Bifidobacterium animalis subsp. Lactis with antioxidant effect and gastrointestinal fluid tolerance and application of bifidobacterium animalis subsp. Lactis

Abstract

The invention discloses a bifidobacterium animalis subsp. Lactis reBF17-4 and application thereof, the strain is preserved in the general microbiological center of the China Committee for Culture Collection of Microorganisms on July 3, 2024, and the preservation number of the strain is CGMCC No.31147. The invention further discloses a preparation method of the bifidobacterium animalis subsp. Lactis reBF17-4. The invention further discloses a preparation method of the bifidobacterium animalis subsp. Lactis reBF17-4 and application of the bifidobacterium animalis subsp. Lactis reBF17-4. The bifidobacterium animalis subsp. Lactis reBF17-4 disclosed by the invention has the advantages that the antioxidation effect is relatively strong, the gastrointestinal tract endurance capacity is relatively strong, and the application prospect in the preparation of medicines is huge.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, and in particular to a strain of animal Bifidobacterium lactis subspecies having antioxidant efficacy and tolerance to gastrointestinal fluid and application thereof. Background Art

[0002] Bifidobacterium animalis subsp. lactis is a beneficial microorganism widely used in dairy products and prebiotic products. It has extremely high safety and is listed in the "Generally Recognized As Safe" (GRAS) list of the U.S. Food and Drug Administration (FDA). The European Food Safety Authority (EFSA) has included this subspecies in the presumption of safety list. It is also listed in China's "List of Bacteria Acceptable for Food". It has been widely accepted and used in the food and pharmaceutical industries worldwide.

[0003] Oxidation is the process by which a substance loses electrons. Since the free radicals generated by oxidation reactions contain one or more unpaired electron groups, they will further oxidize other substances, thus leading to a chain reaction and releasing more free radicals. Oxidative metabolic activities in human cells produce reactive oxygen free radicals, including superoxide anions, hydroxyl free radicals, and hydrogen peroxide. The level of endogenous and exogenous free radicals in the body may exceed the balance capacity of its own antioxidant system, resulting in oxidative stress, which in turn causes aging and various diseases, such as degenerative diseases, diabetes, cirrhosis, cancer, Parkinson's disease, and cardiovascular disease. Antioxidants prevent the occurrence of oxidative stress by inhibiting the oxidation process. Supplementing exogenous antioxidants is a direct way to deal with oxidative stress. Since antioxidants are consumed and become ineffective in the process of resisting oxidation, they need to be supplemented regularly. Synthetic antioxidants such as butylated hydroxyanisole and butylated hydroxytoluene can effectively reduce oxidative damage, but their safety is questioned due to damage to the liver and the risk of carcinogenesis. The antioxidant activity of various natural products such as phospholipids, Ganoderma lucidum polysaccharides, and seaweed extracts has been confirmed through model animals such as yeast, nematodes, fruit flies, and higher mammals. However, the separation and purification costs of these natural antioxidant active substances are high, and some of them have certain side effects.

[0004] As an internationally recognized probiotic and the earliest bacterium colonizing the intestines of mammals, Bifidobacterium animalis subsp. lactis plays an important role in maintaining the balance of intestinal microecology, inhibiting the invasion and colonization of pathogenic bacteria, regulating the body's immune ability, and reducing cholesterol levels. It has also shown antioxidant potential and is an important candidate for antioxidant probiotic preparations. At present, most probiotic products sold on the market have the main functions of regulating intestinal health, improving digestion, and relieving diarrhea and constipation, while probiotic products that have both antioxidant and gastrointestinal tolerance are very limited.

[0005] Therefore, there is an urgent need for a Bifidobacterium lactis subspecies with stronger antioxidant and gastrointestinal tolerance. The present invention expands the source of probiotics, and separates and screens Bifidobacterium lactis subspecies with antioxidant properties from the first excrement (called meconium) of a newborn after delivery. According to the life characteristics of a newborn in the early stage of life, the Bifidobacterium lactis subspecies isolated from its meconium may have more antioxidant potential. Summary of the invention

[0006] The present invention aims to solve at least one of the technical problems existing in the prior art to at least a certain extent.

[0007] To this end, the first aspect of the present invention provides an animal Bifidobacterium lactis subspecies (Bifidobacteriumanimalis subsp.lactis) reBF17-4, wherein the animal Bifidobacterium lactis subspecies (Bifidobacteriumanimalis subsp.lactis) reBF17-4 was deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration on July 3, 2024, with a deposit number of CGMCC No.31147.

[0008] The animal Bifidobacterium lactis subspecies provided by the present invention has both antioxidant and gastrointestinal tolerance capabilities, and can be used to prevent and / or alleviate and / or treat related diseases caused by oxidative stress, wherein the related diseases caused by oxidative stress include at least one of degenerative diseases, diabetes, cirrhosis, cancer, Parkinson's disease and cardiovascular diseases; on the other hand, it can maintain intestinal health, wherein the maintenance of intestinal health includes repairing intestinal mucosal damage, regulating the absorption of sodium and water by intestinal cell walls, enhancing intestinal barrier function, inhibiting the formation of colon adenomas and tumors, and at least one of antibacterial or bactericidal effects.

[0009] The second aspect of the present invention provides a fermentation supernatant, wherein the fermentation supernatant comprises the metabolites of the animal Bifidobacterium lactis subsp. reBF17-4 described in the first aspect.

[0010] The third aspect of the present invention provides a bacterial suspension, which includes the animal Bifidobacterium lactis subspecies reBF17-4 described in the first aspect.

[0011] The fourth aspect of the present invention provides use of the animal Bifidobacterium lactis subsp. lactis reBF17-4 described in the first aspect, the fermentation supernatant described in the second aspect, or the bacterial suspension described in the third aspect in the preparation of a medicament for preventing and / or treating related diseases caused by oxidative stress.

[0012] According to an embodiment of the present invention, the related diseases caused by oxidative stress include at least one of degenerative diseases, diabetes, liver cirrhosis, cancer, Parkinson's disease and cardiovascular disease.

[0013] The fifth aspect of the present invention provides use of the animal Bifidobacterium lactis subsp. reBF17-4 described in the first aspect, the fermentation supernatant described in the second aspect, or the bacterial suspension described in the third aspect in the preparation of a medicament for maintaining intestinal health.

[0014] The sixth aspect of the present invention provides a medicine, which comprises at least one of the animal Bifidobacterium lactis subsp. reBF17-4 described in the first aspect, the fermentation supernatant described in the second aspect, and the bacterial suspension described in the third aspect.

[0015] According to an embodiment of the present invention, the drug further contains an excipient and / or a carrier.

[0016] According to an embodiment of the present invention, the excipient includes at least one selected from a binder, a disintegrant, a lubricant, a glidant, a stabilizer, a filler, a diluent and a sustained-release agent.

[0017] According to an embodiment of the present invention, the carrier includes at least one selected from sugars, cellulose and its derivatives, calcium phosphates, alkaline earth metal stearates, vegetable oils, nonionic surfactants, cationic surfactants, anionic surfactants, fatty alcohols and cereal hydrolyzed solids.

[0018] According to an embodiment of the present invention, the dosage form of the drug includes at least one selected from oral liquid, powder, granule, capsule, tablet and pill.

[0019] According to an embodiment of the present invention, when animal Bifidobacterium lactis subsp. lactis reBF17-4 takes effect, it may exist in the form of living cells or in the form of non-living cells.

[0020] According to an embodiment of the present invention, the living cells refer to cells of Bifidobacterium animalis subspecies lactis reBF17-4 that have the ability to metabolize, reproduce or replicate, and the non-living cells refer to cells that do not have the ability to metabolize, reproduce and replicate, including but not limited to dry bacteria (such as freeze-dried powder).

[0021] In addition, the present invention provides a microbial agent, which contains the aforementioned animal Bifidobacterium lactis subsp. reBF17-4, the aforementioned fermentation supernatant or the aforementioned bacterial suspension. The microbial agent of the present invention is used to prevent and / or treat related diseases caused by oxidative stress.

[0022] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention.

[0023] Collection information:

[0024] Strain name: reBF17-4

[0025] Date of deposit: July 3, 2024

[0026] Depository: China General Microbiology Center (CGMCC)

[0027] Deposit number: CGMCC No.31147 Storage address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the colony morphology of animal Bifidobacterium lactis subspecies CGMCC NO.31147 in Example 1.

[0029] Figure 2 This is the basic structure, component prediction and annotation analysis results of the genome of animal Bifidobacterium lactis subspecies CGMCC NO.31147 in Example 2.

[0030] Figure 3 This is a phylogenetic tree comparing the relationship between animal Bifidobacterium lactis subspecies CGMCC NO.31147 and animal Bifidobacterium from other sources in Example 2.

[0031] Figure 4 The scavenging rate of animal Bifidobacterium lactis subspecies CGMCC NO.31147 on DPPH free radicals in Example 3; strains marked with different letters indicate significant differences (p<0.05).

[0032] Figure 5 The scavenging rate of animal Bifidobacterium lactis subspecies CGMCC NO.31147 on hydroxyl radicals in Example 4; those marked with different letters indicate significant differences between the strains (p<0.05).

[0033] Figure 6 The scavenging rate of superoxide anion free radicals by Bifidobacterium animalis subspecies lactis CGMCC NO.31147 in Example 5; strains marked with different letters indicate significant differences (p<0.05).

[0034] Figure 7 The tolerance of animal Bifidobacterium lactis subspecies CGMCC NO.31147 to artificial gastric juice in Example 6; strains marked with different letters indicate significant differences (p<0.05).

[0035] Figure 8The tolerance of animal Bifidobacterium lactis subspecies CGMCC NO.31147 to artificial intestinal fluid in Example 7; strains marked with different letters indicate significant differences (p<0.05).

[0036] Fig. 9 The metabolite profiles of animal Bifidobacterium lactis subspecies CGMCC NO.31147 and positive strain Bb-12 in Example 8. (A) Principal component analysis score diagram of metabolite profiles of animal Bifidobacterium lactis subspecies CGMCC NO.31147 and positive strain Bb-12. (B) Differential analysis of metabolites of animal Bifidobacterium lactis subspecies CGMCC NO.31147 and positive strain Bb-12.

[0037] Fig.10 The difference in metabolites in the culture supernatant of animal Bifidobacterium lactis subspecies CGMCC NO.31147 in Example 8. DETAILED DESCRIPTION

[0038] Embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to be used to explain the present invention, but should not be understood as limiting the present invention.

[0039] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, the meaning of "plurality" is two or more.

[0040] In this document, the terms “contain”, “include” or “comprise” are open expressions, that is, they include the contents specified in the present invention but do not exclude other contents.

[0041] As used herein, the terms "optionally", "optional" or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0042] As used herein, "prevention" and "prevention" are used interchangeably. These terms refer to an approach to obtaining a beneficial or desired result, including but not limited to a prophylactic benefit. To obtain a "prophylactic benefit," fermented Lactobacillus muciniphila or a product containing it may be administered to a subject at risk of developing a particular disease, or to a subject reporting one or more physiological symptoms of a disease, even though a diagnosis of the disease may not have been made.

[0043] As used herein, the terms "treatment" and "alleviation" both refer to the use of drugs to obtain the desired pharmacological and / or physiological effects. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or may be therapeutic in terms of partially or completely curing a disease and / or the adverse effects caused by the disease. "Treatment" as used herein covers diseases in mammals, particularly humans, and includes: (a) preventing the occurrence of a disease or condition in an individual who is susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, such as arresting the progression of the disease; or (c) alleviating the disease, such as alleviating symptoms associated with the disease. "Treatment" as used herein covers any medication that administers a drug or compound to an individual to treat, cure, alleviate, improve, reduce or inhibit an individual's disease, including but not limited to administering a drug containing a compound described herein to an individual in need.

[0044] As used herein, "acceptable in health foods" refers to substances or compositions that can be consumed by humans, which may be adjusted according to the health food requirements of different countries.

[0045] In this article, "pharmaceutically acceptable" means that the substance or composition must be chemically and / or toxicologically compatible with the other ingredients of the formulation and / or the mammals treated therewith. Preferably, "pharmaceutically acceptable" as described in the present invention refers to those approved by federal regulatory agencies or national governments or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopeias for use in animals, particularly humans.

[0046] As used herein, the term "pharmaceutically acceptable carrier" includes any solvent, drug stabilizer, or combination thereof, which are known to those skilled in the art. Except where any conventional carrier is incompatible with the active ingredient, its use in treatment or pharmaceutical compositions is encompassed.

[0047] In this article, the term "pharmaceutically acceptable excipients" may include carbohydrates, including monosaccharides or polysaccharides, such as lactose, sucrose, mannitol and sorbitol; cellulose and its derivatives, such as sodium carboxymethylcellulose, ethyl cellulose and methyl cellulose; calcium phosphates, such as dicalcium phosphate and tricalcium phosphate; sodium sulfate; calcium sulfate; polyvinyl pyrrolidone; polyvinyl alcohol; stearic acid; alkaline earth metal stearates, such as magnesium stearate and calcium stearate; vegetable oils, such as peanut oil, cottonseed oil, sesame oil, olive oil and corn oil; nonionic, cationic and anionic surfactants; ethylene glycol polymers; fatty alcohols; and cereal hydrolyzed solids and other non-toxic compatible fillers, binders, disintegrants, buffers, preservatives, antioxidants, lubricants, colorants and other excipients commonly used in pharmaceutical preparations.

[0048] According to a specific embodiment of the present invention, the present invention proposes a Bifidobacterium animalis subsp. lactis reBF17-4. According to an embodiment of the present invention, the preservation number of the Bifidobacterium animalis subsp. lactis is CGMCC No. 31147. The Bifidobacterium animalis subsp. lactis of the present invention has strong antioxidant efficacy and gastrointestinal tolerance.

[0049] As used herein, "Bifidobacterium animalis subsp. lactis reBF17-4" and "Bifidobacterium animalis subsp. lactis reBF17-4" and "reBF17-4" are synonymous.

[0050] The first aspect of the present invention provides a Bifidobacterium animalis subsp. lactis reBF17-4, wherein the Bifidobacterium animalis subsp. lactis reBF17-4 was deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration on July 3, 2024, with a deposit number of CGMCC No.31147.

[0051] The animal Bifidobacterium lactis subspecies provided by the present invention has both antioxidant and gastrointestinal tolerance capabilities, and can be used to prevent and / or alleviate and / or treat related diseases caused by oxidative stress, wherein the related diseases caused by oxidative stress include at least one of degenerative diseases, diabetes, cirrhosis, cancer, Parkinson's disease and cardiovascular diseases; on the other hand, it can maintain intestinal health, wherein the maintenance of intestinal health includes repairing intestinal mucosal damage, regulating the absorption of sodium and water by intestinal cell walls, enhancing intestinal barrier function, inhibiting the formation of colon adenomas and tumors, and at least one of antibacterial or bactericidal effects.

[0052] Compared with other animal bifidobacterium lactis subspecies, the animal bifidobacterium lactis reBF17-4 of the present invention has a stronger tolerance to artificial gastrointestinal fluid. In artificial gastric fluid (pH=3.0), the survival rate of the strain reBF17-4 of the present invention is as high as 113.66% after being cultured at 37°C for 3 hours; in artificial intestinal fluid (pH=8.0, bile salt concentration 0.3%), the survival rate can still reach 100.66% after being cultured at 37°C for 2 hours, indicating that the strain reBF17-4 has a strong tolerance to artificial gastrointestinal fluid and has the potential to maintain high activity in the gastrointestinal tract and exert its probiotic effect.

[0053] The second aspect of the present invention provides a fermentation supernatant, which comprises the metabolites of the animal Bifidobacterium lactis subspecies reBF17-4 described in the first aspect. The animal Bifidobacterium lactis subspecies reBF17-4 of the present invention has strong antioxidant efficacy and gastrointestinal tolerance.

[0054] It should be noted that “fermentation supernatant” refers to the supernatant after centrifugation of the fermentation broth, which may contain animal Bifidobacterium lactis subsp. reBF17-4 or its metabolites, or both.

[0055] The third aspect of the present invention provides a bacterial suspension, the bacterial suspension comprising the animal Bifidobacterium lactis subsp. reBF17-4 described in the first aspect. The bacterial suspension of the present invention is used in a drug for preventing and / or treating related diseases caused by oxidative stress.

[0056] According to an embodiment of the present invention, the animal Bifidobacterium lactis subspecies reBF17-4 exists in the form of living cells and / or non-living cells.

[0057] As used herein, "living cells" refer to cells that have the ability to metabolize, reproduce or replicate.

[0058] Exemplarily, the living cells may be immobilized cells. Herein, "immobilized cells" refer to Bifidobacterium animalis subsp. lactis reBF17-4 fixed on a carrier, which can carry out life activities such as growth, development, reproduction, inheritance and metabolism within a certain spatial range.

[0059] Herein, "non-living cells" refer to cells that do not have the ability to metabolize, reproduce and replicate, including but not limited to dried bacteria. Exemplarily, the microbial agent is a freeze-dried powder.

[0060] As a specific embodiment, the animal Bifidobacterium lactis subspecies reBF17-4 exists in the form of living cells, dry bacteria, immobilized cells or in any other form.

[0061] As a specific embodiment, the dry bacteria are obtained by freeze-drying the animal Bifidobacterium lactis subspecies reBF17-4.

[0062] The fourth aspect of the present invention provides use of the animal Bifidobacterium lactis subsp. lactis reBF17-4 described in the first aspect, the fermentation supernatant described in the second aspect, or the bacterial suspension described in the third aspect in the preparation of a medicament for preventing and / or treating related diseases caused by oxidative stress.

[0063] According to an embodiment of the present invention, the related diseases caused by oxidative stress include at least one of degenerative diseases, diabetes, liver cirrhosis, cancer, Parkinson's disease and cardiovascular disease.

[0064] The fifth aspect of the present invention provides use of the animal Bifidobacterium lactis subsp. reBF17-4 described in the first aspect, the fermentation supernatant described in the second aspect, or the bacterial suspension described in the third aspect in the preparation of a medicament for maintaining intestinal health.

[0065] According to an embodiment of the present invention, maintaining intestinal health includes at least one of repairing intestinal mucosal damage, regulating the absorption of sodium and water by intestinal cell walls, enhancing intestinal barrier function, inhibiting the formation of colon adenomas and tumors, and antibacterial or bactericidal effects.

[0066] According to an embodiment of the present invention, maintaining intestinal health includes treating and / or preventing related diseases caused by at least one of Staphylococcus aureus, Escherichia coli, Salmonella paratyphi B, Listeria monocytogenes, Shigella dysenteriae, Enterococcus faecalis and Clostridium perfringens.

[0067] The sixth aspect of the present invention provides a medicine, which comprises at least one of the animal Bifidobacterium lactis subsp. reBF17-4 described in the first aspect, the fermentation supernatant described in the second aspect, and the bacterial suspension described in the third aspect.

[0068] According to an embodiment of the present invention, the drug further contains an excipient and / or a carrier.

[0069] According to an embodiment of the present invention, the excipient includes at least one selected from a binder, a disintegrant, a lubricant, a glidant, a stabilizer, a filler, a diluent and a sustained-release agent.

[0070] According to an embodiment of the present invention, the carrier includes at least one selected from sugars, cellulose and its derivatives, calcium phosphates, alkaline earth metal stearates, vegetable oils, nonionic surfactants, cationic surfactants, anionic surfactants, fatty alcohols and cereal hydrolyzed solids.

[0071] According to an embodiment of the present invention, the dosage form of the drug includes at least one selected from oral liquid, powder, granule, capsule, tablet and pill.

[0072] According to an embodiment of the present invention, when animal Bifidobacterium lactis subsp. lactis reBF17-4 takes effect, it may exist in the form of living cells or in the form of non-living cells.

[0073] According to an embodiment of the present invention, the living cells refer to cells of Bifidobacterium animalis subspecies lactis reBF17-4 that have the ability to metabolize, reproduce or replicate, and the non-living cells refer to cells that do not have the ability to metabolize, reproduce and replicate, including but not limited to dry bacteria (such as freeze-dried powder).

[0074] In addition, the present invention provides a microbial agent, which contains the aforementioned animal Bifidobacterium lactis subsp. reBF17-4, the aforementioned fermentation supernatant or the aforementioned bacterial suspension. The microbial agent of the present invention is used to prevent and / or treat related diseases caused by oxidative stress.

[0075] According to an embodiment of the present invention, the present invention proposes a medicine containing animal Bifidobacterium lactis subspecies reBF17-4. Preferably, the medicine of the present application is a live bacterial preparation for the prevention and / or treatment of related diseases caused by oxidative stress. It should be noted that the medicine of the present application can be various preparations of a single active animal Bifidobacterium lactis subspecies reBF17-4, and can also be used in combination with other active ingredients, as long as the activity is not affected by each other; further, the best case of the medicine is that the active ingredients can have functional complementarity or a promoting effect, for example, animal Bifidobacterium lactis subspecies reBF17-4 can be combined with other probiotics to form a composite probiotic tablet, which can be used to prevent and / or treat related diseases caused by oxidative stress on the one hand, and can maintain intestinal health on the other hand, and the maintenance of intestinal health includes repairing intestinal mucosal damage, regulating the absorption of sodium and water in the intestinal cell wall, enhancing intestinal barrier function, inhibiting the formation of colon adenomas and tumors, antibacterial or bactericidal at least one, specifically, it can be determined according to the activity and components of the composite probiotic tablet, which is not limited here. Optionally, the drug further includes a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, an acceptable carrier or excipient in food, or an acceptable carrier or excipient in health food. It should be noted that when preparing a live bacteria preparation, it is usually necessary to add a carrier or excipient, as long as there is no mutual inhibition or adverse side effects between the added carrier or excipient and animal Bifidobacterium lactis subsp. reBF17-4. The drug can be a powder, tablet, beverage, or capsule.

[0076] In addition, the present invention provides a microbial agent, which contains the aforementioned animal Bifidobacterium lactis subsp. reBF17-4, the aforementioned fermentation supernatant or the aforementioned bacterial suspension, and the microbial agent of the present invention is used to prevent and / or treat related diseases caused by oxidative stress. The microbial agent of the present invention is used to prevent and / or treat related diseases caused by oxidative stress. It should be noted that the microbial agent of the present invention can be a microbial liquid agent, including but not limited to fermentation broth, etc., and can also be a microbial solid agent, including but not limited to freeze-dried powder, etc.

[0077] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

[0078] Example 1: Screening and identification of Bifidobacterium animalis subspecies CGMCC NO.31147

[0079] 1. Screening of Bifidobacterium animalis subspecies CGMCC NO.31147

[0080] The strain used in the present invention was isolated from meconium collected from newborns born by cesarean section in the Department of Obstetrics of Peking University Third Hospital in Beijing. The meconium sample was added to a sterile phosphate buffered saline (PBS) buffer and mixed thoroughly. The suspension was diluted with PBS at 10 -6 , 10 -7 , 10 -8 Gradient dilution, 100 μL of each bacterial solution was spread on MRS medium plates containing 0.5% L-cysteine ​​hydrochloride, each dilution was repeated three times, and anaerobic culture was carried out at 37°C for 48 hours. After the culture was completed, a single colony plate with 30-300 colonies was selected from the agar medium, and then a single colony was picked from the plate and continued to be isolated and cultured on MRS liquid medium containing 0.5% L-cysteine ​​hydrochloride for 48 hours to obtain purified bacteria. The obtained strains were all frozen and stored at -80°C in MRS liquid medium containing 60% glycerol.

[0081] 2. CGMCC NO.31147 strain identification

[0082] 1 mL of bacteria cultured for 48 h was taken into a 1.5 mL EP tube, centrifuged at 8000 r / min for 3 min, the supernatant was discarded, 1 mL of sterile water was added, mixed thoroughly, centrifuged (8000 r / min, 3 min), the supernatant was discarded, and the bacterial precipitate was used as a template for strain identification, followed by subsequent colony polymerase chain reaction (PCR), gene fragment amplification using 16S rRNA universal primers, and the product was sent to Shanghai Shenggong Biotechnology Co., Ltd. for nucleic acid sequence determination after identification by 1.2% agarose gel electrophoresis. The base sequence in the determination result was entered into the NCBI database for BLAST sequence comparison, and the results showed that the CGMCC NO.31147 strain sequence from the meconium sample had a 16SrDNA sequence homology of more than 99% with that of animal Bifidobacterium lactis subsp. Figure 1It is the colony morphology of animal Bifidobacterium lactis subspecies CGMCC NO.31147.

[0083] The sequence of strain CGMCC NO.31147 is:

[0084]

[0085] Experimental Example 2: Genomic Features

[0086] The frozen CGMCC NO.31147 was inoculated into MRS medium and cultured anaerobically at 37°C. The inoculation volume was 2% and passed to the next generation every 24 hours. After 2 generations, the bacterial solution in the logarithmic growth phase was collected and mailed to Annoroad Gene Technology (Beijing) Co., Ltd. for whole genome sequencing. The genomic DNA was fragmented to construct a library and sequenced using the second-generation Illumina X Plus platform and paired-end 150bp mode. Each sample also provides Illumina sequencing data of no less than 500x of the genome to obtain a more complete and accurate bacterial completion map.

[0087] The software used in this experiment to predict, assemble, and analyze the whole gene sequence is shown in Table 1.

[0088] Table 1

[0089] Software and database Version use megahit v 1.2.9 Genome assembly checkm v 1.2.2 Genome integrity and contamination test prokka v 1.14.6 Functional annotation interproscan v 5.59_91.0 Functional annotation seqkit v 2.8.0 Calculate genome length orthofinder v 2.5.5 Find homologous genes ggplot v 3.10.0 Drawing Software and database Version use pheatmap v 1.0.12 Drawing ggtree v 3.5.1 Drawing

[0090] The CGMCC NO.31147 gene was compared with databases such as Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), UniProt, and Pfam to obtain gene function annotation results.

[0091] 1. Genome-wide component prediction and functional annotation

[0092] The genome circle map of Bifidobacterium animalis subsp. lactis CGMCC NO.31147 is shown in Figure 2 As shown. The whole genome size is about 1.92Mbp, with an average GC content of 60.49%. The number of CDS is 1561, and non-coding genes include 5 rRNAs, 55 tRNAs and 1 tmRNA. According to the annotation results of Uniprot, Pfam, GO, and KEGG databases, there are 15 genes related to antioxidant function on the genome of CGMCC NO.31147, including genes encoding multiple reductases: alkyl hydroperoxide reductase (ahpC), ferredoxin-NADP reductase, thioredoxin reductase (trxB), methionine sulfoxide reductase (MsrAB), and other antioxidant genes such as metF, nfrA1, hpr, folA, gpr, nrdF2, iolS, nrdE2, proC, etc.

[0093] 2. Phylogenetic Analysis

[0094] Comparative genomics was used to compare and analyze the genomes of other animal Bifidobacterium lactis subspecies that have been published. Figure 3 As shown, by constructing a phylogenetic tree, it was found that animal Bifidobacterium lactis subspecies CGMCC NO.31147 belonged to the same branch as animal Bifidobacterium lactis subspecies from other sources, and animal Bifidobacterium lactis subspecies CGMCC NO.31147 was closest to strains of unknown origin and was close to some strains from feces.

[0095] Example 3: Determination of the ability of Bifidobacterium animalis subspecies CGMCC NO.31147 to scavenge diphenyltrinitrophenylhydrazine free radical (DPPH)

[0096] The neonatal isolates including CGMCC NO.31147 and the animal Bifidobacterium lactis subsp. lactis isolated from commercial products (Chr. Hansen Bb-12 probiotic powder solid beverage, as a positive control) were inoculated into MRS solid medium, incubated anaerobically at 37°C for 48 h, inoculated into MRS liquid medium, incubated anaerobically at 37°C for 24 h, and then centrifuged (8000 r / min, 10 min, 4°C), the cells were collected, and resuspended in sterile 0.9% saline to obtain 1×10 8 CFU / mL bacterial suspension. Take 0.2mL 1×10 8 CFU / mL bacterial suspension was mixed with 0.2mL DPPH (0.2mmol / L, prepared in anhydrous ethanol) in a 2mL EP tube (the control group used an equal volume of PBS solution instead of bacterial suspension), reacted at room temperature in the dark for 30min, centrifuged (8000r / min, 10min, 4℃), and the absorbance at 517nm was measured by an enzyme reader.

[0097] DPPH free radical scavenging rate (%) = (1-A 实验组 / A 对照组 )×100%

[0098] The DPPH free radical scavenging rate was positively correlated with the antioxidant capacity of the strain. Figure 4 As shown, the DPPH free radical scavenging rate of the positive control strain was 22.69%. The DPPH free radical scavenging rates of the six experimental strains ranged from 5.44% to 66.99%, among which the DPPH free radical scavenging rate of animal Bifidobacterium lactis subspecies CGMCC NO.31147 was significantly higher than that of the other five experimental strains (p<0.05), with a scavenging rate of 66.99%. At the same time, compared with the commercialized Bb-12 in the commercial probiotic products, its scavenging rate was significantly increased, 197.31% higher; the DPPH free radical scavenging rate of animal Bifidobacterium lactis subspecies BiBF01-2 was the lowest, at 5.44% (p<0.05).

[0099] Example 4: Determination of the ability of Bifidobacterium animalis lactis subspecies CGMCC NO.31147 to scavenge hydroxyl radicals

[0100] The neonatal isolates including CGMCC NO.31147 and animal Bifidobacterium lactis subsp. lactis isolated from commercial products (Chr. Hansen Bb-12 probiotic powder solid beverage, as a positive control) were inoculated into MRS solid medium, incubated anaerobically at 37°C for 48 h, inoculated into MRS liquid medium, incubated anaerobically at 37°C for 24 h, and centrifuged (8000 r / min, 10 min, 4°C) to collect the cells, resuspended in 0.9% sterile saline, and obtained 1×10 8 CFU / mL bacterial suspension. Take 0.1mL 1×10 8 CFU / mL bacterial suspension, 0.1mL 2.5mmol / L 1,10-phenanthroline, 0.1mL PBS, 0.1mL 2.5mmol / L FeSO 4 , mix well and add 0.1mL 20mmol / LH 2 O 2 (The control group used an equal volume of deionized water instead of 20mmol / LH 2 O 2 In the blank group, an equal volume of blank culture medium was used to replace the bacterial suspension, and an equal volume of deionized water was used to replace 20mmol / L H 2 O 2 ), completely mixed in a 2.0 mL EP tube, placed in a light-proof water bath at 37°C for 1.5 h, and the absorbance at 536 nm was measured using an ELISA reader.

[0101] Hydroxyl free radical scavenging rate (%) = (A 实验组 -A 对照组 ) / (A 对照组 -A 空白组 )×100%

[0102] Hydroxyl free radicals are the most harmful reactive oxygen free radicals that cause lipid peroxidation, DNA damage and oxidative damage of biological molecules. Excessive hydroxyl free radicals can affect the normal function of the body. Therefore, the hydroxyl free radical scavenging ability is an important indicator for evaluating the antioxidant activity of strains. Figure 5 As shown, the hydroxyl radical scavenging rate of the positive control strain was 16.49%. The hydroxyl radical scavenging rates of the 6 experimental strains ranged from 10.77% to 67.29%, showing strain specificity. Among them, the hydroxyl radical scavenging rate of animal Bifidobacterium lactis subspecies BiBF01-2 was the lowest, at 10.77%, while the hydroxyl radical scavenging rate of animal Bifidobacterium lactis subspecies CGMCC NO.31147 was 67.29%, significantly higher than other strains (p<0.05).

[0103] Example 5: Determination of the ability of Bifidobacterium animalis lactis subspecies CGMCC NO.31147 to scavenge superoxide anion free radicals

[0104] The neonatal isolates including CGMCC NO.31147 and animal Bifidobacterium lactis subsp. lactis isolated from commercial products (Chr. Hansen Bb-12 probiotic powder solid beverage, as a positive control) were inoculated into MRS solid medium, incubated anaerobically at 37°C for 48 h, inoculated into MRS liquid medium, incubated anaerobically at 37°C for 24 h, and centrifuged (8000 r / min, 10 min, 4°C) to collect the cells, resuspended in 0.9% sterile saline, and obtained 1×10 8 CFU / mL bacterial suspension. The bacterial suspension was broken with an ultrasonic disruptor under ice bath conditions, centrifuged (8000r / min, 10min, 4℃), and the obtained supernatant was sterilized by a 0.22μm pore size filter membrane to obtain a cell-free extract. Take 0.85mL Tris-HCl solution (50mmol / L, pH=8.2), 0.125mL pyrogallol solution (50mmol / L), add 0.25mL cell-free extract (the control group used an equal volume of PBS solution instead of cell-free extract), mix well, and react in a 25℃ metal bath for 4min, then add 0.025mL HCl solution (8mol / L) to terminate the reaction, and measure the absorbance at 320nm with an enzyme marker.

[0105] Superoxide anion free radical scavenging rate (%) = (1-A 实验组 / A 对照组 )×100%

[0106] Superoxide anion can be used as an important signal molecule for cell apoptosis or cell senescence, so the ability to scavenge superoxide anion free radicals is an important indicator for evaluating the antioxidant activity of strains. Figure 6 As shown, the strains have different abilities to scavenge superoxide anions. The superoxide anion free radical scavenging rates of animal Bifidobacterium lactis subspecies CGMCC NO.31147 and BiB1M03-1 were 7.34% and 8.07%, respectively, which were not significantly different from the commercialized Bb-12 (7.52%, p<0.05), and were higher than the four experimental strains of animal Bifidobacterium lactis subspecies BiBF01-2, BiBF09-6, BiB2F19-4 and reBF26-15.

[0107] Example 6: Evaluation of the tolerance of Bifidobacterium animalis subspecies CGMCC NO.31147 to artificial gastric juice

[0108] Adjust the pH of 0.5% sterile saline to 3.0 with 1mol / L HCl, add 0.01% pepsin, adjust the concentration to 3g / L, and filter with a 0.22μm filter membrane to obtain artificial gastric juice. Animal Bifidobacterium lactis subspecies (Bb-12) and animal Bifidobacterium lactis subspecies strains isolated from commercial products (Chr. Hansen Bb-12 probiotic powder solid beverage) and neonatal samples were inoculated into MRS solid medium, inoculated into MRS liquid medium after anaerobic culture at 37°C for 48h, centrifuged (8000r / min, 10min, 4°C) after anaerobic culture at 37°C for 24h, and the precipitate was resuspended in 1mL sterile PBS solution to obtain a bacterial suspension. 1mL of the bacterial suspension to be tested was inoculated into 9mL artificial gastric juice (pH=3.0), and after 37°C culture for 3h, the bacterial solution was plated in an appropriate dilution gradient, and anaerobically cultured at 37°C for 48h, the number of viable bacteria was determined, and the survival rate was calculated.

[0109] Survival rate (%) = n1 / n0×100%, n1 is the number of viable bacteria (CFU / mL) after treatment with artificial gastric juice; n0 is the number of viable bacteria (CFU / mL) before treatment with artificial gastric juice.

[0110] The low pH value in the host's gastric juice is an important barrier that affects the activity of probiotics after entering the gastrointestinal tract. Therefore, the strain's tolerance to artificial gastric juice is an important indicator for evaluating the strain's activity in the human gastrointestinal tract. Figure 7 As shown, after culturing in an artificial gastric fluid environment for 3 hours, all strains still have a certain activity, the survival rate of the positive strain Bb-12 is 101.29%, and the remaining strains are between 57.24% and 113.66%. Among them, animal Bifidobacterium lactis subspecies CGMCC NO.31147 maintains the highest activity, and there is no significant difference between the two compared with Bb-12 (p<0.05), and the survival rates of the two strains are both higher than 100%, indicating that the animal Bifidobacterium lactis subspecies CGMCC NO.31147 of the present invention has a good tolerance to artificial gastric fluid.

[0111] Example 7: Evaluation of the tolerance of Bifidobacterium animalis subspecies CGMCC NO.31147 to artificial intestinal fluid

[0112] The pH of 0.5% sterile saline was adjusted to 8.0 with 1 mol / L NaOH, 4% pepsin was added to adjust the concentration to 1 g / L, bile salts were added to make the final concentration reach 0.3%, and the mixture was filtered using a 0.22 μm filter membrane to obtain artificial intestinal fluid. Animal Bifidobacterium lactis subsp. (Bb-12) and animal Bifidobacterium lactis subsp. strains isolated from commercial products (Chr. Hansen Bb-12 probiotic powder solid beverage) and neonatal samples were inoculated into MRS solid medium, inoculated into MRS liquid medium after anaerobic culture at 37°C for 48 hours, centrifuged (8000 r / min, 10 min, 4°C) after anaerobic culture at 37°C for 24 hours, and the precipitate was resuspended in 1 mL sterile PBS solution to obtain a bacterial suspension. Inoculate 1 mL of the test bacterial suspension into 9 mL of artificial intestinal fluid (pH=8.0, bile salt concentration 0.2%), culture at 37°C for 2 h, spread the bacterial suspension on plates at appropriate dilution gradients, culture anaerobically at 37°C for 48 h, determine the number of viable bacteria, and calculate the survival rate.

[0113] Survival rate (%) = n 1 / n 0 ×100%,n 1 is the number of viable bacteria after artificial intestinal fluid treatment (CFU / mL); n 0 It is the number of viable bacteria (CFU / mL) before artificial intestinal fluid treatment.

[0114] The normal intestinal bile salt concentration of the human body is 0.03% to 0.3%. Bile salts can change the cell membrane permeability of bifidobacteria or induce DNA damage leading to cell death, resulting in a decrease in the number of active bacteria. Figure 8 As shown, after 2 hours of digestion in artificial intestinal fluid, the survival rate of CGMCC NO.31147 was still as high as 100.66%, showing high tolerance to artificial intestinal fluid. In addition, the survival rate of CGMCC NO.31147 was significantly higher than that of the positive strain Bb-12 (68.25%) and the other three experimental strains (p<0.05).

[0115] Example 8: Metabolomics analysis of Bifidobacterium animalis subspecies CGMCC NO.31147

[0116] Bifidobacterium animalis subsp. lactis (Bb-12) and Bifidobacterium animalis subsp. lactis CGMCC NO.31147 isolated from commercial products (Chr. Hansen Bb-12 probiotic powder solid beverage) and neonatal samples were inoculated into MRS solid culture medium, anaerobically cultured at 37°C for 48 h, inoculated into MRS liquid culture medium, anaerobically cultured at 37°C for 24 h, and centrifuged (8000 r / min, 10 min, 4°C) to obtain the supernatant. Weigh 50±5 mg of supernatant, add grinding beads and 400 μL of extract (methanol: water = 4:1 (v:v)), grind in a frozen tissue grinder for 6 min (-10°C, 50 Hz), extract by low-temperature ultrasound for 30 min (5°C, 40 kHz), centrifuge (13000g, 15 min, 4°C), transfer the supernatant to a liquid phase vial, transfer an appropriate amount of sample to mix as quality control, and use liquid chromatography-mass spectrometry (LC-MS) to determine serum metabolites. ACQUITY UPLC HSS T3 (100 mm × 2.1 mmi.d., 1.8 μm; Waters, Milford, USA) column was used; mobile phase A was 95% water + 5% acetonitrile (containing 0.1% formic acid), mobile phase B was 47.5% acetonitrile + 47.5% isopropanol + 5% water (containing 0.1% formic acid), injection volume was 3 μL, column temperature was 40 ° C. The sample was electrospray ionized, and mass spectrometry signals were collected in positive and negative ion scanning modes. Specific parameters are shown in Table 2.

[0117] Table 2

[0118] describe parameter Scanning range (m / z) 70-1050 Sheath gas flow rate (arb) 60 Auxiliary gas flow rate (arb) 20 Heating temperature(℃) 350 Capillary temperature (℃) 320 Spray voltage (positive mode) (V) 3400 Spray voltage (negative mode) (V) -3000 S-Lens Voltage 70 Collision energy (%) 20,40,60 Resolution (Full MS) 60000 Resolution (MS) 15000

[0119] Non-targeted metabolomics is an omics method in systems biology that can analyze changes in small molecule metabolites to reflect different biological processes in cells. In order to gain a deeper understanding of the metabolism during the cultivation of animal Bifidobacterium lactis subspecies CGMCC NO.31147, LC-MS was used to identify the differences in metabolites in the culture supernatant of animal Bifidobacterium lactis subspecies CGMCC NO.31147 and the positive strain Bb-12. Metabolites with high-quality peak signals were collected, and then the overall characteristics between the two groups of samples were analyzed. Principal component analysis (PCA) was used to perform multivariate statistical analysis on the LC-MS data, such as Fig. 9 As shown in A; the volcano plot reflects the differences in metabolites, such as Fig. 9 As shown in B.

[0120] Principal component analysis (PCA) is a method that uses fewer variables to explain the internal structure of more variables through the concept of dimensionality reduction. Fig. 9As shown in A, the metabolite levels of the supernatant of CGMCC NO.31147 changed significantly compared with the positive strain Bb-12, and the two groups were clearly separated on the PCA graph, indicating that there were significant differences in the metabolites between CGMCC NO.31147 and Bb-12 cultures. Using FC>1.0, p<0.05 and VIP>2 as screening conditions, a total of 108 differential metabolites between CGMCC NO.31147 and Bb-12 were screened. The p value and variation fold value were visualized using a volcano plot, and the results are shown in Figure 1. Fig. 9 B. Among them, the significantly up-regulated differential metabolites are marked with red dots, the significantly down-regulated differential metabolites are marked with blue dots, and the insignificant differential metabolites are marked with gray dots. Among the 108 differential metabolites, 77 were significantly decreased and 31 were significantly increased.

[0121] Among the 31 metabolites significantly upregulated in CGMCC NO.31147 compared with the positive strain Bb-12, 6 metabolites had anti-inflammatory or antioxidant activities, namely, γ-glutamylmethionine, N-ethylmaleimide, Ascorbyl palmitate, Furanodienone, 6-Hydroxyhexanoic Acid, and N-Acetyl-L-Glutamic Acid. The relative contents of these metabolites in the total metabolites were as follows: Fig.10 As shown in Figure 2. For example, ascorbyl palmitate, as an ascorbic acid derivative, has antioxidant function. Among the 77 metabolites that were significantly downregulated in CGMCC NO.31147 compared with the positive strain Bb-12, the relative contents of prostaglandin J2 and adenine in the total metabolites are shown in Figure 2. Fig.10As shown. Maria E. Figueiredo-Pereira et al. found that prostaglandin J2 can induce neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease through multiple pathways such as inducing a positive feedback loop of upregulation of cyclooxygenase-2 (COX-2) in cancer cells and neurons, promoting the transition from acute to chronic inflammation. In addition, high concentrations of adenine can cause metabolic disorders in the body, produce a large number of oxygen free radicals, stimulate oxidative stress reactions, and cause severe damage to the kidneys. It is one of the causes of chronic renal failure, indicating that CGMCC NO.31147 may have the potential to relieve oxidative stress, protect kidney function, and prevent inflammation and neurodegenerative diseases. The results of differential metabolites showed that compared with the positive strain Bb-12, CGMCC NO.31147 can upregulate some metabolites with antioxidant or anti-inflammatory activity, and has a downregulation effect on some metabolites that promote oxidative stress or degenerative diseases, so it has greater antioxidant potential.

[0122] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0123] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A Bifidobacterium animalis subsp. lactis reBF17-4, characterized in that The preservation number of the Bifidobacterium animalis subsp. lactis reBF17-4 is CGMCC No.31147.

2. A fermentation supernatant, characterized in that: The invention comprises the metabolites of the animal Bifidobacterium lactis subspecies reBF17-4 as claimed in claim 1.

3. A bacterial suspension, characterized in that: It comprises the animal Bifidobacterium lactis subspecies reBF17-4 as described in claim 1.

4. Use of the animal Bifidobacterium lactis subsp. reBF17-4 according to claim 1, the fermentation supernatant according to claim 2 or the bacterial suspension according to claim 3 in the preparation of a medicament for preventing and / or treating related diseases caused by oxidative stress; Optionally, the oxidative stress-induced related diseases include at least one of degenerative diseases, diabetes, liver cirrhosis, cancer, Parkinson's disease and cardiovascular disease.

5. Use of the animal Bifidobacterium lactis subsp. reBF17-4 according to claim 1, the fermentation supernatant according to claim 2 or the bacterial suspension according to claim 3 in the preparation of a medicament for maintaining intestinal health.

6. The use according to claim 5, characterized in that The maintenance of intestinal health includes at least one of repairing intestinal mucosal damage, regulating the absorption of sodium and water by intestinal cell walls, enhancing intestinal barrier function, inhibiting the formation of colon adenomas and tumors, and antibacterial or bactericidal effects.

7. The use according to claim 5, characterized in that The maintaining of intestinal health comprises treating and / or preventing diseases related to at least one of Staphylococcus aureus, Escherichia coli, Salmonella paratyphi B, Listeria monocytogenes, Shigella dysenteriae, Enterococcus faecalis and Clostridium perfringens.

8. A drug, characterized in that The drug comprises at least one of the animal Bifidobacterium lactis subspecies reBF17-4 according to claim 1, the fermentation supernatant according to claim 2, and the bacterial suspension according to claim 3.

9. The drug according to claim 8, characterized in that The medicament further contains excipients and / or carriers.

10. The drug according to claim 9, characterized in that The excipients include at least one selected from a binder, a disintegrant, a lubricant, a glidant, a stabilizer, a filler, a diluent and a sustained-release agent.

11. The drug according to claim 9, characterized in that The carrier comprises at least one selected from sugars, cellulose and its derivatives, calcium phosphates, stearic acid alkaline earth metal salts, vegetable oils, nonionic surfactants, cationic surfactants, anionic surfactants, fatty alcohols and cereal hydrolyzed solids.

12. The drug according to claim 8, characterized in that The dosage form of the drug includes at least one selected from oral liquid, powder, granule, capsule, tablet and pill.

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