Hemp seed active peptide with immunological enhancement effect as well as preparation method and application of hemp seed active peptide

Abstract

The invention provides a hemp seed active peptide with an immunological enhancement effect as well as a preparation method and application thereof, and belongs to the technical field of preparation of bioactive peptides. The amino acid sequence of the hemp seed protein source immunological enhancement active peptide provided by the invention is DGDGSGFF. The China-hemp seed protein source immunological enhancement active peptide provided by the invention is separated from China-hemp seed protein for the first time, is a novel China-hemp seed protein source immunological enhancement active peptide, has good immunological enhancement activity, can be used as a natural immunopotentiator in food, health care products and immunological enhancement drugs, and has good application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of bioactive peptide preparation, and specifically relates to a hemp seed active peptide with immune enhancement effect and a preparation method and application thereof. Background Art

[0002] At present, the number of people with low immunity or even immunosuppression due to factors such as unhealthy diet, stress, disease, drugs and lack of exercise is increasing year by year. When the immune system is impaired, the body cannot eliminate invading pathogens in time, which will increase the risk of disease. Although some synthetic drugs are effective in improving the activity of the human immune system, their potential toxicity to human health is worrying.

[0003] Compared with drugs, food-derived immunomodulatory peptides not only have the functions of promoting immunity, preventing infection and cancer, but also have the advantages of being easily absorbed, having low immunogenicity and being non-toxic.

[0004] Hemp seeds are an important source of nutrients, fiber and bioactive compounds, rich in protein (20%-25%), oil (more than 30%) and a large amount of vitamins and minerals. In addition, its protein is mainly composed of hemp kernel globulin and albumin, and is well known for its digestibility and high-quality essential amino acid composition. However, the low water solubility of hemp protein limits its application in the food industry. Therefore, if hemp polypeptide functional foods with immune-enhancing activity are developed through enzymatic hydrolysis, it will be of great significance to the high-value utilization of hemp kernel protein and the prevention and improvement of immunosuppression by dietary hemp polypeptides. Summary of the invention

[0005] In order to achieve high-value utilization of hemp kernel protein and prevent and improve immunosuppression, the present invention provides a hemp kernel active peptide with immune enhancement effect and a preparation method and application thereof.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A hemp seed active peptide with immune enhancement effect, the amino acid sequence of which is DGDGSGFF.

[0008] The method for preparing the hemp seed active peptide having the effect of enhancing immunity comprises the following steps:

[0009] Hemp kernel protein powder is obtained by supercritical extraction combined with three-phase separation, and then the hemp kernel protein powder is enzymatically hydrolyzed by a composite protease to obtain an enzymatic solution; components with a molecular weight less than 1KDa in the enzymatic solution are collected, and separated by polysaccharide gel G-15 column chromatography, the immune enhancing activity of each peak collected liquid is determined, the components with the highest activity are collected, and LC-MS / MS identification is performed, and hemp kernel active peptides are obtained by chemical synthesis.

[0010] In the present invention, the parameters of three-phase separation are: the volume concentration of butanol solution is 50%, the volume concentration of ammonium sulfate solution is 60%, the mass ratio of ammonium sulfate solution to butanol solution is 1:3, and the supercritical extraction parameters are: extraction kettle pressure 30Mpa, temperature: 45°C, separation kettle I: pressure 10Mpa, temperature 60°C, separation kettle II: pressure 4.5Mpa, temperature 60°C, pump frequency: 28Hz; flow rate: 700L / H. The enzymatic hydrolysis step is: adding water to the hemp kernel protein powder to form a suspension with a liquid-to-solid ratio of 16:1mL / g, using flavor protease and papain to perform composite enzymatic hydrolysis on the hemp kernel protein powder, the amount of the flavor protease and papain is based on the weight of protein, preferably adding 5 mg of flavor protease and 5 mg of papain per gram of protein, the enzymatic hydrolysis temperature is preferably 55°C, the enzymatic hydrolysis time is preferably 300min, and the pH is preferably 6.3.

[0011] In the present invention, the dextran gel G-15 column chromatography, the eluent is ultrapure water; the column type is: (diameter) 1.6×100 cm; detection wavelength is 280 nm; sample loading is 5 mL, flow rate is 1.2 mL / min, elution volume is 375 mL, and the volume of peak fraction collected is 7.5 mL / tube.

[0012] An immunopotentiator comprises the hemp seed active peptide having immunopotentiating effect.

[0013] An immune enhancement product comprises the hemp seed active peptide having immune enhancement effect.

[0014] An immune enhancement product comprises the immune enhancer.

[0015] An application of the hemp seed active peptide with immune enhancement effect, and an application of the hemp seed active peptide with immune enhancement effect in the preparation of immune enhancement active products. The products include food, medicine or health care products.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention separates and identifies for the first time a hemp seed active peptide with an amino acid sequence of DGDGSGFF from hemp seed protein hydrolysate. The peptide provided by the present invention has the effect of enhancing immune activity and can be used to prepare foods and medicines with immune activity enhancing function, providing new ideas for the prevention and treatment of patients with immunosuppressive diseases and people with low immune function. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 A flow chart of the test of the present invention;

[0019] Figure 2 is the effect of F1 on cell morphology;

[0020] Figure 3 The effect of F1 on the phagocytic index of neutral red in cells, n = 3;

[0021] Figure 4 is the effect of F1 on IL-1β secretion, n = 9;

[0022] Figure 5 is the effect of F1 on IL-6 secretion, n = 9;

[0023] Figure 6 is the effect of F1 on TNF-α secretion, n = 9;

[0024] Figure 7 The effect of F1 on the changes of RAW264.7 cell cycle;

[0025] Figure 8 This is the molecular docking diagram of hemp seed active peptide DGDGSGFF with TLR2 and TLR4;

[0026] Fig. 9 is the number of fluorescent microspheres remaining in zebrafish after peptide DGDGSGFF treatment, n = 10;

[0027] Fig.10 is the fluorescence intensity of zebrafish T cells after peptide DGDGSGFF treatment, n = 10;

[0028] Fig.11 This is a typical graph of the number of neutrophils in the tail vein of zebrafish after treatment with peptide DGDGSGFF;

[0029] Fig.12 This is a typical graph of the fluorescence intensity of zebrafish tail vein macrophages after peptide DGDGSGFF treatment;

[0030] Fig.13 The relative expression levels of IFN-γ, Rag1, Rag2, IL-12, and TNF-α genes induced by peptide DGDGSGFF, n=3. DETAILED DESCRIPTION

[0031] The technical solution of the present invention will be clearly and completely described below in conjunction with the drawings and embodiments. Obviously, the described embodiments are only part of the embodiments of the invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0032] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0033] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0034] Example 1

[0035] A hemp seed active peptide with immune-enhancing activity, the specific preparation method is as follows:

[0036] 1. Preparation of hemp seed protein hydrolysate

[0037] Hemp seeds are taken, and hemp kernel protein isolate is obtained after being treated by supercritical extraction and three-phase separation technology. Water is added to prepare a suspension with a liquid-to-solid ratio of 16:1 (mL / g), and then enzymolysis is performed with 0.5% of flavor protease and 0.5% of papain, and the enzymolysis conditions are as follows: enzymolysis temperature of 55°C, enzymolysis time of 300min, enzymolysis pH of 6.3, after the enzymolysis is completed, the enzyme is heated at 95°C for 10min to inactivate the enzyme, the enzymolyzate is centrifuged at 8000r / min for 20min and the precipitate is discarded, and the obtained supernatant is the hemp kernel protein hydrolyzate.

[0038] 2. Ultrafiltration classification of hemp seed protein hydrolysate

[0039] Ultrafiltration was performed using ultrafiltration membranes with molecular weight cutoffs of 3KDa and 1KDa. The 3KDa ultrafiltration membrane was connected to the ultrafiltration system, and the enzymatic hydrolyzate was passed through the ultrafiltration system, and the components with molecular weights less than 3KDa were passed through the ultrafiltration membrane and collected. The 1KDa ultrafiltration membrane was connected to the ultrafiltration system, and the components with molecular weights less than 3KDa were passed through the ultrafiltration system, and the components with molecular weights less than 1KDa were passed through the ultrafiltration membrane and collected. The three collected components greater than 3KDa, between 1KDa and 3KDa, and less than 1KDa were subjected to RAW264.7 cell proliferation activity assay:

[0040] RAW264.7 cells were seeded in 96-well culture plates at a cell density of approximately 10 4 cfu / mL, 200μL per well, 5% CO2, incubate at 37℃ for 24h, add hemp polypeptides at concentrations of 100, 250 and 1000μg / mL respectively, and set up a blank control group at the same time, with 3 replicates in each group. After 24h of treatment, add 10μL of CCK-8 solution to each well, incubate in the cell culture incubator for 1h, and measure the OD value at 450nm with an enzyme marker. A0 is the OD value of the blank control group, and A1 is the OD value of the hemp polypeptide treatment group. The RAW264.7 cell proliferation viability of the sample is calculated according to the following formula:

[0041]

[0042] The results showed that the RAW264.7 cell proliferation activity of the fraction with a molecular weight less than 1KDa obtained by ultrafiltration fractionation was the highest.

[0043] 3. Sephadex G-15 column chromatography separation

[0044] The fraction with the highest RAW264.7 cell proliferation activity (molecular weight less than 1KDa) obtained by ultrafiltration fractionation was lyophilized and then passed through a 0.22μm microporous filter membrane. The protein concentration was determined by Bradford method, and the ultrapure water was fixed to 100mg / mL. The AKTAgo protein purifier was separated by a Sephadex G-15 (16×1000mm) column, with a sample volume of 5mL and an ultrapure water elution of 1.2mL / min. Each tube was 7.5mL, and a total of 50 tubes were collected. The RAW264.7 cell proliferation activity of each tube collection fluid was determined (the specific determination method was the same as step 2), and the fraction F1 with the highest RAW264.7 cell proliferation activity was collected and lyophilized for further analysis.

[0045] The results of RAW264.7 cell proliferation activity showed that compared with the blank control group, the relative proliferation rates of 100, 250, and 1000 μg / mL F1 on RAW264.7 cells were 12.72%, 33.80%, and 68.82%, respectively.

[0046] Effect of F1 on RAW264.7 cell morphology: RAW264.7 cells were inoculated in 6-well plates and cultured at 37°C and 5% CO2 for 12 hours. Then, 100, 250 and 1000 μg / mL of F1 and 1 μg / mL of LPS were added as positive controls. A blank control group was also set up, with 3 replicates in each group. After 36 hours of treatment, the cells were observed and photographed under an inverted microscope.

[0047] like Figure 2 As shown in the figure, the effect of F1 on the morphology of RAW264.7 cells showed that the number of cells in the blank control group and the 100μg / mL F1 group was small and the morphology was round. After treatment with 250 and 1000μg / mL F1 and LPS, the number of RAW264.7 cells increased significantly, and with the increase in the concentration of F1, the proportion of cells stimulated to differentiate and form pseudopodia also increased significantly.

[0048] Determination of the effect of F1 on the phagocytic ability of RAW264.7 cells: RAW264.7 cells were inoculated in 96-well plates, with a cell concentration of about 104 cfu / mL, 200 μL per well, and incubated in an incubator for 12 hours. F1 at concentrations of 100, 250, and 1000 μg / mL was added, respectively. A positive control group and a blank control group were set up at the same time, with 3 replicates in each group. After 36 hours of treatment, the A540 of the samples was determined according to the instructions of the Neutrophil Proliferation and Cytotoxicity Detection Kit.

[0049] like Figure 3As shown in the figure, the effect of F1 on the phagocytic ability of RAW264.7 cells showed that compared with the blank control group, F1 enhanced the phagocytic ability of RAW264.7 macrophages to neutral red in a dose-dependent manner. After RAW264.7 cells were treated with 100, 250 and 1000 μg / mL of F1, the cell phagocytic index increased by 39%, 76% and 133%, respectively (p<0.01).

[0050] Effect of F1 on NO, IL-1β, IL-6 and TNF-α secretion of RAW264.7 cells: RAW264.7 cells were inoculated in 96-well culture plates, with a cell concentration of about 104 cfu / mL, 200 μL per well, 5% CO2, 37°C incubation for 24 h, and F1 at concentrations of 100, 250 and 1000 μg / mL was added, respectively, and a blank control group was set up at the same time, with 9 replicates in each group. After 36 h of treatment, the NO level in the supernatant and the secretion of cytokines IL-1β, IL-6 and TNF-α were determined according to the respective kit methods.

[0051] like Figure 4-6 As shown in the figure, the effect of F1 on the secretion of NO, IL-1β, IL-6 and TNF-α of RAW264.7 cells showed that the addition of 100, 250 and 1000 μg / mL of F1 increased the release level of NO (p<0.01) and the secretion of L-1β and IL-6 (p<0.05) of macrophages RAW264.7 in a dose-dependent manner, but was weaker than the LPS group. The secretion of TNF-α in the LPS group was significantly higher than that in the blank control group and the 100 and 250 μg / mL F1 groups. When the concentration of F1 was 1000 μg / mL, the secretion of TNF-α in the culture supernatant reached 1226.75 pg / mL, even exceeding the 1169.27 pg / mL of the LPS group (p<0.05).

[0052] Determination of the effect of F1 on the cell cycle of RAW264.7: RAW264.7 cells were inoculated in T25 culture flasks and incubated for 12 hours. Hemp active peptides at concentrations of 100, 250 and 1000 μg / mL were added, and positive control group and blank control group were set up at the same time, with 3 parallels in each group. After 36 hours of treatment, the cells were washed twice with PBS and centrifuged at 1000 rpm for 3 minutes to precipitate the cells. About 1 mL of 70% ethanol precooled in an ice bath was added, gently blown evenly, and fixed at 4°C for 2 hours; centrifuged at 1000 rpm for 3 minutes, and the cells were resuspended in 1 mL of precooled PBS, and the supernatant was discarded after centrifugation. 10 μL RNaseA (50×) solution and 500 μL propidium iodide stain (20×) were added to each tube of cell sample, incubated at 37°C in the dark for 30 minutes, and detected by flow cytometry.

[0053] like Figure 7As shown in the figure, the effect of F1 on the cell cycle of RAW264.7 cells showed that the percentage of G0 / G1 phase cells in the total number of cells increased after LPS and F1 were applied to RAW264.7 cells for 36 hours. Compared with the control group, the proportion of cells in the G0 / G1 phase increased by 24.39%, 42.3% and 56.76% after treatment with 100, 250 and 1000 μg / mL concentrations of F1, respectively (p<0.01). At the same time, it was found that the proportion of cells in the S phase and G2 / M phase in the total number of cells decreased, but the difference was not statistically significant (p>0.05). See Table 1.

[0054] Table 1

[0055]

[0056] 4. LC-MS / MS mass spectrometry sequencing

[0057] The components obtained in step 3 were subjected to mass spectrometry sequencing (commissioned to Shanghai Zhongke New Life Co., Ltd.), and after database retrieval, 9 amino acid sequences with relative abundance > 1% were obtained, namely DGDGSGFF, MPEDVIAN, SYNNGDQQ, DGDQPLVT, WSYNNG, SYNNGDQQL, ADWVYN, NNGDSPLVL, and DWVYNNG, a total of 9 dominant peptides of hemp seed polypeptides.

[0058] 5. Molecular docking

[0059] The PDB structures of pattern recognition receptors TLR2 (ID: 1FYW) and TLR4 (ID: 5IJD) were obtained from the RCSB website, and the water molecules and ligands of the receptor proteins were removed using PyMOL2.6.0 software. The energy of small molecule peptides was minimized using the MM2 force field of Chem3D 20.0 software, and docking was performed using AutoDockVina software.

[0060] like Figure 8 As shown in Table 2, the molecular docking results showed that among the nine candidate peptides, the peptide DGDGSGFF had the strongest binding affinity to TLR2 and TLR4, with binding energies of -10.166 and -10.569 kcal / moL, respectively.

[0061] Table 2

[0062]

[0063] Example 2

[0064] 1. Chemical synthesis

[0065] After the hemp seed active peptide DGDGSGFF was chemically synthesized (commissioned to Nanjing Jiepeptide Biotechnology Co., Ltd. for synthesis), its immune enhancement activity was determined.

[0066] 2. Verification of in vivo activity in zebrafish

[0067] Detection of the effect of DGDGSGFF on the phagocytic function of immunosuppressive zebrafish macrophages: According to the random grouping method, wild-type AB strain zebrafish with normal development 3 days after fertilization were divided into normal control group, model control group (vinorelbine injection), positive control group (125μg / mL Ganoderma lucidum spore powder) and hemp seed active peptide DGDGSGFF low (62.5μg / mL), medium (125μg / mL), and high (250μg / mL) experimental groups, with 30 fish in each group. In a 6-well plate, 10 zebrafish were randomly selected from each group and photographed under a fluorescence microscope to analyze the number of remaining fluorescent microspheres in the zebrafish body and evaluate the efficacy of hemp seed active peptide in promoting macrophage phagocytosis.

[0068] like Fig. 9 As shown in the figure, the effect of DGDGSGFF on the phagocytic function of immunosuppressed zebrafish macrophages showed that compared with the normal control group, the number of fluorescent microspheres remaining in the zebrafish in the model control group was significantly reduced (p<0.01), indicating that the macrophage phagocytic function reduction model was successfully established. Compared with the model control group, 62.5 and 125μg / mL of hemp seed active peptides increased the phagocytic effect of macrophages by 21.8% and 46.2%, respectively, but the difference was not significant (p>0.05), but the promotion effect of 250μg / mL of hemp seed active peptides reached 37.1% (p<0.05).

[0069] Detection of the effect of DGDGSGFF on the number of immune cells in immunosuppressive zebrafish: Transgenic T cell red fluorescent zebrafish, neutrophil green fluorescent zebrafish, and transgenic macrophage green fluorescent zebrafish were placed in 6-well plates and treated at 28°C for 48 hours. Ten zebrafish were randomly selected from each experimental group and photographed under a fluorescence microscope. The fluorescence intensity of each cell in the zebrafish cloaca to the tail vein was counted, and the T cells, neutrophils, and macrophages in the xenotransplanted zebrafish were quantified.

[0070] like Figure 10-12As shown in the figure, the effect of DGDGSGFF on the number of immune cells in immunosuppressive zebrafish showed that the fluorescence intensity of T cells in the model control group decreased from 21214.4 in the normal control group to 10570.2 in the model control group, a decrease of 50.2% (p<0.01). The fluorescence intensity was significantly increased after treatment with 125 and 250μg / mL hemp seed immune peptide, by 56.5% and 50.85% respectively (p<0.05); vinorelbine caused a decrease in neutrophils in the tail of zebrafish, and the difference was statistically significant compared with the normal control group (p<0.01), indicating that vinorelbine successfully caused certain immune damage to zebrafish. In the positive control Ganoderma lucidum spore powder group, the number of neutrophils increased by 43.8% (p<0.01). In the mass concentration range of 62.5-250μg / mL of hemp seed active peptide, the number of neutrophils gradually increased with the increase of the administration concentration, showing a dose-dependent manner. Compared with the model control group, the neutrophil growth rate of 250μg / mL hemp seed active peptides has reached 46.8% (p<0.01), returning to normal levels; after treatment with vinorelbine, the fluorescence intensity of zebrafish tail vein macrophages was significantly reduced (p<0.01), significantly inhibiting the formation of macrophages in the xenotransplanted zebrafish model. Similar to the results of T cells and neutrophils, the number of macrophages in the body was significantly improved after the action of 125 and 250μg / mL hemp active peptides and Ganoderma lucidum spore powder, increasing by 21.6%, 26.1% and 18.5% respectively (p<0.01).

[0071] Effect of DGDGSGFF on the changes in the content of IFN-γ, IL-12, TNF-α, Rag1 and Rag2 in immunosuppressive zebrafish: At the end of the experiment, extract the total RNA of zebrafish. Take 2 μg of total RNA of zebrafish samples (A260 / A280=1.8-2.2) and synthesize 20 μL cDNA according to the instructions of the cDNA first-strand synthesis kit. β-actin was used as an internal reference, and the relative expression levels of β-actin, IFN-γ, Rag1, Rag2, IL-12 and TNF-α genes in zebrafish were detected by qRT-PCR.

[0072] like Fig.13As shown in the figure, the effect of DGDGSGFF on the changes in the contents of IFN-γ, IL-12, TNF-α, Rag1 and Rag2 in immunosuppressed zebrafish showed that the relative expression of zebrafish IFN-γ, Rag1, Rag2, IL-12 and TNF-α genes in the model group was significantly reduced compared with the blank control group (p<0.05). After the administration of 62.5 μg / mL of hemp active peptide, the expression of IFN-γ, Rag1, Rag2, IL-12 and TNF-α genes was well improved, increasing to 3.70 times, 8.00 times, 9.89 times, 2.40 times and 3.58 times that of the control group, respectively, with significant differences ((p<0.05 or p<0.01).

[0073] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A hemp seed active peptide with immune enhancement effect, characterized in that: The amino acid sequence of the hemp seed polypeptide with immune enhancement effect is DGDGSGFF.

2. A method for preparing hemp seed active peptide with immune enhancement effect, characterized in that: The following steps are involved: Step 1: Processing hemp kernels by supercritical extraction and three-phase separation technology to obtain hemp kernel protein powder, and then hydrolyzing the hemp kernel protein powder by composite protease to obtain enzymolysis solution; Step 2: Collect the components with molecular weight less than 1KDa in the enzymatic hydrolyzate, perform polysaccharide gel G-15 column chromatography to separate them, determine the immunoenhancing activity of each peak collection solution, collect the components with the highest activity, perform LC-MS / MS identification, and obtain hemp seed active peptides by chemical synthesis.

3. The preparation method according to claim 2, characterized in that: The supercritical extraction technical parameters are: the pressure of the extraction kettle is 30Mpa, and the temperature is 45°C; the pressure of the separation kettle I is 10Mpa, and the temperature is 60°C; the pressure of the separation kettle II is 4.5Mpa, and the temperature is 60°C; the pump frequency is 28Hz; the flow rate is 700L / H; the parameters of the three-phase separation technology are: the volume concentration of the butanol solution is 50%, the volume concentration of the ammonium sulfate solution is 60%, and the mass ratio of the sulfate solution to the butanol solution is 1:

3.

4. The preparation method according to claim 2, characterized in that: The composite protease comprises flavor protease and papain, and the dosage of the flavor protease and papain is calculated by protein weight, and 5 mg of flavor protease and 5 mg of papain are added per gram of protein; the enzymolysis temperature is 55°C, the enzymolysis time is 300 minutes, and the pH is 6.3; after the enzymolysis is completed, the enzyme is heated at 95°C for 10 minutes to kill the enzyme, the enzymolysis product is centrifuged, the precipitate is discarded, and the obtained supernatant is the enzymolysis solution.

5. The preparation method according to claim 2, characterized in that: The parameters of the polysaccharide gel G-15 column chromatography separation are: the eluent is ultrapure water; the column shape is: diameter φ=1.6×100 cm; the detection wavelength is 280 nm; the sample load is 5 mL, the flow rate is 1.2 mL / min, the elution volume is 375 mL, and the volume of the peak component collection is 7.5 mL / tube.

6. An immunopotentiator, characterized in that: The immunopotentiator comprises the hemp seed active peptide with immunopotentiating effect as claimed in claim 1.

7. An immune enhancement product, characterized in that: The immune enhancement product comprises the hemp seed active peptide with immune enhancement effect as claimed in claim 1.

8. An immune enhancement product, characterized in that: The immunoenhancing product comprises the immunoenhancing agent according to claim 6.

9. An application of the hemp seed active peptide with immune enhancement effect as claimed in claim 1, characterized in that: The application of the hemp seed active peptide with immune enhancement effect in the preparation of immune enhancement active products.

10. The use according to claim 9, characterized in that: The products include food, medicine or health products.

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