Probiotics with uric acid degrading function and application thereof

Abstract

The invention relates to a method for screening probiotics with a uric acid degradation function. The invention also relates to application of the probiotics screened by the method in preparation of drugs or health care products for degrading uric acid, especially for treating or preventing hyperuricemia or gout.

Description

Technical Field

[0001] This invention relates to the field of microbial technology. More specifically, this invention relates to probiotics with uric acid-degrading function, their screening methods, and their applications. Background Technology

[0002] Uric acid is the final product of the degradation of exogenous purines in food. Excessive deposition in the body can lead to hyperuricemia and gout. In recent years, the global prevalence of hyperuricemia has been rising year by year, adding to the social burden. At present, there are various treatments for hyperuricemia and gout, but the treatment effects are not good (Jasvinder A.Singh,Angelo Gaffo.Goutepidemiology and comorbidities[J].Seminars in Arthritis and Rheumatism,2020,50(3):11-16). The most important part of treatment is to effectively control the risk factors of hyperuricemia. For arthritis that may have acute attacks, continuous uric acid-lowering treatment should be carried out. The ultimate goal of treatment is to cure arthritis that may have acute attacks, prevent recurrent arthritis, and delay or stop the formation of tophi and kidney stones (Riches PL, Wright A.F., Ralston SH. Recent insights into the pathogenesis of hyperuricaemia andgout[J]. Human Molecular Genetics, 2009, 18(2): 177-184.). There are two ways to treat hyperuricemia: non-pharmacological treatment and pharmacological treatment. Non-pharmacological treatment mainly includes reducing the intake of purine-rich foods, but the effect is usually limited and insufficient to significantly reduce the uric acid level in the patient's body. Therefore, the most important treatment is still pharmacological treatment. There are two main types of drugs that can lower uric acid: uric acid inhibitors (such as allopurinol) and uric acid excretion drugs (such as sulfapyrone) (Pascart T, Richette P. Investigational drugs for hyperuricemia, an update on recent developments[J]. Expert Opinion on Investigational Drugs, 2018, 27(5):437-444). However, pharmacological treatment can have side effects such as gastrointestinal discomfort, allergic conjunctivitis, and rash, which limits its clinical application. When the condition is more serious, such as infection, severe joint deformity, or nerve compression, surgical treatment should be adopted. Therefore, it is very necessary to develop new safe and effective treatment methods (Liu Huimin. Screening, genomic characteristics and application research of lactic acid bacteria purine-degrading strains[D]. Yangzhou University, 2021).

[0003] Currently, there are many probiotic health products on the market, such as those from brands like "Renhe," "By-Health," "Life-Space," and "Jiangzhong." Their main functions are to promote intestinal peristalsis, nourish the gut, regulate the gut microbiota, and aid digestion and immunity. They also have functions such as regulating vaginal microbiota and protecting women's intimate health. However, no health product currently has the function of regulating uric acid levels in the human body. Therefore, this invention contributes to filling this gap by helping to regulate the uric acid degradation process in the human body through pharmacological means, thereby reducing the risk of hyperuricemia, gout, and other diseases. Summary of the Invention

[0004] This invention aims to propose probiotics with uric acid-degrading capabilities obtained through bioinformatics methods, and simplifies the process of discovering probiotics with target functions, overcoming the inherent drawbacks of traditional culture methods such as cumbersome operations and long processing times. We obtained a series of probiotics with uric acid-degrading capabilities, providing an application basis for the development of uric acid-degrading functions in probiotic drugs or health products.

[0005] Therefore, according to one aspect of the present invention, a method for screening probiotics with uric acid degradation function is provided, the method comprising the following steps:

[0006] 1) Select probiotics from the candidate probiotics that have xanthine amide hydrolase (XnhA) protein or its homologous protein in their genome;

[0007] 2) Select probiotics obtained in step 1) that possess the uric acid transporter protein (UacT) or its homologous protein in their genome; and

[0008] 3) Conduct a uric acid degradation experiment on the probiotics obtained in step 2) and select those probiotics that have the ability to degrade uric acid.

[0009] In a preferred embodiment, the probiotic is an intestinal probiotic, preferably an edible probiotic.

[0010] In another preferred embodiment, the selection in steps 1) and 2) above is made by homology analysis of protein sequences.

[0011] In another preferred embodiment, the method further includes: 1) predicting the enzyme activity of XnhA, UacT, or their homologous proteins with the assistance of an artificial intelligence model, and selecting probiotics with higher enzyme activity; and / or 2) constructing a phylogenetic tree after homology analysis. In a particularly preferred embodiment, the artificial intelligence model includes TurNuP (available at https: / / turnup.cs.hhu.de) and CLEAN (available at...).

[0012] https: / / clean.platform.moleculemaker.org).

[0013] In another preferred embodiment, the uric acid degradation experiment is conducted in an in vitro simulated intestinal fluid environment, preferably under aerobic or microaerobic conditions.

[0014] In another preferred embodiment, the probiotic is selected from the group consisting of: *Latilactobacillus* sp., *Bacillus* sp., *Pantoea* sp., *Staphylococcus* sp., *Lacticaseibacillus* sp., and *Pediococcus* sp., or a mixture thereof.

[0015] In another preferred embodiment, the probiotic is selected from the group consisting of: *Latilactobacillus curvatus*, *Pediococcus pentosaceus*, *Bacillus subtilis*, *Pantoea sp.*, *Staphylococcus carnosus*, *Lacticaseibacillus paracasei*, *Pediococcus acidilactici*, or mixtures thereof.

[0016] According to one aspect of the invention, the invention provides the use of probiotics in the preparation of pharmaceuticals or health products for degrading uric acid, wherein the probiotics are selected from the group consisting of: Lactobacillus, Bacillus, Pantotheca, Staphylococcus, Lactobacillus and Pediococcus or mixtures thereof, preferably the probiotics can be obtained by screening using the method according to the invention.

[0017] In a preferred embodiment, the probiotic is selected from the group consisting of: *Lactobacillus curvatureii*, *Pediococcus pentosaceus*, *Bacillus subtilis*, *Pantospira*, *Staphylococcus carinatum*, *Lactobacillus casei*, *Pediococcus lactis*, or mixtures thereof, preferably selected from the group consisting of: *Lactobacillus curvatureii* CICC25172, *Pediococcus pentosaceus* CGMCC1.2695, *Bacillus subtilis* CGMCC1.821, *Pantospira* CICC22035, *Staphylococcus carinatum* CICC25173, *Lactobacillus casei* CICC20241, and *Pediococcus lactis* CGMCC1.2696.

[0018] In a preferred embodiment, the drug or health product is used to treat or prevent hyperuricemia or gout, preferably hyperuricemia or gout in mammals (including humans and animals). Attached Figure Description

[0019] The above features and advantages of the present invention will become more apparent from the following detailed description taken in conjunction with the accompanying drawings, wherein:

[0020] Figure 1 This is a complete annotated diagram of the uric acid degradation pathway;

[0021] Figure 2 It is a sequence homology analysis;

[0022] Figure 3 It is a phylogenetic tree about XnhA;

[0023] Figure 4 This demonstrates the strain's ability to degrade uric acid. Detailed Implementation

[0024] Unless otherwise stated, the terms used herein have their general technical meanings as understood by those skilled in the art. (For definitions and terms in this field...)

[0025] In this invention, the term "probiotics" refers to microorganisms that improve the host's microecological balance, thereby enhancing the host's health level and state. Beneficial bacteria or fungi in animals mainly include lactic acid bacteria, bifidobacteria, actinomycetes, and yeasts. In this invention, probiotics are intestinal probiotics that can regulate the balance of intestinal flora, promote nutrient absorption, and maintain intestinal health. All probiotics known in the prior art can be used as candidate probiotics in the screening method of this invention.

[0026] In this invention, the term "uric acid degradation function" specifically refers to the ability of probiotics, particularly intestinal probiotics, to reduce uric acid levels in animals (e.g., in the intestines).

[0027] In this invention, the term "xanthine amidohydrolase (XnhA)" refers to the key enzyme responsible for catalyzing the hydrolysis and ring-opening of xanthine to form 4-ureo-5-imidazolium carboxylic acid (UIC) in the uric acid metabolism pathway (xanthine anaerobic degradation pathway). In one specific embodiment, the Uniport entry of XnhA is A0A160MBB4.

[0028] In this invention, the term "uric acid transporter (UacT)" refers to a polypeptide capable of regulating uric acid transport and thus regulating plasma uric acid levels. In one specific embodiment, the Uniport entry for UacT is Q46821.

[0029] In this invention, the term "homologous protein" refers to a protein with significant similarity in amino acid sequences that performs the same or similar functions in different organisms or within the same organism. The protein sequences for homology analysis can be newly discovered or obtained from public databases such as NCBI's GenBank and UniProt. Bioinformatics tools known in the art can be selected for homology searches; BLAST (Basic Local Alignment Search Tool) is one of the most widely used tools. By analyzing the alignment results, the similarity and possible evolutionary relationships between sequences are assessed, and based on the alignment results, the function of the target protein can be predicted, and its evolutionary relationship can be inferred. For example, MEGA can be used for evolutionary analysis, including the construction of a phylogenetic tree and statistical analysis. Homology analysis allows for the prediction of the function of unknown proteins, especially when they are similar to protein sequences with known functions. In one specific embodiment, XnhA (A0A160MBB4) and UacT (Q46821) can be used as protein sequences with known functions, and a search for homologous proteins can be performed in the aforementioned public databases to obtain probiotics containing these homologous proteins.

[0030] In this invention, the term "probiotics having xanthine amide hydrolase (XnhA) protein or its homologous protein" refers to probiotics that can express XnhA protein or its homologous protein, or whose genome contains a gene encoding XnhA protein or its homologous protein.

[0031] In this invention, the term "probiotics having uric acid transporter protein (UacT) or its homologous protein" refers to probiotics that can express UacT protein or its homologous protein, or whose genome contains a gene encoding UacT protein or its homologous protein.

[0032] In this invention, the term "simulated intestinal fluid" refers to artificial intestinal fluid, also known as synthetic intestinal fluid, which is a solution simulating the components and pH of intestinal fluid. Typically, simulated intestinal fluid mainly consists of phosphates, trypsin, etc., with a pH of approximately 6.8. Those skilled in the art can prepare simulated intestinal fluid according to, for example, the specifications in the Chinese Pharmacopoeia. Alternatively, simulated intestinal fluid can be purchased from commercial sources.

[0033] In a preferred embodiment of the method for screening probiotics with uric acid degradation function according to the present invention, the conserved structural domains of XnhA and UacT protein sequences and the list of edible probiotics are used as input and comparison databases for homology analysis, respectively. The input sequences for the phylogenetic tree are selected from the homology analysis results based on annotation content and similarity level, and a phylogenetic tree is constructed.

[0034] Specifically, to more efficiently identify strains of natural probiotics containing target degrading enzymes, this invention employs more advanced molecular biology tools, unlike traditional target strain discovery techniques which involve significant engineering work, and utilizes the artificial intelligence models TurNuP and CLEAN for assistance. The specific discovery process of this invention is as follows: First, the uric acid degradation pathway is investigated, selecting uric acid transport proteins and the xanthine anaerobic degradation pathway as key points, such as... Figure 1 Furthermore, the key protein sequences XnhA and UacT in the degradation pathway were summarized. Conserved domains of these protein sequences and a list of edible probiotics were used as input and comparison databases for homology analysis, respectively. Input sequences for the phylogenetic tree were selected from the homology analysis results based on annotation content and similarity levels, and a phylogenetic tree was constructed. Finally, seven strains were selected for wet experiments: *Latilactobacillus curvatus*, *Pediococcus pentosaceus*, *Bacillus subtilis*, *Pantoea sp.*, *Staphylococcus carnosus*, *Lacticaseibacillus paracasei*, *Pediococcus acidilactici*, and their mixed strains. Five of these strains belong to the "List of Microbial Strains that Can Be Used in Food" published by the National Health and Safety Commission: *Latilactobacillus curvatus*, *Pediococcus pentosaceus*, *Staphylococcus carnosus*, *Lacticaseibacillus paracasei*, and *Pediococcus acidilactici*. Experimental results showed that all seven strains and their mixtures possessed a certain degree of uric acid degradation ability, although the ability to degrade uric acid differed between microaerobic and aerobic environments. This demonstrates the reliability of the method (mining process) for screening probiotics with uric acid degradation function in this invention, and also serves as an effective verification of the homology analysis and artificial intelligence prediction results.

[0035] This invention completes the process of metabolic pathway investigation and bioinformatics mining, improving the efficiency, reducing costs, and shortening the time for selecting desired probiotics from the database. Natural probiotics have the advantages of high safety and easy transformation, making them the best choice for screening functional probiotics. However, conventional screening methods rely on high-throughput screening, requiring all strains to be cultured and grown on selective media before selecting the desired strains. This process is time-consuming, inefficient, and difficult to obtain the desired results. This invention establishes a bioinformatics-based rational mining strategy, performing homology analysis of core characteristic genes and AI-assisted enzyme activity prediction. Compared to cultivating and screening expected strains from natural probiotics, its advantage lies in targeted analysis and target finding. Homology analysis of core gene sequences can quickly identify the desired strains. This invention uses the uric acid transporter UacT and xanthine amide hydrolase XnhA as targets, with Uniport entries Q46821 and A0A160MBB4, respectively. BLASTP homology analysis is then used to select strains containing both hydrolase and transporter genes from the database of edible probiotics within the policy framework.

[0036] In addition, the uric acid degradation experiment also proved that the seven selected strains and their mixed strains have the ability to degrade uric acid, providing a complete process and reliable approach for discovering strains with uric acid degradation ability from natural probiotics.

[0037] The invention is further illustrated in the following examples. These examples are for illustrative purposes only and are not intended to limit the scope of the invention. All chemicals used in the following reactions are commercially available products unless otherwise specified.

[0038] Example 1

[0039] Note the anaerobic degradation pathway of xanthine

[0040] By using "urate degrading" and "urate transporter" as keywords on the BioCyc website (https: / / www.biocyc.org), a survey was conducted on the uric acid degradation pathway and uric acid transport proteins in organisms. The uric acid transport protein UacT was selected, along with degrading enzymes in the anaerobic degradation pathway of xanthine. The enzymes required for each step of this pathway include xanthine amide hydrolase XnhA, 4-ureido-5-imidazolium carboxylate amide hydrolase XnhB, 4-amino-5-imidazolium carboxylate decarboxylase XnhC, 4-aminoimidazolium deaminase XnhD, and 4-imidazolone amide hydrolase XnhE. Analyzing the key steps of this degradation pathway, xanthine amide hydrolase XnhA, the key protein used in this invention, was identified. Therefore, uric acid transport protein UacT and xanthine amide hydrolase XnhA can be selected as targets for subsequent bioinformatics mining.

[0041] Example 2

[0042] Key protein homology analysis

[0043] The protein sequences of uric acid transporter (UacT) and xanthine amidohydrolase (XnhA) were determined from the Uniport website (https: / / www.uniport.org), with Uniport entries Q46821 and A0A160MBB4, respectively. Then, the protein domains were obtained from the NCBI website (https: / / www.ncbi.nlm.nih.gov) as input for homology analysis. Homology analysis was performed using a BLAST database, employing all known edible probiotics. The complete sequences from the results were exported, and the intersection of the two analyses was taken. The XnhA homology analysis sequence was used on the intersection strains, and tblastn analysis was performed on the intersection strains using both XnhA and UacT sequences to test the prevalence of these two enzymes. If strains within the same genus predominantly possess these two enzymes, strains from that genus can be selected for the experiment. Based on protein annotations and homology analysis similarity, a dozen or so closely annotated sequences were selected from the exported sequences as input for the phylogenetic tree. Homology analysis results were verified using the CLEAN website (https: / / clean.platform.moleculemaker.org), and the selected sequences all had the same EC number as the input sequences, demonstrating high reliability. Next, the TurNuP website (https: / / turnup.cs.hhu.de) was used to predict enzyme catalytic efficiency, and the results were compiled. See [link to detailed steps](link to detailed steps). Figure 2 .

[0044] Example 3

[0045] Constructing a phylogenetic tree

[0046] Eleven selected sequences were used as input for the phylogenetic tree. Multiple sequence alignment was performed in MEGA11 software to calculate the optimal model. The phylogenetic tree was constructed using the maximum likelihood (ML) method and evaluated using the bootstrap test. Finally, the phylogenetic tree was adjusted and refined on the iTOL website (https: / / itol.embl.de) to characterize the evolutionary relationships between different branches (see [phylogenetic tree reference]). Figure 3 ), and selected 7 of them for uric acid degradation experiments.

[0047] Example 4

[0048] Wet test verification

[0049] The selected experimental strains (Latilactobacillus curvatus, CICC number 25172; Pediococcus pentosaceus, CGMCC number 1.2695; Bacillus subtilis, CGMCC number 1.821; Pantoea sp., CICC number 22035; Staphylococcus carnosus, CICC number 25173; Lacticaseibacillus paracasei, CICC number 20241; Pediococcus acidilactici, CGMCC number 1.2696) were purchased from the China General Microbiological Culture Collection Center (CGMCC) and the China Industrial Microbiological Culture Collection Center (CICC). After autoclaving the culture medium, the strain preserved in glycerol was inoculated into the culture medium for subculturing. After culturing at the optimal temperature for 18-24 hours, it was inoculated into a new 3 ml culture medium at an inoculation rate of 2% (V / V). After two consecutive subculturings, the activated third-generation bacterial solution was obtained. OD600 was measured before the formal experiment began.

[0050] Prepare a sufficient amount of 1.17mM uric acid-simulated intestinal fluid (pH 6.8, containing trypsin). Divide the activated bacterial strains into tubes: Group A is set to an aerobic environment, with 0.3ml of bacterial culture and 14.7ml of uric acid-PBS buffer added to a 50ml tube and incubated at the optimal temperature for 30min, with 3 samples per group; Group B is set to a micro-aerobic environment, with 0.3ml of bacterial culture and 14.7ml of uric acid-PBS buffer added to a 15ml tube and incubated at the optimal temperature for 30min, with 3 samples per group.

[0051] Degradation capacity was tested using a uric acid content assay kit (brand: Solarbio, catalog number: 1365, specification: BC1365-100T / 48S):

[0052] 1. Prepare working solution A, working solution B, and blank tubes in 1.5ml centrifuge tubes according to the instructions of the uric acid content test kit, and prepare 6 standard tubes of different gradients in proportion;

[0053] 2. Take 25 μL of each sample from group A and group B and add it to a 1.5 ml centrifuge tube. Then add 75 μL of working solution A to obtain the test tube. Take 25 μL of each sample from group A and group B and add it to a 1.5 ml centrifuge tube. Then add 75 μL of working solution B to obtain the control tube.

[0054] 3. Vortex mix all four groups: blank tube, standard tube, test tube and control tube, and react in a water bath at 37°C for 30 min. Measure the absorbance at 505 nm using a visible light spectrophotometer.

[0055] Finally, data processing was performed: Δdetermination = test tube - control tube, Δstandard = standard tube - blank tube. The uric acid degradation capacity of each strain was calculated according to the formulas in the kit instructions. See the attached results. Figure 4 All seven target strains and the mixed strains showed a certain ability to degrade uric acid, which verified the reliability of the strain discovery and selection process and indicated that the other strains on the evolutionary tree may also have the ability to degrade uric acid.

[0056] Those skilled in the art should understand that although the present invention has been specifically described with reference to the above embodiments, the present invention is not limited to these specific embodiments. Based on the methods and technical solutions taught in this invention, those skilled in the art can make appropriate modifications or improvements without departing from the spirit of the present invention, and the equivalent embodiments obtained therefrom are all within the scope of the present invention.

Claims

1. A method for screening probiotics with uric acid-degrading function, the method comprising the following steps: 1) Select probiotics from the candidate probiotics that contain xanthine amide hydrolase (XnhA) protein or its homologous protein; 2) Select probiotics with uric acid transporter protein (UacT) or its homologous protein from the probiotics obtained in step 1); and 3) Conduct a uric acid degradation experiment on the probiotics obtained in step 2) and select those probiotics that have the ability to degrade uric acid.

2. The method according to claim 1, wherein the probiotic is an intestinal probiotic, preferably an edible probiotic.

3. The method according to claim 1 or 2, wherein the selection in steps 1) and 2) is performed by homology analysis of protein sequences.

4. The method according to any one of claims 1 to 3, wherein the method further comprises: 1) With the assistance of an artificial intelligence model, predict the enzyme activity of XnhA, UacT, or their homologous proteins, and select probiotics with high enzyme activity; and / or 2) After homology analysis, construct a phylogenetic tree.

5. The method according to any one of claims 1 to 4, wherein the uric acid degradation experiment is performed in an in vitro simulated intestinal fluid environment, preferably under aerobic or microaerobic conditions.

6. The method according to any one of claims 1 to 5, wherein the probiotic is selected from the group consisting of: *Latilactobacillus* sp., *Bacillus* sp., *Pantoea* sp., *Staphylococcus* sp., *Lacticaseibacillus* sp., and *Pediococcus* sp., or mixtures thereof.

7. The method of claim 6, wherein the probiotic is selected from the group consisting of: *Latilactobacillus curvatus*, *Pediococcus pentosaceus*, *Bacillus subtilis*, *Pantoea sp.*, *Staphylococcus carnosus*, *Lacticaseibacillus paracasei*, *Pediococcus acidilactici*, or mixtures thereof.

8. The use of probiotics in the preparation of pharmaceuticals or health products for degrading uric acid, wherein the probiotics are selected from the group consisting of: Lactobacillus, Bacillus, Pantotheca, Staphylococcus, Lactobacillus and Pediococcus or mixtures thereof, preferably the probiotics can be obtained by screening according to any one of claims 1 to 7.

9. The application according to claim 8, wherein the probiotic is selected from the group consisting of: *Lactobacillus curvatureii*, *Pediococcus pentosaceus*, *Bacillus subtilis*, *Pantospira*, *Staphylococcus carinatum*, *Lactobacillus casei*, *Pediococcus lactis*, or mixtures thereof, preferably selected from the group consisting of: *Lactobacillus curvatureii* CICC25172, *Pediococcus pentosaceus* CGMCC1.2695, *Bacillus subtilis* CGMCC1.821, *Pantospira* CICC22035, *Staphylococcus carinatum* CICC25173, *Lactobacillus casei* CICC20241, and *Pediococcus lactis* CGMCC1.2696.

10. The application according to claim 8 or 9, wherein the drug or health product is used to treat or prevent hyperuricemia or gout, preferably hyperuricemia or gout in mammals (including humans and animals).

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