Preparation method and application of chicken blood heme peptide

Abstract

The invention discloses a preparation method and application of chicken blood heme peptide, and belongs to the technical field of deep processing of animal blood. The preparation method comprises the following steps: S1, carrying out anticoagulation on chicken blood by adopting an anticoagulant, centrifuging, and collecting blood cells; s2, after the blood cells are washed with normal saline, the blood cells are mixed with a Tris-HCl solution to be split for 1-2 h, centrifugation is conducted, supernate is taken, and lysate is obtained; and S3, mixing the lysate with a protease solution, performing enzymolysis, performing heating enzyme deactivation, and performing centrifugation to obtain supernate, thereby obtaining the chicken blood heme peptide. The preparation method of the chicken blood heme peptide provided by the invention does not use toxic chemical substances, and is safe and environment-friendly; reaction conditions are mild, and the production process is simple and controllable; fresh chicken blood is used, the cost is low, the equipment investment requirement is not high, continuous operation can be achieved, and the method is suitable for industrial production. The method is suitable for preparing food, health care products or feed additives with the effect of improving iron-deficiency anemia.

Description

Technical Field

[0001] The present invention belongs to the technical field of deep processing of animal blood, and relates to a method for preparing heme peptide from chicken blood, specifically a preparation method and application of chicken blood heme peptide. Background Art

[0002] Iron is an essential trace element for the growth and development of the body and the maintenance of health, and is also a necessary metal element for the vast majority of organisms. In the state of iron deficiency in the body, it will not only lead to a decrease in the synthesis of hemoglobin and myoglobin in the body, causing anemia, but also affect the functions of the body's immune, gastrointestinal, and endocrine systems, resulting in various metabolic disorders in the body. At present, most of the iron fortifiers used in China are inorganic irons such as ferrous ion preparations. Although they have the effect of iron supplementation, their utilization rate in the body is low, the side effects are large, and they have a special metallic rust smell.

[0003] As a biological iron, heme peptide can be directly absorbed by intestinal mucosal cells, without producing any digestive tract irritation symptoms, has a high biological utilization rate, and is not affected by the dietary structure. It can better promote the hematopoietic function of the bone marrow and is an ideal drug for treating anemia. At present, the main methods for producing heme peptide include the acetone method, the glacial acetic acid method, the surfactant method, etc. These methods use toxic chemical reagents in the production process, cause great environmental pollution, are harmful to the health of production personnel, and it is also easy to leave harmful substances in the products. At the same time, it is difficult to carry out large-scale production. Summary of the Invention

[0004] The purpose of the present invention is to provide a preparation method and application of chicken blood heme peptide to solve the problems such as the use of toxic chemicals in the current industrial production methods of heme peptide.

[0005] To achieve the above purpose, the technical solution adopted by the present invention is: A preparation method of chicken blood heme peptide, comprising the following steps carried out in sequence: S1. Anticoagulate chicken blood with an anticoagulant, centrifuge, and collect blood cells; S2. After washing the blood cells with physiological saline, mix them with Tris-HCl solution and lyse for 1-2 h, centrifuge and take the supernatant to obtain a lysate; S3. Mix the lysate with a protease solution, carry out enzymatic hydrolysis, heat to inactivate the enzyme, centrifuge and take the supernatant to obtain chicken blood heme peptide.

[0006] As a limitation, the anticoagulant includes ethylenediaminetetraacetic acid (EDTA), sodium citrate or sodium heparin. Preferably, the anticoagulant is sodium citrate.

[0007] As another limitation, the volume ratio of the blood cells to the Tris-HCl solution is 1:2 to 1:5; the pH value of the Tris-HCl solution is 7.0 to 9.0. Preferably, the volume ratio of the blood cells to the Tris-HCl solution is 1:3; the pH value of the Tris-HCl solution is 8.0.

[0008] As the third limitation, the protease includes flavor protease or alkaline protease; the protease addition amount per gram of the substrate is 4000 to 12000 U. Preferably, the protease addition amount per gram of the substrate is 10000 U.

[0009] As a further limitation, the pH value of the flavor protease is 6.0 to 8.0; the pH value of the alkaline protease is 8.5 to 10.5. Preferably, the pH value of the flavor protease is 7.0, and the pH value of the alkaline protease is 10.0.

[0010] As the fourth limitation, for the enzymatic hydrolysis, the temperature is 40 to 60 °C, and the time is 2 to 10 h. Preferably, the enzymatic hydrolysis temperature is 45 to 50 °C, and the time is 4 to 8 h.

[0011] As the fifth limitation, it further includes drying the chicken blood heme peptide.

[0012] As a further limitation, the drying treatment includes spray drying, vacuum drying, or vacuum freeze drying.

[0013] As a further limitation, for the spray drying, the inlet temperature is 120 to 140 °C, and the outlet temperature is 100 °C; for the vacuum drying, the temperature is 80 °C, and the vacuum degree is 0.1 MPa; for the vacuum freeze drying, the temperature is -40 °C, and the vacuum degree is 0.12 mbar.

[0014] The present invention also provides an application of the heme peptide prepared by the above preparation method, and the heme peptide is used to prepare foods, health products, or feed additives with the efficacy of improving iron deficiency anemia.

[0015] Due to the adoption of the above technical solutions, compared with the prior art, the technical progress achieved by the present invention is as follows: ① A preparation method of chicken blood heme peptide provided by the present invention does not use toxic chemical substances, is safe and environmentally friendly; the reaction conditions are mild, and the production process is simple and controllable; fresh chicken blood is used, the cost is low, and the requirements for equipment investment are not high, and it can be continuously operated, which is suitable for industrial production; ② The product prepared by the method for preparing chicken blood heme peptide provided by the present invention has good safety, and its hemolytic property is within the safe range according to the drug concentration-dependent threshold; it has good water solubility and can be dissolved in solutions with a pH value of 6 to 10; it has good temperature stability and is stable at 40 to 80 °C; it can tolerate gastrointestinal digestion and has a good iron supplement effect, and is suitable for preparing foods, health products or feed additives with the efficacy of improving iron deficiency anemia, and has a wide range of applications. Description of the Drawings

[0016] Figure 1 Effect of different anticoagulants on hemoglobin absorbance in Example 1; Figure 2 Effect of different lysis methods on hemoglobin concentration in Example 1; Figure 3 Effect of different proteases on heme content and degree of hydrolysis in Example 1; Figure 4 Effect of enzymatic hydrolysis time of different flavor proteases on degree of hydrolysis in Example 2; Figure 5 Effect of enzymatic hydrolysis time of different alkaline proteases on degree of hydrolysis in Example 3; Figure 6 Effect of enzymatic hydrolysis temperature of different alkaline proteases on degree of hydrolysis in Example 4; Figure 7 Effect of addition amount of different flavor proteases on degree of hydrolysis in Example 5; Figure 8 Effect of addition amount of different alkaline proteases on degree of hydrolysis in Example 6; Figure 9 Effect of alkaline protease solutions with different pH values on degree of hydrolysis in Example 7; Figure 10 Relationship between heme peptide concentration and hemolysis rate in Example 8; Figure 11 Effect of temperature on the stability of iron ion content in heme peptide in Example 8; Figure 12 Effect of pH value on the stability of iron ion content in heme peptide in Example 8; Figure 13 Effect of in vitro digestion on the stability of iron ion content in heme peptide in Example 8; Figure 14 Change in hemoglobin content of rats in each group in Example 8; Figure 15 Iron content in spleen and liver of rats in each group in Example 8; Figure 16 Organ index of rats in each group in Example 8. Detailed Description of the Invention

[0017] The present invention will be further described in detail below through specific embodiments. It should be understood that the described embodiments are only used to explain the present invention and do not limit the present invention.

[0018] Unless otherwise specified, the experimental methods used in the following embodiments are all conventional methods in the art.

[0019] Unless otherwise specified, the materials, reagents, etc. used in the following embodiments can all be obtained from commercial channels.

[0020] Example 1 This example discloses a preparation method of chicken blood heme peptide, which specifically includes the following steps carried out in sequence: S1. Take fresh chicken blood, add sodium citrate according to a volume ratio of 9:1, mix well, carry out anticoagulation treatment, centrifuge at 6000 rpm for 10 min, and collect the sedimented blood cells.

[0021] As a control, replace sodium citrate with EDTA and sodium heparin respectively, centrifuge at 6000 rpm for 10 min, and measure the absorbance value of hemoglobin. The results are as Figure 1 shown; From Figure 1 it can be seen that EDTA, sodium citrate, and sodium heparin can all be used as anticoagulants for chicken blood. Among them, sodium citrate has the best effect, and basically no hemoglobin leaks out.

[0022] S2. Add 5 volumes of physiological saline to the blood cells. After washing and centrifuging multiple times, add a Tris-HCl solution with a pH value of 8.0 that is 3 times the volume of the blood cells, mix and lyse for 1 h, centrifuge and take the supernatant to obtain the lysate.

[0023] As a control, methods such as freeze-thaw method, homogenization method, swelling method, and lysis method were also used to lyse the blood cells respectively. The operations are as follows: Freeze-thaw method: Place the blood cells in a refrigerator at -40 °C, freeze for 20 min, take them out, melt them at room temperature, mix them evenly with physiological saline, centrifuge and take the supernatant to obtain the lysate; Homogenization method: Mix the blood cells and deionized water at a volume ratio of 1:9, then homogenize at 12000 r / min with a high-speed disperser for 60 s, centrifuge and take the supernatant to obtain the lysate; Swelling method: Mix the blood cells and deionized water at a volume ratio of 1:9, start the magnetic stirrer, set the stirring time to 10 min, centrifuge and take the supernatant to obtain the lysate; Use the sodium lauryl sulfate hemoglobin (SLS-Hb) method to measure the hemoglobin concentration in the lysate respectively. The results are as Figure 2 shown; From Figure 2It can be seen that the use of Tris-HCl solution for mixed lysis is most suitable for the lysis of chicken blood red blood cells, and its hemoglobin content is the best.

[0024] S3. Mix the lysis solution with sodium carbonate-sodium bicarbonate buffer in a volume ratio of 1:9. According to the standard of adding 8000 U of alkaline protease per gram of substrate protease, add alkaline protease with a pH value of 10.0, adjust the temperature of the water bath shaker to 50 °C, enzymatically hydrolyze for 4 h. After the enzymatic hydrolysis is completed, heat to 100 °C to inactivate the enzyme for 5 min, centrifuge to obtain the supernatant, and obtain the heme peptide solution.

[0025] As a control, replace the alkaline protease with pepsin, neutral protease, papain, trypsin, flavor protease and acidic protease respectively, and use the colorimetric method to determine the heme content and the formaldehyde titration method to determine the hydrolysis degree of hemoglobin. The results are as Figure 3 shown; From Figure 3 it can be seen that considering both the heme content and the hydrolysis degree of hemoglobin, the enzymatic hydrolysis effects of flavor protease and alkaline protease are better.

[0026] S4. Dry the heme peptide solution by vacuum freeze-drying method at a temperature of -40 °C and a vacuum degree of 0.12 mbar. After vacuum freeze-drying for 24 h, the powdery product of chicken blood heme peptide is obtained.

[0027] Take an appropriate amount of the dried powdery product and use atomic absorption spectrometry to determine its iron content, and the iron content is obtained as 70 mg / 100 g.

[0028] Example 2 This example discloses a preparation method of chicken blood heme peptide, which specifically includes the following steps carried out in sequence: S1. Take fresh chicken blood, add sodium citrate in a volume ratio of 9:1, mix well, carry out anticoagulation treatment, centrifuge at 6000 rpm for 10 min, and collect the sedimented blood cells.

[0029] S2. Add 5 times the volume of physiological saline to the blood cells. After washing and centrifuging multiple times, add Tris-HCl solution with a pH value of 7.0 and a volume 2 times that of the blood cells, mix and lyse for 2 h, centrifuge to obtain the supernatant, and obtain the lysis solution.

[0030] S3. Mix the lysis solution with PBS buffer in a volume ratio of 1:9. According to the standard of adding 10000 U of flavor protease per gram of substrate protease, add flavor protease with a pH value of 6.0, adjust the temperature of the water bath shaker to 50 °C, enzymatically hydrolyze for 2 - 10 h respectively. After the enzymatic hydrolysis is completed, heat to 100 °C to inactivate the enzyme for 5 min, centrifuge to obtain the supernatant, and obtain the heme peptide solution.

[0031] Use the formaldehyde titration method to determine the hydrolysis degree of hemoglobin respectively. The results are asFigure 4 as shown It can be seen from Figure 4 that within the range of 2 - 10 h of enzymatic hydrolysis time, it is suitable for the preparation of heme peptides, and the degree of hydrolysis is 20.63% - 36.30%.

[0032] S4. The heme peptide solution is dried by spray drying (the inlet temperature is 120 °C and the outlet temperature is 100 °C) to obtain the powdery product of chicken blood heme peptide.

[0033] Example 3 This example discloses a method for preparing chicken blood heme peptide, which specifically includes the following steps carried out in sequence: S1. Take fresh chicken blood, add sodium citrate according to a volume ratio of 9:1, mix well, carry out anticoagulation treatment, centrifuge at 6000 rpm for 10 min, and collect the sedimented blood cells.

[0034] S2. Add 5 times the volume of physiological saline to the blood cells. After washing and centrifuging multiple times, add a Tris - HCl solution with a pH value of 9.0 and a volume 5 times that of the blood cells, mix and lyse for 1.5 h, centrifuge and take the supernatant to obtain the lysate.

[0035] S3. Mix the lysate with sodium carbonate - sodium bicarbonate buffer in a volume ratio of 1:9. According to the standard of adding 8000 U of protease per gram of substrate, add alkaline protease with a pH value of 10.0, adjust the temperature of the water bath shaker to 50 °C, and carry out enzymatic hydrolysis for 2 - 10 h respectively. After the enzymatic hydrolysis is completed, heat to 100 °C to inactivate the enzyme for 5 min, centrifuge and take the supernatant to obtain the heme peptide solution.

[0036] The degree of hydrolysis of hemoglobin is measured by the formaldehyde titration method, and the results are as Figure 5 shown It can be seen from Figure 5 that within the range of 2 - 10 h of enzymatic hydrolysis time, it is suitable for the preparation of heme peptides, and the degree of hydrolysis is 16.59% - 20.60%.

[0037] S4. The heme peptide solution is dried by vacuum drying (the temperature is 80 °C and the vacuum degree is 0.1 MPa) to obtain the powdery product of chicken blood heme peptide.

[0038] Example 4 This example discloses a method for preparing chicken blood heme peptide, which specifically includes the following steps carried out in sequence: S1. Take fresh chicken blood, add sodium citrate according to a volume ratio of 9:1, mix well, carry out anticoagulation treatment, centrifuge at 6000 rpm for 10 min, and collect the sedimented blood cells.

[0039] S2. Add normal saline with a volume 5 times that of the blood cells. After washing and centrifuging multiple times, add Tris-HCl solution with a pH of 9.0 and a volume 3 times that of the blood cells, mix and lyse for 1.5 h, then centrifuge and take the supernatant to obtain the lysate.

[0040] S3. Mix the lysate with sodium carbonate-sodium bicarbonate buffer in a volume ratio of 1:9. According to the standard of adding 8000 U of alkaline protease per gram of substrate protease, add alkaline protease with a pH of 10.0, adjust the temperature of the water bath shaker to 40 - 60 °C, enzymatically hydrolyze for 4 h. After the enzymatic hydrolysis is completed, heat to 100 °C to inactivate the enzyme for 5 min, centrifuge and take the supernatant to obtain the heme peptide solution.

[0041] The degree of hydrolysis of hemoglobin was measured by the formaldehyde titration method, and the results are as Figure 6 shown; From Figure 6 it can be seen that the enzymatic hydrolysis temperature in the range of 40 - 60 °C is suitable for the preparation of heme peptides. Among them, under the condition of 45 °C, the degree of hydrolysis is the highest, reaching 23.80%.

[0042] S4. Dry the heme peptide solution by vacuum freeze-drying method at a temperature of -40 °C and a vacuum degree of 0.12 mbar. After vacuum freeze-drying for 24 h, the powdery product of chicken blood heme peptide is obtained.

[0043] Example 5 The preparation method of chicken blood heme peptide disclosed in this example specifically includes the following steps carried out in sequence: S1. Take fresh chicken blood, add sodium citrate according to a volume ratio of 9:1, mix well, carry out anticoagulation treatment, centrifuge at 6000 rpm for 10 min, and collect the sedimented blood cells.

[0044] S2. Add normal saline with a volume 5 times that of the blood cells. After washing and centrifuging multiple times, add Tris-HCl solution with a pH of 9.0 and a volume 3 times that of the blood cells, mix and lyse for 1.5 h, then centrifuge and take the supernatant to obtain the lysate.

[0045] S3. Mix the lysate with the buffer in a volume ratio of 1:9. According to the standard of adding 4000 - 12000 U of flavor protease per gram of substrate protease, add flavor protease with a pH of 8.0, adjust the temperature of the water bath shaker to 50 °C, enzymatically hydrolyze for 8 h. After the enzymatic hydrolysis is completed, heat to 100 °C to inactivate the enzyme for 5 min, centrifuge and take the supernatant to obtain the heme peptide solution.

[0046] The degree of hydrolysis of hemoglobin was measured by the formaldehyde titration method, and the results are as Figure 7 shown; From Figure 7It can be seen that chicken blood heme peptides can be obtained when the addition amount of flavor protease is in the range of 4000 - 12000 U / g, and the degree of hydrolysis is 22.34% - 38.75%. Among them, when the addition amount of flavor protease is 10000 U / g, the degree of hydrolysis reaches 38.75%.

[0047] S4. Dry the heme peptide solution by vacuum freeze-drying at a temperature of -40°C and a vacuum degree of 0.12 mbar. After 24 hours of vacuum freeze-drying, the powdery product of the chicken blood heme peptide is obtained.

[0048] Example 6 This example discloses a preparation method of chicken blood heme peptide, which specifically includes the following steps carried out in sequence: S1. Take fresh chicken blood, add sodium citrate according to a volume ratio of 9:1, mix well, carry out anticoagulation treatment, centrifuge at 6000 rpm for 10 min, and collect the sedimented blood cells.

[0049] S2. Add 5 times the volume of physiological saline to the blood cells. After washing and centrifuging multiple times, add a Tris-HCl solution with a pH value of 9.0 that is 3 times the volume of the blood cells, mix and lyse for 2 h, centrifuge and take the supernatant to obtain the lysate.

[0050] S3. Mix the lysate with sodium carbonate-sodium bicarbonate buffer in a volume ratio of 1:9. According to the standard of adding 4000 - 12000 U of protease per gram of substrate, add alkaline protease with a pH value of 10.0, adjust the temperature of the water bath shaker to 45°C, enzymatically hydrolyze for 4 h. After the enzymatic hydrolysis is completed, heat to 100°C to inactivate the enzyme for 5 min, centrifuge and take the supernatant to obtain the heme peptide solution.

[0051] The degree of hydrolysis of hemoglobin was measured by the formaldehyde titration method, and the results are as Figure 8 shown; It can be seen from Figure 8 that chicken blood heme peptides can be obtained when the addition amount of alkaline protease is in the range of 4000 - 12000 U / g, and the degree of hydrolysis is 18.34% - 26.85%. Among them, when the optimal addition amount of alkaline protease is 10000 U / g, the degree of hydrolysis reaches 26.25%.

[0052] S4. Dry the heme peptide solution by vacuum freeze-drying at a temperature of -40°C and a vacuum degree of 0.12 mbar. After 24 hours of vacuum freeze-drying, the powdery product of the chicken blood heme peptide is obtained.

[0053] Example 7 This example discloses a preparation method of chicken blood heme peptide, which specifically includes the following steps carried out in sequence: S1. Take fresh chicken blood, add sodium citrate according to a volume ratio of 9:1, mix well, perform anticoagulation treatment, centrifuge at 6000 rpm for 10 min, and collect the sedimented blood cells.

[0054] S2. Add 5 times the volume of normal saline to the blood cells. After washing and centrifuging multiple times, add a Tris-HCl solution with a pH of 9.0 that is 3 times the volume of the blood cells, mix and lyse for 1.5 h, centrifuge and take the supernatant to obtain the lysate.

[0055] S3. Mix the lysate with a sodium carbonate-sodium bicarbonate buffer in a volume ratio of 1:9. Add an alkaline protease with a pH of 8.5 - 10.5 according to the standard of an addition amount of 10000 U per gram of substrate protease. Adjust the temperature of the water bath shaker to 45°C and enzymatically hydrolyze for 4 h. After the enzymatic hydrolysis is completed, heat to 100°C to inactivate the enzyme for 5 min, centrifuge and take the supernatant to obtain the heme peptide solution.

[0056] The degree of hydrolysis of hemoglobin was measured by the formaldehyde titration method, and the results are as Figure 9 shown; It can be Figure 9 seen that when the pH value of the alkaline protease solution is 8.5 - 10.5, the enzymatic hydrolysis effect is better, and the degree of hydrolysis is 13.67% - 26.29%.

[0057] S4. Dry the heme peptide solution by vacuum freeze-drying at a temperature of -40°C and a vacuum degree of 0.12 mbar. After 24 h of vacuum freeze-drying, the powdery product of chicken blood heme peptide is obtained.

[0058] Example 8 This example provides an application of chicken blood heme peptide. The heme peptide prepared in Example 7 (the product obtained by enzymatic hydrolysis with an alkaline protease with a pH of 10.0) is used to prepare a health product with the efficacy of improving iron deficiency anemia, and the related properties of the heme peptide are verified, which specifically include the following steps: (I) Solubility of the product Take a heme standard product (HPLC ≥ 98%) and the powdery product of the heme peptide prepared in Example 7, add 0.1 mol / L NaOH to dissolve, and prepare solutions with the same heme concentration. Adjust the pH values to 2, 4, 6, 8, and 10 respectively. After standing for 20 min, centrifuge and take the supernatant to measure the OD 385 value. The results are shown in Table 1: Table 1 Content of heme in the solution at different pH values pH 2 4 6 8 10 Heme content in the standard product (μg / mL) 1.19±0.05 0.86±0.05 56.15±0.61 64.43±0.45 74.05±0.28 Heme content in the product (μg / mL) 11.67±0.51 3.56±0.16 61.89±0.56 68.09±0.39 71.75±1.01 As can be seen from Table 1, heme peptide and heme standard have strong solubility when the pH value is greater than 6; when the pH value is less than 4, the solubility is small, and the solubility of the heme peptide prepared by the preparation method provided by the present invention is greater than that of the heme standard. The chicken blood heme peptide prepared by the present invention has good water solubility and can be dissolved in a solution with a pH value of 6-10.

[0059] (II) Hemolytic property and stability of the product (1) Hemolytic test Prepare heme peptide solutions with concentrations of 25, 50, 100, 200, and 400 μg / mL respectively with physiological saline, then mix them with the erythrocyte suspension in equal volumes. After incubating at 37 °C for 1 h, centrifuge at 1500 r / min for 5 min, take the supernatant, and measure its absorbance value at OD 540 value. Use 0.1% TritonX-100 with a final concentration as the positive control and physiological saline as the negative control. The results are as Figure 10 shown; As can be seen from Figure 10 the above, the hemolytic property of heme peptide is weak at a certain concentration. According to the drug concentration-dependent threshold analysis table, when the test concentration < 10 μg / mL, the acceptable hemolysis rate ≤ 5%; when the test concentration is 10-100 μg / mL, the acceptable hemolysis rate ≤ 10%; when the test concentration > 100 μg / mL (suprapharmacological concentration), the acceptable hemolysis rate ≤ 20%. Therefore, heme peptide is within the safe and acceptable hemolysis rate range.

[0060] (2) Temperature stability test Prepare a 5 mg / mL heme peptide solution, incubate it in a water bath at 40, 50, 60, 70, and 80 °C for 1 h respectively, cool it to room temperature, and then centrifuge at 5000 r / min for 10 min to investigate the effect of temperature on the iron ions with iron-supplementing function in heme peptide. Use the treatment group at room temperature as the blank control. The results are as Figure 11 shown; As can be seen from Figure 11 the above, the iron ions in heme peptide have good thermal stability.

[0061] (3) pH stability test Prepare a 5 mg / mL heme peptide solution, place it at room temperature for 1 h under the conditions of pH values of 3.0, 5.0, 7.0, 9.0, and 11.0 respectively. After the reaction ends, centrifuge it to investigate the effect of pH on the iron ions in heme peptide. Use the treatment group without adjusting the pH as the blank control. The results are as Figure 12 shown; As can be seen from Figure 12 the above, the iron ions in heme peptide have good stability at different pH values.

[0062] (4)Simulated gastrointestinal digestion The pH value of the heme peptide solution at 5 mg / mL was adjusted to 2.0 with 1 mol / L HCl solution, then 1000 U / mg pepsin was added, and the reaction was carried out in the dark in a constant temperature water bath at 37 °C and 160 r / min for 90 min. After digestion, it was placed in a boiling water bath for 5 min. After cooling, it was centrifuged (5000 r / min, 10 min), and the supernatant was collected to measure the iron ion content in the heme peptide. The treatment group under the pH environment without adding pepsin was used as the control group; The supernatant after 90 min of pepsin digestion was taken, the pH value was adjusted to 7.5 with NaOH, 1000 U / mg trypsin was added, and the digestion was carried out in the dark in a constant temperature water bath at 37 °C and 160 r / min for 90 min. The digestion was terminated by heating in a boiling water bath for 5 min. After the sample was cooled, it was centrifuged at 5000 r / min for 10 min, and the supernatant was collected to measure the iron ion content of the heme peptide solution. The results are as Figure 13 shown; As can be seen from Figure 13 it, the iron ion content in the heme peptide still has good stability after digestion with pepsin and trypsin.

[0063] (III)Iron supplementation effect of the product An iron-deficiency anemia model was established by feeding rats with an iron-deficient diet (using Hb < 100 g / L as the standard for judging iron-deficiency anemia). A normal group (fed with a normal diet and gavaged with physiological saline), an iron-deficient group (fed with an iron-deficient diet and gavaged with an equal amount of physiological saline), a positive group (fed with an iron-deficient diet and gavaged with a succinic acid ferrous solution at 5 mg / kg body weight), and a heme peptide group (fed with an iron-deficient diet and gavaged with an equal amount of heme peptide solution) were set up. Among them, the heme peptide samples were gavaged into iron-deficient anemic rats at 2 mg / kg (low-dose group) and 5 mg / kg (high-dose group) body weight respectively. After the model was successfully established, the initial content of hemoglobin in each group of rats was measured using a kit from Solarbio Science & Technology Co., Ltd. After continuous gavage for 3 weeks, the rats were sacrificed, and the final content of hemoglobin in each group of rats was measured again. The results are Figure 14 shown; As can be seen from Figure 14 it, the hemoglobin content in the low-dose heme peptide group and the high-dose heme peptide group increased with the increase of the heme peptide concentration, and the hemoglobin content of these two groups of rats recovered compared with the initial content. This shows that the heme peptide has a certain iron supplementation effect.

[0064] The iron changes in the liver and spleen tissues of each group of rats were measured. The results are Figure 15 shown; As can be seen from Figure 15It can be seen that the iron content in the liver and spleen tissues of the low-dose heme peptide group and the high-dose heme peptide group increases with the increase of the heme peptide concentration, and the iron content in the liver and spleen tissues of these two groups of rats has recovered compared with the iron-deficient group, indicating that heme peptide has a certain iron-supplementing effect and can improve the iron content in the spleen and liver.

[0065] The organ coefficients of rats in each group were measured, and the results Figure 16 are shown as follows: As Figure 16 can be seen, there were no particularly obvious changes in the liver and kidney coefficients of rats in iron-deficiency anemia symptoms, while the spleen and heart showed splenomegaly, ventricular hypertrophy and dilation. However, with the use of heme peptide, the anemia symptoms of rats were improved to varying degrees, indicating that heme peptide has a certain iron-supplementing effect.

Claims

1. A preparation method of chicken blood heme peptide, characterized in that, It includes the following steps carried out sequentially: S1. Anticoagulate chicken blood with an anticoagulant, centrifuge, and collect blood cells. S2. After washing the blood cells with physiological saline, mix them with Tris-HCl solution and lyse for 1 - 2 h, centrifuge and take the supernatant to obtain the lysate. S3. Mix the lysate with a protease solution, carry out enzymatic hydrolysis, heat to inactivate the enzyme, centrifuge and take the supernatant to obtain chicken blood heme peptide.

2. The preparation method of a chicken blood heme peptide according to claim 1, characterized in that, The anticoagulant includes ethylenediaminetetraacetic acid, sodium citrate or sodium heparin.

3. The preparation method of a chicken blood heme peptide according to claim 1, characterized in that, The volume ratio of the blood cells to the Tris-HCl solution is 1:2 - 1:

5. The pH value of the Tris-HCl solution is 7.0 - 9.

0.

4. A preparation method of chicken blood heme peptide according to any one of claims 1 to 3, characterized in that, The protease includes flavor protease or alkaline protease. The protease addition amount per gram of substrate is 4000 - 12000 U.

5. A method for preparing chicken blood heme peptide according to claim 4, characterized in that, The pH value of the flavor protease is 6.0 - 8.0; the pH value of the alkaline protease is 8.5 - 10.

5.

6. A method for preparing chicken blood heme peptide according to any one of claims 1 to 3, characterized in that, For the enzymatic hydrolysis, the temperature is 40 - 60 °C and the time is 2 - 10 h.

7. A method for preparing chicken blood heme peptide according to any one of claims 1 to 3, characterized in that, It also includes drying the chicken blood heme peptide.

8. The preparation method of a chicken blood heme peptide according to claim 7, characterized in that, The drying treatment includes spray drying, vacuum drying or vacuum freeze drying.

9. The preparation method of a chicken blood heme peptide according to claim 8, characterized in that, For the spray drying, the inlet temperature is 120 - 140 °C and the outlet temperature is 100 °C; for the vacuum drying, the temperature is 80 °C and the vacuum degree is 0.1 MPa; for the vacuum freeze drying, the temperature is -40 °C and the vacuum degree is 0.12 mbar.

10. Use of the heme peptide prepared by the preparation method according to any one of claims 1 to 9, characterized in that, The heme peptide is used to prepare foods, health products or feed additives with the efficacy of improving iron deficiency anemia.

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