Method for preparing probiotic microcapsule based on O1 / O2 / W structure and application

Abstract

The invention provides a method for preparing a probiotic microcapsule based on an O1 / O2 / W. The microcapsule structurally comprises a core material, a middle oil phase and an outer water phase, the core material is composed of probiotics and a first oil phase, and the preparation method of the probiotic microcapsule comprises the following steps that probiotic sludge is prepared and evenly mixed with the first oil phase to prepare a mixture 1, and the mixture 2 is prepared into a mixture 3; mixing the mixture 1 with a second oil phase to obtain a mixture 2, mixing the mixture 2 with an external water phase, dropwise adding into a calcium chloride solution to form microspheres, and shaping. The external water phase is composed of sodium alginate and nanocrystalline cellulose, the film-forming property of sodium alginate can protect probiotics in the oil phase from being influenced by external environment change, and meanwhile, calcium chloride and sodium alginate are subjected to complex reaction to further reinforce the mechanical property of the film, so that moisture in the environment is prevented from entering the microcapsule, and the microcapsule has good stability. The nanocrystal cellulose endows the microcapsule with the effects of targeted precise delivery and gastric acid digestion resistance, and ensures that the probiotics can stably survive in the stomach.

Description

Technical Field

[0001] The invention belongs to the technical field of probiotics, and specifically relates to a method for preparing probiotic microcapsules based on an O1 / O2 / W structure and application thereof. Background Art

[0002] Probiotics are a type of microorganism that can improve the intestinal microbial balance of the host and produce positive benefits to the host. Since the bacteria are highly sensitive to the external environment, they are prone to low survival rate or poor biological activity during production, storage and use, which greatly reduces the probiotic effect of probiotics. For example, in actual application, probiotics are often affected by gastric acid and bile, and their activity decreases or even disappears after entering the human intestine, making them unable to play their role. At present, the market for probiotic preparations is vast, but when the activity of probiotics is higher than 10 6 -10 7 CFU / g will show beneficial physiological effects, so some methods must be used to improve the survival rate of probiotics in the intestine. Microencapsulation technology uses natural or synthetic polymer materials as capsule materials, and wraps the active substance, i.e., the capsule core, through chemical, physical or physicochemical methods to form a microcapsule with a semipermeable or sealed capsule membrane. The application of microencapsulation technology to probiotics, especially non-spore lactic acid bacteria, can better solve the shortcomings of probiotics themselves and improve their resistance to adverse environments. However, studies have shown that microencapsulation does not show a good protective effect on the survival of probiotics in gastric juice. At present, people mainly use three methods to improve the protective effect of microcapsules on live bacteria: the first is to coat the surface of the microcapsule; the second is to use a combination of multiple wall materials; the third is to add prebiotics to the wall material to improve the adaptability of probiotics to adverse growth environments, thereby improving their survival rate. Traditional microencapsulation technology improves the activity of probiotics through W / W emulsions, O / W emulsions, and W / W microcapsules, but the resulting products are still difficult to survive in a liquid environment (with a high water activity) for a long time. The main reason is that traditional microcapsules are prone to rupture in an environment with high water activity, causing the probiotics to be released and inactivated. Therefore, more means are needed to improve the protective effect of microcapsules on live probiotics, such as microcapsule coating technology (also known as microcapsule coating technology, which refers to coating the surface of probiotics after they are microencapsulated to reduce the exposure of probiotics to high oxygen and high pH environments). Although the prior art can also ensure that probiotics have high activity, long-term storage will have a great impact on the activity of probiotics. For example, CN116636616A discloses a stable probiotic nutritional paste and its preparation method and use, wherein the probiotic nutritional paste comprises (1) a core material, which contains probiotics and a first oil phase, (2) an intermediate oil phase layer, which contains a first oil phase, prebiotics, whey protein and medium-chain triglycerides, and (3) an outer water phase layer, which contains vitamins, amino acids, mineral elements and functional additives, and the first oil phase contains fish oil and a surfactant. Although this is more convenient to store than the traditional probiotic preservation method (probiotic liquid and glycerol are mixed in a certain proportion), the probiotic nutritional paste does not require additional glycerol, but it is in a semi-liquid state and cannot guarantee a true oil-water barrier, and cannot completely and effectively isolate air and moisture. After being stored at room temperature for one year, the survival rate of probiotics decreases significantly. Therefore, there is an urgent need for a probiotic product that can both ensure the high activity of probiotics and be preserved for a long time. Summary of the invention

[0003] Technical problems to be solved: In view of the above technical problems, the purpose of the present invention is to provide a method and application for preparing probiotic microcapsules based on O1 / O2 / W structure, wherein the microcapsule structure includes a core material, an intermediate oil phase and an external water phase, wherein the core material is composed of probiotics and a first oil phase, and the steps of preparing the probiotic microcapsules are as follows: preparing probiotic mud, mixing it with the first oil phase to obtain mixture 1, mixing mixture 1 with the second oil phase to obtain mixture 2, and then mixing mixture 2 with the external water phase and dropping it into a calcium chloride solution to form microspheres, which are shaped. The external water phase in the present invention is composed of sodium alginate and nanocrystalline cellulose. The film-forming property of sodium alginate can protect the probiotics in the oil phase from changes in the external environment. At the same time, calcium chloride and sodium alginate undergo a complexation reaction to further strengthen the mechanical properties of the film, prevent moisture in the environment from entering the interior of the microcapsules, and nanocrystalline cellulose gives the microcapsules targeted and precise delivery and resistance to gastric acid digestion, ensuring that the probiotics can survive stably in the stomach.

[0004] Technical solution: A method for preparing probiotic microcapsules based on O1 / O2 / W structure, comprising the following steps: S1. Preparing probiotic slurry; S2. Add the probiotic slurry to the first oil phase and mix well to obtain a mixture 1; S3. The mixture 1 is added to the second oil phase and mixed evenly to obtain a mixture 2; S4. Add mixture 2 to the external aqueous phase and mix evenly, add it dropwise into the calcium chloride solution, filter it, and fix it with liquid nitrogen to obtain probiotic microcapsules. Furthermore, in step S1, the probiotics are one or more of bifidobacteria, lactobacilli, bacillus subtilis, and yeast. Furthermore, the method for preparing the probiotic sludge in step S1 comprises the following steps: Step 1. Probiotics are inoculated into liquid culture medium and subcultured at 30-40°C until the number of viable bacteria is > 1×10 10 CFU / g, culture medium was prepared; Step 2. The culture solution is centrifuged at 0-4°C and 3000-5000 rpm for 5-15 min, the bacterial precipitate is taken, and washed 2-4 times with sterile saline at a volume ratio of 1:(1-3) to obtain probiotic bacterial sludge. Furthermore, in the step S2, the first oil phase is one or more of animal oil, medium-chain triglycerides, and vegetable oil, wherein the animal oil is one or more of fish oil, lard, beef tallow, chicken oil, duck oil, and goose oil, and the vegetable oil is one or more of corn oil, soybean oil, peanut oil, rapeseed oil, and sesame oil, and the weight ratio of the probiotic sludge to the first oil phase is 1:(0.1-0.4); further, in the step S2, the animal oil is fish oil, and the vegetable oil is corn oil. Furthermore, in step S3, the second oil phase is one or more of animal oil, medium-chain triglycerides, and vegetable oil, wherein the animal oil is one or more of fish oil, lard, beef tallow, chicken oil, duck oil, and goose oil, and the vegetable oil is one or more of corn oil, soybean oil, peanut oil, rapeseed oil, and sesame oil, and the amount of the second oil phase added is 10-40%, based on the weight of the probiotic mud; further, in step S3, the animal oil is fish oil, and the vegetable oil is corn oil. Furthermore, in step S4, the external aqueous phase is sodium alginate and nanocrystalline cellulose, the mass concentration of sodium alginate is 1-4%, and the mass concentration of nanocrystalline cellulose is 0.1-0.8%; further, in step S4, the mass concentration of sodium alginate is 1-3%, and the mass concentration of nanocrystalline cellulose is 0.2-0.5%. Furthermore, the amount of the external water phase added in step S4 is 10-30% based on the weight of the probiotic sludge. Furthermore, the mass concentration of the calcium chloride solution in step S4 is 3-8%. Application of the probiotic microcapsules prepared by any of the above methods in the preparation of food, health products and pet diets. Beneficial effects: 1. The present invention prepares probiotic microcapsules based on the O1 / O2 / W structure, and the oil phase is two layers, so that the probiotics are firmly encapsulated in the oil phase, thereby isolating the probiotics from the moisture in the outer water phase, effectively preventing the probiotics from contacting with external water and air to affect the activity, thereby achieving the purpose of long-term survival. Moreover, the activity is still maintained at more than 90% when stored at 4°C for one year, thus overcoming the problem of low activity of probiotic microcapsules during long-term storage. 2. In the present invention, sodium alginate and nanocrystalline cellulose are used in the outer aqueous phase of the probiotic microcapsules. On the one hand, sodium alginate can form a gel network structure in the outer layer of the oil phase, giving the probiotic microcapsules a layer of stable and dense membrane. At the same time, calcium ions can undergo ion exchange with sodium alginate to form a more stable cross-linked network structure with certain strength and toughness, so that the probiotic microcapsules form a uniform sphere; on the other hand, the probiotics can be protected from gastric acid damage and delivered to the intestinal tract in a targeted manner. The nanocrystalline cellulose is wrapped in the membrane structure. Under the acidic conditions of gastric juice, a large number of hydroxyl groups in the nanocrystalline cellulose can form hydrogen bonds, thereby forming a stable network structure on the surface of the probiotic microcapsules, resisting the erosion and degradation of gastric acid, and ensuring that the probiotic microcapsules can survive stably in the stomach of the body and be released in a targeted manner in the intestine. 3. The particle size of the probiotic microcapsules of the present invention can be regulated in the preparation of the microcapsules, so that the particle size of the microcapsules is more uniform and the microcapsules are shaped to a visual shape, thereby avoiding deformation and irregularity of the microcapsules during the traditional freeze-drying process, which makes them difficult to market. In addition, the preparation process is simple and efficient, mild and safe, with readily available raw materials and low cost, and is suitable for large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 This is a picture of probiotic microcapsules prepared using a microencapsulation instrument; Figure 2 This is a diagram of the probiotic microcapsules prepared in Example 1; Figure 3 The staining results of probiotics embedded in corn oil, MCT oil and corn oil / MCT oil two-phase system, wherein a represents FITC staining and unstained corn oil (O1), b represents Nile Red staining and unstained MCT oil (O2); c represents FITC staining, Nile Red staining, FITC and Nile Red simultaneous staining, FITC and Nile Red simultaneous unstained corn oil and MCT oil two-phase system (O1 / O2); Figure 4 Probiotic activity of probiotics encapsulated in corn oil, MCT oil and corn oil / MCT oil dual-phase system. DETAILED DESCRIPTION The present invention will be further described below in conjunction with embodiments, which are explanations of the present invention and are not limited to the following embodiments: The bacterial species used in the present invention: Lactobacillus plantarum WCF, Bacillus subtilis (CAS: 68038-70-0), Bifidobacterium ATCC15703, were all purchased from Chr. Hansen (Beijing) Trading Co., Ltd. Example 1 A method for preparing probiotic microcapsules based on O1 / O2 / W structure comprises the following steps: S1. Add the activated Lactobacillus plantarum WCFS1 into liquid culture medium and culture at 37°C for 24 h until the viable count of the strain reaches 1×10 10 CFU / g, centrifuged at 4°C and 4000 rpm for 10 min to collect the bacterial precipitate, then added 2 times the volume of sterile saline and washed repeatedly for 3 times to prepare probiotic sludge; S2. Take 10 g of probiotic slurry, add 2.5 g of fish oil and mix well, and homogenize with a rotor-stator homogenizer at 5000 rpm for 2 min to obtain a mixture 1; S3. The mixture 1 was added to 3 g of fish oil and mixed evenly, and homogenized with a rotor-stator homogenizer at 5000 rpm for 2 min to obtain a mixture 2; S4. Weigh 2 g of sodium alginate and 0.4 g of nanocrystalline cellulose, add 97.6 g of water and mix, prepare an external aqueous phase with a mass concentration of 2% sodium alginate and a mass concentration of 0.4% nanocrystalline cellulose, add mixture 2 to 2 g of the external aqueous phase and mix well, and homogenize with a rotor-stator homogenizer at 5000 rpm for 2 min to obtain a mixture 3; S5. The mixture 3 is pumped into a microencapsulation apparatus (eg Figure 1 As shown), the third mixture is dripped into a calcium chloride solution with a mass concentration of 5% in the form of droplets with a particle size of about 400 μm by adjusting the frequency, and then placed in a low-temperature environment of liquid nitrogen for shaping to obtain probiotic microcapsules, such as Figure 2 shown. Example 2 A method for preparing probiotic microcapsules based on O1 / O2 / W structure comprises the following steps: S1. Add the activated Bacillus subtilis into liquid culture medium and culture at 37°C for 24 h until the viable count of the strain reaches 1×10 10 CFU / g, centrifuged at 4°C and 4000 rpm for 10 min to collect the bacterial precipitate, then added 2 times the volume of sterile saline and washed repeatedly for 3 times to prepare probiotic sludge; S2. Take 10 g of probiotic slurry, add 3 g of corn oil and mix well, and homogenize with a rotor-stator homogenizer at 5000 rpm for 2 min to obtain a mixture 1; S3. The mixture 1 was added to 3 g of fish oil and mixed evenly, and homogenized with a rotor-stator homogenizer at 5000 rpm for 2 min to obtain a mixture 2; S4. Weigh 2 g of sodium alginate and 0.5 g of nanocrystalline cellulose, add 97.5 g of water and mix, prepare an external aqueous phase with a mass concentration of 2% sodium alginate and a mass concentration of 0.5% nanocrystalline cellulose, add mixture 2 to 3 g of the external aqueous phase and mix well, and homogenize with a rotor-stator homogenizer at 5000 rpm for 2 min to obtain a mixture 3; S5. Mixture 3 is pumped into a microencapsulation apparatus by a vacuum pump, and the third mixture is dripped into a calcium chloride solution with a mass concentration of 6% in the form of droplets with a particle size of about 450 μm by adjusting the frequency, filtered, and placed in a low-temperature environment of liquid nitrogen for shaping, thereby obtaining probiotic microcapsules. Example 3 A method for preparing probiotic microcapsules based on O1 / O2 / W structure comprises the following steps: S1. Add the activated Bifidobacterium ATCC15703 into liquid culture medium and culture at 37°C for 24 h until the viable count of the strain reaches 1×10 10 CFU / g, centrifuged at 4°C and 4000 rpm for 10 min to collect the bacterial precipitate, then added 2 times the volume of sterile saline and washed repeatedly for 3 times to prepare probiotic sludge; S2. Take 10 g of probiotic slurry, add 3.5 g of chicken fat and mix well, homogenize with a rotor-stator homogenizer at 5000 rpm for 2 min to obtain a mixture 1; S3. The mixture 1 was added to 3 g of fish oil and mixed evenly, and homogenized with a rotor-stator homogenizer at 5000 rpm for 2 min to obtain a mixture 2; S4. Weigh 2 g of sodium alginate and 0.4 g of nanocrystalline cellulose, add 97.6 g of water and mix, prepare an external aqueous phase with a mass concentration of 2% sodium alginate and a mass concentration of 0.4% nanocrystalline cellulose, add mixture 2 to 2 g of the external aqueous phase and mix well, and homogenize with a rotor-stator homogenizer at 5000 rpm for 2 min to obtain a mixture 3; S5. Mixture 3 is pumped into a microencapsulation apparatus by a vacuum pump, and the third mixture is dripped into a calcium chloride solution with a mass concentration of 7% in the form of droplets with a particle size of about 450 μm by adjusting the frequency, filtered, and placed in a low-temperature environment of liquid nitrogen for shaping, thereby obtaining probiotic microcapsules. Example 4 A method for preparing probiotic microcapsules based on O1 / O2 / W structure comprises the following steps: S1. Add the activated Lactobacillus plantarum WCFS1 into liquid culture medium and culture at 37°C for 24 h until the viable count of the strain reaches 1×10 10 CFU / g, centrifuged at 4°C and 4000 rpm for 10 min to collect the bacterial precipitate, then added 2 times the volume of sterile saline and washed repeatedly for 3 times to prepare probiotic sludge; S2. Take 10 g of probiotic slurry, add 2.5 g of fish oil and mix well, and homogenize with a rotor-stator homogenizer at 5000 rpm for 2 min to obtain a mixture 1; S3. The mixture 1 was added to 3 g of fish oil and mixed evenly, and homogenized with a rotor-stator homogenizer at 5000 rpm for 2 min to obtain a mixture 2; S4. Weigh 2 g of sodium alginate and 0.6 g of nanocrystalline cellulose, add 97.4 g of water and mix, prepare an external aqueous phase with a mass concentration of 2% sodium alginate and a mass concentration of 0.6% nanocrystalline cellulose, add mixture 2 to 2 g of the external aqueous phase and mix well, and homogenize with a rotor-stator homogenizer at 5000 rpm for 2 min to obtain a mixture 3; S5. The mixture 3 is pumped into a microencapsulation apparatus (eg Figure 1 As shown), the third mixture is dripped into a calcium chloride solution with a mass concentration of 5% in the form of droplets with a particle size of about 400 μm by adjusting the frequency, filtered, and placed in a low-temperature environment of liquid nitrogen for shaping, thereby obtaining probiotic microcapsules. Example 5 A method for preparing probiotic microcapsules based on O1 / O2 / W structure comprises the following steps: S1. Add the activated Bacillus subtilis into liquid culture medium and culture at 37°C for 24 h until the viable count of the strain reaches 1×10 10 CFU / g, centrifuged at 4°C and 4000 rpm for 10 min to collect the bacterial precipitate, then added 2 times the volume of sterile saline and washed repeatedly for 3 times to prepare probiotic sludge; S2. Take 10 g of probiotic slurry, add 3 g of corn oil and mix well, and homogenize with a rotor-stator homogenizer at 5000 rpm for 2 min to obtain a mixture 1; S3. The mixture 1 was added to 3 g of fish oil and mixed evenly, and homogenized with a rotor-stator homogenizer at 5000 rpm for 2 min to obtain a mixture 2; S4. Weigh 2 g of sodium alginate and 0.8 g of nanocrystalline cellulose, add 97.2 g of water and mix, prepare an external aqueous phase with a mass concentration of 2% sodium alginate and a mass concentration of 0.8% nanocrystalline cellulose, add mixture 2 to 3 g of the external aqueous phase and mix well, and homogenize with a rotor-stator homogenizer at 5000 rpm for 2 min to obtain a mixture 3; S5. Mixture 3 is pumped into a microencapsulation apparatus by a vacuum pump, and the third mixture is dripped into a calcium chloride solution with a mass concentration of 6% in the form of droplets with a particle size of about 450 μm by adjusting the frequency, filtered, and placed in a low-temperature environment of liquid nitrogen for shaping, thereby obtaining probiotic microcapsules. Example 6 A method for preparing probiotic microcapsules based on O1 / O2 / W structure comprises the following steps: S1. Add the activated Bifidobacterium ATCC15703 into liquid culture medium and culture at 37°C for 24 h until the viable count of the strain reaches 1×1010 CFU / g, centrifuged at 4°C and 4000 rpm for 10 min to collect the bacterial precipitate, then added 2 times the volume of sterile saline and washed repeatedly for 3 times to prepare probiotic sludge; S2. Take 10 g of probiotic slurry, add 3.5 g of chicken fat and mix well, homogenize with a rotor-stator homogenizer at 5000 rpm for 2 min to obtain a mixture 1; S3. The mixture 1 was added to 3 g of fish oil and mixed evenly, and homogenized with a rotor-stator homogenizer at 5000 rpm for 2 min to obtain a mixture 2; S4. Weigh 2 g of sodium alginate and 0.6 g of nanocrystalline cellulose, add 97.4 g of water and mix, prepare an external aqueous phase with a mass concentration of 2% sodium alginate and a mass concentration of 0.6% nanocrystalline cellulose, add mixture 2 to 2 g of the external aqueous phase and mix well, and homogenize with a rotor-stator homogenizer at 5000 rpm for 2 min to obtain a mixture 3; S5. Mixture 3 is pumped into a microencapsulation apparatus by a vacuum pump, and the third mixture is dripped into a calcium chloride solution with a mass concentration of 7% in the form of droplets with a particle size of about 450 μm by adjusting the frequency, filtered, and placed in a low-temperature environment of liquid nitrogen for shaping, thereby obtaining probiotic microcapsules. Comparative Example 1 The difference between this comparative example and Example 1 is that the composition of the external aqueous phase is different. The external aqueous phase is a solution with a mass concentration of 2% prepared by functional additives. The formula of the functional additives is 1 part of taurine, 0.6 parts of protease, 0.6 parts of cellulase, 0.6 parts of lipase, 1 part of gallic acid, 1 part of lipoic acid, 2 parts of vitamin B1, 2 parts of vitamin B2, and 2 parts of vitamin B6. A method for preparing a probiotic nutritional paste comprises the following steps: S1. Add the activated Lactobacillus plantarum WCFS1 into liquid culture medium and culture at 37°C for 24 h until the viable count of the strain reaches 1×10 10 CFU / g, centrifuged at 4°C and 4000 rpm for 10 min to collect the bacterial precipitate, then added 2 times the volume of sterile saline and washed repeatedly for 3 times to prepare probiotic sludge; S2. Take 10 g of probiotic slurry, add 2.5 g of fish oil and mix well, and homogenize with a rotor-stator homogenizer at 5000 rpm for 2 min to obtain a mixture 1; S3. The mixture 1 was added to 3 g of fish oil and mixed evenly, and homogenized with a rotor-stator homogenizer at 5000 rpm for 2 min to obtain a mixture 2; S4. Weigh 0.19 g taurine, 0.11 g protease, 0.11 g cellulase, 0.11 g lipase, 0.19 g gallic acid, 0.19 g lipoic acid, 0.37 g vitamin B1, 0.37 g vitamin B2, 0.37 g vitamin B6, add 98 g water and mix to prepare an external aqueous phase with a mass fraction of 2%; S5. Add mixture 2 to 2 g of the external aqueous phase and mix well. Use a rotor-stator homogenizer to homogenize at 5000 rpm for 2 min to prepare a probiotic nutritional paste. Comparative Example 2 The difference between this comparative example and Example 3 is that no nanocrystalline cellulose is added. A method for preparing probiotic microcapsules based on O1 / O2 / W structure comprises the following steps: S1. Add the activated Bifidobacterium ATCC15703 into liquid culture medium and culture at 37°C for 24 h until the viable count of the strain reaches 1×10 10 CFU / g, centrifuged at 4°C and 4000 rpm for 10 min to collect the bacterial precipitate, then added 2 times the volume of sterile saline and washed repeatedly for 3 times to prepare probiotic sludge; S2. Take 10 g of probiotic slurry, add 3.5 g of chicken fat and mix well, homogenize with a rotor-stator homogenizer at 5000 rpm for 2 min to obtain a mixture 1; S3. The mixture 1 was added to 3 g of fish oil and mixed evenly, and homogenized with a rotor-stator homogenizer at 5000 rpm for 2 min to obtain a mixture 2; S4. Weigh 2 g of sodium alginate, add 97.6 g of water and mix, prepare an external aqueous phase with a mass concentration of 2% sodium alginate and a mass concentration of 0.4% nanocrystalline cellulose, add mixture 2 to 2 g of the external aqueous phase and mix well, homogenize with a rotor-stator homogenizer at 5000 rpm for 2 min to obtain a mixture 3; S5. Mixture 3 is pumped into a microencapsulation apparatus by a vacuum pump, and the third mixture is dripped into a calcium chloride solution with a mass concentration of 7% in the form of droplets with a particle size of about 450 μm by adjusting the frequency, filtered, and placed in a low-temperature environment of liquid nitrogen for shaping, thereby obtaining probiotic microcapsules. Performance Testing (1) Particle size The particle sizes of Examples 1 to 6 and Comparative Example 2 were measured using a vernier caliper. (2) Encapsulation efficiency The embedding efficiency of Examples 1-6 and Comparative Examples 1-2 was determined. The specific process was as follows: 1-2 g of the sample was accurately weighed, added to 9 mL of sodium citrate solution (0.05 M), and vigorously shaken at 230 rpm for 30 min to disintegrate the system and release the probiotics, and counted; the probiotics were then continuously diluted with 9 mL of sterile phosphate buffered saline (PBS), shaken thoroughly, and then 10-fold serial dilutions were made, 2-3 appropriate dilution gradients were selected, and 100 μL of each dilution was applied to an agar medium plate, 3 plates were made for each dilution, and then incubated at 37° C. for 48 h. The results were expressed in logarithmic colony forming units (CFU / g). The formula for calculating the embedding efficiency (EE) is: EE (%) = N / N0 × 100%, where N is the number of live cells released from the microcapsules and N0 is the number of live cells concentrated before microencapsulation. Table 1 Particle size and embedding efficiency of Examples 1-6 and Comparative Examples 1-2 Particle size (μm) Embedding efficiency (%) Example 1 0.96±0.041 94.99 Example 2 0.94±0.020 95.31 Example 3 0.93±0.021 95.14 Example 4 0.90±0.041 96.13 Example 5 0.89±0.020 96.24 Example 6 0.92±0.021 95.93 Comparative Example 1 - 75.62 Comparative Example 2 1.02±0.012 87.64 As can be seen from Table 1, the particle sizes of Examples 1-6 are all smaller than those of Comparative Example 2, and the encapsulation efficiencies are all greater than those of Comparative Example 1 and Comparative Example 2, indicating that the method for preparing probiotic microcapsules of the present invention can efficiently encapsulate probiotics without affecting the activity of the probiotics. At the same time, the addition of nanocrystalline cellulose can be adsorbed on the surface of the oil droplets of the second oil phase to form a stable interface structure, thereby efficiently encapsulating the probiotics. (3) Probiotic activity during storage The probiotic activities of the probiotic microcapsules prepared in Examples 1-3 and the probiotic nutrient paste prepared in Comparative Example 1 were measured after being stored at 4° C. for one year. Table 2 Probiotic activity of Examples 1-3 and Comparative Example 1 stored at 4°C for 1 year Initial probiotic activity (CFU / g) Probiotic activity after 1 year storage (CFU / g) Example 1 <![CDATA[8.59×10 10 ]]> <![CDATA[7.73×10 10 ]]> Example 2 <![CDATA[8.43×10 10 ]]> <![CDATA[7.58×10 10 ]]> Example 3 <![CDATA[9.34×10 10 ]]> <![CDATA[8.70×10 10 ]]> Example 4 <![CDATA[8.74×10 10 ]]> <![CDATA[7.83×10 10 ]]> Example 5 <![CDATA[8.72×10 10 ]]> <![CDATA[7.76×10 10 ]]> Example 6 <![CDATA[9.51×10 10 ]]> <![CDATA[8.83×10 10 ]]> Comparative Example 1 <![CDATA[9.16×10 10 ]]> <![CDATA[4.65×10 2 ]]> Comparative Example 2 <![CDATA[9.32×10 10 ]]> <![CDATA[6.52×10 5 ]]> As shown in Table 2, the probiotic activity of the probiotic microcapsules prepared in Examples 1-6 stored at 4°C for one year was 10 times that of Comparative Example 1. 8 times and 10 times of Comparative Example 2 5 times, all showing a very significant exponential growth trend, which can effectively preserve probiotics and maintain high activity of probiotics. At the same time, the initial probiotic activity of Examples 1-6 is not significantly reduced compared with the probiotic activity stored at 4°C for 1 year, indicating that in the method for preparing probiotics of the present invention, the external aqueous phase is sodium alginate and nanocrystalline cellulose, which give the probiotic microcapsule a high-strength and tough membrane structure, which can protect the probiotics in the microcapsule from the external environment and harmful bacteria, ensure that the probiotics in the microcapsule are not greatly affected within 1 year of storage, and retain the initial probiotic activity of the preparation. (4) In vitro simulated gastric digestion test Preparation of artificial gastric juice: Take 3.2g of pepsin, add 800mL of water, then add appropriate amount of dilute hydrochloric acid, adjust pH to 2.5, stir well and add water to make up to 1000mL. Then, before use, preheat the prepared simulated gastric juice in a 37℃ constant temperature box and sterilize it with a 0.22μm filter; Probiotic activity determination: 0.5 g microcapsules were added to a 10 mL Eppendorf tube containing 4.5 mL simulated gastric juice, mixed thoroughly for 15 s, and shaken in a shaker at 37 °C and 200 rpm for 50, 100, and 150 min. At each measurement time, one 10 mL Eppendorf tube was added to 5 mL sodium citrate solution (0.05 M) in each group, and then shaken vigorously at 230 rpm for 30 min to disintegrate the hydrogel network and release the probiotics. The live encapsulated probiotics were counted. The probiotics (10 -1 Up to 10 -9 ), shake thoroughly, then make 10-fold serial dilutions, select 2-3 appropriate dilution gradients, and spread 100 μL of each dilution on Lactobacillus agar plates, make 3 plates for each dilution, and then culture at 37°C for 48h. The results are expressed in logarithmic colony forming units (CFU / mL or CFU / g). Table 3 Probiotic activity of Examples 1-3 and Comparative Example 1 in simulated gastric juice digestion As shown in Table 3, the probiotic activities of Examples 1-3, Comparative Examples 1 and 2 decreased with the prolongation of the residence time of the probiotic microcapsules in the simulated stomach, but the decreasing trends of Comparative Examples 1 and 2 were significantly faster than those of Examples 1-3, and the probiotic activities of Examples 1-3 at a simulated stomach residence time of 150 min were 10 times that of Comparative Example 1. 5 times, showing an exponential growth trend, indicating that the outer aqueous phase of the probiotic microcapsule in the present invention is sodium alginate and nanocrystalline cellulose, which can form a stable and dense gel network in the outermost layer of the intermediate oil phase, giving the probiotic microcapsule a stable membrane structure, protecting the probiotic core material from the interference of the extreme pH and enzymes of simulated gastric juice digestion, and ensuring that the probiotics can exist stably in the gastric juice. (5) In vitro simulated intestinal digestion test Preparation of artificial intestinal fluid: Take 10g trypsin and 6.8g KH2PO4, add appropriate amount of water to dissolve, adjust the pH to 6.8 with NaOH solution, and add water to make up to 1000mL. Before use, preheat the prepared simulated intestinal fluid in a 37℃ constant temperature box and sterilize it with a 0.22μm filter; Probiotic activity determination: 0.5 g microcapsules were added to a 10 mL Eppendorf tube containing 4.5 mL simulated gastric juice, mixed thoroughly for 15 s, shaken at 37 ° C and 200 rpm for 150 min, and then 40 mL simulated intestinal juice was added, and the shaking digestion was continued at 37 ° C and 120 rpm in a shaker for 60 and 150 min; at each measurement time, 1 test tube was taken from each group and added with 5 mL sodium citrate solution (0.05 M), and then vigorously shaken at 230 rpm for 30 min to disintegrate the hydrogel network and release the probiotics, and the live encapsulated probiotics were counted; the probiotics (10 -1 Up to 10 -9 ), shake thoroughly, then make 10-fold serial dilutions, select 2-3 appropriate dilution gradients, and spread 100 μL of each dilution on Lactobacillus agar plates, make 3 plates for each dilution, and then culture at 37°C for 48h. The results are expressed in logarithmic colony forming units (CFU / g). Table 4 Probiotic activity of Examples 1-3, Comparative Example 1 and Comparative Example 2 in simulated gastrointestinal digestion As shown in Table 4, the probiotic activities of Examples 1-3, Comparative Examples 1 and 2 all showed a downward trend as the probiotic microcapsules stayed in the simulated intestinal tract for a longer time, and the probiotic activities of Examples 1-3 were significantly higher than those of Comparative Examples 1 and 2, while the probiotic activities of Comparative Examples 1 and 2 were both lower than 10 6 CFU / g, which cannot produce beneficial effects in the intestine, indicating that sodium alginate and nanocrystalline cellulose are used as the external aqueous phase. On the one hand, sodium alginate can form a gel network structure, so that the probiotic oil phase core material is tightly embedded in it, isolating the adverse factors in the external environment. On the other hand, the hydroxyl groups contained in nanocrystalline cellulose can form stable hydrogen bonds under acidic conditions, and further form a tighter and more stable crystal structure to resist the erosion and degradation of gastric acid and ensure the high activity of probiotics. With the increase of intestinal pH, the probiotic microcapsule structure disintegrates, and is released and exerts its effect in the intestine. (6) FITC staining and Nile Red staining The probiotics embedded in corn oil, MCT oil and corn oil / MCT oil dual-phase system were stained with FITC and Nile Red, and the probiotic activity on the 0th, 30th and 60th days of storage was determined. The staining method is as follows: 0.25g CNCs was added to 0.1M NaOH and magnetically stirred at room temperature to form a dispersion with a concentration of 1wt%; then 0.05g FITC was weighed and added to the dispersion, the entire flask was covered with tin foil to maintain dark conditions, and the reaction was carried out at 25°C for 72h; finally, the reaction mixture was taken out, the pH of the mixture was adjusted to neutral with a 5% sulfuric acid aqueous solution, poured into a dialysis bag, dialyzed in deionized water for one week, and the cellulose nanocrystal fluorescent material was obtained by centrifugal concentration and freeze-drying; the fresh emulsion (100μL) was stained with 20μL containing 0.1% (wt) Nile Red (for coloring the oil phase). Depend on Figure 3 and Figure 4 It can be seen that the probiotic activity of probiotics encapsulated based on the O1 / O2 structure is significantly higher than that of probiotics encapsulated in the oil-free phase and the single-layer oil phase. Because probiotics will die quickly under liquid conditions, in order to maintain high activity of probiotics in liquid state, the probiotics can only be preserved in the oil phase, and the survival rate of probiotics in the two-phase oil phase is significantly improved than that in the single-phase oil phase, especially under long-term storage time (60d). Therefore, a two-layer oil design is adopted to preserve probiotics for a long time under liquid conditions. The above is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any technician familiar with the art can make many possible changes and modifications to the technical solution of the present invention by using the above disclosed methods and technical contents without departing from the spirit and technical solution of the present invention, or modify it into an equivalent embodiment of equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still falls within the scope of protection of the technical solution of the present invention.

Claims

1. A method for preparing probiotic microcapsules based on O1 / O2 / W structure, characterized in that: The following steps are involved: S1. Preparing probiotic slurry; S2. Add the probiotic slurry to the first oil phase and mix well to obtain a mixture 1; S3. The mixture 1 is added to the second oil phase and mixed evenly to obtain a mixture 2; S4. Add mixture 2 to the external aqueous phase and mix evenly, add it dropwise into the calcium chloride solution, filter it, and fix it with liquid nitrogen to obtain probiotic microcapsules.

2. The method for preparing probiotic microcapsules based on O1 / O2 / W structure according to claim 1, characterized in that: In step S1, the probiotics are one or more of bifidobacteria, lactobacilli, bacillus subtilis, and yeast.

3. The method for preparing probiotic microcapsules based on O1 / O2 / W structure according to claim 1, characterized in that: The method for preparing the probiotic sludge in step S1 comprises the following steps: Step 1. Probiotics are inoculated into liquid culture medium and subcultured at 30-40°C until the number of viable bacteria is > 1×10 10 CFU / g, culture medium was prepared; Step 2. The culture solution is centrifuged at 0-4°C and 3000-5000 rpm for 5-15 min, the bacterial precipitate is taken, and washed 2-4 times with sterile saline at a volume ratio of 1:(1-3) to obtain probiotic bacterial sludge.

4. The method for preparing probiotic microcapsules based on O1 / O2 / W structure according to claim 1, characterized in that: In the step S2, the first oil phase is one or more of animal oil, medium-chain triglycerides, and vegetable oil, and the weight ratio of the probiotic sludge to the first oil phase is 1:(0.1-0.4).

5. The method for preparing probiotic microcapsules based on O1 / O2 / W structure according to claim 1, characterized in that: In step S3, the second oil phase is one or more of animal oil, medium chain triglyceride, and vegetable oil, and the amount of the second oil phase added is 10-40%, based on the weight of the probiotic mud.

6. The method for preparing probiotic microcapsules based on O1 / O2 / W structure according to claim 1, characterized in that: In step S4, the external aqueous phase is sodium alginate and nanocrystalline cellulose, the mass concentration of sodium alginate is 1-4%, and the mass concentration of nanocrystalline cellulose is 0.1-0.8%.

7. The method for preparing probiotic microcapsules based on O1 / O2 / W structure according to claim 1, characterized in that: The amount of the external water phase added in step S4 is 10-30% based on the weight of the probiotic mud.

8. The method for preparing probiotic microcapsules based on O1 / O2 / W structure according to claim 1, characterized in that: The mass concentration of the calcium chloride solution in step S4 is 3-8%.

9. Use of the probiotic microcapsules prepared according to the method according to any one of claims 1 to 8 in the preparation of food, health products and pet diets.

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