Bifidobacterium adolescentis strain capable of inhibiting clinical multi-drug-resistant bacteria and having anti-inflammatory activity and application of bifidobacterium adolescentis strain

Abstract

The invention provides a Bifidobacterium adolescentis strain capable of inhibiting clinical multi-drug-resistant bacteria and having anti-inflammatory activity, and an application of the Bifidobacterium adolescentis strain, and belongs to the technical field of functional microorganisms. According to the invention, a strain B24728 resistant to multiple drug-resistant bacteria is separated from feces of Guangxi 100-year old people, and the strain B24728 is comprehensively judged through morphological detection and molecular identification results and belongs to bifidobacterium adolescentis. The bacterial strain B24728 also has a good anti-inflammatory effect. Meanwhile, the strain B24728 is a human-derived strain, has no species rejection reaction, has the potential of colonizing a human body, has no hemolysis, and meets the safety requirement, so that the strain B24728 and the metabolite thereof can be used as food or medicines.

Description

Technical Field

[0001] The present invention belongs to the technical field of functional microorganisms, and specifically relates to a Bifidobacterium adolescentis strain having the activity of inhibiting clinical multidrug-resistant bacteria and anti-inflammatory activity and an application thereof. Background Art

[0002] Clinical multidrug-resistant bacteria are a class of pathogens that are resistant to multiple antibiotics. Their existence poses a huge challenge to global public health. The most common drug-resistant pathogens include methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococci (VRE), carbapenem-resistant Enterobacteriaceae (CRE), multidrug-resistant Pseudomonas aeruginosa, and multidrug-resistant Acinetobacter baumannii. In the face of these drug-resistant pathogens, the commonly used clinical response strategy is to increase the antibiotic dose or use more powerful and newer antibacterial drugs. However, this approach often leads to further enhancement of the drug resistance of pathogens. In this context, it becomes particularly important to find new treatment strategies and drugs.

[0003] In recent years, some new therapies that replace antibiotics have emerged continuously, mainly including probiotics, oligosaccharides, antimicrobial peptides, enzyme preparations, Chinese herbal medicine preparations and acidifiers, among which probiotics have received extensive attention due to their unique advantages. Probiotics are a class of microorganisms that are beneficial to human health and play an important role in maintaining human health. The microorganism currently reported for inhibiting clinical multidrug-resistant bacteria is a lactic acid bacteria strain, for example including plant lactobacillus GS083, plant lactobacillus GS086, plant lactobacillus GS090 and fermented lactobacillus GF110 (CN111534452A). There is no report on the activity of Bifidobacterium adolescentis to inhibit clinical multidrug-resistant bacteria. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a use of Bifidobacterium adolescentis strain B24728 in the preparation of a product for inhibiting clinical drug-resistant bacteria, which has strong inhibitory activity against various types of clinical drug-resistant bacteria.

[0005] The present invention provides an application of a Bifidobacterium adolescentis strain B24728 in preparing a product for inhibiting clinical drug-resistant bacteria. The preservation number of the Bifidobacterium adolescentis strain B24728 is GDMCC No: 65489.

[0006] Preferably, the Bifidobacterium adolescentis strain B24728 also exists in the form of a metabolite of the Bifidobacterium adolescentis strain B24728 and / or a culture of the Bifidobacterium adolescentis strain B24728.

[0007] Preferably, the method for preparing the metabolites of Bifidobacterium adolescentis strain B24728 comprises the following steps:

[0008] The Bifidobacterium adolescentis strain B24728 was inoculated into MPYG liquid medium for anaerobically culturing to obtain a culture;

[0009] The liquid phase of the culture is separated to obtain metabolites of Bifidobacterium adolescentis strain B24728.

[0010] Preferably, the Bifidobacterium adolescentis strain B24728 is also present in the form of a probiotic.

[0011] Preferably, the probiotic comprises at least one of the following dosage forms: aqueous solution, powder and granule.

[0012] Preferably, the clinical drug-resistant bacteria include at least one of the following: Clostridium difficile, Acinetobacter baumannii, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa.

[0013] Preferably, the product includes at least one of the following: medicine, disinfected daily necessities, food, food additives, feed or feed additives.

[0014] The present invention provides the use of the Bifidobacterium adolescentis strain B24728, the culture of the Bifidobacterium adolescentis strain B24728 or the probiotic agent containing the Bifidobacterium adolescentis strain B24728 in the above technical solution in the preparation of anti-inflammatory drugs.

[0015] Preferably, the anti-inflammatory drug comprises at least one of the following dosage forms: tablets, powders, granules, capsules and oral solutions.

[0016] The present invention provides an application of a Bifidobacterium adolescentis strain B24728 in the preparation of a product for inhibiting clinical drug-resistant bacteria, and the deposit number of the Bifidobacterium adolescentis strain B24728 is GDMCC No: 65489. The strain B24728 is isolated from the intestinal flora of centenarians in Guangxi, is a human-derived strain, has no species rejection reaction, has the potential to colonize the human body, and according to the results of its hemolytic test, the bacteria meet the safety requirements, so it can be used as food or medicine. The strain B24728 has a good inhibitory effect on a variety of clinical drug-resistant bacteria, can inhibit the growth of drug-resistant pathogenic strains Escherichia coli, drug-resistant Staphylococcus aureus MRSA, Pseudomonas aeruginosa PAO1, Acinetobacter baumannii HRAB-85, and Clostridium difficile, and can provide a new means for clinical treatment of multi-drug resistant bacterial infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a colony morphology diagram of Bifidobacterium adolescentis strain B24728;

[0018] Figure 2 This is a Gram staining microscopic examination of Bifidobacterium adolescentis strain B24728;

[0019] Figure 3 This is the gel electrophoresis diagram of the 16S rRNA gene of Bifidobacterium adolescentis strain B24728;

[0020] Figure 4 This is the phylogenetic tree of the 16S rRNA gene of Bifidobacterium adolescentis strain B24728;

[0021] Figure 5 This is the antibacterial effect diagram of the antibacterial experiment of Bifidobacterium adolescentis strain B24728;

[0022] Figure 6 This is a graph showing the results of a hemolytic activity test of Bifidobacterium adolescentis strain B24728;

[0023] Figure 7 These are the results of the antibiotic sensitivity test for Bifidobacterium adolescentis strain B24728.

[0024] Biomaterial Deposit Information

[0025] Bifidobacterium adolescentis strain B24728 was deposited on November 15, 2024 in the Guangdong Provincial Microbiological Culture Collection Center, abbreviated as GDMCC, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Institute of Microbiology, with the deposit number GDMCC No: 65489. DETAILED DESCRIPTION

[0026] The invention provides a Bifidobacterium adolescentis strain B24728, with a preservation number of GDMCC No: 65489.

[0027] In the present invention, the strain B24728 is isolated from the intestinal flora of centenarians in Guangxi. The colony morphology of strain B24728 is round with slight protrusions, the colony is milky white, and its edges are smooth and neat. The Gram staining of strain B24728 is blue-purple, and the strain shape is rod-shaped, which is judged to be a Gram-positive bacterium. After 16S rRNA gene detection, the sequencing result of the 16S rRNA gene of strain B24728 is SEQ ID NO: 1, and the 16S rRNA gene sequence comparison result shows that it is similar to Bifidobacterium adolescentis ATCC 15703. T The sequence homology of the strain B24728 was 98.70%. Based on morphological and molecular identification, the strain B24728 belonged to Bifidobacterium adolescentis.

[0028] The present invention provides a metabolite or culture of the Bifidobacterium adolescentis strain B24728.

[0029] The present invention provides a method for preparing the metabolite or culture, comprising the following steps:

[0030] The activated Bifidobacterium adolescentis strain B24728 is inoculated into MPYG liquid medium for anaerobically culturing to obtain a culture;

[0031] The liquid phase of the culture is separated to obtain metabolites of Bifidobacterium adolescentis strain B24728.

[0032] In the present invention, the temperature of the anaerobic culture can be 36-38°C, or 37°C. The time of the anaerobic culture is 20-26h, or 22-24h. The inoculation amount of the inoculation is preferably 4%-6%, or 5%. The culture contains the bacterial cells of Bifidobacterium adolescentis strain B24728 and the metabolites of Bifidobacterium adolescentis strain B24728. The method of separating the liquid phase is preferably centrifugation and filtration. The speed of the centrifugation is preferably 8000-12000rpm, or 10000rpm. The time of the centrifugation is preferably 4-6min, or 5min. The filtration method is preferably membrane filtration. The pore size of the filter membrane is preferably 0.22μm. After collecting the liquid phase, it is preferably further concentrated. The concentration method preferably includes freeze drying. The multiple of the concentration is preferably 1:5-10, or 1:7-8.

[0033] The present invention provides a probiotic preparation, comprising the Bifidobacterium adolescentis strain B24728 or the metabolite or culture.

[0034] In the present invention, the probiotic preferably includes at least one of the following dosage forms: aqueous solution, powder and granule. The effective live bacteria concentration of the Bifidobacterium adolescentis strain B24728 is not less than 1×10 8 CFU / mL, can be 5×10 8 CFU / mL, 10×10 8 CFU / mL, 50×10 8 CFU / mL, 100×10 8 CFU / mL, 500×10 8 CFU / mL. The effective concentration of the metabolite or culture is not less than 10% to 99%, can be 15% to 80%, can also be 20% to 70%, can also be 30% to 50%. The present invention has no special restrictions on the preparation method of the probiotics, and the probiotic preparation method known in the art can be used.

[0035] The present invention provides the use of the Bifidobacterium adolescentis strain B24728, the metabolite or culture or the probiotic in preparing a product for inhibiting clinical drug-resistant bacteria.

[0036] In the present invention, the clinical drug-resistant bacteria preferably include at least one of the following: Clostridium difficile, Acinetobacter baumannii, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa. The Acinetobacter baumannii is represented by Acinetobacter baumannii HRAB-85. The Staphylococcus aureus is represented by resistant Staphylococcus aureus MRSA. The Escherichia coli is represented by resistant Escherichia coli that produces broad-acting beta-lactamase. The Pseudomonas aeruginosa is represented by resistant Pseudomonas aeruginosa PAO1.

[0037] In the embodiment of the present invention, the metabolites of the Bifidobacterium adolescentis strain B24728 were used to carry out in vitro antibacterial experiments. The results showed that the metabolites had good antibacterial effects on Acinetobacter baumannii HRAB-85, resistant Staphylococcus aureus, resistant Escherichia coli, Pseudomonas aeruginosa PAO1 and Clostridium difficile, among which the antibacterial activity against Pseudomonas aeruginosa PAO1 and resistant Staphylococcus aureus MRSA was the strongest. This shows that the Bifidobacterium adolescentis strain B24728 exerts its in vitro antibacterial activity by secreting antibacterial metabolites.

[0038] In the present invention, the product preferably includes medicines and / or disinfection daily necessities. The medicines include oral medicines or external medicines. The dosage forms of the oral medicines are preferably tablets, powders, granules, capsules and oral liquids. The external medicines include sprays, ointments, patches, etc. The disinfection daily necessities preferably include disinfection sprays, disinfection hand gels, disinfection detergents, etc. In addition, the product may also include food additives, feed additives, food or feed, etc., and the antibacterial effect of Bifidobacterium adolescentis strain B24728 and its metabolites is used to inhibit the growth of pathogenic and drug-resistant bacteria in food or feed. The effective live bacterial concentration of Bifidobacterium adolescentis strain B24728 in the product is not less than 1×10 8 CFU / mL, can be 5×10 8 CFU / mL, 10×10 8 CFU / mL, 50×10 8 CFU / mL, 100×10 8 CFU / mL, 500×10 8 CFU / mL. The effective concentration of the metabolite or culture is not less than 10% to 99%, can be 15% to 80%, can also be 20% to 70%, can also be 30% to 50%. The present invention has no special restrictions on the preparation method of the product, and the method well known in the art can be used.

[0039] The present invention provides the use of the Bifidobacterium adolescentis strain B24728, the culture or the probiotic agent in the preparation of anti-inflammatory drugs.

[0040] In the present invention, the anti-inflammatory drug preferably includes at least one of the following dosage forms: tablets, powders, granules, capsules and oral liquids. The present invention has no particular limitation on the preparation method of the anti-inflammatory drug, and any method known in the art can be used.

[0041] In the examples of the present invention, an anti-inflammatory experiment at the cellular level was carried out, and the LPS-induced RAW 264.7 cell inflammation model was used as the experimental object. The metabolites of strain B24728 can effectively reduce the release levels of IL-6 and TNF-α in LPS-induced RAW 264.7 cells, and the reduction degrees are 97.72% and 99.12%, respectively. The results show that the strain B24728 has good anti-inflammatory activity.

[0042] The following is a detailed description of a Bifidobacterium adolescentis strain having clinical multi-drug resistant bacteria inhibition and anti-inflammatory activity and its application provided by the present invention in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0043] The culture medium involved in the embodiment:

[0044] MPYG medium: 5 g tryptone, 3 g peptone, 2 g soy peptone, 1 g polypeptone, 10 g yeast extract, 5 g beef extract, 5 g glucose, 0.5 g maltose, 0.5 g cellobiose, 0.5 g soluble starch, 2 g dipotassium hydrogen phosphate, 0.5 g cysteine, 0.25 g sodium sulfide, 1 mg resazurin, 0.5 mL Tween 80, 0.5 mL glycerol, 1000 mL distilled water, pH 6.6-6.8. When preparing solid culture medium, add 15 g agar and sterilize at 121°C under high pressure for 15 min.

[0045] LB medium: 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, 1000 mL of distilled water, pH 7.0. When preparing solid culture medium, add 15 g of agar and sterilize at 121°C for 15 min.

[0046] Example 1

[0047] Isolation, purification and identification of a strain of Bifidobacterium adolescentis B24728

[0048] (1) The intestinal feces of centenarians in Guangxi were used as samples. After dilution with physiological saline, streaking culture was performed, and a single colony was picked and inoculated into MPYG solid culture medium. The plate was placed in an anaerobic workstation and cultured at 37°C for 2-3 days. After the colonies grew, a single colony was picked and streaked for two generations to obtain strain B24728. A single colony on the final purification plate was picked for Gram staining microscopy. Figure 1 The colonies of bifidobacteria are round with slight protrusions, milky white, and smooth and neat edges. Figure 2 The bacteria are blue-purple in color and rod-shaped, so they are Gram-positive bacteria.

[0049] (2) Identification of 16S rRNA gene of strain B24728: Pick a small amount of purified single colony with a sterile bamboo stick, shake and mix in 50 μL sterile PBS (10 mmol / L, pH 7.4) buffer to prepare a bacterial suspension. Now prepare a 16S rRNA gene amplification system. The system is configured as follows: 1 μL each of primer 27F (5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO: 2) and primer 1492R (5'-CTACGGCTACCTTGTTACGA-3', SEQ ID NO: 3), 12.5 μL of 2×Es TaqMix, and ddH 2 09.5μL was mixed in a PCR tube, and finally 1μL of bacterial suspension was added to a PCR tube containing a 16S rRNA gene amplification system, and mixed, and placed in a PCR amplifier for amplification. The amplification program was 94℃ pre-denaturation for 5min, 94℃ denaturation for 30s, 55℃ annealing for 30s, 72℃ extension for 1min, 30 cycles, and 72℃ final extension for 2min. After the amplification, the amplified liquid was taken for agarose gel electrophoresis (1% agarose gel), with a spot volume of 6μL, a voltage of 115V, and a time of 35min. Finally, a gel imager was used to observe whether there was a band at about 1500bp (see Figure 3T ) has a sequence homology of 98.70%, and the phylogenetic tree is further constructed as Figure 4 It can be seen that this strain is clustered with Bifidobacterium adolescentis, and combined with its physiological and biochemical characteristics, it is preliminarily judged that this strain is Bifidobacterium adolescentis.

[0050] Example 2

[0051] Antibacterial test for inhibiting clinical multi-drug resistant bacteria

[0052] (1) Preparation of pathogenic bacteria liquid: Take out the glycerol tubes of Acinetobacter baumannii HRAB-85, Methicillin-resistant Staphylococcus aureus (MRSA), Extended-Spectrumβ-Lactamases (ESBL)-producingEscherichia coli, and Pseudomonas aeruginosa PAO1 stored at -80°C from a -80°C refrigerator, and apply 50 μL of each tube on LB solid medium, aerobically culture at 37°C for 24 hours, pick a single colony after growth and streak it on LB solid medium, aerobically culture at 37°C for 24 hours, activate two generations in this way to obtain a single colony with good activity. Then pick a single colony and inoculate it into LB liquid medium, aerobically culture it at 37°C for 18 hours to obtain a bacterial liquid.

[0053] (2) Preparation of Clostridium difficile suspension: Take out the glycerol tube of Clostridium difficile stored in the strain bank, apply 100 μL on the blood cell solid culture medium, and culture it anaerobically at 37°C in an anaerobic workstation for 2 days. After growth, pick a single colony and activate it on the blood plate for two generations. Scrape the activated Clostridium difficile from the blood plate, add sterile PBS buffer, and blow it to mix well to obtain Clostridium difficile suspension.

[0054] (3) Preparation of concentrated fermentation supernatant of Bifidobacterium adolescentis strains B24728 and Bifidobacterium adolescentis CICC6070: A single colony of activated Bifidobacterium adolescentis was inoculated into 3 mL of liquid MPYG medium and cultured anaerobically at 37°C for 18 h, and then inoculated into 15 mL of liquid MPYG medium at a rate of 5% (v / v) and cultured anaerobically at 37°C for 24 h to obtain a fermentation broth, the fermentation broth was centrifuged at 10,000 rpm for 5 minutes, and the supernatant was extracted after centrifugation; the supernatant was filtered through a 0.22 μm filter membrane, and the filtered supernatant was freeze-dried and concentrated (5 to 10 times) to obtain a fermentation supernatant concentrate.

[0055] (4) Preparation of culture dishes with indicator bacteria (resistant pathogenic bacteria): Prepare LB culture medium with a 1.7% agar content, sterilize it in an autoclave at 121°C for 15 min and set aside. When the temperature of the solid culture medium drops below 50°C, add 0.5% (v / v) resistant Staphylococcus aureus, shake well, and pour it onto a 90 mm × 90 mm square bacterial culture dish. After cooling and solidifying, punch holes with a hole puncher to obtain a culture dish with resistant Staphylococcus aureus MRSA. Similarly, add bacterial solutions of resistant Escherichia coli, Pseudomonas aeruginosa PAO1, and Acinetobacter baumannii HRAB-85 according to the above method, pour the plates, and punch holes to obtain the corresponding indicator bacteria culture dishes. Obtaining a Clostridium difficile culture dish: prepare a blood cell culture medium with a 1.4% agar content, sterilize it, wait until the temperature drops below 50°C, add 5% sterile defibrinated sheep blood and 0.5% (v / v) Clostridium difficile solution, shake well, pour it onto a 90mm×90mm square bacterial culture dish plate, and after it cools and solidifies, punch holes with a hole puncher to obtain a culture dish with Clostridium difficile.

[0056] (5) Antibacterial test: The culture dishes with indicator bacteria (resistant pathogenic bacteria) mentioned in (4) above were arranged in order, and 150 μL of concentrated fermentation supernatant of Bifidobacterium avium was added to the corresponding holes respectively. The culture dishes containing indicator bacteria of resistant Escherichia coli, resistant Staphylococcus aureus, Pseudomonas aeruginosa PAO1, and Acinetobacter baumannii HRAB-85 were placed in a 37°C incubator for aerobic culture for 18 hours, and the presence of inhibition zones was observed. The culture dishes containing indicator bacteria of Clostridium difficile were placed in a 37°C anaerobic incubator for anaerobically cultured for 2 days, and the presence of inhibition zones was observed. The results are shown in Table 1:

[0057] Table 1 Antibacterial zone diameter of antibacterial experiment

[0058] Pathogen / Group B24728(mm) CICC6070(mm) Drug-resistant Staphylococcus aureus (MRSA) 20.00 18.00 Drug-resistant Escherichia coli 14.50 13.00 Pseudomonas aeruginosa PAO1 17.50 14.50 Acinetobacter baumannii HRAB-85 14.00 13.50 Clostridium difficile 11.00 No effect

[0059] The experimental results are as follows Figure 5 The strain B24728 can inhibit the growth of drug-resistant indicator pathogens used in this experiment, and has the strongest inhibitory effect on drug-resistant Staphylococcus aureus and Pseudomonas aeruginosa PAO1. Compared with the commercially available Bifidobacterium adolescentis CICC6070, the Bifidobacterium adolescentis strain B24728 has a stronger inhibitory effect on pathogenic bacteria.

[0060] Example 3

[0061] Hemolytic test of Bifidobacterium adolescentis strain B24728

[0062] (1) Method: A single colony of the activated strain B24728 was inoculated into 3 mL of liquid MPYG medium and cultured anaerobically at 37°C for 18 h to obtain a Bifidobacterium bacterial suspension. A small amount of the bacterial suspension was inoculated into a blood cell culture medium using the three-zone streak method and placed in an anaerobic workstation for anaerobically culture at 37°C for 24 h. The blood plate was observed for hemolysis around each zone.

[0063] The results are as follows Figure 6 No hemolysis was observed in the blood cells treated with strain B24728, indicating that the bacteria do not produce hemolysin and cannot lyse blood cells, which shows that the bacteria have a certain degree of safety.

[0064] Example 4

[0065] Antibiotic susceptibility test of strain B24728

[0066] (1) Preparation of strain activation solution: A single colony of the activated strain B24728 was inoculated into 3 mL of liquid MPYG medium and cultured anaerobically at 37° C. for 18 h. The culture solution was inoculated into 10 mL of liquid culture medium at 5% (v / v) and cultured anaerobically at 37° C. for 24 h to obtain a bacterial solution of strain B24728.

[0067] (2) Drug sensitivity test: prepare MPYG medium with 1.7% agar content, sterilize it in a high pressure sterilizer at 121℃ for 15 minutes and set aside. When the temperature of the solid culture medium drops below 50℃, add 1% (v / v) bifidobacterium activation solution, shake well, pour it on a 90mm×90mm square bacterial culture plate, and use it after cooling and solidification. Take the square culture plates containing bifidobacteria and stick them with commercially available drug sensitivity sheets, two parallel to each drug sensitivity sheet, and then put the culture plates into an anaerobic workstation at 37℃ for 24 hours to observe the size of the inhibition zone. The results of the inhibition zone are shown in Table 2 and Figure 7 shown.

[0068] Table 2 Antibiotic sensitivity test results of strain B24728

[0069] Drug Name Inhibition zone (mm) Roxithromycin (15 μg / tablet) 31.05 Ceftriaxone (30 μg / tablet) 39.00 Ciprofloxacin (5 μg / tablet) 15.55 Gentamycin (10 μg / tablet) 14.40 Chloramphenicol (30 μg / tablet) 36.45 Tetracycline (30 μg / tablet) 23.00 Ampicillin (10 μg / tablet) 39.20

[0070] The above experiments showed that strain B24728 was most sensitive to roxithromycin, ceftriaxone, chloramphenicol, tetracycline and ampicillin, and less sensitive to ciprofloxacin and gentamicin.

[0071] Example 5

[0072] In vitro anti-inflammatory function test of Bifidobacterium adolescentis strain B24728

[0073] (1) Activation of strains and liquid fermentation: Obtain activated single colonies of Bifidobacterium adolescentis strain B24728 in the manner of Example 1 above, pick out activated single colonies of Bifidobacterium adolescentis strain B24728, inoculate them into 3 mL of liquid MPYG medium and culture them anaerobically at 37°C for 18 h, and continue to inoculate them into 10 mL of liquid MPYG medium at a 5% (v / v) inoculation amount and culture them anaerobically at 37°C for 24 h to obtain fermentation liquid, centrifuge the fermentation liquid at a centrifugation parameter of 10000 rpm for 5 min, and extract the supernatant after centrifugation. The collected supernatant is sterilized by filtration with a 0.22 μm filter membrane for determination of in vitro anti-inflammatory activity.

[0074] (2) Activation and subculture of mouse RAW 264.7 cells: The RAW 264.7 cell cryovials were taken out from the liquid nitrogen tank and slowly thawed in a 37°C water bath. After thawing, the old culture medium was aspirated out and washed with sterile PBS buffer by slowly blowing. Finally, the cells were collected by centrifugation (4°C, 800 rpm) for 5 min. The washing was repeated twice. The washed RAW 264.7 cells were placed in DMEM complete culture medium (containing 10% fetal bovine serum, 80 U / mL penicillin, and 0.08 mg / mL streptomycin) and cultured in an environment of 5% CO. 2 , 37°C, saturated humidity, and cultured in a closed cell culture incubator. The cell growth status was observed every day, and the cells were collected and passaged. The cells in the logarithmic growth phase and in good cell condition were used for subsequent anti-inflammatory experiments.

[0075] (3) RAW264.7 cell seeding plate: discard the old culture medium of the cells in good condition, add an appropriate amount of sterile PBS, blow the cells off the wall of the culture flask, transfer them to a 15 mL centrifuge tube, and centrifuge the cells (4°C, 800 rpm) for 5 min to collect the cells. Repeat this washing twice, add an appropriate amount of DMEM culture medium to the washed cells and blow them to resuspend them. Take an appropriate amount of the resuspended cells and count them with a cell counting plate. Dilute the cells in the original tube appropriately according to the cell number, and inoculate them into a 24-well cell culture plate with 500,000 cells per well in 1 mL for 6 h. Use a cell microscope to observe the cell status and whether the cells are attached to the wall.

[0076] (4) RAW 264.7 cell anti-inflammatory experiment: cells in the wells of the cell plate with good shape and growth adherent to the wall were selected as experimental subjects and divided into four experimental groups, namely, blank control group: 800 μL DMEM culture medium; positive control group: 800 μL DMEM culture medium containing LPS at a final concentration of 50 ng / mL; fermentation supernatant experimental group: 400 μL DMEM culture medium containing LPS at a final concentration of 50 ng / mL + 400 μL bacterial fermentation supernatant; fermentation supernatant control group: 400 μL DMEM culture medium containing LPS at a final concentration of 50 ng / mL + 400 μL fermentation medium. After adding the reagents of each experimental group, the 24-well cell plate was placed in a cell culture incubator and cultured for 16 hours. Finally, the liquid in the well plate was collected and centrifuged to obtain the supernatant for measuring its inflammatory factors.

[0077] (5) Determination of IL-6 and TNF-α concentrations: Kits: Mouse (IL-6) ELISA Detection Kit and Mouse Tumor Necrosis Factor α (TNF-α) ELISA Detection Kit from Jianglai Biological. According to the instructions in the kit, the IL-6 and TNF-α factors in each experimental group were measured.

[0078] The standard curve equation for IL-6 is:

[0079] y=(1.853+0.0086) / [1+(x / 300.98) -1.369 ]-0.0086Formula I;

[0080] R 2 =0.99876, X represents concentration (pg / mL), and Y represents OD value (450nm).

[0081] The standard curve equation for TNF-α is:

[0082] y=(5.328+0.0082) / [1+(x / 1636.41) -0.979 ]-0.0082 Formula II;

[0083] R 2 =0.99984, X represents concentration (pg / mL), and Y represents OD value (450nm).

[0084] The results of anti-inflammatory factor detection are shown in Tables 3 and 4.

[0085] Table 3 Effect of strain B24728 on the concentration of IL-6 secreted by RAW264.7 cells induced by LPS

[0086] Group / concentration (pg / mL) Positive control group Supernatant control Blank control B24728 1 1363.7966 966.1465 43.3389 36.3628 2 1664.8779 898.4219 46.0611 37.7337 3 1930.7394 956.9321 42.3689 41.8406 4 1628.5742 1083.9909 39.2630 38.8678 5 1811.7811 1043.4284 45.0197 38.0463 6 1749.6180 974.8431 40.6680 38.4433 Mean 1691.5646 987.2938 42.7866 38.5491

[0087] Table 4 Effect of strain B24728 on the concentration of TNF-α secreted by RAW264.7 cells induced by LPS

[0088] Group / concentration (pg / mL) Positive control group Supernatant control Blank control B24728 1 105764.1085 107390.8400 1391.1992 1029.9213 2 100807.1450 132427.1290 1653.6613 960.9927 3 124093.1845 128067.0020 1543.8488 946.3560 4 109140.8005 131707.6685 1509.3470 955.1368 5 110116.1815 113508.4950 1349.4589 974.1745 6 11798.4300 126075.5255 1365.8484 1021.1095 Mean 111317.1417 123196.1100 1468.8939 981.2818

[0089] The inhibition rate calculation formula III is shown as follows:

[0090]

[0091] According to the results of relevant inflammatory index detection, the strain B24728 can reduce the release levels of IL-6 and TNF-α in LPS-induced RAW 264.7 cells, and the degree of reduction is 97.72% and 99.12%, respectively. The results show that the strain B24728 has certain in vitro anti-inflammatory function.

[0092] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An application of Bifidobacterium adolescentis strain B24728 in the preparation of a product for inhibiting clinical drug-resistant bacteria, wherein the Bifidobacterium adolescentis strain B24728 is deposited with GDMCC No: 65489.

2. The application according to claim 1, characterized in that: The Bifidobacterium adolescentis strain B24728 also includes metabolites of the Bifidobacterium adolescentis strain B24728 and / or a culture of the Bifidobacterium adolescentis strain B24728.

3. The application according to claim 2, characterized in that: The method for preparing the metabolites of Bifidobacterium adolescentis strain B24728 is characterized by comprising the following steps: The Bifidobacterium adolescentis strain B24728 was inoculated into MPYG liquid medium for anaerobically culturing to obtain a culture; The liquid phase of the culture is separated to obtain metabolites of Bifidobacterium adolescentis strain B24728.

4. The application according to claim 2, characterized in that: The Bifidobacterium adolescentis strain B24728 is also present in the form of a probiotic.

5. The application according to claim 4, characterized in that: The probiotics include at least one of the following dosage forms: aqueous solution, powder and granule.

6. The use according to claim 1, characterized in that: The clinical drug-resistant bacteria include at least one of the following: Clostridium difficile, Acinetobacter baumannii, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa.

7. The use according to claim 1, characterized in that: The product includes at least one of the following: medicine, disinfected daily necessities, food, food additives, feed and feed additives.

8. Use of the Bifidobacterium adolescentis strain B24728, a culture of the Bifidobacterium adolescentis strain B24728, or a probiotic comprising the Bifidobacterium adolescentis strain B24728 according to claim 1 in the preparation of an anti-inflammatory drug.

9. The use according to claim 8, characterized in that: The anti-inflammatory drug comprises at least one of the following dosage forms: tablets, powders, granules, capsules and oral solutions.

Images