Octapeptide, preparation method and application of octapeptide in antibiosis or immunoregulation

Abstract

The invention provides the immunomodulatory octapeptide, the small peptide molecule has a unique structure, high broad-spectrum antibacterial activity, strong pathogenic bacterium inhibition and high purity, the stability of the structure and the antibacterial activity can be maintained in artificial gastric juice, no obvious hemolytic activity exists, and the immunomodulatory octapeptide has a very high application value in livestock breeding. The invention also provides a preparation method and a purity detection method of the immunomodulatory octapeptide.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an octapeptide, a preparation method and application thereof. Background Art

[0002] Lipopeptides or polypeptides are a very important class of compounds in microbial secondary metabolites. They have attracted widespread attention from researchers due to their unique chemical structure and diverse biological activities. Lipopeptides or polypeptides are amino acid polymerization products between amino acids and proteins and are basically synthesized by ribosomal or multi-module non-ribosomal peptide synthetases. Among them, the immunomodulatory octapeptide can be expressed by Brevibacillus laterosporus and is a non-ribosomal peptide secondary metabolite of the octapeptin family. Brevibacillus laterosporus ( Bacillus laterosporus ) is a type of Gram-negative bacillus, with a slender rod-shaped body, strong adaptability, and high temperature resistance. At the same time, this strain can produce a variety of biologically active substances including lipopeptides and polypeptides.

[0003] The emergence of traditional antibiotics has not only led to rapid development of the pharmaceutical industry, but also has very important applications in livestock and poultry farming. However, due to the abuse of traditional antibiotics, the emergence of multi-drug resistant bacteria has seriously affected the development of animal husbandry, and also endangered the ecological environment and human health. In order to solve this problem, it is urgent to develop new antibiotic substitutes for use in animal husbandry. Summary of the invention

[0004] In view of the above problems, the present invention provides an immunomodulatory octapeptide with a unique structure, excellent activity, strong specificity, high purity, high adaptability in vivo, stability and safety.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is as follows: The present invention also provides an octapeptide, the structural formula of which is as follows:

[0006] The above octapeptide is prepared by fermentation of Brevibacillus laterosporus. Preferably, Brevibacillus laterosporus developed by this research group is used. Bacillus laterosporus M811, which is deposited in China Center for Type Culture Collection, deposit date: April 18, 2024, address: Wuhan University, Wuhan, China, deposit number: CCTCC NO: M2024715. This strain is used to prepare immunomodulatory octapeptide to improve the yield, purity, activity, adaptability, etc. of the product.

[0007] The method for preparing the octapeptide is characterized by comprising: (1) Fermenting with Brevibacillus laterosporus to prepare a fermentation liquid; (2) Preparation of crude fermentation extract by salt ion precipitation; (3) The crude fermentation extract is separated and purified by high performance liquid chromatography, acetonitrile is removed by rotary evaporation, and then freeze-dried.

[0008] Preferably, the reverse phase high performance liquid chromatography preparation process in step (3) is: preparative liquid chromatography column, Agilent Zorbax 300SB-C18 PrepHT; mobile phase contains 0.1% trifluoroacetic acid in water (A) and 0.1% trifluoroacetic acid in acetonitrile (B); The elution mode is shown in Table 1: Table 1 Elution gradient for octapeptide analysis Time, min A, % B, % 0 70 30 57 63.4 36.6 58 0 100 64 0 100 65 70 30 67 70 30 The flow rate was 5 mL / min, the column temperature was 30°C, and the detection wavelength was 220 nm.

[0009] Preferably, the process in step (2) is as follows: the fermentation broth obtained in step (1) is adjusted to pH 3.0 using 5 M H2SO4, centrifuged at 8000 rpm for 5 min, and the supernatant is retained. Ammonium sulfate at a concentration of 5 to 30 g / mL is added to the supernatant, slowly stirred for 30 min, and then centrifuged at 8000 rpm for 10 min, the precipitate is collected, and dried at 60°C to obtain a crude octapeptide extract. The immunomodulatory octapeptide can be precipitated and enriched by ammonium sulfate at a specific concentration, and different concentrations of ammonium sulfate can precipitate different types of proteins. In the present invention, adding an optimal concentration of ammonium sulfate to the fermentation broth obtained in step (1) can precipitate and enrich the immunomodulatory octapeptide to the greatest extent to obtain the best recovery rate, and can also remove the interference of foreign proteins with the subsequent preparative liquid chromatography separation and purification steps, thereby improving the purity of the immunomodulatory octapeptide pure product.

[0010] The method for preparing an immunomodulatory octapeptide using Brevibacillus laterosporus specifically comprises the following steps: (1) Brevibacillus laterosporus Bacillus laterosporus M811 was inoculated in NB slant medium for activation, and after culturing at 37°C for 24 h, a single colony was inoculated into a sterilized NB liquid medium shake bottle, and cultured at 37°C and 220 rpm for 24 h to obtain a first-level seed solution. The first-level seed solution was fully mixed and transferred to a second-level seed liquid medium at a 5% inoculation rate, and cultured at 37°C and 220 rpm for 24 h to obtain a second-level seed solution. The second-level seed solution was inoculated into the optimized fermentation medium at a volume ratio of 5% inoculation rate, and cultured at 30°C and 220 rpm for 48 h to obtain a fermentation liquid.

[0011] (2) The fermentation liquid obtained in step (1) was adjusted to pH 3.0, centrifuged at 8000 rpm for 5 min, and the supernatant was retained. 5-30 g / mL of ammonium sulfate powder was added to the supernatant, slowly stirred for 30 min, centrifuged to collect the precipitate and dried at 60°C to obtain a crude immunomodulatory octapeptide extract.

[0012] (3) The crude extract obtained in step (2) was dissolved in the initial mobile phase of a preparative high performance liquid phase to a concentration of 0.1 g / mL, and the supernatant obtained by centrifugation was separated and purified by a preparative high performance liquid chromatograph. The fractions corresponding to the immunomodulatory octapeptide were collected, acetonitrile was removed by rotary evaporation, and then freeze-dried to obtain the pure octapeptide. The purity was determined by an analytical high performance liquid chromatograph.

[0013] Preferably, the secondary seed liquid culture medium in step (1) is: 3 g / L peptone, 16 g / L yeast powder, 2 g / L sucrose, 0.1 g / L magnesium sulfate, pH 6.8±0.1. The fermentation medium is: 15 g / L corn syrup powder, 10 g / L sucrose, 3 g / L ammonium sulfate, 0.3 g / L magnesium sulfate, pH 6.8±0.1.

[0014] The present invention also provides a method for detecting the purity of the octapeptide, comprising: Take 1 mg of the pure octapeptide obtained in step (3) above and place it in a centrifuge tube and dissolve it in 1 mL of ultrapure water. Vortex and shake for 1 min, then filter it through a 0.22 μm filter membrane, and use an analytical high performance liquid chromatograph to detect the purity of the immunomodulatory octapeptide. Preferably, the reversed phase high performance liquid chromatography analysis process is as follows: analytical liquid chromatography column, Sepax Bio-C18 4.6×250 mm 5μm; mobile phase containing 0.1% trifluoroacetic acid in water (A) and 0.1% trifluoroacetic acid in acetonitrile (B); The elution mode is shown in Table 2: Table 2 Elution gradient for octapeptide analysis Time, min A, % B, % 0 80 20 4 80 20 10 50 50 13 40 60 15 0 100 19 0 100 22 80 20 26 80 20 The flow rate was 1 mL / min, the column temperature was 30°C, and the detection wavelength was 220 nm.

[0015] The present invention also provides the use of the above-mentioned octapeptide in antibiotics or immunomodulators, or in the preparation of drugs, reagents, feeds, and feed additives. Preferably, the octapeptide is an anti-Enterobacterial octapeptide, and its use in the preparation of drugs or feed additives for resisting Gram-negative Enterobacteriaceae infection. The Gram-negative Enterobacteriaceae include but are not limited to Escherichia coli, Salmonella, Shigella dysenteriae, and Pseudomonas aeruginosa. Through the screening of antibacterial activity, the octapeptide has good activity in inhibiting pathogenic Enterobacteriaceae such as Escherichia coli, Salmonella, Shigella dysenteriae, and Pseudomonas aeruginosa, and can be used for the development and utilization of antibacterial drugs, leading veterinary drugs, and feed additives for inhibiting related diseases and infections caused by various Gram-negative pathogenic Enterobacteriaceae. Beneficial Effects

[0016] The invention provides an octapeptide having the ability to resist Gram-negative and Gram-positive pathogens. The small peptide molecule has a unique structure, high broad-spectrum antibacterial activity, strong inhibition of pathogens, high purity and strong performance stability.

[0017] 2. The immunomodulatory octapeptide provided by the present invention can maintain the stability of its structure and antibacterial activity in artificial gastric juice and has no obvious hemolytic activity. The immunomodulatory octapeptide has a strong bactericidal effect on both Gram-positive and Gram-negative pathogens, and its bactericidal ability against Escherichia coli and Pseudomonas aeruginosa is significantly stronger than that against Staphylococcus aureus, and has a high application potential in animal husbandry.

[0018] 3. The present invention provides a method for preparing an immunomodulatory octapeptide, and prepares a high-purity octapeptide finished product through multi-stage seed liquid culture, fermentation liquid preparation, fermentation liquid pretreatment, octapeptide extraction and refining, thereby greatly improving the fermentation efficiency and reducing the separation and purification process.

[0019] 4. The present invention utilizes salt ion precipitation-preparative reverse phase high performance liquid chromatography to treat the octapeptide fermentation broth, thereby improving the preparation efficiency and sample purity of the octapeptide and reducing the preparation cost. The purity of the octapeptide prepared by this method is as high as 98.23%. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The Brevibacillus laterosporus in Example 1 of the present invention Bacillus laterosporus M811 strain identification phylogenetic tree; Figure 2 The recovery rate test result of the octapeptide in Example 2 of the present invention is shown in FIG. Figure 3 This is the HPLC preparation diagram of the octapeptide in Example 2 of the present invention; Figure 4 This is a purity test diagram of the octapeptide in Example 2 of the present invention; Figure 5 HPLC preparation diagram of three octapeptides of Examples of the present invention; Figure 6 This is a graph showing the purity of the octapeptide in Example 3 of the present invention; Figure 7 HPLC preparation diagram of octapeptide in Example 4 of the present invention Figure 8 This is a graph showing the purity of the octapeptide in Example 4 of the present invention; Fig. 9 This is the primary mass spectrum of the octapeptide in Example 5 of the present invention; Fig.10 is the secondary mass spectrum of the octapeptide in Example 5 of the present invention; Fig.11 The GC-MS spectrum of fatty acids of the octapeptide hydrolysate in Example 5 of the present invention; Fig.12 The stability results of the octapeptide in Example 7 of the present invention in artificial gastric juice; Fig.13 The hemolytic results of the octapeptide in Example 8 of the present invention. DETAILED DESCRIPTION

[0021] The strain used in the present invention is Brevibacillus laterosporus isolated from the soil of Hewanzi Village, Yongchuan District, Chongqing City (N 29°19′54″, E 105°41′11″). The strain was deposited in the China Center for Type Culture Collection on April 18, 2024, address: Wuhan University, Wuhan, China, with a deposit number of CCTCC NO: M2024715.

[0022] The strain isolated by the present invention has been identified and its morphological characteristics are: the colony is transparent beige with a shiny surface; the bacteria are slender rod-shaped, about 2.0×1.0 μm in size, with flagella around the bacteria, can form oval spores, and are Gram-positive. The 16S rRNA gene of the strain was amplified by PCR, sequenced, and phylogenetic analysis was performed. Figure 1 It was identified as Brevibacillus laterosporus and named Brevibacillus laterosporus Brevibacillus Laterosporus M811.

[0023] The NB culture medium used in the present invention is composed of: 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L sodium chloride, which are purchased from Beijing Solebow Technology Co., Ltd. The corn steep liquor powder, sucrose, and magnesium sulfate culture medium components are all commercially available culture medium components.

[0024] The fermentation and compound separation and purification parameters involved in the present invention are well known to those skilled in the art.

[0025] Example 1 An identification of a Brevibacillus laterosporus strain for preparing an immunomodulatory octapeptide comprises the following steps: (1) DNA extraction Use TSINGKE Plant DNA Extraction Kit (Universal), the specific steps are as follows: 1. Place the Spin Column in the Collection Tube, add 250 μL Buffer BL, and centrifuge at 12,000 rpm for 1 min to activate the silica membrane; 2. Take the dried tissue of the sample (no more than 20 mg), add liquid nitrogen and grind it thoroughly. After grinding, place it in a 1.5 mL centrifuge tube, add 400 μL Buffer gP1, vortex for 1 minute, and place it in a 65°C water bath for 10-30 minutes. During this period, you can take it out and mix it upside down to fully lyse it; 3. Add 150 μL Buffer gP2, vortex for 1 min, and place on ice for 5 min; 4. Centrifuge at 12000 rpm for 5 min and transfer the supernatant to a new centrifuge tube; 5. Add an equal volume of anhydrous ethanol to the supernatant, immediately shake and mix thoroughly, transfer all the liquid into the Spin Column, centrifuge at 12000 rpm for 30 s, and discard the waste liquid; 6. Add 500 μL Buffer Pw (anhydrous ethanol has been added before use) to the Spin Column, centrifuge at 12000 rpm for 30 s, and discard the waste liquid; 7. Add 500 μL Wash Buffer (anhydrous ethanol has been added before use) to the Spin Column, centrifuge at 12000 rpm for 30 s, and discard the waste liquid; 8. Repeat step 7; 9. Place the Spin Column back into the Collection Tube, centrifuge at 12,000 rpm for 2 min, and open the cover to dry for 1 min; 10. Take out the Spin Column and put it into a clean centrifuge tube. Add 50-100 μL TE Buffer (preheat TE Buffer at 65℃) to the center of the adsorption membrane. Incubate at 20-25℃ for 2 min and centrifuge at 12,000 rpm for 2 min.

[0026] (2) PCR amplification 1. Universal primers for bacterial species identification

[0027] 2. The extracted DNA sample was diluted appropriately and used as a PCR template. Amplification was performed using Qingke 1×TSE101 Gold Mix. The components of the amplification system are as follows: 1×TSE101 Gold Mix 45 μL 27F (10P) 2 μL 1492R (10P) 2 μL DNA template 1 μL The above amplification system is amplified according to the following amplification program:

[0028] (3) Electrophoresis detection The amplified PCR product was subjected to agarose gel electrophoresis (2 μL sample + 6 μL bromophenol blue) at 300 V for 12 minutes to obtain the identification gel image. The prepared PCR product was subjected to first-generation sequencing (sequencing primers were 722F / 907R).

[0029] (4) Identification 1. Use ContigExpress to assemble the sequencing results and remove the inaccurate parts at both ends.

[0030] 2. Compare the assembled sequences in the NCBI database (blast.ncbi.nlm.nih.gov).

[0031] 3. In the comparison results, the species with a homology of more than 97% and the highest homology, the highest ranking, and clear species information obtained from NCBI are generally used as reference species for identification. Figure 1 As shown, the 16S sequence comparison results of the identified strain samples are Brevibacillus Laterosporus (or the same genus), so it was named Brevibacillus laterosporus Brevibacillus Laterosporus M811.

[0032] Example 2 The preparation method of the immunomodulatory octapeptide specifically comprises the following steps: (1) Fermentation Brevibacillus laterosporus Bacillus laterosporus M811 was inoculated in NB slant medium and cultured at 37°C for 24 h for activation. A single colony was picked and inoculated into NB liquid medium and cultured at 37°C and 220 rpm for 24 h to obtain the primary seed solution.

[0033] The first-level seed liquid was transferred to the second-level seed liquid medium at a 5% inoculation rate and cultured at 37°C and 220 rpm for 24 h to obtain the second-level seed liquid. The second-level seed liquid medium was: 3 g / L peptone, 16 g / L yeast powder, 2 g / L sucrose, 0.1 g / L magnesium sulfate, pH 6.8±0.1.

[0034] The secondary seed liquid was inoculated into the optimized fermentation medium at a 5% inoculation rate and cultured at 30°C and 220 rpm for 48 h to obtain the fermentation liquid. The fermentation medium was: corn syrup powder 15 g / L, sucrose 10 g / L, ammonium sulfate 3 g / L, magnesium sulfate 0.3 g / L, pH 6.8±0.1.

[0035] (2) Preparation of fermentation crude extract by salt ion precipitation After the fermentation of immunomodulatory octapeptide, the fermentation liquid was adjusted to pH 3.0 with 0.5 M sulfuric acid, centrifuged at 8000 rpm for 5 min to remove the bacteria, and the supernatant was retained. Ammonium sulfate powder with concentrations of 5, 10, 15, 20, 25 and 30 g / mL was slowly added to the supernatant, slowly stirred for 30 min, centrifuged to collect the precipitate and dried at 60°C to obtain the immunomodulatory octapeptide crude extract. The recovery rate of octapeptide precipitated with ammonium sulfate at different concentrations is shown in Figure 2. Figure 2 As shown in the figure, when the amount of ammonium sulfate added was 20 g / mL, the recovery rate of the immunomodulatory octapeptide reached the maximum value, which was 62.93±5.87%. In the subsequent optimization of experimental conditions, 20 g / mL of ammonium sulfate was used to prepare the crude extract of the immunomodulatory octapeptide.

[0036] Recovery rate calculation formula:

[0037] (3) Separation and purification The crude extract was dissolved and diluted to 0.1 g / mL with the initial mobile phase of the preparative liquid phase and then centrifuged. The supernatant was separated and purified using preparative HPLC (Agilent, 1260 Infinity Ⅱ). The preparation process was as follows: preparative liquid chromatography column, Agilent Zorbax 300SB-C18 PrepHT; the mobile phase contained 0.1% trifluoroacetic acid in water (A) and 0.1% trifluoroacetic acid in acetonitrile (B).

[0038] The elution mode is shown in Table 3: Table 3 Elution gradient for octapeptide preparation Time, min A, % B, % 0 80 20 4 72 28 8 66 34 23 66 34 35 62 38 42 59 41 45 0 100 52 0 100 53 80 20 55 80 20 The flow rate was 5 mL / min, the column temperature was 30°C, and the detection wavelength was 220 nm. The chromatogram was prepared as shown in Figure 3 The peak 1 indicated is the chromatographic peak corresponding to the octapeptide.

[0039] The corresponding fractions of the octapeptide were collected, and the acetonitrile was removed by rotary evaporation (Shanghai Yarong RE-3000B rotary evaporator), and then freeze-dried (Millrock box freeze dryer REVO TM ) to obtain pure octapeptide.

[0040] (4) Purity analysis and testing 1 mg of pure octapeptide was dissolved in 1 mL of ultrapure water, vortexed for 1 min, filtered through a 0.22 μm membrane, and analyzed for purity by high performance liquid chromatography (Agilent, 1260 Infinity II). The chromatographic column used for the analysis was SepaxBio-C18 4.6×250 mm 5μm; the mobile phase contained 0.1% trifluoroacetic acid in water (A) and 0.1% trifluoroacetic acid in acetonitrile (B).

[0041] The elution mode is shown in Table 4: Table 4 Elution gradient for octapeptide analysis Time, min A, % B, % 0 80 20 4 80 20 10 50 50 13 40 60 15 0 100 19 0 100 22 80 20 26 80 20 The flow rate was 1 mL / min, the column temperature was 30°C, and the detection wavelength was 220 nm.

[0042] Purity was tested by analytical reverse phase high performance liquid chromatography. Figure 4 The purity of the immunomodulatory octapeptide sample prepared in Example 2 was 68.75%.

[0043] Example 3 The preparation method of the immunomodulatory octapeptide specifically comprises the following steps: (1) The fermentation step is the same as that in Example 1.

[0044] (2) After the fermentation of the immunomodulatory octapeptide, the fermentation liquid was adjusted to pH 3.0 with 0.5 M sulfuric acid, centrifuged at 8000 rpm for 5 min to remove the bacteria, and the supernatant was retained. 20 g / mL ammonium sulfate powder was slowly added to the supernatant, slowly stirred for 30 min, and the precipitate was collected by centrifugation and dried at 60°C to obtain the immunomodulatory octapeptide crude extract.

[0045] (3) Separation and purification The crude extract was dissolved and diluted to 0.1 g / mL with the initial mobile phase of the preparative liquid phase and then centrifuged. The obtained supernatant was separated and purified using preparative HPLC (Agilent, 1260 Infinity II). The preparation process was as follows: preparative liquid chromatography column, Agilent Zorbax 300SB-C18 PrepHT; mobile phases contained 0.1% trifluoroacetic acid in water (A) and 0.1% trifluoroacetic acid in acetonitrile (B); The elution mode is shown in Table 5: Table 5 Elution gradient for octapeptide preparation Time, min A, % B, % 0 70 30 57 63.4 36.6 58 0 100 64 0 100 65 70 30 67 70 30 The flow rate was 5 mL / min, the column temperature was 30°C, and the detection wavelength was 220 nm. The chromatogram was prepared as shown in Figure 5 The peak 1 indicated is the chromatographic peak corresponding to the octapeptide.

[0046] The corresponding fractions of the octapeptide were collected, and the acetonitrile was removed by rotary evaporation (Shanghai Yarong RE-3000B rotary evaporator), and then freeze-dried (Millrock box freeze dryer REVO TM ) to obtain pure octapeptide.

[0047] (4) The purity detection step is the same as the purity detection step in Example 2. Purity detection is performed by analytical reverse phase high performance liquid chromatography. The results are as follows: Figure 6The purity of the immunomodulatory octapeptide sample prepared in Example 3 was 98.23%.

[0048] The test results show that the purity of the immunomodulatory octapeptide can be significantly improved by optimizing the elution gradient and elution time of the mobile phase when separating and purifying the crude fermentation extract of the present invention.

[0049] Example 4 The preparation method of the immunomodulatory octapeptide specifically comprises the following steps: (1) The fermentation step is the same as that in Example 3.

[0050] (2) The steps for preparing the crude extract are the same as those in Example 3.

[0051] (3) Separation and purification The crude extract was dissolved and diluted to 0.1 g / mL with the initial mobile phase of the preparative liquid phase and then centrifuged. The obtained supernatant was separated and purified using preparative HPLC (Agilent, 1260 Infinity II). The preparation process was as follows: preparative HPLC column, Agilent Zorbax 300SB-C18 PrepHT; mobile phases contained 0.1% trifluoroacetic acid in water (A) and 0.1% trifluoroacetic acid in acetonitrile (B); The elution mode is shown in Table 6: Table 6 Elution gradient for octapeptide preparation Time, min A, % B, % 0 70 30 70 62 38 71 0 100 77 0 100 78 70 30 80 70 30 The flow rate was 5 mL / min, the column temperature was 30°C, and the detection wavelength was 220 nm. The chromatogram was prepared as shown in Figure 7 The peak 1 indicated is the chromatographic peak corresponding to the octapeptide.

[0052] The corresponding fractions of the octapeptide were collected, and the acetonitrile was removed by rotary evaporation (Shanghai Yarong RE-3000B rotary evaporator), and then freeze-dried (Millrock box freeze dryer REVO TM ) to obtain pure octapeptide.

[0053] (4) The purity detection step is the same as the purity detection step in Example 2. Purity detection is performed by analytical reverse phase high performance liquid chromatography. The results are as follows: Figure 8 As shown. The purity of the immunomodulatory octapeptide sample prepared in Example 4 is 93.38%. The test results show that in the separation and purification of the crude fermentation extract of the present invention, further increasing the elution time of the mobile phase will reduce the purity of the pure immunomodulatory octapeptide. Therefore, the high-performance liquid phase preparation method constructed in Example 3 is the optimal separation and purification elution phase, elution gradient and elution time for the immunomodulatory octapeptide.

[0054] Example 5 Identification of the molecular structure of the immunomodulatory octapeptide High-resolution mass spectrometry and fatty acid derivatization reactions were used to identify the molecular structure. Figure 9-11 As shown. High-resolution mass spectrometry (HR-ESI-MS) in positive ion mode gave the octapeptide molecular ion peak as m / z 1044.6921 [M+H] + , m / z 522.8500 [M+2H] 2+ and m / z 348.9029 [M+3H] 3+ , its molecular formula is estimated to be C 51 H 89 N 13 O 10 ( Fig. 9 ). Primary and secondary mass spectrometry information and GC-MS analysis of fatty acid derivatization products ( Fig.10 , Fig.11 ) The results show that the prepared sample of the present invention is an octapeptide shown in the following structural formula.

[0055]

[0056] Example 6 Antibacterial activity of immunomodulatory octapeptide Referring to the National Committee for Clinical Laboratory Standards, MH medium (MH broth medium, Mueller Hinton Broth medium, Qingdao Hi-Tech Industrial Park Haibo Biotechnology Co., Ltd.) was used to serially dilute the octapeptide (sample source: Example 3) to concentrations of 512, 256, 128, 64, 32, 16, 8, 4, 2, 1, and 0.5 μg / mL. 50 μL of the diluted octapeptide was added to each well of the 96-well plate, and 50 μL of the octapeptide was resuspended in 2-7×10 5 CFU / mL indicator bacteria solution. Shake and culture for at least 18 h, read the OD value at 600 nm with a microplate reader to obtain the minimum inhibitory concentration of the octapeptide. Five strains of Gram-negative bacteria were selected, namely: Escherichia coli ATCC 25922, Salmonella gallinarum CVCC 534, Shigella dysenteriae CGMCC 1.1869, Pasteurella multocida CVCC 442, Pseudomonas aeruginosa ATCC 27853; 5 strains of Gram-positive bacteria, namely: Enterococcus faecalis ATCC 29212, Bacillus cereus CVCC 4101, Listeria monocytogenesCVCC 3746, Streptococcus dysgalactiae ATCC 35666, Staphylococcus aureus ATCC 43300. Vancomycin and Colistin were used as positive controls. The experimental results are shown in Table 7 below: Table 7 MIC values ​​of octapeptide against 10 indicator bacteria

[0057] The results of the antibacterial activity experiment showed that the octapeptide exhibited antibacterial activity against all indicator bacteria. The MIC value ranged from 4 to 8 μg / mL for anti-Gram-negative bacteria activity. The anti-Gram-positive bacteria activity was weaker, with a MIC value range of 8 to 64 μg / mL. The anti-Gram-negative Enterobacteriaceae activity of the octapeptide was generally better than the anti-Gram-positive bacteria activity. Salmonella gallinarum The MIC value of CVCC 534 strain was 2 μg / mL, which was very close to the positive control Colistin. In summary, the octapeptide has good antibacterial activity against bacteria and has the research and development value of being developed as a drug lead compound and antimicrobial peptide.

[0058] Example 7 Evaluation of the stability of immunomodulatory octapeptide in pepsin solution The stability of the anti-Enterobacterial immunomodulatory octapeptide in gastric protein solution was determined by high performance liquid chromatography and inhibition zone method. According to the Veterinary Pharmacopoeia of the People's Republic of China (2020 edition), 16.4 mL of dilute hydrochloric acid was taken, about 800 mL of water was added, and 10 g of pepsin was shaken well, then transferred to a 1000 mL volumetric flask and diluted to the mark with ultrapure water to obtain artificial gastric juice. The immunomodulatory octapeptide (sample source: Example 3) was dissolved and diluted with artificial gastric juice to a concentration of 200 μg / mL, placed in a 37°C water bath, and samples were taken at 0, 0.5, 1, 2, 4, and 6 h. After the sample was inactivated by 85°C water bath for 5 min, it was centrifuged at 9500 rpm and 4°C for 15 min, the chromatographic peak area of ​​the octapeptide at different time points was detected, and the remaining percentage was calculated.

[0059] Depend on Fig.12 It can be seen that the octapeptide can stably exist in the pepsin solution for 0 to 6 h, and the degradation residual rate is maintained at more than 98.5%, showing excellent stability in the pepsin solution.

[0060] Peptide products have the advantages of low toxicity, strong specificity, high drug activity and low accumulation in the body, and have received extensive attention. However, peptides are highly sensitive to various hydrolases, including intraluminal enzymes from gastrointestinal and pancreatic secretions, bacterial enzymes in the colon, and mucosal enzymes, and most peptides are rapidly degraded in simulated gastric juice. Therefore, oral use of peptides is a huge challenge. In the present invention, the high-purity immunomodulatory octapeptide produced by Brevibacillus laterosporus and prepared by salt precipitation-high performance liquid chromatography exhibits strong resistance to pepsin degradation, laying a foundation for its development as an oral drug and feed additive.

[0061] Example 8 Evaluation of hemolytic activity of immunomodulatory octapeptide Whole blood from pigs was collected intravenously using a blood collection tube containing sodium heparin, and red blood cells were obtained after centrifugation. The blood cells were washed with 10 mM PBS (pH7.3) until the supernatant had no obvious red color. The blood cells were prepared into a 4% suspension with PBS, and 100 μL of 4% blood cells and 100 μL of a dilution of octapeptide solution with a concentration range of 0~512 μg / mL were added to the wells of a 96-well plate, incubated in a 37°C incubator for 1 h, centrifuged at 1500 rpm for 5 min, and the supernatant was transferred to a new 96-well plate. The OD value was detected at 540 nm using an enzyme reader. 10 mM PBS and 0.1% Triton X-100 were used as negative and positive controls, respectively. The hemolyticity of the octapeptide was calculated. The calculation formula is as follows:

[0062] Among them, A 八肽 is the OD value of the octapeptide (sample source: Example 3) with different concentrations added into the blood cell well; A PBS A is the OD value of adding PBS into the blood cell well; 0.1% Triton X-100 The OD value of 0.1% Triton X-100 added to the blood cell well. Fig.13 As shown. As can be seen, the hemolytic rate of the octapeptide in the concentration range of 1-512 μg / mL is 0.201-6.477%, and its variation is within 7%. The results show that the octapeptide has no obvious hemolytic activity.

[0063] Due to the advantages of simple structure, strong ability to kill bacteria and weak induction of bacterial resistance, immunomodulatory peptides or antimicrobial peptides have the potential to become new antimicrobial drugs and feed additives to replace traditional antibiotics. However, immunomodulatory peptides also have certain side effects such as hemolysis and instability. Among them, hemolysis is an important indicator for evaluating the cytotoxicity of immunomodulatory peptides. The hemolytic evaluation showed that the immunomodulatory octapeptide did not show obvious hemolytic activity, indicating that it has the potential to be further developed into a new antimicrobial drug and feed additive.

[0064] In summary, the immunomodulatory octapeptide produced in the present invention has the advantages of good antibacterial activity, stability, low toxicity, etc., and has broad application prospects in the development of new antibacterial drugs and feed additives.

[0065] The above are only embodiments of the present invention. The invention is not limited to the fields involved in this implementation case. The common sense such as the known specific structures and characteristics in the scheme is not described in detail here. The ordinary technicians in the relevant field know all the common technical knowledge in the technical field to which the invention belongs before the application date or priority date, can obtain all the existing technologies in the field, and have the ability to apply the conventional experimental means before that date. The ordinary technicians in the relevant field can improve and implement this scheme in combination with their own abilities under the enlightenment given by this application. Some typical known structures or known methods should not become obstacles for ordinary technicians in the relevant field to implement this application. It should be pointed out that for those skilled in the art, without departing from the structure of the present invention, several deformations and improvements can be made, which should also be regarded as the protection scope of the present invention, which will not affect the effect of the implementation of the present invention and the practicality of the present invention. The protection scope required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to explain the content of the claims.

Claims

1. An octapeptide, characterized in that: The structural formula is shown in (Ⅰ): 。 2. Use of the octapeptide according to claim 1 in antibiotics or immunomodulators, or use of the octapeptide according to claim 1 in the preparation of medicines, reagents, feeds, or feed additives.

3. An antibiotic, immunomodulator, medicine, preparation, reagent, additive or feed, characterized in that: The invention comprises the immunomodulatory octapeptide according to claim 1.

4. The octapeptide according to claim 1 or the antibiotic, immunomodulator, drug, preparation, reagent, additive or feed according to claim 3, characterized in that: It is prepared by fermentation of Brevibacillus laterosporus; the Brevibacillus laterosporus was deposited in the China Center for Type Culture Collection on April 18, 2024, with a preservation number of CCTCC NO: M2024715.

5. The method for preparing the octapeptide according to claim 1, wherein: include: (1) Fermentation of Brevibacillus laterosporus to prepare a fermentation broth; the Brevibacillus laterosporus was deposited in the China Center for Type Culture Collection on April 18, 2024, with a deposit number of CCTCC NO: M2024715; (2) Preparation of crude fermentation extract by salt ion precipitation; (3) The crude fermentation extract was separated and purified by high performance liquid chromatography, acetonitrile was removed by rotary evaporation, and then freeze-dried.

6. The method for preparing an octapeptide according to claim 5, wherein: In step (3), reverse phase high performance liquid chromatography is used, and the separation and purification steps include: a preparative liquid chromatography column, Agilent Zorbax 300SB-C18 PrepHT; a mobile phase containing 0.1% trifluoroacetic acid in water (A) and 0.1% trifluoroacetic acid in acetonitrile (B); The elution pattern is shown in Table 1: Table 1 Elution gradient for octapeptide analysis The flow rate was 5 mL / min, the column temperature was 30 °C, and the detection wavelength was 220 nm.

7. The method for preparing an octapeptide according to claim 5 or 6, wherein: The salt ion precipitation comprises adding ammonium sulfate with a final concentration of 20 g / mL to the supernatant of the fermentation broth, and then centrifuging and drying to obtain the crude octapeptide extract.

8. The method for preparing the immunomodulatory octapeptide prepared by Brevibacillus laterosporus according to claim 1, characterized in that The steps include: (1) Cultivation and fermentation The strain according to claim 1 is inoculated into NB medium for activation, cultured at 37°C for 24 hours, inoculated into a sterilized NB liquid medium shake flask, and cultured at 37°C and 220 rpm for 24 hours to obtain a primary seed solution; After the first-level seed solution is fully mixed, it is transferred to the second-level seed solution culture medium at a 5% inoculum rate and cultured at 37°C and 220 rpm for 24 h to obtain the second-level seed solution; The secondary seed liquid was inoculated into the optimized fermentation medium at a volume ratio of 5%, and cultured at 30°C and 220 rpm for 48 h to obtain the fermentation liquid. (2) Preparation of crude immunomodulatory octapeptide extract The fermentation broth obtained in step (1) was adjusted to a pH of 3.0, centrifuged at 8000 rpm for 5 min, and the supernatant was retained. 20 g / mL ammonium sulfate powder was added to the supernatant, and the mixture was slowly stirred for 30 min to precipitate the immunomodulatory octapeptide. The precipitate was collected by centrifugation and dried at 60°C to obtain a crude immunomodulatory octapeptide extract. (3) Isolation and purification of pure immunomodulatory octapeptide The crude immunomodulatory octapeptide obtained in step (2) was dissolved in the initial mobile phase of preparative high performance liquid phase to a concentration of 0.1 g / mL, and the supernatant obtained by centrifugation was separated and purified by preparative high performance liquid chromatography. The fractions corresponding to the immunomodulatory octapeptide were collected, evaporated to remove acetonitrile, and then freeze-dried to obtain the pure octapeptide.

9. The preparation method according to claim 5, characterized in that: The secondary seed liquid culture medium described in step (1) is: 3 g / L peptone, 16 g / L yeast powder, 2 g / L sucrose, 0.1 g / L magnesium sulfate, pH 6.8±0.

1. The fermentation medium is: 15 g / L corn steep liquor, 10 g / L sucrose, 3 g / L ammonium sulfate, 0.3 g / L magnesium sulfate, pH 6.8±0.

1.

10. The method for detecting an octapeptide according to claim 1, wherein: include: Sample processing: Shake the octapeptide fermentation broth, take out 1 mL with a pipette and place it in a 2 mL centrifuge tube, add 20 μL of 5M sulfuric acid, vortex and shake for 1 min, and centrifuge at 12000 rpm for 5 min; the supernatant is filtered through a 0.22 μm membrane for reversed-phase liquid chromatography analysis; The reversed-phase high-performance liquid chromatography analysis and detection: analytical liquid chromatography column, Sepax Bio-C18 4.6×250 mm 5μm; mobile phase containing 0.1% trifluoroacetic acid in water (A) and 0.1% trifluoroacetic acid in acetonitrile (B); The elution pattern is shown in Table 2: Table 2 Elution gradient for octapeptide analysis The flow rate was 1 mL / min, the column temperature was 30°C, and the detection wavelength was 220 nm.

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