Application of camel milk polypeptide

Abstract

The method comprises the following steps: taking a camel milk powder sample, sieving, extracting with pure water, fermenting to obtain a crude camel milk polypeptide sample, purifying the crude camel milk polypeptide sample, and respectively carrying out ion exchange column chromatography and gel column chromatography separation to obtain an acidic camel milk polypeptide sample with the yield of 12.9%. The camel milk polypeptide sample obtained by the method disclosed by the invention is seen as a single peak through an HPLC (High Performance Liquid Chromatography) liquid phase, so that the camel milk polypeptide sample is high in purity. And detection shows that the polypeptide has excellent cell hypoglycemic activity, and when the polypeptide concentration is greater than 40 [mu] g / mL, the glucose content in cells can be remarkably reduced by 15.7% compared with a blank group. MTT experiments show that when the concentration of the polypeptide reaches 320 [mu] g / mL, the cell survival rate is obviously inhibited. The camel milk polypeptide sample can be used as a raw material for preparing health-care products or foods with hypoglycemic and anti-inflammatory activity.

Description

Technical Field

[0001] This invention relates to the use of a camel milk polypeptide. Background Technology

[0002] Camel milk, as a traditional milk source, has attracted much attention due to its unique nutritional and bioactive components. Compared to cow's and goat's milk, camel milk is rich in lactoferrin, immunoglobulins, lysozyme, and various vitamins (such as vitamin C and vitamin D), while having lower milk fat and lactose content, making it more suitable for people with lactose intolerance. This invention uses camel milk powder, a solid byproduct generated during the deep processing of camel milk. It is rich in milk calcium, lactoferrin, and prebiotics, but due to its rough texture and poor solubility, it is often used as feed or disposed of as waste in traditional processes, resulting in low resource utilization. Therefore, there is an urgent need for a research method that can utilize this resource more efficiently, thereby extending the industrial chain and increasing added value.

[0003] Polypeptides are short-chain amino acid sequences produced from proteins through enzymatic hydrolysis or fermentation, combining the advantages of easy absorption as small molecules with the functionality of large molecules. In recent years, bioactive peptides have become a research hotspot due to their diverse physiological functions, including antioxidant, anti-inflammatory, antibacterial, antihypertensive, and immunomodulatory effects.

[0004] Currently, research on bioactive peptides has become a hot topic, primarily focusing on pharmaceuticals, functional foods, and advanced materials. In the field of peptide drugs, research mainly revolves around three core challenges: the discovery and validation of innovative drug targets, overcoming bottlenecks in in vivo delivery and stability, and the efficient synthesis and production of complex peptide structures. This field is characterized by active research and intense competition both domestically and internationally, and has already yielded a series of authorized patents with clear technical characteristics and potential, providing specific technological pathways.

[0005] Domestic patent application status for camel milk peptides: Patent application CN202511001148.1 discloses camel milk polypeptides with xanthine oxidase inhibitory activity and their applications. Using fresh camel milk as raw material, fat is removed by low-temperature centrifugation. The enzymatic hydrolysate obtained after triple enzymatic hydrolysis with papain, pepsin, and trypsin is passed through an ultrafiltration membrane (molecular weight cutoff of 10 kDa), and the filtrate is freeze-dried to obtain the camel milk polypeptides, which exhibit xanthine oxidase inhibitory activity. The patent application also discloses four small molecule polypeptides with xanthine oxidase inhibitory activity, the sequences of which are shown in any one of SEQ ID NO. 1-4, and these can be used to prepare drugs with uric acid-inhibiting effects. Patent application CN202110687025.3 relates to an oral liquid of camel milk polypeptide nanoparticles with hypoglycemic effects and its preparation method. This composition contains camel milk polypeptides, an oil phase, emulsifiers, stabilizers, antioxidants, thickeners, sweeteners, preservatives, etc. The camel milk polypeptide nanoparticle oral liquid of this invention is transparent and uniform in color, colorless, odorless, has a mild and refreshing taste, slightly sweet aftertaste, uniform texture, no layering or sedimentation, and has a good effect on lowering blood sugar.

[0006] In summary, current advancements in the field of peptide drugs have penetrated to core levels such as molecular design, delivery technology, and manufacturing processes. The aforementioned literature represents specific technological breakthroughs achieved in addressing key issues, laying a solid foundation for subsequent transformation and application. Despite the rapid development in related fields, the following problems still urgently need to be addressed, considering the specific raw materials and objectives of this invention: 1. Low efficiency in the targeted preparation and purification of highly active peptides: Peptides prepared by traditional enzymatic hydrolysis or fermentation methods have complex compositions, resulting in low yields of target active peptides. Efficient separation and purification processes (such as the combined use of ion exchange and gel chromatography) need optimization. 2. Insufficient depth in mechanism research: Many studies only describe the activity phenomena, lacking sufficient elucidation of specific targets, signaling pathways (such as AMPK, NF-κB, etc.), and systemic mechanisms such as gut microbiota. 3. Insufficient high-value utilization of raw materials: Research on processing byproducts such as camel milk powder is lacking, and there is a lack of effective technical pathways to transform them into high-purity products with specific functions (such as hypoglycemic and immunomodulatory effects), resulting in resource waste.

[0007] Based on the above analysis, the necessity and innovation of this invention lie in the first systematic development of a method for preparing high-purity camel milk polypeptides with clear in vitro hypoglycemic activity using camel milk powder, a low-value by-product, as raw material through a specific process, thus achieving the dual goals of high-value utilization and precise preparation of active ingredients. Summary of the Invention

[0008] The purpose of this invention is to provide a use for camel milk polypeptide, which, according to testing, exhibits excellent cellular hypoglycemic and anti-inflammatory activities. The camel milk polypeptide is obtained by dissolving camel milk powder in deionized water, sterilizing it, inoculating it with four fermentation strains, centrifuging, collecting the supernatant, and then purifying the crude camel milk polypeptide through ion exchange column chromatography and gel column chromatography. The camel milk polypeptide obtained by the method of this invention has high purity and exhibits excellent cellular hypoglycemic activity. When the polypeptide concentration is >40 μg / mL, it can significantly reduce the glucose content in cells, decreasing it by 15.7% compared to the control group. MTT assays show that when the polypeptide concentration reaches 320 μg / mL, it significantly inhibits cell viability. This invention also has applications in the preparation of hypoglycemic drug raw materials and anti-inflammatory drugs.

[0009] The use of the camel milk polypeptide described in this invention, specifically its use in the preparation of pharmaceutical raw materials with hypoglycemic activity, is carried out according to the following steps: a. Take camel milk powder, add it to deionized water at a material-to-liquid ratio of 15:1-35:1, and sterilize at 85℃ for 5 hours. b. Inoculate the solution obtained in step a with the fermentation strain Lactobacillus casei L. casei and Lactobacillus fermentum. L. fermentum Thermophilic and acidophilic Bifidobacterium B. thermophilum or Lactobacillus paracasei L. paracasei At a temperature of 37℃, the bacterial density is 8×10⁻⁶. 7 Fermentation was carried out for 8 hours at a concentration of CFU / mL to obtain the fermentation broth; c. Centrifuge the fermentation broth obtained in step b at 4℃ and 7000 rpm for 10 minutes, collect the supernatant, use deionized water as the exchange medium, dialyze and desalt in a dialysis bag for 60 hours, collect the liquid in the dialysis bag, freeze dry to obtain the camel milk polypeptide product, and store at -20℃. d. The product obtained in step c is loaded onto a diethylaminoethyl 650M ion exchange column and eluted sequentially with phosphate buffer solution and 0.1-0.8 mol / L NaCl + phosphate buffer solution at a flow rate of 1.6 mL / min. The purified fraction with phosphate buffer solution as the eluent is collected and dialyzed with pure water in a 2-5 kDa dialysis bag for 2 days. The product is then freeze-dried to obtain crude camel milk polypeptide. e. The crude camel milk polypeptide obtained in step d is separated and purified using a G25 gel column, and then purified a second time with deionized water at a flow rate of 1.0 mL / min. The fraction is collected and freeze-dried to obtain the camel milk polypeptide.

[0010] The use of the camel milk polypeptide in the preparation of anti-inflammatory drugs. Attached Figure Description

[0011] Figure 1 The elution curve of the camel milk polypeptide of the present invention by diethylaminoethyl-650M ion exchange chromatography is shown. Figure 2 The elution curve of the camel milk polypeptide of the present invention is obtained by G-25 gel column chromatography. Figure 3 This is the HPLC chromatogram of the camel milk polypeptide CMP-0a of the present invention; Figure 4 This is a Fourier transform infrared absorption spectrum of the camel milk polypeptide of the present invention. Figure 5 This is a diagram showing the activity of α-amylase and α-glucosidase in camel milk polypeptides of the present invention; Figure 6 The effect of the camel milk polypeptide of this invention on glucose consumption in HEPG2 cells; Figure 7 This invention demonstrates the inhibitory effect of camel milk polypeptide on HEPG2 cells. Figure 8 This invention relates to the effect of camel milk polypeptide on NO release in RAW264.7 cells. Detailed Implementation Example 1

[0012] a. Take camel milk powder, add it to deionized water at a material-to-liquid ratio of 15:1, and sterilize it at 85℃ for 5 hours. b. Inoculate the solution obtained in step a with the fermentation strain Lactobacillus casei, and incubate at 37°C with a bacterial density of 8 × 10⁻⁶. 7 Fermentation was carried out for 8 hours at a concentration of CFU / mL to obtain the fermentation broth; c. Centrifuge the fermentation broth obtained in step b at 4℃ and 7000 rpm for 10 minutes, collect the supernatant, use deionized water as the exchange medium, dialyze and desalt in a dialysis bag for 60 hours, collect the liquid in the dialysis bag, freeze dry to obtain the camel milk polypeptide product, and store at -20℃. d. The product obtained in step c is loaded onto a diethylaminoethyl 650M ion exchange column and eluted sequentially with phosphate buffer solution and 0.1 mol / L NaCl + phosphate buffer solution at a flow rate of 1.6 mL / min. The purified fraction with phosphate buffer solution as the eluent is collected, dialyzed with pure water in a 2 kDa dialysis bag for 2 days, and then freeze-dried to obtain crude camel milk polypeptide. e. The crude camel milk polypeptide obtained in step d is separated and purified using a G25 gel column. A second purification is performed with deionized water at a flow rate of 1.0 mL / min. The fraction is collected, freeze-dried, and the camel milk polypeptide is obtained. Figure 1As shown, it was named CMP-0, with a yield of 12.9%.

[0013] Figure 1 The elution curve of diethylaminoethyl-650M ion exchange chromatography for a homogeneous polypeptide is shown. Figure 2 Elution curves of G-25 gel column chromatography for homogeneous peptides. Example 2

[0014] a. Take camel milk powder, add it to deionized water at a material-to-liquid ratio of 20:1, and sterilize it at 85℃ for 5 hours. b. Inoculate the solution obtained in step a with the fermentation strain Lactobacillus fermentum. L. fermentum At a temperature of 37℃, the bacterial density is 8×10⁻⁶. 7 Fermentation was carried out for 8 hours at a concentration of CFU / mL to obtain the fermentation broth; c. Centrifuge the fermentation broth obtained in step b at 4℃ and 7000 rpm for 10 minutes, collect the supernatant, use deionized water as the exchange medium, dialyze and desalt in a dialysis bag for 60 hours, collect the liquid in the dialysis bag, freeze dry to obtain the camel milk polypeptide product, and store at -20℃. d. The product obtained in step c is loaded onto a diethylaminoethyl 650M ion exchange column and eluted sequentially with phosphate buffer solution and 0.3 mol / L NaCl + phosphate buffer solution at a flow rate of 1.6 mL / min. The purified fraction with phosphate buffer solution as the eluent is collected, dialyzed with pure water in a 3 kDa dialysis bag for 2 days, and then freeze-dried to obtain crude camel milk polypeptide. e. The crude camel milk polypeptide obtained in step d is separated and purified using a G25 gel column. A second purification is performed with deionized water at a flow rate of 1.0 mL / min. The fraction is collected, freeze-dried, and the camel milk polypeptide is obtained. Figure 1 As shown, it was named CMP-1, with a yield of 5.8%. Example 3

[0015] a. Take camel milk powder, add it to deionized water at a material-to-liquid ratio of 30:1, and sterilize it at 85℃ for 5 hours. b. Inoculate the solution obtained in step a with the fermentation strain Bifidobacterium thermophilum and acidophilus. B. thermophilum At a temperature of 37℃, the bacterial density is 8×10⁻⁶. 7 Fermentation was carried out for 8 hours at a concentration of CFU / mL to obtain the fermentation broth; c. Centrifuge the fermentation broth obtained in step b at 4℃ and 7000 rpm for 10 minutes, collect the supernatant, use deionized water as the exchange medium, dialyze and desalt in a dialysis bag for 60 hours, collect the liquid in the dialysis bag, freeze dry to obtain the camel milk polypeptide product, and store at -20℃. d. The product obtained in step c is loaded onto a diethylaminoethyl 650M ion exchange column and eluted sequentially with phosphate buffer solution and 0.6 mol / L NaCl + phosphate buffer solution at a flow rate of 1.6 mL / min. The purified fraction with phosphate buffer solution as the eluent is collected, dialyzed with pure water in a 4 kDa dialysis bag for 2 days, and then freeze-dried to obtain crude camel milk polypeptide. e. The crude camel milk polypeptide obtained in step d is separated and purified using a G25 gel column. A second purification is performed with deionized water at a flow rate of 1.0 mL / min. The fraction is collected, freeze-dried, and the camel milk polypeptide is obtained. Figure 1 As shown, it was named CMP-2, with a yield of 8.4%. Example 4

[0016] a. Take camel milk powder, add it to deionized water at a material-to-liquid ratio of 35:1, and sterilize it at 85℃ for 5 hours. b. Inoculate the solution obtained in step a with the fermentation strain Lactobacillus paracasei. L. paracasei At a temperature of 37℃, the bacterial density is 8×10⁻⁶. 7 Fermentation was carried out for 8 hours at a concentration of CFU / mL to obtain the fermentation broth; c. Centrifuge the fermentation broth obtained in step b at 4℃ and 7000 rpm for 10 minutes, collect the supernatant, use deionized water as the exchange medium, dialyze and desalt in a dialysis bag for 60 hours, collect the liquid in the dialysis bag, freeze dry to obtain the camel milk polypeptide product, and store at -20℃. d. The product obtained in step c is loaded onto a diethylaminoethyl 650M ion exchange column and eluted sequentially with phosphate buffer solution and 0.8 mol / L NaCl + phosphate buffer solution at a flow rate of 1.6 mL / min. The purified fraction with phosphate buffer solution as the eluent is collected, dialyzed with pure water in a 5 kDa dialysis bag for 2 days, and then freeze-dried to obtain crude camel milk polypeptide. e. The crude camel milk polypeptide obtained in step d is separated and purified using a G25 gel column. A second purification is performed with deionized water at a flow rate of 1.0 mL / min. The fraction is collected, freeze-dried, and the camel milk polypeptide is obtained. Figure 1 As shown, it is named CMP-3. Example 5

[0017] The obtained camel milk peptides were analyzed by high-performance liquid chromatography. Liquid chromatography conditions: Column: ZORBAX SB-C18 (2.7 μm, 2.1 mm × 100 mm); Mobile phase A: ultrapure water (containing 0.1% trifluoroacetic acid), B: acetonitrile (containing 0.1% trifluoroacetic acid); Elution program: 0-10 min, with phase B increasing linearly from 5% to 40%; Column temperature: 35℃; Flow rate: 0.3 mL / min; Detection wavelength: 220 nm; Injection volume: 1 μL; Figure 3 As shown, under these chromatographic conditions, CMP-0a exhibits a sharp baseline separation peak at 12.31 min. Integration calculations using a chromatography workstation show that this peak area accounts for 99.2% of the total peak area, indicating that the obtained camel milk polypeptide is a high-purity single component. Example 6

[0018] The obtained camel milk polypeptide was detected by Fourier transform infrared absorption spectroscopy. The infrared absorption spectrum of dried camel milk polypeptides was determined by KBr pellet method, with a measurement range of 4000-400 cm⁻¹. -1 ; The result is as follows Figure 4 As shown: Infrared spectral analysis of the camel milk polypeptide sample revealed characteristic absorption peaks typical of polypeptides / proteins, providing crucial evidence for confirming its amide bond structure and secondary structure. The main characteristic peaks are assigned as follows: The broad, strong absorption peak near 3280 cm⁻¹ belongs to the amide A band, mainly originating from the NH stretching vibration and related to hydrogen bond formation. The weak peak near 3070 cm⁻¹ belongs to the amide B band. The most crucial evidence comes from the amide I and amide II bands: the former is mainly contributed by the C=O stretching vibration and is highly sensitive to the secondary structures of the polypeptide backbone (such as α-helices and β-sheets); the latter is mainly attributed to the coupling of NH bending vibration and CN stretching vibration. In addition, absorption peaks at 1400 cm⁻¹ and 1240 cm⁻¹ are usually observed, belonging to the COO-symmetric stretching vibration and the amide III band (related to CN stretching and NH bending), respectively; the latter is also often used to analyze secondary structures. The fingerprint region of 1200-900 cm⁻¹ contains specific vibrational information of various functional groups (such as CO, CC, etc.) in the amino acid side chains.

[0019] In summary, the characteristic appearance of amide I and II bands in the infrared spectrum directly confirms the presence of the characteristic amide bond (-CO-NH-) structure of the peptide in the sample, laying the material structure foundation for subsequent functional studies. Example 7

[0020] The obtained camel milk peptides were subjected to an α-amylase inhibition rate detection experiment: Take 50 μL of camel milk polypeptide sample solution (prepared with 0.1 M phosphate buffer, pH 6.8) and mix it with 100 μL of α-glucosidase solution (0.5 U / mL), and incubate at 37℃ for 10 minutes; add 50 μL of 5 mM p-nitrobenzene-α-D-glucopyranoside substrate solution, and continue the reaction at 37℃ for 30 minutes; stop the reaction by adding 2 mL of 0.1 M Na2CO3 solution; measure the absorbance at 405 nm and record it as . A sample ; Use buffer solution to replace the sample and measure the absorbance of the enzyme at full activity. A negative ; Buffer solution replaces enzyme solution to determine sample background. A background ; Buffer solution replaces sample and enzyme solution to determine substrate background. A blank ; Inhibition rate (%) = [1−( A negative - A blank ()( A sample - A background )]×100%; All experiments were independently repeated three times, and results are expressed as mean ± standard deviation. GraphPad Prism XX software was used for data processing and graphing. One-way ANOVA was used for inter-group comparisons, and Tukey's test was used for post-hoc multiple comparisons. P < 0.05 was considered statistically significant. Example 8

[0021] The obtained camel milk polypeptide was subjected to an α-glucosidase inhibition rate detection experiment: Take 200 μL of polypeptide solution and 200 μL of α-glucosidase solution (0.3 U / mL), mix well, and react at 37 ℃ for 20 min. Add 200 μL of p-nitrobenzene-α-D-glucopyranoside solution with a concentration of 0.5 mmol / L, mix well, and continue water bath for 30 min. Finally, add 5 mL of 0.1 mol / L Na2CO3 solution, mix well, and react at 37 ℃ for 20 min. Add 200 μL of p-nitrobenzene-α-D-glucopyranoside solution with a concentration of 0.5 mmol / L, mix well, and continue water bath for 30 min. Finally, add 5 mL of 0.1 mol / L Na2CO3 solution to terminate the reaction. Measure the absorbance at 405 nm. The control group does not add any solution, and the blank group uses purified water instead of α-glucosidase solution. Acarbose is used as a positive control. Calculate the inhibition rate according to formula (1): Inhibition rate (%) = A i / A 0 × 100%: In the formula: A 0 Enzyme activity in the blank control group; A i α-glucosidase activity in experimental groups with different concentrations of added samples. Example 9

[0022] The obtained camel milk peptides were used in a cellular glucose-lowering experiment. Cell culture: Heat the water bath to 37°C, thaw the HepG2 cells frozen in liquid nitrogen in the water bath, and slowly shake them in a cross-shaped motion to thaw them as quickly as possible. After complete thawing, transfer the cell suspension to centrifuge tubes in a clean bench and add culture medium. Centrifuge at 1000 rpm for 5 min, remove the supernatant, add culture medium containing 10% fetal bovine serum, mix well to form a cell suspension, and transfer to a culture flask. Incubate in a CO2 incubator at 5% CO2 and 37°C. When the cells have adhered to the wall and grown to 80-90%, remove the culture medium and slowly wash three times with phosphate-buffered saline. Add 0.25% trypsin for 1-2 min to digest the HepG2 cells, passage them, and continue culturing in a CO2 incubator to perform experiments on cells in the logarithmic growth phase. Effects of camel milk peptides on glucose consumption in HepG2 cells: HepG2 cells were used to investigate the effect of camel milk peptide on glucose consumption in normal cells. HepG2 cells were treated with camel milk peptide at concentrations of 20, 40, and 80 μg / mL for 48 h. Culture media from each treatment group were collected to determine glucose content. Culture media from cells cultured without the peptide for 48 h served as a control, and the supernatant was used for analysis. Following the glucose assay kit instructions, 96-well plates were selected, and working solution and samples were added, with three parallel wells for each treatment group. After mixing, the plates were incubated at 37℃ for 15 min. The absorbance and glucose content of each well were measured at 505 nm using a microplate reader, and the glucose concentration in the culture medium was calculated according to the instructions. The results showed that glucose concentration in the culture medium decreased at concentrations of 20 and 40 μg / mL, with a statistically significant decrease at 80 μg / mL (P<0.05), indicating that this concentration significantly promoted glucose consumption. The higher the concentration of added peptide, the more significant the decrease in glucose concentration, showing a dose-response relationship. MTT assay for HepG2 cell viability: Cell viability was determined using the MTT assay. Cells in the logarithmic growth phase were digested, centrifuged, and diluted with complete culture medium to a density of 5 × 10⁶ cells / mL.4 Cells / mL. Add 100 µL of phosphate-buffered saline to each well of the outermost ring of a 96-well plate, and seed the remaining wells with 100 µL of cells per well. Incubate for 24 h in a cell culture incubator. Aspirate the culture medium, wash each well twice with phosphate-buffered saline, and prepare samples of different concentrations (50, 100, 200, 400, 800 µg / mL) using complete culture medium. Add 100 µL to each well of the 96-well plate, with three replicates for each concentration, and include a blank control. After culturing in a cell culture incubator for 24 h, the cell culture medium containing the sample was aspirated, and the cells were rinsed twice with phosphate-buffered saline. 200 µL of LMTT working solution (prepared by using 5 mg / mL phosphate-buffered saline at pH 7.4, then diluted to 0.5 mg / mL with DMEM basal medium; the working solution should be prepared fresh before use) was added to each well and cultured in a cell culture incubator for 4 h. The MTT working solution was then aspirated, and 150 µL of DMSO was added. The plate was shaken for 10 min to allow complete color development, and the absorbance at 570 nm was measured. Cell viability was calculated using the formula.

[0023] Modeling was performed using sample concentrations that showed no toxicity to HepG2 cells. The results showed that high concentrations had an inhibitory effect, and the effect was significant, with a statistically significant difference compared to the blank control (P<0.05), indicating a certain degree of cytotoxicity. Example 10

[0024] The obtained camel milk peptides were subjected to cellular anti-inflammatory experiments. NO level measurement: Logarithmic growth phase cells were digested with trypsin and then subjected to a 1×10⁻⁶ solution. 4 Inoculate 96-well plates at a density of 1 / mL (200 µL / well) and incubate at 37 ℃ with 5% CO2 for 24 h. Set up a blank group (100 μL of culture medium only), a model group (50 μL of 1 μg / mL lipopolysaccharide and 50 μL of culture medium), a positive control group (50 μL of 1 μg / mL lipopolysaccharide and 50 μL of 1-100 μL of hydrocortisone), and a drug treatment group (50 μL of 1 μg / mL lipopolysaccharide and 50 μL of 1-100 μL of the compound). After incubation for another 24 h, collect the supernatant; add 50 µL of standard and sample at 50 µL / well, then add an equal volume of Griess Reagent I and II, and react at room temperature in the dark for 10 min. Measure the absorbance at 570 nm and calculate the NO inhibition rate. NO inhibition rate (%) = [(OD 模型组–OD 给药组 ) / (OD 模型组 –OD 空白组 )]×100%: MTT assay for RAW 264.7 cell viability: Cultured cells in the logarithmic growth phase were digested with 0.25% trypsin and then prepared into a single-cell suspension of 1×10⁴ cells / mL in DMEM medium containing 10% FBS. 200 μL of each suspension was seeded into 96-well plates. The concentrations of the monomeric compounds targeting RAW264.7 cells were 6.25, 12.5, 25.0, 50.0, and 100.0 μmol / L, respectively. After 24 h of cell incubation, 20 μL of MTT (5 mg / mL) was added to each well, and incubation continued for another 4 h. The supernatant was discarded, and 150 μL of dimethyl sulfoxide solution was added to each well to dissolve the formazan crystals within the cells. The absorbance of each well was measured at 570 nm using a microplate reader with low-speed shaking in the dark for 5 min, and the calculation formula was the same as above. To eliminate the interference of the compounds' cytotoxicity on the experimental results, the cell viability of the test samples was detected by the MTT assay. At a compound concentration of 50 μg / mL, the viability of RAW 264.7 cells was greater than 85%. An inflammation model induced by lipopolysaccharide (LPS) in RAW 264.7 cells was used for activity screening, and the results showed that most compounds possessed certain anti-inflammatory activities. Among them, compounds CMP-0 and CMP-1 showed more significant anti-inflammatory effects, with IC50 values ​​of 2.13±0.04 μg / mL and 5.582±0.07 μg / mL, respectively. Table 1 Anti-inflammatory activities of different camel milk peptides Example 11

[0025] After fermentation, camel milk was separated using DEAE-650M anion exchange resin and G-25 gel. The most active CMP-0a fraction was then separated by capillary high-performance liquid chromatography (HPLC) and analyzed by mass spectrometry using a Q Exactive HF-X mass spectrometer (Thermo Fisher) for 60 min. Detection mode: positive ion. The mass-charge ratio of peptides and peptide fragments was collected using the following method: 10 fragment spectra were acquired after each full scan (MS² scan).

[0026] The raw mass spectrometry test files were retrieved from the relevant database using MaxQuant 1.5.5.1 software, and finally the protein identification and quantification results were obtained. Table 2. LC-MS / MS Mass Spectrometry Identification Results

[0027] The sequences are rich in hydrophobic amino acids such as valine (V), leucine (L), proline (P), and phenylalanine (F) (e.g., LFPHASEVVKPQ, VMVPFLQPKVMD), which are usually associated with cell membrane affinity. These structural features suggest that the peptide may possess some cell-penetrating ability or be able to interact with biological membranes, which is crucial for its intracellular regulatory functions. Sequences such as PHASEVVKPQ contain fragments that may form specific secondary structures (such as α-helices), which are often the recognition and binding interfaces between proteins.

[0028] The use of a camel milk polypeptide described in this invention was investigated using camel milk industrial scavenging powder as the research object. Ion exchange resin and gel column were used as separation systems to ensure the accuracy of the research object. Finally, enzyme activity experiments and cell experiments revealed its use in the preparation of hypoglycemic drug raw materials and anti-inflammatory drugs.

[0029] The above embodiments are preferred embodiments of the invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The use of a camel milk polypeptide, characterized in that, The camel milk polypeptide is used in the preparation of pharmaceutical raw materials with hypoglycemic activity, and the specific operation is carried out according to the following steps: a. Take camel milk powder, add it to deionized water at a material-to-liquid ratio of 15:1-35:1, and sterilize at 85℃ for 5 hours. b. Inoculate the solution obtained in step a with the fermentation strain Lactobacillus casei L. casei and Lactobacillus fermentum. L. fermentum Thermophilic and acidophilic Bifidobacterium B. thermophilum or Lactobacillus paracasei L. paracasei At a temperature of 37℃, the bacterial density is 8×10⁻⁶. 7 Fermentation was carried out for 8 hours at a concentration of CFU / mL to obtain the fermentation broth; c. Centrifuge the fermentation broth obtained in step b at 4℃ and 7000 rpm for 10 minutes, collect the supernatant, use deionized water as the exchange medium, dialyze and desalt in a dialysis bag for 60 hours, collect the liquid in the dialysis bag, freeze dry to obtain the camel milk polypeptide product, and store at -20℃. d. The product obtained in step c is loaded onto a diethylaminoethyl 650M ion exchange column and eluted sequentially with phosphate buffer solution and 0.1-0.8 mol / L sodium chloride + phosphate buffer solution at a flow rate of 1.6 mL / min. The purified fraction with phosphate buffer solution as the eluent is collected, dialyzed with pure water in a 2-5 kDa dialysis bag for 2 days, and then freeze-dried to obtain crude camel milk polypeptide. e. The crude camel milk polypeptide obtained in step d is separated and purified using a G25 gel column, and then purified a second time with deionized water at a flow rate of 1.0 mL / min. The fraction is collected and freeze-dried to obtain the camel milk polypeptide.

2. The use as described in claim 1, characterized in that... The use of the camel milk polypeptide in the preparation of anti-inflammatory drugs.

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