A strain of Corynebacterium striatum NMGCS43 and its application

Abstract

The present invention relates to the field of microorganisms, and in particular to a strain of Corynebacterium striatum NMGCS43 and its application. The present invention provides a strain of Corynebacterium striatum NMGCS43, and the preservation number of the Corynebacterium striatum NMGCS43 is CGMCC NO.31063. The supernatant of the strain can effectively inhibit the growth of MRSA, and the supernatant of the strain can also be extracted to obtain L-tryptophan and 6"-O-acetyl genistin, which provides a new way to obtain L-tryptophan and 6"-O-acetyl genistin; at the same time, the present invention first discovered that L-tryptophan and 6"-O-acetyl genistin have the effect of inhibiting the growth of Staphylococcus aureus, providing a new research direction for the research on inhibiting the growth of Staphylococcus aureus.

Description

Technical Field

[0001] The invention relates to the field of microorganisms, in particular to a Corynebacterium striatum NMGCS43 and an application thereof. Background Art

[0002] Staphylococcus aureus is one of the important pathogens that cause infections in the community and inpatients. The nasal cavity is the most common site of Staphylococcus aureus colonization. Under certain conditions, it can migrate to other parts of the body and cause various types of infections. Staphylococcus aureus colonization is considered an important risk factor for Staphylococcus aureus infection. The host immune function, strain virulence and the interaction between the two can all affect the colonization status of Staphylococcus aureus.

[0003] In order to prevent and treat diseases caused by Staphylococcus aureus, the harm of the bacteria can be reduced from two aspects: clinical treatment and environmental prevention. In terms of clinical treatment, antibiotics are mainly used for treatment. However, in recent years, with the increase in the dosage, frequency and range of antibiotics in clinical treatment, the degree of variation of drug-resistant Staphylococcus aureus has increased, and the resistance time has been shortened. The therapeutic effect of the original antibiotics has been greatly reduced, and the number of drug-resistant species of the bacteria has increased and the drug resistance has also increased, resulting in a high infection rate of drug-resistant strains of Staphylococcus aureus. Given that Staphylococcus aureus is widely distributed in the environment, controlling the number and survival of Staphylococcus aureus in the environment is an effective way to prevent infection from the source. Therefore, environmental disinfection is very important to reduce the infection of such pathogenic microorganisms. At present, the disinfection of pathogens in the environment is mainly carried out by chemical disinfectants, and there are also enzymes that inhibit bacteria, and the use of antibacterial microorganisms with antibacterial activity to inhibit Staphylococcus aureus.

[0004] However, common chemical disinfectants such as quaternary ammonium compounds, phenols, aldehydes, oxidants, and guanidines have experienced decades of development, and bacteria have gradually developed resistance to disinfectants. In addition, these chemical reagents need to be extracted, and the preparation process is complex and costly. There is a risk of polluting the environment with waste liquid and chemical byproducts, which is not green and environmentally friendly; the enzymatic antibacterial production process is harsh and complex and the cost is too high in application; and the use of microorganisms with antibacterial activity to inhibit Staphylococcus aureus, that is, "using bacteria to treat bacteria", is an economical and effective method. Other microbial flora that coexist with Staphylococcus aureus in the same part can affect the various biological activities of Staphylococcus aureus through non-direct contact with the bacteria, including Corynebacterium, Propionibacterium, coagulase-negative Staphylococcus, etc. Corynebacterium striata is a Gram-positive rod-shaped bacillus, which is often considered to be one of the normal flora of human skin and mucous membranes. At this stage, no microorganisms with good inhibitory effects on Staphylococcus aureus have been found. In order to enrich microbial resources, the present invention intends to develop a microbial preparation to effectively inhibit the growth of Staphylococcus aureus. Summary of the invention

[0005] The purpose of the present invention is to provide a strain of Corynebacterium striatum NMGCS43 and its application to solve the problems existing in the above-mentioned prior art. The present invention provides a strain of Corynebacterium striatum NMGCS43, the supernatant of which can effectively inhibit the growth of Staphylococcus aureus, and the supernatant of the strain can also extract L-tryptophan and 6″-O-acetyl genistin, which can also be used to inhibit the growth of Staphylococcus aureus.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The invention provides a Corynebacterium striatum NMGCS43, and the preservation number of the Corynebacterium striatum NMGCS43 is CGMCC NO.31063.

[0008] The present invention provides the use of the above-mentioned Corynebacterium striatum NMGCS43 in the preparation of a biological preparation for inhibiting Staphylococcus aureus.

[0009] The present invention provides a biological preparation, wherein the effective ingredients of the biological preparation include the above-mentioned Corynebacterium striatum NMGCS43.

[0010] The present invention provides the use of the above-mentioned Corynebacterium striatum NMGCS43 in the preparation of L-tryptophan and / or 6"-O-acetylgenistin.

[0011] The present invention provides a method for preparing L-tryptophan and 6″-O-acetyl genistin, comprising the following steps:

[0012] After centrifuging the bacterial solution of Corynebacterium truncatum NMGCS43, a supernatant is obtained; the deposit number of Corynebacterium truncatum NMGCS43 is CGMCC NO.31063;

[0013] The supernatant is subjected to adsorption, separation and purification in sequence to obtain the L-tryptophan and the 6″-O-acetyl genistin.

[0014] Preferably, the adsorption comprises the step of adsorbing the supernatant using a macroporous resin.

[0015] Preferably, the separation comprises the steps of detecting by liquid chromatography, collecting a solution having L-tryptophan and no impurity peaks and a solution having 6″-O-acetyl genistin and no impurity peaks;

[0016] The purification comprises the steps of performing liquid chromatography detection on the separated solution, collecting a solution containing L-tryptophan and no impurity peaks and a solution containing 6″-O-acetyl genistin and no impurity peaks.

[0017] Further preferably, the preparation method of the bacterial liquid of Bacillus striatus NMGCS43 comprises picking a single colony of Bacillus striatus into TSB culture medium, placing it in a shaking incubator containing 5% CO2, and shaking and culturing it at 37°C and 220rpm for 10 hours, then transferring the obtained bacterial suspension to fresh TSB broth culture medium with a pipette, sealing it with a disposable breathable sealing film, and placing it in a shaking incubator containing 5% CO2, and shaking and culturing it at 37°C and 220rpm for 48 hours.

[0018] The production of small molecule preparations (L-tryptophan and 6″-O-acetyl genistin) was increased by improving the culture conditions of Corynebacterium striatum NMGCS43 bacterial suspension.

[0019] The present invention provides L-tryptophan and 6″-O-acetyl genistin prepared by the preparation method.

[0020] The present invention provides the use of the above-mentioned L-tryptophan and 6″-O-acetyl genistin in preparing a small molecule inhibitor capable of inhibiting Staphylococcus aureus.

[0021] The present invention provides a small molecule inhibitor, wherein the effective component of the small molecule inhibitor comprises the above-mentioned L-tryptophan and / or 6"-O-acetyl genistin.

[0022] The present invention also preferably provides a method for inhibiting the growth of Staphylococcus aureus for non-therapeutic purposes, comprising the following steps:

[0023] (1) Preparation of plate

[0024] Place an autoclaved silicone gasket (8 mm in diameter × 1.5 mm in thickness) in the center of the culture dish, place the Oxford cup on the gasket, pour TSA culture medium containing 0.8% Tween-80, wait for it to cool and solidify, remove the Oxford cup with sterilized tweezers, and place a blank drug-sensitive paper in the center of the well;

[0025] (2) Plate antibacterial test

[0026] Pick a single colony of Corynebacterium striatum NMGCS43 into TSB culture medium, place it in a shaking incubator containing 5% CO2, and culture it at 37℃, 220rpm for 10 hours. Then prepare a 0.5 McFarland's bacterial suspension, use a pipette to draw up the prepared bacterial suspension, carefully drop it onto the drug-sensitive paper, place it upright in a CO2 constant temperature incubator, and culture it at 37℃ for 48 hours.

[0027] Pick a single colony of Staphylococcus aureus into TSB culture medium, place it in a shaking incubator containing 5% CO2, and culture it at 37℃ and 220rpm for 10 hours. Then prepare a 0.5 McFarland's bacterial suspension, dilute it 100×, and use a disposable cotton swab to evenly spread it on a plate of Corynebacterium streaked that has been grown for 48 hours. Place it in a common constant temperature incubator at 37℃ for 14-18 hours, and observe the inhibition zone.

[0028] The antibacterial effect can be improved by the double-layer punching method of the Oxford cup.

[0029] The present invention discloses the following technical effects:

[0030] The present invention provides a Corynebacterium striatum NMGCS43, the preservation number of the Corynebacterium striatum NMGCS43 is CGMCC NO.31063. The supernatant of the strain can effectively inhibit the growth of MRSA, and the supernatant of the strain can also extract L-tryptophan and 6"-O-acetyl genistin, which provides a new way to obtain L-tryptophan and 6"-O-acetyl genistin; at the same time, the present invention firstly finds that L-tryptophan and 6"-O-acetyl genistin have the effect of inhibiting the growth of Staphylococcus aureus, which provides a new research direction for the research on inhibiting the growth of Staphylococcus aureus. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] 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 work.

[0032] Figure 1 This is a plate inhibition test - an experiment of Corynebacterium taeniae inhibiting Staphylococcus aureus; AH are 8 clinical strains of Corynebacterium taeniae that have significant antibacterial activity against Staphylococcus aureus MRSA N315 strain;

[0033] Figure 2 The experimental results of method 1 in Example 2; wherein AF are 6 strains of Corynebacterium striatum;

[0034] Figure 3 The experimental results of method 2 in Example 2 are shown in FIG. 1 , wherein AD are 4 strains of Corynebacterium striatum ;

[0035] Figure 4 The experimental results of method 3 in Example 2; wherein AH are 8 strains of Corynebacterium striatum;

[0036] Figure 5 The growth colony counts of Staphylococcus aureus N315 under the action of different concentration gradients of Corynebacterium CFCS; A is the positive control of Staphylococcus aureus MRSA N315, BD are the results of the action of 20%, 30% and 50% of the final concentration of Corynebacterium CFCS components on Staphylococcus aureus MRSA N315 strains, E is the colony count of each treatment, compared with the control group, **P < 0.01;

[0037] Figure 6 This is the HPLC spectrum of the crude extract of Corynebacterium striatum CFCS;

[0038] Figure 7 is the HPLC spectrum of L-tryptophan;

[0039] Figure 8 is the HPLC spectrum of 6″-O-acetyl genistin;

[0040] Fig. 9 is the chemical structure diagram of L-tryptophan;

[0041] Fig.10 Schematic diagram of the structure of L-tryptophan;

[0042] Fig.11 The chemical structure diagram of 6″-O-acetyl genistin;

[0043] Fig.12 is a schematic diagram of the structure of 6″-O-acetyl genistin;

[0044] Fig.13 Schematic diagram of key HMBC and 1H-1H COSY related signals in L-tryptophan;

[0045] Fig.14 Schematic diagram of key HMBC and 1H-1H COSY related signals in 6″-O-acetyl genistin;

[0046] Fig.15 The results of the plate antibacterial test of three groups of components separated from the crude extract of Corynebacterium striatum CFCS are shown in Figure 1. Among them, A is the positive control of Staphylococcus aureus, B is L-tryptophan, and C is 6″-O-acetyl genistin.

[0047] Fig.16 The results of the L-tryptophan bacterial quantitative experiment are shown in Figure 1. A is the positive control of Staphylococcus aureus MRSA N315, and B and D are the results of the action of 20%, 30% and 50% final concentration of L-tryptophan on Staphylococcus aureus MRSA N315 strains, respectively. E is the colony count of each treatment, compared with the control group, **P < 0.01;

[0048] Fig.17 Effect of L-tryptophan on the proliferation activity of Staphylococcus aureus MRSA N315-sarA deletion strain and Staphylococcus aureus MRSA N315-△sarA complemented strain; A is the positive control of Staphylococcus aureus MRSA N315-△sarA complemented strain, BD are 50μg / mL, 75μg / mL and 100μg / mL L-tryptophan acting on Staphylococcus aureus N315-△sarA complemented strain respectively; E is the positive control of Staphylococcus aureus MRSA N315-sarA deletion strain, F, G, H are 50μg / mL, 75μg / mL and 100μg / mL L-tryptophan acting on Staphylococcus aureus N315-sarA deletion strain respectively, compared with the positive control group of Staphylococcus aureus MRSA N315-△sarA complemented strain, **P<0.01, n=3;

[0049] Fig.18 The colony counts of MRSA N315-sarA deletion strain and MRSA N315-△sarA complemented strain of Staphylococcus aureus under the action of L-tryptophan; A is the positive control of MRSA N315-△sarA complemented strain of Staphylococcus aureus, and BD are 50μg / mL, 75μg / mL and 100μg / mL L-tryptophan acting on MRSA N315-△sarA complemented strain of Staphylococcus aureus, respectively; E is the positive control of MRSA N315-sarA deletion strain of Staphylococcus aureus, and F, G and H are 50μg / mL, 75μg / mL and 100μg / mL L-tryptophan acting on MRSA N315-sarA deletion strain of Staphylococcus aureus, respectively. Compared with the positive control group of MRSA N315-△sarA complemented strain of Staphylococcus aureus, **P<0.01, n=3;

[0050] Fig.19 The antibacterial effects of different elution fractions are shown in Figure 2; from left to right, they are the positive control, the 50% elution fraction, and the 100% elution fraction.

[0051] Strain deposit information

[0052] Corynebacterium striatum NMGCS43, the strain was deposited in the General Microbiology Center of China Culture Collection Administration on June 24, 2024. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.31063. DETAILED DESCRIPTION

[0053] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0054] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0055] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0056] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0057] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0058] Example 1

[0059] 1. Strain source

[0060] The Corynebacterium striatum used in this study originated from routine specimens sent by the Affiliated Hospital of Inner Mongolia Medical University from February 2020 to February 2022. A total of 20 clinical isolates of Corynebacterium striatum were obtained and stored at -80°C.

[0061] 2. Strain Identification

[0062] (1) The MALDI-TOF mass spectrometry method was used to identify and confirm the Corynebacterium truncatum selected in this example. The following are the basic experimental steps: Take the strain to be tested out of the -80°C refrigerator and resuscitate it at room temperature. Dip the bacterial solution with a sterilized inoculation loop, inoculate it on the blood plate using the three-zone streak method, and place it in a 37°C constant temperature CO2 incubator for overnight culture. The next day, use a disposable inoculation loop to pick up an appropriate amount of fresh monoclonal and spread it evenly on the target plate. After it dries, add 1μL of formic acid solution. After standing and drying at room temperature, use a micropipette to draw 1μL of matrix and drop it into the target point. After standing and drying completely, place the strain to be tested in the MALDI-TOF MS mass spectrometer and test it on the machine. The results showed that the selected clinical strains were all Corynebacterium truncatum.

[0063] (2) The plate inhibition method was used to screen clinical strains of Corynebacterium striata that had significant activity in inhibiting the proliferation of Staphylococcus aureus. The results are shown in Tables 1 and Figure 1 shown.

[0064] Table 1 Inhibition rate of different clinical strains of Corynebacterium striata against Staphylococcus aureus

[0065] strain Inhibition zone (mm) A 18 B 17 C 16 D 16 E 25 F 25 G 32 H 22

[0066] From Table 1 and Figure 1 It can be seen that 8 of the 20 clinical isolates of Corynebacterium striatum had significant antibacterial activity against Staphylococcus aureus MRSAN315 (e.g. Figure 1 Among them, one strain has the best effect ( Figure 1 G in, i.e., Corynebacterium striatum NMGCS43), so this clone strain of Corynebacterium striatum was selected for subsequent experiments.

[0067] In summary, the dominant bacteria screened in step (2) is Corynebacterium striatum, which is named Corynebacterium striatum NMGCS43. The strain was deposited in the General Microbiology Center of China Culture Collection Administration on June 24, 2024. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.31063.

[0068] Example 2 Screening of culture conditions

[0069] 1. Experimental methods

[0070] Method 1: The antibacterial experiment was carried out using the single-layer agar plate method. For specific steps, refer to the literature "Corynebacterium accolens Has Antimicrobial Activity against Staphylococcus aureus and Methicillin-Resistant S.aureus Pathogens Isolated from the Sinonasal Niche of Chronic Rhinosinusitis Patients" (Menberu MA, Liu S, Cooksley C, Hayes AJ, PsaltisAJ, Wormald PJ, Vreugde S.Corynebacterium accolens Has Antimicrobial Activity against Staphylococcus aureus and Methicillin-Resistant S.aureus PathogensIsolated from the Sinonasal Niche of Chronic Rhinosinusitis Patients. Pathogens. 2021Feb 14;10(2):207.doi:10.3390 / pathogens10020207.PMID:33672855; PMCID:PMC7918835);

[0071] Method 2: (1) Preparation of plates: Place a high-pressure sterilized silicone gasket (8 mm in diameter × 1.5 mm in thickness) in the center of an empty culture dish with a diameter of 90 mm, place an Oxford cup on the gasket, pour 10 mL of TSA culture medium, wait for it to cool and solidify, remove the Oxford cup with sterilized tweezers, and place a blank drug-sensitive paper in the center of the hole; (2) Plate antibacterial experiment: Pick a single colony of Corynebacterium striata NMGCS43 into 3 mL of TSB culture medium, place it in a shaking incubator containing 5% CO2, and culture it at 37°C, 220 rpm overnight. The next day, prepare a 0.5 ml suspension of Staphylococcus aureus, use a pipette to draw 20 μL of the prepared bacterial suspension, and carefully drop it into the drug-sensitive paper. Place it upright in a CO2 constant temperature incubator at 37°C and culture it for 48 hours. Pick a single colony of Staphylococcus aureus into 3 mL of TSB culture medium, place it in a shaking incubator containing 5% CO2, and culture it at 37°C, 220 rpm overnight. The next day, prepare 0.5 McBacteria suspension and dilute it 100×. Use a disposable cotton swab to evenly apply it on the plate of Corynebacterium striata that has been grown for 48 hours, place it in a common constant temperature incubator at 37°C, culture for 24 hours, and observe the inhibition zone.

[0072] Method 3: This method is a modified Oxford cup punching method, and the steps are as follows: (1) Preparation of a plate: Place a high-pressure sterilized silicone gasket (diameter 8mm×thickness 1.5mm) in the center of an empty culture dish with a diameter of 90mm, place the Oxford cup on the gasket, pour 10mL TSA culture medium (containing 0.8% Tween-80), wait for it to cool and solidify, remove the Oxford cup with sterilized tweezers, and place a blank drug-sensitive paper in the center of the hole; (2) Plate antibacterial experiment: Pick a single colony of Corynebacterium striata NMGCS43 into 3mL TSB culture medium, place it in a shaking incubator containing 5% CO2, 37°C, 220rpm shaking and culture overnight. The next day, prepare a 0.5 ml suspension of bacteria, use a pipette to draw 20μL of the prepared bacterial suspension, and carefully drop it into the drug-sensitive paper. Place it upright in a CO2 constant temperature incubator at 37°C and culture for 48h. Pick a single colony of Staphylococcus aureus and add it to 3mL TSB culture medium. Place it in a shaking incubator containing 5% CO2, 37℃, 220rpm shaking and culture overnight. The next day, prepare 0.5Merck's suspension and dilute it 100×. Use a disposable cotton swab to evenly apply it on a plate of Corynebacterium striata that has been grown for 48 hours, place it in a normal constant temperature incubator, 37℃, culture for 14 hours, and observe the inhibition zone.

[0073] 2. Experimental results:

[0074] The experimental results are as follows Figure 2-Figure 4 It can be seen that method 3 has the best antibacterial effect, so method 3 is selected as the optimal culture condition.

[0075] Example 2 Inhibitory effect of different concentration gradients of Corynebacterium striata supernatant on Staphylococcus aureus

[0076] (1) Preparation of plates: Place a sterilized silicone gasket (8 mm in diameter × 1.5 mm in thickness) in the center of an empty culture dish with a diameter of 90 mm, place an Oxford cup on the gasket, pour 10 mL of TSA culture medium (containing 0.8% Tween-80), wait for it to cool and solidify, remove the Oxford cup with sterilized tweezers, and place a blank drug-sensitive paper in the center of the hole; (2) Plate antibacterial experiment: Pick a single colony of Corynebacterium striata NMGCS43 and add it to 3 mL of TSB culture medium, place it in a shaking incubator containing 5% CO2, and culture it at 37°C, 220 rpm for 10 hours, then centrifuge it at 7000 rpm for 5 minutes to obtain the supernatant of Corynebacterium striata, i.e., Corynebacterium striata CFCS. Use a pipette to draw 20 uL of the supernatant with a final concentration of 20%, 30%, or 50%, and carefully drop it into the drug-sensitive paper. Place it upright in a CO2 constant temperature incubator at 37°C for 48 hours. Pick a single colony of Staphylococcus aureus and add it to 3mL TSB culture medium. Place it in a shaking incubator containing 5% CO2, and culture it overnight at 37℃ and 220rpm. The next day, prepare 0.5Merck's suspension and dilute it 100×. Use a disposable cotton swab to evenly apply it on a plate of Corynebacterium striata that has been grown for 48 hours, place it in a normal constant temperature incubator, culture it at 37℃ for 18 hours, and observe the inhibition zone. The results are as follows. Figure 5 As shown, different final concentrations of Corynebacterium striatum CFCS all had an inhibitory effect.

[0077] Example 4 Isolation of L-tryptophan or 6″-O-acetyl genistin

[0078] 1. Preparation of Corynebacterium striata NMGCS43 bacterial suspension

[0079] Pick a single colony of Corynebacterium striata NMGCS43 into 5 mL TSB culture medium, place it in a shaking incubator containing 5% CO2, and shake and culture it at 37°C and 220 rpm overnight. The next day, transfer 5 mL of the bacterial suspension to fresh 500 mL TSB broth with a pipette, seal it with a disposable breathable sealing film, and place it in a shaking incubator containing 5% CO2, and shake and culture it at 37°C and 220 rpm for 48 hours.

[0080] 2. Separation and preparation of small molecule components

[0081] 2.1. Centrifugation of bacterial liquid

[0082] The bacterial solution was centrifuged (centrifugal conditions: centrifugal force 7000g, centrifugal time 5min), and after centrifugation, the precipitate was discarded and the supernatant was retained.

[0083] 2.2. Adsorption of pre-treated supernatant by porous adsorption resin

[0084] First, a 50×800mm glass column was filled with a macroporous resin (Mitsubishi HP-20) pre-cleaned with alcohol about 60cm high, and then washed with water until there was no ethanol smell. Then, 4.5L of filtered and clarified fermentation liquid was loaded onto the macroporous adsorption resin. After loading, it was washed with water until the color was lighter, and then eluted with 50% ethanol (2L) and 100% alcohol in turn, and the eluted solution was collected. After rotary evaporation concentration at room temperature, it was freeze-dried.

[0085] 2.3. Reserve 100% alcohol elution fraction after activity test

[0086] Add 100 μL of elution solution to the center of the MH plate and let it stand for 30 minutes to dry. Prepare 0.5 McLaughlin Staphylococcus aureus suspension, dilute 100×, and evenly apply it on the MH plate with a disposable cotton swab. Place it in a constant temperature incubator at 37°C and culture for 14 hours. Observe the antibacterial effect. The results are as follows Fig.19 As shown, the activity of the 100% alcohol elution fraction was higher than that of the 50% ethanol elution fraction, therefore, the 100% alcohol elution fraction was reserved.

[0087] 2.4. Crude separation and preparation of target compounds

[0088] (1) Chromatographic conditions: Hanbang high pressure preparative chromatograph DAC100, computer: HP, chromatographic column: 100×250 mm DAC chromatographic column, filler: YMC C18, 10 μm, wavelength: 210 nm, flow rate: 80 mL / min. Mobile phase A: acetonitrile, mobile phase B: aqueous solution containing 0.1% acetic acid.

[0089] (2) Solution preparation: Dissolve the crude extract (100% alcohol elution fraction) in 45 mL of 80% acetonitrile, filter through a 0.45 μm filter membrane, inject 15 mL / injection, repeat three injections, and the HPLC spectrum is as follows: Figure 6 . Collect the solution in separate bottles, and collect the peaks according to the chromatogram, that is, collect the solution with L-tryptophan and no impurity peaks and the solution with 6"-O-acetylgenistin and no impurity peaks, to obtain L-tryptophan and 6"-O-acetylgenistin ( Figure 7-Figure 8 ), concentrated and then freeze-dried to obtain the active component. Among them, the molecular formula of L-tryptophan is C 11 H 12 N2O2, molecular weight is 204.23, chemical results are as follows Fig. 9 As shown, the structural diagram is as follows Fig.10 As shown; the molecular formula of 6″-O-acetyl genistin is C 23 H 22 O 11 , molecular weight is 474.42, chemical results are as follows Fig.11 As shown, the structural diagram is as follows Fig.12 shown.

[0090] 2.4. Secondary purification and identification of target compounds

[0091] The active component was dissolved in 15 mL of 80% acetonitrile, filtered with a 0.45 μm filter membrane, and injected. The samples were collected in separate bottles, and the peaks were collected according to the chromatogram, i.e., the solution with L-tryptophan and no impurity peaks and the solution with 6″-O-acetyl genistin and no impurity peaks. The separated substances were then tested by hydrogen spectrum, carbon spectrum, DEPT spectrum, 1H-1H COSY spectrum, HSQC spectrum, HMBC spectrum, and NOESY spectrum using a 600 MHz nuclear magnetic resonance instrument, and the identified components were concentrated and freeze-dried for storage. The results are shown in FIG. Figure 13-Figure 14 As shown in Tables 2 to 5.

[0092] Table 2h1 Component 1H & 13C-NMR data

[0093]

[0094] Table 3h1 NMR spectrum data and structural assignment

[0095]

[0096] Table 4h2 Component 1H & 13C-NMR data

[0097]

[0098]

[0099] Table 5h2 NMR spectrum data and structural assignment

[0100]

[0101] Example 3 Results of plate antibacterial test of L-tryptophan and 6″-O-acetyl genistin

[0102] (1) Preparation of plates: Place a sterilized silicone gasket (8 mm in diameter × 1.5 mm in thickness) in the center of an empty culture dish with a diameter of 90 mm, place an Oxford cup on the gasket, pour 10 mL of TSA medium (containing 0.8% Tween-80), wait for it to cool and solidify, remove the Oxford cup with sterilized tweezers, and place a blank drug-sensitive paper in the center of the well;

[0103] (2) Plate antibacterial test: Pick a single colony of Staphylococcus aureus and add it to 3 mL TSB culture medium. Place it in a shaking incubator containing 5% CO2, 37°C, 220 rpm shaking and culture overnight. The next day, prepare 0.5 Staphylococcus aureus suspension, dilute it 100×, then add 100 μL of L-tryptophan or 6″-O-acetyl genistin, place it in a common constant temperature incubator, 37°C, culture for 18 hours, and observe the inhibition zone. The results are as follows: Fig.15 As shown, L-tryptophan and 6″-O-acetyl genistin can effectively inhibit the growth of Staphylococcus aureus.

[0104] Example 4 Bacterial Quantification Experimental Test

[0105] The bacterial quantitative experiment was carried out on L-tryptophan. The experimental steps were the same as in Example 2. The results are as follows: Fig.16 As shown, L-tryptophan can significantly inhibit the growth of Staphylococcus aureus.

[0106] Example 5 Intrinsic Mechanism of L-Tryptophan Inhibiting MRSA Growth

[0107] 1. Experimental methods:

[0108] Homologous recombination was used to construct a strain with sarA deletion in the regulatory gene of Staphylococcus aureus N315 (MRSA N315-sarA deletion strain), and a complementation strain (MRSA N315-△sarA complementation strain) was constructed by restriction ligation. The effect of the active antibacterial components derived from CFCS of Corynebacterium striata on the in vitro proliferation activity of the deletion strain and complementation strain was detected by broth microdilution method.

[0109] 2. Experimental results:

[0110] The results showed that at different concentrations of 50 μg / mL, 75 μg / mL and 100 μg / mL L-tryptophan, L-tryptophan could not inhibit the proliferation of the N315-sarA gene deletion strain of Staphylococcus aureus compared with the control group (P>0.05); compared with the control group, L-tryptophan had a significant inhibitory effect on the proliferation activity of the N315-△sarA complemented strain (P<0.01). The sarA deletion strain can relieve the inhibition of L-tryptophan on the proliferation activity of Staphylococcus aureus ( Fig.17 and Fig.18 ).

[0111] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A strain of Corynebacterium striatum NMGCS43, characterized in that: The deposit number of the Corynebacterium striatum NMGCS43 is CGMCC NO.31063.

2. Use of the Corynebacterium striatum NMGCS43 described in claim 1 in the preparation of a biological agent for inhibiting Staphylococcus aureus.

3. A biological agent, characterized in that: The active ingredient of the biological preparation includes the Corynebacterium striatum NMGCS43 described in claim 1.

4. Use of the Corynebacterium striatum NMGCS43 according to claim 1 in the preparation of L-tryptophan and / or 6"-O-acetylgenistin.

5. A method for preparing L-tryptophan and 6″-O-acetyl genistin, characterized in that: The following steps are involved: After centrifuging the bacterial solution of Corynebacterium truncatum NMGCS43, a supernatant is obtained; the deposit number of Corynebacterium truncatum NMGCS43 is CGMCC NO.31063; The supernatant is subjected to adsorption, separation and purification in sequence to obtain the L-tryptophan and the 6″-O-acetyl genistin.

6. The preparation method according to claim 5, characterized in that: The adsorption comprises the step of using a macroporous resin to adsorb the supernatant.

7. The preparation method according to claim 5, characterized in that: The separation comprises the steps of detecting by liquid chromatography, collecting a solution containing L-tryptophan and no impurity peaks and a solution containing 6″-O-acetyl genistin and no impurity peaks; The purification comprises the steps of performing liquid chromatography detection on the separated solution, collecting a solution containing L-tryptophan and no impurity peaks and a solution containing 6″-O-acetyl genistin and no impurity peaks.

8. Use of L-tryptophan and 6"-O-acetyl genistin prepared by the preparation method according to any one of claims 5 to 7 in the preparation of a small molecule inhibitor for inhibiting Staphylococcus aureus.

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