A probiotic microcapsule and its preparation method

Abstract

This invention discloses a probiotic microcapsule and its preparation method, belonging to the field of microcapsule preparation technology. The preparation method includes the following steps: (1) dissolving Sophora japonica extract in water, stirring, adding sodium alginate, and stirring again to obtain a wall material solution; (2) centrifuging and washing the activated probiotics, preparing a bacterial suspension, mixing it with the wall material solution obtained in step (1), stirring, and obtaining a wall material bacterial suspension; (3) adding the wall material bacterial suspension obtained in step (2) to a calcium chloride solution and letting it stand, then adding the formed gel to a chitosan solution, stirring, washing, and obtaining the final product. The preparation process of this invention is simple and convenient, and the raw materials are inexpensive and easy to obtain, which can effectively reduce the production cost of microcapsules, facilitate large-scale industrial production and application promotion, effectively improve the biocompatibility, biodegradability, mechanical strength and encapsulation rate of microcapsules, and enhance the storage stability and resistance to gastrointestinal fluid stress of microcapsules.

Description

Technical Field

[0001] This invention relates to the field of microcapsule preparation technology, specifically to a probiotic microcapsule and its preparation method. Background Technology

[0002] Lactobacillus paracasei, belonging to the genus Lactobacillus, is a Gram-positive bacterium. It not only influences the human digestive system and enhances immunity by regulating the intestinal microbiota, but also produces bacteriocins to inhibit harmful bacteria in the gut and regulate the intestinal flora. Therefore, it has been included in the list of probiotics and has received widespread attention from researchers both domestically and internationally. However, Lactobacillus paracasei faces challenges during food processing, storage, transportation, and gastrointestinal digestion, including oxygen exposure, relative humidity, temperature, osmotic stress, enzyme activity, and pH changes. Therefore, effective protection of Lactobacillus paracasei is a prerequisite for it to exert its probiotic effects.

[0003] Microencapsulation technology can enhance the resistance of probiotics to adverse environments, thereby extending the shelf life of probiotic products. Currently, methods for encapsulating *Lactobacillus paracasei* include freeze-drying, spray-drying, emulsification, and extrusion. Among these, spray-drying reduces the survival ability and activity of probiotics due to the high temperatures generated by solvent evaporation; freeze-drying is time-consuming and energy-intensive, resulting in higher costs; emulsification produces microcapsules with a liquid core, resulting in small size and uneven shape and size. Extrusion-based microcapsules generally have a uniform particle size distribution and are low-cost and simple to process, making them promising for widespread application in food processing. However, current extrusion encapsulation materials typically include pectin, sodium alginate, and chitosan. While these materials can produce microcapsules with good biocompatibility, biodegradability, mechanical strength, and encapsulation efficiency, the limited material composition leads to poor storage stability and tolerance to adverse environments, which is detrimental to long-term preservation and efficacy.

[0004] In summary, how to prepare a probiotic microcapsule that simultaneously possesses good biocompatibility, biodegradability, mechanical strength, encapsulation efficiency, good storage stability, and strong stress tolerance is one of the key issues that urgently need to be solved in the field of microcapsule preparation technology. Summary of the Invention

[0005] To address the aforementioned technical problems, the present invention aims to provide a probiotic microcapsule and its preparation method, thereby solving the problems of poor storage stability and low stress tolerance of existing probiotic microcapsules.

[0006] The technical solution of the present invention to solve the above-mentioned technical problems is as follows:

[0007] A method for preparing probiotic microcapsules includes the following steps:

[0008] (1) Dissolve Sophora japonica extract in water, stir, add sodium alginate, stir again to obtain wall material solution;

[0009] (2) After centrifuging and washing the activated probiotics, prepare a bacterial suspension, mix it with the wall material solution obtained in step (1), stir, and obtain a wall material bacterial suspension.

[0010] (3) The wall material bacterial suspension obtained in step (2) is added to calcium chloride solution and allowed to stand. Then the formed gel is added to chitosan solution and stirred, washed, and the product is obtained.

[0011] The beneficial effects of this invention are as follows: This invention uses sodium alginate and chitosan as encapsulation materials. The microcapsules prepared by the extrusion method have good encapsulation effects. The sodium alginate / chitosan composite scaffold has good biocompatibility, biodegradability, and mechanical strength. Simultaneously, the co-encapsulation method using Sophora japonica extract and Lactobacillus paracasei effectively improves the storage stability of Lactobacillus paracasei and enhances its tolerance to stress conditions, especially to simulated gastrointestinal fluids. This invention uses the extrusion method to prepare microcapsules, which is simple and convenient to prepare, uses inexpensive and readily available raw materials, effectively reducing the preparation cost of microcapsules and making it suitable for large-scale industrial production and application.

[0012] Further, the Sophora japonica extract in step (1) is prepared by the following method: Sophora japonica powder is mixed with water and boiled in boiling water, ultrasonically treated, then filtered, frozen overnight, and freeze-dried.

[0013] Furthermore, the boiling time is 20-40 minutes; the ultrasonic treatment power is 30-50W and the time is 20-40 minutes; the overnight freezing temperature is -30 to -10℃; and the freeze-drying time is 40-60 hours.

[0014] Preferably, the boiling time is 30 minutes; the ultrasonic treatment power is 40W and the time is 30 minutes; the overnight freezing temperature is -20℃; and the freeze-drying time is 48 hours.

[0015] The beneficial effects of adopting the above-mentioned further technical solution are as follows: This invention extracts Sophora japonica flower extract through wastewater boiling + ultrasound + freeze treatment, and then dries it by freeze drying. This effectively avoids impurities in the Sophora japonica flower extract that could affect the subsequent microcapsule preparation results, while also protecting the large amount of flavonoids and polysaccharides such as rutin and quercetin contained in Sophora japonica flower extract. This provides a variety of substances with prebiotic potential. On the one hand, the addition of Sophora japonica flower extract improves the storage stability and resistance to gastrointestinal fluid stress of Lactobacillus paracasei microcapsules. On the other hand, the large number of nutrients in Sophora japonica flower extract, including a variety of prebiotic potential components such as rutin and Sophora japonica polysaccharides, further enhance the nutritional value of Lactobacillus paracasei microcapsules.

[0016] Further, in step (1), the mass-to-volume ratio of Sophora japonica extract, water and sodium alginate is 0.05-0.2g: 15-25mL: 0.3-0.5g.

[0017] Preferably, in step (1), the mass-to-volume ratio of Sophora japonica extract, water, and sodium alginate is 0.1g:20mL:0.4g.

[0018] Further, in step (1), the stirring speed is 200-500 rpm, the temperature is 20-30℃, and the time is 1-5 min; the stirring speed again is 1000-1500 rpm, the temperature is 20-30℃, and the time is 5-30 min.

[0019] Preferably, in step (1), the stirring speed is 300 rpm, the temperature is 25°C, and the time is 3 min; the stirring speed again is 1200 rpm, the temperature is 25°C, and the time is 20 min.

[0020] The beneficial effects of adopting the above-mentioned further technical solution are as follows: In this invention, sodium alginate is added as one of the encapsulation materials. On the one hand, sodium alginate, as a natural polysaccharide, has good biocompatibility and stability. On the other hand, sodium alginate can be used as a microcapsule wall material and also acts as a cross-linking agent between various wall materials. Through its own activatable sites, it forms covalent bonds with other molecules, including Sophora japonica extract and chitosan, to form a three-dimensional network structure, which greatly improves the compactness of the prepared microcapsules and provides a certain protective effect on the probiotics inside the microcapsules.

[0021] Furthermore, in step (2), the probiotic is Lactobacillus paracasei; the concentration of the bacterial suspension is greater than or equal to 10. 9 CFU / mL; the volume ratio of bacterial suspension to wall material solution is 4-6:15-25.

[0022] Preferably, the volume ratio of probiotics to wall material solution in step (2) is 5:20.

[0023] Furthermore, in step (2), the centrifugation speed is 3000-5000 rpm and the time is 4-6 min; the stirring speed is 200-500 rpm, the temperature is 20-30℃, and the time is 5-30 min.

[0024] Preferably, in step (2), the centrifugation speed is 4000 rpm and the time is 5 min; the stirring speed is 300 rpm, the temperature is 25℃ and the time is 20 min.

[0025] Furthermore, the probiotics are activated at least 3 times in step (2).

[0026] Further, after centrifugation in step (2), the sample is washed with sterile saline at least three times.

[0027] The beneficial effects of adopting the above-mentioned further technical solution are as follows: After the present invention activates Lactobacillus paracasei multiple times, it is mixed with the wall material solution to prepare a wall material bacterial suspension, which ensures the activity of Lactobacillus paracasei in the wall material bacterial suspension and ensures that the Lactobacillus paracasei inside the probiotic microcapsules has stronger initial activity.

[0028] Furthermore, in step (3), the wall material bacterial suspension is added by extrusion; the concentration of calcium chloride solution is 0.4-0.6 mol / L; and the mass fraction of chitosan solution is 0.4-0.6%.

[0029] Preferably, in step (3), the concentration of the calcium chloride solution is 0.5 mol / L; and the mass fraction of the chitosan solution is 0.5%.

[0030] Furthermore, in step (3), the settling time is 20-40 min; the stirring speed is 200-500 rpm, the time is 30-60 min, and the temperature is 20-30℃.

[0031] Preferably, in step (3), the settling time is 30 min; the stirring speed is 300 rpm, the time is 40 min, and the temperature is 25℃.

[0032] Furthermore, the washing method in step (3) is to wash twice with sterile saline.

[0033] The beneficial effects of adopting the above-mentioned further technical solution are as follows: This invention uses an extrusion method to dropwise extrude a wall material bacterial suspension into a calcium chloride solution for microcapsule formation. On the one hand, this method can efficiently prepare a large number of uniformly sized microcapsules. By controlling the size of the droplets, the size, shape, and wall thickness of the microcapsules can be precisely controlled to meet the needs of different applications. On the other hand, compared with other microcapsule preparation methods, this method is low-cost, requires no complex equipment or expensive raw materials, and makes the production of microcapsules more economical and efficient. After the microcapsules are formed, this invention adds a chitosan solution and continues stirring. Through the cross-linking effect of sodium alginate, a composite scaffold with good biocompatibility, biodegradability, and mechanical strength is formed between sodium alginate and chitosan. This not only improves the hardness and elasticity of the prepared microcapsules but also increases the density of the microcapsules, thereby enhancing the protective ability of the microcapsules for the internal probiotics.

[0034] A probiotic microcapsule is prepared using the above-described preparation method.

[0035] The present invention has the following beneficial effects:

[0036] (1) This invention uses sodium alginate and chitosan as encapsulation materials, and introduces Sophora japonica extract and Lactobacillus paracasei for co-encapsulation. On the one hand, the composite scaffold formed by sodium alginate and chitosan effectively improves the biocompatibility, biodegradability, mechanical strength and encapsulation rate of the microcapsules. On the other hand, the addition of Sophora japonica extract effectively improves the storage stability and gastrointestinal stress resistance of Lactobacillus paracasei inside the microcapsules, and further enhances the nutritional value of the microcapsules by adding a large number of prebiotic potential components. At the same time, Sophora japonica extract, sodium alginate and chitosan form a three-dimensional network structure, which further improves the compactness of the microcapsules.

[0037] (2) The present invention uses the extrusion method to prepare microcapsules. The preparation process is simple and convenient, and the raw materials are inexpensive and easy to obtain. This can effectively reduce the production cost of microcapsules and facilitate large-scale industrial production and application. Attached Figure Description

[0038] Figure 1 This is a scanning electron microscope image of the microcapsules in Experiment Example 4. Detailed Implementation

[0039] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0040] Example 1:

[0041] A method for preparing Lactobacillus paracasei microcapsules includes the following steps:

[0042] (1) Preparation of Sophora japonica extract

[0043] Sophora japonica powder and pure water were mixed at a mass ratio of 1:100, boiled in boiling water for 30 minutes, and then ultrasonically treated at 40W for 30 minutes. The resulting solution was filtered, and the filtrate was frozen at -20℃ overnight. Finally, it was freeze-dried for 48 hours to obtain Sophora japonica extract.

[0044] (2) Preparation of bacterial suspension

[0045] The activated Lactobacillus paracasei was centrifuged at 4000 rpm for 5 min, then washed 5 times with sterile physiological saline, and finally resuspended in sterile physiological saline to prepare a concentration of 10. 9 CFU / mL bacterial suspension.

[0046] (3) Preparation of wall material solution

[0047] Dissolve 0.08g of Sophora japonica extract in 20mL of sterile water and stir at 300rpm for 2min at 25℃ on a magnetic stirrer. Then add 0.4g of low-viscosity sodium alginate (Aladdin) to the above solution and adjust the speed to 1200rpm and continue stirring for 20min until the solution is homogeneous to obtain the wall material solution.

[0048] (4) Preparation of microcapsules

[0049] Add 5 mL of the bacterial suspension obtained in step (2) to the wall material solution obtained in step (3), stir at 300 rpm for 20 min at 25°C until the solution is uniformly mixed; then squeeze the uniformly mixed solution dropwise into a sterile 0.5 mol / L CaCl2 solution by extrusion and let it stand for 30 min, then add it to a sterile 0.5% chitosan solution, stir at 300 rpm for 40 min at 25°C using a magnetic stirrer, and finally wash the obtained microcapsule sol twice with sterile physiological saline to obtain Lactobacillus paracasei microcapsules.

[0050] Example 2:

[0051] A method for preparing Lactobacillus paracasei microcapsules includes the following steps:

[0052] (1) Preparation of Sophora japonica extract

[0053] Sophora japonica powder and pure water were mixed at a mass ratio of 1:100, boiled in boiling water for 30 minutes, and then ultrasonically treated at 40W for 30 minutes. The resulting solution was filtered, and the filtrate was frozen at -20℃ overnight. Finally, it was freeze-dried for 48 hours to obtain Sophora japonica extract.

[0054] (2) Preparation of bacterial suspension

[0055] The activated Lactobacillus paracasei was centrifuged at 4000 rpm for 5 min, then washed 5 times with sterile physiological saline, and finally resuspended in sterile physiological saline to prepare a concentration of 10. 9 CFU / mL bacterial suspension.

[0056] (3) Preparation of wall material solution

[0057] Dissolve 0.10g of Sophora japonica extract in 20mL of sterile water and stir at 300rpm for 2min at 25℃ on a magnetic stirrer. Then add 0.4g of low-viscosity sodium alginate to the above solution and adjust the speed to 1200rpm and continue stirring for 20min until the solution is homogeneous to obtain the wall material solution.

[0058] (4) Preparation of microcapsules

[0059] Add 5 mL of the bacterial suspension obtained in step (2) to the wall material solution obtained in step (3), stir at 300 rpm for 20 min at 25°C until the solution is uniformly mixed; then squeeze the uniformly mixed solution dropwise into a sterile 0.5 mol / L CaCl2 solution by extrusion and let it stand for 30 min, then add it to a sterile 0.5% chitosan solution, stir at 300 rpm for 40 min at 25°C using a magnetic stirrer, and finally wash the obtained microcapsule sol twice with sterile physiological saline to obtain Lactobacillus paracasei microcapsules.

[0060] Example 3:

[0061] A method for preparing Lactobacillus paracasei microcapsules includes the following steps:

[0062] (1) Preparation of Sophora japonica extract

[0063] Sophora japonica powder and pure water were mixed at a mass ratio of 1:100, boiled in boiling water for 30 minutes, and then ultrasonically treated at 40W for 30 minutes. The resulting solution was filtered, and the filtrate was frozen at -20℃ overnight. Finally, it was freeze-dried for 48 hours to obtain Sophora japonica extract.

[0064] (2) Preparation of bacterial suspension

[0065] The activated Lactobacillus paracasei was centrifuged at 4000 rpm for 5 min, then washed 5 times with sterile physiological saline, and finally resuspended in sterile physiological saline to prepare a concentration of 10. 9 CFU / mL bacterial suspension.

[0066] (3) Preparation of wall material solution

[0067] Dissolve 0.12g of Sophora japonica extract in 20mL of sterile water and stir at 300rpm for 2min at 25℃ on a magnetic stirrer. Then add 0.4g of low-viscosity sodium alginate to the above solution and adjust the speed to 1200rpm and continue stirring for 20min until the solution is homogeneous to obtain the wall material solution.

[0068] (4) Preparation of microcapsules

[0069] Add 5 mL of the bacterial suspension obtained in step (2) to the wall material solution obtained in step (3), stir at 300 rpm for 20 min at 25°C until the solution is uniformly mixed; then squeeze the uniformly mixed solution dropwise into a sterile 0.5 mol / L CaCl2 solution by extrusion and let it stand for 30 min, then add it to a sterile 0.5% chitosan solution, stir at 300 rpm for 40 min at 25°C using a magnetic stirrer, and finally wash the obtained microcapsule sol twice with sterile physiological saline to obtain Lactobacillus paracasei microcapsules.

[0070] Comparative Example 1:

[0071] A method for preparing Lactobacillus paracasei microcapsules includes the following steps:

[0072] (1) Preparation of bacterial suspension

[0073] The activated Lactobacillus paracasei was centrifuged at 4000 rpm for 5 min, then washed 5 times with sterile physiological saline, and finally resuspended in sterile physiological saline to prepare a concentration of 10. 9 CFU / mL bacterial suspension.

[0074] (2) Preparation of wall material solution

[0075] Add 0.4g of low-viscosity sodium alginate to 20mL of sterile water, stir at 1200rpm for 20min at 25℃ until the solution is homogeneous to obtain the wall material solution.

[0076] (3) Preparation of microcapsules

[0077] Add 5 mL of the bacterial suspension obtained in step (1) to the wall material solution obtained in step (2), stir at 300 rpm for 20 min at 25°C until the solution is uniformly mixed; then squeeze the uniformly mixed solution dropwise into a sterile 0.5 mol / L CaCl2 solution by extrusion and let it stand for 30 min, then add it to a sterile 0.5% chitosan solution, stir at 300 rpm for 40 min at 25°C using a magnetic stirrer, and finally wash the obtained microcapsule sol twice with sterile physiological saline to obtain Lactobacillus paracasei microcapsules.

[0078] Comparative Example 2:

[0079] A method for preparing Lactobacillus paracasei microcapsules includes the following steps:

[0080] (1) Preparation of bacterial suspension

[0081] The activated Lactobacillus paracasei was centrifuged at 4000 rpm for 5 min, then washed 5 times with sterile physiological saline, and finally resuspended in sterile physiological saline to prepare a concentration of 10. 9 CFU / mL bacterial suspension.

[0082] (2) Preparation of wall material solution

[0083] Dissolve 0.10g of inulin in 20mL of sterile water and stir at 300rpm for 2min at 25℃ on a magnetic stirrer. Then add 0.4g of low-viscosity sodium alginate to the above solution and adjust the speed to 1200rpm and continue stirring for 20min until the solution is homogeneous to obtain the wall material solution.

[0084] (3) Preparation of microcapsules

[0085] Add 5 mL of the bacterial suspension obtained in step (1) to the wall material solution obtained in step (2), stir at 300 rpm for 20 min at 25°C until the solution is uniformly mixed; then squeeze the uniformly mixed solution dropwise into a sterile 0.5 mol / L CaCl2 solution by extrusion and let it stand for 30 min, then add it to a sterile 0.5% chitosan solution, stir at 300 rpm for 40 min at 25°C using a magnetic stirrer, and finally wash the obtained microcapsule sol twice with sterile physiological saline to obtain Lactobacillus paracasei microcapsules.

[0086] Experimental Example 1: Embedding Rate

[0087] Accurately weigh 1.0 g of *Lactobacillus paracasei* microcapsules prepared in Examples 1-3 and Comparative Examples 1-2, and dissolve them in 9 mL of decapsulation solution (a mixed solution of 0.2 mol / L sodium bicarbonate and 0.06 mol / L trisodium citrate, pH 8). Stir thoroughly until completely decapsulated, then serially dilute with physiological saline and determine the viable count. Encapsulation efficiency (%) = (Viable count after encapsulation / Viable count before encapsulation) × 100%. The encapsulation efficiency test results are shown in Table 1.

[0088] Table 1 Encapsulation efficiency of microcapsules

[0089]

[0090]

[0091] As can be seen from the results in Table 1 above, the encapsulation rate of the Lactobacillus paracasei microcapsules prepared in the embodiments of the present invention is significantly higher than that of Comparative Example 1, and the encapsulation rate of the Lactobacillus paracasei microcapsules prepared in the present invention is also relatively higher than that of Comparative Example 2, which uses inulin as one of the raw materials.

[0092] Experimental Example 2: Storage Stability

[0093] The Lactobacillus paracasei microcapsules prepared in Examples 1-3 and Comparative Examples 1-2 were stored in a 4°C refrigerator, and their viable bacterial counts were tested every 12 days. The test results are shown in Table 2.

[0094] Table 2 Storage stability of microcapsules (log CFU / g)

[0095] As shown in Table 2, the storage stability of *Lactobacillus paracasei* prepared in Examples 1-3 of this invention is higher than that of Comparative Examples 1 and 2. Example 2 exhibits the best storage stability, significantly higher than the two comparative examples, followed by Examples 3 and 1. Specifically, after 24 days of storage at 4°C, the viable cell counts of Examples 3 and 1 are roughly equivalent to those of Comparative Example 2. However, by day 36, the viable cell counts of Examples 1 and 3 are significantly higher than those of Comparative Example 2. The *Lactobacillus paracasei* microcapsules prepared in Example 2 showed the highest viable cell count among the five experimental groups after 36 days of storage at 4°C, reaching 8.39 log CFU / g, a decrease of only 0.41 log CFU / g compared to day 0, significantly higher than all comparative examples.

[0096] In addition, the Lactobacillus paracasei prepared in Example 2 was subjected to a storage test extended to 48 days. The results showed that the number of live bacteria was still as high as 7.69 log CFU / g, with a decrease of only 1.11 log CFU / g. The number of live bacteria was still sufficient to meet the requirements for probiotics to exert their probiotic effects.

[0097] Experimental Example 3: Simulated Gastrointestinal Fluid Survival Rate

[0098] Preparation of simulated gastric juice (SGF): Adjust the pH of a 9 g / L NaCl solution to 2.0 using 0.1 mol / L HCl solution, and add 0.3% (w / v) pepsin solution (mass ratio 1:3000).

[0099] Group 0d 12d 24d 36d Comparative Example 1 8.68±0.16a 8.18±0.04a 7.29±0.01a 4.54±0.08a Comparative Example 2 8.85±0.02a 8.59±0.01b 8.38±0.05b 7.59±0.07b Example 1 8.67±0.05a 8.62±0.04b 8.34±0.03b 7.81±0.05c Example 2 8.80±0.06a 8.96±0.02d 8.69±0.08c 8.39±0.02d Example 3 8.97±0.02a 8.80±0.01c 8.40±0.06b 7.92±0.08c

[0100] After dissolving evenly, filter through a 0.22-micron filter membrane for sterilization, and refrigerate at 4°C for later use.

[0101] Preparation of simulated small intestinal fluid (SIF): Adjust the pH of the PBS solution to 8.0 with 0.1 mol / L NaOH solution, add 0.1% (w / v) trypsin solution (mass ratio 1:250) and 0.3% (w / v) bile salts (bovine bile salt BR), dissolve evenly, filter sterilize with a 0.22 micron filter membrane, and store at 4°C for later use.

[0102] Simulated continuous gastrointestinal fluid digestion process: 1.0 g of microcapsules were placed in 9 mL of SGF and incubated on a shaker at 37℃ and 100 rpm for 2 h. The simulated gastric fluid was removed using a sterile pipette tip to terminate the SGF digestion reaction. Next, the microcapsules were redispersed in 9 mL of SIF and incubated on a shaker at 37℃ and 100 rpm for 2 h. After digestion, the microcapsules were centrifuged at 4000 rpm for 5 min, and the supernatant was removed to terminate the SIF digestion reaction. Finally, the precipitate was redispersed in 9 mL of sterile encapsulation fluid and serially diluted with sterile physiological saline for viable cell counting. The survival rate (%) of the simulated gastrointestinal fluid was calculated as: (Number of viable cells after simulation / Number of viable cells before simulation) × 100%. The survival rates of the microcapsules after 2 h of simulated gastric fluid and 4 h of simulated continuous gastrointestinal fluid are shown in Table 3.

[0103] Table 3. Survival rate of microcapsules in simulated gastrointestinal fluid

[0104] Group SGF 2h survival rate (%) SIF 2h survival rate (%) Comparative Example 1 80.12±0.77a 61.80±0.33a Comparative Example 2 92.62±0.78bc 75.69±0.14b Example 1 92.59±0.16bc 76.12±0.21b Example 2 93.77±0.03c 80.63±0.24c Example 3 91.95±0.39b 75.78±0.47b

[0105] As shown in Table 3 above, the survival rate of all embodiments was significantly higher than that of Comparative Example 1 after 2 hours of simulated gastric fluid. After simulating continuous gastrointestinal fluid, the survival rate of all embodiments was still significantly higher than that of Comparative Example 1. Among them, the survival rate of Example 2 was the highest, significantly higher than that of other groups, increasing by 30.47% compared with Comparative Example 1 and by 6.53% compared with Comparative Example 2. The results indicate that the Lactobacillus paracasei microcapsules prepared in the embodiments of the present invention can effectively improve the survival rate of Lactobacillus paracasei inside the microcapsules in simulated gastrointestinal fluid.

[0106] Experimental Example 4: Scanning Electron Microscopy Observation

[0107] 1 g each of *Lactobacillus paracasei* microcapsules prepared in Examples 1-3 and Comparative Examples 1-2 were weighed and placed in separate glass bottles. 9 mL of simulated intestinal fluid was added, and the mixtures were incubated at 37°C and 100 rpm on a shaker for 2 hours for digestion. After digestion, the simulated intestinal fluid was removed, and the digested and undigested microcapsules were frozen at -20°C overnight, followed by lyophilization for 24 hours. The lyophilized microcapsules were collected for scanning electron microscopy (SEM) observation. The SEM results are as follows: Figure 1 As shown.

[0108] Depend on Figure 1As can be seen, before simulating intestinal fluid, the structures of Comparative Example 1 and Examples 1-3 were quite similar, but all differed significantly from Comparative Example 2. This is because the microcapsules prepared by adding inulin in Comparative Example 2 had a rougher surface structure and larger pores. After simulating intestinal fluid, the pores of Comparative Example 1 increased, and the surfaces of the microcapsules in Examples 1, 3, and 2 all became rougher. However, the structure of the microcapsules prepared in Example 2 did not change much before and after simulation, remaining relatively smooth and dense. Therefore, the microcapsules prepared in Example 2 were more resistant to simulated gastrointestinal fluid, significantly improving the tolerance and survival rate of Lactobacillus paracasei to simulated gastrointestinal fluid.

[0109] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing probiotic microcapsules, characterized by, The method comprises the following steps: (1) dissolving the sophora extract in water, stirring, adding sodium alginate, stirring again to obtain a wall material solution; (2) centrifuging and washing the activated probiotics to prepare a bacterial suspension, mixing the bacterial suspension with the wall material solution obtained in step (1), and stirring to obtain a wall material bacterial suspension; (3) adding the wall material bacterial suspension obtained in step (2) into a calcium chloride solution, standing, then adding the formed gel into a chitosan solution, stirring, washing, and preparing; The sophora extract in step (1) is prepared by the following method: mixing sophora powder with water, boiling, ultrasonic treatment, then performing suction filtration, freezing overnight, freeze-drying, and preparing; The boiling time is 20-40 min; the ultrasonic treatment power is 30-50 W, and the time is 20-40 min; the freezing temperature is-30~-10 ℃; and the freeze-drying time is 40-60 h; The mass-volume ratio of the sophora extract, water, and sodium alginate in step (1) is 0.05-0.2 g: 15-25 mL: 0.3-0.5 g; The probiotic bacteria in the step (2) is Lactobacillus paracasei; the concentration of the bacterial suspension is greater than or equal to 10 9 CFU / mL; the volume ratio of the bacterial suspension to the wall material solution is 4-6:15-25.

2. The method for preparing probiotic microcapsules according to claim 1, characterized by, The stirring speed in step (1) is 200-500 rpm, the temperature is 20-30 ℃, and the time is 1-5 min; the stirring speed in the second stirring is 1000-1500 rpm, the temperature is 20-30 ℃, and the time is 5-30 min.

3. The method of claim 1, wherein the probiotic microcapsules are prepared by a process comprising the steps of: The centrifugation speed in step (2) is 3000-5000 rpm, and the time is 4-6 min; the stirring speed is 200-500 rpm, the temperature is 20-30 ℃, and the time is 5-30 min.

4. The method of claim 1, wherein the probiotic microcapsules are prepared by a process comprising the steps of: The adding method of the wall material bacterial suspension in step (3) is extrusion; the concentration of the calcium chloride solution is 0.4-0.6 mol / L; and the mass fraction of the chitosan solution is 0.4-0.6%.

5. The method of claim 1, wherein the probiotic microcapsules are prepared by a process comprising the steps of: The standing time in step (3) is 20-40 min; the stirring speed is 200-500 rpm, the time is 30-60 min, and the temperature is 20-30 ℃.

6. A probiotic microcapsule, characterized in that, The method is prepared by any one of claims 1-5.

Images