Screening method of probiotics for inhibiting growth of staphylococcus aureus

Abstract

The invention discloses a screening method of probiotics for inhibiting growth of staphylococcus aureus, and relates to the technical field of microorganisms. According to the screening method, to-be-screened bacteria are subjected to cationic antibacterial peptide resistance screening and staphylococcus aureus resistance screening in sequence, the probiotics with specific stable resistance to staphylococcus aureus are obtained through screening, and the probiotics also have good drug resistance to antibacterial peptides. The screening method is simple in step, and the cation-resistant antibacterial peptide and the probiotics with a good bacteriostatic effect on staphylococcus aureus can be effectively screened.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a method for screening probiotics capable of inhibiting the growth of Staphylococcus aureus. Background Art

[0002] Staphylococcus aureus is a major human pathogen that can cause a wide range of skin and soft tissue infections and is a major cause of bacteremia and device-related infections. At the same time, Staphylococcus aureus has a strong ability to adapt to the environment and can acquire resistance to antibiotics and disinfectants, so it is widely spread in the world's healthcare and community settings.

[0003] At present, the main local treatment for Staphylococcus aureus colonized on the skin is still to use antiseptics or antibiotics, but there is still a risk of incomplete removal and local microbial depletion, which may even increase the risk of reinfection and easily induce drug resistance. Probiotics refer to microorganisms that can have a positive effect on the health of the host at a certain dosage. Compared with antiseptic or antibiotic treatment, probiotic treatment will not induce depletion of symbiotic microbiota, and can also improve skin health, resist pathogen colonization and enhance the host's immune response to pathogens by increasing the diversity of skin surface flora. Therefore, it is necessary to provide a method for screening probiotics that can effectively inhibit the growth of Staphylococcus aureus. Summary of the invention

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention provides a method for screening probiotics that inhibit the growth of Staphylococcus aureus, which can effectively screen and obtain probiotics that are resistant to cationic antimicrobial peptides and have good antibacterial effects on Staphylococcus aureus.

[0005] The present invention also provides probiotics screened by the above screening method.

[0006] The present invention also provides application of the probiotics screened by the above screening method.

[0007] A method for screening probiotics that inhibit the growth of Staphylococcus aureus according to the first aspect of the present invention comprises the following steps: A1. Cationic antimicrobial peptide resistance screening: Preparing a first mixture of the first bacteria to be screened and a cationic antimicrobial peptide solution of different test concentrations, incubating to obtain an incubation solution; placing the incubation solution on a culture plate, and culturing until a single colony grows; A2. Screening for resistance to Staphylococcus aureus: A single colony in the group treated with the highest concentration of cationic antimicrobial peptides in step A1 is selected as the second bacteria to be screened, metabolites of the second bacteria to be screened are obtained, a second mixture of the metabolites and Staphylococcus aureus is prepared, the mixture is incubated, and the survival of the Staphylococcus aureus is detected; and the desired probiotics are screened.

[0008] The screening method according to the embodiment of the present invention has at least the following beneficial effects: The screening method of the embodiment can effectively screen out probiotics that are resistant to cationic antimicrobial peptides and have a good antibacterial effect on Staphylococcus aureus. By first performing cationic antimicrobial peptide screening, the steps are simple and more bacteria can be screened at the same time; then performing Staphylococcus aureus screening can simplify the screening process and speed up the screening. By using a transparent buffer to dilute the antimicrobial peptide with cationic properties, and then mixing the bacteria with the diluted antimicrobial peptide for incubation and quantitative titration on the Columbia blood plate, the screening method not only overcomes the problem of inaccurate measurement results of antimicrobial resistance caused by turbidity after the antimicrobial peptide is mixed with the culture medium, but also can accurately calculate the killing ability of the antimicrobial peptide on bacteria based on the number of bacteria on the plate after culture and the control group without adding antimicrobial peptides, providing a universal and accurate blood plate titration method for measuring the killing ability of antimicrobial peptides on bacteria, which can achieve the effect of continuous screening while obtaining a large amount of data, and effectively combine the measurement data with bacterial screening. This screening method combines the method of measuring bacterial resistance data and the method of screening bacteria with strong drug resistance, providing an experimental method that can accurately measure bacterial resistance and screen for bacteria with strong drug resistance, providing convenience for studying bacterial resistance mechanisms or using bacteria to carry out metabolic activities in an environment with high drug concentrations.

[0009] The bacteria that survived the high concentration of antimicrobial peptides in the experiment were purified, revived, and the drug resistance data was measured again; the bacteria that survived the high concentration of antimicrobial peptides in the measurement experiment can be purified and revived for the next drug resistance data measurement. As the number of repetitions increases, the dilution factor in the experiment should also increase in order to obtain accurate drug resistance data measurement results. By repeating this process, bacteria with strong drug resistance can be obtained, realizing the function of directional screening. The obtained bacteria can be designed to study drug resistance mechanisms and exert physiological functions under local high drug concentrations.

[0010] According to some embodiments of the present invention, the cationic antimicrobial peptide solution is a buffer solution containing cationic antimicrobial peptides.

[0011] According to some embodiments of the present invention, the buffer is a transparent buffer, which is conducive to judging whether the cationic antimicrobial peptide is completely dissolved by light transmittance, and avoids the possibility that a non-transparent buffer may cause different local concentrations of the cationic antimicrobial peptide, thereby affecting the screening results.

[0012] According to some embodiments of the present invention, the transparent buffer is selected from Tris / HCl buffer, phosphate buffer, HEPES buffer or physiological saline.

[0013] According to some embodiments of the present invention, the first bacteria to be screened are pure bacteria or mixed bacteria. The first bacteria to be screened are derived from skin.

[0014] According to some embodiments of the present invention, the first bacteria to be screened is Staphylococcus aureus.

[0015] According to some embodiments of the present invention, in the first mixture, the bacterial density of the first bacteria to be screened is 1×10 4 CFU / mL~4×10 5 CFU / mL. For example: it can be 1×10 4 CFU / mL, 2×10 4 CFU / mL, 4×10 4 CFU / mL, 6×10 4 CFU / mL, 8×10 4 CFU / mL, 1×10 5 CFU / mL, 2.0×10 5 CFU / mL or 4×10 5 CFU / mL.

[0016] According to some embodiments of the present invention, the cationic antimicrobial peptide is LL-37. LL-37 is an antimicrobial peptide secreted by human skin.

[0017] According to some embodiments of the present invention, in the first mixture, the concentration of the antimicrobial peptide is 0.1 μM to 1000 μM. For example, it may be 1000 μM, 800 μM, 600 μM, 400 μM, 200 μM, 100 μM, 80 μM, 60 μM, 40 μM, 20 μM, 10 μM, 5 μM, 2.5 μM, 1.25 μM, 1 μM, 0.5 μM or 0.1 μM.

[0018] According to some embodiments of the present invention, the shaking frequency of the incubation in step A1 is 120 rpm to 240 rpm, for example, 120 rpm, 140 rpm, 160 rpm, 180 rpm, 200 rpm, 220 rpm or 240 rpm.

[0019] According to some embodiments of the present invention, the incubation temperature in step A1 is 25°C to 37°C. For example, it can be 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C or 37°C.

[0020] According to some embodiments of the present invention, the incubation time in step A1 is 30 min to 120 min. For example, it can be 30 min, 40 min, 50 min, 60 min, 70 min, 80 min, 90 min, 100 min, 110 min or 120 min.

[0021] According to some embodiments of the present invention, in step A1, the culture plate is divided into a plurality of regions, one of which corresponds to one sample. The shapes of the regions include but are not limited to grid and fan shapes.

[0022] According to some embodiments of the present invention, the culture plate comprises at least one of a Columbia blood plate, a TSA plate, and an LB plate.

[0023] Since skin probiotics and Staphylococcus aureus have similar properties (including but not limited to similar shapes and colors on solid culture media, similar phenomena after centrifugation in liquid culture media, etc.), and Staphylococcus aureus is more likely to contaminate Staphylococcus epidermidis in experiments and is difficult for technicians in the field to detect, blood plates, separate bench experiments, and separate bagging are used in the screening process to ensure that the subtype of Staphylococcus epidermidis is single and uncontaminated. All operations involving Staphylococcus aureus need to be performed in a separate clean bench, and operations for screening probiotics need to be performed in another clean bench. When the probiotic supernatant is mixed with Staphylococcus aureus, the probiotic supernatant is placed in an EP tube and transferred to the clean bench where Staphylococcus aureus is operated to avoid contamination of the probiotics by Staphylococcus aureus.

[0024] According to some embodiments of the present invention, in step A1, each diluted sample is dropped onto the culture plate in at least three replicates.

[0025] According to some embodiments of the present invention, step A1 includes pre-culture treatment, wherein the pre-culture treatment includes: appropriately diluting the incubation solution and placing it on a culture plate, and placing each diluted sample on the culture plate in at least three replicates.

[0026] According to some embodiments of the present invention, step A1 includes post-culture processing; the post-culture processing includes: counting the colonies of single colonies grown on the culture plate and calculating the killing percentage.

[0027] According to some embodiments of the present invention, the number of times step A1 is repeated is 3 to 4 times. A person skilled in the art can select a suitable number of repetitions as needed, as long as the second bacteria to be screened with the desired properties can be screened. As the number of times step A1 is repeated increases, the dilution factor of the sample also increases.

[0028] According to some embodiments of the present invention, the number of the second bacteria to be screened is at least 1. Those skilled in the art can select a suitable number of strains as needed, for example, 5, 10 or 15 strains.

[0029] According to some embodiments of the present invention, the method for preparing the metabolites of the second bacteria to be screened comprises the following steps: The second bacteria to be screened are cultured to obtain a culture solution; the culture solution is diluted to a desired bacterial density, and the bacterial bodies are removed to obtain the metabolites.

[0030] The preparation method of the metabolites of the embodiment improves the preparation method of the metabolites. When it is used to screen the antibacterial efficacy of skin probiotics, it realizes the acquisition of accurate data in combination with the optimization steps and improves the credibility of the results. Counting and diluting the bacterial culture fluid can eliminate the influence of the bacterial proliferation rate on the concentration of antibacterial substances in the supernatant. Further combined with the drop plate method, detailed and large amounts of antibacterial rate data can be obtained, while it is difficult to obtain detailed data by the 96-well plate method.

[0031] According to some embodiments of the present invention, when preparing the metabolite, the culture time of the second bacteria to be screened is 12 h to 24 h.

[0032] According to some embodiments of the present invention, when preparing the metabolites, the oscillation frequency of the culture of the second bacteria to be screened is 120 rpm to 240 rpm. For example, it can be 120 rpm, 140 rpm, 160 rpm, 180 rpm, 200 rpm, 220 rpm, or 240 rpm.

[0033] According to some embodiments of the present invention, when preparing the metabolites, the culture temperature of the second bacteria to be screened is 25-37° C. For example, it can be 25° C., 26° C., 27° C., 28° C., 29° C., 30° C., 31° C., 32° C., 33° C., 34° C., 35° C., 36° C. or 37° C.

[0034] According to some embodiments of the present invention, when preparing the metabolites, the desired bacterial density is 5×10 6 CFU / mL~4×10 7 CFU / mL. For example, it can be 5×10 6 CFU / mL, 6×10 6 CFU / mL, 7×10 6 CFU / mL, 8×10 6 CFU / mL, 9×10 6 CFU / mL, 1×10 7 CFU / mL, 2×10 7 CFU / mL, 3×10 7CFU / mL or 4×10 7 CFU / mL.

[0035] According to some embodiments of the present invention, when preparing the metabolites, methods for removing bacteria include but are not limited to centrifugation and filtration.

[0036] According to some embodiments of the present invention, when preparing the second mixture, the metabolites of the second bacteria to be screened are mixed with the bacterial suspension of Staphylococcus aureus at a volume ratio of 1:0.9-1.1 to obtain the second mixture.

[0037] According to some embodiments of the present invention, step A2 comprises: sealing the container containing the second mixture (eg, using a sealing bag and / or sealing film) and then incubating.

[0038] According to some embodiments of the present invention, in the second mixture, the bacterial density of Staphylococcus aureus is 1×10 4 CFU / mL~4×10 5 CFU / mL. For example: it can be 1×10 4 CFU / mL, 2×10 4 CFU / mL, 4×10 4 CFU / mL, 6×10 4 CFU / mL, 8×10 4 CFU / mL, 1×10 5 CFU / mL, 2.0×10 5 CFU / mL or 4×10 5 CFU / mL.

[0039] According to some embodiments of the present invention, the incubation temperature in step A2 is 25°C to 37°C. For example, it can be 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C or 37°C.

[0040] According to some embodiments of the present invention, the incubation time in step A2 is 30 min to 120 min. For example, it can be 30 min, 40 min, 50 min, 60 min, 70 min, 80 min, 90 min, 100 min, 110 min or 120 min.

[0041] According to some embodiments of the present invention, step A2 is repeated 5 to 6 times.

[0042] According to some embodiments of the present invention, the criteria for screening the desired probiotics include: the probiotics have a stable ability to kill Staphylococcus aureus.

[0043] A probiotic according to the second aspect of the present invention is obtained by screening using the screening method in the first aspect of the present invention.

[0044] Use of the probiotics according to the embodiment of the second aspect of the third aspect of the present invention in any one of B1) to B3); B1) preparing a product resistant to the antimicrobial peptides described in the first aspect of the embodiment; B2) preparing products that kill and / or inhibit Staphylococcus aureus; B3) Kill and / or inhibit Staphylococcus aureus.

[0045] According to some embodiments of the present invention, the product is selected from a reagent, a kit or a medicine.

[0046] According to some embodiments of the present invention, the use is for non-disease diagnosis and treatment purposes.

[0047] Other features and advantages of the present invention will be set forth in the description which follows, and in part will be apparent from the description, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 Schematic diagram of skin probiotics on Columbia blood agar plates by blood agar titration; Figure 2 The results of the resistance test of different bacteria to LL-37; Figure 3 The inhibitory effect of different bacteria on Staphylococcus aureus. DETAILED DESCRIPTION

[0049] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0050] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0051] In the description of the present invention, if there is a description of first, second, etc., it is only for the purpose of distinguishing technical features, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features or implicitly indicating the order of the indicated technical features.

[0052] When a numerical range is disclosed herein, the above range is considered to be continuous and includes the minimum and maximum values ​​of the range, as well as every value between such minimum and maximum values. Further, when a range refers to an integer, every integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be combined. In other words, unless otherwise indicated, all ranges disclosed herein should be understood to include any and all subranges included therein.

[0053] "And / or" is used to indicate that one or both of the stated situations may occur, for example, A and / or B includes (A and B) and (A or B).

[0054] TSB liquid medium (tryptic soy casein broth): trypticase 17.0 g / L, sodium chloride 5.0 g / L, soybean papain digest 3.0 g / L, potassium dihydrogen phosphate 2.5 g / L, glucose 2.5 g / L, pH = 7.3±0.2.

[0055] TSA solid culture medium (Soybean casein agar medium): tryptone 15.0 g / L, soytone 5.0 g / L, sodium chloride 5.0 g / L, agar 15.0 g / L, pH=7.3±0.2.

[0056] Columbia blood plate: casein pancreatic digest 10.0 g / L, heart pancreatic digest 3.0 g / L, corn starch 1.0 g / L, meat gastric digest 5.0 g / L, yeast extract powder 5.0 g / L, sodium chloride 5.0 g / L, agar 20.0 g / L, 5% sterile defibrinated sheep blood. Columbia blood plate is used for the culture of fastidious and non-fastidious bacteria and the identification of hemolytic bacteria.

[0057] Skin probiotics: skin bacteria purchased from ATCC official website, number ATCC 27844.

[0058] LL-37: A polypeptide secreted from the surface of human skin, belonging to the cathelicidin family peptide. LL-37 is the C-terminal cleavage product of the cathelicidin peptide Hcap-18, which can directly kill bacteria, fungi and viruses. The amino acid sequence is LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES.

[0059] Diluted LL-37 solution: Use PBS to dilute the 1 mM LL-37 stock solution (solvent is PBS) to concentrations of 40, 20, 10, 5, and 2.5 μM, respectively, to obtain diluted LL-37 solution.

[0060] Staphylococcus aureus: ATCC 6538. Its recovery method is as follows: Take 100 μL of primary Staphylococcus aureus solution and dilute it into a 1 mL EP tube. Take 200 μL of the diluted bacterial solution and spread it on the Columbia blood plate. Wrap it with a sealing film and invert it in a 37°C incubator for 12 h. Take out the plate for observation. Yellow colonies can be seen on the Columbia blood plate, and there is an obvious hemolysis ring around the colonies. Pick multiple colonies on the Columbia blood plate and add them to a 20 mL EP tube. Incubate at 160 rpm and 37°C for 24 h to obtain the bacterial culture solution.

[0061] Example 1 This embodiment provides a method for screening skin probiotics that inhibit the growth of Staphylococcus aureus.

[0062] The screening object of this screening method is skin probiotics (Staphylococcus aureus ATCC 27844). According to the characteristics of skin probiotics on different culture media and blood plates, and according to their growth characteristics, a set of culture schemes most suitable for the growth and recovery of skin probiotics is obtained. The recovery and activation method of Staphylococcus aureus ATCC 27844 is as follows: Dilute 100 μL of the primary bacterial solution into 1 mL of TSB liquid medium, take 200 μL of the diluted bacterial solution and spread it on the Columbia blood plate, wrap it with a sealing film and invert it in a 37°C incubator for 12 h. Take out the plate for observation, and you can see that there are white small colonies on the Columbia blood plate, and there is no hemolysis ring around the colonies. Pick a few colonies and add them to 20 mL20 mL TSB medium, and culture them at 160 rpm and 37°C for 24 h to obtain bacterial culture solution.

[0063] The bacterial culture solution was diluted 2, 4, 8, 16, 32, and 64 times by half-dilution method, and 100 μL of the diluted culture solution was placed in a 96-well plate to measure the absorbance OD of the bacterial solution. 600 At the same time, dilute the bacterial culture to 10 5 , 10 6 , 10 7 After 100 μL of the diluted culture solution was spread on a TSA plate, wrapped with a sealing film and inverted for 12 h at 37°C. A standard curve of absorbance and bacterial count of skin probiotics in TSB medium was then established based on the measured absorbance and bacterial count on the plate.

[0064] The steps of the method for screening probiotics that inhibit the growth of Staphylococcus aureus are as follows: S1. LL-37 drug resistance screening (blood plate titration method): (1) Preparation of diluted skin probiotic culture solution.

[0065] Diluted skin probiotic culture medium: Add 100 μL of revived and activated bacterial culture medium into 20 mL of TSB liquid medium and culture at 37°C and 160 rpm for 12 h until the logarithmic growth phase. Dilute the bacterial solution with PBS to a bacterial density of 2.0×10 5 CFU / mL, and the diluted skin probiotic culture fluid was obtained.

[0066] (2) Take 500 μL of diluted skin probiotic culture medium and mix with different concentrations of LL-37 solution in 2 mL EP tubes, and incubate with shaking at 37°C (based on the final concentration of LL-37, record as: 20 μM group, 10 μM group, 5 μM group, 2.5 μM group, 1.25 μM group); replace the LL-37 solution with an equal volume of PBS as the control group. After incubation for 30 min, the incubation solutions of the 20 μM group and 10 μM group were diluted by one-fold, and the incubation solutions of the 5 μM group, 2.5 μM group, 1.25 μM group, and blank group were diluted by ten-fold.

[0067] (3) Divide the Columbia blood plate into six equal parts, each of which is a separate sector. Take 20 μL of the incubation solution diluted in step (2) (which is helpful to control the number of bacteria on the blood plate within a reasonable range and facilitate counting), and drop it into three sectors respectively to ensure the accuracy of the results. Then open the Columbia blood plate and place it close to the alcohol lamp to dry the droplets; after drying, a circular area with a border can be observed in the center of each sector. Wrap the Columbia blood plate with sealing film and incubate it upside down at 37°C for 24 h. Use a colony counter to measure the number of bacteria in each sector on the Columbia blood plate and calculate the killing percentage.

[0068] Killing percentage = (average number of colonies in the control group - average number of colonies in the experimental group) / average number of colonies in the control group × 100%; Among them, the experimental groups refer to 20 μM group, 10 μM group, 5 μM group, 2.5 μM group, and 1.25 μM group.

[0069] S2, LL-37 resistance cycle screening: Pick the single colony of skin probiotics that survive on the sector with the highest LL-37 final concentration, and repeat step S1 to screen for skin probiotics that are resistant to LL-37. The details are as follows: (1) Use a 200 μL pipette tip to pick a single, spherical, white, medium-sized colony from the 20 μM group on the Columbia blood agar plate in step S1 and place it in 20 mL TSB liquid culture medium for 12 h. Take 100 μL of bacterial culture medium and add it to 20 mL TSB liquid culture medium. Incubate at 37°C and 160 rpm for 12 h until the logarithmic growth phase. Dilute the bacterial culture with PBS to a bacterial density of 2.0×10 5 CFU / mL, and the diluted skin probiotic culture fluid was obtained.

[0070] (2) Take 500 μL of diluted skin probiotic culture medium and different concentrations of LL-37 solution (40 μM, 20 μM, 10 μM, 5 μM, 2.5 μM) in 2 mL EP tubes, and incubate at 37°C with shaking (based on the final concentration of LL-37, recorded as: 20 μM group, 10 μM group, 5 μM group, 2.5 μM group, 1.25 μM group); replace the LL-37 solution with an equal volume of PBS as the control group. After incubation for 30 min, the incubation solutions of the 20 μM group and the 10 μM group were diluted ten-fold, and the incubation solutions of the 5 μM group, the 2.5 μM group, the 1.25 μM group, and the blank group were diluted twenty-fold.

[0071] (3) Divide the Columbia blood plate into six equal parts, each of which is a separate sector. Take 20 μL of the diluted incubation solution and drop it into three sectors respectively, that is, repeat each sample three times to ensure the accuracy of the results (schematic diagram as shown in the figure) Figure 1 ). The Columbia blood plate was then opened and placed close to an alcohol lamp to dry the droplets; after drying, a circular area with a border was observed in the center of each sector. The Columbia blood plate was wrapped with a sealing film and incubated upside down at 37°C for 24 hours. The number of bacteria in each sector on the Columbia blood plate was determined using a colony counter and the killing percentage was calculated.

[0072] (4) Repeat steps (1) to (3); when repeating step (2), the incubation solutions of the 20 μM group, 10 μM group, 5 μM group, 2.5 μM group, 1.25 μM group, and control group are diluted twenty-fold, respectively.

[0073] If the LL-37 resistance test experiment is further repeated, those skilled in the art can adjust the dilution multiple of step (2) as needed to facilitate obtaining a single colony in step (3).

[0074] S3, Staphylococcus aureus resistance screening: (1) Preparation of bacterial supernatant and indicator Staphylococcus aureus culture solution.

[0075] Bacterial supernatant: Pick a single colony of uniform size and shape from the 20 μM group on the Columbia blood plate in step S2 and place it in 1 mL TSB liquid medium. Culture it at 37°C and 160 rpm for 24 h. Take 100 μL of bacterial culture medium in a 96-well plate and measure the OD on a microplate reader. 600 , and the bacterial culture solution was diluted with TSB medium to the same bacterial concentration (1×10 7 CFU / mL) to reduce the possible impact of bacterial growth rate on metabolite concentration and maintain the relative consistency of antibacterial components and bacterial concentration in the bacterial supernatant, thereby eliminating the impact of different bacterial growth rates on the evaluation of the inhibitory effect on Staphylococcus aureus. After sufficient suspension, the diluted bacterial culture was centrifuged at 12,000 rpm for 5 min, the supernatant was taken, and filtered through a 0.22 μm water filter to obtain the bacterial supernatant with skin probiotics removed.

[0076] Staphylococcus aureus indicator culture: inoculate 200 μL of Staphylococcus aureus culture medium into 20 mL of TSB liquid culture medium and culture at 37°C and 160 rpm for 12 h to obtain a Staphylococcus aureus indicator culture in the logarithmic growth phase.

[0077] (2) Mix 100 μL of bacterial supernatant with 100 μL of Staphylococcus aureus indicator solution. The final concentration of Staphylococcus aureus is 1×10 5 CFU / mL. Add to 96-well cell culture plate 1 as the experimental group; mix 100 μL of sterile PBS with 100 μL of Staphylococcus aureus indicator bacteria solution and add to 96-well cell culture plate 2 as the positive control group; add 200 μL of TSB culture medium to 96-well cell culture plate 3 as the negative control group. Wrap the three 96-well plates with sealed bags to avoid contamination. After the three 96-well plates were placed at 37°C for 8 h, the absorbance value (OD) of each sample was measured using an enzyme marker. 600 ) were measured and the inhibition rate was calculated.

[0078] Inhibition rate = (absorbance of positive control group - absorbance of experimental group) / (absorbance of positive control group - absorbance of negative control group).

[0079] At the same time, refined operations such as separate table culture and separate plate culture can be used to avoid Staphylococcus aureus contaminating the control group and the probiotic culture medium.

[0080] (3) Select the strain with the highest antibacterial rate in the test results of step (2) and repeat steps (1) to (2); repeat 5 times in total.

[0081] (4) Select the strain with the most stable antibacterial effect and the highest antibacterial rate for bacterial subtype identification.

[0082] The strains obtained by screening were named Staphylococcus hominis 621 (abbreviated as Sh 621) was subjected to 16s bidirectional assay and gyrB bidirectional assay. The sequence obtained by 16s bidirectional assay is shown in SEQ ID NO: 1; the sequence obtained by gyrB bidirectional assay is shown in SEQ ID NO: 2.

[0083] GGGGGGTGCTATACATGCAAGTCGAGCGAACAGACGAGGAGCTTGCTCCTTTGACGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTACCTATAAGACTGGGATAACTTCGGGAAACCGGAGCTAATACCGGATAATATTTCGAACCGCATGGTTCGATAGTGAAAGATGGCTTTGCTATCACTTATAGATGGACCTGCGCCGTATTAGCTAGTTGGTAAGGTAACGGCTTACCAAGGCAACGATACGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGGCGAAAGCCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTCTTCGGATCGTAAAACTCTGTTATTAGGGAAGAACAAACGTGTAAGTAACTGTGCACGTCTTGACGGTACCTAATCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCGTAGGCGGTTTTTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGAAAACTTGAGTGCAGAAGAGGAAAGTGGAATTCCATGTGTAGCGGTGAAATGCGCAGAGATATGGAGGAACACCAGTGGCGAAGGCGACTTTCTGGTCTGTAACTGACGCTGATGTGCGAAAGCGTGGGGATCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGACCCGCCAAGCGGTGGAGCATGTGGTTAATTCGAAGCACGCGAAAACCTTACCAATCTTGACTCCTTTGACCTTCTAAG(SEQID NO: 1);

[0084] Data comparison in the NCBI database revealed that the similarity between Sh 621 and Staphylococcus hominis strain 6-421 16S ribosomal RNA gene, partial sequence was 99.18% in 16s determination and 99.60% in gyrB determination.

[0085] Test example 1. After Sh 621, Staphylococcus aureus ATCC 27844 and Staphylococcus aureus were cultured to the logarithmic growth phase, they were diluted with PBS to a bacterial density of 2.0×10 5 CFU / mL, and the diluted bacterial solution was obtained. 100 μL of the diluted bacterial solution was mixed with 100 μL of LL-37 solution of different concentrations (solvent was PBS) to a final concentration of LL-37 of 20 μM, 10 μM, 5 μM, 2.5 μM, and 1.25 μM, respectively, and incubated at 37°C and 160 rpm for 30 min as the experimental group; the LL-37 solution was replaced with an equal volume of PBS and treated in the same way as the control group. After incubation, the Columbia blood plate was divided into six parts, each of which was a separate sector. After the incubation solution of each group was appropriately diluted, 20 μL was taken and dropped into three sectors respectively. After the droplets dried, they were incubated at 37°C for 24 h. The number of bacteria in each sector on the Columbia blood plate was determined using a colony counter, and the survival rate was calculated.

[0086] Survival rate = average number of surviving colonies in the experimental group / average number of surviving colonies in the control group × 100%.

[0087] The results are as follows Figure 2 shown.

[0088] Sh 621 has very stable resistance to LL-37; while human Staphylococcus ATCC 27844 has very poor and unstable resistance to LL-37; Staphylococcus aureus has almost no resistance to LL-37. At the same LL-37 concentration, taking 10 μM as an example, the screened human Staphylococcus has a survival rate of 90% in this environment, the unscreened human Staphylococcus has only a survival rate of about 20%, and the survival rate of Staphylococcus aureus is below 1%. This shows that the screening method of the present invention can effectively screen out probiotics with stable resistance to LL-37.

[0089] 2. (1) Randomly pick 5 single colonies of uniform size and shape (named Normal Sh 1~Normal Sh 5) on the Columbia blood agar plate containing revived and activated human Staphylococcus aureus ATCC 27844. Inoculate Sh 621 and Normal Sh 1~Normal Sh 5 into 1 mL TSB liquid culture medium respectively and culture at 37℃ and 160 rpm for 24 h. Take 100 μL of bacterial culture medium in a 96-well plate and measure the OD on an ELISA reader. 600 According to the measured absorbance, the bacterial culture solution was diluted to 1×10 7 CFU / mL, after sufficient suspension, the diluted bacterial culture was centrifuged at 12000rpm for 5 min, the supernatant was taken and filtered through a 0.22 μm water filter to obtain the bacterial supernatant after removing bacteria.

[0090] Staphylococcus aureus indicator culture: inoculate 200 μL of Staphylococcus aureus culture medium into 20 mL of TSB liquid culture medium and culture at 37°C and 160 rpm for 12 h to obtain a Staphylococcus aureus indicator culture in the logarithmic growth phase.

[0091] (2) Mix 100 μL of bacterial supernatant with 100 μL of indicator Staphylococcus aureus (the final concentration of Staphylococcus aureus is 1×10 5 CFU / mL), added to 96-well cell culture plate 1 as the experimental group; 100 μL sterile PBS was mixed with 100 μL of Staphylococcus aureus indicator bacteria solution and added to 96-well cell culture plate 2 as the positive control group; 200 μL of TSB medium was added to 96-well cell culture plate 3 as the negative control group. The three 96-well plates were wrapped in sealed bags to avoid contamination. After the three 96-well plates were placed at 37℃ for 8 h, the absorbance value (OD600) of each sample was measured using an enzyme marker, and the inhibition rate was calculated.

[0092] The results are as follows Figure 3 shown.

[0093] It can be seen that the Sh 621 obtained after screening has an inhibitory effect of more than 70% on Staphylococcus aureus, while the unscreened Normal Sh 1~Normal Sh 5 have only an inhibitory effect of about 40% on Staphylococcus aureus.

[0094] The embodiments of the present invention are described in detail above in conjunction with the embodiments, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge scope of ordinary technicians in the relevant technical field without departing from the purpose of the present invention.

Claims

1. A method for screening probiotics that inhibit the growth of Staphylococcus aureus, characterized in that: The following steps are involved: A1. Cationic antimicrobial peptide resistance screening: Preparing a first mixture of the first bacteria to be screened and a cationic antimicrobial peptide solution of different test concentrations, incubating to obtain an incubation solution; placing the incubation solution on a culture plate, and culturing until a single colony grows; A2. Screening for resistance to Staphylococcus aureus: A single colony in the group treated with the highest concentration of cationic antimicrobial peptides in step A1 is selected as the second bacteria to be screened, metabolites of the second bacteria to be screened are obtained, a second mixture of the metabolites and Staphylococcus aureus is prepared, the mixture is incubated, and the survival of the Staphylococcus aureus is detected; and the desired probiotics are screened.

2. The screening method according to claim 1, characterized in that In the first mixture, the bacterial density of the first bacteria to be screened is 1×10 4 CFU / mL~4×10 5 CFU / mL; And / or, in the first mixture, the concentration of the cationic antimicrobial peptide is 0.1 μM~1000 μM.

3. The screening method according to claim 1, characterized in that The cationic antimicrobial peptide is LL-37.

4. The screening method according to claim 1, characterized in that In step A1, the culture plate is divided into several areas, and each area corresponds to one sample.

5. The screening method according to claim 4, characterized in that The shape of the area is selected from a grid shape and a fan shape.

6. The screening method according to claim 1, characterized in that The method for preparing the metabolites of the second bacteria to be screened comprises the following steps: The second bacteria to be screened are cultured to obtain a culture solution; the culture solution is diluted to a desired bacterial density, and the bacterial bodies are removed to obtain the metabolites.

7. The screening method according to claim 1, characterized in that In the second mixture, the bacterial density of Staphylococcus aureus is 1×10 4 CFU / mL~4×10 5 CFU / mL.

8. A probiotic, characterized in that: Obtained by screening according to any one of claims 1 to 7.

9. Use of the probiotics according to claim 8 in any one of items B1) to B3); B1) preparing a product resistant to the cationic antimicrobial peptides described in claim 1; B2) preparing products that kill and / or inhibit Staphylococcus aureus; B3) Kill and / or inhibit Staphylococcus aureus.

10. The use according to claim 9, characterized in that: The product is selected from a reagent, a kit or a medicine.

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