Camel milk-derived immunoactive peptide for adjuvant therapy of tumors as well as extraction method and application of camel milk-derived immunoactive peptide

Abstract

The invention relates to the technical field of biological active peptides, in particular to camel milk-derived immunoactive peptide for adjuvant therapy of tumors and an extraction method and application thereof.The camel milk-derived immunoactive peptide comprises 32 peptide fragments, the sequences of the peptide fragments are shown as SEQ ID NO: 1-SEQ ID NO: 32, and the camel milk-derived immunoactive peptide is obtained according to the following method: culturing probiotics including lactic acid bacteria and saccharomycetes; the lactic acid bacteria are lactobacillus kefiranofaciens, lactobacillus acidophilus and lactobacillus pentosus, and the saccharomycetes are issatchenkia orientalis; preparing fermented camel milk, and inoculating probiotics into the sterilized and degreased camel milk to obtain the fermented camel milk; and extracting and separating the biological active peptide in the obtained fermented camel milk to obtain the camel milk-derived immunoactive peptide for adjuvant therapy of tumors. The camel milk-derived immunocompetent peptide can be rapidly and effectively extracted from fresh camel milk, and the camel milk-derived immunocompetent peptide is identified and subjected to target determination, so that it is verified that the camel milk-derived immunocompetent peptide has a proliferation effect on RAW264.7 cells, and the camel milk-derived immunocompetent peptide can be applied to prevention or / and antitumor drugs.

Description

Technical Field

[0001] The invention relates to the technical field of bioactive peptides, in particular to a camel milk-derived immune active peptide for assisting in treating tumors, and an extraction method and application thereof. Background Art

[0002] Bioactive peptides, as a peptide compound with special benefits for physiological health, are small, specific, and easily absorbed fragments separated from proteins. They are usually composed of 2 to 50 amino acids and have a molecular mass of less than 10kDa. They have various biological properties, such as antihypertensive, antioxidant, immunomodulatory, antibacterial, anti-inflammatory, and anti-diabetic effects.

[0003] Dairy products are considered an important source of bioactive peptides. Fermented camel milk is a dairy beverage made from fresh camel milk using traditional Xinjiang fermentation bacteria. It's used in folk medicine to treat diseases like tuberculosis and is also often used as an important adjunct to other conditions like cancer.

[0004] Chinese patent publication CN119613491A discloses a camel milk active peptide and its use in the preparation of drugs and edible products for treating and / or preventing diabetes. The present invention obtains two polypeptide compounds from papain hydrolysis products of fresh camel milk. Biological activity evaluation revealed that these polypeptide compounds exhibit significant protein tyrosine phospholipase 1B (PTP1B) inhibitory activity, enhance insulin receptor sensitivity, and promote glucose uptake in hepatocytes. These compounds have potential as drug candidates for the treatment of type 2 diabetes, providing a new material foundation for the development of innovative drugs for the treatment of type 2 diabetes and innovative hypoglycemic drugs such as PTP1B inhibitors.

[0005] The Chinese patent document with authorization announcement number CN104430849B discloses a multi-bacteria fermented skim camel milk for assisting anti-diabetes and a production method thereof. The multi-bacteria fermented skim camel milk is obtained by adding Lactobacillus helveticus fermentation agent, Lactobacillus plantarum fermentation agent, Lactobacillus paracasei fermentation agent, Lactobacillus paracasei subspecies firmus fermentation agent, Lactobacillus mucosa fermentation agent, Lactobacillus Harbini fermentation agent, Lactobacillus hilarii fermentation agent, Lactobacillus pentosus fermentation agent, Lactobacillus rhamnosus fermentation agent, Lactococcus lactis fermentation agent, Issaffron orientalis fermentation agent, Kluyveromyces marxianus fermentation agent, Pichia pastoris fermentation agent and Candida ethanolicus fermentation agent to skim camel milk and fermenting.

[0006] Chinese patent publication CN118955611A discloses a method for preparing hypoglycemic peptides from camel milk. Molecular docking was used to screen for endogenous camel milk peptide sequences, including AGF and MPL, that inhibit DPP-IV and SGLT-2. In vitro experiments with HepG2 hepatocytes demonstrated that the camel milk peptides increased insulin uptake and utilized glucose for glycogen synthesis. In vivo experiments also demonstrated that the camel milk peptides had a moderately beneficial effect on insulin-resistant mice and also inhibited DPP-IV and SGLT-2 expression.

[0007] The camel milk active peptides or fermented camel milk-derived bioactive peptides mentioned in the above-mentioned public documents are all used for preventing diabetes, assisting in anti-diabetes and lowering blood sugar, etc.

[0008] The study "Isolation and Identification of Immunoreactive Peptides from Xinjiang Fermented Camel Milk" (Zhang Yi, Yu Lan, Xiao Xueyun, Tubbs Manar, and Xinhua Nabi, "China Dairy Industry," Vol. 43, No. 11, 2015 (Total Issue 300)) published results showing that fermented camel milk has anti-inflammatory effects and can promote the expression of INF-γ cytokines in mouse splenic lymphocytes. This study isolated and isolated immunomodulatory milk-derived active peptides from laboratory-produced fermented camel milk and analyzed their structures using high-performance liquid chromatography-mass spectrometry.

[0009] The results of the study published in "Extraction, Isolation and Immunomodulatory Activity Evaluation of Bioactive Peptides from Fermented Camel Milk" (Bahti Urazbek, Liu Hongmei, Xinhua Nabi, "China Dairy Industry" Vol. 42, No. 4, 2014 (Total No. 281)) show that fermented camel milk has anti-inflammatory effects, which may be related to its immunomodulatory effects. The development of immunology has made it increasingly clear that specific natural functional factors in food can regulate immune function and resist chronic inflammation and damage.

[0010] It can be seen that traditional fermented dairy products are beneficial to human health, which is related to their rich content of probiotics and bioactive peptides. Using microbial fermentation to produce bioactive peptides, probiotic fermented dairy products can effectively enhance protein degradation in dairy products, increase the levels of peptides and free amino acids in fermented dairy products, and improve the quality of fermented milk.

[0011] However, the aforementioned public literature only mentions the anti-inflammatory and immune-regulating effects of fermented camel milk-derived bioactive peptides. Detailed information regarding the peptide segments, composition, molecular weight, and identification of these fermented camel milk-derived immunoactive peptides is unclear. This leads to complex preparation and extraction methods and uncertainty about their efficacy when used as antibacterial and anti-inflammatory drugs, a problem that urgently needs to be addressed. Summary of the Invention

[0012] The present invention provides a camel milk-derived immunoactive peptide for assisting in the treatment of tumors, as well as an extraction method and application thereof, which overcomes the deficiencies of the above-mentioned prior art and can effectively solve the problems of the existing fermented camel milk-derived bioactive peptides, such as complex preparation methods, unclear identification of peptide segments, composition, and molecular weight, which limit their use as antibacterial and anti-inflammatory drugs.

[0013] One of the technical solutions of the present invention is achieved through the following measures: a camel milk-derived immune active peptide for auxiliary treatment of tumors, including 32 peptide segments, the sequences of the 32 peptide segments are shown as SEQ ID NO: 1 to SEQ ID NO: 32 respectively.

[0014] The following is a further optimization and / or improvement of one of the above-mentioned technical solutions: The above was extracted according to the following method: S1, culture probiotics, including lactic acid bacteria and yeasts, the lactic acid bacteria are Lactobacillus kumiss, Lactobacillus caucasus, and Lactobacillus pentosus, and the yeast is Issaffron orientalis; S2, preparing fermented camel milk, inoculating probiotics into sterilized skimmed camel milk to obtain fermented camel milk; S3, extracting and separating the bioactive peptides from the fermented camel milk to obtain camel milk-derived immune active peptides for auxiliary treatment of tumors.

[0015] In the above step S1, the specific operations of culturing probiotics include: S101, placing a single Lactobacillus kumiss strain, a Lactobacillus caucasus strain, and a Lactobacillus pentosus strain in MRS solid culture medium, coating and streaking the medium, and then culturing the medium in a 37° C. incubator for 48 hours to obtain Lactobacillus kumiss strains, Lactobacillus caucasus strains, and Lactobacillus pentosus strains; S102, inoculating a single Lactobacillus kumiss-like strain, a Lactobacillus caucasus strain, and a Lactobacillus pentosus strain into an MRS broth liquid culture medium, culturing in a constant temperature incubator at 37° C. for 24 hours, activating the second to third generations, and obtaining lactic acid bacteria; S103, streaking a single strain of I. orientalis on Sabouraud agar, and culturing the culture in a 37°C constant temperature incubator for 72 hours to obtain an I. orientalis strain; S104, picking a single strain of Issachus orientalis and placing it into a malt extract liquid culture medium, culturing it in a constant temperature incubator at 35° C. for 24 hours, activating it for 2 to 3 generations, and obtaining a yeast.

[0016] In the above step S2, the specific operations of preparing fermented camel milk include: S201, centrifuging the fresh camel milk at a speed of 3000 rpm for 15 minutes, repeating this process 2 to 3 times, and discarding the upper layer of fat to obtain fat-free camel milk; S202, centrifuging the camel milk after the fat is discarded at a speed of 3000 r / min for 15 minutes, repeating 2 to 3 times, discarding the biomacromolecules at the bottom, collecting the supernatant, and obtaining skimmed camel milk; S203, pasteurizing the skimmed camel milk at 95° C. for 5 to 10 minutes to obtain sterilized skimmed camel milk; S204, inoculating lactic acid bacteria and yeast into sterilized skimmed camel milk, fermenting in a constant temperature incubator at 37° C. for 12 hours, and then cooling to room temperature to obtain fermented camel milk.

[0017] In the above step S204, the inoculation weight ratio of lactic acid bacteria to yeast is 1 to 3:1, and the total inoculation weight of lactic acid bacteria and yeast is 0.5% to 2% of the weight of the sterilized skimmed camel milk.

[0018] In the above step S3, the specific operations of extracting and separating the bioactive peptides from the fermented camel milk include: S301, centrifuging the fermented camel milk at 3000 rpm for 15 minutes, repeating 2 to 3 times, collecting the supernatant to obtain a fermented camel milk supernatant; S302, ultrafiltration of the fermented camel milk supernatant to obtain an ultrafiltrate; S303, concentrating the ultrafiltrate to obtain a concentrated solution; S304, the concentrated solution is placed in a sterile culture dish and frozen at -20°C, and then freeze-dried for 24 hours to obtain a crude peptide freeze-dried powder; S305, separating the crude peptide freeze-dried powder using a Sephadex G-50 gel chromatography column to obtain camel milk-derived immune active peptides for adjuvant treatment of tumors.

[0019] In the above step S302, a membrane separation device equipped with a spiral ultrafiltration membrane with a cut-off capacity of 30 kDa is used for ultrafiltration, the ultrafiltration temperature is 40° C., and the ultrafiltration pressure is 0.1 MPa.

[0020] In the above step S303 , the specific operation of concentrating the ultrafiltrate includes: using a rotary evaporator to concentrate at a concentration ratio of 10:1, and setting the temperature of the rotary evaporator to 48° C. and the pressure to 0.8 KPa.

[0021] The second technical solution of the present invention is achieved by the following measures: A method for extracting camel milk-derived immunoactive peptides for auxiliary treatment of tumors is carried out according to the following steps: S1, culture probiotics, including lactic acid bacteria and yeasts, the lactic acid bacteria are Lactobacillus kumiss, Lactobacillus caucasus, and Lactobacillus pentosus, and the yeast is Issaffron orientalis; S2, preparing fermented camel milk, inoculating probiotics into sterilized skimmed camel milk to obtain fermented camel milk; S3, extracting and separating the bioactive peptides from the fermented camel milk to obtain camel milk-derived immune active peptides for auxiliary treatment of tumors.

[0022] The third technical solution of the present invention is achieved through the following measures: application of a camel milk-derived immune active peptide for auxiliary treatment of tumors in the preparation of preventive and / or anti-tumor drugs.

[0023] The present invention can quickly and effectively extract camel milk-derived immune active peptides from fresh camel milk, identify and target them, and further verify that they have a proliferation effect on RAW264.7 cells, and can be used in preventive and / or anti-tumor drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Attachment Figure 1 This is a curve diagram of the components obtained by gel chromatography separation in Example 11 of the present invention.

[0025] Attachment Figure 2-A This is a diagram of the GO analysis results in Example 12 of the present invention.

[0026] Attachment Figure 2-B This is the enrichment of the KEGG analysis results in Example 12 of the present invention.

[0027] Attachment Figure 3 This is a diagram of the molecular dynamics simulation results in Example 12 of the present invention.

[0028] Attachment Figure 4-A This is a diagram of the interaction between protein and bioactive peptide 2 / STAT3 during the molecular dynamics process in Example 12 of the present invention.

[0029] Attachment Figure 4-B This is a diagram of the interaction between protein and bioactive peptide 5 / CASP3 during the molecular dynamics process in Example 12 of the present invention.

[0030] Attachment Figure 5 This is a graph showing the effects of the crude peptide, component 1 and component 2 in Example 12 of the present invention on the proliferation rate of RAW264.7 macrophages.

[0031] Attachment Figure 6 This is a diagram showing the effect of the crude peptide in Example 12 of the present invention on the morphology of RAW264.7 macrophages.

[0032] Attachment Figure 7 This is a diagram showing the effect of point 1 on the morphology of RAW264.7 macrophages in Example 12 of the present invention.

[0033] Attachment Figure 8 This is a diagram showing the effect of component 2 in Example 12 of the present invention on the morphology of RAW264.7 macrophages. DETAILED DESCRIPTION

[0034] The present invention is not limited to the following embodiments, and specific implementation methods can be determined based on the technical solutions of the present invention and actual conditions. Unless otherwise specified, the various chemical reagents and chemicals mentioned in the present invention are all chemical reagents and chemicals commonly known in the prior art; unless otherwise specified, the percentages in the present invention are all percentages by mass; unless otherwise specified, the solutions in the present invention are all aqueous solutions with water as the solvent, for example, hydrochloric acid solution is an aqueous hydrochloric acid solution; normal temperature and room temperature in the present invention generally refer to temperatures between 15°C and 25°C, and are generally defined as 25°C.

[0035] The present invention will be further described below in conjunction with the embodiments: Example 1: The camel milk-derived immunoactive peptide for assisting in the treatment of tumors comprises 32 peptide segments, and the sequences of the 32 peptide segments are shown in SEQ ID NO: 1 to SEQ ID NO: 32, respectively.

[0036] Example 2: As an optimization of the above example, the following method was used to extract: S1, culture probiotics, including lactic acid bacteria and yeasts, the lactic acid bacteria are Lactobacillus kumiss, Lactobacillus caucasus, and Lactobacillus pentosus, and the yeast is Issaffron orientalis; S2, preparing fermented camel milk, inoculating probiotics into sterilized skimmed camel milk to obtain fermented camel milk; S3, extracting and separating the bioactive peptides from the fermented camel milk to obtain camel milk-derived immune active peptides for auxiliary treatment of tumors.

[0037] Example 3: As an optimization of the above example, in step S1, the specific operation of culturing probiotics includes: S101, placing a single Lactobacillus kumiss strain, a Lactobacillus caucasus strain, and a Lactobacillus pentosus strain in MRS solid culture medium, coating and streaking the medium, and then culturing the medium in a 37° C. incubator for 48 hours to obtain Lactobacillus kumiss strains, Lactobacillus caucasus strains, and Lactobacillus pentosus strains; S102, inoculating a single Lactobacillus kumiss-like strain, a Lactobacillus caucasus strain, and a Lactobacillus pentosus strain into an MRS broth liquid culture medium, culturing in a constant temperature incubator at 37° C. for 24 hours, activating the second to third generations, and obtaining lactic acid bacteria; S103, streaking a single strain of I. orientalis on Sabouraud agar, and culturing the culture in a 37°C constant temperature incubator for 72 hours to obtain an I. orientalis strain; S104, picking a single strain of Issachus orientalis and placing it into a malt extract liquid culture medium, culturing it in a constant temperature incubator at 35° C. for 24 hours, activating it for 2 to 3 generations, and obtaining a yeast.

[0038] Example 4: As an optimization of the above example, in step S2, the specific operations of preparing fermented camel milk include: S201, centrifuging the fresh camel milk at a speed of 3000 rpm for 15 minutes, repeating this process 2 to 3 times, and discarding the upper layer of fat to obtain fat-free camel milk; S202, centrifuging the camel milk after the fat is discarded at a speed of 3000 r / min for 15 minutes, repeating 2 to 3 times, discarding the biomacromolecules at the bottom, collecting the supernatant, and obtaining skimmed camel milk; S203, pasteurizing the skimmed camel milk at 95° C. for 5 to 10 minutes to obtain sterilized skimmed camel milk; S204, inoculating lactic acid bacteria and yeast into sterilized skimmed camel milk, fermenting in a constant temperature incubator at 37° C. for 12 hours, and then cooling to room temperature to obtain fermented camel milk.

[0039] Example 5: As an optimization of the above example, in step S204, the inoculation weight ratio of lactic acid bacteria to yeast is 1 to 3:1, and the total inoculation weight of lactic acid bacteria and yeast is 0.5% to 2% of the weight of sterilized skimmed camel milk.

[0040] Example 6: As an optimization of the above example, in step S3, the specific operation of extracting and separating the bioactive peptides from the fermented camel milk includes: S301, centrifuging the fermented camel milk at 3000 rpm for 15 minutes, repeating 2 to 3 times, collecting the supernatant to obtain a fermented camel milk supernatant; S302, ultrafiltration of the fermented camel milk supernatant to obtain an ultrafiltrate; S303, concentrating the ultrafiltrate to obtain a concentrated solution; S304, the concentrated solution is placed in a sterile culture dish and frozen at -20°C, and then freeze-dried for 24 hours to obtain a crude peptide freeze-dried powder; S305, separating the crude peptide freeze-dried powder using a Sephadex G-50 gel chromatography column to obtain camel milk-derived immune active peptides for adjuvant treatment of tumors.

[0041] Example 7: As an optimization of the above example, in step S302, a membrane separation device equipped with a rolled ultrafiltration membrane with a cutoff of 30 kDa is used for ultrafiltration, the ultrafiltration temperature is 40°C, and the ultrafiltration pressure is 0.1 MPa.

[0042] Example 8: As an optimization of the above example, in step S303, the specific operation of concentrating the ultrafiltrate includes: using a rotary evaporator to concentrate at a concentration ratio of 10:1, and setting the rotary evaporator temperature to 48°C and the pressure to 0.8 KPa.

[0043] Example 9: The method for extracting the camel milk-derived immunoreactive peptide for adjuvant tumor treatment is carried out according to the following steps: S1, culture probiotics, including lactic acid bacteria and yeasts, the lactic acid bacteria are Lactobacillus kumiss, Lactobacillus caucasus, and Lactobacillus pentosus, and the yeast is Issaffron orientalis; S2, preparing fermented camel milk, inoculating probiotics into sterilized skimmed camel milk to obtain fermented camel milk; S3, extracting and separating the bioactive peptides from the fermented camel milk to obtain camel milk-derived immune active peptides for auxiliary treatment of tumors.

[0044] Example 10: Use of the camel milk-derived immune active peptide for auxiliary treatment of tumors in the preparation of preventive and / or anti-tumor drugs.

[0045] Example 11: The camel milk-derived immunoreactive peptides for adjuvant tumor treatment include 32 peptide segments, the sequences of which are shown in SEQ ID NO: 1 to SEQ ID NO: 32, respectively, and are extracted according to the following method: S1, cultivation of probiotics: Individual strains of Lactobacillus kumiss, Lactobacillus caucasus, and Lactobacillus pentosus were selected and placed in MRS solid culture medium for coating and streaking, and then cultured in a 37°C constant temperature incubator for 48 hours. Subsequently, individual strains were selected and inoculated into MRS broth liquid culture medium, and cultured in a 37°C constant temperature incubator for 24 hours to activate the second to third generations, thereby obtaining lactic acid bacteria for later use. A single strain of I. orientalis was streaked onto Sabouraud agar and incubated in a 37°C incubator for 72 hours. After 72 hours, a single strain was placed into a malt wort liquid medium and incubated in a 35°C incubator for 24 hours to activate two to three generations of yeast for later use. S2, preparing fermented camel milk: The fresh camel milk was centrifuged at 3000 r / min for 15 min, and the upper fat layer was discarded to obtain fat-free camel milk; The fat-removed camel milk was centrifuged at 3000 r / min for 15 minutes, and the process was repeated 2 to 3 times. The biomacromolecules at the bottom were discarded, and the supernatant was collected. The skimmed camel milk was pasteurized (95°C, 5 to 10 minutes) to obtain sterilized skimmed camel milk. Lactic acid bacteria and yeast were inoculated into sterilized skimmed camel milk at an inoculation weight ratio of 3:1, with the total inoculation weight of the lactic acid bacteria and yeast being 2% of the weight of the sterilized skimmed camel milk. The inoculated sterilized skimmed camel milk was placed in a constant temperature incubator at 37°C for 12 hours, and after the fermentation was completed, it was cooled to room temperature to obtain the fermented camel milk for later use. S3, extracting and separating bioactive peptides from fermented camel milk: Centrifuging the fermented camel milk at 3000 r / min for 15 min, repeating 2 to 3 times, collecting the supernatant to obtain the fermented camel milk supernatant; The fermented camel milk supernatant was prepared by installing a spiral ultrafiltration membrane with a cut-off of 30 kDa in a membrane separation device to ultrafilter the fermented camel milk at a temperature of 40°C and a pressure of 0.1 MPa to obtain an ultrafiltrate. The obtained ultrafiltrate was concentrated using a rotary evaporator at a concentration ratio of 10:1, and the rotary evaporator temperature was set to 48°C and the pressure was set to 0.8KPa to obtain a concentrated solution; The concentrated solution was placed in a sterile culture dish and placed in a -20°C refrigerator for freezing and then freeze-dried for 24 hours to obtain a crude peptide freeze-dried powder which was stored at -20°C. Sephadex G-50 gel chromatography column was used to separate the crude peptides: Place 15g of dextran gel G-50 in a 1000mL beaker and soak in 1000mL of distilled water for 24 hours to allow it to swell and remove impurities such as bubbles, dust, and fine particles from the gel. After swelling is complete, remove impurities and the upper layer of water by decanting. Rinse the gel repeatedly with plenty of distilled water until the gel is free of impurities. Seal the beaker with plastic wrap and set aside. Take a 110 cm long, 1.5 cm inner diameter glass chromatography column, clean it, and fix it vertically on a rack. Close the valve and first pour about 1 / 3 of distilled water into the column. Then, add a small amount of distilled water to the treated Sephadex G-50 to make a homogenous slurry. Use a glass rod to guide the flow and slowly and evenly pour it into the chromatography column. At the same time, open the valve at the bottom of the chromatography column and maintain an appropriate flow rate to allow the Sephadex G-50 particles in the chromatography column to settle evenly until the sedimentation is complete. Then, a large amount of distilled water was used for repeated elution. After the column was loaded, the column was equilibrated for 24 hours. Take the crude peptide freeze-dried powder and add appropriate amount of distilled water to dissolve it to prepare a solution with a concentration of 0.5g / mL for use. Centrifuge at 3000r / min for 5min, collect the supernatant for use, and prepare it immediately; Use a pipette to draw the distilled water from the top of the dextran gel in the chromatography column. At the same time, open the valve to allow the distilled water to flow out. When the distilled water is just flush with the top of the gel, close the valve. Take 5% of the volume of the chromatography column as the sample load (the first sample load is 1% to 2% of the column volume), open the valve to allow the sample to penetrate into the chromatography column, use distilled water as the eluent for elution, and maintain the flow rate at 0.5 mL / min. Continuously add eluent to keep the gel from being exposed to the liquid surface. Collect the effluent from the beginning of elution, collect one tube for every 3 ml, and use a UV spectrophotometer to detect the effluent of each tube at 214 nm (such as Figure 1 As shown), the effluents with the same peak time were combined, placed in a -20°C refrigerator for freezing and forming, and then freeze-dried. The obtained components 1 and 2 (i.e., the camel milk-derived immune active peptides for the auxiliary treatment of tumors) were stored at -20°C.

[0046] Example 12: Component 1 and component 2 obtained in Example 11 (i.e., the camel milk-derived immune active peptides for adjuvant tumor treatment) were identified, their targets determined, molecular dynamics simulations performed, and cell verification performed.

[0047] 1. Identification results of camel milk-derived immunoreactive peptides for adjuvant tumor treatment: HPLC-MS / MS identification yielded 32 peptides from components 1 and 2, primarily derived from whey acidic protein (WAP), α-lactalbumin (α-LA), and peptidoglycan recognition protein 1 (PGLYRP1). The 32 peptides identified from components 1 and 2 by HPLC-MS / MS are shown in Table 1, as the closer the mass spectrometry data matches the protein database, the higher the score.

[0048] 2. Target determination and molecular dynamics simulation of camel milk-derived immunoactive peptides for adjuvant tumor therapy: (1) Acquisition of camel milk-derived immunoactive peptides for adjuvant treatment of tumors and intersectional targets of diseases The 2D structures of seven camel milk-derived immunoreactive peptides derived from PGLYRP1 were mapped using MarvinSketch 22.11 software. Target prediction for these peptides was performed using the Super PRED database, with the results including protein name and Uniport ID. The corresponding gene names were searched for in the Uniport database based on the Uniport IDs. Duplicates were then pooled and removed, resulting in a total of 222 potential targets for these peptides. Using the keywords "immunomodulatory" and "inflammation," the GeneCards and OMIM-GENE-MAP databases were used to screen for immunomodulatory and anti-inflammatory disease-related targets (with a score of >10 in the GeneCards database). Target information meeting these requirements was then obtained, and duplicates were merged and removed, resulting in 866 targets related to immunomodulation and 2598 targets related to anti-inflammatory diseases.

[0049] Using an online Venn diagram-based platform, we identified 66 intersecting targets between camel milk-derived immunoreactive peptides and immunomodulatory and anti-inflammatory diseases. These targets were then imported into the String database for analysis. The resulting data were then imported into Cytoscape 3.9.0 software, where the Network Analyzer tool was used to evaluate and screen 18 core targets (shown in Table 2). The results showed that many of these key intersecting targets were related to immunomodulation.

[0050] (2) GO and KEGG enrichment analysis The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and gene ontology (GO) functional enrichment analysis of key targets at the intersection of camel milk-derived immune active peptides and diseases were performed using the David database to obtain key target function and pathway analysis. The key targets and KEGG pathway enrichment analysis results at the intersection of camel milk-derived immune active peptides and diseases were collected and imported into Cytoscape 3.9.0 software to construct a "camel milk-derived immune active peptide-disease-target-pathway" relationship network diagram (such as Figure 2-A 、 Figure 2-BGO analysis indicated that its bioactive peptides may be involved in biological processes such as inflammation and apoptosis. KEGG pathways such as lipids and atherosclerosis, cancer pathways, Kaposi's sarcoma-associated herpes virus infection, hepatitis B, tumor necrosis factor signaling pathways, human immunodeficiency virus type 1 infection, and apoptosis are associated with inflammatory diseases, viral infections, and tumors, all of which involve immune responses.

[0051] (3) Molecular docking The top four proteins with the highest degree values, STAT3, HSP90AA1, CASP3, and PIK3CA, were selected as receptors and active peptides for molecular docking using Autodock vina. Molecular docking technology was used to verify the interaction between the predicted camel milk-derived immune active peptides and targets. The 2D structure of the peptide was converted into a 3D structure using MarvinSketch 22.11. The three-dimensional structure of the key target protein was obtained using PDB. Then, AutoDockTool1.5.7 and PyMOL 2.4.0 software were used to remove water molecules, add hydrogen, and set the flexible bond of the small molecule ligand to be rotatable. Finally, the docking process was performed using AutoDockVina 1.1.2, and the molecular binding energy was calculated. The results are shown in Table 3. It is generally believed that the binding energy (affinity) is <-5.5 kcal·mol -1 The results in Table 3 show that the binding energy of all molecules to the target is less than -5.5 kcal·mol -1 , indicating that camel milk-derived immune active peptides have good binding activity with key targets.

[0052] (4) Molecular dynamics simulation According to the molecular docking results, almost all camel milk-derived immunoactive peptides interact with STAT3, and some camel milk-derived immunoactive peptides interact with CASP3, and the binding energy of camel milk-derived immunoactive peptide 2 / STAT3 and camel milk-derived immunoactive peptide 3 / CASP3 is the best. Therefore, camel milk-derived immunoactive peptide 2 / STAT3 and camel milk-derived immunoactive peptide 3 / CASP3 were selected for molecular dynamics simulation and the binding free energy was calculated. Molecular dynamics simulation is an important method to simulate molecular systems and properties by computer. Desmond software was used to perform molecular dynamics simulation on the docking complex of camel milk-derived immunoactive peptide and protein target. Adding a reasonable amount of Na + and Cl − ions to keep the system neutral and achieve a final generation phase of 100ns. The molecular dynamics simulation process is as follows: Input complex parameters → Establish equilibrium system → Minimize system energy → Heat up the system, balance the system → Molecular dynamics simulation → Generate data and analyze The molecular dynamics simulation results are as follows Figure 3 As shown, Figure 3 A is the root mean square deviation (RMSD) result during the molecular dynamics process, and B is the root mean square fluctuation (RMSF) result during the molecular dynamics process.

[0053] Figure 3 The results showed that the RMSD of camel milk-derived immunoactive peptides and proteins fluctuated greatly in the early stage and moved violently, and fluctuated less in the later stage and tended to be stable. Camel milk-derived immunoactive peptides fluctuated more than proteins, which may be caused by the excessive flexibility of camel milk-derived immunoactive peptides. The RMSFs of the two complexes were relatively low, and the RMSFs of most protein sequences were below 2Å, indicating that the protein system was still relatively stable. The interactions between proteins and ligands can be divided into four categories, namely hydrogen bonds, hydrophobic bridges, ionic bridges and water bridges. Among them, hydrogen bonds (H bonds) play an important role in the binding of proteins and ligands. The results are as follows. Figure 4-A and 4-B As shown. In the molecular dynamics stage, the number of hydrogen bonds in the camel milk-derived immunoreactive peptide 2 / STAT3 and camel milk-derived immunoreactive peptide 5 / CASP3 systems is relatively large. Figure 4-A Camel milk-derived immune active peptide 2 / STAT3, Figure 4-B In the middle is camel milk-derived immunoreactive peptide 5 / CASP3. The superimposed histograms are normalized over the course of the trajectory: for example, a value of 0.7 means that a specific interaction is maintained for 70% of the simulation time.

[0054] The binding energy of camel milk-derived immunoreactive peptide 2 / STAT3 is -70.5876±15.72 kcal·mol -1 The binding energy of camel milk-derived immunoreactive peptide 5 / CASP3 is -64.1431±11.06 kcal·mol -1 Negative values indicate that the two molecules have a higher binding affinity for the target protein. The high binding energy of the two molecules indicates that they have good binding strength and good stability, which is consistent with the results in Figure 4 above.

[0055] 3. Cell Verification of Bioactive Peptides: Take RAW264.7 macrophages growing in the logarithmic phase, first transfer most of the upper culture medium into a 15 mL sterile centrifuge tube, and use a pipette to blow the attached cells with the remaining culture medium to blow the attached cells off, and then draw the cell suspension in the cell bottle into a 15 mL sterile centrifuge tube, centrifuge at 1000 r / min for 5 minutes, discard the supernatant, add RPMI1640 culture medium, and gently blow to make a cell suspension and count the cells. RAW264.7 macrophages were cultured at 1×10 per well. 5 Cells were seeded in 96-well plates, with 100 μL per well, and incubated in a CO2 incubator for 24 hours. A blank control group (RPMI-1640 medium) and camel milk-derived immunoreactive peptide groups (crude peptide, component 1, and component 2, prepared in RPMI-1640 medium at concentrations of 0.1 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 0.75 mg / mL, and 1 mg / mL, respectively) were established. Each group was plated in triplicate, with three replicates. After 24 hours of incubation in the incubator, 10 μL of CCK-8 solution was added to each well and incubated in the incubator for an additional 2 hours. OD values at 450 nm were measured using a multifunctional enzyme-linked immunosorbent assay (ELISA) reader. This procedure was repeated three times.

[0056] The effects of different doses of bioactive peptides on the proliferation of RAW264.7 macrophages are shown in Table 4. The effects of crude peptide, component 1 and component 2 on the proliferation rate of RAW264.7 macrophages are shown in Table 4. Figure 5 As shown in A, B and C.

[0057] Table 4 and Figure 5 The results showed that compared with the blank control group, the treatment groups with different doses had a certain proliferation-promoting effect on RAW264.7 cells ( p <0.001), and at lower concentrations, it had a good proliferative effect on RAW264.7 cells. This result indicates that crude peptides, fraction 1, and fraction 2 extracted from compound probiotic fermented camel milk have a proliferative effect on RAW264.7 cells.

[0058] Prepare RAW264.7 macrophage suspension with the same steps as above. RAW264.7 macrophages were cultured at 1×10 5The cells were seeded in a 24-well plate and cultured in a CO2 incubator for 24 hours. The culture medium was discarded, and a blank control group (RPMI-1640 culture medium) and a camel milk-derived immune active peptide group (divided into crude peptide, component 1, and component 2, with concentrations of 0.1 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 0.75 mg / mL, and 1 mg / mL, prepared using RPMI-1640 culture medium as a solvent, and filtered using a sterile filter membrane with a pore size of 0.22 μm) were set up, with 1 mL each. The 24-well plate was placed in a CO2 incubator and cultured for another 24 hours. After 24 hours, the culture medium was discarded, and the cells were washed three times with PBS buffer, and then observed and images were collected under an inverted microscope. The results are as follows: Figure 6 、 Figure 7 、 Figure 8 shown.

[0059] Among them, Figure 6 Among them, A is the blank control group, B is the 0.1 mg / mL crude peptide treatment group, C is the 0.25 mg / mL crude peptide treatment group, D is the 0.5 mg / mL crude peptide treatment group, E is the 0.75 mg / mL crude peptide treatment group, and F is the 1 mg / mL crude peptide treatment group; exist Figure 7 A is the blank control group, B is the 0.1 mg / mL component 1 treatment group, C is the 0.25 mg / mL component 1 treatment group, D is the 0.5 mg / mL component 1 treatment group, E is the 0.75 mg / mL component 1 treatment group, and F is the 1 mg / mL component 1 treatment group; exist Figure 8 Among them, A is the blank control group, B is the 0.1 mg / mL component 2 treatment group, C is the 0.25 mg / mL component 2 treatment group, D is the 0.5 mg / mL component 2 treatment group, E is the 0.75 mg / mL component 2 treatment group, and F is the 1 mg / mL component 2 treatment group; Figure 6 、 Figure 7 、 Figure 8 The results showed that compared with the blank group, the cell number and cell volume increased in the groups treated with different doses of crude peptide, component 1 and component 2.

[0060] In summary, the present invention can quickly and effectively extract camel milk-derived immunoactive peptides from fresh camel milk, identify and target them, and further verify that they have a proliferative effect on RAW264.7 cells, and can be used in preventive and / or anti-tumor drugs.

[0061] The above technical features constitute the embodiments of the present invention, which have strong adaptability and implementation effect. Non-essential technical features can be added or removed according to actual needs to meet the requirements of different situations.

Claims

1. A camel milk-derived immunoactive peptide for adjuvant treatment of tumors, characterized in that It comprises 32 peptide segments, and the sequences of the 32 peptide segments are shown as SEQ ID NO: 1 to SEQ ID NO: 32 respectively.

2. The camel milk-derived immunoreactive peptide for adjuvant treatment of tumors according to claim 1, characterized in that Extracted as follows: S1, culture probiotics, including lactic acid bacteria and yeasts, the lactic acid bacteria are Lactobacillus kumiss, Lactobacillus caucasus, and Lactobacillus pentosus, and the yeast is Issaffron orientalis; S2, preparing fermented camel milk, inoculating probiotics into sterilized skimmed camel milk to obtain fermented camel milk; S3, extracting and separating the bioactive peptides from the fermented camel milk to obtain camel milk-derived immune active peptides for auxiliary treatment of tumors.

3. The camel milk-derived immunoreactive peptide for adjuvant treatment of tumors according to claim 2, characterized in that In step S1, the specific operations of culturing probiotics include: S101, placing a single Lactobacillus kumiss strain, a Lactobacillus caucasus strain, and a Lactobacillus pentosus strain in MRS solid culture medium, coating and streaking the medium, and then culturing the medium in a 37° C. incubator for 48 hours to obtain Lactobacillus kumiss strains, Lactobacillus caucasus strains, and Lactobacillus pentosus strains; S102, inoculating a single Lactobacillus kumiss-like strain, a Lactobacillus caucasus strain, and a Lactobacillus pentosus strain into an MRS broth liquid culture medium, culturing in a constant temperature incubator at 37° C. for 24 hours, activating the second to third generations, and obtaining lactic acid bacteria; S103, streaking a single strain of I. orientalis on Sabouraud agar, and culturing the culture in a 37°C constant temperature incubator for 72 hours to obtain an I. orientalis strain; S104, picking a single strain of Issachus orientalis and placing it into a malt extract liquid culture medium, culturing it in a constant temperature incubator at 35° C. for 24 hours, activating it for 2 to 3 generations, and obtaining a yeast.

4. The camel milk-derived immunoreactive peptide for adjuvant treatment of tumors according to claim 2 or 3, characterized in that In step S2, the specific operations of preparing fermented camel milk include: S201, centrifuging the fresh camel milk at a speed of 3000 rpm for 15 minutes, repeating this process 2 to 3 times, and discarding the upper layer of fat to obtain fat-free camel milk; S202, centrifuging the camel milk after the fat is discarded at a speed of 3000 r / min for 15 minutes, repeating 2 to 3 times, discarding the biomacromolecules at the bottom, collecting the supernatant, and obtaining skimmed camel milk; S203, pasteurizing the skimmed camel milk at 95° C. for 5 to 10 minutes to obtain sterilized skimmed camel milk; S204, inoculating lactic acid bacteria and yeast into sterilized skimmed camel milk, fermenting in a constant temperature incubator at 37° C. for 12 hours, and then cooling to room temperature to obtain fermented camel milk.

5. The camel milk-derived immunoreactive peptide for adjuvant treatment of tumors according to claim 4, characterized in that In step S204, the inoculation weight ratio of lactic acid bacteria to yeast is 1 to 3:1, and the total inoculation weight of lactic acid bacteria and yeast is 0.5% to 2% of the weight of the sterilized skimmed camel milk.

6. The camel milk-derived immunoreactive peptide for adjuvant treatment of tumors according to any one of claims 2 to 5, characterized in that In step S3, the specific operations of extracting and separating the bioactive peptides from the fermented camel milk include: S301, centrifuging the fermented camel milk at 3000 rpm for 15 minutes, repeating 2 to 3 times, collecting the supernatant to obtain a fermented camel milk supernatant; S302, ultrafiltration of the fermented camel milk supernatant to obtain an ultrafiltrate; S303, concentrating the ultrafiltrate to obtain a concentrated solution; S304, the concentrated solution is placed in a sterile culture dish and frozen at -20°C, and then freeze-dried for 24 hours to obtain a crude peptide freeze-dried powder; S305, separating the crude peptide freeze-dried powder using a Sephadex G-50 gel chromatography column to obtain camel milk-derived immune active peptides for adjuvant treatment of tumors.

7. The camel milk-derived immunoreactive peptide for adjuvant treatment of tumors according to claim 6, characterized in that In step S302, a membrane separation device equipped with a spiral ultrafiltration membrane with a cut-off of 30 kDa is used for ultrafiltration, the ultrafiltration temperature is 40°C, and the ultrafiltration pressure is 0.1 MPa.

8. The camel milk-derived immunoreactive peptide for adjuvant treatment of tumors according to claim 6 or 7, characterized in that In step S303 , the specific operation of concentrating the ultrafiltrate includes: using a rotary evaporator to concentrate the ultrafiltrate at a concentration ratio of 10:1, and setting the temperature of the rotary evaporator to 48° C. and the pressure to 0.8 KPa.

9. A method for extracting camel milk-derived immunoactive peptides for adjuvant tumor treatment according to any one of claims 1 or 3 to 8, characterized in that Follow the steps below: S1, culture probiotics, including lactic acid bacteria and yeasts, the lactic acid bacteria are Lactobacillus kumiss, Lactobacillus caucasus, and Lactobacillus pentosus, and the yeast is Issaffron orientalis; S2, preparing fermented camel milk, inoculating probiotics into sterilized skimmed camel milk to obtain fermented camel milk; S3, extracting and separating the bioactive peptides from the fermented camel milk to obtain camel milk-derived immune active peptides for auxiliary treatment of tumors.

10. Use of the camel milk-derived immunoactive peptide for adjuvant treatment of tumors according to any one of claims 1 to 8 in the preparation of preventive and / or anti-tumor drugs.

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