Microencapsulated probiotic product, preparation method and application in hygienic products

Abstract

The invention discloses a microencapsulated probiotic product, a preparation method and application of the microencapsulated probiotic product in hygienic products, and relates to the technical field of probiotic microencapsulation. According to the preparation method disclosed by the invention, the probiotics are embedded by adopting the modified sodium alginate as a wall material, the sodium alginate is modified by adopting the succinic anhydride and then is grafted with the whey protein, and the shell cross-linking constructed by adopting the sodium alginate modified by the succinic anhydride is tighter, so that glycerol can be effectively prevented from permeating into an inner layer; the chitosan-whey protein shell is modified by succinic anhydride, so that carboxyl on the chitosan-whey protein shell is increased, more whey protein can be grafted, the whey protein grafted on the chitosan-whey protein shell can fix glycerol in gaps of the whey protein, and the whey protein can be cross-linked with the chitosan-whey protein shell on the outermost layer, so that the chitosan-whey protein shell can be crosslinked. The overall crosslinking strength of the microcapsule is improved, so that the microcapsule is not easy to collapse and break.

Description

Technical Field

[0001] The present invention relates to the technical field of probiotic microencapsulation, in particular to a microencapsulated probiotic product, a preparation method and application in sanitary products. Background Art

[0002] Probiotics are important physiological bacteria in the human intestine and have multiple physiological functions, including improving human intestinal function, reducing intestinal diseases, promoting the absorption of nutrients, alleviating lactose intolerance, lowering cholesterol, regulating the immune system and improving antioxidant capacity. It is precisely because probiotics have many functions that are beneficial to human health that their application range is becoming wider and wider; However, probiotics are very sensitive to changes in the external environment such as pH and temperature, which greatly limits their preservation and application. Probiotics can effectively treat gastrointestinal diseases. However, their beneficial effects can only be seen when their number exceeds a certain content. Therefore, improving their survival ability is crucial to expanding their application. Therefore, the present application uses a double-layer microcapsule encapsulation method to encapsulate them. First, a polymer with pH responsiveness is used to encapsulate them once, so that they have the ability to release at a specific pH. After that, a layer of phase change material is adsorbed on the outer surface of the microcapsule formed by the first encapsulation, and a layer of wall material is wrapped on its outer surface to enable it to resist temperature changes, thereby improving its survival ability. Summary of the invention

[0003] The purpose of the present invention is to provide a microencapsulated probiotic product, a preparation method and application in sanitary products to solve the problems existing in the prior art.

[0004] In order to solve the above technical problems, the present invention provides the following technical solutions: A method for preparing a microencapsulated probiotic product, wherein the double-layer probiotic microcapsule comprises the following preparation steps: S1. The activated probiotic solution and the modified sodium alginate solution are uniformly mixed in a mass ratio of 1:6-8 to obtain a mixed solution; the mixed solution is uniformly added dropwise to a calcium chloride solution of 10-20 times the mass of the mixed solution at a rate of 1-1.5 mL / min. After the addition is completed, the mixture is allowed to stand for 30-35 min at a temperature of 25-35 ° C. After standing, the filtered product is washed with deionized water for 3-5 times, and then washed with sterile saline with a mass fraction of 0.85-0.9% for 2-3 times. After washing, the product is placed at -15--20 ° C, pre-frozen for 24-30 hours, and then transferred to a vacuum freeze dryer. After drying for 48-56 hours, a microcapsule freeze-dried powder is obtained; S2. Weigh the microcapsule freeze-dried powder and whey protein in a mass ratio of 1:6, add the microcapsule freeze-dried powder and whey protein into deionized water 10-12 times the mass of the microcapsule freeze-dried powder, stir for 30-40 minutes at a temperature of 35-40°C, add 0.95% sorbitan oleate by mass, and then add 0.5-0.8 times the mass of the microcapsule freeze-dried powder glycerol, stir for 2-2.5 hours to prepare a microcapsule mixed solution; add the microcapsule mixed solution dropwise to a 3% chitosan solution at a speed of 0.5-1 mL / min, stir and mix evenly, then add 8-10 times the mass of the microcapsule freeze-dried powder and a 1.2-1.5% sodium sulfate aqueous solution, ultrasonicate for 10-15 minutes, centrifuge to remove the lower precipitate after ultrasonication, wash with deionized water and anhydrous ethanol for 3-5 times respectively, freeze-dry after washing to prepare a double-layer probiotic microcapsule.

[0005] As an optimization, the concentration of Bifidobacterium bifidum in the activated probiotic solution in S1 is 10 9~10 CFU / mL.

[0006] As an optimization, the modified sodium alginate solution in S1 is prepared by the following steps: adding the modified sodium alginate to deionized water with a mass of 33 to 35 times that of the modified sodium alginate, stirring evenly at a temperature of 65 to 70°C, sterilizing and filtering with a 0.22 μm filter membrane, and preparing a modified sodium alginate solution for use.

[0007] As an optimization, the calcium chloride solution in S1 includes the following preparation method: adding calcium chloride to deionized water with a mass dissolution of 48 to 50 times the calcium chloride, stirring evenly, and sterilizing under high pressure for 15 to 20 minutes at a temperature of 120 to 125° C. to prepare a calcium chloride solution for use.

[0008] As an optimization, the modified sodium alginate includes the following preparation steps: adjusting the pH of a 0.2 mol / L MES buffer solution to 6.5 with a 0.3 mol / L sodium chloride solution; adding the grafted sodium alginate to the pH-adjusted MES buffer solution with a mass of 42 to 45 times that of the grafted sodium alginate, stirring evenly, then sequentially adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, stirring for 20 to 22 hours at a temperature of 25 to 35° C., adding whey protein with a mass of 0.8 to 1.2 times that of the grafted sodium alginate, stirring for another 10 to 15 minutes, then dialyzing and purifying, and then freeze-drying for 48 to 56 hours to obtain the modified sodium alginate.

[0009] As an optimization, the mass ratio between the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and the grafted sodium alginate is 0.6-0.8.

[0010] As an optimization, the mass ratio between the N-hydroxysuccinimide and the grafted sodium alginate is 0.2-0.25.

[0011] As an optimization, the grafted sodium alginate includes the following preparation steps: adding sodium alginate to deionized water with a mass of 32 to 34 times that of the sodium alginate, adjusting the pH to 7.5 to 8.5 with a hydrogen peroxide solution with a mass fraction of 10 to 15%, then adding succinic anhydride with a mass of 0.08 to 0.09 times that of the sodium alginate, stirring the reaction at a temperature of 45 to 55° C. for 2 to 3 hours, then dialyzing and purifying, and freeze-drying with a vacuum freeze dryer for 48 to 56 hours to obtain the grafted sodium alginate.

[0012] A microencapsulated probiotic product is prepared by the method for preparing a microencapsulated probiotic product according to any one of claims 1 to 8.

[0013] A microencapsulated probiotic product prepared by any one of the above methods is used in sanitary products.

[0014] Compared with the prior art, the beneficial effects achieved by the present invention are: The present application firstly uses modified sodium alginate as a wall material to embed probiotics, and then uses succinic anhydride to modify the sodium alginate and then grafts whey protein. The shell constructed by the sodium alginate modified by succinic anhydride is more tightly cross-linked, which can effectively prevent glycerol from penetrating into the inner layer. Moreover, since the carboxyl groups on the alginate are increased after modification by succinic anhydride, more whey protein can be grafted. Then, the grafted whey protein can also fix glycerol in the gaps of the whey protein. Then, the whey protein can be cross-linked with the outermost chitosan-whey protein shell, so that the overall cross-linking strength of the microcapsule is increased, making it not easy to collapse and break. The inner wall of the microcapsule constructed by sodium alginate modified with succinic anhydride is smoother and denser, which can effectively prevent the overflow of glycerol. The grafted whey protein fixes the glycerol on the periphery, which allows the glycerol to effectively absorb external heat as a phase change material, preventing the inactivation of probiotics caused by external temperature changes. The grafted whey protein can also provide a certain supporting capacity and participate in cross-linking in the subsequent preparation process of the outermost wall material, so that the overall performance of the final microcapsule is improved. In addition, since sodium alginate contains a large number of carboxyl groups, it is not easy to swell under acidic conditions, making the microcapsule acid-resistant. The outermost layer does not use whey protein alone as the wall material mainly because the mechanical properties of the outer shell constructed with whey protein alone are poor, and it is difficult to prevent the diffusion of fat-soluble substances when encapsulating them, which increases the oil content on the surface of the microcapsule powder and thus loses the ability to resist temperature changes. Therefore, the use of chitosan to jointly construct an external barrier can effectively resist external environmental changes and pressure. DETAILED DESCRIPTION

[0015] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0016] Example 1

[0017] S1. Grafting sodium alginate includes the following preparation steps: adding sodium alginate to deionized water with a mass of 32 times that of sodium alginate, adjusting the pH to 7.5 with a 10% by mass hydrogen peroxide solution, then adding succinic anhydride with a mass of 0.08 times that of sodium alginate, stirring and reacting for 2 hours at a temperature of 45°C, then dialyzing and purifying, and freeze-drying with a vacuum freeze dryer for 48 hours to obtain grafted sodium alginate; S2, modified sodium alginate includes the following preparation steps: adjusting the pH of 0.2 mol / L MES buffer solution to 6.5 with 0.3 mol / L sodium chloride solution; adding grafted sodium alginate to the pH-adjusted MES buffer solution of 42 times the mass of grafted sodium alginate, stirring evenly, then adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in sequence, stirring for 20 hours at a temperature of 25°C, adding whey protein of 0.8 times the mass of grafted sodium alginate, stirring for another 10 minutes, then dialyzing and purifying, and freeze-drying for 48 hours to obtain modified sodium alginate; the mass ratio between 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and grafted sodium alginate is 0.6; the mass ratio between N-hydroxysuccinimide and grafted sodium alginate is 0.2; S3. The activated probiotic solution and the modified sodium alginate solution were mixed uniformly at a mass ratio of 1:6 to obtain a mixed solution; the mixed solution was uniformly added dropwise to a calcium chloride solution 10 times the mass of the mixed solution at a rate of 1 mL / min. After the addition was completed, the mixture was allowed to stand for 30 min at 25°C. After standing, the mixture was filtered. The filtered product was washed with deionized water for 3 times, and then washed twice with sterile saline with a mass fraction of 0.85%. After washing, the mixture was placed at -15°C, pre-frozen for 24 hours, and then transferred to a vacuum freeze dryer. After drying for 48 hours, microcapsule freeze-dried powder was obtained; the concentration of Bifidobacterium bifidum in the activated probiotic solution was 10 9CFU / mL; the modified sodium alginate solution is prepared by the following steps: adding the modified sodium alginate to deionized water with a mass of 33 times that of the modified sodium alginate, stirring evenly at a temperature of 65°C, using a 0.22μm filter membrane for sterilization filtration, and preparing a modified sodium alginate solution for standby use; the calcium chloride solution in S1 includes the following preparation method: adding calcium chloride to deionized water with a mass of 48 times that of the calcium chloride, stirring evenly, and sterilizing under high pressure for 15 minutes at a temperature of 120°C to prepare a calcium chloride solution for standby use; S4. Weigh the microcapsule freeze-dried powder and whey protein in a mass ratio of 1:6, add the microcapsule freeze-dried powder and whey protein into deionized water 10 times the mass of the microcapsule freeze-dried powder, stir for 30 minutes at 35°C, add 0.95% sorbitan oleate by mass, and then add 0.5 times the mass of the microcapsule freeze-dried powder of glycerol, stir for 2 hours to prepare a microcapsule mixed solution; add the microcapsule mixed solution dropwise to the 3% chitosan solution at a speed of 0.5mL / min, stir and mix evenly, then add 1.2% sodium sulfate aqueous solution 8 times the mass of the microcapsule freeze-dried powder, ultrasonicate for 10 minutes, centrifuge to remove the lower precipitate after ultrasonication, wash it with deionized water and anhydrous ethanol for 3 times respectively, freeze-dry after washing to prepare a double-layer probiotic microcapsule.

[0018] Example 2

[0019] S1. Grafting sodium alginate includes the following preparation steps: adding sodium alginate to deionized water with a mass of 33 times that of sodium alginate, adjusting the pH to 7.5 with a 12% by mass hydrogen peroxide solution, then adding succinic anhydride with a mass of 0.085 times that of sodium alginate, stirring and reacting for 2.5 hours at a temperature of 50°C, then dialyzing and purifying, and freeze-drying with a vacuum freeze dryer for 52 hours to obtain grafted sodium alginate; S2. Modified sodium alginate includes the following preparation steps: adjusting the pH of 0.2 mol / L MES buffer solution to 6.5 with 0.3 mol / L sodium chloride solution; adding grafted sodium alginate to the pH-adjusted MES buffer solution with a mass of 43 times that of grafted sodium alginate, stirring evenly, then adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in sequence, stirring for 21 hours at a temperature of 30°C, adding whey protein with a mass of 0.9 times that of grafted sodium alginate, stirring for another 12 minutes, then dialyzing and purifying, and freeze-drying for 52 hours to obtain modified sodium alginate; the mass ratio between 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and grafted sodium alginate is 0.7; the mass ratio between N-hydroxysuccinimide and grafted sodium alginate is 0.25; S3. The activated probiotic solution and the modified sodium alginate solution were mixed evenly at a mass ratio of 1:7 to obtain a mixed solution; the mixed solution was uniformly added dropwise at a rate of 1.2 mL / min to a calcium chloride solution 15 times the mass of the mixed solution; after the addition was completed, the mixture was allowed to stand for 35 min at 30°C, and filtered after the standing was completed. The filtered product was washed with deionized water 4 times, and then washed twice with sterile saline with a mass fraction of 0.85%. After washing, it was placed at -20°C, pre-frozen for 28 hours, and then transferred to a vacuum freeze dryer. After drying for 52 hours, microcapsule freeze-dried powder was obtained; the concentration of Bifidobacterium bifidum in the activated probiotic solution was 10 10 CFU / mL; the modified sodium alginate solution is prepared by the following steps: adding the modified sodium alginate to deionized water with a mass of 34 times that of the modified sodium alginate, stirring evenly at a temperature of 70°C, using a 0.22μm filter membrane for sterilization filtration, and preparing a modified sodium alginate solution for standby use; the calcium chloride solution in S1 includes the following preparation method: adding calcium chloride to deionized water with a mass of 50 times that of the calcium chloride, stirring evenly, and sterilizing under high pressure for 15 minutes at a temperature of 125°C to prepare a calcium chloride solution for standby use; S4. Weigh the microcapsule freeze-dried powder and whey protein in a mass ratio of 1:6, add the microcapsule freeze-dried powder and whey protein into deionized water 11 times the mass of the microcapsule freeze-dried powder, stir for 35 minutes at 35°C, add 0.95% sorbitan oleate by mass, and then add 0.7 times the mass of the microcapsule freeze-dried powder of glycerol, stir for 2 hours to prepare a microcapsule mixed solution; add the microcapsule mixed solution dropwise to the 3% chitosan solution at a speed of 0.8 mL / min, stir and mix evenly, then add 1.3% sodium sulfate aqueous solution 9 times the mass of the microcapsule freeze-dried powder, ultrasonicate for 12 minutes, centrifuge to remove the lower layer of precipitate after ultrasonication, wash it with deionized water and anhydrous ethanol for 4 times respectively, freeze-dry after washing to prepare double-layer probiotic microcapsules.

[0020] Example 3

[0021] S1. Grafted sodium alginate includes the following preparation steps: adding sodium alginate to deionized water with a mass of 34 times that of sodium alginate, adjusting the pH to 8.5 with a 15% by mass hydrogen peroxide solution, then adding succinic anhydride with a mass of 0.09 times that of sodium alginate, stirring and reacting for 3 hours at a temperature of 55°C, then dialyzing and purifying, and freeze-drying with a vacuum freeze dryer for 56 hours to obtain grafted sodium alginate; S2, modified sodium alginate includes the following preparation steps: adjusting the pH of 0.2 mol / L MES buffer solution to 6.5 with 0.3 mol / L sodium chloride solution; adding grafted sodium alginate to the pH-adjusted MES buffer solution with a mass of 45 times that of grafted sodium alginate, stirring evenly, then adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in sequence, stirring for 22 hours at a temperature of 35°C, adding whey protein with a mass of 1.2 times that of grafted sodium alginate, stirring for another 15 minutes, then dialyzing and purifying, and freeze-drying for 56 hours to obtain modified sodium alginate; the mass ratio between 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and grafted sodium alginate is 0.8; the mass ratio between N-hydroxysuccinimide and grafted sodium alginate is 0.25; S3. The activated probiotic solution and the modified sodium alginate solution were mixed uniformly at a mass ratio of 1:8 to obtain a mixed solution; the mixed solution was uniformly added dropwise to a calcium chloride solution 20 times the mass of the mixed solution at a rate of 1.5 mL / min. After the addition was completed, the mixture was allowed to stand for 35 min at a temperature of 35°C. After standing, the mixture was filtered. The filtered product was washed with deionized water for 5 times, and then washed with sterile saline with a mass fraction of 0.9% for 3 times. After washing, the mixture was placed at -20°C, pre-frozen for 30 hours, and then transferred to a vacuum freeze dryer. After drying for 56 hours, microcapsule freeze-dried powder was obtained; the concentration of Bifidobacterium bifidum in the activated probiotic solution was 10 10 CFU / mL; the modified sodium alginate solution is prepared by the following steps: adding the modified sodium alginate to deionized water with a mass of 35 times that of the modified sodium alginate, stirring evenly at a temperature of 70°C, using a 0.22μm filter membrane for sterilization filtration, and preparing a modified sodium alginate solution for standby use; the calcium chloride solution in S1 includes the following preparation method: adding calcium chloride to deionized water with a mass of 50 times that of the calcium chloride, stirring evenly, and sterilizing under high pressure at a temperature of 125°C for 20 minutes to prepare a calcium chloride solution for standby use; S4. Weigh the microcapsule freeze-dried powder and whey protein in a mass ratio of 1:6, add the microcapsule freeze-dried powder and whey protein to deionized water 12 times the mass of the microcapsule freeze-dried powder, stir for 40 minutes at a temperature of 40°C, add 0.95% sorbitan oleate by mass, and then add 0.8 times the mass of the microcapsule freeze-dried powder glycerol, stir for 2.5 hours to prepare a microcapsule mixed solution; add the microcapsule mixed solution dropwise to a 3% chitosan solution at a speed of 1 mL / min, stir and mix evenly, then add a 1.5% sodium sulfate aqueous solution 10 times the mass of the microcapsule freeze-dried powder, ultrasonicate for 15 minutes, centrifuge to remove the lower precipitate after ultrasonication, wash it with deionized water and anhydrous ethanol for 5 times respectively, freeze-dry after washing to prepare a double-layer probiotic microcapsule; Example 4

[0022] The difference from Example 2 is only in step S2: the modified sodium alginate includes the following preparation steps: adjusting the pH of 0.2 mol / L MES buffer solution to 6.5 with 0.3 mol / L sodium chloride solution; adding sodium alginate to the pH-adjusted MES buffer solution with a mass of 43 times that of sodium alginate, stirring evenly, then adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in sequence, stirring for 21 hours at a temperature of 30°C, adding whey protein with a mass of 0.9 times that of sodium alginate, stirring for another 12 minutes, then dialyzing and purifying, and freeze-drying for 52 hours to obtain modified sodium alginate; the mass ratio between 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and sodium alginate is 0.7; the mass ratio between N-hydroxysuccinimide and sodium alginate is 0.25; Example 5

[0023] The only difference from Example 2 is step S3: the activated probiotic solution and the sodium alginate solution are mixed uniformly at a mass ratio of 1:7 to obtain a mixed solution; the mixed solution is uniformly added dropwise to a calcium chloride solution 15 times the mass of the mixed solution at a speed of 1.2 mL / min. After the addition is completed, the mixture is allowed to stand for 35 min at a temperature of 30°C. After standing, the filtered product is washed 4 times with deionized water, and then washed twice with sterile saline with a mass fraction of 0.85%. After washing, it is placed at -20°C, pre-frozen for 28 hours, and then transferred to a vacuum freeze dryer. After drying for 52 hours, a microcapsule freeze-dried powder is obtained; the concentration of Bifidobacterium bifidum in the activated probiotic solution is 10 10CFU / mL; the sodium alginate solution is prepared by the following steps: adding sodium alginate to deionized water with a mass of 34 times that of the sodium alginate, stirring evenly at a temperature of 70°C, using a 0.22μm filter membrane for sterilization filtration, and preparing a sodium alginate solution for standby use; the calcium chloride solution in S1 includes the following preparation method: adding calcium chloride to deionized water with a mass of 50 times that of the calcium chloride, stirring evenly, and sterilizing under high pressure for 15 minutes at a temperature of 125°C to prepare a calcium chloride solution for standby use; Example 6

[0024] The only difference from Example 2 is step S3: the activated probiotic solution and the grafted sodium alginate solution are mixed uniformly at a mass ratio of 1:7 to obtain a mixed solution; the mixed solution is uniformly added dropwise to a calcium chloride solution 15 times the mass of the mixed solution at a rate of 1.2 mL / min. After the addition is completed, the mixture is allowed to stand for 35 min at a temperature of 30°C. After standing, the filtered product is washed 4 times with deionized water, and then washed twice with sterile saline with a mass fraction of 0.85%. After washing, it is placed at -20°C, pre-frozen for 28 hours, and then transferred to a vacuum freeze dryer. After drying for 52 hours, a microcapsule freeze-dried powder is obtained; the concentration of Bifidobacterium bifidum in the activated probiotic solution is 10 10 CFU / mL; the grafted sodium alginate solution is prepared by the following steps: adding the grafted sodium alginate to deionized water with a mass of 34 times that of the grafted sodium alginate, stirring evenly at a temperature of 70°C, using a 0.22μm filter membrane for sterilization filtration, and preparing a grafted sodium alginate solution for standby use; the calcium chloride solution in S1 includes the following preparation method: adding calcium chloride to deionized water with a mass of 50 times that of the calcium chloride, stirring evenly, and sterilizing under high pressure for 15 minutes at a temperature of 125°C to prepare a calcium chloride solution for standby use; Example 7

[0025] The only difference from Example 2 is that step S4 is not performed, and the microcapsule freeze-dried powder prepared in step S3 is used as the final product.

[0026] Comparative Example 1 0.5 g of the probiotic microcapsules prepared in Examples 1 to 7 were added to 4.5 mL of simulated gastric fluid, stirred evenly, and shaken in a shaker at 200 r / min for 120 min at 37 ° C. After the treatment, centrifuged, the supernatant was discarded, the precipitate was collected, 4.5 mL of PBS buffer with pH 7.4 was added to the precipitate, and shaken at 37 ° C until the probiotic microcapsules in the precipitate were completely disintegrated to obtain a bacterial suspension, and then the viable bacteria of the bacterial suspension were counted. According to the viable bacteria count result, the survival rate of the probiotics was calculated.

[0027] Table 1 Experimental data on survival rate of probiotics in gastric juice simulation

[0028] From the comparison of the experimental data in Table 1, it can be found that Examples 1 to 3 all maintain a good survival rate and are originally higher than Examples 4 to 7. It can be expected that it will decrease in Examples 4 and 5, mainly because the internal wall material uses sodium alginate without grafting succinic anhydride to directly graft whey protein or unmodified sodium alginate, resulting in the final wall material not being tightly sealed. In addition to being easily mixed with glycerol, it will also cause the inner wall to be unable to effectively control the release of probiotics after the outer wall material is ruptured, resulting in a large number of probiotics being released into the gastric juice and ruptured and dying; although whey protein is not grafted in Example 6, it is more tightly sealed and not easy to disperse in the gastric juice, so its survival rate is significantly different. Example 7 is roughly the same as Example 6, but it is also expected that its survival rate will decrease due to the lack of the outermost wall material.

[0029] Comparative Example 2 0.5 g of the probiotic microcapsules prepared in Examples 1 to 7 were placed in a constant temperature box at 40 ° C. and allowed to stand for 5 h. Then, they were transferred to 4.5 mL of PBS buffer at pH 7.4 and shaken at 37 ° C. until the probiotic microcapsules in the precipitate were completely disintegrated to obtain a bacterial suspension. The live bacteria in the bacterial suspension were counted, and the survival rate of the probiotics was calculated based on the live bacteria count results.

[0030] Table 2 Experimental data on survival rate of temperature-sensitive probiotics

[0031] From the comparison of experimental data in Table 2, it can be found that Examples 1 to 3 all maintain good survival rates and are higher than Examples 5 to 7. It can be expected that it will decrease in Example 7, mainly because the lack of glycerol as a phase change material makes it more sensitive to temperature changes. However, it is speculated that the sensitivity to temperature changes in 5 and 6 is due to the fact that whey protein is not grafted, so that there is less glycerol attached to its outer surface, resulting in a decrease in the final resistance to temperature changes. This can also be proved from Example 4, because although the survival ability of the grafted whey protein in Example 4 in gastric juice decreases, its resistance to temperature changes is still significantly better than that of Examples 5 to 7.

[0032] It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above and that the invention can be implemented in other specific forms without departing from the spirit or essential features of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations within the meaning and scope of the equivalent elements of the claims be included in the invention. Any marking in a claim should not be considered as limiting the claim to which it relates.

Claims

1. A method for preparing a microencapsulated probiotic product, characterized in that: The double-layer probiotic microcapsule comprises the following preparation steps: S1. The activated probiotic solution and the modified sodium alginate solution are uniformly mixed in a mass ratio of 1:6-8 to obtain a mixed solution; the mixed solution is uniformly added dropwise to a calcium chloride solution of 10-20 times the mass of the mixed solution at a rate of 1-1.5 mL / min. After the addition is completed, the mixture is allowed to stand for 30-35 min at a temperature of 25-35 ° C. After standing, the mixture is filtered, and the filtered product is washed with deionized water for 3-5 times, and then washed with sterile saline with a mass fraction of 0.85-0.9% for 2-3 times. After washing, the mixture is placed at -15--20 ° C, pre-frozen for 24-30 hours, and then transferred to a vacuum freeze dryer. After drying for 48-56 hours, a microcapsule freeze-dried powder is obtained; S2. Weigh the microcapsule freeze-dried powder and whey protein in a mass ratio of 1:6, add the microcapsule freeze-dried powder and whey protein into deionized water 10-12 times the mass of the microcapsule freeze-dried powder, stir for 30-40 minutes at a temperature of 35-40°C, add 0.95% sorbitan oleate by mass, and then add 0.5-0.8 times the mass of the microcapsule freeze-dried powder glycerol, stir for 2-2.5 hours to prepare a microcapsule mixed solution; add the microcapsule mixed solution dropwise to a 3% chitosan solution at a speed of 0.5-1 mL / min, stir and mix evenly, then add 8-10 times the mass of the microcapsule freeze-dried powder and a 1.2-1.5% sodium sulfate aqueous solution, ultrasonicate for 10-15 minutes, centrifuge to remove the lower precipitate after ultrasonication, wash with deionized water and anhydrous ethanol for 3-5 times respectively, freeze-dry after washing to prepare a double-layer probiotic microcapsule.

2. The method for preparing a microencapsulated probiotic product according to claim 1, characterized in that: The concentration of Bifidobacterium bifidum in the activated probiotic solution in S1 is 10 9~10 CFU / mL.

3. The method for preparing a microencapsulated probiotic product according to claim 1, characterized in that: The modified sodium alginate solution in S1 is prepared by the following steps: adding the modified sodium alginate to deionized water with a mass of 33 to 35 times that of the modified sodium alginate, stirring evenly at a temperature of 65 to 70° C., sterilizing and filtering with a 0.22 μm filter membrane, and preparing a modified sodium alginate solution for standby use.

4. The method for preparing a microencapsulated probiotic product according to claim 1, characterized in that: The calcium chloride solution in S1 includes the following preparation method: adding calcium chloride to deionized water with a mass dissolution of 48 to 50 times of calcium chloride, stirring evenly, and sterilizing under high pressure for 15 to 20 minutes at a temperature of 120 to 125° C. to prepare a calcium chloride solution for use.

5. The method for preparing a microencapsulated probiotic product according to claim 1, characterized in that: The modified sodium alginate comprises the following preparation steps: adjusting the pH value of a 0.2 mol / L MES buffer solution to 6.5 with a 0.3 mol / L sodium chloride solution; adding the grafted sodium alginate to the pH-adjusted MES buffer solution with a mass of 42 to 45 times that of the grafted sodium alginate, stirring evenly, then sequentially adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, stirring for 20 to 22 hours at a temperature of 25 to 35° C., adding whey protein with a mass of 0.8 to 1.2 times that of the grafted sodium alginate, stirring for another 10 to 15 minutes, then dialyzing and purifying, and then freeze-drying for 48 to 56 hours to obtain the modified sodium alginate.

6. The method for preparing a microencapsulated probiotic product according to claim 5, characterized in that: The mass ratio between the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and the grafted sodium alginate is 0.6-0.

8.

7. The method for preparing a microencapsulated probiotic product according to claim 5, characterized in that: The mass ratio between the N-hydroxysuccinimide and the grafted sodium alginate is 0.2-0.

25.

8. The method for preparing a microencapsulated probiotic product according to claim 5, characterized in that: The grafted sodium alginate comprises the following preparation steps: adding sodium alginate to deionized water with a mass of 32 to 34 times that of the sodium alginate, adjusting the pH to 7.5 to 8.5 with a hydrogen peroxide solution with a mass fraction of 10 to 15%, then adding succinic anhydride with a mass of 0.08 to 0.09 times that of the sodium alginate, stirring and reacting for 2 to 3 hours at a temperature of 45 to 55° C., then dialyzing and purifying, and freeze-drying with a vacuum freeze dryer for 48 to 56 hours to obtain the grafted sodium alginate.

9. A microencapsulated probiotic product, characterized in that: The microencapsulated probiotic product is prepared by the method for preparing the microencapsulated probiotic product according to any one of claims 1 to 8.

10. A microencapsulated probiotic product prepared by any one of claims 1 to 8, characterized in that: Used in hygiene products.

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