A strain of Bifidobacterium breve BX-18 and its application

Abstract

The present invention provides a strain of Bifidobacterium breve BX-18 and its application, and belongs to the field of biotechnology. The deposit number of Bifidobacterium breve (Bifidobacterium breve) BX-18 provided by the present invention is CGMCCNo.28446. The Bifidobacterium breve BX-18 can synthesize active substances such as amino acids and metabolites thereof, benzene derivatives, flavonoids, and its metabolites have broad-spectrum antibacterial activity and have good antibacterial effect on foodborne pathogens or spoilage bacteria. The present invention provides a new idea for the in-depth study and utilization of probiotics with functional active substances, which has huge research and application value.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a strain of Bifidobacterium breve BX-18 and an application thereof. Background Art

[0002] As probiotic research deepens, a growing number of researchers are focusing on the potential of inactivated probiotics or their lysates to modulate immunity, alleviate allergies, regulate gastrointestinal function, provide antioxidant and anti-inflammatory benefits, lower blood sugar levels, prevent dental caries, fight tumors, and lower blood pressure. These products, for example, have shown significant benefits in treating irritable bowel syndrome, allergic diseases, and periodontitis. Active products synthesized from probiotics offer advantages such as safety, stability, and ease of storage and transportation, holding broad application prospects in the food and medical sectors.

[0003] Bifidobacterium is a commensal bacterial species in the human gut. Many strains isolated from the human gut have demonstrated health benefits, such as alleviating lactose intolerance, lowering serum cholesterol levels, improving gastrointestinal discomfort such as diarrhea and constipation, and even alleviating symptoms associated with diseases such as cancer. Therefore, the preparation, evaluation, and development of active metabolites of Bifidobacterium breve can more precisely target regulatory pathways, improve host health, and enhance the effectiveness of probiotics. However, there are currently no reports on the inhibitory effects of Bifidobacterium breve against foodborne pathogens or spoilage bacteria. Summary of the Invention

[0004] In view of the defects in the prior art, the object of the present invention is to provide a strain of Bifidobacterium breve BX-18 that has a good antibacterial effect on foodborne pathogens or spoilage bacteria.

[0005] The purpose of the present invention is achieved through the following technical solutions:

[0006] The present invention provides a strain of Bifidobacterium breve BX-18. The preservation number of the Bifidobacterium breve BX-18 is CGMCC No. 28446.

[0007] The present invention provides a microbial preparation comprising the Bifidobacterium breve BX-18 and / or a metabolically active product of the Bifidobacterium breve BX-18 described in the above technical solution.

[0008] Preferably, the metabolically active products include intracellular metabolically active products and / or extracellular metabolically active products;

[0009] The intracellular metabolic active products include any one or more of amino acids, amino acid metabolites, benzene derivatives, coenzymes, nucleotides, flavonoids and aldehydes, ketones and esters.

[0010] Preferably, the amino acids and / or amino acid metabolites include any one or more of L-aspartic acid, panthenol, N-palmitoylglycine, L-lysine, valacyclovir, tryptophanase and L-glutamine;

[0011] The benzene derivatives include any one or more of glucoside, topotecan hydrochloride, 3,8'-biflavone, retigabine, eprosartan, sulfoxaflor, troxipad, probucol, 2,4-di-tert-butylphenol, pterostilbene, tetrahydrocurcumin, bifonazole, ivacaftor, dopamine, pimozide, coniferin, baicalin and leonurine;

[0012] The coenzyme includes menadione and / or calcium D-pantothenate;

[0013] The nucleotides include flavin mononucleotide;

[0014] The flavonoids include any one or more of robinin, cyperiflavonoids, ginkgolide, dihydrodaidzein, naringenin, nateglinide and 5-deoxyarapine glycoside;

[0015] The aldehydes, ketones and esters include cefoxitin sodium and / or saponin D.

[0016] The present invention provides a method for preparing the microbial preparation described in the above technical solution, comprising:

[0017] Cultivating the Bifidobacterium breve BX-18 in a culture medium to obtain a fermentation broth containing the Bifidobacterium breve BX-18 and metabolically active products of the Bifidobacterium breve BX-18, thereby obtaining a microbial preparation;

[0018] After inactivating the fermentation broth, the sterile culture broth is taken out and dried to obtain a microbial preparation including extracellular metabolic active products of Bifidobacterium breve BX-18;

[0019] After the fermentation bacterial liquid is inactivated, the bacterial bodies are crushed and dried to obtain a microbial preparation containing intracellular metabolic active products of Bifidobacterium breve BX-18.

[0020] Preferably, the culture includes anaerobic culture; the culture temperature is 37-41° C.; the culture time is 12-16 hours; and the culture pH value is 5.4-7.0.

[0021] The present invention provides the use of the Bifidobacterium breve BX-18 described in the above technical solution, the microbial preparation described in the above technical solution, or the microbial preparation prepared by the preparation method described in the above technical solution in the preparation of edible products and / or medical and sanitary products.

[0022] Preferably, the medical and sanitary products include products for inhibiting pathogens and / or treating diseases caused by pathogens.

[0023] Preferably, the pathogens include any one or more of Listeria monocytogenes, Escherichia coli, Salmonella, Bacillus cereus, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus mutans, Actinobacillus actinomycetemcomitans, Fusobacterium nucleatum, Porphyromonas gingivalis, Trichophyton rubrum, Candida albicans and Pseudomonas nicotianae.

[0024] Preferably, the edible product and / or medical and sanitary product contains components: any one or more of amino acids, amino acid metabolites, benzene derivatives, coenzymes, nucleotides, flavonoids and aldehydes, ketones and esters.

[0025] Preferably, the amino acids and / or amino acid metabolites include any one or more of L-aspartic acid, panthenol, N-palmitoylglycine, L-lysine, valacyclovir, tryptophanase and L-glutamine;

[0026] The benzene derivatives include any one or more of glucoside, topotecan hydrochloride, 3,8'-biflavone, retigabine, eprosartan, sulfoxaflor, troxipad, probucol, 2,4-di-tert-butylphenol, pterostilbene, tetrahydrocurcumin, bifonazole, ivacaftor, dopamine, pimozide, coniferin, baicalin and leonurine;

[0027] The coenzyme includes menadione and / or calcium D-pantothenate;

[0028] The nucleotides include flavin mononucleotide;

[0029] The flavonoids include any one or more of robinin, cyperiflavonoids, ginkgolide, dihydrodaidzein, naringenin, nateglinide and 5-deoxyarapine glycoside;

[0030] The aldehydes, ketones and esters include cefoxitin sodium and / or saponin D.

[0031] Beneficial effects of the present invention

[0032] The present invention provides a strain of Bifidobacterium breve BX-18, and the deposit number of the Bifidobacterium breve BX-18 is CGMCC No.28446. The Bifidobacterium breve BX-18 can synthesize active substances such as amino acids and metabolites thereof, benzene derivatives, flavonoids, and its metabolites have broad-spectrum antibacterial activity and have good antibacterial effects on food-borne pathogens or spoilage bacteria. The present invention shows through the results of the examples that the metabolites of the Bifidobacterium breve BX-18 have good antibacterial effects on Listeria monocytogenes, Escherichia coli, Salmonella, Bacillus cereus, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus mutans, Actinobacillus actinomycetemcomitans, Fusobacterium nucleatum, Porphyromonas gingivalis, Trichophyton rubrum, Candida albicans and Pseudomonas nicotianae. The present invention provides a new idea for the in-depth study and utilization of probiotics with functional active substances, and has huge research and application value.

[0033] Biological deposit information

[0034] Bifidobacterium breve BX-18 was deposited on September 12, 2023 at the General Microbiology Center of China Culture Collection Administration, located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, Institute of Microbiology, Chinese Academy of Sciences, with the deposit number CGMCC No. 28446. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0036] Figure 1 This is the result of blast alignment of 16S rDNA of Bifidobacterium breve BX-18 with NCBI database;

[0037] Figure 2 This is a classification and weight distribution diagram of functional substances in the intracellular metabolic active products of Bifidobacterium breve BX-18;

[0038] Figure 3 This is a graph showing the inhibitory effect of different concentrations of the supernatant of the extracellular metabolic active products of Bifidobacterium breve BX-18 on Listeria monocytogenes, Escherichia coli, Salmonella, Bacillus cereus and Pseudomonas aeruginosa;

[0039] Figure 4This is a graph showing the inhibitory effect of different concentrations of supernatant of dry matter of extracellular metabolic active products of Bifidobacterium breve BX-18 on Staphylococcus aureus, Streptococcus mutans, Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis polymorpha subspecies of Fusobacterium nucleatum. DETAILED DESCRIPTION

[0040] The present invention provides a strain of Bifidobacterium breve BX-18. The preservation number of the Bifidobacterium breve BX-18 is CGMCC No. 28446.

[0041] In the present invention, the Bifidobacterium breve BX-18 is a strain isolated from the feces of a centenarian in Enshi, Hubei in 2018. The present invention classifies and identifies the strain BX-18 with the help of 16S rDNA, and ultimately determines that the strain BX-18 belongs to Bifidobacterium breve. In the present invention, the 16S rDNA gene sequence of the Bifidobacterium breve BX-18 is shown in SEQID NO.1. In the present invention, the Bifidobacterium breve BX-18 is a Gram-positive anaerobic bacterium; the Bifidobacterium breve BX-18 has no flagella, does not move, does not form spores, and the bacteria are V or Y-shaped; the colonies of the Bifidobacterium breve BX-18 are milky white or white, and have a soft texture. In the present invention, the Bifidobacterium breve BX-18 can be cultured on RCM medium; the optimal growth temperature of the Bifidobacterium breve BX-18 is 37-41° C., the growth pH value is 5.4-7.0, and the optimal pH value is 6.5-7.0.

[0042] The present invention provides a microbial preparation comprising the Bifidobacterium breve BX-18 and / or a metabolically active product of the Bifidobacterium breve BX-18 described in the above technical solution. In the present invention, the metabolically active product may include an intracellular metabolically active product and / or an extracellular metabolically active product.

[0043] In the present invention, when the microbial preparation includes the Bifidobacterium breve BX-18 and / or the metabolic activity products of the Bifidobacterium breve BX-18, the microbial preparation can be used as a functional microbial preparation; the functional microbial preparation can be a powder, tablet, capsule, oral liquid dosage form, granule or other, etc.; the functional microbial preparation may also include other pharmaceutically acceptable excipients.

[0044] In the present invention, the intracellular metabolic active products include amino acids, amino acid metabolites, benzene derivatives, coenzymes, nucleotides, flavonoids and aldehydes and ketones; amino acids and / or amino acid metabolites include L-aspartic acid, panthenol, N-palmitoylglycine, L-lysine, valacyclovir, tryptophanase and L-glutamine; the benzene derivatives include glucoside, topotecan hydrochloride, 3,8'-biflavonoid, retigabine, eprosartan, sulfoxaflor, troxifen, thiazolinone ... The present invention relates to a novel ...

[0045] In the present invention, the extracellular metabolic active product has a broad-spectrum antibacterial activity and has a good antibacterial effect on foodborne pathogens or spoilage bacteria; it has a good antibacterial effect on Listeria monocytogenes, Escherichia coli, Salmonella, Bacillus cereus, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus mutans, Actinobacillus actinomycetemcomitans, Fusobacterium nucleatum and Porphyromonas gingivalis.

[0046] The Bifidobacterium breve BX-18 strain provided by the present invention can synthesize active ingredients such as amino acids and their metabolites, benzene derivatives, flavonoids, and broad-spectrum antibacterial substances, demonstrating its potential for intestinal regulation. This strain has a clear origin and can be used in conventional foods, health foods, and biopharmaceuticals, facilitating industrial production and possessing broad application prospects.

[0047] The present invention provides a method for preparing the microbial preparation described in the above technical solution, comprising:

[0048] Cultivating the Bifidobacterium breve BX-18 in a culture medium to obtain a fermentation broth containing the Bifidobacterium breve BX-18, thereby obtaining a microbial preparation;

[0049] After obtaining the fermentation broth containing the Bifidobacterium breve BX-18, the present invention can inactivate the fermentation broth, take out the sterile culture solution and dry it to obtain a microbial preparation including the extracellular metabolic active products of Bifidobacterium breve BX-18.

[0050] After obtaining the fermentation broth containing the Bifidobacterium breve BX-18, the present invention can inactivate the fermentation broth, crush the bacteria, and dry them to obtain a microbial preparation containing intracellular metabolic active products of Bifidobacterium breve BX-18.

[0051] Before culturing the Bifidobacterium breve BX-18 in a culture medium, the Bifidobacterium breve BX-18 is preferably activated and cultured. The present invention can use RCM liquid culture medium to activate and culture the Bifidobacterium breve BX-18; the temperature of the activation culture can be 37 to 41°C. In a specific embodiment of the present invention, the temperature of the activation culture can be 37°C, 38°C, 39°C, 40°C or 41°C; the time of the activation culture can be 12 to 24 hours. In a specific embodiment of the present invention, the time of the activation culture can be 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours or 24 hours; the activation culture is performed anaerobic culture. After activation culture is completed, the present invention preferably inoculates the activated cultured bacterial solution into a seed culture medium for seed culture to obtain a Bifidobacterium breve BX-18 seed solution. The seed culture temperature may be 37-41°C. In specific embodiments of the present invention, the seed culture temperature may be 37°C, 38°C, 39°C, 40°C, or 41°C. The seed culture duration may be 12-16 hours. In specific embodiments of the present invention, the seed culture duration may be 12 hours, 13 hours, 14 hours, 15 hours, or 16 hours. The seed culture is performed anaerobically. After the seed culture is completed, a Bifidobacterium breve BX-18 seed solution is obtained. After obtaining the Bifidobacterium breve BX-18 seed solution, the present invention preferably performs morphological observation of the obtained Bifidobacterium breve BX-18 seed solution to ensure bacterial purity. After ensuring that a pure Bifidobacterium breve BX-18 seed solution is obtained, the present invention preferably expands the Bifidobacterium breve BX-18 seed solution in a culture medium to obtain a fermentation solution containing the Bifidobacterium breve BX-18. In the present invention, the expanded culture can be cultured using RCM liquid culture medium; the pH during the expanded culture can be 5.4-7.0, and in a specific embodiment of the present invention, the initial pH of the expanded culture can be 6.50±0.02; the pH during the expanded culture process can be (5.50-6.00)±0.02; the temperature of the expanded culture can be 37-41°C, and in a specific embodiment of the present invention, the temperature of the expanded culture can be 37°C, 38°C, 39°C, 40°C or 41°C; the time of the expanded culture can be 12-24h, and in a specific embodiment of the present invention, the time of the expanded culture can be 12h, 13h, 14h, 15h, 16h, 17h, 18h, 19h, 20h, 21h, 22h, 23h or 24h. In the present invention, the expanded culture stage uses sterile nitrogen to maintain a pressure of 0.02-0.03MPa, and the stirring speed is set to 100rpm. The expanded culture of the present invention is preferably cultured to the fermentation end point to obtain a fermentation bacterial liquid containing the Bifidobacterium breve BX-18.

[0052] After obtaining the fermentation liquid containing the Bifidobacterium breve BX-18, the fermentation liquid can be directly used as a microbial preparation, or the bacterial cells and the fermentation liquid of the fermentation liquid can be separated and the bacterial cells and the fermentation liquid can be used as microbial preparations respectively.

[0053] After obtaining a fermentation broth containing Bifidobacterium breve BX-18, the present invention inactivates the fermentation broth, removes the sterile culture medium, and dries it to obtain the extracellular metabolically active products of Bifidobacterium breve BX-18. The present invention does not specifically limit the inactivation method; conventional inactivation methods in the art may be employed. In a specific embodiment of the present invention, the inactivation method may include sterilizing and inactivating the fermentation broth by maintaining it at 90°C for 15 minutes. After obtaining the inactivated fermentation broth, the present invention may separate the fermentation broth from the bacteria by centrifugation, with the resulting supernatant being the fermentation broth or sterile culture medium. The present invention preferably dries the sterile culture medium to obtain the extracellular metabolically active products of Bifidobacterium breve BX-18. The present invention does not specifically limit the drying method; conventional drying methods in the art may be employed. In a specific embodiment of the present invention, the drying method may be spray drying; the inlet air temperature for the spray drying process may be 160-180°C, and the outlet air temperature may be 70-80°C; the drying process may be performed until the product has a moisture content of ≤5.0%.

[0054] After obtaining a fermentation broth containing Bifidobacterium breve BX-18, the present invention inactivates the fermentation broth, then disrupts and dries the cells to obtain intracellular metabolically active products of Bifidobacterium breve BX-18. The present invention does not specifically limit the inactivation method; conventional inactivation methods in the art may be employed. In a specific embodiment of the present invention, the inactivation method may include sterilizing and inactivating the fermentation broth by maintaining the fermentation broth at 90°C for 15 minutes. After obtaining the inactivated fermentation broth, the present invention may separate the fermentation broth from the cells by centrifugation. The present invention collects the cells to prepare intracellular metabolically active products of Bifidobacterium breve BX-18. After collecting the cells, the present invention may wash them. In a specific embodiment of the present invention, the washing may be performed using sterile purified water; the washing may be performed three times. After washing, the present invention preferably freeze-dries the wet cells obtained by washing to obtain a dried cell sample. The present invention does not specifically limit the vacuum freeze-drying method; conventional vacuum freeze-drying methods in the art may be employed.

[0055] After obtaining the dried bacterial cell sample, the present invention can disrupt the obtained dried bacterial cell sample. The present invention does not specifically limit the disruption method; conventional bacterial cell disruption methods in the art can be used. In a specific embodiment of the present invention, the disruption method preferably includes: homogenizing the dried bacterial cell sample with a methanol-water solution, and separating the resulting homogenate supernatant and a homogenate precipitate. In a specific embodiment of the present invention, the volume fraction of methanol in the methanol-water solution can be 70%; the methanol-water solution can be pre-cooled at -20°C; and the mass-to-volume ratio of the dried bacterial cell sample to the methanol-water solution can be (48-52) mg:500 μL. In a specific embodiment of the present invention, the homogenization can be repeated four times; the homogenization power can be 30 Hz; and each homogenization time can be 30 seconds. After homogenization, the present invention shakes the resulting homogenate mixture and then allows it to stand to obtain a homogenate system. In a specific embodiment of the present invention, the shaking time can be 5 minutes; the standing time can be performed on ice; and the standing time can be 15 minutes. After obtaining the homogenate system, the present invention preferably separates the homogenate system to obtain a homogenate supernatant and a homogenate precipitate. The present invention does not specifically limit the separation method; any conventional separation method in the art may be employed. In a specific embodiment of the present invention, the separation method may be centrifugation; the centrifugation temperature may be 4°C; the centrifugation speed may be 12,000 rpm; and the centrifugation time may be 10 minutes. After obtaining the homogenized precipitate, the present invention may mix the homogenized precipitate with an ethyl acetate-methanol solution, and then collect the homogenized precipitate supernatant. In a specific embodiment of the present invention, the volume ratio of ethyl acetate to methanol in the ethyl acetate-methanol solution may be 1:3. After obtaining the mixture of the homogenized precipitate and the ethyl acetate-methanol solution, the present invention may oscillate the resulting mixture and then allow it to stand to obtain a homogenized precipitate system. In a specific embodiment of the present invention, the oscillation time may be 5 minutes; the standing time may be performed on ice; and the standing time may be 15 minutes. After obtaining the homogenized precipitate system, the present invention separates the homogenized precipitate system to obtain a homogenized precipitate supernatant. In a specific embodiment of the present invention, the separation method for the homogenized precipitate system is the same as that for the homogenized system and is not further described here. After obtaining the homogenate precipitation supernatant, the present invention preferably mixes the homogenate supernatant and the homogenate precipitation supernatant to obtain a solution containing intracellular metabolic active products of Bifidobacterium breve BX-18; the solution containing intracellular metabolic active products of Bifidobacterium breve BX-18 is dried to obtain crude intracellular metabolic active products of Bifidobacterium breve BX-18. The present invention does not particularly limit the drying method, and conventional drying methods in the art can be used. In a specific embodiment of the present invention, the drying method can be concentrated drying.After obtaining the crude intracellular metabolically active products of Bifidobacterium breve BX-18, the present invention further comprises purifying the crude intracellular metabolically active products of Bifidobacterium breve BX-18. The purification method may include mixing the crude intracellular metabolically active products of Bifidobacterium breve BX-18 with a methanol-water solution and then sonicating the mixture. The volume fraction of methanol in the methanol-water solution may be 70%, the power of the sonication may be 180 W / L, and the sonication duration may be 3 minutes. After the sonication is completed, the present invention preferably centrifuges the sonicated product. The centrifugation temperature may be 4°C, the speed of the centrifugation may be 12,000 rpm, and the centrifugation duration may be 3 minutes. The supernatant obtained by centrifugation contains the intracellular metabolically active products of Bifidobacterium breve BX-18. The supernatant containing the intracellular metabolically active products of Bifidobacterium breve BX-18 can be directly used for metabolomics analysis. In the present invention, the obtained intracellular metabolically active products of Bifidobacterium breve BX-18 can be directly used as a microbial preparation.

[0056] The present invention provides the use of the Bifidobacterium breve BX-18 described in the above technical solution, the microbial preparation described in the above technical solution, or the microbial preparation prepared by the preparation method described in the above technical solution in the preparation of edible products and / or medical and sanitary products.

[0057] In the present invention, the edible products and / or medical and sanitary products can be used to prepare products for inhibiting pathogens and / or treating diseases caused by pathogens. In the present invention, the pathogens can include any one or two or more of Listeria monocytogenes, Escherichia coli, Salmonella, Bacillus cereus, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus mutans, Actinobacillus actinomycetemcomitans, Fusobacterium nucleatum, Porphyromonas gingivalis, Trichophyton rubrum, Candida albicans, and Pseudomonas nicotinicum. In the present invention, the edible products and / or medical and sanitary products include any one or two or more of amino acids, amino acid metabolites, benzene derivatives, coenzymes, nucleotides, flavonoids, and aldehydes, ketones, and esters. In the present invention, the amino acid and / or amino acid metabolite may include any one or more of L-aspartic acid, panthenol, N-palmitoylglycine, L-lysine, valacyclovir, tryptophanase and L-glutamine; the benzene derivative may include glucoside, topotecan hydrochloride, 3,8'-biflavonoid, retigabine, eprosartan, sulfoxaflor, troxipad, probucol, 2,4-di-tert-butylphenol, pterostilbene, tetrahydrocurcumin, biphenyl Any one or more of benzylazole, ivacaftor, dopamine, pimozide, coniferin, baicalin and leonurine; the coenzyme may include menadione and / or D-calcium pantothenate; the nucleotide includes flavin mononucleotide; the flavonoids may include any one or more of robinin, cypermethrin, ginkgolide, dihydrodaidzein, naringenin, nateglinide and 5-deoxyarapine; the aldehydes, ketones and esters may include cefoxitin sodium and / or saponin D.

[0058] In the present invention, any one or more of the Bifidobacterium breve BX-18, the intracellular metabolic active products of the Bifidobacterium breve BX-18, and the extracellular metabolic active products of the Bifidobacterium breve BX-18 can be added to the food base of the edible product and / or medical and sanitary product. In the present invention, any one or more of the Bifidobacterium breve BX-18, the intracellular metabolic active products of the Bifidobacterium breve BX-18, and the extracellular metabolic active products of the Bifidobacterium breve BX-18 can be added in the form of a dry powder or other forms, for example, including in the form of a liquid preparation.

[0059] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0060] In the following examples, unless otherwise specified, the culture of Bifidobacterium breve BX-18 was performed anaerobicly.

[0061] Example 1

[0062] Origin, Isolation and Identification of Bifidobacterium breve BX-18

[0063] Bifidobacterium breve BX-18 was isolated from the feces of a centenarian in Enshi, Hubei Province in 2018. The specific isolation method is:

[0064] The fecal samples collected from the elderly were smeared on site to isolate the strains. The samples were first diluted in multiples and 10 -5 and 10 -6 Evenly spread 200 μL of the dilution gradient sample onto a plate of RCM solid culture medium. Place the sample in an anaerobic jar along with an anaerobic gas-producing bag and bring it back to the laboratory for anaerobically incubation at 37°C for 72–96 hours. Select individual colonies of varying morphology, size, and color and inoculate them into liquid culture medium. Incubate in a 37°C incubator for 24–36 hours. Once the strain has grown well, perform Gram staining and microscopic examination to obtain strain BX-18, which is then preserved.

[0065] Strain BX-18 is a Gram-positive bacterium observed under a microscope. It has no flagella, does not move, does not form spores, and has a V or Y-shaped body. The colony diameter on RCM solid culture medium is 2 to 3 mm, and the colonies are milky white or white with a soft texture.

[0066] The DNA sample of the isolated strain BX-18 was extracted and the sample was identified by 16S rDNA molecular identification by Shanghai Meiji Biopharmaceutical Technology Co., Ltd. The molecular sequence was compared with the NCBI database Blast. Figure 1 As shown, the strain BX-18 was confirmed to be Bifidobacterium breve, and its 16S rDNA sequence was shown as SEQ ID No.1.

[0067]

[0068] Example 2

[0069] Preparation and analysis of intracellular metabolic active products of Bifidobacterium breve BX-18

[0070] 1 Preparation method

[0071] 1.1 Activation of Strain: Inoculate the test strain, frozen and stored at -80°C, into RCM liquid medium (adjusted to pH 6.5) and perform activation culture at 37°C under anaerobic conditions. After activation, use a pipette to inoculate the test strain at a 2% inoculum into seed medium (fresh RCM liquid medium, adjusted to pH 6.5). Continue anaerobic culture at 37°C for 12-16 hours. To ensure the purity of the test strain, observe the microbial morphology during passage.

[0072] 1.2 Expanded Culture: The above seed solution was inoculated at a rate of 5% (V / V) in RCM medium for expanded culture. After inoculation, ammonia water or sodium hydroxide was added to adjust the initial pH of the fermentation liquid to 6.50±0.02. Sterile nitrogen was maintained at a pressure of 0.02-0.03 MPa. The stirring speed was set at 100 rpm and fermentation was started. The culture temperature was 37±0.2°C. During the culture process, if the pH value of the culture system was lower than 5.5, ammonia water was added through the automatic alkali addition system to maintain a constant pH of (5.50-6.00)±0.02.

[0073] 1.3 Inactivation of bacteria: ferment to the end point (ie, the fermentation process will monitor OD 600 Value, if OD within half an hour 600 The fermentation broth was sterilized at 90℃ for 15min.

[0074] 1.4 Centrifugal washing: The inactivated fermentation broth was placed in a centrifuge for centrifugal separation (rotation speed 14000 rpm) and washed three times with sterile purified water.

[0075] 1.5 Freeze-drying: The wet cells obtained after three washes were vacuum freeze-dried.

[0076] 1.6 Active Ingredient Assay: Weigh 50 ± 2 mg of the freeze-dried sample into a 2 mL centrifuge tube. Add 500 μL of a 70% methanol-water solution pre-cooled at -20°C. Homogenize the weighed sample with the methanol-water solution four times, 30 seconds each time, at 30 Hz. After homogenization, shake for 5 minutes and let stand on ice for 15 minutes. Centrifuge at 4°C, 12,000 rpm, for 10 minutes. Pipette 400 μL of the supernatant into another centrifuge tube. Add 500 μL of an ethyl acetate / methanol solution (with a volume ratio of ethyl acetate to methanol of 1:3 (v / v)) to the pellet in the original centrifuge tube. Shake for 5 minutes and let stand on ice for 15 minutes. Centrifuge at 4°C, 12,000 rpm, for 10 minutes. Remove 400 μL of the supernatant. Combine the two supernatants and concentrate to dryness. After concentration and drying, 100 μL of 70% methanol aqueous solution was added to the dried product, and ultrasonication was performed at 180 W / L for 3 min. The product was centrifuged at 4°C and 12,000 rpm for 3 min, and 60 μL of the supernatant was aspirated into the corresponding inner liner tube for metabolome detection and analysis.

[0077] The intracellular active products of Bifidobacterium breve BX-18 were detected by targeted metabolomics. The metabolomics detection and analysis results are shown in Table 1 and Figure 2 The potential functions of the intracellular active products of Bifidobacterium breve BX-18 are shown in Table 2.

[0078] Table 1 Detection results of intracellular active products of Bifidobacterium breve BX-18

[0079]

[0080]

[0081]

[0082]

[0083] Table 2 Potential functions of intracellular active products of Bifidobacterium breve BX-18

[0084]

[0085]

[0086] From Table 1, Table 2 and Figure 1 It can be analyzed that the intracellular active products of Bifidobacterium breve BX-18 have been identified through metabolomics detection technology, and a total of 37 functional metabolites (relative content> 0.01 mg / g) have been identified. Among them, the functional metabolites with higher content are mainly concentrated in active substances such as amino acids and their metabolites, benzene and its derivatives, and flavonoids, with a specific proportion of Figure 2 shown.

[0087] Among them, more and more studies on amino acids and their metabolites have confirmed that certain amino acids in the body are not only substrates for protein synthesis, but they can also regulate many life activities in the animal body through the biological activities of themselves and their metabolites, such as regulating nutrient metabolism, providing energy for the body, maintaining the homeostasis of the body's internal environment, synthesizing nitric oxide, polyamines, glutathione, nucleic acid hormones and neurotransmitters to affect nerves and endocrine, regulate cell gene expression and signal transduction, immunity, antioxidant, anti-stress and other functions, and these regulatory effects ultimately affect the growth and development, production performance and health of animals. The amino acids and their metabolites with relatively high content in the intracellular active products of Bifidobacterium breve BX-18 of the present application include L-aspartic acid, which has an immune-enhancing effect, panthenol, which has anti-inflammatory and antioxidant effects, and L-lysine, which has an antibacterial effect on Escherichia coli, Staphylococcus aureus, Trichophyton rubrum and Candida albicans.

[0088] Benzene series, or aromatic organic compounds, are a general term for benzene and its derivatives. The present application describes a novel nonionic surfactant with comprehensive performance, including glucosides, a potent intracellular active product of Bifidobacterium breve BX-18, which exhibits strong broad-spectrum antimicrobial activity. Topotecan hydrochloride exhibits anticancer activity, while 3,8'-biflavone plays a significant role in softening blood vessels, lowering blood lipids, and preventing cardiovascular and cerebrovascular diseases.

[0089] Due to their unique chemical structure, flavonoids have many important physiological and biochemical effects on mammals and other cell types and are active ingredients in many Chinese herbal medicines. It has been reported that moderate intake of flavonoids can reduce the incidence of diseases such as cancer, tumors, cardiovascular disease, lipid peroxidation, and osteoporosis. The intracellular active product of Bifidobacterium breve BX-18 in this application contains a relatively high content of robinin, which has anti-inflammatory effects, cyperitone, which has protective effects on the liver and kidneys, and ginkgolide, which has anti-inflammatory, anti-cancer, anxiolytic, anti-atherosclerotic, and neuroprotective and hepatoprotective effects.

[0090] In summary, the intracellular active products of Bifidobacterium breve BX-18 have regulatory and improvement effects on the body and have potential application value as health foods, functional foods and pharmaceutical preparations.

[0091] Example 3

[0092] Preparation and testing of extracellular active products of Bifidobacterium breve BX-18

[0093] 1.1 Spray Drying: The sterile culture solution separated after cell extraction of Bifidobacterium breve BX-18 in Example 2 (step 1.4) was spray dried (inlet air temperature 160-180°C, exhaust air temperature 70-80°C) to prepare an extracellular active product, maintaining the product moisture content below 5.0%, to obtain a spray-dried powder of the extracellular metabolite of Bifidobacterium breve BX-18.

[0094] 1.2 Antibacterial activity test: The spray-dried powder of the extracellular metabolites of Bifidobacterium breve BX-18 was reconstituted with sterile deionized water to prepare extracellular metabolite solutions with dry matter contents of 0%, 10%, 20%, 30%, and 40%, respectively.

[0095] Pathogenic bacteria were selected from Listeria monocytogenes (CICC21633), Escherichia coli (CICC24106), Salmonella enterica (CICC21483), Bacillus cereus (CICC10468), Pseudomonas aeruginosa (CICC21636), Staphylococcus aureus (CICC21600), Streptococcus mutans (CICC10438), Fusobacterium nucleatum (CICC10468), and Streptococcus mutans (CICC10438). 25586 TM ), Porphyromonas gingivalis ( 33277 TM ), Actinobacillus actinomycetemcomitans ( 29522 TM ). Among them, Fusobacterium nucleatum, Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans were purchased from Shanghai Baolu Biotechnology Co., Ltd.; the remaining pathogenic bacteria were purchased from the China Industrial Microbiology Culture Collection Center (CICC). The above-mentioned pathogenic bacteria were activated and subcultured, and the second-generation culture fluid was taken and counted, and the number of viable bacteria in the second-generation culture fluid of the pathogenic bacteria was counted. Among them, Listeria monocytogenes, Escherichia coli, Salmonella, Bacillus cereus, Pseudomonas aeruginosa, and Staphylococcus aureus were activated and subcultured using NB nutrient broth medium. Streptococcus mutans was activated and subcultured using BHI brain heart infusion broth medium. Actinobacillus actinomycetemcomitans, Fusobacterium nucleatum, and Porphyromonas gingivalis were activated and subcultured using TSB tryptone soy broth medium.

[0096] Take the second generation pathogenic bacteria and the corresponding culture medium, and dilute them in a 2.0mL EP tube until the concentration is 10 6CFU / mL is reserved. Use a disposable syringe to extract the metabolite solution of Bifidobacterium breve BX-18 with different dry matter contents, filter through a 0.22μm filter membrane, and mix 150μL of the gradient diluted pathogenic bacteria liquid and the BX-18 extracellular metabolite solution with different dry matter contents. Then, inject into a 100-well honeycomb culture plate, with three replicates for each sample.

[0097] The honeycomb culture plate was placed in an automatic growth curve instrument. Anaerobic bacteria were sealed with liquid paraffin on the honeycomb culture plate. The culture time was set to 24 hours and the culture temperature was 37°C for cultivation. The BX-18 extracellular metabolite solution with a dry matter content of 0% was used as the control group and its specific growth rate was used as the baseline. The BX-18 metabolite solution with a dry matter content of 10%, 20%, 30%, and 40% was used as the experimental group. The specific growth rate was plotted and the OD was measured. 600 , measure three parallels each time, record the readings, and calculate the specific growth rate, μ, using the following formula:

[0098]

[0099] Where x is the culture time, y is the bacterial density corresponding to the culture time, y0 is the initial bacterial density, a is the maximum bacterial density, and Wmax is the maximum specific growth rate.

[0100] The effects of extracellular metabolites of Bifidobacterium breve BX-18 on the specific growth rates of Listeria monocytogenes, Escherichia coli, Salmonella, Bacillus cereus, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus mutans, Actinobacillus actinomycetemcomitans, Fusobacterium nucleatum, and Porphyromonas gingivalis are shown in Tables 3 and Figures 3-4 As shown. Figure 3 The inhibitory effects of dried supernatants of Bifidobacterium breve BX-18 at different concentrations on Listeria monocytogenes, Escherichia coli, Salmonella, Bacillus cereus and Pseudomonas aeruginosa were shown. Figure 4 The inhibitory effect of dried supernatant of Bifidobacterium breve BX-18 at different concentrations on Staphylococcus aureus, Streptococcus mutans, Actinobacillus actinomycetemcomitans, Fusobacterium nucleatum polymorpha subsp. and Porphyromonas gingivalis. Figure 3 and Figure 4 The dry matter content is the concentration of the dried supernatant of different concentrations of Bifidobacterium breve BX-18.

[0101] Table 3 Inhibitory effects of extracellular metabolites of Bifidobacterium breve BX-18 on different pathogens

[0102] Dry matter concentration 0％ 10％ 20％ 30％ 40％ Listeria monocytogenes <![CDATA[0.124±0.005 a ]]> <![CDATA[0.114±0.007 b ]]> <![CDATA[0.070±0.003 c ]]> <![CDATA[0.070±0.004 c ]]> <![CDATA[0.059±0.005 d ]]> salmonella <![CDATA[0.126±0.004 a ]]> <![CDATA[0.125±0.002 a ]]> <![CDATA[0.118±0.003 b ]]> <![CDATA[0.071±0.006 c ]]> <![CDATA[0.054±0.004 d ]]> Escherichia coli <![CDATA[0.161±0.004 a ]]> <![CDATA[0.082±0.006 b ]]> <![CDATA[0.013±0.008 e ]]> <![CDATA[0.065±0.004 c ]]> <![CDATA[0.037±0.003 d ]]> Bacillus cereus <![CDATA[0.152±0.004 a ]]> <![CDATA[0.105±0.004 b ]]> <![CDATA[0.106±0.005 b ]]> <![CDATA[0.043±0.005 c ]]> <![CDATA[0.035±0.004 d ]]> Pseudomonas aeruginosa <![CDATA[0.141±0.005 a ]]> <![CDATA[0.128±0.004 b ]]> <![CDATA[0.033±0.004 c ]]> <![CDATA[0.032±0.005 c ]]> <![CDATA[0.011±0.004 e ]]> Staphylococcus aureus <![CDATA[0.132±0.007 a ]]> <![CDATA[0.093±0.003 b ]]> <![CDATA[0.042±0.005 d ]]> <![CDATA[0.065±0.004 c ]]> <![CDATA[0.041±0.005 d ]]> Streptococcus mutans <![CDATA[0.118±0.005 a ]]> <![CDATA[0.034±0.005 b ]]> <![CDATA[0.035±0.002 b ]]> <![CDATA[0.011±0.004 c ]]> <![CDATA[0.015±0.005 d ]]> Actinobacillus actinomycetemcomitans <![CDATA[0.131±0.006 a ]]> <![CDATA[0.019±0.003 b ]]> <![CDATA[0.009±0.001 c ]]> <![CDATA[0.002±0.000 d ]]> <![CDATA[0.002±0.000 d ]]> Fusobacterium nucleatum <![CDATA[0.133±0.005 a ]]> <![CDATA[0.038±0.005 b ]]> <![CDATA[0.033±0.005 c ]]> <![CDATA[0.009±0.003 d ]]> <![CDATA[0.031±0.004 c ]]> Porphyromonas gingivalis <![CDATA[0.098±0.001 a ]]> <![CDATA[0.097±0.004 a ]]> <![CDATA[0.051±0.004 b ]]> <![CDATA[0.037±0.004 c ]]> <![CDATA[0.022±0.003 d ]]>

[0103] From Table 3, Figures 3-4It was found that the extracellular metabolites of Bifidobacterium breve BX-18 had a significant antibacterial effect on Listeria monocytogenes, Escherichia coli, Salmonella, Bacillus cereus, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus mutans, Actinobacillus actinomycetemcomitans, Fusobacterium nucleatum, and Porphyromonas gingivalis.

[0104] Initial concentration of Listeria monocytogenes was 1.80×10 6 CFU / mL, the specific growth rate of the experimental groups with BX-18 metabolite solution dry matter content of 10%, 20%, 30%, and 40% was lower than that of the control group with dry matter content of 0%, and the minimum inhibitory concentration of BX-18 metabolite solution against Listeria monocytogenes was 10%.

[0105] Initial concentration of E. coli: 2.00×10 6 CFU / mL, the specific growth rate of the experimental groups with BX-18 supernatant dry matter content of 10%, 20%, 30%, and 40% was lower than that of the control group with dry matter content of 0%, and the minimum inhibitory concentration of BX-18 metabolite solution against Escherichia coli was 10%.

[0106] Initial concentration of Salmonella was 1.44×10 6 CFU / mL, the specific growth rate of the experimental groups with BX-18 supernatant dry matter content of 10%, 20%, 30%, and 40% was lower than that of the control group with dry matter content of 0%, and the minimum inhibitory concentration of BX-18 metabolite solution against Salmonella was 20%.

[0107] The initial concentration of Bacillus cereus was 7.00×10 6 CFU / mL, the specific growth rate of the experimental groups with BX-18 supernatant dry matter content of 10%, 20%, 30%, and 40% was lower than that of the control group with dry matter content of 0%, and the minimum inhibitory concentration of BX-18 metabolite solution against Bacillus cereus was 10%.

[0108] The initial concentration of Pseudomonas aeruginosa was 1.35×10 6 CFU / mL, the specific growth rate of the experimental groups with BX-18 supernatant dry matter content of 10%, 20%, 30%, and 40% was lower than that of the control group with dry matter content of 0%, and the minimum inhibitory concentration of BX-18 metabolite solution against Pseudomonas aeruginosa was 10%.

[0109] Initial concentration of Staphylococcus aureus: 5.60×10 6 CFU / mL, the specific growth rate of the experimental groups with BX-18 supernatant dry matter content of 10%, 20%, 30%, and 40% was lower than that of the control group with dry matter content of 0%, and the minimum inhibitory concentration of BX-18 metabolite solution against Staphylococcus aureus was 10%.

[0110] Initial concentration of Streptococcus mutans was 1.83×10 6 CFU / mL, the specific growth rate of the experimental groups with BX-18 supernatant dry matter content of 10%, 20%, 30%, and 40% was lower than that of the control group with dry matter content of 0%, and the minimum inhibitory concentration of BX-18 metabolite solution against Streptococcus mutans was 10%.

[0111] The initial concentration of Actinobacillus actinomycetemcomitans was 8.3×10 7 CFU / mL, the specific growth rate of the experimental groups with BX-18 supernatant dry matter content of 10%, 20%, 30%, and 40% was lower than that of the control group with dry matter content of 0%, and the minimum inhibitory concentration of BX-18 metabolite solution against Actinobacillus actinomycetemcomitans was 10%.

[0112] The initial concentration of Fusobacterium nucleatum subsp. polymorphum was 5.3×10 8 CFU / mL, the specific growth rate of the experimental groups with BX-18 supernatant dry matter content of 10%, 20%, 30%, and 40% was lower than that of the control group with dry matter content of 0%, and the minimum inhibitory concentration of BX-18 metabolite solution against Fusobacterium nucleatum polymorpha subspecies was 10%.

[0113] The initial concentration of Porphyromonas gingivalis was 3.1×10 8 CFU / mL, the specific growth rate of the experimental groups with BX-18 supernatant dry matter content of 10%, 20%, 30%, and 40% was lower than that of the control group with dry matter content of 0%. It can be concluded that the minimum inhibitory concentration of BX-18 metabolite solution against Porphyromonas gingivalis is 20%.

[0114] According to the antibacterial test results of this example, it can be seen that the extracellular active products of Bifidobacterium breve BX-18 have an inhibitory effect on foodborne pathogens and spoilage bacteria, and have application value as a biological antibacterial agent.

[0115] In summary, Bifidobacterium breve BX-18 can synthesize functional active substances such as amino acids and their derivatives, benzene and its derivatives, and flavonoids within its cells. The products synthesized extracellularly have broad-spectrum antibacterial activity, effectively inhibiting pathogenic bacteria and food-borne spoilage bacteria. This invention provides a new approach for the in-depth research and utilization of probiotics containing functional active substances, and has enormous research and application value.

[0116] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A strain of Bifidobacterium breve ( Bifidobacterium breve ) Use of BX-18 or a microbial preparation thereof in the preparation of edible products and / or medical and sanitary products, wherein the medical and sanitary products are products for inhibiting pathogens and / or treating diseases caused by pathogens; the pathogens are any one or two or more of Listeria monocytogenes, Streptococcus mutans, Actinobacillus actinomycetemcomitans, Fusobacterium nucleatum, Porphyromonas gingivalis, Trichophyton rubrum, Candida albicans, and Pseudomonas nicotianae; the deposit number of Bifidobacterium breve BX-18 is CGMCC No. 28446.

2. The application according to claim 1, characterized in that The microbial preparation includes the Bifidobacterium breve BX-18 and / or the extracellular metabolic active product of the Bifidobacterium breve BX-18; the extracellular metabolic active product is prepared by inactivating the fermentation bacteria liquid of Bifidobacterium breve BX-18, taking the sterile culture liquid and drying it.

3. The use according to claim 1 or 2, characterized in that: The preparation method of the microbial preparation comprises: Cultivating the Bifidobacterium breve BX-18 in a culture medium to obtain a microbial preparation containing the cells of the Bifidobacterium breve BX-18; The fermentation liquid obtained by culture is inactivated, and then the sterile culture liquid is taken out and dried to obtain a microbial preparation including the extracellular metabolic active products of Bifidobacterium breve BX-18.

4. The application according to claim 3, characterized in that The culture includes anaerobic culture; the culture temperature is 37-41° C.; the culture time is 12-16 hours; and the pH value of the culture is 5.4-7.0.

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