embedding.

[0036] Survival rate determination 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 1g of the prepared enteric-coated lactic acid bacteria capsules and add them to 9mL of simulated gastric fluid that has been preheated (37°C) and filter-sterilized. After culturing at 37°C and 120 r / min for 2h, count the live bacteria on the plate and calculate the survival rate; then transfer to 9mL of simulated intestinal fluid and continue digestion for 8h, and then determine the survival rate; the simulated intestinal fluid contains 1mg / mL trypsin and 0.3% bile salts, and the pH value is adjusted to 6.8 with 0.1mol / L sodium hydroxide.

[0037] The test results are as follows:

[0038] According to the data in the above table, we can clearly draw the following conclusions: Compared with Examples 1-3, the embedding rate and survival rate of the products obtained in Comparative Examples 1 and 2 are both reduced, indicating that the addition of the complex in the present invention is beneficial to improving the embedding rate and survival rate; as the amount of complex added increases, the viscosity of the mixed solution increases, resulting in a worse magnetic stirring effect, thereby affecting the emulsification process, and ultimately manifested as a significant decrease in the embedding rate. It can be seen that the performance of the composite wall material solution prepared by the present invention is affected by the component ratio thereof, and by selecting the component ratio within the said range, a composite wall material solution with excellent performance can be prepared.

[0039] Compared with Examples 1-3, the embedding rate and survival rate of the product obtained in Comparative Example 3 were both reduced, which indicates that compared with the unmodified feruloyl oligosaccharide, the modified feruloyl oligosaccharide prepared by the present invention has amphiphilicity, is more conducive to forming a stable complex, and improves the embedding efficiency and biocompatibility of the carrier.

[0040] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

Claims

1. A method for preparing an enteric-coated lactic acid bacteria capsule, characterized in that: The steps include: Step S1: activating and centrifuging the lactic acid bacteria in sequence to obtain lactic acid bacteria mud, and then resuspending the lactic acid bacteria in sterile physiological saline to obtain a bacterial suspension; Step S2: dispersing rice protein in distilled water to obtain a rice protein suspension, adjusting the pH of the system to 8-9, keeping warm at 40-50° C. for 15-30 min, adding α-chymotrypsin, reacting until the degree of hydrolysis is 4%, inactivating the enzyme, centrifuging, collecting the supernatant, dialyzing, and freeze-drying to obtain a rice protein hydrolyzate; The rice protein hydrolysate is dispersed in distilled water to obtain a rice protein hydrolysate solution, the pH of the system is adjusted to 4-5, laccase is added, stirred for 2-4 hours, the loaded feruloyl oligosaccharide is added, the reaction is continued for 22-24 hours, the enzyme is inactivated and then centrifuged, and a complex is obtained after dialyzing and freeze-drying; Mix the sodium alginate, the composite and distilled water evenly, add calcium carbonate, and swell for 22-24 hours to obtain a composite wall material solution; Step S3: Evenly mix the composite wall material solution and the bacterial suspension, add them into soybean oil containing an emulsifier, stir evenly to form a uniform water-in-oil emulsion, add glacial acetic acid, stir evenly, let stand for 1-2 hours, obtain a precipitate, centrifuge and separate, and obtain enteric lactic acid bacteria capsules.

2. The method for preparing an enteric-coated lactic acid bacteria capsule according to claim 1, characterized in that: In step S1, the specific preparation process of the lactic acid bacteria sludge is as follows: the lactic acid bacteria are inoculated into an MRS liquid culture medium, cultured at a constant temperature of 37°C for 24 hours, activated twice, and then inoculated into an MRS liquid culture medium with an inoculum amount of 1%, cultured at a constant temperature of 37°C for 24 hours, centrifuged at 4°C and 10,000 rpm for 10 minutes, and the supernatant is removed to obtain the lactic acid bacteria sludge.

3. The method for preparing an enteric-coated lactic acid bacteria capsule according to claim 1, characterized in that: In step S2, the composite wall material solution includes the following raw materials in mass concentrations: 3-5% sodium alginate, 5-10% composite material, 1-2% calcium carbonate, and the remainder is distilled water.

4. The method for preparing an enteric-coated lactic acid bacteria capsule according to claim 1, characterized in that: The preparation method of the supported feruloyl oligosaccharide is as follows: Step (1): mixing feruloyl oligosaccharide and distilled water uniformly, stirring at 70-80° C. for 2-3 hours, hydrating at 3-5° C. for 10-12 hours to obtain a feruloyl oligosaccharide solution, adjusting the pH value to 8-9 with sodium hydroxide, adding octenyl succinic anhydride, reacting at 40-50° C. for 1-2 hours, adjusting the pH value of the solution to 5.5-6.0 with hydrochloric acid, cooling to room temperature, centrifuging, washing, dialyzing, and freeze-drying to obtain modified feruloyl oligosaccharide; Step (2): uniformly mix the modified feruloyl oligosaccharide and distilled water, hydrate at 3-5°C for 10-12 hours to obtain a modified feruloyl oligosaccharide solution, add quercetin, homogenize at 40-50°C for 5-10 minutes, centrifuge, collect the supernatant, dialyze, and freeze-dry to obtain the loaded feruloyl oligosaccharide.

5. The method for preparing an enteric-coated lactic acid bacteria capsule according to claim 4, characterized in that: In the step (1), the mass ratio of feruloyl oligosaccharide, distilled water and octenyl succinic anhydride is 1:(35-45):(1-2).

6. The method for preparing an enteric-coated lactic acid bacteria capsule according to claim 1, characterized in that: In the step S2, the mass of the loaded feruloyl oligosaccharide is 0.5-1.0 times the mass of the rice protein hydrolysate.

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

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

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

10. An enteric-coated lactic acid bacteria capsule prepared according to the preparation method according to any one of claims 1 to 9.

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