Composition of human intestinal flora of lactobacillus rhamnosus and dendrobium officinale polysaccharide and application thereof in preparation of reagent for preventing, improving and / or treating hyperuricemia

Abstract

This invention belongs to the field of biotechnology, and particularly relates to a composition comprising *Lactobacillus rhamnosus*, a human-derived intestinal flora, and polysaccharides from *Dendrobium officinale*, and its application in the preparation of reagents for the prevention, improvement, and / or treatment of hyperuricemia. The composition comprises *Lactobacillus rhamnosus* (… Lacticaseibacillus rhamnosus The composition comprises TG037 or its culture and / or cell fragments, and Dendrobium officinale polysaccharide. The preservation number for Lactobacillus rhamnosus TG037 is CCTCC NO: M 2026064. In this composition, Lactobacillus rhamnosus TG037 and Dendrobium officinale polysaccharide form an organic whole. They work synergistically to achieve highly effective intervention for hyperuricemia and its complications through multiple, multi-target mechanisms, including reducing uric acid production, promoting uric acid excretion, exerting anti-inflammatory and repair functions, and regulating the microecology. This composition has significant application prospects and clinical translational value.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and particularly relates to a composition of human-derived intestinal native bacteria Lactobacillus rhamnosus and Dendrobium officinale polysaccharide, and its application in the preparation of reagents for the prevention, improvement and / or treatment of hyperuricemia. Background Technology

[0002] Hyperuricemia is a common metabolic disease caused by purine metabolism disorders and / or impaired uric acid excretion. It is closely related to the occurrence of various diseases such as gout, chronic kidney disease, and cardiovascular disease. Commonly used first-line uric acid-lowering drugs include xanthine oxidase inhibitors (alopurinol, febuxostat) and uricosuric agents (benzbromarone). While these drugs can effectively lower blood uric acid levels, they generally have safety risks such as hepatotoxicity, nephrotoxicity, and serious skin adverse reactions, limiting their feasibility for long-term use.

[0003] In recent years, utilizing probiotics to regulate the gut microbiota has become a potential strategy for the intervention of hyperuricemia. Previous reports have indicated that some lactic acid bacteria strains possess the activity to degrade purine nucleosides or inhibit xanthine oxidase, exhibiting certain uric acid-lowering functions. However, existing strains suffer from problems such as low purine nucleoside degradation efficiency or narrow spectrum of action, relatively simple functional mechanisms, and a lack of multi-target synergistic effects, resulting in less than ideal uric acid-lowering effects in vivo and significant limitations. Summary of the Invention

[0004] Based on addressing the problems of low purine nucleoside degradation efficiency, narrow spectrum of action, relatively simple functional mechanisms, and lack of multi-target synergistic effects in the application of existing probiotic agents for the intervention of hyperuricemia, the inventors of this invention, through extensive and in-depth research and numerous experiments, creatively discovered a strain of *Lactobacillus rhamnosus* TG037 with highly efficient ability to degrade multiple purine nucleosides. This *Lactobacillus rhamnosus* TG037 can prevent, improve, and / or treat hyperuricemia through multiple mechanisms, including purine degradation, inhibition of xanthine oxidase activity, and regulation of intestinal flora structure. The study also explored the effects of polysaccharides extracted from Dendrobium officinale on acidosis. Further research creatively revealed that polysaccharides not only act as prebiotics to directly promote the growth and proliferation of *Lactobacillus rhamnosus* TG037, enhancing its activity and colonization in the intestinal environment, but also, through the synergistic effect between the polysaccharides and *Lactobacillus rhamnosus* TG037, endowed the resulting composition with bioactivity not present when either ingredient acts alone. Firstly, the combined action of Dendrobium officinale polysaccharides and *Lactobacillus rhamnosus* TG037 can… It precisely targets uric acid transport-related signaling pathways, exhibiting both significant upregulation of the uric acid efflux transporter ABCG2 in kidney tissue and significant downregulation of the uric acid reabsorption transporter GLUT9 in jejunal tissue. Furthermore, the combined action of *Dendrobium officinale* polysaccharide and *Lactobacillus rhamnosus* TG037 can significantly reverse the effects of β-alanine metabolism, cysteine ​​and methionine metabolism, glycine, serine and threonine metabolism, phenylalanine, tyrosine and tryptophan biosynthesis signaling pathways, fatty acid degradation signaling pathways, and fatty acid elongation signaling pathways. Furthermore, the combined effects of Dendrobium officinale polysaccharides and Lactobacillus rhamnosus TG037 can significantly reduce kidney tissue inflammation induced by hyperuricemia and significantly reduce the levels of pro-inflammatory factors TNF-α, IL-1β, and IL-6 in kidney tissue. Through multiple mechanisms of action, including inhibiting kidney tissue inflammation, targeted regulation of uric acid transport processes, adjustment of intestinal flora structure, and improvement of metabolic homeostasis imbalance, this invention can effectively intervene in, improve, and / or treat hyperuricemia and its complications. Based on this, the technical solution of this invention was obtained.

[0005] Specifically, the first objective of this invention is to provide a composition comprising Dendrobium officinale polysaccharide and Lactobacillus rhamnosus (…). Lacticaseibacillus rhamnosus TG037 or its culture and / or cell fragments; wherein the preservation number of the Lactobacillus rhamnosus TG037 is CCTCC NO: M 2026064.

[0006] In this invention, the culture of Lactobacillus rhamnosus TG037 refers to the culture medium obtained by culturing a pure strain of Lactobacillus rhamnosus TG037 under certain culture conditions; the culture specifically includes, but is not limited to, one or more of live bacterial cells, intracellular active components, and extracellular metabolic secretions.

[0007] In this invention, the cell fragments of Lactobacillus rhamnosus TG037 refer to the cell structure of Lactobacillus rhamnosus TG037 that has been disrupted by physical, chemical or biological lysis methods, thereby releasing the intracellular contents; the cell fragments specifically include, but are not limited to, one or more of the following: cell fragments, intracellular soluble active components, membrane structure fragments and extracellular metabolites.

[0008] In this invention, the composition of Dendrobium officinale polysaccharide and Lactobacillus rhamnosus TG037 is an organic whole. The synergistic effect of the two can achieve comprehensive and efficient intervention on hyperuricemia and its complications through multiple and multi-target mechanisms such as reducing uric acid production, promoting uric acid excretion, anti-inflammatory repair, and regulating microecology. It has excellent application prospects in the research and development of safe and effective uric acid-lowering products.

[0009] Furthermore, the neutral sugar content of the Dendrobium officinale polysaccharide is 50%~99.999%. In some specific embodiments, the neutral sugar content of the Dendrobium officinale polysaccharide can be 50%, 55%, 58%, 60%, 63.89%, 65%, 70%, 80%, 90%, 95%, 99.999%, or any value between them.

[0010] Furthermore, the addition ratio of *Dendrobium officinale* polysaccharide to *Lactobacillus rhamnosus* TG037 is (100~1000) mg: (1×10⁻⁶) mg. 8 ~1×10 11 CFU. In some specific embodiments, the addition ratio of Dendrobium officinale polysaccharide and Lactobacillus rhamnosus TG037 can be 100mg:1×10 8 CFU, 200mg: 1×10 8 CFU, 400mg: 5×10 9 CFU, 1000mg: 8×10 10 CFU, 1000mg: 1×10 11 CFU or any value between them.

[0011] Furthermore, the preparation of the Dendrobium officinale polysaccharide includes: mixing Dendrobium officinale powder with water for extraction to obtain a crude polysaccharide extract; and purifying the crude polysaccharide extract to obtain the Dendrobium officinale polysaccharide.

[0012] Further, the ratio of Dendrobium officinale powder to water is (0.1~10)g:(20~100)mL. In some specific embodiments, the ratio of Dendrobium officinale powder to water is specifically 0.1g:20mL, 1g:20mL, 5g:50mL, 7g:100mL, 10g:100mL, or any value between them.

[0013] Further, the extraction treatment temperature is 85℃~100℃, and the time is 1h~4h. In some specific embodiments, the extraction treatment temperature can be 85℃, 88℃, 90℃, 95℃, 98℃, 100℃ or any value between them; the extraction treatment time can be 1h, 1.5h, 2h, 3h, 4h or any value between them.

[0014] Furthermore, the purification process includes deproteinization, alcohol precipitation, and freeze-drying. In this invention, the deproteinization, alcohol precipitation, and freeze-drying processes are conventional techniques used in polysaccharide extraction. Those skilled in the art can make appropriate selections according to actual needs, and are not particularly limited thereto.

[0015] In this invention, the composition further includes pharmaceutically or food-grade excipients. In some specific embodiments, specific examples of pharmaceutically or food-grade excipients include, but are not limited to, one or more of solvents, preservatives, solubilizers, preservatives, and emulsifiers.

[0016] A second object of the present invention is to provide the use of the above-described composition in the preparation of agents for the prevention, improvement and / or treatment of hyperuricemia or its complications.

[0017] In this invention, the reagent refers to a class of substances that can prevent, improve and / or treat hyperuricemia or its complications, and specific examples include, but are not limited to, pharmaceuticals.

[0018] In this invention, the complications of hyperuricemia specifically refer to a series of target organ damage, organic lesions and combined metabolic diseases caused by long-term blood uric acid concentration exceeding the physiological saturation threshold. Specific examples include, but are not limited to, one or more of the following: gouty arthritis, acute uric acid nephropathy, chronic uric acid interstitial nephropathy, uric acid kidney stones and metabolic syndrome.

[0019] Further, the reagent is used for one or more of the following purposes: (1) reducing serum uric acid levels; (2) reducing serum creatinine levels; (3) reducing serum urea nitrogen levels; (4) reducing the expression levels of pro-inflammatory factors TNF-α, IL-1β and / or IL-6 in kidney tissue; (5) upregulating the expression of uric acid efflux transporter ABCG2 in kidney tissue; (6) downregulating the expression of intestinal uric acid reabsorption transporter GLUT9; and (7) regulating the structure of intestinal flora.

[0020] A third object of the present invention is to provide the use of the above-described composition in the preparation of agents for the prevention, improvement, and / or treatment of kidney injury. Specifically, the kidney injury is caused by hyperuricemia.

[0021] In this invention, the reagent refers to a class of substances that can prevent, improve and / or treat kidney damage caused by hyperuricemia, and specific examples include, but are not limited to, pharmaceuticals.

[0022] A fourth objective of this invention is to provide the use of the above-described composition in the preparation of agents for the prevention, improvement, and / or treatment of metabolic homeostasis imbalances. More specifically, the metabolic homeostasis imbalance is caused by hyperuricemia, and the metabolic homeostasis imbalance includes one or more of the following abnormally expressed metabolic pathways: (1) amino acid metabolism-related signaling pathways; (2) fatty acid metabolism-related signaling pathways.

[0023] In this invention, the reagent refers to a class of substances that can prevent, improve and / or treat metabolic homeostasis imbalance caused by hyperuricemia, and specific examples include, but are not limited to, pharmaceuticals.

[0024] Furthermore, the amino acid metabolism-related signaling pathways include one or more of the following metabolic pathways: (1) β-alanine metabolic pathway; (2) cysteine ​​and methionine metabolic pathway; (3) glycine, serine and threonine metabolic pathway; and (4) phenylalanine, tyrosine and tryptophan biosynthesis signaling pathway.

[0025] Furthermore, the fatty acid metabolism-related signaling pathways include one or more of the following metabolic pathways: (1) fatty acid degradation signaling pathway; (2) fatty acid elongation signaling pathway; (3) fatty acid metabolism pathway.

[0026] A fifth objective of the present invention is to provide the use of the above-described composition in the preparation of reagents that improve and / or enhance the renal capacity to excrete uric acid.

[0027] In this invention, the reagent refers to a class of substances that can improve and / or enhance the kidney's ability to excrete uric acid, and specific examples include, but are not limited to, pharmaceuticals.

[0028] A sixth objective of the present invention is to provide the use of the above composition in the preparation of a reagent that enhances the expression of the uric acid efflux transporter ABCG2 in kidney tissue.

[0029] In this invention, the reagent refers to a class of substances that can increase the expression of uric acid efflux transporter ABCG2 in kidney tissue, and specific examples include, but are not limited to, pharmaceuticals.

[0030] Biological Preservation The Lactobacillus rhamnosus provided by this invention ( Lacticaseibacillus rhamnosus TG037, deposited on January 12, 2026, accession number CCTCC NO: M 2026064, deposited at China Center for Type Culture Collection, Wuhan University, Wuhan, China. Attached Figure Description

[0031] Figure 1 This is a colony morphology diagram of Lactobacillus rhamnosus TG037 provided in Example 2 of the present invention (blood agar plate, Staphylococcus aureus on the left and Lactobacillus rhamnosus TG037 on the right). Figure 2 The figure shows the experimental results of the gastrointestinal fluid resistance test of Lactobacillus rhamnosus TG037 provided in Example 3 of this invention. Figure 3 The figure shows the experimental results of the bile salt tolerance test of Lactobacillus rhamnosus TG037 provided in Example 3 of this invention; Figure 4 This is one of the experimental results of the in vitro degradation ability of purine nucleosides of Lactobacillus rhamnosus TG037 provided in Example 3 of the present invention (before adding the bacterial cells). Figure 5 The second figure shows the experimental results of the in vitro degradation ability of purine nucleosides by Lactobacillus rhamnosus TG037 provided in Example 3 of this invention (after adding bacterial cells and culturing for 2 hours). Figure 6 This is a diagram showing the experimental results of ROS level detection in Caco-2 cells after treating an LPS-induced Caco-2 cell model with Lactobacillus rhamnosus TG037, as provided in Example 4 of this invention (compared to the blank control group). # P<0.05, ## P<0.01, ### P<0.001; compared with the cell model group, * P<0.05, ** P<0.01, *** P<0.001); Figure 7This is a graph showing the experimental results of detecting the transmembrane resistance level in Caco-2 cells after treating an LPS-induced Caco-2 cell model with Lactobacillus rhamnosus TG037, as provided in Example 4 of this invention (compared to the blank control group). # P<0.05, ## P<0.01, ### P<0.001; compared with the cell model group, * P<0.05, ** P<0.01, *** P<0.001); Figure 8 This is a diagram showing the experimental results of FITC-glucan permeability detection in Caco-2 cells after treating an LPS-induced Caco-2 cell model with Lactobacillus rhamnosus TG037, as provided in Example 4 of this invention (compared to the blank control group). # P<0.05, ## P<0.01, ### P<0.001; compared with the cell model group, * P<0.05, ** P<0.01, *** P<0.001); Figure 9 This is one of the experimental results of the test on the growth and proliferation promoting effect of Dendrobium officinale polysaccharide on Lactobacillus rhamnosus TG037 provided in Example 4 of this invention (compared with the MRS group). # P<0.05, ## P<0.01, ### P<0.001); Figure 10 Figure 2 shows the experimental results of the test on the growth and proliferation promoting effect of Dendrobium officinale polysaccharide provided in Example 4 of this invention on Lactobacillus rhamnosus TG037; Figure 11 The figure shows the experimental results of testing serum uric acid levels in hyperuricemic mice after treatment with the combined use of Dendrobium officinale polysaccharide and Lactobacillus rhamnosus TG037 provided in Example 5 of this invention (compared to the CON group). # P<0.05, ## P<0.01, ### P<0.001; compared with the MOD group, * P<0.05, ** P<0.01, *** P<0.001; compared with the MIX group, & P<0.05, && P<0.01, &&& P<0.001); Figure 12 The figure shows the experimental results of serum creatinine levels in hyperuricemic mice after treatment with the combined use of Dendrobium officinale polysaccharide and Lactobacillus rhamnosus TG037 provided in Example 5 of this invention (compared to the CON group). # P<0.05, ## P<0.01, ### P<0.001; compared with the MOD group, * P<0.05, ** P<0.01, *** P<0.001; compared with the MIX group, & P<0.05, && P<0.01, &&& P<0.001); Figure 13 The figure shows the experimental results of testing serum urea nitrogen levels in hyperuricemic mice after treatment with the combined use of Dendrobium officinale polysaccharide and Lactobacillus rhamnosus TG037 provided in Example 5 of this invention (compared to the CON group). # P<0.05, ## P<0.01, ### P<0.001; compared with the MOD group, * P<0.05, ** P<0.01, *** P<0.001; compared with the MIX group, & P<0.05, && P<0.01, &&& P<0.001); Figure 14 HE staining of kidney tissue of mice with hyperuricemia after treatment with the combined use of Dendrobium officinale polysaccharide and Lactobacillus rhamnosus TG037 provided in Example 5 of this invention (scale bar is 100 μm). Figure 15 The figure shows the experimental results of detecting TNF-α levels in the kidney tissue of hyperuricemic mice after treatment with the combined use of Dendrobium officinale polysaccharide and Lactobacillus rhamnosus TG037 provided in Example 5 of this invention (compared to the CON group). # P<0.05, ## P<0.01, ### P<0.001; compared with the MOD group, * P<0.05, ** P<0.01, *** P<0.001; compared with the MIX group, & P<0.05, && P<0.01, &&& P<0.001); Figure 16 The figure shows the experimental results of detecting IL-1β levels in the kidney tissue of hyperuricemic mice after treatment with the combined use of Dendrobium officinale polysaccharide and Lactobacillus rhamnosus TG037 provided in Example 5 of this invention (compared to the CON group). # P<0.05, ## P<0.01, ### P<0.001; compared with the MOD group, * P<0.05, ** P<0.01, *** P<0.001; compared with the MIX group, & P<0.05, && P<0.01, &&& P<0.001); Figure 17 The figure shows the experimental results of detecting IL-6 levels in the kidney tissue of hyperuricemic mice after treatment with the combined use of Dendrobium officinale polysaccharide and Lactobacillus rhamnosus TG037 provided in Example 5 of this invention (compared to the CON group). # P<0.05, ## P<0.01, ### P<0.001; compared with the MOD group, * P<0.05, ** P<0.01, *** P<0.001; compared with the MIX group, & P<0.05, && P<0.01, &&& P<0.001); Figure 18 The figure shows the experimental results of detecting the relative level of ABCG2 mRNA in the kidney tissue of hyperuricemic mice after treatment with the combined use of Dendrobium officinale polysaccharide and Lactobacillus rhamnosus TG037 provided in Example 5 of this invention (compared to the CON group). # P<0.05, ## P<0.01, ### P<0.001; compared with the MOD group, * P<0.05, ** P<0.01, *** P<0.001; compared with the MIX group, & P<0.05, && P<0.01, &&& P<0.001); Figure 19The figure shows the experimental results of detecting the relative level of NTP1 mRNA in the kidney tissue of hyperuricemic mice after treatment with the combined use of Dendrobium officinale polysaccharide and Lactobacillus rhamnosus TG037 provided in Example 5 of this invention (compared to the CON group). # P<0.05, ## P<0.01, ### P<0.001; compared with the MOD group, * P<0.05, ** P<0.01, *** P<0.001; compared with the MIX group, & P<0.05, && P<0.01, &&& P<0.001); Figure 20 The figure shows the experimental results of detecting the relative level of ABCG2 mRNA in the liver tissue of hyperuricemic mice after treatment with the combined use of Dendrobium officinale polysaccharide and Lactobacillus rhamnosus TG037 provided in Example 5 of this invention (compared to the CON group). # P<0.05, ## P<0.01, ### P<0.001; compared with the MOD group, * P<0.05, ** P<0.01, *** P<0.001; compared with the MIX group, & P<0.05, && P<0.01, &&& P<0.001); Figure 21 The figure shows the experimental results of detecting the relative level of GLUT9 mRNA in the liver tissue of hyperuricemic mice after treatment with the combined use of Dendrobium officinale polysaccharide and Lactobacillus rhamnosus TG037 provided in Example 5 of this invention (compared to the CON group). # P<0.05, ## P<0.01, ### P<0.001; compared with the MOD group, * P<0.05, ** P<0.01, *** P<0.001; compared with the MIX group, & P<0.05, && P<0.01, &&& P<0.001); Figure 22The figure shows the experimental results of detecting the relative level of NTP1 mRNA in the liver tissue of hyperuricemic mice after treatment with the combined use of Dendrobium officinale polysaccharide and Lactobacillus rhamnosus TG037 provided in Example 5 of this invention (compared to the CON group). # P<0.05, ## P<0.01, ### P<0.001; compared with the MOD group, * P<0.05, ** P<0.01, *** P<0.001; compared with the MIX group, & P<0.05, && P<0.01, &&& P<0.001); Figure 23 The figure shows the experimental results of detecting the relative level of ABCG2 mRNA in the jejunal tissue of hyperuricemic mice after treatment with the combined use of Dendrobium officinale polysaccharide and Lactobacillus rhamnosus TG037 provided in Example 5 of this invention (compared to the CON group). # P<0.05, ## P<0.01, ### P<0.001; compared with the MOD group, * P<0.05, ** P<0.01, *** P<0.001; compared with the MIX group, & P<0.05, && P<0.01, &&& P<0.001); Figure 24 The figure shows the experimental results of detecting the relative level of GLUT9 mRNA in the jejunal tissue of hyperuricemic mice after treatment with the combined use of Dendrobium officinale polysaccharide and Lactobacillus rhamnosus TG037 provided in Example 5 of this invention (compared to the CON group). # P<0.05, ## P<0.01, ### P<0.001; compared with the MOD group, * P<0.05, ** P<0.01, *** P<0.001; compared with the MIX group, & P<0.05, && P<0.01, &&& P<0.001); Figure 25The figure shows the experimental results of detecting the relative level of NTP1 mRNA in the jejunal tissue of hyperuricemic mice after treatment with the combined use of Dendrobium officinale polysaccharide and Lactobacillus rhamnosus TG037 provided in Example 5 of this invention (compared to the CON group). # P<0.05, ## P<0.01, ### P<0.001; compared with the MOD group, * P<0.05, ** P<0.01, *** P<0.001; compared with the MIX group, & P<0.05, && P<0.01, &&& P<0.001); Figure 26 The diagram shows the abundance of intestinal flora at the genera level in mice treated with the combined use of Dendrobium officinale polysaccharide and Lactobacillus rhamnosus TG037 in Example 5 of this invention. Figure 27 Box plot of genus-level differences in intestinal flora in mice treated with the combined use of Dendrobium officinale polysaccharide and Lactobacillus rhamnosus TG037 provided in Example 5 of this invention; Figure 28 The metabolite clustering heatmap of mice feces after treating hyperuricemic mice with the combined use of Dendrobium officinale polysaccharide and Lactobacillus rhamnosus TG037 provided in Example 5 of this invention. Figure 29 This is a differential metabolite pathway enrichment map of CON group and MOD group mice provided in Example 5 of this invention; Figure 30 This is a differential metabolite pathway enrichment map of the MOD group and TG037 group mice provided in Example 5 of this invention; Figure 31 This is a differential metabolite pathway enrichment map of MOD group and MIX group mice provided in Example 5 of this invention; Figure 32 This is a differential metabolite pathway enrichment map of the MOD group and DOP group mice provided in Example 5 of this invention. Detailed Implementation

[0032] The embodiments of the present invention are described in detail below. These embodiments are intended to explain the present invention and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0033] Example 1 This embodiment illustrates a method for preparing Dendrobium officinale polysaccharides, specifically including: S1. Take dried Dendrobium officinale medicinal material, pulverize it and pass it through a 60-mesh sieve. Then, defatt it with petroleum ether and decolorize it with 95% ethanol to obtain Dendrobium officinale powder.

[0034] S2. At a material-to-liquid ratio of 1:60 (g / mL), Dendrobium officinale powder and distilled water were mixed and extracted in a 90℃ water bath for 2.5 hours. The mixture was then filtered, and the precipitate was extracted three times. The filtrates were combined and concentrated under reduced pressure to obtain a crude polysaccharide extract.

[0035] S3. The crude polysaccharide extract was deproteinized 8 times using Sevage reagent (including n-butanol and chloroform in a volume ratio of 1:4). The supernatant was centrifuged and 4 times the volume of 95% ethanol was added. The mixture was precipitated at 4°C overnight. The precipitate was collected by centrifugation and freeze-dried at -20°C for 24 hours to obtain Dendrobium officinale polysaccharide.

[0036] The polysaccharides from Dendrobium officinale prepared in this example were determined using the phenol-sulfuric acid method. The results showed that the neutral sugar content was 63.89% and the extraction rate was 26.84%.

[0037] Example 2 This embodiment illustrates the isolation, identification, and preservation of *Lactobacillus rhamnosus* TG037, specifically including: 1. Isolation of bacterial strains: Fecal samples taken from a healthy adult volunteer in Fujian, China, were serially diluted using sterile PBS buffer (10 mM, pH 7.4) to obtain a dilution factor of 10. -2 ~10 -7 Diluent; take an appropriate amount of diluent and spread it on an MRS solid medium plate (Guangdong Huankai Microbial Technology Co., Ltd., catalog number 027312), and incubate it under anaerobic conditions at 37℃ for 24 hours to obtain single colonies; pick single colonies and perform multiple streak purification cultures to obtain pure cultures of the strain.

[0038] 2. Identification of the strain: Genomic DNA was extracted from the pure culture using a bacterial genomic DNA extraction kit (purchased from Sangon Biotech) according to the instructions. Then, PCR amplification was performed on the DNA using the primers shown in Table 1. The PCR amplification products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results were analyzed and identified, confirming the strain as *Lactobacillus rhamnosus*. Lacticaseibacillus rhamnosus ), named TG037, Figure 1 This is a colony morphology diagram of Lactobacillus rhamnosus TG037 on a blood agar plate.

[0039] Table 1.

[0040] Depend on Figure 1 The results show that Lactobacillus rhamnosus TG037 does not undergo hemolysis on blood agar plates, does not exhibit strong invasiveness or cytotoxicity, and has a high safety profile.

[0041] 3. Preservation of the strain: Lactobacillus rhamnosus TG037 was deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2026064 and deposit date of January 12, 2026.

[0042] The preparation of the seed culture used in the following examples included: inoculating a single clone of *Lactobacillus rhamnosus* TG037 into MRS liquid medium (Guangdong Huankai Microbial Technology Co., Ltd., catalog number 027312) using a disposable inoculation loop, and culturing under anaerobic conditions at 37°C to obtain OD. 600 Seed liquid with a value of 0.8 to 1.0.

[0043] Example 3 This embodiment is used to illustrate the relevant performance of Lactobacillus rhamnosus TG037 provided in Example 2, specifically including: 1. Resistance to gastrointestinal fluid: (1) At an inoculation rate of 1%, seed culture was inoculated into simulated gastric fluid (Regen Biotech, catalog number CZ0213, pH=2.5), and digested under anaerobic culture conditions at 37℃ for 3 hours to obtain gastric fluid-treated culture medium; 20 μL of gastric fluid-treated culture medium was spread on MRS solid medium plates (each dilution was biologically replicated 3 times), and cultured under anaerobic culture conditions at 37℃ for 48 hours until single colonies formed, the number of viable bacteria was counted, and the survival rate was calculated according to formula (1). The results are as follows. Figure 2 As shown.

[0044] Survival rate = N t / N0×100% formula (1) In formula (1), Nt is the number of viable bacteria in the culture medium after 3 hours of simulated gastric digestion, in CFU / mL; N0 is the number of viable bacteria in the culture medium after 0 hours of simulated gastric digestion, in CFU / mL.

[0045] (2) At an inoculation rate of 1%, seed culture was inoculated into simulated intestinal fluid (Regen Biotech, catalog number CZ0201, pH=8.0), and digested under anaerobic culture conditions at 37℃ for 3 hours to obtain intestinal fluid-treated culture medium; 20 μL of intestinal fluid-treated culture medium was spread on MRS solid medium plates (each dilution was biologically replicated 3 times), and cultured under anaerobic culture conditions at 37℃ for 48 hours until single colonies formed. The viable number was counted and the survival rate was calculated according to formula (2). The results are as follows: Figure 2As shown.

[0046] Survival rate = N t / N0×100% formula (2) In equation (2), N t The number of viable bacteria in the culture medium after 3 hours of simulated intestinal digestion is expressed in CFU / mL; N0 is the number of viable bacteria in the culture medium after 0 hours of simulated intestinal digestion is expressed in CFU / mL.

[0047] Depend on Figure 1 The results show that after digestion in simulated gastric juice at pH 2.5 for 3 hours, the survival rate of Lactobacillus rhamnosus TG037 was 18.49% ± 1.86%, and after digestion in simulated intestinal juice at pH 8.0 for 3 hours, the survival rate was 63.54 ± 11.75%, indicating good tolerance to gastrointestinal juice.

[0048] 2. Bile salt tolerance: (1) Add bile salt to MRS liquid culture medium at final concentrations of 0.1%, 0.2% and 0.3% to obtain bile salt solution.

[0049] (2) At an inoculation rate of 1%, seed culture was inoculated into bile salt solution and digested under anaerobic culture conditions at 37°C for 3 hours to obtain bile salt-treated culture medium; 20 μL of bile salt-treated culture medium was spread on MRS solid medium plates (each dilution was biologically replicated 3 times) and cultured under anaerobic culture conditions at 37°C for 48 hours until single colonies formed. The viable number was counted and the survival rate was calculated according to formula (3). The results are as follows: Figure 3 As shown.

[0050] Survival rate = N t / N0×100% formula (3) In equation (3), N t The number of viable bacteria in the culture medium after 3 hours of bile salt digestion is expressed in CFU / mL; N0 is the number of viable bacteria in the culture medium after 0 hours of bile salt digestion is expressed in CFU / mL.

[0051] Depend on Figure 3 The results show that the survival rate of Lactobacillus rhamnosus TG037 was 27.54% after digestion in 1% bile salt solution for 3 hours.

[0052] Depend on Figure 2 and 3 The results show that Lactobacillus rhamnosus TG037 has good resistance to gastrointestinal fluids and bile salts, and can colonize well in the intestine.

[0053] 3. In vitro degradation ability of purine nucleosides: (1) Centrifuge 50 mL of seed culture to collect bacterial cells, then resuspend the bacterial cells in 1.5 mL of PBS buffer (10 mM, pH=7.4) containing adenosine, guanosine and inosine. After culturing under anaerobic conditions at 37℃ for 2 h, centrifuge to collect the supernatant. HPLC is used to detect the concentration of purine nucleosides in the supernatant. The degradation rate is calculated according to formula (4). The results are as follows: Figure 4 and 5 And as shown in Table 2.

[0054] Degradation rate = (C0-C1) / C0×100% Equation (4) In formula (4), C0 is the concentration of each purine nucleoside before the addition of bacterial cells, in μg / mL; C1 is the concentration of each purine nucleoside after culturing for 2 hours after the addition of bacterial cells, in μg / mL.

[0055] Table 2.

[0056] Depend on Figure 4 and 5 As shown in Table 2, Lactobacillus rhamnosus TG037 exhibits excellent in vitro degradation capabilities for adenosine, guanosine, and inosine.

[0057] 4. Xanthine oxidase inhibitory activity: (1) Take 50 mL of seed culture and centrifuge at 10000 r / min for 10 min to collect the bacterial cells; resuspend the bacterial cells in 2 mL of PBS buffer (10 mM, pH=7.4) and sonicate them under ice bath conditions (power 300 W, sonication for 3 s, interval 10 s, cumulative sonication for 20 min); then centrifuge at 4℃ and 7000 r / min for 10 min to collect the supernatant. The supernatant obtained is the cell-free extract of bacterial cells.

[0058] (2) Mix 20 µL of cell-free bacterial extract with 20 µL of xanthine oxidase solution at a concentration of 0.1 U / mL, then add 140 µL of PBS buffer (10 mM, pH=7.4) and 20 µL of xanthine substrate solution at a concentration of 10 mmol / L. Measure the absorbance of the reaction system at 293 nm at the beginning of the reaction and at 10 min of the reaction. Calculate the xanthine oxidase inhibition rate according to formula (5). Use 20 µL of allopurinol instead of cell-free bacterial extract as a positive control.

[0059] Equation (5) In equation (5), A s0 With A s These represent the absorbance values ​​of the sample group at min 0 and min 10 of the reaction, respectively; A b0 With Ab The absorbance values ​​of the blank control group at min 0 and min 10 of the reaction are respectively represented.

[0060] Table 3.

[0061] 5. Antioxidant activity: (1) According to 1×10 5 The seeding volume was 200 µL / well. Caco-2 cells were seeded into Transwell chambers (Corning, catalog number 3413) containing MEM medium (NEAA, Wuhan Pronosei Life Sciences Co., Ltd., catalog number PM150410) containing 20% ​​fetal bovine serum and 100 U / mL penicillin-streptomycin solution (Beyotime Biotechnology, catalog number C0222). Then, LPS (Sigma, catalog number L2630) was added to a final concentration of 50 µg / mL and cultured at 37℃ and 5% CO2 for 24 h to construct an LPS-induced Caco-2 cell model.

[0062] (2) At an inoculation rate of 1% (v / v), seed culture was added to the culture medium containing the Caco-2 cell model and cultured at 37℃ and 5% CO2 for 4 h. The bacterial culture was then aspirated and the cells were washed with MEM medium containing 100 U / mL penicillin-streptomycin. ROS levels were detected using the DCFH-DA fluorescent probe (Beyotime Biotechnology, catalog number S0033S) according to the instructions. Transmembrane resistance was detected using an epithelial / endothelial transmembrane resistance meter. FITC-glucan permeability was detected using FITC-glucan (4kDa, Aladdin, catalog number F491425) according to the instructions. Caco-2 cells without any treatment were used as a blank control, Caco-2 cells treated only with LPS were used as the cell model group, and 5 mM 5-ASA (Invivochem, catalog number 89-57-6) was added as a positive control. The results are as follows: Figures 6-8 As shown.

[0063] Depend on Figures 6-8 The results show that treating LPS-induced Caco-2 cell models with Lactobacillus rhamnosus TG037 can significantly inhibit LPS-induced ROS generation, restore cell transmembrane resistance, and reduce FITC-glucan permeability, demonstrating potential bioactivity in anti-oxidative stress and repairing the intestinal physical barrier.

[0064] Example 4 This embodiment illustrates the promoting effect of Dendrobium officinale polysaccharide provided in Example 1 on the growth and proliferation of Lactobacillus rhamnosus TG037, specifically including: (1) Dendrobium officinale polysaccharide (DOP) was added to MRS liquid culture medium (Guangdong Huankai Microbial Technology Co., Ltd., catalog number 027312) according to the final concentrations of 0.8%, 1.6%, 2.4%, 3.2% and 4.0% to obtain Dendrobium officinale polysaccharide culture medium.

[0065] (2) At an inoculation rate of 1%, seed liquid was inoculated into Dendrobium officinale polysaccharide medium and cultured at 37℃ for 24 h under anaerobic conditions. The absorbance of the culture medium at 600 nm was measured. Pure MRS liquid medium was used as a blank control (MRS), and inulin medium (Shanghai Maclean Technology Co., Ltd., catalog number F750187) was used as a positive control (POS). The growth rate was calculated according to formula (6). The results are as follows. Figure 9 and 10 As shown.

[0066] The growth rate is calculated as (A1-A0) / A1×100% (Equation 6). In equation (6), A1 is the OD of the experimental group or the positive control group. 600 Value, A0 is the OD value of the blank control group. 600 value.

[0067] Depend on Figure 9 and 10 The results show that, compared with the blank control group, the addition of Dendrobium officinale polysaccharide can promote the growth and proliferation of Lactobacillus rhamnosus TG037. When the amount of Dendrobium officinale polysaccharide added is 2.4%, the growth rate reaches 65.09%, which has a very good growth and proliferation promoting effect.

[0068] Example 5 This embodiment illustrates the therapeutic effect of the combined use of Dendrobium officinale polysaccharide provided in Example 1 and Lactobacillus rhamnosus TG037 provided in Example 2 on hyperuricemia, specifically including: 1. Eight-week-old male C57BL / 6J mice, weighing approximately 20g, were used as experimental animals. After one week of acclimatization under standard dietary conditions, they were randomly divided into 6 groups for the following procedures: (1) Control group (CON, n=7): The body was given a dose of 300 mg / kg, and was given an intraperitoneal injection of physiological saline once a day and fed standard ordinary feed for 3 weeks. (2) Model group (MOD, n=7): potassium oxonate was injected intraperitoneally once a day at a dose of 300 mg / kg and fed a high-purine diet (prepared by adding 0.2% adenine to a standard ordinary diet) for 3 weeks. (3) Allopurinol group (ALL, n=7): Based on the treatment of the model group mice, allopurinol was administered by gavage once a day at a dose of 5 mg / kg for 3 weeks; (4) Dendrobium officinale polysaccharide group (DOP, n=7): Based on the treatment of the model group mice, Dendrobium officinale polysaccharide was administered by gavage once a day at a dose of 5 mg / kg for 3 weeks. (5) Lactobacillus rhamnosus group (TG037, n=7): Based on the treatment of the model group mice, according to 5×10 9 The dosage was CFU / animal, and the seed liquid was administered by gavage once a day for 3 weeks. (6) Combined drug administration group (MIX, n=7): Based on the treatment of the model group mice, mice were given 5 mg / kg and 5 × 10 mg / kg of the combined drug. 9 The dosage was CFU / animal, and Dendrobium officinale polysaccharide and seed liquid were administered by gavage once a day for 3 weeks.

[0069] 2. After gavage on the last day, mice were fasted for 12 hours but allowed free access to water. Blood samples were then collected from the eyeballs. The blood samples were allowed to stand and centrifuged. The supernatant was collected, and the serum uric acid, creatinine, and blood urea nitrogen levels in each group of mice were measured using a UA assay kit (Nanjing Jiancheng Bioengineering Institute, catalog number C012-1-1), a CREA assay kit (Shenzhen Mindray Animal Medical Co., Ltd., catalog number C011-2-1), and a BUN assay kit (Shenzhen Mindray Animal Medical Co., Ltd., catalog number C013-2-1) according to the manufacturer's instructions. The results are as follows: Figures 11-13 As shown.

[0070] Depend on Figures 11-13 The results showed that, compared with the model group mice, the combined treatment group mice had a 41.82% reduction in serum uric acid level, a 55.06% reduction in serum creatinine level, and a 51.18% reduction in serum urea nitrogen level, which were not significantly different from the normal group mice. Moreover, the therapeutic effect of the combined administration of Dendrobium officinale polysaccharide and Lactobacillus rhamnosus TG037 was significantly better than that of Dendrobium officinale polysaccharide alone or Lactobacillus rhamnosus TG037 alone (p<0.05).

[0071] 3. After taking blood samples from the eyeballs, the mice were sacrificed, and kidney tissue, liver tissue, intestinal contents, and jejunum tissue were collected to obtain kidney tissue samples, liver tissue samples, and jejunum tissue samples.

[0072] 4. After taking some kidney tissue samples, fixation with 10% paraformaldehyde, paraffin embedding, sectioning, dewaxing with xylene, hydration with alcohol, and staining with hematoxylin and eosin (HE), the samples were photographed and observed under a microscope. The results are shown in Figure 14.

[0073] Depend on Figure 14The results show that the kidney tissue of the model group mice exhibited obvious glomerular atrophy, renal tubular dilation, and inflammatory cell infiltration; while the kidney pathological damage of the mice in the combined drug administration group was significantly reduced.

[0074] 5. Take 0.05g of kidney tissue and add it to 0.45mL of pre-cooled physiological saline. Homogenize the tissue using a tissue homogenizer. Centrifuge and collect the supernatant. Use the TNF-α detection kit (Ruixin Biotech, catalog number RX202412M), IL-1β detection kit (Ruixin Biotech, catalog number RX203063M), and IL-6 detection kit (Ruixin Biotech, catalog number RX203049M) according to the manufacturer's instructions to detect the levels of TNF-α, IL-1β, and IL-6 in the supernatant. The results are as follows: Figures 15-17 As shown.

[0075] Depend on Figures 15-17 The results show that, compared with the model group, the combined drug administration group reduced the levels of inflammatory factors TNF-α by 39.22%, IL-1β by 46.10%, and IL-6 by 27.97% in the kidney tissue of mice, which is better than the Dendrobium officinale polysaccharide group and Lactobacillus rhamnosus group.

[0076] 6. RNA was extracted from selected kidney, liver, and jejunum tissue samples using an RNA extraction kit (Nanjing Novizan Biotechnology, catalog number RC112-01) according to the manufacturer's instructions. RNA samples were then subjected to qPCR to obtain the mRNA levels of genes related to the uric acid transport regulatory signaling pathway. The relative mRNA levels were calculated using the control group as a baseline. The results are shown below. Figures 18-25 As shown.

[0077] Depend on Figures 18-25 The results showed that, compared with the model group, the mRNA expression level of ABCG2, the renal uric acid efflux transporter, was significantly increased in the kidney tissue of mice in the combined drug administration group. The mRNA expression level of sodium-dependent phosphotransporter NTP1 was also increased in the kidney tissue, liver tissue, and jejunum tissue. However, the mRNA expression level of GLUT9, the uric acid reabsorption transporter, was significantly downregulated in the jejunum tissue. The combined administration of Dendrobium officinale polysaccharide and Lactobacillus rhamnosus TG037 synergistically enhanced the renal uric acid clearance capacity.

[0078] 7. RNA was extracted from the intestinal contents using an RNA extraction kit according to the instructions, yielding RNA samples. The RNA samples were then subjected to 16S rRNA sequencing, and the results are as follows: Figure 26 and 27 As shown.

[0079] Depend on Figure 26 and 27The results show that, compared with the model group, the combined administration of Dendrobium officinale polysaccharide and Lactobacillus rhamnosus TG037 in the combined treatment group can effectively intervene in the dysbiosis caused by hyperuricemia and significantly increase the... Lactobacillus , Lachnospiraceae , Prevotellaceae The relative abundance of beneficial bacteria, and reduced Bilophila The relative abundance of potentially harmful bacteria.

[0080] 8. Mouse feces were collected for LC-MS / MS non-targeted metabolomics analysis, and the results are as follows: Figures 28-32 As shown.

[0081] Depend on Figures 28-32 The results show that, compared with the model group, the combined administration of Dendrobium officinale polysaccharide and Lactobacillus rhamnosus TG037 in the combined administration group can effectively restore multiple disordered metabolic pathways such as amino acid metabolism and fatty acid metabolism caused by hyperuricemia.

[0082] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention without departing from the principles and spirit of the present invention.

Claims

1. A composition, characterized in that, The composition includes Dendrobium officinale polysaccharide and Lactobacillus rhamnosus (…). Lacticaseibacillus rhamnosus TG037 or its culture and / or cell fragments; The preservation number of Lactobacillus rhamnosus TG037 is CCTCCNO: M 2026064.

2. The composition according to claim 1, characterized in that, The composition includes one or more of the following technical features: (1) The neutral sugar content of the Dendrobium officinale polysaccharide is 50%~99.999%; (2) The addition ratio of Dendrobium officinale polysaccharide and Lactobacillus rhamnosus TG037 is (100~1000) mg: (1×10 8 ~1×10 11 CFU; (3) The preparation of Dendrobium officinale polysaccharide includes: mixing Dendrobium officinale powder with water for extraction to obtain crude polysaccharide extract; and purifying the crude polysaccharide extract to obtain Dendrobium officinale polysaccharide.

3. The composition according to claim 2, characterized in that, The preparation of the Dendrobium officinale polysaccharide includes one or more of the following technical features: (1) The addition ratio of Dendrobium officinale powder to water is (0.1~10)g:(20~100)mL; (2) The extraction treatment is carried out at a temperature of 85℃~100℃ for 1h~4h; (3) The purification process includes deproteinization, alcohol precipitation and freeze drying.

4. Use of the composition according to any one of claims 1 to 3 in the preparation of reagents for the prevention, improvement and / or treatment of hyperuricemia or its complications.

5. The application according to claim 4, characterized in that, The reagent is used for one or more of the following purposes: (1) Lower serum uric acid levels; (2) Reduce serum creatinine levels; (3) Reduce serum urea nitrogen levels; (4) Reduce the expression levels of pro-inflammatory factors TNF-α, IL-1β and / or IL-6 in kidney tissue; (5) Upregulates the expression of ABCG2, the uric acid efflux transporter, in kidney tissue; (6) Downregulate the expression of GLUT9, the intestinal uric acid reabsorption transporter; (7) Regulate the structure of intestinal flora.

6. The use of the composition according to any one of claims 1 to 3 in the preparation of agents for the prevention, improvement, and / or treatment of kidney injury, characterized in that, The kidney damage was caused by hyperuricemia.

7. The use of the composition according to any one of claims 1 to 3 in the preparation of agents for preventing, improving, and / or treating metabolic homeostasis imbalances, characterized in that, The metabolic homeostasis imbalance is caused by hyperuricemia, and the metabolic homeostasis imbalance includes one or more of the following abnormally expressed metabolic pathways: (1) Amino acid metabolism-related signaling pathways; (2) Fatty acid metabolism-related signaling pathways.

8. The application according to claim 7, characterized in that, The amino acid metabolism-related signaling pathways include one or more of the following metabolic pathways: (1) β-alanine metabolic pathway; (2) cysteine ​​and methionine metabolic pathway; (3) glycine, serine and threonine metabolic pathway; (4) phenylalanine, tyrosine and tryptophan biosynthesis signaling pathway; The fatty acid metabolism-related signaling pathways include one or more of the following metabolic pathways: (1) fatty acid degradation signaling pathway; (2) fatty acid elongation signaling pathway; (3) fatty acid metabolism pathway.

9. Use of the composition according to any one of claims 1 to 3 in the preparation of a reagent for improving and / or enhancing the renal capacity to excrete uric acid.

10. Use of the composition according to any one of claims 1 to 3 in the preparation of a reagent for increasing the expression of the uric acid efflux transporter ABCG2 in kidney tissue.

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