Black-bone chicken liver hydrolyzed peptide capable of improving ulcerative colitis as well as preparation method and application of black-bone chicken liver hydrolyzed peptide

Abstract

The invention discloses black-bone chicken liver hydrolyzed peptide capable of improving ulcerative colitis as well as a preparation method and application of the black-bone chicken liver hydrolyzed peptide, and belongs to the technical field of biological medicines. The preparation method of the black-bone chicken liver hydrolyzed peptide comprises the following steps: preparing the black-bone chicken liver into meat paste, mixing the meat paste with water to obtain a liquid material, heating the liquid material, adding alkaline protease to carry out enzymolysis, carrying out enzyme deactivation, centrifugally collecting supernate, and freeze-drying to obtain the black-bone chicken liver hydrolyzed peptide. According to the invention, a formaldehyde titration method is adopted to detect the degree of hydrolysis, SDS-PAGE electrophoresis and Tricine-SDS-PAGE electrophoresis are adopted to detect the molecular weight, LC-MS / MS is adopted to carry out sequence analysis, a database is adopted to predict the biological function of a peptide fragment, an in vitro experiment is adopted to detect the antioxidant and antihypertensive ability of the black-bone chicken liver peptide, and a mouse experiment is adopted to detect the colitis improvement effect of the black-bone chicken liver peptide. The result shows that the black-bone chicken liver peptide prepared by the invention has the advantages of resisting oxidation, reducing blood pressure and improving ulcerative colitis.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to a black chicken liver hydrolyzed peptide capable of improving ulcerative colitis, and a preparation method and application thereof. Background Art

[0002] Ulcerative colitis (UC) is a recurrent inflammatory bowel disease. As one of the main types of inflammatory bowel disease, UC is mainly characterized by weight loss, abdominal pain, diarrhea, and blood in the stool. Conventional UC intervention drugs include corticosteroids, 5-aminosalicylic acid, immunosuppressants, and antibiotics, but their effects are limited and accompanied by a series of side effects. The toxicity caused by long-term use will also limit their efficacy. Therefore, it is crucial to explore safe and effective ways to prevent or improve UC.

[0003] Chicken liver is an edible byproduct of chicken slaughter, rich in protein, calcium, phosphorus, iron, zinc, vitamin A, B vitamins, etc. However, fresh chicken liver is cheap and has a special smell, so it is rarely eaten directly. Most of it is used as animal feed or even directly discarded. Only a small amount of it is processed into braised liver and chicken liver paste, resulting in a large waste of high-quality protein food resources. Therefore, the high-value utilization of chicken liver resources is of great practical significance.

[0004] China's patent publication CN118685483A discloses a method for preparing a black chicken peptide with blood-tonifying function. The black chicken peptide product prepared by the invention has the advantages of high oligopeptide content, light bitterness, astringency and saltiness, good antioxidant activity, high purity and good blood-tonifying effect. The current research on black chicken peptides mainly focuses on anti-oxidation, immune regulation and blood-tonifying, and there is a lack of relevant preparation methods for black chicken liver peptides and their application in improving colitis. Summary of the invention

[0005] The purpose of the present invention is to provide a hydrolyzed black chicken liver peptide that can be used to improve ulcerative colitis, and a preparation method and application thereof, so as to solve the problems existing in the above-mentioned prior art. The hydrolyzed black chicken liver peptide prepared by the present invention has the advantages of anti-oxidation, lowering blood pressure and improving ulcerative colitis.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] Technical solution 1: A method for preparing hydrolyzed peptides from black-bone chicken liver, comprising the step of enzymatically hydrolyzing the black-bone chicken liver using alkaline protease.

[0008] The black-bone chicken comes from the Luoyeyang black-bone chicken in Shaanxi.

[0009] Furthermore, the preparation method comprises the following steps: making minced meat of black chicken liver and mixing it with water, heating the liquid material, adding alkaline protease for enzymolysis, inactivating the enzyme and collecting the supernatant by centrifugation, and freeze-drying to obtain the black chicken liver hydrolyzed peptide.

[0010] The heating temperature is 55°C.

[0011] Furthermore, the black-bone chicken liver is made into minced meat and mixed with water, and the minced meat is adjusted to a mass concentration of 30% by adding water.

[0012] Furthermore, the addition amount of the alkaline protease is 1% of the mass of the minced meat in the liquid material.

[0013] Furthermore, the enzymolysis is carried out at 55° C. for 1 hour.

[0014] Furthermore, the enzyme inactivation is carried out at 90° C. for 15 minutes.

[0015] Furthermore, the centrifugation is carried out at 8000 r / min at 4°C for 10 min.

[0016] Technical solution 2: The black chicken liver hydrolyzed peptide prepared by the preparation method.

[0017] Technical solution three: Use of the black-bone chicken liver hydrolyzed peptide in the preparation of products with antioxidant activity and blood pressure lowering effects.

[0018] Technical solution 4: Application of the black-bone chicken liver hydrolyzed peptide in the preparation of a product having the effect of improving ulcerative colitis.

[0019] The products include functional foods and medicines.

[0020] The present invention discloses the following technical effects:

[0021] The invention obtains black chicken liver hydrolyzed peptides (black chicken liver peptides) by enzymatic hydrolysis of Lueyang black chicken liver, detects the degree of hydrolysis by formaldehyde titration, detects the molecular weight by SDS-PAGE electrophoresis and Tricine-SDS-PAGE electrophoresis, performs sequence analysis by LC-MS / MS, predicts the biological function of peptide segments by using a database, detects the antioxidant and blood pressure lowering abilities of the black chicken liver peptides by in vitro experiments, and detects the effect of the black chicken liver peptides on improving colitis by using mouse experiments.

[0022] The results show that the prepared black chicken liver peptide is light yellow, and the surface is loose and porous; the present invention adopts formaldehyde titration method for detection, and the results show that the black chicken liver has reached a high degree of hydrolysis in 1 hour, and the amino nitrogen content in the black chicken liver peptide obtained by hydrolysis for 1 hour reaches 70.43% of that in hydrolysis for 4 hours; the present invention adopts SDS-PAGE electrophoresis and Tricine-SDS-PAGE electrophoresis to detect that the molecular weight of black chicken liver is between 20-120kDa, and the molecular weight of black chicken liver peptide is between 4.1-6.5kDa, and as the hydrolysis time increases, the molecular weight range of the peptide segment decreases; the present invention identifies and analyzes the sequence of black chicken liver peptide based on LC-MS / MS mass spectrometry detection method, and finds that the number of peptide segments after hydrolysis of black chicken liver is 6637, and analyzes the amino acid composition ratio of black chicken liver peptide, and finds that black chicken liver peptide contains 20 kinds of amino acids, including 9 kinds of essential amino acids, accounting for 42.7%, and has high nutritional value to human body; the present invention Ming also found that black chicken liver peptide had 487 peptides with antibacterial activity, 156 peptides with antihypertensive activity, and 2 peptides with anticancer activity through comparison and prediction of BioPepDB database; antioxidant activity detection found that compared with black chicken liver, the black chicken liver peptide prepared by the present invention had enhanced scavenging ability for DPPH free radicals and ABTS free radicals, and the scavenging rate of black chicken liver peptide for DPPH free radicals was 80.92%, and the scavenging rate for ABTS free radicals reached 95.83%; the present invention also found that black chicken liver peptide had ACE2 inhibitory activity through antihypertensive ability detection, and the inhibition rate was 12.35%; the results of mouse colon length, DAI score and colon section showed that black chicken liver peptide had the effect of alleviating ulcerative colitis; the colon section results of the present invention showed that black chicken liver peptide could significantly reduce the mouse colon tissue damage induced by sodium dextran sulfate; serum inflammatory factor detection showed that black chicken liver peptide had an improving effect on the level of intestinal inflammation induced by DSS. The above results show that the black chicken liver peptide prepared by the present invention has the advantages of anti-oxidation, lowering blood pressure and improving ulcerative colitis. Furthermore, using it to prepare products for preventing or improving ulcerative colitis is of great significance for improving the living conditions of patients with ulcerative colitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0024] Figure 1 This is the surface condition of black chicken liver peptide;

[0025] Figure 2 The amino acid composition of black chicken liver peptide;

[0026] Figure 3 The antioxidant capacity (DPPH free radical and ABTS free radical) of black chicken liver and black chicken liver peptide is compared, where BL is black chicken liver; BLP is black chicken liver peptide;

[0027] Figure 4 The colon length statistics of mice in each group;

[0028] Figure 5 DAI scores of mice in each group;

[0029] Figure 6 HE staining of mouse colon tissue (50 μm), where NC is the normal group (A); DSS is the enteritis model group (B); ASA is the group given 40 mg / kg 5-aminosalicylic acid by intragastric administration (C); BLPL is the group given 100 mg / kg black chicken liver peptide by intragastric administration (D); BLPH is the group given 300 mg / kg black chicken liver peptide by intragastric administration (E);

[0030] Figure 7 The expression of various inflammatory factors in mouse serum, among which A is IL-6; B is IL-1β; C is TNF-α; and D is IL-10. DETAILED DESCRIPTION

[0031] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0032] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0033] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0034] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0035] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0036] The present invention is further described below by way of specific examples. Unless otherwise specified, the various chemical reagents used in the examples of the present invention are obtained through conventional commercial channels.

[0037] Alkaline protease was purchased from Dongheng Huadao Biotechnology Co., Ltd., with a unit enzyme activity of 200,000 U / g;

[0038] Example 1

[0039] 1. Preparation of black-bone chicken liver hydrolyzed peptide (black-bone chicken liver peptide)

[0040] Use a meat grinder to grind black chicken liver (from Lueyang black chicken in Lueyang County, Shaanxi Province) into minced meat, add water to adjust the mass concentration to 30%, heat the liquid to 55°C, add 1% alkaline protease by mass of minced meat, natural pH, enzymolysis at 55°C for 1h, inactivate enzyme at 90°C for 15min, centrifuge at 8000r / min for 10min at 4°C, collect the supernatant, filter and retain the filtrate, vacuum freeze-dry the filtrate to obtain black chicken liver peptide. The obtained black chicken liver peptide is light yellow, with a crisp and porous surface ( Figure 1 ).

[0041] 2. Determination of physical and chemical properties

[0042] 2.1 Detection of hydrolysis degree of black-bone chicken liver by formaldehyde potentiometric titration

[0043] According to the method of step 1, the enzymatic hydrolyzate (i.e., filtrate) of black chicken liver peptide after enzymatic hydrolysis for 1h and 4h was prepared respectively. Take 15mL of enzymatic hydrolyzate respectively, adjust the pH to 8.2 with 0.05mol NaOH standard solution, add 5mL formaldehyde, and then titrate to pH9.2 with 0.05mol NaOH standard solution, and record the sodium hydroxide consumption V1. Use water as a blank control, record the sodium hydroxide consumption V2, and calculate the mass fraction of amino nitrogen by the following formula: X (mg / g) = [(V1-V2) × C × 14 × (50 / 15)] / 0.2, where X is the mass fraction of amino nitrogen; C is the mass molar concentration of NaOH; V1 and V2 are the volumes of sodium hydroxide consumed. The results show that the black chicken liver has reached a high degree of hydrolysis in 1h, and the amino nitrogen content in the black chicken liver peptide obtained by hydrolysis for 1h reaches 70.43% of that of hydrolysis for 4h.

[0044] 2.2 Molecular weight detection

[0045] SDS-PAGE electrophoresis detection: Mix black chicken liver (BL) and black chicken liver peptide (BLP) with 5× protein loading buffer at a ratio of 4:1, boil for 10 min and perform SDS-PAGE electrophoresis detection. Prepare SDS-PAGE gel according to Table 1, with a marker loading volume of 4 μL and a sample loading volume of 6 μL. First, use a constant voltage of 80V, and adjust the voltage to 120V when the gel is separated until the electrophoresis ends. Use Coomassie Brilliant Blue to decolorize until the target band is displayed.

[0046] Table 1 SDS-PAGE gel recipe

[0047]

[0048]

[0049] Tricine-SDS-PAGE electrophoresis detection: Mix the black chicken liver peptide with 2× protein loading buffer at a ratio of 1:1, boil for 10 minutes, and perform Tricine-SDS-PAGE electrophoresis detection. Prepare Tricine-SDS-PAGE gel according to Table 2, with a Marker loading volume of 5 μL and a sample loading volume of 10 μL. Use a constant current of 20 mA first, and adjust to 30 mA when the separation gel is reached. Use Coomassie Brilliant Blue to decolorize until the target band is displayed.

[0050] Preparation of black-bone chicken liver (BL): Use a meat grinder to grind the black-bone chicken liver into minced meat, add water to adjust the mass concentration to 30%, heat the liquid to 55°C, natural pH, treat at 55°C for 1h, treat at 90°C for 15min, centrifuge at 8000r / min for 10min at 4°C, collect the supernatant, filter and retain the filtrate, and vacuum freeze-dry the filtrate to obtain black-bone chicken liver.

[0051] Electrophoresis detection steps of black chicken liver: (1) Mix black chicken liver and 5× protein loading buffer at a ratio of 4:1, boil for 10 minutes for SDS-PAGE electrophoresis detection, the sample volume is 6μL, first use 80V constant voltage, and adjust the voltage to 120V when the separation gel is reached, and electrophoresis is completed. Use Coomassie Brilliant Blue to decolorize until the target band appears. (2) Mix black chicken liver and 2× protein loading buffer at a ratio of 1:1, boil for 10 minutes for Tricine-SDS-PAGE electrophoresis detection, the sample volume is 10μL, first use 20mA constant current, and adjust the voltage to 30mA when the separation gel is reached, and electrophoresis is completed. Use Coomassie Brilliant Blue to decolorize until the target band appears.

[0052] Table 2 Tricine-SDS-PAGE gel formula

[0053]

[0054] The results show that the molecular weight of black chicken liver protein detected by SDS-PAGE electrophoresis and Tricine-SDS-PAGE electrophoresis is between 20-120 kDa, and the molecular weight of black chicken liver peptide is between 4.1-6.5 kDa. As the hydrolysis time increases, the molecular weight range of the peptide segment decreases.

[0055] 3. Statistics of peptide identification results and analysis of amino acid composition ratio

[0056] The analytical column was 75 μm id×25 cm, NanoViper C18 1.9 μm, 100A, mobile phase A was 0.1% FA, mobile phase B was 0.1% FA, 80% CAN, flow rate was 600 nL / min, and the analysis time for each component was 35 min. Specific chromatographic conditions are shown in Table 3.

[0057] Table 3 Liquid chromatography conditions

[0058]

[0059] The full mass spectrometry scan range is 100-1500m / z, the primary mass spectrometry resolution is set to 60000, AGC is Custom, MaximumIT: Custom; the secondary mass spectrometry resolution is set to Resolution: 15000, AGC is Custom, MaximumIT: Custom, Cycletime: 2s, and the peptide fragmentation collision energy is set to 32 to generate mass spectrometry detection raw data (raw). The raw file of mass spectrometry acquisition is retrieved by the software database to obtain the identification results and the number of peptides for each sample. Each amino acid has different physical and chemical properties and corresponding functions. For peptides, the amino acid sequence composition determines the physical and chemical properties and functions of the peptides.

[0060] The results showed that the number of peptides after hydrolysis of black chicken liver was 6637 based on the LC-MS / MS mass spectrometry detection method. Figure 2 ) found that black chicken liver peptide contains 20 kinds of amino acids, including 9 essential amino acids, accounting for 42.7%, which has high nutritional value to the human body.

[0061] 4. Peptide function prediction

[0062] Use BioPepDB database to predict peptide functions. The peptide sequences obtained by peptide analysis are used for function prediction in the BioPepDB database. Click Get Result in Prediction to predict the function of the corresponding peptide.

[0063] The results showed that through comparison and prediction of the BioPepDB database, black chicken liver peptide was found to have 487 peptides with antibacterial activity, 156 peptides with antihypertensive activity, and 2 peptides with anticancer activity.

[0064] 5. Antioxidant activity test of black-bone chicken liver and black-bone chicken liver peptide

[0065] For details on the preparation of black chicken liver (BL), please refer to "2.2 Molecular Weight Detection"; for details on the preparation of black chicken liver peptide (BLP), please refer to "1. Preparation of Black Chicken Liver Hydrolyzed Peptide (Black Chicken Liver Peptide)".

[0066] 5.1 Determination of DPPH free radical scavenging rate: The total antioxidant capacity (T-AOC) detection kit (DPPH method) of Shanghai Biotech was used to detect the DPPH free radical scavenging rate of black chicken liver and filtered black chicken liver hydrolyzed peptide filtrate. Preheat the microplate reader for 30 minutes and adjust the wavelength to 515nm. React in a 1.5mL centrifuge tube, and the sample addition method is shown in Table 4. After the addition is completed, mix thoroughly, react at room temperature in the dark for 20 minutes, and take 200μL to a 96-well plate to measure the absorbance at 515nm. The calculation formula is as follows: DPPH free radical scavenging rate (%) = (A 空白 -A 测定 ) / A 空白 ×100%.

[0067] Table 4 DPPH method sample addition method

[0068]

[0069]

[0070] 5.2 ABTS free radical scavenging rate determination

[0071] The total antioxidant capacity (T-AOC) detection kit (ABTS method) of Shanghai Biotech was used to detect the ABTS free radical scavenging rate of black chicken liver and filtered black chicken liver hydrolyzed peptide filtrate. The microplate reader was preheated for 30 minutes and the wavelength was adjusted to 734nm. The reaction was carried out in a PCR tube, and the sample addition method was shown in Table 5. After the sample addition was completed, it was fully mixed, allowed to react for 20 minutes, and transferred to a 96-well plate to measure the absorbance at 734nm. The calculation formula is as follows: ABTS free radical scavenging rate (%) = (A 空白 -A 测定 ) / A 空白 ×100%.

[0072] Table 5 ABTS method sample addition method

[0073]

[0074] Antioxidant activity tests showed that compared with black chicken liver, black chicken liver peptide had enhanced scavenging ability for DPPH free radicals and ABTS free radicals. The scavenging rate of black chicken liver peptide for DPPH free radicals was 80.92%, and the scavenging rate for ABTS free radicals was 95.83% ( Figure 3 ).

[0075] 6. Blood pressure lowering activity detection

[0076] The angiotensin-converting enzyme 2 (ACE2) inhibitor screening kit of Biyuntian was used to detect the antihypertensive activity of the hydrolyzed peptides.

[0077] The results showed that black chicken liver peptide has ACE2 inhibitory activity, with an inhibition rate of 12.35%. The specific detection steps are as follows:

[0078] 6.1 Preparation of test solution

[0079] Take 20 mg of black chicken liver hydrolyzed peptide powder and add 200 μL Assay Buffer to prepare a 10 mg / mL solution.

[0080] 6.2 Preparation of positive control

[0081] Take 1 μL of 0.5 mM positive control inhibitor mLN-4760 and add it to 999 μL Assay Buffer to prepare a 50 nM positive control inhibitor mLN-4760.

[0082] 6.3 Sample determination

[0083] 6.3.1 Preparation of Assay Reagent

[0084] Prepare an appropriate amount of Assay Reagent according to the number of samples (including relevant controls). Take 10 μL of ACE2 Enzyme and add it to 1.85 mL of Assay Buffer and mix thoroughly.

[0085] 6.3.2 Sample addition

[0086] Use a 96-well blackboard to set up each group, and add the detection reagents and samples in order according to Table 6. After adding the sample to be tested, mix well. Set up 3 replicate wells for each sample. Quickly add 2μL of Substrate to each well on ice and mix well. Incubate at 37℃ in the dark for 10 minutes and then use enzyme labeling for fluorescence measurement. The excitation wavelength is 325nm and the emission wavelength is 393nm. The average fluorescence value of each sample well and blank control well can be recorded as RFU, respectively. 空白对照 、RFU 100％酶活性对照 、RFU 阳性对照 and RFU 样品The calculation formula of the inhibition percentage of the sample is as follows: Inhibition rate (%) = (RFU 100％酶活性对照 -RFU 样品 ) / (RFU 100％酶活性对照 -RFU 空白对照 )×100%.

[0087] Table 6 Sample addition method for antihypertensive activity detection

[0088]

[0089] 7. Animal Experimentation

[0090] 7.1 Grouping and modeling

[0091] All 5-week-old C57 mice were divided into 5 groups after one week of adaptive culture, with 10 mice in each group: normal group (NC): free drinking water + purified water; model group (M): 3.5% DSS + purified water; positive control group (ASA): 3.5% DSS + oral gavage 40mg / kg 5-aminosalicylic acid; low-concentration dose group (BLPL): 3.5% DSS + 100mg / kg black chicken liver hydrolyzed peptide; high-concentration dose group (BLPH): 3.5% DSS + 300mg / kg black chicken liver hydrolyzed peptide. During the feeding period, the mice were weighed regularly every day and the blood in the stool was observed, and the disease activity index (DAI) score was recorded. After the end of the drug administration, the mice were killed by dislocation of the neck, and blood, colon and colon contents were collected, and the colon length was measured.

[0092] 7.2 Weight loss rate, colon length and DAI score of mice

[0093] During the experiment, the mice were weighed before gavage every day. The average of the three scores based on weight loss was the final DAI score of the mice, and the specific details are shown in Table 7. The colon length of each mouse was measured after the mice were killed. The weight loss rate was calculated according to the following formula. Weight loss rate (%) = [mouse weight at this time point (g) - mouse weight before modeling (g)] / mouse weight before modeling (g) × 100%.

[0094] Table 7 DAI scoring criteria

[0095]

[0096] Colon shortening is another significant feature of dextran sulfate sodium-induced colon inflammation. The NC and ASA groups had the longest colon length, about 8.0 cm; the DSS group had the shortest length, only about 4.5 cm. Compared with the DSS group, the colon length of the BLPL and BLPH groups increased to about 6.5 cm. It can be seen that there was no significant difference in the effect of different doses of black chicken liver peptide on colon length ( Figure 4 ).

[0097] The DAI score showed that there was no significant difference in the scores between the groups in the first 4 days, but the score gap began to widen from the 5th day. The NC group remained at a low level, the DSS group scored much higher than the other groups, and the ASA group, BLPL group and BLPH group had similar levels in the last 3 days, indicating that black chicken liver peptide did have a relieving effect on colitis in mice ( Figure 5 ).

[0098] 7.3 Colonic Histopathological Observation

[0099] The colon tissue fixed in 4% paraformaldehyde solution was obtained, dehydrated, embedded, and sliced. Then, HE was used for staining and the slices were observed under an optical microscope to analyze the pathological damage.

[0100] The results of colon section showed that the mucosal layer of colon tissue in the DSS group was thinner, the crypt structure almost completely disappeared, and the inflammatory cell infiltration was severe; the mucosal layer of the BLPL group and the BLPH group was thicker, the crypt structure was relatively complete, and the inflammatory cells were relatively few. Compared with the BLPL group, the colon tissue structure in the BLPH group was more complete and the inflammation level was lower. This shows that black chicken liver peptide can significantly reduce the colon tissue damage induced by sodium dextran sulfate in mice ( Figure 6 ).

[0101] 7.4 Determination of serum inflammatory factors

[0102] An appropriate amount of colon sample was taken, pre-cooled saline was added at a solid-liquid ratio of 1:9, the homogenate was centrifuged and the supernatant was collected, and the contents of pro-inflammatory factors IL-6, IL-1β, TNF-α and IL-10 as well as the anti-inflammatory factor IL-10 were determined according to the instructions of the mouse interleukin IL-6, IL-1β, TNF-α and IL-10 ELISA quantitative detection kit (Shanghai ELISA Biotechnology Co., Ltd.).

[0103] Serum inflammatory factor detection showed that compared with the NC group, the levels of pro-inflammatory factors IL-6, IL-1β and TNF-α, and anti-inflammatory factor IL-10 in the DSS group were significantly increased, indicating that the inflammatory response in the DSS group was severe. Compared with the DSS group, the levels of inflammatory factors in the BLPL group and the BLPH group decreased, indicating that black chicken liver peptide has an improving effect on DSS-induced enteritis ( Figure 7 ).

[0104] The results of the above experiments show that based on the length of mouse colon, DAI score and colon section results, it can be concluded that the black chicken liver peptide prepared by the present invention has the effect of alleviating ulcerative colitis.

[0105] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing hydrolyzed peptides from black-bone chicken liver, characterized in that: The method comprises the step of enzymatically hydrolyzing the black-bone chicken liver by utilizing alkaline protease.

2. The preparation method according to claim 1, characterized in that: The preparation method comprises the following steps: preparing black chicken liver into minced meat and mixing it with water, heating the obtained liquid material, adding alkaline protease for enzymolysis, inactivating the enzyme, centrifuging and collecting the supernatant, and freeze-drying to obtain the black chicken liver hydrolyzed peptide.

3. The preparation method according to claim 2, characterized in that: The method comprises preparing minced meat from the black-bone chicken liver and mixing the minced meat with water, wherein the minced meat is adjusted to a mass concentration of 30% by adding water.

4. The preparation method according to claim 2, characterized in that: The addition amount of the alkaline protease is 1% of the mass of the minced meat in the feed liquid.

5. The preparation method according to claim 2, characterized in that: The enzymolysis was carried out at 55°C for 1 hour.

6. The preparation method according to claim 2, characterized in that: The enzyme inactivation step is to inactivate the enzyme at 90° C. for 15 min.

7. The preparation method according to claim 2, characterized in that: The centrifugation was carried out at 8000 r / min at 4°C for 10 min.

8. The black-bone chicken liver hydrolyzed peptide prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the black-bone chicken liver hydrolyzed peptide according to claim 8 in the preparation of a product having antioxidant activity and blood pressure lowering effect.

10. Use of the hydrolyzed peptide from black-bone chicken liver according to claim 8 in preparing a product having the effect of improving ulcerative colitis.

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