A method for preparing microcapsules containing probiotics

Abstract

The present invention belongs to the technical field of probiotic microcapsules, and specifically relates to a method for preparing microcapsules for embedding probiotics. The present invention first cultivates activated probiotics, prepares them into a probiotic suspension, and obtains a core material solution; then adds modified sodium alginate and whey protein into deionized water, disperses them uniformly by ultrasonication, and obtains a wall material solution after sterilization; finally, the core material solution and the wall material solution are evenly mixed, added into a calcium chloride solution by extrusion, and allowed to stand to form a gel, and then the gel is added into a modified carboxymethyl chitosan solution for stirring, washing, and drying to obtain microcapsules; the present invention uses modified sodium alginate, whey protein, and modified carboxymethyl chitosan as embedding materials, and realizes efficient embedding of probiotics. The prepared microcapsules have good mechanical properties, good tolerance in simulated gastric juice, simulated intestinal juice, and at 4°C, and can maintain a high survival rate and stability of the probiotics in the microcapsules.

Description

Technical Field

[0001] The invention belongs to the technical field of probiotic microcapsules, and particularly relates to a method for preparing microcapsules for embedding probiotics. Background Art

[0002] Probiotics are a type of active microorganisms that colonize in the human body and change the composition of the flora in a certain part. They have multiple functions such as maintaining the balance of intestinal flora, inhibiting the growth of pathogenic bacteria, enhancing the body's immunity, and preventing diseases. They are beneficial to human health and are widely used in food areas such as fermented products, dairy products, and milk beverages. Probiotics have harsh living conditions and are extremely sensitive to oxygen, gastric acid, enzymes, bile salts and other environmental factors. They are easily killed during processing, storage, digestion, etc., which leads to the inability of probiotic products to fully exert their efficacy in the human body. At present, probiotics are mainly microencapsulated through encapsulation technology to improve the stability of probiotics, reduce the death of probiotics, and increase their survival rate, so that they can smoothly reach the intestinal tract to exert their effects, which is beneficial to improving the bioavailability of probiotics.

[0003] A Chinese patent with application number 202010351092.3 discloses a probiotic microcapsule and its preparation method and application. The probiotics are encapsulated by three layers of materials. The first layer of encapsulation material includes whey protein and carrageenan, which can provide multiple protections for the sclerotia and are conducive to the growth of probiotics; the second layer of encapsulation material includes pectin and xanthan gum, which can reduce the impact of gastric juice on probiotics, thereby increasing the survival rate of probiotics when passing through gastric juice; the third layer of coating raw materials is enteric-soluble substances with the characteristics of sealing, resistance to gastric acid and resistance to digestive enzymes, which can make the outermost layer of the microcapsule degrade in the intestinal tract; although the use of multi-layer materials has achieved ideal encapsulation of probiotics, the preparation method involved is complicated and not conducive to industrial production.

[0004] A Chinese patent with application number 202110483840.8 discloses a colon-targeted probiotic multi-layer embedded microcapsule and its preparation method and application. Sodium alginate / prebiotics / probiotics are used as the core layer, sodium alginate / pectin is used as the shell wall material, and soy protein isolate is used as the outermost wall material. Multilayer microcapsules loaded with active substances are prepared by a coaxial electrostatic spraying device. This method has the advantages of simple and mild preparation conditions and easy large-scale production. Although the wall materials pectin and soy protein isolate have a certain improvement effect on the porosity of the microcapsules formed by sodium alginate and improve the stability of the probiotic microcapsules, there is still a lot of room for improvement in its embedding rate, and there is no improvement on the defects of the low mechanical strength of the microcapsules formed by sodium alginate.

[0005] Therefore, there is an urgent need to improve the encapsulation materials of probiotics, especially sodium alginate, in order to improve their mechanical properties and the density of microcapsules and obtain probiotic microcapsules with more stable performance. Summary of the invention

[0006] In view of the deficiencies in the prior art, the present invention provides a method for preparing microcapsules for embedding probiotics. Modified sodium alginate, whey protein and modified carboxymethyl chitosan are used as embedding materials to achieve efficient embedding of probiotics. The prepared microcapsules have good mechanical properties and good tolerance in simulated gastric juice, simulated intestinal juice and at 4°C, and can maintain a high survival rate and stability of the probiotics in the microcapsules.

[0007] The technical solution adopted by the present invention is:

[0008] A method for preparing microcapsules for embedding probiotics comprises the following steps:

[0009] S1, culturing the activated probiotics to prepare a probiotic suspension to obtain a core material solution;

[0010] S2, adding modified sodium alginate and whey protein into deionized water, dispersing them evenly by ultrasonication, and obtaining a wall material solution after sterilization;

[0011] S3, mixing the core material solution and the wall material solution evenly, adding them into a calcium chloride solution by extrusion, standing to form a gel, then adding the gel into a modified carboxymethyl chitosan solution, stirring, washing, and drying to obtain microcapsules;

[0012] Furthermore, the probiotics in step S1 are any one or a mixture of Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus casei, Bifidobacterium lactis, and Bifidobacterium; the concentration of the probiotic suspension is 1.0×10 9 -1.0×10 10 CFU / mL.

[0013] Furthermore, in step S2, the concentration of the modified sodium alginate is 2-3wt%; the concentration of the whey protein is 1-1.5wt%; and the ultrasonic conditions are: the ultrasonic power is 300-400W, and the ultrasonic treatment is performed for 10-15min at room temperature.

[0014] Furthermore, in step S3, the volume ratio of the core material solution to the wall material solution is 1:4-5; the concentration of the calcium chloride solution is 1-1.5wt%; the concentration of the modified carboxymethyl chitosan solution is 0.5-0.6wt%; and the stirring conditions are: the stirring speed is 300-400rpm, and the stirring is carried out at room temperature for 20-30min.

[0015] In the technical scheme of the present invention, the preparation method of the modified sodium alginate is as follows: deionized water and sodium alginate are added to a reaction container, stirred evenly, 0.1-0.2 mol / L sodium hydroxide solution is used to adjust the pH to 8.5-9, the temperature is raised to 40-50°C, epichlorohydrin is slowly added dropwise, the temperature is raised to 70-80°C, the temperature is reacted for 6-7h, 4-phenylbenzylamine is added, the temperature is raised to 70-80°C, the temperature is reacted for 3-4h, and after the reaction is completed, the temperature is naturally lowered to room temperature, acetic acid is used to adjust the pH to 4-4.5, acetone is added, the mixture is soaked for 12h, filtered, the filter residue is repeatedly washed with acetone, and the modified sodium alginate is obtained after drying.

[0016] Furthermore, the mass ratio of the sodium alginate, epichlorohydrin, 4-phenylbenzylamine and deionized water is 1:0.3-0.4:0.5-0.6:25-30.

[0017] In the technical scheme of the present invention, the preparation method of modified carboxymethyl chitosan is as follows: add anhydrous tetrahydrofuran and polyethylene glycol monomethyl ether into a reaction container, stir evenly, protect with nitrogen, add sodium hydride, heat to 60-70°C, react for 3-4h, add chlorinated carboxymethyl chitosan, continue to react for 6-7h, and after the reaction is completed, naturally cool to room temperature, remove tetrahydrofuran, wash the product with acetone, and dry to obtain modified carboxymethyl chitosan.

[0018] Furthermore, the mass ratio of the polyethylene glycol monomethyl ether, sodium hydride, chloride-carboxymethyl chitosan and anhydrous tetrahydrofuran is 1:0.2-0.3:1.5-2:20-25.

[0019] In the technical scheme of the present invention, the preparation method of the chlorinated-carboxymethyl chitosan is as follows: 4-phenylbenzylamine and N,N'-carbonyldiimidazole are added to acetonitrile, stirred at room temperature for 5-6 hours, most of the acetonitrile is removed, and a modified liquid is obtained for standby use; in addition, a 50-60wt% ethanol solution and carboxymethyl chitosan are added to a reaction container, stirred evenly, and epichlorohydrin is slowly added dropwise. After the addition is completed, the temperature is raised to 50-60°C, the reaction is carried out for 4-5 hours, the modified liquid is added, and the reaction is continued for 6-8 hours. After the reaction is completed, the temperature naturally returns to room temperature, the pH is adjusted to neutral, acetone is added for precipitation, and the filter residue is filtered, and the filter residue is repeatedly washed with acetone, and the chlorinated-carboxymethyl chitosan is obtained after drying.

[0020] Furthermore, the mass ratio of the carboxymethyl chitosan, epichlorohydrin and 50-60wt% ethanol solution is 1:0.4-0.5:25-30; the molar amount of the 4-phenylbenzylamine and N,N'-carbonyldiimidazole is the same as the molar amount of epichlorohydrin.

[0021] The present invention also provides a probiotic-embedded microcapsule, which is prepared by the above-mentioned probiotic-embedded microcapsule preparation method.

[0022] The present invention has the following beneficial effects:

[0023] In order to obtain microcapsules with more stable performance for embedding probiotics, the present invention modifies the embedding material. On the one hand, the active group hydroxyl contained in sodium alginate is used to modify it, and it is reacted with epichlorohydrin and 4-phenylbenzylamine in sequence to graft the rigid double benzene ring group into the modified sodium alginate. The rigid double benzene ring group can play a good supporting role and help to improve the mechanical properties of the gel formed. In the embedding process, the present invention uses the modified sodium alginate and whey protein in combination. The emulsifying effect of the whey protein helps the wall material solution to form a uniform and stable embedding of the probiotics, and its good film-forming property can also improve the porous characteristics of the gel. On the other hand, the present invention uses the active group amino contained in carboxymethyl chitosan to modify it. , first react with epichlorohydrin and 4-phenylbenzylamine, and then graft with polyvinyl alcohol monomethyl ether to obtain a modified carboxymethyl chitosan containing a rigid double benzene ring group and a polyvinyl alcohol monomethyl ether side chain in the structure. The modified carboxymethyl chitosan and the gel are further cross-linked by the polyvinyl alcohol monomethyl ether side chain with a surface active effect, which helps the modified carboxymethyl chitosan and the gel to be more fully and evenly cross-linked, thereby improving the porous characteristics of the gel and forming a denser three-dimensional network structure, thereby improving the mechanical properties of the microcapsule. The rigid double benzene ring group can also synergistically act with the rigid double benzene ring group grafted in the modified sodium alginate structure to achieve the effect of jointly improving the mechanical properties of the microcapsule, thereby improving the survival rate and stability of the probiotics in the microcapsule.

[0024] The present invention uses modified sodium alginate, whey protein and modified carboxymethyl chitosan as embedding materials to achieve efficient embedding of probiotics. The prepared microcapsules have good mechanical properties and good tolerance in simulated gastric juice, simulated intestinal juice and at 4°C, and can maintain a high survival rate and stability of the probiotics in the microcapsules. DETAILED DESCRIPTION

[0025] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. In the absence of conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other.

[0026] In the technical scheme of the present invention, all chemical reagents in the following embodiments and comparative examples are commercially available, among which sodium alginate CAS No. 9005-38-3 (molecular weight 20 kDa, G unit content 60-75%), carboxymethyl chitosan CAS No. 83512-85-0 (molecular weight 8.5 kDa, deacetylation degree = 90%, carboxymethylation degree = 85%), polyethylene glycol monomethyl ether CAS No. 9004-74-4 (relative molecular weight 1000) are all pharmaceutical grade reagents; whey protein CAS No. 84082-5 1-9 (food grade); 4-phenylbenzylamine CAS No. 712-76-5, epichlorohydrin CAS No. 106-89-8, N,N'-carbonyldiimidazole CAS No. 530-62-1, sodium hydride CAS No. 7646-69-7, sodium hydroxide CAS No. 1310-73-2, acetic acid CAS No. 64-19-7, acetone CAS No. 67-64-1, acetonitrile CAS No. 75-05-8, tetrahydrofuran CAS No. 3003-84-7, ethanol CAS No. 64-17-5, all of which are analytical grade reagents.

[0027] In the technical scheme of the present invention, the preparation method of chlorinated-carboxymethyl chitosan is as follows: 4-phenylbenzylamine and N,N'-carbonyldiimidazole are added to acetonitrile, stirred at room temperature for 6 hours, most of the acetonitrile is removed, and a modified liquid is obtained for standby use; in addition, a 60wt% ethanol solution and carboxymethyl chitosan are added to a reaction container, stirred evenly, and epichlorohydrin is slowly added dropwise. After the addition is completed, the temperature is raised to 60°C, the reaction is carried out for 5 hours, the modified liquid is added, and the reaction is continued for 8 hours. After the reaction is completed, the temperature is naturally lowered to room temperature, the pH is adjusted to neutral, and acetone is added for precipitation. Filtering, repeatedly washing the filter residue with acetone, and drying to obtain chlorinated carboxymethyl chitosan; the prepared modified carboxymethyl chitosan has strong fluorescence absorption under ultraviolet light (256nm); the substitution degree of 4-phenylbenzylamine structure in chlorinated carboxymethyl chitosan is 13.5% as determined by elemental analysis; the mass ratio of carboxymethyl chitosan, epichlorohydrin, and 60wt% ethanol solution is 1:0.5:30; the molar amount of 4-phenylbenzylamine and N,N'-carbonyldiimidazole is the same as the molar amount of epichlorohydrin.

[0028] Example 1

[0029] A method for preparing microcapsules for embedding probiotics comprises the following steps:

[0030] S1. Cultivate the activated probiotics to prepare a probiotic suspension to obtain a core material solution; the probiotics are Lactobacillus plantarum; the concentration of the probiotic suspension is 1.0×10 10 CFU / mL;

[0031] S2, adding modified sodium alginate and whey protein into deionized water, dispersing them evenly by ultrasonication, and obtaining a wall material solution after sterilization; the concentration of modified sodium alginate is 3wt%; the concentration of whey protein is 1.5wt%; the ultrasonic conditions are: ultrasonic power is 300W, and ultrasonication is carried out at room temperature for 15min;

[0032] S3. The core material solution and the wall material solution are mixed evenly, added into the calcium chloride solution by extrusion, allowed to stand to form a gel, and then the gel is added into the modified carboxymethyl chitosan solution for stirring, and microcapsules are obtained after washing and drying; the volume ratio of the core material solution to the wall material solution is 1:5; the concentration of the calcium chloride solution is 1.3wt%; the concentration of the modified carboxymethyl chitosan solution is 0.6wt%; the stirring conditions are: the stirring speed is 300rpm, and the stirring is carried out at room temperature for 30min.

[0033] The preparation method of modified sodium alginate is as follows: deionized water and sodium alginate are added to a reaction container, stirred evenly, 0.2 mol / L sodium hydroxide solution is used to adjust the pH to 9, the temperature is raised to 40°C, epichlorohydrin is slowly added dropwise, the reaction is completed for 7 hours, 4-phenylbenzylamine is added, the temperature is continued to be raised to 80°C, the reaction is carried out for 3 hours, and after the reaction is completed, the temperature is naturally reduced to room temperature, acetic acid is used to adjust the pH to 4, acetone (the volume is the same as the deionized volume) is added, soaking for 12 hours, filtering, repeatedly washing the filter residue with acetone, and drying to obtain modified sodium alginate; the mass ratio of sodium alginate, epichlorohydrin, 4-phenylbenzylamine, and deionized water is 1:0.4:0.6:25.

[0034] The preparation method of modified carboxymethyl chitosan is as follows: add anhydrous tetrahydrofuran and polyethylene glycol monomethyl ether into a reaction container, stir evenly, protect with nitrogen, add sodium hydride, heat to 70°C, react for 3.5 hours, add chlorinated carboxymethyl chitosan, continue to react for 7 hours, and after the reaction is completed, naturally cool to room temperature, remove tetrahydrofuran, wash the product with acetone, and obtain modified carboxymethyl chitosan after drying; the prepared modified carboxymethyl chitosan has strong fluorescence absorption under ultraviolet lamp (256nm); the mass ratio of polyethylene glycol monomethyl ether, sodium hydride, chlorinated carboxymethyl chitosan and anhydrous tetrahydrofuran is 1:0.2:1.5:25.

[0035] Example 2

[0036] A method for preparing microcapsules for embedding probiotics comprises the following steps:

[0037] S1. Cultivate the activated probiotics to prepare a probiotic suspension to obtain a core material solution; the probiotics are Lactobacillus casei; the concentration of the probiotic suspension is 1.0×10 9 CFU / mL;

[0038] S2, adding modified sodium alginate and whey protein into deionized water, dispersing them evenly by ultrasonication, and obtaining a wall material solution after sterilization; the concentration of modified sodium alginate is 2.5wt%; the concentration of whey protein is 1wt%; the ultrasonic conditions are: ultrasonic power is 350W, and ultrasonication is carried out for 10 minutes at room temperature;

[0039] S3. The core material solution and the wall material solution are mixed evenly, added into the calcium chloride solution by extrusion, allowed to stand to form a gel, and then the gel is added into the modified carboxymethyl chitosan solution for stirring, and microcapsules are obtained after washing and drying; the volume ratio of the core material solution to the wall material solution is 1:4.5; the concentration of the calcium chloride solution is 1.5wt%; the concentration of the modified carboxymethyl chitosan solution is 0.5wt%; the stirring conditions are: the stirring speed is 400rpm, and the stirring is carried out at room temperature for 20min.

[0040] The preparation method of modified sodium alginate is as follows: add deionized water and sodium alginate into a reaction container, stir evenly, adjust the pH to 8.5 with a 0.1 mol / L sodium hydroxide solution, heat to 45°C, slowly drop epichlorohydrin, react for 6 hours after the drop is complete, then add 4-phenylbenzylamine, continue to heat to 70°C, react for 4 hours, and after the reaction is completed, naturally return to room temperature, adjust the pH to 4.5 with acetic acid, add acetone (the volume is the same as the deionized volume), soak for 12 hours, filter, repeatedly wash the filter residue with acetone, and obtain modified sodium alginate after drying; the mass ratio of sodium alginate, epichlorohydrin, 4-phenylbenzylamine, and deionized water is 1:0.35:0.5:30.

[0041] The preparation method of modified carboxymethyl chitosan is as follows: add anhydrous tetrahydrofuran and polyethylene glycol monomethyl ether into a reaction container, stir evenly, protect with nitrogen, add sodium hydride, heat to 60°C, react for 4 hours, add chlorinated carboxymethyl chitosan, continue to react for 6.5 hours, naturally cool to room temperature after the reaction is completed, remove tetrahydrofuran, wash the product with acetone, and obtain modified carboxymethyl chitosan after drying; the prepared modified carboxymethyl chitosan has strong fluorescence absorption under ultraviolet lamp (256nm); the mass ratio of polyethylene glycol monomethyl ether, sodium hydride, chlorinated carboxymethyl chitosan and anhydrous tetrahydrofuran is 1:0.3:2:23.

[0042] Example 3

[0043] A method for preparing microcapsules for embedding probiotics comprises the following steps:

[0044] S1. Cultivate the activated probiotics to prepare a probiotic suspension to obtain a core material solution; the probiotics are Bifidobacterium lactis; the concentration of the probiotic suspension is 1.0×10 10 CFU / mL;

[0045] S2, adding modified sodium alginate and whey protein into deionized water, dispersing them evenly by ultrasonication, and obtaining a wall material solution after sterilization; the concentration of modified sodium alginate is 2wt%; the concentration of whey protein is 1.3wt%; the ultrasonic conditions are: ultrasonic power is 400W, and ultrasonication is carried out for 12min at room temperature;

[0046] S3. The core material solution and the wall material solution are mixed evenly, added into the calcium chloride solution by extrusion, allowed to stand to form a gel, and then the gel is added into the modified carboxymethyl chitosan solution for stirring, and microcapsules are obtained after washing and drying; the volume ratio of the core material solution to the wall material solution is 1:4; the concentration of the calcium chloride solution is 1wt%; the concentration of the modified carboxymethyl chitosan solution is 0.55wt%; the stirring conditions are: the stirring speed is 350rpm, and the stirring is carried out at room temperature for 25min.

[0047] The preparation method of modified sodium alginate is as follows: deionized water and sodium alginate are added to a reaction container, stirred evenly, 0.2 mol / L sodium hydroxide solution is used to adjust the pH to 8.8, the temperature is raised to 50°C, epichlorohydrin is slowly added dropwise, the reaction is completed for 6.5 hours, 4-phenylbenzylamine is added, the temperature is continued to be raised to 75°C, the reaction is carried out for 3.5 hours, and after the reaction is completed, the temperature is naturally reduced to room temperature, acetic acid is used to adjust the pH to 4.3, acetone (the volume is the same as the deionized volume) is added, soaking for 12 hours, filtering, repeatedly washing the filter residue with acetone, and drying to obtain modified sodium alginate; the mass ratio of sodium alginate, epichlorohydrin, 4-phenylbenzylamine, and deionized water is 1:0.3:0.55:28.

[0048] The preparation method of modified carboxymethyl chitosan is as follows: add anhydrous tetrahydrofuran and polyethylene glycol monomethyl ether into a reaction container, stir evenly, protect with nitrogen, add sodium hydride, heat to 65°C, react for 3 hours, add chlorinated carboxymethyl chitosan, continue to react for 6 hours, and after the reaction is completed, naturally cool to room temperature, remove tetrahydrofuran, wash the product with acetone, and obtain modified carboxymethyl chitosan after drying; the prepared modified carboxymethyl chitosan has strong fluorescence absorption under ultraviolet lamp (256nm); the mass ratio of polyethylene glycol monomethyl ether, sodium hydride, chlorinated carboxymethyl chitosan and anhydrous tetrahydrofuran is 1:0.25:1.6:20.

[0049] Example 4

[0050] A method for preparing microcapsules for embedding probiotics comprises the following steps:

[0051] S1. The activated probiotics are cultured and prepared into a probiotic suspension to obtain a core material solution; the probiotics are a mixture of the same amount of Lactobacillus acidophilus and Bifidobacterium; the concentration of the probiotic suspension is 1.0×10 9 CFU / mL;

[0052] S2, adding modified sodium alginate and whey protein into deionized water, dispersing them evenly by ultrasonication, and obtaining a wall material solution after sterilization; the concentration of modified sodium alginate is 2.5wt%; the concentration of whey protein is 1.5wt%; the ultrasonic conditions are: ultrasonic power is 300W, and ultrasonication is carried out for 10min at room temperature;

[0053] S3. The core material solution and the wall material solution are mixed evenly, added into the calcium chloride solution by extrusion, allowed to stand to form a gel, and then the gel is added into the modified carboxymethyl chitosan solution for stirring, and microcapsules are obtained after washing and drying; the volume ratio of the core material solution to the wall material solution is 1:5; the concentration of the calcium chloride solution is 1.5wt%; the concentration of the modified carboxymethyl chitosan solution is 0.5wt%; the stirring conditions are: the stirring speed is 400rpm, and the stirring is carried out at room temperature for 30min.

[0054] The preparation method of modified sodium alginate is as follows: add deionized water and sodium alginate into a reaction container, stir evenly, adjust the pH to 8.6 with a 0.1 mol / L sodium hydroxide solution, heat up to 50°C, slowly drop epichlorohydrin, react for 7 hours after the drop is complete, then add 4-phenylbenzylamine, continue to heat up to 80°C, react for 4 hours, and after the reaction is completed, naturally return to room temperature, adjust the pH to 4.2 with acetic acid, add acetone (the volume is the same as the deionized volume), soak for 12 hours, filter, repeatedly wash the filter residue with acetone, and obtain modified sodium alginate after drying; the mass ratio of sodium alginate, epichlorohydrin, 4-phenylbenzylamine, and deionized water is 1:0.4:0.5:30.

[0055] The preparation method of modified carboxymethyl chitosan is as follows: add anhydrous tetrahydrofuran and polyethylene glycol monomethyl ether into a reaction container, stir evenly, protect with nitrogen, add sodium hydride, heat to 60°C, react for 3 hours, add chlorinated carboxymethyl chitosan, continue to react for 6 hours, and after the reaction is completed, naturally cool to room temperature, remove tetrahydrofuran, wash the product with acetone, and obtain modified carboxymethyl chitosan after drying; the prepared modified carboxymethyl chitosan has strong fluorescence absorption under ultraviolet lamp (256nm); the mass ratio of polyethylene glycol monomethyl ether, sodium hydride, chlorinated carboxymethyl chitosan and anhydrous tetrahydrofuran is 1:0.2:2:20.

[0056] Comparative Example 1

[0057] Compared with Example 1, sodium alginate is used in step S2.

[0058] A method for preparing microcapsules for embedding probiotics comprises the following steps:

[0059] S1. Cultivate the activated probiotics to prepare a probiotic suspension to obtain a core material solution; the probiotics are Lactobacillus plantarum; the concentration of the probiotic suspension is 1.0×10 10 CFU / mL;

[0060] S2, adding sodium alginate and whey protein into deionized water, ultrasonically dispersing them uniformly, and sterilizing them to obtain a wall material solution; the concentration of sodium alginate is 3wt%; the concentration of whey protein is 1.5wt%; the ultrasonic conditions are: ultrasonic power is 300W, and ultrasonication is carried out at room temperature for 15min;

[0061] S3. The core material solution and the wall material solution are mixed evenly, added into the calcium chloride solution by extrusion, allowed to stand to form a gel, and then the gel is added into the modified carboxymethyl chitosan solution for stirring, and microcapsules are obtained after washing and drying; the volume ratio of the core material solution to the wall material solution is 1:5; the concentration of the calcium chloride solution is 1.3wt%; the concentration of the modified carboxymethyl chitosan solution is 0.6wt%; the stirring conditions are: the stirring speed is 300rpm, and the stirring is carried out at room temperature for 30min.

[0062] The preparation method of modified carboxymethyl chitosan is the same as that in Example 1.

[0063] Comparative Example 2

[0064] Compared with Example 1, chlorinated carboxymethyl chitosan is used in step S3.

[0065] A method for preparing microcapsules for embedding probiotics comprises the following steps:

[0066] S1. Cultivate the activated probiotics to prepare a probiotic suspension to obtain a core material solution; the probiotics are Lactobacillus plantarum; the concentration of the probiotic suspension is 1.0×10 10 CFU / mL;

[0067] S2, adding modified sodium alginate and whey protein into deionized water, dispersing them evenly by ultrasonication, and obtaining a wall material solution after sterilization; the concentration of modified sodium alginate is 3wt%; the concentration of whey protein is 1.5wt%; the ultrasonic conditions are: ultrasonic power is 300W, and ultrasonication is carried out at room temperature for 15min;

[0068] S3. The core material solution and the wall material solution are mixed evenly, added into the calcium chloride solution by extrusion, allowed to stand to form a gel, and then the gel is added into the chloride-carboxymethyl chitosan solution for stirring, and microcapsules are obtained after washing and drying; the volume ratio of the core material solution to the wall material solution is 1:5; the concentration of the calcium chloride solution is 1.3wt%; the concentration of the chloride-carboxymethyl chitosan solution is 0.6wt%; the stirring conditions are: the stirring speed is 300rpm, and the stirring is carried out at room temperature for 30min.

[0069] The preparation method of modified sodium alginate is the same as that in Example 1.

[0070] Comparative Example 3

[0071] Compared with Example 1, carboxymethyl chitosan is used in step S3.

[0072] A method for preparing microcapsules for embedding probiotics comprises the following steps:

[0073] S1. Cultivate the activated probiotics to prepare a probiotic suspension to obtain a core material solution; the probiotics are Lactobacillus plantarum; the concentration of the probiotic suspension is 1.0×10 10 CFU / mL;

[0074] S2, adding modified sodium alginate and whey protein into deionized water, dispersing them evenly by ultrasonication, and obtaining a wall material solution after sterilization; the concentration of modified sodium alginate is 3wt%; the concentration of whey protein is 1.5wt%; the ultrasonic conditions are: ultrasonic power is 300W, and ultrasonication is carried out at room temperature for 15min;

[0075] S3. The core material solution and the wall material solution are mixed evenly, added into the calcium chloride solution by extrusion, allowed to stand to form a gel, and then the gel is added into the carboxymethyl chitosan solution for stirring, and microcapsules are obtained after washing and drying; the volume ratio of the core material solution to the wall material solution is 1:5; the concentration of the calcium chloride solution is 1.3wt%; the concentration of the carboxymethyl chitosan solution is 0.6wt%; the stirring conditions are: the stirring speed is 300rpm, and the stirring is carried out at room temperature for 30min.

[0076] The preparation method of modified sodium alginate is the same as that in Example 1.

[0077] Comparative Example 4

[0078] Compared with Example 1, sodium alginate is used in step S2, and carboxymethyl chitosan is used in step S3.

[0079] A method for preparing microcapsules for embedding probiotics comprises the following steps:

[0080] S1. Cultivate the activated probiotics to prepare a probiotic suspension to obtain a core material solution; the probiotics are Lactobacillus plantarum; the concentration of the probiotic suspension is 1.0×10 10 CFU / mL;

[0081] S2, adding sodium alginate and whey protein into deionized water, ultrasonically dispersing them uniformly, and sterilizing them to obtain a wall material solution; the concentration of sodium alginate is 3wt%; the concentration of whey protein is 1.5wt%; the ultrasonic conditions are: ultrasonic power is 300W, and ultrasonication is carried out at room temperature for 15min;

[0082] S3. The core material solution and the wall material solution are mixed evenly, added into the calcium chloride solution by extrusion, allowed to stand to form a gel, and then the gel is added into the carboxymethyl chitosan solution for stirring, and microcapsules are obtained after washing and drying; the volume ratio of the core material solution to the wall material solution is 1:5; the concentration of the calcium chloride solution is 1.3wt%; the concentration of the carboxymethyl chitosan solution is 0.6wt%; the stirring conditions are: the stirring speed is 300rpm, and the stirring is carried out at room temperature for 30min.

[0083] 1. Microcapsule particle size and mechanical properties test

[0084] The particle size and mechanical properties of the microcapsules prepared in Examples 1-4 and Comparative Examples 1-4 were tested. The particle size of the microcapsules was measured using a vernier caliper. The particle sizes of 20 microcapsules were randomly tested and the average value was taken. The microcapsules were placed on an analytical balance and gradually pressurized on the front side with appropriate weights until they broke. The pressure displayed on the balance suddenly changed from small to large and then suddenly became small. The maximum front pressure at this time, i.e., the mechanical strength of the microcapsule, was recorded. 20 microcapsules were randomly tested and the average value was taken. The results are shown in Table 1.

[0085] Table 1 Microcapsule particle size and mechanical properties results

[0086] sample Particle size (mm) Mechanical strength (N) Example 1 3.08±0.02 7.5 Example 2 3.10±0.04 7.4 Example 3 3.13±0.07 7.3 Example 4 3.06±0.05 7.4 Comparative Example 1 2.95±0.06 5.3 Comparative Example 2 3.18±0.08 5.4 Comparative Example 3 2.98±0.05 5.1 Comparative Example 4 3.21±0.10 4.0

[0087] From the results in Table 1, it can be seen that the microcapsules prepared in Examples 1-4 of the present invention are spherical in shape, with an average particle size of about 3.1 mm, which meets the standards of microcapsules, and their mechanical strength is much greater than that of Comparative Examples 1-4, indicating that they have good mechanical strength; By comparing Example 1 with Comparative Examples 1-4, the wall material solution in Example 1 of the present invention contains modified sodium alginate, and the rigid double benzene ring group grafted in the modified sodium alginate structure can play a good supporting role, which is helpful to improve the mechanical properties of its gel formation, and the present invention adopts modified carboxymethyl chitosan The sugars are further cross-linked. On the one hand, the polyethylene glycol monomethyl ether side chains with surface activity grafted into the modified carboxymethyl chitosan structure help the modified carboxymethyl chitosan to cross-link with the gel more fully and evenly, thereby improving the porous properties of the gel and forming a denser three-dimensional network structure, thereby improving the mechanical properties of the microcapsules. On the other hand, the rigid double benzene ring groups grafted into the modified carboxymethyl chitosan structure can also synergize with the rigid double benzene ring groups grafted into the modified sodium alginate structure to achieve the effect of jointly improving the mechanical properties of the microcapsules.

[0088] 2. Microcapsule embedding rate, survival rate in different environments and stability test

[0089] The microcapsules prepared in Examples 1-4 and Comparative Examples 1-4 were tested for embedding efficiency, survival rate in simulated gastric fluid and simulated intestinal fluid, and stability.

[0090] Embedding rate test: Take 1g of each microcapsule and dissolve it in 9mL of encapsulation solution (a mixed solution of 0.2mol / L sodium bicarbonate and 0.06mol / L trisodium citrate at pH 8), decapsulate it, wash it with physiological saline, and determine the number of viable bacteria by plate counting method. Embedding rate (%) = number of viable bacteria after embedding / number of viable bacteria before embedding × 100%.

[0091] Survival rate test in simulated gastric juice: adjust the pH of 9g / L NaCl solution to 2.0 with 0.1mol / L hydrochloric acid, and then add 0.3% (w / v) pepsin solution (mass ratio is 1:3000) to obtain simulated gastric juice, add 1g microcapsule to 9mL simulated gastric juice, mix thoroughly, shake in a shaker at 200r / min at 37°C for 4h, wash with physiological saline after decapsulation, and determine the number of viable bacteria by plate counting method, survival rate (%) = number of viable bacteria after simulated gastric juice treatment / number of viable bacteria before treatment × 100%.

[0092] Survival rate test in simulated intestinal fluid: adjust the pH value of PBS solution to 8.0 with 0.1 mol / L NaOH solution, add 0.1% (w / v) trypsin solution (mass ratio is 1:250) and 0.3% (w / v) sodium cholate, dissolve evenly, filter and sterilize to obtain simulated intestinal fluid, add 1g microcapsule to 9mL simulated intestinal fluid, mix thoroughly, bathe at 37℃ for 3h, wash with physiological saline after decapsulation, and determine the number of viable bacteria by plate counting method, survival rate (%) = number of viable bacteria after treatment with simulated intestinal fluid / number of viable bacteria before treatment × 100%.

[0093] Stability test: The microcapsules were stored at 4°C for 90 days. After being opened, they were washed with physiological saline and the number of viable bacteria was determined by plate counting method. The survival rate (%) = number of viable bacteria after storage / number of viable bacteria before storage × 100%.

[0094] The above test results are shown in Table 2.

[0095] Table 2 Microcapsule embedding rate, survival rate in different environments and stability test results

[0096]

[0097]

[0098] As shown in Table 2, the embedding rate, the survival rate in simulated gastric juice and simulated intestinal juice, and the survival rate after 90 days of storage at 4°C of the microcapsules prepared in Examples 1-4 of the present invention are much higher than those in Comparative Examples 1-4, indicating that the microcapsules prepared in the present invention not only achieve efficient embedding of probiotics, but also enable them to maintain a high survival rate under different environments; by comparing Example 1 with Comparative Examples 1-4, in Example 1, modified sodium alginate and whey protein are first used to embed the probiotics, and the emulsification effect of whey protein helps the wall material solution to form a uniform and stable embedding of the probiotics, and its good film-forming property can also improve the porous properties of the gel, and then modified carboxymethyl chitosan is used for further cross-linking, which improves the compactness of the microcapsules, thereby obtaining microcapsules with good mechanical properties, enhancing the tolerance of the microcapsules in simulated gastric juice, simulated intestinal juice and at 4°C, and improving the survival rate and stability of the probiotics in the microcapsules.

[0099] Although the embodiments of the present application have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present application, and that the scope of the present application is defined by the appended claims and their equivalents.

Claims

1. A method for preparing microcapsules for embedding probiotics, characterized in that: The steps include: S1, culturing the activated probiotics to prepare a probiotic suspension to obtain a core material solution; S2, adding modified sodium alginate and whey protein into deionized water, dispersing them evenly by ultrasonication, and obtaining a wall material solution after sterilization; S3, mixing the core material solution and the wall material solution evenly, adding them into a calcium chloride solution by extrusion, standing to form a gel, then adding the gel into a modified carboxymethyl chitosan solution, stirring, washing, and drying to obtain microcapsules; The preparation method of the modified sodium alginate in step S2 is as follows: add deionized water and sodium alginate to a reaction container, stir evenly, adjust the pH to 8.5-9 with a 0.1-0.2 mol / L sodium hydroxide solution, raise the temperature to 40-50° C., slowly drop epichlorohydrin, react for 6-7 hours after the drop is complete, then add 4-phenylbenzylamine, continue to heat to 70-80° C., react for 3-4 hours, and after the reaction is completed, naturally cool to room temperature, adjust the pH to 4-4.5 with acetic acid, add acetone, soak for 12 hours, filter, repeatedly wash the filter residue with acetone, and dry to obtain the modified sodium alginate; The preparation method of the modified carboxymethyl chitosan in step S3 is as follows: add anhydrous tetrahydrofuran and polyethylene glycol monomethyl ether to a reaction container, stir evenly, protect with nitrogen, add sodium hydride, heat to 60-70° C., react for 3-4 hours, add chlorinated carboxymethyl chitosan, continue to react for 6-7 hours, and after the reaction is completed, naturally cool to room temperature, remove tetrahydrofuran, wash the product with acetone, and dry to obtain modified carboxymethyl chitosan; The preparation method of the chlorinated-carboxymethyl chitosan is as follows: 4-phenylbenzylamine and N,N'-carbonyldiimidazole are added to acetonitrile, stirred at room temperature for 5-6 hours, and most of the acetonitrile is removed to obtain a modified liquid for standby use; in addition, a 50-60wt% ethanol solution and carboxymethyl chitosan are added to a reaction container, stirred evenly, and epichlorohydrin is slowly added dropwise. After the addition is completed, the temperature is raised to 50-60°C, reacted for 4-5 hours, the modified liquid is added, and the reaction is continued for 6-8 hours. After the reaction is completed, the temperature is naturally lowered to room temperature, the pH is adjusted to neutral, acetone is added for precipitation, and the filter residue is filtered, and the filter residue is repeatedly washed with acetone, and the chlorinated-carboxymethyl chitosan is obtained after drying.

2. The method for preparing a probiotic-encapsulated microcapsule according to claim 1, characterized in that: In the preparation method of chlorinated-carboxymethyl chitosan, the mass ratio of carboxymethyl chitosan, epichlorohydrin and 50-60wt% ethanol solution is 1:0.4-0.5:25-30; the molar amount of 4-phenylbenzylamine and N,N'-carbonyldiimidazole is the same as the molar amount of epichlorohydrin.

3. The method for preparing a microcapsule for embedding probiotics according to claim 1, characterized in that: In the preparation method of modified sodium alginate, the mass ratio of sodium alginate, epichlorohydrin, 4-phenylbenzylamine and deionized water is 1:0.3-0.4:0.5-0.6:25-30.

4. The method for preparing a microcapsule for embedding probiotics according to claim 1, characterized in that: In the preparation method of modified carboxymethyl chitosan, the mass ratio of polyethylene glycol monomethyl ether, sodium hydride, chloride-carboxymethyl chitosan and anhydrous tetrahydrofuran is 1:0.2-0.3:1.5-2:20-25.

5. The method for preparing a microcapsule for embedding probiotics according to claim 1, characterized in that: The probiotics in step S1 are any one or a mixture of Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus casei, and Bifidobacterium lactis; the concentration of the probiotic suspension is 1.0×10 9 -1.0×10 10 CFU / mL.

6. The method for preparing a probiotic-embedded microcapsule according to claim 1, characterized in that: In step S2, the concentration of the modified sodium alginate is 2-3wt%; the concentration of the whey protein is 1-1.5wt%; and the ultrasonic conditions are: the ultrasonic power is 300-400W, and the ultrasonic treatment is performed at room temperature for 10-15 minutes.

7. The method for preparing a probiotic-embedded microcapsule according to claim 1, characterized in that: In step S3, the volume ratio of the core material solution to the wall material solution is 1:4-5; the concentration of the calcium chloride solution is 1-1.5wt%; the concentration of the modified carboxymethyl chitosan solution is 0.5-0.6wt%; and the stirring conditions are: stirring speed is 300-400rpm, and stirring is carried out at room temperature for 20-30min.

8. A microcapsule for embedding probiotics, characterized in that: The microcapsule is prepared by the method for preparing probiotic-embedded microcapsules according to any one of claims 1 to 7.

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