Polypeptide as well as preparation method and application thereof in preparation of anti-inflammatory products

Abstract

The invention relates to the technical field of biology, and particularly discloses a polypeptide, a preparation method thereof and application of the polypeptide in preparation of anti-inflammatory products. The amino acid sequence of the polypeptide disclosed by the invention is as shown in SEQ ID NO. 1 or SEQ ID NO. 2. The polypeptide disclosed by the invention has high cell safety and a remarkable inhibition effect on inflammation, can be used for preparing anti-inflammatory drugs or products for preventing diseases related to inflammation, can also be compounded with other component additives for use, can also be used in compatibility with cosmetics for use, can also be used as a feed additive for livestock and poultry production, and is wide in application range.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a polypeptide, a preparation method thereof, and application thereof in the preparation of anti-inflammatory products. Background Art

[0002] Inflammation, particularly chronic inflammation, has been shown to be closely linked to numerous diseases in both animal and human health. While traditional anti-inflammatory drugs can alleviate inflammatory symptoms, they are often accompanied by significant toxic side effects, limiting their long-term use. Therefore, the development of natural products with excellent safety and high anti-inflammatory properties has become a research hotspot.

[0003] Probiotics secrete a variety of functional products during their metabolic processes, including bioactive peptides. These peptides, with their diverse structures and rich functionalities, have been widely used in antimicrobial, anti-inflammatory, antioxidant, and immunomodulatory fields. For example, antimicrobial peptides (such as bacteriocins) produced by probiotics can effectively inhibit bacterial growth by disrupting pathogen cell membranes and interfering with protein or nucleic acid synthesis. Some immunomodulatory peptides can target immune-related signaling pathways such as Toll-like receptors (TLRs) and NOD receptors to regulate the host immune response. Furthermore, certain peptides possess physiological functions such as scavenging free radicals, strengthening the intestinal barrier, and promoting tissue repair, demonstrating significant potential for disease prevention and control and the development of functional products.

[0004] In anti-inflammatory activity studies, RAW 264.7 cells, a monocyte-macrophage cell line derived from Balb / c mice, are a classic in vitro model for studying the anti-inflammatory mechanisms of natural products due to their high sensitivity to inflammatory stimuli such as lipopolysaccharide (LPS) produced by Gram-negative bacteria. LPS stimulation activates classic inflammatory pathways such as TLR4 / NF-κB, triggering the release of inflammatory mediators such as NO, TNF-α, IL-6, and PGE2. Therefore, this model is frequently used for the initial screening of anti-inflammatory substances and for mechanistic research.

[0005] Compared with traditional synthetic anti-inflammatory drugs, naturally derived anti-inflammatory active peptides have the advantages of low toxicity, high targeting, and strong biodegradability. They are considered to be safe and efficient anti-inflammatory alternatives and are particularly suitable for development as functional products or new natural medicines. Although the current research on active peptides is constantly deepening and their application prospects in anti-inflammatory and antibacterial fields are broad, the relevant research on probiotic-derived anti-inflammatory peptides is still in its infancy, especially in terms of functional sequence screening, structural feature analysis and mechanism of action. In addition, existing studies have mostly focused on the intervention of whole bacteria of probiotics or the overall extraction of metabolites, lacking the systematic separation and verification of specific peptide components with anti-inflammatory functions. Therefore, the development of probiotic anti-inflammatory peptides with clear sources, stable structures, safety and non-toxicity will not only help fill the gaps in the research of natural anti-inflammatory substances, but also provide a theoretical basis and technical support for the development of new anti-inflammatory functional factors. Summary of the Invention

[0006] One of the purposes of the present invention is to provide a novel polypeptide with high safety and ideal anti-inflammatory effect.

[0007] The present invention provides a polypeptide, the amino acid sequence of which is shown in SEQ ID NO.1 or SEQ ID NO.2.

[0008] The present invention separates and obtains a polypeptide with ideal anti-inflammatory efficacy and no cytotoxicity from the co-fermentation product of two new bacterial strains. The polypeptide has a short peptide chain and is easy to prepare, thus enriching the types of products with anti-inflammatory efficacy.

[0009] Those skilled in the art should understand that adding tag sequences to both ends of a polypeptide for purposes such as easy purification and polypeptide labeling is a conventional technical means in the art and does not affect the inherent functions and activities of the polypeptide itself. Therefore, the polypeptide derivatives obtained by adding tag sequences to both ends of the polypeptide shown in SEQ ID NO.1 or SEQ ID NO.2 are also within the scope of protection of the present invention.

[0010] Based on the anti-inflammatory amino acid sequence provided by the present invention, the gene sequence encoding the polypeptide thereof also falls within the scope of protection of the present invention.

[0011] The present invention also provides a method for preparing the above polypeptide, which comprises isolating the polypeptide from the solid component obtained by solid-state fermentation of Bacillus amyloliquefaciens CAU X1 and Bacillus coagulans B2 using HPLC-MS / MS technology; The deposit number of the Bacillus amyloliquefaciens CAU X1 is CGMCC No. 26769; the deposit number of the Bacillus coagulans B2 is CGMCC No. 26767.

[0012] In the preparation method of the present invention, the fermentation culture medium comprises 25-40 parts of corn flour, 35-45 parts of bran powder and 15-24 parts of soybean meal powder.

[0013] Preferably, the fermentation medium consists of 38% corn flour, 42% bran flour and 20% soybean meal flour.

[0014] The polypeptide of the present invention can be obtained by fermentation of two specific bacterial strains, or can be prepared by conventional polypeptide chemical synthesis methods in the art.

[0015] The present invention also provides the use of the above polypeptide or the polypeptide prepared by the preparation method in the preparation of anti-inflammatory products.

[0016] The present invention also provides a cosmetic comprising the above polypeptide or the polypeptide prepared by the preparation method.

[0017] The present invention also provides an anti-inflammatory drug, which comprises the above polypeptide or the polypeptide prepared by the preparation method and pharmaceutically acceptable excipients.

[0018] The present invention also provides an animal feed or feed additive, which comprises the above polypeptide or the polypeptide prepared by the preparation method.

[0019] The present invention also provides the use of the polypeptide or the polypeptide prepared by the preparation method in livestock and poultry breeding.

[0020] The present invention also provides the use of the above polypeptide or the polypeptide prepared by the preparation method in preparing products that inhibit LPS-induced NO release, overexpression of cytokine IL-6 and / or overexpression of cytokine TNF-α.

[0021] The polypeptide of the present invention can reduce the secretion of inflammatory factors IL-6 and TNF-α and inhibit LPS-induced NO release, thereby achieving anti-inflammatory effects. The polypeptide of the present invention can be applied to a variety of fields with anti-inflammatory needs, with high safety and a wide range of applications.

[0022] The present invention also provides the use of the above polypeptide or the polypeptide prepared by the preparation method in preparing products that competitively bind to TLR4 / MD2 receptors or inhibit IκB and P65 protein phosphorylation. The amino acid sequence of the polypeptide is as shown in SEQ ID NO.1.

[0023] The polypeptide of the present invention as shown in SEQ ID NO.1 can compete with LPS for binding to TLR4 / MD2 receptor and significantly downregulate the phosphorylation levels of P65 and IκB, key proteins in the NF-κB signaling pathway.

[0024] The beneficial effects of the present invention are at least: The present invention is the first to obtain an anti-inflammatory peptide through solid-state fermentation of two probiotic strains, Bacillus amyloliquefaciens CAU X1 and Bacillus coagulans B2. The peptide has high cellular safety and a significant inhibitory effect on inflammation.

[0025] The anti-inflammatory polypeptide of the present invention has a significant effect on regulating the generation and development of inflammation and has the efficacy of alleviating inflammation. It can be used to prepare anti-inflammatory drugs or products for preventing the occurrence of inflammation-related diseases. It can also be used in combination with other component additives, can also be used in combination with cosmetics, and can also be used as a feed additive in livestock and poultry production. It has a wide range of applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Schematic diagram of the process for obtaining the anti-inflammatory polypeptide of the present invention.

[0027] Figure 2 This is the HPLC-MS / MS chart after enrichment of the polypeptide of the present invention.

[0028] Figure 3 Schematic diagram of the tertiary structure of M7 (upper figure) and W10 (lower figure) binding to TLR4 / MD2 receptor in the examples of the present invention.

[0029] Figure 4 These are the safety evaluation results of the six polypeptides on cells in the examples of the present invention.

[0030] Figure 5 The results of the effects of the six peptides on the NO content in LPS-induced RAW 264.7 cells in the present invention are shown in Figure 2. #### indicates P < 0.0001 compared with the blank control (Ctrl) group. Represents P < 0.001 compared with the LPS group, Represents P < 0.0001 compared with the LPS group.

[0031] Figure 6 The results of the effects of M7 and W10 on the levels of IL-6 and TNF-α in LPS-induced RAW 264.7 cells in the present invention are shown in Figure 2. #### indicates P < 0.0001 compared with the blank control (Ctrl) group. Represents P < 0.01 compared with the LPS group, Represents P < 0.0001 compared with the LPS group.

[0032] Figure 7The results of the effect of M7 on LPS-induced NF-κB signaling pathway protein phosphorylation in RAW 264.7 cells in the present invention are as follows. # represents P < 0.05 compared with the blank control (Ctrl) group, #### represents P < 0.0001 compared with the blank control (Ctrl) group, Represents P < 0.0001 compared with the LPS group. DETAILED DESCRIPTION

[0033] The preferred embodiments of the present invention will be described in detail below with reference to the examples. It should be understood that the following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0034] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods. Unless otherwise specified, the materials, reagents, etc. used in the following examples are all commercially available or prepared according to conventional methods in the art.

[0035] Example 1 This example provides a new anti-inflammatory peptide, the process diagram of which is shown in FIG. Figure 1 , the specific method is as follows: 1. The present invention has isolated and preserved two new strains of bacteria, Bacillus amyloliquefaciens CAU X1 and Bacillus coagulans B2.

[0036] Bacillus amyloliquefaciens CAU X1 was deposited on March 7, 2023, at the General Microbiology Center of China Culture Collection Administration (CGMCC, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China) and was named Bacillus amyloliquefaciens Bacillus amyloliquefaciens , the deposit number is CGMCC No. 26769.

[0037] Bacillus coagulans B2 is deposited in the General Microbiology Center of China Culture Collection Administration (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China. The deposit number is CGMCC No. 26767, the deposit date is March 7, 2023, and the classification name is Bacillus coagulans. Bacillus coagulans .

[0038] 2. Prepare seed stocks of Bacillus amyloliquefaciens CAU X1 and Bacillus coagulans B2, mix them in a 1:1 ratio, and inoculate 8% of the mixture into a solid-state fermentation medium composed of 38% corn flour, 42% bran flour, and 20% soybean meal. After fermentation, extract with sterile water overnight, filter, and freeze-dry to a powder for future use.

[0039] 3. Dissolve the lyophilized powder after extraction and enrich it using ultrafiltration tubes with specifications of 10kDa and 30kDa. The enriched solution is identified by HPLC-MS / MS. The following steps are included: After reduction and alkylation, the appropriate amount of trypsin (Promega) was added for enzymatic digestion. The hydrolyzate was desalted, lyophilized, and reconstituted in 0.1% formic acid for HPLC analysis. Mass spectrometry was performed using a Q Exactive™ Hybrid Quadrupole-Orbitrap™ Mass Spectrometer. Samples were injected at a flow rate of 600 nL / min onto an Acclaim PepMap RPLC C18, 5 μm, 100Å (300 μm × 5 mm), followed by an Acclaim PepMap RPLC C18, 1.9 μm, 100Å (150 μm × 150 mm). Gradient elution conditions were identical for both columns. Mobile phase A consisted of 0.1% formic acid in water, and mobile phase B consisted of 0.1% formic acid in acetonitrile (84% acetonitrile in water). Elution conditions were: 0–2 min, 4%B–8%B; 2–45 min, 8%B–28%B; 45–55 min, 28%B–40%B; 55–56 min, 40%B–95%B; 56–60 min, 95%B, with the sum of mobile phases A and B equaling 100%. Samples were separated by chromatography and analyzed by mass spectrometry. Detection was positive ionization, with a parent ion scan range of 300–1800 m / z, a primary mass spectrometer resolution of 70,000 at 200 m / z, an AGC (automatic gain control) target of 1e6, a maximum IT of 50 ms, and a dynamic exclusion time of 30.0 s. The mass-to-charge ratios of peptides and peptide fragments were collected using the following method: 20 fragmentation spectra (MS2 scans) were collected after each full scan, with the MS2 activation type being HCD, the isolation window being 2 m / z, the secondary mass spectrometry resolution being 17,500 at 200 m / z, the normalized collision energy being 27 eV, and the underfill being 0.1%. The total ion current is shown in Figure 1. Figure 2 shown.

[0040] 4. The mass spectrometry raw files were searched against the Uniport database using MaxQuant 1.6.14, yielding 67 peptide sequence matches. The bioactivity and physicochemical properties of the peptides were predicted using online databases. The following online search tools were used: peptide hydrophobicity evaluation (https: / / pepdraw.com / ), bioactivity scoring (http: / / distilldeep.ucd.ie / PeptideRanker / ), toxicity evaluation (https: / / webs.iiitd.edu.in / raghava / toxinpred / design.php), and peptide solubility evaluation (https: / / pepcalc.com / ). All peptides were screened in silico, ultimately identifying six peptides with excellent bioactivity, water solubility, and non-toxicity. As shown in Table 1, the activities of M7, D8, and W10, all in silico, were above 0.75, indicating excellent solubility and non-toxicity. The remaining 61 peptides with lower activity were not included in this table and were not included in the screening.

[0041] The polypeptide M7 is shown in SEQ ID NO. 1, and its sequence is: Met Pro Lys Tyr Pro Tyr Arg (MPKYPYR); The polypeptide D8 is shown in SEQ ID NO. 2, and its sequence is: Asp Gly Gln Met Met Met Met Lys (DGQMMMMK); The polypeptide W10 is shown in SEQ ID NO. 3, and its sequence is: Trp Ser Arg Gly Met Lys Pro Ile PheArg (WSRGMKPIFR); The polypeptide C15 is shown in SEQ ID NO. 4, and its sequence is: Cys Trp Tyr Glu Glu Phe Asn Tyr IleTyr Val Phe Asn Leu Arg (CWYEEFNYIYVFNLR); The polypeptide M9 is shown in SEQ ID NO. 5, and its sequence is: Met Lys Tyr His Pro Lys Met Gly Arg (MKYHPKMGR); The polypeptide P10 is shown in SEQ ID NO. 6, and its sequence is: Pro Asn Trp Leu Met Lys Gln Met GlnLys (PNWLMKQMQK).

[0042] Table 1 Peptide computer screening results and their physicochemical properties Note: Score: peptide credibility score; Length: number of amino acids in the peptide; m / z: mass-to-charge ratio of the peptide; z: number of charges carried by the peptide; RT: liquid chromatography retention time (min); Mass: peptide molecular weight (Da); ppm: error between the detected molecular weight and the theoretical molecular weight of the identified peptide.

[0043] 5. Docking of the tertiary structure of peptides with TLR4 / MD2: The online docking tool HPEPDOCK 2.0 was used to perform peptide-protein docking and predict the scores of the six peptides in Table 1, including peptides M7 and W10, with the TLR4 / MD2 crystal structure. The ranking of the docking models and the docking energy score were recorded, and the anti-inflammatory activity of the peptides was predicted accordingly. The lower the docking energy value, the stronger the affinity of the peptide to the TLR4 / MD2 receptor. Table 2 lists the 10 prediction models for each peptide, and the docking energy scores during the molecular docking process were statistically analyzed. The results show that the docking energy of the four peptides M7, W10, C15 and M9 are generally lower than that of D8 and P10, indicating that the former may have a stronger binding affinity with the receptor. The specific results of the docking of M7 and W10 with the complex are shown in Figure 3 Table 3 shows that M7 forms five hydrogen bonds with arginines ARG at positions 90 and 132, and glutamate GLU at position 122 of the TLR4 / MD2 complex. W10 binds to threonine THR at position 357, tyrosine TYR at position 375, aspartic acid ASP at position 403, histidine HIS at position 424, and aspartic acid ASP at position 426 on TLR4 / MD2, respectively. Notably, the binding site of M7 is within the hydrophobic pocket of MD2, partially overlapping with the LPS binding site on MD2, suggesting that M7 can compete with LPS for binding to the TLR4 / MD2 receptor.

[0044] Table 2 Docking energy scores of six peptides including M7 and TLR4 / MD2 complex Table 3 Distances between W10 and M7 and TLR4 / MD2 residues 6. Entrust Shanghai Jier Biochemical Technology Co., Ltd. to synthesize all the peptides in Table 1 using the solid phase method.

[0045] 7. Effects of the six peptides in Table 1, including M7 and W10, on cell viability Sample Preparation: Add 1 mL of PBS to each of the six peptides listed in Table 1 (10 mg of each, including M7 and W10) to obtain a 10 mg / mL stock solution. Filter through a 0.22 μm filter and store at -80°C until ready for use. Upon use, dilute the stock solution to a gradient of concentrations (1, 10, 50, and 100 μg / mL) using DMEM.

[0046] Frozen mouse macrophages (RAW 264.7) were removed from liquid nitrogen and thawed in a 37°C waterbath. The cells were transferred to sterile centrifuge tubes, centrifuged, and the supernatant discarded. Fresh DMEM (10% FBS-containing 1% penicillin / streptomycin) was added to evenly disperse the cells. The culture dish was incubated at 37°C in a 5% CO2 incubator for 24 hours. The culture medium for RAW 264.7 cells requires regular medium replacement and subculture. The procedure is as follows: discard the supernatant, rinse twice with an appropriate amount of PBS (pH 7.4) (approximately 20 seconds each time), then add fresh culture medium and pipette adherent cells. Distribute the cells evenly and plate them for later use. Cells in the logarithmic growth phase were seeded into 96-well plates, with 30,000 cells per well. Except for the blank control group, 100 μL of 0, 1, 10, 50, and 100 μg / mL of each peptide solution was added to each well and incubated in a 37°C, 5% CO2 incubator for 24 hours. 10 μL of CCK8 reagent was added to each well and incubated for 2 hours. During this time, the OD450 was measured to prevent the assay from exceeding the measurement range.

[0047] Calculate cell viability: Cell viability (%) = [A(drug added) - A(blank)] / [A(0 drug added) - A(blank)] × 100; A (drug added): absorbance of wells with cells, CCK-8 solution, and drug solution; A (blank): absorbance of wells with culture medium and CCK-8 solution but no cells; A(0 drug addition): absorbance of the wells containing cells, CCK-8 solution, and no drug solution.

[0048] Cell viability, also known as cell proliferation activity or cytotoxic activity, is a measure of the effect of drugs on the activity of target cells. Figure 4 It can be seen that the viability of RAW 264.7 cells was maintained at 90% when the concentration of six polypeptides including polypeptides M7 and W10 was 1-100 μg / mL, indicating that the polypeptides were absolutely safe for macrophages, and the safe concentration was 100 μg / mL.

[0049] 8. Study on the anti-inflammatory activity of the six peptides in Table 1, including M7 and W10 (1) Establishing an inflammation model An inflammatory model was established by treating RAW 264.7 cells with LPS as follows: 400,000 cells per well of a 24-well plate were plated in logarithmic growth phase DMEM (containing 1% penicillin / streptomycin) and cultured for 24 hours. After 24 hours, the culture medium was aspirated and replaced with serum-free DMEM for 4 hours. Except for the blank control group, 100 μL of 0 and 50 μg / mL peptide solutions were added to each well and incubated in a 37°C, 5% CO2 incubator for 6 hours. Subsequently, 100 μL of 100 ng / mL LPS solution was added to all wells except the blank control group for 24 hours to stimulate RAW 264.7 cells. The cell supernatant was collected in a 24-well plate, and then 1 mL of RIPA lysis buffer was added to collect the cell protein. The supernatant protein was collected by centrifugation at 12000g for 15 min, and the loading buffer was added and boiled for 15 min to denature the protein. The cell supernatant and protein were stored in a -80℃ refrigerator.

[0050] (2) Determination of NO Standard curve preparation: Prepare standards in DMEM solution at concentrations of 0, 1, 2, 5, 10, 20, 40, 60, and 100 µM. Assay steps: Take 50 µL of cell supernatant or standard, first add 50 µL of Griess reagent A, then add 50 µL of Griess reagent B to the same well. Gently tap the bottom of a 96-well plate to mix the solution. Incubate at room temperature in the dark for 10 minutes. Measure absorbance at 540 nm on a microplate reader, and calculate NO content based on a sodium nitrite standard curve. Calculate NO content in the cell supernatant using the standard curve.

[0051] NO is a key inflammatory mediator synthesized by inducible nitric oxide synthase (iNOS) in macrophages during inflammatory responses. Lipopolysaccharide (LPS) stimulation of macrophages significantly induces iNOS expression, thereby promoting the production of significant amounts of NO. Measuring NO levels can serve as a sensitive indicator of the intensity of the inflammatory response.

[0052] See the results Figure 5 As can be seen, LPS-stimulated cells significantly increased NO content (P<0.0001). When 50 μg / mL M7 or W10 were added, NO content was significantly reduced (P<0.0001). The NO content of cells pretreated with M7 was only 1 / 3 of that of cells stimulated with LPS alone, and the NO content of cells pretreated with W10 was 1 / 2 of that of cells stimulated with LPS alone. The other homologous peptides were less effective in reducing NO than M7 and W10, indicating that M7 and W10 have better anti-inflammatory effects and can inhibit LPS-induced NO release and suppress inflammation.

[0053] 9. Effects of M7 and W10 on IL-6 and TNF-α The cell supernatant obtained in step (1) Establishing an Inflammation Model was used to detect the IL-6 and TNF-α levels in the supernatant using an ELISA kit according to the instructions of the Invitrogen kit. The specific steps are as follows: 1) Sample addition: Add 100 μL of a 2-fold serially diluted standard to the standard wells. Add 100 μL of standard / sample dilution buffer to well 0. Add 100 μL of the cell supernatant to be tested to the sample wells.

[0054] 2) Incubation: Seal the plate with sealing film and incubate at 37°C for 90 min; 3) Washing: Discard the supernatant, spin dry, and add 300 μL of wash buffer to each well. Soak for 1.5 minutes, then remove the wash buffer and pat dry. Repeat 5 times.

[0055] 4) Add biotinylated detection antibody: Add 100 μL of biotinylated antibody working solution to each well, incubate at 37°C for 60 min, discard the solution, and wash the plate five times (same steps as above).

[0056] 5) Add substrate for color development: Add 100 μL of enzyme color development substrate TMB to each well and incubate at 37°C in the dark for 15 min.

[0057] 6) Add stop solution: Add 50 μL of stop solution to each well and measure the OD value of each well at a wavelength of 450 nm using a microplate reader within 5 minutes.

[0058] 7) Calculation: Calculate the IL-6 and TNF-α contents according to the standard curve.

[0059] See the results Figure 6 As shown in the results, compared with the blank group, the IL-6 content (34 pg / mL) and TNF-α content (413 pg / mL) of RAW 264.7 cells stimulated by LPS were significantly increased, while the IL-6 content in the W10 and M7 pretreatment groups was significantly reduced to 27 pg / mL and 24 pg / mL, respectively. Regarding the proinflammatory cytokine TNF-α content, W10 reduced it to 341 pg / mL, while M7 reduced it to 128 pg / mL. These results indicate that W10 and M7 can significantly inhibit the overexpression of cytokines (IL-6 and TNF-α) induced by LPS, thereby suppressing the inflammatory response, with M7 having a superior inhibitory effect to W10.

[0060] 10. Effect of M7 on protein phosphorylation in the NF-κB signaling pathway The cell proteins obtained in the step “(1) Establishing an Inflammation Model” were used to detect the phosphorylation of IKK, IκB, and P65 proteins, using β-actin as the internal reference protein. The steps for Western-blot protein detection are as follows: 1) Preparation of protein gel: Prepare 12% SDS-PAGE gel, 2) Sample loading: Add 10 μL of protein sample to each lane and run the stacking gel at a constant voltage of 60 V for 30 min and the separating gel at a constant voltage of 150 V for 60 min.

[0061] 3) Transfer: After the run, place the gel at the target protein band on a methanol-activated PVDF membrane (0.22 μm) to create a "sponge-filter paper-gel-membrane-filter paper-sponge" transfer sandwich. Transfer at a constant current of 200-300 mA for 2 h.

[0062] 4) Blocking: Block the transferred strips in 5% skim milk for at least 1 hour. Wash three times with TBST, 10 minutes each time.

[0063] 5) Primary antibody incubation: Place in diluted p-IKK, p-IκB, or p-P65 primary antibody solution and incubate overnight at 4°C.

[0064] 6) Secondary antibody incubation: Wash three times with TBST, then place in secondary antibody solution and incubate at room temperature for 1 h.

[0065] 7) Development: After washing three times with TBST, ECL developer was added and imaging was performed in a chemiluminescence imaging system.

[0066] Image J software was used to analyze the grayscale values ​​of the bands. The experimental results were as follows: Figure 7 The results showed that compared with the blank control group, the expression of p-IKK, p-IκB, and p-P65 proteins in the LPS group was significantly increased, indicating that the cells were inflammatory; while the expression levels of p-IκB and p-P65 proteins in the M7 pretreatment group were significantly lower than those in the LPS group, indicating that M7 inhibited the phosphorylation of IκB and P65 proteins, inhibited the NF-κB signaling pathway, and thus suppressed inflammation.

[0067] In summary, the two peptides M7 and W10 of the present invention have short amino acid sequences, simple structures, convenient synthesis, safety, and non-toxicity. They inhibit the expression of inflammatory factors, and M7, in particular, effectively inhibits the activation of the NF-κB signaling pathway. This has a significant preventive and therapeutic effect on inflammatory diseases caused by future microbial infections.

[0068] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A polypeptide, characterized in that The amino acid sequence is shown in SEQ ID NO.1 or SEQ ID NO.

2.

2. The method for preparing the polypeptide according to claim 1, characterized in that: The polypeptide is separated from the solid fraction obtained by solid-state fermentation of Bacillus amyloliquefaciens CAU X1 and Bacillus coagulans B2 using HPLC-MS / MS technology; The deposit number of the Bacillus amyloliquefaciens CAU X1 is CGMCC No. 26769; the deposit number of the Bacillus coagulans B2 is CGMCC No. 26767.

3. The preparation method according to claim 2, characterized in that The fermentation culture medium comprises 25-40 parts of corn flour, 35-45 parts of bran flour and 15-24 parts of soybean meal flour.

4. Use of the polypeptide according to claim 1 or the polypeptide prepared by the preparation method according to claim 2 or 3 in the preparation of anti-inflammatory products.

5. A cosmetic, characterized in that: The invention comprises the polypeptide according to claim 1 or the polypeptide prepared by the preparation method according to claim 2 or 3.

6. An anti-inflammatory drug, characterized in that The invention comprises the polypeptide according to claim 1 or the polypeptide prepared by the preparation method according to claim 2 or 3, and pharmaceutically acceptable excipients.

7. An animal feed or feed additive, characterized in that The invention comprises the polypeptide according to claim 1 or the polypeptide prepared by the preparation method according to claim 2 or 3.

8. Use of the polypeptide according to claim 1 or the polypeptide prepared by the preparation method according to claim 2 or 3 in livestock and poultry breeding.

9. Use of the polypeptide according to claim 1 or the polypeptide prepared by the preparation method of claim 2 or 3 in preparing a product for inhibiting LPS-induced NO release, overexpression of cytokine IL-6 and / or overexpression of cytokine TNF-α.

10. Use of the polypeptide according to claim 1 or the polypeptide prepared by the preparation method of claim 2 or 3 in preparing a product that competitively binds to TLR4 / MD2 receptors or a product that inhibits the phosphorylation of IκB or P65 proteins, wherein the amino acid sequence of the polypeptide is as shown in SEQ ID NO. 1.

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