An octapeptide, a preparation method thereof and applications thereof in antibacterial or immunomodulation

Abstract

The present invention provides an immunomodulatory octapeptide. This small peptide molecule has a unique structure, has high broad-spectrum antibacterial activity, strong inhibition of pathogenic bacteria and high purity, can maintain the stability of its structure and antibacterial activity in artificial gastric juice, and has no obvious hemolytic activity, and has high application value in livestock breeding. The present invention also provides a preparation method and a purity detection method for the immunomodulatory octapeptide.

Description

Technical Field

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

[0002] Lipopeptides or polypeptides are very important compounds among microbial secondary metabolites. Due to their unique chemical structures and diverse biological activities, they have attracted wide attention from researchers. Lipopeptides or polypeptides are amino acid polymerization products between amino acids and proteins and are basically synthesized by ribosomes 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. The cells are slender rods, with strong adaptability and the property of being heat-resistant. At the same time, this strain can produce a variety of bioactive substances including lipopeptides and polypeptides.

[0003] The emergence of traditional antibiotics has not only enabled the rapid development of the pharmaceutical industry but also has very important applications in the livestock and poultry breeding industry. However, due to the abuse of traditional antibiotics, the emergence of multi-drug resistant bacteria has seriously affected the development of the livestock industry and also endangered the ecological environment and human health. To solve this problem, it is urgent to develop new antibiotic alternative products for application in the livestock industry. 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, and stability and safety.

[0005] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] The present invention also provides an octapeptide, the structural formula of which is as follows:

[0007]

[0008] The above octapeptide is prepared by fermentation with Brevibacillus laterosporus. Preferably, it is Brevibacillus laterosporus Bacillus laterosporus M811 developed by our research group. It is preserved in the China Center for Type Culture Collection, with the preservation date: April 18, 2024, address: Wuhan University, Wuhan, China, and the preservation number is CCTCC NO: M2024715. The use of this strain for preparing the immunomodulatory octapeptide has improved the yield, purity, activity, adaptability, etc. of the product.

[0009] The preparation method of the above octapeptide is characterized by including:

[0010] (1)Ferment Bacillus brevis to prepare the fermentation broth;

[0011] (2)Prepare the crude fermentation extract by salt ion precipitation;

[0012] (3)Separate and purify the crude fermentation extract by high performance liquid chromatography, remove acetonitrile by rotary evaporation, and freeze-dry.

[0013] Preferably, the reverse-phase high performance liquid chromatography preparation process in step (3) is as follows: preparative liquid chromatography column, Agilent Zorbax 300SB-C18 PrepHT; mobile phase: water containing 0.1% trifluoroacetic acid (A) and acetonitrile containing 0.1% trifluoroacetic acid (B);

[0014] The elution mode is shown in Table 1:

[0015] Table 1 Octapeptide analysis elution gradient table

[0016] Time, min A, % B, % 0 70 30 57 63.4 36.6 58 0 100 64 0 100 65 70 30 67 70 30

[0017] Flow rate 5 mL / min, column temperature 30 °C, detection wavelength 220 nm.

[0018] Preferably, the process in step (2) is as follows: use 5 M H 2 SO 4 Adjust the fermentation broth obtained in step (1) to pH 3.0, centrifuge at 8000 rpm for 5 min, and retain the supernatant. Add ammonium sulfate with a concentration of 5 - 30 g / mL to the supernatant, slowly stir for 30 min, then centrifuge at 8000 rpm for 10 min, collect the precipitate, and dry it at 60 °C to obtain the crude octapeptide extract. The immunomodulatory octapeptide can be enriched by precipitation with a specific concentration of ammonium sulfate, and different concentrations of ammonium sulfate can precipitate different kinds of proteins. In the present invention, adding the 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 at the same time, it can also remove the interference of impurity proteins on the subsequent preparative liquid chromatography separation and purification step, and improve the purity of the pure immunomodulatory octapeptide.

[0019] A method for preparing an immunomodulatory octapeptide using Bacillus brevis specifically includes the following steps:

[0020] (1)Bacillus brevis Bacillus laterosporusM811 was activated by inoculating it into an NB slant medium. After culturing for 24 h at 37 °C, a single colony was taken and inoculated into a sterilized NB liquid medium shake flask. It was cultured for 24 h at 37 °C and 220 rpm to obtain the primary seed liquid. After thoroughly mixing the primary seed liquid, it was transferred to the secondary seed liquid medium according to an inoculation amount of 5%, and cultured for 24 h at 37 °C and 220 rpm to obtain the secondary seed liquid. The secondary seed liquid was inoculated into the optimized fermentation medium at an inoculation amount of 5% by volume and cultured for 48 h at 30 °C and 220 rpm to obtain the fermentation broth.

[0021] (2) The pH value of the fermentation broth obtained in step (1) was adjusted to 3.0, centrifuged at 8000 rpm for 5 min, and the supernatant was retained. Ammonium sulfate powder at 5 - 30 g / mL was added to the supernatant, slowly stirred for 30 min, and the precipitate was collected by centrifugation and dried at 60 °C to obtain the crude extract of immunomodulatory octapeptide.

[0022] (3) The crude extract obtained in step (2) was dissolved to a concentration of 0.1 g / mL with the initial mobile phase of preparative high performance liquid chromatography. The supernatant obtained by centrifugation was separated and purified by a preparative high performance liquid chromatograph. The fraction corresponding to the immunomodulatory octapeptide was collected, the 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.

[0023] Preferably, the secondary seed liquid medium in step (1) is: peptone 3 g / L, yeast powder 16 g / L, sucrose 2 g / L, magnesium sulfate 0.1 g / L, pH 6.8 ± 0.1. The fermentation medium is: corn steep liquor powder 15 g / L, sucrose 10 g / L, ammonium sulfate 3 g / L, magnesium sulfate 0.3 g / L, pH 6.8 ± 0.1.

[0024] The present invention also provides a method for detecting the purity of the above octapeptide, including:

[0025] 1 mg of the pure octapeptide obtained in step (3) above was placed in a centrifuge tube, dissolved with 1 mL of ultrapure water, vortexed for 1 min, then passed through a 0.22 μm filter membrane, and the purity of the immunomodulatory octapeptide was detected by an analytical high performance liquid chromatograph. Preferably, the reverse phase high performance liquid chromatography analysis process is as follows: analytical liquid chromatography column, Sepax Bio-C18 4.6×250 mm 5μm; mobile phase: water containing 0.1% trifluoroacetic acid (A) and acetonitrile containing 0.1% trifluoroacetic acid (B);

[0026] The elution mode is shown in Table 2:

[0027] Table 2 Octapeptide analysis elution gradient table

[0028] 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

[0029] Flow rate: 1 mL / min, column temperature: 30 °C, detection wavelength: 220 nm.

[0030] The present invention also provides the application of the above octapeptide in antibiotics or immunomodulators, or in the preparation of drugs, reagents, feeds, and feed additives. Preferably, the octapeptide is an anti-enterobacter octapeptide, and its application in the preparation of drugs or feed additives against Gram-negative enterobacter infections. The Gram-negative enterobacteria include, but are not limited to, Escherichia coli, Salmonella, Shigella, and Pseudomonas aeruginosa. Through the screening of antibacterial activity, this octapeptide has good activity in inhibiting pathogenic enterobacteria such as Escherichia coli, Salmonella, Shigella, 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 enterobacteria. Beneficial effects

[0031] The present invention provides an octapeptide with antibacterial activity against Gram-negative and positive pathogenic bacteria. The small peptide molecule has a unique structure, high broad-spectrum antibacterial activity, strong inhibition of pathogenic bacteria, high purity, and strong performance stability.

[0032] 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. This immunomodulatory octapeptide has strong bactericidal effects on both Gram-positive and negative pathogenic bacteria, and its bactericidal ability against Escherichia coli and Pseudomonas aeruginosa is significantly stronger than that against Staphylococcus aureus, showing high application potential in livestock breeding.

[0033] 3. The present invention provides a preparation method of the immunomodulatory octapeptide. Through processes such as multi-stage seed liquid culture, fermentation broth preparation, fermentation broth pretreatment, octapeptide extraction and purification, high-purity octapeptide products are prepared, which effectively improves the fermentation efficiency and shortens the separation and purification process.

[0034] 4. The present invention combines salt ion precipitation and preparative reverse-phase high-performance liquid chromatography to treat the octapeptide fermentation broth, which improves the preparation efficiency and sample purity of the octapeptide and reduces the preparation cost. The purity of the octapeptide prepared by this method is as high as 98.23%. Description of the drawings

[0035] Figure 1 For Bacillus brevis in Example 1 of the present invention Bacillus laterosporus Phylogenetic tree of the identification of strain M811;

[0036] Figure 2 For the recovery rate detection results of the octapeptide in Example 2 of the present invention;

[0037] Figure 3 For the HPLC preparation diagram of the octapeptide in Example 2 of the present invention;

[0038] Figure 4 It is the purity detection chart of the octapeptide in Example 2 of the present invention;

[0039] Figure 5 It is the HPLC preparation chart of the octapeptide in Example 3 of the present invention;

[0040] Figure 6 It is the purity detection chart of the octapeptide in Example 3 of the present invention;

[0041] Figure 7 It is the HPLC preparation chart of the octapeptide in Example 4 of the present invention

[0042] Figure 8 It is the purity detection chart of the octapeptide in Example 4 of the present invention;

[0043] Figure 9 It is the first-order mass spectrum chart of the octapeptide in Example 5 of the present invention;

[0044] Figure 10 It is the second-order mass spectrum chart of the octapeptide in Example 5 of the present invention;

[0045] Figure 11 It is the GC-MS spectrum of the fatty acid of the hydrolysis product of the octapeptide in Example 5 of the present invention;

[0046] Figure 12 The stability result of the octapeptide in artificial gastric juice in Example 7 of the present invention;

[0047] Figure 13 The hemolytic result of the octapeptide in Example 8 of the present invention. Detailed implementation manners

[0048] 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″). This strain was deposited at the China Center for Type Culture Collection on April 18, 2024. Address: Wuhan University, Wuhan, China. The deposit number is CCTCC NO: M2024715.

[0049] The strain isolated in the present invention was identified, and its morphological characteristics are as follows: The colony is transparent and light yellow, with a shiny surface; The cells are slender rod-shaped, about 2.0×1.0 μm in size, with peritrichous flagella, and can form oval spores, and are Gram-positive. The 16S rRNA gene of this strain was amplified by PCR, sequenced and phylogenetically analyzed, as Figure 1 shown. It was identified as Brevibacillus laterosporus and named Brevibacillus laterosporus Brevibacillus Laterosporus M811.

[0050] The NB medium used in the present invention is composed of: 10 g / L of Tryptone, 5 g / L of yeast extract, and 10 g / L of sodium chloride, purchased from Beijing Solarbio Science & Technology Co., Ltd. The components of corn steep liquor powder, sucrose, and magnesium sulfate are all commercially available medium components.

[0051] The fermentation and compound separation and purification parameters involved in the present invention are well-known to those skilled in the art.

[0052] Example 1

[0053] Identification of a Brevibacillus laterosporus strain for preparing immunomodulatory octapeptide specifically includes the following steps:

[0054] (1) DNA extraction

[0055] Use the TSINGKE Plant DNA Extraction Kit (universal type), and the specific steps are as follows:

[0056] 1. Place the Spin Column in the Collection Tube, add 250 μL of Buffer BL, and centrifuge at 12000 rpm for 1 min to activate the silica gel membrane;

[0057] 2. Take the dry tissue of the sample (not more than 20 mg), add liquid nitrogen and grind thoroughly. After grinding, place it in a 1.5 mL centrifuge tube, add 400 μL of Buffer gP1, vortex for 1 min, and incubate in a water bath at 65 °C for 10 - 30 min. During this period, it can be taken out and inverted to mix evenly to fully lyse;

[0058] 3. Add 150 μL of Buffer gP2, vortex for 1 min, and incubate on ice for 5 min;

[0059] 4. Centrifuge at 12000 rpm for 5 min, and transfer the supernatant to a new centrifuge tube;

[0060] 5. Add an equal volume of absolute ethanol to the supernatant, immediately shake well, transfer all the liquid into the Spin Column, centrifuge at 12000 rpm for 30 s, and discard the waste liquid;

[0061] 6. Add 500 μL of 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;

[0062] 7. Add 500 μL of 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;

[0063] 8. Repeat the operation steps in Step 7;

[0064] 9. Place the Spin Column back into the Collection Tube, centrifuge at 12000 rpm for 2 min, and open the lid to air dry for 1 min;

[0065] 10. Take out the Spin Column and place it into a clean centrifuge tube. Add 50 - 100 μL of TE Buffer (preheated TE Buffer at 65 °C) to the center of the adsorption membrane, place it at 20 - 25 °C for 2 min, and centrifuge at 12000 rpm for 2 min.

[0066] (2)Amplification of PCR

[0067] 1. Universal primers for bacterial species identification

[0068]

[0069] 2. Dilute the extracted DNA sample appropriately and use it as the PCR template. Amplify it with Tsingke 1×TSE101 Gold Mix. The components of the amplification system are as follows:

[0070] 1×TSE101 Gold Mix 45 μL 27F (10P) 2 μL 1492R (10P) 2 μL DNA template 1 μL

[0071] The above amplification system is amplified according to the following amplification program:

[0072]

[0073] (3)Detection by electrophoresis

[0074] Perform agarose gel electrophoresis on the amplified PCR product (2 μL sample + 6 μL bromophenol blue) at 300 V for 12 minutes to obtain the identification gel image. Perform first-generation sequencing on the prepared PCR product (the sequencing primers are 722F / 907R).

[0075] (4)Identification

[0076] 1. Use ContigExpress to assemble the sequencing results and remove the inaccurate parts at both ends.

[0077] 2. Align the assembled sequence in the NCBI database (blast.ncbi.nlm.nih.gov).

[0078] 3. In the alignment results, generally use the species with a homology of more than 97% obtained by alignment on NCBI, the highest homology, the top-ranked, and clear species information as the reference species for identification. As Figure 1 shown, the alignment result of the 16S sequence of the identified strain sample is Brevibacillus Laterosporus(or belonging to the same genus), so it is named Brevibacillus laterosporus Brevibacillus Laterosporus M811

[0079] Example 2

[0080] A method for preparing immunomodulatory octapeptide, specifically including the following steps:

[0081] (1) Fermentation

[0082] Inoculate Brevibacillus laterosporus Bacillus laterosporus M811 on the NB slant medium and culture it at 37°C for 24 h for activation. Pick a single colony and inoculate it into the NB liquid medium, and culture it at 37°C and 220 rpm for 24 h to obtain the primary seed liquid.

[0083] Transfer the primary seed liquid to the secondary seed liquid medium according to an inoculation amount of 5%, and culture it at 37°C and 220 rpm for 24 h to obtain the secondary seed liquid. The secondary seed liquid medium is: peptone 3 g / L, yeast powder 16 g / L, sucrose 2 g / L, magnesium sulfate 0.1 g / L, pH 6.8±0.1.

[0084] Inoculate the secondary seed liquid into the optimized fermentation medium according to an inoculation amount of 5%, and culture it at 30°C and 220 rpm for 48 h to obtain the fermentation broth. The fermentation medium is: corn steep powder 15 g / L, sucrose 10 g / L, ammonium sulfate 3 g / L, magnesium sulfate 0.3 g / L, pH 6.8±0.1.

[0085] (2) Preparation of fermentation crude extract by salt ion precipitation

[0086] Adjust the pH value of the fermentation broth after the fermentation of immunomodulatory octapeptide to 3.0 with 0.5 M sulfuric acid, centrifuge at 8000 rpm for 5 min to remove the thallus, and retain the supernatant. Slowly add ammonium sulfate powder with concentrations of 5, 10, 15, 20, 25, and 30 g / mL to the supernatant, stir slowly for 30 min, centrifuge to collect the precipitate and dry it at 60°C to obtain the crude extract of immunomodulatory octapeptide. The recovery rates of octapeptide precipitated by ammonium sulfate at different concentrations are as Figure 2 shown. It can be seen from the figure that when the addition amount of ammonium sulfate is 20 g / mL, the recovery rate of immunomodulatory octapeptide reaches the maximum value, which is: 62.93±5.87%. In the subsequent optimization of experimental conditions, 20 g / mL of ammonium sulfate is used to prepare the crude extract of immunomodulatory octapeptide.

[0087] Recovery rate calculation formula:

[0088]

[0089] (3) Separation and purification

[0090] The crude extract was dissolved and diluted to 0.1 g / mL with the initial mobile phase of preparative liquid chromatography and then centrifuged. The obtained supernatant was separated and purified by preparative high performance liquid chromatography (Agilent, 1260 Infinity Ⅱ). The preparation process was as follows: preparative liquid chromatography column, Agilent Zorbax 300SB-C18 PrepHT; the mobile phase consisted of water containing 0.1% trifluoroacetic acid (A) and acetonitrile containing 0.1% trifluoroacetic acid (B).

[0091] The elution mode was as shown in Table 3:

[0092] Table 3 Elution gradient table for octapeptide preparation

[0093] 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

[0094] The flow rate was 5 mL / min, the column temperature was 30 °C, and the detection wavelength was 220 nm. The preparative chromatogram was as shown Figure 3 in the figure. The marked peak 1 was the chromatographic peak corresponding to the octapeptide.

[0095] The fractions corresponding to the octapeptide were collected, and the acetonitrile was removed by rotary evaporation (Shanghai Yarong RE-3000B rotary evaporator) and then freeze-dried (Millrock box-type freeze dryer REVO TM ) to obtain the pure octapeptide.

[0096] (4) Purity analysis and detection

[0097] 1 mg of the pure octapeptide was dissolved in 1 mL of ultrapure water, vortexed for 1 min, filtered through a 0.22 μm membrane, and then analyzed for purity using a high performance liquid chromatograph (Agilent, 1260 Infinity Ⅱ). The chromatographic column used for the analysis was SepaxBio-C18 4.6×250 mm 5μm; the mobile phase consisted of water containing 0.1% trifluoroacetic acid (A) and acetonitrile containing 0.1% trifluoroacetic acid (B).

[0098] The elution mode was as shown in Table 4:

[0099] Table 4 Elution gradient table for octapeptide analysis

[0100] 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

[0101] The flow rate was 1 mL / min, the column temperature was 30 °C, and the detection wavelength was 220 nm.

[0102] Purity detection was performed using analytical reversed-phase high performance liquid chromatography, and the results were as shown Figure 4 in the figure. The purity of the immunomodulatory octapeptide sample prepared in Example 2 was 68.75%.

[0103] Example 3

[0104] Preparation method of immunomodulatory octapeptide, specifically including the following steps:

[0105] (1) The fermentation step is the same as the fermentation step in Example 1.

[0106] (2) After the fermentation of immunomodulatory octapeptide is completed, the pH value of the fermentation broth is adjusted to 3.0 with 0.5 M sulfuric acid, and the bacteria are removed by centrifugation at 8000 rpm for 5 min, and the supernatant is retained. Ammonium sulfate powder at 20 g / mL is slowly added to the supernatant, and it is slowly stirred for 30 min, and the precipitate is collected by centrifugation and dried at 60 °C to obtain the crude extract of immunomodulatory octapeptide.

[0107] (3) Isolation and purification

[0108] The crude extract is dissolved and diluted to 0.1 g / mL with the initial mobile phase of preparative liquid chromatography and then centrifuged. The obtained supernatant is separated and purified by preparative high performance liquid chromatography (Agilent, 1260 Infinity Ⅱ). The preparation process is as follows: preparative liquid chromatography column, Agilent Zorbax 300SB-C18 PrepHT; mobile phase: water containing 0.1% trifluoroacetic acid (A) and acetonitrile containing 0.1% trifluoroacetic acid (B);

[0109] The elution mode is shown in Table 5:

[0110] Table 5 Elution gradient table for octapeptide preparation

[0111] Time, min A, % B, % 0 70 30 57 63.4 36.6 58 0 100 64 0 100 65 70 30 67 70 30

[0112] The flow rate is 5 mL / min, the column temperature is 30 °C, and the detection wavelength is 220 nm. The preparative chromatogram is as Figure 5 shown. The marked peak 1 is the chromatographic peak corresponding to the octapeptide.

[0113] Collect the fractions corresponding to the octapeptide, remove acetonitrile by rotary evaporation (Shanghai Yarong RE-3000B type rotary evaporator), and then freeze-dry (Millrock box-type freeze dryer REVO TM ) to obtain the pure octapeptide product.

[0114] (4) The purity detection step is the same as the purity detection step in Example 2. The purity is detected by analytical reversed-phase high performance liquid chromatography, and the result is as Figure 6 shown. The purity of the immunomodulatory octapeptide sample prepared in Example 3 is 98.23%.

[0115] The test results show that by optimizing the elution gradient and elution time of the mobile phase during the separation and purification of the fermentation crude extract of the present invention, the purity of the pure immunomodulatory octapeptide can be significantly improved.

[0116] Example 4

[0117] Preparation method of immunomodulatory octapeptide, specifically including the following steps:

[0118] (1) The fermentation step is the same as the fermentation step in Example 3.

[0119] (2) The crude extract preparation step is the same as the crude extract preparation step in Example 3.

[0120] (3) Separation and purification

[0121] The crude extract is dissolved and diluted to 0.1 g / mL with the initial mobile phase of preparative liquid chromatography and then centrifuged. The obtained supernatant is separated and purified using preparative high-performance liquid chromatography (Agilent, 1260 Infinity Ⅱ). The preparation process is as follows: preparative liquid chromatography column, Agilent Zorbax 300SB-C18 PrepHT; mobile phase: water containing 0.1% trifluoroacetic acid (A) and acetonitrile containing 0.1% trifluoroacetic acid (B);

[0122] The elution mode is shown in Table 6:

[0123] Table 6 Elution gradient table for octapeptide preparation

[0124] Time, min A, % B, % 0 70 30 70 62 38 71 0 100 77 0 100 78 70 30 80 70 30

[0125] The flow rate is 5 mL / min, the column temperature is 30 °C, and the detection wavelength is 220 nm. The preparative chromatogram is as shown in Figure 7 The marked peak 1 is the chromatographic peak corresponding to the octapeptide.

[0126] Collect the fractions corresponding to the octapeptide, remove acetonitrile by rotary evaporation (Shanghai Yarong RE-3000B type rotary evaporator), and then freeze-dry (Millrock box-type freeze dryer REVO TM ) to obtain the pure octapeptide.

[0127] (4) The purity detection step is the same as the purity detection step in Example 2. Purity detection is carried out using analytical reverse-phase high-performance liquid chromatography, and the results are as shown in Figure 8 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 fermentation crude extract described in the present invention, further increasing the elution time of the mobile phase will instead reduce the purity of the pure immunomodulatory octapeptide. Therefore, the high-performance liquid chromatography preparation method constructed in Example 3 is the optimal separation and purification eluent phase, elution gradient, and elution time for the immunomodulatory octapeptide.

[0128] Example 5

[0129] Identification of the molecular structure of immunomodulatory octapeptide

[0130] High-resolution mass spectrometry and fatty acid derivatization reaction are used for molecular structure identification, and the results are as shown in Figures 9 - 11As shown. The high-resolution mass spectrometry (HR-ESI-MS) in the positive ion mode gives the molecular ion peak of the octapeptide as m / z 1044.6921 [M+H] + 、 m / z 522.8500 [M+2H] 2+ and m / z 348.9029 [M+3H] 3+ , and its molecular formula is speculated to be C 51 H 89 N 13 O 10 ( Figure 9 ). The information of the first-order and second-order mass spectrometry and the results of the GC-MS analysis of the fatty acid derivatives ( Figure 10 , Figure 11 ) prove that the prepared sample of the present invention is the octapeptide shown in the following structural formula.

[0131]

[0132] Example 6

[0133] Antibacterial Activity of the Immunomodulatory Octapeptide

[0134] Referring to the National Committee for Clinical Laboratory Standards, the concentration of the octapeptide (sample source: Example 3) was successively diluted by serial dilution to 512, 256, 128, 64, 32, 16, 8, 4, 2, 1, and 0.5 μg / mL using MH medium (MH broth medium, Mueller Hinton Broth medium, Haibo Biotechnology Co., Ltd., Qingdao High-tech Industrial Park). 50 μL of the diluted octapeptide was added to each well of a 96-well plate, and 50 μL of the indicator bacteria suspension resuspended in MH at 2 - 7×10 5 CFU / mL was added to each well. The mixture was shaken and cultured for at least 18 h, and the OD value was read at 600 nm using an enzyme-linked immunosorbent assay reader to obtain the minimum inhibitory concentration of the octapeptide. Among them, 5 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 were selected, namely: Enterococcus faecalis ATCC 29212, Bacillus cereus CVCC 4101, Listeria monocytogenes CVCC 3746, Streptococcus dysgalactiaeATCC 35666, Staphylococcus aureus ATCC 43300. Vancomycin and Colistin were used as positive controls respectively. The experimental results are shown in Table 7 below:

[0135] Table 7 MIC values of octapeptide inhibiting 10 indicator bacteria

[0136]

[0137] The results of the antibacterial activity experiment showed that the octapeptide exhibited antibacterial activity against all the indicator bacteria. For the anti - Gram - negative bacteria activity, the MIC value range was 4 - 8 μg / mL. The anti - Gram - positive bacteria activity was relatively weak, and the MIC value range was 8 - 64 μg / mL. Generally, the anti - Gram - negative enterobacteria activity of the octapeptide was superior to the anti - Gram - positive bacteria activity. Among them, the MIC value of the octapeptide against Salmonella gallinarum the CVCC 534 strain was 2 μg / mL, which was very close to the positive control Colistin. In summary, this octapeptide has good antibacterial activity against bacteria and has the research and development value of being developed as a drug lead compound and an antibacterial peptide.

[0138] Example 7

[0139] Stability evaluation of the immunomodulatory octapeptide in pepsin solution

[0140] The stability of the anti - enterobacteria immunomodulatory octapeptide in pepsin solution was determined by high - performance liquid chromatography and the inhibition zone method. According to the "Chinese Veterinary Pharmacopoeia" (2020 edition), take 16.4 mL of dilute hydrochloric acid, add about 800 mL of water and 10 g of pepsin, shake well, transfer to a 1000 mL volumetric flask and make up to the mark with ultrapure water to obtain artificial gastric juice. Dissolve and dilute the immunomodulatory octapeptide (sample source: Example 3) with artificial gastric juice to a concentration of 200 μg / mL, place it in a water bath at 37 °C, and take samples at 0, 0.5, 1, 2, 4, and 6 h time points respectively. After inactivating the protease by heating the sample in a water bath at 85 °C for 5 min, centrifuge at 9500 rpm and 4 °C for 15 min, detect the chromatographic peak area of the octapeptide at different time points, and calculate the remaining percentage.

[0141] It can be seen from Figure 12 that the octapeptide can stably exist in the pepsin solution within 0 - 6 h of treatment, and the degradation remaining rate remains above 98.5%, showing excellent stability in the pepsin solution.

[0142] Polypeptide products have the advantages of low toxicity and side effects, strong specificity, high drug activity, and being not easily accumulated in the body, and have received extensive attention. However, polypeptides are highly sensitive to various hydrolases, including luminal enzymes from gastrointestinal and pancreatic secretions, bacterial enzymes in the colon, and mucosal enzymes, etc. Most polypeptides are rapidly degraded in simulated gastric juice. Therefore, oral use of polypeptides is a huge challenge. In the present invention, the highly purified immunomodulatory octapeptide produced by Brevibacillus laterosporus and prepared by salt precipitation-high performance liquid chromatography exhibits strong pepsin-resistant degradation performance, laying a foundation for its development as an oral drug and feed additive.

[0143] Example 8

[0144] Hemolytic evaluation of immunomodulatory octapeptide

[0145] Collect porcine whole blood by vein using a blood collection tube containing sodium heparin, and obtain red blood cells after centrifugation. Wash the blood cells with 10 mM PBS (pH 7.3) until the supernatant is not significantly red. Prepare a 4% suspension of blood cells with PBS. Take 100 μL of 4% blood cells and 100 μL of a two-fold diluted octapeptide solution with a concentration range of 0 - 512 μg / mL and add them into the wells of a 96-well plate. Incubate in a 37°C incubator for 1 h, centrifuge at 1500 rpm for 5 min, take the supernatant and transfer it to a new 96-well plate, and detect the OD value at 540 nm using an enzyme-linked immunosorbent assay (ELISA) reader. Use 10 mM PBS and 0.1% Triton X-100 as negative and positive controls respectively. Calculate the hemolytic rate of the octapeptide. The calculation formula is as follows:

[0146]

[0147] Among them, A 八肽 is the OD value of adding octapeptides with different concentrations (sample source: Example 3) into the blood cell wells; A PBS is the OD value of adding PBS into the blood cell wells; A 0.1% Triton X-100 is the OD value of adding 0.1% Triton X-100 into the blood cell wells. The results are as Figure 13 shown. As can be seen, the hemolytic rate of the octapeptide is 0.201 - 6.477% in the concentration range of 1 - 512 μg / mL, and its change is within 7%. The results show that the octapeptide has no obvious hemolytic activity.

[0148] Due to the advantages of immune regulatory polypeptides or antimicrobial peptides, such as simple structure, strong bactericidal ability against bacteria, and weak phenomenon of inducing bacteria to produce drug resistance, they have the potential to become new antibacterial drugs and feed additives to replace traditional antibiotics. However, immune regulatory polypeptides also have certain side effects such as hemolytic activity and instability. Among them, hemolytic activity is an important index for evaluating the cytotoxicity of immune regulatory polypeptides. The hemolytic activity evaluation shows that the immune regulatory octapeptide does not show obvious hemolytic activity, indicating its potential for further development into new antibacterial drugs and feed additives.

[0149] In summary, the immune regulatory octapeptide produced in the present invention has advantages such as good antibacterial activity, stability, and low toxicity, and has broad application prospects in the development of new antibacterial drugs and feed additives.

[0150] The above are only the embodiments of the present invention. The invention is not limited to the fields involved in this embodiment. Common knowledge such as specific structures and characteristics known in the art is not described in detail here. Those of ordinary skill in the art know all the common technical knowledge in the technical field to which the invention belongs before the application date or the priority date, can know all the existing technologies in this field, and have the ability to apply the conventional experimental means before this date. Those of ordinary skill in the art can, under the inspiration given by this application, combine their own abilities to improve and implement this solution. Some typical well-known structures or well-known methods should not become obstacles for those of ordinary skill in the art to implement this application. It should be noted 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, and these will not affect the implementation effect and the practicality of the present invention. The protection scope required by this application should be based on the content of its claims, and the specific implementation manners described 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 as claimed in claim 1 in the preparation of antibiotics, feed or feed additives.

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

4. The octapeptide according to claim 1 or the antibiotic, feed or feed additive according to claim 3, characterized in that: It is prepared by fermentation with Brevibacillus Laterosporus M811; the Brevibacillus Laterosporus M811 was deposited in the China Center for Type Culture Collection on April 25, 2024, with a deposit number of CCTCC NO: M2024715.

5. The method for preparing the octapeptide according to claim 1, characterized in that: include: (1) Fermentation broth is prepared by fermenting Brevibacillus Laterosporus M811; Brevibacillus Laterosporus M811 was deposited in China Center for Type Culture Collection on April 25, 2024, with the deposit number of CCTCC NO: M2024715; (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.

6. The method for preparing the octapeptide according to claim 5, characterized in that: In the method (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 mode is shown in Table 1: 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, characterized in that: The salt ion precipitation comprises adding ammonium sulfate with a final concentration of 20 g / mL to the supernatant of the fermentation broth, and obtaining the crude octapeptide extract through centrifugation and drying.

8. The method for detecting octapeptide according to claim 1, characterized in that: 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 5M sulfuric acid, vortex 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 analysis; Reversed-phase HPLC analysis and detection: Analytical HPLC column, Sepax Bio-C18 4.6×250 mm 5μm; 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 2: The flow rate was 1 mL / min, the column temperature was 30°C, and the detection wavelength was 220 nm.

Images