CN117731697A

CN117731697A - Lactobacillus, microbial inoculum and application thereof

Info

Publication number: CN117731697A
Application number: CN202311690646.2A
Authority: CN
Inventors: 朱永亮; 张莹; 陆敏
Original assignee: Suzhou Preyson Biotechnology Co ltd
Current assignee: Suzhou Preyson Biotechnology Co ltd
Priority date: 2023-12-11
Filing date: 2023-12-11
Publication date: 2024-03-22

Abstract

The invention discloses lactobacillus, a microbial agent and application thereof, and relates to the technical field of microorganisms. The lactobacillus plantarum provided by the invention has the effect of obviously reducing the uric acid content in blood, has higher activity in the alimentary canal nutrition environment, and has better development and utilization prospects for treating or preventing and improving hyperuricemia and gout, gout complications or kidney injury caused by the hyperuricemia. In addition, the lactobacillus plantarum has the advantage of good strains for degrading uric acid and uric acid metabolic precursors.

Description

Lactobacillus, microbial inoculum and application thereof

Technical Field

The invention relates to the technical field of microorganisms, in particular to lactobacillus, a microbial inoculum and application thereof.

Background

Hyperuricemia (HUA) is currently the "fourth highest" following hypertension, hyperlipidemia, hyperglycemia, and is a disease caused by excessive uric acid production or insufficient excretion in the human body, and persistent Hyperuricemia can lead to gout and a series of complications. Uric acid is the final metabolite of human purine compounds, and purine metabolic disorders lead to hyperuricemia. Xanthine is a precursor of uric acid synthesis, and reduces uric acid synthesis by reducing xanthine synthesis. While other products are produced from the purine precursor to reduce xanthine production and ultimately uric acid production. 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 the test method are only purine precursor substrates, and the purine precursor substrates can synthesize purine nucleosides, nucleotides and other substances, and the screening mode is that a reaction system only contains the purine precursor substrates, and no other nutrition conditions exist; since other nutrients such as carbon source amino acids and the like are contained in the intestinal tract, the strain may preferably utilize these nutrients rather than the purine precursor, and thus may not have an effect in a practically rich intestinal nutrient environment.

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

Disclosure of Invention

The invention aims to provide lactobacillus plantarum, a microbial inoculum and application thereof for providing a new solution for preventing and/or treating hyperuricemia and gout, gout complications or kidney injury caused by the hyperuricemia. The lactobacillus plantarum provided by the invention can degrade uric acid and/or uric acid metabolic precursors.

The invention is realized in the following way:

in a first aspect, the present invention provides the use of lactobacillus plantarum (Lactiplantibacillus plantarum) or a culture thereof for the manufacture of a medicament for the prevention and/or treatment of hyperuricemia, and gout, gout complications or kidney damage caused by hyperuricemia, the lactobacillus plantarum being selected from at least one bacterial strain of the group consisting of:

(1) The 16S rDNA sequence of Lactobacillus plantarum has at least 98% sequence identity with the 16S rDNA sequence present in the strain that was preserved in CCTCC No. M2023444 at 3.2023;

(2) The whole genome sequence of Lactobacillus plantarum has a genome average nucleotide similarity (Average Nucleotide Identity, ANI) of at least 97.5% with the sequence shown in NCBI sequence number JAWNBD000000000, or the whole genome sequence of Lactobacillus plantarum has a genome average nucleotide similarity (ANI score) of at least 97.5% with a strain with a preservation number of CCTCC NO: M2023444, which is preserved in CCTCC at 3 months of 2023;

the lactobacillus plantarum is a live strain, a dry strain or an inactivated strain of lactobacillus plantarum;

the culture is at least one of dead bacteria, cell broken material, fermentation supernatant and fermentation precipitate of lactobacillus plantarum.

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 3400433bp in full length, gc content was 44.29%, and contained 3337 genes. The strain was analyzed for whole genome sequencing data using GTDBTK software, and the closest reference strain was Lactiplantibacillus plantarum DSM 20174, with an ANI value of 98.8%, by FASTANI algorithm. When the ANI value is greater than 95, then 2 are considered to be the same species, and thus the strain is identified as Lactobacillus plantarum and designated PRS-49 with NCBI number JAWNBD000000000.

The study shows that lactobacillus plantarum PRS-49 has a remarkable effect on reducing blood uric acid content, and one skilled in the art can expect that lactobacillus plantarum which has at least 98% sequence identity with the sequence shown in SEQ ID No.1 also has the same or similar effect and can reduce blood uric acid content. The experiment of simulating artificial gastric juice and artificial intestinal juice shows that the lactobacillus plantarum provided by the invention still has higher bacterial strain survival rate in the alimentary canal nutrition environment (gastric juice and intestinal juice), and the viable count is not obviously reduced. The result indicates that the lactobacillus plantarum 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 gout, gout complications or kidney injury caused by hyperuricemia.

The results of in vitro uric acid and uric acid metabolism precursor degradation experiments on the lactobacillus plantarum PRS-49 show that the lactobacillus plantarum PRS-49 has good uric acid and uric acid metabolism precursor degradation strain advantages.

By comparing and analyzing uric acid synthesis pathway related enzyme coding genes in the genome of lactobacillus plantarum PRS-49 bacteria, the result shows that the lactobacillus plantarum PRS-49 does not contain EC1.17.1.4, EC1.17.3.2, EC2.4.2.1, EC3.5.4.3, so that the strain cannot generate xanthine from purine substrates. By comparison, the lactobacillus plantarum PRS-49 contains EC2.4.2.8 genes, and can generate other products from substrates for generating xanthine, so that the generation of xanthine is further reduced. Whereas the deletion of EC3.1.3.5 renders guanine nucleotides unavailable, again reducing xanthine production.

Wherein EC3.1.3.5 is 5 '-nucleotidase, and can act on adenosine (inosine) -5' -phosphate. Catalyzing hydrolysis of nucleoside-5' -monophosphate to produce nucleoside and inorganic phosphate.

EC3.5.4.3 is a guanine deaminase that catalyzes the hydrolytic deamination of guanine to produce xanthine and ammonia.

EC2.4.2.8 is hypoxanthine guanine phosphoribosyl transferase, which catalyzes the conversion of 5-phosphoribosyl-1-pyrophosphate and 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 generate uric acid and superoxide anion, and is one of main sources of active oxygen.

EC2.4.2.1 it is Purine Nucleoside Phosphorylase (PNP).

The above-mentioned cell disruption product includes, but is not limited to, a cell disruption product obtained by at least one of the following means: ultrasonic, mechanical disruption, enzymatic, physical or chemical treatment.

The fermentation supernatant is prepared by the following preparation method: inoculating Lactobacillus plantarum, culturing, and removing thallus.

The fermentation sediment is prepared by the following preparation method: inoculating lactobacillus plantarum, culturing, centrifuging or naturally settling, and removing supernatant to obtain fermented precipitate.

In a preferred embodiment of the invention, the lactobacillus plantarum is a gram positive bacterium;

the whole genome sequence of lactobacillus plantarum has at least 97.5% of genome-average nucleotide similarity (ANI score) with that shown in NCBI serial number JAWNBD000000000, or the whole genome sequence of lactobacillus plantarum has at least 97.5% of genome-average nucleotide similarity (ANI score) with that of a strain with the preservation number CCTCC NO: M2023444, which is preserved in CCTCC for 3 months of 2023;

in an alternative embodiment, the whole genome sequence of lactobacillus plantarum has at least 99% genomic average nucleotide similarity (ANI score) to the sequence shown in NCBI sequence number JAWNBD000000000, or the whole genome sequence of lactobacillus plantarum has at least 99% genomic average nucleotide similarity (ANI score) to the strain with the accession number cctccc No. M2023444 deposited with CCTCC at 3 months of 2023; the method comprises the steps of carrying out a first treatment on the surface of the

In an alternative embodiment, the whole genome sequence of lactobacillus plantarum has a genome-average nucleotide similarity (ANI score) of at least 99.5% to the sequence shown in NCBI sequence number JAWNBD000000000, or the whole genome sequence of lactobacillus plantarum has a genome-average nucleotide similarity (ANI score) of at least 99.5% to the strain with accession number cctccc No. M2023444, which was deposited on CCTCC for 3 months of 2023.

In an alternative embodiment, the 16S rDNA sequence of Lactobacillus plantarum has at least 99.5% sequence identity with the sequence set forth in SEQ ID NO.1.

In an alternative embodiment, the Lactobacillus plantarum has a preservation number of CCTCCNO: M2023444, and is preserved in China center for type culture Collection with a preservation date of 2023, 3, and 31.

In an alternative embodiment, the lactobacillus plantarum is at least one of a concentrate, a paste, a dry, a liquid, a dilution, and a broken up product of lactobacillus plantarum.

The lactobacillus plantarum dried product of the present invention includes, but is not limited to, spray-dried products, freeze-dried products, vacuum-dried products, roller-dried products.

In a preferred embodiment of the application of the present invention, the gout complications are selected from at least one of gouty arthritis, tophus, joint deformity, urinary tract stones, gouty kidney disease, gouty kidney failure and cardiovascular disease.

In an alternative embodiment, the kidney injury is acute kidney injury or chronic kidney injury. Renal injury includes, but is not limited to: tubular epithelial cells undergo vacuolation, apoptosis and shedding.

In a preferred embodiment of the use of the invention, the medicament is formulated for oral administration or injection administration. Injection administration includes, but is not limited to, subcutaneous injection, intramuscular injection, intravenous injection, and intradermal injection.

In a preferred embodiment of the invention, the medicament 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 titrilex 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 invention, the pharmaceutical dosage form is a tablet, pill, powder, suspension, gel, emulsion, cream, granule, nanoparticle, capsule, suppository, injection or spray.

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 an alternative embodiment, the medicament is a solid pharmaceutical formulation, such as a lyophilized bacterial powder, a granular formulation, or the like.

In a preferred embodiment of the use of the invention, the medicament is 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 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 second aspect, the invention also provides lactobacillus plantarum with a preservation number of CCTCC NO: M2023444, which is preserved in China center for type culture Collection, and the preservation date is 2023 and 31 days.

The lactobacillus plantarum PRS-49 can still ensure the survival rate of 99.48 percent after being incubated for 3 hours in an artificial gastric juice environment with the pH value of 3.0, and has the advantage of strong acid resistance. In simulated intestinal juice with pH of 6.8, the lactobacillus plantarum PRS-49 can still ensure the survival rate of 99.79 percent within 3 hours, the viable count is not obviously reduced, and the intestinal juice has good intestinal juice tolerance. The survival rate of lactobacillus plantarum PRS-49 incubated for 3 hours at the concentration of 0.2% of bovine bile salt is 100.00%, which shows that the bile salt tolerance capability of the bacterium provided by the invention is stronger.

In a third aspect, the invention also provides a microbial inoculum or culture comprising the lactobacillus plantarum described above;

in an alternative embodiment, the form of the microbial agent or culture is liquid, solid or semi-solid.

In an alternative embodiment, the amount of Lactobacillus plantarum in the solid microbial agent or culture is at least 5X 10 ⁹ CFU/g; the lactobacillus plantarum is present in the liquid microbial inoculum or culture in an amount of at least 5×10 ⁹ CFU/mL。

In a fourth aspect, the invention also provides a composition comprising lactobacillus plantarum as described above.

In an alternative embodiment, the composition is a pharmaceutical composition.

In an alternative embodiment, 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.

In an alternative embodiment, the composition is a food product.

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

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 plantarum provided by the invention can be prepared into general drinks and foods of dairy products and fermented milk, and can be used as a starter (starters) for manufacturing dairy products such as yoghurt and cheese. 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.

In a fifth aspect, the present invention also provides a method for culturing lactobacillus plantarum, comprising: inoculating lactobacillus plantarum on a culture medium for culture;

in an alternative embodiment, the culturing means is at least one of anaerobic culture and facultative anaerobic culture.

The invention has the following beneficial effects:

the lactobacillus plantarum PRS-49 is separated and purified, has the effect of obviously reducing the uric acid content in blood, and a person skilled in the art can expect that lactobacillus plantarum which has at least 98 percent of sequence identity with the sequence shown in SEQ ID NO.1 also has the same or similar effect and can reduce the uric acid content in blood. The experiment of simulating artificial gastric juice and artificial intestinal juice shows that the lactobacillus plantarum provided by the invention still has higher bacterial strain survival rate in the alimentary canal nutrition environment (gastric juice and intestinal juice), the viable count is not obviously reduced, and the bacterial strain has good bile salt resistance. The lactobacillus plantarum which has at least 98 percent sequence identity with the sequence shown in SEQ ID NO.1 is predicted to have higher activity in the alimentary canal nutrition environment, and has better development and utilization prospects for treating or preventing and improving hyperuricemia, gout complications or kidney injury caused by the hyperuricemia.

Lactobacillus plantarum having at least 98% sequence identity with the sequence shown in SEQ ID NO.1 has the advantage of good strain degradation of uric acid and uric acid metabolic precursors.

Preservation description

Preservation address: chinese, wuhan, university of Wuhan

Preservation date: 2023, 3, 31

Strain name: lactobacillus plantarum PRS-49

Authentication features: lactiplantibacillus plantarum PRS-49

Preservation mechanism: china center for type culture Collection

The preservation organization is abbreviated as: CCTCC (cctccc)

Accession numbers of the preservation center: CCTCC NO: M2023444

The identification result is as follows: 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 strain morphology graph and gram staining result graph;

FIG. 2 is a graph showing the results of genomic analysis of 6 enzyme genes and PRS-49 associated with the xanthine synthesis pathway;

FIG. 3 is the blood uric acid levels of mice after dosing.

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 et al, 1984); animal cell culture (Animal Cell Culture) (r.i. freshney, 1987); methods of enzymology (Methods in Enzymology) (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 Vectors 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, inc., 1987), PCR: polymerase chain reaction (PCR: the Polymerase Chain Reaction, inc., 1994), and methods of contemporary immunology (Current Protocols in Immunology) (J.E.Coligan et al, 1991), each of which is expressly incorporated herein by reference.

The fastANI algorithm was used to calculate the ANI values between species whole genome data by GTDBTK software. 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. BLAST holonomics Basic Local Alignment Search Tool, a search tool based on a local alignment algorithm, is a tool software commonly used in bioinformatics, and can compare an input nucleic acid or protein sequence with a known sequence in a database to obtain information such as sequence similarity, so as to determine 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.

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

Example 1

The embodiment provides a lactobacillus plantarum, and the separation and identification processes are as follows:

1. isolation of Lactobacillus plantarum

Collecting feces of healthy people, performing 10-time gradient dilution by using PBS under aseptic conditions, selecting three concentration gradients, streaking and inoculating on MRS agar medium (purchased from Qingdao sea Bo biotechnology Co., ltd., product number: HB 0396), performing three repetitions of each gradient, simultaneously using PBS as a blank control, performing anaerobic culture in a constant temperature incubator at 37 ℃ for 24-72 hours, then picking colonies of different forms for gram staining microscopy, performing 2 times streaking and purification in the MRS medium, and performing staining microscopy again to observe bacterial forms and whether purification is performed.

The purified strain was designated PRS-49.

The identification method of the strain PRS-49 is as follows:

(1) Morphological characteristics: as shown in FIG. 1, colonies appeared milky, round, clean-edged, opaque, smooth on MRS solid plates.

(2) The result of gram staining is shown in FIG. 1, and PRS-49 is a gram positive bacterium.

(3) Extracting genome from the separated PRS-49, and sequencing and identifying 16S rDNA thereof, wherein the sequence of the used primer is as follows:

27F(SEQ ID NO.2)：agagtttgatcctggctcag

1429R(SEQ ID NO.3)：ggttaccttgttacgactt

the primer is used for amplifying and sequencing the 16S rDNA gene sequence of the strain PRS-49, and the PCR amplified product is sent to Shanghai biological engineering (Shanghai) Inc. for sequencing, and the 16S rDNA nucleotide sequence of the strain PRS-49 is shown as SEQ ID NO.1.

The physiological and biochemical properties are shown in Table 1.

TABLE 1 PRS-49 physiological and biochemical identification results of strains

Glycerol	v	Mannitol (mannitol)	+	D-raffinose	+
						Erythritol	—	Sorbitol	+	Starch	—
D-ArabicSugar	—	alpha-methyl-D-mannosides	—	Liver sugar (glycogen)	—
						L-arabinose	+	alpha-methyl-D-glucoside	—	Xylitol	—
D-ribose	+	N-acetyl-glucosamine	+	D-gentiobiose	+
						D-xylose	—	Amygdalin	+	D-melezitose	+
L-xylose	—	Arbutin	+	D-lyxose	—
						D-ribitol/adonitol	—	Radix Schefflerae Arboricolae	+	D-tagatose	—
beta-methyl-D-xylosides	—	Salicin	+	D-fucose	—
						D-galactose	+	D-cellobiose	+	L-fucose	—
D-glucose	+	D-maltose	+	D-arabitol	—
						D-fructose	+	D-lactose	+	L-arabitol	—
D-mannose	+	D-melibiose	+	Potassium gluconate	+
						L-sorbose	—	D-sucrose	+	2-Keto-gluconate Potassium	—
L-rhamnose	v	D-trehalose	+	5-Keto-Potassium gluconate	—
						Dulcitol	—	Inulin	+
Inositol (inositol)	—	D-melezitose	+

Note that: +: positive; -: negative; v: weak positive

Further, it was subjected to whole genome sequencing: the extracted whole genome was sent to Suzhou gold only Biotechnology Co., ltd for second generation sequencing, the whole genome sequence was shown as NCBI sequence number JAWNBD000000000, and the obtained sequence result was compared with reference strain Lactiplantibacillus plantarumDSM 20174 in NCBI database, and was identified as Lactobacillus plantarum, with an ANI value of 98.9%.

The lactobacillus plantarum (Lactiplantibacillus plantarum) PRS-49 is preserved in China Center for Type Culture Collection (CCTCC) No. M2023444, and the preservation date is 2023, 3 and 31.

Experimental example 1

The test example carries out drug resistance detection on the strain PRS-49.

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 glycerol bacteria frozen at-80 ℃ to an MRS agar culture medium plate for streak activation, culturing at 37 ℃ for 24-48 hours, picking plate colonies, dispersing into the MRS broth culture medium, fully mixing uniformly to ensure that the OD of bacterial liquid is equal to that of the strain ₆₂₅ =0.16 to 0.2, the number of corresponding viable bacteria is about 3×10 ⁸ cfu/mL is inoculated into LSM broth culture medium (LSM culture medium is formed by compounding 90% IST+10% MRS+0.3g/L L-cysteine according to the inoculum size of 0.1% (v/v), and the LSM broth culture medium is purchased from Shanghai Rui Chu Biotechnology Co., qingdao high-tech industrial park Haibo Biotechnology Co., guoguo Chemie Co., ltd.) respectively, and is uniformly mixed, and 100 mu L of bacterial liquid is taken into a 96-well plate of plus antibiotics.

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 plantarum PRS-49 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 threshold ECOFF of microbial susceptibility to different antibiotics. If the MIC value is greater than ECOFF, the strain is indicated as resistant to the antibiotic, and if the MIC value is less than or equal to ECOFF, the strain is indicated as sensitive to the antibiotic.

TABLE 2MIC values and drug sensitivity results

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

The results are shown in Table 2, and lactobacillus plantarum PRS-49 is mainly resistant to clindamycin; is sensitive to tetracycline, clindamycin, ampicillin, chloramphenicol, kanamycin, gentamicin, and streptomycin.

Experimental example 2

The experimental example is subjected to experimental evaluation on the digestive tract environmental tolerance characteristics of lactobacillus plantarum PRS-49.

1 simulation of Artificial gastric juice (Simulated Gastric Fluid, SGF) experiment

(1) Artificial gastric juice: 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;

(2) Collecting bacteria: the strain is statically cultured for 8-10 h at 37 ℃ to reach logarithmic growth phase, bacterial liquid is split-packed in a 50mL sterile EP tube, centrifuged at 4000rpm at room temperature for 5min, the supernatant is discarded, the bacterial cells are resuspended by PBS, and the OD of the bacterial liquid is adjusted ₆₀₀ The number of viable bacteria was about 2X 10 ⁹ CFU/mL followed by 15mL withoutThe bacteria EP tube is respectively added with 6mL of bacteria liquid with the OD value adjusted, the supernatant is removed by centrifugation (the conditions are the same as above), bacterial mud is collected for standby, and the 1-pipe PBS is used for resuspension as a control group;

(3) 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 evenly mixed, incubated and cultured for 3 hours, and the coating count is carried out.

(4) 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 different time of artificial gastric juice action at different pH values. Calculating a survival rate formula: survival = A2/a1×100% formula: 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.

The results are shown in Table 3, and show that the lactobacillus plantarum PRS-49 can still ensure the survival rate of 99.48% after being incubated for 3 hours in an artificial gastric juice environment with the pH value of 3.0, and has strong acid resistance.

TABLE 3 Lactobacillus plantarum PRS-49 acid and bile salt tolerance data

2. Simulated artificial intestinal juice (SIF) -SimulatedIntestinalFluid (SIF) experiment

(1) Artificial intestinal juice: taking 6.8g of monopotassium phosphate, adding 500mL of water to dissolve the monopotassium phosphate, and adjusting the pH value to 6.8 by using 0.1mol/L sodium hydroxide solution; another 10g of pancreatin (Soxhinbao trypsin, 1:250) was dissolved by adding water in an appropriate amount, and the two solutions were mixed, diluted to 1000mL with water, filtered and sterilized.

(2) The bacteria collecting method is the same as the artificial gastric juice (SimulatedGastricFluid, SGF) simulating experiment;

(3) Incubating and culturing artificial intestinal juice: 8mL of artificial intestinal juice is taken and respectively added into 4 15mL centrifuge tubes containing bacterial mud, and the mixture is blown and evenly mixed, incubated and cultivated for 3 hours, and the viable count is measured, and the plate counting method is the same as that of the simulated gastric juice (SGF) experiment.

As shown in Table 3, in the simulated intestinal juice at pH6.8, the Lactobacillus plantarum PRS-49 survived well in 3 hours without significant decrease in viable count.

3. Experiment for simulating human body internal bile salt environment

(1) Preparing a ox gall salt culture medium: weighing 2g/L (0.2%) of ox gall salt (OXOID, LP 0055) and inoculating to MRS liquid culture medium, and autoclaving for use;

(2) The strain was collected as in the simulated artificial gastric fluid (SimulatedGastricFluid, SGF) experiment above.

(3) Incubation and culture of ox gall salt: respectively adding 3ml MRS broth containing 0.2% ox gall salt into a centrifuge tube containing bacterial sludge sediment, and incubating and culturing for 3h;

(4) Plate coating count: performing 10-time gradient dilution on the incubated bacterial liquid, selecting three proper dilution factors, uniformly absorbing 100uL of dilution liquid, performing flat plate coating, setting 3 parallel concentration dilution liquids, and culturing at 37 ℃;

(5) 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%. A1 is the number of viable bacteria (CFU/mL) of bacterial liquid in 0% ox gall salt culture medium solution for 0 h; a2 is the number of viable bacteria (CFU/mL) incubated for 3 h.

As shown in Table 3, the survival rate of the strain was 100.00% when incubated for 3 hours at a concentration of 0.2% of bovine bile salt, and the bile salt tolerance of the strain was strong.

In conclusion, the experimental result of the experimental example shows that the lactobacillus plantarum PRS-49 can still keep better survival rate in gastric juice environment with lower pH value, and can still ensure the survival rate of 99.48% after being incubated for 3 hours in artificial gastric juice environment with pH value of 3.0; the survival rate of 99.79% can be ensured after the artificial intestinal juice with the pH value of 6.8 is incubated for 3 hours; the survival rate of 100.00% can be ensured after 0.3% of ox gall salt is incubated for 3 hours, and the gall salt tolerance capability is strong. Therefore, the lactobacillus plantarum PRS-49 has good application prospect in preparing oral medicines or foods.

Experimental example 3

In this experimental example, the genome of Lactobacillus plantarum PRS-49 was analyzed, and 6 enzyme genes (shown in FIG. 2) related to the uric acid synthesis pathway were compared with the genome of Lactobacillus plantarum PRS-49.

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
		EC2.4.2.1	Without any means for
EC2.4.2.8	orf_03060
		EC3.1.3.5	Without any means for
EC3.5.4.3	Without any means for

From the BLAST alignment results, none of the 4 genes producing xanthines (EC1.17.1.4, EC1.17.3.2, EC2.4.2.1, EC3.5.4.3) was matched in the whole genome data, thus making the strain incapable of producing xanthines. The 2 genes that simultaneously produced uric acid (EC1.17.1.4, EC1.17.3.2) were also not matched, and the strain was also unable to produce uric acid.

The EC2.4.2.8 gene was aligned and found to have 1 copy in the genome, and intermediates that produced xanthines could be used to produce other products. The deletion of EC3.1.3.5 renders guanine nucleotides unavailable, as well as reduces xanthine production.

Experimental example 4

In this experimental example, lactobacillus plantarum PRS-49 was used to react with part of uric acid metabolic precursors and examine the degradation ability of these substances in vitro.

Inoculating Lactobacillus plantarum PRS-49 stored in glycerol tube into MRS culture medium, culturing at 37deg.C to OD ₆₀₀ 1.2, the cultured fermentation broth was placed in a centrifuge tube for 10min at 4000rpm per 2ml, and the supernatant was discarded to recover the cells.

30mg of hypoxanthine, guanosine and inosine were dissolved in 100ml of PBS buffer having pH 7.0, respectively, to prepare reaction solutions.

Adding the above reaction solution into centrifuge tubes containing thallus precipitate (equal volume of PBS is used for replacing thallus in control group), adding 0.75ml of the reaction solution into each tube, suspending thallus, shaking at 37deg.C for incubation for 60min, adding 40 microliter of 0.1mol/L HClO at the end of incubation ₄ The reaction was stopped and the supernatant was centrifuged at 10000rpm for 15min for further analysis.

Substrates (inosine, guanosine, inosine) in the supernatant were detected. Analysis was performed by HPLC chromatography under the following conditions: by KH ₂ PO ₄ (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 uric acid metabolic precursors (5 total) was further calculated based on the ratio of peak areas of the HPLC experimental group to the control group.

Degradation rate = (1-experimental group peak area/control group peak area) ×100%

The experimental results are shown in the following table

Substrate(s)	Degradation rate
		Hypoxanthine (inosine)	42.95％
Guanine nucleotide	1.29％
		Guanosine (guanosine)	31.57％
Inosine	20.55％
		Inosinic acid	18.81％

Experimental results show that the lactobacillus plantarum PRS-49 has good degradation effect on partial uric acid metabolic precursors, and hypoxanthine is more remarkable. As described in Experimental example 3, gene EC2.4.2.8 was found in the entire genome of PRS-49, and EC2.4.2.8 could convert hypoxanthine to inosine and guanine to guanine nucleotides, thereby facilitating degradation of hypoxanthine.

Experimental example 5

The lactobacillus plantarum PRS-49 is tested in a mice model of hyperuricemia, and the application of the lactobacillus plantarum PRS-49 in treating or preventing hyperuricemia is verified.

The experimental method comprises the following steps:

(1) Sample preparation: centrifuging the lactobacillus plantarum PRS-49 strain suspension cultured in example 1, discarding supernatant, and adjusting the concentration of the strain to about 1×10 viable count with sterile physiological saline ⁹ CFU/mL。

(2) Animal experiment: the mice selected in this experimental example were female Balb/c mice, 6-8 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:

the first day of molding began administration was started for 7 days, once a day.

Experimental results:

experimental results were analyzed using GraphPad Prism 8 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. 3, uric acid levels in blood were significantly elevated in mice of the model group (P < 0.01) compared to the control group, indicating successful modeling.

As can be seen from FIG. 3, the Lactobacillus plantarum PRS-49 of the present example significantly improved blood uric acid levels in hyperuricemia mice, and the blood uric acid levels in the probiotic treated group were reduced by 21.06% (blood uric acid was reduced by 3.03. Mu.g/mL, i.e., 18.0. Mu. Mol/L) as compared to the model group.

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 plantarum (Lactiplantibacillus plantarum) or a culture thereof for the manufacture of a medicament for the prevention and/or treatment of hyperuricemia, and gout, gout complications or kidney damage caused by hyperuricemia, characterized in that the lactobacillus plantarum is selected from the group consisting of at least one bacterial strain of the following:

(1) The 16S rDNA sequence of the lactobacillus plantarum has at least 98 percent sequence identity with the 16S rDNA sequence existing in a strain which is preserved in CCTCC NO: M2023444 in 3.2023 and 31.d;

(2) The whole genome sequence of the lactobacillus plantarum has at least 97.5 percent of genome average nucleotide similarity (ANI score) with a sequence shown as NCBI serial number JAWNBD000000000, or has at least 97.5 percent of genome average nucleotide similarity (ANI score) with a strain which is preserved in CCTCC at 3-month 31 of 2023 and has the preservation number of CCTCC NO: M2023444;

the lactobacillus plantarum is a live strain, a dry strain or an inactivated strain of the lactobacillus plantarum;

the culture is at least one of dead bacteria, cell broken matters, fermentation supernatant and fermentation sediment of the lactobacillus plantarum.

2. The use according to claim 1, wherein the lactobacillus plantarum is a gram positive bacterium;

preferably, the whole genome sequence of lactobacillus plantarum has a genome-average nucleotide similarity (ANI score) of at least 97.5% with that shown in NCBI sequence number JAWNBD000000000, or the whole genome sequence of lactobacillus plantarum has a genome-average nucleotide similarity (ANI score) of at least 97.5% with a strain with a accession number cctccc No. M2023444, which is deposited with CCTCC at 3 months 31 of 2023;

preferably, the whole genome sequence of lactobacillus plantarum has at least 99% genome-average nucleotide similarity (ANI score) with the sequence shown in NCBI sequence number JAWNBD000000000, or the whole genome sequence of lactobacillus plantarum has at least 99% genome-average nucleotide similarity (ANI score) with the strain with the accession number cctccc No. M2023444, which is preserved in CCTCC at 3 months 31 of 2023;

preferably, the whole genome sequence of lactobacillus plantarum has a genome-average nucleotide similarity (ANI score) of at least 99.5% to the sequence shown in NCBI sequence number JAWNBD000000000, or the whole genome sequence of lactobacillus plantarum has a genome-average nucleotide similarity (ANI score) of at least 99.5% to a strain with a accession number cctccc No. M2023444, which is preserved in CCTCC at 3 months of 2023;

preferably, the 16S rDNA sequence of Lactobacillus plantarum has at least 99.5% sequence identity with the sequence set forth in SEQ ID NO.1.

3. The use according to claim 1, wherein the lactobacillus plantarum has a preservation number of cctccc No. M2023444, and is preserved in the chinese collection for typical cultures, with a preservation date of 2023, 3 and 31 days;

preferably, the lactobacillus plantarum is at least one of a concentrate, a paste, a dried product, a liquid product, a diluted product and a crushed product of the lactobacillus plantarum.

4. The use according to any one of claims 1-3, wherein the gout complication is selected from at least one of gouty arthritis, tophus, joint deformity, urinary tract stones, gouty kidney disease, gouty kidney failure, and cardiovascular disease;

preferably, the kidney injury is an acute kidney injury or a chronic kidney injury.

5. The use according to any one of claims 1-3, wherein the medicament is formulated for oral administration or injection administration.

6. The use according to any one of claims 1-3, wherein the medicament further comprises a pharmaceutically acceptable adjuvant;

preferably, the medicament is for decomposing purine compounds;

preferably, the purine compounds are selected from uric acid and/or uric acid metabolic precursors;

preferably, the uric acid metabolism precursor is selected from at least one of a purine nucleotide, a purine nucleoside, a purine base, an oligonucleotide containing a purine base, and a polynucleotide;

preferably, the uric acid metabolism precursor is selected from at least one of guanine, inosine, xanthosine, and guanosine.

7. The lactobacillus plantarum is characterized in that the lactobacillus plantarum is preserved with the preservation number of CCTCCNO: M2023444, and is preserved in China center for type culture Collection, and the preservation date is 2023 and 31.

8. A microbial agent or culture, characterized in that it comprises the lactobacillus plantarum of claim 7;

preferably, the bacterial agent or culture is in the form of a liquid, solid or semi-solid.

9. A composition comprising the lactobacillus plantarum of claim 7;

preferably, the composition is a pharmaceutical composition;

preferably, the composition is a vaccine composition;

preferably, the composition is a food product.

10. A method of culturing lactobacillus plantarum according to claim 7, comprising: inoculating the lactobacillus plantarum on a culture medium for culture;

preferably, the culture means is at least one of anaerobic culture and facultative anaerobic culture.