Daily ration feed for constructing hyperuricemia animal model as well as preparation method and application of daily ration feed

Abstract

The invention relates to a daily ration feed for constructing a hyperuricemia animal model as well as a preparation method and application thereof, and the daily ration feed comprises the following components in percentage by mass: 30%-35% of corn, 10%-12% of wheat middling, 20%-25% of wheat, 10%-15% of soybean meal, 1%-2% of a high-fat substance, 2%-3% of a premix, 1%-2% of stone powder, 0.2%-0.3% of choline chloride, 0.9%-1.4% of feed-grade minerals, 4%-9% of soybean hulls, 1%-5% of chicken meal and 2%-3% of uric acid. The composition also comprises 0.05%-0.15% of a fusobacterium nucleatum subsp. Multiforme freeze-dried powder. According to the daily ration feed, different raw material combinations and inactivated fusobacterium nucleatum subsp.polymorpha are mixed to prepare the daily ration feed, a hyperuricemia mouse model is constructed through feed feeding, and the comprehensiveness and stability of model pathological characteristics are improved through exogenous uric acid supplementation and a fusobacterium nucleatum subsp.polymorpha inflammation regulation and uric acid axis mode; the method has the advantages that mouse stress is reduced, operation is simpler and more convenient, the built hyperuricemia mouse model is higher in modeling rate, the model is more stable and safer, the modeling process is quicker, and the health problems such as renal failure metabolism can be effectively avoided in the modeling process.

Description

Technical Field

[0001] This invention relates to the field of experimental animal model technology, and more particularly to the field of constructing animal models of hyperuricemia, specifically to a dietary feed for constructing animal models of hyperuricemia, its preparation method, and its application. Background Technology

[0002] Hyperuricemia ( Hyperuricemia Hyperuricemia (HUA) is a chronic metabolic disease caused by purine metabolism disorders, which can be divided into primary and secondary types. Primary hyperuricemia is mainly caused by decreased uric acid excretion or increased uric acid production, while secondary hyperuricemia is caused by certain systemic diseases or the use of certain drugs that inhibit uric acid excretion. In recent years, with rapid economic development and changes in human dietary structure, the traditional diet mainly composed of vegetables and carbohydrates has shifted to a diet rich in purines, such as meat, dairy products, and high-protein foods, leading to a year-on-year increase in the incidence of hyperuricemia.

[0003] Numerous epidemiological and genetic studies have shown that hyperuricemia plays an independent role in the risk of coronary artery disease, heart failure, chronic kidney disease, and cardiovascular death.

[0004] The main clinical symptom of hyperuricemia is elevated blood uric acid. When it develops into gout, it can present with acute arthritis, tophi, and other clinical manifestations. Accompanying symptoms often include obesity, type 2 diabetes, dyslipidemia, hypertension, and arteriosclerosis. Currently, the main drugs used clinically to treat hyperuricemia include allopurinol, febuxostat, and probenecid, which primarily work by inhibiting uric acid production or promoting its excretion to improve hyperuricemia. However, long-term use of these drugs commonly has side effects, such as inducing acute kidney disease, chronic kidney disease, and hepatotoxicity. To make the treatment of hyperuricemia more effective and rational, further in-depth research is needed on the pathogenesis of hyperuricemia, drug screening, and treatment methods.

[0005] Existing animal models mostly induce hyperuricemia by adding adenine or yeast powder to the feed, but they have problems such as long modeling time and unstable pathological characteristics. In particular, they ignore the key role of gut microbiota dysbiosis in hyperuricemia and lack simulation of the "microbiota-inflammation-uric acid metabolism" axis. Therefore, it is still necessary to study a mouse model that is closer to the human hyperuricemia process in terms of more stable pathological manifestations, shorter modeling time, and better modeling effect.

[0006] There is currently no effective solution to the problems of side effects, long modeling time, and poor modeling results when constructing hyperuricemia models using drugs. Summary of the Invention

[0007] The purpose of this invention is to address at least one deficiency in the existing technology by providing a dietary feed for constructing an animal model of hyperuricemia, its preparation method, and its application. By feeding mice with this dietary feed, the model achieves comprehensive improvement in the pathological characteristics and stability of the model through "exogenous uric acid supplementation + regulation of the inflammation-uric acid axis by Fusobacterium nucleatum polymorphum subsp.," resulting in good modeling effect, strong stability, and balanced nutrition. This effectively improves the modeling effect and success rate of hyperuricemia and avoids health problems such as renal failure and metabolism during the modeling process. It effectively solves the problems of side effects, long modeling time, and poor modeling effect in related technologies that use drugs to construct hyperuricemia models.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect of the present invention is to provide a diet for constructing an animal model of hyperuricemia, comprising, by weight percentage: 30%~35% corn, 10%~12% wheat middlings, 20%~25% wheat, 10%~15% soybean meal, 1%~2% high-fat substances, 2%~3% premix, 1%~2% limestone, 0.2%~0.3% choline chloride, 0.9%~1.4% feed-grade minerals, 4%~9% soybean hulls, 1%~5% chicken meal, and 2%~3% uric acid.

[0009] Further, by weight percentage, it includes the following components: 33%~34% corn, 11%~12% wheat middlings, 23%~23.5% wheat, 13%~14% soybean meal, 2.5%~3% premix, 1%~2% high-fat substances, 1%~1.5% limestone, 0.2%~0.3% choline chloride, 1.3%~1.4% feed-grade minerals, 4%~5% soybean hulls, 3%~4% chicken meal, and 2%~3% uric acid.

[0010] Furthermore, the diet feed also includes 0.05% to 0.15% freeze-dried powder of *Fusobacterium nucleatum* subsp. *polymorpha*, preferably 0.1% to 0.15% freeze-dried powder of *Fusobacterium nucleatum* subsp. *polymorpha*.

[0011] Furthermore, by weight percentage, the diet consists of 33.66% corn, 11.74% wheat middlings, 23.00% wheat, 13.7% soybean meal, 2.74% premix, 1.57% high-fat substances, 1.08% limestone powder, 0.2% choline chloride, 1.37% feed-grade minerals, 4.89% soybean hulls, 3.91% chicken meal, 2% uric acid, and 0.15% freeze-dried powder of Fusobacterium nucleatum polymorpha.

[0012] Furthermore, the polymorphic subspecies of *Fusobacterium nucleatum* (… Fasobacterium nucleatum subsp.polymorphumIt is named AK17, with accession number CCTCC NO: M2026008, accession date January 4, 2026, and deposited by China Center for Type Culture Collection.

[0013] Furthermore, the concentration of the *Fusobacterium nucleatum* polymorphic subspecies is 1 × 10⁻⁶. 8 ~1×10 9 CFU / kg; preferably 5×10 8 CFU / kg.

[0014] Furthermore, the *Fusobacterium nucleatum* polymorphic subspecies is 100% inactivated.

[0015] Furthermore, the premix includes amino acids and vitamins; wherein the amino acids include methionine, lysine, leucine, isoleucine, valine, threonine, and tryptophan; and the vitamins include vitamin B2, vitamin B3, vitamin B5, vitamin B6, and biotin.

[0016] Further, in the diet feed, the premix contains, by weight percentage, 0.45%~0.49% methionine, 0.54%~0.58% lysine, 0.41%~0.45% leucine, 0.35%~0.39% isoleucine, 0.32%~0.36% valine, 0.35%~0.40% threonine, and 0.11%~0.15% tryptophan; and / or, Furthermore, in the diet feed, the vitamins in the premix include, by mass percentage, 0.014%~0.017% vitamin B2, 0.024%~0.027% vitamin B3, 0.006%~0.009% vitamin B5, 0.007%~0.010% vitamin B6, and 0.001%~0.002% biotin.

[0017] Further, the high-fat substance includes at least one selected from palm oil, butter, lard, anhydrous butter, margarine, soybean oil, and corn oil. Preferably, the high-fat substance is soybean oil.

[0018] Further, the feed-grade mineral includes at least one of magnesium oxide, ferric citrate, zinc carbonate, manganese carbonate, copper carbonate, magnesium chloride, and dicalcium phosphate. Preferably, the feed-grade mineral is a mixture of magnesium oxide, sodium chloride, and dicalcium phosphate.

[0019] A second aspect of the present invention is to provide a method for preparing a diet feed as described in the first aspect, comprising the following steps: S1. First, culture a strain of Fusobacterium nucleatum polymorpha, then inactivate it to prepare freeze-dried powder of Fusobacterium nucleatum polymorpha; S2. Weigh out corn, wheat middlings, wheat, soybean meal, and soybean hulls according to the predetermined mass percentages, mix and crush them to obtain the first mixture; S3. Mix the chicken powder with the first mixture according to the predetermined mass percentage to obtain the second mixture, and pre-cook it. After cooling, pulverize it to obtain the pulverized mixture. S4. Mix the pulverized mixture with the premix, stone powder, soybean oil, choline chloride, uric acid, high-fat substances, and freeze-dried powder of Fusobacterium nucleatum polymorpha according to the predetermined mass percentage to obtain the third mixture; S5. Condition, granulate and dry the third mixture to obtain the diet feed.

[0020] Further, in step S1, the culture of Fusobacterium nucleatum polymorphic subspecies includes the following steps: picking Fusobacterium nucleatum polymorphic subspecies and inoculating it into Columbia blood agar medium, and culturing it in an anaerobic environment at 37°C for 30-50 h; preferably, culturing it at 37°C for 40-48 h.

[0021] Further, in step S1, the inactivation includes the following steps: collecting colonies, washing with sterile physiological saline 2-4 times, and adjusting the bacterial concentration to 1×10⁻⁶. 8 ~1×10 9 CFU / kg, inactivated by moist heat sterilization or gamma ray irradiation; preferably, the bacterial concentration is adjusted to 5×10⁻⁶. 8 CFU / mL, when using moist heat sterilization, inactivate at 121℃ for 20 min.

[0022] Furthermore, when using gamma-ray irradiation for inactivation, the irradiation intensity is 25~35 kGy, the irradiation time is 15~30 min, and the ambient temperature is controlled at 20~30℃ during the irradiation process.

[0023] In one specific embodiment, the preparation of freeze-dried powder of *Fusobacterium nucleatum* subsp. *polymorphum* includes the following steps: The preserved strain of *Fusobacterium nucleatum* subsp. *pylori* was inoculated onto Columbia blood agar medium (containing 5% sheep blood) and incubated anaerobicly at 37°C for 48 h. Colonies were then collected, washed three times with sterile physiological saline, and the bacterial concentration was adjusted to 1×10⁻⁶. 11CFU / mL, with an ambient temperature controlled at 20~30℃, sterilized by moist heat (121℃, 20 min) or by γ-ray irradiation at an intensity of 25~35 kGy for 15~30 min. After inactivation, 100% inactivation was verified by plate counting (plate count colony count = 0). Add a cryoprotectant (by mass ratio, glycerol:sucrose:skim milk powder = 1:2:3) to the inactivated bacterial solution. The amount of cryoprotectant added is 10%~15% of the volume of the bacterial solution. After vortexing and mixing, pre-cool at 4℃ for 30~60 min for freeze-drying treatment. Dispense the pre-cooled bacterial solution into sterile freeze-drying bottles, 5~10 mL per bottle, and place them in a freeze dryer. Freeze at -40℃~-50℃ for 2~4 h to completely freeze the bacterial solution. Maintain a vacuum of 10~30 Pa and gradually raise the temperature to -10~0℃ for 12~18 h to remove most of the free water. Maintain a vacuum of 5~15 Pa and raise the temperature to 20~25℃ for 4~6 h to remove bound water. After freeze-drying, quickly seal the freeze-drying bottles and store them at 4℃ to obtain freeze-dried powder of Fusobacterium nucleatum polymorpha (moisture content ≤3%).

[0024] Furthermore, the diet feed is mixed as a whole using a stainless steel trough mixer, with a mixing temperature of 20~30℃; preferably 25℃.

[0025] Furthermore, in step S2, the mixing time for preparing the first mixture is 2 to 4 minutes; preferably 3 minutes.

[0026] Furthermore, in step S3, the mixing time for preparing the second mixture is 6 to 10 minutes; preferably 8 minutes.

[0027] Furthermore, in step S4, the mixing time for preparing the third mixture is 12-18 min; preferably 15 min.

[0028] Furthermore, in step S5, the granulation is performed using a single-screw extrusion granulation machine.

[0029] Further, in step S5, the drying is carried out in a sampling hot air circulating oven at a temperature of 53~57℃ and a humidity of 18%~22% for 22~26 hours; preferably at 55℃ and 20% humidity for 24 hours.

[0030] The third aspect of the present invention is to provide a method for constructing an animal model of hyperuricemia, wherein mice are fed a diet as described in any of the first aspects for four weeks, with free access to food and water, and a light and dark cycle during the feeding period, and the feeding environment temperature is 20~25°C and the humidity is 40%~70%.

[0031] A fourth aspect of the present invention is to provide the use of a diet as described in the first aspect, or a preparation method as described in the second aspect, or a method as described in the third aspect, in the construction of a mouse model of hyperuricemia.

[0032] Furthermore, the success rate of constructing a mouse model of hyperuricemia was 87.5%–100%.

[0033] Furthermore, in the constructed hyperuricemic mouse model, serum uric acid and xanthine oxidase levels were increased.

[0034] The present invention adopts the above technical solution and has the following technical effects compared with the prior art: First, this invention innovatively proposes a mouse diet formulation that uses different combinations of raw materials mixed with inactivated Fusobacterium nucleatum polymorpha subsp. 1, to prepare a diet. By feeding mice with this diet, a mouse model of hyperuricemia can be constructed. The constructed hyperuricemia mouse model has a higher success rate, is more stable and safer, and the modeling process is faster. During the modeling process, health problems such as renal failure and metabolism can be effectively avoided. According to the model indicators required, the construction method of this invention can be used to construct a hyperuricemia mouse model with automated and customized feeding methods, which has important application significance and research value.

[0035] Secondly, live bacteria intervention may colonize and multiply in the mouse intestine, causing non-targeted effects such as intestinal flora imbalance and mucosal inflammation, and even interfering with model stability. This invention uses inactivated Fusobacterium nucleatum polymorpha subsp., which is highly safe, avoids the risk of live bacteria colonization, and completely eliminates its proliferative capacity, retaining only functional active ingredients such as cell wall components (such as lipopolysaccharide LPS and fimbriae protein) and nucleic acid fragments. This allows it to exert regulatory effects through intestinal mucosal immune or metabolic pathways while avoiding the infection risks associated with live bacteria. At the same time, the physicochemical properties of inactivated bacteria are stable, and their active ingredients (such as antigenic epitopes and metabolism-related molecules) are not easily degraded, facilitating batch-to-batch quality control. By adjusting the concentration of inactivated bacteria added to the feed, the intake of mice can be precisely controlled, reducing experimental errors and improving data repeatability.

[0036] In addition, adding appropriate long-term intervention to the feed can reduce stress in mice. Compared with gavage administration, adding inactivated bacteria to the feed can achieve free-feeding intervention, which is simpler to operate and can avoid stress damage to mice caused by gavage (stress may affect uric acid metabolism-related indicators). This method is especially suitable for combined intervention with high-fat and high-sugar model diets, which can simulate the pathophysiological scenario of clinical "diet-microbiota" interaction and fit the pathogenesis research of hyperuricemia.

[0037] The polymorphic subspecies of *Fusobacterium nucleatum* involved in this invention is classified as *Fusobacterium nucleatum* polymorphic subspecies (… Fasobacterium nucleatum subsp.polymorphum ), Latin literary name Fasobacterium nucleatum subsp.polymorphum Hereafter referred to as AK17, it was deposited at the China Center for Type Culture Collection on January 4, 2026, with accession number CCTCC NO: M2026008, and the deposit address is Wuhan University, Wuhan, Hubei Province, China. Attached Figure Description

[0038] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this invention, illustrate exemplary embodiments of the invention and are for illustrative purposes only, and do not constitute an undue limitation of the invention. In the drawings: Figure 1 This is a graph showing the changes in body weight of mice in each group in one embodiment of the present invention; Figure 2 This is a graph showing the uric acid levels of mice in each group at week 4 in one embodiment of the present invention; Figure 3 This is a graph showing the creatinine levels of mice in each group at week 4 in one embodiment of the present invention. Figure 4 This is a graph showing the xanthine oxidase levels of mice in each group during week 4 in one embodiment of the present invention; Figure 5 This is a graph showing the results of blood urea nitrogen levels in each group of mice at week 4 in one embodiment of the present invention. Detailed Implementation

[0039] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental materials in the following embodiments that do not specify their source are all commercially available raw materials. The equipment used in each step of the following embodiments is conventional equipment. If there is no corresponding national standard, it is carried out according to general international standards, conventional conditions, or conditions recommended by the manufacturer. Unless otherwise stated, all parts are parts by weight, and all percentages are percentages by mass. Unless otherwise defined or stated, all professional and scientific terms used in the present invention have the same meaning as those skilled in the art. In addition, any methods and materials similar or equivalent to those described can be applied to the methods of the present invention.

[0040] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but this is not intended to limit the scope of the invention.

[0041] Unless otherwise specified, all products in this example are readily available through commercial purchase.

[0042] In some embodiments of the present invention, a diet for constructing an animal model of hyperuricemia is provided, comprising, by weight percentage, the following components: 30%–35% corn, 10%–12% wheat middlings, 20%–25% wheat, 10%–15% soybean meal, 1%–2% high-fat substances, 2%–3% premix, 1%–2% limestone powder, 0.2%–0.3% choline chloride, 0.9%–1.4% feed-grade minerals, 4%–9% soybean hulls, 1%–5% chicken meal, 2%–3% uric acid, and 0.05%–0.15% freeze-dried powder of *Fusobacterium nucleatum* subsp. *polymorpha*; wherein the freeze-dried powder of *Fusobacterium nucleatum* subsp. *polymorpha* is added to the diet in the form of 100% inactivated freeze-dried bacterial powder, and the *Fusobacterium nucleatum* subsp. *polymorpha* (… Fasobacterium nucleatum subsp.polymorphum It is named AK17, with accession number CCTCCNO: M2026008, accession date January 4, 2026, and deposited by China Center for Type Culture Collection.

[0043] The premix comprises amino acids and vitamins, including, by weight percentage: 0.45%~0.49% methionine, 0.54%~0.58% lysine, 0.41%~0.45% leucine, 0.35%~0.39% isoleucine, 0.32%~0.36% valine, 0.35%~0.40% threonine, 0.11%~0.15% tryptophan, 0.014%~0.017% vitamin B2, 0.024%~0.027% vitamin B3, 0.006%~0.009% vitamin B5, 0.007%~0.010% vitamin B6, and 0.001%~0.002% biotin.

[0044] The high-fat substance includes at least one of palm oil, butter, lard, anhydrous butter, margarine, soybean oil, and corn oil. Preferably, the high-fat substance is soybean oil.

[0045] The feed-grade minerals include at least one of magnesium oxide, ferric citrate, zinc carbonate, manganese carbonate, copper carbonate, magnesium chloride, and dicalcium phosphate. Preferably, the feed-grade minerals are a mixture of magnesium oxide, sodium chloride, and dicalcium phosphate.

[0046] In the formulation of this invention: corn provides the core energy source, ensuring palatability and feed intake, and avoiding interference from high carbohydrates in the model; wheat middlings supplement dietary fiber and protein, aiding pelleting and balancing absorption and intestinal motility; wheat provides synergistic energy supply and contains non-starch polysaccharides that can regulate the intestinal microenvironment; soybean meal provides high-quality plant protein and essential amino acids to meet the metabolic needs of animal growth; chicken meal supplements animal protein and essential amino acids, improves palatability, promotes animal growth and development, and improves physiological performance; soybean oil provides unsaturated fatty acids, helps the absorption of fat-soluble vitamins, and controls basal fat intake; high-fat substances simulate the characteristics of a high-fat diet, promote the absorption of uric acid precursors, and do not trigger uric acid production. Secondary metabolic abnormalities; limestone powder supplements calcium, regulates feed pH, and maintains physiological functions; choline chloride participates in fat metabolism, prevents fatty liver, and has no uric acid interference; feed-grade minerals supplement essential minerals such as magnesium and iron, ensuring basal metabolism; soybean hulls act as dietary fiber to regulate the intestines and prevent excessive obesity in mice; exogenous uric acid core modeling factor stably reaches the high uric acid threshold without acute toxicity; premix provides essential amino acids and vitamins, ensuring nutritional balance and maintaining model stability; freeze-dried powder of Fusobacterium nucleatum polymorpha regulates the "microbiota-inflammation-uric acid axis," improving the modeling rate (87.5%~100%), and the inactivation treatment balances safety and function.

[0047] In some embodiments of the present invention, a method for preparing a diet feed is also provided, comprising the following steps: (1) Inoculate the preserved strain of *Fusobacterium nucleatum* polymorpha onto Columbia blood agar medium (containing 5% sheep blood), and incubate in an anaerobic environment (85% N2 + 10% H2 + 5% CO2) at 37°C for 30-50 h. Collect colonies, wash 2-4 times with sterile physiological saline, and adjust the bacterial concentration to 1×10⁻⁶. 8 ~1×10 9 CFU / mL, completely inactivated by moist heat sterilization (121℃, 20 min) or γ-ray irradiation (plate count = 0), and stored at 4℃ for later use.

[0048] (2) The whole mixture is made by using a stainless steel trough mixer. The corn, wheat middlings, wheat, soybean meal and soybean hulls are weighed according to the predetermined mass percentage of the formula and mixed for 2-4 min and then crushed to obtain the first mixture. Then, the chicken meal is mixed with the first mixture for 6-10 min according to the predetermined mass percentage of the formula to obtain the second mixture. The mixture is pre-cured, cooled and crushed to obtain the crushed mixture. The crushed mixture is mixed with premix, stone powder, soybean oil, choline chloride, uric acid, high-fat substances and freeze-dried Fusobacterium nucleatum polymorpha subsp. 12-18 min according to the predetermined mass percentage of the formula to obtain the third mixture. Then, the mixture is conditioned, granulated and sampled by a single screw extruder. The mixture is dried in a hot air circulating oven at 50-55℃ and 18%-22% humidity for 22-26 h to obtain the diet feed.

[0049] The present invention will be described by way of example below.

[0050] Example 1 - Isolation, screening and identification of Fusobacterium nucleatum polymorphic subspecies This embodiment relates to the isolation, screening, and identification of a specific polymorphic subspecies of Fusobacterium nucleatum, and specifically includes the following steps: 1. Separation Fecal samples were taken from patients with clinical hyperuricemia, serially diluted, and spread on Columbia blood agar medium (containing 5% sheep blood). The samples were then cultured in an anaerobic environment (85% N2 + 10% H2 + 5% CO2) at 37°C and pH 7.1 ± 0.2 for 46–48 h. Circular colonies with hemolytic zones were picked for purification culture.

[0051] 2. Filtering The isolated strains were screened by Gram staining and microscopic examination, and Gram-negative fusiform bacteria were selected.

[0052] 3. Identification (1) Preliminary preparation 1) The selected target strains were inoculated into Fusobacterium-specific anaerobic culture medium (containing blood, brain and heart extract, yeast extract, vitamin K1, and heme), and cultured in an anaerobic incubator at 37°C, 80% N2 + 10% CO2 + 10% H2 for 48 h to obtain pure cultures.

[0053] 2) Preparation of bacterial suspension: Take fresh pure cultured colonies and adjust the turbidity to McFarland 0.5 with sterile physiological saline for later use.

[0054] (2) Carbohydrate fermentation experiment 1) Prepare a Fusobacterium fermentation medium containing glucose, lactose, and sucrose (final concentration of each carbon source is 1%, with phenol red indicator added), sterilize it, and dispense it into test tubes.

[0055] 2) Inoculate each fermentation tube with 0.1 mL of the above bacterial suspension, and set up a blank control with sterile culture medium. Incubate anaerobically at 37°C for 24-48 h and observe the color change of the culture medium.

[0056] The results showed that the target strain could grow normally in glucose, lactose and sucrose fermentation media, and the media changed from red to yellow (phenol red indicator changes color when it comes into contact with acid). The blank control showed no color change, indicating that the strain can ferment glucose, lactose and sucrose, which is a typical carbon source utilization characteristic of Fusobacterium nucleatum polymorpha subspecies.

[0057] (3) Detection of characteristic metabolites 48 h of anaerobic pure culture was used to detect metabolites by gas chromatography (GC): after centrifugation, acidification, and extraction, the culture was injected into the gas chromatograph. Acetic acid and propionic acid standards were used as references. The chromatographic conditions were set (capillary column 30 mm × 0.25 mm, column temperature 120 ℃, FID detector) to detect the target metabolites.

[0058] Gas chromatography analysis showed that characteristic metabolites of acetic acid and propionic acid were detected in the pure culture of this strain, and the peak area of ​​the products matched the standard by more than 99%. Other non-characteristic metabolites of Fusobacterium, such as butyric acid and valeric acid, were not detected, which is consistent with the metabolic characteristics of Fusobacterium nucleatum polymorphic subspecies.

[0059] (4) Identification by 16S rRNA gene sequencing 1) DNA Extraction: Total DNA was extracted from the pure culture strain using a bacterial genomic DNA extraction kit. DNA purity and concentration were determined by agarose gel electrophoresis (1%) and a nucleic acid quantification instrument to ensure OD... 260 / OD 280 Between 1.8 and 2.0.

[0060] 2) PCR amplification: PCR amplification was performed using universal primers for Fusobacterium (27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; 1492R: 5'-GGTTACCTTGTTACGACTT-3'). The amplification system was 25 μL (containing 1 μL DNA template, 1 μL each of forward and reverse primers, 2.5 U Taq enzyme, and 2 μL dNTPs). The amplification program was as follows: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 1 min, 30 cycles, and a final extension at 72℃ for 10 min.

[0061] 3) Product detection and sequencing: After the PCR products are verified by 1% agarose gel electrophoresis, they are sent to a professional sequencing company for bidirectional sequencing to obtain the 16S rRNA gene sequence.

[0062] 4) Sequence alignment: The obtained sequences were submitted to the GenBank database, and the BLAST software was used to perform homology comparison with the standard strain sequences of each subspecies of Fusobacterium nucleatum (Polymorphic subspecies, Fusobacterium nucleatum subspecies, and Fusobacterium viniferum subspecies) in the database to construct a phylogenetic tree.

[0063] The results are as follows: A 16S rRNA gene-specific fragment of approximately 1500 bp was successfully amplified, and a clear gene sequence without any impurities was obtained by sequencing. The homology between this strain and the standard strain of Fusobacterium nucleatum polymorphum subsp. spp. in GenBank (accession number: NR_117629.1) was 99.8%, while the homology with Fusobacterium nucleatum subsp. spp. and Fusobacterium viniferum subsp. spp. was <98.5%. Phylogenetic analysis showed that this strain clustered with the standard strain of Fusobacterium nucleatum polymorphum subsp. spp. in the same evolutionary branch, indicating the closest phylogenetic relationship.

[0064] As can be seen from the above, the biochemical characteristics of the isolated and screened strains in this embodiment, such as carbohydrate fermentation characteristics, characteristic metabolites, acid and bile salt tolerance, are highly consistent with the standard characteristics of *Fusobacterium nucleatum* subsp. *polymorphic*. Further verification of its taxonomic position was achieved through 16S rRNA gene sequencing homology and phylogenetic analysis, ultimately identifying the strain as *Fusobacterium nucleatum* subsp. *polymorphic*. Fasobacterium nucleatum subsp.polymorphum ), and named it AK17.

[0065] Example 2 - Biological characteristics of Fusobacterium nucleatum polymorphic subspecies This embodiment uses the polymorphic subspecies of Fusobacterium nucleatum preserved in Example 1 as the experimental object to study its related biological characteristics.

[0066] 1. Acid resistance test The AK17 strain was inoculated into thioethanol hydrochloric acid liquid medium at pH 2.5, 3.0, 3.5, 4.0, and 7.0, and anaerobically cultured at 37°C for 24 h. The OD of the bacterial culture was then measured. 600 The values ​​are shown in Table 1. The OD values ​​of the bacterial culture were determined using a spectrophotometer. 600 The values ​​were set up in 3 replicates for each group, and the results are expressed as mean ± standard deviation. The results show that the strain can grow normally in the pH range of 3.0 to 7.0 and still survives at pH 2.5, indicating that it has strong acid tolerance and can adapt to the acidic environment of mouse intestine.

[0067] Table 1 OD of bacterial culture under different pH conditions 600 value 2. Adhesion ability test Fusobacterium nucleatum polymorphic subsp. (standard strain / clinical isolate) was inoculated into thioglycolate liquid medium containing vitamin K1 and heme chloride, and cultured anaerobically at 37°C until mid-log (OD100). 600 =0.5~0.8); centrifuge at 4000 rpm for 5 min, wash twice with PBS, resuspend, and adjust the bacterial concentration to 1×10⁻⁶. 8CFU / mL, for later use. Caco-2 intestinal epithelial cells (purchased from the Cell Bank of the Chinese Academy of Sciences, catalog number TCHU 104, cultured in DMEM high-glucose medium containing 10% fetal bovine serum, and passaged in a 37℃, 5% CO2 incubator) were selected and cultured in DMEM medium containing 10% fetal bovine serum until the logarithmic growth phase. The cell concentration was then adjusted to 1×10⁻⁶ cells / mL. 5 Cells / mL; 100 μL of the bacterial suspension was inoculated into each well of a 96-well cell culture plate and cultured at 37°C with 5% CO2 for 24 h until the cells were fully adhered. The cell supernatant in the 96-well plate was discarded, and the cells were gently washed once with PBS. 100 μL of the above bacterial suspension was added to each well and the cells were co-cultured anaerobically at 37°C for 2 h. Three parallel wells were set up, and a blank control group (only sterile culture medium was added, without bacterial suspension) was also set up. Unadhered bacterial suspension in the wells was discarded, and the cells were gently washed three times with PBS (to avoid intestinal epithelial cell detachment). 50 μL of cell lysis buffer was added to each well, and the cells were lysed at room temperature for 10 min. The cells were thoroughly mixed by pipetting. The lysis buffer was serially diluted 10-fold, and 100 μL of the diluted solution was spread on anaerobic blood agar plates containing vitamin K1 and heme chloride. The cells were anaerobically cultured at 37°C for 48-72 h and the colony count was then performed. The results are shown in Table 2. As can be seen from Table 2, Fusobacterium nucleatum polymorpha subsp. can effectively adhere to intestinal epithelial cells, with an average adhesion rate of 29.2±3.5%. No bacteria were detected in the blank control group, which confirms that the method is accurate and specific in quantification and can effectively reflect the adhesion ability of this strain to intestinal epithelial cells.

[0068] Table 2 Adhesion Ability Test Results 3. Metabolic characteristics test The metabolites of purine substances fermented by strain AK17 were detected by high performance liquid chromatography. The results are shown in Tables 3, 4 and 5.

[0069] Table 3. Purine metabolite content (mg / L) in fermentation broth of strain AK17 Table 4. Detection results of target enzyme activity in the fermentation broth of strain AK17 (U / mL) Table 5 HPLC Detection Method Validation Parameters The results show that this strain can efficiently decompose adenine and guanine to produce uric acid precursors such as hypoxanthine and xanthine. At the same time, it secretes purine nucleoside phosphorylase and xanthine dehydrogenase with enzyme activities of (12.5±1.8) U / mL and (8.6±1.3) U / mL, respectively, providing an enzymatic basis for its regulation of uric acid metabolism.

[0070] Example 3 This embodiment relates to a specific method for preparing a daily feed diet, including the following steps: (1) Preparation of freeze-dried powder of Fusobacterium nucleatum subsp. polymorphum: The preserved strain of Fusobacterium nucleatum subsp. polymorphum was inoculated into Columbia blood agar medium (containing 5% sheep blood), and cultured in an anaerobic environment (85% N2 + 10% H2 + 5% CO2) at 37℃ for 48 h. After collecting the colonies, the bacterial solution was washed three times with sterile physiological saline and the bacterial concentration was adjusted to 5 × 10⁻⁶. 8 The bacterial culture was sterilized at CFU / mL using moist heat sterilization (121℃, 20 min) or γ-ray irradiation to completely inactivate the bacteria. 0.1 mL of the inactivated bacterial culture was spread onto Columbia blood agar medium and anaerobic cultured for 48 h. After confirming no viable bacterial growth (plate count = 0), the bacteria were lyophilized to obtain Fusobacterium nucleatum polymorpha subsp. lumbricoides lyophilized powder. The powder was then sealed in a package and stored at 4℃ for later use.

[0071] (2) Prepare the daily feed according to the following five formulas, and the preparation methods are the same; Blank group It includes the following ingredients by weight: 34.98% corn, 12.06% wheat middlings, 23.04% wheat, 13.86% soybean meal, 0.48% methionine, 0.56% lysine, 0.42% leucine, 0.36% isoleucine, 0.34% valine, 0.39% threonine, 0.13% tryptophan, 0.015% vitamin B2, 0.026% vitamin B3, 0.009% vitamin B5, 0.008% vitamin B6, 0.002% biotin, 1.57% soybean oil, 1.08% limestone powder, 0.2% choline chloride, 0.21% feed-grade sodium chloride, 0.13% feed-grade magnesium oxide, 1.03% dicalcium phosphate, 4.9% soybean hulls, and 4.2% chicken meal.

[0072] Feed composition A It includes the following ingredients by weight percentage: 33.71% corn, 11.76% wheat middlings, 23.03% wheat, 13.72% soybean meal, 0.48% methionine, 0.56% lysine, 0.42% leucine, 0.36% isoleucine, 0.34% valine, 0.39% threonine, 0.13% tryptophan, 0.015% vitamin B2, 0.026% vitamin B3, 0.009% vitamin B5, 0.008% vitamin B6, 0.002% biotin, 1.57% soybean oil, 1.08% limestone powder, 0.2% choline chloride, 0.21% feed-grade sodium chloride, 0.13% feed-grade magnesium oxide, 1.03% dicalcium phosphate, 4.9% soybean hulls, 3.92% chicken meal, and 2% uric acid.

[0073] Feed composition BIt contains the following ingredients by weight: 33.70% corn, 11.75% wheat middlings, 23.02% wheat, 13.71% soybean meal, 0.48% methionine, 0.56% lysine, 0.42% leucine, 0.36% isoleucine, 0.34% valine, 0.39% threonine, 0.13% tryptophan, 0.015% vitamin B2, 0.026% vitamin B3, 0.009% vitamin B5, 0.008% vitamin B6, 0.002% biotin, 1.57% soybean oil, 1.08% limestone powder, 0.2% choline chloride, 0.21% feed-grade sodium chloride, 0.13% feed-grade magnesium oxide, 1.02% dicalcium phosphate, 4.9% soybean hulls, 3.92% chicken meal, 2% uric acid, and 0.05% freeze-dried Fusobacterium nucleatum subsp. polymorpha.

[0074] Feed composition C It comprises the following ingredients by weight percentage: 33.68% corn, 11.75% wheat middlings, 23.01% wheat, 13.71% soybean meal, 0.48% methionine, 0.56% lysine, 0.42% leucine, 0.36% isoleucine, 0.34% valine, 0.39% threonine, 0.13% tryptophan, 0.015% vitamin B2, 0.026% vitamin B3, 0.009% vitamin B5, 0.008% vitamin B6, 0.002% biotin, 1.57% soybean oil, 1.08% limestone powder, 0.2% choline chloride, 0.21% feed-grade sodium chloride, 0.13% feed-grade magnesium oxide, 1.0% dicalcium phosphate, 4.9% soybean hulls, 3.92% chicken meal, 2% uric acid, and 0.1% freeze-dried Fusobacterium nucleatum subsp. polymorpha.

[0075] Feed composition D It contains the following ingredients by weight percentage: 33.67% corn, 11.75% wheat middlings, 23.00% wheat, 13.7% soybean meal, 0.48% methionine, 0.56% lysine, 0.42% leucine, 0.36% isoleucine, 0.34% valine, 0.39% threonine, 0.13% tryptophan, 0.015% vitamin B2, 0.026% vitamin B3, 0.009% vitamin B5, 0.008% vitamin B6, 0.002% biotin, 1.57% soybean oil, 1.08% limestone powder, 0.2% choline chloride, 0.21% feed-grade sodium chloride, 0.13% feed-grade magnesium oxide, 1.0% dicalcium phosphate, 4.89% soybean hulls, 3.91% chicken meal, 2% uric acid, and 0.15% freeze-dried Fusobacterium nucleatum subsp. polymorpha.

[0076] (3) Preparation of diet feed: The whole mixture is carried out using a stainless steel trough-type mixer. Weigh corn, wheat middlings, wheat, soybean meal, and soybean hulls according to the predetermined mass percentage and mix them for 3 min, then crush to obtain the first mixture. Then mix chicken powder with the first mixture according to the predetermined mass percentage for 8 min to obtain the second mixture, and conduct pre-cooking, and after cooling, crush to obtain the crushed mixture. Mix the crushed mixture with premix, limestone powder, soybean oil, choline chloride, feed-grade magnesium oxide, feed-grade sodium chloride, calcium hydrogen phosphate, uric acid, and freeze-dried powder of Fusobacterium nucleatum polymorphum subspecies according to the predetermined mass percentage for 15 min to obtain the third mixture. Then use a single-screw extrusion noodle machine for conditioning, granulation, and sample in a hot air circulation oven at a temperature of 55 °C and a humidity of 20% for 24 h of drying to obtain the diet feed.

[0077] Example 4 - Construction of a hyperuricemia mouse model This example relates to a specific method for constructing a hyperuricemia mouse model, which uses the feed prepared in Example 3 to verify the effects of uric acid and freeze-dried powder of Fusobacterium nucleatum polymorphum subspecies in the feed formula on model construction. The specific steps include: 1. Experimental materials: Mice: 32 adult male C57BL / 6 mice (4 weeks old) were purchased from Sibei Mansion (Beijing) Biotechnology Co., Ltd., license number: SCXK (Beijing) 2019-0010.

[0078] 2. Experimental group settings: (1) Blank group: Use the blank group feed in Example 3 as the control group.

[0079] (2) Experimental group 1: Feed with feed composition A in Example 3.

[0080] (3) Experimental group 2: Feed with feed composition B in Example 3.

[0081] (4) Experimental group 3: Feed with feed composition C in Example 3.

[0082] (5) Experimental group 4: Feed with feed composition D in Example 3.

[0083] 3. Experiment: (1) Adaptive feeding: Place the mice in steel cages, control the environmental conditions: temperature is 22 °C + 2 °C, humidity is 60% - 80%, light / dark cycle is 12 h, such as light period: 07:00 - 19:00; dark period: 19:00 - 7:00, conduct adaptive feeding for one week, with free diet and free drinking water.

[0084] (2) After the adaptation period, the weight of the mice was recorded and they were randomly divided into cages (4 mice per cage). The mice were randomly divided into a blank group (fed control diet), experimental group 1 (fed control diet + 2% uric acid), experimental group 2 (fed control diet + 2% uric acid + 0.05% *Fusobacterium nucleatum* subsp. *polymorpha* diet), experimental group 3 (fed control diet + 2% uric acid + 0.1% *Fusobacterium nucleatum* subsp. *polymorpha* diet), and experimental group 4 (fed control diet + 2% uric acid + 0.15% *Fusobacterium nucleatum* subsp. *polymorpha* diet), with 8 mice in each group. The mice were fed for four weeks under controlled environmental conditions: temperature 23℃±2°C, humidity 40%~70%, and a 12-hour light / dark cycle (light period: 07:00~19:00; dark period: 19:00~7:00). The mice had free access to food and water. The mice's mental state was observed during the feeding period. After the start of the experiment, the weight of the mice was measured at a fixed time every week (Monday). The weight results are as follows: Figure 1 As shown.

[0085] 4. Verify the model After feeding for week 4, eight male C57BL / 6 mice were selected from each group. After a 12-hour fast, the mice were anesthetized. Immediately after anesthesia, blood was collected by enucleation, centrifugation, and serum separation. Biochemical tests were performed according to the kit instructions. Specifically, uric acid (UA) content was determined using a kit (CAT: bc1365); blood urea nitrogen (BUN) content was determined using a kit (CAT: bc1535); creatinine (Cr) content was determined using a kit (CAT: bc4910); and xanthine oxidase (XOD) activity was determined using a kit (CAT: bc1095). All kits were purchased from Beijing Solarbio Biotechnology Co., Ltd. (Beijing, China). The results of the UA content determination are as follows: Figure 2 As shown, the creatinine (Cr) content detection results are as follows: Figure 3 As shown; xanthine oxidase (XOD) detection results are as follows. Figure 4 As shown; the results of the blood urea nitrogen (BUN) content determination are as follows. Figure 5 As shown.

[0086] 5. Results Analysis Depend on Figure 1 It can be seen that the body weight of mice in each group showed a clear gradient trend within 4 weeks of the experiment. The control group had the highest average body weight, at 23.75 g. The body weight of mice in the experimental groups decreased to some extent. As the intensity of the combined treatment with uric acid and Fusobacterium nucleatum polymorpha increased, the average body weight of mice decreased. The mice in the 0.1% Fusobacterium nucleatum polymorpha supplement group had the lowest body weight, at 21.81 g. No individual mice showed a sharp drop in body weight, indicating that the experimental treatment did not produce significant acute toxicity to the mice.

[0087] Depend on Figure 2As shown in Table 6, the serum UA in all mice in the blank group was within the normal range (106.37~114.89 μmol / L), and there were no individuals with hyperuricemia. In the control group with only 2% uric acid, 3 out of 8 mice developed hyperuricemia and 5 did not, with an actual modeling rate of 37.5%. The UA range of the individuals with hyperuricemia was 420.78~429 μmol / L, and the UA range of the individuals without hyperuricemia was 366.97~381.99 μmol / L, showing individual differences in borderline hyperuricemia.

[0088] In experimental groups 1-4, which were fed a 2% uric acid diet and treated with different concentrations of *Fusobacterium nucleatum* subsp. *p.*, all mice had serum UA levels ≥420 μmol / L, with model success rates of 87.5%, 100%, and 100%, respectively. Among these groups, experimental group 3 (0.1% *Fusobacterium nucleatum* subsp. *p.*) showed the highest UA levels in the model individuals, ranging from 626.93 to 675.58 μmol / L. Experimental group 4 (0.15% *Fusobacterium nucleatum* subsp. *p.*) showed slightly lower UA levels than experimental group 3 (480.87-510.2 μmol / L), but significantly higher than experimental group 2 (0.05% *Fusobacterium nucleatum* subsp. *p.*), with UA levels ranging from 400.2 to 438.6 μmol / L.

[0089] Table 6 Success rate of hyperuricemia model in mice Depend on Figure 2 , 3 It was found that the serum UA and XOD levels in each group of mice showed a highly consistent gradient, specifically: Experimental group 3 (0.1% *Fusobacterium nucleatum* subsp. *p.*) > Experimental group 4 (0.15% *Fusobacterium nucleatum* subsp. *p.*) > Experimental group 2 (0.05% *Fusobacterium nucleatum* subsp. *p.*) > Experimental group 1 (2% uric acid) > Control group. In experimental group 3, the average serum UA and XOD levels were significantly higher than in other groups; in experimental group 4, the average serum UA and XOD levels were slightly lower than in experimental group 3 but significantly higher than in experimental group 2; in experimental group 2, the average serum UA and XOD levels were significantly higher than in experimental group 1 (which only had uric acid added); in experimental group 1, the average serum UA and XOD levels were slightly higher than in the normal control group.

[0090] Depend on Figure 4 It can be seen that the serum Cr and BUN levels of mice in each group showed a slight upward trend with the increase of treatment intensity, but all were within the physiological safety range of C57BL / 6 mice. Experimental group 3 was the group with the highest treatment intensity, and its average serum Cr and BUN levels were 43.12 μmol / L and 23.46 mg / dL, respectively. No individual showed signs of kidney damage with Cr > 46 μmol / L or BUN > 25 mg / dL.

[0091] As shown above, the content of *Fusobacterium nucleatum* subsp. *polymorpha* has a significant enhancing effect on uric acid-induced hyperuricemia, and this effect is clearly concentration-dependent. Within the experimental concentration range of 0.05%–0.15%, the serum UA and XOD levels in mice initially increased and then decreased with increasing strain concentration. The 0.1% concentration group (experimental group 3) showed the best enhancing effect, while the 0.15% concentration group (experimental group 4) showed a slight metabolic tolerance effect. It is possible that low concentrations of *Fusobacterium nucleatum* subsp. *polymorpha* can regulate the intestinal flora structure of mice, inhibit uric acid excretion pathways, or promote purine metabolism, thereby synergistically increasing serum UA levels with uric acid-containing diets. High concentrations of *Fusobacterium nucleatum* subsp. *polymorpha* may induce compensatory regulation of the intestinal flora or cause slight stimulation of the intestinal mucosa, leading to adaptive adjustments in the body's metabolic pathways, ultimately resulting in a decrease in the increase of UA.

[0092] In summary, the feed formulation of this invention, while ensuring sufficient nutritional supply to mice, establishes a hyperuricemia model by adding uric acid and *Fusobacterium nucleatum* subsp. *polymorphum*. Serum UA and XOD levels are highly positively correlated, with the formulation promoting uric acid production by upregulating XOD activity. Furthermore, the optimal concentration of *Fusobacterium nucleatum* subsp. *polymorphum* was determined to be 0.1%. This provides a stable and reliable animal model for research on the interaction mechanism between hyperuricemia and gut microbiota. The modeling method of this invention results in a higher success rate, greater stability, greater safety, and faster modeling process for hyperuricemia mice. The experimental mice can be scientifically fed using automated and customized feeding methods according to the model indicators required, making it a valuable tool for constructing hyperuricemia mouse models for research purposes.

[0093] The above description is merely a preferred embodiment of the present invention and does not limit the implementation and protection scope of the present invention. Those skilled in the art should realize that any equivalent substitutions and obvious changes made based on the description and illustrations of the present invention should be included within the protection scope of the present invention.

Claims

1. A dietary diet for constructing an animal model of hyperuricemia, characterized in that, By weight percentage, it includes the following components: 30%~35% corn, 10%~12% wheat middlings, 20%~25% wheat, 10%~15% soybean meal, 1%~2% high-fat substances, 2%~3% premix, 1%~2% limestone powder, 0.2%~0.3% choline chloride, 0.9%~1.4% feed-grade minerals, 4%~9% soybean hulls, 1%~5% chicken meal, and 2%~3% uric acid.

2. The diet feed according to claim 1, characterized in that, The diet also includes 0.05% to 0.15% freeze-dried powder of Fusobacterium nucleatum polymorphum subsp. *nucleatum*, by weight percentage.

3. The diet feed according to claim 2, characterized in that, The polymorphic subspecies of *Fusobacterium nucleatum* ( Fasobacterium nucleatum subsp.polymorphum Named AK17, with accession number CCTCC NO: M2026008, accession date January 4, 2026, and deposited at the China Center for Type Culture Collection; and / or, The concentration of the polymorphic subspecies of *Fusobacterium nucleatum* was 1 × 10⁻⁶. 8 ~1×10 9 CFU / kg; and / or, The *Fusobacterium nucleatum* polymorphic subspecies was 100% inactivated.

4. The diet feed according to claim 1, characterized in that, The premix comprises amino acids and vitamins; wherein the amino acids include methionine, cystine, lysine, leucine, isoleucine, valine, threonine, and tryptophan; and the vitamins include vitamin A, vitamin D, vitamin B2, vitamin B3, vitamin B5, vitamin B6, biotin, and vitamin B1. 10 Vitamin B 12 Vitamin K, Vitamin E; and / or, The high-fat substances include at least one of palm oil, tallow, lard, anhydrous butter, margarine, soybean oil, and corn oil; and / or, The feed-grade minerals include at least one of magnesium oxide, ferric citrate, zinc carbonate, manganese carbonate, copper carbonate, magnesium chloride, and dicalcium phosphate.

5. The diet feed according to claim 4, characterized in that, In the diet feed, the premix contains, by weight percentage, 0.41%–0.45% methionine, 0.51%–0.55% lysine, 0.34%–0.37% leucine, 0.29%–0.32% isoleucine, 0.27%–0.31% valine, 0.32%–0.37% threonine, and 0.07%–0.11% tryptophan; and / or, In the diet feed, the vitamins in the premix include 0.006%~0.009% vitamin B2, 0.013%~0.016% vitamin B3, 0.004%~0.007% vitamin B5, 0.003%~0.006% vitamin B6, and 0.001%~0.002% biotin.

6. A method for preparing a dietary feed as described in any one of claims 2 to 5, characterized in that, Includes the following steps: S1. First, culture a strain of Fusobacterium nucleatum polymorpha, then inactivate it to prepare freeze-dried powder of Fusobacterium nucleatum polymorpha; S2. Weigh out corn, wheat middlings, wheat, soybean meal, and soybean hulls according to the predetermined mass percentages, mix and crush them to obtain the first mixture; S3. Mix the chicken powder with the first mixture according to the predetermined mass percentage to obtain the second mixture, and pre-cook it. After cooling, pulverize it to obtain the pulverized mixture. S4. Mix the pulverized mixture with the premix, stone powder, soybean oil, choline chloride, feed-grade sodium chloride, feed-grade magnesium oxide, dicalcium phosphate, uric acid, and freeze-dried powder of Fusobacterium nucleatum polymorpha according to the predetermined mass percentage to obtain the third mixture. S5. Condition, granulate and dry the third mixture to obtain the diet feed.

7. The preparation method according to claim 6, characterized in that, In step S1, the culture of *Fusobacterium nucleatum* subsp. *polymorpha* includes the following steps: picking *Fusobacterium nucleatum* subsp. *polymorpha* and inoculating it onto Columbia blood agar medium, and culturing it anaerobically at 37°C for 30-50 h; and / or, In step S1, the inactivation includes the following steps: collecting bacterial colonies, washing them 2-4 times with sterile physiological saline, and adjusting the bacterial concentration to 1×10⁻⁶. 8 ~1×10 9 CFU / kg, inactivated by moist heat sterilization or gamma ray irradiation; and / or The diet feed is mixed as a whole using a stainless steel trough mixer at a mixing temperature of 20~30℃; and / or, In step S2, the mixing time for preparing the first mixture is 2-4 min; and / or, In step S3, the mixing time for preparing the second mixture is 6-10 min; and / or, In step S4, the mixing time for preparing the third mixture is 12-18 min; and / or, In step S5, the granulation is performed using a single-screw extrusion granulation machine; and / or, In step S5, the drying is carried out in a hot air circulating oven at a temperature of 50~55℃ and a humidity of 18%~22% for 22~26 hours.

8. A method for constructing an animal model of hyperuricemia, characterized in that, Mice were fed the diet as described in any one of claims 1 to 5 for four weeks, with free access to food and water. During the feeding period, there was a light and dark cycle, and the feeding environment temperature was 20 to 25°C and the humidity was 40% to 70%.

9. The application of a diet as described in any one of claims 1 to 5, a preparation method as described in any one of claims 6 to 7, or the method as described in claim 8 in the construction of a mouse model with high uric acid.

10. The application according to claim 9, characterized in that, When constructing a mouse model of hyperuricemia, the success rate was 87.5%–100%; and / or, The constructed mouse model of hyperuricemia showed increased serum uric acid and xanthine oxidase levels.

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