Neonatal-derived bifidobacterium animalis subsp. Lactis and application thereof

Abstract

The invention discloses bifidobacterium animalis subsp. Lactis derived from newborns and application of the bifidobacterium animalis subsp. Lactis. The bacterial strain is named as Bifidobacterium animalis subsp. Lactis reBF24-1, is preserved in the China General Microbiological Culture Collection Center on July 23, 2024, and has the preservation number of CGMCC (China General Microbiological Culture Collection Center) No.31316. The invention further discloses a preparation method of the bifidobacterium animalis subsp. Lactis reBF24-1, and the bifidobacterium animalis subsp. Lactis reBF24-1 can be used for preparing the bifidobacterium animalis subsp. Lactis reBF24-1. The bifidobacterium animalis subsp. Lactis is screened from excreta of first defecation of local healthy newborns in Beijing within 24-48 hours after birth, and the new strain has antioxidant capacity and excellent gastrointestinal tract endurance capacity and intestinal tract colonization capacity, can remarkably relieve intestinal tract oxidative stress and is used for preparing medicines for maintaining gastrointestinal tract health.

Description

Technical Field

[0001] The invention relates to the field of microorganisms, and in particular to a strain of animal Bifidobacterium lactis subspecies derived from a newborn and application thereof. Background Art

[0002] Bifidobacterium animalis subsp. lactis is a type of obligate anaerobic, non-spore-producing Gram-positive bacteria, which belongs to the genus Bifidobacterium and subsp. lactis in systematic taxonomy. This species was first isolated from the feces of healthy breastfed infants and is one of the main types of Bifidobacteria in the infant intestine. It has a long history of safe consumption.

[0003] Oxidative stress is one of the main causes of many chronic human diseases. Due to the imbalance between the body's oxidative capacity and antioxidant capacity, excessive free radicals accumulate in tissues and cells, inducing oxidative damage, which can cause diabetes, aging, obesity, Parkinson's syndrome, and cancer. Studies have shown that in order to adapt to the sudden change from the intrauterine hypoxic environment to the extrauterine hyperoxic environment, the fetus needs to rely on the lungs to complete autonomous breathing. In this process, mitochondrial respiration may be accompanied by a large amount of reactive oxygen species, which increases the level of oxidative stress in the baby's body and promotes the ability of the intestinal flora to resist oxygen stress. Meconium, as the excrement of the first bowel movement after the baby is born, retains the most primitive intestinal microorganisms in the early stage of life. The number of bifidobacteria in the infant's intestine is also richer than that of adults. Studies have found that the initial intestinal components can significantly alleviate oxidative stress and have long-term effects on the health of the body.

[0004] At present, domestic patents have been published for animal bifidobacteria with antioxidant functions, which are widely used. Common sources of isolation are breast milk or the intestines of healthy infants of a certain age. The commercial strain BB-12 was originally isolated from dairy products, mainly used to regulate intestinal flora and improve immunity. CN201510018144.4 discloses an oxygen-tolerant animal Bifidobacterium lactis subspecies BZ11 that can lower cholesterol isolated from a fragrant pig source, and CN202010766121.2 discloses an anti-aging strain animal Bifidobacterium 952 isolated from the intestines of centenarians. Due to the regional, ethnic and strain specificity of probiotics, the wide applicability and effect of the above strains in antioxidant applications are limited. The strain in this patent application is uniquely derived from neonatal meconium, which is usually the first defecation within 24 to 48 hours after the baby is born. Compared with animal-derived strains, the colonization effect in the human intestine may be better, but there are few reports in China.

[0005] Therefore, the present invention uses the commercial strain BB-12 as a positive control, separates and screens the intestinal initial carrying strain animal Bifidobacterium lactis subsp. animalis, which is suitable for the local population and has anti-oxidative stress, from the meconium of healthy newborns in Beijing. This will help broaden the selection range of antioxidant probiotic products. 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] Therefore, in the first aspect of the present invention, the present invention proposes an animal Bifidobacterium subsp. lactis reBF24-1, and the animal Bifidobacterium subsp. lactis reBF24-1 was deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration on July 23, 2024, with a deposit number of CGMCC No.31316.

[0008] Collection information:

[0009] Strain name: reBF24-1

[0010] Classification and nomenclature: Bifidobacterium animalis subsp. lactis

[0011] Storage date: July 23, 2024

[0012] Depository: China National Microbiological Culture Collection Administration General Microbiology Center

[0013] Deposit number: CGMCC No.31316

[0014] Storage address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101

[0015] The animal Bifidobacterium lactis subspecies provided by the present invention is screened from the excrement of the first defecation of healthy local newborns in Beijing within 24 to 48 hours after birth. The new strain has antioxidant ability, excellent gastrointestinal tolerance and intestinal colonization ability, can significantly alleviate intestinal oxidative stress, and is used to prepare drugs for maintaining gastrointestinal health.

[0016] In a second aspect of the present invention, the present invention provides a bacterial suspension. According to an embodiment of the present invention, the bacterial suspension includes the aforementioned Bifidobacterium animalis subsp. lactis reBF24-1.

[0017] Those skilled in the art will appreciate that the features and advantages described above for Bifidobacterium animalis subsp. lactis reBF24-1 are also applicable to the bacterial suspension and will not be described in detail herein.

[0018] In a third aspect of the present invention, the present invention provides a microbial agent. According to an embodiment of the present invention, the microbial agent comprises: the aforementioned Bifidobacterium animalis subsp. lactis reBF24-1 or the aforementioned bacterial suspension.

[0019] Those skilled in the art will appreciate that the features and advantages described above for Bifidobacterium animalis subsp. lactis reBF24-1 are also applicable to this microbial agent and will not be described in detail here.

[0020] In a fourth aspect of the present invention, the present invention provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition comprises: at least one of the aforementioned animal Bifidobacterium lactis subsp. reBF24-1, the aforementioned bacterial suspension, or the aforementioned microbial agent. The pharmaceutical composition according to an embodiment of the present invention can be used to develop drugs for maintaining gastrointestinal health, preventing or treating gastrointestinal diseases.

[0021] In the fifth aspect of the present invention, the present invention proposes the use of the aforementioned animal Bifidobacterium lactis subspecies reBF24-1, the aforementioned bacterial suspension, the aforementioned microbial agent or the aforementioned pharmaceutical composition in the preparation of a drug, which has the purpose of maintaining gastrointestinal health.

[0022] Those skilled in the art will appreciate that the features and advantages described above for Bifidobacterium animalis subsp. lactis reBF24-1, bacterial suspension, microbial agent, and pharmaceutical composition are also applicable to this use and will not be described in detail herein.

[0023] In a sixth aspect of the present invention, the present invention provides an antioxidant product. According to an embodiment of the present invention, the antioxidant product comprises: at least one of the aforementioned Bifidobacterium animalis subsp. lactis reBF24-1, the aforementioned bacterial suspension, the aforementioned microbial agent, or the aforementioned pharmaceutical composition.

[0024] Those skilled in the art will appreciate that the features and advantages described above for Bifidobacterium animalis subsp. lactis reBF24-1, bacterial suspension, microbial agent, and pharmaceutical composition are also applicable to the antioxidant product and will not be described in detail herein.

[0025] In a seventh aspect of the present invention, a method for improving the antioxidant activity of a sample is provided. According to an embodiment of the present invention, the method comprises: contacting the sample with at least one of the aforementioned Bifidobacterium animalis subsp. lactis reBF24-1, the aforementioned bacterial suspension, the aforementioned microbial agent, the aforementioned pharmaceutical composition, or the aforementioned antioxidant product.

[0026] Those skilled in the art will appreciate that the features and advantages described above for Bifidobacterium animalis subsp. lactis reBF24-1, bacterial suspension, microbial agent, pharmaceutical composition, and antioxidant product are also applicable to this method and will not be described in detail herein.

[0027] In the eighth aspect of the present invention, the present invention proposes the use of the aforementioned animal Bifidobacterium lactis subsp. reBF24-1, the aforementioned bacterial suspension, the aforementioned microbial agent or the aforementioned antioxidant product in the preparation of a product for antioxidant use.

[0028] Those skilled in the art will appreciate that the features and advantages described above for animal Bifidobacterium lactis subsp. reBF24-1, bacterial suspension, microbial agent, and antioxidant preparation are also applicable to this application and will not be described in detail here.

[0029] 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0031] Figure 1 The basic composition, component prediction and annotation analysis results of the CGMCC No.31316 genome in Example 1 of the present invention;

[0032] Figure 2 This is a phylogenetic tree comparing the relationship between CGMCC No.31316 and Bifidobacteria from other animal sources in Example 1 of the present invention;

[0033] Figure 3 The clearance rates of CGMCC No.31316 and other animal Bifidobacterium lactis subspecies on DPPH in Example 2 of the present invention, wherein BB-12 was used as a positive control group, and CGMCC No.31316 and other reference strains were used as experimental groups, and a, b, c, and d indicated significant differences among different strains (P<0.05);

[0034] Figure 4 The scavenging rates of CGMCC No.31316 and other animal Bifidobacterium lactis subspecies on hydroxyl radicals in Example 3 of the present invention, wherein BB-12 was used as a positive control group, and CGMCC No.31316 and other reference strains were used as experimental groups, and a, b, c, and d represent significant differences among different strains (P<0.05);

[0035] Figure 5This is the evaluation of the simulated gastrointestinal fluid tolerance of CGMCC No.31316 in Example 4 of the present invention, wherein BB-12 is used as a positive control group;

[0036] Figure 6 This is an evaluation of the self-aggregation ability of CGMCC No. 31316 in Example 5 of the present invention, wherein BB-12 is used as a positive control group. DETAILED DESCRIPTION

[0037] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0038] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention but not excluding other contents.

[0039] 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.

[0040] In this article, the term "MRS medium" is a selective medium specifically used for culturing lactic acid bacteria. It is commonly used in the identification and research of lactic acid bacteria. It can support the growth of lactic acid bacteria and help inhibit the growth of other non-target microorganisms.

[0041] The present invention provides animal Bifidobacterium lactis subsp. reBF24-1, bacterial suspension, microbial agent and use thereof, pharmaceutical composition, antioxidant product and method for improving antioxidant activity of sample, which will be described in detail below.

[0042] Bacteria

[0043] The present invention proposes a Bifidobacterium animalis subsp. lactis reBF24-1. According to an embodiment of the present invention, the Bifidobacterium animalis subsp. lactis reBF24-1 was deposited in the General Microbiological Center of the China National Committee for the Preservation of Microorganisms on July 23, 2024, with a deposit number of CGMCC No. 31316. The strain is a brand-new isolate, which has been deposited in the "General Microbiological Center of the China National Committee for the Preservation of Microorganisms" on July 23, 2024, with a deposit number of CGMCC No. 31316, and a deposit address of: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101.

[0044] According to an embodiment of the present invention, the 16s rDNA of the Bifidobacterium animalis subsp. lactis reBF24-1 has a nucleotide sequence as shown in SEQ ID NO: 1 or having 99% homology thereto.

[0045] The animal Bifidobacterium lactis subspecies reBF24-1 provided by the present invention is screened from the excrement of the first defecation of healthy local newborns in Beijing within 24 to 48 hours after birth, has antioxidant ability and excellent gastrointestinal tolerance and intestinal colonization ability, can significantly alleviate intestinal oxidative stress, and is used for preparing drugs for maintaining gastrointestinal health.

[0046] Herein, the terms "Bifidobacterium animalis subsp. lactis reBF24-1" and "Bifidobacterium animalis subsp. lactis reBF24-1", "reBF24-1", "CGMCC No. 31316" are synonymous.

[0047] Compared with commercial animal Bifidobacterium BB-12 and other animal Bifidobacterium lactis strains screened from the same batch, the animal Bifidobacterium lactis subspecies reBF24-1 according to the embodiment of the present invention has significantly improved DPPH radical scavenging rate and significantly enhanced hydroxyl radical scavenging rate.

[0048] bacterial suspension

[0049] The present invention provides a bacterial suspension. According to an embodiment of the present invention, the bacterial suspension includes the aforementioned Bifidobacterium animalis subsp. lactis reBF24-1.

[0050] As used herein, the term "bacterial suspension" refers to a suspension containing cells of a microorganism (Bifidobacterium animalis subsp. lactis reBF24-1).

[0051] Those skilled in the art will appreciate that the features and advantages described above for Bifidobacterium animalis subsp. lactis reBF24-1 are also applicable to the bacterial suspension and will not be described in detail herein.

[0052] Microbial Agents

[0053] The present invention provides a microbial agent. According to an embodiment of the present invention, the microbial agent includes the aforementioned Bifidobacterium animalis subsp. lactis reBF24-1 or the aforementioned bacterial suspension.

[0054] Those skilled in the art will appreciate that the characteristics and advantages described above for Bifidobacterium animalis subsp. lactis reBF24-1 and bacterial suspension are also applicable to the microbial agent and will not be described in detail here.

[0055] In this article, the term "microbial agent" can refer to liquid microbial agents, including but not limited to fermentation broth, etc.; it can also refer to solid microbial agents, including but not limited to freeze-dried powder, etc.

[0056] In the microbial agent of the present invention, the Bifidobacterium animalis subspecies lactis reBF24-1 exists in the form of living cells and / or non-living cells.

[0057] As used herein, the term "living cell" refers to a cell that has the ability to metabolize, reproduce or replicate. Exemplarily, the living cell may be an immobilized cell. As used herein, "immobilized cell" refers to a living cell that is fixed on a carrier and can carry out life activities such as growth, development, reproduction, inheritance and metabolism within a certain spatial range.

[0058] In this article, the term "non-living cells" refers to cells that do not have the ability to metabolize, reproduce and replicate, including but not limited to dry bacteria. Exemplarily, the microbial agent is a freeze-dried powder.

[0059] In some specific embodiments, the Bifidobacterium animalis subsp. lactis reBF24-1 exists in the form of living cells, dried bacteria, immobilized cells or in any other form.

[0060] In some specific embodiments, the Bifidobacterium animalis subsp. lactis reBF24-1 is obtained by freeze-drying.

[0061] Pharmaceutical composition

[0062] The present invention provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition comprises at least one of the aforementioned animal Bifidobacterium lactis subsp. reBF24-1, the aforementioned bacterial suspension, or the aforementioned microbial agent. The pharmaceutical composition according to an embodiment of the present invention can be used to develop drugs for maintaining gastrointestinal health, preventing or treating gastrointestinal diseases.

[0063] According to an embodiment of the present invention, the pharmaceutical composition further comprises an excipient and / or a carrier.

[0064] According to an embodiment of the present invention, the excipient includes but is not limited to: at least one of a binder, a disintegrant, a lubricant, a glidant, a stabilizer, a filler, a diluent, and a sustained-release agent. Exemplarily, pharmaceutically specific acceptable excipients are well known in the art; examples of diluents may include but are not limited to: calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, corn starch, powdered sugar and / or a combination thereof.

[0065] 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.

[0066] According to an embodiment of the present invention, the dosage form of the pharmaceutical composition includes at least one selected from oral liquid, powder, granule, capsule, tablet, and pill. The pharmaceutical composition disclosed in the present invention can be prepared by methods well known in the pharmaceutical field.

[0067] Pharmaceutical use

[0068] The present invention proposes the use of the aforementioned animal Bifidobacterium lactis subspecies reBF24-1, the aforementioned bacterial suspension, the aforementioned microbial agent or the aforementioned pharmaceutical composition in the preparation of a drug, and the drug has the use of maintaining gastrointestinal health.

[0069] Those skilled in the art will appreciate that the features and advantages described above for Bifidobacterium animalis subsp. lactis reBF24-1, bacterial suspension, microbial agent, and pharmaceutical composition are also applicable to this use and will not be described in detail herein.

[0070] According to some optional embodiments of the present invention, the aforementioned maintenance of gastrointestinal health includes: repairing gastrointestinal mucosal damage, regulating gastrointestinal flora, alleviating oxidative stress, alleviating inflammation, inhibiting the formation of colorectal tumors, moisturizing the intestines and promoting bowel movements, and preventing and / or treating at least one of gastrointestinal diseases caused by pathogenic bacteria infection.

[0071] Antioxidant products

[0072] The present invention provides an antioxidant product. According to an embodiment of the present invention, the antioxidant product comprises: at least one of the aforementioned Bifidobacterium animalis subsp. lactis reBF24-1, the aforementioned bacterial suspension, the aforementioned microbial agent or the aforementioned pharmaceutical composition.

[0073] Those skilled in the art will appreciate that the features and advantages described above for Bifidobacterium animalis subsp. lactis reBF24-1, bacterial suspension, and microbial agent are also applicable to the antioxidant product and will not be described in detail herein.

[0074] method

[0075] The present invention provides a method for improving the antioxidant activity of a sample. According to an embodiment of the present invention, the method comprises: contacting the sample with at least one of the aforementioned Bifidobacterium animalis subsp. lactis reBF24-1, the aforementioned bacterial suspension, the aforementioned microbial agent, the aforementioned pharmaceutical composition, and the aforementioned antioxidant product.

[0076] Those skilled in the art will appreciate that the features and advantages described above for Bifidobacterium animalis subsp. lactis reBF24-1, bacterial suspension, microbial agent, pharmaceutical composition, and antioxidant product are also applicable to this method and will not be described in detail herein.

[0077] According to an embodiment of the present invention, the sample is a biological sample or a cell culture medium.

[0078] According to an embodiment of the present invention, the biological sample contains cells, including but not limited to: blood samples, tissue samples, bone marrow samples, saliva samples, urine samples, stool samples, cerebrospinal fluid samples, alveolar lavage fluid, cell culture, biopsy samples, amniotic fluid samples, semen samples, and cervical smears.

[0079] Product preparation and application

[0080] The present invention proposes the use of the aforementioned animal Bifidobacterium lactis subspecies reBF24-1, the aforementioned bacterial suspension, the aforementioned microbial agent or the aforementioned antioxidant product in the preparation of a product, and the product is used for antioxidant.

[0081] Those skilled in the art will appreciate that the features and advantages described above for animal Bifidobacterium lactis subsp. reBF24-1, bacterial suspension, microbial agent, and antioxidant preparation are also applicable to this application and will not be described in detail here.

[0082] According to an embodiment of the present invention, the product includes but is not limited to: medicine, food, health care product, feed, cosmetics, biological sample detection reagent, cell culture medium, biological sample detection reagent, cell culture medium.

[0083] According to an embodiment of the present invention, the biological sample contains cells, including but not limited to: blood samples, tissue samples, bone marrow samples, saliva samples, urine samples, stool samples, cerebrospinal fluid samples, alveolar lavage fluid, cell culture, biopsy samples, amniotic fluid samples, semen samples, and cervical smears.

[0084] 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.

[0085] Example 1: Isolation and identification of strains and genome characteristics of CGMCC No.31316

[0086] 1. Sample source

[0087] The strain used in the present invention is separated from meconium of newborns delivered by caesarean section in the obstetrics department of Peking University Third Hospital in Beijing.

[0088] 2. Isolation and Purification of Strains

[0089] Meconium was collected from newborns delivered by cesarean section in the Department of Obstetrics of Peking University Third Hospital in Beijing. The meconium samples were added to sterile phosphate buffered saline (PBS) buffer and mixed thoroughly. -6 , 10 -7 , 10 -8 Gradient dilution, 100uL of each gradient bacterial solution was spread on MRS medium plates containing nystatin and mupirocin, each dilution was repeated three times, and anaerobic culture was carried out at 37°C for 48h. After the culture was completed, a single colony plate with 50-150 colonies was selected from the agar medium, and then a single colony was picked from the plate and streaked on MRS medium containing nystatin and mupirocin for 48h to obtain purified strains, which were numbered as reBF24-1, BiBF01-2, BiBF07-5, BiBF09-6, BiBF10-4, BiBF12-4, Bi2BF19-4, reBF20-3, BiBF23-20, and reBF26-8.

[0090] 3. Identification of strains

[0091] The bacterial suspension cultured for 24 hours was used as a template for bacterial identification, and then colony polymerase chain reaction (PCR) was performed. Gene fragments were amplified using 16S rRNA universal primers. The products were identified by 1.0% agarose gel electrophoresis and sent to Shanghai Sangon Biotechnology Co., Ltd. for nucleic acid sequence determination. The base sequences in the determination results were entered into the NCBI database for BLAST sequence comparison. The results showed that the sequences of reBF24-1, BiBF01-2, BiBF07-5, BiBF09-6, BiBF10-4, BiBF12-4, Bi2BF19-4, reBF20-3, BiBF23-20, and reBF26-8 strains from meconium samples had a homology of more than 99% with the 16SrDNA sequence of animal Bifidobacterium lactis subsp.

[0092] According to the results of the determination of the ability to scavenge DPPH free radicals and the ability to scavenge hydroxyl free radicals, the sample named reBF24-1 was deposited for microbial preservation. The strain was deposited in the General Microbiological Center of China Microbiological Culture Collection Administration on July 23, 2024, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 31316 (strain "reBF24-1" is hereinafter referred to as "CGMCC No. 31316").

[0093] The 16s rDNA sequencing result of Bifidobacterium animalis subsp. lactis reBF24-1 is shown in SEQ ID NO:1.

[0094] CTTAGACGGCTCCCCCACAAGGGTCGGGCCACCGGCTTCGGGTGCTACCCACTTTCATGACTTGACGGGCGGTGTGTACAAGGCCCGGGAACGCATTCACCGCGGCGTTGCTGATCCGCGATTACTAGCGACTCCGCCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGAGACCGGTTTTCAGCGATCCGCCCCACGTCACCGTGTCGCACCGCGTTGTACCGGCCATTGTAGCATGCGTGAAGCCCTGGACGTAAGGGGCATGATGATCTGACGTCATCCCCACCTTCCTCCGAGTTGACCCCGGCGGTCCCACATGAGTTCCCGGCATCACCCGCTGGCAACATGCGGCGAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACGACCATGCACCACCTGTGAACCGGCCCCGAAGGGAAACCGTGTCTCCACGGCGATCCGGCACATGTCAAGCCCAGGTAAGGTTCTTCGCGTTGCATCGAATTAATCCGCATGCTCCGCCGCTTGTGCGGGCCCCCGTCAATTTCTTTGAGTTTTAGCCTTGCGGCCGTACTCCCCAGGCGGGATGCTTAACGCGTTGGCTCCGACACGGGACCCGTGGAAAGGGCCCCACATCCAGCATCCACCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTGACGGCCCAGAGACCTGCCTTCGCCATTGGTGTTCTTCCCGATATCTACACATTCCACCGTTACACCGGGAATTCCAGTCTCCCCTACCGCACTCCAGCCCGCCCGTACCCGGCGCAGATCCACCGTTAGGCGATGGACTTTCACACCGGACGCGACGAACCGCCTACGAGCCCTTTACGCCCAATAAATCCGGATAACGCTCGCACCCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGGTGCTTATTCGAACAATCCACTCAACACGGCCGAAACCGTGCCTTGCCCTTGAACAAAAGCGGTTTACAACCCGAAGGCCTCCATCCCGCACGCGGCGTCGCTGCATCAGGCTTGCGCCCATTGTGCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGGCCGTATCTCAGTCCCAAGTGGCCGGTCACCCTCT CAGGCCGGCTACCCGTCCAACGCCTTGGTGGCCATCACCCCGCCAACAAGCTGATAGGACGCGACCCCATCCATGCCGCAAAAGCATTTCCCACCCCACCATGCGATGGAGCGGAGCATCCGGTATTACCACCCGTTTCCAGGAGCTATTCCGGTGCACAGGGCAGGTTGGTCACGCATTACTCACCCGTTCGCCACTCTCACCCCGACAGCAAGCTGCCAGGGATCCCGTTCGACTTGCA(SEQ ID NO:1).

[0095] 4. CGMCC No.31316 Whole Genome Sequencing

[0096] The frozen CGMCC No.31316 was inoculated into MRS medium and cultured anaerobically at 37°C. The inoculation volume was passed to the next generation every 24h at 2%. 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 sequencing was carried out using the second-generation Illumina X Plus platform, and a 150bp DNA fragment was selected to construct a pair-end sequencing library. Each sample also provided Illumina sequencing data of no less than 500x of the genome to obtain a more complete and accurate bacterial completion map. The software used in this experiment for prediction, assembly, analysis, and other operations on the whole gene sequence is shown in Table 1.

[0097] Table 1 Software used for whole genome sequence analysis

[0098]

[0099] The CGMCC No.31316 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.

[0100] 5. Genome-wide component prediction and functional annotation

[0101] The whole genome circle map of CGMCC No.31316 is as follows Figure 1 shown.

[0102] The results showed that the whole genome size was about 1.92Mbp, with an average GC content of 60.49%. The number of CDS was 1552, and non-coding genes included 4 rRNAs, 53 tRNAs and 1 tmRNA. According to the annotation results of Uniprot, Pfam, GO, and KEGG databases, there were 15 genes related to antioxidant function on the chromosome of CGMCC No.31316, including genes encoding multiple reductases: alkyl hydroperoxide reductase (ahpC), ferredoxin-NADP reductase, thioredoxin reductase (trxB), methionine sulfoxide reductase (MsrAB), and other antioxidant genes metF, nfrA1, hpr, folA, gpr, nrdF2, oilS, nrdE2, proC, etc.

[0103] 6. Phylogenetic Analysis

[0104] Comparative genomics was used to compare and analyze the genomes of other animal Bifidobacterium lactis subspecies that have been published. By constructing a phylogenetic tree, it was found that CGMCC No.31316 was in the same branch as animal Bifidobacterium lactis subspecies from other sources, and CGMCC No.31316 was closest to strains of unknown origin and close to some fecal strains ( Figure 2 ).

[0105] Example 2: Determination of the ability to scavenge DPPH free radicals

[0106] 2,2-Diphenyl-1-trinitrohydrazine (DPPH) is a stable free radical and is widely used in the determination of biological antioxidant capacity.

[0107] CGMCC No.31316 was inoculated into MRS solid medium and cultured at 37°C for 48h. After the strain was activated for two generations, it was inoculated into MRS liquid medium at a volume fraction of 2% and incubated in a 37°C constant temperature incubator for 24h. The cells were collected by centrifugation at 4°C and 8000g for 10min. The centrifuged cells were washed three times with phosphate buffered saline (PBS) at pH 7.40 and resuspended in PBS to adjust the bacterial count to 10. 8 CFU / mL, and obtain the bacteria to be tested. Take 1mL of bacterial suspension and add 1mL of 1,1-diphenyl-2-trinitrophenylhydrazine (1,1-diphenyl-2-picrylhydrazil, DPPH) anhydrous ethanol solution (0.2mmol / L) and mix evenly. React at room temperature in the dark for 20min, centrifuge at 8000g for 10min, collect the supernatant, and measure its absorbance value at a wavelength of 517nm. The calculation formula is as follows:

[0108]

[0109] Where:

[0110] A0 is the absorbance value when the sample is replaced by an equal volume of anhydrous ethanol; A1 is the absorbance value of the experimental group; A2 is the absorbance value when the DPPH ethanol solution is replaced by an equal volume of anhydrous ethanol.

[0111] The results are as follows Figure 3 shown.

[0112] The results showed that the DPPH free radical scavenging rate of CGMCC No.31316 reached 42.59%, which was significantly better than the commercially available probiotic product Bifidobacterium animalis BB-12 (Chr. Hansen, Denmark) (22.69%, P<0.05) and the other 8 strains of Bifidobacterium animalis lactis (the DPPH scavenging rates ranged from 8.56% to 34.80%, all lower than CGMCC No.31316).

[0113] Example 3: Determination of the ability to scavenge hydroxyl radicals

[0114] Hydroxyl radical (·OH) is a cleavage product formed by O2 receiving two electrons and two protons to form hydrogen peroxide (H2O2) and then further reduced. Hydroxyl radicals can degrade DNA, damage cell membranes and polysaccharide compounds. It is the most important free radical that can cause lipid peroxidation and damage to various human cells. It is also one of the most active and aggressive free radicals. Therefore, the scavenging ability of hydroxyl radicals is an effective indicator for evaluating antioxidant activity.

[0115] CGMCC No.31316 was inoculated into MRS solid medium and cultured at 37°C for 48h. After the strain was activated for two generations, it was inoculated into MRS liquid medium at a volume fraction of 2% and incubated in a 37°C constant temperature incubator for 24h. The cells were collected by centrifugation at 4°C and 8000g for 10min. The centrifuged cells were washed three times with PBS buffer at pH 7.40 and resuspended in PBS to adjust the bacterial count to 10. 8 CFU / mL, and obtain the test bacteria. Mix 1mL of o-phenanthroline (2.5mmol / L), 1mL of PBS (pH7.4), and 0.5mL of the test strain evenly, then add 1mL of FeSO4 solution (2.5mmol / L) and 0.5mL of H2O2 (20mmol / L), and place in a 37℃ water bath for 1h, and measure its absorbance at a wavelength of 536nm. The hydroxyl radical scavenging rate is calculated as follows:

[0116]

[0117] Where:

[0118] A0 is the absorbance value when an equal volume of water replaces the sample; A1 is the absorbance value when an equal volume of water replaces the sample and H2O2 respectively; A2 is the absorbance value containing the sample and H2O2.

[0119] The results are as follows Figure 4 shown.

[0120] The results showed that the hydroxyl radical scavenging ability of different strains was significantly different. Compared with the positive control commercial strain BB-12, the hydroxyl radical scavenging rate of CGMCC No.31316 was significantly increased (P<0.05). Specifically, the hydroxyl radical scavenging rate of CGMCC No.31316 was higher than 40%, while the hydroxyl radical scavenging rate of the positive control commercial strain BB-12 was only 16.49%.

[0121] Example 4: Evaluation of tolerance to artificial simulated gastrointestinal fluid

[0122] 1. Evaluation of tolerance to artificial simulated gastric juice

[0123] Tolerating the human digestive tract environment is one of the important conditions for probiotics to colonize, grow and exert their effects in the intestines. Probiotics need to pass through the gastrointestinal environment and maintain a certain number of live bacteria to exert their specific probiotic properties.

[0124] After the strains with better vitality were cultured anaerobically at 37°C for 24 hours, they were centrifuged at 8000g for 10 minutes, washed twice with PBS, and the supernatant was discarded to collect the cells. The cell concentration was adjusted to 10 8CFU / mL bacterial suspension. Dissolve pepsin (1:10000) in sterilized saline (0.5% w / v), adjust the pH to 3.0, so that the final concentration is 3g / L, and sterilize with a 0.22μm microporous filter to prepare simulated gastric juice. Inoculate 1mL of the test bacterial suspension into 9mL of artificial gastric juice at pH3.0. After incubation at 37°C for 3h, take the bacterial solution and spread it on the plate in an appropriate dilution gradient, culture anaerobically at 37°C for 48h, determine the number of viable bacteria, and calculate the survival rate. The survival rate calculation formula is as follows:

[0125]

[0126] Where:

[0127] n1 is the number of viable bacteria after treatment (CFU / mL); n2 is the initial number of viable bacteria of the strain (CFU / mL).

[0128] The results are as follows Figure 5 shown.

[0129] The results showed that the survival rate of CGMCC No.31316 in a strong acid environment was over 90%, which was comparable to the positive control BB-12. This indicates that CGMCC No.31316 has a strong tolerance to artificial simulated gastric juice and can pass through the gastric juice smoothly and enter the small intestine in a live bacterial state.

[0130] 2. Evaluation of tolerance to artificial simulated intestinal fluid

[0131] The normal concentration of bile salts in the human intestine 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.

[0132] Dissolve 10 g of trypsin (1:250) in sterilized saline (0.5% w / v), adjust the pH to 8.0 with NaOH to a concentration of 1 g / L, add bile salts to a final concentration of 0.2%, and sterilize with a 0.22 μm microporous filter to prepare simulated intestinal fluid. 8 The test bacterial suspension of CFU / mL was inoculated into 9mL of simulated intestinal fluid at pH 8.0 and incubated at 37°C. After 2 hours, the bacterial suspension was plated at an appropriate dilution gradient and cultured anaerobically at 37°C for 48 hours. The number of viable bacteria was determined and the survival rate was calculated. The survival rate calculation formula is as follows:

[0133]

[0134] Where:

[0135] n1 is the number of viable bacteria (CFU / mL) after strain treatment; n2 is the initial number of viable bacteria (CFU / mL) of the strain.

[0136] The results are as follows Figure 5 shown.

[0137] The results showed that after digestion in simulated intestinal fluid (pH 8.0, bile salt concentration 0.2%) for 2 hours, the survival rate of CGMCC No.31316 was about 28.42%, which was lower than the positive control BB-12. This indicates that the bile salt tolerance of bifidobacteria, the dominant bacteria in the intestine of newborn infants, is weak and the survival rate is low. Nevertheless, this does not mean that the probiotics are not effective in practical applications, because the overall effect of probiotics is also affected by many other factors, including the colonization ability of the strain, the interaction with other intestinal flora, and individual differences in the host.

[0138] Example 5: Surface Adhesion Evaluation

[0139] Self-aggregation refers to the phenomenon that bacteria aggregate into clusters due to rapid reproduction in large numbers. The ability of self-aggregation is one of the factors that affect whether bacteria can successfully adhere to intestinal epithelial cells to form biofilm flora. In addition to being conducive to colonization in the intestine, strains with high self-aggregation ability are in an advantageous position when competing for cell matrix-host binding sites, and can effectively prevent pathogenic bacteria from binding to intestinal epithelial cells.

[0140] Take 4 mL of bacterial resuspension and measure its absorbance at 600 nm as the absorbance of the whole bacterial resuspension, then place it at 37 ° C. After 2h, 4h, 6h and 8h of treatment, take the supernatant and transfer it to another clean test tube, and measure its absorbance at 600 nm. Perform 3 independent experiments. The formula for calculating the autoaggregation ability is as follows:

[0141]

[0142] The results are shown in Table 2. Figure 6 shown.

[0143] Table 2 Determination of self-aggregation ability

[0144]

[0145] The results showed that the self-aggregation rate of CGMCC No.31316 and the positive control BB-12 at four time points increased with the extension of time. Compared with the positive control BB-12, the self-aggregation rate of CGMCC No.31316 was always higher than that of BB-12 in the time period from 2 hours to 8 hours; especially in 4 hours and 6 hours, the self-aggregation rate of CGMCC No.31316 was significantly higher than that of BB-12, which were 18.10% and 19.36% respectively, while the data of BB-12 at the same time points were 15.08% and 17.60%. This shows that CGMCC No.31316 has a stronger self-aggregation ability, which helps it to colonize and function more effectively in the intestinal environment.

[0146] 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.

[0147] 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 reBF24-1, characterized in that The animal Bifidobacterium lactis subspecies reBF24-1 was deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration on July 23, 2024, with the deposit number CGMCC No.31316.

2. The animal Bifidobacterium lactis subspecies reBF24-1 according to claim 1, characterized in that The 16s rDNA of the Bifidobacterium animalis subsp. lactis reBF24-1 has a nucleotide sequence as shown in SEQ ID NO: 1 or a nucleotide sequence having 99% homology thereto.

3. A bacterial suspension, characterized in that It comprises the animal Bifidobacterium lactis subspecies reBF24-1 as described in claim 1.

4. A microbial agent, characterized in that: include: The animal Bifidobacterium lactis subsp. reBF24-1 according to claim 1 or the bacterial suspension according to claim 3.

5. A pharmaceutical composition, characterized in that include: At least one of the animal Bifidobacterium lactis subsp. reBF24-1 according to claim 1, the bacterial suspension according to claim 3, or the microbial agent according to claim 4.

6. The pharmaceutical composition according to claim 5, characterized in that The pharmaceutical composition further comprises an excipient and / or a carrier.

7. Use of the animal Bifidobacterium lactis subsp. reBF24-1 according to claim 1, the bacterial suspension according to claim 3, the microbial agent according to claim 4, or the pharmaceutical composition according to claim 5 or 6 in the preparation of a drug, wherein the drug has the purpose of maintaining gastrointestinal health.

8. The use according to claim 7, characterized in that The maintenance of gastrointestinal health includes: repairing gastrointestinal mucosal damage, regulating gastrointestinal flora, alleviating oxidative stress, alleviating inflammation, inhibiting the formation of colorectal tumors, moisturizing the intestines and facilitating bowel movements, and preventing and / or treating at least one of gastrointestinal diseases caused by pathogenic bacteria infection.

9. An antioxidant product, characterized in that: include: At least one of the animal Bifidobacterium lactis subsp. reBF24-1 according to claim 1, the bacterial suspension according to claim 3, the microbial agent according to claim 4, or the pharmaceutical composition according to claim 5 or 6.

10. A method for improving the antioxidant activity of a sample, characterized in that: include: contacting the sample with at least one of the Bifidobacterium animalis subsp. lactis reBF24-1 according to claim 1, the bacterial suspension according to claim 3, the microbial agent according to claim 4, the pharmaceutical composition according to claim 5 or 6, or the antioxidant product according to claim 9; Optionally, the sample is a biological sample or a cell culture medium; Optionally, the biological sample comprises cells, including but not limited to: blood sample, tissue sample, bone marrow sample, saliva sample, urine sample, stool sample, cerebrospinal fluid sample, bronchoalveolar lavage fluid, cell culture, biopsy sample, amniotic fluid sample, semen sample, cervical smear.

11. Use of the animal Bifidobacterium lactis subsp. reBF24-1 according to claim 1, the bacterial suspension according to claim 3, the microbial agent according to claim 4 or the antioxidant product according to claim 9 in preparing a product, wherein the product is used for antioxidant; Optionally, the product includes but is not limited to: medicine, food, health care products, feed, cosmetics, reagents for biological sample detection, cell culture medium, reagents for biological sample detection, cell culture medium; Optionally, the biological sample comprises cells, including but not limited to: blood sample, tissue sample, bone marrow sample, saliva sample, urine sample, stool sample, cerebrospinal fluid sample, bronchoalveolar lavage fluid, cell culture, biopsy sample, amniotic fluid sample, semen sample, cervical smear.

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