Lactobacillus paracasei and its application in probiotic delivery system for reducing blood pressure

Abstract

The present application belongs to the technical field of probiotic preparation, and particularly relates to a lactobacillus paracasei and application of the lactobacillus paracasei in a probiotic delivery system for reducing blood pressure. The lactobacillus paracasei provided by the present application is screened from traditional fermented dairy products in Tibet, and is verified to have good ability of reducing high blood pressure. The probiotic delivery system constructed by the present application utilizes the strain, and through oxidation modification of astragalus polysaccharide, the oxidized astragalus polysaccharide can form a gel with carboxymethyl chitosan having an amino group through a Schiff base reaction, and form a compact microsphere structure of a cross-gel network in a sodium alginate-metal ion hydrogel system, so as to realize embedding of the lactobacillus paracasei and improve the probiotic delivery efficiency. Meanwhile, the prebiotic characteristics of the astragalus polysaccharide can effectively enhance the functional characteristics of the probiotic in reducing high blood pressure. Therefore, the present application can provide a new idea and method for microbial treatment of high blood pressure, and provide a theoretical basis for development of medicines or health products for reducing high blood pressure.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of probiotic preparations, and particularly relates to a lactobacillus paracasei and application thereof in a probiotic delivery system for reducing blood pressure. BACKGROUND

[0002] In recent years, as the most common chronic disease, hypertension seriously affects the physical health and quality of life of patients, and has become an important public health problem worldwide. The pathogenesis of hypertension is very complex and is regulated by multiple factors. Endothelial dysfunction (ED) is one of the widely recognized pathogenesis of hypertension. Although there are currently many drugs for the treatment of hypertension, such as amlodipine besylate tablets, valsartan capsules, etc., long-term use of these drugs will have adverse effects on the body. Therefore, it is crucial to seek a safer and more effective treatment method for the treatment of hypertension.

[0003] Probiotics are beneficial active microorganisms that can colonize in the human intestinal tract and change the composition of the flora in a specific part of the host. Existing studies have found that some probiotics can relieve hypertension by regulating vascular oxidative stress, restoring endothelial cell function, and remodeling the intestinal microenvironment. Therefore, probiotics are expected to bring new treatment options and drug choices for the treatment of hypertension. However, due to the complexity of the gastrointestinal environment in the human body, the activity of probiotics is easily affected, and it is difficult for them to fully play a role in the body. In recent years, microcapsules, hydrogels and other embedding carriers have emerged as effective ways to protect the activity of probiotics and achieve probiotic delivery. However, for the treatment of hypertension, the existing probiotic delivery system still has the problems of low delivery efficiency, poor functional characteristics of probiotics, and difficulty in effective application to hypertension.

[0004] Therefore, how to construct a new probiotic delivery system that can embed probiotics, effectively improve the delivery efficiency and functional characteristics of probiotics, and achieve the purpose of significantly improving the treatment of hypertension has become a technical problem that needs to be solved in the field. SUMMARY

[0005] In order to overcome the deficiencies of the prior art, the purpose of the present application is to provide a lactobacillus paracasei and application thereof in a probiotic delivery system for reducing blood pressure. The lactobacillus paracasei provided by the present application is screened from traditional fermented dairy products in Tibet, and it has been verified that it has good ability to reduce blood pressure. The probiotic delivery system constructed by the present application using the strain can embed probiotics and improve the delivery efficiency of probiotics, and at the same time, the prebiotic properties of astragalus polysaccharide can effectively enhance the functional characteristics of probiotics in reducing blood pressure.

[0006] In order to achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0007] A Lactobacillus paracasei, the Lactobacillus paracasei is named Lactobacillus paracasei (Lactobacillus paracasei) Lacticaseibacillus paracasei ) M11-7, the preservation unit is Guangdong Microbial Culture Collection Center, the preservation number is GDMCC NO: 66511, and the preservation date is June 13, 2025.

[0008] The above-mentioned Lactobacillus paracasei is applied to the preparation of a product for preventing and treating hypertension.

[0009] A probiotic delivery system for reducing blood pressure, a preparation method of the probiotic delivery system for reducing blood pressure, comprising the following steps:

[0010] (1) mixing an oxidized astragalus polysaccharide solution, a carboxymethyl chitosan solution and a sodium alginate solution to obtain a ternary mixed solution;

[0011] (2) mixing a bacterial suspension of Lactobacillus paracasei and the ternary mixed solution to obtain a probiotic suspension; wherein the bacterial suspension of Lactobacillus paracasei adopts Lactobacillus paracasei (Lactobacillus paracasei) Lacticaseibacillus paracasei ) M11-7, the preservation unit is Guangdong Microbial Culture Collection Center, the preservation number is GDMCC NO: 66511, and the preservation date is June 13, 2025;

[0012] (3) adding the probiotic suspension into a metal ion solution to form a gel ball, and then solidifying to obtain the probiotic delivery system for reducing blood pressure.

[0013] Preferably, in step (1), the mass concentration of the oxidized astragalus polysaccharide solution is 0.6%~1.0% w / v; the mass concentration of the carboxymethyl chitosan solution is 0.4%~0.6% w / v; and the concentration of the sodium alginate solution is 1.5%~2.5% w / v.

[0014] Preferably, in step (1), the oxidized astragalus polysaccharide used in the oxidized astragalus polysaccharide solution is prepared by an oxidation reaction with astragalus polysaccharide and sodium periodate as raw materials; and the mass ratio of the astragalus polysaccharide and the sodium periodate is 5:(2~3).

[0015] Preferably, in step (2), the effective bacteria concentration in the bacterial suspension of Lactobacillus paracasei is 10 10 ~10 11 CFU / mL; the bacterial suspension of Lactobacillus paracasei is prepared by inoculating Lactobacillus paracasei in MRS liquid medium and incubating at 36~38℃ for 15~36h.

[0016] Preferably, in step (2), the volume ratio of the Lactobacillus paracasei bacterial suspension and the ternary mixture is 1: (8-12), more preferably 1:9.

[0017] Preferably, in step (3), the metal ion solution is one of ZnCl2 solution, CaCl2 solution and FeCl3 solution; and the concentration of the metal ion solution is 1.0%-3.0% w / v.

[0018] Preferably, in step (3), the solidification time is 30-90 min.

[0019] The application of the blood pressure-lowering probiotic delivery system is the application of the blood pressure-lowering probiotic delivery system in the preparation of a product for preventing and treating hypertension; and the product is a drug.

[0020] The technical solution of the present application has the following comprehensive advantages and beneficial effects:

[0021] The Lactobacillus paracasei provided by the present application is screened from traditional fermented dairy products in Tibet and has been verified to have good blood pressure-lowering ability. The probiotic delivery system constructed by the strain is used to oxidize and modify astragalus polysaccharide, so that the oxidized astragalus polysaccharide can quickly form a gel with carboxymethyl chitosan having an amino group through a Schiff base reaction and form a dense microsphere structure of a cross-gel network in a sodium alginate-metal ion hydrogel system, thereby embedding the Lactobacillus paracasei and improving the probiotic delivery efficiency. Meanwhile, the astragalus polysaccharide can enhance the functional properties and effects of the probiotic in lowering blood pressure.

[0022] Meanwhile, the stability and biocompatibility of the probiotic delivery system are verified through in-vitro resistance and safety experiments, a spontaneous hypertension rat model is constructed, the effects of the probiotic delivery system in lowering blood pressure are revealed, and the molecular mechanism of the alleviation of vascular endothelial dysfunction is analyzed. Therefore, the present application can provide new ideas and methods for microbial treatment of hypertension and provide a theoretical basis for the development of corresponding drugs for preventing and treating hypertension. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 is a total peak area graph of short-chain fatty acids produced by different strains in the present application;

[0024] Figure 2 is a hydrogen peroxide tolerance determination result of different strains in the present application;

[0025] Figure 3 is an antioxidant capacity determination result of different strains in the present application;

[0026] Figure 4Growth curves of L. paracasei M11-7 in different pH and at different time points;

[0027] Figure 5 Growth curves of L. paracasei M11-7 in different culture media;

[0028] Figure 6 Effect of oxidized polysaccharide concentration and metal ion on the swelling rate of hydrogel microspheres;

[0029] Figure 7 Effect of sodium alginate concentration, carboxymethyl chitosan mass fraction, zinc chloride mass fraction and fixing time on the swelling rate of hydrogel microspheres;

[0030] Figure 8 Appearance and morphology of Alg hydrogel microspheres, Alg-OGP hydrogel microspheres and Alg-OGP-CMC hydrogel microspheres;

[0031] Figure 9 Infrared spectra of OGP raw material, Alg raw material, CMC raw material and Alg-OGP-CMC hydrogel microspheres;

[0032] Figure 10 SEM morphology of Alg hydrogel microspheres, Alg-OGP hydrogel microspheres and Alg-OGP-CMC hydrogel microspheres;

[0033] Figure 11 Effect of bile salt treatment on the activity of Alg hydrogel microspheres, Alg-OGP hydrogel microspheres and Alg-OGP-CMC hydrogel microspheres;

[0034] Figure 12 Effect of simulated gastric fluid treatment and simulated intestinal fluid treatment on the activity of Alg hydrogel microspheres, Alg-OGP hydrogel microspheres and Alg-OGP-CMC hydrogel microspheres;

[0035] Figure 13 Effect of storage conditions on the survival rate of Alg hydrogel microspheres, Alg-OGP hydrogel microspheres and Alg-OGP-CMC hydrogel microspheres;

[0036] Figure 14 Effect of Alg-OGP-CMC hydrogel microspheres on the activity of Caco-2 cells;

[0037] Figure 15 Change of blood pressure of hypertensive rats in the application;

[0038] Figure 16 Change of ROS content in abdominal aorta of hypertensive rats in the application;

[0039] Figure 17 The results of the contraction factor ET-1, NO and the antioxidant enzymes SOD and T-AOC of the hypertensive rats;

[0040] Figure 18 The PI3K / Akt signaling pathway gene expression amount in the application;

[0041] Figure 19 The PI3K-Akt pathway related protein expression amount in the application;

[0042] Figure 20 The influence of the hydrogel microspheres on the intestinal flora abundance of the rats in the application;

[0043] Figure 21 The influence of the hydrogel microspheres on the intestinal SCFAs content of the rats in the application. DETAILED DESCRIPTION

[0044] In order to make the purpose, technical scheme and advantages of the application more clear, the application will be clearly and completely explained by combining with specific examples.

[0045] The biological preservation information involved in the following embodiments of the application is as follows:

[0046] Preservation name: Lactobacillus paracasei M11-7 (Lactobacillus paracasei M11-7) Lacticaseibacillus paracasei ); preservation number: GDMCC NO: 66511; preservation unit: Guangdong Microbial Culture Collection Center; preservation address: 5th floor, Building 59, 100 Middle Martyrs Road, Guangzhou; preservation date: June 13, 2025.

[0047] In the embodiments of the application, the sodium alginate and carboxymethyl chitosan come from Aladdin Reagent (Shanghai) Co., Ltd.; the astragalus polysaccharide comes from Shaanxi Yinao Biological Technology Co., Ltd.; the MRS broth medium and the sugar-free MRS broth medium come from Qingdao Haibo Biological Technology Co., Ltd. The experimental animals used in the application are spontaneous hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats used for normal blood pressure control, which are purchased from Beijing Vito Lihua Co., Ltd. Human colorectal adenocarcinoma cells (Caco-2) are purchased from Shanghai Genomed Biotech Co., Ltd. Other raw materials not mentioned are conventional materials that can be obtained through market channels.

[0048] Example 1

[0049] This example provides a Lactobacillus paracasei M11-7 (Lactobacillus paracasei M11-7) Lacticaseibacillus paracasei ); preservation number: GDMCC NO: 66511; preservation unit: Guangdong Microbial Culture Collection Center; preservation date: June 13, 2025.

[0050] The isolation and identification process of Lactobacillus paracasei M11-7 involved in this embodiment is as follows: Traditional fermented dairy products from Tibet were used as the isolation sample. The sample was placed in a sterile operating table, and sterile physiological saline was added at a ratio of 1:10 (v / v). The mixture was thoroughly shaken to prepare a suspension, which was then serially diluted (10⁻⁶ oz / v). -1 Up to 10 -6 Take 0.1 mL of suspension at each dilution and spread it onto MRS solid agar plates, with three replicates for each dilution. After spreading, place the plates in an anaerobic incubator at 37°C and incubate for 48-72 h, observing colony growth. Once colonies have grown, select single colonies of different morphological characteristics (such as size, shape, color, edge regularity, and surface smoothness). Pick a single colony with an inoculation loop and inoculate it into MRS liquid agar, incubate anaerobically at 37°C for 24 h, then streak it onto MRS solid agar. Repeat the purification process 3-4 times to obtain pure cultures. Subsequently, evaluate the beneficial properties of the selected strains, including acid tolerance and bile salt tolerance, and extract genomic DNA from the strains. Perform PCR amplification using universal primers for the bacterial 16S rRNA gene. Sequencing the amplified products and comparing their homology with known sequences in the GenBank database will construct a phylogenetic tree to determine the taxonomic position of the strains. The comparison results showed that the isolated strain had 100% homology with Lactobacillus paracasei, and was therefore identified as Lactobacillus paracasei. In this invention, it was named Lactobacillus paracasei M11-7.

[0051] Example 2

[0052] This embodiment provides a probiotic delivery system for lowering blood pressure, the preparation method of which includes the following steps:

[0053] (1) Mix the oxidized astragalus polysaccharide solution, carboxymethyl chitosan solution, and sodium alginate solution to obtain a ternary mixture;

[0054] (2) The bacterial suspension of Lactobacillus paracasei is mixed with the ternary mixture to obtain a probiotic suspension; wherein, the Lactobacillus paracasei used in the bacterial suspension of Lactobacillus paracasei is the Lactobacillus paracasei of Example 1;

[0055] (3) The probiotic suspension is added to the metal ion solution to form gel balls, and then solidified to obtain the probiotic delivery system for lowering blood pressure, denoted as Alg-OGP-CMC. Its specific process and parameters are the same as those in Experiment Example 4.

[0056] The technical effects of this invention are illustrated below with specific experimental examples. SPSS one-way ANOVA was used to analyze the means of multiple groups of samples, and the results are expressed as mean ± standard deviation. Different lowercase letters indicate significant differences between different groups. p<0.05, the same lowercase letter indicates that the difference between different groups is not significant ( p >0.05).

[0057] Experimental Example 1: Screening of bacterial strains with antihypertensive effects

[0058] Seventeen strains identified through laboratory screening were selected as test strains. Their short-chain fatty acid production and antioxidant capacity were measured to evaluate their potential application in lowering blood pressure. The test strains were isolated and screened from different samples using this invention, and then identified using 16S rDNA. The isolation and identification methods for control strains, except for *Lactobacillus paracasei* M11-7, were similar to those for *Lactobacillus paracasei* M11-7, and will not be described in detail here. The sample sources and identification results of each experimental strain involved in this invention are shown in Table 1.

[0059] Table 1. Isolation source and identification results of strains

[0060]

[0061] After numbering each of the selected test strains, they were activated and cultured to prepare experimental strains for subsequent testing. The preparation method for the experimental strains was as follows: *L. paracasei* M11-7 strain was inoculated into MRS liquid medium at a 3% v / v inoculum and cultured at 37°C for 18 h, followed by streak culturing for 48 h. A single colony was picked and cultured for another 18 h. The resulting bacterial culture was then mixed with a 75% v / v glycerol aqueous solution at a ratio of 3:1 (v / v), stored in cryovials, and preserved in a refrigerator to obtain the experimental strains for subsequent use. The culture methods for other test strains to be screened in the experiment were the same.

[0062] 1.1 Determination of short-chain fatty acids

[0063] Short-chain fatty acids (SCFAs) are the final products of anaerobic gut microbiota fermentation of dietary fiber, sugar alcohols, etc. Studies have shown that probiotics producing SCFAs are beneficial in lowering blood pressure levels in hypertensive rats and hypertensive individuals and restoring gut microbiota imbalance caused by hypertension. The peak area of ​​short-chain fatty acids is generally positively correlated with their content, thus reflecting the probiotic strain's ability to produce SCFAs. This invention uses gas chromatography to detect and screen probiotics that produce high levels of short-chain fatty acids by measuring the peak areas of acetic acid, propionic acid, butyric acid, valeric acid, isovaleric acid, and hexanoic acid. The experimental procedure is as follows:

[0064] ① Seventeen experimental bacterial strains were inoculated into 4 mL of MRS liquid medium at a ratio of 3 v / v% and incubated at 37℃ for 24 h to obtain the test bacterial solutions. 2 mL of each test bacterial solution was taken and acidified with 0.4 mL of 50% v / v sulfuric acid solution, and mixed thoroughly. Chromatographically pure dichloromethane (1:1 v / v) was then added for extraction. The lower extract was filtered through a 0.22 μm organic filter membrane, and the filtered liquid was placed in a sample vial for gas chromatography analysis. ② Preparation of standard curve solutions: Accurately measure the mixed standard stock solution, add dichloromethane for stepwise dilution, and prepare a series of standard curve solutions with acetic acid concentrations of 1, 5, 20, 25, 37.5, and 50 mg / mL; propionic acid and butyric acid concentrations of 0.6, 3, 12, 15, 22.5, and 30 mg / mL; and isovaleric acid, valeric acid, and hexanoic acid concentrations of 0.4, 2, 8, 10, 15, and 20 mg / mL. ③ Separation was performed using a DB-FFAP gas chromatography column. The injection port temperature was 250℃; the flow rate was 2 mL / min; the carrier gas was nitrogen; the injection volume was 1 μL; the split ratio was 15:1; the FID temperature was 250℃; and the sample was loaded and analyzed using an autosampler.

[0065] The total peak area of ​​short-chain fatty acids produced by different strains is shown in the figure below. Figure 1 As shown. Figure 1 Among them, the nine probiotic strains with higher total peak areas of short-chain fatty acids were L. paracasei M11-7, L. paracasei 100-1, L. gasseri JM1, L. plantarum 4-10, L. plantarum 18-5, L. rhamnosus 20-37, L. paracasei JY091, L. reuteri J1, and L. acidophilus 11975. Therefore, these nine probiotic strains were selected for subsequent antioxidant experiments.

[0066] 1.2 Antioxidant Capacity Determination

[0067] Sample pretreatment: Seventeen experimental bacterial strains were inoculated into 4 mL of MRS liquid medium at a ratio of 3 v / v% and incubated at 37℃ for 24 h to obtain the test bacterial suspension. Supernatant preparation (CFS): The test bacterial suspension cultured for 24 h was centrifuged at 8000 r / min for 10 min at 4℃. The supernatant was then filtered through a 0.22 μm organic filter membrane to obtain the fermentation supernatant (CFS). Intact cell (IC) preparation: The centrifuged bacterial cells were washed 2-3 times with PBS buffer, resuspended in PBS buffer, and the cell density was adjusted to OD=1.0 (1×10⁻⁶). 9CFU / mL indicates intact cells (IC). Preparation of cell-free extract (CFE): Add 175 μL (1 mg / mL) of lysozyme to the above bacterial suspension, incubate at 37°C for 30 min, then sonicate on ice (200 W, 10 min, working time 6 s, interval 9 s), centrifuge (4°C, 10000 r / min, 10 min), and collect the supernatant, which is the cell-free extract (CFE).

[0068] (1) Hydrogen peroxide tolerance test

[0069] The H2O2 tolerance of probiotics refers to their ability to survive and grow in an environment containing hydrogen peroxide. In environments such as the gut, probiotics may face oxidative stress damage from host immune cells or other microorganisms; probiotics with strong tolerance can better exert their beneficial functions. This ability is crucial for the colonization and function of probiotics in complex environments such as the gut. Therefore, this invention, using LGG (Lactobacillus rhamnosus GG) as the standard strain, determined the hydrogen peroxide tolerance of probiotics among the nine strains screened above. The specific testing procedure was as follows: the activated strains were inoculated at a rate of 3 v / v% into MRS medium containing H2O2 (final hydrogen peroxide concentration of 0 or 1.0 mmol / L), anaerobically cultured at 37°C for 8 h, the absorbance of the bacterial solution was measured, and the H2O2 tolerance rate was calculated to screen for strains with high H2O2 tolerance. The results of the hydrogen peroxide tolerance tests for different strains are shown below. Figure 2 As shown.

[0070] Depend on Figure 2 It was found that the hydrogen peroxide tolerance of the four strains L. paracasei M11-7, L. paracasei100-1, L. gasseri JM1, and L. plantarum 4-10 was higher than that of LGG. Therefore, these four probiotic strains were selected for subsequent antioxidant capacity determination.

[0071] (2) Determination of antioxidant capacity of probiotic strains

[0072] The antioxidant capacity of probiotic strains refers to their ability to neutralize free radicals, reduce oxidative stress, and protect host cells from oxidative damage. The method for determining antioxidant capacity is as follows:

[0073] ① Determination of hydroxyl radical scavenging rate: Take 1 mL of o-phenanthroline (2.5 mmol / L) into a centrifuge tube, add 1 mL of PBS (pH=7.4, 0.02 mol / L), 1 mL of 2.5 mmol / L FeSO4 solution, 1 mL of 20 mmol / L H2O2 and 1 mL of the sample to be tested (CFS, IC or CFE) in sequence, and incubate at 37℃ for 1.5 h. The absorbance at 536 nm is measured as As; then, the absorbance Ap is measured by replacing 1 mL of sample with 1 mL of distilled water; the absorbance Ab is measured by replacing 1 mL of H2O2 with 1 mL of distilled water. Then, the hydroxyl radical scavenging rate is calculated.

[0074] ② Determination of DPPH free radical scavenging rate: Take 1 mL of sample (CFS, IC or CFE), add 2 mL of 0.2 mmol / L DPPH-ethanol solution, mix well, and react in the dark at room temperature for 30 min. Use distilled water and anhydrous ethanol as blanks to zero the absorbance at 517 nm and calculate the DPPH free radical scavenging rate.

[0075] ③ Determination of iron reducing power: Take 0.5 mL of sample (CFS, IC or CFE), add 0.5 mL of 0.2 mol / L PBS (pH=6.6) solution and 0.5 mL of 1% (w / w) potassium ferricyanide, mix well, and incubate in a water bath at 50℃ for 20 min. After rapid cooling, add 0.5 mL of 10% (w / v) trichloroacetic acid, centrifuge at 3500 r / min for 10 min, take 1 mL of the supernatant and mix it with 0.1% (w / w) FeCl3. Let it stand at 37℃ for 10 min, and measure its absorbance at 700 nm to calculate the reducing power.

[0076] ④ Determination of total antioxidant (T-AOC) capacity: The total antioxidant (T-AOC) capacity of the sample was determined according to the method of the kit from Nanjing Jiancheng Bioengineering Institute.

[0077] Figure 3 These are the results of antioxidant capacity determination of different strains in this invention; where (a) is the scavenging rate of hydroxyl radicals; (b) is the scavenging rate of DPPH radicals; (c) is the iron reducing power; and (d) is the total antioxidant capacity. Figure 3 It was found that all four strains exhibited scavenging abilities against different free radicals in the CFS, IC, and CFE treatment groups, but the scavenging abilities varied among the strains. The free radical scavenging rate of the L. paracasei M11-7 group was higher than that of the other three probiotic strains, demonstrating that L. paracasei M11-7 has a strong antioxidant effect. Therefore, L. paracasei M11-7 was selected as the target strain for the subsequent construction of the probiotic delivery system.

[0078] Experimental Example 2: Characteristics of L. paracasei M11-7 strain

[0079] The acid tolerance of probiotic strains is a key characteristic for evaluating their survival and function in the gastrointestinal environment. This experiment assessed the acid tolerance of L. paracasei M11-7 using MRS media at different pH levels. The acid tolerance was determined as follows: L. paracasei M11-7 cultured for 24 hours in Example 1 was inoculated into MRS liquid medium at a ratio of 3 v / v%. The pH of the medium was adjusted to 1.0, 2.0, 3.0, and 6.0, with untreated medium serving as a control. The cultures were incubated at 37°C for 3 hours. The viable cell count was calculated using the dilution plating method, and the bacterial survival rate was determined to assess the strain's acid tolerance.

[0080] Meanwhile, the growth curve can reflect the growth characteristics of the strain and provide a preliminary basis for the subsequent culture time and bacterial concentration of the experimental strain. The procedure for determining the growth curve is as follows: the experimental strain L. paracasei M11-7 was inoculated into MRS medium at a ratio of 3v / v%, and then cultured in a constant temperature incubator at 37℃. Every 2 hours, 5 mL of fermentation broth was taken and used as a blank control with uninoculated MRS medium to plot the growth curve.

[0081] Figure 4 The growth of L. paracasei M11-7 under different pH conditions (A) and its growth curves at different time points (B) are shown. Figure 4 As shown in Figure A, *L. paracasei* M11-7 showed no significant growth in MRS media at pH 1.0 and 2.0; however, bacterial growth was observed in MRS media at pH 3.0, and it grew well in MRS media at pH 4.0, indicating that this strain can tolerate pH 4.0 acidity well. Therefore, environments below pH 3.0 significantly inhibited the growth of *L. paracasei* M11-7, making it necessary to encapsulate it to improve its utilization in the gastrointestinal tract (Figure A). Furthermore, after 4 hours of culture, the strain rapidly grew into the logarithmic phase. Between 12 and 16 hours, the growth rate decreased but still maintained a certain pace. After 16 hours, the growth gradually stabilized, entering the stationary phase. Therefore, the optimal time to collect *L. paracasei* M11-7 bacterial suspension is between 16 and 20 hours, which falls between the late logarithmic and early stationary phases, ensuring the required bacterial concentration while maintaining some growth activity (Figure B).

[0082] Experiment 3: Structural characterization of oxidized astragalus polysaccharide and its effect on the growth of L. paracasei M11-7

[0083] 3.1 Determination of aldehyde content and oxidation degree of oxidized astragalus polysaccharide (OGP)

[0084] Oxidized Astragalus Polysaccharide (OGP) was prepared by periodate oxidation. The specific preparation process was as follows: 2.5 g of Astragalus polysaccharide (GP) was dissolved in 100 mL of distilled water. When GP was completely dissolved, NaIO4 was added at a ratio of NaIO4:GP (1:5, 2:5, 3:5, and 4:5). The mixture was stirred in the dark for 8 hours. Excess ethylene glycol was added to quench unreacted NaIO4, and the mixture was stirred for 1 hour. Then, 4 times the volume of ethanol was added to initiate the precipitation reaction. The mixture was incubated overnight at 4°C. The solution was purified by thorough dialyzing with distilled water (with a cutoff of 3500 Da) for 24 hours, with the water changed at least twice daily during dialysis. After dialysis, the product was pre-frozen and then freeze-dried to obtain oxidized Astragalus polysaccharide (OGP). 0.1 g of OGP was mixed with 25 mL of hydroxylamine hydrochloride-methyl solution, titrated, and the pH was measured. Titration was stopped when the solution changed from red to yellow, and the pH was approximately 5.0. The aldehyde concentration was calculated based on the volume of NaOH solution consumed (ΔV). The aldehyde concentration of each sample was measured three times and the average value was taken. The results are shown in Table 2.

[0085] Table 2. Aldehyde content and degree of oxidation of oxidized polysaccharides

[0086]

[0087] Table 2 shows that the aldehyde content of Astragalus polysaccharide increases with increasing sodium periodate content, but the increase in aldehyde content becomes less significant with further increases in sodium periodate content. This may be because the high concentration of aldehyde groups in OGP leads to the formation of hemiacetals. Hemiacetals are formed between the aldehyde and hydroxyl groups in the monomer unit to prevent further oxidation. Therefore, to ensure the aldehyde concentration, OGP with a NaIO4:GP mass ratio of 2:5 and 3:5 was selected for further screening and preparation of hydrogel microspheres.

[0088] 3.2 Fourier Transform Infrared Spectroscopy (FT-IR) Analysis of Oxidized Astragalus Polysaccharide (OGP)

[0089] The OGP lyophilized sample (NaIO4∶GP=3∶5) prepared above was ground, mixed with KBr powder at a ratio of 1:100, compressed into tablets, and then subjected to Fourier transform infrared scanning. The wavenumber range was 400-4000 cm⁻¹. -1 The resolution is 4cm. -1 Analysis of the infrared spectrum of the obtained OGP shows that the stretching vibration absorption peak of the C=O bond in the aldehyde group is mainly in the 1700-1750 cm⁻¹ range. -1 At 1735cm -1An absorption peak was observed, but it was not prominent. This may be because, after oxidation, the aldehyde group in the polysaccharide exists as a hemiacetal, resulting in no obvious characteristic in the infrared spectrum. However, the aldehyde content indicates that the Astragalus polysaccharide has been successfully oxidized.

[0090] 3.3. Oxidized Astragalus Polysaccharide on L. paracasei The influence of M11-7 growth

[0091] To verify the potential prebiotic properties of OGP (NaIO4∶GP=3:5), the utilization of OGP as a carbon source by *L. paracasei* M11-7 was determined. Glucose in MRS medium was replaced with OGP as the sole carbon source, and *L. paracasei* M11-7 was cultured in vitro. MRS medium and MRS medium with fructooligosaccharides (FOS) as the sole carbon source were used as controls for growth curve plotting. The growth curve was determined by replacing glucose in the MRS medium with OGP, keeping other components unchanged, and sterilizing at 121℃ for 15 min to obtain OGP medium. The growth curve of *L. paracasei* M11-7 was determined using the turbidimetric method. The *L. paracasei* M11-7 experimental strain was inoculated into the above OGP medium at an inoculum size of 3 v / v%, and then cultured in a constant temperature incubator at 37℃. 5 mL of fermentation broth was collected every 4 h for OD analysis. 600 The growth curves of *L. paracasei* M11-7 were plotted to determine the OGP utilization of *L. paracasei* M11-7 and to observe its utilization of OGP. The growth curves of *L. paracasei* M11-7 in different culture media are shown below. Figure 5 As shown.

[0092] Depend on Figure 5 It can be seen that *L. paracasei* M11-7 can grow normally in other available carbon source media. Although the viable cell density of *L. paracasei* M11-7 cultured with OGP as the sole carbon source is lower than that in MRS medium, it is still on the same order of magnitude as the viable cell density cultured with the common prebiotic FOS as the carbon source. This indicates that OGP can be utilized by probiotics and has potential prebiotic characteristics.

[0093] Experimental Example 4: Preparation and Process Screening of Hydrogel Microspheres

[0094] The preparation process of Alg-OGP-CMC hydrogel microspheres is as follows: ① The experimental strain L. paracasei M11-7 was activated and cultured in MRS liquid medium at 37℃ for 18h, then the bacterial cells were collected and activated and cultured again for 18h. Finally, the bacterial cells were collected by centrifugation (6000r, 10min, 4℃), and the bacterial cells were washed twice with NaCl solution (0.9%, w / v) to remove the MRS medium. The collected live bacteria were resuspended in 0.9% NaCl solution, and the plate count was determined to 10⁻⁶ using the plate counting method. 10 -10 11 ① Obtain a bacterial suspension containing colony-forming units (CFU / mL) for later use. ② Disperse 80 mg of OGP (NaIO4∶GP=3:5) in 10 mL of deionized water to obtain an OGP solution (concentration 0.8% w / v). Separately, disperse 100 mg of carboxymethyl chitosan (CMC) in 20 mL of deionized water to obtain a CMC solution (concentration 0.5% w / v). Then, mix the above OGP solution and CMC, and then add 0.6 mL of 2% (w / v) sodium alginate (Alg) solution and mix well to obtain an Alg-OGP-CMC mixed solution. ③ Mix 3.4 mL of bacterial suspension with the 30.6 mL Alg-OGP-CMC mixed solution obtained above to form a viscous probiotic suspension. Using a 10 mL syringe, drip the above mixed solution into a 2% (w / v) metal ion (ZnCl2) solution at a constant rate through a sterile medical syringe (0.3×30 mm needle) to form gel spheres. Then, place them in a refrigerator at 4℃ for 1 hour to solidify. Filter the unreacted metal solution with deionized water, keeping the distance between the syringe and the hardening solution 15 cm. After solidification, filter out the gel microspheres with a filter screen and rinse their surface thoroughly with 0.9% sterile physiological saline to remove surface bacteria, obtaining hydrogel microspheres, designated Alg-OGP-CMC, for later use.

[0095] Meanwhile, the following control groups were set up: (i) The CMC solution in step ② above was omitted, and the remaining conditions were the same as in the preparation process of Alg-OGP-CMC hydrogel microspheres. The prepared hydrogel microspheres were denoted as Alg-OGP. (ii) The CMC solution and OGP solution in step ② above were omitted, and the remaining conditions were the same as in the preparation process of Alg-OGP-CMC hydrogel microspheres. The prepared hydrogel microspheres were denoted as Alg.

[0096] 4.1 Single-factor experimental optimization of gel microsphere embedding process

[0097] Using swelling performance as an indicator, experiments were conducted to determine the effects of different oxidized polysaccharides (NaIO4∶GP=3:5 or 2:5) and metal ions (ZnCl2, CaCl2, FeCl3), as well as different OGP concentrations (0.4%, 0.6%, 0.8%, 1.0% w / v), Alg concentrations (1.0%, 1.5%, 2.0%, 2.5% w / v), CMC concentrations (0.3%, 0.4%, 0.5%, 0.6% w / v), fixed times (15, 30, 60, 90 min), and Zn... 2+ The effect of ion concentration (1.0%, 2.0%, 3.0%, 4.0% w / v) on swelling ratio was investigated. The swelling ratio was determined as follows: After preparing dried hydrogel beads, the initial mass M0 was recorded, and each gel sample was immersed in deionized water. At regular intervals, the gel microspheres were removed, dried, weighed, and the mass Ms was recorded. The swelling ratio was then calculated using the formula... Calculate the swelling ratio. The results are as follows:

[0098] When the mass fraction of metal ions was 2.0%, the concentration of CMC was 0.5% w / v, and the fixation time was 30 min, the effects of oxidized polysaccharide concentration and metal ion type on the swelling rate of hydrogel microspheres were investigated. The results are as follows: Figure 6 As shown. By Figure 6 It can be seen that, comparing the swelling rates of OGP prepared with NaIO4:GP mass fraction ratios of 2:5 and 3:5 and different metal ions at different OGP concentrations, the swelling rate is largest when the OGP concentration is 0.8% for both the different mass fraction ratios and the different metals. Therefore, the OGP concentration of 0.8% was selected for further comparison (Figure a). When the OGP concentration is 0.8%, the mass fraction ratio is 3:5, and the metal ion is Zn... 2+ At this point, the swelling ratio of the hydrogel microspheres is the largest (Figure b). Therefore, when preparing the hydrogel microspheres, a mass fraction ratio of 3:5, an OGP concentration of 0.8%, and Zn metal ions were selected. 2+ Cross-linking occurs.

[0099] When the concentration of OGP was 0.80%, the mass fraction of ZnCl2 was 2.0%, the concentration of CMC was 0.5%, and the fixed time was 30 min, the effect of different sodium alginate concentrations on the swelling rate was investigated. The results are as follows: Figure 7 As shown in Figure A, the swelling ratio of the microspheres is maximized when the sodium alginate concentration is 2.0%. When the Alg solution concentration exceeds 2.0%, the viscosity of the system becomes too high, making extrusion of the hydrogel microspheres difficult, resulting in non-uniform shapes and reduced swelling efficiency. Therefore, an Alg solution concentration of 2.0% was chosen.

[0100] Furthermore, when the OGP concentration was 0.80% w / v, the ZnCl2 mass fraction was 2.0%, the Alg concentration was 2.0% w / v, and the fixation time was 30 min, the effect of the carboxymethyl chitosan mass fraction on the swelling rate was investigated. The results are as follows: Figure 7 As shown in Figure B, the swelling rate is maximized when the concentration of CMC is 0.5% w / v.

[0101] Furthermore, when the concentration of OGP was 0.80% w / v, the concentration of CMC was 0.5% w / v, the concentration of Alg was 2.0% w / v, and the fixation time was 30 min, the effect of different metal ion concentrations on the swelling ratio was investigated. The results are as follows: Figure 7 As shown in Figure C, the swelling ratio gradually increases as the ZnCl2 solution concentration increases from 1.0% to 2.0%, possibly due to the increasing concentration of Zn in the system. 2+ As the concentration increases, more Alg in the system binds, forming a dense gel structure; the swelling ratio reaches its maximum when the ZnCl2 solution concentration is 2.0%. When the ZnCl2 solution concentration is higher than 2.0%, the extruded droplets float on the surface of the fixative, making it difficult to form gel microspheres. Therefore, a ZnCl2 solution concentration of 2.0% is chosen for the preparation of hydrogel microspheres.

[0102] Furthermore, when the concentration of oxidized polysaccharide was 0.80% w / v, the concentration of chitosan was 0.5% w / v, the concentration of sodium alginate was 2.0% w / v, and the mass fraction of Zn ions was 2.0%, the effect of a fixed time on the swelling ratio was investigated, and the results are as follows. Figure 7 As shown in Figure D, the swelling ratio of the hydrogel spheres continuously increases as the fixation time increases from 15 min to 60 min, but stops increasing with further increases in fixation time. This indicates that when the fixation time reaches 60 min, the hydrogel microspheres are fully cross-linked, the wall thickness reaches its maximum, and the swelling ratio reaches its maximum. Therefore, a fixation time of 60 min is chosen for the preparation of hydrogel microspheres.

[0103] 4.2 Response Surface Optimization of Gel Microsphere Encapsulation Process

[0104] Single-factor experiments revealed that OGP concentration (0.6, 0.8, 1.0 w / v%), CMC concentration (0.4, 0.5, 0.6 w / v%), Alg concentration (1.5, 2.0, 2.5 w / v%), and fixed time (0.5, 1.0, 1.5 h) significantly affected the swelling rate of the hydrogel beads. These four factors were selected as target variables, and a response surface methodology was designed with the swelling rate of the gel microspheres as the response value to determine the optimal encapsulation process for the gel microspheres. Based on the response surface methodology results, a quadratic regression analysis was performed using Design-Expert 13 software. The regression equation was used to predict the influence of each factor on the response value R. The results are as follows: R = 205.04 - 10.03*A - 1.97*B - 5.29*C + 7.80*D - 14.71*AB + 1.45*AC + 5.60*AD - 2.58*BC + 1.48*BD - 1.03*CD - 31.68*A² - 29.56*B² - 30.43*C² - 15.52*D². According to the variance results, the regression equation model term P < 0.01, and the lack-of-fit term P = 0.2030 > 0.05. This indicates that the established quadratic regression model is significant, and the lack-of-fit term is not significant, indicating that the established regression equation fits well. The correlation coefficient R² = 0.9962, indicating that the changes in the response value can be explained by this model.

[0105] Based on the established model, the optimal preparation conditions for gel microspheres were predicted to be 0.772% OGP, 0.501% CMC, 1.953% Alg, and a curing time of 1.115 h. Under these conditions, the swelling ratio of the gel microspheres was 206.885%. Based on actual conditions, the following conditions were determined: 0.772% OGP, 0.501% CMC, 1.953% Alg, and a curing time of 1.115 h. Three repeated experiments were conducted, and the swelling ratio of the gel microspheres under these conditions was 206.096%. The verified value was very close to the predicted value, indicating that the optimized gel microsphere preparation process is feasible.

[0106] Experimental Example 5: Structural Characterization and Performance Testing of Hydrogel Microspheres

[0107] 5.1 Particle size determination

[0108] Thirty hydrogel microspheres (Alg, Alg-OGP, and Alg-OGP-CMC) prepared in Example 4 were randomly selected for observation of their morphology. They were divided into three groups of ten microspheres each, and the particle size was measured. The results are expressed as d (mm). The morphology of each group of hydrogel microspheres is shown below. Figure 8 As shown, Figure 8In the figures, (a) represents the Alg group; (b) represents the Alg-OGP group; and (c) represents the Alg-OGP-CMC group. All three groups of hydrogel microspheres are opaque and uniform in size. Except for the Alg group, which has white hydrogel microspheres, the other two groups are darker in color. The particle size analysis results show that the average particle size of the Alg group is 0.585±0.10 mm, the average particle size of the Alg-OGP group is 0.597±0.07 mm, and the average particle size of the Alg-OGP-CMC group is 0.687±0.07 mm, indicating that the addition of OGP increases the particle size of the hydrogel microspheres to some extent.

[0109] 5.2 Fourier Transform Infrared Spectroscopy (FT-IR) Analysis

[0110] The lyophilized hydrogel microsphere powder (Alg-OGP-CMC), along with OGP, Alg, and CMC raw materials, were mixed with potassium bromide at a mass ratio of 1:100 and then compressed into tablets. The sample tablets were scanned and tested against a blank KBr background. The power supply voltage was 220V, the frequency was 20Hz, and the scanning range was 500-4000 cm⁻¹. -1 Infrared spectra of OGP raw materials, Alg raw materials, CMC raw materials, and Alg-OGP-CMC hydrogel microspheres are shown below. Figure 9 As shown.

[0111] 3600cm -1 and 3200cm -1 The strong absorption peaks between them correspond to the stretching vibrations of the hydroxyl and amino groups, at 2920 cm⁻¹. -1 The weak band recorded at this point is related to CH tensile vibration. At 1735 cm⁻¹ -1 The relatively strong absorption peak recorded at this point indicates the presence of a carbonyl group. For example... Figure 9 As shown, in the FT-IR plot of OGP, at 1735cm -1 A characteristic absorption peak is observed at 3417 cm⁻¹, which weakens within the gel spheres. This indicates that OGP and CMC undergo chemical cross-linking, thus reducing the carbonyl content. The characteristic absorption of CMC appears at 3417 cm⁻¹. -1 Location, 1418cm -1 and 1604cm -1 1061cm -1 2917cm -1 and 1310cm -1 In Alg, the main characteristic peak is located at 3422 cm⁻¹. -1 Location, 2917cm -1 1417cm -1 and 1613cm -1Compared to the FT-IR spectra of CMC and Alg, the infrared absorbance of -COO-, -OH, and -NH2 in the hydrogel microspheres shifted to 1320 cm⁻¹. -1 At a lower wavenumber, overlapping absorption peaks of CN stretching vibration and NH bending vibration were observed, demonstrating a strong intermolecular interaction between CMC and Alg, enhancing structural stability. The characteristic absorption peak of -COO- is at 1613 cm⁻¹. -1 1417cm -1 The weakening indicates that Na + Zn 2+ Replace and use -COO- crosslinking.

[0112] 5.3 Determination of the texture of gel microspheres

[0113] The textural properties of hydrogel beads (Alg, Alg-OGP, Alg-OGP-CMC) were measured using a TA-XT texture analyzer. Microspheres were uniformly spread on 50 mm culture dishes, and tests were performed using a 25 mm probe in TPA mode. The test conditions were: the hydrogel beads were compressed twice at a speed of 40 m / min to 25% of their original height, with a thixotropic force of 5 g. The results of the determination of the textural properties of the hydrogel microspheres are shown in Table 3.

[0114] Table 3. Determination of the textural properties of hydrogel microspheres

[0115]

[0116] The gel strength of hydrogel microspheres is closely related to the protective effect of probiotics. As shown in Table 3, hydrogel microspheres exhibit higher hardness, cohesiveness, adhesiveness, and chewiness compared to other samples, making them more beneficial for protecting the core material. The addition of OGP and CMC improves the hardness and elasticity of the hydrogel beads. This is mainly because the interaction between CMC and OGP molecules promotes the aggregation and rearrangement of the internal network of the hydrogel.

[0117] 5.4 Observation of the microstructure of gel microspheres

[0118] Images were captured using a field emission scanning electron microscope (FET), with secondary electron resolutions of 15 kV, 1.0 nm; and 1 kV, 1.3 nm, at a maximum accelerating voltage of 30 kV, to observe the microstructure. The SEM morphology of the hydrogel microspheres is shown below. Figure 10 As shown, (a) is the Alg group; (b) is the Alg-OGP group; and (c) is the Alg-OGP-CMC group. Figure 10As shown, the hydrogel microspheres lost moisture during the freeze-drying process, resulting in uneven wrinkles and even cracks on the surface of the hydrogel microspheres. However, compared with Figures (a) and (b), Figure (c) has fewer wrinkles and a more compact and full structure. This may be because OGP fills the gaps in the gel structure, which is more conducive to resisting the stress of the external environment on the bacteria, giving the hydrogel microspheres the potential to improve the survival rate of bacteria in harsh environments.

[0119] 5.5 Determination of the embedding efficiency of hydrogel microspheres

[0120] The Alg-OGP-CMC hydrogel spheres were placed in a sterile sodium citrate solution and ruptured in a shaker at 37°C. They were then spread on MRS agar medium and incubated for 48 hours. Colony counts were performed using the plate count method, and the encapsulation efficiency (EE) of the hydrogel spheres was calculated using the following formula. In the formula, EE represents the encapsulation efficiency; N represents the number of live cells released from the hydrogel spheres (log CFU / mL); and N0 represents the number of unencapsulated live cells (log CFU / mL). The results of the encapsulation efficiency determination of the gel microspheres are shown in Table 4.

[0121] Table 4. Determination of encapsulation efficiency of hydrogel microspheres

[0122]

[0123] Based on the data in Table 4 and the formula above, the encapsulation rate of the hydrogel microspheres was determined to be 91.40%, indicating that the encapsulation effect of the hydrogel microspheres was good.

[0124] 5.6 Stability of hydrogel microspheres

[0125] 5.6.1. Tolerance to bile salts

[0126] Tolerance to bile salt environments is a necessary indicator for evaluating the protective effect of probiotics. Hydrogel microspheres (Alg, Alg-OGP, Alg-OGP-CMC) were mixed with sterile bile salt solutions at concentrations of 0.2%, 0.4%, and 0.6% w / v at a ratio of 1:9 (m / v), respectively, and treated in a shaker at 37℃ and 180 r / min for 2 h. Samples were taken at 0.5 h intervals, and the microspheres were disrupted with sodium citrate solution. Viable cell counts were determined using the plate count method. Separately, 1 mL of L. paracasei M11-7 bacterial culture cultured for 24 h in Example 1 (hereinafter the same) was treated under the same conditions as a blank control group for viable cell count. The effect of bile salt treatment on the activity of hydrogel microspheres is as follows: Figure 11 As shown.

[0127] Depend on Figure 11It can be seen that, under the same bile salt concentration, the free cell group, Alg group, Alg-OGP group, and Alg-OGP-CMC group treated with bile salt solutions at concentrations of 0.2%, 0.4%, and 0.6% w / v showed that, among the four experimental groups, the free cell group had the lowest survival rate, while the Alg-OGP-CMC group had the highest survival rate of L. paracasei M11-7, with survival rates of 90.08%, 77.57%, and 61.03% in different concentrations of bile salt solutions, respectively.

[0128] 5.6.2 Simulated gastric juice tolerance test

[0129] Hydrogel microspheres (Alg, Alg-OGP, Alg-OGP-CMC) were mixed with gastric juice solution at pH 1.5 at a mass-to-volume ratio of 1:9. The samples were then treated in a shaker at 37℃ and 180 rpm for 120 min. Samples were taken at 0, 30, 60, 90, and 120 min. The hydrogel microspheres were completely broken up with sodium citrate solution, and the viable bacteria count in the capsules was determined by plate count. 1 mL of L. paracasei M11-7 bacterial culture was treated under the same conditions as a blank control for viable bacteria counting. The simulated gastric juice (SGF) was prepared by adding 0.20% (w / v) NaCl and 0.32% (w / v) pepsin to 0.70% (v / v) HCl solution and mixing thoroughly. To approximate the gastric juice environment, the pH of the solution was adjusted to 2.00 ± 0.02 with 1.0 mol / L hydrochloric acid before use. The ratio of digestive fluid to gel microspheres was 9:1. 1g of gel microspheres was weighed and added to 10ml of SGF. The mixture was shaken continuously at 180r / min for 2h at 37℃. During this process, the pH of the system was kept constant at 2.0 using 0.1mol / L hydrochloric acid.

[0130] The effect of simulated gastric juice treatment on the activity of hydrogel microspheres, such as Figure 12 As shown in Figure A, under simulated gastric fluid treatment, the survival rate of each group decreased with increasing treatment time. The survival rate of the probiotic culture showed a rapid decline, with all bacteria becoming inactive after 90 minutes. After 2 hours of simulated gastric fluid treatment, the survival rate of the Alg group was 75.73%, the Alg-OGP group was 84.07%, and the Alg-OGP-CMC group was 91.13%. Compared with the free bacteria group, all other groups were able to resist the influence of simulated gastric fluid on probiotic activity, thus ensuring the probiotics could exert their beneficial effects to a certain extent.

[0131] 5.6.3 Simulated enteric coating test

[0132] Undissolved hydrogel microspheres after treatment with artificial gastric fluid were removed and placed in artificial intestinal fluid. The solubility of the hydrogel microspheres in the artificial intestinal fluid was determined by plate count method. During the experiment, the hydrogel microspheres treated with artificial gastric fluid for 2 hours were filtered, washed, and collected. The collected microspheres were added to test tubes containing 9 mL of artificial intestinal fluid and incubated at 37℃ and 180 rpm in a water bath with shaking. Samples were taken at 0, 30, 60, 90, and 120 min. The hydrogel microspheres were completely broken up with sodium citrate solution, and the viable bacteria count in the capsules was determined by plate count method. The method for preparing simulated intestinal fluid (SIF) is as follows: After gastric digestion, the pH of the system is adjusted to 7 with 1 mol / L NaOH solution. Then, 0.70% (w / v) ox bile salts, 0.50% (w / v) trypsin, 0.40% (w / v) lipase and 0.80% (w / v) CaCl2 are added and mixed to prepare simulated SIF. The pH is then adjusted to 7.50 ± 0.02 with 0.1 mol / L NaOH. The mixture is shaken continuously at 180 r / min at 37℃ for 2 h. During this process, the pH of the reaction system is kept constant at 7.50 using 2 mol / L NaOH solution.

[0133] The effect of simulated intestinal fluid treatment on the activity of hydrogel microspheres, such as Figure 12 As shown in Figure B, the release rate of probiotics from the microspheres was fastest after 30 minutes of treatment in artificial intestinal fluid. The release rate gradually slowed down over the next 120 minutes, and by 150 minutes, the probiotics embedded in the three groups of gel microspheres were almost fully released, with the Alg-OGP-CMC group showing the best release effect. The results indicate that the prepared hydrogel microspheres can effectively protect the bacteria from the gastric fluid environment and promote their effective release in the intestine.

[0134] 5.6.4 Storage stability of gel microspheres

[0135] Storage stability is an important indicator of shelf life. Hydrogel microspheres (Alg, Alg-OGP, and Alg-OGP-CMC) were placed in sterile PBS buffer and stored at 4°C and 25°C for 60 days. Samples were taken every 10 days, and the hydrogel microspheres were broken up with sodium citrate solution. The viable bacteria count in the hydrogel microspheres was determined by plate count method. Figure 13 The effect of storage conditions on the survival rate of hydrogel microspheres: (a) Changes in cell viability when stored at 4°C; (b) Changes in cell viability when stored at 25°C.

[0136] Depend on Figure 13It was found that the survival rate of *L. paracasei* M11-7 in the Alg group, Alg-OGP-CMC group, and Alg-OGP group all decreased with increasing storage time. Compared with free cells, microspheres improved the viability of probiotics under different storage conditions. After storage at 4℃ for 2 weeks, the cell viability in the Alg group, Alg-OGP group, and Alg-OGP-CMC group were 7.7, 7.91, and 9.00 log CFU·g, respectively. -1 After storage at 25℃ for 2 weeks, the cell viability in the Alg group, Alg-OGP group, and Alg-OGP-CMC group were 7.21, 7.55, and 8.00 log CFU·g, respectively. -1 Meanwhile, after 60 days of storage at 4℃ and 25℃, the Alg-OGP-CMC group showed less loss of viable bacteria compared to the Alg-OGP group and the Alg group. This indicates that the polysaccharide composite hydrogel microspheres possess prebiotic properties, providing nutritional support for probiotic growth. By promoting probiotic proliferation, they effectively slow down the rate of decline in survival rate, enhancing the stability of probiotics during storage. Furthermore, the addition of OGP strengthens the protective effect of the hydrogel microspheres against environmental damage to L. paracasei M11-7.

[0137] Experimental Example 6: Cellular Experiment Evaluation of Hydrogel Microspheres

[0138] 6.1 Cytotoxicity assay

[0139] The effect of hydrogel microspheres (Alg-OGP-CMC) on human intestinal epithelial cells (Caco-2) was determined using the CCK-8 assay. Cells were digested with trypsin and seeded in 96-well plates (1 × 10⁶ cells per well). 5 Cells were incubated for 24 hours, and then culture medium soaked in gel microspheres for 24 hours was added. The effect on cell growth was then measured. The results are as follows: Figure 14 As shown.

[0140] Depend on Figure 14 It can be seen that the cell viability of the control group was close to 100%, indicating that the Caco-2 cells were of normal viability, and the cells were evenly distributed among the groups, with no significant difference in number or activity. p >0.05). After co-culturing cells with different concentrations of gel extract, the cell viability was 99.00±0.10%, 98.41±0.10%, 97.61±0.02%, 96.93±0.04%, and 96.50±0.03%, respectively, maintaining high activity. There was no significant difference from the normal level, indicating that the gel microspheres had no significant toxic effect on Caco-2 cells.

[0141] 6.2 Study on the effect of gel microspheres on vascular dysfunction in hypertensive rats

[0142] Animal grouping and model establishment: The animal experiment was approved by the Animal Welfare and Ethics Committee of Northeast Agricultural University (No.: NEAUEC2024 04 34). Forty spontaneously hypertensive (SHR) male rats and eight age-matched normotensive (WKY) male rats, approximately 16 weeks old and weighing 200-250g, were selected and housed at a temperature of 22±2℃ and a relative humidity of 60±5%, maintaining a 12h light / dark cycle. All rats were administered the medication by gavage once daily. After one week of acclimatization feeding with normal diet and free access to water, the SHR rats were randomly divided into 5 groups (n=8 per group): model group (SHR), free bacteria group (M11-7), empty gel microsphere group (AOC), gel microsphere group (AOC-M), and positive control group (PC). WKY rats served as the blank control group (NC). The specific methods of gavage are shown in Table 5, and gavage was performed continuously for 6 weeks. During the 6-week intervention experiment, the rats had free access to food and water. Blood pressure was measured weekly, and changes in rat blood pressure were recorded. The preparation method of the hollow gel microspheres was basically the same as that of Alg-OGP-CMC in Experiment 4, except that step ① and the bacterial suspension in step ③ were omitted, while the rest of the process was the same.

[0143] Table 5. Grouping and Gavage Procedures in Animal Experiments

[0144]

[0145] 6.2.1 Measurement of rat blood pressure: Blood pressure of rats in each group was measured weekly using tail cuff plethysmography. Each rat was measured three times, and the mean was taken to ensure data accuracy. Hypertension is often accompanied by erectile dysfunction (ED). Weekly blood pressure measurements in each group were used to evaluate the effect of M11-7 hydrogel microspheres on alleviating ED. The results of blood pressure changes in hypertensive rats are shown below. Figure 15 As shown, (a) shows the changes in systolic blood pressure in hypertensive rats; (b) shows the changes in diastolic blood pressure in hypertensive rats.

[0146] like Figure 15 As shown, the systolic blood pressure (SBP) of WKY rats in the NC group remained relatively stable at 104.9 ± 1.98 mmHg, and the diastolic blood pressure (DBP) remained relatively stable at 82.08 ± 2.98 mmHg. In the SHR group, both SBP and DBP were consistently significantly higher than those in the control group. p <0.05. Before sample intervention, there was no significant difference in blood pressure among the SHR rat groups ( p>0.05). Six weeks after administration, compared with the SHR group, the blood pressure levels in the M11-7 group, AOC group, AOC-M group, and PC group were significantly lower, especially in the AOC-M group. With the increase of administration time, both SBP and DBP in the administered groups continued to decrease. After 6 weeks of continuous administration, the antihypertensive effect of the AOC-M group was similar to that of the PC group, with no significant difference between the two groups. p >0.05, but still significantly higher than the control group ( p <0.05). This indicates that the M11-7-loaded hydrogel microspheres Alg-OGP-CMC can effectively reduce blood pressure in hypertensive mice.

[0147] 6.2.2 Sample Collection: After the final gavage, rats were fasted overnight but allowed free access to water for 12 hours. Phenobarbital was injected into the groin, and blood was collected. Serum was collected after centrifugation and stored at -80°C. The rats were euthanized by cervical dislocation. The mesenteric aorta was harvested, and connective tissue, mesenteric veins, and adhering fat were removed. The aorta was rapidly frozen in liquid nitrogen and maintained at -80°C. A portion was fixed with 4% paraformaldehyde and stored at 4°C for pathological analysis. Another portion was used for ROS detection and Western blotting analysis. The remaining tissues were rapidly frozen at -80°C to prevent RNA degradation. Cecal contents and feces were rapidly frozen in liquid nitrogen and stored at -80°C for subsequent biochemical index and intestinal flora determination in rats.

[0148] 6.2.3. ROS assay of rat aorta: Cells were resuspended and stained in phenol red-free medium containing 10 μmol / L DCFH-DA and incubated at 37°C for 30 min. After staining, the cells were treated with endothelial microparticles (EMPs) suspension for 4 h, and then detected by flow cytometry with 488 nm laser excitation and data read at 535 nm (FL1 channel).

[0149] Flow cytometry is a commonly used technique for detecting tissue ROS levels. Using the DCFH-DA fluorescent probe, intracellular ROS levels can be quantitatively detected, and the oxidative stress status of tissue cells can be assessed by the positive cell rate (i.e., the proportion of ROS-positive cells). Excessive ROS is a major cause of oxidative stress and one of the main causes of erectile dysfunction (ED). Changes in the ROS-positive cell rate in the abdominal aorta of hypertensive rats are shown below. Figure 16 As shown. Figure 16 As shown, the ROS positivity rate in the SHR group was significantly higher than that in the NC group (as indicated by the data). p <0.05 indicates excessive ROS expression in rats. After sample intervention, the ROS expression levels in the AOC-M group, M11-7 group, and AOC group were all significantly reduced ( p<0.05), especially the AOC-M group, showed the most significant effect. This indicates that the M11-7-loaded gel microspheres exhibit good ROS scavenging ability and can alleviate oxidative stress in the aortic endothelium, which is consistent with the biochemical results.

[0150] 6.2.4 Pathological Analysis of Vascular Histopathology

[0151] (1) H&E staining: After the experiment, rats were euthanized by phenobarbital anesthesia, and abdominal aortic tissue was collected. The samples were fixed at room temperature, dehydrated by ethanol gradient, embedded in paraffin, sectioned, and dewaxed for subsequent analysis. Using the H&E staining kit, the tissue sections could be directly stained with H&E and observed under an optical microscope at a magnification of 200×. H&E staining was used to examine the degree of lesions in the abdominal aorta of rats. The H&E staining results of the abdominal aorta of hypertensive rats showed that the aortic vascular structure of the NC group rats was intact, the endothelial cells were flat, and there were no obvious proliferating or sloughed necrotic cells, indicating that the selected NC group WKY rats were in a normal physiological state. In the SHR group, the aortic wall thickness of the rats was significantly increased, the inner wall was rough and damaged, and a large number of vascular endothelial cells were sloughed off. After sample intervention, compared with the SHR group, the AOC group, M11-7 group, and AOC-M group showed reduced vascular endothelial cell sloughing and a lower degree of vascular lesions.

[0152] (2) Sirius Red Staining: Abdominal aortic sections were washed three times with PBS for 5 min each time. Then, staining was performed according to the instructions of the Sirius Red staining kit. The sections were stained with Sirius Red staining solution for 60 min, washed twice with acidified water working solution (acidified water concentrate: distilled water = 1:100), and rinsed with tap water for 1 min to remove excess dye. Finally, the sections were dehydrated and cleared with graded alcohol and dimethylformaldehyde, and mounted with neutral resin. Images were captured using a fluorescence microscope. Sirius Red staining can visually show the distribution and content of collagen fibers. Sirius Red staining of the abdominal aorta of hypertensive rats showed that the SHR group had increased red and yellow collagen fiber deposition and thickened blood vessels. Compared with the SHR group, the drug intervention group had reduced red and yellow collagen fiber deposition. Pathological analysis results showed that M11-7 loaded gel microspheres could alleviate collagen fiber deposition and the number of perivascular inflammation-related cells caused by vascular endothelial impairment.

[0153] (3) MASSON staining: Abdominal aortic tissue was cut into 4 μm thick sections and stained with MASSON. Images were captured by fluorescence microscopy. Collagen fibers were observed to be blue; muscle fibers, cytoplasm, cellulose, keratin, and erythrocytes were observed to be red. Vascular endothelial dysfunction can cause tissue fibrosis, and Masson staining is a commonly used method for qualitatively assessing collagen fiber content. Through Masson staining, collagen fibers were stained blue or green, and muscle fibers were stained red. The results of MASSON staining of the aorta in hypertensive rats showed that the NC group had intact vascular structure with no fibrosis deposition, while the SHR group had collagen fibers stained blue, indicating increased collagen fiber deposition. Intervention in the M11-7 group, AOC group, and AOC-M group all reduced collagen fiber deposition, with the AOC-M group showing a more significant effect, similar to the positive control group. These results indicate that hydrogel microspheres can reduce the level of collagen accumulation or fibrosis around the aorta and the number of perivascular inflammation-related cells, thereby improving vascular endothelial dysfunction.

[0154] 6.2.5 Detection of vasoconstrictor factors and antioxidant enzymes

[0155] Endothelial dysfunction is a crucial pathological basis of hypertension, and endothelin-1 (ET-1) and nitric oxide (NO) are key antagonistic molecules secreted by endothelial cells. The pathological effects of vascular endothelial dysfunction mainly involve inhibiting the synthesis of the vasodilator NO and increasing the release of the vasoconstrictor ET-1. Therefore, NO and ET-1 levels were measured in rat serum. Simultaneously, ET-1 increases the activity of NADPH oxidase, producing ROS to inhibit NO synthesis, further leading to oxidative stress damage. Therefore, changes in oxidative stress indicators (SOD, T-AOC) in the body were analyzed. Superoxide dismutase (SOD) is a metabolic product of oxidative stress and endothelial damage. SOD can effectively inhibit ROS generation and reduce oxidative stress. The role of total antioxidant capacity (T-AOC) in the body's defense system is mainly to maintain the dynamic balance of reactive oxygen species in the internal environment, clearing excess reactive oxygen species and maintaining a relatively stable redox balance in the body.

[0156] After centrifuging the serum samples, the supernatant serum was collected and stored at -80℃ for later use. Following the instructions of the relevant ELISA kits, the levels of vasoconstrictor 1 (ET-1), vasodilator 1 (NO), and antioxidant enzymes (SOD, T-AOC) in the serum of rats in each group were measured. The results are as follows: Figure 17 As shown in Figures a-d.

[0157] Depend on Figure 17 As shown in Figures a and b, compared with the NC group, the NO content in the SHR group was significantly decreased, while the ET-1 content was significantly increased. p<0.05), which leads to decreased vasomotor capacity and promotes collagen deposition, consistent with pathological analysis results. After intervention, the NO content in the plasma of rats showed varying degrees of increase, while the ET-1 content showed varying degrees of decrease, especially with no significant difference in NO content between the AOC-M group and the PC group. This indicates that M11-7 hydrogel microspheres can improve vascular protection by balancing the ET-1 / NO level in plasma, effectively alleviating vascular endothelial dysfunction. Figure 17 As shown in Figures c and d, compared with the NC group, the expression levels of SOD and T-AOC in the SHR group were significantly reduced. p <0.05 indicates significant oxidative stress in the vascular endothelium. Compared with the SHR group, all intervention groups showed varying degrees of increased SOD and T-AOC expression levels, especially the AOC-M group ( p <0.05), indicating that M11-7 loaded gel microspheres can alleviate ROS-induced oxidative damage to vascular endothelium by increasing the activity of antioxidant enzymes in hypertensive rats.

[0158] Experimental Example 7: Study on the signaling pathway regulating vascular endothelial dysfunction

[0159] 7.1 mRNA expression test

[0160] (1) Extraction of total RNA from the mesenteric aorta: RNA extraction was performed using an RNA extraction kit. Aortic tissue was collected, flash-frozen in liquid nitrogen, and rapidly ground into powder. The powder was then transferred to enzyme-free centrifuge tubes and extracted in the dark according to the Simply P kit instructions. The extracted total RNA was collected in enzyme-free centrifuge tubes, and the integrity of the RNA was detected by agarose gel electrophoresis. (2) RNA purity detection: The purity and concentration of the extracted RNA were determined using a nucleic acid protein concentration analyzer. When its OD value was... 260 / 280 When the value is in the range of 1.8-2.1, it indicates that the extracted RNA is of good quality and can be used for subsequent experiments. (3) Reverse transcription reaction: RNA was reverse transcribed into cDNA according to the operating procedure of the Prime Script™ RT kit. The reverse transcription reaction system was prepared according to the kit instructions. The system was first reacted at 37 ℃ for 15 min, and then at 85 ℃ for 5 s to inactivate the reverse transcriptase. The cDNA product after the reaction was frozen at -20 ℃ for subsequent experiments. Gene-specific primers were designed using Primer 5.0 software, and the primer sequences of the target gene were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The Real-time RT-PCR reaction system of cDNA was prepared according to the instructions of the TB Green® Premix Ex Taq™ II kit, and QuantStudio was used. ®3. Real-time RT-PCR reaction was performed using the system. Using β-actin as an internal reference gene for comparison, 2... -ΔΔCt The method involves determining the relative expression level of the target gene, with each group repeated at least three times.

[0161] In this experiment, RT-PCR was used to determine the expression levels of related genes (p-PI3K, PI3K, p-Akt, Akt, p-eNOS, Nrf2, HO-1) to explore the alleviating effect of hydrogel microspheres on vascular endothelial dysfunction. β-actin was used as an internal reference gene, and the gene expression of p-PI3K, PI3K, p-Akt, Akt, p-eNOS, Nrf2, and HO-1 was also measured. The results of gene expression levels in the PI3K / Akt signaling pathway are shown below. Figure 18 As shown. Figure 18 In the figures, a~g show the relative gene expression levels of PI3K, p-pi3k, Akt, p-Akt, eNOS, Nrf2, and HO-1, respectively.

[0162] Depend on Figure 18 It can be seen that, compared with the NC group rats, the p-PI3K, PI3K, p-Akt, Akt, and eNOS genes in the SHR group rats were significantly reduced. p <0.05, and the expression of Nrf2 gene, which is related to antioxidant enzymes, was significantly reduced ( p <0.05), indicating increased expression of the oxidation-related HO-1 gene. Compared with the SHR group, the relative mRNA expression levels of p-PI3K, PI3K, p-Akt, Akt, p-eNOS, Nrf2, and HO-1 were significantly increased in the PC, M11-7, AOC, and AOC-M groups. p The increase was <0.05% in the AOC-M group, indicating that all intervention groups significantly inhibited oxidative stress by altering the expression levels of related mRNAs, with hydrogel microspheres showing a better effect. Therefore, hydrogel microspheres can improve vascular endothelial dysfunction through the expression of PI3K / Akt-related genes.

[0163] 7.2 Western blotting detection of the PI3K / Akt signaling pathway

[0164] This experiment detected protein expression in the PI3K / Akt / eNOS signaling pathway using Western blot. First, total protein was extracted, and its concentration was accurately determined using a BCA protein assay kit. Subsequently, SDS-PAGE electrophoresis was performed to separate the proteins, followed by membrane transfer, overnight incubation with primary antibody, 2 hours incubation with secondary antibody, and color development. The results were then quantitatively analyzed using ImageJ software. The expression results of PI3K-Akt pathway-related proteins are shown below. Figure 19As shown in Figures a-c, the relative expression levels of p-PI3K / PI3K protein, p-Akt / Akt protein, and p-eNOS protein are respectively displayed.

[0165] like Figure 19 As shown, the protein expression levels in the mesenteric aorta of the SHR group were significantly lower than those of the normal group (p-PI3K / PI3K ratio, p-Akt / Akt ratio, and p-eNOS). p <0.05%, the intervention group increased the expression levels of these proteins ( p <0.05. Compared with the SHR group, the AOC-M group showed significantly upregulated p-PI3K / PI3K, p-Akt / Akt ratios, and Akt and p-eNOS expression levels ( p <0.05), compared with SHR, AOC, and M11-7, the p-PI3K / PI3K and p-Akt / Akt ratios in the AOC-M group were significantly increased ( p <0.05, p-eNOS protein expression level significantly increased ( p <0.05). This indicates that the hydrogel microspheres can lower blood pressure by activating the PI3K / Akt pathway, regulating vascular endothelial factors, and restoring endothelium-dependent relaxation.

[0166] 7.3 Changes in the gut microbiota of rats

[0167] After euthanizing the rats, the cecal contents were removed and aliquoted. DNA was extracted using a fecal DNA extraction kit, with the V3 and V4 regions of the 16S rDNA serving as the target sequences for amplification. Agarose gel electrophoresis was used for detection, and sequencing was performed using an Illumina MiSeq sequencer. The composition of the gut microbiota, based on changes at the phylum and genus levels, was analyzed. The results are as follows: Figure 20 As shown. Figure 20 In the figure, Figure a shows the gut microbiota composition analysis based on changes at the phylum level; Figure b shows the gut microbiota composition analysis based on the genus level.

[0168] Figure 20 The results showed that the probiotic-loaded hydrogel microspheres Alg-OGP-CMC effectively reduced Bacteroides ( Bacteroides The abundance of Bifidobacteria (Bifidobacteria) increased. Bifidobacterium ) and Lactobacillus ( Lactobacillus The abundance of ) indicates that it can regulate the intestinal flora by promoting the proliferation of beneficial bacteria and inhibiting the growth of pathogenic bacteria.

[0169] 7.4 Determination of Short-Chain Fatty Acid Content

[0170] SCFAs are the main metabolites produced by the intestinal flora of the colon through the fermentation of indigestible polysaccharides. Existing research indicates that SCFAs are involved in the regulation of blood pressure. This experiment measured the changes in the content of acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid in rat feces. The test procedure was as follows: rat feces were weighed, pretreated, and prepared into a 10% suspension. Crotonic acid metaphosphate solution was added to the suspension, and the mixture was frozen for 24 hours. After thawing, the mixture was centrifuged, impurities were removed, and the supernatant was filtered and analyzed. The results of the effect of hydrogel microspheres on the content of SCFAs in the rat intestine are as follows. Figure 21 As shown. Figure 21 In the figures, a~f represent the contents of acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid, respectively.

[0171] from Figure 21 It can be seen that, compared with the NC group rats, the SHR group rats had significantly lower levels of acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid. p <0.05%. In the PC, M11-7, AOC, and AOC-M groups, compared with the SHR group, the contents of acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid were significantly increased ( p The levels were <0.05%, showing a significant difference, with the AOC-M group exhibiting the most significant effect. This indicates that both probiotics and hydrogel microspheres can promote the production of short-chain fatty acids, which have a significant effect on vasodilation and thus lowering blood pressure, but the hydrogel microspheres loaded with probiotics showed a better effect.

[0172] In summary, the above results demonstrate the application potential of Lactobacillus paracasei M11-7 in the treatment of hypertension. Furthermore, a probiotic delivery system based on Lactobacillus paracasei M11-7 has been successfully constructed, and it has been shown to effectively alleviate vascular dysfunction caused by spontaneous hypertension. Therefore, this invention can provide theoretical and technical reference for the treatment of hypertension.

Claims

1. A probiotic delivery system for lowering blood pressure, characterized in that, The preparation method of the blood pressure-lowering probiotic delivery system includes the following steps: (1) Mix the oxidized astragalus polysaccharide solution, carboxymethyl chitosan solution, and sodium alginate solution to obtain a ternary mixture; the oxidized astragalus polysaccharide used in the oxidized astragalus polysaccharide solution is prepared by oxidizing astragalus polysaccharide and sodium periodate as raw materials; the mass ratio of astragalus polysaccharide to sodium periodate is 5:(2~3); (2) The bacterial suspension of *Lactobacillus paracasei* is mixed with the ternary mixture to obtain a probiotic suspension; wherein, the *Lactobacillus paracasei* used in the bacterial suspension is named *Lactobacillus paracasei* (… Lacticaseibacillus paracasei M11-7, deposited at Guangdong Provincial Center for Microbial Culture Collection, accession number GDMCC NO: 66511, deposited on June 13, 2025; (3) The probiotic suspension is added to the metal ion solution to form gel balls, and then solidified to obtain the probiotic delivery system for lowering blood pressure.

2. The probiotic delivery system for lowering blood pressure according to claim 1, characterized in that, In step (1), the mass concentration of the oxidized astragalus polysaccharide solution is 0.6%~1.0%w / v; the mass concentration of the carboxymethyl chitosan solution is 0.4%~0.6%w / v; and the concentration of the sodium alginate solution is 1.5%~2.5%w / v.

3. The probiotic delivery system for lowering blood pressure according to claim 1 or 2, characterized in that, In step (2), the effective bacterial concentration in the Lactobacillus paracasei suspension is 10. 10 ~10 11 CFU / mL; The bacterial suspension of Lactobacillus paracasei is prepared by inoculating Lactobacillus paracasei into MRS liquid medium and incubating at 36-38°C for 15-36 hours.

4. The probiotic delivery system for lowering blood pressure according to claim 1 or 2, characterized in that, In step (2), the volume ratio of the Lactobacillus paracasei suspension to the ternary mixture is 1:(8~12).

5. The probiotic delivery system for lowering blood pressure according to claim 1 or 2, characterized in that, In step (3), the metal ion solution is one of ZnCl2 solution, CaCl2 solution, and FeCl3 solution; the concentration of the metal ion solution is 1.0%~3.0%w / v.

6. The probiotic delivery system for lowering blood pressure according to claim 1 or 2, characterized in that, In step (3), the curing time is 30~90 min.

7. The application of a probiotic delivery system for lowering blood pressure as described in any one of claims 1 to 6, characterized in that, The application is the use of a probiotic delivery system for lowering blood pressure in the preparation of products for the prevention and treatment of hypertension; the product is a pharmaceutical product.

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