CN117899126A

CN117899126A - Lactobacillus reuteri, composition and application thereof in uric acid reduction and weight losing

Info

Publication number: CN117899126A
Application number: CN202410080900.5A
Authority: CN
Inventors: 朱永亮; 刘丹
Original assignee: Suzhou Preyson Biotechnology Co ltd
Current assignee: Suzhou Preyson Biotechnology Co ltd
Priority date: 2024-01-19
Filing date: 2024-01-19
Publication date: 2024-04-19

Abstract

The invention discloses lactobacillus reuteri, a composition and application thereof in uric acid reduction and weight losing, and relates to the technical field of high uric acid and gout prevention and treatment. The lactobacillus reuteri screened by the invention has the effect of obviously reducing the uric acid content in blood, and can be used for preventing and/or treating hyperuricemia, and gout, gout complications and kidney injury caused by the hyperuricemia. The lactobacillus reuteri screened by the invention also has the effects of reducing the weight of mammals and reducing the blood sugar of the mammals, and can be used for preventing and/or treating diabetes and obesity. The lactobacillus reuteri still has higher activity in the alimentary canal nutrition environment, and has better development and utilization prospects for preventing and treating obesity, diabetes, hyperuricemia, gout complications or kidney injury caused by hyperuricemia.

Description

Lactobacillus reuteri, composition and application thereof in uric acid reduction and weight losing

Technical Field

The invention relates to the technical field of prevention and treatment of hyperuricemia and gout, in particular to lactobacillus reuteri, a composition and application thereof in uric acid reduction and weight reduction.

Background

Hyperuricemia (Hyperuricemia, HUA) is currently the fourth highest following hypertension, hyperlipidemia and hyperglycemia, is a disease caused by excessive uric acid generation or insufficient excretion in a human body, and can lead to diseases such as gout, kidney stones and the like, and is also a risk factor of three highs (hyperglycemia, hyperlipidemia and hypertension) to cause a series of metabolic complications. The medicine for treating hyperuricemia is the most commonly used means in clinic, has quick response and short period, but has side effects, is easy to cause anaphylactic reaction and can increase the physical burden of patients to different degrees, so a product which is safe and effective for treating hyperuricemia and has no toxic or side effects is urgently needed.

In recent years, probiotics play a remarkable role in regulating intestinal health of a human body, about 30% of uric acid in the human body is directly discharged from the intestinal tract, and intestinal probiotics play an important role in uric acid reduction, and some probiotics have been demonstrated to be capable of relieving hyperuricemia by producing metabolites affecting purine decomposition and uric acid production, and have the potential of becoming a new method for clinically treating hyperuricemia.

Chinese patent 202011066042.7 discloses a probiotic strain for reducing purine and uric acid, a composition and application thereof, and lactobacillus plantarum KLpl-3 shows obvious effect of reducing blood uric acid in a murine model of hyperuricemia. However, the screening conditions of this test method are only purine precursor substrates, and may not be effective in a practically rich enteral nutrition environment. Moreover, the bile salt tolerance experiments disclosed in the patent show that the strain is intolerant to bile salts with the concentration of 0.2% and above, and the number of viable bacteria is reduced by 1-2 orders of magnitude. The continuous effectiveness of the strain is questionable in the environment of complex intestinal tracts.

In view of this, the present invention has been made.

Disclosure of Invention

The invention aims to provide lactobacillus reuteri, a composition and application thereof in uric acid reduction and weight reduction so as to solve the technical problems.

The invention is realized in the following way:

In a first aspect, the present invention provides the use of lactobacillus reuteri (Limosilactobacillus reuteri) for the manufacture of a medicament for the prophylaxis and/or treatment of at least one disease or condition selected from the group consisting of:

diabetes, obesity and obesity related diseases, hyperuricemia, gout, complications of gout and kidney injury caused by hyperuricemia;

The 16S rDNA sequence of lactobacillus reuteri has the accession number GDMCC No: the 16S rDNA sequence present in the M2023591 deposited lactobacillus reuteri strain has at least 99% sequence identity;

and/or lactobacillus reuteri whole genome sequence and accession number GDMCC No: the lactobacillus reuteri strain deposited with M2023591 has a genome-average nucleotide similarity (ANI score) of at least 97.5%.

The inventor separates, screens and purifies a strain from the feces of healthy people. The isolated strain was subjected to whole genome sequencing using Illumina PE150 platform and genome assembly using SPAdes software, the strain was 1881471bp in full length, had gc content of 38.69%, and contained 1910 genes. The analysis of the whole genome sequencing data of the strain using GTDBTK software, the closest reference strain was Limosilactobacillus reuteri (GCF_ 000016825.1) by FASTANI algorithm, the ANI value was 96.22%, and when the ANI value was greater than 95, 2 were considered to be the same species, so the strain was identified as Lactobacillus reuteri and designated PRS-156.

By comparing and analyzing uric acid synthesis pathway related enzyme coding genes in the genome of the Lactobacillus reuteri PRS-156 bacterium, the result shows that the Lactobacillus reuteri PRS-156 does not contain EC1.17.1.4, EC1.17.3.2, EC3.5.4.3, so that the strain can reduce the conversion of purine substrates into xanthines. Also contains EC2.4.2.8, and can convert xanthine which is a precursor compound of uric acid into other products without producing uric acid.

Wherein EC3.5.4.3 is guanine deaminase, which catalyzes the hydrolytic deamination of guanine to produce xanthine and ammonia.

EC2.4.2.8 is a hypoxanthine guanine phosphoribosyl transferase that catalyzes the conversion of 5-phosphoribosyl-1-pyrophosphate with hypoxanthine, guanine or 6-mercaptopurine to the corresponding 5' -mononucleotide and pyrophosphate.

EC1.17.1.4 is xanthine dehydrogenase.

EC1.17.3.2 is xanthine oxidase, can catalyze xanthine to oxidize to generate uric acid and superoxide anion, and is one of main sources of active oxygen.

In a second aspect, the invention also provides lactobacillus reuteri, which is deposited under accession number GDMCCNo: m2023591 deposited Lactobacillus reuteri strain.

In a third aspect, the invention also provides a composition comprising lactobacillus reuteri as described above.

In a fourth aspect, the invention also provides a method for culturing lactobacillus reuteri, which comprises the following steps: inoculating the lactobacillus reuteri, and culturing under the condition;

the above-mentioned condition culture is anaerobic culture.

The invention has the following beneficial effects:

The lactobacillus reuteri screened by the invention has the effect of obviously reducing the uric acid content in blood, and can be used for preventing and/or treating hyperuricemia, and gout, gout complications and kidney injury caused by the hyperuricemia.

The lactobacillus reuteri screened by the invention also has the effects of reducing the weight of mammals and reducing the blood sugar of the mammals, and can be used for preventing and/or treating diabetes and obesity.

The lactobacillus reuteri provided by the invention still has higher bacterial strain survival rate in alimentary canal nutrition environment (gastric juice and intestinal juice) through simulating artificial gastric juice and artificial intestinal juice experiments, and the viable count is not obviously reduced. The result indicates that the lactobacillus reuteri provided by the invention still has higher activity in the alimentary canal nutrition environment, and has better development and utilization prospects for treating or preventing and improving hyperuricemia, gout and gout complications or kidney injury caused by the hyperuricemia.

Preservation description

Preservation address: chinese, wuhan, university of Wuhan

Preservation date: 2023, 04, 23

Strain name: lactobacillus reuteri PRS-156

Latin name: limosilactobacillus reuteri PRS-156

Preservation mechanism: china center for type culture Collection

The preservation organization is abbreviated as: CCTCC (cctccc)

Accession numbers of the preservation center: CCTCC NO: M2023591

Identification result: survival of

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a colony morphology of Lactobacillus reuteri PRS-156;

FIG. 2 is a gram-staining microscopy chart of Lactobacillus reuteri PRS-156;

FIG. 3 is a uric acid level standard graph;

FIG. 4 is a graph showing statistical results of blood uric acid levels in hyperuricemia mice fed 7d Lactobacillus reuteri PRS-156;

FIG. 5 is a graph showing the statistical results of the percentage of body weight change of obese model mice fed 28d Lactobacillus reuteri PRS-156;

FIG. 6 is a graph showing the statistical results of body weight of obese model mice fed Lactobacillus reuteri PRS-156 at 28 d;

FIG. 7 is a graph of blood glucose statistics of obese model mice fed Lactobacillus reuteri PRS-156 at 28 d;

FIG. 8 is a flow chart of uric acid synthesis and metabolism.

Detailed Description

Reference now will be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. Indeed, it will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment can be used on another embodiment to yield still a further embodiment.

Unless otherwise indicated, practice of the present invention will employ conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the ability of a person skilled in the art. This technique is well explained in the literature, as is the case for molecular cloning: laboratory Manual (Molecular Cloning: A Laboratory Manual), second edition (Sambrook et al, 1989); oligonucleotide Synthesis (Oligonucleotide Synthesis) (M.J.Gait, eds., 1984); animal cell Culture (ANIMAL CELL Culture) (r.i. freshney, 1987); methods of enzymology (Methods in Enzymology) (academic Press Co., ltd. (ACADEMIC PRESS, inc.)), experimental immunology handbook (Handbook of Experimental Immunology) (D.M.Weir and C.C. Blackwell, inc.), gene transfer vectors for mammalian cells (GENE TRANSFER vector for MAMMALIAN CELLS) (J.M.Miller and M.P.Calos, inc., 1987), methods of contemporary molecular biology (Current Protocols in Molecular Biology) (F.M.Ausubel et al, 1987), polymerase chain reaction (PCR: the Polymerase Chain Reaction) (Mullis et al, 1994), and methods of contemporary immunology (Current Protocols in Immunology) (J.E.Coligan et al, 1991), each of which are expressly incorporated herein by reference.

The inter-species genome-wide data ANI values were calculated by GTDBTK software using fastANI algorithm. Average nucleotide similarity (Average Nucleotide Identity, ANI) is an indicator of the relatedness of two genomes at the nucleotide level. ANI is defined as the average base similarity between homologous fragments of two microbial genomes, characterized by a higher discrimination between closely related species. The BLAST is known as Basic Local ALIGNMENT SEARCH Tool, namely a search Tool based on a Local alignment algorithm, is a Tool software commonly used in bioinformatics, and can be used for comparing an input nucleic acid or protein sequence with a known sequence in a database to obtain information such as sequence similarity and the like, so as to judge the source or evolutionary relationship of the sequence.

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Diabetes, obesity, hyperuricemia, gout complications and kidney injury caused by hyperuricemia;

The inventor separates and purifies a strain of bacteria from the feces of healthy donors, and the strain is identified as gram positive bacillus, which does not form spores and is not motile. The colonies appeared round raised, opaque, white, smooth in surface. It was incubated at 37℃under isothermal anaerobic conditions in MRS medium for about 12h to the end of the log.

The 16s rDNA gene sequence of the strain is amplified and tested, and compared with the lactobacillus reuteri strain in Genebank, the similarity rate reaches 99.79 percent. The strain is further subjected to whole genome sequencing, the obtained sequence result is searched and subjected to similarity comparison by using FASTANI, the ANI value is 96.22%, and the sequencing result is identified as lactobacillus reuteri.

Furthermore, it will be readily apparent to those skilled in the art that the strain provided by the present invention is suitable for use in obesity-related diseases, including at least one of the following diseases: cardiovascular disease, hyperlipidemia, insulin resistance syndrome, obesity-related gastroesophageal reflux disease, and steatohepatitis.

In other embodiments, the "obesity-related diseases" described above may be selected from the following diseases: overeating (overeating), binge eating, bulimia, hypertension, diabetes, elevated plasma insulin concentrations, insulin resistance, hyperlipidemia, metabolic syndrome, insulin resistance syndrome, obesity-related gastroesophageal reflux disease, atherosclerosis, hypercholesterolemia, hyperuricemia, lower back pain, cardiac hypertrophy and left ventricular hypertrophy, lipodystrophy, nonalcoholic steatohepatitis, cardiovascular disease, and polycystic ovary syndrome, as well as those subjects with these obesity-related disorders including those desiring weight loss.

The three major types of diabetes are type 1 diabetes (T1D), type 2 diabetes (T2D), and Gestational Diabetes (GDM). Type 1 diabetes is caused by autoimmune damage or idiopathic causes, and is characterized by absolute destruction of islet function, and occurs in children and adolescents, where insulin therapy is necessary to achieve satisfactory efficacy, otherwise life threatening. Type 2 diabetes is a multifactorial syndrome characterized by abnormal carbohydrate/fat metabolism, and generally includes hyperglycemia, hypertension, and cholesterol abnormalities. Type 2 diabetes is caused by the fact that insulin cannot effectively act (has a small amount of binding to a receptor), and therefore, it is necessary to examine not only fasting blood glucose but also blood glucose 2 hours after meal, and it is particularly desirable to examine islet function. Diabetes during pregnancy has two conditions, one of which is a condition that has been diagnosed with diabetes before pregnancy, called "diabetes mellitus combined pregnancy"; another type is diabetes mellitus in which the metabolism of sugar before pregnancy is normal or potential sugar tolerance is reduced, and only occurs or is diagnosed in gestation, also called as "Gestational Diabetes Mellitus (GDM)", and more than 80% of pregnant women with diabetes mellitus are GDM.

It should be noted that the application of the above pharmaceutical use to diabetes includes, but is not limited to, treatment or prevention of type 1 diabetes (T1D), type 2 diabetes (T2D), and Gestational Diabetes (GDM).

In a preferred embodiment of the invention, the 16S rDNA sequence and accession number GDMCC No of Lactobacillus reuteri: the 16S rDNA sequence present in the M2023591 deposited lactobacillus reuteri strain has at least 99%, or at least 99.5%, or at least 99.8%, or at least 99.9%, or 100% sequence identity.

The whole genome sequence of lactobacillus reuteri and the preservation number are GDMCC No: the lactobacillus reuteri strain deposited with M2023591 has a genome-average nucleotide similarity (ANI score) of at least 98%, at least 98.65%, or at least 99%, or at least 99.9%, or 100%.

In a preferred embodiment of the invention, the 16S rDNA sequence and accession number GDMCC No of Lactobacillus reuteri: the 16SrDNA sequence present in the M2023591 deposited lactobacillus reuteri strain has at least 99.5% sequence identity;

And/or lactobacillus reuteri whole genome sequence and accession number GDMCC No: the lactobacillus reuteri strain deposited with M2023591 has a genome-average nucleotide similarity (ANI score) of at least 98%.

In an alternative embodiment, lactobacillus reuteri is deposited under accession number GDMCC No: m2023591 deposited Lactobacillus reuteri.

In a preferred embodiment of the present invention, the lactobacillus reuteri is at least one of a concentrate, a paste, a dry, a liquid and a dilution of lactobacillus reuteri.

The lactobacillus reuteri dry matter of the present invention includes, but is not limited to, spray-dried matter, freeze-dried matter, vacuum-dried matter, roller-dried matter.

Furthermore, it will be readily apparent to those skilled in the art that a live, dry or inactivated strain of lactobacillus reuteri is used for the preparation of at least one of concentrate, paste, dry, liquid and dilution.

When lactobacillus reuteri is a liquid, the amount of lactobacillus reuteri included in the liquid is at least 5×10 ⁹ CFU/mL, and when lactobacillus reuteri is a solid, the amount of lactobacillus reuteri included in the solid is at least 5×10 ⁹ CFU/g.

Lactobacillus reuteri is a culture of lactobacillus reuteri, including but not limited to: at least one of dead bacteria of lactobacillus reuteri, cell debris, fermentation supernatant and fermentation precipitate.

In a preferred embodiment of the invention for use, the medicament is for use in any one of the following:

(1) Lowering blood uric acid in a mammal;

(2) Reducing the weight of the mammal;

(3) Lowering blood glucose in a mammal;

(4) Is used for decomposing purine compounds.

Purine compounds are components constituting nucleic acids, and are supplied to the body via the de novo (de novo) synthesis pathway, the salvage synthesis pathway, and nuclear proteins in the diet, and unnecessary purine compounds are metabolically excreted in the liver. Uric acid is the final metabolite of purine compounds in humans, higher primates, birds, reptiles, and the like.

The purine compounds in the present specification refer to compounds having a purine skeleton. As typical examples of purine compounds, purine nucleotides (adenylate, deoxyadenylate, guanylate, deoxyguanylate), purine nucleosides (adenosine, deoxyadenosine, guanylate, deoxyguanylate), purine bases (adenine, guanine), oligonucleotides and polynucleotides containing purine bases can be cited. The purine bases may constitute a plurality of biological components such as ATP, GTP, cAMP, cGMP and coenzyme A, FAD, NAD in addition to nucleic acids. In the present specification, as long as the purine skeleton is present, all of such biological components are contained in the purine compound.

Purine compounds in the living body are metabolized into uric acid. The metabolic pathway from purine compounds to uric acid is well known. AMP is converted to adenosine by 5' -nucleotidase, which is converted to hypoxanthine by inosine. GMP is converted to guanine by 5' -nucleotidase and then to guanosine. Hypoxanthine is metabolized to xanthine by xanthine oxidase and guanine is metabolized to xanthine by guanine deaminase, and xanthine is further converted to uric acid by xanthine oxidase.

The purine compounds decomposing ability in the present invention means an ability to decompose at least one purine compound, regardless of whether the decomposition product has a purine skeleton. That is, the ability to decompose a certain purine compound into a compound having no purine skeleton and the ability to decompose a certain purine compound into another purine compound (a compound having a purine skeleton) are all the purine compound decomposing ability of the present invention.

In an alternative embodiment, the purine compounds are selected from uric acid and/or uric acid metabolic precursors;

in an alternative embodiment, the uric acid metabolism precursor is selected from at least one of a purine nucleotide, a purine nucleoside, a purine base, an oligonucleotide comprising a purine base, and a polynucleotide.

In an alternative embodiment, the uric acid metabolism precursor is selected from at least one of guanine, inosine, xanthosine, and guanosine.

In a preferred embodiment of the invention, the medicament further comprises pharmaceutically acceptable excipients.

In a preferred embodiment of the use of the invention, the medicament is formulated for oral administration or injection administration.

In a preferred embodiment of the invention, the composition further comprises pharmaceutically acceptable excipients.

In a preferred embodiment of the present invention, the pharmaceutically acceptable excipients are selected from at least one of fillers, disintegrants, lubricants, flavoring agents, binders, suspending agents and fragrances.

Pharmaceutically acceptable excipients include, but are not limited to: pharmaceutically acceptable carriers, auxiliary substances or solvents. Pharmaceutically acceptable excipients include various organic or inorganic carriers and/or auxiliary materials, as they are commonly used for pharmaceutical purposes, in particular for solid pharmaceutical formulations. Examples include: excipients, for example sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate, calcium carbonate; binders, such as cellulose, methylcellulose, hydroxypropyl cellulose, polypropylene pyrrolidone, gelatin, acacia, polyethylene glycol, sucrose, starch; disintegrants, for example starch, hydrolyzed starch, carboxymethyl cellulose calcium salt, hydroxypropyl starch, sodium starch glycolate, sodium bicarbonate, calcium phosphate, calcium citrate; lubricants, such as magnesium stearate, talc, sodium lauryl sulfate; perfumes such as citric acid, menthol, glycine, orange powder; preservatives, such as sodium benzoate, sodium bisulphite, parabens (e.g. methyl parahydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate, butyl parahydroxybenzoate); stabilizers such as citric acid, sodium citrate, acetic acid and polycarboxylic acids from the titriplex series, such as diethylenetriamine pentaacetic acid (DTPA); suspending agents, such as methylcellulose, polyvinylpyrrolidone, aluminum stearate; a dispersing agent; diluents, such as water, organic solvents; waxes, fats and oils, such as beeswax, cocoa butter; polyethylene glycol; white vaseline, etc.

In a preferred embodiment of the use of the present invention, the dosage form of the above-mentioned drug is selected from any one of injection, tablet, granule, capsule, emulsion, suspension, elixir, syrup, powder and tablet.

In an alternative embodiment, the medicament is a liquid pharmaceutical formulation (e.g., as one of an injection), such as a solution, suspension, and gel, typically containing a liquid carrier, such as water and/or a pharmaceutically acceptable organic solvent. In addition, such liquid formulations may also contain pH adjusting agents, emulsifying or dispersing agents, buffering agents, preservatives, wetting agents, gelling agents (e.g., methylcellulose), dyes, and/or flavoring agents, e.g., as defined above. The drugs may be isotonic, i.e. they may have the same osmotic pressure as blood. The isotonicity of the drug may be adjusted by using sodium chloride and other pharmaceutically acceptable agents such as dextrose, maltose, boric acid, sodium tartrate, propylene glycol and other inorganic or organic soluble materials. The viscosity of the liquid composition may be adjusted by a pharmaceutically acceptable thickener such as methyl cellulose. Other suitable thickening agents include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomers, and the like. The preferred concentration of thickener depends on the agent selected.

In a preferred embodiment of the use of the invention, the composition is a pharmaceutical composition, a vaccine composition or a food product.

When the composition is a vaccine composition, the vaccine composition further comprises an adjuvant.

The term "adjuvant" refers to a substance capable of modifying or enhancing an immune response to an antigen. In other words, the immune response to an antigen may be higher or different in the presence of an adjuvant than when an adjuvant is not present (including when the response is modified, e.g., a subset of T cells activated in the presence of an adjuvant is different than a subset activated in the absence of an adjuvant). The adjuvant is for example selected from lipid adjuvants.

The type and class of foods produced by the lactobacillus reuteri provided by the present invention are not limited, and the foods may be functional foods, specific health foods, and nursing foods, and may be dairy products such as snack foods, lactobacillus beverages, cheese, yogurt, and the like, seasonings, and the like.

Food products include, but are not limited to, drinking food products. The form of the food or beverage is not limited, and any form of food or beverage that can be circulated in general, such as solid, liquid, fluid food, jelly, tablet, granule, capsule, etc., may be used. The production of the above-mentioned foods and drinks can be carried out by a method which is routine to those skilled in the art. In the production of the food or drink, saccharides, proteins, fats, dietary fibers, vitamins, trace metals essential to living bodies (manganese sulfate, zinc sulfate, magnesium chloride, potassium carbonate, etc.), flavors, and other complexes may be added as long as the growth of lactic acid bacteria is not inhibited.

The lactobacillus reuteri provided by the invention can be prepared into general drinks and foods of dairy products and fermented milk, and can be used as a primer (starters) for manufacturing dairy products such as yoghurt or cheese and the fermented milk. When the lactobacillus of the genus lactobacillus of the present invention and the lactobacillus of the genus lactobacillus of the present invention are used as primers, other microorganisms may be mixed as long as they do not interfere with the growth and propagation of the lactobacillus and lactobacillus orotate of the present invention and do not interfere with the production of dairy products. For example, the primer may be used in combination with Lactobacillus delbrueckii subspecies bulgaricus, streptococcus thermophilus (Streptococcus thermophilus), lactobacillus acidophilus, etc., which are mainly species of lactic acid bacteria for yogurt, or may be used in combination with a strain commonly used for yogurt or cheese. The production of the dairy product or fermented milk using the above starter can be carried out according to a conventional method. For example, the above starter is mixed with milk or dairy products cooled after heating, mixing, homogenizing and sterilizing, and the mixture is fermented and cooled to obtain pure yogurt.

The above-mentioned condition culture is anaerobic culture. For example anaerobic cultivation at 36-37 ℃.

The features and capabilities of the present invention are described in further detail below in connection with the examples.

Example 1

This example shows the isolation, screening and identification of Lactobacillus reuteri.

(1) Isolation and screening of lactobacillus: a fresh, healthy donor fecal CBS sample 1mL was taken and placed in PBS containing 9mL of sterile anaerobic L-cysteine (0.3 g/L) and shaken for 1 minute. 0.1mL of the culture solution was dropped onto the modified tomato juice agar medium, streaked, and then placed in an anaerobic incubator to be cultured at 37℃for 96 hours. Single colonies were picked on modified tomato juice dishes and anaerobically cultured at 37℃for 72h for gram staining.

The morphology is observed under a microscope, and microscopic examination is carried out on gram-positive bacillus, which indicates that the bacillus is successfully separated and purified, single bacterial colony is selected to be inoculated into MRS broth, and anaerobic culture is carried out for 72 hours. And judging whether to purify or not according to the morphological characteristics of the plate colony and the dyeing characteristics, the size, the shape and the distribution condition of the liquid culture dyeing mirror observation thalli. If the bacteria are not pure, the steps are continued, and the separation and passage are repeated for a plurality of times until a purified strain is obtained, which is named as PRS-156.

Characteristics of cells (fig. 1): gram-positive staining, no sporulation, and no motility.

Colony characterization (fig. 2): it is round, opaque, white and smooth.

Growth characteristics: culturing in MRS medium at 37deg.C under isothermal anaerobic condition for about 12 hr to end of logarithm.

(2) Molecular biological identification of lactobacillus

The obtained strain PRS-156 is identified by utilizing 16S rDNA sequencing and physiological and biochemical identification, and the sequence 27F of the 16S rDNA universal primer is as follows: 5'-AGAGTTTGATCCTGGCTCAG-3',1492R is: 5'-GGTTACCTTGTTACGACTT-3'.

The 16S rDNA gene sequence of the strain PRS-156 was amplified and sequenced, and the PCR amplified product was sent to Shanghai Biotechnology (Shanghai) Biotechnology Co., ltd. For sequencing, the nucleotide sequence of the 16S rDNA of the strain PRS-156 was as set forth in Seq ID No: 1. The similarity of the 16s rDNA gene comparison with lactobacillus reuteri strains in Genebank is up to 99.79%.

Further, whole genome sequencing is carried out, the extracted whole genome is sent to Jin Weizhi Biotechnology Inc. of Suzhou for second generation sequencing, the obtained sequence result is compared with a reference strain Limosilactobacillus reuteri (GCF_ 000016825.1) in NCBI database, and the lactobacillus reuteri is identified, wherein the ANI value is 96.22%, and the lactobacillus reuteri is frozen at-80 ℃ for later use.

The strain PRS-156 is identified as a strain of Lactobacillus reuteri by combining with physiological and biochemical identification results, and is named as the Lactobacillus reuteri PRS-156, and the physiological and biochemical properties are shown in Table 1.

TABLE 1 PRS-156 physiological and biochemical identification results of strains

Note that: +: a positive result; -: negative results; v: weak positive.

Example 2

This example was conducted for drug resistance detection of the strain isolated in example 1.

The determination was carried out using a micro broth dilution method with reference to the standard specified in the ISO 10932 document. According to the concentration ranges and solvents of the respective antibiotics in Table 2, the corresponding antibiotic mother liquor was prepared and then filtered with a 0.22 μm filter membrane. And (3) continuously diluting for nine times by adopting a double dilution method to obtain ten continuous double concentration gradient antibiotic solutions with corresponding concentrations, and respectively adding the antibiotic solutions into 2-11 columns of sterile 96-well plates according to the sequence from low concentration to high concentration, wherein 100 mu L of antibiotic solution is added into each well.

Inoculating frozen glycerol bacteria at the temperature of minus 80 ℃ to an MRS agar culture medium plate for streak activation, culturing for 24-48 hours at the temperature of 37 ℃, picking out plate colonies, dispersing the plate colonies into the MRS broth culture medium, fully mixing the plate colonies, enabling the OD ₆₀₀ = 0.16-0.2 of bacterial liquid to correspond to the viable bacteria number of about 3 multiplied by 10 ⁸ CFU/mL, inoculating the bacterial liquid into LSM broth culture medium with the concentration of 2 times according to the inoculum size of 0.1% (v/v), mixing the bacterial liquid uniformly, and taking 100 mu L of bacterial liquid into a 96-well plate of plus antibiotic.

Column 1 of the 96-well plate was a positive control, containing no antibiotic only the experimental strain and medium, column 12 of the 96-well plate was added with 100 μl of sterile water and 100 μl of 2-fold concentration LSM medium as a negative control. And (3) placing the 96-well plate at the constant temperature of 37 ℃ for culture for 48 hours, wherein the lowest antibiotic concentration of the strain without visible growth is the MIC of the strain for antibiotics.

The results of the lactobacillus reuteri antibiotic resistance analysis are shown in table 2, and the resistance phenotype of the strain to antibiotics is analyzed according to the judgment of the european food safety agency (EFSA (2012)) on the sensitivity threshold ECOFF of the microorganism to different antibiotics. If the MIC value is greater than ECOFF, it is indicated that the strain is resistant to this antibiotic, and if the MIC value is less than or equal to ECOFF, it is indicated that the strain is sensitive to this antibiotic.

TABLE 2MIC values and drug sensitivity results

Note that: "S" means sensitivity and "R" means resistance.

As a result, the PRS-156 strain of example 1 was mainly resistant to tetracycline, ampicillin, chloramphenicol, kanamycin, and drug; is sensitive to erythromycin, clindamycin, gentamicin and streptomycin.

Example 3

This example examined the digestive tract environmental tolerance characteristics of Lactobacillus reuteri PRS-156.

(1) Simulated artificial gastric fluid (Simulated Gastric Fluid, SGF) experiment

The preparation method of the artificial gastric juice comprises the following steps: 2.0g NaCl and 3.2g pepsin (Soy bao pepsin, 1:3000, marked as 800-2500 activity units in each mg) are taken, 7.0mL of 37% diluted hydrochloric acid and pure water are added for dissolution and volume fixing to 1000mL, and the pH value of the solution is 1.2. Adjusting pH to 3.0 (simulating human postprandial intestinal juice pH and mouse fasting intestinal juice pH), filtering, and sterilizing;

Collecting bacteria for standby: the strain is statically cultured for 8-10 h at 37 ℃ to reach logarithmic growth phase, bacterial liquid is split into 50mL sterile EP pipes, after centrifugation is carried out at 4000rpm at room temperature for 5min, supernatant is discarded, PBS is used for resuspension of bacterial bodies, OD ₆₀₀ value of bacterial liquid is regulated to obtain bacterial number of about 2X 10 ⁹ CFU/mL, then 15mL sterile EP pipes are taken, 6mL bacterial liquid with OD value regulated is respectively added, centrifugation (same as above) is carried out, bacterial mud is collected for standby, PBS is used for resuspension as a control group;

Incubating and culturing artificial gastric juice: 6mL of the artificial gastric juice with the pH value of 3 is added into a 15mL centrifuge tube containing bacterial mud, and the mixture is blown and mixed uniformly, and incubated for 3h for coating counting. After incubation for 3 hours, the number of viable bacteria is still kept at an order of magnitude, which indicates that the strain has stronger acid resistance;

Calculating the gastric acid tolerance of bacteria: and (3) counting the plates, recording the colony numbers of the plates, and performing data processing to obtain gastric juice tolerance results of the strain after the artificial gastric juice with different pH values acts for different time. Calculating a survival rate formula: survival = A2/a1×100%. Wherein: a1 is the number of viable bacteria (CFU/mL) of artificial gastric juice incubated for 0h at different pH values; a2 is the number of viable bacteria (CFU/mL) incubated for 3h in artificial gastric juice at different pH.

As a result, as shown in Table 3, the survival rate of the strain was calculated to be 45.24%, and the acid resistance was strong.

TABLE 3PRS-217-156 (Lactobacillus reuteri) acid and bile salt tolerance data

(2) Experiment for simulating human body internal bile salt environment

Firstly preparing a ox gall salt culture medium according to the following steps: weighing 2g/L (0.2%) of ox gall salt, adding into the solution of target bacteria culture medium (MRS broth, etc.), and sterilizing under high pressure for use;

then collecting the strain, wherein the strain collecting step is the same as SGF experiment;

then, the ox gall salt is incubated, and the incubation and culture steps are as follows: respectively adding 3mL of MRS broth containing 0.2% of ox gall salt into 16 centrifuge tubes of bacterial sediment collected after washing, and incubating and culturing for 3h;

Finally, plate coating counting is carried out: carrying out 10-time gradient dilution on bacterial solutions of different concentration ox gall salt incubation time points, selecting three proper dilution factors, uniformly absorbing 100uL of dilution liquid, carrying out flat plate coating, setting 3 parallel concentration dilution liquids, and culturing at 37 ℃ for 3 hours; and (3) counting the plates, recording the colony number of each plate, and performing data processing to obtain the cholate tolerance condition of the strain.

The calculated survival rate formula is as follows: survival = A2/a1×100% formula: a1 is the number of viable bacteria (CFU/mL) of bacterial liquid in 0.2% ox gall salt culture medium solution for 0 h; a2 is the number of viable bacteria (CFU/mL) incubated for various times.

The results are shown in Table 3, and in summary, the results show that the Lactobacillus reuteri PRS-156 can still keep better survival rate in gastric juice environment with lower pH value, and can still ensure 45.24% survival rate after incubation for 3 hours in artificial gastric juice environment with pH value of 3.0; the survival rate at 0.2% ox gall salt concentration was 46.25%.

Example 4

In this example, in vitro enzyme activity assay of urate oxidase was performed.

(1) Uric acid standard curve determination

Uric acid was measured in a buffer by ultraviolet spectrophotometry, and absorbance at 293nm was measured directly. Uric acid standard solutions of 0, 10. Mu.M, 25. Mu.M, 50. Mu.M, 100. Mu.M, 250. Mu.M, 500. Mu.M, 750. Mu.M and 1000. Mu.M were prepared first, and uric acid concentration standard curves were drawn, see FIG. 3. Subsequent appropriate dilutions were performed for the measurement to determine uric acid concentration.

(2) Enzyme activity determination method

Lactobacillus reuteri PRS-156 was inoculated into liquid MRS medium and cultured at 37 ℃ for 20h to obtain a bacterial suspension. The bacterial suspension was centrifuged at 8000r/min at 4℃for 5min to collect the strain, which was washed 1 time in HEPES (pH 7.0,0.50 mM) buffer. The bacterial suspension OD ₆₀₀ was controlled to be about 1.0, 1mM uric acid was added to induce a reaction, and the test tube was incubated at 37℃for 60min for sampling detection. After centrifugation at 10000rpm for 3min, the concentration of UA in the supernatant was determined spectrophotometrically.

(3) Enzyme activity calculation formula

The amount of enzyme required to catalyze the decomposition of 1. Mu. Mol of uric acid per minute at 30℃and pH8.0 was defined as 1 enzyme activity unit.

The enzyme activity was calculated according to the following formula:

Wherein, vt: total volume (4 mL), vs: bacterial suspension volume (3.6 mL), t: reaction time (60 min), K: uric acid standard curve slope, df: dilution factor; OD _blank is a blank control group incubated with uric acid alone, and OD _test is the absorbance actually tested.

The enzyme activity of urate oxidase produced by Lactobacillus reuteri PRS-156 was determined to be 3.87U/mL.

Example 5

The present experimental example was conducted by analyzing the genome of Lactobacillus reuteri PRS-156, and comparing the 4 enzyme genes related to the uric acid synthesis pathway with the genome of Lactobacillus reuteri PRS-156.

Blastn selects and downloads the nucleic acid sequence of IMG database related gene to compare, the parameter selection e-value is less than 1e-05, bit-score is greater than 50, identity is greater than 97%, coverage is greater than 90%.

Gene enzyme numbering	Strain genome Gene numbering
		EC1.17.1.4	Without any means for
EC1.17.3.2	Without any means for
		EC3.5.4.3	Without any means for
EC2.4.2.8	orf_00777，orf_01764

From the BLAST alignment results, none of the 3 xanthine-producing genes (EC1.17.1.4, EC1.17.3.2, EC3.5.4.3) were matched in the whole genome data, resulting in a reduction in xanthine production by the strain. The 2 genes (EC1.17.1.4, EC1.17.3.2) that simultaneously produced uric acid also did not match, nor did the strain produce uric acid.

Alignment EC2.4.2.8 genes found that there were 2 copies in the genome that could generate other products from intermediates that produced xanthines. Specifically, EC2.4.2.8 can produce inosine and guanine from inosine. A uric acid synthesis and metabolism flow chart is shown with reference to fig. 8.

Example 6

In this example, experiments were performed to decompose purine compounds.

Lactobacillus reuteri PRS-156 stored in glycerol tubes was inoculated in MRS medium, cultured at 37 ℃ until OD ₆₀₀ was 1.0, and the cultured broth was centrifuged at 4000rpm for 10min with each 2ml in a centrifuge tube, and the supernatant was discarded to recover the cells.

30Mg of guanosine, inosinic acid, and guanosine, and inosine were each dissolved in 100ml of PBS buffer at pH 7.0, to prepare reaction solutions.

The reaction solutions were added into centrifuge tubes containing bacterial cell sediment (the control group uses equal volume of PBS instead of bacterial cells), 0.75ml of the reaction solution was added into each tube, after bacterial cells were resuspended, the cells were incubated at 37℃with shaking for 60min, the reaction was terminated by adding 40. Mu.l of 0.1mol/L HClO ₄ at the end of incubation, and the supernatant was centrifuged at 10000rpm for 15min for further analysis.

The substrates (hypoxanthine, inosinic acid, guanine nucleotides) in the supernatant were detected. Analysis was performed by HPLC chromatography under the following conditions: KH2PO4 (20 mmol/L, pH 6.0) solution: methanol=95:5 as mobile phase, the column temperature is 25 ℃, the flow rate is 1.2ml/min, and the elution time is 30min. Agilent 1290 liquid chromatograph, eclipse XDB-C18, 150×4.6mm, and DAD as detector were used. The degradation rate of the strain on purine compounds was further calculated based on the ratio of peak areas of the HPLC experimental group to the control group.

The experimental results are shown in the following table:

Substrate(s)	0H	1H	Degradation rate
				Inosinic acid	29.08	6.37	78.09％
Guanosine (guanosine)	29.58	0.0	100％
				Guanine nucleotide	32.65	34.65	0.00％
Inosine	29.63	0.0	100％
				Hypoxanthine (inosine)	31.54	27.01	14.36％

Experimental results show that the lactobacillus reuteri PRS-156 has good degradation effects on inosinic acid, inosine and guanosine.

Example 7

This example tested lactobacillus reuteri in example 1 in a mouse model of hyperuricemia, demonstrating its use in the treatment or prevention of hyperuricemia.

The experimental method comprises the following steps:

(1) Sample preparation: the cultured lactobacillus reuteri PRS-156 bacterial suspension is centrifuged to remove the supernatant, and the concentration of the bacterial solution is regulated by sterile physiological saline to the viable count of about 1X 10 ⁹ CFU/mL.

(2) Animal experiment: the mice selected in this experiment were female Balb/c mice, 7-8 weeks old, and had a weight range of 18-22g, purchased from Shanghai Ling Biotechnology Co.

The mouse hyperuricemia model is used for verifying the drug effect, and the model construction method is as follows:

Mice were acclimatized for one week before the start of the experiment. Mice were weighed before molding began and randomly grouped according to body weight. Control mice were lavaged with 200. Mu.l of physiological saline daily and injected intraperitoneally with 200. Mu.l of 0.5% CMC-Na. Model and treatment mice were lavaged with 75mg/kg adenine (MCE, HY-B0152) daily, while being intraperitoneally injected with 250mg/kg Potassium oxazinate (MCE, HY-17511) daily. The molding lasts for 7 days, mice are fed intermittently for 16 hours after the molding on the 7 th day, and blood samples are collected on the 8 th day to detect uric acid content.

The experimental groupings were as follows:

Experimental results:

The experimental results were analyzed using GRAPHPAD PRISM software. Experimental data are expressed as mean ± SEM. P <0.05 indicates that the difference is statistically significant. Wherein P <0.05; * P <0.01; * P <0.001.

As can be seen from fig. 4, uric acid levels in blood were significantly elevated in mice of the model group (P < 0.001) compared to the control group, indicating successful modeling.

As can be seen from FIG. 4, the Lactobacillus reuteri PRS-156 of the present example significantly improved the blood uric acid level of hyperuricemia mice, which was reduced by 53.08% (by 2.93. Mu.g/mL) compared to the model group.

Example 8

This example tested lactobacillus reuteri in example 1 in a mouse model of high uric acid to verify its use in the treatment or prevention of obesity.

The experimental method comprises the following steps:

(2) Animal experiment: the mice selected in this experiment were female C57BL/6 mice, 7-8 weeks old, and had a body weight range of 18-22g, purchased from Shanghai Ling Biotechnology Co.

The mouse obesity model is used for verifying the drug effect, and the model construction method comprises the following steps:

Mice were acclimatized for one week before the start of the experiment. Control mice were fed maintenance diet and model mice were fed high fat diet (D12492, synergistic, production lot number: 20230207) for 12 weeks to induce obesity models. The weight of the mice is weighed before the drug intervention is started, 16 obese model mice with the weight which exceeds the average weight of the control group by 20 percent are selected, and the weight is divided into 2 groups according to the average weight, namely a model group and a PRS-156 intervention group respectively. Control and model mice were lavaged 200 microliters per day with PBS, and PRS-156 intervention groups were lavaged PRS-1561X 109CFU/200 microliters per day. For a total of 28 days. Intervention day 28 mice were tested for blood glucose using a glucometer.

The experimental groupings were as follows:

Experimental results:

As shown in FIG. 5, the PRS-156 dry prognosis significantly inhibited high fat diet-induced weight gain in mice.

As can be seen from fig. 6, on day 28 of intervention, the mice in the model group had significantly increased body weight (P < 0.001) compared to the control group, confirming successful modeling, and PRS-156-intervention group had significantly decreased body weight (P < 0.01) compared to the model group.

As can be seen from fig. 7, on day 28 of intervention, mice had significantly higher blood glucose than the control group (P < 0.001), and PRS-156 of the intervention group had significantly lower blood glucose than the model group (P < 0.01).

The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of lactobacillus reuteri (Limosilactobacillus reuteri) for the manufacture of a medicament for the prevention and/or treatment of at least one disease or condition selected from the group consisting of:

The 16S rDNA sequence and the preservation number of the lactobacillus reuteri are GDMCC No: the 16S rDNA sequence present in the M2023591 deposited lactobacillus reuteri strain has at least 99% sequence identity;

And/or, the whole genome sequence and the preservation number of the lactobacillus reuteri are GDMCC No: the lactobacillus reuteri strain deposited with M2023591 has a genome-average nucleotide similarity (ANI score) of at least 97.5%.

2. The use according to claim 1, wherein the lactobacillus reuteri has a 16SrDNA sequence and accession number GDMCC No: the 16S rDNA sequence present in the M2023591 deposited lactobacillus reuteri strain has at least 99.5% sequence identity;

And/or, the whole genome sequence and the preservation number of the lactobacillus reuteri are GDMCC No: the lactobacillus reuteri strain deposited with M2023591 has a genome-average nucleotide similarity (ANI score) of at least 98%;

Preferably, the lactobacillus reuteri is a strain with accession number GDMCC No: m2023591 deposited Lactobacillus reuteri.

3. The use according to claim 1, wherein the lactobacillus reuteri is at least one of a concentrate, a paste, a dry, a liquid and a dilution of the lactobacillus reuteri.

4. The use according to claim 1, wherein the medicament is for any one of the following:

(1) Lowering blood uric acid in a mammal;

(2) Reducing the weight of the mammal;

(3) Lowering blood glucose in a mammal;

(4) Is used for decomposing purine compounds.

5. The use according to claim 4, wherein the medicament further comprises pharmaceutically acceptable excipients;

Preferably, the medicament is formulated for oral administration or injection administration.

6. Lactobacillus reuteri, characterized in that it has a deposit number GDMCC No: m2023591 deposited Lactobacillus reuteri strain.

7. A composition comprising lactobacillus reuteri according to claim 6.

8. The composition of claim 7, wherein the composition further comprises a pharmaceutically acceptable adjuvant.

9. The composition of claim 7, wherein the composition is a pharmaceutical composition, a vaccine composition, or a food product.

10. A method for culturing lactobacillus reuteri, comprising the steps of: inoculating the lactobacillus reuteri of claim 6, and performing conditional culture;

Preferably, the conditioned culture is an anaerobic culture.