Bacitracin a-producing bacillus subtilis var. asporus sg18 and application thereof

Abstract

This invention belongs to the field of bio-fermentation technology and discloses a strain of Bacillus paralicheniformis SG18 that produces bacitracin A and its application. This strain, Bacillus paralicheniformis SG18, was deposited at the Guangdong Provincial Microbial Culture Collection Center on March 30, 2026, with accession number GDMCC No:68015. The invention describes the application of Bacillus paralicheniformis SG18 in the fermentation preparation of bacitracin A. This invention solves the problem of relatively single bacitracin A producing strains. Starting with the fermentation supernatant of strain SG18, it provides a complete separation and purification process for bacitracin A, achieving a monomer purity of over 95%. This method does not rely on commercially available crude extracts, establishing a complete chain from complex fermentation broth to high-purity monomers. The prepared bacitracin A exhibits excellent antibacterial effects against various Gram-positive pathogens (such as Staphylococcus aureus).

Description

Technical Field

[0001] This invention belongs to the field of bio-fermentation technology, specifically relating to a Bacillus paralichrysiformis that produces bacitracin A, as well as a method and application for purifying and preparing high-purity bacitracin A. Background Technology

[0002] Bacitracin is a class of cyclic polypeptide antibiotics mainly produced by the fermentation of Bacillus licheniformis and Bacillus subtilis. These antibacterial substances exhibit inhibitory activity against a variety of Gram-positive bacteria, some Gram-negative bacteria, molds, and yeasts. Given their advantages, such as non-absorption in the animal intestine and low likelihood of developing drug resistance in vivo, bacitracin has broad application prospects in veterinary clinical treatment, topical antibacterial drugs, and agricultural biological control. Currently reported bacitracin families mainly include various derivatives such as A, B, C, D, and E. Among them, bacitracin A, formed by the dehydration condensation of 12 amino acids, is a core component with high antibacterial activity in the bacitracin family and has extremely high development value.

[0003] Due to its unique cyclic structure, the chemical synthesis of bacitracin A is difficult and costly; therefore, it is currently mainly produced through microbial fermentation. While technologies for the fermentation preparation and purification of bacitracin A have been disclosed, limitations remain in the selection of fermentation strains and the matching of downstream purification processes. Regarding production strains, existing production hosts are concentrated in *Bacillus licheniformis*. For example, patent CN115948319B discloses an engineered bacterium that knocks out the kipR gene and its preparation method and application; patent CN114874950B discloses a method for improving bacitracin production by *Bacillus licheniformis*; and patent CN113817652B discloses a *Bacillus licheniformis* strain CPL618 and its screening and application. Although there have been attempts in the industry to explore other strains, such as the patent document number CN121592534A which discloses a Sonora desert Bacillus Y2 that produces high levels of bacitracin, overall, the existing fermentation strain resources are relatively limited.

[0004] In terms of separation and purification, existing extraction processes struggle to balance the pretreatment of complex systems with the high purity of the final product. For example, patent CN116003507A discloses an extraction-back-extraction method for separating and purifying bacitracin, which uses the eluent as the separation source and yields a product with a purity of only 79% or higher. Patent CN113248571B discloses a method for extracting fermentation broth using a heat-responsive polymer, but the purity of the extracted bacitracin from the powder is only 57%.

[0005] Due to the limitations of existing technologies in the above-mentioned stages, the current bacitracin A preparation system mainly faces the following problems that urgently need to be solved: (1) The producing strain is singular, lacking novel host resources with independent genetic status. (2) Existing separation technologies generally suffer from poor selectivity, easy degradation and loss of target products, and low monomer purity, which makes it difficult to meet the needs of high-value-added applications. At the same time, some extraction processes heavily rely on a large amount of highly toxic organic reagents, which not only cause environmental pollution and solvent residues, but also easily lead to irreversible decay of the bioavailability of the polypeptide product. Therefore, it is urgent in this field to introduce a novel bacitracin A producing strain and develop a separation and purification process that can directly obtain high-purity monomers from its fermentation broth. Summary of the Invention

[0006] To address the current issues of relatively limited strains producing bacitracin A, poor selectivity in purification processes, and low product purity, this invention provides a novel bacitracin A-producing strain, identified by whole-genome sequencing as a strain of Bacillus paralicheniformis. It also provides a method for isolating, purifying, and preparing high-purity bacitracin A using the fermentation supernatant of this strain.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a Bacillus paralicheniformis SG18 strain that produces bacitracin A. This strain is Bacillus paralicheniformis SG18, which was deposited at the Guangdong Provincial Center for Microbial Culture Collection on March 30, 2026, with accession number GDMCC No: 68015.

[0009] A second aspect of the present invention provides the application of the aforementioned Bacillus paralichrysiformis SG18 in the fermentation preparation of bacitracin A.

[0010] Preferably, after fermenting and culturing the SG18 strain to obtain the fermentation broth, the fermentation supernatant is taken and successively subjected to macroporous adsorption resin elution, DEAE anion exchange chromatography, HLB extraction column desalting, and reversed-phase C18-H chromatography column separation to prepare high-purity bacitracin A.

[0011] Preferably, the preparation of the high-purity bacitracin A includes the following steps:

[0012] (1) Inoculate SG18 strain into LB medium for fermentation culture, and collect fermentation supernatant by centrifugation;

[0013] (2) The fermentation supernatant obtained in step (1) is loaded onto a macroporous adsorption resin for elution. The eluent containing antibacterial activity is collected and concentrated by rotary evaporation to obtain crude extract.

[0014] (3) Load the crude extract obtained in step (2) onto a DEAE anion exchange column, use gradient elution, and collect the active target peak components;

[0015] (4) The active target peak components obtained in step (3) are desalted by passing them through an HLB extraction column, and the eluent rich in active components is collected and combined by rotary evaporation to obtain the HLB active components.

[0016] (5) The active component obtained in step (4) is purified and separated by a high performance liquid chromatography system using a reversed phase C18-H column, the target peak component is collected, and after drying, high purity bacitracin A is obtained.

[0017] Preferably, the fermentation conditions in step (1) are: a culture temperature of 28-30℃, an inoculum size of 1-5%, a shaking speed of 150-200 rpm, and a fermentation time of 32-48 h; the centrifugation conditions are centrifugation at 8000-10000 rpm for 10-15 min.

[0018] Preferably, the macroporous adsorption resin in step (2) is AB-8 type; the elution is carried out using a methanol aqueous solution with a volume fraction of 70%-90%.

[0019] Preferably, the elution conditions of the DEAE anion exchange column in step (3) are as follows: the proportion of equilibration solution A is 80-100%, and the proportion of eluent B is 0-20%B; the composition of equilibration solution A is 20 mM Tris-HCl, pH 8.10-8.30, and the composition of eluent B is 20 mM Tris-HCl + 1M NaCl, pH 8.10-8.30.

[0020] Preferably, the specific process of HLB extraction column desalting in step (4) is as follows: after loading the active component, collect the methanol eluent with a volume fraction of 60%-100%, and evaporate it at room temperature to obtain the HLB active component.

[0021] Preferably, in step (5), the mobile phase system of the reversed-phase C18-H column is an organic phase: aqueous phase = 63:37, the elution flow rate is 1 mL / min, the monitoring wavelength is 220 nm, and the column temperature is 40℃; the organic phase is a mixture of methanol: acetonitrile = 27: 2, and the aqueous phase is phosphate buffer. Specifically, the phosphate buffer is a mixture of 0.2 M dipotassium hydrogen phosphate solution and 0.2 M potassium dihydrogen phosphate solution in a ratio of 1:4.5, and then the resulting solution is diluted with deionized water at a ratio of 1:3.

[0022] A third aspect of the present invention provides the use of bacitracin A prepared by the above method in the preparation of products that inhibit Gram-positive pathogenic bacteria.

[0023] Preferably, the Gram-positive pathogenic bacteria include Staphylococcus aureus; the product includes veterinary clinical treatment drugs, topical antibacterial drugs, or agricultural biological control agents.

[0024] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0025] (1) This invention solves the problem of relatively single strains for bacitracin A production, provides a new host resource for bacitracin preparation, and lays a solid material foundation for the optimization of downstream fermentation substrate.

[0026] (2) This invention uses the fermentation supernatant of the bacterial strain as a starting point and develops a process for the separation and purification of bacitracin A, and the purity of the obtained monomer reaches more than 95%. This method does not rely on commercial crude extracts and opens up a complete chain from complex fermentation broth to high-purity monomers.

[0027] (3) The prepared bacitracin A has a good antibacterial effect against a variety of Gram-positive pathogens (such as Staphylococcus aureus).

[0028] This invention provides a strain for producing bacitracin A, named Bacillus paralicheniformis SG18, which was deposited on March 30, 2026, at the Guangdong Provincial Microbial Culture Collection Center, with accession number GDMCC No: 68015, located at Institute of Microbiology, Guangdong Academy of Sciences, Guangzhou, China, 510070, China. Attached Figure Description

[0029] Figure 1 This image shows the colony morphology of B. paralicheniformis SG18 on an LB plate.

[0030] Figure 2 Phylogenetic tree of B. paralicheniformis SG18.

[0031] Figure 3 (A) The assembly pathway diagram of the nutrient synthesis gene cluster and NRPS module predicted based on the whole genome of B. paralicheniformis SG18; (B) The structural analysis diagram of the nutrient NRPS predicted based on the whole genome mining of antiSMASH.

[0032] Figure 4The inhibitory effect of fermentation supernatant of B. paralicheniformis SG18 on Staphylococcus aureus was investigated.

[0033] Figure 5 The inhibitory effect of B. paralicheniformis SG18 fermentation supernatant on Staphylococcus aureus after gradient elution with AB-8 macroporous adsorption resin was investigated.

[0034] Figure 6 This study investigated the separation of active crude protein from B. paralicheniformis SG18 on a DEAE anion exchange column and the antibacterial effects of each component against Staphylococcus aureus.

[0035] Figure 7 The inhibitory effect of DEAE active components on Staphylococcus aureus after gradient elution using an HLB extraction column.

[0036] Figure 8 (A) The separation of HLB active components on a reversed-phase C18-H column and the antibacterial effect of each component on Staphylococcus aureus; (B) The purity detection of the active components after separation on the C18-H column.

[0037] Figure 9 The first-order mass spectrum (A) and MALDI-TOF spectrum (B) of the active component separated by the C18-H column are shown.

[0038] Figure 10 (A) The bacitracin synthesis gene cluster and NRPS module assembly pathway predicted based on the whole genome of B. paralicheniformis SG18; (B) The bacitracin NRPS structure analysis diagram predicted based on the whole genome mining of antiSMASH.

[0039] Figure 11 The active antibacterial substance was verified to be bacitracin A by secondary mass spectrometry analysis; (A) Secondary mass spectrum; (B) Fracture location inferred from fragment ions. Detailed Implementation

[0040] The present invention will be further described in detail below with reference to specific embodiments, but the implementation of the present invention is not limited thereto. For test methods not specifically noted, conventional test techniques can be referred to.

[0041] 1. Acquisition and identification of production strains

[0042] The *Bacillus paralichrysum* of this invention was isolated by the following method: 25 g of Fujian pickled bamboo shoot sample was weighed and dissolved in 225 mL of sterile physiological saline, and after thorough mixing, 10... -1Diluent. The above diluent was heated in a 70°C water bath for 20 min to kill non-spore-forming vegetative bacteria. After cooling to room temperature, it was serially diluted with sterile physiological saline to prepare 10... -2 10 -3 10 -4 10 -5 10 -6 10 -7 The sample dilution solution. Select 10... -5 10 -6 10 -7 Three gradient dilutions were prepared, with 100 μL of each diluted solution evenly spread onto LB agar plates. The plates were then inverted and incubated at 28°C for 24–48 h. After single colonies of varying morphologies had grown on the plates, single colonies exhibiting typical Bacillus characteristics were picked using an inoculation loop and subjected to repeated streaking purification on fresh LB agar plates until a pure culture was obtained.

[0043] Morphological identification of the strain: The strain produced in this invention was identified and named Bacillus paralicheniformis SG18 (hereinafter referred to as SG18). Its colonies are milky white, with a rough and moist surface and irregular edges (see...). Figure 1 ).

[0044] Molecular biological identification: Phylogenetic tree constructed based on the full-length 16S rRNA gene sequence (see...) Figure 2 The results showed that the Bootstrap support rate of the core node was as high as 97%, indicating that strain SG18 is closely related to *Bacillus paralichrysogenus*. Given the high homology between *Bacillus paralichrysogenus* and *Bacillus licheniformis*, this invention further combines whole-genome sequencing analysis as a key basis for species identification. Existing literature confirms that the Fengycin synthesis gene cluster is specifically present in the genome of *Bacillus paralichrysogenus*, while it is completely absent in the genomes of all currently known *Bacillus licheniformis* strains, and can serve as a key molecular marker to distinguish the two. Through mining and analysis of the whole-genome sequence of SG18, such as... Figure 3 As shown, this invention successfully predicted information related to fibroblasts in the SG18 genome. Based on morphological, molecular biological, and whole-genome characteristics, the strain was confirmed to be *Bacillus paralichrysogenus*.

[0045] The 16S rDNA sequence of the SG18 strain is as follows:

[0046]

[0047] The LB liquid medium contains the following components per 1000 mL: 10 g peptone, 5 g sodium chloride, 1 g glucose, 5 g yeast extract, and the remainder is deionized water, with a pH of 7.0 ± 0.2. It is sterilized at 121℃ for 15 min. For the solid medium, 0.75-1.5% agar powder is also added.

[0048] 2. Obtaining fermentation supernatant of production strain and determining its antibacterial activity

[0049] (1) Obtaining fermentation supernatant: 100 μL of SG18 glycerol mixed bacterial culture was inoculated into 5 mL of LB liquid medium and activated by shaking at 180 rpm at 30℃ for 12 h, and then transferred for activation once more. The activated SG18 was inoculated into LB liquid medium at a 2% inoculation rate and cultured at 30℃ and 180 rpm with shaking. After 48 h, the culture was centrifuged at 10000 rpm for 10 min and filtered through a 0.22 μm filter membrane for sterilization, and the supernatant was collected for analysis.

[0050] (2) Preparation of antibacterial plates: Staphylococcus aureus was cultured in LB liquid medium at 37°C and 180 rpm until OD. 600 Approximately 1.0, then diluted to OD using LB liquid medium. 600 The value was 0.5. Subsequently, the diluted bacterial solution was inoculated into LB solid medium cooled to 45-50℃ at a ratio of 160 μL bacterial solution / 100 mL medium. After thorough mixing, the solution was poured into plates and allowed to solidify before use.

[0051] (3) Antibacterial activity determination: The antibacterial activity of SG18 supernatant was determined by the agar plate diffusion method. After sterilizing the 8mm punch by flame, holes were punched in the antibacterial plate described in (2); the culture medium block in the hole was removed with a sterile toothpick, and then 100 μL of the SG18 fermentation supernatant described in (1) was injected into the hole. After drying and diffusion in a clean bench, the plate was incubated in a 37℃ incubator for 12-18 h, and the diameter of the inhibition zone was observed and measured. Figure 4 As shown, the fermentation supernatant of SG18 has good antibacterial activity against Gram-positive bacteria such as Staphylococcus aureus.

[0052] 3. Crude extraction of antibacterial substances from the fermentation supernatant of the production strain

[0053] The fermentation supernatant of the producing strain was obtained as described in 2(1), and crude protein was extracted by elution with AB-8 macroporous adsorption resin. The specific operation was as follows: 55 g of AB-8 macroporous adsorption resin was weighed and soaked in methanol overnight for later use; the column was packed the next day, and the methanol was replaced to remove impurities. The relevant parameters of the chromatographic column were as follows: diameter 2.8 cm, column height 13.5 cm, column volume approximately 83 cm³.3 Subsequently, an HL-2B digital display constant flow pump was connected in series. After the column stabilized, SG18 fermentation supernatant, 5% methanol, 30% methanol, 50% methanol, and 90% methanol were loaded sequentially, and the corresponding eluates were collected for antibacterial activity testing. Figure 5 As shown, the uncoated flow-through solution and the 5%, 30%, and 50% methanol eluents showed no antibacterial activity, while antibacterial activity was detected in the 90% methanol eluent. This indicates that the active component can be adsorbed by the AB-8 macroporous adsorption resin and requires a high concentration of methanol solution for elution, suggesting that the active substance has low polarity. Finally, the active component was rotary evaporated (45°C) to remove methanol and concentrate, obtaining the crude antibacterial extract.

[0054] 4. Separation of crude extract on DEAE anion exchange column

[0055] Based on the crude extract, gradient elution separation was performed using the AKTA protein separation system on a DEAE anion exchange column, with a monitoring wavelength of 220 nm. Equilibration buffer A consisted of 20 mM Tris-HCl, pH 8.10-8.30, and eluent B consisted of 20 mM Tris-HCl + 1 M NaCl, pH 8.10-8.30. The loading volume was 8 mL of crude extract solution, and the flow rate was 1 mL / min. The gradient elution conditions were: 0% B (i.e., 100% A), 0-20% B (i.e., 80% A) for 30 min, and 100% B (i.e., 0% A). The eluent fractions at each stage were collected, with 8-10 mL of eluent automatically collected from each tube, and its antibacterial activity was measured. Figure 6 As shown, peaks eluted from tubes 2-8 are from 0% B, peaks eluted from tubes 9-18 are from 0-20% B, and peaks eluted from tubes 19-20 are from 100% B. Testing revealed that only tubes 11 and 12 exhibited significant antibacterial activity, indicating that the active component was located at the circled peak positions. Therefore, the eluents from tubes 11 and 12 were combined to identify the DEAE active component, and subsequent desalting treatment was performed.

[0056] 5. DEAE active components were eluted using an HLB extraction column gradient elution.

[0057] The DEAE active component was desalted using an HLB extraction column. The HLB extraction column was fixed, and 10-15 mL of 100% methanol and 10-15 mL of 5% methanol were slowly added sequentially to remove residues and equilibrate the column. The DEAE active component solution was then gently loaded onto the column, and the fraction not adhering to the column was collected. Subsequently, elution was performed by adding 30% methanol, 60% methanol, and 100% methanol in fractions. Each eluted fraction was collected, and its antibacterial activity was measured. Figure 7As shown, no antibacterial activity was detected in the uncoated flow-through and the 30% methanol eluent, while activity was detected in the 60% and 100% methanol eluents. This indicates that the antibacterial substance was adsorbed by the HLB column and could be eluted by high concentrations of methanol. The 60% and 100% methanol eluents were combined, and the methanol was removed by rotary evaporation at room temperature to obtain the HLB active component.

[0058] 6. Separation of HLB active components on a reversed-phase C18-H column

[0059] The above-mentioned HLB active components will be further purified using a C18 column in a semi-preparative liquid chromatography system. The specific steps are as follows:

[0060] (1) Preparation of mobile phase: First, prepare 0.2 M dipotassium hydrogen phosphate mother liquor and potassium dihydrogen phosphate mother liquor, then mix them at a ratio of dipotassium hydrogen phosphate mother liquor: potassium dihydrogen phosphate mother liquor = 1: 4.5. Then, dilute the resulting solution with deionized water at a ratio of 1:3 to obtain an aqueous phase. For the organic phase, prepare it by mixing methanol and acetonitrile at a ratio of 27: 2.

[0061] (2) Chromatographic conditions: The chromatographic column was C18-H (250 mm × 4.6 mm, 5 μm, Amethyst), the column temperature was 40℃, the detection wavelength was 220 nm, the flow rate was 1.0 mL / min, the injection volume was 100 μL, and the ratio between the organic phase and the aqueous phase was set to 63:37 by the HPLC mixing valve for isogradient elution.

[0062] like Figure 8 As shown in Figure A, the component with a retention time of 21.34 min exhibited antibacterial activity. Subsequent purity testing of this component revealed only one well-symmetrical peak (…). Figure 8 B) indicates that the purified substance has a high purity, and the purity is approximately 95.26% obtained by integrating the peak area.

[0063] 7. Identification of purified active components by LC-MS / MS and MALDI-TOF

[0064] The C18-H purified fraction was placed in a vacuum drying oven to evaporate the organic solvent, followed by desalting and freeze-drying to obtain a purified powder. After dissolving in ultrapure water, it was identified by LC-MS / MS and MALDI-TOF mass spectrometry. Figure 9 Primary mass spectrometry analysis of A revealed a mother ion peak with a mass-to-charge ratio of 1422.7585, suggesting it is [M+H]. + Calculations show that the molecular mass of the target substance is 1421.7585 Da; the mass-to-charge ratio of the multi-charged ion peak is 711.8828, suggesting it is [M+2H]. 2+The calculated molecular mass is 1421.7656 Da. Another peak with a mass-to-charge ratio of 386.1790 is presumed to be background impurities introduced by the system solvent / pipeline.

[0065] Figure 9 MALDI-TOF analysis of B revealed a signal peak at m / z of 1460.8710, which is inferred to be [M+K]. + The molecular weight was deduced to be 1421.9073 Da; the signal peak with m / z of 1444.9055 was deduced to be [M+Na]. + The molecular weight is inferred to be 1421.91565 Da; the signal peak at 1422.932 is inferred to be [M+H]. + The estimated molecular weight is 1421.932 Da. In summary, the molecular weight of the active component is inferred to be M = 1421 Da.

[0066] 8. Identify active components by combining whole genome information of the production strain.

[0067] The molecular weight of the active substance was determined to be 1421 Da. To further clarify the active substance, the whole genome of SG18 was sequenced, and the whole genome sequence of this strain was uploaded to the antiSMASH online prediction (https: / / antismash.secondarymetabolites.org) database to predict the ribosomal synthetic peptides and non-ribosomal synthetic metabolites and their biosynthetic gene clusters contained in the genome. Figure 10 A shows that SG18 contains a gene cluster encoding bacitracin. NRPS / PKS monomer structure prediction was performed on this gene cluster. Figure 10 B), combined with secondary mass spectrometry ( Figure 11 A), suggesting that the antibacterial active component is bacitracin A. Specifically, L-Ile and L-Cys at positions 1 and 2 undergo dehydration condensation to form a thiazole ring, and L-Asn at position 12 and L-Lys at position 6 connect to form a seven-membered ring (A). Figure 11 B).

[0068] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications and substitutions based on the technical solutions and inventive concepts provided by the present invention should be covered within the scope of protection of the present invention.

Claims

1. A *Bacillus paralichrysiformis* SG18 producing bacitracin A, characterized in that, The strain is Bacillus paralicheniformis SG18, which was deposited at the Guangdong Provincial Center for Microbial Culture Collection on March 30, 2026, with accession number GDMCC No: 68015.

2. The application of Bacillus paralichrysiformis SG18 as described in claim 1 in the fermentation preparation of bacitracin A.

3. The application according to claim 2, characterized in that, The strain described in claim 1 was fermented to obtain a fermentation broth. The fermentation supernatant was then subjected to macroporous adsorption resin elution, DEAE anion exchange chromatography, HLB extraction column desalting, and reversed-phase C18-H chromatography column separation to prepare high-purity bacitracin A.

4. The application according to claim 3, characterized in that, Includes the following steps: (1) The strain described in claim 1 is inoculated into LB medium for fermentation culture, and the fermentation supernatant is collected by centrifugation; (2) The fermentation supernatant obtained in step (1) is loaded onto a macroporous adsorption resin for elution. The eluent containing antibacterial activity is collected and concentrated by rotary evaporation to obtain crude extract. (3) Load the crude extract obtained in step (2) onto a DEAE anion exchange column, use gradient elution, and collect the active target peak components; (4) The active target peak components obtained in step (3) are desalted by passing them through an HLB extraction column, and the eluent rich in active components is collected and combined by rotary evaporation to obtain the HLB active components. (5) The active component obtained in step (4) is purified and separated by a high performance liquid chromatography system using a reversed phase C18-H column, the target peak component is collected, and after drying, high purity bacitracin A is obtained.

5. The application according to claim 3 or 4, characterized in that, The fermentation conditions in step (1) are as follows: the culture temperature is 28-30℃, the inoculum size is 1-5%, the shaking speed is 150-200 rpm, and the fermentation time is 32-48 h; the centrifugation conditions are centrifugation at 8000-10000 rpm for 10-15 min.

6. The application according to claim 3 or 4, characterized in that, The macroporous adsorption resin mentioned in step (2) is AB-8 type; the elution is carried out using a methanol aqueous solution with a volume fraction of 70%-90%.

7. The application according to claim 3 or 4, characterized in that, The elution conditions of the DEAE anion exchange column in step (3) are as follows: the proportion of equilibration solution A is 80-100%, and the proportion of eluent B is 0-20%B; the composition of equilibration solution A is 20 mM Tris-HCl, pH 8.10-8.30, and the composition of eluent B is 20 mM Tris-HCl + 1M NaCl, pH 8.10-8.

30.

8. The application according to claim 3 or 4, characterized in that, The specific process of HLB extraction column desalting in step (4) is as follows: after loading the active component, collect the methanol eluent with a volume fraction of 60%-100%, and evaporate it at room temperature to obtain the HLB active component.

9. The application according to claim 3 or 4, characterized in that, The mobile phase system of the reversed-phase C18-H column in step (5) is organic phase: aqueous phase = 63: 37, the elution flow rate is 1 mL / min, the monitoring wavelength is 220 nm, and the column temperature is 40℃; the organic phase is a mixture of methanol: acetonitrile = 27: 2, and the aqueous phase is phosphate buffer.

10. The use of bacitracin A prepared according to any one of claims 2-9 in the preparation of products that inhibit Gram-positive pathogenic bacteria.

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