Small-molecular bacteriostatic active component as well as preparation method and application thereof

Abstract

The invention discloses a micromolecular bacteriostatic active component as well as a preparation method and application thereof. Bacteriostatic active molecular substances generated by metabolism of bacillus subtilis are separated, the structures of the bacteriostatic active molecular substances are different from those of existing known compounds through analysis, and further analysis shows that the obtained micromolecular bacteriostatic active components have broad-spectrum bacteriostatic ability and have the biological characteristics of high temperature resistance, acid resistance, protease resistance and the like.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and in particular to a small molecule antibacterial active ingredient and a preparation method and use thereof. Background Art

[0002] The misuse of antibiotics in animal feed may lead to antibiotic residues and bacterial resistance in animal products, which is harmful to humans. Probiotics are small molecule polypeptide substances with biological activity. Studies have found that probiotics can improve the growth performance of the body, enhance the body's immunity and improve the structure of intestinal microbial flora. They can also regulate meat quality and treat diseases. And as a new type of feed additive, probiotics can replace the use of antibiotics in production, solve the problem of antibiotic residues, and protect environmental safety and human health.

[0003] Bacillus subtilis (BS), as an important probiotic, can secrete a variety of secondary metabolites with antibacterial properties. Previous studies have found that Bacillus subtilis BS-21 has antibacterial activity molecules including bacillibactin, subtilosin A, bacilysin, fengycin, surfactin, and zwittermicin A as antimicrobial peptides, and bacillaene as a polyketide. For details, see Chinese patent application CN116024146A, the contents of which are incorporated herein by reference in their entirety.

[0004] However, the direct addition of Bacillus subtilis as a feed additive cannot guarantee the stability of the growth environment of Bacillus subtilis. It is easily affected by factors such as feed quality and storage conditions, which affects the growth effect of Bacillus subtilis and further affects the antibacterial activity of Bacillus subtilis. Therefore, it is currently necessary to solve the problem of purifying the active substances of Bacillus subtilis to ensure the stability of the active substances and facilitate the further identification and development of new antibacterial active ingredients. Summary of the invention

[0005] In order to solve at least some of the problems in the prior art, the present invention provides a small molecule antibacterial active ingredient and a preparation method and use thereof. Specifically, the present invention includes the following contents.

[0006] In a first aspect of the present invention, a small molecule antibacterial active ingredient is provided, which is extracted from the fermentation supernatant of Bacillus subtilis by a method comprising the following steps:

[0007] (1) adding a protein precipitant to the fermentation supernatant to obtain a precipitate, and further extracting the precipitate with an alcohol solvent to obtain a supernatant containing alcohol-soluble components;

[0008] (2) concentrating the supernatant containing the alcohol-soluble component to obtain a precipitate, and dissolving it in water to obtain an aqueous solution;

[0009] (3) Purifying the aqueous solution through an anion column and a cation column in sequence to obtain the small molecule antibacterial active ingredient.

[0010] In certain embodiments, according to the small molecule antibacterial active ingredient of the present invention, wherein, in the step (1), before adding the protein precipitant, the step further comprises centrifuging the fermentation supernatant to remove impurities;

[0011] Add ammonium sulfate to a concentration of 65-75% under stirring at a temperature of 0-10°C to precipitate the protein; or

[0012] The temperature of the alcohol solvent extraction is 0-10°C, and after the extraction, a supernatant containing alcohol-soluble components is obtained by further centrifugal separation.

[0013] In certain embodiments, according to the small molecule antibacterial active ingredient of the present invention, in the step (2), the supernatant containing the alcohol-soluble component is concentrated at 25-50° C. to obtain a precipitate, and the water-soluble precipitate is further filtered to obtain an aqueous solution.

[0014] In certain embodiments, according to the small molecule antibacterial active ingredient of the present invention, in step (3), the anion column purification conditions include:

[0015] a. Elution and collection of equilibrium solution QA: flow rate 0.5-2.5 ml / min, elution 6-8 column volumes, during which samples are collected according to the UV signal peak. When the UV value tends to be flat, stop collecting and continue to balance the system to wash the UV flow-through peak back to the baseline;

[0016] b. Elution and collection with eluent QB: flow rate 0.5-2.5 ml / min, select gradient target QB from 0% to 100% to start elution, and collect the flow-through peak components that are not bound to the anion column.

[0017] In certain embodiments, according to the small molecule antibacterial active ingredient described in the present invention, in step (3), the cationic column purification conditions include: using spA buffer to balance the system for 6-8 column volumes, setting the gradient target spB to start elution from 0% to 100%, and the elution peak begins to appear when the target spB reaches 20-30%, and collecting the corresponding components is the purified small molecule antibacterial active ingredient.

[0018] In certain embodiments, the small molecule antibacterial active ingredient according to the present invention further comprises a desalting step.

[0019] In certain embodiments, according to the small molecule antibacterial active ingredient of the present invention, the molecular weight of the small molecule antibacterial active ingredient is 424.20789 Da.

[0020] The second aspect of the present invention provides a method for preparing a small molecule antibacterial active ingredient.

[0021] The third aspect of the present invention provides an antibacterial method, which comprises the step of using the small molecule antibacterial active ingredient of the present invention.

[0022] The fourth aspect of the present invention provides use of the small molecule antibacterial active ingredient of the present invention in the preparation of an antibacterial agent.

[0023] The present invention provides a new small molecule antibacterial active substance with an accurate molecular weight of 424.20789, which is identified by ultra-high performance liquid chromatography-mass spectrometry chromatography experiments as having a different structure from currently known compounds. At the same time, analysis has found that the small molecule antibacterial active substance extracted by the present invention is an antibacterial substance with a broad-spectrum antibacterial ability and biological characteristics such as high temperature resistance, acid resistance and protease resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The colony morphology of Bacillus subtilis BS21 in Example 1 is shown.

[0025] Figure 2 The graph shows the antibacterial activity of the supernatant of Bacillus subtilis BS21 in Example 2.

[0026] Figure 3 The graph shows the antibacterial activity of the supernatant of Bacillus subtilis BS21 after ammonium sulfate precipitation in Example 2.

[0027] Figure 4 The flow diagram of the anion column purification of the AKTA protein purifier in Example 3 is shown.

[0028] Figure 5 The antibacterial activity graph of the anion column elution sample in Example 3 is shown.

[0029] Figure 6 The purification flow diagram of the AKTA protein purifier cation column in Example 3 is shown.

[0030] Figure 7 The antibacterial activity graph of the cationic column elution sample in Example 3 is shown.

[0031] Figure 8 The graph showing the antibacterial activity comparison of the antibacterial substance of Bacillus subtilis BS21 before and after purification in Example 3 is shown.

[0032] Fig. 9The graph showing the acid resistance of the antibacterial substance after purification in Example 4 is shown.

[0033] Fig.10 The graph showing the high temperature resistance of the antibacterial substance after purification in Example 4 is shown.

[0034] Fig.11 The graph showing the protease resistance test of the antibacterial substance after purification in Example 4 is shown.

[0035] Fig.12 The liquid chromatography HPLC purification chart in Example 5 is shown.

[0036] Fig.13 The antibacterial activity graph of the HPLC product in Example 5 is shown.

[0037] Fig.14 The figure shows the precise molecular weight of the antibacterial substance after purification by ultra-high performance liquid chromatography (UPLC) experimental analysis in Example 6.

[0038] Fig.15 A comparison chart of the antibacterial activity of the fermentation products of Bacillus subtilis BS21 at different time periods in Example 7 is shown. DETAILED DESCRIPTION

[0039] 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.

[0040] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that the upper and lower limits of the scope and each intermediate value therebetween are specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and 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.

[0041] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which the invention belongs. 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 related to the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0042] Small molecule antibacterial active ingredients

[0043] In one aspect of the present invention, a small molecule antibacterial active ingredient is provided, which is extracted from the fermentation supernatant of Bacillus subtilis by a method comprising the following steps:

[0044] (1) adding a protein precipitant to the fermentation supernatant to obtain a precipitate, and further extracting the precipitate with an alcohol solvent to obtain a supernatant containing alcohol-soluble components;

[0045] (2) concentrating the supernatant containing the alcohol-soluble component to obtain a precipitate, and dissolving it in water to obtain an aqueous solution;

[0046] (3) Purifying the aqueous solution through an anion column and a cation column in sequence to obtain the small molecule antibacterial active component. The separation and purification method of the present invention is described in detail below.

[0047] In the present invention, the small molecule antibacterial active ingredients are derived from the metabolites of Bacillus subtilis BS-21. The classification name of Bacillus subtilis BS-21 is Bacillus subtilis. The strain has been deposited in the General Microbiological Center of China Microbiological Culture Collection Committee (referred to as CGMCC, address No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101) on October 28, 2022, with the deposit number of CGMCC No. 25977. Previous studies have isolated and identified the following biomolecules with antibacterial activity, including:

[0048] (1) Bacillibactin, with a molecular weight of 882.78 and a molecular formula of C 39 H 42 N 6 O 18 , CAS number is 95536-41-7;

[0049] (2) Subtilosin A, with a molecular weight of 3015.44 and a molecular formula of C 129 H 208 N 36 O 41 S 3 , CAS number is 98914-01-3;

[0050] (3) Bacilysin, with a molecular weight of 270.282 and a molecular formula of C 12 H 18 N 2 O 5 , CAS number is 29393-20-2;

[0051] (4) Fengycin, with a molecular weight of 1463.71 and a molecular formula of C 12 H 18 N2 O 5 , CAS number is 102577-03-7;

[0052] (5) Surfactin, with a molecular weight of 1036.34 and a molecular formula of C 53 H 93 N 7 O 13 , CAS number is 24730-31-2;

[0053] (6) Zwittermicin A, with a molecular weight of 396.397 and a molecular formula of C 13 H 28 N 6 O 8 , CAS number is 155547-95-8;

[0054] (7) Bacillaene, with a molecular weight of 580.8 and a molecular formula of C 34 H 48 N 2 O 6 , Pubchem CID: 25144999.

[0055] Through in-depth research, the present invention has found that a small molecule antibacterial active ingredient with excellent stability and antibacterial (sometimes also called "antibacterial") properties can be obtained by separation and purification by a specific method, wherein the molecular weight of the small molecule antibacterial active ingredient is 424.20789Da.

[0056] In step (1) of the present invention, the fermentation supernatant is a culture of Bacillus subtilis BS21 in an optimized fermentation medium, and the optimized fermentation medium includes corn flour with a concentration of 1-10%, preferably 1-5% (e.g., 1, 2, 3, 4, 5%) as a suitable carbon source; soybean meal with a concentration of 1-10%, preferably 1-5% (e.g., 1, 2, 3, 4, 5%) as a suitable carbon source and NaCl with a concentration of 0.1-2%, preferably 0.1-1% (e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1%) as a suitable inorganic salt. Inoculate Bacillus subtilis BS21 with a suitable inoculum amount (preferably 1-10%, such as 1-5%), and then culture at 150-400 rpm (preferably 200-300 rpm, and also preferably 200-250 rpm) and 28-35° C. (preferably 30-35° C.) for 12-72 h (preferably 18-30 h).

[0057] The supernatant after fermentation is added with a protein precipitant to obtain precipitation. The protein precipitant can be any known substance capable of precipitating protein components, such as ammonium sulfate. In order to collect the supernatant, the culture is preferably centrifuged at 4000-5000 rpm (preferably 4200-4700 rpm, such as 4200, 4300, 4400, 4500, 4600, 4700 rpm) at low temperature (preferably 1-4° C.) for 20-60 min (preferably 30-50 min, also preferably 40-50 min, such as 40, 42, 44, 46, 48, 50 min), so that the effective active substance is present in the supernatant and impurities are removed. Ammonium sulfate particles are added to the supernatant while stirring at low temperature (preferably 1-4° C.) to make the final concentration reach 50-80%, preferably 70-80%, such as 71, 72, 73, 74, 75, 76, 77, 78, 79, 80%. The stirring time is 8-16h, preferably 9-15h, and also preferably 10-15h, such as 10, 11, 12, 13, 14, 15h. Further, by high-speed centrifugation, preferably 6000-10000rpm, also preferably 7000-9000rpm (such as 7000, 7500, 8000, 8500, 9000rpm) at low temperature (preferably 1-4°C) for 20-60min (preferably 30-50min, also preferably 40-50min, such as 40, 42, 44, 46, 48, 50min) to separate the precipitate and the supernatant. Preferably, the obtained precipitate is further resuspended and washed with an ammonium sulfate solution of the same concentration to further remove impurities.

[0058] The precipitate obtained above is added with an appropriate amount of alcohol solvent and mixed, and the alcohol solvent and protein are combined and precipitated in a low temperature environment, and separated from other components with good solubility. The alcohol solvent includes but is not limited to methanol, ethanol, isopropanol, etc. The mixed sample is centrifuged at 10000-15000 rpm, preferably 11000-14000 rpm, and preferably 12000-13000 rpm (e.g. 12000, 12200, 12400, 12600, 12800, 13000 rpm) at low temperature for 5-20 min, preferably 6-18 min, and preferably 10-18 min, such as 10, 12, 14, 16, 18 min.

[0059] In step (2) of the present invention, the spin-dried precipitate obtained by concentrating the supernatant at 28-35°C, preferably 30-35°C (e.g. 30, 31, 32, 33, 34, 35°C) using a centrifugal dryer is then dissolved in sterile water and filtered for sterilization.

[0060] Step (3) of the present invention is a metabolite purification step, and the buffer system used in the present invention comprises:

[0061] I. First equilibrium buffer: Tris buffer with a pH of 7.5-8.5 (preferably 7.6-8.4, such as 7.6, 7.8, 8.0, 8.2, 8.4), and a concentration of 10-30 mM, preferably 15-25 mM, and further preferably 20-25 mM, such as 20, 21, 22, 23, 24, 25 mM;

[0062] II. First elution buffer: Tris buffer with a pH of 7.5-8.5 (preferably 7.6-8.4, for example 7.6, 7.8, 8.0, 8.2, 8.4), a concentration of 10-30 mM, preferably 15-25 mM, and preferably 20-25 mM, for example 20, 21, 22, 23, 24, 25 mM, 0.5-2 M NaCl, preferably 0.8-1.2 M, for example 0.8, 0.9, 1.0, 1.1, 1.2 M;

[0063] III. A second equilibrium buffer: a pH of 5.0-5.5, for example 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, a concentration of 10-30 mM, preferably 15-25 mM, and preferably 20-25 mM, for example 20, 21, 22, 23, 24, 25 mM MES buffer;

[0064] IV. Second elution buffer: pH 5.0-5.5, for example 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, concentration 10-30mM, preferably 15-25mM, also preferably 20-25mM, for example 20, 21, 22, 23, 24, 25mM MES buffer, 0.5-2M NaCl, preferably 0.8-1.2M, for example 0.8, 0.9, 1.0, 1.1, 1.2M.

[0065] In a preferred embodiment of step (3), the metabolites are first purified using an anion column to obtain an active substance fraction that is not bound to the anion column. The anion chromatography medium is not particularly limited. In a specific embodiment, the filler used in the anion column is HITRAP Q HP, 5 mL (17-1154-01), and the first equilibration buffer is used to balance the column at a flow rate of 0.8-1.5 ml / min, preferably 0.8-1.2 ml / min (e.g., 0.8, 0.9, 1.0, 1.1, 1.2 ml / min). Subsequently, the first equilibration buffer is used to elute 6-8 column volumes at the above flow rate, and the UV flow-through peak is collected according to the flow-through UV peak. Then, the first eluent described herein is used to start elution at the above flow rate from 0% to 100%, and each elution peak is collected. Finally, the present invention determines through an antibacterial activity test that the active substance is located in the flow-through peak and the active substance is not bound to the anion column.

[0066] After collecting the sample flowing through the anion exchange column, further collect the cation column elution sample through the cation column purification step. The cation chromatography medium is not particularly limited. In a specific embodiment, the filler used in the cation column is HITRAPSP HP, 5mL (17-1152-01), and the flow rate of the second equilibration buffer is 0.8-1.5ml / min, preferably 0.8-1.2ml / min (e.g. 0.8, 0.9, 1.0, 1.1, 1.2ml / min). Specifically, the cation column is equilibrated with the second equilibration buffer for 6-8 column volumes, and then the second elution buffer is used to perform gradient elution from 0-100%, and an elution peak begins to appear at 25%, and the elution peak is collected, and the small molecule active substance and antibacterial activity are identified.

[0067] The small molecule active substance of the present invention has significantly improved antibacterial activity, acid resistance (still maintains high antibacterial activity when the pH is lower than 4), heat resistance (still maintains high antibacterial activity at a temperature higher than 60°C, for example, 60-90°C), and protease resistance (still maintains high antibacterial activity after protease treatment).

[0068] In order to further reduce the salt concentration, it is preferred to further perform a desalting step on the purified metabolite. Desalting can be performed using methods known in the art, such as HPLC-based methods. Specific parameters are not particularly limited and can be adjusted as needed.

[0069] In a preferred embodiment, mobile phases A and B are pure water and acetonitrile, respectively; flow rate 1-5 ml / min, for example 1, 2, 3, 4, 5 ml / min; high pressure limit 200-500 bar, for example 200, 300, 400, 500 bar; extraction and discharge speed 100-500 μl / min, for example 100, 200, 300, 400, 500 μl / min; injection volume 100-300 μl, preferably 200 μl; peak width greater than 0.0063 min (40 Hz); slit 4 nm; signal collection wavelength 220 nm-360 nm; using a ZORBAX SB-C18 chromatographic column.

[0070] In a specific embodiment, the collection retention time is 12-13min, especially 12.407min, the organic phase acetonitrile is 56%, the UV peak height is 679.9mAU, and the peak area is 4346. After the main substance peak is collected, it is further concentrated at 40-70°C, preferably 50-65°C (e.g. 50, 55, 60, 65°C) to remove the organic solvent acetonitrile.

[0071] Identification of metabolites can be carried out by methods known in the art, for example, mass spectrometry, liquid chromatography-mass spectrometry, mass spectrometry-liquid chromatography-gas chromatography, nuclear magnetic resonance, surface enhanced Raman spectroscopy, etc. can be used to determine the molecular weight, structure and other properties of the metabolites. The parameters involved in the determination method and the equipment used can be adjusted according to actual needs and are not particularly limited.

[0072] Preparation method of small molecule antibacterial active ingredient

[0073] One aspect of the present invention provides a large-scale or industrialized production method of small molecule antibacterial active ingredients, which includes high-density culture of Bacillus subtilis BS-21 and purification and extraction of the product based on the above purification and extraction method, wherein the raw materials for high-density culture include, based on a 50L fermentation tank, 2-10%, preferably 2-5%, most preferably 3.4% corn flour; 2-10%, preferably 2-5%, most preferably 2.89% soybean meal and 0.1-1, preferably 0.1-0.8, most preferably 0.5% NaCl.

[0074] Inoculate Bacillus subtilis BS21 with a suitable inoculum size (e.g. 1-10%, preferably 1-5%), and then culture at 150-400 rpm (preferably 200-300 rpm, also preferably 200-350 rpm, for example 200, 250, 300, 350 rpm) at 28-35° C. (preferably 30-35° C., for example 30, 31, 32, 33, 34, 35° C.) for 12-72 h (preferably 24-28 h, for example 24, 25, 26, 27, 28 h).

[0075] use

[0076] In one aspect of the present invention, the use of the small molecule antibacterial active ingredient of the present invention in the preparation of an antibacterial agent is provided. The small molecule antibacterial active ingredient of the present invention can be directly applied or can be mixed with any suitable carrier known in the art to prepare an antibacterial agent.

[0077] In a preferred embodiment, the small molecule antibacterial active ingredient of the present invention can be used in at least one of the following situations:

[0078] (1) preparing antibacterial drugs or compositions;

[0079] (2) Used as feed additives;

[0080] (3) Used in veterinary drug production;

[0081] (4) Used for animal production in livestock and poultry farms.

[0082] Example 1

[0083] This example shows the cultivation of Bacillus subtilis BS21.

[0084] The initial culture medium for Bacillus subtilis is LB medium, and the culture steps are as follows:

[0085] The preserved strain was inoculated onto LB solid medium and cultured at 37°C for 12-18 hours.

[0086] After the culture was completed, the colony morphology was observed ( Figure 1 ), the microbiological characteristics of Bacillus subtilis BS21 are as follows: the color is milky white, opaque, the colony surface is rough, and round protrusions are present. Pick a single bacterium that grows well on LB solid medium and shake culture it in LB medium for 12 hours at 220 rpm and 37°C.

[0087] Example 2

[0088] This example shows the separation and purification of metabolite supernatant after fermentation of Bacillus subtilis BS21.

[0089] Bacillus subtilis BS21 was inoculated into sterilized LB medium, cultured at 37°C with shaking for 24 hours, and then taken out for use.

[0090] Step 1: Optimize and expand the culture

[0091] The culture medium was optimized in the laboratory, and corn flour was selected as the appropriate carbon source with a concentration of 2%, soybean meal as the appropriate nitrogen source with a concentration of 1.7%, and NaCl as the appropriate inorganic salt with a concentration of 0.5%.

[0092] Step 2: Transfer into 5 L of optimized fermentation medium at 1%, culture at 220 rpm and 30°C with shaking for 24 h, and set aside.

[0093] Step 3: Precipitation of active substances with ammonium sulfate

[0094] 5L of fermentation culture product was centrifuged at 4500rpm and 4℃ for 45min, and the supernatant was collected. The effective active substance was determined to be present in the supernatant by antibacterial experiment ( Figure 2 ), and then centrifuged to remove impurities. The obtained supernatant was stirred at 4°C and ammonium sulfate particles were added to make the final concentration reach 75%, and stirred for 12 hours to precipitate the target substance.

[0095] The precipitate and the supernatant were separated by centrifugation at 8000 rpm and 4°C for 45 min in a large volume high-speed centrifuge. The precipitate was resuspended and washed with an ammonium sulfate solution of the same concentration to further remove impurities for subsequent purification.

[0096] Step 4: Extraction of active substances with ethanol

[0097] Slowly add 50 ml of anhydrous ethanol to the precipitate obtained in the above steps and gently stir to mix so that the ethanol is fully in contact with the sample. Place the sample in a low temperature environment (4°C) to allow the ethanol and protein to combine and precipitate, and separate from other components with better solubility.

[0098] The ethanol mixed sample obtained in the previous step was centrifuged at 13000rpm and 4℃ for 15min, and the supernatant was carefully obtained (without touching the precipitate). The ethanol supernatant was concentrated using a centrifugal dryer in V-AL mode at 30℃, and the spin-dried precipitate was dissolved in sterile water and filtered using a 0.22μm filter. The obtained sample was stored at 4℃ and tested for antibacterial activity ( Figure 3 ) for subsequent experiments.

[0099] Example 3

[0100] This example demonstrates the purification of Bacillus subtilis BS21 metabolites.

[0101] The purification process was performed using an AKTA pure purifier, and the steps are as follows.

[0102] Step 1: Preparation of working buffer

[0103] The working buffer used in the purification process needs to be prepared in advance. After preparation, it should be stirred evenly on a magnetic stirrer and filtered twice using a 0.22μm water filter membrane. After filtration, it should be stored in a refrigerator at 4 degrees. The configuration details are as follows:

[0104] 1) Equilibration buffer QA 1 L: 20 mM Tris, pH = 8.0;

[0105] 2) Elution buffer QB 500 ml: 20 mM Tris, 1 M NaCl, pH = 8.0;

[0106] 3) Equilibration buffer spA 1 L: 20 mM MES, pH = 5.0;

[0107] 4) Elution buffer spB 500 ml: 20 mM MES, 1 M NaCl, pH = 5.0;

[0108] 5) Regeneration buffer 500 ml: 0.5 M NaOH.

[0109] Step 2: Collect the sample from the anion exchange column using the AKTA pure purification instrument

[0110] (1) Pump wash: First, place the A1 pipe inlet into the equilibration buffer QA, and place the B1 pipe inlet into the elution buffer QB, and start automatic washing.

[0111] (2) Install the anion exchange column: In the manual instructions system, set the flow rate to 1 ml / min and set the pressure sensor alarm. After the installation is complete, proceed to the next step.

[0112] (3) Column equilibration: Use equilibration buffer QA and keep the flow rate at 1 ml / min. When the values ​​of UV, COND and pH tend to be stable, the sample obtained in step 4 of Example 2 can be prepared for loading.

[0113] (4) Sample loading: Due to the large sample volume, a pump was used for sample loading. The setting conditions were flow rate 1 ml / min and end-timer 50 min.

[0114] (5) Elution and collection of equilibrium solution QA: Set the conditions to a flow rate of 1 ml / min and elution for 6-8 column volumes. During this period, start collecting samples according to the UV signal peak. When the UV value tends to be flat, stop collecting. Continue to balance the system and wash the UV flow-through peak back to the baseline.

[0115] (6) Elution with eluent QB and collection, setting the flow rate to 1 ml / min, selecting the gradient target B from 0% to 100% to start elution, not to avoid loss of active substances, and collecting each elution peak ( Figure 4 ).

[0116] (7) Clean the pump and remove the chromatography column: Place ddH 2 In O, start the pumpwash function to flush pumps A and B and the entire pipeline. Then put the A1 and B1 inlets into 20% ethanol, and do the same to fill the entire pipeline with ethanol. Set a slow flow rate (0.5ml / min) for the system, set the system protection pressure, first remove the lower end joint of the column, and immediately screw on the plug without stopping. The second step is to remove the upper end joint of the column and screw on the upper end plug. The whole process prevents bubbles from entering the chromatography column. Prepare for the next experiment.

[0117] (8) The collected flow-through peak and elution peak samples were tested for antibacterial activity. The results showed that the active substance existed in the flow-through that did not bind to the anion column, indicating that the active substance did not bind to the anion column. Figure 5 4-8 are flow-through peak samples, and 25-27 are elution peak samples).

[0118] Step 3: Collect the elution sample from the cation column using the AKTA protein purifier

[0119] Continue to use the AKTA protein purifier for subsequent purification steps. The method is similar to step 2. Place the A1 pipe inlet in the equilibrium buffer spA, and place the B1 pipe inlet in the elution buffer spB. The flow rate is still set to 1ml / min. After the system is balanced, the cation exchange column needs to be installed on the machine. When the values ​​of UV, COND, and pH tend to be flat, you can load the sample. For sample volumes exceeding 10ml, choose to use pump A for loading.

[0120] When the anion flow-through material obtained in step 2 of this embodiment is loaded to 5 ml, a relatively high flow-through UV peak still appears. All samples under this UV peak must be collected until the UV value tends to be flat. Use spA buffer to balance the system for about 6-8 column volumes, set the gradient target spB from 0% to 100% to start elution, and when target B reaches 25%, an elution peak with a UV value significantly higher than that of the anion column begins to appear. Use an AKTA collector to collect 1 ml per tube ( Figure 6 After testing the antibacterial activity, it was found that the extracted Bacillus active substance was present in the elution peak of the cationic column, indicating that the elution peak had antibacterial activity ( Figure 7 ), and the UV peak is relatively single, judging that the extracted substance is relatively pure.

[0121] The Oxford cup method was used to detect the antibacterial activity during the experiment. The antibacterial experiment was carried out on the typical strain Escherichia coli K88. The results showed that this method can be used to simply, quickly and cost-effectively separate small molecules with antibacterial activity from Bacillus subtilis BS21.

[0122] After the purification is completed, the A1 and B1 pumps need to be placed in pure water, and then all pipes and ion adsorption columns need to be cleaned. After removing the columns, the entire system needs to be filled with 20% ethanol.

[0123] In addition, the antibacterial activity of the purified product significantly exceeded that of the fermentation broth supernatant, which was increased by about 40% ( Figure 8 ).

[0124] Example 4

[0125] This example shows the antibacterial activity tolerance test of the purified metabolite of Bacillus subtilis BS21.

[0126] The Oxford cup method was used to detect the antibacterial activity during the experiment, and an antibacterial experiment was carried out on the typical bacterial strain Escherichia coli K88.

[0127] Step 1: Acid resistance test

[0128] The purified small molecule product was adjusted to pH 2, 3, and 4 using concentrated hydrochloric acid, and then treated in a water bath at 37°C for 1 hour. The antibacterial activity was detected using the Oxford cup method. The results showed that the antibacterial activity remained basically unchanged after treatment with different concentrations of acid, indicating that the small molecule was not affected by the acidic environment ( Fig. 9 ), can withstand the gastric acid environment.

[0129] Step 2: Heat resistance test

[0130] The purified small molecule product was placed in a metal bath at 60, 70, 80, and 90°C for 20 minutes, cooled to room temperature, and the antibacterial activity was detected by the Oxford cup method. The results showed that the antibacterial activity remained basically unchanged after treatment at different temperatures, indicating that the small molecule was not affected by temperature ( Fig.10 ).

[0131] Step 3: Protease resistance test

[0132] The purified small molecule product was treated with trypsin and pepsin at a working concentration of 1 mg / ml at 37°C for 1 hour, and the antibacterial activity was detected using the Oxford cup method. The results showed that after treatment with the two proteases, the antibacterial activity remained at 98%, indicating that the small molecule can withstand trypsin and pepsin ( Fig.11 ).

[0133] Example 5

[0134] This example shows the desalting of the AKTA purified product of Bacillus subtilis BS21 metabolites.

[0135] The metabolites of Bacillus subtilis BS21 obtained by AKTA purification, according to the experimental results, the target is stored in 20mM MES pH5.0, 160mM NaCl buffer. Considering that the salt concentration is too high, it will affect subsequent applications. Therefore, the high-performance liquid chromatography (HPLC) experimental method is used for desalination, and at the same time, the purpose of further purifying the active molecule can be achieved. After that, the freeze-dried product can be reconstituted with water. The experimental steps are as follows:

[0136] Step 1: HPLC desalting purification

[0137] Desalting was performed using an Agilent 1260 Infinity II liquid chromatography system. The operating parameters were as follows: mobile phases A and B were pure water and acetonitrile, respectively; flow rate 3.000 ml / min; high pressure limit 400.00 bar; extraction and discharge speed 300 μl / min; injection volume 200 μl; peak width greater than 0.0063 min (40 Hz); slit 4 nm; signal collection wavelength 220-360 nm; and a ZORBAX SB-C18 column.

[0138] The results are as follows Fig.12At a retention time of 12.407 min, when the organic phase was 56%, a main substance peak with a UV peak height of 679.9 mAU appeared, with a peak area of ​​4346, accounting for 45.348% of the total volume. The desalting effect was successfully achieved and further purified into a single substance. Two clustered impurity peaks appeared at retention times of 3.319 min and 9.470 min, respectively, with peak heights of less than 60 mAU, and were all collected for subsequent verification.

[0139] Step 2: Removal of organic phase

[0140] According to the results of the previous experiment, the acetonitrile content of the organic phase was 56% when the main substance peak was collected. The substance obtained in step 2 was concentrated to less than half of the original volume at 60°C using a centrifugal dryer in V-HV mode to ensure that all acetonitrile was removed, and then all was collected in one tube for later use.

[0141] Step 3: Concentrate the sample by vacuum freeze drying

[0142] The active substance dissolved in water in step 2 is made into solid powder by a freeze dryer (LGJ-10C) to better maintain the stability of the substance.

[0143] All freeze-dried samples were dissolved at 100 mg / ml and the results showed that the substance purified by HPLC still had strong antibacterial activity ( Fig.13 3); and Fig.13 1 is the first stage of the collection of impurity peaks, which has no antibacterial activity; Fig.13 The 2 is the second segment impurity peak group. Although it is active, its composition is relatively complex and its content is low, making its subsequent use difficult.

[0144] Example 6

[0145] This example shows the identification of isolated and purified Bacillus subtilis BS21 metabolites.

[0146] The active substance purified in Example 5 was extracted and analyzed using the American Thermo Fisher Q Exactive Focus combined quadrupole liquid chromatography-mass spectrometry (UPLC).

[0147] The operating parameters were as follows: Scan type: complete mass spectrometry; Mass-to-charge range: 133.4 to 2000 m / z; Resolution: 70000; Polarity: positive / negative; AGC target: 1*10 6 ; Scan time: 200ms; Sheath gas flow: 2L / min; Spray Voltage: 3.5V; Capillary temperature: 320℃; S-Lens RF level: 55; Auxiliary gas heater temperature: 0℃.

[0148] The mass spectrometry results showed that the exact size of the substance was 424.20789 ( Fig.14 ), and when it was identified by mass spectrometry, it was found that its structure was different from the structures of the compounds currently known in the database. Since it is a completely new compound, its structure cannot be confirmed at present.

[0149] Example 7

[0150] This example shows high-density fermentation culture in a 50 L fermenter.

[0151] A 50 L fermenter was used for high-density culture, and the culture raw materials were adjusted as follows: corn meal 3.4%, soybean meal 2.89%, and NaCl 0.5%.

[0152] 500 ml of seed liquid was inoculated in a 50L fermentation tank. The fermentation conditions were 220 rpm, 30°C, and pH 7.0. The fermentation products were collected at 24h, 28h, and 32h, respectively. Escherichia coli K88 and Staphylococcus aureus CVCC1882 were used as indicator bacteria, and the diameter of the inhibition zone of the fermentation supernatant was used as an indicator of the antibacterial activity, providing a basis for the subsequent large-scale purification of active substances. The results showed that ( Fig.15 ), the fermentation broth between 24h-28h has higher antibacterial activity, and the subsequent purification can select the fermentation broth during this period for large-scale extraction.

[0153] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A small molecule antibacterial active ingredient, characterized in that: It is obtained by extracting the supernatant of Bacillus subtilis fermentation through a method comprising the following steps: (1) adding a protein precipitant to the fermentation supernatant to obtain a precipitate, and further extracting the precipitate with an alcohol solvent to obtain a supernatant containing alcohol-soluble components; (2) concentrating the supernatant containing the alcohol-soluble component to obtain a precipitate, and dissolving it in water to obtain an aqueous solution; (3) Purifying the aqueous solution through an anion column and a cation column in sequence to obtain the small molecule antibacterial active ingredient.

2. The small molecule antibacterial active ingredient according to claim 1, characterized in that: In the step (1), before adding the protein precipitant, the method further comprises centrifuging the fermentation supernatant to remove impurities; Add ammonium sulfate to a concentration of 65-75% under stirring at a temperature of 0-10°C to precipitate the protein; or The temperature of the alcohol solvent extraction is 0-10°C, and after the extraction, a supernatant containing alcohol-soluble components is obtained by further centrifugal separation.

3. The small molecule antibacterial active ingredient according to claim 1, characterized in that: In the step (2), the supernatant containing the alcohol-soluble component is concentrated at 25-50° C. to obtain a precipitate, and the water-soluble precipitate is further filtered to obtain an aqueous solution.

4. The small molecule antibacterial active ingredient according to claim 1, characterized in that: In step (3), the anion column purification conditions include: a. Elution and collection of the first equilibrium solution: flow rate 0.5-2.5 ml / min, elution 6-8 column volumes, during which samples are collected according to the UV signal peak. When the UV value tends to be flat, stop collecting and continue to balance the system to wash the UV flow-through peak back to the baseline; b. Elution and collection with the first eluent: flow rate 0.5-2.5 ml / min, gradient elution from 0% to 100%, and collection of the flow-through peak components that are not bound to the anion column.

5. The small molecule antibacterial active ingredient according to claim 1, characterized in that: In step (3), the cationic column purification conditions include: using the second equilibrium liquid to equilibrate the system for 6-8 column volumes, using the second eluent to start gradient elution from 0% to 100%, and the elution peak begins to appear when it reaches 20-30%, and the corresponding components are collected as the purified small molecule antibacterial active ingredients.

6. The small molecule antibacterial active ingredient according to claim 5, characterized in that: A desalting step is further included.

7. The small molecule antibacterial active ingredient according to claim 6, characterized in that: The molecular weight of the small molecule antibacterial active ingredient is 424.20789 Da.

8. A method for preparing the small molecule antibacterial active ingredient according to any one of claims 1 to 7.

9. A bacteriostatic method, characterized in that: The method comprises the step of using the small molecule antibacterial active ingredient according to any one of claims 1 to 7.

10. Use of the small molecule antibacterial active ingredient according to any one of claims 1 to 7 in the preparation of an antibacterial agent.

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