Bifidobacterium longum subsp. longum strain for reducing uric acid and application thereof

Abstract

The present application belongs to the field of microbial technology, and particularly relates to a longissima subspecies of bifidobacterium longum for reducing uric acid and application thereof. The longissima subspecies of bifidobacterium longum provided by the present application is named bifidobacterium longum longissima YBT228, and the preservation number is CGMCC NO.35417. It is verified that the longissima subspecies of bifidobacterium longum has good uric acid reducing function. Further, the double-layer microgel preparation prepared by the longissima subspecies of bifidobacterium longum breaks through the technical bottleneck of traditional probiotic preparation in terms of gastric acid tolerance, targeted release and multi-function integration, significantly improves the uric acid reducing effect, and shows more significant curative effect, so as to provide a safe and effective new microbial treatment scheme for the prevention and treatment of hyperuricemia and its complications, and provide a theoretical basis and technical foundation for the development of drugs for hyperuricemia.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, and particularly relates to a longissimus bifidobacterium longum subsp. and application thereof for reducing uric acid. BACKGROUND

[0002] At present, with the popularity of high purine diet mode and the prevalence of metabolic syndrome, the global prevalence of hyperuricemia continues to rise. Uric acid, as the end product of purine metabolism, its excessive accumulation can cause gout, chronic kidney disease, cardiovascular disease and other complications. The existing clinical treatment of hyperuricemia mainly depends on xanthine oxidase inhibitors (such as allopurinol) and uric acid excretion drugs (such as benzbromarone), but these drugs have side effects such as liver and kidney toxicity, allergic reactions, and long-term use can easily lead to drug resistance, which limits their further application. Therefore, it is urgent to develop natural, non-toxic and side-effect-free products to prevent and treat hyperuricemia.

[0003] In recent years, the theory of gut-metabolic axis provides a new idea for the prevention and treatment of hyperuricemia. Studies have shown that specific probiotics can regulate uric acid metabolism through multiple mechanisms such as degradation of endogenous purines in the gut, competitive inhibition of xanthine oxidase activity, and regulation of uric acid transporter expression. However, single active probiotics and existing probiotic composite preparations face key problems such as high inactivation rate in gastric acid, low intestinal colonization efficiency, and lack of efficient uric acid-lowering function. In particular, special probiotic preparations for bidirectional regulation of uric acid synthesis and excretion are still blank.

[0004] Therefore, how to develop a uric acid-lowering probiotic and its composite preparation, so as to break through the technical bottleneck of traditional probiotics and their preparations in terms of gastric acid tolerance, targeted release and anti-uric acid multiple functions, can provide a safe and effective new microbial treatment for the prevention and treatment of hyperuricemia and its complications. SUMMARY

[0005] In order to overcome the shortcomings of the prior art, the purpose of the present application is to provide a longissimus bifidobacterium longum subsp. and application thereof for reducing uric acid. The longissimus bifidobacterium longum subsp. provided by the present application has good uric acid-lowering effect. The double-layer microgel embedding system prepared by using the longissimus bifidobacterium longum subsp. can not only effectively reduce the level of uric acid, but also relieve the increase of inflammatory factor level and liver and kidney damage caused by high uric acid, and is suitable for being developed and applied as a drug for treating hyperuricemia.

[0006] To achieve the above object, the first aspect of the present application provides a long double Bifidobacterium longum subsp. Longum YBT228, which is named as Bifidobacterium longum subsp. Longum YBT228, is preserved in the China General Microbiological Culture Collection Center on July 28, 2025, and has a preservation number of CGMCC NO.35417.

[0007] The second aspect of the present application provides the use of the above-mentioned Bifidobacterium longum subsp. Longum YBT228 in the preparation of a double-layer microgel preparation for reducing uric acid.

[0008] The third aspect of the present application provides a preparation method of a double-layer microgel preparation for reducing uric acid, comprising the following steps:

[0009] (1) thiol-modified chitosan is obtained by thiol-modifying chitosan, and a thiol-modified chitosan solution is obtained by dissolving the thiol-modified chitosan;

[0010] In addition, the Bifidobacterium longum subsp. Longum is activated and cultured to obtain a bacterial suspension, and the bacterial suspension is centrifuged to obtain bacterial bodies. The Bifidobacterium longum subsp. Longum is named as Bifidobacterium longum subsp. Longum YBT228, is preserved in the China General Microbiological Culture Collection Center on July 28, 2025, and has a preservation number of CGMCC NO.35417.

[0011] (2) the bacterial bodies are resuspended in the thiol-modified chitosan solution for co-incubation, and then centrifuged to obtain single-layer microcapsules;

[0012] (3) the single-layer microcapsules are resuspended in a mixed solution containing gellan gum and shellac, and then subjected to gas shearing to obtain droplets, which are then injected into a solidification liquid for solidification, thereby obtaining the double-layer microgel preparation for reducing uric acid.

[0013] As a preferred scheme, in step (1), the preparation method of the thiol-modified chitosan comprises the following steps:

[0014] (a) L-cysteine hydrochloride monohydrate is dissolved in an HCl aqueous solution to obtain a Cys solution; N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride are added to the Cys solution under stirring, and the mixture is stirred for 1-3 h at room temperature to obtain an activated Cys solution; chitosan is dissolved in an HCl aqueous solution to obtain a chitosan solution;

[0015] (b) drop the chitosan solution obtained in step (a) into the activated Cys solution under stirring, then adjust the pH of the system to 5-6, and stir the reaction at room temperature for 4-8 h; after the reaction is completed, the generated product is subjected to dialysis and freeze-drying, thereby obtaining the thiolated chitosan;

[0016] The mass ratio of the L-cysteine hydrochloride monohydrate, N-hydroxysuccinimide, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and chitosan is (0.8-1.2):(1.1-1.5):(3-3.5):1.

[0017] As a preferred solution, in step (1), the process of activating the culture is as follows: inoculate Bifidobacterium longum subsp. longum in mMRS liquid medium at a inoculation amount of 1-3% V / V for subculture, then centrifuge to obtain bacterial slurry, and then resuspend the bacterial slurry in PBS to obtain a bacterial suspension.

[0018] As a preferred solution, in step (1), the concentration of the thiolated chitosan solution is 1-1.4 g / mL; and the effective bacterial concentration in the bacterial suspension is (0.8-1.2) × 10 8 CFU / mL.

[0019] As a preferred solution, in step (2), the amount of the thiolated chitosan solution corresponding to 0.8-1.2 mL of bacterial bodies obtained after centrifugation of the bacterial suspension is 0.8-1.2 mL; and the co-incubation is mixed by shaking at 36-38°C for 20-60 min.

[0020] As a preferred solution, in step (3), the mass concentration of gellan gum in the mixed solution is 0.8-1.2% w / v, and the mass concentration of shellac is 0.8-1.2% w / v; the amount of the mixed solution corresponding to 0.8-1.2 mL of the bacterial suspension prepared is 0.8-1.2 mL; the gas flow rate of the gas shearing is 1-2 L / min; and the solidification liquid is a calcium chloride solution with a concentration of 1-5 wt%.

[0021] The fourth aspect of the present application provides a uric acid-lowering double-layer microgel preparation prepared by the above preparation method.

[0022] The fifth aspect of the present application provides the application of the above uric acid-lowering double-layer microgel preparation, and the application is the application of the uric acid-lowering double-layer microgel preparation in the preparation of a drug for preventing and treating hyperuricemia.

[0023] The technical solution of the present application has the following comprehensive advantages and beneficial effects:

[0024] The longissimus bifidobacterium longum subsp. YBT228 provided by the application is verified to have good uric acid reducing function, and its action mechanism comprises: reducing the uric acid level in serum by inhibiting the activity of xanthine oxidase and the expression of uric acid transporter protein; reducing the serum IL-1beta and TNF-alpha levels, and relieving the systemic inflammation caused by hyperuricemia; reducing the serum creatinine, urea nitrogen, aspartate aminotransferase and alanine aminotransferase levels, and improving the liver and kidney damage.

[0025] Further, the uric acid reducing double-layer microgel preparation provided by the application is prepared by first preparing a single-layer microcapsule (YBT228@CS-SH) by using a layer-by-layer self-assembly technology, and then preparing a double-layer microgel preparation (YBT228@CS-SH / GS) with a core-shell structure by using a gas shearing technology. The double-layer microgel preparation not only breaks through the technical bottlenecks of traditional probiotic preparations in terms of gastric acid tolerance, targeted release and multi-function integration, but also significantly improves the uric acid reducing effect, and shows more significant curative effect.

[0026] Therefore, the bifidobacterium longum longissimus subsp. and the double-layer microgel preparation thereof provided by the application can provide a safe and effective microbial treatment new scheme for the prevention and treatment of hyperuricemia and its complications, and can also provide a theoretical basis and technical foundation for the drug development of hyperuricemia. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 is the embedding rate determination result of different microgel preparations in the application;

[0028] Figure 2 is the survival rate determination result of different microgel preparations in the application in simulated gastric juice and simulated intestinal juice;

[0029] Figure 3 is the influence of different microgel preparations in the application on the uric acid content in serum and urine of rats;

[0030] Figure 4 is the influence of different microgel preparations in the application on the xanthine oxidase (XOD) activity of rats;

[0031] Figure 5 is the influence of different microgel preparations in the application on the urea nitrogen and creatinine content in serum of rats;

[0032] Figure 6 is the influence of different microgel preparations in the application on the IL-1beta and TNF-alpha inflammatory factor levels in serum of rats;

[0033] Figure 7 is the influence of different microgel preparations in the application on the aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels in liver of rats;

[0034] Figure 8 The influence of different microgel preparations in the present application on the relative expression amount of URAT1 and GLUT9 mRNA of rats. DETAILED DESCRIPTION

[0035] In order to make the objects, technical solutions and advantages of the present application clearer, the present application will be clearly and completely described below in combination with specific embodiments. The special terms used below are the same as the understanding of the ordinary skilled person in the art, unless otherwise explicitly defined. Among them, “room temperature” generally refers to 15-35℃.

[0036] The biological preservation information related to the present application is as follows:

[0037] Bifidobacterium longum subsp. Longum YBT228, which is preserved in the China General Microbiological Culture Collection Center, has a preservation date of July 28, 2025, a preservation number of CGMCC NO. 35417, and a preservation address of No. 3, Beichen West Road, Chaoyang District, Beijing.

[0038] In the embodiments of the present application, chitosan (CS) with a molecular weight of 200KDa and a degree of deacetylation of ≥90% is used. L-cysteine hydrochloride monohydrate (Cys), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC·HCl), and N-hydroxysuccinimide (NSH) are purchased from Macklin Biotechnology Co., Ltd. Gellan gum (GG) is purchased from Macklin Biotechnology Co., Ltd. with a model number of G821481. Shellac (SC, i.e., lac) is purchased from Shanghai Yuanye Biotechnology Co., Ltd. with a model number of S30507.

[0039] In the embodiments of the present application, the Trizol reagent for total RNA extraction is from the Thermo Fisher Scientific brand. The first strand cDNA synthesis SuperMix kit is from Shanghai Yixing Biotechnology Co., Ltd. The TNF-α and IL-1β kits are from Beijing Chenglin Biotechnology Co., Ltd. The ALT and AST detection kits are from Nanjing Jiancheng Bioengineering Institute. The MK3 type enzyme label instrument is from the United States Thermoelectric Company. The ABI 7500 type real-time fluorescence quantitative PCR instrument is from the United States Applied Biosystems Company. The uric acid kit, urea nitrogen kit, and creatinine kit are from Beckman Coulter Experimental System (Suzhou) Co., Ltd. In addition, the gas shearing process is carried out by using a gas shearing microfluidic device, which is purchased from Shanghai Pengzan Biotechnology Co., Ltd. and is a device for preparing uniform droplets on the market in the art, and the structure and use method thereof are not particularly described in the present application. Among them, the examples and comparative examples prepared by gas shearing have a diameter of the embedding preparation between 200-300 μm. Other raw materials and equipment not mentioned are materials or equipment that can be obtained through conventional channels.

[0040] In the following embodiments of the present application, the mMRS liquid medium can be prepared according to the formula or obtained through the market channel. The preparation method of the mMRS medium is as follows: 5 g of tryptone, 2.5 g of proteose peptone, 2.5 g of yeast extract powder, 2.5 g of sodium acetate, 10 g of glucose, 1 g of diammonium hydrogen citrate, 1 g of dipotassium hydrogen phosphate trihydrate, 0.5 g of Tween 80, 0.125 g of manganese sulfate, 0.290 g of magnesium sulfate, 2.5 g of beef extract, and 0.25 g of L-cysteine hydrochloride are dissolved in 500 mL of deionized water, stirred until completely dissolved, sterilized at 121°C for 15 min, and used.

[0041] Example 1

[0042] This example provides a long bifidobacterium longum subsp. that can reduce uric acid, which is named Bifidobacterium longum subsp. Longum YBT228, which is preserved in the China General Microbiological Culture Collection Center, and the preservation date is July 28, 2025, and the preservation number is CGMCC NO. 35417.

[0043] The separation and identification process of the above-mentioned long bifidobacterium longum subsp. is as follows:

[0044] Bifidobacterium longum subsp. longum YBT228 was isolated from traditional dairy products in Xinjiang. Isolation process: fresh dairy products were transported to the laboratory at low temperature, and 1 mL sample was gradient diluted, and 100 μL was taken and coated on Bifidobacterium agar plate. The agar plate was placed in an anaerobic tank at 37°C and cultured for 48-72h. All isolates were isolated and purified by streak plate, and Bifidobacterium was isolated according to the colony morphology of the strain growing on mMRS plate and microscopic observation. The colony morphology of Bifidobacterium on agar plate was translucent or opaque white or milky white, the surface was moist and smooth convex round, and the gram staining was positive. Microscopic observation showed that the morphology was "Y" type, "V" type or rod with blunt round ends. The colonies meeting the above requirements were picked for pure culture and identified by 16S rRNA sequence analysis. After 16S rRNA sequence identification, it was confirmed as Bifidobacterium longum subsp. longum, named Bifidobacterium longum subsp. longum YBT228.

[0045] The 16S rRNA sequence of Bifidobacterium longum subsp. longum YBT228 is as follows (such as SEQ ID No. 1):

[0046] TGCGAGGCAGGTCTCTGGGCCGTTACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGATGCTGGATGTGGGGCCCGTTCCACGGGTTCCGTGTCGGAGCTAACGCGTTAAGCATCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGAAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGCGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGCTTGACATGTTCCCGACGGTCGTAGAGATACGGCTTCCCTTCGGGGCGGGTTCACAGGTGGTGCATGGTCGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGCCCCGTGTTGCCAGCGGATTATGCCGGGAACTCACGGGGGACCGCCGGGGTTAACTCGGAGGAAGGTGGGGATGACGTCAGATCATCATGCCCCTTACGTCCAGGGCTTCACGCATGCTACAATGGCCGGTACAACGGGATGCGACGCGGCGACGCGGAGCGGATCCCTGAAAACCGGTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGGCGGAGTCGCTAGTAATCGCGAATCAGCAACGTCGCGGTGAATGCGTTCCCGGGCCTTGTACACACCGCCCGTCAAGTCATGAAAGTGGGCAGCACCCGAAGCCGGTGGCCTAACCCCTTGTGGGATGGAGCCGTCTAAGGTGAGGCTCGTGATTGGGACTAAGTCGTAACAAGGTAGCCGTACCGGAAGGTGCGGCTGACTCACCACTCCCCTTAGAGT.

[0047] The example also provides a double-layer microgel formulation (YBT228@CS-SH / GS) prepared by a method comprising the following steps:

[0048] (1) Preparation of thiolated chitosan (CS-SH)

[0049] 1 g of L-cysteine hydrochloride monohydrate (Cys) was dissolved in 20 mL of 1 wt% HCl aqueous solution to obtain a Cys solution; 1.3 g of N-hydroxysuccinimide (NSH) and 3.3 g of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC•HCl) were added to the Cys solution under stirring, and the mixture was stirred at room temperature for 2 h to obtain an activated Cys solution;

[0050] Subsequently, 1 g of chitosan (CS) was dissolved in 80 mL of 1 wt% HCl aqueous solution under stirring until completely dissolved to obtain a chitosan solution;

[0051] The chitosan solution was added dropwise to the activated Cys solution under stirring, and the pH of the mixture was adjusted to 5.5±0.2 using a 1.0 M NaOH solution. The mixture was then stirred at room temperature for 6 h under a nitrogen atmosphere. After the reaction, the product was dialyzed against ultrapure water for 3 days and then freeze-dried to obtain a freeze-dried powder, which was thiolated chitosan (denoted as CS-SH). Subsequently, the CS-SH was prepared into a CS-SH solution with a concentration of 1.2 g / mL using 1 wt% HCl aqueous solution, and the solution was ready for use.

[0052] (2) Preparation of single-layer microcapsules (YBT228@CS-SH)

[0053] Bifidobacterium longum YBT228 was inoculated into mMRS liquid medium at an inoculation amount of 2% (V / V) and anaerobically cultured at 37°C for two generations, followed by subculture for 20 h. Then, the YBT228 bacterial solution obtained by culture was centrifuged at 6000 r / min for 10 min at 4°C, washed twice with PBS, resuspended in PBS, and adjusted to an effective bacterial concentration of 1×10 8 CFU / mL to obtain a bacterial suspension. Then, 10 mL of the bacterial suspension was centrifuged and resuspended in 10 mL of a CS-SH solution with a concentration of 1.2 g / mL. The mixture was shaken at 37°C for 30 min, centrifuged again, and washed twice with PBS. Finally, the single-layer microcapsules with CS-SH adsorbed on the surface of the YBT228 bacterial bodies were obtained by centrifugation, and were denoted as YBT228@CS-SH.

[0054] (3) Preparation of double-layer microgel preparation (YBT228@CS-SH / GS)

[0055] According to the concentrations of gellan gum (GG) and shellac (SC), the corresponding amounts of gellan gum and shellac were dissolved in sterile deionized water to obtain a mixed solution containing 1% (w / v) gellan gum and 1% (w / v) shellac;

[0056] The YBT228@CS-SH prepared in the above step was resuspended in 1 mL of a mixed solution containing 1% (w / v) gellan gum and 1% (w / v) shellac and mixed uniformly to obtain a mixture solution; and a microgel preparation was prepared by using a gas shearing technique. The specific process was as follows: the mixture solution was injected into the coaxial syringe of the gas shearing microfluidic device, the gas flow rate was controlled to be 1.2 L / min, so that the mixture solution was converted into uniform droplets under the shearing force generated by the nitrogen flow, and then the droplets were injected into a collection tank containing a 2 wt% calcium chloride solution (CaCl2) for solidification, thereby preparing a double-layer microgel preparation with a core-shell structure, denoted as YBT228@CS-SH / GS.

[0057] Comparative Example 1

[0058] The comparative example provides a bacterial suspension, and the preparation method thereof includes the following steps:

[0059] Bifidobacterium longum YBT228 was inoculated into mMRS liquid medium at an inoculation amount of 2% (V / V), and was subcultured for 20 h after being anaerobically cultured at 37°C for two generations; then the YBT228 bacterial liquid obtained by the culture was centrifuged at 6000 r / min for 10 min at 4°C to obtain bacterial bodies, the bacterial bodies were washed twice with PBS, and then the bacterial bodies were resuspended in PBS, and the effective bacterial concentration was adjusted to 1×10 8 CFU / mL to obtain a bacterial suspension, denoted as YBT228.

[0060] Comparative Example 2

[0061] The comparative example provides a single-layer microcapsule preparation (YBT228@CS-SH), and the preparation method thereof is different from that of Example 1 in that step (3) is omitted, and the rest of the process is the same as that of Example 1. The single-layer microcapsule obtained by the preparation is denoted as YBT228@CS-SH.

[0062] Comparative Example 3

[0063] The comparative example provides a single-layer microcapsule preparation (YBT228@GS), and the preparation method thereof is different from that of Example 1 in that the microgel does not contain thiolated chitosan, and the rest of the process is the same as that of Example 1. The specific preparation process includes the following steps:

[0064] Bifidobacterium longum YBT228 was inoculated into mMRS liquid medium at an inoculation amount of 2% (V / V), and was subcultured for 20 h after being anaerobically cultured at 37°C for two generations; then the YBT228 bacterial liquid obtained by the culture was centrifuged at 6000 r / min for 10 min at 4°C to obtain bacterial bodies, the bacterial bodies were washed twice with PBS, and then the bacterial bodies were resuspended in PBS, and the effective bacterial concentration was adjusted to 1×10 8 CFU / mL to obtain a bacterial suspension.

[0065] Take 1 mL of bacterial suspension, centrifugation to obtain YBT228 bacterial slurry; the YBT228 bacterial slurry was resuspended in 1 mL of mixed solution containing 1% (w / v) gellan gum (GG) and 1% (w / v) shellac (SC) and mixed uniformly to obtain a mixture solution; and then a microgel preparation was prepared by gas shearing technology. The specific process is as follows: the above mixture solution is injected into the coaxial syringe of the gas shearing microfluidic device, and the gas flow rate is controlled at 1.2 L / min, so that the mixture solution is converted into uniform droplets under the shearing force generated by the nitrogen flow, and then the droplets are injected into a collection tank containing 2wt% calcium chloride solution (CaCl2) for solidification, thereby preparing a single-layer microcapsule preparation of the comparative example, denoted as YBT228@GS.

[0066] Comparative Example 4

[0067] This comparative example provides a double-layer microgel preparation (YBT228@CS / GS), the difference between its preparation method and that of Example 1 is that the chitosan is not modified by thiol group, and the rest of the process is the same as Example 1. The specific preparation process includes the following steps:

[0068] (1) Preparation of chitosan (CS) solution

[0069] Chitosan (CS) was configured into a CS solution with a concentration of 1.2 g / mL for standby use.

[0070] (2) Preparation of single-layer microcapsules (YBT228@CS)

[0071] According to the inoculation amount of 2% (V / V), Bifidobacterium longum YBT228 was inoculated into mMRS liquid medium, and after two generations of anaerobic culture at 37°C, it was subcultured for 20 h; then the YBT228 bacterial liquid obtained by culture was centrifuged at 4°C for 10 min at 6000 r / min, washed twice with PBS, and then the bacterial body was resuspended in PBS, and the effective bacterial concentration was adjusted to 1×10 8 CFU / mL to obtain a bacterial suspension. Then 10 mL of bacterial suspension was resuspended in 10 mL of CS solution with a concentration of 1.2 g / mL after centrifugation, and the resulting mixture was shaken at 37°C for 30 min, then centrifuged and washed twice with PBS, and finally centrifuged to obtain single-layer microcapsules with CS adsorbed on the surface of YBT228 bacterial body, denoted as YBT228@CS.

[0072] (3) Preparation of microgel preparation (YBT228@CS / GS)

[0073] The YBT228@CS prepared in the above step was resuspended in 1 mL of a mixed solution containing 1% (w / v) gellan gum (GG) and 1% (w / v) shellac (SC) and mixed uniformly to obtain a mixed solution; and a microgel preparation was prepared by using a gas shearing technique. The specific process was as follows: the mixed solution was injected into the coaxial syringe of the gas shearing microfluidic device, the gas flow rate was controlled at 1.2 L / min, so that the mixed solution was converted into uniform droplets under the shearing force generated by the nitrogen flow, and then the droplets were injected into a collection tank containing a 2wt% calcium chloride solution (CaCl2) for solidification, thereby preparing the microgel preparation of the comparative example, which was recorded as YBT228@CS / GS.

[0074] Comparative Example 5

[0075] The comparative example provided a double-layer microgel preparation (YBT228@CS-SH / GG), and the difference between its preparation method and that of Example 1 was that the mixed solution containing 1% (w / v) gellan gum (GG) and 1% (w / v) shellac (SC) in step (3) was replaced by a solution containing 2% (w / v) gellan gum (GG), and the rest of the process was the same as that of Example 1. The double-layer microgel preparation prepared in the comparative example was recorded as YBT228@CS-SH / GG.

[0076] The application effect of the technical scheme of the present application is described below in combination with specific experimental examples. The test data obtained in the experiments were subjected to statistical analysis, and the single-factor variance analysis method (One-way ANOVA) was used to test the differences between groups. According to the standard of P<0.05, it was determined whether the difference between groups had statistical significance. In the figures, * represents P<0.05, ** represents P<0.01, *** represents P<0.001, and **** represents P<0.0001.

[0077] Experimental Example I, Determination of Encapsulation Rate

[0078] The encapsulation rate is an important indicator for evaluating the encapsulation capacity of the encapsulation method. In this experiment, the encapsulation rate of the microgel preparation was tested to investigate the encapsulation effect of different encapsulation systems on YBT228. The test method of the encapsulation rate was as follows:

[0079] (i) N0 test: the amount of YBT228 used was the same (1×10 8CFU) conditions, N0 is the initial colony count before embedding. For YBT228@CS-SH, the embedding principle is that the positive charged polysaccharide is attracted to the negative charge on the surface of the bacteria, and the initial colony count of the unembedded bacteria is the colony count of the single bacterial suspension; during the test, YBT228@CS-SH was diluted with physiological saline (0.9% NaCl solution) and then coated on mMRS agar medium, and the colony count was performed after anaerobic culture at 37°C for 48 h, and the viable bacterial count was recorded as N0. For YBT228@GS, YBT228@CS / GS, YBT228@CS-SH / GG and YBT228@CS-SH / GS, since the process of embedding involves centrifugation of the bacterial suspension to obtain bacterial slurry or resuspension of YBT228@CS and YBT228@CS-SH in a 1% (w / v) gel and 1% shellac (w / v) mixed solution or a 2% (w / v) gel mixed solution, the initial colony count is the colony count of the resuspended mixed solution; during the test, YBT228 bacterial suspension or resuspended mixed solution was diluted and then coated on mMRS agar medium, and the colony count was performed after anaerobic culture at 37°C for 48 h, and the viable bacterial count was recorded as N0.

[0080] (ii) N1 test: under the same dosage of YBT228 (1×10 8 CFU) conditions, the microgel preparations prepared in Comparative Examples 2-5 (YBT228@CS-SH, YBT228@GS, YBT228@CS / GS, YBT228@CS-SH / GG) and Example 1 (YBT228@CS-SH / GS) were placed in 9.9 mL of 0.5% sodium citrate solution, incubated at 37°C for 10 min, vortexed until the microgel preparation was completely dissolved, then diluted and coated on mMRS agar medium, and then the plate was placed in an anaerobic incubator at 37°C for 48 h, and the colony count was performed, and the viable bacterial count of YBT228 in each embedding system was recorded as N1.

[0081] The formula for calculating the embedding rate is: embedding rate = (N1 / N0) × 100%. The test results of the embedding rate are shown in Table 2. Figure 1

[0082] As shown in Table 2. Figure 1 ​As shown, YBT228@CS-SH microgel preparation utilizes the positive charge of CS-SH to adsorb the surface by mutual attraction with the negative charge of YBT228 surface. The layer-by-layer attraction of the charge has no significant effect on the growth of the bacterial body, so the embedding rate of YBT228@CS-SH reaches 99.23%. Further, the embedding rates of YBT228@GS, YBT228@CS / GS, YBT228@CS-SH / GG, and YBT228@CS-SH / GS are 81.23%, 88.97%, 94.87%, and 96.73%, respectively. The reason for the decrease in embedding rate is that there is a YBT228 bacterial body overflow phenomenon in the gel formation process. However, compared with YBT228@GS, the embedding rate of YBT228@CS / GS is significantly improved (P<0.01), indicating that the introduction of CS can interact with SC and GG, thereby further improving the embedding rate. Compared with YBT228@GS and YBT228@CS / GS, the embedding rates of YBT228@CS-SH / GG and YBT228@CS-SH / GS are significantly improved (P<0.05). Therefore, in general, YBT228@CS-SH / GG and YBT228@CS-SH / GS core-shell double-layer microgels show good embedding capacity.

[0083] Experimental Example Two, Evaluation of Anti-gastrointestinal Ability

[0084] When probiotics pass through the human gastrointestinal environment, they are usually subjected to stress from gastric acid and bile salts, causing bacterial inactivation and a sharp drop in the number of live bacteria reaching the colon. Therefore, in vitro simulation of gastrointestinal fluid digestion is an important indicator of the protective ability of embedding materials. The determination process of the anti-gastrointestinal ability is as follows:

[0085] (i) Preparation of simulated gastric fluid: 0.32 g of pepsin and 0.2 g of NaCl were dissolved in 100 mL of sterile water, and the pH value was adjusted to 2.0 to obtain the simulated gastric fluid. Preparation of simulated intestinal fluid: 0.68 g of KH2PO4, 1 g of trypsin, and 0.3 g of bile salt were dissolved in 100 mL of sterile water, and the pH was adjusted to 6.8 to obtain the simulated intestinal fluid.

[0086] (ii) Under the condition of the same amount of YBT228 (1×10 8 CFU), the bacterial suspension of Comparative Example 1, the microgel preparations prepared in Comparative Examples 2-5 (YBT228@CS-SH, YBT228@GS, YBT228@CS / GS, YBT228@CS-SH / GG), and the microgel preparation prepared in Example 1 (YBT228@CS-SH / GS) were resuspended in an equal amount of simulated gastric fluid and incubated for 2 h. Then 10 μL of the incubated liquid was serially diluted and plated on mMRS agar medium, and after anaerobic culture at 37°C for 48 h, colony counting was performed.

[0087] Similarly, the amount of YBT228 used was the same (1×10⁻⁶). 8 Under CFU conditions, the bacterial suspension of Comparative Example 1, the microgel preparations (YBT228@CS-SH, YBT228@GS, YBT228@CS / GS, YBT228@CS-SH / GG) prepared by Comparative Examples 2-5, and the microgel preparation (YBT228@CS-SH / GS) prepared in Example 1 were incubated together in an equal volume of simulated intestinal fluid for 4 h. Then, 10 μL of the incubated liquid was continuously diluted and spread on mMRS agar medium, and cultured anaerobically at 37°C for 48 h before colony counting.

[0088] The number of viable YBT228 cells after simulating gastric fluid was recorded as N2, and the number of viable YBT228 cells after simulating intestinal fluid was recorded as N3. The survival rates of simulated gastric fluid and simulated intestinal fluid were calculated using the following formulas: Simulated gastric fluid survival rate = (logN2 / logN1) × 100%; Simulated intestinal fluid survival rate = (logN3 / logN1) × 100%. Where N1 is the N1 of each embedding group in Experimental Example 1, and N0 is used during YBT228 testing. The survival rate determination results for simulated gastric fluid and simulated intestinal fluid are as follows: Figure 2 As shown. Figure 2 In the figure, (a) shows the results of the simulated gastric fluid test, and (b) shows the results of the simulated intestinal fluid test.

[0089] Depend on Figure 2 It was found that the survival rate of YBT228 bacterial suspension decreased to 49.23% and 36.93% after digestion with simulated gastric and intestinal juices, respectively. The survival rates of the five encapsulated microgel formulations after digestion with simulated gastric juices were as follows: YBT228@CS-SH / GS (95.43%) > YBT228@CS-SH / GG (89.93%) > YBT228@CS / GS (84.30%) > YBT228@GS (74.07%) > YBT228@CS-SH (61.8%). After simulated intestinal digestion, the survival rates were as follows: YBT228@CS-SH / GS (89.63%) > YBT228@CS-SH / GG (90.41%) > YBT228@CS (81.13%) > GS; YBT228@GS (65.90%) > YBT228@CS-SH (40.57%). The results indicate that YBT228@CS / GS, YBT228@CS-SH / GG, and YBT228@CS-SH / GS exhibited good probiotic protection capabilities. This may be because the outer layer of the core-shell microgel is composed of acid-resistant polysaccharides, which effectively protects the internal probiotics in the acidic environment of the stomach. Furthermore, the core-shell bilayer structure of the microgel further enhances its protective ability against probiotics.

[0090] Since there was no significant difference in the survival rate of YBT228@CS / GS, YBT228@CS-SH / GG, and YBT228@CS-SH / GS after simulating intestinal fluid, YBT228, YBT228@CS / GS, YBT228@CS-SH / GG, and YBT228@CS-SH / GS were subsequently selected for animal experiments.

[0091] Experiment Example 3: Animal Experiment

[0092] 3.1 Animal Experiment Design

[0093] The experimental animals were male Sprague Dawley rats, weighing 180-220g, housed in a monitored environment (temperature: 22-25°C, relative humidity: 45-55%, 12h light / dark cycle), with free access to standard diet and sterile water. After one week of acclimatization, they were randomly divided into: blank group, model group, YBT228 group, YBT228@CS / GS group, YBT228@CS-SH / GG group, and YBT228@CS-SH / GS group, with 8 rats in each group.

[0094] Except for the control group, rats in other groups were administered hypoxanthine and potassium oxonate solution by gavage daily (500 mg / kg / bw hypoxanthine and 500 mg / kg / bw potassium oxonate were dissolved in 0.5 wt% sodium carboxymethyl cellulose (CMC-Na) solution), with a gavage dose of 500 mg / kg / bw for 7 consecutive days.

[0095] After one week of continuous modeling, rats in the treatment group continued to be administered hypoxanthine and potassium oxonate solution by gavage. Simultaneously, 0.5 h after hypoxanthine and potassium oxonate administration, they were given YBT228, YBT228@CS / GS, YBT228@CS-SH / GG, and YBT228@CS-SH / GS, respectively, for three weeks. The control group, however, was only administered 0.5 wt% CMC-Na aqueous solution by gavage. The gavage dose in the treatment group was based on the original effective bacterial load of YBT228 used during preparation, maintaining a concentration of 1 × 10⁻⁶. 8 CFU / day. During gavage, each treatment group dissolved YBT228 and various encapsulation systems in PBS solution and administered the solution according to the corresponding dosage.

[0096] After the experiment, all animals were moved to metabolic cages the day before sacrifice to collect urine for 24 hours, and serum samples were collected from the retroorbital venous plexus of rats via capillary tubes 1 hour after the last administration. Liver and kidney tissues were collected after euthanasia. The right kidney was fixed in 4% paraformaldehyde, and the left kidney and liver samples were stored at -80°C until further analysis. Specific experimental methods are shown in Table 1.

[0097] Table 1. Grouping and Feeding Methods of Rats

[0098]

[0099] 3.2, Determination of uric acid content in rats

[0100] Increased uric acid synthesis or impaired excretion can lead to increased uric acid content in the blood, resulting in hyperuricemia. The enzyme-linked immunosorbent assay (ELISA) kit was used to quantitatively detect the uric acid (UA) level in rat serum and urine. The kit was equilibrated to room temperature before the experiment. According to the kit operation process, the reaction system was constructed, incubated, washed and developed, and finally the absorbance at 450 nm was measured by the enzyme label instrument. Combined with the standard curve, the UA concentration in serum and urine was calculated. The uric acid content in rat serum and urine is shown in Figure 3 Figure 3 (a) is the result of uric acid content in serum; (b) is the result of uric acid content in urine.

[0101] As shown in (a) of Figure 3 , after treatment with xanthine and potassium oxonate, the uric acid content in the plasma of the model group was significantly higher than that of the control group (P<0.0001), indicating that the hyperuricemia model was successful. After treatment with YBT228 bacterial suspension, the plasma uric acid level was reduced and had a significant difference (P<0.01), indicating that YBT228 can promote the metabolism of uric acid by regulating metabolic pathways, and can be used as a potential strain to alleviate hyperuricemia. After treatment with YBT228@CS-GS, YBT228@CS-SH / GG and YBT228@CS-SH / GS, the serum uric acid level was further reduced compared with the YBT228 group. This may be due to the further improvement of the number of live bacteria of YBT228 reaching the colon after embedding treatment. And due to the introduction of CS-SH, the intestinal retention rate of YBT228 was further improved. In addition, compared with YBT228@CS-GS, YBT228@CS-SH / GG and YBT228@CS-SH / GS significantly reduced the serum uric acid content (P<0.05, P<0.01). In addition, compared with YBT228@CS-SH / GG and YBT228@CS-SH / GS, YBT228@CS / GS has limited ability to alleviate the uric acid level in blood and urine, which may be due to the fact that the inner chitosan is not thiolated, and cannot effectively improve the intestinal retention of YBT228 embedded in the inner layer.

[0102] Correspondingly, as shown in (b) of Figure 3 ​As shown in (b), YBT228 promotes uric acid metabolism, leading to a further increase in uric acid levels in urine. After administration of YBT228@CS-SH / GG and YBT228@CS-SH / GS, the urinary uric acid levels increased to 1.10 and 1.23 times that of YBT228@CS-GS, respectively, with a significant difference between the two (P < 0.01). This indicates that the YBT228@CS-SH / GG and YBT228@CS-SH / GS encapsulation systems significantly enhance the uric acid metabolism capacity of YBT228 due to the presence of the inner CS-SH layer. Therefore, YBT228@CS-SH / GG and YBT228@CS-SH / GS were selected for further investigation.

[0103] 3.3 Determination of Xanthine Oxidase (XOD) Activity in Rats

[0104] XOD can catalyze the oxidation of hypoxanthine and xanthine to uric acid, thereby increasing the uric acid content in the blood and liver. Therefore, inhibiting the activity of XOD in plasma and liver is one of the common methods for controlling hyperuricemia. In this experiment, the activity of xanthine oxidase (XOD) in rat plasma and liver was determined spectrophotometrically: the reaction system contained 1.0 mM xanthine substrate, 0.1 M phosphate buffer (pH 7.4), and an appropriate amount of enzyme solution (final volume 1 mL). After starting the reaction at 37℃, the change in uric acid production at 290 nm was recorded every 30 seconds for 5 minutes. The results were based on the molar extinction coefficient of uric acid (ε = 12.3 mM). -1 cm -1 Enzyme activity was calculated, with one unit of enzyme activity (U) defined as the production of 1 μmol of uric acid per minute. The results of the rat xanthine oxidase (XOD) activity assay are shown below. Figure 4 As shown. Figure 4 In the table, (a) shows the results of XOD activity measurement in plasma; (b) shows the results of XOD activity measurement in liver.

[0105] like Figure 4 As shown in (a) and (b), compared with the model group, YBT228@CS-SH / GS significantly reduced XOD activity in plasma and liver (P < 0.001, P < 0.0001), which is consistent with the aforementioned reduction in plasma uric acid content by YBT228@CS-SH / GS. Furthermore, as... Figure 4 As shown in (a), compared with YBT228@CS-SH / GG, the XOD activity in plasma was further reduced after YBT228@CS-SH / GS treatment with a significant difference (P<0.05). This indicates that the combined use of shellac and gellan gum wall materials can further reduce the XOD activity in plasma compared with the use of gellan gum alone.

[0106] 3.4, Determination of urea nitrogen and creatinine content in rat serum

[0107] Hyperuricemia is closely related to renal failure and liver failure. Urea nitrogen and creatinine are key indicators of renal function, and the failure of renal function is usually accompanied by the increase of these two indicators in serum. In the experiment, a fully automatic biochemical analyzer was used, and the detection parameters were set according to the instructions of the kit: creatinine was detected by picric acid method (wavelength 510 nm), and urea nitrogen was detected by urease-glutamate dehydrogenase method (wavelength 340 nm). Standard, quality control and serum samples to be tested were loaded, and the instrument automatically completed sample loading, mixing, incubation and colorimetric analysis, and directly output concentration data. The determination results of urea nitrogen and creatinine content in rat serum are shown in Figure 5 Figure 5 Fig. 3 shows the determination results of urea nitrogen and creatinine content in rat serum, wherein (a) is the determination result of urea nitrogen, and (b) is the determination result of creatinine.

[0108] As shown in Figure 5 compared with the model group, the levels of urea nitrogen and creatinine in serum were significantly reduced after YBT228 administration (P<0.01, P<0.05), indicating that YBT228 not only promotes the metabolism of uric acid, but also relieves the damage of uric acid to the kidney. Compared with YBT228, the levels of urea nitrogen and creatinine in serum were further reduced after YBT228@CS-SH / GS administration, and there was a significant difference (P<0.001, P<0.01), indicating that this embedding method can better exert the function of YBT228. In addition, compared with YBT228@CS-SH / GG, YBT228@CS-SH / GS significantly reduced the content of urea nitrogen and creatinine in serum (P<0.05), indicating that the co-embedding of gellan gum and shellac can relieve the harm of hyperuricemia. Therefore, the double-layer gel preparation YBT228@CS-SH / GS prepared by the composite embedding system of YBT228 and gellan gum and shellac has great potential to relieve hyperuricemia and induced symptoms.

[0109] 3.5, Determination of inflammatory markers in rats

[0110] ​Previous studies have shown that excess uric acid in the blood can activate NLRP3 inflammasome, promote the release of pro-inflammatory factors such as IL-1β and TNF-α, and trigger inflammatory response. Therefore, the levels of IL-1β and TNF-α in plasma were evaluated. During the experiment, the rat serum samples were added to a 6-well plate, sealed with adhesive tape, and incubated at 37°C for 90 min. Then, biotinylated antibodies were added to the wells, incubated for 60 min, and then the color developing substrate in the kit was added to the wells, avoiding light for 10-15 min. Then, the termination solution was added and mixed for 10 min. The enzyme-labeled instrument was used for double-wavelength detection to determine the optical density values at 450 nm (maximum absorption wavelength) and 570 nm (reference wavelength). The determination results of IL-1β and TNF-α inflammatory factor levels in rat serum are shown in Figure 6 . Figure 6 Among them, (a) is the determination result of IL-1β; (b) is the determination result of TNF-α.

[0111] As shown in Figure 6 , compared with the blank group, the levels of IL-1β and TNF-α in the serum of the model group increased to 11.7 pg / mL and 61.7 pg / ML, respectively. After administration of YBT228, YBT228@CS-SH / GG and YBT228@CS-SH / GS, the levels of IL-1β in the serum decreased to 9.8, 8.8 and 7.4 pg / mL, respectively. The levels of TNF-α in the serum decreased to 57.4, 54.8 and 53.2 pg / mL, respectively. This indicates that YBT228 and its embedding system can further reduce the levels of pro-inflammatory factors by reducing the levels of uric acid in plasma.

[0112] 3.6, Determination of rat glutamic transaminase (AST) and glutathione transaminase (ALT) activity

[0113] Glutamic transaminase (AST) and glutathione transaminase (ALT) mainly exist in the cytoplasm of liver cells and are commonly used indicators for evaluating liver function damage in clinical practice. During the experiment, enzyme-linked immunosorbent assay (ELISA) kit was used to quantitatively detect the activities of AST and ALT in serum. Before the experiment, the kit was equilibrated to room temperature. According to the operation procedure of the kit, the reaction system was constructed, incubated, washed and developed, and finally the absorbance at 450 nm was measured by the enzyme-labeled instrument. Combined with the standard curve, the activities of AST and ALT in serum were calculated. The determination results of glutamic transaminase (AST) and glutathione transaminase (ALT) in rat liver are shown in Figure 7 . Figure 7 Among them, (a) is the determination result of ALT, and (b) is the determination result of AST.

[0114] As shown in Figure 7As shown, compared with the control group, the levels of ALT and AST in the liver of the model group increased to 55.1 and 113.1 U / L, respectively. This indicated that the liver was damaged due to hyperuricemia. After intervention by YBT228, YBT228@CS-SH / GG and YBT228@CS-SH / GS, the levels of ALT in the liver decreased to 49.5, 45.7 and 41.6 U / L, respectively, and the levels of AST in the liver decreased to 107.5, 104.4 and 99.0 U / L, respectively. The results showed that YBT228 could effectively alleviate the damage of uric acid to the liver by reducing the level of uric acid in the plasma. In particular, compared with YBT228@CS-SH / GG, YBT228@CS-SH / GS further reduced the levels of ALT and AST in the liver, and there was a significant difference (P<0.05), indicating that the embedding system of YBT228@CS-SH / GS further alleviated the damage of hyperuricemia to the liver due to the combined use of shellac and gellan gum.

[0115] 3.7, RT-qPCR detection of kidney tissue URAT1, GLUT9 gene expression

[0116] Uric acid transporter (URAT1) is mainly responsible for the reabsorption of uric acid, and glucose transporter 9 (GLUT9) is a uric acid transporter, both of which play a key role in maintaining blood uric acid levels, and uric acid excretion can usually be promoted by inhibiting the functions of URAT1 and GLUT9. During the experiment, total RNA of the kidney was extracted by Trizol reagent, and was reverse transcribed into cDNA using Hifair® III First-Strand cDNA Synthesis SuperMix reagent. The primer sequences of the target genes were designed and synthesized, and quantitative real-time polymerase chain reaction (RT-qPCR) was performed using Hieff® qPCR SYBR Green Master Mix on a System 7500 instrument. Glycerolaldehyde 3-phosphate dehydrogenase (GAPDH) was used as an endogenous control, and the reaction conditions were as follows: 3 min, 95°C; 5 s, 95°C; annealing for 1 min, 60°C; and repeating 40 times. 2 -△△Ct The relative expression amounts of rat URAT1 and GLUT9 mRNA were obtained by analysis. The primer sequences of the target genes used are shown in Table 2.

[0117] Table 2, primer sequences

[0118]

[0119] The results of determination of the relative expression amounts of rat URAT1 and GLUT9 mRNA are shown in Table 3. Figure 8 Figure 8 Table 3, determination results of the relative expression amounts of rat URAT1 and GLUT9 mRNA Figure 8 Among them, (a) is the relative expression amount of URAT1 mRNA; (b) is the relative expression amount of GLUT9 mRNA.

[0120] As shown in Figure 8 Compared with the control group, the expression levels of URAT1 and GLUT9 mRNA in the model group were significantly increased after treatment with hypoxanthine and potassium oxonate (P<0.0001, P<0.0001). Compared with the model group, the expression levels of URAT1 and GLUT9 mRNA were significantly reduced after YBT228 intervention (P<0.01, P<0.001), indicating that the mechanism of YBT228 in relieving hyperuricemia is to reduce the expression levels of URAT1 and GLUT9 mRNA, thereby promoting the excretion of uric acid and relieving the damage caused by uric acid. In addition, compared with YBT228, YBT228@CS-SH / GS further reduces the expression levels of URAT1 and GLUT9 mRNA and has a significant difference (P<0.0001, P<0.001), and YBT228@CS-SH / GG is better than YBT228@CS-SH / GS (P<0.0001, P<0.05). It is indicated that YBT228@CS-SH / GS can further improve the effect of YBT228 in relieving hyperuricemia, and can be used as a potential preparation for relieving hyperuricemia.

[0121] According to the above results, it can be known that the double-layer microgel preparation YBT228@CS-SH / GS has the most excellent uric acid-lowering effect, which indicates that the combined use of thiolated chitosan, shellac and gellan gum in the embedding system of YBT228 can maximize the relief of the harm of hyperuricemia and synergistically improve the ability of YBT228 to relieve hyperuricemia.

[0122] From the above, it can be known that by designing and comparing the application potential of YBT228@CS-SH, YBT228@GS two single-layer embedding systems and YBT228@CS / GS, YBT228@CS-SH / GG, YBT228@CS-SH / GS three double-layer embedding systems in hyperuricemia, it is confirmed that the YBT228@CS / GS, YBT228@CS-SH / GG, YBT228@CS-SH / GS double-layer core-shell microgel can effectively improve the survival rate of YBT228 in simulated gastrointestinal fluid. In addition, compared with YBT228@CS / GS and YBT228@CS-SH / GG, YBT228@CS-SH / GS can exert the best uric acid-lowering effect, which can not only effectively reduce the uric acid level, but also relieve the increase of inflammatory factor level and liver and kidney damage caused by high uric acid. From the mechanism of action, the mechanism of microgel preparation in relieving hyperuricemia is to inhibit the activity of XOD in plasma and liver, and to inhibit the expression levels of URAT1 and GLUT9 mRNA to promote the excretion of uric acid.

[0123] Therefore, the double-layer microgel preparation YBT228@CS-SH / GS provided by the application can provide a safe probiotic preparation for relieving hyperuricemia in the future, and has a wide application prospect in the drug research and development of hyperuricemia.

Claims

1. A method of preparing a dual-layer microgel formulation for lowering uric acid, characterized by, The method comprises the following steps: (1) thiol-modified chitosan is prepared by modifying chitosan, and a thiol-modified chitosan solution is obtained after the thiol-modified chitosan is dissolved; Another Bifidobacterium longum subsp. longum is activated and cultured to obtain a bacterial suspension; the bacterial suspension is centrifuged to obtain bacterial bodies; wherein the Bifidobacterium longum subsp. longum is named Bifidobacterium longum subsp. longum (ATCC 15697) Bifidobacterium longumsubsp.Longum ) YBT228, which is preserved in the China General Microbiological Culture Collection Center on July 28, 2025, and has a preservation number of CGMCC NO. 35417. (2) the bacteria are resuspended in the thiol-modified chitosan solution for co-incubation, and then centrifuged to obtain single-layer microcapsules; (3) the single-layer microcapsules are resuspended in a mixed solution containing gellan gum and shellac, and then subjected to gas shearing to obtain droplets, and then the droplets are injected into a solidification liquid for solidification, thereby obtaining the uric acid-lowering double-layer microgel preparation.

2. The method of preparing a hypouricemic bi-layer microgel formulation according to claim 1, wherein the bi-layer microgel formulation is prepared by the following steps of: In step (1), the preparation method of the thiol-modified chitosan comprises the following steps: (a) L-cysteine hydrochloride monohydrate is dissolved in an HCl aqueous solution to obtain a Cys solution; under stirring, N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride are added to the Cys solution, and the mixture is stirred at room temperature for 1-3 hours to obtain an activated Cys solution; chitosan is dissolved in an HCl aqueous solution to obtain a chitosan solution; (b) the chitosan solution obtained in step (a) is added dropwise to the activated Cys solution under stirring, and then the pH of the system is adjusted to 5-6, and the mixture is stirred at room temperature for 4-8 hours; after the reaction is completed, the product is dialyzed and freeze-dried to obtain the thiol-modified chitosan; wherein the mass ratio of L-cysteine hydrochloride monohydrate, N-hydroxysuccinimide, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and chitosan is (0.8-1.2):(1.1-1.5):(3-3.5):

1.

3. The method of preparing a hypouricemic bi-layer microgel formulation according to claim 1, wherein the bi-layer microgel formulation is prepared by the following steps of: In step (1), the process of the activated culture is as follows: Bifidobacterium longum subsp. longum is inoculated into mMRS liquid medium at a inoculation amount of 1-3% V / V for subculture, and then centrifuged to obtain bacterial slurry, and the bacterial slurry is resuspended in PBS to obtain a bacterial suspension.

4. The method of preparing a hypouricemic bi-layer microgel formulation according to claim 1, wherein the bi-layer microgel formulation is prepared by the following steps of: In step (1), the concentration of the thiolated chitosan solution is 1-1.4 g / mL; the effective bacteria concentration in the bacteria suspension is (0.8-1.2)×10 8 CFU / mL.

5. The method for preparing the uric acid-lowering bilayer microgel formulation according to claim 1, characterized in that, In step (2), 0.8-1.2 mL of the bacterial suspension is centrifuged, and the amount of the thiol-modified chitosan solution used is 0.8-1.2 mL; the co-incubation is performed by mixing and shaking at 36-38℃ for 20-60 minutes.

6. The method of preparing a hypouricemic bi-layer microgel formulation according to claim 1, wherein the bi-layer microgel formulation is prepared by the following steps of: In step (3), in the mixed solution, the mass concentration of gellan gum is 0.8-1.2% w / v, and the mass concentration of shellac is 0.8-1.2% w / v; the amount of the mixed solution used for 0.8-1.2 mL of the bacterial suspension to prepare the single-layer microcapsules is 0.8-1.2 mL; the gas flow rate of the gas shearing is 1-2 L / min; and the solidification liquid is a calcium chloride solution with a concentration of 1-5 wt%.

7. A uric acid-lowering double-layer microgel preparation prepared by the preparation method of any one of claims 1-6.

8. Use of the uric acid-lowering double-layer microgel preparation of claim 7 in the preparation of a medicament for treating hyperuricemia.

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