Calcium alginate microspheres containing chitosan oligosaccharide coated bifidobacterium and caffeic acid phenethyl ester nano-enzyme and preparation method of calcium alginate microspheres

Abstract

The invention belongs to the field of pharmaceutical preparations. The invention relates to a calcium alginate microsphere containing chitosan oligosaccharide coated bifidobacterium and caffeic acid phenethyl ester nano-enzyme and a preparation method of the calcium alginate microsphere. The calcium alginate microspheres containing the chitosan oligosaccharide coated bifidobacterium and the caffeic acid phenethyl ester nano-enzyme prepared by the preparation method disclosed by the invention can be used for remarkably reducing the uric acid level of rats with gouty arthritis and alleviating the joint swelling of the rats with gouty arthritis.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical preparations. It relates to calcium alginate microspheres containing chitosan oligosaccharide-coated Bifidobacterium (B / COS) and caffeic acid phenylethyl ester nanozyme (NC-B), and their preparation method (hereinafter referred to as: calcium alginate microspheres containing B / COS and NC-B in this patent). The calcium alginate microspheres containing chitosan oligosaccharide-coated Bifidobacterium and caffeic acid phenylethyl ester nanozyme include chitosan oligosaccharide-coated Bifidobacterium, caffeic acid phenylethyl ester nanozyme, and calcium alginate microspheres, wherein the chitosan oligosaccharide-coated Bifidobacterium and caffeic acid phenylethyl ester nanozyme are carried within the calcium alginate microspheres; the caffeic acid phenylethyl ester nanozyme (NC-B) includes caffeic acid phenylethyl ester and cerium nickel nanozyme, wherein caffeic acid phenylethyl ester is carried within the cerium nickel nanozyme. Background Technology

[0002] Gouty arthritis is an inflammatory joint disease caused by the deposition of monosodium urate crystals within and around the joints, triggering the release of pro-inflammatory cytokines. The main clinical manifestations are recurrent episodes of joint redness, swelling, pain, and functional impairment. In severe cases, tophi formation, joint deformities, and kidney damage may occur. Currently, drug treatment for gouty arthritis is based on both controlling excessive inflammation during acute attacks and preventing attacks by lowering urate levels. First-line clinical treatment for acute gouty arthritis attacks involves nonsteroidal anti-inflammatory drugs (NSAIDs), colchicine, or corticosteroids, or a combination of both. However, this approach requires long-term use, has limited efficacy, and carries toxic side effects. Caffeic acid phenethyl ester (CA) is a flavonoid isolated from propolis. It has uric acid-lowering effects, is generally non-toxic, and has protective effects on the liver and kidneys. However, its poor water solubility, low stability, and low bioavailability limit its application. Cerium nanozymes possess catalase-like and superoxide dismutase-like activities, reducing reactive oxygen species levels at inflammatory sites. Nickel can be used in Ni... 3+ and Ni 2+ The conversion between these components has the potential to consume reactive oxygen species. Probiotics (such as Bifidobacteria) are live microorganisms that can play a role in the defense and recovery from intestinal pathogen infections, but they are easily destroyed in the stomach. Chitosan oligosaccharides are degraded only in the colon due to the presence of degrading enzymes such as chitosanase. Bifidobacteria are a dominant group in the gastrointestinal microbiota and are considered probiotics. Sodium alginate can cross-link with calcium ions to form hydrogel microspheres for drug delivery.

[0003] A review of patents and literature reveals the following: there are currently no research reports on chitosan oligosaccharide-coated Bifidobacterium (B / COS), no research reports on cerium-nickel nanozymes, no reports on caffeic acid phenethyl ester carried in cerium-nickel nanozymes (NC-B), no reports on calcium alginate microspheres containing NC-B, no research reports on any formulations containing NC-B, no reports on calcium alginate microspheres containing B / COS, no research reports on any formulations containing B / COS, no reports on calcium alginate microspheres containing both Bifidobacterium and NC-B, no reports on calcium alginate microspheres containing both B / COS and NC-B, and no research reports on any formulations containing both B / COS and NC-B. Summary of the Invention

[0004] The technical problem to be solved by this invention is to provide calcium alginate microspheres containing B / COS and NC-B, and a method for preparing the same. Calcium alginate microspheres containing B / COS and NC-B overcome the drawback of poor stability in calcium alginate (CA). The calcium alginate microspheres containing B / COS and NC-B provided by this invention can significantly reduce uric acid levels in rats with gouty arthritis and also significantly alleviate joint swelling in these rats. This study provides a novel formulation of CA that can be used to treat gouty arthritis and related diseases.

[0005] The calcium alginate microspheres containing B / COS and NC-B provided by the present invention are characterized in that they comprise bifidobacteria coated with chitosan oligosaccharide, caffeic acid phenylethyl ester nanozyme, and calcium alginate microspheres, wherein the bifidobacteria coated with chitosan oligosaccharide and the caffeic acid phenylethyl ester nanozyme are carried within the calcium alginate microspheres; the caffeic acid phenylethyl ester nanozyme comprises caffeic acid phenylethyl ester and cerium nickel nanozyme, wherein caffeic acid phenylethyl ester is carried within the cerium nickel nanozyme.

[0006] The chitosan oligosaccharide-coated Bifidobacterium and caffeic acid phenethyl ester nanozyme are carried in calcium alginate microspheres; the B / COS and NC-B-containing calcium alginate microspheres comprise the following components in the following mass ratio: 250-1000 parts of cerium nitrate hexahydrate, 100-400 parts of polyvinylpyrrolidone, 1-4 parts of nickel chloride hexahydrate, 50-200 parts of caffeic acid phenethyl ester, 30-120 parts of albumin, and Bifidobacterium... Bacillus 250-3000 parts, chitosan oligosaccharide 10-120 parts, sodium alginate 150-600 parts, calcium chloride 600-2400 parts, ultrapure water 1: 10000-40000 parts, ultrapure water 2: 10000-40000 parts, ultrapure water 3: 1000-4000 parts, ultrapure water 4: 10000-40000 parts, ultrapure water 5: 5000-20000 parts, ultrapure water 6: 5 00-2000 parts, ultrapure water 7 is 10000-40000 parts, ultrapure water 8 is 250-3000 parts, ultrapure water 9 is 10000-40000 parts, ultrapure water 10 is 7500-30000 parts, phosphate buffer 1 is 250-3000 parts, phosphate buffer 2 is 250-3000 parts, phosphate buffer 1 and phosphate buffer 2 are the same type of buffer, both with pH 7.4, the Bifidobacterium includes one of Bifidobacterium adolescentis, Bifidobacterium bifidum, and Bifidobacterium longum; the preparation method of calcium alginate microspheres containing B / COS and NC-B provided by the present invention includes the following steps: (1) Preparation method of caffeic acid phenethyl ester nanozyme: Cerium nitrate hexahydrate and polyvinylpyrrolidone are dissolved in ethylene glycol, and 1M hydrochloric acid aqueous solution is added under vigorous stirring, and stirred for 30-60 minutes. After a period of time, heat at 160-180 ℃ for 2-5 h, cool to room temperature, centrifuge to collect the precipitate, and wash with ultrapure water 1 to obtain solid A; disperse solid A in ultrapure water 2 to form liquid B, and add ammonia to adjust the pH to 9-11 to form liquid C; dissolve nickel chloride hexahydrate in ultrapure water 3 to form liquid D; add liquid D to liquid C, stir in a water bath at 50-90 ℃ for 2-5 h, centrifuge to collect the precipitate, wash with ultrapure water 4 and dry to obtain cerium-nickel nanozyme; dissolve phenethyl caffeate in methanol to form liquid E; disperse the cerium-nickel nanozyme in ultrapure water 5 to form liquid F; dissolve albumin in ultrapure water 6 to form liquid G; add liquid E to liquid F and stir for 12-24 h; centrifuge to collect the precipitate, wash with ultrapure water 7, and add to liquid G and let stand for 12-24 hours. h, centrifuge to collect the precipitate and obtain caffeic acid phenylethyl ester nanozyme; (2) Preparation method of chitosan oligosaccharide-coated Bifidobacterium: dissolve chitosan oligosaccharide in ultrapure water 8 to form liquid H, disperse Bifidobacterium in phosphate buffer 1 to form bacterial suspension, and adjust the concentration of bacterial suspension to 1.0E+08~1.0E+09 CFU / mL, after thoroughly mixing liquid H with bacterial suspension by vortexing, let stand for 30-60 min, centrifuge and wash, discard the supernatant, and disperse the lower precipitate in phosphate buffer 2 to obtain chitosan oligosaccharide-coated Bifidobacterium; (3) Preparation method of calcium alginate microspheres containing chitosan oligosaccharide-coated Bifidobacterium and caffeic acid phenylethyl ester nanozyme: dissolve sodium alginate in ultrapure water 9 to obtain sodium alginate solution, dissolve calcium chloride in ultrapure water 10 to obtain calcium chloride solution, add caffeic acid phenylethyl ester nanozyme obtained in step (1) and chitosan oligosaccharide-coated Bifidobacterium obtained in step (2) to sodium alginate solution, stir and mix evenly to obtain liquid I, then add liquid I to liquid paraffin containing 1%-5% Span 80, stir at 150-350 rpm for 1-2 h, then add calcium chloride solution dropwise, stir for 1-2 h. h, centrifuge to collect the lower precipitate, wash the precipitate with 0.9% sodium chloride solution to obtain calcium alginate microspheres containing chitosan oligosaccharide-coated Bifidobacteria and caffeic acid phenylethyl ester nanozyme.

[0007] The cerium-nickel nanozyme prepared in this invention has a spherical morphology. Figure 1 ).

[0008] The calcium alginate microspheres containing B / COS and NC-B prepared in this invention were observed under an inverted microscope, showing a bright-field pattern of the microspheres (…). Figure 2 a) and Bifidobacterium fluorescence image ( Figure 2 b) After superposition, the fluorescence overlaps within the microspheres ( Figure 2 c) indicates that B / COS successfully loaded calcium alginate microspheres.

[0009] The swelling results of the calcium alginate microspheres containing B / COS and NC-B prepared in this invention in simulated gastrointestinal fluids showed that almost no swelling occurred in simulated gastric fluid, but the swelling rate increased significantly in simulated small intestinal fluid and simulated colonic fluid. Once in simulated small intestinal fluid, the swelling rate could reach 480% in the first hour and 1100% within 4 hours. When entering simulated colonic fluid, the swelling rate further increased. Figure 3 Due to the property that the outer shell of the microspheres is stable in acidic environments but disintegrates in weakly alkaline environments, the carrier can be successfully delivered to the small intestine.

[0010] The calcium alginate microspheres containing B / COS and NC-B provided by this invention, when administered in vivo to male SD rats with a gouty arthritis model, can reduce serum uric acid levels. Figure 4 The serum uric acid levels of calcium alginate microspheres containing B / COS and NC-B were close to those of the normal group, and the effect was stronger than that of free drugs and other treatment groups, indicating that calcium alginate microspheres containing B / COS and NC-B can significantly reduce serum uric acid levels.

[0011] The calcium alginate microspheres containing B / COS and NC-B provided by this invention significantly reduced ankle joint swelling in male SD rats with a gouty arthritis model, with a faster swelling reduction rate than the free drug group and other treatment groups. Figure 5 It recovered to normal within 96 hours.

[0012] The calcium alginate microsphere delivery system containing B / COS and NC-B provided by this invention offers an effective therapeutic approach for the delivery of small-molecule drugs to treat hyperuricemia, gout, and related diseases. We are the first to dope cerium nanozymes with nickel, forming cerium-nickel nanozymes, enabling the doping of cerium... 4+ / Ce 3+ and Ni 3+ / Ni 2+For coexistence, nickel doping can significantly improve the enzyme-like activity of cerium nanozymes. Cerium-nickel nanozymes have a hollow structure, thus they can carry poorly soluble drugs such as hydroxylamine (CA), improving CA's solubility and stability, and exerting a synergistic therapeutic effect (the nanozymes exert enzyme-like activity to scavenge reactive oxygen species, while CA exerts anti-inflammatory and uric acid-lowering effects, synergistically contributing to the therapeutic effect). Sodium alginate is an anionic natural polysaccharide extracted from brown algae. Its structure and properties can mimic the natural polysaccharide matrix in organisms (such as glycosaminoglycans in the extracellular matrix). It can also form a bio-tissue-like hydrogel structure by cross-linking with calcium ions. In this invention, B / COS and NC-B are encapsulated in calcium alginate microspheres formed by sodium alginate and calcium ions (calcium alginate microspheres are water-insoluble three-dimensional network gel microspheres with degradability, biocompatibility, and pH sensitivity, enabling controlled delivery, protection, and targeted drug release). The ion gelation process does not require high temperatures, making it suitable for encapsulating sensitive components such as probiotics. The network structure of the calcium alginate hydrogel microspheres in this invention can encapsulate drugs that are easily degraded by gastric acid (such as probiotics), preventing their inactivation in the stomach. Simultaneously, the microspheres can slow down the drug release rate, reducing rapid metabolism and excretion in the intestines, significantly improving drug bioavailability. The calcium alginate microspheres containing B / COS and NC-B described in this invention can release drugs in stages, meeting the needs of different drugs to exert their effects at different targets and improving therapeutic efficacy. This invention uses calcium alginate as the shell material of the formulation. The shell is stable under acidic conditions, thus maintaining the integrity of the formulation in the stomach without releasing the drug. After the formulation reaches the intestines, the calcium alginate shell material disintegrates in a weakly alkaline environment, thus gradually releasing NC-B and B / COS in the small intestine and other intestinal tracts. Some NC-B can exert enzyme-like activity in the small intestine and other intestinal tracts using cerium-nickel nanozymes, removing reactive oxygen species (ROS). CA is released from NC-B and inhibits uric acid production by inhibiting xanthine oxidase and promotes uric acid excretion by inhibiting urate transporter 1, thereby reducing intestinal uric acid levels. CA can also inhibit the synthesis of prostaglandins and leukotrienes, thus exerting anti-inflammatory effects. Some NC-B can be absorbed from the intestines into the bloodstream and reach the site of gouty arthritis in the blood or through blood / lymphatic circulation, where it exerts its effects of removing ROS, reducing uric acid levels, and anti-inflammation in the blood and at the site of arthritis. Bifidobacteria (B / COS) coated with chitosan oligosaccharides are specifically released into the colon because chitosan oligosaccharides are degraded only in the colon (due to the presence of degrading enzymes such as chitosanase in the colon). This allows them to colonize stably and improve the gut microbiota structure, reducing harmful bacteria that produce purine metabolic intermediates, thereby decreasing the substrate supply for hepatic xanthine oxidase and indirectly reducing uric acid production. The short-chain fatty acids produced by Bifidobacteria can also provide energy for intestinal epithelial cells, improve intestinal barrier function, and reduce inflammatory responses.The calcium alginate microspheres containing B / COS and NC-B prepared in this invention, through synergistic drug action, can not only significantly reduce uric acid levels in rats with gouty arthritis, but also significantly alleviate joint swelling in these rats. The calcium alginate microspheres containing B / COS and NC-B prepared in this invention can be used for the treatment of gouty arthritis and related diseases.

[0013] This invention differs from previously reported CA delivery carriers and preparation processes. Other CA delivery systems include liposomes, inorganic nanoparticles, polymer nanoparticles, and nanomicelles. The calcium alginate microspheres containing B / COS and NC-B in this invention represent a novel biomimetic drug delivery system. This invention is the first to use oral biomimetic microspheres to co-deliver multiple active ingredients (CA, cerium-nickel nanozymes, and Bifidobacteria) for the treatment of gouty arthritis. This invention is also the first to synthesize cerium-nickel nanozymes, the first to load CA into cerium-nickel nanozymes to form NC-B, the first to use calcium alginate microspheres to deliver NC-B, the first to use calcium alginate microspheres to deliver Bifidobacteria and NC-B, and the first to use calcium alginate microspheres to deliver B / COS and NC-B. Attached Figure Description

[0014] Figure 1 Transmission electron microscopy image of the cerium-nickel nanozyme prepared in this invention; scale bar is 50 nm.

[0015] Experimental conditions: The morphology of cerium-nickel nanozymes was determined using transmission electron microscopy.

[0016] The results showed that the cerium-nickel nanozyme prepared by this invention has a spherical shape.

[0017] Figure 2 Bright-field fluorescence image of calcium alginate microspheres containing B / COS and NC-B prepared in this invention; scale bar is 50 μm. Figure 2 a is the bright-field plot of calcium alginate microspheres containing B / COS and NC-B. Figure 2 b is the fluorescence image of calcium alginate microspheres containing B / COS and NC-B. Figure 2 c is the overlay of bright-field and fluorescence images of calcium alginate microspheres containing B / COS and NC-B.

[0018] Experimental conditions: Calcium alginate microspheres containing B / COS and NC-B were prepared after labeling Bifidobacterium with FITC. B / COS was then encapsulated in the microspheres, and the presence of fluorescence in the microspheres was observed using an inverted fluorescence microscope.

[0019] The results showed that when the bright-field and fluorescence images of calcium alginate microspheres containing B / COS and NC-B were superimposed, the fluorescence (emitted by FITC-labeled Bifidobacteria) overlapped within the microspheres, indicating that B / COS was successfully loaded into the calcium alginate microspheres.

[0020] Figure 3The swelling rate of the calcium alginate microspheres containing B / COS and NC-B prepared in this invention in simulated gastrointestinal fluid is shown.

[0021] Experimental conditions: Microspheres (M0) were placed in centrifuge tubes and weighed (M1). Simulated gastric, small intestinal, and colonic fluids were added, and the mixture was continuously shaken at 100 rpm at 37 °C. After centrifugation, the supernatant was removed, excess liquid was removed with filter paper, and the samples were weighed at the corresponding time points (M). t ), where M0 represents the initial mass of the microsphere, using the equation: Expansion (%) = [(M t [-M1) / M0]×100; Determine the swelling ratio of the microspheres.

[0022] The results showed that calcium alginate microspheres containing B / COS and NC-B exhibited almost no swelling in simulated gastric fluid, but significantly increased swelling rates in simulated small intestinal and colonic fluids. Upon entering the simulated small intestinal fluid, the swelling rate reached 480% at 1 hour and 1100% within 4 hours; upon entering the simulated colonic fluid, the swelling rate further increased. Data in the figure are expressed as mean ± standard deviation (n=3).

[0023] Figure 4 The figure shows the uric acid level in rats after oral administration of the calcium alginate microspheres containing B / COS and NC-B of this invention.

[0024] Experimental conditions: Thirty male SD rats were randomly divided into 5 groups (n=6 per group): normal group, model group, CA group, NC-B calcium alginate microsphere group, and B / COS and NC-B calcium alginate microsphere groups. Except for the normal group, the other groups were administered hypoxanthine by gavage at 500 mg / kg, followed by intraperitoneal injection of potassium oxonate at 300 mg / kg for 7 consecutive days to establish a hyperuricemia model. On day 8, 25 mg / mL sodium urate crystal suspension was injected into the joint cavity on the lateral side of the right ankle joint of the SD rats to establish a rat model of gouty arthritis under hyperuricemia. Except for the normal and model groups, the other groups were subsequently administered CA or various preparations containing the same dose of CA by gavage daily. After treatment, blood was collected, and the uric acid level in the blood was measured using a uric acid assay kit.

[0025] The results showed that in vivo administration of calcium alginate microspheres containing B / COS and NC-B to a rat model of gouty arthritis significantly reduced serum uric acid levels, with a stronger effect than that of free drug and other treatment groups. This indicates that calcium alginate microspheres loaded with NC-B and B / COS can reduce serum uric acid levels. Data in the figure are expressed as mean ± standard deviation (n=6). (Compared with the normal group, ***p<0.001; compared with the model group, ###p<0.001; compared with the CA group, $p<0.05, $$$p<0.001).

[0026] Figure 5 Ankle swelling-time curve in rats with gouty arthritis, prepared using calcium alginate microspheres containing B / COS and NC-B according to this invention.

[0027] Experimental conditions: Thirty male SD rats were randomly divided into 5 groups (n=6 per group): normal group, model group, CA group, NC-B calcium alginate microsphere group, and B / COS and NC-B calcium alginate microsphere groups. Except for the normal group, the other groups were administered hypoxanthine by gavage at 500 mg / kg, followed by intraperitoneal injection of potassium oxonate at 300 mg / kg for 7 consecutive days to establish a hyperuricemia model. On day 8, 25 mg / mL sodium urate crystal suspension was injected into the joint cavity on the lateral side of the right ankle joint of the SD rats to establish a rat model of gouty arthritis under hyperuricemia. Except for the normal and model groups, the other groups were subsequently administered CA or a preparation containing the same dose of CA by gavage daily. Ankle diameter was measured in each rat in each group at different time points.

[0028] The results showed that after in vivo administration of B / COS and NC-B calcium alginate microspheres, ankle joint swelling was significantly reduced, and the joint swelling subsided faster than in the free drug group and other treatment groups. The joints had returned to normal after 96 hours, while the model group showed the most significant joint swelling compared to other groups. Detailed Implementation

[0029] To further illustrate the present invention and its advantages, the following specific embodiments are provided. It should be understood that these embodiments are for illustrative purposes only and not as limiting the scope of the invention.

[0030] Example 1:

[0031] The microspheres containing B / COS and NC-B alginate calcium spheres consist of chitosan oligosaccharide-coated Bifidobacterium longum, caffeic acid phenylethyl ester nanozyme, and calcium alginate microspheres. The mass ratio of each component in the B / COS and NC-B calcium alginate calcium spheres is as follows: 250 parts cerium nitrate hexahydrate, 100 parts polyvinylpyrrolidone, 1 part nickel chloride hexahydrate, 50 parts caffeic acid phenylethyl ester, 30 parts albumin, 250 parts Bifidobacterium longum, 10 parts chitosan oligosaccharide, 150 parts sodium alginate, 600 parts calcium chloride, 10,000 parts ultrapure water 1, 10,000 parts ultrapure water 2, and 100 parts ultrapure water 3. 0 parts, ultrapure water 4 is 10000 parts, ultrapure water 5 is 5000 parts, ultrapure water 6 is 500 parts, ultrapure water 7 is 10000 parts, ultrapure water 8 is 250 parts, ultrapure water 9 is 10000 parts, ultrapure water 10 is 7500 parts, phosphate buffer 1 is 250 parts, phosphate buffer 2 is 250 parts. Phosphate buffer 1 and phosphate buffer 2 are the same type of buffer, and both have a pH of 7.4.

[0032] Preparation method: (1) Preparation method of caffeic acid phenethyl ester nanozyme: Cerium nitrate hexahydrate and polyvinylpyrrolidone were dissolved in ethylene glycol. Under vigorous stirring, 1M hydrochloric acid aqueous solution was added. After stirring for 30 min, the mixture was heated at 160 °C for 3 h. After cooling to room temperature, the precipitate was collected by centrifugation and washed with ultrapure water 1 to obtain solid A. Solid A was dispersed in ultrapure water 2 to form liquid B, and ammonia was added dropwise to adjust the pH to 9 to form liquid C. Nickel chloride hexahydrate was dissolved in ultrapure water 3 to form liquid D. Liquid D was added to liquid C, stirred in a water bath at 50 °C for 2 h, the precipitate was collected by centrifugation, washed with ultrapure water 4 and dried to obtain cerium nickel nanozyme. Caffeic acid phenethyl ester was dissolved in methanol to form liquid E. Cerium nickel nanozyme was dispersed in ultrapure water 5 to form liquid F. Albumin was dissolved in ultrapure water 6 to form liquid G. Liquid E was added to liquid F and stirred for 12 h. The precipitate was collected by centrifugation and washed with ultrapure water 7. The precipitate was added to liquid G and placed for 24 h. h, centrifuge to collect the precipitate and obtain caffeic acid phenylethyl ester nanozyme; (2) Preparation method of chitosan oligosaccharide-coated Bifidobacterium longum: dissolve chitosan oligosaccharide in ultrapure water 8 to form liquid H, disperse Bifidobacterium longum in phosphate buffer 1 to form bacterial suspension, adjust the concentration of bacterial suspension to 1.0E+08 CFU / mL, vortex and shake the liquid H and bacterial suspension to mix thoroughly, and place for 30 h. min, centrifuge and wash, discard the supernatant and disperse the lower precipitate in phosphate buffer 2 to obtain chitosan oligosaccharide-coated Bifidobacterium longum; (3) Preparation method of calcium alginate microspheres containing chitosan oligosaccharide-coated Bifidobacterium longum and caffeic acid phenylethyl ester nanozyme: dissolve sodium alginate in ultrapure water 9 to obtain sodium alginate solution, dissolve calcium chloride in ultrapure water 10 to obtain calcium chloride solution, add caffeic acid phenylethyl ester nanozyme obtained in step (1) and chitosan oligosaccharide-coated Bifidobacterium longum obtained in step (2) to sodium alginate solution, stir and mix evenly to obtain liquid I, then add liquid I to liquid paraffin containing 5% Span 80, stir at 150 rpm for 1 h, then add calcium chloride solution dropwise, stir for 2 h, centrifuge and collect the lower precipitate, wash the precipitate with 0.9% sodium chloride solution to obtain calcium alginate microspheres containing chitosan oligosaccharide-coated Bifidobacterium longum and caffeic acid phenylethyl ester nanozyme.

[0033] Example 2:

[0034] The microspheres contain B / COS and NC-B calcium alginate, including *Bifidobacterium adolescentis*, caffeic acid phenylethyl ester nanozyme, and calcium alginate microspheres. The mass ratio of each component in the B / COS and NC-B calcium alginate microspheres is as follows: 625 parts cerium nitrate hexahydrate, 250 parts polyvinylpyrrolidone, 1.8 parts nickel chloride hexahydrate, 137.5 parts caffeic acid phenylethyl ester, 68 parts albumin, 1250 parts *Bifidobacterium adolescentis*, 20 parts chitosan oligosaccharide, 525 parts sodium alginate, 2100 parts calcium chloride, 25000 parts ultrapure water 1, 25000 parts ultrapure water 2, and 17 parts ultrapure water 3. 50 parts, ultrapure water 4 is 25,000 parts, ultrapure water 5 is 13,750 parts, ultrapure water 6 is 1,133 parts, ultrapure water 7 is 25,000 parts, ultrapure water 8 is 500 parts, ultrapure water 9 is 35,000 parts, ultrapure water 10 is 26,250 parts, phosphate buffer 1 is 1,250 parts, phosphate buffer 2 is 1,250 parts. Phosphate buffer 1 and phosphate buffer 2 are the same type of buffer, both with a pH of 7.4.

[0035] Preparation method: (1) Preparation method of caffeic acid phenethyl ester nanozyme: Cerium nitrate hexahydrate and polyvinylpyrrolidone were dissolved in ethylene glycol. Under vigorous stirring, 1M hydrochloric acid aqueous solution was added. After stirring for 30 min, the mixture was heated at 170 °C for 3 h. After cooling to room temperature, the precipitate was collected by centrifugation and washed with ultrapure water 1 to obtain solid A. Solid A was dispersed in ultrapure water 2 to form liquid B, and ammonia was added dropwise to adjust the pH to 10 to form liquid C. Nickel chloride hexahydrate was dissolved in ultrapure water 3 to form liquid D. Liquid D was added to liquid C, stirred in a water bath at 90 °C for 2 h, the precipitate was collected by centrifugation, washed with ultrapure water 4 and dried to obtain cerium nickel nanozyme. Caffeic acid phenethyl ester was dissolved in methanol to form liquid E. Cerium nickel nanozyme was dispersed in ultrapure water 5 to form liquid F. Albumin was dissolved in ultrapure water 6 to form liquid G. Liquid E was added to liquid F and stirred for 12 h. The precipitate was collected by centrifugation and washed with ultrapure water 7. The precipitate was added to liquid G and placed for 12 h. h, centrifuge to collect the precipitate and obtain caffeic acid phenylethyl ester nanozyme; (2) Preparation method of chitosan oligosaccharide-coated Bifidobacterium adolescentis: dissolve chitosan oligosaccharide in ultrapure water 8 to form liquid H, disperse Bifidobacterium adolescentis in phosphate buffer 1 to form bacterial suspension, adjust the concentration of bacterial suspension to 1.0E+08 CFU / mL, vortex and shake the liquid H and bacterial suspension to mix thoroughly, and place for 60 h. min, centrifuge and wash, discard the supernatant and disperse the lower precipitate in phosphate buffer 2 to obtain chitosan oligosaccharide-coated Bifidobacterium adolescentis; (3) Preparation method of calcium alginate microspheres containing chitosan oligosaccharide-coated Bifidobacterium adolescentis and caffeic acid phenylethyl ester nanozyme: dissolve sodium alginate in ultrapure water 9 to obtain sodium alginate solution, dissolve calcium chloride in ultrapure water 10 to obtain calcium chloride solution, add caffeic acid phenylethyl ester nanozyme obtained in step (1) and chitosan oligosaccharide-coated Bifidobacterium adolescentis obtained in step (2) to sodium alginate solution, stir and mix evenly to obtain liquid I, then add liquid I to liquid paraffin containing 4% Span 80, stir at 200 rpm for 1 h, then add calcium chloride solution dropwise, stir for 1 h, centrifuge and collect the lower precipitate, wash the precipitate with 0.9% sodium chloride solution to obtain calcium alginate microspheres containing chitosan oligosaccharide-coated Bifidobacterium adolescentis and caffeic acid phenylethyl ester nanozyme.

[0036] Example 3:

[0037] The microspheres contain B / COS and NC-B calcium alginate, including *Bifidobacterium longum*, caffeic acid phenylethyl ester nanozyme, and calcium alginate microspheres. The mass ratio of each component in the B / COS and NC-B calcium alginate microspheres is as follows: 437.5 parts cerium nitrate hexahydrate, 175 parts polyvinylpyrrolidone, 1.5 parts nickel chloride hexahydrate, 112.5 parts caffeic acid phenylethyl ester, 45 parts albumin, 750 parts *Bifidobacterium longum*, 80 parts chitosan oligosaccharide, 450 parts sodium alginate, 1800 parts calcium chloride, and 17500 parts ultrapure water 1, 17500 parts ultrapure water 2, and 1 part ultrapure water 3. 500 parts, ultrapure water 4 is 17500 parts, ultrapure water 5 is 11250 parts, ultrapure water 6 is 750 parts, ultrapure water 7 is 17500 parts, ultrapure water 8 is 2000 parts, ultrapure water 9 is 30000 parts, ultrapure water 10 is 22500 parts, phosphate buffer 1 is 750 parts, phosphate buffer 2 is 750 parts. Phosphate buffer 1 and phosphate buffer 2 are the same type of buffer, both with a pH of 7.4.

[0038] Preparation method: (1) Preparation method of caffeic acid phenethyl ester nanozyme: Cerium nitrate hexahydrate and polyvinylpyrrolidone were dissolved in ethylene glycol. Under vigorous stirring, 1M hydrochloric acid aqueous solution was added. After stirring for 30 min, the mixture was heated at 180 ℃ for 2 h. After cooling to room temperature, the precipitate was collected by centrifugation and washed with ultrapure water 1 to obtain solid A. Solid A was dispersed in ultrapure water 2 to form liquid B, and ammonia was added dropwise to adjust the pH to 9 to form liquid C. Nickel chloride hexahydrate was dissolved in ultrapure water 3 to form liquid D. Liquid D was added to liquid C, stirred in a water bath at 70 ℃ for 3 h, the precipitate was collected by centrifugation, washed with ultrapure water 4 and dried to obtain cerium nickel nanozyme. Caffeic acid phenethyl ester was dissolved in methanol to form liquid E. Cerium nickel nanozyme was dispersed in ultrapure water 5 to form liquid F. Albumin was dissolved in ultrapure water 6 to form liquid G. Liquid E was added to liquid F and stirred for 15 h. The precipitate was collected by centrifugation and washed with ultrapure water 7. The precipitate was added to liquid G and placed for 24 h. h, centrifuge to collect the precipitate and obtain caffeic acid phenylethyl ester nanozyme; (2) Preparation method of chitosan oligosaccharide-coated Bifidobacterium longum: dissolve chitosan oligosaccharide in ultrapure water 8 to form liquid H, disperse Bifidobacterium longum in phosphate buffer 1 to form bacterial suspension, adjust the concentration of bacterial suspension to 1.0E+08 CFU / mL, vortex and shake the liquid H and bacterial suspension to mix thoroughly, and place for 40 min, centrifuge and wash, discard the supernatant and disperse the lower precipitate in phosphate buffer 2 to obtain chitosan oligosaccharide-coated Bifidobacterium longum; (3) Preparation method of calcium alginate microspheres containing chitosan oligosaccharide-coated Bifidobacterium longum and caffeic acid phenylethyl ester nanozyme: dissolve sodium alginate in ultrapure water 9 to obtain sodium alginate solution, dissolve calcium chloride in ultrapure water 10 to obtain calcium chloride solution, add caffeic acid phenylethyl ester nanozyme obtained in step (1) and chitosan oligosaccharide-coated Bifidobacterium longum obtained in step (2) to sodium alginate solution, stir and mix evenly to obtain liquid I, then add liquid I to liquid paraffin containing 3% Span 80, stir at 350 rpm for 1 h, then add calcium chloride solution dropwise, stir for 2 h, centrifuge and collect the lower precipitate, wash the precipitate with 0.9% sodium chloride solution to obtain calcium alginate microspheres containing chitosan oligosaccharide-coated Bifidobacterium longum and caffeic acid phenylethyl ester nanozyme.

[0039] Example 4:

[0040] The product contains B / COS and NC-B calcium alginate microspheres, including Bifidobacterium bifidum, caffeic acid phenylethyl ester nanozyme, and calcium alginate microspheres. The mass ratio of each component in the B / COS and NC-B calcium alginate microspheres is as follows: 375 parts cerium nitrate hexahydrate, 150 parts polyvinylpyrrolidone, 2.8 parts nickel chloride hexahydrate, 162.5 parts caffeic acid phenylethyl ester, 90 parts albumin, 2750 parts Bifidobacterium bifidum, 60 parts chitosan oligosaccharide, 375 parts sodium alginate, 1500 parts calcium chloride, 15000 parts ultrapure water 1, 15000 parts ultrapure water 2, and 27 parts ultrapure water 3. 50 parts, ultrapure water 4 is 15000 parts, ultrapure water 5 is 16250 parts, ultrapure water 6 is 1500 parts, ultrapure water 7 is 15000 parts, ultrapure water 8 is 1500 parts, ultrapure water 9 is 25000 parts, ultrapure water 10 is 18750 parts, phosphate buffer 1 is 2750 parts, phosphate buffer 2 is 2750 parts. Phosphate buffer 1 and phosphate buffer 2 are the same type of buffer, both with a pH of 7.4.

[0041] Preparation method: (1) Preparation method of caffeic acid phenethyl ester nanozyme: Cerium nitrate hexahydrate and polyvinylpyrrolidone were dissolved in ethylene glycol. Under vigorous stirring, 1M hydrochloric acid aqueous solution was added. After stirring for 30 min, the mixture was heated at 160 ℃ for 4 h. After cooling to room temperature, the precipitate was collected by centrifugation and washed with ultrapure water 1 to obtain solid A. Solid A was dispersed in ultrapure water 2 to form liquid B, and ammonia was added to adjust the pH to 11 to form liquid C. Nickel chloride hexahydrate was dissolved in ultrapure water 3 to form liquid D. Liquid D was added to liquid C, stirred in a water bath at 90 ℃ for 2 h, the precipitate was collected by centrifugation, washed with ultrapure water 4 and dried to obtain cerium nickel nanozyme. Caffeic acid phenethyl ester was dissolved in methanol to form liquid E. Cerium nickel nanozyme was dispersed in ultrapure water 5 to form liquid F. Albumin was dissolved in ultrapure water 6 to form liquid G. Liquid E was added to liquid F and stirred for 12 h. The precipitate was collected by centrifugation and washed with ultrapure water 7. The precipitate was added to liquid G and placed for 12 h. h, centrifuge to collect the precipitate and obtain caffeic acid phenylethyl ester nanozyme; (2) Preparation method of chitosan oligosaccharide-coated Bifidobacterium bifidum: dissolve chitosan oligosaccharide in ultrapure water 8 to form liquid H, disperse Bifidobacterium bifidum in phosphate buffer 1 to form bacterial suspension, adjust the concentration of bacterial suspension to 1.0E+09 CFU / mL, vortex and shake the liquid H and bacterial suspension to mix thoroughly, and place for 60 min, centrifuge and wash, discard the supernatant and disperse the lower precipitate in phosphate buffer 2 to obtain chitosan oligosaccharide-coated Bifidobacterium bifidum; (3) Preparation method of calcium alginate microspheres containing chitosan oligosaccharide-coated Bifidobacterium bifidum and caffeic acid phenylethyl ester nanozyme: dissolve sodium alginate in ultrapure water 9 to obtain sodium alginate solution, dissolve calcium chloride in ultrapure water 10 to obtain calcium chloride solution, add caffeic acid phenylethyl ester nanozyme obtained in step (1) and chitosan oligosaccharide-coated Bifidobacterium bifidum obtained in step (2) to sodium alginate solution, stir and mix evenly to obtain liquid I, then add liquid I to liquid paraffin containing 3% Span 80, stir at 350 rpm for 1 h, then add calcium chloride solution dropwise, stir for 1 h, centrifuge and collect the lower precipitate, wash the precipitate with 0.9% sodium chloride solution to obtain calcium alginate microspheres containing chitosan oligosaccharide-coated Bifidobacterium bifidum and caffeic acid phenylethyl ester nanozyme.

[0042] Example 5:

[0043] The microspheres contain B / COS and NC-B calcium alginate, including *Bifidobacterium longum*, caffeic acid phenylethyl ester nanozyme, and calcium alginate microspheres. The mass ratio of each component in the B / COS and NC-B calcium alginate microspheres is as follows: 750 parts cerium nitrate hexahydrate, 300 parts polyvinylpyrrolidone, 3.3 parts nickel chloride hexahydrate, 175 parts caffeic acid phenylethyl ester, 83 parts albumin, 1500 parts *Bifidobacterium longum*, 30 parts chitosan oligosaccharide, 187 parts sodium alginate, 750 parts calcium chloride, 30,000 parts ultrapure water 1, 30,000 parts ultrapure water 2, and 325 parts ultrapure water 3. 0 parts, ultrapure water 4 is 30,000 parts, ultrapure water 5 is 17,500 parts, ultrapure water 6 is 1,383 parts, ultrapure water 7 is 30,000 parts, ultrapure water 8 is 750 parts, ultrapure water 9 is 12,467 parts, ultrapure water 10 is 9,375 parts, phosphate buffer 1 is 1,500 parts, phosphate buffer 2 is 1,500 parts. Phosphate buffer 1 and phosphate buffer 2 are the same type of buffer, both with a pH of 7.4.

[0044] Preparation method: (1) Preparation method of caffeic acid phenethyl ester nanozyme: Cerium nitrate hexahydrate and polyvinylpyrrolidone were dissolved in ethylene glycol. Under vigorous stirring, 1M hydrochloric acid aqueous solution was added. After stirring for 30 min, the mixture was heated at 180 ℃ for 3 h. After cooling to room temperature, the precipitate was collected by centrifugation and washed with ultrapure water 1 to obtain solid A. Solid A was dispersed in ultrapure water 2 to form liquid B, and ammonia was added dropwise to adjust the pH to 10 to form liquid C. Nickel chloride hexahydrate was dissolved in ultrapure water 3 to form liquid D. Liquid D was added to liquid C, stirred in a water bath at 50 ℃ for 4 h, the precipitate was collected by centrifugation, washed with ultrapure water 4 and dried to obtain cerium nickel nanozyme. Caffeic acid phenethyl ester was dissolved in methanol to form liquid E. Cerium nickel nanozyme was dispersed in ultrapure water 5 to form liquid F. Albumin was dissolved in ultrapure water 6 to form liquid G. Liquid E was added to liquid F and stirred for 24 h. The precipitate was collected by centrifugation and washed with ultrapure water 7. The precipitate was added to liquid G and placed for 12 h. h, centrifuge to collect the precipitate and obtain caffeic acid phenylethyl ester nanozyme; (2) Preparation method of chitosan oligosaccharide-coated Bifidobacterium longum: dissolve chitosan oligosaccharide in ultrapure water 8 to form liquid H, disperse Bifidobacterium longum in phosphate buffer 1 to form bacterial suspension, adjust the concentration of bacterial suspension to 1.0E+09 CFU / mL, vortex and shake the liquid H and bacterial suspension to mix thoroughly, and place for 30 h. min, centrifuge and wash, discard the supernatant and disperse the lower precipitate in phosphate buffer 2 to obtain chitosan oligosaccharide-coated Bifidobacterium longum; (3) Preparation method of calcium alginate microspheres containing chitosan oligosaccharide-coated Bifidobacterium longum and caffeic acid phenylethyl ester nanozyme: dissolve sodium alginate in ultrapure water 9 to obtain sodium alginate solution, dissolve calcium chloride in ultrapure water 10 to obtain calcium chloride solution, add caffeic acid phenylethyl ester nanozyme obtained in step (1) and chitosan oligosaccharide-coated Bifidobacterium longum obtained in step (2) to sodium alginate solution, stir and mix evenly to obtain liquid I, then add liquid I to liquid paraffin containing 3% Span 80, stir at 150 rpm for 2 h, then add calcium chloride solution dropwise, stir for 2 h, centrifuge and collect the lower precipitate, wash the precipitate with 0.9% sodium chloride solution to obtain calcium alginate microspheres containing chitosan oligosaccharide-coated Bifidobacterium longum and caffeic acid phenylethyl ester nanozyme.

[0045] Example 6:

[0046] The microspheres contain B / COS and NC-B calcium alginate, including *Bifidobacterium adolescentis*, caffeic acid phenylethyl ester nanozyme, and calcium alginate microspheres. The mass ratio of each component in the B / COS and NC-B calcium alginate microspheres is as follows: 687.5 parts cerium nitrate hexahydrate, 275 parts polyvinylpyrrolidone, 2.3 parts nickel chloride hexahydrate, 87.5 parts caffeic acid phenylethyl ester, 53 parts albumin, 1750 parts *Bifidobacterium adolescentis*, 50 parts chitosan oligosaccharide, 487 parts sodium alginate, 1950 parts calcium chloride, and 27,500 parts ultrapure water 1, 27,500 parts ultrapure water 2, and 2... 250 parts, ultrapure water 4 is 27,500 parts, ultrapure water 5 is 8,750 parts, ultrapure water 6 is 883 parts, ultrapure water 7 is 27,500 parts, ultrapure water 8 is 1,250 parts, ultrapure water 9 is 32,467 parts, ultrapure water 10 is 24,375 parts, phosphate buffer 1 is 1,750 parts, phosphate buffer 1 is 1,750 parts, phosphate buffer 1 and phosphate buffer 2 are the same type of buffer, both with pH 7.4.

[0047] Preparation method: (1) Preparation method of caffeic acid phenethyl ester nanozyme: Cerium nitrate hexahydrate and polyvinylpyrrolidone were dissolved in ethylene glycol. Under vigorous stirring, 1M hydrochloric acid aqueous solution was added. After stirring for 30 min, the mixture was heated at 170 ℃ for 3 h. After cooling to room temperature, the precipitate was collected by centrifugation and washed with ultrapure water 1 to obtain solid A. Solid A was dispersed in ultrapure water 2 to form liquid B, and ammonia was added dropwise to adjust the pH to 9 to form liquid C. Nickel chloride hexahydrate was dissolved in ultrapure water 3 to form liquid D. Liquid D was added to liquid C, stirred in a water bath at 70 ℃ for 2 h, the precipitate was collected by centrifugation, washed with ultrapure water 4 and dried to obtain cerium nickel nanozyme. Caffeic acid phenethyl ester was dissolved in methanol to form liquid E. Cerium nickel nanozyme was dispersed in ultrapure water 5 to form liquid F. Albumin was dissolved in ultrapure water 6 to form liquid G. Liquid E was added to liquid F and stirred for 12 h. The precipitate was collected by centrifugation and washed with ultrapure water 7. The precipitate was added to liquid G and placed for 20 h. h, centrifuge to collect the precipitate and obtain caffeic acid phenylethyl ester nanozyme; (2) Preparation method of chitosan oligosaccharide-coated Bifidobacterium adolescentis: dissolve chitosan oligosaccharide in ultrapure water 8 to form liquid H, disperse Bifidobacterium adolescentis in phosphate buffer 1 to form bacterial suspension, adjust the concentration of bacterial suspension to 1.0E+09 CFU / mL, vortex and shake the liquid H and bacterial suspension to mix thoroughly, and place for 50 min, centrifuge and wash, discard the supernatant and disperse the lower precipitate in phosphate buffer 2 to obtain chitosan oligosaccharide-coated Bifidobacterium adolescentis; (3) Preparation method of calcium alginate microspheres containing chitosan oligosaccharide-coated Bifidobacterium adolescentis and caffeic acid phenylethyl ester nanozyme: dissolve sodium alginate in ultrapure water 9 to obtain sodium alginate solution, dissolve calcium chloride in ultrapure water 10 to obtain calcium chloride solution, add caffeic acid phenylethyl ester nanozyme obtained in step (1) and chitosan oligosaccharide-coated Bifidobacterium adolescentis obtained in step (2) to sodium alginate solution, stir and mix evenly to obtain liquid I, then add liquid I to liquid paraffin containing 4% Span 80, stir at 200 rpm for 1 h, then add calcium chloride solution dropwise, stir for 2 h, centrifuge and collect the lower precipitate, wash the precipitate with 0.9% sodium chloride solution to obtain calcium alginate microspheres containing chitosan oligosaccharide-coated Bifidobacterium adolescentis and caffeic acid phenylethyl ester nanozyme.

[0048] Example 7:

[0049] The microspheres contain B / COS and NC-B calcium alginate, including *Bifidobacterium adolescentis*, caffeic acid phenylethyl ester nanozyme, and calcium alginate microspheres. The mass ratio of each component in the B / COS and NC-B calcium alginate microspheres is as follows: 562.5 parts cerium nitrate hexahydrate, 225 parts polyvinylpyrrolidone, 1.3 parts nickel chloride hexahydrate, 125 parts caffeic acid phenylethyl ester, 38 parts albumin, 500 parts *Bifidobacterium adolescentis*, 110 parts chitosan oligosaccharide, 412 parts sodium alginate, 1650 parts calcium chloride, and 22,500 parts ultrapure water 1, 22,500 parts ultrapure water 2, and 1 part ultrapure water 3. 250 parts, ultrapure water 4 is 22,500 parts, ultrapure water 5 is 12,500 parts, ultrapure water 6 is 633 parts, ultrapure water 7 is 22,500 parts, ultrapure water 8 is 2,750 parts, ultrapure water 9 is 27,467 parts, ultrapure water 10 is 20,625 parts, phosphate buffer 1 is 500 parts, phosphate buffer 2 is 500 parts. Phosphate buffer 1 and phosphate buffer 2 are the same type of buffer, both with a pH of 7.4.

[0050] Preparation method: (1) Preparation method of caffeic acid phenethyl ester nanozyme: Cerium nitrate hexahydrate and polyvinylpyrrolidone were dissolved in ethylene glycol. Under vigorous stirring, 1M hydrochloric acid aqueous solution was added. After stirring for 30 min, the mixture was heated at 180 °C for 2 h. After cooling to room temperature, the precipitate was collected by centrifugation and washed with ultrapure water 1 to obtain solid A. Solid A was dispersed in ultrapure water 2 to form liquid B, and ammonia was added dropwise to adjust the pH to 10 to form liquid C. Nickel chloride hexahydrate was dissolved in ultrapure water 3 to form liquid D. Liquid D was added to liquid C, stirred in a water bath at 90 °C for 2 h, the precipitate was collected by centrifugation, washed with ultrapure water 4 and dried to obtain cerium nickel nanozyme. Caffeic acid phenethyl ester was dissolved in methanol to form liquid E. Cerium nickel nanozyme was dispersed in ultrapure water 5 to form liquid F. Albumin was dissolved in ultrapure water 6 to form liquid G. Liquid E was added to liquid F and stirred for 12 h. The precipitate was collected by centrifugation and washed with ultrapure water 7. The precipitate was added to liquid G and placed for 12 h. h, centrifuge to collect the precipitate and obtain caffeic acid phenylethyl ester nanozyme; (2) Preparation method of chitosan oligosaccharide-coated Bifidobacterium adolescentis: dissolve chitosan oligosaccharide in ultrapure water 8 to form liquid H, disperse Bifidobacterium adolescentis in phosphate buffer 1 to form bacterial suspension, adjust the concentration of bacterial suspension to 1.0E+08 CFU / mL, vortex and shake the liquid H and bacterial suspension to mix thoroughly, and place for 30 h. min, centrifuge and wash, discard the supernatant and disperse the lower precipitate in phosphate buffer 2 to obtain chitosan oligosaccharide-coated Bifidobacterium adolescentis; (3) Preparation method of calcium alginate microspheres containing chitosan oligosaccharide-coated Bifidobacterium adolescentis and caffeic acid phenylethyl ester nanozyme: dissolve sodium alginate in ultrapure water 9 to obtain sodium alginate solution, dissolve calcium chloride in ultrapure water 10 to obtain calcium chloride solution, add caffeic acid phenylethyl ester nanozyme obtained in step (1) and chitosan oligosaccharide-coated Bifidobacterium adolescentis obtained in step (2) to sodium alginate solution, stir and mix evenly to obtain liquid I, then add liquid I to liquid paraffin containing 5% Span 80, stir at 350 rpm for 1 h, then add calcium chloride solution dropwise, stir for 2 h, centrifuge and collect the lower precipitate, wash the precipitate with 0.9% sodium chloride solution to obtain calcium alginate microspheres containing chitosan oligosaccharide-coated Bifidobacterium adolescentis and caffeic acid phenylethyl ester nanozyme.

[0051] Example 8:

[0052] The product contains B / COS and NC-B calcium alginate microspheres, including Bifidobacterium bifidum, caffeic acid phenylethyl ester nanozyme, and calcium alginate microspheres. The mass ratio of each component in the B / COS and NC-B calcium alginate microspheres is as follows: cerium nitrate hexahydrate 312.5 parts, polyvinylpyrrolidone 125 parts, nickel chloride hexahydrate 3.5 parts, caffeic acid phenylethyl ester 150 parts, albumin 75 parts, Bifidobacterium bifidum 2250 parts, chitosan oligosaccharide 100 parts, sodium alginate 337 parts, calcium chloride 1350 parts, ultrapure water 1 12500 parts, ultrapure water 2 12500 parts, and ultrapure water 3 35 parts. 00 parts, ultrapure water 4 is 12500 parts, ultrapure water 5 is 15000 parts, ultrapure water 6 is 1250 parts, ultrapure water 7 is 12500 parts, ultrapure water 8 is 2500 parts, ultrapure water 9 is 22467 parts, ultrapure water 10 is 16875 parts, phosphate buffer 1 is 2250 parts, phosphate buffer 2 is 2250 parts. Phosphate buffer 1 and phosphate buffer 2 are the same type of buffer, and both have a pH of 7.4.

[0053] Preparation method: (1) Preparation method of caffeic acid phenethyl ester nanozyme: Cerium nitrate hexahydrate and polyvinylpyrrolidone were dissolved in ethylene glycol. Under vigorous stirring, 1M hydrochloric acid aqueous solution was added. After stirring for 30 min, the mixture was heated at 170 °C for 3 h. After cooling to room temperature, the precipitate was collected by centrifugation and washed with ultrapure water 1 to obtain solid A. Solid A was dispersed in ultrapure water 2 to form liquid B, and ammonia was added to adjust the pH to 9 to form liquid C. Nickel chloride hexahydrate was dissolved in ultrapure water 3 to form liquid D. Liquid D was added to liquid C, stirred in a water bath at 70 °C for 2 h, the precipitate was collected by centrifugation, washed with ultrapure water 4 and dried to obtain cerium nickel nanozyme. Caffeic acid phenethyl ester was dissolved in methanol to form liquid E. Cerium nickel nanozyme was dispersed in ultrapure water 5 to form liquid F. Albumin was dissolved in ultrapure water 6 to form liquid G. Liquid E was added to liquid F and stirred for 24 h. The precipitate was collected by centrifugation and washed with ultrapure water 7. The precipitate was added to liquid G and placed for 12 h. h, centrifuge to collect the precipitate and obtain caffeic acid phenylethyl ester nanozyme; (2) Preparation method of chitosan oligosaccharide-coated Bifidobacterium bifidum: dissolve chitosan oligosaccharide in ultrapure water 8 to form liquid H, disperse Bifidobacterium bifidum in phosphate buffer 1 to form bacterial suspension, adjust the concentration of bacterial suspension to 1.0E+09 CFU / mL, vortex and shake the liquid H and bacterial suspension to mix thoroughly, and place for 50 min, centrifuge and wash, discard the supernatant and disperse the lower precipitate in phosphate buffer 2 to obtain chitosan oligosaccharide-coated Bifidobacterium bifidum; (3) Preparation method of calcium alginate microspheres containing chitosan oligosaccharide-coated Bifidobacterium bifidum and caffeic acid phenylethyl ester nanozyme: dissolve sodium alginate in ultrapure water 9 to obtain sodium alginate solution, dissolve calcium chloride in ultrapure water 10 to obtain calcium chloride solution, add caffeic acid phenylethyl ester nanozyme obtained in step (1) and chitosan oligosaccharide-coated Bifidobacterium bifidum obtained in step (2) to sodium alginate solution, stir and mix evenly to obtain liquid I, then add liquid I to liquid paraffin containing 3% Span 80, stir at 150 rpm for 2 h, then add calcium chloride solution dropwise, stir for 1 h, centrifuge and collect the lower precipitate, wash the precipitate with 0.9% sodium chloride solution to obtain calcium alginate microspheres containing chitosan oligosaccharide-coated Bifidobacterium bifidum and caffeic acid phenylethyl ester nanozyme.

[0054] Example 9:

[0055] The product contains B / COS and NC-B calcium alginate microspheres, including Bifidobacterium bifidum, caffeic acid phenylethyl ester nanozyme, and calcium alginate microspheres. The mass ratio of each component in the B / COS and NC-B calcium alginate microspheres is as follows: 500 parts cerium nitrate hexahydrate, 200 parts polyvinylpyrrolidone, 2 parts nickel chloride hexahydrate, 100 parts caffeic acid phenylethyl ester, 60 parts albumin, 1000 parts Bifidobacterium bifidum, 40 parts chitosan oligosaccharide, 300 parts sodium alginate, 1200 parts calcium chloride, and 20,000 parts ultrapure water 1, 20,000 parts ultrapure water 2, and 2000 parts ultrapure water 3. The buffer solutions are as follows: 4 is 20,000 parts ultrapure water, 5 is 10,000 parts ultrapure water, 6 is 1,000 parts ultrapure water, 7 is 20,000 parts ultrapure water, 8 is 1,000 parts ultrapure water, 9 is 20,000 parts ultrapure water, 10 is 15,000 parts ultrapure water, 1 is 1,000 parts phosphate buffer 1, and 2 is 1,000 parts phosphate buffer 2. 1 and 2 phosphate buffers are the same type of buffer solution with a pH of 7.4.

[0056] Preparation method: (1) Preparation method of caffeic acid phenethyl ester nanozyme: Cerium nitrate hexahydrate and polyvinylpyrrolidone were dissolved in ethylene glycol. Under vigorous stirring, 1M hydrochloric acid aqueous solution was added. After stirring for 30 min, the mixture was heated at 160 ℃ for 3 h. After cooling to room temperature, the precipitate was collected by centrifugation and washed with ultrapure water 1 to obtain solid A. Solid A was dispersed in ultrapure water 2 to form liquid B, and ammonia was added dropwise to adjust the pH to 10 to form liquid C. Nickel chloride hexahydrate was dissolved in ultrapure water 3 to form liquid D. Liquid D was added to liquid C, stirred in a water bath at 70 ℃ for 3 h, the precipitate was collected by centrifugation, washed with ultrapure water 4 and dried to obtain cerium nickel nanozyme. Caffeic acid phenethyl ester was dissolved in methanol to form liquid E. Cerium nickel nanozyme was dispersed in ultrapure water 5 to form liquid F. Albumin was dissolved in ultrapure water 6 to form liquid G. Liquid E was added to liquid F and stirred for 24 h. The precipitate was collected by centrifugation and washed with ultrapure water 7. The precipitate was added to liquid G and placed for 24 h. h, centrifuge to collect the precipitate and obtain caffeic acid phenylethyl ester nanozyme; (2) Preparation method of chitosan oligosaccharide-coated Bifidobacterium bifidum: dissolve chitosan oligosaccharide in ultrapure water 8 to form liquid H, disperse Bifidobacterium bifidum in phosphate buffer 1 to form bacterial suspension, adjust the concentration of bacterial suspension to 1.0E+08 CFU / mL, vortex and shake the liquid H and bacterial suspension to mix thoroughly, and place for 30 min, centrifuge and wash, discard the supernatant and disperse the lower precipitate in phosphate buffer 2 to obtain chitosan oligosaccharide-coated Bifidobacterium bifidum; (3) Preparation method of calcium alginate microspheres containing chitosan oligosaccharide-coated Bifidobacterium bifidum and caffeic acid phenylethyl ester nanozyme: dissolve sodium alginate in ultrapure water 9 to obtain sodium alginate solution, dissolve calcium chloride in ultrapure water 10 to obtain calcium chloride solution, add caffeic acid phenylethyl ester nanozyme obtained in step (1) and chitosan oligosaccharide-coated Bifidobacterium bifidum obtained in step (2) to sodium alginate solution, stir and mix evenly to obtain liquid I, then add liquid I to liquid paraffin containing 5% Span 80, stir at 150 rpm for 1 h, then add calcium chloride solution dropwise, stir for 2 h, centrifuge and collect the lower precipitate, wash the precipitate with 0.9% sodium chloride solution to obtain calcium alginate microspheres containing chitosan oligosaccharide-coated Bifidobacterium bifidum and caffeic acid phenylethyl ester nanozyme.

[0057] Example 10:

[0058] The product contains B / COS and NC-B calcium alginate microspheres, including *Bifidobacterium adolescentis*, caffeic acid phenylethyl ester nanozyme, and calcium alginate microspheres. The mass ratio of each component in the B / COS and NC-B calcium alginate microspheres is as follows: 812.5 parts cerium nitrate hexahydrate, 325 parts polyvinylpyrrolidone, 2.5 parts nickel chloride hexahydrate, 75 parts caffeic acid phenylethyl ester, 98 parts albumin, 2500 parts *Bifidobacterium adolescentis*, 90 parts chitosan oligosaccharide, 262 parts sodium alginate, 1050 parts calcium chloride, and 32500 parts ultrapure water 1, 32500 parts ultrapure water 2, and 25 parts ultrapure water 3. 00 parts, ultrapure water 4 is 32500 parts, ultrapure water 5 is 7500 parts, ultrapure water 6 is 1633 parts, ultrapure water 7 is 32500 parts, ultrapure water 8 is 2250 parts, ultrapure water 9 is 17467 parts, ultrapure water 10 is 13125 parts, phosphate buffer 1 is 2500 parts, phosphate buffer 2 is 2500 parts. Phosphate buffer 1 and phosphate buffer 2 are the same type of buffer, both with a pH of 7.4.

[0059] Preparation method: (1) Preparation method of caffeic acid phenethyl ester nanozyme: Cerium nitrate hexahydrate and polyvinylpyrrolidone were dissolved in ethylene glycol. Under vigorous stirring, 1M hydrochloric acid aqueous solution was added. After stirring for 30 min, the mixture was heated at 170 °C for 3 h. After cooling to room temperature, the precipitate was collected by centrifugation and washed with ultrapure water 1 to obtain solid A. Solid A was dispersed in ultrapure water 2 to form liquid B, and ammonia was added dropwise to adjust the pH to 10 to form liquid C. Nickel chloride hexahydrate was dissolved in ultrapure water 3 to form liquid D. Liquid D was added to liquid C, stirred in a water bath at 50 °C for 2 h, the precipitate was collected by centrifugation, washed with ultrapure water 4 and dried to obtain cerium nickel nanozyme. Caffeic acid phenethyl ester was dissolved in methanol to form liquid E. Cerium nickel nanozyme was dispersed in ultrapure water 5 to form liquid F. Albumin was dissolved in ultrapure water 6 to form liquid G. Liquid E was added to liquid F and stirred for 12 h. The precipitate was collected by centrifugation and washed with ultrapure water 7. The precipitate was added to liquid G and placed for 24 h. h, centrifuge to collect the precipitate and obtain caffeic acid phenylethyl ester nanozyme; (2) Preparation method of chitosan oligosaccharide-coated Bifidobacterium adolescentis: dissolve chitosan oligosaccharide in ultrapure water 8 to form liquid H, disperse Bifidobacterium adolescentis in phosphate buffer 1 to form bacterial suspension, adjust the concentration of bacterial suspension to 1.0E+08 CFU / mL, vortex and shake the liquid H and bacterial suspension to mix thoroughly, and place for 50 min, centrifuge and wash, discard the supernatant and disperse the lower precipitate in phosphate buffer 2 to obtain chitosan oligosaccharide-coated Bifidobacterium adolescentis; (3) Preparation method of calcium alginate microspheres containing chitosan oligosaccharide-coated Bifidobacterium adolescentis and caffeic acid phenylethyl ester nanozyme: dissolve sodium alginate in ultrapure water 9 to obtain sodium alginate solution, dissolve calcium chloride in ultrapure water 10 to obtain calcium chloride solution, add caffeic acid phenylethyl ester nanozyme obtained in step (1) and chitosan oligosaccharide-coated Bifidobacterium adolescentis obtained in step (2) to sodium alginate solution, stir and mix evenly to obtain liquid I, then add liquid I to liquid paraffin containing 4% Span 80, stir at 150 rpm for 2 h, then add calcium chloride solution dropwise, stir for 2 h, centrifuge and collect the lower precipitate, wash the precipitate with 0.9% sodium chloride solution to obtain calcium alginate microspheres containing chitosan oligosaccharide-coated Bifidobacterium adolescentis and caffeic acid phenylethyl ester nanozyme.

[0060] Example 11:

[0061] The product contains B / COS and NC-B calcium alginate microspheres, including *Bifidobacterium adolescentis*, caffeic acid phenylethyl ester nanozyme, and calcium alginate microspheres. The mass ratio of each component in the B / COS and NC-B calcium alginate microspheres is as follows: 875 parts cerium nitrate hexahydrate, 350 parts polyvinylpyrrolidone, 3 parts nickel chloride hexahydrate, 62.5 parts caffeic acid phenylethyl ester, 105 parts albumin, 2000 parts *Bifidobacterium adolescentis*, 70 parts chitosan oligosaccharide, 225 parts sodium alginate, 900 parts calcium chloride, and 35,000 parts ultrapure water (1, 2, and 300 parts respectively). 0 parts, ultrapure water 4 is 35000 parts, ultrapure water 5 is 6250 parts, ultrapure water 6 is 1750 parts, ultrapure water 7 is 35000 parts, ultrapure water 8 is 1750 parts, ultrapure water 9 is 15000 parts, ultrapure water 10 is 11250 parts, phosphate buffer 1 is 2000 parts, phosphate buffer 2 is 2000 parts. Phosphate buffer 1 and phosphate buffer 2 are the same type of buffer, both with a pH of 7.4.

[0062] Preparation method: (1) Preparation method of caffeic acid phenethyl ester nanozyme: Cerium nitrate hexahydrate and polyvinylpyrrolidone were dissolved in ethylene glycol. Under vigorous stirring, 1M hydrochloric acid aqueous solution was added. After stirring for 30 min, the mixture was heated at 160 ℃ for 5 h. After cooling to room temperature, the precipitate was collected by centrifugation and washed with ultrapure water 1 to obtain solid A. Solid A was dispersed in ultrapure water 2 to form liquid B, and ammonia was added dropwise to adjust the pH to 11 to form liquid C. Nickel chloride hexahydrate was dissolved in ultrapure water 3 to form liquid D. Liquid D was added to liquid C, stirred in a water bath at 90 ℃ for 2 h, the precipitate was collected by centrifugation, washed with ultrapure water 4 and dried to obtain cerium nickel nanozyme. Caffeic acid phenethyl ester was dissolved in methanol to form liquid E. Cerium nickel nanozyme was dispersed in ultrapure water 5 to form liquid F. Albumin was dissolved in ultrapure water 6 to form liquid G. Liquid E was added to liquid F and stirred for 24 h. The precipitate was collected by centrifugation and washed with ultrapure water 7. The precipitate was added to liquid G and placed for 12 h. h, centrifuge to collect the precipitate and obtain caffeic acid phenylethyl ester nanozyme; (2) Preparation method of chitosan oligosaccharide-coated Bifidobacterium adolescentis: dissolve chitosan oligosaccharide in ultrapure water 8 to form liquid H, disperse Bifidobacterium adolescentis in phosphate buffer 1 to form bacterial suspension, adjust the concentration of bacterial suspension to 1.0E+09 CFU / mL, vortex and shake the liquid H and bacterial suspension to mix thoroughly, and place for 60 min, centrifuge and wash, discard the supernatant and disperse the lower precipitate in phosphate buffer 2 to obtain chitosan oligosaccharide-coated Bifidobacterium adolescentis; (3) Preparation method of calcium alginate microspheres containing chitosan oligosaccharide-coated Bifidobacterium adolescentis and caffeic acid phenylethyl ester nanozyme: dissolve sodium alginate in ultrapure water 9 to obtain sodium alginate solution, dissolve calcium chloride in ultrapure water 10 to obtain calcium chloride solution, add caffeic acid phenylethyl ester nanozyme obtained in step (1) and chitosan oligosaccharide-coated Bifidobacterium adolescentis obtained in step (2) to sodium alginate solution, stir and mix evenly to obtain liquid I, then add liquid I to liquid paraffin containing 4% Span 80, stir at 300 rpm for 1 h, then add calcium chloride solution dropwise, stir for 1 h, centrifuge and collect the lower precipitate, wash the precipitate with 0.9% sodium chloride solution to obtain calcium alginate microspheres containing chitosan oligosaccharide-coated Bifidobacterium adolescentis and caffeic acid phenylethyl ester nanozyme.

[0063] Example 12:

[0064] The microspheres contain B / COS and NC-B calcium alginate, including *Bifidobacterium longum*, caffeic acid phenylethyl ester nanozyme, and calcium alginate microspheres. The mass ratio of each component in the B / COS and NC-B calcium alginate microspheres is as follows: 1000 parts cerium nitrate hexahydrate, 400 parts polyvinylpyrrolidone, 4 parts nickel chloride hexahydrate, 200 parts caffeic acid phenylethyl ester, 120 parts albumin, 3000 parts *Bifidobacterium longum*, 120 parts chitosan oligosaccharide, 600 parts sodium alginate, 2400 parts calcium chloride, and 40000 parts ultrapure water (1, 2, and 3). 0 parts, ultrapure water 4 is 40,000 parts, ultrapure water 5 is 20,000 parts, ultrapure water 6 is 2,000 parts, ultrapure water 7 is 40,000 parts, ultrapure water 8 is 3,000 parts, ultrapure water 9 is 40,000 parts, ultrapure water 10 is 30,000 parts, phosphate buffer 1 is 3,000 parts, phosphate buffer 2 is 3,000 parts. Phosphate buffer 1 and phosphate buffer 2 are the same type of buffer, both with a pH of 7.4.

[0065] Preparation method: (1) Preparation method of caffeic acid phenethyl ester nanozyme: Cerium nitrate hexahydrate and polyvinylpyrrolidone were dissolved in ethylene glycol. Under vigorous stirring, 1M hydrochloric acid aqueous solution was added. After stirring for 30 min, the mixture was heated at 180 °C for 3 h. After cooling to room temperature, the precipitate was collected by centrifugation and washed with ultrapure water 1 to obtain solid A. Solid A was dispersed in ultrapure water 2 to form liquid B, and ammonia was added dropwise to adjust the pH to 10 to form liquid C. Nickel chloride hexahydrate was dissolved in ultrapure water 3 to form liquid D. Liquid D was added to liquid C, stirred in a water bath at 70 °C for 2 h, the precipitate was collected by centrifugation, washed with ultrapure water 4 and dried to obtain cerium nickel nanozyme. Caffeic acid phenethyl ester was dissolved in methanol to form liquid E. Cerium nickel nanozyme was dispersed in ultrapure water 5 to form liquid F. Albumin was dissolved in ultrapure water 6 to form liquid G. Liquid E was added to liquid F and stirred for 24 h. The precipitate was collected by centrifugation and washed with ultrapure water 7. The precipitate was added to liquid G and placed for 24 h. h, centrifuge to collect the precipitate and obtain caffeic acid phenylethyl ester nanozyme; (2) Preparation method of chitosan oligosaccharide-coated Bifidobacterium longum: dissolve chitosan oligosaccharide in ultrapure water 8 to form liquid H, disperse Bifidobacterium longum in phosphate buffer 1 to form bacterial suspension, adjust the concentration of bacterial suspension to 1.0E+08 CFU / mL, vortex and shake the liquid H and bacterial suspension to mix thoroughly, and place for 30 h. min, centrifuge and wash, discard the supernatant and disperse the lower precipitate in phosphate buffer 2 to obtain chitosan oligosaccharide-coated Bifidobacterium longum; (3) Preparation method of calcium alginate microspheres containing chitosan oligosaccharide-coated Bifidobacterium longum and caffeic acid phenylethyl ester nanozyme: dissolve sodium alginate in ultrapure water 9 to obtain sodium alginate solution, dissolve calcium chloride in ultrapure water 10 to obtain calcium chloride solution, add caffeic acid phenylethyl ester nanozyme obtained in step (1) and chitosan oligosaccharide-coated Bifidobacterium longum obtained in step (2) to sodium alginate solution, stir and mix evenly to obtain liquid I, then add liquid I to liquid paraffin containing 1% Span 80, stir at 150 rpm for 1 h, then add calcium chloride solution dropwise, stir for 2 h, centrifuge and collect the lower precipitate, wash the precipitate with 0.9% sodium chloride solution to obtain calcium alginate microspheres containing chitosan oligosaccharide-coated Bifidobacterium longum and caffeic acid phenylethyl ester nanozyme.

Claims

1. A calcium alginate microsphere containing chitosan oligosaccharide-coated Bifidobacterium and caffeic acid phenylethyl ester nanozyme, characterized in that, The invention comprises chitosan oligosaccharide-coated Bifidobacterium, caffeic acid phenylethyl ester nanozyme, and calcium alginate microspheres, wherein the chitosan oligosaccharide-coated Bifidobacterium and the caffeic acid phenylethyl ester nanozyme are carried in the calcium alginate microspheres; the caffeic acid phenylethyl ester nanozyme comprises caffeic acid phenylethyl ester and cerium nickel nanozyme, wherein caffeic acid phenylethyl ester is carried in the cerium nickel nanozyme. The calcium alginate microspheres containing chitosan oligosaccharide-coated Bifidobacteria and caffeic acid phenylethyl ester nanozyme comprise the following components, wherein phosphate buffer 1 and phosphate buffer 2 are the same type of buffer, both with a pH of 7.4, and the mass ratio of each component is as follows: The aforementioned Bifidobacteria include one of Bifidobacterium adolescentis, Bifidobacterium bifidum, and Bifidobacterium longum. The preparation method of calcium alginate microspheres containing chitosan oligosaccharide-coated Bifidobacterium and caffeic acid phenylethyl ester nanozyme includes the following steps: (1) Preparation method of caffeic acid phenylethyl ester nanozyme: Cerium nitrate hexahydrate and polyvinylpyrrolidone are dissolved in ethylene glycol. Under vigorous stirring, 1M hydrochloric acid aqueous solution is added. After stirring for 30-60 min, the mixture is heated at 160-180 ℃ for 2-5 h. After cooling to room temperature, the precipitate is collected by centrifugation and washed with ultrapure water 1 to obtain solid A; solid A is dispersed in ultrapure water 2 to form liquid B, and ammonia water is added dropwise to adjust the pH to 9-11 to form liquid C; nickel chloride hexahydrate is dissolved in ultrapure water 3 to form liquid D; liquid D is added to liquid C, and the mixture is stirred in a water bath at 50-90 ℃ for 2-5 h. h, centrifuge to collect the precipitate, wash with ultrapure water 4 and dry to obtain cerium nickel nanozyme; dissolve caffeic acid phenethyl ester in methanol to form liquid E, disperse cerium nickel nanozyme in ultrapure water 5 to form liquid F, dissolve albumin in ultrapure water 6 to form liquid G, add liquid E to liquid F and stir for 12-24 h, centrifuge to collect the precipitate and wash with ultrapure water 7, add to liquid G and place for 12-24 h, centrifuge to collect the precipitate and obtain caffeic acid phenethyl ester loaded nanozyme; (2) Preparation method of chitosan oligosaccharide-coated Bifidobacterium: dissolve chitosan oligosaccharide in ultrapure water 8 to form liquid H, disperse Bifidobacterium in phosphate buffer 1 to form bacterial suspension, adjust the bacterial suspension concentration to 1.0E+08~1.0E+09 CFU / mL, vortex and shake liquid H and bacterial suspension to mix thoroughly, and place for 30-60 h. min, centrifuge and wash, discard the supernatant and disperse the lower precipitate in phosphate buffer 2 to obtain chitosan oligosaccharide-coated bifidobacteria; (3) Preparation method of calcium alginate microspheres containing chitosan oligosaccharide-coated bifidobacteria and caffeic acid phenylethyl ester nanozyme: dissolve sodium alginate in ultrapure water 9 to obtain sodium alginate solution, dissolve calcium chloride in ultrapure water 10 to obtain calcium chloride solution, add caffeic acid phenylethyl ester nanozyme obtained in step (1) and chitosan oligosaccharide-coated bifidobacteria obtained in step (2) to sodium alginate solution, stir and mix evenly to obtain liquid I, then add liquid I to liquid paraffin containing 1%-5% Span 80, stir at 150-350 rpm for 1-2 h, then add calcium chloride solution dropwise, stir for 1-2 h, centrifuge and collect the lower precipitate, wash the precipitate with 0.9% sodium chloride solution to obtain calcium alginate microspheres containing chitosan oligosaccharide-coated bifidobacteria and caffeic acid phenylethyl ester nanozyme.

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