Bifidobacterium breve BX-18 and application thereof

Abstract

The invention provides bifidobacterium breve BX-18 and application thereof, and belongs to the technical field of biology. The preservation number of the Bifidobacterium breve BX-18 provided by the invention is CGMCC (China General Microbiological Culture Collection Center) No.28446. The Bifidobacterium breve BX-18 provided by the invention has the advantages that the preservation number is CGMCC No.28446; the bifidobacterium breve BX-18 can be used for synthesizing amino acids and metabolites thereof, benzene derivatives, flavonoids and other active substances, and the metabolites of the bifidobacterium breve BX-18 have broad-spectrum antibacterial activity and have a good antibacterial effect on food-borne pathogenic bacteria or putrefying bacteria. The invention provides a brand new thought for deep research and utilization of probiotics with functional active substances, and has huge research and application values.

Description

Technical Field

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

[0002] With the in-depth advancement of probiotic research, more and more scholars are focusing on the research of inactivated probiotics or their lysates in regulating immunity, allergy relief, gastrointestinal function regulation, anti-oxidation, anti-inflammation, as well as hypoglycemia, dental caries prevention, anti-tumor, and blood pressure reduction. For example, they have a significant improvement effect on irritable bowel syndrome, allergic diseases, and periodontitis. The active products synthesized by probiotics have the advantages of safety, stability, and easy storage and transportation, and have broad application prospects in the fields of food and medicine and health.

[0003] Bifidobacterium is part of the symbiotic bacteria in the human intestine. Many isolates from the human intestine have shown health benefits, such as relieving 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, through the preparation, evaluation and development of active metabolites of Bifidobacterium breve, it is possible to act more accurately on regulatory targets, improve host health in a targeted manner, and enhance the utilization effect of probiotics. However, there are currently no reports on the inhibitory effect of Bifidobacterium breve on 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 which has a good antibacterial effect on foodborne pathogens or spoilage bacteria.

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

[0006] The invention provides a strain of Bifidobacterium breve BX-18, and 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, cypermethrin, ginkgolide, dihydrodaidzein, naringenin, nateglinide and 5-deoxyarabinoside;

[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 liquid containing the Bifidobacterium breve BX-18 and metabolically active products of the Bifidobacterium breve BX-18, thereby obtaining a microbial preparation;

[0018] After the fermentation bacterial liquid is inactivated, the sterile culture liquid is taken out and dried to obtain a microbial preparation including an extracellular metabolic active product 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 including 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, cypermethrin, ginkgolide, dihydrodaidzein, naringenin, nateglinide and 5-deoxyarabinoside;

[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 pathogenic bacteria or spoilage bacteria. The present invention shows through the results of embodiments 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 great 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 drawings required for use in the embodiments will be briefly introduced below. 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 creative labor.

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

[0037] Figure 2 This is the classification and density 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 dry matter 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 Fusobacterium nucleatum polymorpha subspecies Porphyromonas gingivalis. DETAILED DESCRIPTION

[0040] The invention provides a strain of Bifidobacterium breve BX-18, and 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 finally 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 bacterial body is V or Y-shaped; the colony of the Bifidobacterium breve BX-18 is milky white or white, and has 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 the metabolic activity products of the Bifidobacterium breve BX-18 described in the above technical solution. In the present invention, the metabolic activity products may include intracellular metabolic activity products and / or extracellular metabolic activity products.

[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 others; the functional microbial preparation can 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, ketones and esters; 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'-biflavonoids, retigabine, eprosartan, sulfoxaflor, troxifen The invention relates to a novel novel novel novel pharmaceutical composition comprising the following: pait, probucol, 2,4-di-tert-butylphenol, pterostilbene, tetrahydrocurcumin, bifonazole, ivacaftor, dopamine, pimozide, coniferin, baicalin and leonurine; the coenzyme comprises menadione and calcium D-pantothenate; the nucleotide comprises flavin mononucleotide; the flavonoid comprises robinin, cypermethrin, ginkgolide, dihydrodaidzein, naringenin, nateglinide and 5-deoxyleucoderma glycoside; the aldehydes, ketones and esters comprise cefoxitin sodium and saponin D.

[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 provided by the present invention can synthesize active ingredients such as amino acids and their metabolites, benzene derivatives, flavonoids, broad-spectrum antibacterial substances, and has potential value in intestinal regulation. The strain has a clear source, can be applied to common food, health food and biopharmaceuticals, is easy to industrialize, and has 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 liquid 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 and dry the bacteria, and obtain a microbial preparation including 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, and 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 24h, and in a specific embodiment of the present invention, the time of the activation culture can be 12h, 13h, 14h, 15h, 16h, 17h, 18h, 19h, 20h, 21h, 22h, 23h or 24h; the activation culture is anaerobic. After the activation culture is completed, the present invention preferably inoculates the activated cultured bacterial liquid into the seed culture medium for seed culture to obtain Bifidobacterium breve BX-18 seed liquid; the temperature of the seed culture can be 37-41°C, and in a specific embodiment of the present invention, the temperature of the seed culture can be 37°C, 38°C, 39°C, 40°C or 41°C; the time of the seed culture can be 12-16h, and in a specific embodiment of the present invention, the time of the seed culture can be 12h, 13h, 14h, 15h or 16h; the seed culture is anaerobic. After the seed culture is completed, the Bifidobacterium breve BX-18 seed liquid is obtained. After obtaining the Bifidobacterium breve BX-18 seed liquid, in order to ensure the purity of the bacteria, the present invention preferably performs morphological observation on the obtained Bifidobacterium breve BX-18 seed liquid. After ensuring that the pure Bifidobacterium breve BX-18 seed liquid is obtained, the present invention preferably expands the Bifidobacterium breve BX-18 seed liquid in the culture medium to obtain a fermented bacterial liquid containing the Bifidobacterium breve BX-18. In the present invention, the expanded culture can be cultured in 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 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 end point of fermentation to obtain a fermentation 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 the fermentation liquid containing the short bifidobacterium BX-18, the present invention inactivates the fermentation liquid, takes the sterile culture liquid and dries it to obtain the extracellular metabolic active products of short bifidobacterium BX-18. The present invention does not specifically limit the inactivation method, and any conventional inactivation method in the art can be used. In a specific embodiment of the present invention, the inactivation method can be to sterilize and inactivate the fermentation liquid by keeping it at 90°C for 15 minutes. After obtaining the inactivated fermentation liquid, the present invention can separate the fermentation liquid from the bacteria by centrifugation, and the obtained supernatant is the fermentation liquid or the sterile culture liquid. The present invention preferably dries the sterile culture liquid to obtain the extracellular metabolic active products of short bifidobacterium BX-18. The present invention does not specifically limit the drying method, and any conventional drying method in the art can be used. In a specific embodiment of the present invention, the drying method can be spray drying; the inlet air temperature of the spray drying can be 160-180°C, and the exhaust air temperature can be 70-80°C; the drying can be dried to a moisture content of the product of ≤5.0%.

[0054] After obtaining the fermentation liquid containing the Bifidobacterium breve BX-18, the present invention inactivates the fermentation liquid and then crushes and dries the bacterial cells to obtain the intracellular metabolic active products of Bifidobacterium breve BX-18. The present invention does not specifically limit the inactivation method, and any conventional inactivation method in the art can be used. In a specific embodiment of the present invention, the inactivation method can be to sterilize and inactivate the fermentation liquid by keeping it at 90°C for 15 minutes. After obtaining the inactivated fermentation liquid, the present invention can separate the fermentation liquid from the bacterial cells by centrifugation. The present invention collects the bacterial cells and prepares the intracellular metabolic active products of Bifidobacterium breve BX-18. After collecting the bacterial cells, the present invention can wash the bacterial cells. In a specific embodiment of the present invention, the washing can be carried out with sterile purified water; the number of washings can be 3 times; after washing is completed, the present invention preferably performs vacuum freeze drying on the wet bacterial cells obtained by washing to obtain a dry bacterial sample. The present invention does not specifically limit the vacuum freeze drying method, and any conventional vacuum freeze drying method in the art can be used.

[0055] After obtaining the dried bacterial sample, the present invention can crush the obtained dried bacterial sample. The present invention does not specifically limit the crushing method, and any conventional bacterial crushing method in the art can be used. In a specific embodiment of the present invention, the crushing method preferably includes: mixing the dried bacterial sample with a methanol aqueous solution to obtain a homogenate, and separating to obtain a homogenate supernatant and a homogenate precipitate. In a specific embodiment of the present invention, the volume fraction of methanol in the methanol aqueous solution can be 70%; the methanol aqueous solution can be a methanol aqueous solution precooled at -20°C; the mass volume ratio of the dried bacterial sample to the methanol aqueous solution can be (48-52) mg:500 μL. In a specific embodiment of the present invention, the number of homogenizations can be 4 times; the power of the homogenization can be 30 Hz; and the time of each homogenization can be 30 s. After the homogenization is completed, the present invention oscillates the obtained homogenate mixture and then allows it to stand to obtain a homogenate system. In a specific embodiment of the present invention, the oscillation time can be 5 min; the standing can be performed on ice; and the standing time can be 15 min. 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, and any conventional separation method in the art can be adopted. In a specific embodiment of the present invention, the separation method can be centrifugation; the centrifugation temperature can be 4°C; the centrifugation speed can be 12000rpm; and the centrifugation time can be 10min. After obtaining the homogenate precipitate, the present invention can mix the homogenate precipitate with the ethyl acetate methanol solution, and then take the homogenate precipitate supernatant. In a specific embodiment of the present invention, the volume ratio of ethyl acetate and methanol in the ethyl acetate methanol solution can be 1:3; after obtaining the mixture of the homogenate precipitate and the ethyl acetate methanol solution, the present invention can oscillate the obtained mixture and then let it stand to obtain a homogenate precipitate system. In a specific embodiment of the present invention, the oscillation time can be 5min; the standing can be performed on ice; and the standing time can be 15min. After obtaining the homogenate precipitate system, the present invention separates the homogenate precipitate system to obtain a homogenate precipitate supernatant. In a specific embodiment of the present invention, the separation method of the homogenate precipitate system is the same as the separation method of the homogenate system, which will not be repeated 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 specifically limit the drying method, and any conventional drying method in the art can be used. In a specific embodiment of the present invention, the drying method can be concentrated drying.After obtaining the intracellular metabolic active products of crudely extracted Bifidobacterium breve BX-18, the present invention further comprises purifying the intracellular metabolic active products of crudely extracted Bifidobacterium breve BX-18; the purification method may include mixing the intracellular metabolic active products of crudely extracted Bifidobacterium breve BX-18 with methanol aqueous solution and then ultrasonicating; the volume fraction of methanol in the methanol aqueous solution may be 70%; the power of the ultrasonication may be 180W / L; and the ultrasonication time may be 3min. After the ultrasonication is completed, the present invention preferably centrifuges the ultrasonic product; the centrifugation temperature may be 4°C; the centrifugation speed may be 12000rpm; and the centrifugation time may be 3min. The supernatant obtained by centrifugation contains the intracellular metabolic active products of Bifidobacterium breve BX-18. The obtained supernatant containing the intracellular metabolic active products of Bifidobacterium breve BX-18 can be directly used for metabolomics analysis. In the present invention, the obtained intracellular metabolic 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 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. In the present invention, the edible products and / or medical and sanitary products include any one 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-deoxyselenoside; 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 in the edible products and / or medical and sanitary products. 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 dry powder or in other forms, for example, including 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 in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope 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] Source, Isolation and Identification of Bifidobacterium breve BX-18

[0063] Bifidobacterium breve BX-18 was isolated from the feces of centenarians in Enshi, Hubei 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 -6 200 μL of the dilution gradient sample was evenly spread on the RCM solid culture medium plate, and after evenly spreading, it was placed in an anaerobic tank together with the anaerobic gas production bag and brought back to the laboratory for anaerobically cultured at 37°C for 72 to 96 hours in an anaerobic workstation (80% nitrogen, 10% hydrogen, 10% carbon dioxide). Single colonies with different morphology, size, and color were picked and inoculated into liquid culture medium and cultured in a 37°C constant temperature incubator for 24 to 36 hours. After the strain grew well, Gram staining and microscopic examination were performed to obtain strain BX-18, and the isolated strain was preserved.

[0065] The 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 shape. The colony diameter on RCM solid culture medium is 2 to 3 mm, and the colonies are milky white or white and have a soft texture.

[0066] The DNA sample of strain BX-18 was extracted and the sample was identified by 16S rDNA molecule by Shanghai Meiji Biopharmaceutical Technology Co., Ltd. The molecular sequence was compared with the NCBI database Blast. Figure 1 As shown, strain BX-18 was confirmed to be Bifidobacterium breve, and its 16S rDNA sequence was shown in 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 strains: The test strains stored frozen at -80°C were inoculated into RCM liquid medium (the pH value of the medium was adjusted to 6.5), and activated under 37°C constant temperature anaerobic conditions. After activation, the test bacteria were inoculated into the seed medium (fresh RCM liquid medium, pH value adjusted to 6.5) with a pipette at a 2% inoculation rate, and then cultured continuously at 37°C constant temperature anaerobic conditions for 12 to 16 hours. To ensure the purity of the test bacteria, the microbial morphology during the passage was observed.

[0072] 1.2 Expanded culture: The above seed solution is inoculated in RCM medium at a rate of 5% (V / V) for expanded culture. After the inoculation, add ammonia water or sodium hydroxide to adjust the initial pH of the fermentation liquid to 6.50±0.02, maintain the pressure of sterile nitrogen at 0.02-0.03MPa, set the stirring speed to 100rpm, and start fermentation. The culture temperature is 37±0.2℃. During the culture process, when the pH value in the culture system is lower than 5.5, add ammonia water 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 (i.e. the fermentation process will monitor OD 600 Value, if OD within half an hour 600 The value had no significant change, reaching the fermentation end point, and 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 detection: Weigh 50±2mg of the freeze-dried sample and add it to a 2mL centrifuge tube, and add 500μL of 70% methanol aqueous solution precooled at -20℃. After adding the weighed sample to the methanol aqueous solution, homogenize it 4 times, 30s each time, 30Hz. After homogenization, oscillate for 5min and let it stand on ice for 15min. Centrifuge at 4℃, 12000r / min for 10min, and draw 400μL of supernatant into another centrifuge tube. Add 500μL of ethyl acetate / methanol solution to the original centrifuge tube precipitate, in which the volume ratio of ethyl acetate and methanol is (V / V) 1:3, oscillate for 5min, and let it stand on ice for 15min. Centrifuge at 4℃, 12000rmp for 10min, and take 400μL of 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 r / min 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 results of metabolomics detection and analysis 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 some amino acids in the body are not only used as substrate raw materials 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, anti-oxidation, anti-stress and other functions, and these regulatory effects ultimately affect the growth and development, production performance and health of animals. The relatively high content of amino acids and their metabolites 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, that is, aromatic organic compounds, are the general term for benzene and its derivatives. The glucoside in the intracellular active product of Bifidobacterium breve BX-18 of the present application has strong broad-spectrum antibacterial activity, a new type of non-ionic surfactant with comprehensive performance, topotecan hydrochloride has anticancer activity, and 3,8'-biflavonoids have a significant effect in softening blood vessels, lowering blood lipids and preventing the formation of cardiovascular and cerebrovascular diseases.

[0089] Flavonoids have many important physiological and biochemical effects on mammals and other types of cells due to their unique chemical structure, and are the active ingredients of many Chinese herbal medicines. It is reported that the moderate intake of flavonoids can reduce the incidence of diseases such as cancer, tumors, cardiovascular diseases, lipid peroxidation and osteoporosis. The relatively high content of robinin in the intracellular active products of the present application Bifidobacterium breve BX-18 has an anti-inflammatory effect, cyperflavonoids have a protective effect on the liver and kidneys, and ginkgolides have anti-inflammatory, anti-cancer, antianxiety drug-like, anti-atherosclerotic and anti-atherosclerotic, neuroprotective and hepatoprotective effects.

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

[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 the bacterial 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, and the moisture content of the product was kept below 5.0% to obtain a spray-dried powder of the extracellular metabolites 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 China Industrial Microbiological Culture Collection Center (CICC). The above pathogenic bacteria were activated and subcultured, and the second-generation culture fluid was taken and counted to count the number of viable bacteria in the second-generation culture fluid of the pathogenic bacteria. 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 for later use. Use a disposable syringe to extract the metabolite solution of Bifidobacterium breve BX-18 with different dry matter contents, filter it with a 0.22μm filter membrane, mix 150μL of the gradient diluted pathogenic bacteria liquid and the BX-18 extracellular metabolite solution with different dry matter contents, and then inject it 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. The 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 determined. 600 , measure three parallels each time, record the readings, and calculate the specific growth rate, i.e., μ, using the following formula:

[0098]

[0099] In the formula, 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 the 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; Figure 4 The inhibitory effects of dried supernatants 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 product of the supernatant of different concentrations of Bifidobacterium breve BX-18.

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

[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 can be found that the extracellular metabolites of Bifidobacterium breve BX-18 have significant antibacterial effects 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 having a dry matter content of 10%, 20%, 30%, and 40% was lower than that of the control group with a dry matter content of 0%. It can be obtained that the minimum inhibitory concentration of BX-18 metabolite solution against Listeria monocytogenes is 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: 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] Initial concentration of Bacillus cereus: 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%. It can be obtained that the minimum inhibitory concentration of BX-18 metabolite solution against Bacillus cereus is 10%.

[0108] 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%. It can be obtained that the minimum inhibitory concentration of BX-18 metabolite solution against Pseudomonas aeruginosa is 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%. It can be obtained that the minimum inhibitory concentration of BX-18 metabolite solution against Staphylococcus aureus is 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] 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%. It can be obtained that the minimum inhibitory concentration of BX-18 metabolite solution against Actinobacillus actinomycetemcomitans is 10%.

[0112] 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%. It can be obtained that the minimum inhibitory concentration of BX-18 metabolite solution against Fusobacterium nucleatum polymorphum subspecies is 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 obtained 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, 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 in the cell, and the extracellular synthetic products of the cell have broad-spectrum antibacterial activity, which can effectively inhibit pathogenic bacteria and food-borne spoilage bacteria. The present invention provides a new idea for the in-depth research and utilization of probiotics with functional active substances, and has great research and application value.

[0116] Although the above embodiment describes the present invention in detail, 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 protection scope of the present invention.

Claims

1. A strain of Bifidobacterium breve BX-18, characterized in that: The deposit number of the Bifidobacterium breve BX-18 is CGMCC No.28446.

2. A microbial preparation, characterized in that: The invention comprises the Bifidobacterium breve BX-18 according to claim 1 and / or a metabolically active product of the Bifidobacterium breve BX-18.

3. The microbial preparation according to claim 2, characterized in that: The metabolically active products include intracellular metabolically active products and / or extracellular metabolically active products; 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.

4. The microbial preparation according to claim 3, characterized in that: 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; 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; The coenzyme includes menadione and / or calcium D-pantothenate; The nucleotides include flavin mononucleotide; The flavonoids include any one or more of robinin, cypermethrin, ginkgolide, dihydrodaidzein, naringenin, nateglinide and 5-deoxyarabinoside; The aldehydes, ketones and esters include cefoxitin sodium and / or saponin D.

5. The method for preparing the microbial preparation according to any one of claims 2 to 4, characterized in that: include: Cultivating the Bifidobacterium breve BX-18 in a culture medium to obtain a fermentation liquid containing the Bifidobacterium breve BX-18 and metabolically active products of the Bifidobacterium breve BX-18, thereby obtaining a microbial preparation; After the fermentation bacterial liquid is inactivated, the sterile culture liquid is taken out and dried to obtain a microbial preparation including an extracellular metabolic active product of Bifidobacterium breve BX-18; After the fermentation bacterial liquid is inactivated, the bacterial bodies are crushed and dried to obtain a microbial preparation including intracellular metabolic active products of Bifidobacterium breve BX-18.

6. The preparation method according to claim 5, characterized in that: 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.

7. Use of the Bifidobacterium breve BX-18 according to claim 1, the microbial preparation according to claims 2 to 4, or the microbial preparation prepared by the preparation method according to claim 5 or 6 in the preparation of edible products and / or medical and sanitary products.

8. The use according to claim 7, characterized in that: The medical and sanitary products include products for inhibiting pathogens and / or treating diseases caused by pathogens; 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.

9. The use according to claim 7, characterized in that: 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.

10. The use according to claim 9, characterized in that: 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; 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; The coenzyme includes menadione and / or calcium D-pantothenate; The nucleotides include flavin mononucleotide; The flavonoids include any one or more of robinin, cypermethrin, ginkgolide, dihydrodaidzein, naringenin, nateglinide and 5-deoxyarabinoside; The aldehydes, ketones and esters include cefoxitin sodium and / or saponin D.

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