Method for improving iron binding capacity of ovotransferrin through moderate enzymolysis

Abstract

The invention discloses a method for improving the iron binding capacity of ovotransferrin through moderate enzymolysis, and belongs to the technical field of food biology. The method comprises the following steps: (1) preparing raw materials: preparing natural ovotransferrin into a protein solution; (2) enzymolysis modification of ovotransferrin: setting the pH value of a protein solution system to a specific pH value, and performing stirring hydrolysis to prepare a hydrolyzed protein solution; and (3) enzyme inactivation: adjusting the pH value of the hydrolyzed protein solution to 7-9, and carrying out heating and water bath treatment to realize enzyme inactivation so as to obtain an enzymatic hydrolysate, namely the high-iron-binding active ovotransferrin enzymatic hydrolysate. According to the method disclosed by the invention, an ovotransferrin solution is subjected to proper enzymolysis reaction under the conditions of specific protease, specific pH, specific temperature, specific time and enzyme / substrate mass ratio, so that the ovotransferrin enzymolysis product with high iron binding capacity and oxidation resistance is prepared.

Description

Technical Field

[0001] This invention relates to the field of food biotechnology, and specifically to a method for improving the iron-binding capacity of ovotransferrin through appropriate enzymatic hydrolysis. Background Technology

[0002] Iron deficiency and iron-deficiency anemia are global public nutritional problems, with infants, adolescents, women of childbearing age and pregnant women, and the elderly being high-risk groups. Dietary non-heme iron constitutes a high proportion and is easily affected by inhibitory factors such as phytic acid, polyphenols, and dietary fiber. Combined with increased physiological iron requirements in some populations, this often leads to insufficient intake or limited absorption and utilization. While widely used inorganic iron salts and some organic complexes are low-cost and have good formulation compatibility, their bioavailability is easily interfered with by inhibitory factors. They may also promote food oxidation and deterioration, cause gastrointestinal discomfort, and interact with formulation ingredients, leading to quality problems such as precipitation and discoloration, thus limiting their application in high-quality foods.

[0003] Against this backdrop, ovotransferrin (OVT), a naturally occurring food-derived protein, has emerged as a potential candidate for iron carriers. This protein comprises approximately 10%-15% of egg white protein and consists of two homologous domains, N- and C-. Each domain can efficiently and affinityly bind Fe with the participation of carbonate (supplyed by bicarbonate) as a cooperating anion. 3+ It forms a closed binding pocket. Its source is natural and its food safety is high. It can produce an antibacterial effect through iron isolation, and it is also expected to serve as a carrier to improve the steady-state transport and utilization of non-heme iron, and reduce the pro-oxidation and flavor burden.

[0004] However, natural ovotransferrin still has limitations in its applications: its iron-binding capacity is significantly dependent on pH and co-ligands, and it easily releases iron in the low pH environment of the stomach. Ovotransferrin has poor thermal stability, undergoing significant denaturation at neutral pH (approximately 60-65℃), and is prone to conformational disruption, aggregation, or site masking during processing, resulting in a significant reduction in iron-binding capacity. Furthermore, ovotransferrin is susceptible to competition from multivalent metals and interference from components such as polyphenols and phytic acid, making it difficult to simultaneously achieve "high capacity, high stability, and controlled release" of iron in complex food / digestive environments. Moreover, among methods for improving ovotransferrin performance, physical methods can easily lead to irreversible denaturation or aggregation under improper processing conditions; chemical methods may introduce solvent residue problems and regulatory uncertainties; and composite carrier methods increase the complexity of raw material preparation and processes. Therefore, there is an urgent need to find a mild, safe, controllable, and simple ovotransferrin modification scheme that significantly improves the iron-binding capacity and stability of ovotransferrin while avoiding chemical residues, ensuring its stability during processing / storage, and moderate release during digestion.

[0005] Currently, among existing technologies, patent CN 118515751 A (A method for rapid and efficient preparation of highly active ovotransferrin) discloses a method for rapidly and efficiently separating ovotransferrin from egg white through mild heat treatment. This method is simple, has a large processing capacity, and has good potential for industrial application. However, during the preparation process, ovotransferrin undergoes slight denaturation upon heating, and its iron-binding capacity is slightly lower than that of natural ovotransferrin, with the iron ion binding capacity generally not exceeding 80%.

[0006] Patent CN 118638213 B (A method for extracting highly soluble ovotransferrin from egg white) discloses a method that utilizes the cavitation effect, molecular polarization effect, and molecular reciprocating motion generated by ultrasound combined with a high-voltage alternating electric field to disrupt the protein complex, fully expose ovotransferrin, and promote its structural extension, which facilitates the binding of ovotransferrin to Fe. 3+ The proposed solution has high requirements for equipment and is complex to operate, making it difficult to promote. Furthermore, the highest iron-bonding capacity it achieves is only 90.72%.

[0007] Furthermore, patent CN 120796428 A (A method for preparing highly immunomodulatory ovotransferrin hydrolysates based on an adult gastrointestinal simulated digestion system) discloses methods for preparing infant and adult simulated digestion products of ovotransferrin. However, its research focus and evaluation system are all centered on the immunomodulatory activity of ovotransferrin after hydrolysis, without specifically exploring or quantitatively characterizing its iron-binding capacity. This invention finds that the adult simulated digestion product of ovotransferrin undergoes a continuous and deep enzymatic hydrolysis process of "gastric digestion-intestinal digestion," resulting in excessive disruption of its protein three-dimensional structure. This leads to severe damage to the specific spatial conformations necessary for maintaining iron ion chelation (such as the iron-binding pocket structure of N- and C-). Combined with boiling inactivation and small molecule dialysis, this is detrimental to the retention and stabilization of polydentate coordination motifs such as His / Tyr / Asp(Glu), making it difficult to form Fe... 3+ The stable enclosure and favorable microenvironment for the synergistic entry of anions provide a stable environment. Therefore, although the simulated digestion process described in this invention can enhance immune activity, it cannot simultaneously maintain the efficient iron transport and binding function of the ovotransferrin core, nor can it provide technical guidance for improving the iron-binding capacity of ovotransferrin, thus failing to meet the needs for preparing highly active iron carrier functional ingredients. Summary of the Invention

[0008] Technical issues In existing technologies, the iron-binding capacity of ovotransferrin is generally between 70% and 80%. Even if it can be increased to 90%, the methods used require a large amount of equipment and complex procedures. Therefore, there is an urgent need to find a mild, simple, safe, and controllable modification scheme for ovotransferrin to effectively improve its iron-binding capacity.

[0009] Technical content To address the aforementioned problems in existing technologies, this invention provides a method for enhancing the iron-binding capacity of ovotransferrin through moderate enzymatic hydrolysis. This method is simple to operate, highly controllable, and low-cost. The entire process is conducted under food-grade conditions and requires no organic reagents. By selecting a specific protease, the ovotransferrin solution is subjected to controlled and moderate enzymatic hydrolysis under specific pH, temperature, time, and enzyme / substrate mass ratio conditions, thereby obtaining an ovotransferrin hydrolysate with high iron-binding and antioxidant capabilities.

[0010] To achieve the above objectives, the technical solution adopted by the present invention is as follows: This invention provides a method for improving the iron-binding capacity of ovotransferrin through appropriate enzymatic hydrolysis, the method comprising the following steps: (1) Raw material preparation: Dissolve ovotransferrin in buffer solution to prepare protein solution; (2) Enzymatic modification of ovotransferrin: After adjusting the pH of the protein solution system obtained in step (1), protease was added for hydrolysis to obtain the hydrolyzed protein solution. (3) Enzyme inactivation: After adjusting the pH of the hydrolyzed protein solution obtained in step (2), the protein solution is heated to obtain the enzymatic hydrolysis product, which is the ovotransferrin with high ferric ion binding capacity.

[0011] In one embodiment of the present invention, in step (1), ovotransferrin is prepared from egg white by heat treatment and centrifugation, with a purity >90%.

[0012] Specifically, the preparation process of ovotransferrin is as follows: adjust the pH of egg white to 5.5-6.5, stir for 1-3 hours, centrifuge and collect the supernatant, then adjust the pH of the supernatant to 7.5-8.5, and then adsorb it through a weakly acidic cation exchange resin for 1-3 hours. After that, adjust the pH of the supernatant after resin adsorption to 6.5-7.5, heat treat it at 50-70℃ for 30-90 minutes, and finally centrifuge and collect the precipitate to obtain ovotransferrin.

[0013] In one embodiment of the present invention, in step (1), the buffer solution is a glycine-hydrochloric acid buffer solution with a pH of 1.5-3.

[0014] In one embodiment of the present invention, in step (1), the mass fraction of ovotransferrin in the protein solution is 2-6% (w / w).

[0015] In one embodiment of the present invention, in step (2), the pH is adjusted to 1.5-2.5.

[0016] In one embodiment of the present invention, in step (2), the protease is selected from alkaline protease, neutral protease, trypsin, and pepsin.

[0017] Preferably, in step (2), the protease is pepsin.

[0018] In one embodiment of the present invention, in step (2), the amount of protease added is 500-1000 U / mg substrate; the substrate is ovotransferrin.

[0019] Preferably, in step (2), the amount of protease added is 700-800 U / mg substrate; the substrate is ovotransferrin.

[0020] In one embodiment of the present invention, in step (2), the hydrolysis time is 80-160 min.

[0021] Preferably, in step (2), the hydrolysis time is 90-150 min.

[0022] In one embodiment of the present invention, in step (2), the hydrolysis temperature is 35-45°C.

[0023] In one embodiment of the present invention, in step (3), the pH is adjusted to 7-9.

[0024] In one embodiment of the present invention, in step (3), the temperature of the heat treatment is 90-100°C.

[0025] Preferably, in step (3), the temperature of the heat treatment is 93-97℃.

[0026] In one embodiment of the present invention, the heating treatment time in step (3) is 8-12 min.

[0027] Preferably, in step (3), the heating treatment time is 9-11 min.

[0028] In one embodiment of the present invention, the reagent used to adjust the pH is an alkaline or acidic solution with a concentration of 0.1~5 mol / L; the solvent is water; the alkaline includes one or more of sodium hydroxide, sodium carbonate, sodium bicarbonate, potassium hydroxide, potassium carbonate, and potassium bicarbonate; the acid includes one or more of hydrochloric acid, acetic acid, citric acid, and fruit acid.

[0029] The present invention also provides an ovoferritin hydrolysate with high ferric ion binding capacity, wherein the ovoferritin hydrolysate is prepared by the following method: S1. Dissolve ovotransferrin in buffer solution to prepare a protein solution; S2. After adjusting the pH of the protein solution system obtained in step S1, add pepsin and stir to hydrolyze, thus obtaining the hydrolyzed protein solution. S3. After adjusting the pH of the hydrolyzed protein solution obtained in step S2, heat it at 93-97°C to obtain the enzymatic hydrolysis product, which is the egg-transferase hydrolysate with high ferric ion binding capacity.

[0030] Furthermore, the iron ion binding capacity of the egg-transferase hydrolysate with high iron ion binding capacity is greater than 90%.

[0031] Furthermore, in step S1, ovotransferrin is prepared from egg white by heat treatment and centrifugation, with a purity >90%.

[0032] Specifically, the preparation process of ovotransferrin is as follows: adjust the pH of egg white to 5.5-6.5, stir for 1-3 hours, centrifuge and collect the supernatant, then adjust the pH of the supernatant to 7.5-8.5, and then adsorb it through a weakly acidic cation exchange resin for 1-3 hours. After the pH of the supernatant after resin adsorption is adjusted to 6.5-7.5, heat-treat it at 50-70℃ for 30-90 minutes, and finally centrifuge and collect the precipitate to obtain ovotransferrin.

[0033] In one embodiment of the present invention, in step S1, the buffer solution is a glycine-hydrochloric acid buffer solution with a pH of 1.5-3.

[0034] In one embodiment of the present invention, in step S1, the mass fraction of ovotransferrin in the protein solution is 2-6% (w / w).

[0035] In one embodiment of the present invention, in step S2, the pH is adjusted to 1.5-2.5.

[0036] Furthermore, in step S2, the hydrolysis time is 80-160 min.

[0037] Preferably, in step S2, the hydrolysis time is 90-150 min.

[0038] Furthermore, in step S2, the hydrolysis temperature is 35-45℃.

[0039] Furthermore, in step S2, the amount of protease added is 500-1000 U / mg substrate; the substrate is ovotransferrin.

[0040] Preferably, in step S2, the amount of protease added is 700-800 U / mg substrate; the substrate is ovotransferrin.

[0041] Furthermore, in step S3, the pH is adjusted to 7-9.

[0042] Furthermore, in step S3, the heat treatment time is 8-12 minutes.

[0043] Preferably, in step S3, the heating treatment time is 9-11 min.

[0044] Furthermore, the reagent used to adjust the pH is an alkaline or acidic solution with a concentration of 0.1~5 mol / L; the solvent is water; the alkaline includes one or more of sodium hydroxide, sodium carbonate, sodium bicarbonate, potassium hydroxide, potassium carbonate, and potassium bicarbonate; the acid includes one or more of hydrochloric acid, acetic acid, citric acid, and fruit acid.

[0045] The egg-transferase hydrolysate with high ferric ion binding capacity provided by this invention can be used in the food or health product fields.

[0046] Beneficial effects (1) The modified product obtained by moderate enzymatic hydrolysis of ovotransferrin in this invention has significantly improved iron-binding activity, with the iron binding rate reaching up to ≥95% and the degree of hydrolysis (DH) >20%. The process uses food-grade protease, which is mild and safe. The reaction conditions are easy to control, avoiding the risk of residual chemical reagents. Moreover, the entire process does not require organic reagents, making it green and environmentally friendly. (2) In this invention, enzymatic hydrolysis selectively exposes and optimizes iron coordination-related sites, thereby improving the iron-coordination response of ovotransferrin to Fe. 3+ The affinity and retention rate of the protein are significantly improved, the antioxidant capacity is significantly enhanced, and the proportion of free iron is reduced, thereby reducing the risk of product oxidation and effectively improving the storage stability, color and flavor of ovotransferrin-related products. (3) The moderate enzymatic hydrolysis used in this invention may produce small peptides that are easier to absorb, while maintaining iron binding sites, which can improve the bioavailability of iron and thus enhance its potential bioavailability. The modification process is simple, has a short cycle, low energy consumption, and controllable labor costs, making it easy to achieve industrial production. Attached Figure Description

[0047] Figure 1 This is a SEM microstructure diagram of the iron-binding active oocyte transferase hydrolysis product obtained in Example 1 of the present invention. Detailed Implementation

[0048] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0049] Source of raw materials Alkaline protease, neutral protease, trypsin, and pepsin were all purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. The pepsin activity was 3000 U / mg; the alkaline protease activity was 2.0 × 10⁻⁶. 5 U / g; neutral protease activity was 5.0 × 10⁻⁶ U / g. 4U / g; trypsin activity was 2500 U / mg.

[0050] Oval transferrin preparation process: (1) Pretreatment of egg white and resin adsorption: Adjust the pH of egg white to 6.0, stir at room temperature for 2 hours, and collect the supernatant of egg white by centrifugation; adjust the pH of supernatant to 8.0, and then perform static adsorption for 120 minutes by weak acid cation exchange resin; (2) Preparation of ovotransferrin: After adjusting the pH of the egg white solution after resin adsorption to 7.0, heat-treat at 60°C for 60 min, and the precipitate obtained after centrifugation is the ovotransferrin raw material used in this invention.

[0051] Test methods 1. Determination of degree of hydrolysis Reagent preparation: OPA colorimetric working solution (80 mg OPA, 3.81 g sodium tetraborate decahydrate, 100 mg SDS, 88 mg DTT dissolved in 100 mL ultrapure water, protected from light, and stored at 4℃ for a short period of time), L-serine standard stock solution 2 mmol / L, cuvette path length 1 cm.

[0052] Sample preparation: Take the OVT enzymatic hydrolysis solution, centrifuge for 15 min, and collect the supernatant. Dilute with ultrapure water as needed (usually 1:100) to prepare the test sample, and record the dilution factor N.

[0053] Establishment of standard curve: Take 400 μL of L-serine standard solutions of different concentrations (0, 0.05, 0.10, 0.20, 0.40, 0.80, 1.20, 1.60, 2.00 mmol / L) and add them to 3.0 mL of OPA working solution. Vortex mix and react at room temperature in the dark for 2 min. Use ultrapure water as a blank and measure the absorbance A340 at 340 nm to establish an L-serine concentration-absorbance standard curve. Record the slope and intercept (R²≥0.995).

[0054] Sample determination: Mix 400 μL of the diluted sample solution with an equal volume of 3.0 mL of OPA working solution, incubate at room temperature in the dark for 2 min, and then read the value at 340 nm. Calculate the equivalent serine concentration C (mmol / L) of the sample from the standard curve. Each sample should be tested in at least three parallel trials, and the average value should be taken. If the reading exceeds the linear range, adjust the dilution factor N and repeat the test. Degree of hydrolysis (DH) is calculated using the following formula:

[0055] Where C is the converted serine concentration, calculated by substituting the sample absorbance value into the standard curve (mmol / L); N is the sample dilution factor; V is the volume of the hydrolyzed protein solution (L); m is the mass of the protein participating in hydrolysis (g); α is 1.0, β is 0.4, and htot Take 8.

[0056] 2. Iron ion binding ability Take 1.0 mL of the prepared OVT enzymatic hydrolysis solution and add it to 3.0 mL of deionized water and mix well. Add 0.10 mL of 1.0 mmol / L FeSO4 solution and 0.20 mL of 5.0 mmol / L o-phenanthroline solution sequentially, vortex thoroughly, and react at 25℃ for 20 min. Measure the absorbance of the reaction solution at 465 nm and record it as A1. Prepare a control tube using deionized water instead of the OVT enzymatic hydrolysis sample solution (all other conditions are the same), and measure the absorbance, recording it as A0. Calculate the iron binding rate using the following formula:

[0057] 3. Iron ion saturation The OVT enzymatic hydrolysate was dissolved in a 20 mmol / L Tris-HCl buffer solution pre-equilibrated to pH 8.0, and NaHCO3 was added to a final concentration of 100 mmol / L to prepare a 2% (w / w) OVT solution. An equal volume of the protein solution was mixed with a 20 mmol / L FeCl3·6H2O solution, incubated at 25°C for 10 min, and the absorbance (A) at 465 nm was measured. When iron saturation was 1%, the corresponding absorbance was 0.57. Iron saturation was calculated using the following formula:

[0058] 4. DPPH free radical scavenging ability The prepared OVT enzymatic hydrolysis product was prepared into a solution with a concentration of 5 mg / mL. 1 mL of the test solution was taken, and 0.5 mL of 0.2 mmol / L DPPH was added. The mixture was thoroughly mixed and reacted in the dark for 30 min. After centrifugation for 10 min, the absorbance A1 was measured at 517 nm using 95% ethanol as a reference. An equal volume of anhydrous ethanol was used as a control, with an absorbance value of A0. The calculation formula is as follows:

[0059] 5. Hydroxyl radical scavenging ability The prepared OVT enzymatic hydrolysis product was prepared into a solution with a concentration of 5 mg / mL. 1 mL of the test solution was taken, and 1 mL of 9 mmol / L ferrous sulfate, 1 mL of 9 mmol / L ethanol-based salicylic acid solution, and 1 mL of 9 mmol / L hydrogen peroxide were added sequentially to initiate the reaction. After shaking and mixing, the solution was placed in a 37℃ constant temperature water bath for 30 min. Using distilled water as a blank, the absorbance A1 was measured at 510 nm. Using distilled water instead of hydrogen peroxide, the absorbance A2 was measured. Using distilled water instead of the protein solution, the absorbance A0 was measured. The calculation formula is as follows:

[0060] 6. Bitterness sensory evaluation level Evaluation conditions: The evaluation was conducted in a sensory room at 22 ± 2℃ and free of odors, by 15 judges who had received basic training. Judges were advised to avoid consuming spicy or caffeinated beverages before the evaluation. Judges underwent basic taste training according to GB / T 16291.1-2012 and were pre-trained on the bitterness intensity of a 0-100 linear scale using a quinine sulfate standard solution to ensure standardized scoring.

[0061] Sample preparation: The obtained OVT enzymatic hydrolysis product was redissolved in deionized water at 1.0% (w / v), the pH was adjusted to 6.8 ± 0.1, and the sample was kept at 37℃ to remove air bubbles; the sample was dispensed into 10 mL / cup odorless plastic cups, and the samples were presented in random order using a three-digit random number code.

[0062] Evaluation Method: A single-sample sequential method was used. Each reviewer took one sample, held it in their mouth or chewed it lightly for 5-10 seconds, and then spat it out. Immediately afterwards, they recorded the bitterness intensity of the sample on a 0-100 linear scale, where 0 represents "no bitterness" and 100 represents "extremely bitterness." Between samples, the reviewer rinsed their mouth thoroughly with warm water at least twice, with an interval of 60-90 seconds to eliminate any residual influence from the previous sample. Each sample was evaluated twice, and the average of the two scores was taken as the reviewer's result for that sample.

[0063] Scoring and Grading: The average bitterness intensity values ​​of all reviewers for the same sample are further averaged to obtain the overall bitterness score for that sample. Based on the overall score, the bitterness level is divided into three grades. (1) Overall score ≤20: It is considered that there is basically no bitterness, which is the "no bitterness" level; (2) 20 < overall score ≤ 40: It is considered that there is a slight bitterness but the overall taste is acceptable, which is the "slight bitterness" level; (3) Overall score > 40: The bitterness is considered obvious and affects the taste, and is classified as "obvious bitterness".

[0064] When the overall score is ≤20, the sample is considered to be qualified in terms of bitterness; when the overall score is between 20 and 40, it is considered to be basically acceptable; when the overall score is >40, it is considered to be unqualified.

[0065] Results presentation: The report shows the bitterness level corresponding to the sensory score of bitterness for each sample.

[0066] Example 1 A method for enhancing the iron-binding capacity of ovotransferrin through appropriate enzymatic hydrolysis specifically includes the following steps: (1) Raw material preparation: Dissolve ovotransferrin in glycine-hydrochloric acid buffer (30 mmol / L, pH 2.0±0.2) to prepare a 4% (w / w) protein solution system; (2) Enzymatic modification of ovotransferrin: The pH of the protein solution reaction system obtained in step (1) was adjusted to 2.0 using 1 mol / L NaOH or HCl aqueous solution. Pepsin was selected, and the enzyme-to-substrate mass ratio (E / S) was 1:4. The protein was hydrolyzed at 40°C for 120 min with constant temperature stirring (800 r / min) to obtain the protein hydrolysate. The pH of the system was dynamically controlled using 1 mol / L NaOH or HCl aqueous solution throughout the hydrolysis process. (3) Enzyme inactivation: After adjusting the pH of the protease hydrolysate obtained in step (2) to 7.0 with 1 mol / L NaOH or HCl aqueous solution, heat it to 95℃ and treat it in a water bath for 10 min. The resulting enzymatic hydrolysate is the high iron-binding active oocyte transferase hydrolysate.

[0067] Example 2 The enzyme to substrate mass ratio (E / S) in step (2) of Example 1 was changed to 1:3, and other conditions were the same as in Example 1.

[0068] Example 3 The enzyme to substrate mass ratio (E / S) in step (2) of Example 1 was changed to 1:6, and other conditions were the same as in Example 1.

[0069] Example 4 The enzymatic hydrolysis temperature in step (2) of Example 1 was changed to 35°C, while other conditions remained the same as in Example 1.

[0070] Example 5 The enzymatic hydrolysis temperature in step (2) of Example 1 was changed to 45°C, while other conditions remained the same as in Example 1.

[0071] Example 6 The enzymatic hydrolysis time in step (2) of Example 1 was changed to 90 min, and other conditions were the same as in Example 1.

[0072] Example 7 The enzymatic hydrolysis time in step (2) of Example 1 was changed to 150 min, and other conditions were the same as in Example 1.

[0073] Example 8 The enzymatic hydrolysis pH value in step (2) of Example 1 was changed to 1.5, and other conditions were the same as in Example 1.

[0074] Example 9 The enzymatic hydrolysis pH value in step (2) of Example 1 was changed to 2.5, and other conditions were the same as in Example 1.

[0075] Example 10 The heat treatment temperature in step (3) of Example 1 was changed to 93°C, and other conditions were the same as in Example 1.

[0076] Example 11 The heat treatment temperature in step (3) of Example 1 was changed to 97°C, and other conditions were the same as in Example 1.

[0077] Example 12 The heat treatment time in step (3) of Example 1 was changed to 8 min, and other conditions were the same as in Example 1.

[0078] Example 13 The heat treatment time in step (3) of Example 1 was changed to 12 min, and other conditions were the same as in Example 1.

[0079] Compare with Example 1 The enzymatic digestion in step (2) of Example 1 was cancelled, and the original conditions of ovotransferrin were maintained. Other conditions were the same as in Example 1.

[0080] Compare with Example 2 In step (2) of Example 1, the protease was changed to an alkaline protease, the enzymatic hydrolysis pH was 8.0, the enzymatic hydrolysis temperature was 55℃, and other conditions were the same as in Example 1.

[0081] Compare with Example 3 In step (2) of Example 1, the protease was changed to a neutral protease, the enzymatic hydrolysis pH was 6.5, the enzymatic hydrolysis temperature was 50°C, and other conditions were the same as in Example 1.

[0082] Compare with Example 4 In step (2) of Example 1, the protease was changed to trypsin, the enzymatic hydrolysis pH was 9.0, the enzymatic hydrolysis temperature was 37°C, and other conditions were the same as in Example 1.

[0083] Compare with Example 5 The enzyme to substrate mass ratio (E / S) in step (2) of Example 1 was changed to 1:2, and other conditions were the same as in Example 1.

[0084] Compare with Example 6 The enzyme-to-substrate mass ratio (E / S) in step (2) of Example 1 was changed to 1:8, and other conditions were the same as in Example 1.

[0085] Compare with Example 7 The enzymatic hydrolysis temperature in step (2) of Example 1 was changed to 25°C, while other conditions remained the same as in Example 1.

[0086] Compare with Example 8 The enzymatic hydrolysis temperature in step (2) of Example 1 was changed to 55°C, while other conditions remained the same as in Example 1.

[0087] Compare with Example 9 The enzymatic hydrolysis time in step (2) of Example 1 was changed to 30 min, and other conditions were the same as in Example 1.

[0088] Compare with Example 10 The enzymatic hydrolysis time in step (2) of Example 1 was changed to 210 min, and other conditions were the same as in Example 1.

[0089] Compare with Example 11 The pH value in step (2) of Example 1 was changed to 3.0, and other conditions were the same as in Example 1.

[0090] Compare with Example 12 The heat treatment temperature in step (3) of Example 1 was changed to 70°C, and other conditions were the same as in Example 1.

[0091] Compare with Example 13 The heat treatment temperature in step (3) of Example 1 was changed to 90°C, and other conditions were the same as in Example 1.

[0092] Compare with Example 14 The heat treatment temperature in step (3) of Example 1 was changed to 100°C, and other conditions were the same as in Example 1.

[0093] Compare with Example 15 The heat treatment time in step (3) of Example 1 was changed to 20 min, and other conditions were the same as in Example 1.

[0094] Compare with Example 16 The preparation process of OVT adult hydrolysate is carried out in accordance with the patent CN 120796428 A, as follows: (1) Dissolve ovotransferrin in buffer solution to prepare a 4% (w / w) protein solution system; (2) The pH of the protein solution reaction system obtained in step (1) was adjusted to 2.0 using 1 mol / L NaOH or HCl aqueous solution. Pepsin was selected, and the enzyme-to-substrate mass ratio (E / S) was 1:4. The protein solution was hydrolyzed for 120 min at 40℃ with constant temperature stirring (800 r / min) to obtain the hydrolyzed protein solution. The pH of the system was dynamically adjusted using 1 mol / L NaOH or HCl aqueous solution throughout the hydrolysis process. (3) Then add bile salts (10 mM) and 5 mL of trypsin (100 U / mL) to the hydrolyzed protein solution obtained in step (2), adjust the pH to 7.0 and incubate at 37°C for 2 h; (4) Heat the hydrolyzed protein solution obtained in step (3) to 95°C and treat it in a water bath for 10 min. The resulting enzymatic hydrolysis product is the egg transferase hydrolysis product.

[0095] The degree of hydrolysis, iron ion binding capacity, iron ion saturation, DPPH radical scavenging rate, and hydroxyl radical scavenging rate of Examples 1-13 and Control Examples 1-16 were determined. The results are shown in Table 1. Table 1 Summary of Results for Examples and Control Examples

[0096] The results above show that after ovotransferrin is prepared into a protein solution, it is enzymatically modified by a specific protease under specific conditions, and then inactivated by heating at a specific temperature to obtain an ovotransferrin enzymatic hydrolysate with high iron-binding capacity and high antioxidant capacity.

[0097] The sample obtained in Example 1 of the table has the best overall performance. Compared with the control Example 1 without enzymatic hydrolysis, the iron binding capacity and antioxidant index are significantly improved, indicating that under the conditions of "moderate enzymatic hydrolysis + controlled heat treatment", the iron coordination-related sites can be effectively exposed and stabilized, and the pro-oxidative effects caused by free iron can be reduced.

[0098] Results of Examples 1 and Control Examples 2-4: In Control Examples 2-4, the degree of hydrolysis of ovotransferrin was similar to that in Example 1 after replacing the protease with alkaline protease, neutral protease, and trypsin, respectively. However, in Control Example 2, compared with Example 1, after replacing pepsin with alkaline protease, the iron-binding capacity and iron saturation decreased by 12.39% and 10.88%, respectively, and the DPPH and hydroxyl radical scavenging rates also decreased by 11.67% and 12.23%, respectively. In Control Example 3, after using neutral protease to hydrolyze ovotransferrin, the iron-binding capacity and iron saturation decreased by 12.66% and 12.20%, respectively, and the DPPH and hydroxyl radical scavenging rates decreased by 12.76% and 12.52%, respectively. In Control Example 4, after using trypsin, the iron-binding capacity and iron saturation decreased by 10.38% and 12.31%, respectively, and the DPPH and hydroxyl radical scavenging rates also decreased by 11.99% and 10.81%, respectively. The degree of hydrolysis in the three control groups was similar to that in Example 1, but their iron-binding capacity and free radical scavenging capacity were significantly lower. This indicates that iron-binding performance is not solely determined by the degree of hydrolysis. The cleavage sites and peptide distribution of alkaline / neutral / trypsin at their respective optimal pH and temperature are not conducive to the simultaneous "exposure and retention" of His-Asp / Glu-Tyr motifs involved in coordination, making it difficult to form a stable multidentate coordination microenvironment. At the same time, alkaline or mismatched conditions easily lead to partial protein denaturation and aggregation / site masking, reducing Fe. 3+The complexation stability and mass transfer accessibility are reduced, increasing the movable iron pool, thus resulting in a simultaneous decrease in iron binding capacity and free radical scavenging capacity.

[0099] In contrast, in Comparative Examples 5-15, when key parameters such as enzyme / substrate ratio, temperature, time, or pH deviated from the range set by this invention, the relevant indicators deteriorated to varying degrees. For example, in Comparative Example 15, the iron binding capacity and iron saturation decreased to 57.11% and 53.07%, respectively. This demonstrates that insufficient or excessive enzymatic hydrolysis, as well as mismatches in process parameters exceeding the range, can all disrupt the iron coordination-related microenvironment and the macroscopic structure of proteins, thereby weakening iron loading and antioxidant properties.

[0100] The results of Examples 1-3 show that the enzyme-to-substrate ratio (E / S) significantly affects the modification effect of ovotransferrin. When the E / S ratio is controlled between 1:3 and 1:6, ovotransferrin is moderately hydrolyzed, exposing key residues that bind to iron ions while maintaining favorable peptide length and spatial structure, resulting in a high iron-binding capacity. In Control Example 5, under the same conditions, the E / S ratio was increased to 1:2, resulting in a significantly higher enzyme dosage. The protein was over-hydrolyzed, and some continuous coordination fragments were cleaved or re-aggregated, leading to a reduction in effective sites for iron binding. The iron-binding capacity decreased by 15.67% compared to Example 1. In Control Example 6, the opposite occurred. The E / S ratio was reduced to 1:8, resulting in a lower enzyme dosage and insufficient hydrolysis. Larger protein domains and aggregates remained, and many potential coordination sites were not fully exposed. Iron ions could not easily access these sites, and therefore the iron-binding capacity was also significantly lower than that of Examples 1-3 with moderate enzyme dosage.

[0101] In Control Example 8, the enzymatic hydrolysis temperature was higher than that in Example 1. This may be because the increased temperature induced thermal loosening and early aggregation of ovotransferrin, partially burying the coordinating residues (His, Tyr, Asp / Glu), restricting diffusion pathways, and inhibiting the effective cleavage trajectory of pepsin, making it difficult to form favorable coordination peptide combinations. Therefore, the iron loading and antioxidant indicators decreased significantly. In contrast, the lower enzymatic hydrolysis temperature in Control Example 7 resulted in insufficient catalytic efficiency, limited exposure of deep sites, and the retained large-scale aggregates increased mass transfer resistance and interfacial permeation resistance, reducing the number of available effective sites and thus leading to lower iron binding and saturation. Regarding the enzymatic hydrolysis time, the shorter hydrolysis time in Control Example 9 meant that the peptides were not fully cleaved and rearranged, making it difficult to form stable multi-point coordinating sequences and suitable surface pore structures. Many potential coordination sites remained buried, resulting in insufficient iron loading and weak free radical scavenging ability. Conversely, the longer hydrolysis time in Control Example 10 led to excessive cleavage of ovotransferrin, with the key fragments required for multidentate coordination being cut too short, making it difficult to stabilize Fe. 3+- Carbonate complexes, along with the short peptides generated, are prone to secondary aggregation or non-ideal competition with metal ions, resulting in a decrease in the occupancy and retention of low-affinity sites. Adjusting the pH of the enzymatic digestion showed that the pH of control example 11 was too high, altering the ionization state of the coordinating residues (His, Tyr, Asp / Glu, etc.) and the carbonate / bicarbonate ratio. This affected the protease's cleavage specificity and peptide length / sequence distribution, making it difficult to maintain the integrity of the multidentate coordination motif while "fully exposing" it. This manifested as a simultaneous decrease in iron-binding capacity / saturation and free radical scavenging ability.

[0102] The results of Examples 10-13 show that, under reasonable heat treatment conditions, the iron binding capacity and antioxidant index remained high and stable. In contrast, the iron ion binding capacity and iron saturation of Control Examples 12 and 15 were only about 69.28% / 64.40% and 57.11% / 53.07%, respectively, and the DPPH and hydroxyl radical scavenging rates were also significantly lower, showing a significant overall performance inferior to the Example group. It is noteworthy that although the degree of hydrolysis in Control Example 15 was similar to that in the Examples, the performance difference was still significant, further illustrating that the iron binding capacity is not solely determined by the degree of hydrolysis, but rather depends more on whether the enzyme cleavage sites and peptide distribution are conducive to the formation of a stable multidentate coordination structure and good interfacial accessibility. The poor performance of Control Example 12 was mainly attributed to insufficient thermal driving force provided by the heat-induced treatment temperature, making it difficult to induce favorable structural rearrangement of enzymatically digested peptides and lock them into stable coordination conformations. During cooling, loosely structured peptides were prone to non-specific aggregation or revert to a disordered state, resulting in key coordination sites (such as His, Asp, etc.) being spatially shielded or conformationally distorted, thus significantly reducing the binding efficiency and stability of iron ions. Control Example 15 was mainly affected by excessive heat treatment. Prolonged heating induced protein aggregation and disulfide bond rearrangement, which shielded key coordination residues such as His / Tyr / Asp / Glu and restricted diffusion, and interfered with the co-entry of carbonate ions, resulting in a decrease in the occupancy and retention rate of low-affinity sites. Ultimately, this manifested as a significant reduction in iron ion binding capacity, iron saturation, and free radical scavenging capacity.

[0103] Examples 10 and 11 show that controlling enzyme inactivation at approximately 95°C for a short time can stably achieve high iron-binding capacity, while maintaining high DPPH and hydroxyl scavenging rates without bitterness. In contrast, the iron-binding capacity of Control Examples 13 and 14 is significantly reduced (binding capacity / saturation is only about 84.62% / 80.59% and 86.33% / 81.02%, respectively). This is because when the heat treatment temperature is too low, the thermal driving force is insufficient, the enzyme activity termination and structural "fixation" are incomplete, and mild hydrolysis is still likely to occur during cooling, resulting in a loose conformation and failure to fully dissipate weak aggregation and short-range interactions. When the heat treatment temperature is too high, thermally induced irreversible aggregation and disulfide bond rearrangement occur in the ovoferroprotein hydrolysate, expanding hydrophobic / β-structure interactions, causing key coordinating residues to be spatially shielded, and Fe 3+ The efficiency of cooperating anions entering and stabilizing the binding site decreased. Furthermore, in Control Example 16, iron binding and saturation were only 82.90% / 78.54%, and a distinct bitter taste was observed. The two-stage digestion and continuous enzymatic hydrolysis across pH continued until termination, and the combined action of bile salts and trypsin further cleaved the protein into shorter peptides. This not only made it difficult to retain the peptide backbone required for stable polydentate coordination but also easily produced bitter peptides. Even with considerable antioxidant properties, it was difficult to meet the core objective of high iron binding capacity.

[0104] The sensory evaluation results of bitterness show that the samples prepared within the pepsin hydrolysis conditions defined in this invention, as well as the unhydrolyzed control example 1, all had average bitterness scores below 20 on the 0-100 linear scale, and were classified as "no bitterness". Conversely, the average scores of controls 2, 3, 4, 5, 10, 12, and 16 were all above 40, falling into the "obvious bitterness" range, and showing significant differences compared to the corresponding examples. Controls 2, 3, and 4 were hydrolyzed using alkaline protease, neutral protease, and trypsin under neutral-alkaline pH conditions, which easily cleaved adjacent peptide bonds of hydrophobic amino acids, generating peptides rich in Val, Leu, and Ile. Short hydrophobic peptides containing certain residues significantly enhance bitterness. In Control Examples 5 and 10, the enzyme / substrate ratio was increased or the hydrolysis time was extended in the same pepsin system, exceeding the preferred range of the present invention, which also resulted in the enrichment of bitter peptides. In Control Example 12, the enzyme inactivation temperature was only 70°C, and the residual enzyme activity continued to act during cooling and storage, making it difficult to control the actual degree of hydrolysis, resulting in increased bitterness and large batch-to-batch fluctuations. The obvious bitterness in Control Example 16 was closely related to its continuous hydrolysis across pH: after hydrolysis of the gastric segment, treatment with pancreatic enzymes / bile salts further cleaved the protein into shorter peptides enriched with hydrophobic residues (such as Val, Leu, and Ile). These peptides retained bitterness even after subsequent treatment. At the same time, residual hydrolysis occurred during the pH transition and temperature rise transition stages of the two reactions, which also caused the accumulation of bitter peptides.

[0105] The egg-transferase hydrolysate prepared within the scope given in this invention exhibits high iron-binding capacity, while its DPPH and hydroxyl radical scavenging abilities are significantly enhanced. Compared to Example 1, the control examples show a significant overall lag: although the degree of hydrolysis in Control Examples 2 and 3 is close to 22%, their iron-binding capacity decreases by 12.39% and 12.66%, respectively. Several controls deviating from the conditions of this invention (changes in enzyme / substrate ratio, temperature, time, or pH) all caused varying degrees of degradation. Among them, the iron-binding capacity of Control Example 8 decreased by 32.29% compared to Example 1, the DPPH and hydroxyl radical scavenging rates of Control Example 10 decreased by 43.75% and 37.12%, respectively, and the iron-binding capacity and iron saturation of Control Examples 12 and 15 decreased by 25.92% and 27.97%, and 38.09% and 39.30%, respectively, due to thermal inactivation conditions exceeding the parameter range involved in this invention. The iron-binding capacity and iron saturation of Control Examples 13 and 14 decreased by 10.58% and 11.78% and 8.87% and 11.35%, respectively, indicating that both excessively low and excessively high inactivation temperatures are detrimental to the exposure and stability of coordination sites. Control Example 16 showed a 12.30% and 13.83% decrease in iron-binding capacity and iron saturation compared to Example 1, and exhibited a noticeable bitter taste, suggesting that this process could not meet the goal of "high iron binding." These comparative results indicate that the system in the examples is significantly superior to the controls in terms of iron binding, iron saturation, and free radical scavenging. Further comparisons show that iron binding capacity is not determined solely by the degree of hydrolysis, but is closely related to the enzyme cleavage sites and peptide distribution: Example 6 achieved a high iron binding capacity of 91.38% and a high iron saturation of 89.26% at a degree of hydrolysis of 18.67%; conversely, Control Example 2 only achieved an iron binding capacity of 82.81% at a degree of hydrolysis of 22.17%. This invention demonstrates that the enzymes and conditions selected in this invention are more conducive to the moderate exposure and retention of His / Tyr / Asp(Glu) and other residues involved in polydentate coordination, thereby forming stable Fe³⁺. + By coordinating the microenvironment and reducing the movable iron pool, the bitterness score of the resulting enzymatic hydrolysate is maintained at the "no bitterness" level while maintaining a high iron binding capacity and antioxidant performance. In contrast, the control samples that deviate from the conditions of this invention generally show obvious bitterness and are difficult to meet the requirements for food applications. This invention comprehensively demonstrates the dual advantages of this invention in terms of function and flavor.

[0106] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. A ferric transferase hydrolysate with high ferric ion binding capacity, characterized in that, The iron-binding capacity of the egg transferase hydrolysate is greater than 90%. The egg transferase hydrolysate was prepared by the following method: S1. Dissolve ovotransferrin in buffer solution to prepare a protein solution; S2. After adjusting the pH of the protein solution system obtained in step S1 to 1.5-2.5, add pepsin and stir to hydrolyze, thus obtaining a hydrolyzed protein solution; the amount of pepsin added is 500-1000 U / mg substrate; the substrate is ovotransferrin. S3. After adjusting the pH of the hydrolyzed protein solution obtained in step S2 to 7-9, heat it at 93-97℃ to obtain the enzymatic hydrolysis product, which is the egg-transferase hydrolysate with high ferric ion binding capacity.

2. The egg transferase hydrolysate according to claim 1, characterized in that, In step S1, ovotransferrin is prepared from egg white by heat treatment and centrifugation, with a purity >90%.

3. The oocyte transferrin hydrolysate according to claim 1, characterized in that, In step S1, the buffer solution is a glycine-hydrochloric acid buffer solution with a pH of 1.5-3.

4. The egg transferase hydrolysate according to claim 1, characterized in that, In step S1, the mass fraction of ovotransferrin in the protein solution is 2-6%.

5. The oocyte transferrin hydrolysate according to claim 1, characterized in that, In step S2, the hydrolysis time is 80-160 min; the hydrolysis temperature is 35-45℃.

6. The oocyte transferrin hydrolysate according to claim 5, characterized in that, In step S2, the hydrolysis time is 90-150 min.

7. The ovoferritin hydrolysate according to claim 1, characterized in that, In step S2, the amount of protease added is 700-800 U / mg substrate; the substrate is ovotransferrin.

8. The ovoferritin hydrolysate according to claim 1, characterized in that, In step S3, the heating treatment time is 8-12 minutes.

9. The oocyte transferrin hydrolysate according to claim 1, characterized in that, The reagent used to adjust the pH is an alkaline or acidic solution with a concentration of 0.1~5 mol / L; the solvent is water; the alkaline includes one or more of sodium hydroxide, sodium carbonate, sodium bicarbonate, potassium hydroxide, potassium carbonate, and potassium bicarbonate; the acid includes one or more of hydrochloric acid, acetic acid, citric acid, and fruit acid.

10. The application of the ovoferrotransferase hydrolysate with high ferric ion binding capacity as described in any one of claims 1-9 in the food or health product field.

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