Uric acid-lowering probiotic-phellinus igniarius compound and preparation method thereof

Abstract

This invention discloses a uric acid-lowering probiotic-Sanghuang compound and its preparation method, specifically involving the use of Mythical Pig Host-Associated Lactobacillus (Sanghuang Compound). Ligilactobacillus saerimneri The compound preparation was obtained by combining *Lactobacillus 6-2* with extracts of *Phellinus linteus*. *Lactobacillus 6-2*, associated with the Mythical Pig Host, was deposited on December 1, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36865. The extract of *Phellinus linteus* was obtained through alcohol extraction, water extraction, filtration, centrifugation, and vacuum concentration. The combination of these two preparations exhibits a good uric acid-lowering effect and can simultaneously improve liver and kidney damage induced by hyperuricemia. Combined use can also improve hyperuricemia by regulating the intestinal flora structure. Therefore, the compound preparation prepared in this invention can be used to prepare drugs for the prevention or treatment of uric acid, opening up a new avenue for the comprehensive development and utilization of probiotics and *Phellinus linteus*.

Description

Technical Field

[0001] This invention relates to the field of functional foods, specifically to a uric acid-lowering probiotic-Sanghuang compound and its preparation method. Background Technology

[0002] Hyperuricemia (HUA) is a type of metabolic disorder, and it has now become the "fourth high" after the traditional "three highs" of high blood sugar, high blood pressure, and high blood lipids. In recent years, unreasonable dietary structure and unhealthy lifestyle have become the main causes of hyperuricemia and gout. Epidemiological data in China show that the overall prevalence of HUA in my country continues to rise, and the age of onset is shifting younger. Hyperuricemia can also lead to chronic kidney disease, kidney failure, and in some cases, even endanger the patient's life and quality of life. Therefore, hyperuricemia has become a major hidden danger to the health of the Chinese people and an important public health issue. Based on this, the prevention and treatment strategies for hyperuricemia have become a research focus of great interest. Existing drugs for treating hyperuricemia have certain drawbacks: some traditional drugs have been on the market for a long time and may have serious adverse effects during use. In the treatment of hyperuricemia, common drugs such as allopurinol and benzbromarone have definite uric acid-lowering effects, but long-term use may cause various adverse reactions such as liver and kidney damage and hypersensitivity reactions. Due to various limitations in the use of drugs, it is very important to find safer and more affordable new ways to lower uric acid.

[0003] Sanghuang, also known as Sangchen or Husunyan, is a type of fungus with important medicinal value in my country and is internationally recognized as one of the best medicinal macrofungi with anti-cancer effects. Modern pharmacological studies indicate that Poplar Sanghuang (Sanghuangporus vaninii, SV) contains various active ingredients, including polysaccharides, flavonoids, polyphenols, terpenes, and amino acids and other secondary metabolites. These active ingredients enable Sanghuang to exert antioxidant, anti-tumor, hypoglycemic, lipid-regulating, and anti-inflammatory pharmacological effects, making it a high-value edible and medicinal fungus. Probiotics, on the other hand, are microorganisms that can benefit the host and are often added to food as additives or used directly as supplements to maintain the balance of intestinal microbiota.

[0004] The goal of improving hyperuricemia is to lower serum uric acid levels, thereby reducing the damage caused by high concentrations of uric acid to various organs. Hyperuricemia develops as a metabolic disorder that gradually worsens without timely control. Intervening in the production, metabolism, and excretion of uric acid through probiotics is an effective and efficient method. Furthermore, probiotics can enhance the body's resistance to hyperuricemia damage by reducing inflammatory responses and regulating gut microbiota imbalance. Therefore, developing a probiotic-Sanghuang compound preparation provides a new approach and technological direction for the efficient synergistic utilization and high-value development of microbial resources and Sanghuang medicinal resources. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a uric acid-lowering probiotic-Sanghuang compound and its preparation method.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a strain of *Lactobacillus mythologius* host-associated with pigs, 6-2, which was deposited on December 1, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36865, and classified as *Lactobacillus mythologius* host-associated with pigs (…). Ligilactobacillus saerimneri ).

[0007] In a second aspect, the present invention provides a microbial agent containing *Lactobacillus mythologius* host-associated 6-2 as described in claim 1.

[0008] The specific method for preparing the above-mentioned microbial inoculant involves inoculating *Lactobacillus mythologius* host-associated 6-2 into MRS medium, activating and culturing it at 33–40°C for 20–24 hours, centrifuging after incubation, and adding sterile physiological saline to the resulting precipitate to obtain a sample containing 10⁻⁶ viable bacteria. 7 ~10 9 CFU / g bacterial suspension.

[0009] Thirdly, the present invention provides the use of the aforementioned Mythical Pig Host-Associated Lactobacillus 6-2 or the aforementioned microbial agents in the preparation of drugs for the prevention or treatment of uric acid.

[0010] Fourthly, the present invention provides a method for preparing poplar mulberry extract, comprising the following steps: (1) Weigh the dried, crushed and sieved fruiting bodies of Populus euphratica, add 50-70% ethanol at a material-to-liquid ratio of 1:25-35, extract with ultrasonic-assisted water bath for 1-2 h, add 50-70% ethanol at the same volume as the first material-to-liquid ratio to the filter residue and extract with ultrasonic for 1-2 h, combine the two filtrates, centrifuge at 4000-5000 rpm for 10-20 min and collect the supernatant; (2) Dry the filter residue obtained in step (1), add ultrapure water at a material-to-liquid ratio of 1:40-60, extract in an ultrasonic-assisted water bath for 1-2 h, and then extract in a boiling water bath for 1-2 h. Add ultrapure water to the filter residue obtained by filtration at a material-to-liquid ratio of 1:40-60 and boil in a water bath for 1-2 h. After combining the two filtrates, collect the filtrate by coarse filtration through a filter cloth, centrifugation and vacuum filtration. (3) The supernatant obtained in step (1) and the filtrate obtained in step (2) are mixed, concentrated under reduced pressure and freeze-dried to obtain poplar mulberry extract.

[0011] Furthermore, in steps (1) and (2), the ultrasonic parameters are 40–50 kHz, 300–400 W, and the water bath temperature is 50–70 °C.

[0012] Fifthly, the present invention also provides a poplar mulberry extract prepared by the above preparation method.

[0013] In a sixth aspect, the present invention provides a compound preparation, which is prepared by mixing the above-mentioned microbial agent with the above-mentioned poplar mulberry extract.

[0014] Furthermore, the mass ratio of the microbial inoculant to the poplar mulberry extract is 1:1 to 3.

[0015] In a seventh aspect, the present invention provides the use of the above-described compound preparation in the preparation of a medicament for the prevention or treatment of uric acid.

[0016] Compared with the prior art, the advantages and positive effects of the present invention are as follows: This invention screened and isolated a strain of *Lactobacillus mythologica* associated with the host of *Pleurotus ostreatus*. Ligilactobacillus saerimneri Lactobacillus 6-2, a strain associated with the mythical pig host, possesses uric acid-lowering properties. When this strain is combined with Phellinus linteus extract, the efficacy of the compound is greater than that of its individual components, resulting in a good uric acid-lowering effect. Furthermore, it effectively improves liver damage induced by hyperuricemia while also alleviating kidney damage induced by hyperuricemia. In addition, this invention has found that combined administration can regulate the intestinal flora structure of hyperuricemic rats, increasing the abundance of beneficial bacteria and the content of beneficial metabolites, inhibiting the growth and colonization of related pathogens, reducing inflammatory responses, and maintaining intestinal homeostasis to improve the condition of hyperuricemic rats. Therefore, the compound preparation prepared in this invention can be used to prepare drugs for the prevention or treatment of uric acid, opening up a new avenue for the comprehensive development and utilization of probiotics and Phellinus linteus. Attached Figure Description

[0017] Figure 1 The colony morphology (A) and Gram staining results (B) of strain 6-2 are shown.

[0018] Figure 2 Phylogenetic tree of strain 6-2.

[0019] Figure 3 Serum biochemical indicators of rats in each group.

[0020] Figure 4 H&E staining was performed on liver sections of rats from each group (scale bar: 50 μm; 200×).

[0021] Figure 5 H&E staining was performed on kidney sections from rats in each group (scale bar: 50 μm; 200 ×).

[0022] Figure 6 The base peak chromatograms of *Phyllanthus linteus* in (A) positive ion mode and (B) negative ion mode are shown.

[0023] Figure 7 The effects of each group on the composition of the gut microbiota at the (A) phylum and (B) genus levels in HUA rats were investigated.

[0024] Figure 8 PCA score plots for rat serum samples from each group, where A1 and A2 represent ESI. + ESI - MC vs NC; B1, B2: ESI + ESI - LS vs MC; C1, C2: ESI + ESI - SV vs MC; D1, D2: ESI + ESI - LS+SV vs MC.

[0025] Figure 9 OPLS-DA score plots for rat serum samples from each group, A1-A2: ESI + ESI - MC vs NC; B1-B2: ESI + ESI - LS vs MC; C1-C2: ESI + ESI - SV vs MC; D1-D2: ESI + ESI - LS+SV vs MC.

[0026] Figure 10 Volcano diagrams showing the differential metabolites among the rat groups; (A) MC vs NC; (B) LS vs MC; (C) SV vs MC; (D) LS+SV vs MC.

[0027] Figure 11 Bubble plots showing the enrichment of differentially metabolites in the KEGG pathway among the groups; (A) MC vs NC; (B) LS vs MC.

[0028] Figure 12 Bubble plots showing the enrichment of differential metabolites in the KEGG pathway among the groups; (A) SV vs MC; (B) LS+SV vs MC. Detailed Implementation

[0029] To make the content of this invention easier to understand, the technical solution of this invention will be further described below with reference to specific embodiments, but this invention is not limited thereto.

[0030] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, all reagents and materials used in the following examples are commercially available products.

[0031] In the examples below, both Myth Pig Lactobacillus 6-2 and strain 6-2 are Myth Pig host-associated lactobacilli ( Ligilactobacillus saerimneri 6-2 was deposited on December 1, 2025 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36865.

[0032] Example 1: Screening, Isolation and Identification of Strains Fecal samples from healthy individuals were thawed after being frozen at -80°C. Then, in a sterile laminar flow hood, the fecal samples were mixed with 60% glycerol at a 1:1 volume ratio to obtain a fecal suspension. One mL of the fecal suspension was serially diluted using sterile 0.9% saline solution, and 10... -5 Up to 10 -7 Three dilution gradients of 100 μL each were inoculated onto MRS agar plates containing 20 g / L calcium carbonate (10.0 g peptone, 8.0 g beef extract, 4.0 g yeast extract, 20.0 g glucose, 2.0 g dipotassium hydrogen phosphate, 2.0 g triammonium citrate, 0.2 g magnesium sulfate, 0.04 g manganese sulfate, 1.0 g Tween-80, 5.0 g sodium acetate, 20 g calcium carbonate, 14.0 g agar). The plates were spread evenly and incubated at 37°C for 48 h. Colonies with clear calcium dissolution zones were selected for streak plating for purification until homogeneous pure cultures conforming to typical lactic acid bacteria characteristics were obtained. The cultures were then divided into three groups of samples. -5 10 -6 10 -7The strains isolated from the three dilutions were numbered 5-N, 6-N, and 7-N respectively (N represents the isolate number). The purified single colonies were inoculated into MRS liquid medium (10.0 g peptone, 8.0 g beef extract, 4.0 g yeast extract, 20.0 g glucose, 2.0 g dipotassium hydrogen phosphate, 2.0 g triammonium citrate, 0.2 g magnesium sulfate, 0.04 g manganese sulfate, 1.0 g Tween-80, 5.0 g sodium acetate, and 20 g calcium carbonate). After incubation at 37°C for 24 h, the culture was subcultured, and finally, the glycerol stock tubes were stored at -80°C. Each bacterial strain was inoculated into MRS medium at a 2% inoculum and cultured at 37℃ for 17-20 h. After centrifugation at 7000 r / min for 10 min, the supernatant was collected, filtered through a sterile 0.22 μm filter membrane, and then inactivated at 95℃ for 10 min to obtain the fermentation supernatant for each strain. The resulting bacterial sludge was washed with PBS buffer after centrifugation, and the process was repeated twice by centrifugation and washing, followed by resuspending in PBS to obtain a concentration of 1×10⁻⁶. 9 A bacterial suspension of CFU / mL was prepared. A 0.1 U / mL xanthine oxidase solution was prepared using PBS solution at pH 7.5. The concentration of xanthine oxidase was determined by reacting the enzyme in a water bath at 25°C for 30 min, and then using a solution of 1×10⁻⁶ CFU / mL. 9 The inhibitory effects of bacterial suspensions (CFU / mL) and 0.1 mL fermentation supernatant on uric acid production in a 0.2 mL system containing 0.1 U / mL xanthine oxidase to convert 2 mmol / L xanthine were assessed. Strawberry 6-2, exhibiting the best overall xanthine oxidase inhibition rate, was selected. Its bacterial suspension showed a 10.91% inhibition rate against xanthine oxidase, while its metabolites showed a 58.41% inhibition rate. Strawberry 6-2 was streaked onto MRS agar and incubated at 37°C for 24-48 h. Colony morphology was observed, and colony color, size, shape, and edge regularity were recorded. Single colonies were Gram-stained. Bacterial DNA was extracted according to the steps provided in the bacterial genomic DNA extraction kit. PCR and sequencing were performed by Sangon Biotech (Shanghai) Co., Ltd. The 16S rDNA gene sequence (SEQ ID NO.1) was determined and compared with known sequences using BLAST on the NCBI website, and a phylogenetic tree was constructed using MEGA11.0 software.

[0033] The colony morphology and Gram staining results of strain 6-2 are as follows: Figure 1 As shown, the colonies are round, white, small, and have neat edges. Under microscopic Gram staining, they appear purple and rod-shaped, indicating that this strain is a Gram-positive bacillus. BLAST analysis of the sequencing results in the NCBI database showed that strain 6-2... Ligilactobacillus saerimneri GPV03Clustering together, and combining colony morphology and Gram staining results, strain 6-2 can be identified as *Lactobacillus mythologosa*, a host-associated bacterium of pig mythology. Ligilactobacillus saerimneri ), phylogenetic tree such Figure 2 As shown. This strain 6-2 was deposited on December 1, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 36865.

[0034] Example 2

[0035] (1) Preparation of Poplar Phellinus linteus extract: Weigh 1 kg of dried and pulverized Phellinus linteus fruiting bodies, add 50% ethanol at a material-to-liquid ratio of 1:25 (v / w), and extract with ultrasound (ultrasound parameters: 40 kHz, 300 W) in a 55℃ water bath for 1 h. After filtration, add 50% ethanol at the same volume as the first material-to-liquid ratio and extract with ultrasound at 55℃ for 1 h. Combine the two filtrates, centrifuge the obtained filtrate at 4000 rpm for 10 min, filter the supernatant and collect the alcohol extract. Dry the alcohol extract residue and weigh it. Add ultrapure water at a material-to-liquid ratio of 1:40 (v / w) according to the latest weight, extract with ultrasound (ultrasound parameters: 40 kHz, 300 W) in a 55℃ water bath for 1 h, and then extract with boiling water at 100℃ for 2 h before filtration. A second extraction was performed by adding 40 times the volume of ultrapure water to the filter residue and boiling it in a water bath for 2 hours. The two filtrates were combined and subjected to coarse filtration through a filter cloth, centrifugation at 4000 r / min for 10 min, and vacuum filtration. The collected filtrate was the water extract. The poplar alcohol extract and water extract were mixed, concentrated under reduced pressure, and freeze-dried to obtain poplar mulberry extract (a dark yellow powder).

[0036] (2) Preparation of Mythical Pig Host-Associated Lactobacillus suspension: Mythical Pig Host-Associated Lactobacillus 6-2 preserved in glycerol was aseptically inoculated into MRS liquid medium, activated and cultured at 35℃ for 20 h, centrifuged at 7000 r / min for 10 min, and sterile physiological saline was added to the precipitate to obtain a viable count of 10. 7 CFU / g bacterial suspension.

[0037] (3) Preparation of probiotic-Sanghuang compound: The poplar Sanghuang extract obtained in step (1) and the bacterial suspension obtained in step (2) are mixed in a mass ratio of 1:2 (mixed and shaken well) to obtain the uric acid-lowering probiotic-Sanghuang compound.

[0038] Example 3

[0039] (1) Preparation of Poplar Phellinus linteus extract: Weigh 1 kg of dried and pulverized Phellinus linteus fruiting bodies, add 60% ethanol at a material-to-liquid ratio of 1:30 (v / w), and extract with ultrasound (ultrasound parameters: 45 kHz, 300 W) in a 60℃ water bath for 1 h. After filtration, add 60% ethanol at the same volume as the first material-to-liquid ratio and extract with ultrasound at 60℃ for 1 h. Combine the two filtrates, centrifuge the obtained filtrate at 4500 rpm for 15 min, filter the supernatant and collect the alcohol extract. Dry the alcohol extract residue and weigh it. Add ultrapure water at a material-to-liquid ratio of 1:50 (v / w) according to the latest weight, extract with ultrasound (ultrasound parameters: 45 kHz, 300 W) in a 60℃ water bath for 1 h, and then continue to extract with boiling water at 100℃ for 2 h before filtration. A second extraction was performed by adding 50 times the volume of ultrapure water to the filter residue and boiling it in a water bath for 2 hours. The two filtrates were combined and subjected to coarse filtration through a filter cloth, centrifugation at 4500 rpm for 15 minutes, and vacuum filtration to collect the filtrate, which was the water extract. The alcohol extract and water extract were mixed, concentrated under reduced pressure, and freeze-dried to obtain the poplar mulberry extract.

[0040] (2) Preparation of Mythical Pig Host-Associated Lactobacillus suspension: Mythical Pig Host-Associated Lactobacillus 6-2 was inoculated into MRS liquid medium under aseptic conditions, activated and cultured at 37℃ for 22 h, centrifuged at 7000 r / min for 10 min, and sterile physiological saline was added to the precipitate to obtain a viable count of 10. 8 CFU / g bacterial suspension.

[0041] (3) Preparation of probiotic-Sanghuang compound: The poplar Sanghuang extract obtained in step (1) and the bacterial suspension obtained in step (2) are compounded at a mass ratio of 1:2 to obtain the uric acid-lowering probiotic-Sanghuang compound.

[0042] Example 4

[0043] (1) Preparation of Poplar Phellinus linteus extract: Weigh 1 kg of dried and pulverized Phellinus linteus fruiting bodies, add 70% ethanol at a material-to-liquid ratio of 1:35 (v / w), and extract with ultrasound (ultrasound parameters: 50 kHz, 400 W) in a 70℃ water bath for 2 h. After filtration, add 70% ethanol at the same volume as the first material-to-liquid ratio and extract with ultrasound at 70℃ for 2 h. Combine the two filtrates, centrifuge the obtained filtrate at 5000 rpm for 20 min, filter the supernatant and collect the ethanol extract. Dry the ethanol extract residue and weigh it. Add ultrapure water at a material-to-liquid ratio of 1:60 (v / w) according to the latest weight, extract with ultrasound (ultrasound parameters: 50 kHz, 400 W) in a 70℃ water bath for 2 h, and then continue to extract with boiling water at 100℃ for 2 h before filtration. A second extraction was performed by adding 60 times the volume of ultrapure water to the filter residue and boiling it in a water bath for 2 hours. The two filtrates were combined and subjected to coarse filtration through a filter cloth, centrifugation at 5000 r / min for 10 min, and vacuum filtration. The collected filtrate was the water extract. The poplar alcohol extract and water extract were mixed, concentrated under reduced pressure, and freeze-dried to obtain poplar linteus extract.

[0044] (2) Preparation of Mythical Pig Host-Associated Lactobacillus suspension: Mythical Pig Host-Associated Lactobacillus 6-2 was inoculated into MRS liquid medium under aseptic conditions, activated and cultured at 40℃ for 24 h, centrifuged at 7000 r / min for 10 min, and sterile physiological saline was added to the precipitate to obtain a viable count of 10. 9 CFU / g bacterial suspension.

[0045] (3) Preparation of probiotic-Sanghuang compound: The poplar Sanghuang extract obtained in step (1) and the bacterial suspension obtained in step (2) are compounded at a mass ratio of 1:3 to obtain the uric acid-lowering probiotic-Sanghuang compound.

[0046] Application Example 1 The raw materials and reagents involved in this application example are as follows: Example 2: Poplar mulberry extract and Mythical pig host-associated Lactobacillus suspension prepared; SPF-grade male SD rats, 200-220g (from Wu's Animal Trade Co., Ltd., Minhou County, Fuzhou City, Fujian Province); Sodium carboxymethyl cellulose: 800-1200 mpa.s, USP grade, Shanghai Aladdin Biochemical Technology Co., Ltd.; Potassium oxonate, potassium oxonate: Shanghai Yuanye Biotechnology Co., Ltd.

[0047] The experimental methods involved in this application example are as follows: Dosage and route of administration: The recommended human dose of *Pinus linteus* extract is 15-20 g / day. The dose for rats is 5-6 times the human dose, which is 320 mg / kg / day. The concentration of *Lactobacillus suis* suspension in pig hosts was 10... 8CFU / g. The route of administration and volume are oral, 10 ml / kg, once daily.

[0048] Experimental Methods: Sixty rats were randomly divided into four groups: a control group (NC), a model group (MC), and four treatment groups: LS (Lactobacillus mythologius host-associated suspension), SV (Poplar mulberry extract), and LS+SV, with 12 rats in each group. All groups were provided with adequate drinking water and a basal diet. Except for the control group, all groups were administered potassium oxonate-hypoxanthine suspension by gavage daily for 7 consecutive days at a dose of 500 mg / kg potassium oxonate and 500 mg / kg hypoxanthine. Intervention began on day 8. One hour after the potassium oxonate-hypoxanthine suspension gavage, the control and model groups were administered 0.5% sodium carboxymethyl cellulose solution by gavage, while the treatment groups received the dosages listed in Table 1. This treatment was continued for 3 weeks.

[0049] The 0.5% sodium carboxymethyl cellulose (CMC-Na) solution: Accurately weigh 5 g of CMC-Na, dissolve it in a small amount of ultrapure water, transfer it to a 1000 mL volumetric flask, and sonicate it at approximately 40°C until dissolved. Make up to volume to prepare a homogeneous suspension. Dispense the required amount for each day's experiment, seal the flasks with sealing film, and store at -20°C for later use. Thaw naturally at room temperature or in a 37°C water bath 1 hour before each experiment, avoiding repeated freeze-thaw cycles.

[0050] Potassium oxonate-hypoxanthine (PO+HX) suspension: Accurately weigh appropriate amounts of potassium oxonate and hypoxanthine, dissolve them in 0.5% CMC-Na aqueous solution to prepare a homogeneous suspension, dispense into daily required amounts, seal with sealing film, and store at -20℃ for later use. Pretreatment before use is the same as for sodium carboxymethyl cellulose.

[0051] Sample solution: Accurately weigh the Phellinus linteus extract, dissolve the sample in ultrapure water, prepare the corresponding bacterial solution and Phellinus linteus extract 1 hour before the experiment, and prepare and use them fresh every day. After the Phellinus linteus extract and bacterial solution are prepared separately, shake them well before gavage and administer directly.

[0052] Table 1 Grouping and Dosing Design

[0053] After all drug administration procedures were completed, serum samples were collected from mice in each group to detect the levels of uric acid (UA), blood urea nitrogen (BUN), creatinine (Cr), and xanthine oxidase (XOD). Liver and kidney tissues from mice in each group were also collected and paraffin-embedded sections were prepared for histopathological analysis. Experimental results are expressed as mean ± standard deviation. Statistical analysis was performed using SPSS 27.0, and graphs were plotted using OriginPro 2024. One-way ANOVA was used to analyze differences between groups, with P < 0.05 considered statistically significant.

[0054] See results Figure 3 Compared with the NC group, the MC group had significantly higher levels of UA, BUN, and Cr. P <0.05), indicating that the combination of potassium oxonate and hypoxanthine successfully induced hyperuricemia. Compared with the MC group, the LS, SV, and LS+SV groups showed significant differences in UA and Cr levels after three weeks of administration. P <0.05), while LS and LS+SV have a significant impact on BUN levels ( P <0.05), significant recovery was achieved at all serum levels. Furthermore, serum XOD activity decreased significantly after intervention ( P <0.05), indicating that the treatment group can reduce uric acid production by inhibiting XOD activity.

[0055] Figure 4 The H&E staining results of rat liver sections showed that some hepatocytes in the NC group exhibited vacuolar degeneration, indicating a tendency towards fatty liver disease. This phenomenon may be related to the good health of the control group rats during the experiment, leading to rapid weight gain due to free feeding. Histological analysis showed that the hepatocyte nuclei in this group were regularly shaped (round or oval), the hepatic cords were neatly arranged, the sinusoidal structure was normal, and filled red blood cells were visible. In contrast, the hepatocytes in the MC group were significantly larger, with a large accumulation of lipid droplets causing marginal nuclear displacement, accompanied by disordered hepatic cord structure. Notably, the boundaries between hepatocytes in this group were blurred, but the red blood cell filling persisted, suggesting that hyperuricemia may induce liver damage by interfering with lipid metabolism pathways. After three weeks of drug intervention, both the LS and SV groups showed varying degrees of liver tissue improvement. The LS group showed reduced hepatocyte vacuolation but persistent red blood cell filling; the SV group maintained good sinusoidal structure in addition to reduced vacuolation; and the liver histological characteristics of the LS+SV combined intervention group were closest to those of the NC group, showing a significant reduction in fat vacuoles and a neat arrangement of hepatic cords. These results confirm that the combined use of LS and SV can effectively improve liver damage induced by hyperuricemia.

[0056] Kidney tissue section H&E staining as shown Figure 5As shown, the kidney tissue of rats in the NC group had normal morphology, intact renal cortex structure, tightly and regularly arranged renal tubular and collecting duct cells, plump and evenly distributed glomerular endothelial cells, and clear and rounded glomerular structure. The tissue sections were uniformly pink in color, and no abnormal deposits were observed in the lumen. In contrast, rats in the MC group showed obvious pathological changes in their kidneys, including glomerular atrophy, renal tubular dilation, and disordered cell arrangement. Some renal tubular cells showed vacuolar degeneration, suggesting that hyperuricemia caused damage to the kidney tissue structure. After three weeks of drug intervention, the pathological changes in the kidneys of all treatment groups improved. The degree of renal tubular dilation was reduced in the LS and SV groups, the renal tubular cast structure became more complete, and the cell arrangement was more tightly arranged than in the MC group, but some renal tubules that had not fully recovered still existed; at the same time, the glomerular atrophy was also alleviated. The improvement effect was most significant in the LS+SV combined intervention group, with a significant reduction in renal tubular dilation, only occasional glomerular atrophy and vacuolar cells, regular renal tubular arrangement, clear renal capsule structure, and renal tubular interstitial space basically returned to normal. These results indicate that LS, SV, and LS+SV combined therapy can all alleviate hyperuricemia-induced kidney damage to varying degrees, with the LS+SV combined intervention showing the best effect.

[0057] In summary, the combined effects of potassium oxonate and hypoxanthine-induced hyperuricemia can cause abnormalities in serum parameters in rats and lead to tissue lesions in the liver and kidneys. However, after 3 weeks of intervention with a combination of Mythical Pig host-associated Lactobacillus suspension and / or Populus euphratica extract, these effects can be prevented and improved to varying degrees.

[0058] UPLC-MS / MS Analysis of Phellinus linteus The extract of *Pinus linteus* (SV) was analyzed using ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS), and its base peak chromatogram (BPC) is shown in Figure 6. According to the primary mass spectrometry, 16,951 and 14,677 compounds were matched in positive and negative ion modes, respectively. Further identification using secondary mass spectrometry revealed a total of 2,092 compounds (1,316 positive ions and 776 negative ions), mainly including 147 terpenoids (7.0%), 141 organic oxygen compounds (6.7%), 130 carboxylic acids and their derivatives (6.2%), 94 benzenes and their derivatives (4.4%), 68 steroids and their derivatives (3.2%), 65 flavonoids and isoflavones (3%), and 34 phenolic compounds (1.6%). The main compounds in the SV fraction are shown in Table 2.

[0059] Table 2. Major compounds (partial list) in *Poplar mulberry fungus*

[0060] Application Example 2 After the intervention experiment in Example 1, 0.2-0.5 g of intestinal contents were collected from rats and placed in centrifuge tubes containing extraction lysis buffer. The samples were homogenized at 60 Hz for pretreatment. Nucleic acid was extracted from the pretreated intestinal contents samples of each group using the MagBeads FastDNA Kit for Soil. The extracted DNA was subjected to 0.8% agarose gel electrophoresis to determine molecular size, and quantified using Nanodrop. The V3-V4 regions of the samples were amplified using specific primers and an Applied Biosystems 2720 PCR instrument. The PCR products were detected, purified, and quantified. Sequencing libraries were constructed using the TruSeq Nano DNA LT Library Prep Kit. After the library quality was verified, the mixed library was diluted to the optimal sequencing concentration, and paired-end sequencing was performed.

[0061] The top ten phyla in terms of relative abundance at the phylum level were selected for statistical analysis, such as... Figure 7 As shown in Figure A, compared with the NC group, the intestinal flora structure of rats in the MC group was significantly altered, with a decrease in the relative abundance of Firmicutes and a significant increase in the abundance of Bacteroidota. Furthermore, the abundance of Actinobacteriota decreased in the MC group, while the abundance of Verrucomicrobiota significantly increased. Notably, Verrucomicrobiota was positively correlated with plasma phenylacetylglutamine (PAGln) levels, and elevated PAGln levels could enhance the pro-inflammatory effects of neutrophils, potentially exacerbating the inflammatory response. After intervention with LS, SV, and LS+SV, the intestinal flora structure of rats in each group showed varying degrees of recovery. Specifically, the abundance of Actinobacteriota increased, with the SV group recovering to levels close to those of the NC group. Simultaneously, the abundance of Verrucomicrobiota in all treatment groups showed a significant decreasing trend. Moreover, compared with the NC and MC groups, the abundance of Bacteroidota further increased in the treatment groups, suggesting that drug intervention may promote the proliferation of Bacteroidota. These results indicate that interventions with LS, SV, and LS+SV can regulate the gut microbiota structure in hyperuricemic rats, with the combined interventions of SV and LS+SV showing particularly significant improvement in microbiota composition.

[0062] A chart of the top 20 genera in terms of abundance at the genus level for each group of rats is shown below. Figure 7 As shown in Figure B, the gut microbiota structure of the MC group showed significant changes compared to the NC group. In the MC group... Lactobacillus, Romboutsia, Bacteroides Abundance decreased, Muribaculum The abundance increased. Although after LS intervention, Lactobacillus The abundance has decreased, but Vibrio butyricum spp. ButyrivibrioAbundance increased significantly. This genus can inhibit pathogen growth by producing butyrate through the degradation of hemicellulose and pectin. After SV intervention, Lactobacillus Abundance recovered to levels close to those of the NC group, while Romboutsia Abundance increased. Studies have shown that... Lactobacillus It can competitively inhibit the colonization of conditionally pathogenic bacteria and hinder pathogen adhesion, and exert its antibacterial effect by secreting metabolites such as organic acids, bacteriocins, and reactive oxygen species. After intervention with LS, SV, and LS+SV, the results were similar to those of the single-drug groups. Bacteroides and Alloprevotella The abundance of all samples increased significantly. Bacteroides It can regulate the synthesis and secretion of key hormones in enteroendocrine L cells, which participate in glucose homeostasis regulation, enhance intestinal barrier function, and exert anti-inflammatory effects; while Alloprevotella This can inhibit the secretion of inflammatory factors in pathogenic Th17 cells. This suggests that intervention with LS, SV, and LS+SV may improve the condition of HUA rats by increasing the abundance of beneficial bacteria and the content of beneficial metabolites in the gut microbiota, inhibiting the growth and colonization of related pathogens, reducing the body's inflammatory response, and maintaining intestinal homeostasis.

[0063] Application Example 3 This study employed non-targeted metabolomics methods, processing serum samples from experimental animals collected in Example 1 before analysis. Various analytical software and websites were used to analyze the data and identify metabolites. The identified metabolites were then analyzed to interpret the biological significance of the results.

[0064] Figure 8 The PCA results for serum samples from each group of rats showed significant differences between the NC and MC groups, indicating a significant separation of serum metabolic profiles between normal and hyperuricemic rats. This result confirms that hypoxanthine and potassium oxonate-induced hyperuricemia successfully altered the serum metabolic profiles of rats, causing differences in serum metabolites. LS, SV, and LS+SV interventions modulated the composition of rat serum metabolites. PCA is an unsupervised dimensionality reduction method that projects data based solely on the variance of the data itself, failing to utilize known group information. When inter-group differences are smaller than within-group variations, PCA often struggles to clearly distinguish between groups. OPLS-DA, as a supervised method, maximizes inter-group differences and significantly improves classification performance by introducing group label information, as shown in the results. Figure 9 As shown, OPLS-DA indicated that 4 weeks after the successful establishment of the HUA model, it could cause significant differences in the serum metabolic profile of rats; and LS, SV, and LS+SV treatment for 3 weeks could significantly interfere with the serum metabolic profile of HUA rats.

[0065] Based on the VIP of differentially metabolites, combined with FC and t-test significance analysis, differentially metabolites were screened using conditions such as P<0.05, VIP>1, FC<0.65 or>1. Figure 10 Volcano plots of differentially expressed metabolites were generated among the rat groups. 398 differentially expressed metabolites were identified between the MC and NC groups, with 271 upregulated and 127 downregulated. 179 differentially expressed metabolites were identified between the LS and MC groups, with 48 upregulated and 131 downregulated. 171 differentially expressed metabolites were identified between the SV and MC groups, with 76 upregulated and 95 downregulated. 222 differentially expressed metabolites were identified between the LS+SV and MC groups, with 66 upregulated and 156 downregulated.

[0066] Differential metabolites were input into the KEGG database, and the results of characterizing the affected metabolic pathways were as follows: Figure 11 and Figure 12 As shown. For the first five pathways in the LS and MC groups (P < 0.05), they were analyzed together. Significant changes were observed in glycine, serine, and threonine metabolism, the pentose phosphate pathway, and sulfur metabolism between the MC and NC groups. Between the LS and MC groups, differentially metabolites were mainly annotated and enriched in the glucagon signaling pathway, central carbon metabolism in cancer, glycolysis / gluconeogenesis pathway, glycine, serine, and threonine metabolism, and the pentose phosphate pathway. Between the SV and MC groups, significant changes were observed in sulfur metabolism, the prolactin signaling pathway, and the intestinal immune network IgA production signaling pathway. Between the LS+SV and MC groups, differentially metabolites were mainly annotated and enriched in leishmaniasis, choline metabolism in cancer, and the intestinal immune network IgA production signaling pathway.

[0067] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. A strain of *Lactobacillus 6-2* associated with a mythical pig host, characterized by: The aforementioned *Lactobacillus mythologica* host-associated 6-2 was deposited on December 1, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36865, and classified as *Lactobacillus mythologica* host-associated 6-2. Ligilactobacillus saerimneri ).

2. A microbial inoculant, characterized in that: The microbial agent contains Mythical Pig Host-Associated Lactobacillus 6-2 as described in claim 1.

3. The method for preparing the microbial inoculant as described in claim 2, characterized in that: Specifically, *Lactobacillus mythologica* host-associated 6-2 was inoculated into MRS liquid medium and cultured at 33–40°C for 20–24 hours. After culture, the culture was centrifuged, and sterile physiological saline was added to the precipitate to obtain a sample containing 10⁻⁶ viable bacteria. 7 ~10 9 CFU / g bacterial suspension.

4. The use of the Mythical Pig Host-Associated Lactobacillus 6-2 as described in claim 1 or the microbial agent as described in claim 2 in the preparation of drugs for the prevention or treatment of uric acid.

5. A method for preparing an extract of *Poplar mulberry bark*, characterized in that: Includes the following steps: (1) Weigh the dried, crushed and sieved fruiting bodies of Populus euphratica, add 50-70% ethanol at a material-to-liquid ratio of 1:25-35, extract with ultrasonic-assisted water bath for 1-2 h, add 50-70% ethanol at the same volume as the first material-to-liquid ratio to the filter residue and extract with ultrasonic for 1-2 h, combine the two filtrates, centrifuge at 4000-5000 rpm for 10-20 min and collect the supernatant; (2) Dry the filter residue obtained in step (1), add ultrapure water at a material-to-liquid ratio of 1:40-60, extract in an ultrasonic-assisted water bath for 1-2 h, and then extract in a boiling water bath for 1-2 h. Add ultrapure water to the filter residue obtained by filtration at a material-to-liquid ratio of 1:40-60 and boil in a water bath for 1-2 h. After combining the two filtrates, collect the filtrate by coarse filtration through a filter cloth, centrifugation and vacuum filtration. (3) The supernatant obtained in step (1) and the filtrate obtained in step (2) are mixed, concentrated under reduced pressure and freeze-dried to obtain poplar mulberry extract.

6. The preparation method according to claim 5, characterized in that: In steps (1) and (2), the ultrasonic parameters are 40-50 kHz, 300-400 W, and the water bath temperature is 50-70℃.

7. A poplar mulberry extract, characterized in that: It is prepared by the method described in claim 5.

8. A compound preparation, characterized in that: The compound preparation is prepared by mixing the microbial agent according to claim 2 with the poplar mulberry extract according to claim 7.

9. The compound preparation according to claim 8, characterized in that: The mass ratio of microbial inoculant to poplar mulberry extract is 1:1 to 3.

10. The use of the compound preparation as described in claim 8 in the preparation of a medicament for the prevention or treatment of uric acid.

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