Multifunctional bovine hemoglobin composite material, preparation method and application thereof in dye wastewater treatment

Abstract

A kind of multifunctional bovine hemoglobin composite material, preparation method and application thereof belong to the technical field of environmental dye wastewater treatment.The hemoglobin in the blood of slaughtered cattle is used as enzyme-like material, macroporous adsorption resin HPD-400 is used as immobilized carrier, and the hemoglobin and macroporous adsorption resin HPD-400 are immobilized by physical adsorption method through π-π interaction.In the absence of H2O2, the composite material only plays a single adsorption function, and can realize the adsorption and surface enrichment of dyes;in the presence of H2O2, the dye molecules adsorbed by the composite material migrate to the active center of hemoglobin by diffusion, hemoglobin exhibits peroxidase-like activity to oxidize H2O2 to produce free radicals to degrade dye molecules, and the removal efficiency of dyes is improved, showing the dual-function characteristics of adsorption and degradation, and having potential application value in the repair of dye wastewater and other related environmental water bodies.

Description

Technical Field

[0001] This invention belongs to the field of environmental dye wastewater treatment technology, specifically relating to a multifunctional bovine hemoglobin composite material, its preparation method, and its application in dye wastewater treatment. Background Technology

[0002] Dyes are widely used in various industries such as textiles, paints, paper, cosmetics, and rubber. Because most synthetic dyes contain a large number of chromogenic groups and complex, stable aromatic structures, direct discharge without effective treatment can disrupt the ecological balance of aquatic environments and pose potential carcinogenic, teratogenic, and mutagenic effects on humans and other organisms. Therefore, the efficient removal of organic dyes from water bodies is a current research hotspot in water treatment. The purification of dye pollutants in water bodies mainly employs physical adsorption, chemical oxidation degradation, and biological / enzymatic decomposition methods. Among these, adsorption is the most researched and reported method, suitable for removing highly toxic or difficult-to-biodegrade substances from wastewater. Compared to other treatment methods, adsorption is recognized as an important technology for dye wastewater treatment due to its simple operation, low cost, wide applicability, and the ability to recycle adsorbents. However, adsorption can only enrich dye molecules in water and cannot completely remove them; once adsorption saturation is reached, the adsorbent loses its effectiveness. The synergistic effect of enzymatic treatment and adsorption treatment for dyes has shown potential for effective decolorization.

[0003] The development of enzyme catalysts for large-scale industrial applications must prioritize cost and sustainability. With the continued growth in global beef market demand, the slaughter volume of cattle is only increasing. However, aside from a small portion being scientifically and effectively utilized, the vast majority of slaughter blood is discarded directly as agricultural waste. In mammals, hemoglobin accounts for 97% of the dry weight of red blood cells. It is a conjugated protein with heme as a cofactor and iron porphyrin as its active center, capable of catalyzing the production of free radicals from hydrogen peroxide to oxidize macrocyclic aromatic organic compounds. Therefore, based on the enzyme-like activity of hemoglobin, utilizing slaughter blood to degrade dye pollutants in wastewater achieves both waste resource utilization and safe removal of toxic pollutants from wastewater, truly achieving waste-to-waste treatment. However, the poor stability, short shelf life, non-recyclability, and high sensitivity to process conditions of natural hemoglobin limit its large-scale application in the treatment of dye wastewater.

[0004] Enzyme immobilization facilitates catalyst recovery and improves enzyme activity stability, meeting the basic requirements for developing economically sustainable enzymatic processes. Enzyme immobilization mainly includes physical and chemical methods. Physical adsorption is an inexpensive and easily implemented reversible immobilization method that fixes enzymes onto the surface or pores of an adsorption carrier. Its preparation conditions are mild, and the enzyme is immobilized on the carrier by weak forces (such as van der Waals forces, electrostatic interactions, π-π forces, hydrogen bonds, etc.). These forces do not chemically modify the enzyme, largely ensuring its biocatalytic activity. Reported enzyme immobilization carriers include carbon nanomaterials, biochar, graphene, polymers, nanosilicon, magnetic mesoporous silica, and titanium dioxide. However, most carriers are not suitable for widespread application due to their complex manufacturing process and high production costs. Macroporous adsorption resins are artificially synthesized porous three-dimensional organic polymer adsorbents with advantages such as large specific surface area, strong adsorption capacity, acid and alkali resistance, and high stability. As an inexpensive and readily available immobilization carrier, they are widely used in enzyme immobilization research.

[0005] Given the current situation, developing novel bio-enzyme composite materials that meet the potential of actual industrial applications is crucial for the effective treatment of dye wastewater. Summary of the Invention

[0006] To improve the treatment effect of actual dye wastewater, this invention develops a multifunctional bovine hemoglobin composite material and its preparation method, using hemoglobin from slaughtered bovine blood as an enzyme-like material and macroporous adsorption resin HPD-400 as an immobilization carrier. This multifunctional bovine hemoglobin composite material can both adsorb and enrich dyes and biodegrade them, showing broad prospects in the field of dye wastewater treatment.

[0007] This invention utilizes a physical adsorption method to immobilize hemoglobin with macroporous adsorption resin HPD-400 through π-π interactions. The immobilized hemoglobin exhibits significantly higher stability than free hemoglobin in terms of temperature, pH, storage, salt, and organic solvents, overcoming the limitation of the non-recoverable nature of free hemoglobin. Driven by electrostatic and π-π interactions, dye molecules adsorb within the pores of the composite material and gradually reach adsorption saturation. In the presence of H₂O₂, the peroxidase-like activity of the immobilized hemoglobin decomposes H₂O₂, generating free radicals that degrade dye pollutants, thus improving dye removal efficiency.

[0008] The preparation method of a multifunctional bovine hemoglobin composite material according to the present invention comprises the following steps:

[0009] (1) Extraction of bovine hemoglobin: Take 3-5% sodium citrate solution (unless otherwise specified, all solutions described in this invention are aqueous solutions) and add it to 150-250 mL of anticoagulated bovine slaughter blood. The volume ratio of sodium citrate solution to bovine slaughter blood is 1:8-10. Mix thoroughly by gently inverting the solution 5-10 times. Filter the mixed solution through 3-5 layers of gauze to remove blood clots, and then centrifuge at 800-1500g for 10-30 min at 2-8℃. Wash the resulting red blood cell precipitate with pre-cooled isotonic saline at 2-4℃ and centrifuge 2-6 times. Then add 2-5 mM PBS buffer (pH 10-1500) to the precipitate. 7.2 The volume ratio of red blood cell pellet to PBS buffer is 1:1-5. The red blood cells are lysed in a cell sonicator under ice bath conditions, with the sonic power being 30-50% of the rated power of the sonicator and the pulse interval ratio being 1-4:1. The resulting red blood cell lysate is centrifuged at 8000-15000g for 15-60 min at 2-8℃. The supernatant is collected to obtain a hemoglobin solution with a hemoglobin concentration of 25-30 mg / mL.

[0010] (2) Pretreatment of macroporous adsorption resin HPD-400: Add 3 to 8 times the mass of anhydrous ethanol to HPD-400 and soak for 3 to 5 hours. Then drain the soaking solution. Add 3 to 8 times the mass of distilled water to the resin soaked in anhydrous ethanol and wash repeatedly until the eluent is not turbid when an equal amount of water is added. Scan the eluent with a UV-2700 ultraviolet-visible spectrometer until no elution peak is detected.

[0011] (3) Preparation of Hb@HPD-400: Take 50-150 mg of HPD-400 pretreated in step (2) and a certain volume of hemoglobin solution extracted in step (1) and add it to 50 mL of 2-5 mM PBS buffer (pH 7.2). The final concentration of hemoglobin is 0.1-1.0 mg / mL. Then place it in a shaker at 50-150 rpm and incubate at 15-35℃ for 10-30 h. After the adsorption reaches equilibrium, place it on gauze to filter and collect the precipitate. Wash the precipitate repeatedly with distilled water until the supernatant is clear and transparent. Then dry the washed precipitate at room temperature for 3-6 h to obtain Hb@HPD-400, which is the multifunctional bovine hemoglobin composite material of the present invention.

[0012] The bovine hemoglobin composite material with multiple functions described in this invention is prepared by the above method.

[0013] The bovine hemoglobin composite material described in this invention has multiple applications in dye wastewater treatment.

[0014] The multifunctional bovine hemoglobin composite material constructed in this invention performs only a single adsorption function in the absence of H2O2, achieving pollutant adsorption and surface enrichment driven by π-π interactions and electrostatic interactions. In the presence of H2O2, dye molecules adsorbed by the composite material migrate to the active sites of hemoglobin through diffusion. Hemoglobin then exhibits peroxidase-like activity to oxidize H2O2, generating free radicals that degrade the dye molecules. Simultaneously, the adsorption sites of the composite material undergo in-situ regeneration, demonstrating a dual-functionality of adsorption and degradation.

[0015] Compared with the prior art, the present invention has the following advantages:

[0016] (1) The bovine hemoglobin composite material involved in this invention has hemoglobin that uses bovine slaughter blood as a natural enzyme source. It is green and environmentally friendly, widely available, and low in cost. It combines the conversion and reuse of waste resources with water environment management, opening up green and clean production technology, which is conducive to the development of a circular economy.

[0017] (2) The bovine hemoglobin composite material with multiple functions described in this invention has a simple preparation process, easy-to-control conditions, easy-to-achieve reaction conditions, low equipment requirements, and can quickly achieve solid-liquid separation by gauze filtration, which greatly reduces production costs and is conducive to industrial production.

[0018] (3) The method for treating dyes with the multifunctional bovine hemoglobin composite material described in this invention is simple and has a certain removal effect on both anionic and cationic dyes. However, it has a stronger adsorption and degradation capacity for cationic dyes, making it a potential excellent material for environmental remediation. Attached Figure Description

[0019] Figure 1 This is the UV-Vis absorption spectrum of bovine hemoglobin in Example 1. As can be seen from the figure, bovine hemoglobin has four obvious absorption peaks. Among them, 280 nm in the UV region is the characteristic absorption peak of protein, and 415 nm, 541 nm and 576 nm in the visible region are the characteristic absorption peaks of porphyrin, which correspond to the Soret band, β band and α band respectively.

[0020] Figure 2 Figure 1 shows the color changes of the resin before and after HPD-400 fixation of bovine hemoglobin in Example 1. Figure (A) is the color of HPD-400 itself, and Figure (B) is the color of HPD-400 after fixation of bovine hemoglobin.

[0021] Figure 3This is a bar chart showing the effects of different pH values ​​(A), bovine hemoglobin solution concentrations (B), HPD-400 addition amounts (C), and immobilization times (D) on the loading capacity and immobilization efficiency of bovine hemoglobin in Example 1.

[0022] Figure 4 This is the Hb@HPD-400 peroxidase activity verification curve from Example 1;

[0023] Figure 5 This is a comparison chart of the stability of Hb@HPD-400 and free bovine hemoglobin in Example 1, including temperature stability curve (A), salt solution tolerance curve (B), organic solvent tolerance curve (C), storage stability curve (D), and a bar chart of the stability of Hb@HPD-400 after a number of operations (E).

[0024] Figure 6 The curves show the changes over time in the adsorption and degradation performance of Hb@HPD-400 on the cationic dye methylene blue (MB, Figure A) and the anionic dye methyl orange (MO, Figure B) in Example 1.

[0025] Figure 7 This is a bar graph showing the effect of Hb@HPD-400 on the removal rate of methylene blue (MB) in different adsorption and degradation cycles in Example 1;

[0026] Figure 8 In Example 1, stock solutions of seven cationic dyes were prepared using real water obtained from tap water (TW) and river water (RW) to simulate real dye wastewater. The effects of Hb@HPD-400 on the removal rates of the seven cationic dyes in distilled water (DW), tap water (TW), and river water (RW) environments were compared. Detailed Implementation

[0027] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are based on the technical solution of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

[0028] Example 1

[0029] This embodiment provides a method for preparing a multifunctional bovine hemoglobin composite material, the required reagents of which are as follows:

[0030] Bovine hemoglobin was extracted from bovine blood collected from Changchun Haoyue Slaughterhouse. The macroporous adsorption resin HPD-400 was purchased from Shanghai Yuanye Biotechnology Co., Ltd. TMB (3,3',5,5'-tetramethylbenzidine), methylene blue (MB), ethyl violet (EV), methyl violet (MV), toluidine blue (TB), rhodamine B (RhB), and methyl orange (MO) were all purchased from Shanghai Aladdin Reagent Co., Ltd. Crystal violet (CV) and auramine O (AO) were purchased from Beijing Solarbio Biotechnology Co., Ltd. River water was collected from the Haihe River in Tianjin.

[0031] The specific synthesis method is as follows:

[0032] 1. Extraction of bovine hemoglobin:

[0033] Add 4% sodium citrate solution (sodium citrate solution: bovine slaughter blood = 1:9, v / v) to 200 mL of bovine slaughter blood for anticoagulation, and gently invert 6 times to mix. Filter the mixture through 3 layers of gauze, and then centrifuge at 1000g for 20 min at 4℃. Wash the precipitate with pre-cooled isotonic saline at 4℃ and centrifuge 3 times. Then add 3.75 mM PBS buffer (pH 7.2, red blood cells: PBS buffer = 1:3, v / v), and place it in an ultrasonic disruptor on ice to lyse the red blood cells. The ultrasonic power is 45% of the rated power of the ultrasonic disruptor, and the pulse interval ratio is 2:1. Centrifuge the red blood cell lysate at 12000g for 35 min at 4℃, and collect 360 mL of hemoglobin solution with a concentration of 28.3 mg / mL.

[0034] 2. Pretreatment of HPD-400:

[0035] Add 5 times the mass of anhydrous ethanol to HPD-400 and soak for 4 hours, then release the soaking solution; then add 5 times the mass of distilled water to the resin and wash repeatedly until the eluent is not cloudy when an equal amount of water is added, and scan the eluent with ultraviolet spectroscopy until no elution peak is detected.

[0036] 3. Preparation of Hb@HPD-400:

[0037] Take 100 mg of HPD-400 pretreated in step (2) and 0.714 mL of hemoglobin solution extracted in step (1) and add it to 50 mL of 3.75 mM PBS buffer (pH 7.2) (the final concentration of hemoglobin is 0.4 mg / mL). Then place it in a shaker at 100 rpm and incubate at 25 °C for 16 h. After reaching adsorption equilibrium, filter the precipitate with gauze and wash the precipitate repeatedly with distilled water until the supernatant is clear and transparent. Then dry the precipitate at room temperature for 4 h to obtain 125 mg of Hb@HPD-400, which is the multifunctional bovine hemoglobin composite material of the present invention.

[0038] Example 2

[0039] This embodiment characterizes the multifunctional bovine hemoglobin composite material obtained in Example 1 using ultraviolet spectroscopy. The ultraviolet spectral results from Example 1 are as follows: Figure 1 As shown in the figure, bovine hemoglobin has four distinct absorption peaks. The 280 nm peak in the ultraviolet region is a characteristic absorption peak for proteins, while the 415 nm, 541 nm, and 576 nm peaks in the visible region are characteristic absorption peaks for porphyrins. The peak near 415 nm is the Soret band, representing information about the quaternary structure of hemoglobin. The two peaks near 541 nm and 576 nm are the β band and α band, respectively, representing hemoglobin subunits and the heme pocket (the heme pocket is a randomly coiled globular molecule formed between two α-helices in the tertiary structure of hemoglobin. The surface of the globular molecule has hydrophilic side chains, and the interior has hydrophobic side chains, forming a pocket in which heme is located; this is called the heme pocket).

[0040] Example 3

[0041] This embodiment observes the color change of the resin before and after preparation of a multifunctional bovine hemoglobin composite material in Example 1. The experimental results are as follows: Figure 2 As shown in the figure, the color of the resin changed from white to brown after bovine hemoglobin fixation, indicating that bovine hemoglobin was successfully adsorbed and fixed in the pores of HPD-400.

[0042] This embodiment aims to obtain optimized process parameters for immobilizing bovine hemoglobin using macroporous adsorption resin. A systematic experiment was conducted to evaluate the effects of different pH values, bovine hemoglobin solution concentrations, HPD-400 dosage, and immobilization time on the amount and immobilization rate of bovine hemoglobin in the macroporous adsorption resin@hemoglobin composite material involved in this invention. The measurement conditions were as follows: 50–150 mg of pretreated HPD-400 and a certain volume of bovine hemoglobin solution were added to 50 mL of 3.75 mM PBS buffer (pH 7.2), maintaining a final bovine hemoglobin concentration of 0.1–1.0 mg / mL. The pH of the system was adjusted to 4–9 using 0.1 M HCl and NaOH, and then placed in a shaker at 25°C and 100 rpm. The absorbance at 280 nm was monitored over 24 hours using a UV-2700 UV-Vis spectrophotometer, and the amount of bovine hemoglobin immobilized within a specified time was calculated using the following formula (Loading capacity: q). t (mg g -1 Immobilization efficiency (Immobilization rate (%)):

[0043]

[0044]

[0045] Where C0 and C t The values ​​represent the bovine hemoglobin concentrations in the solution at the initial and specific time points, respectively. m is the mass of HPD-400, and V is the total volume of the bovine hemoglobin solution and the PBS buffer solution. The experimental results are as follows: Figure 3 As shown, the optimized process parameters for preparing Hb@HPD-400 are as follows: the pH value of the fixed system is 7.0, the final concentration of bovine hemoglobin is 0.4 mg / mL, the amount of HPD-400 added is 0.1 g, and the fixation time is 16 h.

[0046] Example 4

[0047] This embodiment demonstrates the dual adsorption and degradation properties of the multifunctional bovine hemoglobin composite material involved in this invention by measuring the peroxidase-like activity of the Hb@HPD-400 complex in Example 1. The measurement conditions were as follows: 1 mL of reaction system contained TMB (2.5 mM), H2O2 (10 mM), Hb@HPD-400 (10 mg), and acetate buffer solution (pH = 4.0, 0.2 M). The substrate conversion of this reaction system within 5 min was monitored using a UV-2700 UV-Vis spectrophotometer in the wavelength range of 500–800 nm. The experimental results are as follows: Figure 4 As shown, when Hb@HPD-400, H2O2, and TMB are present in the reaction system (top curve), the concentration of oxidized TMB increases continuously over time, exhibiting a significant characteristic absorption peak at 652 nm. When only Hb@HPD-400+TMB, H2O2+TMB, and TMB monomer are present in the system, no oxidized TMB is generated (the three curves almost completely overlap). Therefore, in the presence of H2O2, the Hb@HPD-400 composite material can exert peroxidase-like activity to catalyze the generation of free radicals from H2O2 to oxidize TMB.

[0048] Example 5

[0049] This embodiment examines the advantages of a macroporous adsorption resin as a hemoglobin immobilization carrier material by evaluating the stability of a multifunctional bovine hemoglobin composite material from Example 1. The determination process was as follows: 1 mL of a reaction system containing TMB (2.5 mM), H2O2 (10 mM), Hb@HPD-400 (10 mg) / free hemoglobin solution (1 mg), and acetate buffer solution (pH = 4.0, 0.2 M) was incubated at different temperatures (45, 55, 65, 75, 85, and 95 °C) for 1 h to evaluate the thermal stability of Hb@HPD-400; different concentrations of NaCl solution (0, 100, 200, 300, 40) were added to the same reaction system. The tolerance of Hb@HPD-400 to salt and organic reagents was investigated by incubating with 0, 500, 600 and 700 mM) and different volume fractions of ethanol (0, 5, 10, 15, 20, 25, 30 and 35%) for 1 h. Hb@HPD-400 and free hemoglobin solution were stored at room temperature for 56 days. Every 7 days, 10 mg of Hb@HPD-400 / 1 mg of free hemoglobin solution was added to the above reaction system to evaluate the storage stability of the composite material involved in this example. 10 mg of Hb@HPD-400 was added to the above reaction system and the reaction was repeated 8 times to evaluate the reusability of the composite material involved in this example. The absorbance at 652 nm was monitored using a UV-2700 ultraviolet-visible spectrometer, and the residual enzyme activity involved in this example was calculated by formula (3):

[0050]

[0051] Where A0 is the absorbance of the oxidation product in the blank group, and A1 is the absorbance of the oxidation product after different treatments. The experimental results are as follows: Figure 5 As shown, Hb@HPD-400 exhibits better residual enzyme activity than free hemoglobin under conditions of high temperature, high salt, long-term storage, and the presence of organic reagents. After being reused 8 times, Hb@HPD-400 still retains more than 50% of its residual enzyme activity. This bovine hemoglobin composite material has good heat resistance, salt and organic reagent tolerance, storage stability, and reusability, and has broad application potential in the field of large-scale environmental remediation.

[0052] Example 6

[0053] An adsorption and degradation performance test of a multifunctional bovine hemoglobin composite material for the cationic dye methylene blue (MB) and the anionic dye methyl orange (MO) was conducted. This example utilizes the macroporous adsorption resin@hemoglobin composite material with dual adsorption and degradation functions described in Example 1 to evaluate the application potential of this composite material in the field of dye wastewater treatment through adsorption and degradation tests on the cationic dye methylene blue (MB) and the anionic dye methyl orange (MO). The experimental conditions were as follows: 50 mL of pH 7.2 PBS buffer (containing 20 mg / L dye), 80 mg Hb@HPD-400 were placed in a shaker and shaken for 1.5 h until adsorption / desorption equilibrium was reached. Then, 64 mM H2O2 was added to carry out the dye degradation reaction. The absorbance values ​​of methylene blue and methyl orange at 664 nm and 464 nm, respectively, were monitored using a UV-2700 UV-Vis spectrophotometer over 0.5, 1, 1.5, 2, 3, 4, and 5 hours. The experimental results are as follows: Figure 6 As shown, the adsorption rate of Hb@HPD-400 for MB is about 40%, and the total removal rate is about 89%; the adsorption rate of Hb@HPD-400 for MO is about 35%, and the total removal rate is about 45%. Compared with the anionic dye methyl orange, this macroporous adsorption resin@hemoglobin composite material has better adsorption and degradation performance for the cationic dye methylene blue.

[0054] Example 7

[0055] To evaluate the reusability of the multifunctional bovine hemoglobin composite material involved in this invention in the treatment of actual dye wastewater, this embodiment conducted a six-cycle adsorption degradation test on the cationic dye methylene blue using Hb@HPD-400. After each test, the composite material was collected by gauze filtration, and then washed three times each with 10% ethanol and distilled water. The absorbance of methylene blue was detected at 664 nm using a UV-2700 ultraviolet-visible spectrometer, and its removal rate was calculated. The results are as follows. Figure 7 As shown, after six adsorption degradation tests of methylene blue dye by Hb@HPD-400, the removal rate of methylene blue remained above 70%, indicating that the macroporous adsorption resin@hemoglobin composite material synthesized in Example 1 has a stable structure and good reusability, and can be used as an inexpensive and excellent material for the remediation of dye-affected aquatic environments.

[0056] Example 8

[0057] This embodiment simulates actual dye wastewater by preparing stock solutions of different cationic dyes using real water obtained from a natural water system, further evaluating the dye removal capacity of a multifunctional bovine hemoglobin composite material involved in Example 1 in actual wastewater. The experimental conditions were as follows: 20 mg / L solutions of methylene blue, crystal violet, ethyl violet, rhodamine B, methyl violet, auramine O, and toluidine blue were prepared from distilled water (DW), tap water (TW), and river water (RW), respectively. Then, 100 mg of Hb@HPD-400 was added, and the mixture was shaken on a shaker for 1.5 h until adsorption / desorption equilibrium was reached. 64 mM H2O2 was added to initiate the dye degradation reaction. The absorbance values ​​at 664 nm, 588 nm, 596 nm, 554 nm, 580 nm, 436 nm, and 630 nm were measured using a UV-2700 UV-Vis spectrophotometer over 3 h. The experimental results are as follows: Figure 8 As shown, compared with distilled water, the macroporous adsorption resin@hemoglobin composite material has a lower removal rate for different cationic dyes in tap water and river water environments; except for methylene blue and toluidine blue, Hb@HPD-400 has a removal rate of more than 60% for the other five cationic dyes in real water samples, and some even reach 90%, the difference is related to the chemical structure of the dyes themselves.

Claims

1. A method for preparing a multifunctional bovine hemoglobin composite material, comprising the following steps: (1) Extraction of bovine hemoglobin: Add 3-5% sodium citrate solution to the bovine slaughter blood for anticoagulation. The volume ratio of sodium citrate solution to bovine slaughter blood is 1:8-10. Mix by gently inverting 5-10 times. Filter the mixed solution through 3-5 layers of gauze to remove blood clots. Then centrifuge at 800-1500g for 10-30min at 2-8℃. Wash the obtained red blood cell precipitate with pre-cooled isotonic saline at 2-4℃ and centrifuge 2-6 times. Then add 2-5mM PBS buffer at pH 7.2 to the precipitate. The volume ratio of red blood cell precipitate to PBS buffer is 1:1-5. Sonicate the solution under ice bath. Centrifuge the obtained red blood cell lysate at 8000-15000g for 15-60min at 2-8℃. Collect the supernatant to obtain the hemoglobin solution. (2) Pretreatment of macroporous adsorption resin HPD-400: Add 3 to 8 times the mass of anhydrous ethanol to HPD-400 and soak for 3 to 5 hours. Then drain the soaking solution. Add 3 to 8 times the mass of distilled water to the resin soaked in anhydrous ethanol and wash repeatedly until the eluent is not turbid when an equal amount of water is added. Scan the eluent with a UV-Vis spectrophotometer until no elution peak is detected. (3) Preparation of Hb@HPD-400: Take 50-150 mg of HPD-400 pretreated in step (2) and a certain volume of hemoglobin solution extracted in step (1) and add it to 50 mL of PBS buffer at 2-5 mM and pH 7.

2. The final concentration of hemoglobin is 0.1-1.0 mg / mL. Then place it in a shaker at 50-150 rpm and incubate at 15-35℃ for 10-30 h. After the adsorption reaches equilibrium, place it on gauze to filter and collect the precipitate. Wash the precipitate repeatedly with distilled water until the supernatant is clear and transparent. Then dry the washed precipitate at room temperature for 3-6 h to obtain Hb@HPD-400, which is the multifunctional bovine hemoglobin composite material.

2. The method for preparing a multifunctional bovine hemoglobin composite material as described in claim 1, characterized in that: Ultrasonic disruption involves placing red blood cells in an ultrasonic cell disruptor to lyse them, where the ultrasonic power is 30-50% of the ultrasonic disruptor's rated power, and the pulse interval ratio is 1-4:

1.

3. A multifunctional bovine hemoglobin composite material, characterized in that: It is prepared by the method described in claim 1 or 2.

4. The application of the multifunctional bovine hemoglobin composite material according to claim 3 in dye wastewater treatment.

5. The application of the multifunctional bovine hemoglobin composite material as described in claim 4 in dye wastewater treatment, characterized in that: The dye is either the cationic dye methylene blue or the anionic dye methyl orange.

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