Bacillus subtilis and purification method of high-efficiency broad-spectrum antibacterial lipopeptide thereof

Abstract

The application belongs to the technical field of bioengineering, and provides a bacillus subtilis strain named ZCK-1 strain, which has been preserved in the China General Microbiological Culture Collection Center, located at No. 1, Xili Beichen, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the postal code is 100101, and the preservation number is CGMCC NO. 30620; the purification method of the high-efficiency broad-spectrum antibacterial lipopeptide is adopted, the obtained antibacterial lipopeptide has high-efficiency broad-spectrum antibacterial capacity, and reliable technical parameters are provided for the large-scale industrial production of the antibacterial lipopeptide of the bacillus subtilis.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and more specifically, to a method for purifying Bacillus subtilis and its highly efficient broad-spectrum antimicrobial lipopeptides. Background Technology

[0002] The clinical application of antibiotics faces a series of challenges, including bacterial resistance and drug side effects. The increasing prevalence of resistant strains, such as methicillin-resistant Staphylococcus aureus (MRSA) and superbugs (such as vancomycin-resistant enterococci, VRE), and Streptococcus pneumoniae, complicates treatment-associated infections. Furthermore, antibiotics can trigger severe allergic reactions and gut microbiota dysbiosis, leading to problems such as diarrhea in patients.

[0003] The widespread use of antibiotics in agriculture and animal husbandry has led to antibiotic resistance, drug residues, animal health problems, and increased economic costs. Resistant strains and cross-resistance not only threaten animal health but also spread to humans through the food chain. Drug residues impact food safety and the environment; overuse of antibiotics causes animal health problems and drug dependence, increases production and treatment costs, and is subject to market regulations.

[0004] Traditionally, the process of isolating and purifying antimicrobial lipopeptides or antibiotics from bacterial fermentation broth usually includes the following main steps: (1) Fermentation, ① Culture medium preparation: Cultivate bacterial strains that produce antimicrobial peptides or antibiotics using appropriate culture medium (such as nutrient broth); ② Inoculation and cultivation: Inoculate the seed culture into the fermenter and ferment under suitable temperature and stirring conditions, usually for 24-72 hours until the maximum yield of antimicrobial substances is obtained. (2) Solid-liquid separation, ① Centrifugation: After fermentation, remove bacterial cells and other solid impurities by high-speed centrifugation, retaining the supernatant containing the target antimicrobial peptides or antibiotics; ② Filtration: Sometimes microfiltration or ultrafiltration is used to further remove cell debris and larger particles. (3) Preliminary purification, ① Solvent extraction: Extract the antimicrobial peptides or antibiotics from the aqueous phase using organic solvents (such as ethyl acetate, methanol, ethanol, etc.). This process can be carried out by liquid-liquid extraction; ② Precipitation: Sometimes salting out (such as ammonium sulfate precipitation) or organic solvent precipitation (such as ethanol precipitation) is used to precipitate proteins and other impurities, thereby preliminarily purifying the antimicrobial peptides or antibiotics. (4) Fine purification: ① Ion exchange chromatography: Based on the charge properties of antimicrobial peptides or antibiotics, separation and purification are carried out using ion exchange resins; ② Gel filtration chromatography (molecular sieve chromatography): Separation is carried out based on molecular size; ③ Reversed-phase high-performance liquid chromatography (RP-HPLC): High-efficiency separation and purification is carried out using differences in hydrophobicity. (5) Concentration and drying: ① Rotary evaporation: The solvent is evaporated under reduced pressure using a rotary evaporator to concentrate the antimicrobial substance. ② Lyophilization: The concentrate is lyophilized to obtain the antimicrobial peptide or antibiotic in dry powder form.

[0005] The limitations of traditional processes include: ① Numerous steps: Traditional processes typically involve multiple steps, making the process complex and time-consuming; ② Low efficiency: Some steps may lead to the loss of antibacterial substances, affecting the final yield and purity; ③ High cost: The use of large amounts of organic solvents and specialized equipment increases production costs; ④ Environmental issues: The use of organic solvents and waste disposal have a certain impact on the environment.

[0006] To overcome the limitations of traditional processes, modern research and industrial production are constantly developing new separation and purification methods, such as macroporous resin adsorption, supercritical fluid extraction, and membrane separation technology, to improve efficiency, reduce costs, and minimize environmental impact. To address the problems associated with antibiotics and preservatives, an effective approach is to develop novel antimicrobial substances with new mechanisms of action to bypass existing drug resistance mechanisms; simultaneously, using probiotics to purify antimicrobial substances can replace antibiotics, reducing dependence on them; furthermore, biopreservatives obtained through bio-fermentation, such as lactobacillus and natamycin, are being applied, as these preservatives exhibit good safety and preservative effects. Summary of the Invention

[0007] The purpose of this invention is to address the shortcomings of existing technologies by proposing a purification method for Bacillus subtilis and its highly efficient broad-spectrum antibacterial lipopeptides.

[0008] The specific technical solution is as follows: Bacillus subtilis, named strain ZCK-1, which has been deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, postal code: 100101, with accession number CGMCC NO.30620.

[0009] Furthermore, the 16S rRNA sequence of the strain has been uploaded to NCBIGenBank, with the sequence number UB14134909 ZCK-1PP079112.

[0010] Furthermore, the complete genome sequence of the strain has been uploaded to NCBIGenBank, with the sequence number NO.JBGOPS000000000.1.

[0011] Another object of the present invention is to provide a purification method for preparing highly efficient broad-spectrum antibacterial lipopeptides of Bacillus subtilis as described above, comprising the following steps:

[0012] Step 1: Prepare seed solution

[0013] The ZCK-1 strain was inoculated onto a nutrient agar plate containing Staphylococcus aureus and cultured at 33-37℃ for 22-25 hours. The colony with the largest inhibition zone was picked from the culture plate and inoculated into 300 mL of nutrient broth. The culture was then carried out at 34-36℃ on a shaker at 175-185 rpm for 16-18 hours to prepare a seed culture.

[0014] Step 2: Inoculation and Culture

[0015] The seed culture was inoculated into nutrient broth medium at an inoculation rate of 5%, and cultured in a shaker at 177-183 rpm for 24 h at a temperature of 34-36℃ to obtain the fermentation broth of strain ZCK-1.

[0016] Step 3: Centrifugation and supernatant collection

[0017] Centrifuge the fermentation broth at 8000 rpm for 15 min to remove bacterial precipitate and retain the supernatant;

[0018] Step 4: Macroporous resin adsorption

[0019] Macroporous resin was added to the supernatant at a mass-to-volume ratio of 2.7%; the mixture was then incubated at 35°C and 180 rpm for 24 hours for adsorption.

[0020] Step 5: Cleaning and Desorption

[0021] After natural sedimentation, pour off the supernatant, retain the macroporous resin, wash the macroporous resin twice with distilled water, then add 95% anhydrous ethanol to the macroporous resin and incubate it on a shaker at 195-205 rpm for 23-25 ​​h at a temperature of 33-36℃; then sonicate for 1 h to improve the desorption effect.

[0022] Step 6: Collection and Treatment of Eluent

[0023] Collect the eluent, centrifuge at 8000 rpm for 15 min to remove residual bacteria and impurities, and retain the supernatant;

[0024] Step 7: Vacuum rotary drying

[0025] The supernatant was placed in a vacuum rotary evaporator and dried at 58°C to obtain a purified antimicrobial lipopeptide.

[0026] The detailed technical solution of the present invention is as follows: In step one, in the preparation of seed liquid, strain ZCK-1 is inoculated onto a nutrient agar plate containing Staphylococcus aureus and cultured at 35°C for 24 hours. After that, the colony with the largest inhibition zone is picked from the culture plate and inoculated into 300 mL of nutrient broth. The culture is then carried out at 35°C on a shaker at 180 rpm for 16-18 hours to prepare the seed liquid.

[0027] In the detailed technical solution of the present invention, in step two, inoculation and culture, the seed liquid is inoculated into the nutrient broth culture medium at an inoculation amount of 5%, and cultured in a shaker at 180 rpm for 24 hours at a temperature of 35°C to obtain the fermentation broth of strain ZCK-1.

[0028] In the detailed technical solution of this invention, in step five, cleaning and desorption, after natural sedimentation, the supernatant is poured out, the macroporous resin is retained, the macroporous resin is washed twice with distilled water, and then 95% anhydrous ethanol is added to the macroporous resin. The resin is then cultured and adsorbed in a shaker at 200 rpm for 24 hours at 35°C. Finally, it is ultrasonically vibrated for 1 hour to improve the desorption effect.

[0029] In the technical solution of the present invention, the antimicrobial lipopeptide obtained by the purification method of the Bacillus subtilis high-efficiency broad-spectrum antimicrobial lipopeptide is used to be sensitive to enzymes including proteinase K, papain and lipase.

[0030] In the technical solution of the present invention, the antimicrobial lipopeptides obtained by the purification method of the Bacillus subtilis high efficiency broad-spectrum antimicrobial lipopeptides are resistant to high temperature. The antimicrobial lipopeptides are treated at 100℃ for 30 min and the antimicrobial activity is not reduced. The antimicrobial lipopeptides are treated with high pressure steam at 121℃ for 20 min and the antimicrobial activity is still 67%.

[0031] In the technical solution of the present invention, the antimicrobial lipopeptides obtained by the purification method of the Bacillus subtilis high-efficiency broad-spectrum antimicrobial lipopeptides are resistant to strong acids and strong alkalis; wherein the antimicrobial lipopeptides retain more than 91% of their antimicrobial activity after being treated with pH 2-4 for 6 hours, and retain more than 77% of their antimicrobial activity after being treated with pH 11-12 for 6 hours.

[0032] In the technical solution of the present invention, the antimicrobial lipopeptide obtained by the purification method of the Bacillus subtilis high-efficiency broad-spectrum antimicrobial lipopeptide is evaluated in accordance with GB15193.3 "Food Safety Toxicology Evaluation Procedures and Methods - Acute Oral Toxicity Test"; the results of the acute oral toxicity test show that the ZCK-1 antimicrobial lipopeptide is practically non-toxic.

[0033] In the technical solution of the present invention, the antimicrobial lipopeptide obtained by the purification method of the Bacillus subtilis high-efficiency broad-spectrum antimicrobial lipopeptide is applied in the medical field; the antimicrobial lipopeptide is prepared into an antimicrobial drug or antimicrobial agent, which serves as a natural antimicrobial agent for the prevention and treatment of pathogenic bacterial infections;

[0034] Antimicrobial lipopeptides are used for the prevention and treatment of clinical pathogenic bacterial infections, exhibiting broad-spectrum antibacterial activity against most Gram-positive bacteria and a small number of Gram-negative bacteria. The Gram-positive bacteria include Corynebacterium striatum, Streptococcus pneumoniae, Staphylococcus schlegelii, Streptococcus group B, methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus, Enterococcus faecalis, and Staphylococcus epidermidis. They exhibit significant antibacterial activity, particularly against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE). The Gram-negative bacteria include Moraxella catarrhalis, Proteus vulgaris, extended-spectrum β-lactamase Escherichia coli, Escherichia coli, and Salmonella.

[0035] In the technical solution of the present invention, the antimicrobial lipopeptides obtained by the purification method of Bacillus subtilis high efficiency broad spectrum antimicrobial lipopeptides are applied in the field of agriculture and animal husbandry to prevent and treat pathogenic bacterial infections in livestock and poultry and improve the health level of livestock and poultry.

[0036] Antimicrobial lipopeptides are formulated into feed additives for use as antimicrobial agents in feed or poultry drinking water to prevent and treat bacterial infections in livestock and poultry, improve their disease resistance, reduce antibiotic use, ensure the safety of livestock products, and reduce antibiotic residues.

[0037] The purification method of Bacillus subtilis and its highly efficient broad-spectrum antibacterial lipopeptides of the present invention has the following advantages compared with the prior art:

[0038] 1) Highly effective broad-spectrum antibacterial ability

[0039] The purification method for highly efficient and broad-spectrum antimicrobial lipopeptides from Bacillus subtilis yielded antimicrobial lipopeptides that exhibited significant antimicrobial activity against all commonly tested clinical Gram-positive cocci, streptococci, and bacilli, and also showed significant antimicrobial effects against some Gram-negative bacteria. Specifically, it showed significant antimicrobial activity against clinical strains of Gram-positive bacteria such as Streptococcus pneumoniae, Corynebacterium streptococcus, Staphylococcus schrenckii, Streptococcus group B, Staphylococcus epidermidis, Staphylococcus aureus, Enterococcus faecalis, methicillin-resistant Staphylococcus aureus, and vancomycin-resistant Enterococci. Among these, it showed significant antimicrobial activity against Corynebacterium streptococcus, Streptococcus pneumoniae, and Staphylococcus schrenckii. The antibacterial activity against cocci was the strongest, with minimum inhibitory concentrations (MICs) of 0.22 mg / mL, 0.11 mg / mL, and 0.11 mg / mL, respectively. Furthermore, the antimicrobial lipopeptides exhibited significant antibacterial activity against multidrug-resistant strains such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci. They also showed relatively strong antibacterial activity against Gram-negative bacteria such as Moraxella catarrhalis and Proteus vulgaris, with an inhibitory concentration of 0.45 mg / mL against Moraxella catarrhalis. However, they showed weaker antibacterial activity against extended-spectrum β-lactamase Escherichia coli, Escherichia coli, and Salmonella.

[0040] 2) Innovative and simple separation and purification methods

[0041] This invention provides an innovative and simple method for purifying highly efficient broad-spectrum antimicrobial lipopeptides. The method includes steps such as bacterial fermentation, macroporous resin adsorption, ethanol desorption, and vacuum drying and rotary evaporation, which can effectively separate and purify antimicrobial lipopeptides in the fermentation broth of strain ZCK-1. This method is not only simple and practical to operate, but also has high yield and purity, and retains the high activity and stability of antimicrobial lipopeptides, laying a certain foundation for large-scale industrial production.

[0042] 3) The antimicrobial lipopeptides obtained by the purification method of Bacillus subtilis with high efficiency and broad spectrum provided by the present invention have fine, tasteless, and light yellow powder that is easily soluble in water and has strong activity. Furthermore, the separation and purification scheme was optimized by single-factor experiments and professional mathematical statistics experiments, which significantly improved its yield, purity and activity. Thus, reliable technical parameters were provided for the large-scale industrial production of antimicrobial lipopeptides of Bacillus subtilis. Attached Figure Description

[0043] Figure 1 The following is a molecular identification assay of Bacillus subtilis ZCK-1 16S rRNA provided in this embodiment of the invention. (A: PCR electrophoresis pattern, B: phylogenetic tree).

[0044] Figure 2 The following is a characteristic map of the chromosome genome of Bacillus subtilis ZCK-1 provided in this embodiment of the invention.

[0045] Figure 3 The following is a feature map of the whole genome sequence of Bacillus subtilis ZCK-1 provided in this embodiment of the invention.

[0046] Figure 4 The antimicrobial lipopeptides obtained by the method for isolating and purifying antimicrobial lipopeptides from the fermentation broth of strain ZCK-1 provided in this embodiment of the invention. (A: Properties, B: Antimicrobial activity detection).

[0047] Figure 5 The embodiments of this invention provide the HPLC detection results of antibacterial lipopeptides and the comparative analysis results of their active components with surfactantin standards.

[0048] Figure 6 The embodiments of this invention provide the detection results of the sensitivity of antimicrobial lipopeptides to various enzymes.

[0049] Figure 7 The temperature sensitivity detection results of the antimicrobial lipopeptide provided in the embodiments of the present invention.

[0050] Figure 8 The acid-base sensitivity detection results of the antibacterial lipopeptides provided in the embodiments of the present invention.

[0051] Figure 9 The antimicrobial lipopeptide provided in this embodiment of the invention has been tested for its antimicrobial activity against clinically infected strains.

[0052] Figure 10 Electron micrographs of the antimicrobial lipopeptide acting on the cell membrane of Staphylococcus aureus provided in this embodiment of the invention.

[0053] Figure 11 Electrophoresis diagram of the antimicrobial lipopeptide acting on the genomic DNA of Staphylococcus aureus provided in this embodiment of the invention.

[0054] Figure 12 The oral safety evaluation results of the antimicrobial lipopeptides provided in the embodiments of the present invention.

[0055] Figure 13 The interaction response surface plots (A: response surface plot, B: contour plot) of macroporous resin dosage and elution ethanol concentration provided in the embodiments of the present invention.

[0056] Figure 14 The interaction response surface plots (A: response surface plot, B: contour plot) of macroporous resin dosage and rotary evaporation temperature provided in the embodiments of the present invention.

[0057] Figure 15The interaction response surface plots of elution ethanol concentration and rotary evaporation temperature provided in the embodiments of the present invention (A: response surface plot, B: contour plot). Detailed Implementation

[0058] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without inventive effort are within the scope of protection of this invention.

[0059] Example 1

[0060] Bacillus subtilis, named strain ZCK-1, was deposited on May 14, 2024, at the China General Microbiological Culture Collection Center (CGMCC) and entered into the catalogue; address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, postal code: 100101, accession number: CGMCCNO.30620.

[0061] Furthermore, the 16S rRNA sequence of the strain has been uploaded to NCBIGenBank, with the sequence number UB14134909 ZCK-1PP079112.

[0062] Furthermore, the complete genome sequence of the strain has been uploaded to NCBIGenBank, with the sequence number NO.JBGOPS000000000.1.

[0063] Example 2

[0064] This embodiment provides a method for separating and purifying antimicrobial lipopeptides from the fermentation broth of Bacillus subtilis as described in Example 1 above, comprising the following steps:

[0065] Step 1: Prepare seed solution

[0066] The ZCK-1 strain was inoculated onto a nutrient agar plate containing Staphylococcus aureus and cultured at 33°C for 22 hours. After that, the colony with the largest inhibition zone was picked from the culture plate and inoculated into 300 mL of nutrient broth. The culture was then carried out at 34°C on a shaker at 175 rpm for 16 hours to prepare a seed culture.

[0067] Step 2: Inoculation and Culture

[0068] The seed culture was inoculated into nutrient broth medium at an inoculation rate of 5%, and cultured in a shaker at 177 rpm for 24 h at 34℃ to obtain the fermentation broth of strain ZCK-1.

[0069] Step 3: Centrifugation and supernatant collection

[0070] Centrifuge the fermentation broth at 8000 rpm for 15 min to remove bacterial precipitate and retain the supernatant;

[0071] Step 4: Macroporous resin adsorption

[0072] Macroporous resin was added to the supernatant at a mass-to-volume ratio of 2.7%; the mixture was then incubated at 35°C and 180 rpm for 24 hours for adsorption.

[0073] Step 5: Cleaning and Desorption

[0074] After natural sedimentation, the supernatant was poured off, and the macroporous resin was retained. The macroporous resin was washed twice with distilled water, and then 95% anhydrous ethanol was added to the macroporous resin. The resin was incubated at 33°C and 195 rpm in a shaker for 23 hours for adsorption. Then, it was ultrasonically vibrated for 1 hour to improve the desorption effect.

[0075] Step 6: Collection and Treatment of Eluent

[0076] Collect the eluent, centrifuge at 8000 rpm for 15 min to remove residual bacteria and impurities, and retain the supernatant;

[0077] Step 7: Vacuum rotary drying

[0078] The supernatant was placed in a vacuum rotary evaporator and dried at 58°C to obtain a purified extract of the novel antibacterial lipopeptide.

[0079] Example 3

[0080] This embodiment provides a method for isolating and purifying antimicrobial lipopeptides from the fermentation broth of Bacillus subtilis described in Example 1 above, comprising the following steps:

[0081] Step 1: Prepare seed solution

[0082] The ZCK-1 strain was inoculated onto a nutrient agar plate containing Staphylococcus aureus and cultured at 37°C for 25 hours. After that, the colony with the largest inhibition zone was picked from the culture plate and inoculated into 300 mL of nutrient broth. The culture was then carried out at 36°C and 185 rpm for 18 hours to prepare the seed culture.

[0083] Step 2: Inoculation and Culture

[0084] The seed culture was inoculated into nutrient broth medium at an inoculation rate of 5%, and cultured in a shaker at 183 rpm for 24 h at 36℃ to obtain the fermentation broth of strain ZCK-1.

[0085] Step 3: Centrifugation and supernatant collection

[0086] Centrifuge the fermentation broth at 8000 rpm for 15 min to remove bacterial precipitate and retain the supernatant;

[0087] Step 4: Macroporous resin adsorption

[0088] Macroporous resin was added to the supernatant at a mass-to-volume ratio of 2.7%; the mixture was then incubated at 35°C and 180 rpm for 24 hours for adsorption.

[0089] Step 5: Cleaning and Desorption

[0090] After natural sedimentation, the supernatant was poured off, and the macroporous resin was retained. The macroporous resin was washed twice with distilled water, and then 95% anhydrous ethanol was added to the macroporous resin. The resin was incubated in a shaker at 205 rpm for 25 h at 36 °C. Then, it was ultrasonically vibrated for 1 h to improve the desorption effect.

[0091] Step 6: Collection and Treatment of Eluent

[0092] Collect the eluent, centrifuge at 8000 rpm for 15 min to remove residual bacteria and impurities, and retain the supernatant;

[0093] Step 7: Vacuum rotary drying

[0094] The supernatant was placed in a vacuum rotary evaporator and dried at 58°C to obtain a purified extract of the novel antibacterial lipopeptide.

[0095] Example 4

[0096] This embodiment provides a method for isolating and purifying antimicrobial lipopeptides from the fermentation broth of Bacillus subtilis described in Example 1 above, comprising the following steps:

[0097] Step 1: Prepare seed solution

[0098] The ZCK-1 strain was inoculated onto a nutrient agar plate containing Staphylococcus aureus and cultured at 35°C for 24 hours. After that, the colony with the largest inhibition zone was picked from the culture plate and inoculated into 300 mL of nutrient broth. The culture was then carried out at 35°C and 180 rpm for 16-18 hours to prepare a seed culture.

[0099] Step 2: Inoculation and Culture

[0100] The seed culture was inoculated into nutrient broth medium at an inoculation rate of 5%, and cultured in a shaker at 180 rpm for 24 h at 35℃ to obtain the fermentation broth of strain ZCK-1.

[0101] Step 3: Centrifugation and supernatant collection

[0102] Centrifuge the fermentation broth at 8000 rpm for 15 min to remove bacterial precipitate and retain the supernatant;

[0103] Step 4: Macroporous resin adsorption

[0104] Macroporous resin was added to the supernatant at a mass-to-volume ratio of 2.7%; the mixture was then incubated at 35°C and 180 rpm for 24 hours for adsorption.

[0105] Step 5: Cleaning and Desorption

[0106] After natural sedimentation, the supernatant was poured off, and the macroporous resin was retained. The macroporous resin was washed twice with distilled water, and then 95% anhydrous ethanol was added to the macroporous resin. The resin was incubated at 35°C and 200 rpm in a shaker for 24 hours for adsorption. Then, it was ultrasonically vibrated for 1 hour to improve the desorption effect.

[0107] Step 6: Collection and Treatment of Eluent

[0108] Collect the eluent, centrifuge at 8000 rpm for 15 min to remove residual bacteria and impurities, and retain the supernatant;

[0109] Step 7: Vacuum rotary drying

[0110] The supernatant was placed in a vacuum rotary evaporator and dried at 58°C to obtain a purified extract of the novel antibacterial lipopeptide.

[0111] The method for isolating and purifying antimicrobial lipopeptides from Bacillus subtilis and its fermentation broth has the following advantages compared with existing technologies:

[0112] (1) Highly effective broad-spectrum antibacterial ability

[0113] The antimicrobial lipopeptides obtained by isolating and purifying Bacillus subtilis fermentation broth showed significant antimicrobial activity against all common clinical Gram-positive cocci, streptococci, and bacilli tested, and also exhibited significant antimicrobial effects against some Gram-negative bacteria. Specifically, they showed significant antimicrobial activity against clinical strains of Gram-positive bacteria such as Corynebacterium striatum, Streptococcus pneumoniae, Staphylococcus schlegelii, Streptococcus group B, Staphylococcus epidermidis, Staphylococcus aureus, Enterococcus faecalis, methicillin-resistant Staphylococcus aureus, and vancomycin-resistant Enterococci. Staphylococcus stearothermiae exhibited the strongest antibacterial activity, with minimum inhibitory concentrations (MICs) of 0.22 mg / mL, 0.11 mg / mL, and 0.11 mg / mL, respectively. Furthermore, the antimicrobial lipopeptides showed significant antibacterial activity against multidrug-resistant strains such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci. They also showed relatively strong antibacterial activity against Gram-negative bacteria such as Moraxella catarrhalis and Proteus vulgaris, with an inhibitory concentration of 0.45 mg / mL against Moraxella catarrhalis. However, they exhibited weaker antibacterial activity against extended-spectrum β-lactamase Escherichia coli (ESC) and ESC. vulgaris.

[0114] (2) Innovative and simple separation and purification methods

[0115] This invention provides an innovative and simple method for separating and purifying antimicrobial lipopeptides from fermentation broth. The method includes steps such as bacterial fermentation, macroporous resin adsorption, ethanol desorption, and vacuum drying and rotary evaporation, which can effectively separate and purify antimicrobial lipopeptides from the fermentation broth of strain ZCK-1. This method is not only simple and practical to operate, but also optimized to achieve high yield and purity, while retaining the high activity and stability of the antimicrobial lipopeptides, thus laying a foundation for large-scale industrial production.

[0116] 3) The antimicrobial lipopeptides obtained by the method for separating and purifying antimicrobial lipopeptides from the fermentation broth of Bacillus subtilis provided by the present invention are fine, odorless, and pale yellow powders that are easily soluble in water and have strong activity. Furthermore, the separation and purification scheme was optimized by single-factor experiments and professional mathematical statistical experiments, which significantly improved its yield, purity, and activity. Thus, reliable technical parameters were provided for the large-scale industrial production of antimicrobial lipopeptides from Bacillus subtilis.

[0117] Example 5

[0118] This embodiment is an addition based on embodiment 4:

[0119] The antimicrobial lipopeptides obtained by the method of isolating and purifying antimicrobial lipopeptides from Bacillus subtilis fermentation broth are sensitive to enzymes including proteinase K, papain and lipase.

[0120] Example 6

[0121] This embodiment is an addition based on embodiment 4:

[0122] The antimicrobial lipopeptides obtained by the method of isolating and purifying antimicrobial lipopeptides from Bacillus subtilis fermentation broth are resistant to high temperatures. Specifically, the antimicrobial lipopeptides showed no significant decrease in antimicrobial activity after treatment at 100℃ for 30 min, and retained 67% of their antimicrobial activity after high-pressure steam treatment at 121℃ for 20 min.

[0123] Example 7

[0124] This embodiment is an addition based on embodiment 4:

[0125] The antimicrobial lipopeptides obtained by the method of separating and purifying antimicrobial lipopeptides from the Bacillus subtilis fermentation broth are resistant to strong acids and strong alkalis; the antimicrobial lipopeptides retain more than 91% of their antimicrobial activity after treatment at pH 2-4 for 6 hours, and more than 77% of their antimicrobial activity after treatment at pH 11-13 for 6 hours.

[0126] Example 8

[0127] This embodiment is an addition based on embodiment 4:

[0128] The antimicrobial lipopeptide obtained by the method of isolating and purifying antimicrobial lipopeptides from the Bacillus subtilis fermentation broth was evaluated according to GB15193.3 "Food Safety Toxicology Evaluation Procedures and Methods - Acute Oral Toxicity Test"; the results of the acute oral toxicity test showed that the antimicrobial lipopeptide was practically non-toxic.

[0129] Example 9

[0130] This embodiment is an addition based on embodiment 4:

[0131] The antimicrobial lipopeptides obtained by the method of isolating and purifying antimicrobial lipopeptides from Bacillus subtilis fermentation broth are applied in the medical field; the antimicrobial lipopeptides are prepared into antimicrobial drugs or antimicrobial agents, which serve as natural antimicrobial agents for the prevention and treatment of pathogenic bacterial infections.

[0132] Antimicrobial lipopeptides are used for the prevention and treatment of clinical pathogenic bacterial infections, exhibiting broad-spectrum antibacterial activity against both Gram-positive and Gram-negative bacteria. The Gram-positive bacteria include *Streptococcus pneumoniae*, *Corynebacterium striatum*, *Staphylococcus schlegelii*, *Streptococcus group B*, *Staphylococcus epidermidis*, *Staphylococcus aureus*, *Enterococcus faecalis*, methicillin-resistant *Staphylococcus aureus*, and vancomycin-resistant *Enterococcus*. They exhibit significant antibacterial activity, particularly against methicillin-resistant *Staphylococcus aureus* (MRSA) and vancomycin-resistant *Enterococcus* (VRE). The Gram-negative bacteria include *Moraxella catarrhalis*, *Proteus vulgaris*, extended-spectrum β-lactamase *Escherichia coli*, and *Escherichia coli*.

[0133] Example 10

[0134] This embodiment is an addition based on embodiment 4:

[0135] The antimicrobial lipopeptides obtained by the method of separating and purifying antimicrobial lipopeptides from Bacillus subtilis fermentation broth are applied in the field of agriculture and animal husbandry to prevent and treat pathogenic bacterial infections in livestock and poultry and improve the health of livestock and poultry.

[0136] Antimicrobial lipopeptides are formulated into feed additives for use as antimicrobial agents in feed or poultry drinking water to prevent and treat bacterial infections in livestock and poultry, improve their disease resistance, reduce antibiotic use, ensure the safety of livestock products, and reduce antibiotic residues.

[0137] Example 11

[0138] This invention provides a method for isolating and purifying antimicrobial lipopeptides from Bacillus subtilis strain ZCK-1 and its fermentation broth. The invention will be described in detail below with reference to the accompanying drawings.

[0139] The steps of strain screening and isolation methods are as follows:

[0140] (1) Antagonistic dominant strains were screened using hydrolyzed casein (MH) agar plates containing mixed indicator bacteria. Staphylococcus aureus (ATCC 25923) in the logarithmic growth phase was used as the indicator bacteria, and a standard bacterial suspension with a turbidity of 0.5 McFarland (1.5 × 10⁻⁶) was prepared using sterile physiological saline. 8 (CFU / mL). Add the standard bacterial suspension at a ratio of 5% to sterile MH agar solution at approximately 45°C, mix immediately, and pour into sterile Petri dishes to prepare MH agar plates containing mixed indicator bacteria.

[0141] (2) Isolation of the target strain

[0142] Methods: Soil samples were collected, homogenized, and then serially diluted 10-fold. 10 samples were taken from each sample. -1 10 -2 and 10 -3 0.1 mL of each diluted sample was added to the surface of two MH agar plates containing mixed indicator bacteria. The samples were spread evenly using a sterile L-shaped glass rod. After incubation at 35°C for 24 hours, the presence of a transparent inhibition zone around the colonies was observed. Colonies with a large transparent inhibition zone were selected as the target strain for pure culture and preservation.

[0143] Results: An antagonistic strain with a large inhibition zone, namely strain ZCK-1, was screened using agar plates containing the indicator bacterium (Staphylococcus aureus ATCC 25923).

[0144] Example 12

[0145] Identification of ZCK-1 strain

[0146] (1) 16S rRNA identification assay

[0147] Methods: Total DNA from strain ZCK-1 was purified (using the CW0552S bacterial genomic DNA purification kit, Kangwei Century). Using this DNA as a template, PCR amplification was performed using universal 16S rRNA primers (27F-1492r, Shanghai Jierui Biotechnology Co., Ltd.). The PCR system consisted of: 1 μL DNA template, 1 μL each primer, 25 μL 2x Es Taq MasterMix (Dye), and double-distilled water to a final volume of 50 μL.

[0148] The PCR program was as follows: 94℃ for 5 minutes, 94℃ for 30 seconds, 50℃ for 30 seconds, 72℃ for 100 seconds, for 30 cycles. The final cycle was 72℃ for 7 minutes. PCR products were analyzed by 1.2% agarose gel electrophoresis and gene sequencing. Sequences were compared for homology in the NCBI database, and a phylogenetic tree was constructed using MEGA11 software.

[0149] Results: Specific 16S rRNA electrophoresis bands were obtained by PCR amplification (see attached image). Figure 1 A) The chromosome gene sequence length is 1433 bp. Sequence alignment with NCBI showed that this strain belongs to the same branch as several Bacillus subtilis strains, exhibiting the highest homology (see appendix). Figure 1 B), therefore it was identified as Bacillus subtilis.

[0150] (2) Whole genome sequencing and analysis

[0151] Methods: Genomic DNA of strain ZCK-1 was purified using the magnetic bead method (Sangon Biotech, China), and Hieff was employed. MaxUp IIDNA Library Prep Kit for Constructing DNA libraries. (In Illumina) TMWhole-genome sequencing was performed on a next-generation sequencing platform (Sangon Biotech, China). Sequencing data was assembled using SPAdes version 3.5.0 to obtain the whole genome sequence of *B. subtilis* ZCK-1. The genome sequence was aligned to the nt database using NCBI Blast+, and homologous strain information was obtained based on matching scores. Furthermore, a phylogenetic tree was constructed based on 16S rRNA and SNPs, and Average Nucleotide Identity (ANI) and DNA-DNA hybridization (DDH) values ​​were calculated for identification. In addition, the *B. subtilis* ZCK-1 genome sequence was uploaded to antiSMASH (version 7.1.0) for online analysis to comprehensively predict secondary metabolite synthesis gene clusters. Simultaneously, the genome sequence was aligned to the VFDB database, and gene annotations were combined with their corresponding virulence factor functional annotations. Annotations with an E-value less than 5 were selected as reliable alignment results.

[0152] Results: The total genome length of strain ZCK-1 was 4,043,370 bp, with a G+C content of 43.73%. The genome predicted 4127 coding genes, including 4041 CDS genes, 76 tRNA genes, and 9 rRNA genes (see appendix). Figure 2 A).

[0153] ② Based on the protein sequence alignment results, 3115 genes were annotated in the COG database, of which the category "for general function prediction only" was the most numerous, with 345 genes (see appendix). Figure 2 B).

[0154] ③ By comparing genomes with the NR database, it was found that 43.72% of Bacillus subtilis sequences were homologous to ZCK-1 (see appendix). Figure 3 A), and possesses multiple unique genes (see appendix). Figure 3 B). Phylogenetic trees constructed based on 16S rRNA and core single-copy genes showed that ZCK-1 clustered with Bacillus subtilis, with bootstrap support values ​​all below 70%. However, phylogenetic trees based on genomic SNPs showed that when ZCK-1 clustered with B. subtilis CV16 GCA_014879275.1, the bootstrap support value was 100% (see Appendix). Figure 3 C), and the ANI and DDH of ZCK-1 and GCA_014879275.1 were ≥98.86% and ≥94.3%, respectively. Therefore, ZCK-1 was identified as Bacillus subtilis.

[0155] ④ Using AntiSMASH online analysis of the ZCK-1 genome, six secondary metabolite gene clusters were predicted, including the antimicrobial lipopeptides fengycin and surfactin synthesized via the non-ribosomal peptide synthase (NRPS) pathway, Subtilosin A synthesized via the ribosomal peptide synthase (RiPP) pathway, bacillaene and bacillibactin synthesized via the polyketide synthase (PKS) pathway, and the dipeptide compound bacilysin. These secondary metabolites possess antimicrobial activity.

[0156] ⑤ By comparing the genome of strain ZCK-1 with the Virulence Factors of Pathogenic Bacteria (VFDB) database, 15 E values ​​less than 10 were predicted. -5 The virulence factor genes were mostly ATP-dependent proteins, NADP-dependent phosphoglucuronide dehydrogenase, flagellar biosynthetic proteins, and γ-glutamyl transpeptidase, but no key genes for bacterial toxin synthesis were found. Therefore, from a genetic perspective, the fermentation products of strain ZCK-1 do not contain any known pathogenic bacterial toxins.

[0157] In summary, this strain has been identified as a novel Bacillus subtilis strain and named ZCK-1. ZCK-1 is deposited at the China General Microbiological Culture Collection Center (DGMCC), with accession number 30620; its 16S rRNA sequence has been uploaded to NCBI GenBank, with accession number UB14134909 ZCK-1PP079112. The complete genome sequence of the strain has also been uploaded to the National Center for Biotechnology Information (NCBI) GenBank, with accession number NO. JBGOPS000000000.1. The ZCK-1 genome predicts the presence of various antimicrobial substances, including antimicrobial lipopeptides, in its secondary metabolites. Furthermore, genomic analysis indicates that the metabolites of this bacterium are non-pathogenic.

[0158] Example 13

[0159] Isolation, purification and analysis of antimicrobial lipopeptides in fermentation broth of strain ZCK-1

[0160] (1) Initial methods for separation and purification

[0161] Antimicrobial lipopeptides in the fermentation broth of strain ZCK-1 were separated and purified using a combination of macroporous resin adsorption and vacuum drying rotary evaporation. The specific steps are as follows:

[0162] ① Seed liquid inoculation: Inoculate the strain seed liquid at a rate of 3% into the nutrient broth medium and incubate at 35℃ and 180rpm for 24 hours.

[0163] ② Centrifugation: Centrifuge the fermentation broth at 8000 rpm for 15 minutes to remove bacterial precipitate and take the fermentation supernatant.

[0164] ③ Resin adsorption: Add 3% macroporous resin XAD 16N to the fermentation supernatant and adsorb at 35℃ and 180rpm for 24 hours.

[0165] ④ Washing and elution: After natural sedimentation, discard the supernatant and wash the resin twice with distilled water. Then elute with anhydrous ethanol, desorb at 35℃ and 200 rpm for 24 hours, and sonicate for 1 hour.

[0166] ⑤ Collection and drying: Collect the eluent and centrifuge at 8000 rpm for 15 minutes, then take the supernatant. Place the supernatant in a vacuum rotary evaporator and evaporate the water at 60℃ to obtain a light yellow powder, which is the antimicrobial lipopeptide.

[0167] (2) Antibacterial activity assay

[0168] The antibacterial activity of the purified antimicrobial lipopeptides against Staphylococcus aureus (ATCC 25923) was detected using the agar perforation diffusion method. The specific steps are as follows:

[0169] ① Sample preparation: Prepare the antibacterial lipopeptide solution to a concentration of 100 mg / mL.

[0170] ② Control settings: Penicillin sodium solution was used as a positive control, and sterile physiological saline was used as a negative control.

[0171] ③ Punching holes: Use a 6mm diameter punch to evenly punch holes on the surface of the mixed bacteria MH agar plate to set up test holes and negative control holes.

[0172] ④ Sample addition: Add 100 μL of antibacterial lipopeptide solution to each well and let stand for 30 minutes.

[0173] ⑤ Incubation: Place the plate at a constant temperature of 35℃ for 24 hours.

[0174] ⑥ Results: Observe the size of the inhibition zone around the well and measure the diameter of the inhibition zone.

[0175] (3) Protein content determination method

[0176] The protein content of the antimicrobial lipopeptide (100 mg / mL) was determined using the quinoline carboxylic acid (BCA) method. The protein purity of the purified antimicrobial lipopeptide was calculated according to Formula 1.

[0177] Formula 1:

[0178]

[0179] (4) Method for detecting the purity of antimicrobial lipopeptides: The purity of antimicrobial lipopeptides was detected and analyzed by C18 reversed-phase high-performance liquid chromatography (HPLC). The specific steps are as follows:

[0180] A high-performance liquid chromatography (HPLC) system (Shimadzu LC-20AT) and a Welch XB-C18 reversed-phase column were used. Chromatographic conditions: column temperature 30℃, mobile phase: acetonitrile and water, flow rate: 1.0 mL / min, detection gradient: 10%–90% (acetonitrile), detection wavelength: 214 nm, injection volume: 6 μL.

[0181] Results: By using macroporous resin XAD 16N adsorption and vacuum drying rotary evaporation technology, a fine, odorless, and highly water-soluble pale yellow powder was obtained, as shown in the attached image. Figure 4 As shown in Figure A, this is the purified antibacterial lipopeptide. It exhibits strong antibacterial activity, as shown in the attached figure. Figure 4 As shown in Figure B, the protein content, determined by BCA method, was 19.74 mg / mL. C18 reversed-phase high-performance liquid chromatography analysis showed only one main peak and 2-3 significantly different low peaks in the purified antimicrobial lipopeptide. Furthermore, comparison with Surfactin standard revealed that the peak exhibiting antibacterial activity overlapped with the Surfactin standard peak. Therefore, it is inferred that the purified product is an antimicrobial lipopeptide containing Surfactin (see Appendix). Figure 5 ).

[0182] Example 14

[0183] Sensitivity test of antimicrobial lipopeptides to various enzymes

[0184] Methods: Proteinase K, papain, trypsin, pepsin, catalase, and lipase were dissolved and diluted in distilled water to concentration gradients of 20 mg / mL, 10 mg / mL, and 5 mg / mL, respectively. Antimicrobial lipopeptides were dissolved in distilled water to a concentration of 50 mg / mL, filtered through a 0.22 μm bacterial membrane, and dispensed into EP tubes (500 μL per tube). The experimental group was divided into 6 groups, each containing 3 different enzyme concentrations, with 2 replicates for each concentration. The corresponding enzyme concentrations were added to each tube to achieve final enzyme concentrations of 10 mg / mL, 5 mg / mL, and 2.5 mg / mL for each group. The blank control group consisted of two equal volumes of enzyme-free antimicrobial lipopeptide solutions of equal concentration. The reaction temperature was set at 35 °C, and the reaction time was 2 hours. After the reaction, each tube was heated at 80 °C for 8 minutes to inactivate the enzymes. Subsequently, Staphylococcus aureus (ATCC 25923) was used as an indicator bacterium, and its antibacterial activity was tested by a well-diffusion test on MH agar plates. The mean diameter of the inhibition zone in the test wells and control wells was observed and measured.

[0185] Results: See attached. Figure 6As shown, the antibacterial activity of the antimicrobial lipopeptide decreased significantly with increasing concentrations of papain, proteinase K, and pepsin, while the effects of trypsin, pepsin, and catalase were not significant. After lipase treatment, the antimicrobial lipopeptide solution lost its antimicrobial activity at all concentrations. Therefore, it can be further inferred that the purified antimicrobial substance is an antimicrobial lipopeptide.

[0186] Example 15

[0187] Temperature sensitivity test of antimicrobial lipopeptides

[0188] Methods: Antimicrobial lipopeptides were dissolved in distilled water to a concentration of 50 mg / mL, filtered through a 0.22 μm bacterial filter, and dispensed into EP tubes, 500 μL per tube. The samples were divided into 8 groups, each with a set treatment temperature: 40℃, 50℃, 60℃, 70℃, 80℃, 90℃, 100℃, and 121℃ (autoclaving). Two replicates were prepared for each temperature. The treatment time at 40℃ to 100℃ was 30 minutes, and the autoclaving time at 121℃ was 20 minutes. Two untreated antimicrobial lipopeptide solutions served as a control group. Antimicrobial activity was assessed using the same method.

[0189] Results: See attached. Figure 7 As shown, the diameter of the inhibition zone of the antimicrobial lipopeptide only began to decrease slightly after treatment at 80℃ for 30 minutes, but this reduction was not significant; after treatment at 100℃ for 30 minutes, the diameter of the inhibition zone decreased significantly, but not significantly; and after high-pressure treatment at 121℃ for 20 minutes, the antimicrobial activity decreased significantly, but still retained 67% of the antimicrobial activity. These results indicate that the antimicrobial lipopeptide possesses strong heat resistance, consistent with the heat resistance characteristics of natural antimicrobial lipopeptides reported in the literature.

[0190] Example 16

[0191] pH sensitivity test of antimicrobial lipopeptides

[0192] Methods: Antimicrobial lipopeptides were dissolved in distilled water at a concentration of 50 mg / mL. After sterilization through a 0.22 μm filter membrane, the solutions were aliquoted into EP tubes, 500 μL per tube. The samples were divided into 14 groups, each adjusted to a pH value from pH 1 to pH 14 using either a strong acid or a strong base, with two replicates per group. After treatment at 4°C for 6 hours, the pH of all samples was adjusted back to pH 7. The unadjusted antimicrobial lipopeptides served as a blank control group. The antimicrobial activity was assessed using the same method as described above.

[0193] Results: See attached. Figure 8As shown, after treatment with antimicrobial lipopeptides for 6 hours under different pH conditions, the diameter of the inhibition zone remained unchanged from pH 7 to pH 10, indicating no impact on antimicrobial activity. Under slightly acidic conditions (pH 2 to 4), the diameter of the inhibition zone decreased slightly, but more than 91% of the antimicrobial activity was still retained. Under slightly alkaline conditions (pH 11 to 13), the diameter of the inhibition zone decreased significantly, but more than 77% of the antimicrobial activity was still retained. These results indicate that antimicrobial lipopeptides possess strong acid and alkali resistance.

[0194] Example 17

[0195] Antibacterial activity analysis and determination of minimum inhibitory concentration

[0196] Qualitative antibacterial assay method: Agar diffusion method was used. Antimicrobial lipopeptides were dissolved in sterile physiological saline, with a 2-fold dilution gradient of protein concentration from 56.32 mg / mL to 1.76 mg / mL. Logarithmic-phase clinical bacteria were adjusted to 0.5 McFarland turbidity and evenly spread onto MH plates, then perforated with a 6 mm punch. Sterile physiological saline (negative control) was added to the central well, and the diluted antimicrobial lipopeptide solution was added to the surrounding six wells, 100 μL per well. After standing for 30 minutes, the plates were incubated at 35°C for 24 hours, and the inhibition zones were observed and measured.

[0197] Quantitative antibacterial assay: The minimum inhibitory concentration (MIC) was determined using the microbroth dilution method. Antimicrobial lipopeptides were also serially diluted 2-fold, with protein concentrations ranging from 56.32 mg / mL to 0.055 mg / mL. Log-phase clinical bacteria were adjusted to 0.5 McFarland turbidity and diluted 1:100 with MH broth to 1×10^6 CFU / mL. In 96-well plates, 100 μL of serially diluted antimicrobial lipopeptide solution was added to the first 10 wells of each row, with well 11 serving as a negative control and well 12 as a positive control. Then, 100 μL of standard bacterial solution was added to the first 11 wells of each row, with two replicates per strain. After incubation at 35°C for 24 hours, bacterial growth was observed, and the lowest concentration at which no growth occurred was used to determine the MIC. Finally, the antimicrobial lipopeptide concentration was converted to a protein concentration.

[0198] Results: See attached. Figure 9 As shown, the antimicrobial lipopeptides exhibited inhibitory activity against all tested Gram-positive pathogens, particularly *Corynebacterium striatum* isolated from urine and nasal secretions (see Appendix). Figure 9 A) Streptococcus pneumoniae (see appendix) Figure 9 B), and Staphylococcus stearotherm isolated from blood (see Appendix). Figure 9 C), with the largest inhibition zone and a MIC as low as 0.11 mg / mL (see appendix). Figure 9 I). It is worth noting that the antimicrobial lipopeptide is effective against methicillin-resistant Staphylococcus aureus (MRSA) (see Appendix). Figure 9 D) and vancomycin-resistant enterococci (VRE) (see appendix) Figure 9It showed significant antibacterial activity against multidrug-resistant strains such as E), with a MIC as low as 3.59 mg / mL. The antimicrobial lipopeptide exhibited relatively weak antibacterial activity against Gram-negative pathogens, but showed activity against Moraxella catarrhalis isolated from sputum (see Appendix). Figure 9 F) and Proteus vulgaris isolated from urine (see Appendix) Figure 9 G) showed significant antibacterial activity, with a MIC as low as 0.45 mg / mL. Furthermore, the antimicrobial lipopeptide also exhibited a small inhibition zone against extended-spectrum β-lactamase Escherichia coli (ESBLs E. coli) (see Appendix). Figure 9 H). However, it has no inhibitory activity against other Gram-negative pathogens such as Salmonella and Klebsiella pneumoniae.

[0199] In summary, antimicrobial lipopeptides possess highly efficient and broad-spectrum antimicrobial activity, including against multidrug-resistant strains such as MRSA and VRE.

[0200] Example 18

[0201] The disruptive effect of antimicrobial lipopeptides on the cell membrane of Staphylococcus aureus

[0202] Methods: Staphylococcus aureus ATCC 25923 was inoculated into BL liquid medium and cultured at 37℃ with shaking until mid-log growth. Equal volumes of bacterial suspension were divided into three groups and mixed with antimicrobial lipopeptide solutions of 0×MIC (blank control), 1×MIC, and 4×MIC concentrations, respectively, and incubated at 37℃ for 2 hours. After incubation, the bacterial pellet was collected by centrifugation at 8000 rpm for 5 minutes. The bacterial pellet was washed with pre-cooled PBS, then fixed with 2.5% (v / v) glutaraldehyde solution at room temperature for 2 hours, followed by overnight fixation at 4℃. The fixed samples were then dehydrated sequentially with gradients of ethanol (30%, 50%, 70%, 80%, 100%), with each concentration set for 10 minutes. After dehydration, the samples were subjected to gradient displacement with isoamyl acetate (50%, 100%), with each displacement lasting 10 minutes. Finally, the samples were lyophilized, gold-plated, and observed using a scanning electron microscope (TESCAN VEGA3, China).

[0203] Results: Scanning electron microscopy revealed that antimicrobial lipopeptide treatment produced significant dose-dependent changes in the cell membrane of Staphylococcus aureus ATCC 25923 (see Appendix). Figure 10The control group (0×MIC) exhibited typical staphylococcal morphology, with smooth, intact surfaces and clear cell boundaries. The low-concentration group (1×MIC) showed significant cell membrane damage: bacterial cells exhibited surface depressions and irregular contractions, with visible transmembrane pores and signs of cytoplasmic leakage. The high-concentration group (4×MIC) showed severe cell lysis: most bacterial cells presented as completely collapsed cystic structures with wrinkled surfaces, and residual cell fragments exhibiting a typical "crater" disintegration morphology.

[0204] Example 19

[0205] The disruptive effect of antimicrobial lipopeptides on the genomic DNA of Staphylococcus aureus

[0206] Methods: The damaging effect of antimicrobial lipopeptides on the genomic DNA of Staphylococcus aureus ATCC 25923 was assessed by agarose gel electrophoresis. Logarithmically growing bacterial culture was used to extract intact genomic DNA using a bacterial genomic DNA extraction kit (CW05525, Kangwei Century, China). DNA purity (A260 / A280 ratio) was determined using a NanoDrop OneC micro-spectrophotometer (Thermo Scientific, USA). 40 μL of DNA solution was mixed with a series of concentrations of antimicrobial lipopeptides (0.25×, 0.5×, 1×, 2×, 4×, 6×MIC) in sterile EP tubes, with a 0×MIC blank control group. The mixtures were incubated at 35℃ for 1 h and 3 h. Electrophoresis was performed on a 1% agarose gel in 1×TAE buffer at a constant voltage of 100 V for 40 min.

[0207] Results: Agarose gel electrophoresis results showed that antimicrobial lipopeptide treatment led to significant dose-time-dependent degradation of Staphylococcus aureus genomic DNA (see Appendix). Figure 11 The blank control group (0×MIC) showed characteristic bands of intact genomic DNA without obvious tailing. When the antimicrobial lipopeptide treatment concentration was ≥1×MIC, the electrophoretic bands began to migrate downwards, indicating a decrease in DNA molecular weight. The high-concentration groups (4×-6×MIC) showed typical DNA degradation characteristics: the bands shifted downwards, darkened, and increased diffusion, indicating that large DNA molecules were broken into small fragments. Notably, the high-concentration group treated for 3 hours exhibited a "comet tail" migration morphology, indicating severe DNA fragmentation.

[0208] Example 20

[0209] Acute toxicity test method for oral administration of antimicrobial lipopeptides in animals

[0210] Methods: To investigate the safety of oral antimicrobial lipopeptides, Kunming mice were used. Different doses of the antimicrobial lipopeptide solution were administered via gavage, and the median lethal dose (LD50) was determined. Mice were randomly divided into 6 groups of 10 mice each, with half male and half female. The experimental groups were further divided into groups I, II, III, IV, and V based on doses of 8 mg, 40 mg, 200 mg, 1000 mg, and 5000 mg per kilogram of body weight (kg·BW), with a gavage dose of 25 μL / g for all groups. The control group received sterile saline orally. Mice were observed for 7 consecutive days after gavage, and toxic signs, mortality, weight gain, and food intake were recorded daily. After 7 days, blood samples were collected for complete blood count, liver function, and kidney function tests. Mice were euthanized by dislocation, and pathological examination and organ weighing were performed. Liver and kidney tissues were collected for pathological examination. Finally, the LD50 was calculated, and the toxicity of the antimicrobial lipopeptide was graded according to GB15193.3 "Procedures and Methods for Food Safety Toxicology Evaluation—Acute Oral Toxicity Test".

[0211] Results: During the 7-day observation period, no mice died or showed any clinical symptoms, and their behavior was no different from that of the normal control group. The mice exhibited normal drinking and feeding behavior, and their body weight increased with increasing doses of the antimicrobial lipopeptide (see Appendix). Figure 12 A) indicates that antimicrobial lipopeptides may have a growth-promoting effect. Complete blood count indicators showed no significant changes, and liver and kidney function were essentially normal, indicating that oral administration of antimicrobial lipopeptides would not cause hemolysis. Organ indices (heart, liver, kidney, lung, and spleen) showed no significant differences, and no obvious pathological changes were observed in liver and kidney tissue cells (see appendix). Figure 12 B). The review results indicate that oral antimicrobial lipopeptides showed no acute toxicity in mice at high doses, demonstrating high safety and providing a scientific basis for their safety in practical applications.

[0212] Example 21

[0213] Optimization of antibacterial lipopeptide isolation and purification methods

[0214] Antimicrobial lipopeptides were initially separated and purified by macroporous resin adsorption combined with vacuum rotary evaporation drying. To further improve the protein yield and antimicrobial activity of the antimicrobial lipopeptides, we optimized this method using single-factor experiments and the PB-BBD response surface methodology. The specific steps are as follows:

[0215] (1) Single-factor experiment

[0216] In the purification process of antimicrobial lipopeptides, five main factors affect the yield and protein activity of antimicrobial lipopeptides: seed culture inoculum amount, macroporous resin addition amount, elution ethanol concentration, eluent centrifugation speed, and rotary evaporation temperature. The purpose of the single-factor experiments was to investigate the effect of each factor at different levels on the yield and protein activity of antimicrobial lipopeptides.

[0217] ① Seed liquid inoculation test: The seed liquid inoculation amount was set to 1%, 2%, 3%, 4%, 5%, and 6%. Other conditions were kept consistent: 500 mL of fermentation broth with NB, 4% macroporous resin added, 100% elution ethanol concentration, 200 mL of eluent, centrifugation speed of 4000 rpm, and rotary evaporation temperature of 60℃.

[0218] ② Macroporous resin addition test: The macroporous resin addition was set at 1%, 2%, 3%, 4%, 5%, and 6%. Other conditions remained the same: seed liquid inoculation amount 5%, fermentation broth 500mL NB, elution ethanol concentration 100%, eluent 200mL, centrifugation speed 4000rpm, and rotary evaporation temperature 60℃.

[0219] ③ Eluent concentration test: The elution ethanol concentrations were set to 50%, 60%, 70%, 80%, 90%, 95%, and 100%. Other conditions remained the same: seed culture inoculation amount 5%, fermentation broth 500mL NB, macroporous resin addition amount 4%, eluent 200mL, centrifugation speed 4000rpm, and rotary evaporation temperature 60℃.

[0220] ④ Centrifugation speed test of eluent: The centrifugation speed was set to 0 rpm, 2000 rpm, 4000 rpm, 6000 rpm, 8000 rpm, and 10000 rpm, with a duration of 15 minutes for each. Other conditions remained the same: seed culture inoculum volume 5%, fermentation broth 500 mL NB, macroporous resin addition 4%, elution ethanol concentration 100%, eluent 200 mL, and rotary evaporation temperature 60℃.

[0221] ⑤ Rotary evaporation temperature test: The rotary evaporation temperatures were set at 40℃, 50℃, 60℃, 70℃, 80℃, and 90℃. Other conditions remained consistent: seed culture inoculum 5%, fermentation broth 500mL NB, macroporous resin addition 4%, elution ethanol concentration 100%, eluent 200mL, and centrifugation speed 4000rpm.

[0222] (2) PB experimental design to screen significant factors

[0223] Based on the results of single-factor experiments, two PB experimental schemes with high and low levels were designed. Using professional mathematical statistics software, the PB design was employed to investigate the factors that had the most significant impact on the combined evaluation index of protein yield and antibacterial activity among five factors: seed culture inoculum amount, macroporous resin addition amount, elution ethanol concentration, eluent centrifugation speed, and rotary evaporation temperature.

[0224] (3) Optimization of process using BBD response surface methodology

[0225] ① BBD Response Surface Design: Based on the PB experimental results, three factors that significantly affected protein yield and antibacterial activity were selected as variables in the response surface model. Each variable was assigned three levels: high, medium, and low (represented by 1, 0, and -1, respectively). Using professional statistical software, 17 experimental schemes were designed and conducted under fixed inoculum size (5%) and elution buffer centrifugation speed (8000 rpm). Finally, the combined evaluation of protein yield and antibacterial activity (Y) was used as the response value for analysis.

[0226] ② Response surface model establishment and analysis: Using professional mathematical statistics software, the BBD response surface method was used to design and optimize the purification process parameters, establish the response surface model, and draw response surface interaction diagrams and contour maps for analysis.

[0227] ③ Validation test: Under optimized conditions (inoculum amount 5%, macroporous resin XAD 16N addition amount 2.7%, elution ethanol concentration 94.5%, eluent centrifugation speed 8000 rpm, rotary evaporation temperature 58℃), antimicrobial lipopeptides were purified, and various performance indicators were measured. The test was conducted in triplicate.

[0228] (4) Methods for testing the antimicrobial lipopeptide properties

[0229] After purifying the antimicrobial lipopeptides through various optimized design experiments, the weight of the dry powder, its antibacterial activity, and protein content need to be determined. The yield of antimicrobial lipopeptides in the fermentation broth (g / L) is calculated using Formula 2, the protein yield in the fermentation broth (g / L) is calculated using Formula 3, and finally, the combined evaluation index of protein yield and antibacterial activity in the fermentation broth is calculated using Formula 4.

[0230] Formula 2: Fermentation broth dry powder yield (g / L) = Extracted dry powder weight (g) / Fermentation broth volume (L)

[0231] Formula 3:

[0232] Formula 4: Joint evaluation index = protein yield in fermentation broth (g / L);

[0233] Optimization of experimental results: Based on the quadratic regression equation of the response value (inhibition zone diameter * protein yield) data model, professional mathematical statistics software was used to optimize the purification process parameters of ZCK-1 antibacterial lipopeptides, as shown in the attached figure. Figure 13 , Figure 14 , Figure 15 As shown, the optimal parameters were obtained: XAD 16N addition of 2.664% (approximately 2.7%), elution ethanol concentration of 94.469% (approximately 95%), and rotary evaporation temperature of 58.318℃ (58%). Under these conditions, the response value (inhibition zone diameter * protein yield) was 3.945.

[0234] Example 21

[0235] Application of antimicrobial lipopeptides in the prevention and treatment of clinical pathogens

[0236] 1. Background

[0237] The incidence of clinical pathogens is very high. Due to the emergence of multidrug-resistant strains, traditional antibiotics are becoming increasingly ineffective in prevention and treatment. Therefore, there is an urgent need to develop new antibacterial agents and apply them in clinical practice to alleviate the serious threat posed by drug-resistant strains to clinical care.

[0238] The antimicrobial lipopeptides isolated and purified from the fermentation broth of Bacillus subtilis strain ZCK-1 provided by this invention have been found to have a broad-spectrum and highly effective ability to inhibit clinical pathogens.

[0239] 2. Implementation Steps

[0240] The antibacterial activity of antimicrobial lipopeptides against 18 common clinical bacteria and fungi was determined using qualitative and quantitative methods, thereby clarifying the antimicrobial spectrum of antimicrobial lipopeptides against clinical pathogens.

[0241] (1) Qualitative determination: The semi-quantitative qualitative detection method of agar plate diffusion was adopted. Specific procedures: ① Reactivate the clinically infected bacterial strain, culture it to the logarithmic growth phase, take 1-3 colonies, grind them evenly in 5 ml of sterile physiological saline, adjust to 0.5 McFarland turbidity, prepare a standard bacterial solution, and inoculate it onto the surface of an MH plate using a sterile cotton swab; ② Dissolve the antimicrobial lipopeptide in sterile physiological saline, and dilute it 2-fold to a protein concentration gradient of 56.32 mg / mL to 1.76 mg / mL; ③ On the MH plate with the strain already coated, punch holes evenly with a 6 mm diameter punch: add sterile physiological saline to the central negative control well; add 100 μL of the diluted antimicrobial lipopeptide solution to each of the surrounding 6 test wells; ④ After the plate stands for 10 minutes, transfer it to a 35℃ incubator and incubate for 24 hours. Observe and measure the diameter of the inhibition zone.

[0242] (2) Quantitative determination: The minimum inhibitory concentration (MIC) was quantitatively determined using the microbroth dilution method. Specific procedures: ① Prepare diluted antimicrobial lipopeptides: The antimicrobial lipopeptides were also diluted 2-fold, with protein concentrations ranging from 56.32 mg / mL to 0.055 mg / mL. ② Prepare standard bacterial solutions: Clinically infected strains grown to the logarithmic phase were prepared into 0.5 McFarland turbidity standard bacterial solutions, which were then diluted 1:100 with MH broth to obtain a standard bacterial solution with a concentration of 1×10⁶ CFU / mL. ③ 96-well micro-susceptibility plate loading: 100 μL was added to each well. The first 10 wells of each row were loaded with decreasing concentrations of antimicrobial lipopeptide solution. Well 11 was used as a negative control, with MH broth added. Well 12 was used as a positive control, with only 200 μL of MH broth added. Afterward, the corresponding standard bacterial solutions were added to wells 1 to 11 of each row. Each strain was repeated twice. ④ Incubation: After sealing the antimicrobial susceptibility plate, incubate at 35℃ for 24 hours. ⑤ Determine MIC: Observe the bacterial growth in each well, and determine the lowest dilution of the drug solution that shows no bacterial growth (i.e., no precipitate in the well) as the minimum inhibitory concentration.

[0243] (3) Method for determining the minimum bactericidal concentration: The determination of the minimum bactericidal concentration (MBC) is based on the determination of the MIC. Take 100 μl of the liquid from the MIC well, the first two concentration wells, and the last drug concentration well, spread it on the surface of an MH plate, and incubate at 35°C for 24 h; repeat twice for each well. Observe the colony growth, and the lowest dilution with an average of less than 5 colonies is taken as the MBC.

[0244] 3. Application Effects

[0245] To clarify the antimicrobial spectrum of antimicrobial lipopeptides against clinical pathogens, this study tested 36 strains of 18 clinically infectious bacteria using qualitative and quantitative antimicrobial assays. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the antimicrobial lipopeptides against these strains were determined.

[0246] (1) Results of the assay of the antimicrobial lipopeptide's antibacterial activity against clinical Gram-positive infectious bacteria

[0247] As shown in Table 1, the antimicrobial lipopeptides exhibited antibacterial activity against 20 common Gram-positive bacteria of 9 species causing clinical infections, but the antibacterial activity varied. Qualitative experiments showed that the ZCK-1 antimicrobial lipopeptide exhibited the strongest antibacterial activity against two strains of Corynebacterium striatum isolated from urine, with inhibition zones exceeding 36 mm in diameter. Due to the large size of the inhibition zones, only a small number of wedge-shaped bacterial films were visible at the edge of the agar plate. Secondly, the ZCK-1 antimicrobial lipopeptide showed strong antibacterial activity against two strains of Streptococcus pneumoniae isolated from nasal secretions, with inhibition zones of 34 mm in diameter. Thirdly, the inhibition zone diameter against two strains of Staphylococcus schleiferi isolated from blood was 30 mm. Furthermore, the ZCK-1 antimicrobial lipopeptide showed significant antibacterial activity against Staphylococcus epidermidis, Staphylococcus aureus, and Enterococcus. Of particular note is that the antimicrobial lipopeptide exhibited significant antibacterial activity against four strains of methicillin-resistant Staphylococcus aureus (MRSA) and one strain of vancomycin-resistant Enterococcus (VRE). Compared to common Staphylococcus aureus, ZCK-1 showed a larger inhibition zone diameter and stronger antibacterial activity against the four MRSA strains.

[0248] Quantitative determination results: The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were used to quantitatively detect antibacterial activity. Higher MIC and MBC values ​​correlated with lower antibacterial activity, showing a negative correlation. Table 1 shows the comparison of MIC values ​​of antimicrobial lipopeptides against Gram-positive bacteria. The three bacteria with the strongest antibacterial activity were: *Streptococcus pneumoniae* (MIC 0.11 mg / mL, MBC 0.22 mg / mL); *Staphylococcus stearothermiae* (MIC 0.11 mg / mL, MBC 0.22 mg / mL); and *Corynebacterium striatum* (MIC 0.22 mg / mL, MBC 0.45 mg / mL). The antibacterial activity of other bacteria was, in descending order, that of *Streptococcus group B*, *Enterococcus*, *Staphylococcus aureus*, and *Staphylococcus epidermidis*. The quantitative antibacterial experiment results largely validated the qualitative antibacterial experiment results.

[0249] Table 1. Results of Antimicrobial Lipopeptide Activity Inhibiting Clinical Gram-positive Bacteria

[0250]

[0251] Note: MIC represents the minimum inhibitory concentration; MBC represents the minimum bactericidal concentration.

[0252] (2) Results of the assay of the antimicrobial lipopeptide's antibacterial activity against clinical Gram-negative infectious bacteria

[0253] As shown in Table 2, the antimicrobial lipopeptides exhibited significant antibacterial activity against Moraxella catarrhalis (two strains isolated from sputum), a Gram-negative coccus. The inhibition zone diameter reached 22 mm, and the highest MIC was 0.45 mg / mL, indicating that the antimicrobial lipopeptides had a strong antibacterial effect against Moraxella catarrhalis.

[0254] Among Gram-negative bacilli, the antimicrobial lipopeptide exhibited significant antibacterial activity against *Proteus vulgaris* isolated from urine, with an inhibition zone diameter of 12 mm and a MIC of 1.8 mg / mL, demonstrating a strong antibacterial effect. Furthermore, five strains of *Ultra-Broad Spectrum β-Lactamase Escherichia coli* (ESBLsE. coli) and one strain of *Proteus vulgaris* showed small, transparent inhibition zones around the high-concentration wells, indicating that the antimicrobial lipopeptide possesses certain antibacterial activity against these strains.

[0255] Table 2. Results of Antimicrobial Lipopeptide Inhibition of Clinical Gram-negative Bacteria Activity

[0256]

[0257] Note: MIC represents the minimum inhibitory concentration; MBC represents the minimum bactericidal concentration; - indicates no inhibition zone; -- indicates not detected.

[0258] Oral application effects of antibacterial lipopeptides

[0259] 1. Background

[0260] In animal husbandry, animal health is key to increasing production, and feed additives play an important role in enhancing animal immunity and reducing disease. Research has found that Bacillus subtilis ZCK-1 can produce antimicrobial lipopeptides with antibacterial activity.

[0261] 2. Implementation Steps

[0262] (1) Strain screening and fermentation: Bacillus subtilis ZCK-1, which can produce antimicrobial lipopeptides, was isolated from the soil environment.

[0263] (2) Isolation and purification of antimicrobial lipopeptides: The strain produces high levels of antimicrobial lipopeptides through a specific fermentation process, followed by purification and drying to produce antimicrobial lipopeptides.

[0264] (3) Oral administration test in mice: Kunming mice were randomly divided into 6 groups of 10 mice each, with half males and half females. The experimental groups and the control group were treated separately. The control group was administered sterile saline by gavage; the experimental groups were divided into groups I, II, III, IV, and V according to the dosage of 8 mg, 40 mg, 200 mg, 1000 mg, and 5000 mg per kilogram of body weight (kg·BW), and the gavage dose for all mice was 25 μg / g. The mice were fasted for 12 hours before gavage and resumed eating after gavage.

[0265] (4) Observation and recording: Mice were observed for 7 consecutive days after gavage. The occurrence of toxic signs and death was recorded daily, and the weight gain of mice was measured.

[0266] 3. Application Effects

[0267] During the 7-day observation period, no abnormalities were observed in the food and water intake of mice in any of the treatment groups, but the average weight gain varied among the groups. (See attached image) Figure 12 As shown in Figure A, compared with the control group, the average weight gain of mice increased with increasing ZCK-1 antimicrobial lipopeptide dosage, meaning the weight gain in the high-dose group was significantly higher than that in the low-dose group. The experimental results indicate that ZCK-1 antimicrobial lipopeptide may promote mouse growth and accelerate weight gain. This suggests that the application of feed supplemented with antimicrobial lipopeptides in animal husbandry may significantly improve animal immunity, inhibit the growth of harmful intestinal bacteria, thereby improving animal health and increasing breeding yield and quality.

[0268] At the same time, the use of this antibacterial lipopeptide feed additive also reduces the dependence on chemical antibiotics, which is conducive to the green and sustainable development of the aquaculture industry.

[0269] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions, and improvements made by those skilled in the art within the scope of the technology disclosed in the present invention, and within the spirit and principles of the present invention, should be included within the scope of protection of the present invention. Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Bacillus subtilis, characterized in that, The strain was named ZCK-1 and has been deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, postal code: 100101, with accession number CGMCC NO.30620.

2. A method for purifying and producing highly efficient broad-spectrum antibacterial lipopeptides of Bacillus subtilis as described in claim 1, characterized in that, Includes the following steps: Step 1: Prepare seed solution The ZCK-1 strain was inoculated onto a nutrient agar plate containing Staphylococcus aureus and cultured at 33-37 ℃ for 22-25 h. After that, the colony with the largest inhibition zone was picked from the culture plate and inoculated into 300 mL of nutrient broth. The culture was then carried out at 34-36 ℃ on a shaker at 175-185 rpm for 16-18 h to prepare a seed culture. Step 2: Inoculation and Culture The seed culture was inoculated into nutrient broth medium at an inoculation rate of 5%, and cultured in a shaker at 177-183 rpm for 24 h at a temperature of 34-36 ℃ to obtain the fermentation broth of strain ZCK-1. Step 3: Centrifugation and supernatant collection Centrifuge the fermentation broth at 8000 rpm for 15 min to remove bacterial precipitate and retain the supernatant; Step 4: Macroporous resin adsorption Macroporous resin was added to the supernatant at a mass-to-volume ratio of 2.7%; the mixture was then incubated at 35 °C on a shaker at 180 rpm for 24 h to allow adsorption. Step 5: Cleaning and Desorption After natural sedimentation, pour off the supernatant, retain the macroporous resin, wash the macroporous resin twice with distilled water, then add 95% anhydrous ethanol to the macroporous resin and incubate it in a shaker at 195-205 rpm for 23-25 ​​h at a temperature of 33-36 ℃; then sonicate for 1 h to improve the desorption effect. Step 6: Collection and Treatment of Eluent Collect the eluent, centrifuge at 8000 rpm for 15 min to remove residual bacteria and impurities, and retain the supernatant; Step 7: Vacuum rotary drying The supernatant was placed in a vacuum rotary evaporator and dried at 58 °C to obtain a purified antimicrobial lipopeptide.

3. The purification method for highly efficient broad-spectrum antibacterial lipopeptides according to claim 2, characterized in that, In step one, the ZCK-1 strain was inoculated onto a nutrient agar plate containing Staphylococcus aureus and cultured at 35 °C for 24 h. After that, the colony with the largest inhibition zone was picked from the culture plate and inoculated into 300 mL of nutrient broth. The culture was then carried out at 35 °C on a shaker at 180 rpm for 16-18 h to prepare the seed culture.

4. The purification method for highly efficient broad-spectrum antibacterial lipopeptides according to claim 2, characterized in that, In step two, inoculation and culture, the seed culture was inoculated into the nutrient broth medium at an inoculation rate of 5%, and cultured in a shaker at 180 rpm for 24 h at a temperature of 35 ℃ to obtain the fermentation broth of strain ZCK-1.

5. The purification method for highly efficient broad-spectrum antibacterial lipopeptides according to claim 2, characterized in that, In step five, washing and desorption, after natural sedimentation, the supernatant is poured off, and the macroporous resin is retained. The macroporous resin is washed twice with distilled water, and then 95% anhydrous ethanol is added to the macroporous resin. The resin is then incubated for 24 h at 35 ℃ on a shaker at 200 rpm. Finally, it is ultrasonically vibrated for 1 h to improve the desorption effect.

6. The purification method for highly efficient broad-spectrum antibacterial lipopeptides according to claim 2, characterized in that, The antimicrobial lipopeptides obtained by the purification method of Bacillus subtilis highly efficient broad-spectrum antimicrobial lipopeptides are sensitive to enzymes including proteinase K, papain and lipase.

7. The purification method for highly efficient broad-spectrum antibacterial lipopeptides according to claim 2, characterized in that, The antimicrobial lipopeptides obtained by the purification method of Bacillus subtilis high efficiency and broad spectrum antimicrobial lipopeptides are resistant to high temperature. The antimicrobial lipopeptides do not show a decrease in antimicrobial activity after being treated at 100℃ for 30 min; and the antimicrobial lipopeptides retain 67% of their antimicrobial activity after being treated with high pressure steam at 121℃ for 20 min.

8. The purification method for highly efficient broad-spectrum antibacterial lipopeptides according to claim 2, characterized in that, The antimicrobial lipopeptides obtained by the purification method of Bacillus subtilis high efficiency and broad spectrum antimicrobial lipopeptides are resistant to strong acids and strong alkalis; the antimicrobial lipopeptides retain more than 91% of their antimicrobial activity after treatment with pH 2-4 for 6 h, and more than 71% of their antimicrobial activity after treatment with pH 11-12 for 6 h.

9. The purification method for highly efficient broad-spectrum antibacterial lipopeptides according to claim 2, characterized in that, The antimicrobial lipopeptides obtained by the purification method of Bacillus subtilis high efficiency and broad spectrum antimicrobial lipopeptides are applied in the medical field; the antimicrobial lipopeptides are prepared into antimicrobial drugs or antimicrobial agents, which are used as natural antimicrobial agents to prevent and treat pathogenic bacterial infections. Antimicrobial lipopeptides are used for the prevention and treatment of clinical pathogen infections, exhibiting broad-spectrum antibacterial activity against both Gram-positive and Gram-negative bacteria. The Gram-positive bacteria include Corynebacterium striatum, Streptococcus pneumoniae, Staphylococcus schlegelii, Streptococcus group B, methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus, Enterococcus faecalis, and Staphylococcus epidermidis; the Gram-negative bacteria include Moraxella catarrhalis, Proteus vulgaris, extended-spectrum β-lactamase Escherichia coli, Escherichia coli, and Salmonella.

10. The purification method for highly efficient broad-spectrum antibacterial lipopeptides according to claim 2, characterized in that, The antimicrobial lipopeptides obtained by the purification method of Bacillus subtilis high efficiency and broad spectrum antimicrobial lipopeptides are applied in the field of agriculture and animal husbandry to prevent and treat pathogenic bacterial infections in livestock and poultry and improve the health level of livestock and poultry. Antimicrobial lipopeptides are formulated into feed additives for use as antimicrobial agents in feed or poultry drinking water to prevent and treat bacterial infections in livestock and poultry, improve their disease resistance, reduce antibiotic use, ensure the safety of livestock products, and reduce antibiotic residues.

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