A composite adsorbent material for enriching phosphopeptides of egg yolk high-phosphoprotein and its preparation method

Abstract

This invention relates to the field of biomedical technology, specifically to a composite adsorption material for enriching phosphopeptides of egg yolk high-phosphoproteins and its preparation method. The material comprises, from the inside out: Zr 4+ and La 3+ A bimetallic-doped TiO2 matrix, an EDTA passivation layer, and an amino-functionalized layer were co-doped. The preparation process included: synthesizing the bimetallic-doped TiO2 matrix via a sol-gel method, passivating and coating it with EDTA solution after two-step calcination, and then grafting the amino layer using APTES / acetic acid catalysis. This material exhibits both high specific surface area and specific binding capacity, achieving an adsorption capacity of 67.5 mg / g. After 10 cycles, the capacity retention rate is >92.8%, enabling efficient enrichment of high-purity PPPs, making it suitable for the industrial production of functional foods and pharmaceuticals.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a composite adsorption material for enriching egg yolk high-phosphoprotein phosphopeptides and its preparation method. Background Technology

[0002] Phosvitin phosphopeptides (PPPs), as enzymatic hydrolysis products of phosphoprotein, are rich in phosphatidylserine residues, which can effectively promote the absorption of metal ions such as calcium, iron, and zinc. Furthermore, the calcium metabolism induced by PPPs does not require vitamin D, making them valuable for applications in functional foods and nutritional fortifiers. With the increasing demand for natural functional peptides in the food industry, the technology for efficiently enriching PPPs has become a research hotspot. Methods for enriching PPPs mainly include ethanol calcium precipitation, membrane separation, ion exchange resin methods, and adsorption material methods. While ethanol calcium precipitation is simple to operate, it requires large amounts of organic reagents, easily causing environmental pollution. Membrane separation suffers from severe membrane fouling, requiring frequent cleaning and maintenance, and its separation efficiency is limited by membrane pore size. Ion exchange resin methods are difficult to adapt to continuous industrial production due to complex pretreatment and regeneration steps.

[0003] In recent years, adsorption methods have attracted attention due to their advantages such as high specificity and convenient operation. The extraction and purification of egg yolk high-phosphoprotein phosphopeptides (PPPs) has been a key research focus in many fields, including the food and fermentation industries. In existing technologies, Su Yujie et al. (“Study on the Purification Performance of Egg Yolk High-phosphoprotein Phosphopeptides by Titanium Dioxide”, Su Yujie et al., Food and Fermentation Industries, Vol. 40, No. 12, pp. 26-31, Publication Date: December 31, 2014) disclosed a method for enriching egg yolk high-phosphoprotein phosphopeptides. This method involves preparing a polypeptide solution with an initial concentration of 10-80 mg / mL from crude egg yolk hydrated polypeptides. A certain amount of this polypeptide solution is then placed in a centrifuge tube, and TiO2 is added for adsorption. After adsorption, a series of operations including centrifugation, washing, and elution are performed to obtain the elution supernatant containing PPPs. However, single TiO2 adsorbents still have some limitations: First, their adsorption performance is greatly affected by environmental factors such as pH and ionic strength, and non-specific adsorption is prone to occur in complex systems; second, the mechanical strength and reusability of TiO2 materials need to be improved, and the adsorption capacity tends to decrease after long-term cycling. Furthermore, for high-concentration crude peptide systems exceeding 20 mg / mL, the adsorption saturation threshold of existing TiO2 materials is low, making it difficult to further improve the unit volume treatment efficiency. To address this, Chinese invention patent CN 105018553B proposes using mesoporous / macroporous titanium dioxide (TiO2) as an adsorbent. Utilizing the specific affinity of TiO2 for phosphate groups, combined with the high specific surface area and excellent pore characteristics of the mesoporous / macroporous structure, it achieves highly efficient enrichment of PPPs, with an adsorption capacity of 42-53 mg / g and a short operating cycle, which is beneficial for industrial applications. The preparation method of this mesoporous / macroporous titanium dioxide (TiO2) is as follows: citric acid is dissolved in ethanol and stirred, then an ammonia solution is added and stirred continuously. Then, tetrabutyl titanate is added dropwise to the above solution while stirring, and then allowed to stand. The precipitate is then washed with water and anhydrous ethanol in sequence, the washed material is dried, and finally the above material is placed in a muffle furnace for calcination. For every 0.3g of citric acid, 18mL of ammonia solution with a mass concentration of 25%-28% and 5mL of tetrabutyl titanate are added.

[0004] However, the aforementioned existing technologies still have some shortcomings in practical applications. For example, the selectivity and binding efficiency of the materials used in key steps such as adsorption and elution for PPPs need to be improved, resulting in low overall extraction and purification efficiency, and the product yield and purity are difficult to meet the growing needs of industrial production and scientific research.

[0005] Therefore, developing a composite adsorption material with high specificity, high adsorption capacity, strong environmental adaptability and good repeatability is of great significance for improving the enrichment efficiency of PPPs and reducing the cost of industrial production. Summary of the Invention

[0006] To address the aforementioned technical problems, the present invention aims to provide a composite adsorbent material for enriching egg yolk high-phosphoprotein phosphopeptides and its preparation method. The adsorbent material possesses both high specific surface area and specific binding capacity, enabling efficient enrichment of high-purity PPPs, and is suitable for industrial production of functional foods and pharmaceuticals.

[0007] To achieve the above-mentioned technical effects, the present invention adopts the following technical solution:

[0008] First, this invention provides a composite adsorbent material for enriching phosphopeptides of egg yolk high-phosphoproteins, which comprises, from the inside out: Zr 4+ and La 3+ A co-doped mesoporous-macroporous TiO2 matrix; an EDTA passivation layer covering the surface of the matrix; and an amino-functionalized layer grafted onto the passivation layer.

[0009] Preferably, in the composite adsorbent material, the Zr / Ti molar ratio is 5-10% and the La / Ti molar ratio is 1-3%. More preferably, the Zr / Ti molar ratio is 8% and the La / Ti molar ratio is 2%.

[0010] Preferably, the loading of the EDTA passivation layer is 0.05-0.2 mmol / g, and the loading of the amino functionalized layer is 0.5-1.2 mmol / g.

[0011] Secondly, the present invention also provides a method for preparing a composite adsorbent material for enriching egg yolk high-phosphoprotein phosphopeptides, comprising the following steps:

[0012] S1: Zr 4+ / La 3+ Preparation of co-doped mesoporous-macroporous TiO2 matrix:

[0013] Citric acid was dissolved in anhydrous ethanol, stirred, and then ammonia was added. Zirconium nitrate and lanthanum nitrate were then added, and the Zr / Ti molar ratio was controlled at 5-10% and the La / Ti molar ratio at 1-3%. After stirring, tetrabutyl titanate was added dropwise. The mixture was allowed to stand to form a gel. After washing and drying, the co-doped TiO2 matrix was obtained by calcination.

[0014] S2: EDTA passivation layer coating

[0015] The matrix obtained in step S1 was dispersed in EDTA solution at a solid-liquid ratio of 1:20-1:50 g / mL, shaken at 50-70℃ for 1-3 h, washed until neutral, and then dried.

[0016] S3: Amino-functionalized layer grafting:

[0017] The material obtained in step S2 was dispersed in anhydrous ethanol containing APTES and acetic acid at a solid-liquid ratio of 1:40-1:60 g / mL, refluxed at 45-55℃ for 3-5 h, washed and dried to obtain the composite adsorbent material.

[0018] Preferably, the calcination in S1 is carried out in two steps: first, calcination at 300-400℃ for 0.5-1.5h, and then calcination at 500-600℃ for 1.5-2.5h.

[0019] Preferably, in S1, the amount of citric acid is 0.3g, the amount of anhydrous ethanol is 18-22mL, the amount of ammonia is 16-20mL, the amount of tetrabutyl titanate is 4-6mL, and the dropping rate is 1-3mL / min.

[0020] Preferably, the concentration of the EDTA solution is 0.05-0.2M and the pH is 5.0-6.0.

[0021] Preferably, the volume fraction of APTES in S3 is 3-7%, and the volume fraction of acetic acid is 0.3-0.7%.

[0022] Thirdly, the present invention also provides an application of the composite adsorption material provided in the first aspect above in the enrichment of yellow high-phosphoprotein phosphopeptides.

[0023] Furthermore, the application includes the following steps:

[0024] (1) Egg yolk protein powder was partially dephosphorized by alkaline method and then enzymatically hydrolyzed to obtain crude polypeptide solution;

[0025] (2) Under pH conditions of 4.0-6.0, the crude polypeptide solution is brought into contact with the composite adsorbent material for adsorption;

[0026] (3) Use a buffer solution with pH 8.0-9.0 to elute the adsorbed phosphopeptides.

[0027] Preferably, step (1) specifically comprises:

[0028] Egg yolk high-phosphorus protein powder was dissolved in an alkaline buffer solution with a pH of 8.0-10.0 to prepare a protein solution with a concentration of 10-50 mg / mL. Alkaline phosphatase was added to the protein solution for partial dephosphorylation, with an ALP addition of 100-500 U / g protein. The solution was incubated at 37-45℃ with shaking for 1-4 hours. The pH of the dephosphorylated protein solution was adjusted to 7.5-9.0, and trypsin or chymotrypsin was added for enzymatic hydrolysis, with the enzyme addition being 1-5% of the protein mass. The solution was incubated at 37-50℃ with shaking for 4-12 hours. After the enzymatic hydrolysis was completed, the reaction system was heated in a boiling water bath for 5-15 minutes to terminate the hydrolysis. After cooling, the solution was centrifuged (8000-12000 rpm, 10-20 min) or filtered, and the supernatant was collected as the crude polypeptide solution.

[0029] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0030] The composite adsorbent material provided by this invention significantly enhances the enrichment performance of egg yolk high-phosphoprotein phosphopeptides (PPPs) through a synergistic innovation of a triple structure:

[0031] First, with Zr 4+ / La 3+ Using co-doped mesoporous-macroporous TiO2 as the matrix, and by controlling the Zr / Ti molar ratio to 5-10% and the La / Ti molar ratio to 1-3%, the pore structure and surface active sites are optimized through bimetallic synergistic effects, resulting in a material with a specific surface area as high as 218.2 μm². 2 / g, breaking through the bottleneck of existing TiO2 materials in processing high-concentration crude peptide systems, and at the same time increasing the adsorption capacity to 67.5mg / g, which is more than 30% higher than that of single metal doping;

[0032] Based on this, an EDTA passivation layer with a loading of 0.05-0.2 mmol / g was introduced to effectively suppress metal ion dissolution and non-specific adsorption, ensuring that the material maintains high selectivity in complex systems. This successfully solved the stability problem of continuous industrial production, achieving a capacity retention rate of over 92.8% after 10 cycles of use.

[0033] Furthermore, by constructing an amino-functionalized outer layer with a loading of 0.5-1.2 mmol / g, the electrostatic effect is used to enhance the specific capture of phosphopeptides. Combined with an alkaline partial dephosphorization pretreatment process, the purity of the enriched product is increased by more than 40%, and the loss of active peptides is avoided by using the mild elution conditions of pH 8.0-9.0 buffer.

[0034] Ultimately, the accompanying two-step calcination process (pre-calcination at 300-400℃ combined with crystallization at 500-600℃) and controllable functionalization process (passivation of EDTA at pH 5.0-6.0 and catalytic grafting of amino groups with APTES / acetic acid) ensure high reproducibility of material performance, achieving a significant advantage of increasing unit processing efficiency by 50% and reducing overall cost by more than 30% compared to existing technologies. Detailed Implementation

[0035] The following embodiments are only used to illustrate the technical solutions of the present invention more clearly, and are therefore only examples and should not be used to limit the scope of protection of the present invention.

[0036] Those skilled in the art will understand that the present invention can be practiced even without certain specific details. In some other embodiments, methods, means, apparatus, and steps well known to those skilled in the art have not been described in detail in order to highlight the spirit of the invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Unless otherwise specified, all units used in this specification are International Standard Units (SI), and all numerical values ​​and ranges appearing in this invention should be understood to include systematic errors unavoidable in industrial production.

[0037] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials, reagents, or instruments used, unless otherwise specified by the manufacturer, are all commercially available; and the conditions not specified in the examples are all performed under conventional conditions or conditions recommended by the manufacturer. Furthermore, this invention does not limit the source of the raw materials used; unless otherwise specified, the raw materials used in this invention are all commercially available products commonly found in this technical field. Unless otherwise specified, the "ratio" referred to in the following examples refers to a ratio of parts by mass.

[0038] Example 1

[0039] The purpose of this embodiment is to provide a composite adsorption material for enriching egg yolk high-phosphoprotein phosphopeptides and its preparation method. The specific preparation method is as follows:

[0040] S1: Zr 4+ / La 3+ Preparation of co-doped mesoporous-macroporous TiO2 matrix

[0041] Weigh 0.3g of citric acid, add 20mL of anhydrous ethanol, place on a magnetic stirrer and stir at 300r / min for 15min until completely dissolved. Slowly add 18mL of 28% ammonia solution to the above solution and continue stirring for 10min to form a homogeneous mixture.

[0042] 0.489 g of zirconium nitrate (Zr(NO3)4·5H2O) and 0.123 g of lanthanum nitrate (La(NO3)3·6H2O) were weighed and added to the mixture. The mixture was stirred for 20 min to dissolve the zirconium nitrate (Zr / Ti molar ratio was 8% and La / Ti molar ratio was 2%). Then, 5 mL of tetrabutyl titanate was added dropwise through a constant pressure dropping funnel at a rate of 2 mL / min, while maintaining a stirring speed of 400 r / min. After the addition was complete, stirring was continued for 1 h to obtain a pale white turbid liquid. The turbid liquid was transferred to a 50 mL beaker and allowed to stand at room temperature for 24 h to form a pale yellow gel. The gel was washed with deionized water until the pH of the filtrate reached 7.0, and then washed three times with anhydrous ethanol (20 mL each time). The washed gel was then dried in a vacuum drying oven at 60 °C for 12 h to obtain a dry gel powder. The dry gel powder was transferred to a ceramic boat and placed in a muffle furnace for two-step calcination: first, the temperature was increased to 350℃ at a rate of 5℃ / min and held for 1 hour; second, the temperature was increased to 550℃ at a rate of 5℃ / min and held for 2 hours. After natural cooling to room temperature, Zr was obtained. 4+ / La 3+ Co-doped mesoporous-macroporous TiO2 matrix for use.

[0043] S2: EDTA passivation layer coating

[0044] Prepare 50 mL of 0.1 M EDTA solution, adjust the pH to 5.5 with 0.1 M HCl and stir well. Weigh 1 g of the co-doped TiO2 matrix prepared in step S1 and add it to the above solution (solid-liquid ratio 1:50 g / mL). Transfer the mixture to a constant temperature water bath shaker and set the temperature to 60 °C and the shaking frequency to 200 r / min for 2 h. After the reaction, wash with deionized water by centrifugation (4000 r / min, 5 min) until the pH of the supernatant is 7.0. Collect the solid and dry it in a 60 °C forced-air drying oven for 8 h to obtain an intermediate product with an EDTA loading of approximately 0.12 mmol / g.

[0045] S3: Amino-functionalized layer grafting

[0046] Measure 40 mL of anhydrous ethanol, add 2 mL of APTES (5% by volume) and 0.2 mL of acetic acid (0.5% by volume), stir for 10 min to form a functionalized modified solution, weigh 0.8 g of the intermediate product obtained in step S2 and add it to the modified solution (solid-liquid ratio 1:50 g / mL), transfer it to a 50 mL round bottom flask connected to a reflux condenser, and place it in a constant temperature water bath at 45 °C for reflux for 4 h (magnetic stirring speed 250 r / min).

[0047] After the reaction was completed, the product was washed three times by centrifugation with anhydrous ethanol (4000 r / min, 5 min). The product was then dried in a vacuum drying oven at 60 °C for 6 h to obtain the target composite adsorbent material (labeled as Zr / La-TiO2@EDTA@NH2) with an amino loading of about 0.8 mmol / g.

[0048] Furthermore, this embodiment also provides a method for enriching egg yolk high-phosphoprotein phosphopeptides using the aforementioned composite adsorbent material, as detailed below:

[0049] (1) Sample pretreatment and enzymatic digestion:

[0050] Weigh 100 mg of egg yolk high-phosphorus protein powder and dissolve it in 10 mL of 0.1 M Tris-HCl buffer (pH 8.5) to prepare a 10 mg / mL protein solution. Add alkaline phosphatase (ALP) to a final concentration of 300 U / g protein and incubate at 40°C with a shaker for 2.5 hours for partial dephosphorylation. Adjust the pH of the dephosphorylated solution to 8.2, add trypsin (enzyme / substrate mass ratio 2%), and incubate at 37°C with shaking for 8 hours for enzymatic digestion.

[0051] After enzymatic hydrolysis, the enzyme was inactivated by heating in a boiling water bath for 10 minutes. After cooling to room temperature, the mixture was centrifuged at 12,000 rpm for 15 minutes, and the supernatant was collected to obtain a crude polypeptide solution.

[0052] (2) Adsorption and enrichment:

[0053] The crude peptide solution was adjusted to pH 5.0 with 0.1 M HCl and then diluted 5-fold with 0.2 M HAc-NaAc buffer at pH 5.0 to obtain the sample solution to be enriched.

[0054] Weigh 20 mg of the composite adsorbent Zr / La-TiO2@EDTA@NH2 prepared in this example and fill it into a solid-phase extraction empty column (3 mL volume). Activate and equilibrate the column sequentially with 2 mL methanol, 3 mL ultrapure water, and 3 mL of 0.2 M HAc-NaAc buffer at pH 5.0.

[0055] Take 1 mL of the sample solution to be enriched prepared in step (1) (containing approximately 2 mg of peptide) and load it at a flow rate of 0.5 mL / min. After loading, wash with 3 mL of 0.2 M HAc-NaAc buffer at pH 5.0 at a flow rate of 1 mL / min to remove unadsorbed impurities, collect and discard the eluent.

[0056] (3) Elution and collection:

[0057] The adsorbed phosphopeptides were eluted with 2 mL of 0.2 M NH4HCO3 buffer (pH 8.5) containing 0.5 M NaCl at a flow rate of 0.3 mL / min. The elution was repeated once, and the two eluates were combined to obtain an enriched phosphopeptide solution. After freeze-drying, a white powdery enriched phosphopeptide product was obtained.

[0058] Example 2

[0059] Referring to Example 1, the purpose of this example is to provide a composite adsorbent material for enriching egg yolk high-phosphoprotein phosphopeptides and its preparation method. The specific preparation method is as follows:

[0060] S1: Zr 4+ / La 3+ Preparation of co-doped mesoporous-macroporous TiO2 matrix

[0061] Weigh 0.3 g of citric acid and add it to 18 mL of anhydrous ethanol. Stir at 250 rpm for 20 min until completely dissolved. Add 16 mL of 28% ammonia and stir for 15 min to form a mixture. Then add 0.294 g of zirconium nitrate and 0.074 g of lanthanum nitrate (Zr / Ti molar ratio 6%, La / Ti molar ratio 1.5%) and stir for 30 min to dissolve. Measure 4 mL of tetrabutyl titanate and add it dropwise to the mixture at a rate of 1 mL / min, stirring at 350 rpm. Continue stirring for 1.5 h after addition. Let stand at room temperature for 20 h to form a gel. Wash with deionized water until neutral, then wash twice with anhydrous ethanol. Dry under vacuum at 50 °C for 10 h to obtain a dry gel powder. Place the dry gel powder in a muffle furnace and heat to 300 °C at a rate of 4 °C / min, hold for 1.5 h, then continue heating to 500 °C at a rate of 4 °C / min, hold for 2.5 h, and cool to obtain a co-doped TiO2 matrix.

[0062] S2: EDTA passivation layer coating

[0063] Prepare 30 mL of 0.08 M EDTA solution and adjust the pH to 5.0. Weigh 1 g of matrix and add it to the solution (solid-liquid ratio 1:30 g / mL). Shake in a 50 °C water bath for 3 h (frequency 180 r / min). After centrifugation and washing until neutral, dry in a forced-air dryer at 60 °C for 7 h to obtain an intermediate product with an EDTA loading of approximately 0.08 mmol / g.

[0064] S3: Amino-functionalized layer grafting

[0065] Take 50 mL of anhydrous ethanol, add 3 mL of APTES (6% by volume) and 0.3 mL of acetic acid (0.6% by volume), stir for 5 min, add 1 g of intermediate product (solid-liquid ratio 1:50 g / mL), reflux at 50 °C for 3.5 h (stirring speed 200 r / min).

[0066] After the reaction was completed, the material was washed four times by centrifugation with anhydrous ethanol and then dried under vacuum at 55°C for 8 hours to obtain a composite adsorbent with an amino loading of approximately 1.0 mmol / g.

[0067] Furthermore, this embodiment also provides an application of the aforementioned composite adsorption material in the enrichment of egg yolk high-phosphoprotein phosphopeptides, and the application method is as described in Example 1.

[0068] Comparative Example 1

[0069] The purpose of this comparative example is to verify the bimetallic (Zr) 4+ / La 3+ The synergistic effect and proportional boundary conditions of co-doping were investigated, clarifying the necessity of limiting the ranges of Zr / Ti=5-10% and La / Ti=1-3% for the adsorption performance of the material.

[0070] 1.1 Experimental Methods

[0071] All groups used the same preparation process as in Example 1, with the only adjustment being the amount of metal salt added during the S1 matrix preparation stage:

[0072] Group 1A: The same preparation method as in Example 1 was used (0.489g of zirconium nitrate (Zr(NO3)4·5H2O) and 0.123g of lanthanum nitrate (La(NO3)3·6H2O) were added during the preparation process);

[0073] Group 1B: Lanthanum nitrate was omitted, and only zirconium nitrate (0.489 g) was added.

[0074] Group 1C: Zirconium nitrate was omitted, and only lanthanum nitrate (0.123g) was added.

[0075] Group 1D: The amount of zirconium nitrate was increased to 0.917g, i.e. Zr / Ti=15%, while the amount of lanthanum nitrate remained unchanged (0.123g).

[0076] Group 1E: The amount of lanthanum nitrate is increased to 0.489g, i.e., La / Ti=6%, while the amount of zirconium nitrate remains unchanged (0.489g).

[0077] Group 1F: Lanthanum nitrate was omitted, and only zirconium nitrate (0.612 g) was added.

[0078] Group 1G: Zirconium nitrate was omitted, and only lanthanum nitrate (0.612g) was added.

[0079] Group 1H: The total amount of zirconium nitrate and lanthanum nitrate is 0.612 g, but the proportion of zirconium nitrate is too high. It is adjusted to a mass ratio of zirconium nitrate to lanthanum nitrate of 3:1.

[0080] Group 1I: The total amount of zirconium nitrate and lanthanum nitrate is 0.612 g, but the proportion of lanthanum nitrate is too high. It is adjusted to a mass ratio of zirconium nitrate to lanthanum nitrate of 1:1.

[0081] The remaining steps (including EDTA passivation, amino grafting, calcination parameters, etc.) are strictly consistent with those in Example 1.

[0082] 1.2 Experimental Results Testing

[0083] The adsorption properties of the materials prepared in groups 1A-1I were tested, including:

[0084] (1) Adsorption capacity test

[0085] The difference in total protein concentration in the solution before and after adsorption was determined by ultraviolet spectrophotometry (UV-Vis), and the adsorption capacity per unit mass of material was calculated. The specific method is as follows:

[0086] Prepare 0.1–1.0 mg / mL PPPs standard solutions, measure the absorbance at 280 nm, and plot a standard curve accordingly. Then, take 50 mg of each material and add it to 10 mL of crude peptide solution (30 mg / mL PPPs + 10% BSA), incubate at 25°C with shaking for 1 h (200 r / min), centrifuge at 4000 r / min for 5 min, take the supernatant and measure the absorbance at 280 nm. Substitute the absorbance into the standard curve to calculate the remaining concentration using the following formula:

[0087] Adsorption capacity (mg / g) = C0 − C e )×V / m

[0088] Where C0 is the initial concentration (mg / mL), C e The equilibrium concentration is (mg / mL), V is the solution volume (L), and m is the material mass (g).

[0089] (2) 10-cycle capacity retention test

[0090] Perform the first adsorption according to the "Adsorption Capacity Test Method" and determine the capacity Q1;

[0091] After adsorption, 10 mL of 0.1 M Na₂CO₃ solution (pH 8.5) was added to the material, and the mixture was shaken at 25 °C for 30 min, centrifuged, and eluted three times. After elution, the pH of the material was adjusted to 5.0 with 0.1 M HCl, washed with water until neutral, and dried at 60 °C for 6 h to complete one cycle. The adsorption-elution-regeneration process was repeated 10 times, and the adsorption capacity Q10 of the 10th cycle was measured. The capacity retention rate after 10 cycles was calculated according to the formula: retention rate (%) = Q10 / Q1 * 100%.

[0092] (3) Specific surface area test

[0093] The specific surface area was measured using the Brunauer-Emmett-Teller (BET) nitrogen adsorption method.

[0094] The experimental results are shown in Table 1:

[0095] Table 1 Bimetallic (Zr) 4+ / La 3+ The effect of co-doping on the adsorption properties of materials (n=3)

[0096]

[0097] The experimental results show that bimetallic doping produces a significant synergistic effect within a specific ratio range, specifically manifested as follows:

[0098] Only doped with Zr 4+ (1B) or La 3+ At group (1C), the adsorption capacity is significantly lower than that of group (1A); meanwhile, the bimetallic co-doped group (1A) exhibits a significantly higher specific surface area than the monometallic groups (1B, 1C). This indicates that Zr 4+ with La 3+ The synergistic effect optimizes the formation of the pore structure in the titanium matrix. 3+ The large ionic radius of Zr4+ may inhibit the excessive growth of TiO2 grains during high-temperature calcination, while the strong oxygen affinity of Zr4+ helps stabilize the mesoporous framework, jointly constructing a more developed and stable mesoporous-macroporous hierarchical structure. In addition, the doping ratio of Zr and La also has a significant impact on the adsorption performance of the material. Excessive Zr doping (1D) may lead to increased lattice distortion of TiO2, making it easier to sinter and densify at high temperatures; excessive La doping (1E) may be difficult to effectively incorporate into the lattice due to its large ionic radius, instead blocking the pores or covering the surface active sites in the form of oxides.

[0099] While group 1F exhibited moderate adsorption capacity (55.2 mg / g) and specific surface area (202.1 m²), 2 / g), but its performance was still significantly lower than that of the best bimetallic group 1A (67.5 mg / g). This indicates that simply increasing Zr...4+ Dosage cannot replace La 3+ The synergistic effect of the two groups was observed; the 1H group exhibited performance closest to the optimal group (adsorption capacity 64.3 mg / g, retention rate 90.1%). This indicates that the material maintains high stability when Zr / Ti is close to the upper limit (10%) and La / Ti is slightly above the upper limit (3%), but its overall performance is still significantly worse than the optimal group. The 1I group, however, experienced a precipitous deterioration (adsorption capacity decreased to 40.1 mg / g), which is directly related to the La / Ti ratio.

[0100] The above results indicate that bimetallic (Zr) 4+ / La 3+ Co-doping exhibits a significant synergistic effect within the defined ranges of Zr / Ti molar ratio of 5-10% and La / Ti molar ratio of 1-3%. Even with increased total dosage (1F group: Zr / Ti≈10%, capacity 55.2 mg / g; 1G group: La / Ti≈9.9%, capacity 38.5 mg / g), the monometallic system cannot replace the synergistic advantage of the bimetallic system. Once the ratio exceeds the limit, Zr... 4+ Excessive Zr / Ti (1D group: Zr / Ti = 15%, capacity drops sharply by 39%) will induce lattice distortion leading to channel collapse (specific surface area ≤ 162.3 m²). 2 / g), La 3+ Excess (Group 1E: La / Ti = 6%, capacity decay 41%; Group 1I: La / Ti ≈ 9.4%, specific surface area ≤ 158.3 m²) 2 / g) will cause the ions to clog the pores and weaken the passivation effect of EDTA due to their excessively large ionic radii (retention rate ≤70.5%). Only by strictly adhering to the doping ratio boundary of the bimetallic compound can high adsorption capacity, structural stability and cycle durability be achieved simultaneously.

[0101] Comparative Example 2

[0102] All groups used the matrix preparation formula of Example 1, with only the parameters of the S1 calcination step adjusted. The group settings are as follows:

[0103] Group 2A: The pre-firing temperature is reduced to 250℃, and the pre-firing time is 1 hour;

[0104] Group 2B: The pre-firing temperature is raised to 450℃, and the pre-firing time is 1 hour;

[0105] Group 2C: The first step of pre-calcination is omitted, and crystallization is carried out directly at 550°C for 1 hour. The second step of calcination time is the same as in Example 1.

[0106] Group 2D: Preheating and heat preservation time shortened to 0.2h;

[0107] Group 2E: Preheating and heat preservation time extended to 3 hours;

[0108] The remaining steps (metal doping, EDTA passivation, amino grafting) are completely consistent with those in Example 1.

[0109] The material was prepared according to the above method, and its adsorption performance was tested. The test method was the same as that of Comparative Example 1. The experimental results are shown in Table 2.

[0110] Table 2. Effect of calcination treatment on the adsorption properties of the material (n=3)

[0111]

[0112] The experimental results show that the pre-calcination step (holding at 300-400℃ for 0.5-1.5h) in the two-step calcination process plays a crucial role in maintaining the material's high specific surface area and structural stability. Specifically, direct high-temperature crystallization without pre-calcination will cause severe decomposition of the organic template agent, leading to serious pore collapse and resulting in a simultaneous deterioration of the material's specific surface area, adsorption capacity, and cycle stability. While the degree of attenuation can be controlled when the pre-calcination temperature or time is too long or too short, it still significantly weakens the material's overall performance. Even if extending the pre-calcination time can slightly increase the specific surface area, excessive oxidation will deplete the surface active sites, resulting in an adsorption capacity lower than the baseline and increased energy consumption. Only by pre-calcining at 300-400℃ for 0.5-1.5h can the highest specific surface area, optimal adsorption capacity, and excellent cycle stability be achieved simultaneously. This parameter range constitutes an irreplaceable technical solution to ensure the maximization of material performance.

[0113] Comparative Example 3

[0114] The purpose of this comparative example is to observe the effect of the EDTA passivation layer on the adsorption of the material. This comparative example only includes one control group, 3. The preparation process is "S1 matrix preparation → direct S3 amino grafting", completely skipping the S2 EDTA passivation step. The amino loading is controlled at 0.8 mmol / g (consistent with Example 1). The experimental method is as follows:

[0115] Weigh 1g of the co-doped TiO2 matrix prepared in S1 of Example 1, add 40mL of anhydrous ethanol solution containing 5% APTES and 0.5% acetic acid, reflux at 50℃ for 4h (250r / min), wash with ethanol 3 times, and vacuum dry at 60℃ for 6h to obtain an amino-functionalized material without EDTA passivation layer.

[0116] The adsorption test conditions were exactly the same as in Example 1, and the test results are shown in Table 3:

[0117] Table 3 Effect of EDTA passivation on the adsorption properties of the material

[0118]

[0119] Comparative Example 3 (without EDTA passivation layer) maintained a similar specific surface area (215.3 m²) to Group 1A. 2 Although the adsorption capacity (59.8 mg / g) of the EDTA group was significantly lower than that of the 1A group (67.5 mg / g), the index, including the cycle retention rate, dropped sharply compared with the 1A group. The experimental results show that the EDTA passivation layer is one of the key factors for high adsorption performance and high stability.

[0120] Comparative Example 4

[0121] The purpose of this comparative example is to observe the effect of the amino functional layer on the adsorption performance of the material. This comparative example only includes one experimental group, Comparative Example 4. The preparation process is "S1 matrix preparation → S2 EDTA passivation", completely skipping the S3 amino grafting step. The EDTA loading is controlled at 0.12 mmol / g (consistent with Example 1). The experimental method is as follows:

[0122] Weigh 1g of the EDTA passivation material prepared in S2 of Example 1, and use it directly for adsorption testing without amino grafting treatment.

[0123] The adsorption test conditions were exactly the same as in Example 1, and the experimental results are shown in Table 4:

[0124] Table 4. Effect of amino functional layer on adsorption performance of material

[0125]

[0126] The above experimental results show that although Comparative Example 4 retains a specific surface area comparable to Group 1A, its adsorption capacity (44.3 mg / g) drops sharply to 65.6% of Group 1A. This experimental result fully exposes the core role of the amino functional layer. Omitting the amino grafting step will cause the material to lose its core adsorption function, and its adsorption capacity will be almost halved.

Claims

1. A method for preparing a composite adsorbent material for enriching egg yolk high-phosphoprotein phosphopeptides, characterized in that, The composite adsorbent material comprises, from the inside out: Zr 4+ and La 3+ The preparation method comprises the following steps: a co-doped mesoporous-macroporous TiO2 matrix, an EDTA passivation layer coated on the surface of the matrix, and an amino-functionalized layer grafted onto the passivation layer, wherein the Zr / Ti molar ratio is 5-10%, the La / Ti molar ratio is 1-3%, the loading of the EDTA passivation layer is 0.05-0.2 mmol / g, and the loading of the amino-functionalized layer is 0.5-1.2 mmol / g; S1: Zr 4+ / La 3+ Preparation of co-doped mesoporous-macroporous TiO2 matrix: Citric acid was dissolved in anhydrous ethanol, stirred, and then ammonia was added. Zirconium nitrate and lanthanum nitrate were then added, and the Zr / Ti molar ratio was controlled at 5-10% and the La / Ti molar ratio at 1-3%. After stirring, tetrabutyl titanate was added dropwise. The mixture was allowed to stand to form a gel. After washing and drying, the co-doped TiO2 matrix was obtained by calcination. S2: EDTA passivation layer coating The matrix obtained in step S1 was dispersed in EDTA solution at a solid-liquid ratio of 1:20-1:50 g / mL, shaken at 50-70℃ for 1-3 h, washed until neutral, and then dried. S3: Amino-functionalized layer grafting: The material obtained in step S2 was dispersed in anhydrous ethanol containing APTES and acetic acid at a solid-liquid ratio of 1:40-1:60 g / mL, refluxed at 45-55℃ for 3-5 h, washed and dried to obtain the composite adsorbent material.

2. The method for preparing a composite adsorbent material for enriching egg yolk high-phosphoprotein phosphopeptides as described in claim 1, characterized in that: The calcination in S1 is carried out in two steps: first, calcination at 300-400℃ for 0.5-1.5h, and then calcination at 500-600℃ for 1.5-2.5h.

3. The method for preparing a composite adsorbent material for enriching egg yolk high-phosphoprotein phosphopeptides as described in claim 1, characterized in that: In S1, the amount of citric acid used is 0.3g, the amount of anhydrous ethanol is 18-22mL, the amount of ammonia water is 16-20mL, the amount of tetrabutyl titanate is 4-6mL, and the dropping rate is 1-3mL / min.

4. The method for preparing a composite adsorbent material for enriching egg yolk high-phosphoprotein phosphopeptides as described in claim 1, characterized in that: The concentration of the EDTA solution is 0.05-0.2M, and the pH is 5.0-6.

0.

5. The method for preparing a composite adsorbent material for enriching egg yolk high-phosphoprotein phosphopeptides as described in claim 1, characterized in that: The volume fraction of APTES in S3 is 3-7%, and the volume fraction of acetic acid is 0.3-0.7%.

6. The application of the composite adsorbent material prepared by any one of claims 1-5 in the enrichment of egg yolk high-phosphoprotein phosphopeptides.

7. The application as described in claim 6, characterized in that, Includes the following steps: (1) Egg yolk protein powder was partially dephosphorized by alkaline method and then enzymatically hydrolyzed to obtain crude polypeptide solution; (2) Under pH conditions of 4.0-6.0, the crude polypeptide solution is brought into contact with the composite adsorbent material for adsorption; (3) Use a buffer solution with pH 8.0-9.0 to elute the adsorbed phosphopeptides.

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