Preparation method of iron-chelated ovotransferrin and iron supplement product of iron-chelated ovotransferrin
The combined use of ultrasonic and high-pressure pulse electric fields with PEG precipitation and iron chelation enhances the bioavailability and taste of iron supplements, addressing taste and absorption issues in existing supplements.
Patent Information
- Application Number
- CN202510558525.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-29
- Publication Date
- 2025-07-15
AI Technical Summary
The current iron supplements have low absorption and utilization rate in the body and have taste problems such as gastrointestinal irritation and fishy smell. Traditional iron supplements have low bioavailability and absorption efficiency, and complex operation, resulting in loss of iron chelated egg transferrin.
Egg transferrin was extracted by ultrasonic composite high-voltage pulsed electric field treatment combined with isoelectric point precipitation method and PEG precipitation method, and then chelated with ferrous sulfate. Two ultrasonic composite high-voltage pulsed electric field treatments were used to improve the iron chelation rate, and finally, iron chelated egg transferrin was prepared by vacuum freeze-drying.
It improves the absorption rate and bioavailability of iron chelated egg transferrin, improves the taste, reduces fishy smell and bitter taste, ensures the stable release and efficient absorption of iron elements in the body, and avoids losses caused by complex operations.
Smart Images
Figure CN120309718A_ABST
Abstract
Description
Technical Field
[0001] This application belongs to the field of protein preparation, and relates to a preparation method and application of iron-chelated ovotransferrin. Background Art
[0002] Usually, iron agents are often used to prevent and treat iron deficiency anemia. Currently, iron supplementation products on the market mainly include iron agents, iron-containing food additives, and iron supplements, etc. Common ordinary iron agents such as ferrous sulfate, ferrous gluconate, and ferrous lactate, although having a high iron content, have a large irritation to mucous membranes and the gastrointestinal tract, reducing the absorption and utilization rate in the body, and having a metallic rust taste. Research on transferrin iron preparations shows that ovotransferrin combined with iron ions can be used as a biological iron supplement. It is easily utilized by tissues and organs, can promote nucleic acid and protein metabolism at the same time, reduce the side effects caused by oxidation in the body, improve the body's immunity, and has a protective effect on cell damage in the body. Therefore, it can be absorbed and utilized as a beneficial component of the body.
[0003] However, although peptide-based iron has advantages in bioavailability and absorption efficiency, they are usually accompanied by taste problems such as fishy smell and bitterness, which severely restrict the market acceptance of related products. At the same time, traditional iron supplements, especially in the form of inorganic iron, may cause gastrointestinal discomfort such as nausea, constipation or diarrhea, and mood instability, etc., and their bioavailability and absorption efficiency are relatively low.
[0004] CN 110204609 discloses a method for industrial extraction of ovotransferrin and its protein iron product. It mainly uses high-voltage pulsed electric field-assisted treatment of egg white to reduce viscosity and surface tension. The electric field molecular polarization effect induces more charged polar groups to be exposed, and the charged molecules generate repeated violent movements. This kind of movement destroys the fibrous structure of ovomucin, disassembles the complex formed by ovomucin and ovomucoid, lysozyme and other soluble proteins, thins the thick egg white, further reduces the viscosity, and the molecular dispersion plays a good auxiliary separation effect, which helps the subsequent ultrafiltration separation and improves the protein yield; by changing to chelation of ferrous ions with ovotransferrin in egg white.
[0005] But after chelation, a large number of operations are carried out, such as two ethanol treatments, two ultrafiltrations, etc., resulting in the loss of iron-chelated ovotransferrin again. Summary of the Invention
[0006] The purpose of this application is to provide a preparation method of iron-chelated ovotransferrin for the deficiencies of the prior art. The method includes the following steps: (1) Pretreatment of egg white: Treat the egg white solution by ultrasonic combined with high-voltage pulsed electric field to thin the thick egg white, further reduce the viscosity, and disperse the molecules, avoiding adding water to dilute the egg white solution in the subsequent process, which helps to improve production efficiency; (2)Removal of ovomucin and ovalbumin: Remove ovomucin and ovalbumin by isoelectric point precipitation combined with PEG precipitation method. Adjust the pH value of the egg white obtained in step (1) to 3.0 - 5.5, add PEG 4000 - 10000 to 1 - 6%, stir at room temperature, perform centrifugation, and collect the egg white supernatant; (3)Preparation of ovotransferrin: Prepare the crude ovotransferrin by isoelectric point precipitation combined with PEG precipitation method. Adjust the pH value of the egg white supernatant obtained in step (2) to 6.0 - 7.5, add PEG 4000 - 10000 to 9 - 18%, stir at room temperature, perform centrifugation, and collect the precipitate; (4)Separation of crude ovotransferrin: Separate the impurity proteins in the crude ovotransferrin by PEG precipitation method. Dissolve the precipitate obtained in step (3) with Tris-HCl, add PEG 1000 - 6000 to 5 - 12%, stir at room temperature, then perform centrifugation, and collect the precipitate; (5)Purification of crude ovotransferrin: After dissolving the precipitate obtained in step (4) with Tris-HCl, perform separation and purification by anion exchange chromatography to obtain high-purity ovotransferrin; (6)Pretreatment of iron-chelated ovotransferrin: Mix high-purity ovotransferrin and ferrous sulfate according to the mass ratio, and prepare a mixed system with the concentration of ovotransferrin being 2.5 - 9.5 mg / mL in a solution containing NaHCO3; (7)Preparation of iron-chelated ovotransferrin: Subject the mixed system obtained in step (6) to ultrasonic combined with high-voltage pulsed electric field treatment, stir and react for 0.5 - 2.0 h, filter to remove impurities, and obtain iron-chelated ovotransferrin by vacuum freeze-drying.
[0007] As a preferred embodiment of the present application, in step (1), the conditions for ultrasonic combined with high-voltage pulsed electric field treatment are as follows: the ultrasonic power is 200 - 300 W, the conditions for the high-voltage pulsed electric field are a field strength of 15 - 25 kV / cm, the number of pulses is 6 - 12, and the treatment time for ultrasonic combined with high-voltage pulsed electric field is 5 - 10 min. For example, the conditions for ultrasonic combined with high-voltage pulsed electric field treatment are: the ultrasonic power is 250 W, the conditions for the high-voltage pulsed electric field are a field strength of 20 kV / cm, the number of pulses is 8, and the treatment time for ultrasonic combined with high-voltage pulsed electric field is 8 min.
[0008] As a preferred embodiment of the present application, in steps (2) and (3), adjust the pH to 3.0 - 5.5 with a citric acid solution.
[0009] As a preferred embodiment of the present application, in steps (2), (3), and (4), stir at room temperature for more than 0.2 h, for example, 1 h. There is no need for low temperature and no need to dilute the egg white, which helps to improve production efficiency.
[0010] As a preferred embodiment of the present application, in steps (2), (3), and (4), the conditions for centrifugation are centrifugation at a rotation speed of 7500 - 12000 rpm for 15 - 45 minutes.
[0011] As a preferred embodiment of the present application, in steps (4) and (5), the crude ovotransferrin is dissolved in a Tris - HCl buffer solution with a pH of 7 - 9.
[0012] As a preferred embodiment of the present application, in step (5), separation is carried out using a Q Sepharose FF chromatographic column, elution is performed with a Tris - HCl buffer solution with a pH of 7 - 9 containing 0, 0.06 - 0.12, 0.25 - 0.5, and 2 mol / L sodium chloride, the eluate with 0.06 - 0.12 mol / L sodium chloride is collected, and freeze - drying is carried out to obtain high - purity ovotransferrin.
[0013] As a preferred embodiment of the present application, ovotransferrin and ferrous sulfate are mixed in a molar ratio of 1:(0.5 - 5), for example, 1:2.
[0014] As a preferred embodiment of the present application, the pH of the NaHCO3 solution is 4.0 - 9.0, and further, it is preferably adjusted with a citric acid solution.
[0015] As a preferred embodiment of the present application, the concentration of NaHCO3 in the NaHCO3 solution is 25 - 125 mmol / L.
[0016] As a preferred embodiment of the present application, in step (7), the conditions for ultrasonic combined with high - voltage pulsed electric field treatment are: the ultrasonic power is 200 - 300 W, the conditions for the high - voltage pulsed electric field are a field strength of 15 - 25 kV / cm, the number of pulses is 6 - 12, and the treatment time for ultrasonic combined with high - voltage pulsed electric field is 5 - 10 minutes. For example, the conditions for ultrasonic combined with high - voltage pulsed electric field treatment are: the ultrasonic power is 250 W, the conditions for the high - voltage pulsed electric field are a field strength of 20 kV / cm, the number of pulses is 8, and the treatment time for ultrasonic combined with high - voltage pulsed electric field is 8 minutes.
[0017] As a preferred embodiment of the present application, in step (7), stirring reaction is carried out at room temperature for 0.5 - 2.0 h.
[0018] As a preferred embodiment of the present application, freeze - drying is carried out under the conditions of a vacuum degree of 1 - 999 Pa, a temperature of - 80°C to - 30°C, and a drying time of 5 - 17 h to obtain iron - chelated ovotransferrin.
[0019] The present application also provides a product with iron - supplementing efficacy and a preparation method, and the method includes the following steps: It is prepared by mixing iron-chelated ovotransferrin with pharmaceutical excipients to obtain a medicinal iron supplement. For example, 10-20 parts of iron-chelated ovotransferrin and 80-90 parts of the pharmaceutical excipient starch.
[0020] and / or It is prepared by mixing iron-chelated ovotransferrin with food additives to obtain an iron supplement nutritional supplement. For example, it is prepared by mixing with egg membrane peptide, whey protein powder, taurine, vitamin C, fructooligosaccharide, milk powder, and compound probiotics. The proportion of iron-chelated ovotransferrin is 30-60 parts, the proportion of egg membrane peptide is 8-15 parts, the proportion of whey protein powder is 10-25 parts, the proportion of taurine is 8-15 parts, the proportion of vitamin C is 5-10 parts, the proportion of fructooligosaccharide is 5-15 parts, the proportion of milk powder is 10-15, and the compound probiotics is 1-8 parts.
[0021] The beneficial technical effects of this application are as follows: The combined use of ultrasonic and high-voltage pulsed electric field can fully unfold ovotransferrin, which helps to improve the iron chelation rate. The innovative chelation technology of ovotransferrin and iron ions effectively improves the absorption rate and bioavailability of iron elements. The iron supplement function benefits from the chelation and compounding of ovotransferrin and ferrous sulfate, and Fe 2+ The relatively high digestion retention rate effectively ensures the stable release and efficient absorption of iron elements in the body. This application uses ultrasonic combined with high-voltage pulsed electric field twice. The first time is to improve the extraction efficiency by treating egg white solution with ultrasonic combined with high-voltage pulsed electric field, and the second time is for better chelation with Fe 2+ ; and it can make the iron-chelated ovotransferrin basically without loss.
[0022] This product has good palatability and does not have the fishy and bitter tastes of peptide iron supplements. Description of the Drawings
[0023] Figure 1 Shows the effect of pH on the iron-binding ability of ovotransferrin.
[0024] Figure 2 Shows the effect of NaHCO3 on the iron-binding ability of ovotransferrin.
[0025] Figure 3 Shows the DPPH free radical scavenging ability of ovotransferrin (OVT1) and iron-chelated ovotransferrin (OVT2). Detailed Embodiments
[0026] For the convenience of understanding by those skilled in the art, the following further illustrates the present application with reference to embodiments. The content mentioned in the embodiments does not limit the present application. Example 1
[0027] (1)Pretreatment of egg white: Treat the egg white solution with ultrasonic combined with high-voltage pulsed electric field to make the thick egg white thinner, further reduce the viscosity, disperse the molecules, avoid diluting the egg white solution with water in the subsequent process, and help improve production efficiency. The treatment conditions of ultrasonic combined with high-voltage pulsed electric field are as follows: the ultrasonic power is 250 W, the conditions of high-voltage pulsed electric field are field strength 20 kV / cm, pulse number 8, and the treatment time of ultrasonic combined with high-voltage pulsed electric field is 8 min.
[0028] (2)Removal of ovomucin and ovalbumin: Remove ovomucin and ovalbumin by isoelectric point precipitation combined with PEG precipitation method. Adjust the pH value of the egg white obtained in step (1) to 5, add PEG 10000 to 4%, stir at room temperature for 1 h, then centrifuge at 8000 rpm for 40 min, and collect the egg white supernatant.
[0029] (3)Preparation of ovotransferrin: Prepare the crude ovotransferrin by isoelectric point precipitation combined with PEG precipitation method. Adjust the pH value of the egg white supernatant obtained in step (2) to 6.5, add PEG 10000 to 15%, stir at room temperature for 1 h, then centrifuge at 8000 rpm for 20 min, and collect the precipitate.
[0030] (4)Separation of crude ovotransferrin: Separate the impurity proteins in the crude ovotransferrin by PEG precipitation method. Dissolve the precipitate obtained in step (3) with Tris-HCl with pH 8.5, add PEG 6000 to 10%, stir at room temperature for 1 h, then centrifuge at 8000 rpm for 20 min, and collect the precipitate.
[0031] (5)Purification of crude ovotransferrin: After dissolving the precipitate obtained in step (4) with Tris-HCl with pH 8.5, perform separation purification, sterilization, drying, etc. with Q Sepharose FF anion exchange chromatography to obtain high-purity ovotransferrin. Separate with a Q Sepharose FF chromatographic column, elute with Tris-HCl buffer solution with pH 7-9 containing 0, 0.08, 0.35, and 2 mol / L sodium chloride, collect the eluate with 0.08 mol / L sodium chloride, and freeze-dry to obtain high-purity ovotransferrin. (6)Pretreatment of iron-chelated ovotransferrin: Mix high-purity ovotransferrin and ferrous sulfate at a molar ratio of 1:2, dissolve in a solution with pH 8.0 containing 100 mmol / L NaHCO3, and prepare a mixed system with the concentration of ovotransferrin being 5 mg / mL.
[0032] Preparation of iron-chelated ovotransferrin: The mixed system obtained in step (1) was subjected to ultrasonic combined with high-voltage pulsed electric field treatment, and stirred for reaction for 1 h, filtered to remove impurities, and vacuum freeze-dried to obtain iron-chelated ovotransferrin. The conditions for ultrasonic combined with high-voltage pulsed electric field treatment were as follows: the ultrasonic power was 250 W, the conditions for high-voltage pulsed electric field were a field strength of 20 kV / cm, the number of pulses was 8, and the treatment time for ultrasonic combined with high-voltage pulsed electric field was 8 min. Iron-chelated ovotransferrin was obtained by freeze-drying under the conditions of a vacuum degree of 1 - 999 Pa, a temperature of -80 °C to -30 °C, and a drying time of 5 - 17 h. Example 2
[0033] Based on Example 1, the difference lies in that it was dissolved in a solution with a pH of 6.5 and containing 100 mmol / L NaHCO3 to prepare a mixed system with an ovotransferrin concentration of 5 mg / mL; other conditions were the same as in Example 1. Example 3
[0034] Based on Example 1, the difference lies in that it was dissolved in a solution with a pH of 7.0 and containing 100 mmol / L NaHCO3 to prepare a mixed system with an ovotransferrin concentration of 5 mg / mL; other conditions were the same as in Example 1. Example 4
[0035] Based on Example 1, the difference lies in that it was dissolved in a solution with a pH of 7.5 and containing 100 mmol / L NaHCO3 to prepare a mixed system with an ovotransferrin concentration of 5 mg / mL; other conditions were the same as in Example 1. Example 5
[0036] Based on Example 1, the difference lies in that it was dissolved in a solution with a pH of 8.5 and containing 100 mmol / L NaHCO3 to prepare a mixed system with an ovotransferrin concentration of 5 mg / mL; other conditions were the same as in Example 1. Example 6
[0037] Based on Example 1, the difference lies in that it was dissolved in a solution with a pH of 8.0 and containing 75 mmol / L NaHCO3 to prepare a mixed system with an ovotransferrin concentration of 5 mg / mL; other conditions were the same as in Example 1. Example 7
[0038] Based on Example 1, the difference lies in that it was dissolved in a solution with a pH of 8.0 and containing 125 mmol / L NaHCO3 to prepare a mixed system with an ovotransferrin concentration of 5 mg / mL; other conditions were the same as in Example 1. Example 8
[0039] On the basis of Example 1, the difference is that the treatment time of the second ultrasonic combined with high-voltage pulsed electric field is 6 min; the others are the same as in Example 1. Example 9
[0040] On the basis of Example 1, the difference is that the treatment time of the second ultrasonic combined with high-voltage pulsed electric field is 10 min; the others are the same as in Example 1. Example 10
[0041] On the basis of Example 1, the difference is that the treatment time of the second ultrasonic combined with high-voltage pulsed electric field is 10 min; the others are the same as in Example 1.
[0042] Comparative Example 1 On the basis of Example 1, the difference is that step (7) of ultrasonic combined with high-voltage pulsed electric field treatment is omitted; that is, the obtained mixed system is directly stirred and reacted for 1 h, filtered to remove impurities, and vacuum freeze-dried to obtain iron-chelated ovotransferrin. The others are the same as in Example 1.
[0043] Comparative Example 2 On the basis of Example 1, the difference is that the ultrasonic combined with high-voltage pulsed electric field treatment in step (1) is omitted; the others are the same as in Example 1.
[0044] The product was subjected to in vitro simulated gastrointestinal digestion to determine the Fe 2+ retention rate and IL-2 promotion rate.
[0045] Table 1 In vitro simulated gastrointestinal digestion Fe 2+ retention rate and IL-2 promotion rate (drug administration group - model group) sample <![CDATA[Fe 2+ Retention rate (%)]]> IL-2 (pg / mL) ferrous sulfate 9.6 / iron-chelated ovotransferrin 35.3 7.1 Example 1 34.9 10.9 Example 2 34.6 10.1 Example 3 34.7 10.6 Example 4 34.7 10.3 Example 5 35.2 10.1 Example 6 34.3 10.3 Example 7 34.5 10.1 Example 8 34.8 10.3 Example 9 34.3 10.5 Example 10 34.9 10.6 Comparative Example 1 30.1 8.2 Comparative Example 2 32.1 7.9 From Table 1 and the attached Figures 1-3 experimental data, it can be seen that when the pH is neutral and slightly alkaline (6.5 - 8.5), the iron-binding ability of ovotransferrin is stronger; when the NaHCO3 concentration is 75 - 125 mM / L, the iron-binding ability of ovotransferrin is stronger; as the concentration of ovotransferrin increases, its ability to scavenge DPPH free radicals gradually increases; at the same concentration, the ability of iron-chelated ovotransferrin to scavenge DPPH free radicals is stronger than that of ovotransferrin; the in vitro simulated gastrointestinal digestion Fe 2+ retention rate of iron-chelated ovotransferrin is the highest, followed by Example 1, and the IL-2 promotion rate of Example 1 is the highest.
[0046] Those skilled in the art can understand that, unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as the general understanding of those of ordinary skill in the art to which this application belongs. It should also be understood that terms such as those defined in a general dictionary should be understood to have a meaning consistent with the meaning in the context of the prior art, and will not be interpreted in an idealized or overly formal sense unless specifically defined as here.
[0047] It should be understood that the detailed description of the technical solutions of this application by means of the preferred embodiments above is illustrative rather than restrictive. Those of ordinary skill in the art can modify the technical solutions recorded in each embodiment on the basis of reading the specification of this application, or make equivalent replacements for some of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of each embodiment of this application.
Claims
1. A preparation method of iron-chelated ovotransferrin, characterized in that, It includes the following steps: (1) Pretreatment of egg white: Treat the egg white solution by ultrasonic combined with high-voltage pulsed electric field; (2) Removal of ovomucin and ovalbumin: Remove ovomucin and ovalbumin by isoelectric point precipitation combined with PEG precipitation method. Adjust the pH value of the egg white obtained in step (1) to 3.0 - 5.5, add PEG 4000 - 10000 to 1 - 6%, stir at room temperature, perform centrifugation, and collect the egg white supernatant; (3) Preparation of ovotransferrin: Prepare the crude ovotransferrin by isoelectric point precipitation combined with PEG precipitation method. Adjust the pH value of the egg white supernatant obtained in step (2) to 6.0 - 7.5, add PEG 4000 - 10000 to 9 - 18%, stir at room temperature, perform centrifugation, and collect the precipitate; (4) Separation of crude ovotransferrin: Separate the impurity proteins in the crude ovotransferrin by PEG precipitation method. Dissolve the precipitate obtained in step (3) with Tris-HCl, add PEG 1000 - 6000 to 5 - 12%, stir and then perform centrifugation, and collect the precipitate; (5) Purification of crude ovotransferrin: After dissolving the precipitate obtained in step (4) with Tris-HCl, perform separation and purification by anion exchange chromatography to obtain high-purity ovotransferrin; (6) Pretreatment of iron-chelated ovotransferrin: Mix high-purity ovotransferrin and ferrous sulfate according to the mass ratio, and prepare a mixed system with the concentration of ovotransferrin being 2.5 - 9.5 mg / mL in a NaHCO₃ solution; (7) Preparation of iron-chelated ovotransferrin: Perform ultrasonic combined with high-voltage pulsed electric field treatment on the mixed system obtained in step (6), stir and react, filter to remove impurities, and obtain iron-chelated ovotransferrin by vacuum freeze-drying.
2. The method according to claim 1, characterized in that, In step (1) and / or step (7), the conditions of ultrasonic combined with high-voltage pulsed electric field treatment are: the ultrasonic power is 200 - 300 W, the conditions of high-voltage pulsed electric field are the field strength of 15 - 25 kV / cm, the number of pulses is 6 - 12, and the treatment time of ultrasonic combined with high-voltage pulsed electric field is 5 - 10 min; for example, the conditions of ultrasonic combined with high-voltage pulsed electric field treatment are: the ultrasonic power is 250 W, the conditions of high-voltage pulsed electric field are the field strength of 20 kV / cm, the number of pulses is 8, and the treatment time of ultrasonic combined with high-voltage pulsed electric field is 8 min.
3. The method according to claim 1, characterized in that, As a preferred embodiment of the present application, ovotransferrin and ferrous sulfate are mixed according to the molar ratio of 1:(0.5 - 5), for example, 1:
2.
4. The method according to claim 1, wherein The pH of the NaHCO₃ solution is 4.0 - 9.
0. Further, it is preferably adjusted with a citric acid solution.
5. The method according to claim 1, characterized in that, The concentration of NaHCO₃ in the NaHCO₃ solution is 25 - 125 mmol / L.
6. The method according to claim 1, characterized in that, In step (7), stir and react at room temperature for 0.5 - 2.0 h.
7. The method according to claim 1, characterized in that, Obtain iron-chelated ovotransferrin by freeze-drying under the conditions of a vacuum degree of 1 - 999 Pa, a temperature of -80°C to -30°C, and a drying time of 5 - 17 h.
8. Use of the iron-chelated ovotransferrin prepared by the method according to any one of claims 1 - 7 in the preparation of iron agent products.
9. An iron agent drug with iron supplement efficacy and a preparation method thereof, the method comprising the following steps: Mix iron-chelated ovotransferrin with pharmaceutical excipients to prepare a medicinal iron supplement; for example, 10-20 parts of iron-chelated ovotransferrin and 80-90 parts of pharmaceutical excipient starch.
10. An iron agent health food with iron supplement and immunity enhancement efficacy and a preparation method thereof, the method comprising the following steps: Mix iron-chelated ovotransferrin with food excipients to prepare an iron supplement nutritional supplement; for example, mix it with egg membrane peptide, whey protein powder, taurine, vitamin C, fructooligosaccharide, milk powder, and compound probiotics. The proportion of iron-chelated ovotransferrin is 30-60 parts, the proportion of egg membrane peptide is 8-15 parts, the proportion of whey protein powder is 10-25 parts, the proportion of taurine is 8-15 parts, the proportion of vitamin C is 5-10 parts, the proportion of fructooligosaccharide is 5-15 parts, the proportion of milk powder is 10-15 parts, and the proportion of compound probiotics is 1-8 parts.