Endothelium corneum gigeriae galli oligopeptide and application thereof in improving weakness of

Abstract

The invention discloses endothelium corneum gigeriae galli oligopeptide and application thereof in improving weakness of the spleen and the stomach, and belongs to the technical field of active peptides, and the amino acid sequence of the endothelium corneum gigeriae galli oligopeptide is one or more of Glu-Asp-Leu-Glu-Ser-Glu-Lys, Leu-Glu-Glu-Glu-Thr-Arg and Lys-Glu-Glu-Leu-Glu-Arg. On one hand, the endothelium corneum gigeriae galli peptide can promote proliferation and migration of gastric mucosa cells and reduce inflammatory response of the cells, so that the effect of repairing gastric mucosa is achieved; on the other hand, by adjusting secretion of motilin, the exercise ability of the stomachs of the rats with weakness of the spleen and the stomach is improved, the gastric emptying rate is increased, food retention is prevented, the stability of the environment in the gastrointestinal tract is maintained, and recovery of spleen and stomach functions is promoted. The endothelium corneum gigeriae galli small peptide prepared by the invention can be used for developing functional foods or biological medicines for improving spleen and stomach functions and digestion and absorption, and has a good application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of active peptides, and particularly relates to an application of chicken gizzard peptide in improving spleen and stomach weakness. Background Art

[0002] Spleen and stomach deficiency can manifest in a variety of negative gastrointestinal symptoms, including slowed gastrointestinal motility, decreased digestive juice secretion, and reduced digestive capacity, leading to loss of appetite. Food cannot be digested and emptied promptly, accumulating in the stomach or intestines, causing abdominal distension and discomfort. The gastrointestinal mucosa is relatively sensitive, with reduced tolerance to stimuli such as food, temperature, and emotions. Even minor dietary inappropriateness (such as overeating, eating too fast, or consuming spicy, greasy, or cold foods), abdominal cold, or emotional fluctuations can trigger gastrointestinal spasms. Long-term spleen and stomach deficiency slows gastric epithelial cell turnover, reducing mucus and bicarbonate secretion, making it easier for gastric acid to break through the barrier and damage the gastric mucosa, increasing the risk of gastric ulcers. This affects normal gastric acid secretion, potentially leading to indigestion due to insufficient secretion or mucosal damage due to excessive secretion. Furthermore, it is closely linked to long-term chronic gastritis and immune dysfunction in the gastric mucosa.

[0003] Endothelial gizzard of chicken is the dried gizzard inner wall of the domestic chicken (Gallus gallus domesticus Brisson), a pheasant family animal. It is sweet and flat in nature, and enters the spleen, stomach, small intestine, and bladder meridians. It has abundant sources and was listed in the catalog of medicine and food homology in 2002. It has the effects of invigorating the stomach and promoting digestion, astringing spermatorrhea to stop nocturnal emission, and relieving stranguria and dispelling phlegm. It often appears in the traditional Chinese medicine prescriptions for the treatment of gastrointestinal diseases. Endothelial gizzard of chicken contains more than 70% protein, and its ability to increase gastric juice secretion and gastrointestinal digestion is speculated to be related to the protein component. However, the application of Endothelial gizzard of chicken mostly adopts traditional Chinese medicine preparation method, and the research report that the protein component therein is enzymolyzed to obtain functional Endothelial gizzard peptide is relatively rare. Therefore, the development of active small peptide products for Endothelial gizzard protein can expand its application in improving spleen and stomach function and dyspepsia. Summary of the Invention

[0004] The present invention aims to provide an application of chicken gizzard lining oligopeptide in the preparation of a functional food or medicine for improving spleen and stomach deficiency. The chicken gizzard lining oligopeptide of the present invention promotes gastric mucosal cell proliferation and migration, reduces inflammatory response, and simultaneously increases the secretion of motilin and gastrin to improve gastrointestinal motility, thereby ultimately achieving the effect of improving spleen and stomach function.

[0005] In a first aspect, according to the chicken glucoside peptide in some embodiments of the present application, the amino acid sequence is shown as SEQ ID NO.1.

[0006] In a second aspect, the method for preparing the chicken glucoside peptide according to the first aspect of some embodiments of the present application comprises: S1. Take chicken gizzard powder and add it into pure water to obtain a solution.

[0007] S2. Add compound protease to the solution for enzymatic hydrolysis, and inactivate the enzyme after enzymatic hydrolysis.

[0008] S3. Take the supernatant, separate and purify it using Sephadex LH-20 column chromatography, and collect the chromatographic peak with a retention time of 6 to 9 minutes.

[0009] S4. Further purification was performed using a C18 RP-HPLC reverse phase column, and the chromatographic peak with a retention time of 5 to 6 minutes was collected to obtain chicken gizzard peptide.

[0010] In step S2, the mass ratio of the composite protease is alkaline protease: trypsin: neutral protease = 1:2:1, wherein the enzymatic hydrolysis temperature is 50° C., the enzymatic hydrolysis pH is 8.5, and the enzymatic hydrolysis time is 4 h.

[0011] In step S3, the mobile phase was 30% methanol by volume, the flow rate was 0.5 mL / min, and the absorbance of the eluent was measured at 280 nm.

[0012] The chromatographic conditions in step S4 are as follows: mobile phase A is water containing 0.1% trifluoroacetic acid by volume, mobile phase B is acetonitrile, and the gradient elution conditions are: 0-10 min, 5% B, 10-20 min, 5%-15% B, 20-30 min, 15% B-30% B, 30-40 min, 30% B-40% B, and the flow rate is 0.8 mL / min.

[0013] According to the preparation method of chicken gizzard peptide described in the first aspect in some embodiments of the present application, the particle size of the chicken gizzard powder in step S1 is 10-50 μm.

[0014] According to the preparation method of chicken gizzard peptide described in the first aspect in some embodiments of the present application, the washed and dried chicken gizzard in step S1 is crushed by a grinder to obtain chicken gizzard powder.

[0015] According to the preparation method of chicken gizzard peptide described in the first aspect in some embodiments of the present application, in step S1, chicken gizzard powder is added to 10 to 20 times the volume of pure water to obtain a solution.

[0016] According to the preparation method of chicken gizzard peptide described in the first aspect in some embodiments of the present application, in step S1, chicken gizzard powder is added to 10 to 20 times the volume of pure water and homogenized to obtain a solution.

[0017] According to the preparation method of chicken glucoside peptide described in the first aspect in some embodiments of the present application, the homogenization speed in step S1 is 10000 r / min and the homogenization time is 2 min.

[0018] According to the preparation method of chicken gizzard peptide described in the first aspect in some embodiments of the present application, the mass of the composite protease in step S2 is 1% to 3% of the mass of chicken gizzard powder.

[0019] According to the preparation method of chicken gizzard peptide described in the first aspect in some embodiments of the present application, after enzymatic hydrolysis in step S2, the enzyme is inactivated, the mixture is centrifuged, and the supernatant is collected.

[0020] According to the preparation method of chicken gizzard peptide described in the first aspect in some embodiments of the present application, after enzymatic hydrolysis in step S2, the enzyme is inactivated for 15 minutes, the mixture is centrifuged at 8000 rpm for 10 minutes, and the supernatant is collected.

[0021] On the third side, according to the chicken glucoside peptide in some embodiments of the present application, the amino acid sequence is shown as SEQ ID NO.2.

[0022] In a fourth aspect, according to the method for preparing chicken glucoside peptide according to the third aspect of some embodiments of the present application, the method comprises: S1. Take chicken gizzard powder and add it into pure water to obtain a solution.

[0023] S2. Add compound protease to the solution for enzymatic hydrolysis, and inactivate the enzyme after enzymatic hydrolysis.

[0024] S3. Take the supernatant, separate and purify it using Sephadex LH-20 column chromatography, and collect the chromatographic peak with a retention time of 6 to 9 minutes.

[0025] S4. Further purification was performed using a C18 RP-HPLC reverse phase column, and the chromatographic peak with a retention time of 8 to 9 minutes was collected to obtain chicken gizzard peptide.

[0026] In step S2, the mass ratio of the composite protease is alkaline protease: trypsin: neutral protease = 1:2:1, wherein the enzymatic hydrolysis temperature is 50° C., the enzymatic hydrolysis pH is 8.5, and the enzymatic hydrolysis time is 4 h.

[0027] In step S3, the mobile phase was 30% methanol by volume, the flow rate was 0.5 mL / min, and the absorbance of the eluent was measured at 280 nm.

[0028] The chromatographic conditions in step S4 are as follows: mobile phase A is water containing 0.1% trifluoroacetic acid by volume, mobile phase B is acetonitrile, and the gradient elution conditions are: 0-10 min, 5% B, 10-20 min, 5%-15% B, 20-30 min, 15% B-30% B, 30-40 min, 30% B-40% B, and the flow rate is 0.8 mL / min.

[0029] According to the preparation method of chicken gizzard peptide described in the third aspect in some embodiments of the present application, the particle size of the chicken gizzard powder in step S1 is 10-50 μm.

[0030] According to the preparation method of chicken gizzard peptide described in the third aspect in some embodiments of the present application, the washed and dried chicken gizzard in step S1 is crushed by a grinder to obtain chicken gizzard powder.

[0031] According to the preparation method of chicken gizzard peptide described in the third aspect in some embodiments of the present application, in step S1, chicken gizzard powder is added to 10 to 20 times the volume of pure water to obtain a solution.

[0032] According to the preparation method of chicken gizzard peptide described in the third aspect in some embodiments of the present application, in step S1, chicken gizzard powder is added to 10 to 20 times the volume of pure water and homogenized to obtain a solution.

[0033] According to the preparation method of chicken glucoside peptide described in the third aspect in some embodiments of the present application, the homogenization speed in step S1 is 10000 r / min and the homogenization time is 2 min.

[0034] According to the preparation method of chicken gizzard peptide described in the third aspect in some embodiments of the present application, the mass of the composite protease in step S2 is 1% to 3% of the mass of chicken gizzard powder.

[0035] According to the preparation method of chicken gizzard peptide described in the third aspect in some embodiments of the present application, after enzymatic hydrolysis in step S2, the enzyme is inactivated, the mixture is centrifuged, and the supernatant is collected.

[0036] According to the preparation method of chicken gizzard peptide described in the third aspect in some embodiments of the present application, after enzymatic hydrolysis in step S2, the enzyme is inactivated for 15 minutes, the mixture is centrifuged at 8000 rpm for 10 minutes, and the supernatant is collected.

[0037] In the fifth aspect, according to the chicken glucoside peptide in some embodiments of the present application, the amino acid sequence is shown as SEQ ID NO.3.

[0038] In a sixth aspect, according to the method for preparing the chicken glucoside peptide according to the fifth aspect of some embodiments of the present application, the method comprises: S1. Take chicken gizzard powder and add it into pure water to obtain a solution.

[0039] S2. Add compound protease to the solution for enzymatic hydrolysis, and inactivate the enzyme after enzymatic hydrolysis.

[0040] S3. Take the supernatant, separate and purify it using Sephadex LH-20 column chromatography, and collect the chromatographic peak with a retention time of 6 to 9 minutes.

[0041] S4. Further purification was performed using a C18 RP-HPLC reverse phase column, and the chromatographic peak with a retention time of 16 to 17 minutes was collected to obtain chicken gizzard peptide.

[0042] In step S2, the mass ratio of the composite protease is alkaline protease: trypsin: neutral protease = 1:2:1, wherein the enzymatic hydrolysis temperature is 50° C., the enzymatic hydrolysis pH is 8.5, and the enzymatic hydrolysis time is 4 h.

[0043] In step S3, the mobile phase was 30% methanol by volume, the flow rate was 0.5 mL / min, and the absorbance of the eluent was measured at 280 nm.

[0044] The chromatographic conditions in step S4 are as follows: mobile phase A is water containing 0.1% trifluoroacetic acid by volume, mobile phase B is acetonitrile, and the gradient elution conditions are: 0-10 min, 5% B, 10-20 min, 5%-15% B, 20-30 min, 15% B-30% B, 30-40 min, 30% B-40% B, and the flow rate is 0.8 mL / min.

[0045] According to the preparation method of chicken gizzard peptide described in the fifth aspect in some embodiments of the present application, the particle size of the chicken gizzard powder in step S1 is 10-50 μm.

[0046] According to the preparation method of chicken gizzard peptide described in the fifth aspect in some embodiments of the present application, the washed and dried chicken gizzard in step S1 is crushed by a grinder to obtain chicken gizzard powder.

[0047] According to the preparation method of chicken gizzard peptide described in the fifth aspect in some embodiments of the present application, in step S1, chicken gizzard powder is added to 10 to 20 times the volume of pure water to obtain a solution.

[0048] According to the preparation method of chicken gizzard peptide described in the fifth aspect in some embodiments of the present application, in step S1, chicken gizzard powder is added to 10 to 20 times the volume of pure water and homogenized to obtain a solution.

[0049] According to the preparation method of chicken glucoside peptide described in the fifth aspect in some embodiments of the present application, the homogenization speed in step S1 is 10000 r / min and the homogenization time is 2 min.

[0050] According to the preparation method of chicken gizzard peptide described in the fifth aspect in some embodiments of the present application, the mass of the composite protease in step S2 is 1% to 3% of the mass of chicken gizzard powder.

[0051] According to the preparation method of chicken gizzard peptide described in the fifth aspect in some embodiments of the present application, after enzymatic hydrolysis in step S2, the enzyme is inactivated, the mixture is centrifuged, and the supernatant is collected.

[0052] According to the preparation method of chicken gizzard peptide described in the fifth aspect in some embodiments of the present application, after enzymatic hydrolysis in step S2, the enzyme is inactivated for 15 minutes, the mixture is centrifuged at 8000 rpm for 10 minutes, and the supernatant is collected.

[0053] In a seventh aspect, the chicken gizzard peptide described in any one of the first to sixth aspects is used in the preparation of a drug for improving spleen and stomach deficiency, a drug for repairing gastric mucosa, or a drug for regulating motilin, or in the preparation of food or functional food.

[0054] The present invention has the beneficial effects of: on the one hand, the chicken's gizzard lining oligopeptide of the present invention can promote the proliferation and migration of gastric mucosal cells and reduce cellular inflammatory reactions, thereby achieving the effect of repairing the gastric mucosa. On the other hand, it can improve the motility of the stomach of rats with spleen and stomach deficiency by regulating the secretion of motilin, increase the gastric emptying rate, prevent food accumulation, maintain the stability of the gastrointestinal environment, and promote the recovery of spleen and stomach function. The chicken's gizzard lining oligopeptide prepared by the present invention can be used as a functional food or biopharmaceutical to improve spleen and stomach function and digestion and absorption, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 . Changes in the proliferation rate of GES-1 cells.

[0056] Figure 2 . Changes in GES-1 cell migration rate.

[0057] Figure 3 . Food intake, gastric emptying rate and serum MLT content in rats.

[0058] Figure 4 . TNF-α and IL-6 levels in rat serum. DETAILED DESCRIPTION

[0059] The present invention will be further described below with reference to the accompanying drawings and embodiments. It should be noted that the embodiments do not limit the scope of protection claimed in the present invention.

[0060] The chicken's gizzard lining oligopeptide of the present invention is selected from any one of the first chicken's gizzard lining oligopeptide, the second chicken's gizzard lining oligopeptide, and the third chicken's gizzard lining oligopeptide.

[0061] The first chicken gizzard oligopeptide SEQ ID NO: 1: Glu-Asp-Leu-Glu-Ser-Glu-Lys.

[0062] The second Gallus gallus endometrium oligopeptide SEQ ID NO: 2: Leu-Glu-Glu-Glu-Glu-Thr-Arg.

[0063] The third chicken gizzard oligopeptide SEQ ID NO: 3: Lys-Glu-Glu-Leu-Glu-Arg.

[0064] The amino acid sequence of the first chicken gizzard lining oligopeptide of the present invention is shown in SEQ ID NO: 1; The amino acid sequence of the second chicken gizzard oligopeptide of the present invention is shown in SEQ ID NO: 2; The amino acid sequence of the third chicken gizzard lining oligopeptide of the present invention is shown in SEQ ID NO: 3.

[0065] The present invention also provides a method for preparing the chicken's gizzard lining oligopeptide, comprising the following steps: S1. Grind the washed and dried chicken gizzard lining into a particle size of 10-50 μm using a grinder; S2. Take an appropriate amount of chicken gizzard powder, add 10-20 times the volume of pure water, and homogenize at 10,000 rpm for 1-2 minutes; S3, add 1-3% of the weight of chicken gizzard powder composite protease for enzymolysis, the composite protease includes alkaline protease: trypsin: neutral protease = 1:2:1, 50 ℃ pH 8.5 enzymolysis 4h, after enzymolysis enzyme inactivation 15min, 8000rpm centrifugation 10min, take the supernatant; S4. Separation and purification were performed using Sephadex LH-20 column chromatography with a mobile phase of 30% methanol at a flow rate of 0.5 mL / min. The absorbance of the eluent was measured at 280 nm, and the chromatographic peak with a retention time of 6 to 9 minutes was collected. S5. Further purification was carried out using a C18 RP-HPLC reverse phase column. The chromatographic conditions were as follows: mobile phase A was water containing 0.1% trifluoroacetic acid by volume, mobile phase B was acetonitrile, and the gradient elution conditions were: 0-10 min, 5% B, 10-20 min, 5%-15% B, 20-30 min, 15% B-30% B, 30-40 min, 30% B-40% B, with a flow rate of 0.8 mL / min. The chromatographic peaks with retention times of 5-6, 8-9, and 16-17 min were collected to obtain three chicken gizzard oligopeptide components C1, C2, and C3, respectively.

[0066] S6. Amino acid sequence determination was performed using high performance liquid chromatography-tandem mass spectrometry, and the amino acid sequence was determined to be: Glu-Asp-Leu-Glu-Ser-Glu-Lys, Leu-Glu-Glu-Glu-Thr-Arg, Lys-Glu-Glu-Leu-Glu-Arg.

[0067] The supernatant in step S3 is concentrated and freeze-dried to obtain chicken gizzard oligopeptide powder A1.

[0068] Furthermore, in the above step S4, the supernatant is separated and purified. In one embodiment, the chicken gizzard oligopeptide powder A1 can be dissolved in water to prepare a solution with a concentration of 1-30 mg / mL (preferably 10 mg / mL), which is then separated and purified by Sephadex LH-20 column chromatography.

[0069] The chromatographic peak with a retention time of 6 to 9 minutes collected in step S4 was freeze-dried to obtain the small molecule active peptide B1.

[0070] Furthermore, in the above step S5, the chromatographic peak with a retention time of 6 to 9 min collected in step S4 is further purified using a C18 RP-HPLC reverse phase column. In one solution, the above small molecule active peptide B1 can also be dissolved in water to prepare a solution with a concentration of 1 to 30 mg / mL (preferably 10 mg / mL) for further purification using a C18 RP-HPLC reverse phase column.

[0071] Example 1: Preparation of Gallus Gallus Gallus Ternifolia Oligopeptides C1, C2, and C3, the preparation method comprising: S1. Take the washed and dried chicken's gizzard lining and grind it into a particle size of 50 μm using a grinder; S2. Take 100g of chicken gizzard powder, add 20 times the volume of pure water, and homogenize at 10000r / min for 2 minutes; S3, weigh alkaline protease 0.75g, trypsin 1.5g, neutral protease 0.75g add into chicken's gizzard lining material liquid and carry out enzymolysis, 50 DEG C of pH8.5 enzymolysis 4h, enzyme inactivation 15min after enzymolysis, 8000rpm centrifugation 10min, take supernatant; S4. Separation and purification were performed using Sephadex LH-20 column chromatography with a mobile phase of 30% methanol at a flow rate of 0.5 mL / min. The absorbance of the eluent was measured at 280 nm, and the chromatographic peak with a retention time of 6 to 9 minutes was collected. S6. Further purification was carried out using a C18 RP-HPLC reverse phase column. The chromatographic conditions were as follows: mobile phase A was water containing 0.1% trifluoroacetic acid by volume, mobile phase B was acetonitrile, and the gradient elution conditions were: 0-10 min, 5% B, 10-20 min, 5%-15% B, 20-30 min, 15% B-30% B, 30-40 min, 30% B-40% B, with a flow rate of 0.8 mL / min. The chromatographic peaks with retention times of 5-6, 8-9, and 16-17 min were collected to obtain three chicken gizzard oligopeptide components C1, C2, and C3, respectively.

[0072] S7. The amino acid sequences of the three oligopeptide components from Gallus gallus domesticus obtained were determined by high performance liquid chromatography-tandem mass spectrometry and Denovo de novo sequencing. The amino acid sequences were determined to be: C1: Glu-Asp-Leu-Glu-Ser-Glu-Lys (Score: 96); C2: Leu-Glu-Glu-Glu-Thr-Arg (Score: 91); C3: Lys-Glu-Glu-Leu-Glu-Arg (Score: 98); The peptide scores indicate that the above sequence determination has a high degree of credibility.

[0073] Comparative Example 1: Preparation of Chicken Gizzard Oligopeptide Powder A1, the preparation method comprises: S1. Take the washed and dried chicken's gizzard lining and grind it into a particle size of 50 μm using a grinder; S2. Take 100g of chicken gizzard powder, add 20 times the volume of pure water, and homogenize at 10000r / min for 2 minutes; S3, weigh alkaline protease 0.75g, trypsin 1.5g, neutral protease 0.75g add into chicken's gizzard lining material liquid and carry out enzymolysis, 50 DEG C of pH8.5 enzymolysis 4h, enzyme inactivation 15min after enzymolysis, 8000rpm centrifugation 10min, take supernatant; S4. The supernatant is concentrated and freeze-dried to obtain chicken gizzard oligopeptide powder A1.

[0074] Comparative Example 2: Preparation of small molecule active peptide B1 from Gallus gallus endometrium, the preparation method comprising: S1. Take the washed and dried chicken's gizzard lining and grind it into a particle size of 50 μm using a grinder; S2. Take 100g of chicken gizzard powder, add 20 times the volume of pure water, and homogenize at 10000r / min for 2 minutes; S3, weigh alkaline protease 0.75g, trypsin 1.5g, neutral protease 0.75g add into chicken's gizzard lining material liquid and carry out enzymolysis, 50 DEG C of pH8.5 enzymolysis 4h, enzyme inactivation 15min after enzymolysis, 8000rpm centrifugation 10min, take supernatant; S4. Sephadex LH-20 column chromatography was used for separation and purification. The mobile phase was 30% methanol by volume, the flow rate was 0.5 mL / min, the absorbance of the eluent was measured at 280 nm, the chromatographic peaks with a retention time of 6 to 9 minutes were collected, and the small molecule active peptide B1 was obtained by freeze-drying.

[0075] Experimental Example 1: Effects of Chicken Gizzard Oligopeptides on the Proliferation and Migration of Human Gastric Mucosal Cells GES-1 The experiment set up control group, A1 group, B1 group, C1 group, C2 group and C3 group. Chicken gluconate peptide in each group was dissolved in 0.01 mol / L PBS buffer (pH 7.4) into 50 mg / L dilution solution.

[0076] Each group of GES-1 cells was seeded into a 96-well plate. After the cells adhered to the wall, the culture medium was discarded and fresh culture medium and 20 μL of each sample dilution were added. The cells were incubated in an incubator for 48 hours. Fresh culture medium and CCK-8 reagent were added at 0, 24, and 48 hours, and the cells were incubated in an incubator for another 30 minutes. The absorbance of each well was measured and recorded at 450 nm on a microplate reader, and the results were used to draw a proliferation curve. The results are shown in Figure 2. Figure 1 As shown, there was no significant difference in cell proliferation rate between the A1 component and the control group (P < 0.05); the B1, C1, C2, and C3 components showed a significant effect of promoting cell proliferation compared with the control group (P < 0.05), among which the promoting effect of the C1, C2, and C3 components was higher than that of the B1 component (P < 0.05).

[0077] GES-1 cells were diluted to a cell suspension concentration of 5 x 105 cells / mL and seeded into a 6-well plate. When the cells filled the 6-well plate, a scratch was made with a 100 μL pipette tip. 80 μL of DMEM culture medium and 20 μL of sample diluent were added and the plate was incubated in an incubator. Images were acquired under a microscope at 0, 24, and 48 hours. The healing area was measured and calculated, and a statistical bar graph was plotted. The results are shown in Figure 2. Figure 2 As shown, the cell migration rates of groups C1, C2, and C3 were significantly higher than those of groups A1 and B1, showing a higher healing-promoting effect.

[0078] Experimental Example 2: Interventional Effect of Chicken Gizzard Oligopeptide on Rats with Functional Dyspepsia of Spleen and Stomach Deficiency Type A comprehensive modeling method (iodoacetamide gavage, hunger and satiety disturbances, and strenuous swimming) was used to establish a spleen-stomach deficiency-type FD rat model. Rats were gavaged daily with 0.2 mL of a 2% sucrose solution containing 0.1% iodoacetamide. A control group received the same dose of 2% sucrose solution. The rats were forced to swim to exhaustion daily and fed every other day. All rats received normal drinking water during the modeling period. The modeling continued for four weeks. After four weeks, two rats were randomly selected weekly. Body weight, food intake, gastric emptying rate, and the presence of inflammatory cell infiltration as determined by gastric mucosal pathological observation were used as modeling criteria. Thirty-five healthy male Sprague-Dawley rats were divided into control, model, A1, B1, C1, C2, and C3 groups (5 rats per group). All experimental groups, except the control and model groups, were given the drug at a dose of 2 g / kg / day for two consecutive weeks.

[0079] Two weeks later, rats were fasted for 24 hours and gavage administered with 2 mL of a semisolid nutrient paste containing charcoal powder (10 g sodium carboxymethylcellulose, 16 g milk powder, 8 g starch, 8 g sugar, and 2 g activated charcoal powder, dissolved in 250 mL of distilled water and mixed to make 300 mL, approximately 300 g of black semisolid paste). 0.5 hour later, rats were anesthetized with an intraperitoneal injection of 3% sodium pentobarbital at 5 mL / kg. The entire stomach was removed and weighed, then opened along the greater curvature and thoroughly rinsed with 0.9% NaCl. The net stomach mass was then weighed and the gastric emptying rate was calculated as [1 - (total stomach mass - net stomach mass) / administered paste mass] x 100%. Blood was collected from the abdominal aorta and centrifuged to obtain serum. Serum levels of motilin (MLT) and the inflammatory factor TNF-α were determined using enzyme-linked immunosorbent assay (ELISA) kits according to the kit instructions.

[0080] Effects of chicken gizzard oligopeptides on promoting gastric emptying in model rats Figure 3 As shown, compared with the control group, the model group rats had significantly reduced food intake and a significantly decreased gastric emptying rate (P < 0.05). Compared with the model group, the rats in each experimental group had a significantly increased food intake and a significantly improved gastric emptying rate (P < 0.05), with the C1, C2, and C3 experimental groups showing the most significant effect. Furthermore, the C1, C2, and C3 experimental groups also had the most significant effect on increasing the level of motilin (MLT) in the serum of the model rats (P < 0.05). At the same dose, the three chicken gizzard lining oligopeptides provided by the present invention have a superior effect on improving gastric motility and increasing appetite than the crude enzymatic hydrolysate of chicken gizzard lining.

[0081] Effects of chicken gizzard oligopeptides on serum inflammatory factors TNF-α and IL-6 in model rats Figure 4 As shown in the results, the levels of inflammatory factors in the serum of the model group rats were significantly increased compared with the control group (P < 0.05), indicating that long-term spleen and stomach weakness caused a certain inflammatory response; the chicken gizzard lining crude enzymatic hydrolysate A1 group had no effect on improving the inflammatory level (P > 0.05); the C1, C2, and C3 components showed a better effect than the B1 component in alleviating the inflammatory response of the model rats (P < 0.05).

[0082] Combined with the effects of the above embodiments, it can be seen that the chicken gizzard oligopeptide provided by the present invention has the effects of improving gastric motility, relieving gastric inflammation, promoting gastric mucosal repair and improving spleen and stomach function, and has good application prospects in the fields of functional foods, biomedicine and the like.

[0083] The invention discloses an application of chicken gizzard peptide in improving spleen and stomach deficiency, belonging to the field of active peptide technology. The chicken gizzard peptide is one or more of the short peptides Glu-Asp-Leu-Glu-Ser-Glu-Lys, Leu-Glu-Glu-Glu-Thr-Arg, and Lys-Glu-Glu-Leu-Glu-Arg isolated and extracted from chicken gizzard. The chicken gizzard peptide of the invention can, on the one hand, promote the proliferation and migration of gastric mucosal cells and reduce cellular inflammatory reactions, thereby achieving the effect of repairing the gastric mucosa; on the other hand, it can improve the motility of the stomach of rats with spleen and stomach deficiency by regulating the secretion of motilin, increase the gastric emptying rate, prevent food accumulation, maintain the stability of the gastrointestinal environment, and promote the recovery of spleen and stomach function. The chicken gizzard peptide prepared by the invention can be used to develop functional foods or biopharmaceuticals that improve spleen and stomach function and digestion and absorption, and has good application prospects.

[0084] Finally, it should be noted that the above examples are only some specific embodiments of the present invention, and all derivatives that can be directly derived or associated with the content disclosed by a person skilled in the art should be considered to be within the scope of protection of the present invention.

Claims

1. A chicken gizzard peptide, characterized in that: The amino acid sequence is shown in SEQ ID NO.

1.

2. The method for preparing the chicken glucoside peptide according to claim 1, wherein include S1. Take chicken gizzard powder and add it to pure water to obtain a solution; S2. adding a composite protease to the solution for enzymatic hydrolysis, and then inactivating the enzyme; S3. The supernatant was separated and purified by Sephadex LH-20 column chromatography, and the chromatographic peak with a retention time of 6 to 9 min was collected; S4. Further purification was performed using a C18 RP-HPLC reverse phase column, and the peak with a retention time of 5 to 6 min was collected to obtain chicken gizzard peptide; In step S2, the mass ratio of the composite protease is alkaline protease: trypsin: neutral protease = 1:2:1, wherein the enzymatic hydrolysis temperature is 50° C., the enzymatic hydrolysis pH is 8.5, and the enzymatic hydrolysis time is 4 h; In step S3, the mobile phase is 30% methanol by volume, the flow rate is 0.5 mL / min, and the absorbance of the eluent is measured at 280 nm; The chromatographic conditions in step S4 are as follows: mobile phase A is water containing 0.1% trifluoroacetic acid by volume, mobile phase B is acetonitrile, and the gradient elution conditions are: 0-10 min, 5% B, 10-20 min, 5%-15% B, 20-30 min, 15% B-30% B, 30-40 min, 30% B-40% B, and the flow rate is 0.8 mL / min.

3. The preparation method according to claim 2, characterized in that Preferably, the particle size of the chicken's gizzard lining powder in step S1 is 10 to 50 μm; Preferably, the washed and dried chicken's gizzard lining in step S1 is crushed with a grinder to obtain chicken's gizzard lining powder; Preferably, in step S1, the chicken's gizzard powder is added to 10 to 20 times the volume of pure water to obtain a solution; More preferably, in step S1, the chicken's gizzard powder is added to 10 to 20 times the volume of pure water and homogenized to obtain a solution; Preferably, the homogenization speed in step S1 is 10000 r / min and the homogenization time is 2 min; Preferably, the mass of the composite protease in step S2 is 1% to 3% of the mass of the chicken gizzard powder; Preferably, in step S2, after enzymatic hydrolysis, the enzyme is inactivated, the mixture is centrifuged, and the supernatant is collected; More preferably, after enzymatic hydrolysis in step S2, the enzyme is inactivated for 15 minutes, the mixture is centrifuged at 8000 rpm for 10 minutes, and the supernatant is collected.

4. A chicken gizzard peptide, characterized in that: The amino acid sequence is shown in SEQ ID NO.

2.

5. The method for preparing the chicken gizzard peptide according to claim 4, wherein: include S1. Take chicken gizzard powder and add it to pure water to obtain a solution; S2. adding a composite protease to the solution for enzymatic hydrolysis, and then inactivating the enzyme; S3. The supernatant was separated and purified by Sephadex LH-20 column chromatography, and the chromatographic peak with a retention time of 6 to 9 min was collected; S4. Further purification was performed using a C18 RP-HPLC reverse phase column, and the peak with a retention time of 8 to 9 min was collected to obtain chicken gizzard peptide; In step S2, the mass ratio of the composite protease is alkaline protease: trypsin: neutral protease = 1:2:1, wherein the enzymatic hydrolysis temperature is 50° C., the enzymatic hydrolysis pH is 8.5, and the enzymatic hydrolysis time is 4 h; In step S3, the mobile phase is 30% methanol by volume, the flow rate is 0.5 mL / min, and the absorbance of the eluent is measured at 280 nm; The chromatographic conditions in step S4 are as follows: mobile phase A is water containing 0.1% trifluoroacetic acid by volume, mobile phase B is acetonitrile, and the gradient elution conditions are: 0-10 min, 5% B, 10-20 min, 5%-15% B, 20-30 min, 15% B-30% B, 30-40 min, 30% B-40% B, and the flow rate is 0.8 mL / min.

6. The preparation method according to claim 5, characterized in that Preferably, the particle size of the chicken's gizzard lining powder in step S1 is 10 to 50 μm; Preferably, the washed and dried chicken's gizzard lining in step S1 is crushed with a grinder to obtain chicken's gizzard lining powder; Preferably, in step S1, the chicken's gizzard powder is added to 10 to 20 times the volume of pure water to obtain a solution; More preferably, in step S1, the chicken's gizzard powder is added to 10 to 20 times the volume of pure water and homogenized to obtain a solution; Preferably, the homogenization speed in step S1 is 10000 r / min and the homogenization time is 2 min; Preferably, the mass of the composite protease in step S2 is 1% to 3% of the mass of the chicken gizzard powder; Preferably, in step S2, after enzymatic hydrolysis, the enzyme is inactivated, the mixture is centrifuged, and the supernatant is collected; More preferably, after enzymatic hydrolysis in step S2, the enzyme is inactivated for 15 minutes, the mixture is centrifuged at 8000 rpm for 10 minutes, and the supernatant is collected.

7. A chicken gizzard peptide, characterized in that: The amino acid sequence is shown in SEQ ID NO.

3.

8. The method for preparing the chicken gizzard peptide according to claim 7, characterized in that: include S1. Take chicken gizzard powder and add it to pure water to obtain a solution; S2. adding a composite protease to the solution for enzymatic hydrolysis, and then inactivating the enzyme; S3. The supernatant was separated and purified by Sephadex LH-20 column chromatography, and the chromatographic peak with a retention time of 6 to 9 min was collected; S4. Further purification was performed using a C18 RP-HPLC reverse phase column, and the peak with a retention time of 16 to 17 min was collected to obtain chicken gizzard peptide; In step S2, the mass ratio of the composite protease is alkaline protease: trypsin: neutral protease = 1:2:1, wherein the enzymatic hydrolysis temperature is 50° C., the enzymatic hydrolysis pH is 8.5, and the enzymatic hydrolysis time is 4 h; In step S3, the mobile phase is 30% methanol by volume, the flow rate is 0.5 mL / min, and the absorbance of the eluent is measured at 280 nm; The chromatographic conditions in step S4 are as follows: mobile phase A is water containing 0.1% trifluoroacetic acid by volume, mobile phase B is acetonitrile, and the gradient elution conditions are: 0-10 min, 5% B, 10-20 min, 5%-15% B, 20-30 min, 15% B-30% B, 30-40 min, 30% B-40% B, and the flow rate is 0.8 mL / min.

9. The preparation method according to claim 8, characterized in that Preferably, the particle size of the chicken's gizzard lining powder in step S1 is 10 to 50 μm; Preferably, the washed and dried chicken's gizzard lining in step S1 is crushed with a grinder to obtain chicken's gizzard lining powder; Preferably, in step S1, the chicken's gizzard powder is added to 10 to 20 times the volume of pure water to obtain a solution; More preferably, in step S1, the chicken's gizzard powder is added to 10 to 20 times the volume of pure water and homogenized to obtain a solution; Preferably, the homogenization speed in step S1 is 10000 r / min and the homogenization time is 2 min; Preferably, the mass of the composite protease in step S2 is 1% to 3% of the mass of the chicken gizzard powder; Preferably, in step S2, after enzymatic hydrolysis, the enzyme is inactivated, the mixture is centrifuged, and the supernatant is collected; More preferably, after enzymatic hydrolysis in step S2, the enzyme is inactivated for 15 minutes, the mixture is centrifuged at 8000 rpm for 10 minutes, and the supernatant is collected.

10. Use of the chicken gizzard peptide according to any one of claims 1, 4 and 7 in the preparation of a medicament for improving spleen and stomach deficiency, or a medicament for repairing gastric mucosa, or a medicament for regulating motilin; or in the preparation of a food or a functional food.

Images