Preparation method and application of black chicken peptide with blood-tonifying function

Abstract

The present invention discloses a preparation method and application of a black chicken peptide with a blood-enriching function, and belongs to the field of biomedical technology. The method comprises the following steps: (1) heat-treating the black chicken, and cutting the meat into minced meat with bones; the heat treatment is steam heat treatment, and the conditions of the steam heat treatment are: temperature 100-120°C, time 1-5 hours; (2) mixing the minced meat obtained in step (1) with water to obtain a liquid material, adding a mixed enzyme for enzymolysis, and obtaining an enzymolysis solution, wherein the mixed enzyme is a mixture of neutral protease and bromelain, and the mass ratio of neutral protease to bromelain is 0.1-10:0.01-10; (3) the enzymolysis solution obtained in step (2) is subjected to a post-processing process to obtain the black chicken peptide. The black chicken peptide product prepared by the present invention has the advantages of high oligopeptide content, light bitterness, astringency and saltiness, good antioxidant activity, high purity, and good blood-enriching effect.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to a preparation method and application of a black-bone chicken peptide with blood-enriching function. Background Art

[0002] Among various tonic medicines, blood tonic medicines occupy an important proportion. At present, blood tonic products are mainly divided into products with "porphyrin iron" as raw materials and products with "medicine and food as raw materials". The efficacy classification is mainly concentrated in the simple blood tonic category, the Qi and blood tonic category, and the menstrual regulation and beauty category.

[0003] Iron is synthesized together with protein into hemoglobin. If protein is deficient, anemia will occur even if iron is sufficient. As a traditional nourishing food and medicinal material, black-bone chicken has been gaining increasing attention. Black-bone chicken contains 20 kinds of amino acids, 27 kinds of trace elements and a variety of fatty acids. It is a typical raw material of medicinal and edible origin. It can provide a high-quality source of protein for the human body, especially in enhancing physical strength, replenishing blood, treating diabetes and gynecological diseases. At the same time, eating black-bone chicken can nourish yin and kidney, delay aging, and has obvious effects on women's iron deficiency anemia.

[0004] Black-bone chicken has good curative and nourishing effects, but the production of black-bone chicken products is still mainly crude processing, lacking in-depth processing and utilization. Traditional black-bone chicken products mainly include black-bone chicken essence, black-bone chicken oral liquid, black-bone chicken Baifeng pills, etc., which have low human absorption rate. The biological enzymatic hydrolysis method is used to release the peptides with physiological functions in the black-bone chicken protein and prepare them into biologically active peptides with physiological functions, which are very suitable for people who need to nourish blood.

[0005] Chinese patent CN109055470A discloses a method for preparing an antioxidant chicken protein polypeptide, comprising the following steps: step 1: take fresh chicken breast and chicken skeleton as raw materials, wash and remove blood from the chicken; step 2: add water and cook; step 3: use a colloid mill to crush the cooked chicken breast and chicken skeleton; step 4: weigh three proteases; step 5: adjust the pH value and temperature, and add the weighed protease; step 6: after the enzymatic reaction is completed, heat and kill the enzyme to obtain an enzymatic solution; step 7: filter and ultrafilter the enzymatic solution; step 8: add the filtrate obtained in step 7 to a mixing tank, concentrate it, and obtain a concentrated solution; step 9: dry the concentrated solution to obtain an antioxidant chicken protein polypeptide. The method for preparing the antioxidant chicken protein polypeptide described in the invention can decompose chicken protein into polypeptides, which have good flavor, rich nutrition, and high antioxidant activity.

[0006] At present, the research on the efficacy of black chicken peptide mainly focuses on anti-oxidation, immune regulation, blood pressure lowering and other aspects. At present, there is still a lack of extraction technology for black chicken peptide that can simultaneously improve the blood-enriching effect and flavor. Summary of the invention

[0007] In view of this, the purpose of the present invention is to provide a preparation method and application of black chicken peptide with blood-enriching function. The prepared black chicken peptide product has the advantages of high oligopeptide content, light bitterness, astringency and saltiness, good antioxidant activity, high purity and good blood-enriching effect.

[0008] To achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows:

[0009] In one aspect, the present invention provides a method for preparing a black-bone chicken peptide having a blood-tonifying function, comprising the following steps:

[0010] (1) heat-treating the black-bone chicken and cutting the meat and bones into minced meat; the heat-treating is steam heat-treating, and the conditions of the steam heat-treating are: temperature 100-120° C., time 1-5 hours;

[0011] (2) mixing the minced meat obtained in step (1) with water to obtain a liquid material, adding a mixed enzyme for enzymolysis to obtain an enzymolysis solution, wherein the mixed enzyme is a mixture of neutral protease and bromelain, and the mass ratio of neutral protease to bromelain is 0.1-10:0.01-10;

[0012] (3) The enzymatic hydrolysate obtained in step (2) is subjected to a post-treatment process to obtain the black-bone chicken peptide.

[0013] Preferably, in step (1), the steam heat treatment is carried out at a temperature of 100-120° C. and for 2-3 hours.

[0014] Preferably, in step (2), the water is 1-3 times the weight of the minced meat, more preferably 1 time.

[0015] Preferably, in step (2), the conditions for the enzymatic hydrolysis are: temperature 45-65°C, pH 5-8, and time 4-8 hours. Further preferably, the conditions for the enzymatic hydrolysis are: temperature 50-60°C, pH 6-7, and enzymatic hydrolysis 5-7 hours. Further preferably, the conditions for the enzymatic hydrolysis are: temperature 54°C, pH 6.3-6.5, and time 5-6 hours.

[0016] Preferably, in step (2), the mass ratio of the neutral protease to bromelain is 3:1.

[0017] Further preferably, in step (2), the amount of the neutral protease added is 0.1-10% of the solid content of the liquid material, and the amount of the bromelain added is 0.01-10% of the solid content of the liquid material.

[0018] More preferably, in step (2), the amount of the neutral protease added is 0.18% of the solid content of the liquid material, and the amount of the bromelain added is 0.06% of the solid content of the liquid material.

[0019] Preferably, in step (3), the post-treatment process includes enzyme inactivation, cooling, filtering, concentration and drying.

[0020] Further preferably, the filtering method is to use a ceramic membrane for filtering. Further preferably, the pore size of the ceramic membrane is 20-200 nm. Even more preferably, the pore size of the ceramic membrane is 200 nm.

[0021] Further preferably, the filtration pressure is 0.16-0.2Mpa.

[0022] Further preferably, the concentration and drying comprises the following steps:

[0023] The filtrate was concentrated to obtain a concentrate, and the concentrate was dried.

[0024] More preferably, the method comprises the following steps:

[0025] The filtrate was concentrated under vacuum to obtain a concentrate, and the concentrate was dried.

[0026] More preferably, the method comprises the following steps:

[0027] The filtrate was concentrated to 35% under vacuum to obtain a concentrate, which was then spray-dried.

[0028] In another aspect, the present invention provides black-bone chicken peptide prepared by the above preparation method.

[0029] In another aspect, the present invention provides the use of the black-bone chicken peptide in the preparation of blood-enriching medicines.

[0030] In another aspect, the present invention provides an application of a composite enzyme in the preparation of black-bone chicken peptide, wherein the composite enzyme consists of a neutral protease and bromelain.

[0031] Preferably, the mass ratio of the neutral protease to bromelain is 0.1-10:0.01-10, more preferably 3:1.

[0032] Preferably, in the application, the addition amount of the neutral protease is 0.1-10% of the solid content of the liquid material, and the addition amount of the bromelain is 0.01-10% of the solid content of the liquid material.

[0033] More preferably, in the application, the amount of the neutral protease added is 0.18% of the solid content of the liquid, and the amount of the bromelain added is 0.06% of the solid content of the liquid.

[0034] The beneficial effects of the present invention are:

[0035] (1) The steam heating treatment of the present invention can not only reduce the content of free amino acids in the enzymatic hydrolyzate and enhance the flavor of the black chicken peptide powder, but also increase the content and functional activity of oligopeptides in the black chicken peptide.

[0036] (2) The present invention takes into account the protein properties and amino acid composition, selects neutral protease and bromelain for synergistic enzymolysis, and optimizes the specific ratio and dosage, thereby significantly improving the efficacy and quality of black chicken peptide.

[0037] (3) The present invention improves the extraction process of black chicken peptide for its blood-tonifying function. The obtained black chicken peptide has the advantages of high oligopeptide content, light bitterness, astringency and saltiness, good antioxidant activity, high purity and good blood-tonifying effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Schematic diagram of the effect of different pre-treatment conditions on the flavor of black chicken peptide solution;

[0039] Figure 2 It is a schematic diagram of the hemoglobin content in rat whole blood;

[0040] Figure 3 for rat Na + K + -ATPase activity diagram;

[0041] Figure 4 Rat Ca 2+ Mg 2+ -ATPase activity diagram;

[0042] Figure 5 This is the result diagram of rat erythropoietin (EPO). DETAILED DESCRIPTION

[0043] The following non-limiting examples can enable those skilled in the art to more fully understand the present invention, but do not limit the present invention in any way. The following content is only an exemplary description of the scope of the present invention, and those skilled in the art can make various changes and modifications to the invention of the present invention based on the disclosed content, and they should also fall within the scope of the present application.

[0044] The present invention is further described below by way of specific examples. The various chemical reagents used in the examples of the present invention are obtained through conventional commercial channels unless otherwise specified. Unless otherwise specified, the contents described below are all weight contents. Unless otherwise specified, it is understood that the process is carried out at room temperature.

[0045] In the following embodiments,

[0046] Neutral protease was purchased from Genenco Biotechnology Co., Ltd., model number FoodPro PNL, unit enzyme activity of 1600-1900 AOZ / g;

[0047] Bromelain was purchased from Angel Enzyme Co., Ltd., with the product number BR-50 and a unit enzyme activity of 1 million U / g;

[0048] Alcalase was purchased from Novozymes Biopharmaceuticals Co., Ltd., with the product number of Alcalase 2.4L and a unit enzyme activity of 580,000-650,000 DU / g;

[0049] Papain was purchased from Beijing Solebow Technology Co., Ltd., with the product number P8150 and the unit enzyme activity of 800,000 U / g;

[0050] Ceramic membranes were purchased from Hefei Yuwang Membrane Engineering Technology Co., Ltd., with pore size filters of 50 nm and 200 nm.

[0051] Example 1

[0052] The black-bone chicken was chopped with a chopper and steamed in a heating device at a temperature of 100°C for 2 hours. After cooling, the heat-treated black-bone chicken was minced with bones and meat using a meat grinder, and 1 times the amount of water was added. The liquid was heated to 54°C, and neutral protease with a solid content of 0.18% and bromelain 0.06% were added at a pH of 6.5. The enzyme was hydrolyzed for 5 hours, and the enzyme was inactivated. The liquid was cooled to room temperature and filtered with a ceramic membrane with a separation pore size of 200nm. The effective filtration area of ​​the ceramic membrane was 0.2m 2 , the working pressure is 0.16-0.2Mpa, the filtrate is retained, the liquid material is vacuum-concentrated to 35%, and the concentrate is spray-dried.

[0053] Example 2

[0054] The black-bone chicken was chopped with a chopper and steamed in a heating device at a temperature of 110°C for 2 hours. After cooling, the heat-treated black-bone chicken was minced with bones and meat using a meat grinder, and 1 times the amount of water was added. The liquid was heated to 54°C, and neutral protease with a solid content of 0.18% and 0.06% bromelain were added. The pH was 6.5, and the enzymolysis was performed for 5 hours to inactivate the enzyme. The liquid was cooled to room temperature and filtered with a ceramic membrane with a separation pore size of 200nm. The effective filtration area of ​​the ceramic membrane was 0.2m 2 , the working pressure is 0.16-0.2Mpa, the filtrate is retained, the liquid material is vacuum-concentrated to 35%, and the concentrate is spray-dried.

[0055] Example 3

[0056] The black-bone chicken was chopped with a chopper and steamed in a heating device at a temperature of 120°C for 2 hours. After cooling, the heat-treated black-bone chicken was minced with bones and meat using a meat grinder, and 1 times the amount of water was added. The liquid was heated to 54°C, and neutral protease with a solid content of 0.18% and 0.06% bromelain were added. The pH was 6.5, and the enzymolysis was performed for 5 hours to inactivate the enzyme. The liquid was cooled to room temperature and filtered with a ceramic membrane with a separation pore size of 200nm. The effective filtration area of ​​the ceramic membrane was 0.2m 2 , the working pressure is 0.16-0.2Mpa, the filtrate is retained, the liquid material is vacuum-concentrated to 35%, and the concentrate is spray-dried.

[0057] Example 4

[0058] The black-bone chicken was chopped with a chopper and steamed in a heating device at a temperature of 110°C for 2 hours. After cooling, the heat-treated black-bone chicken was minced with bones and meat using a meat grinder, and 1 times the amount of water was added. The liquid was heated to 54°C, and neutral protease with a solid content of 0.18% and 0.06% bromelain were added. The pH was 6.5, and the enzymolysis was performed for 6 hours to inactivate the enzyme. The liquid was cooled to room temperature and filtered with a ceramic membrane with a separation pore size of 200nm. The effective filtration area of ​​the ceramic membrane was 0.2m 2 , the working pressure is 0.16-0.2Mpa, the filtrate is retained, the liquid material is vacuum-concentrated to 35%, and the concentrate is spray-dried.

[0059] Example 5

[0060] The black-bone chicken was chopped with a chopper and steamed in a heating device at a temperature of 110°C for 3 hours. After cooling, the heat-treated black-bone chicken was minced with bones and meat using a meat grinder, and 1 times the amount of water was added. The liquid was heated to 54°C, and a neutral protease with a solid content of 10% and 0.01% bromelain were added. The pH was 6.5, and the enzymolysis was performed for 6 hours to inactivate the enzyme. The liquid was cooled to room temperature and filtered with a ceramic membrane with a separation pore size of 200nm. The effective filtration area of ​​the ceramic membrane was 0.2m 2 , the working pressure is 0.16-0.2Mpa, the filtrate is retained, the liquid material is vacuum-concentrated to 35%, and the concentrate is spray-dried.

[0061] Example 6

[0062] The black-bone chicken was chopped with a chopper and steamed in a heating device at a temperature of 120°C for 2 hours. After cooling, the heat-treated black-bone chicken was minced with bones and meat using a meat grinder, and 1 times the amount of water was added. The liquid was heated to 54°C, and a neutral protease with a solid content of 0.1% and 10% bromelain were added. The pH was 6.5, and the enzymolysis was performed for 6 hours to inactivate the enzyme. The liquid was cooled to room temperature and filtered with a ceramic membrane with a separation pore size of 200nm. The effective filtration area of ​​the ceramic membrane was 0.2m 2 , the working pressure is 0.16-0.2Mpa, the filtrate is retained, the liquid material is vacuum-concentrated to 35%, and the concentrate is spray-dried.

[0063] Example 7

[0064] The black-bone chicken was chopped with a chopper and steamed in a heating device at a temperature of 110°C for 2 hours. After cooling, the heat-treated black-bone chicken was minced with bones and meat in a meat grinder, and 1 times the amount of water was added. The liquid was heated to 54°C, and neutral protease with a solid content of 0.18% and 0.06% bromelain were added. The pH was 6.5, and the enzymolysis was performed for 6 hours to inactivate the enzyme. The liquid was cooled to room temperature and filtered with a ceramic membrane with a separation pore size of 50nm. The effective filtration area of ​​the ceramic membrane was 0.2m 2 , the working pressure is 0.16-0.2Mpa, the filtrate is retained, the liquid material is vacuum-concentrated to 35%, and the concentrate is spray-dried.

[0065] Comparative Example 1

[0066] The black-bone chicken was chopped with a chopper, minced into minced meat with a meat grinder, and then boiled in a heating device at 50°C for 2 hours. When the liquid material was heated to 54°C, enzymolysis was performed, and the enzymolysis and subsequent treatment steps were the same as in Example 1.

[0067] Comparative Example 2

[0068] The black-bone chicken was chopped with a chopper, minced into minced meat with a meat grinder, and then boiled in a heating device at 70°C for 2 hours. When the liquid material was cooled to 54°C, enzymolysis was performed, and the enzymolysis and subsequent treatment steps were the same as in Example 1.

[0069] Comparative Example 3

[0070] The black-bone chicken was chopped with a chopper, minced into minced meat with a meat grinder, and then boiled in a heating device at 90°C for 2 hours. When the liquid material was cooled to 54°C, it was enzymolyzed. The enzymolysis and subsequent treatment steps were the same as in Example 1.

[0071] Comparative Example 4

[0072] The black-bone chicken was chopped with a chopper, minced into minced meat with a meat grinder, and then boiled in a heating device at 70°C for 2 hours. When the liquid material was cooled to 54°C, enzymolysis was performed, and the enzymolysis and subsequent treatment steps were the same as in Example 1.

[0073] Comparative Example 5

[0074] Compared with Example 1, only 0.24% of the neutral protease of the solid content of the liquid was added during enzymolysis, and the enzymolysis was carried out for 6 hours. The remaining steps were the same as in Example 1.

[0075] Comparative Example 6

[0076] Compared with Example 1, only 0.24% of the solid content of bromelain in the liquid was added during enzymolysis, and the enzymolysis was performed for 6 hours. The remaining steps were the same as in Example 1.

[0077] Comparative Example 7

[0078] Compared with Example 1, 0.18% alkaline protease and 0.06% papain were added to the liquid solid content during enzymolysis, and the enzymolysis was carried out for 6 hours. The remaining steps were the same as in Example 1.

[0079] Comparative Example 8

[0080] Compared with Example 1, the ceramic membrane filtration is replaced by multi-stage stratified extraction (adding water three times, standing and settling after each addition of water, the feed liquid will be stratified, and the clear liquid is collected) for oil-water separation, and the remaining steps are the same as Example 1.

[0081] Comparative Example 9

[0082] Compared with Example 1, the oil-water separation was performed by replacing the ceramic membrane filtration with centrifugal separation (4000 g, 10 min), and the remaining steps were the same as in Example 1.

[0083] Comparative Example 10

[0084] Compared with Example 1, 0.002% of neutral protease and 0.238% of bromelain were added to the liquid solid content, and the remaining steps were the same as in Example 1.

[0085] The parameters of each comparative example and embodiment are compared as follows:

[0086] Table 1.

[0087]

[0088] Results

[0089] 1. Determination of physical and chemical properties

[0090] 1.1 Determination of protein content

[0091] The Kjeldahl nitrogen determination method (GB / T5009.5-2003) was used.

[0092] Protein recovery rate = N1 / N2×100%

[0093] Where: N1 is the total nitrogen content of the enzymatic supernatant, g; N2 is the total nitrogen content of the raw material, g.

[0094] 1.2 Determination of oligopeptide content

[0095] The method in Appendix B of GB / T22492 is adopted.

[0096] 1.3 Determination of free amino acid content

[0097] The content of free amino acids in the supernatant was determined by formaldehyde potentiometric titration.

[0098] 2. Determination of antioxidant activity

[0099] 2.1 Determination of reducing power

[0100] Use deionized water to prepare a certain protein concentration of saury hydrolysate, take 2mL of each sample and add it to a mixture of 2mL0.2mol / L phosphate buffer (pH6.6) and 2mL1% potassium ferricyanide solution, keep it at 50°C for 20min, then add 2mL10% TCA, and then centrifuge it at 3000r / min for 10min, take 2mL of the supernatant and react with 2mL distilled water and 0.4mL0.1% ferric chloride, and measure its absorbance OD700 at 700nm after 10min.

[0101] 2.2 Determination of DPPH free radical scavenging ability

[0102] Accurately weigh 7.92 mg of DPPH free radicals, dissolve it in 95% ethanol and make up to 100 mL to prepare a 0.2 mmol DPPH ethanol solution. Use deionized water to prepare a saury hydrolyzate with a certain protein concentration. Take 2.0 mL of the sample and mix it with 2.0 mL of DPPH ethanol solution and shake it well. After leaving it at room temperature for 30 minutes, measure the absorbance at 517 nm, which is counted as Ai; take 2.0 mL of each sample solution and control solution and mix it with 2.0 mL of 95% ethanol solution and shake it well to measure the absorbance at 517 nm, and the reading is counted as Aj; take 2 mL of DPPH ethanol solution and add 2 mL of distilled water, mix it and shake it well to measure the absorbance at 517 nm, and the reading is counted as Ac; the blank is 2 mL of 95% ethanol and add 2 mL of distilled water to zero. All measured values ​​are the average of three times, and the calculation formula for the scavenging rate R of DPPH free radicals is:

[0103] R=[1-(Ai-Aj) / Ac]×100%

[0104] 2.3 Determination of Oxidative Radical Absorbance Capacity (ORAC Method)

[0105] Add 20 μL of the sample to be tested to each microwell of the 96-well fluorescence plate (dilute the sample to be tested with 75 mmol / L phosphate buffer appropriately, and the concentration of each sample to be tested is 0.04-0.06 mg / mL), then add 120 μL of 70 nmol / L fluorescein (FL), and keep it at 37°C for 15 min. Then, use a multichannel pipette to quickly add 60 μL of 12 mmol / L AAPH (azobisisobutylamidine hydrochloride) to each well to start the reaction. Place the microplate in a microplate reader at 37°C for continuous measurement with an excitation wavelength of 485 nm and an emission wavelength of 520 nm. Measure the fluorescence intensity of each well once every 2 min, and set the measurement time until the fluorescence decays to the baseline, that is, 2 h. The fluorescence intensity was recorded as f0, f1, f2, ⋯, f100, respectively. The absolute fluorescence intensity data fn recorded at different time points of each microwell were compared with the initial fluorescence intensity f0 and converted into relative fluorescence intensity. The area under the fluorescence quenching curve (AUCsample) value was calculated according to the formula AUC=f1 / f0+f2 / f0+f3 / f0+,⋯,fi / f0+⋯f100 / f0. Then, the antioxidant effects of different concentrations of Trolox (water-soluble vitamin E) and samples relative to the blank control were calculated according to the formula netAUC=AUCsample−AUCblank, that is, the net AUC value, where AUCblank is the AUC value of the free radical action control in the absence of antioxidants.

[0106] 3. Sensory evaluation test of electronic tongue flavor

[0107] 3.1 Sample pretreatment:

[0108] The sample for electronic tongue measurement must be clear liquid and must not contain organic reagents. The fat content must not be higher than 5% and the alcohol content must be less than 20%.

[0109] 3.2 Sensor preparation:

[0110] Taste sensor (S), reference solution (30 mM KCl + 0.3 mM tartaric acid), internal solution (3.33 M KCl saturated with silver chloride), positive and negative sensors (positive), disposable gloves, 100 μL pipette, 100 mL beaker.

[0111] 3.3 Sensor activation:

[0112] Before use, the sensor needs to be soaked in the relevant solution for at least 24 hours; the electrode is soaked in 3.3 M potassium chloride solution, and the taste sensor is soaked in reference solution.

[0113] 3.4 Assemble the electronic tongue, install the taste sensor, place the sample and reference solution, and use the Setup of measurement program to perform measurements and export data.

[0114] 4. Research on the blood-tonifying and blood-nourishing effects based on animal level

[0115] 4.1 Materials and Equipment

[0116] 4.1.1 Raw materials: black chicken peptide samples of Comparative Example 4-Comparative Example 10 and Example 4-Example 7.

[0117] 4.1.2 Experimental animals: 6-week-old specific pathogen-free (SPF) grade Kunming (KM) female rats were purchased from Beijing Spike Laboratory Animal Technology Co., Ltd., weighing (160±2) g, and kept at a temperature of (20±2)°C with normal day and night changes. During the experiment, the animals were allowed to eat and drink freely.

[0118] 4.1.3 Materials and reagents: Hemoglobin test solution, ATPase activity detection kit, Nanjing Jiancheng Bioengineering Institute; BCA protein concentration determination kit, Beyotime Biotechnology Institute; sodium heparin anticoagulant tube, Pfizer Medical Devices Co., Ltd.; cyclophosphamide, Jiangsu Shengdi Pharmaceutical Co., Ltd.; EPO enzyme-linked immunosorbent assay kit, Blueprint Biotechnology Co., Ltd.

[0119] 4.1.4 Instruments and equipment: ELISA reader, Beckman, USA; Millipore Elix15 water purifier, Merck Chemical Co., Ltd.; EL104 electronic balance, Mettler-Toledo Instrument (Shanghai) Co., Ltd.; 80-2 centrifugal precipitator, Jintan Medical Instrument Factory, Jiangsu Province; 79-1 magnetic heating stirrer, Guohua Electric Co., Ltd.; WB-10L1 constant temperature water bath, Memmert, Germany.

[0120] 4.2 Experimental methods:

[0121] 4.2.1 Experimental design

[0122] The experimental animals were randomly divided into blank group, model group and experimental group (fed with black chicken peptide prepared by different processes) after adaptive feeding for 3 days. The rats in the experimental group were given 0.616g / (kg·bw) black chicken peptide for 7 days, and the rats in the model group and blank group were given an equal amount of pure water for 7 days. From the 8th day, except for the blank group, the bone marrow blood deficiency model was established for the rats in the other groups, that is, intraperitoneal injection of cyclophosphamide 80mg / kg, once a day, and 1mL of blood was drawn through the orbit for 3 consecutive days; the blank group was injected with an equal amount of normal saline to complete the modeling. At the same time as the modeling, the experimental group should continue to be gavaged with 0.616g / (kg·bw) black chicken peptide once a day for 7 consecutive days, and blood was drawn from the orbit of all rats on the 7th day; gavage was continued for 7 days, and blood was drawn again on the 21st day, for a total of 21 days.

[0123] 4.2.2 Hemoglobin detection

[0124] The whole blood of mice was collected by using anticoagulation tubes, and the hemoglobin content was measured according to the hemoglobin kit purchased from Nanjing Jiancheng.

[0125] 4.2.3 ATPase activity assay

[0126] Wound area measurement: The rats were laid flat and photographed at the same height using an industrial high-definition camera and the initial wound measurement was performed.

[0127] 4.2.4 Erythropoietin Detection

[0128] The mouse blood samples were centrifuged at 3000 r / min and 4°C for 10 minutes to obtain serum. The hematopoietic function factor EPO content in the serum was determined using an enzyme-linked immunosorbent assay kit according to the manufacturer's instructions.

[0129] 4.2.5 Measurement of body weight changes

[0130] The first body weight was measured before the injection of cyclophosphamide on the 8th day; the second body weight was measured after fasting for 12 hours at the end of the 21st day after oral gavage, and the weight difference was calculated.

[0131] 4.2.6 Organ index determination

[0132] Rats were killed by cervical dislocation, and the liver, kidney and spleen tissues of the rats were collected and weighed to calculate the organ index. The calculation formula was: organ index (mg / g) = organ mass (mg) / body weight (g).

[0133] 4.2.7 Immunoproliferation assay

[0134] Rats were killed by cervical dislocation and immersed in 75% alcohol. The spleen was then isolated under a sterile environment. The spleen was cut into pieces and ground with scissors. During the grinding process, it was continuously rinsed with PBS and passed through a 200-mesh nylon sieve. The filtrate was collected. Centrifuged at 1200r / min for 5min, the supernatant was discarded, 2mL of red blood cell lysis buffer was added to treat the cells for 2 minutes, and washed twice with PBS. The spleen cells without red blood cells were resuspended in RPMI-1640 medium supplemented to obtain a spleen cell suspension. 100 microliters of cell suspension (2×10 6 Cells were added to 96-well Costar plates with 5 μg / mL of concanavalin A (ConA) (5 μg / mL) or 10 μg / mL of lipopolysaccharide (LPS) (10 μg / mL) as well as black chicken peptide. ConA-only or LPS-only culture medium was selected as control, respectively. The cells were then cultured in a humidified incubator at 37 ˚C with 5% carbon dioxide for 72 hours. Cell Counting Kit-8 (WST-8, Beyotime, China) was used to measure cell proliferation according to the manufacturer's protocol, and the absorbance was measured at 450 nm after another 4 hours of culture. The data were expressed as stimulation index (SI) and calculated according to the following formula:

[0135] .

[0136] 4.2.8 Data Statistical Analysis

[0137] Origin2021 statistical software was used to process the data, and the experimental results were expressed as mean ± standard deviation (Mean ± SE). The inter-group t-value test was used, and p < 0.05 indicated a significant difference.

[0138] 5. Experimental results

[0139] 5.1 Effects of different pretreatment conditions on the preparation of black chicken peptide

[0140] 5.1.1 Degradation and enzymatic hydrolysis of long fibers under different pretreatment conditions

[0141] The degradation and enzymatic hydrolysis of long fibers pretreated under different conditions are shown in Table 2.

[0142] Table 2. Degradation and enzymatic hydrolysis of long fibers under different pretreatment conditions

[0143]

[0144] The results show that in Comparative Example 1, the long fibers are not degraded at all or slightly degraded. Although the long fibers in Comparative Example 3 are basically degraded, the free amino acid content in the supernatant is high, which is unfavorable to the flavor of black chicken peptide. In Comparative Example 2, the muscle fibers are degraded, but the free amino acid content will also increase after 2 hours of treatment. However, steam heating is used in the embodiment to reduce the dissolution of free amino acids. At the same time, the first heating and then mincing adopted in Examples 1-3 can further reduce the free amino acids retained in the enzymatic hydrolysis sample. Therefore, the processing methods of Examples 1-3 can not only reduce the content of free amino acids in the supernatant, but also achieve the purpose of complete degradation of chicken fiber. The content of oligopeptides in the black chicken peptides prepared by Examples 1-3 is also higher than that in Comparative Examples 1-3.

[0145] 5.1.2 Effects of different pretreatment conditions on the flavor of black chicken peptide solution

[0146] The bitterness, astringency and saltiness in the raw materials will reduce the user's compliance. Figure 1 As shown, the results show that the bitterness, astringency and saltiness of the black chicken peptides prepared in Comparative Examples 1 to 3 are more prominent than those of the black chicken peptides prepared in Examples 1 to 3, and the richness value is also higher.

[0147] 5.1.3 Effects of different pretreatment conditions on the antioxidant activity of black-bone chicken peptide

[0148] The effects of different pretreatment conditions on the antioxidant activity of black chicken peptide are shown in Table 3.

[0149] Table 3. Effects of different pretreatment conditions on the antioxidant activity of black chicken peptides

[0150]

[0151] The results show that the reducing power of Examples 1 to 3 is stronger than that of the comparative example, the scavenging ability of Examples 1 to 3 of DPPH free radicals is stronger than that of Comparative Example 1 to 3, and the ORAC value is also higher than that of Comparative Example 1 to 3. Among them, the ORAC value of Examples 1 to 3 reaches more than 850 μmolTE / g, the reducing power is also about 0.7, and the DPPH scavenging ability is also significantly stronger than that of the comparative example group, indicating that the antioxidant activity of the black chicken peptide treated by steam heating is stronger than that of the black chicken peptide obtained by boiling and heating.

[0152] In summary, steam heating treatment is more suitable for the preparation of black chicken peptide than boiling heating treatment. It not only improves the flavor of black chicken peptide, but also increases the content of oligopeptides in black chicken peptide, while ensuring the biological activity of peptide.

[0153] 5.2 Effects of different preparation processes on the preparation of black-bone chicken peptide

[0154] 5.2.1 Analysis of the physicochemical results of black chicken peptides obtained by different preparation processes

[0155] Black-bone chicken peptides were prepared by enzymatic hydrolysis of black-bone chicken according to different pretreatment schemes. The protein purity, peptide content and weight-average molecular weight were used as evaluation indicators to compare the enzymatic hydrolysis effects of different preparation processes.

[0156] Table 4. Protein purity, peptide content and molecular weight of black chicken peptides prepared by different processes

[0157]

[0158] As can be seen from Table 4, in the 11 groups of samples, the protein purity is distributed between 84.36% and 91.23%; the peptide content is distributed between 75.36% and 86.81%; and the weight average molecular weight is distributed between 424.51 and 654.53u. On the whole, although there are certain differences between the comparative examples and the various groups of the embodiments, small molecule peptides are the main components; at the same time, the protein purity and peptide content of the comparative examples 4 to 10 are lower than those of the embodiment group, and the weight average molecular weight is higher than that of the embodiment group, while the differences between the embodiments are not large, and small molecule peptides are more conducive to rapid absorption by the human body, so the black chicken peptide in the embodiment is more conducive to absorption by the human body.

[0159] 5.2.2 Hemoglobin detection

[0160] like Figure 2 As shown (the same letters between the groups in the figure indicate no significant difference between the groups, and different letters indicate significant difference between the groups), compared with the blank group, the hemoglobin content of the model group was 74.67% of the blank group, and the hemoglobin content decreased significantly (p<0.05). Compared with the model group, the hemoglobin content of each comparative example and embodiment increased to varying degrees. The increase in hemoglobin content of each group in the comparative example was basically the same, with no significant difference (p>0.05); the increase in hemoglobin content of each group in the embodiment was basically the same, with no significant difference (p>0.05). Among them, the hemoglobin content of Example 5 and Example 6 was 98.76% and 97.36% of the blank group. The above results show that after steam heating treatment, the black chicken peptide obtained by using neutral protease and bromelain for enzymatic hydrolysis and then oil-water separation by ceramic membrane can restore the blood deficiency of rats by increasing the hemoglobin content. There is no significant difference in increasing the hemoglobin content among the black chicken peptides of different embodiments.

[0161] 5.2.3 ATPase activity

[0162] like Figure 3 and Figure 4 As shown in the figure (the same letters between the data groups in the figure indicate that there is no significant difference between the groups, and different letters indicate that there is a significant difference between the groups), compared with the blank group, the model group Na+ K + -ATPase activity and Ca 2+ Mg 2+ -ATPase activity decreased significantly (p<0.05). After oral administration of black chicken peptides prepared by different processes, Na + K + -ATPase activity and Ca 2+ Mg 2+ -ATPase activity was improved compared with the model group, which was 15.86% and 33.04% of the blank group, respectively, indicating that chicken peptide can alleviate blood deficiency by restoring ATPase activity. Among them, the effects of Examples 4 to 7 were relatively significant (p<0.05), and the Na + K + -ATPase activity and Ca 2+ Mg 2+ -ATPase activity levels were 5.18 and 2.70 times higher than those of the model group. + K + -ATPase activity and Ca 2+ Mg 2+ -ATPase activity is higher; from the comparison of comparative examples 5-7 with Example 4, it can be seen that the neutral protease and bromelain compound group is better than the single enzyme enzymatic hydrolysis group, and is also better than other double enzyme compound groups; from the comparison of comparative examples 8-9 with Example 4, it can be seen that the ceramic membrane is more conducive to retaining the active ingredients in the black chicken peptide; compared with comparative examples 4-9, Na + K + -ATPase activity and Ca 2+ Mg 2+ The above results show that the best black chicken peptide with blood-tonifying effect can be obtained after steam heating treatment, enzymatic hydrolysis with neutral protease and bromelain, and oil-water separation with ceramic membrane.

[0163] 5.2.4 Erythropoietin Detection

[0164] like Figure 5As shown (the same letters between the data groups in the figure indicate that there is no significant difference between the groups, and different letters indicate that there is a significant difference between the groups), compared with the blank group, the EPO of the model group rats was significantly reduced (p<0.05), which was 60.34% of the blank group. The EPO content of each experimental group was increased to varying degrees compared with the model group, among which there was no significant difference between the comparative examples 4-7 and the model group (p>0.05), and there was a significant difference between the EPO content of comparative examples 8 and 9 and the model group, which was 1.34 times and 1.30 times of the model group, respectively, indicating that the black chicken peptide of the experimental group can significantly restore the ability of blood deficiency in rats. On the other hand, there are significant differences between Examples 4-7 and the model group, and there is no significant difference between them and the blank group. There is no significant difference between the black chicken peptides of different embodiments, indicating that Examples 4-7 have good recovery effects on blood deficiency in rats. The above results show that black chicken peptide can alleviate blood deficiency by restoring EPO, and Examples 4-7 have significant effects.

[0165] 5.2.5 Measurement of body weight changes

[0166] The results are shown in Table 5 (in the table, a, b, and c marks represent significant differences. Specifically, the same letters between groups of data indicate no significant difference between the groups, and different letters indicate significant difference between the groups (p < 0.05).

[0167] Table 5. Changes in rat body weight during two measurements

[0168]

[0169] After 21 days of feeding, the weight of the rats in the blank group increased significantly, and the weight of the rats in the model group decreased significantly, and there was a significant difference between the two (p<0.05); the weight changes of each comparative example and the embodiment were improved compared with the model group, and the weight changes of the four groups of rats in comparative examples 8-10 and examples 4-7 were significantly different from those in the model group (p<0.05). The above results show that after steam heating treatment, the black chicken peptide prepared by the combined enzymatic hydrolysis of neutral protease and bromelain can better maintain the weight of mice, and the oil-water separation method has little effect on the maintenance of the weight of the black chicken peptide, among which the weight of the rats in comparative examples 8 and 9, and examples 5 and 7 are relatively stable, with the best effect.

[0170] 5.2.6 Organ index determination

[0171] After the experiment, the rats were killed and the weight of the rats' kidneys, livers and spleens, i.e., the organ indexes, were measured. The organ indexes of the rats are shown in Table 6 (in the table, a, b, c, and d marks represent the results of significant differences, specifically: the same letters between the data groups indicate that there is no significant difference between the groups, and different letters indicate that there is a significant difference between the groups, p < 0.05).

[0172] Table 6. Rat organ index (mg / g)

[0173]

[0174] It can be seen that the renal index of the rats in the model group has no significant difference compared with the blank group (p>0.05), and the renal index of each comparative group and the embodiment group has no significant difference compared with the blank group and the model group, but the index of embodiment 4 is the highest and the recovery effect is the best. The liver index of the rats in the model group has no significant difference with the blank group (p>0.05), but there is a significant difference with embodiment 4-embodiment 7 (p<0.05), and there is no significant difference between the embodiments, indicating that embodiment 4-embodiment 7 has a good recovery effect on the liver. There is a significant difference between the spleen index of the rats in the model group and the blank group and embodiment 4-embodiment 7 (p<0.05), among which embodiment 4-embodiment 7 has no significant difference with the blank group, indicating that embodiment 4-embodiment 7 has a good effect of restoring the spleen of blood-deficient rats. In summary, there is no significant difference between the groups in the renal index, but the effect of embodiment 4 group is the best in the liver index and spleen index.

[0175] 5.2.7 Determination of immune indicators

[0176] After the rats were killed, the spleen was taken to prepare a cell suspension for cell culture, and ConA and LPS were added to induce the spleen cells to differentiate into T cells and B cells, and then the optical density (OD value) was detected at 490nm, and the stimulation index (SI) was calculated. The OD value of the rats in the blank group was used as a benchmark, and the OD values ​​of the rats in the 6 experimental groups (the rats in the model group had died and did not participate in the experiment) were measured, and their SI values ​​were calculated according to the formula. The rat T cell stimulation index is shown in Table 7 (in the table, a and b marks represent the results of significant differences, specifically: the same letters between the data of each group indicate that there is no significant difference between the groups, and different letters indicate that there is a significant difference between the groups p < 0.05).

[0177] Table 7. Rat cell stimulation index

[0178]

[0179] The results show that there is no significant difference between the comparative example group and the embodiment group (p>0.05), but there are significant differences between each comparative example group and the embodiment group (p<0.05). At the same time, it can be seen that the T cell stimulation index of Examples 4-7 is relatively high, which are 1.13, 1.17, 1.11 and 1.09 respectively, among which the stimulation index of Example 5 is the highest. As shown in Table 7, there is little difference in the B cell stimulation index between each comparative example group and each embodiment group. Similarly, there is a significant difference between each comparative example group and the embodiment group (p<0.05). The B cell stimulation index of Examples 4-7 is relatively high, which are 1.11, 1.18, 1.16 and 1.15 respectively, among which the stimulation index of Example 5 is the highest. In summary, Example 5 has the best differentiation effect and the best effect on restoring blood deficiency; at the same time, Examples 4, 6 and 7 also have a good effect of restoring blood deficiency.

[0180] Table 8 summarizes the effects of black chicken peptides in various regimens on the blood-enriching and blood-nourishing function of rats.

[0181] Table 8. Comprehensive evaluation results of various indicators of black chicken peptide in various comparative examples and examples in nourishing blood

[0182]

[0183]

[0184] Comprehensively considering all the evaluation indicators, Example 5 and Example 7 scored the highest. The samples of Example 4-Example 7 have the most significant effects on improving the five direct indicators of hemoglobin content, ATPase activity, EPO content, liver index, and spleen index, while Example 5 also has a good effect on cellular immunity, and Example 7 also has a good effect on improving body weight ability. Comparing Comparative Example 4 with Example 4, it can be seen that the steam heating treatment is better than the boiling heating group; comparing Comparative Example 5-Comparative Example 7 with Example 4, it can be seen that when the pre-treatment method is the same, the black chicken peptide prepared by enzymatic hydrolysis of neutral protease and bromelain has better blood-tonifying activity; Comparative Example 8-Comparative Example 10 is compared with Example 4, and it can be seen that ceramic membrane filtration can better retain the active ingredients in the black chicken peptide and thus exert a better blood-tonifying effect. Based on the above results, the heating method and enzymatic hydrolysis process of the pre-treatment have a greater impact on the efficacy of the product. The blood-tonifying and nourishing functions of the black chicken peptides of Example 5 and Example 7 are the strongest, and the blood-tonifying functions of the black chicken peptides of Example 4 and Example 7 are relatively strong.

Claims

1. A method for preparing black-bone chicken peptide with blood-enriching function, characterized in that: The following steps are involved: (1) heat-treating the black-bone chicken and cutting the meat and bones into minced meat; the heat-treating is steam heat-treating, and the conditions of the steam heat-treating are: temperature 100-120° C., time 1-5 hours; (2) mixing the minced meat obtained in step (1) with water to obtain a liquid material, adding a mixed enzyme for enzymolysis to obtain an enzymolysis solution, wherein the mixed enzyme is a mixture of neutral protease and bromelain, and the mass ratio of neutral protease to bromelain is 0.1-10:0.01-10; (3) The enzymatic hydrolysate obtained in step (2) is subjected to a post-treatment process to obtain the black-bone chicken peptide.

2. The preparation method according to claim 1, characterized in that: In step (1), the steam heat treatment is carried out at a temperature of 100-120° C. and for 2-3 hours.

3. The preparation method according to claim 1, characterized in that: In step (2), the conditions for enzymatic hydrolysis are: temperature 45-65°C, pH 5-8, and time 4-8 hours.

4. The preparation method according to claim 1, characterized in that: In step (2), the mass ratio of the neutral protease to bromelain is 3:

1.

5. The preparation method according to claim 1, characterized in that: In step (2), the amount of the neutral protease added is 0.1-2% of the solid content of the liquid material, and the amount of the bromelain added is 0.01-2% of the solid content of the liquid material.

6. The preparation method according to claim 5, characterized in that In step (2), the amount of the neutral protease added is 0.18% of the solid content of the liquid material, and the amount of the bromelain added is 0.06% of the solid content of the liquid material.

7. The preparation method according to claim 1, characterized in that In step (2), the conditions for enzymatic hydrolysis are: temperature 50-60°C, pH 6-7, and enzymatic hydrolysis for 5-7 hours.

8. The preparation method according to claim 1, characterized in that: In step (3), the post-treatment process includes enzyme inactivation, cooling, filtration, concentration and drying; the filtration method is to use a ceramic membrane for filtration.

9. The preparation method according to claim 8, characterized in that: The conditions for the ceramic membrane filtration are: the pore size of the ceramic membrane is 200 nm; and the filtration pressure is 0.16-0.2 Mpa.

10. Use of the black chicken peptide prepared by the preparation method according to any one of claims 1 to 9 in the preparation of blood-tonifying drugs.

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