Preparation method of blood protein polypeptide rich in heme iron
Through low temperature extraction and ultrasonic assisted technology, optimization of enzymatic decomposition and iron binding conditions, and adopting efficient purification and concentration technology, problems such as low extraction efficiency and incomplete enzymatic decomposition in the preparation process of blood protein polypeptides in the prior art have been solved, and efficient, safe and economical preparation of heme iron-rich hemoprotein polypeptides is achieved.
Patent Information
- Application Number
- CN202510127353.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-04
- Publication Date
- 2025-05-30
AI Technical Summary
The preparation method for the heme iron-rich heme polypeptide in the prior art has low extraction efficiency, incomplete enzymatic decomposition, low iron release efficiency, complex purification process, high production cost and possible hygiene and safety problems.
Low-temperature extraction and ultrasonic assisted technology are used to improve the extraction rate of hemoglobin, special enzymes are selected for enzymatic decomposition and optimize the enzymatic decomposition conditions, improve the bioavailability of iron through iron ion binding and reduction treatment, and use efficient purification technologies such as ultrafiltration and affinity chromatography to optimize the concentration and drying process to ensure the purity and safety of the product.
It improves the extraction rate of hemoglobin and the biological activity of the peptide, improves the bioavailability of iron, reduces production costs, improves the purity and safety of the product, and meets the nutritional supplement needs.
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biotechnology, and particularly to a method for preparing hemoglobin polypeptide rich in heme iron. Background Art
[0002] The method for preparing hemoglobin polypeptide rich in heme iron generally includes the following steps: First, hemoglobin in animal blood is separated and extracted. Usually, pig blood, bovine blood or other sources rich in hemoglobin are selected. The extraction process can adopt methods such as solution extraction and centrifugal separation to separate hemoglobin from red blood cells. Then, through enzymatic hydrolysis technology or acid-base hydrolysis, etc., hemoglobin is decomposed into smaller polypeptide fragments, and these polypeptide fragments retain the binding structure of heme iron. In order to improve the bioavailability of heme iron, it is usually necessary to further purify the hemoglobin polypeptide, remove impurities, and ensure its iron-rich characteristics. Finally, advanced ultrafiltration, freeze-drying and other technologies are used to concentrate and dry the hemoglobin polypeptide to obtain the final hemoglobin polypeptide powder or liquid preparation rich in heme iron, which is widely used in nutritional supplements, medicine and the food industry. The core of this method lies in maintaining the effectiveness of heme iron through enzymatic hydrolysis and purification of hemoglobin, so as to provide a good iron source;
[0003] In the prior art, although the method for preparing hemoglobin polypeptide rich in heme iron has achieved certain results to a certain extent, there are still the following several disadvantages:
[0004] Low extraction efficiency: The traditional hemoglobin extraction process usually relies on methods such as solution extraction or centrifugal separation, but these methods have a low utilization rate of blood and may cause partial degradation or loss of hemoglobin, affecting the purity and efficacy of the final product;
[0005] Incomplete enzymatic hydrolysis process: Hemoglobin may be affected by incomplete hydrolysis or non-specific degradation during the enzymatic hydrolysis process, resulting in some of the obtained polypeptide fragments not being rich in heme iron or having low biological activity. This limits the bioavailability and iron absorption efficiency of the prepared hemoglobin polypeptide;
[0006] Low iron release efficiency: The absorption of heme iron in the body is affected by multiple factors, including the binding mode of iron and the structure of the polypeptide. If heme iron fails to effectively bind to the polypeptide or its release is inhibited during the preparation process, the final bioavailability of heme iron is low and its nutritional value may not be fully exerted;
[0007] Complex purification process: The purification process of hemoglobin polypeptide is usually relatively complex and requires multiple steps of filtration, centrifugation and chromatography, etc. These steps not only increase the preparation cost, but also may cause product loss and residue of impurities, affecting the quality and safety of the final product;
[0008] High production cost: Due to the cumbersome processes of extraction, enzymatic hydrolysis, purification, etc., and the need for a large amount of animal blood raw materials, the production cost of the existing technology is relatively high, which limits its large-scale application. In addition, materials such as equipment and enzyme preparations involved in the production process may also increase the overall cost;
[0009] There may be health and safety issues: Since animal blood is used as a raw material, there may be a risk of pathogenic microorganisms. If the sanitary conditions during the production process cannot be effectively controlled, it may affect the safety of the final product and the health of consumers;
[0010] The processing process has a great impact on the polypeptide structure: During the preparation of blood protein polypeptides, factors such as high temperature, acid-base conditions, the type and dosage of enzymes may affect the spatial structure and activity of polypeptides. The structural change may lead to a weakened binding ability of heme iron, thus affecting its nutritional value and biological efficacy.
[0011] Therefore, we propose a method for preparing blood protein polypeptides rich in heme iron. Summary of the Invention
[0012] To achieve the above object, the present invention provides the following technical solution: A method for preparing blood protein polypeptides rich in heme iron, comprising the following steps:
[0013] S1: Optimize blood source and pretreatment
[0014] S1.1: Select high-quality blood source: Use fresh disease-free animal blood (such as pig blood, bovine blood or chicken blood), and ensure the safety of the blood source to avoid contamination by pathogenic microorganisms. Frozen or liquid blood meeting the quality standards can be selected;
[0015] S1.2: Pretreatment: Store the blood using low-temperature refrigeration to avoid denaturation or degradation of hemoglobin during storage. Then, separate the red blood cells in the blood by the freeze centrifugation method and remove impurities such as plasma to obtain pure red blood cells.
[0016] S2: Efficient hemoglobin extraction
[0017] S2.1: Low-temperature extraction: To avoid the destruction of hemoglobin by high temperature, extract the red blood cells in a low-temperature environment (4°C). Use a buffer solution (such as phosphate buffer solution) to rupture the red blood cells and release hemoglobin. Further break the cells by ultrasonic treatment or high-pressure homogenization to improve the extraction rate of hemoglobin;
[0018] S2.2: Centrifugal separation: The extract is centrifuged to remove cell debris and other impurities, and the supernatant rich in hemoglobin is retained. By centrifuging multiple times, the purity of hemoglobin can be further improved.
[0019] S3: Enzymatic Hydrolysis Optimization
[0020] S3.1: Select the appropriate enzyme species: Use enzymes specifically targeting hemoglobin, such as pepsin, trypsin, or modified enzyme preparations. These enzymes can efficiently hydrolyze hemoglobin, releasing hemoglobin polypeptides rich in heme iron. The selected enzyme should have high selectivity to avoid damaging the heme iron-binding part;
[0021] S3.2: Optimize enzymatic hydrolysis conditions: Adjust the temperature, pH value, and time of the enzymatic hydrolysis reaction. Generally, the enzymatic hydrolysis temperature should be controlled between 35 - 45 °C, and the pH value should be controlled between 6 - 8 to maximize the enzyme activity and hemoglobin hydrolysis efficiency. The enzymatic hydrolysis time should be controlled between 4 - 8 hours to avoid inactivation of polypeptides due to over-hydrolysis;
[0022] S3.3: End of reaction and neutralization: After the reaction is completed, stop the enzymatic hydrolysis reaction by heating or acid-base neutralization to ensure the stability of hemoglobin polypeptides.
[0023] S4: Binding and Optimization of Heme Iron
[0024] S4.1: Optimization of iron ion binding: Add an iron ion source (such as iron salt) to the polypeptide solution after enzymatic hydrolysis to promote the binding of heme iron. By adjusting the pH value and temperature conditions, optimize the iron ion binding efficiency. Generally, the pH value is controlled between 6.5 - 7.5, and the temperature is maintained at 30 - 40 °C to promote the effective binding of iron and the stability of polypeptides;
[0025] S4.2: Reduction treatment of iron: To improve the bioavailability of iron, a reducing agent (such as ascorbic acid) can be used to reduce ferric iron to ferrous iron, enhancing the absorption and utilization of heme iron.
[0026] S5: Purification and Separation
[0027] S5.1: Multi-step purification: Further purify hemoglobin polypeptides using ultrafiltration technology or affinity chromatography technology. The ultrafiltration membrane can separate polypeptides and low-molecular-weight impurities according to molecular weight to ensure the purity of the final product. Affinity chromatography further removes non-target proteins by the binding of hemoglobin polypeptides to specific ligands, improving the iron binding rate;
[0028] S5.2: Dialysis to remove impurities: Use dialysis to remove residual low-molecular-weight substances, salts, and unbound iron ions to further improve the purity and safety of hemoglobin polypeptides.
[0029] S6: Concentration and Drying
[0030] S6.1: Concentration: Concentrate the hemoglobin polypeptide solution using low-temperature evaporation or freeze concentration technology to remove the solvent, making the final product more concentrated and rich in heme iron;
[0031] S6.2: Drying: Use freeze-drying or spray-drying techniques to convert the concentrated solution into powder for easy storage and transportation. These drying techniques can effectively maintain the activity of the polypeptide and the binding state of heme iron.
[0032] S7: Final product quality inspection and packaging
[0033] S7.1: Quality inspection: Conduct quality inspection on the final product to ensure that the content, purity, and biological activity of its heme iron meet the standards. Detect the composition of the polypeptide and the binding of iron through techniques such as high-performance liquid chromatography (HPLC) and spectrophotometer;
[0034] S7.2: Packaging: Package the final heme iron-rich blood protein polypeptide powder or liquid preparation to ensure that it is not affected by factors such as moisture and oxidation during transportation and storage. Use sealed packaging and antioxidant materials to extend the shelf life of the product.
[0035] Preferably, in step S1, select a blood supplier with reliable sources to ensure that the blood comes from healthy animals. Common sources include pig blood, bovine blood, or chicken blood. It is best to choose the blood of disease-free animals. The blood can be provided in frozen or fresh liquid form. Frozen blood needs to be thawed in advance and used quickly to avoid repeated freezing and thawing. Pour the animal blood into a clean container and use refrigeration technology (4°C) to store it to ensure that hemoglobin does not denature. Separate the red blood cells in the blood by low-speed centrifugation (about 2000 rpm, 5 - 10 minutes). The supernatant after centrifugation contains plasma and should be discarded. Collect the precipitated red blood cells and remove most of the plasma. Rinse the red blood cells with physiological saline 2 - 3 times to remove the remaining plasma and other impurities to obtain a relatively pure red blood cell precipitate.
[0036] Preferably, in step S2, suspend the separated red blood cells in phosphate buffer (PBS, pH 7.4) to ensure that the pH of the buffer matches the most stable pH of hemoglobin. Place the suspension at 4°C and perform ultrasonic disruption in an ultrasonic cleaner. The ultrasonic frequency is 20 - 25 kHz, the power is 200 - 300 W, and each treatment lasts for 5 - 10 minutes to avoid hemoglobin denaturation caused by temperature rise. After ultrasonic disruption, perform centrifugation using a high-speed centrifuge (about 12000 rpm, 10 - 15 minutes) to precipitate red blood cell fragments and macromolecular impurities to obtain the supernatant (hemoglobin solution). Transfer the supernatant to another container as the preliminary extract of hemoglobin.
[0037] Preferably, in step S3, a suitable enzyme is selected according to the target for the hydrolysis of hemoglobin, such as pepsin (e.g., Pepsin) or trypsin (e.g., Trypsin). These enzymes can specifically hydrolyze the peptide bonds in hemoglobin, releasing hemoglobin polypeptides. The hemoglobin solution and the enzyme are mixed in a certain ratio. Usually, the mass ratio of hemoglobin to the enzyme is 100:1. Adjust the pH value of the enzyme solution to the pH of the optimal enzyme activity (for example, the pH value of pepsin is 1.5 - 2.5), control the temperature between 35 - 45 °C, and perform temperature control to prevent the inactivation of the enzyme due to excessive temperature. Usually, the reaction time is set to 4 - 8 hours, and the hydrolysis effect is monitored according to the reaction progress. After the enzymatic hydrolysis reaction is completed, the enzyme activity is stopped by heating to 80 °C and maintaining for 5 - 10 minutes, preventing...
[0038] Preferably, in step S4, an iron source (such as ferric chloride or ferrous sulfate) is added to the polypeptide solution after enzymatic hydrolysis to provide iron ions. The concentration of iron ions is usually 10 - 20 times the mass of hemoglobin to ensure that sufficient iron ions can bind to the polypeptide. The solution temperature is controlled to 30 - 40 °C, and the pH value is adjusted to 6.5 - 7.5 to ensure that the iron ions bind to the heme part of the hemoglobin polypeptide within this pH range. To improve the bioavailability of iron, a reducing agent (such as ascorbic acid or citric acid) can be added to reduce ferric iron to ferrous iron, enhancing the iron absorption capacity. The addition amount of the reducing agent can be controlled at 0.5 - 1.0% (w / w), and the reaction is carried out while maintaining the temperature at about 30 °C.
[0039] Preferably, in step S5, ultrafiltration: The hemoglobin polypeptide solution is concentrated using an ultrafiltration membrane (molecular weight cut-off value of 5 - 10 kDa) to remove low-molecular substances and solvents. Affinity chromatography: Using an affinity chromatography column (such as taking advantage of the affinity of hemoglobin polypeptide binding to specific metal ions), further remove impurities and retain the iron-rich hemoglobin polypeptide. This step helps to separate and improve the iron-binding effect. The solution purified by ultrafiltration and affinity chromatography is dialyzed, and a low-molecular-weight dialysis bag is used to remove salts, unbound iron ions, and small-molecular impurities. Dialysis can be carried out at 4 °C for 12 - 24 hours to ensure complete removal of impurities.
[0040] Preferably, in step S6, low-temperature evaporation concentration or freeze concentration technology (such as using a rotary evaporator or a refrigerated centrifuge) is used to remove excess water. Concentrate to a solid content of 30 - 50% in the hemoglobin polypeptide solution, and use freeze-drying technology (lyophilizer) to convert the concentrated solution into a powder form. The freeze-drying process is carried out at a low temperature, which can effectively maintain the structure and activity of heme iron and polypeptide. Spray drying equipment can also be used to spray the polypeptide solution into fine droplets and quickly evaporate the water in hot air to form dry powder.
[0041] Preferably, in step S7, high performance liquid chromatography (HPLC) is used to analyze the components of the blood protein polypeptide, confirm the binding of heme iron, and a spectrophotometer is used to measure the content of heme iron to ensure compliance with the standards. Microbiological testing is performed on the finished product to ensure the absence of harmful bacteria, heavy metals and other contaminants. Sealed glass bottles or aluminum bags are used for packaging to minimize oxygen and humidity inside the package, avoid oxidation and moisture, and desiccants can be placed in the packaging bag to ensure that the product remains dry during transportation and storage.
[0042] Compared with the prior art, the present invention provides a preparation method of a blood protein polypeptide rich in heme iron, which has the following beneficial effects:
[0043] The preparation method of the blood protein polypeptide rich in heme iron improves the extraction rate of hemoglobin and reduces the degradation of hemoglobin through low-temperature extraction and ultrasonic-assisted technology. By adjusting the enzymatic hydrolysis and iron ion binding conditions, it ensures that heme iron can be fully bound, improves the bioavailability of iron, and uses high-efficiency purification technologies such as ultrafiltration and affinity chromatography to remove impurities, ensuring the purity and safety of the final product. The extraction, enzymatic hydrolysis, purification and other links are optimized, improving production efficiency, reducing the consumption of raw materials and energy. Through precise iron ion reduction treatment and optimized structural design of the blood protein polypeptide, the final product has higher biological activity and absorption efficiency, meeting the nutritional supplement requirements. Detailed implementation manners
[0044] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.
[0045] Embodiment
[0046] An embodiment of a preparation method of a blood protein polypeptide rich in heme iron
[0047] A preparation method of a blood protein polypeptide rich in heme iron, comprising the following steps:
[0048] S1: Optimize blood source and pretreatment
[0049] S1.1: Select high-quality blood source: Use fresh disease-free animal blood (such as pig blood, bovine blood or chicken blood), and ensure the safety of the blood source to avoid contamination by pathogenic microorganisms. Frozen or liquid blood meeting the quality standards can be selected;
[0050] S1.2: Pretreatment: The blood is stored using cryogenic refrigeration to avoid denaturation or degradation of hemoglobin during storage. Then, the red blood cells in the blood are separated by cryogenic centrifugation, and impurities such as plasma are removed to obtain pure red blood cells.
[0051] S2: High-efficiency hemoglobin extraction
[0052] S2.1: Low-temperature extraction: To avoid the destruction of hemoglobin by high temperature, the red blood cells are extracted in a low-temperature environment (4°C). A buffer solution (such as phosphate buffer) is used to rupture the red blood cells and release hemoglobin. The cells are further disrupted by ultrasonic treatment or high-pressure homogenization to improve the extraction rate of hemoglobin;
[0053] S2.2: Centrifugal separation: The extract is centrifuged to remove cell debris and other impurities, and the supernatant rich in hemoglobin is retained. By centrifuging multiple times, the purity of hemoglobin can be further improved.
[0054] S3: Enzymatic hydrolysis optimization
[0055] S3.1: Select the appropriate enzyme species: Enzymes specifically for hemoglobin, such as pepsin, trypsin, or modified enzyme preparations, are used. These enzymes can efficiently hydrolyze hemoglobin and release hemoglobin polypeptides rich in heme iron. The selected enzyme should have high selectivity to avoid damage to the heme iron-binding part;
[0056] S3.2: Optimization of enzymatic hydrolysis conditions: The temperature, pH value, and time of the enzymatic hydrolysis reaction are adjusted. Usually, the enzymatic hydrolysis temperature should be controlled between 35 - 45°C, the pH value is controlled between 6 - 8 to maximize the enzyme activity and hemoglobin hydrolysis efficiency. The enzymatic hydrolysis time should be controlled between 4 - 8 hours to avoid inactivation of the polypeptide due to over-hydrolysis;
[0057] S3.3: End of reaction and neutralization: After the reaction is completed, the enzymatic hydrolysis reaction is stopped by heating or acid-base neutralization to ensure the stability of the hemoglobin polypeptide.
[0058] S4: Binding and optimization of heme iron
[0059] S4.1: Optimization of iron ion binding: An iron ion source (such as iron salt) is added to the polypeptide solution after enzymatic hydrolysis to promote the binding of heme iron. By adjusting the pH value and temperature conditions, the binding efficiency of iron ions is optimized. Usually, the pH value is controlled between 6.5 - 7.5, and the temperature is maintained at 30 - 40°C to promote the effective binding of iron and the stability of the polypeptide;
[0060] S4.2: Reduction treatment of iron: To improve the bioavailability of iron, a reducing agent (such as ascorbic acid) can be used to reduce ferric iron to ferrous iron, enhancing the absorption and utilization of heme iron.
[0061] S5: Purification and Separation
[0062] S5.1: Multi-step Purification: Ultrafiltration technology or affinity chromatography technology is used to further purify the blood protein polypeptide. The ultrafiltration membrane can separate polypeptides and low-molecular impurities according to molecular weight to ensure the purity of the final product. Affinity chromatography further removes non-target proteins by the binding of hemoglobin polypeptide to specific ligands, improving the iron-binding rate;
[0063] S5.2: Dialysis to Remove Impurities: Dialysis method is used to remove residual low-molecular substances, salts and unbound iron ions to further improve the purity and safety of the blood protein polypeptide.
[0064] S6: Concentration and Drying
[0065] S6.1: Concentration: The blood protein polypeptide solution is concentrated by low-temperature evaporation or freeze concentration technology to remove the solvent, making the final product more concentrated and rich in heme iron;
[0066] S6.2: Drying: Freeze drying or spray drying technology is used to convert the concentrated solution into powder for easy storage and transportation. These drying technologies can effectively maintain the activity of the polypeptide and the binding state of heme iron.
[0067] S7: Final Product Quality Inspection and Packaging
[0068] S7.1: Quality Inspection: The final product is subjected to quality inspection to ensure that the content, purity and biological activity of its heme iron meet the standards. High performance liquid chromatography (HPLC), spectrophotometer and other technologies are used to detect the composition of the polypeptide and the iron-binding situation;
[0069] S7.2: Packaging: The final heme iron-rich blood protein polypeptide powder or liquid preparation is packaged to ensure that it is not affected by moisture, oxidation and other factors during transportation and storage. Sealed packaging and antioxidant materials are used to extend the shelf life of the product.
[0070] Specifically, in step S1, a blood supplier with reliable sources is selected to ensure that the blood comes from healthy animals. Common sources include pig blood, bovine blood or chicken blood. It is best to choose the blood of disease-free animals. The blood can be provided in frozen or fresh liquid form. Frozen blood needs to be thawed in advance and used quickly to avoid repeated freezing and thawing. Pour the animal blood into a clean container and use refrigeration technology (4°C) to store it to ensure that hemoglobin does not denature. The red blood cells in the blood are separated by low-speed centrifugation (about 2000 rpm, 5 - 10 minutes). The supernatant after centrifugation contains plasma and should be discarded. The precipitated red blood cells are collected and most of the plasma is removed. The red blood cells are rinsed with physiological saline 2 - 3 times to remove the residual plasma and other impurities, obtaining a relatively pure red blood cell precipitate.
[0071] Specifically, in step S2, the separated red blood cells are suspended in phosphate buffered saline (PBS, pH 7.4) to ensure that the pH of the buffer matches the most stable pH of hemoglobin. The suspension is placed at 4°C and sonicated in an ultrasonic cleaner. The ultrasonic frequency is 20 - 25 kHz, the power is 200 - 300 W, and each treatment lasts for 5 - 10 minutes to avoid hemoglobin denaturation caused by temperature rise. After sonication, centrifugation is performed using a high-speed centrifuge (about 12000 rpm, 10 - 15 minutes) to precipitate red blood cell fragments and macromolecular impurities, obtaining a supernatant (hemoglobin solution). The supernatant is transferred to another container as the preliminary extract of hemoglobin.
[0072] Specifically, in step S3, according to the target, a suitable enzyme is selected for the hydrolysis of hemoglobin, such as pepsin (e.g., Pepsin) or trypsin (e.g., Trypsin). These enzymes can specifically hydrolyze the peptide bonds in hemoglobin, releasing hemoglobin polypeptides. The hemoglobin solution is mixed with the enzyme in a certain ratio, usually the mass ratio of hemoglobin to the enzyme is 100:1. The pH value of the enzymatic hydrolysis solution is adjusted to the pH of the optimal enzyme activity (for example, the pH value of pepsin is 1.5 - 2.5), the temperature is controlled between 35 - 45°C, and temperature control is carried out to prevent the enzyme from being inactivated due to excessive temperature. Usually, the reaction time is set to 4 - 8 hours, and the hydrolysis effect is monitored according to the reaction progress. After the enzymatic hydrolysis reaction is completed, the enzyme activity is stopped by heating to 80°C and maintaining for 5 - 10 minutes.
[0073] Specifically, in step S4, an iron source (such as ferric chloride or ferrous sulfate) is added to the hydrolyzed polypeptide solution to provide iron ions. The concentration of iron ions is usually 10 - 20 times the mass of hemoglobin to ensure that sufficient iron ions can bind to the polypeptide. The solution temperature is controlled at 30 - 40°C, and the pH value is adjusted to 6.5 - 7.5 to ensure that the iron ions bind to the heme part of the hemoglobin polypeptide within this pH range. To improve the bioavailability of iron, a reducing agent (such as ascorbic acid or citric acid) can be added to reduce ferric iron to ferrous iron and enhance the iron absorption capacity. The addition amount of the reducing agent can be controlled at 0.5 - 1.0% (w / w), and the reaction is carried out while maintaining the temperature at about 30°C.
[0074] Specifically, in step S5, ultrafiltration: The blood protein polypeptide solution is concentrated using an ultrafiltration membrane (with a molecular weight cut-off value of 5 - 10 kDa) to remove low-molecular substances and solvents. Affinity chromatography: Using an affinity chromatography column (such as utilizing the affinity of hemoglobin polypeptide to bind with specific metal ions), impurities are further removed, and the iron-rich blood protein polypeptide is retained. This step helps to separate and improve the iron-binding effect. The solution purified by ultrafiltration and affinity chromatography is dialyzed using a low-molecular-weight dialysis bag to remove salts, unbound iron ions, and small-molecule impurities. Dialysis can be carried out at 4°C for 12 - 24 hours to ensure complete removal of impurities.
[0075] Specifically, in step S6, low-temperature evaporation concentration or freeze concentration technology (such as using a rotary evaporator or a refrigerated centrifuge) is used to remove excess water. The solution is concentrated until the solid content of the blood protein polypeptide solution is 30 - 50%. The concentrated solution is converted into a powder form using freeze-drying technology (a freeze dryer). The freeze-drying process is carried out at a low temperature, which can effectively maintain the structure and activity of heme iron and polypeptides. A spray drying device can also be used to spray the polypeptide solution into fine droplets, and the water is rapidly evaporated in hot air to form a dry powder.
[0076] Specifically, in step S7, high-performance liquid chromatography (HPLC) is used to analyze the components of the blood protein polypeptide, confirm the binding situation of its heme iron, a spectrophotometer is used to measure the content of heme iron to ensure it meets the standards, microbial detection is carried out on the finished product to ensure there are no harmful bacteria, heavy metals and other pollutants, and sealed glass bottles or aluminum bags are used for packaging to minimize the oxygen and humidity inside the package, avoiding oxidation and moisture. A desiccant can be placed in the packaging bag to ensure the product remains dry during transportation and storage.
[0077] Through the above technical solutions, in the present invention, through low-temperature extraction and ultrasonic-assisted technology, the extraction rate of hemoglobin is increased, and the degradation of hemoglobin is reduced. By adjusting the enzymatic hydrolysis and iron ion binding conditions, it is ensured that heme iron can be fully bound, improving the bioavailability of iron. Using high-efficiency purification technologies such as ultrafiltration and affinity chromatography, impurities are removed, ensuring the purity and safety of the final product. The extraction, enzymatic hydrolysis, purification and other links are optimized, improving production efficiency and reducing the consumption of raw materials and energy. Through precise iron ion reduction treatment and optimized structural design of blood protein polypeptide, the final product has higher biological activity and absorption efficiency, meeting the nutritional supplement requirements.
[0078] Although the embodiments of the present invention have been shown and described, for those of ordinary skill in the art, it can be understood that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.
Claims
1. A method for preparing a hemoglobin polypeptide rich in heme iron, characterized in that: The following steps are involved: S1: Optimizing blood source and pretreatment S1.1: Choose a high-quality blood source: Use fresh disease-free animal blood (such as pig blood, cow blood or chicken blood), and ensure the safety of the blood source to avoid contamination by pathogenic microorganisms. You can choose frozen or liquid blood that meets quality standards; S1.2: Pretreatment: Use low temperature refrigeration to preserve blood to prevent hemoglobin from denaturing or degrading during storage. Then, use frozen centrifugation to separate red blood cells from the blood and remove impurities such as plasma to obtain pure red blood cells. S2: Efficient hemoglobin extraction S2.1: Low temperature extraction: To avoid the damage of high temperature to hemoglobin, the red blood cells are extracted in a low temperature environment (4°C). A buffer solution (such as phosphate buffer) is used to break the red blood cells to release hemoglobin. Ultrasonic treatment or high pressure homogenization is used to further break the cells to increase the extraction rate of hemoglobin; S2.2: Centrifugation: The extract is centrifuged to remove cell debris and other impurities, retaining the hemoglobin-rich supernatant. The purity of hemoglobin can be further improved by multiple centrifugations. S3: Enzymatic hydrolysis optimization S3.1: Choose the right enzyme: Use enzymes that are specific for hemoglobin, such as pepsin, trypsin, or modified enzyme preparations, which can efficiently hydrolyze hemoglobin and release heme iron-rich hemoglobin polypeptides. The selected enzyme should be highly selective to avoid damage to the heme iron binding moiety; S3.2: Optimization of enzymatic hydrolysis conditions: Adjust the temperature, pH value and time of the enzymatic hydrolysis reaction. Generally, the enzymatic hydrolysis temperature should be controlled between 35-45°C and the pH value should be controlled between 6-8 to maximize the activity of the enzyme and the efficiency of hemoglobin hydrolysis. The enzymatic hydrolysis time should be controlled between 4-8 hours to avoid excessive hydrolysis leading to polypeptide inactivation; S3.3: End of reaction and neutralization: After the reaction is completed, the enzymatic reaction is stopped by heating or acid-base neutralization to ensure the stability of the blood protein polypeptide. S4: Binding and Optimization of Heme Iron S4.1: Optimization of iron ion binding: Add an iron ion source (such as iron salt) to the enzymatically hydrolyzed polypeptide solution to promote the binding of heme iron. Optimize the binding efficiency of iron ions by adjusting the pH value and temperature conditions. Usually the pH value is controlled between 6.5-7.5 and the temperature is maintained at 30-40°C to promote the effective binding of iron and the stability of the polypeptide; S4.2: Iron reduction treatment: In order to improve the bioavailability of iron, reducing agents (such as ascorbic acid) can be used to reduce trivalent iron to divalent iron, thereby enhancing the absorption and utilization of heme iron. S5: Purification and Isolation S5.1: Multi-step purification: Ultrafiltration or affinity chromatography is used to further purify the blood protein polypeptides. Ultrafiltration membranes can separate peptides and low-molecular impurities according to molecular weight to ensure the purity of the final product. Affinity chromatography further removes non-target proteins and increases the iron binding rate through the binding of hemoglobin peptides to specific ligands; S5.2: Dialysis to remove impurities: Use dialysis to remove residual low molecular weight substances, salts and unbound iron ions to further improve the purity and safety of blood protein polypeptides. S6: Concentration and drying S6.1: Concentration: Use low temperature evaporation or freeze concentration technology to concentrate the hemoglobin polypeptide solution and remove the solvent, so that the final product is more concentrated and rich in heme iron; S6.2: Drying: Freeze drying or spray drying techniques are used to convert the concentrate into powder for easy storage and transportation. These drying techniques can effectively maintain the activity of the polypeptide and the binding state of heme iron. S7: Finished product quality inspection and packaging S7.1: Quality testing: The final product is tested to ensure that the content, purity and biological activity of heme iron meet the standards. The composition of the peptide and the binding of iron are tested by high performance liquid chromatography (HPLC), spectrophotometer and other technologies; S7.2: Packaging: The final heme iron-rich hemoglobin polypeptide powder or liquid preparation shall be packaged to ensure that it is not affected by moisture, oxidation, etc. during transportation and storage. Use sealed packaging and antioxidant materials to extend the shelf life of the product.
2. The method for preparing a hemoglobin iron-rich blood protein polypeptide according to claim 1, characterized in that: in the step S1, a blood supplier with a reliable source is selected to ensure that the blood comes from healthy animals. Common sources include pig blood, cow blood or chicken blood, preferably disease-free animal blood, and the blood can be provided in frozen or fresh liquid form. Frozen blood needs to be thawed in advance and used quickly to avoid repeated freezing and thawing. Pour the animal blood into a clean container and store it using refrigeration technology (4°C) to ensure that hemoglobin does not denature. Separate the red blood cells from the blood by low-speed centrifugation (about 2000rpm, 5-10 minutes). The supernatant after centrifugation contains plasma and should be discarded. Collect the precipitated red blood cells and remove most of the plasma. Rinse the red blood cells 2-3 times with saline to remove residual plasma and other impurities to obtain a relatively pure red blood cell precipitate.
3. The method for preparing a heme iron-rich blood protein polypeptide according to claim 1, characterized in that: In step S2, the separated red blood cells are suspended in phosphate buffer (PBS, pH 7.4), ensuring that the pH of the buffer matches the most stable pH of hemoglobin, and the suspension is placed at 4°C and ultrasonically crushed in an ultrasonic cleaning machine. The ultrasonic frequency is 20-25kHz, the power is 200-300W, and each treatment lasts 5-10 minutes to avoid temperature rise leading to hemoglobin denaturation. After ultrasonic crushing, a high-speed centrifuge (about 12000rpm, 10-15 minutes) is used for centrifugation to precipitate red blood cell fragments and macromolecular impurities to obtain a supernatant (hemoglobin solution), and the supernatant is transferred to another container as a preliminary extract of hemoglobin.
4. The method for preparing a heme iron-rich blood protein polypeptide according to claim 1, characterized in that: In the step S3, according to the target, a suitable enzyme is selected to hydrolyze hemoglobin, such as pepsin (such as Pepsin) or trypsin (such as Trypsin). These enzymes can specifically hydrolyze the peptide bonds in hemoglobin, release hemoglobin polypeptides, and mix the hemoglobin solution with the enzyme in a certain proportion, usually the mass ratio of hemoglobin to the enzyme is 100:
1. The pH value of the enzymolysis solution is adjusted to the pH of the optimal activity of the enzyme (for example, the pH value of pepsin is 1.5-2.5), the temperature is controlled between 35-45 ° C, and temperature control is performed to prevent excessive temperature from causing enzyme inactivation. The reaction time is usually set to 4-8 hours, and the hydrolysis effect is monitored according to the progress of the reaction. After the enzymolysis reaction is completed, the activity of the enzyme is stopped by heating to 80 ° C and maintaining for 5-10 minutes to prevent excessive hydrolysis, and the pH is adjusted to neutral using dilute hydrochloric acid or sodium hydroxide solution to ensure the stability of the polypeptide.
5. The method for preparing a heme iron-rich blood protein polypeptide according to claim 1, characterized in that: In step S4, an iron source (such as ferric chloride or ferrous sulfate) is added to the polypeptide solution after enzymatic hydrolysis to provide iron ions. The concentration of iron ions is usually 10-20 times the mass of hemoglobin to ensure that sufficient iron ions can bind to the polypeptide, the solution temperature is controlled to 30-40°C, the pH value is adjusted to 6.5-7.5, and the iron ions are ensured to bind to the heme part of the hemoglobin polypeptide within this pH range. In order to improve the bioavailability of iron, a reducing agent (such as ascorbic acid or citric acid) can be added to reduce trivalent iron to divalent iron to enhance the absorption capacity of iron. The amount of reducing agent added can be controlled at 0.5-1.0% (w / w), and the temperature is kept at about 30°C for reaction.
6. The method for preparing a heme iron-rich blood protein polypeptide according to claim 1, characterized in that: Ultrafiltration in step S5: using an ultrafiltration membrane (molecular weight cutoff value is 5-10kDa) to concentrate the blood protein polypeptide solution to remove low molecular weight substances and solvents, affinity chromatography: using an affinity chromatography column (such as using the affinity of the hemoglobin polypeptide to bind to a specific metal ion) to further remove impurities and retain iron-rich blood protein polypeptides. This step helps to separate and improve the binding effect of iron. The solution purified by ultrafiltration and affinity chromatography is dialyzed, and a low molecular weight dialysis bag is used to remove salt, unbound iron ions and small molecular impurities. The dialysis can be performed at 4°C for 12-24 hours to ensure that the impurities are completely removed.
7. The method for preparing a heme iron-rich blood protein polypeptide according to claim 1, characterized in that: In step S6, low temperature evaporation concentration or freeze concentration technology (such as using a rotary evaporator or a refrigerated centrifuge) is used to remove excess water. The solid content of the hemoprotein polypeptide solution is concentrated to 30-50%, and the concentrated solution is converted into a powder form using freeze drying technology (freeze dryer). The freeze drying process is carried out at low temperatures, which can effectively maintain the structure and activity of heme iron and polypeptides. Spray drying equipment can also be used to spray the polypeptide solution into fine droplets, and the water is quickly evaporated in hot air to form a dry powder.
8. The method for preparing a heme iron-rich blood protein polypeptide according to claim 1, characterized in that: In step S7, high performance liquid chromatography (HPLC) is used to analyze the components of the hemoprotein polypeptide to confirm the binding of the heme iron, a spectrophotometer is used to measure the content of the heme iron to ensure that it meets the standards, microbiological testing is performed on the finished product to ensure that there are no pollutants such as harmful bacteria and heavy metals, and sealed glass bottles or aluminum bags are used for packaging to ensure that the oxygen and humidity in the package are minimized to avoid oxidation and moisture. A desiccant can be placed in the packaging bag to ensure that the product remains dry during transportation and storage.