A microencapsulated probiotic product, method of preparation and use in hygiene products

Abstract

The application discloses a kind of microencapsulated probiotic products, preparation method and application in sanitary products, it is related to probiotic microencapsulation technical field.The application uses modified sodium alginate as wall material to embed probiotics, after using succinic anhydride to modify sodium alginate, then grafting whey protein, the shell of the sodium alginate modified by succinic anhydride is more closely crosslinked, which can effectively prevent glycerol from penetrating into the inner layer, and because the carboxyl on it will be more after being modified by succinic anhydride, more whey protein can be grafted, and the whey protein grafted on it can also fix glycerol in the gap of whey protein, and the whey protein can be crosslinked with the outermost chitosan-whey protein shell, so that the overall crosslinking strength of microcapsule is increased, so that it is not easy to collapse and break.

Description

TECHNICAL FIELD

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

[0002] Probiotics are important physiological bacteria in the human intestinal tract, and have various physiological functions, including improving intestinal function, reducing intestinal diseases, promoting the absorption of nutrients, alleviating lactose intolerance, reducing cholesterol, regulating the immune system and improving antioxidant capacity, etc. Because probiotics have many functions that are beneficial to human health, their application range is becoming wider and wider.

[0003] However, probiotics are very sensitive to changes in external environment such as pH and temperature, which greatly limits their preservation and application. Probiotics can effectively treat gastrointestinal diseases, but only when their number exceeds a certain content can their beneficial effects be seen. Therefore, improving their survival ability is crucial for expanding their application. Therefore, the present application uses a double-layer microcapsule wrapping method to embed them, first using a polymer with pH response ability to embed them once, so that they have the ability to release at a specific pH, and then adsorbing a layer of phase change material on the outer surface of the microcapsule formed by the first embedding and wrapping a layer of wall material on the outer surface, so that they have the ability to resist temperature changes, thereby improving their survival ability. SUMMARY

[0004] The purpose of the present application is to provide a microencapsulated probiotic product, a preparation method and application in sanitary products to solve the problems in the prior art.

[0005] In order to solve the above technical problems, the present application provides the following technical scheme:

[0006] A preparation method of a microencapsulated probiotic product, the double-layer probiotic microcapsule comprising the following preparation steps:

[0007] S1, mix the activated probiotic solution and the modified sodium alginate solution uniformly according to a mass ratio of 1:6-8 to obtain a mixed solution; uniformly drop the mixed solution into a calcium chloride solution at a speed of 1-1.5 mL / min, the mass of the calcium chloride solution being 10-20 times that of the mixed solution; after the dropping is completed, place the solution at a temperature of 25-35℃ for 30-35 min; after the placement is completed, filter the solution; wash the filtered product with deionized water for 3-5 times, then wash it with sterile normal saline with a mass fraction of 0.85-0.9% for 2-3 times; after the washing is completed, place the product at a temperature of-15--20℃ for 24-30 h, then transfer it to a vacuum freeze-drying machine; dry it for 48-56 h to obtain a microcapsule freeze-dried powder;

[0008] S2. Weigh the lyophilized microcapsule powder and whey protein at a mass ratio of 1:6. Add the lyophilized microcapsule powder and whey protein to 10-12 times the mass of the lyophilized microcapsule powder in deionized water. Stir for 30-40 minutes at 35-40℃. Then add 0.95% sorbitan oleate and 0.5-0.8 times the mass of the lyophilized microcapsule powder in glycerol. Stir for 2-2.5 hours to prepare a microcapsule mixture solution. Add the microcapsule mixture solution dropwise at a rate of 0.5-1 mL / min to a 3% chitosan solution. Stir until well mixed. Then add 8-10 times the mass of the lyophilized microcapsule powder in a 1.2-1.5% sodium sulfate aqueous solution. Sonicate for 10-15 minutes. After sonication, centrifuge and collect the lower precipitate. Wash with deionized water and anhydrous ethanol 3-5 times respectively. After washing, freeze-dry to obtain double-layer probiotic microcapsules.

[0009] The modified sodium alginate includes the following preparation steps: 0.2 mol / L MES buffer solution is adjusted to pH 6.5 using 0.3 mol / L sodium chloride solution; grafted sodium alginate is added to 42-45 times its mass of the pH-adjusted MES buffer solution, stirred until homogeneous, then 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide are added sequentially. The mixture is stirred at 25-35°C for 20-22 hours, then 0.8-1.2 times its mass of whey protein is added, and the mixture is stirred for another 10-15 minutes. After dialysis purification and freeze-drying for 48-56 hours, modified sodium alginate is obtained.

[0010] The grafted sodium alginate comprises the following preparation steps: sodium alginate is added to deionized water at a mass of 32-34 times that of sodium alginate, and the pH is adjusted to 7.5-8.5 with a hydrogen peroxide solution at a mass of 10-15%. Then, succinic anhydride at a mass of 0.08-0.09 times that of sodium alginate is added. The mixture is stirred and reacted at a temperature of 45-55°C for 2-3 hours. After purification by dialysis, the mixture is freeze-dried in a vacuum freeze dryer for 48-56 hours to obtain grafted sodium alginate.

[0011] As an optimization, the concentration of Bifidobacterium bifidum in the activated probiotic solution in S1 is 10. 9~10 CFU / mL.

[0012] As an optimization, the modified sodium alginate solution in S1 is prepared by the following steps: adding modified sodium alginate to deionized water at a mass of 33 to 35 times that of modified sodium alginate, stirring evenly at a temperature of 65 to 70°C, and filtering with a 0.22 μm filter membrane for sterilization to prepare a modified sodium alginate solution for later use.

[0013] As an optimization, the calcium chloride solution in S1 is prepared by the following method: calcium chloride is added to deionized water at a mass of 48 to 50 times that of calcium chloride, stirred evenly, and then sterilized by autoclaving at a temperature of 120 to 125°C for 15 to 20 minutes to prepare a calcium chloride solution for later use.

[0014] As an optimization, the mass ratio between 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and grafted sodium alginate is 0.6~0.8.

[0015] As an optimization, the mass ratio between the N-hydroxysuccinimide and the grafted sodium alginate is 0.2~0.25.

[0016] A microencapsulated probiotic product is prepared using the method described above for preparing microencapsulated probiotic products.

[0017] Microencapsulated probiotic products are used in hygiene products.

[0018] Compared with the prior art, the beneficial effects achieved by the present invention are:

[0019] This application first uses modified sodium alginate as a wall material to encapsulate probiotics. The sodium alginate is modified with succinic anhydride and then grafted with whey protein. The shell constructed by the sodium alginate modified with succinic anhydride has a tighter cross-linking, which can effectively prevent glycerol from penetrating into the inner layer. Furthermore, the modification with succinic anhydride increases the number of carboxyl groups on it, thereby allowing more whey protein to be grafted. The whey protein grafted on it can also fix glycerol in the gaps of the whey protein. In addition, the whey protein can cross-link with the outermost chitosan-whey protein shell, which increases the overall cross-linking strength of the microcapsule and makes it less prone to collapse and breakage.

[0020] The inner wall of the microcapsule constructed with sodium alginate modified with succinic anhydride is smoother and denser, which can effectively prevent glycerol from leaking out. The grafted whey protein fixes the glycerol on the outside, which can effectively absorb external heat as a phase change material, preventing the probiotics from being inactivated due to external temperature changes. In addition, the grafted whey protein can also provide a certain degree of support and participate in cross-linking in the subsequent preparation of the outermost wall material, which improves the overall performance of the final microcapsule. Furthermore, because sodium alginate contains a large number of carboxyl groups, it is not easy to swell under acidic conditions, which makes the microcapsule acid-resistant.

[0021] The outermost layer does not use whey protein alone as the wall material mainly because whey protein alone has poor mechanical properties and is difficult to prevent the diffusion of fat-soluble substances when encapsulating them. This increases the oil content on the surface of the microcapsule powder, thus losing its ability to resist temperature changes. Therefore, using chitosan to jointly construct the outer barrier can effectively resist external environmental changes and pressure. Detailed Implementation

[0022] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0023] Example 1

[0024] S1. The preparation steps of grafted sodium alginate are as follows: Sodium alginate is added to deionized water at a mass of 32 times that of sodium alginate, and the pH is adjusted to 7.5 with 10% hydrogen peroxide solution. Then, succinic anhydride at a mass of 0.08 times that of sodium alginate is added. The mixture is stirred and reacted at a temperature of 45°C for 2 hours. After dialysis purification, the mixture is freeze-dried in a vacuum freeze dryer for 48 hours to obtain grafted sodium alginate.

[0025] S2. Modified sodium alginate comprises the following preparation steps: The pH of a 0.2 mol / L MES buffer solution is adjusted to 6.5 using a 0.3 mol / L sodium chloride solution; grafted sodium alginate is added to the pH-adjusted MES buffer solution at 42 times its mass, and stirred until homogeneous. Then, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide are added sequentially. The mixture is stirred at 25°C for 20 hours, followed by the addition of whey protein at 0.8 times its mass, and stirring for another 10 minutes. After dialysis purification and freeze-drying for 48 hours, modified sodium alginate is obtained. The mass ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to grafted sodium alginate is 0.6; the mass ratio of N-hydroxysuccinimide to grafted sodium alginate is 0.2.

[0026] S3. The activated probiotic solution and the modified sodium alginate solution were mixed evenly at a mass ratio of 1:6 to obtain a mixed solution. The mixed solution was added dropwise at a rate of 1 mL / min to a calcium chloride solution with a mass of 10 times that of the mixed solution. After the addition was complete, the mixture was allowed to stand at 25℃ for 30 min. After standing, it was filtered. The filtered product was washed three times with deionized water and then twice with sterile physiological saline with a mass fraction of 0.85%. After washing, it was placed at -15℃ for pre-freezing for 24 h and then transferred to a vacuum freeze dryer for drying for 48 h to obtain microcapsule lyophilized powder. 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: Add modified sodium alginate to 33 times its mass of deionized water, stir evenly at 65℃, and filter sterilize using a 0.22μm filter membrane to prepare the modified sodium alginate solution for later use; The calcium chloride solution in S1 is prepared by the following method: Add calcium chloride to 48 times its mass of deionized water, stir evenly, and autoclave at 120℃ for 15 minutes to prepare the calcium chloride solution for later use;

[0027] S4. Weigh the lyophilized microcapsule powder and whey protein at a mass ratio of 1:6. Add the lyophilized microcapsule powder and whey protein to 10 times the mass of the lyophilized microcapsule powder in deionized water. Stir for 30 min at 35℃. Then add 0.95% sorbitan oleate and 0.5 times the mass of the lyophilized microcapsule powder in glycerol. Stir for 2 h to prepare a microcapsule mixture solution. Add the microcapsule mixture solution dropwise at a rate of 0.5 mL / min to a 3% chitosan solution. Stir until well mixed. Then add 8 times the mass of the lyophilized microcapsule powder in a 1.2% sodium sulfate aqueous solution. Sonicate for 10 min. After sonication, centrifuge and collect the lower precipitate. Wash three times with deionized water and anhydrous ethanol, respectively. After washing, freeze-dry to obtain double-layer probiotic microcapsules.

[0028] Example 2

[0029] S1. The preparation steps of grafted sodium alginate are as follows: Sodium alginate is added to deionized water at a mass of 33 times that of sodium alginate, and the pH is adjusted to 7.5 with 12% hydrogen peroxide solution. Then, succinic anhydride at a mass of 0.085 times that of sodium alginate is added. The mixture is stirred and reacted at 50°C for 2.5 hours. After dialysis purification, the mixture is freeze-dried in a vacuum freeze dryer for 52 hours to obtain grafted sodium alginate.

[0030] S2. Modified sodium alginate includes the following preparation steps: The pH of a 0.2 mol / L MES buffer solution is adjusted to 6.5 using a 0.3 mol / L sodium chloride solution; grafted sodium alginate is added to the pH-adjusted MES buffer solution at 43 times its mass, and stirred until homogeneous. Then, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide are added sequentially. The mixture is stirred at 30°C for 21 hours, followed by the addition of whey protein at 0.9 times its mass, and stirring for another 12 minutes. After dialysis purification and freeze-drying for 52 hours, modified sodium alginate is obtained. The mass ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to grafted sodium alginate is 0.7; the mass ratio of N-hydroxysuccinimide to grafted sodium alginate is 0.25.

[0031] S3. The activated probiotic solution and modified sodium alginate solution were mixed evenly at a mass ratio of 1:7 to obtain a mixed solution. The mixed solution was added dropwise at a rate of 1.2 mL / min to a calcium chloride solution with a mass of 15 times that of the mixed solution. After the addition was complete, the mixture was allowed to stand at 30℃ for 35 min. After standing, it was filtered. The filtered product was washed four times with deionized water and then twice with sterile physiological saline with a mass fraction of 0.85%. After washing, it was placed at -20℃ for pre-freezing for 28 h and then transferred to a vacuum freeze dryer for drying for 52 h to obtain microcapsule lyophilized powder. 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: Add modified sodium alginate to 34 times its mass of deionized water, stir evenly at 70℃, and filter sterilize using a 0.22μm filter membrane to prepare the modified sodium alginate solution for later use; The calcium chloride solution in S1 is prepared by the following method: Add calcium chloride to 50 times its mass of deionized water, stir evenly, and autoclave at 125℃ for 15 minutes to prepare the calcium chloride solution for later use;

[0032] S4. Weigh the lyophilized microcapsule powder and whey protein at a mass ratio of 1:6. Add the lyophilized microcapsule powder and whey protein to 11 times the mass of the lyophilized microcapsule powder in deionized water. Stir at 35℃ for 35 min, then add 0.95% sorbitan oleate and 0.7 times the mass of the lyophilized microcapsule powder in glycerol. Stir for 2 h to prepare a microcapsule mixture solution. Add the microcapsule mixture solution dropwise at a rate of 0.8 mL / min to a 3% chitosan solution and stir until homogeneous. Then add 9 times the mass of the lyophilized microcapsule powder in a 1.3% sodium sulfate aqueous solution. Sonicate for 12 min. After sonication, centrifuge and collect the lower precipitate. Wash with deionized water and anhydrous ethanol four times each. After washing, freeze-dry to obtain double-layer probiotic microcapsules.

[0033] Example 3

[0034] S1. The preparation steps of grafted sodium alginate are as follows: Sodium alginate is added to deionized water at a mass of 34 times that of sodium alginate, and the pH is adjusted to 8.5 with 15% hydrogen peroxide solution. Then, succinic anhydride at a mass of 0.09 times that of sodium alginate is added. The mixture is stirred and reacted at 55°C for 3 hours. After dialysis purification, the mixture is freeze-dried in a vacuum freeze dryer for 56 hours to obtain grafted sodium alginate.

[0035] S2. Modified sodium alginate includes the following preparation steps: The pH of a 0.2 mol / L MES buffer solution is adjusted to 6.5 using a 0.3 mol / L sodium chloride solution; grafted sodium alginate is added to the pH-adjusted MES buffer solution at 45 times its mass, and stirred until homogeneous. Then, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide are added sequentially. The mixture is stirred at 35°C for 22 hours, followed by the addition of whey protein at 1.2 times its mass, and stirring for another 15 minutes. After dialysis purification and freeze-drying for 56 hours, modified sodium alginate is obtained. The mass ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to grafted sodium alginate is 0.8; the mass ratio of N-hydroxysuccinimide to grafted sodium alginate is 0.25.

[0036] S3. The activated probiotic solution and modified sodium alginate solution were mixed evenly at a mass ratio of 1:8 to obtain a mixed solution. The mixed solution was added dropwise at a rate of 1.5 mL / min to a calcium chloride solution with a mass of 20 times that of the mixed solution. After the addition was complete, the mixture was allowed to stand at 35℃ for 35 min. After standing, the mixture was filtered. The filtered product was washed 5 times with deionized water and then 3 times with sterile physiological saline with a mass fraction of 0.9%. After washing, the mixture was placed at -20℃ for pre-freezing for 30 h and then transferred to a vacuum freeze dryer for drying for 56 h to obtain microcapsule lyophilized powder. 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: Add modified sodium alginate to 35 times its mass of deionized water, stir evenly at 70℃, and filter sterilize using a 0.22μm filter membrane to prepare the modified sodium alginate solution for later use; The calcium chloride solution in S1 is prepared by the following method: Add calcium chloride to 50 times its mass of deionized water, stir evenly, and autoclave at 125℃ for 20 minutes to prepare the calcium chloride solution for later use;

[0037] S4. Weigh the microcapsule lyophilized powder and whey protein at a mass ratio of 1:6. Add the microcapsule lyophilized powder and whey protein to 12 times the mass of the microcapsule lyophilized powder in deionized water. Stir at 40℃ for 40 min, then add 0.95% sorbitan oleate and 0.8 times the mass of the microcapsule lyophilized powder in glycerol. Stir for 2.5 h to prepare a microcapsule mixture solution. Add the microcapsule mixture solution dropwise at a rate of 1 mL / min to a 3% chitosan solution and stir until homogeneous. Then add 10 times the mass of the microcapsule lyophilized powder in a 1.5% sodium sulfate aqueous solution. Sonicate for 15 min. After sonication, centrifuge and collect the lower precipitate. Wash with deionized water and anhydrous ethanol 5 times each. After washing, freeze-dry to obtain double-layer probiotic microcapsules.

[0038] Example 4

[0039] The difference from Example 2 lies only in step S2: the modified sodium alginate includes the following preparation steps: The pH of a 0.2 mol / L MES buffer solution is adjusted to 6.5 using a 0.3 mol / L sodium chloride solution; sodium alginate is added to the pH-adjusted MES buffer solution at 43 times its mass, stirred until homogeneous, and then 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide are added sequentially. After stirring at 30°C for 21 hours, whey protein at 0.9 times its mass is added, and the mixture is stirred for another 12 minutes. After dialysis purification and freeze-drying for 52 hours, modified sodium alginate is obtained; the mass ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to sodium alginate is 0.7; the mass ratio of N-hydroxysuccinimide to sodium alginate is 0.25.

[0040] Example 5

[0041] The only difference from Example 2 is step S3: the activated probiotic solution and sodium alginate solution are mixed evenly at a mass ratio of 1:7 to obtain a mixed solution; the mixed solution is added dropwise at a rate of 1.2 mL / min to a calcium chloride solution with a mass of 15 times that of the mixed solution. After the addition is complete, the mixture is allowed to stand for 35 min at 30°C. After standing, it is filtered, and the filtered product is washed four times with deionized water, and then washed twice with sterile physiological saline with a mass fraction of 0.85%. After washing, it is placed at -20°C for pre-freezing for 28 h, and then transferred to a vacuum freeze dryer for drying for 52 h to obtain microcapsule lyophilized powder; the concentration of Bifidobacterium bifidum in the activated probiotic solution is 10. 10CFU / mL; The sodium alginate solution is prepared by the following steps: Sodium alginate is added to 34 times its mass of deionized water, stirred evenly at 70℃, and then filtered sterilly using a 0.22μm filter membrane to prepare the sodium alginate solution for later use; The calcium chloride solution in S1 is prepared by the following method: Calcium chloride is added to 50 times its mass of deionized water, stirred evenly, and then autoclaved at 125℃ for 15 minutes to prepare the calcium chloride solution for later use;

[0042] Example 6

[0043] The only difference from Example 2 is step S3: the activated probiotic solution and the grafted sodium alginate solution are mixed evenly at a mass ratio of 1:7 to obtain a mixed solution; the mixed solution is added dropwise at a rate of 1.2 mL / min to a calcium chloride solution with a mass of 15 times that of the mixed solution. After the addition is complete, the mixture is allowed to stand for 35 min at 30°C. After standing, it is filtered, and the filtered product is washed four times with deionized water and then twice with sterile physiological saline with a mass fraction of 0.85%. After washing, it is placed at -20°C for pre-freezing for 28 h, and then transferred to a vacuum freeze dryer for drying for 52 h to obtain microcapsule lyophilized powder; 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: Add the grafted sodium alginate to 34 times its mass of deionized water, stir evenly at 70℃, and filter sterilly using a 0.22μm filter membrane to prepare the grafted sodium alginate solution for later use; The calcium chloride solution in S1 is prepared by the following method: Add calcium chloride to 50 times its mass of deionized water, stir evenly, and then autoclave at 125℃ for 15 minutes to prepare the calcium chloride solution for later use;

[0044] Example 7

[0045] The only difference from Example 2 is that step S4 is omitted, and the microcapsule freeze-dried powder prepared in step S3 is used as the final product.

[0046] Comparative Example 1

[0047] Take 0.5g of the probiotic microcapsules prepared in Examples 1-7 and add them to 4.5mL of simulated gastric fluid. After stirring evenly, shake in a shaker at 200r / min for 120min at 37℃. After treatment, centrifuge, discard the supernatant, collect the precipitate, add 4.5mL of PBS buffer (pH 7.4) to the precipitate, and shake at 37℃ until the probiotic microcapsules in the precipitate completely disintegrate to obtain a bacterial suspension. Then, count the viable bacteria in the bacterial suspension and calculate the survival rate of the probiotics based on the viable bacteria count results.

[0048] Table 1. Experimental data on the survival rate of probiotics simulated by gastric juice.

[0049] No. Survival rate of probiotic bacteria (%) 1 86.90 2 87.25 3 87.36 4 65.22 5 63.12 6 78.33 7 71.56

[0050] A comparison of the experimental data in Table 1 reveals that Examples 1-3 maintained good survival rates, which were higher than those in Examples 4-7. In Examples 4 and 5, a decrease was expected, primarily because the internal wall material used ungrafted sodium alginate directly grafted with whey protein or unmodified sodium alginate, resulting in a loose seal. This not only facilitated mixing with glycerol but also prevented effective control of probiotic release after the outer wall material ruptured, leading to a large number of probiotics being released into the gastric juice and dying. Although Example 6 did not graft whey protein, its seal was relatively tight, making it less prone to dispersion in gastric juice, thus resulting in a significantly different survival rate. Example 7 was roughly the same as Example 6, but the lack of an outermost wall material led to a predictable decrease in survival rate.

[0051] Comparative Example 2

[0052] Take 0.5g of the probiotic microcapsules prepared in Examples 1-7 and place them in a constant temperature incubator. Let them stand for 5 hours at 40°C. Then transfer them to 4.5mL of PBS buffer at pH 7.4 and shake at 37°C until the probiotic microcapsules in the precipitate are completely disintegrated to obtain a bacterial suspension. Then count the viable bacteria in the bacterial suspension and calculate the survival rate of the probiotics based on the viable bacteria count results.

[0053] Table 2. Experimental data on the survival rate of temperature-sensitive probiotics

[0054] No. Survival rate of probiotic bacteria (%) 1 98.90 2 97.25 3 97.36 4 95.22 5 83.12 6 84.33 7 76.56

[0055] A comparison of the experimental data in Table 2 reveals that Examples 1-3 maintained good survival rates, which were higher than those in Examples 5-7. A decrease was expected in Example 7, primarily due to the lack of glycerol as a phase change material, making it more sensitive to temperature changes. However, the sensitivity to temperature changes in Examples 5 and 6 is presumably due to the absence of whey protein grafting, resulting in less glycerol adhering to its outer surface and consequently a decreased resistance to temperature changes. This is also evidenced in Example 4, where although the survival rate of whey protein grafted in gastric juice decreased, its resistance to temperature changes was still significantly better than that in Examples 5-7.

[0056] 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 invention can be implemented in other specific forms without departing from its spirit or essential characteristics. 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. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No markings in the claims should be construed as limiting the scope of the claims.

Claims

1. A method for preparing a microencapsulated probiotic product, characterized in that, The preparation steps for the double-layer probiotic microcapsules are as follows: S1. Mix the activated probiotic solution and the modified sodium alginate solution evenly at a mass ratio of 1:6~8 to obtain a mixed solution. Add the mixed solution dropwise at a rate of 1~1.5mL / min to a calcium chloride solution with a mass of 10~20 times that of the mixed solution. After the addition is complete, let it stand for 30~35min at a temperature of 25~35℃. After standing, filter the solution. Wash the filtered product with deionized water 3~5 times, and then wash it with sterile physiological saline with a mass fraction of 0.85~0.9% 2~3 times. After washing, place it at -15~-20℃ for pre-freezing for 24~30h, and then transfer it to a vacuum freeze dryer for drying for 48~56h to obtain microcapsule lyophilized powder. S2. Weigh the lyophilized microcapsule powder and whey protein at a mass ratio of 1:

6. Add the lyophilized microcapsule powder and whey protein to 10-12 times the mass of the lyophilized microcapsule powder in deionized water. Stir for 30-40 minutes at 35-40℃. Then add 0.95% sorbitan oleate and 0.5-0.8 times the mass of the lyophilized microcapsule powder in glycerol. Stir for 2-2.5 hours to prepare a microcapsule mixture solution. Add the microcapsule mixture solution dropwise at a rate of 0.5-1 mL / min to a 3% chitosan solution. Stir until well mixed. Then add 8-10 times the mass of the lyophilized microcapsule powder in a 1.2-1.5% sodium sulfate aqueous solution. Sonicate for 10-15 minutes. After sonication, centrifuge and collect the lower precipitate. Wash with deionized water and anhydrous ethanol 3-5 times respectively. After washing, freeze-dry to obtain double-layer probiotic microcapsules. The modified sodium alginate includes the following preparation steps: 0.2 mol / L MES buffer solution is adjusted to pH 6.5 using 0.3 mol / L sodium chloride solution; grafted sodium alginate is added to 42-45 times its mass of the pH-adjusted MES buffer solution, stirred until homogeneous, then 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide are added sequentially. The mixture is stirred at 25-35°C for 20-22 hours, then 0.8-1.2 times its mass of whey protein is added, and the mixture is stirred for another 10-15 minutes. After dialysis purification and freeze-drying for 48-56 hours, modified sodium alginate is obtained. The grafted sodium alginate comprises the following preparation steps: sodium alginate is added to deionized water at a mass of 32-34 times that of sodium alginate, and the pH is adjusted to 7.5-8.5 with a hydrogen peroxide solution at a mass of 10-15%. Then, succinic anhydride at a mass of 0.08-0.09 times that of sodium alginate is added. The mixture is stirred and reacted at a temperature of 45-55°C for 2-3 hours. After purification by dialysis, the mixture is freeze-dried in a vacuum freeze dryer for 48-56 hours to obtain grafted sodium alginate. The mass ratio between 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and grafted sodium alginate is 0.6~0.

8. The mass ratio of N-hydroxysuccinimide to grafted sodium alginate is 0.2 to 0.

25.

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

4. The method for preparing microencapsulated probiotic products according to claim 1, characterized in that, The calcium chloride solution in S1 is prepared by the following method: calcium chloride is added to deionized water at a mass of 48 to 50 times that of calcium chloride, stirred evenly, and then sterilized by autoclaving at a temperature of 120 to 125°C for 15 to 20 minutes to prepare a calcium chloride solution for later use.

5. A microencapsulated probiotic product, characterized in that: The microencapsulated probiotic product was prepared using the method described in any one of claims 1 to 4.

6. The microencapsulated probiotic product according to claim 5, characterized in that, It is used in hygiene products.