Hydrogel complexing agent and application thereof in separation of main protein of royal jelly

Abstract

The invention discloses a hydrogel complexing agent and application thereof in separation of main protein of royal jelly. The hydrogel complexing agent comprises a poly (N-isopropylacrylamide) hydrogel matrix, a network topology structure is formed in the poly (N-isopropylacrylamide) hydrogel, and the pore diameter of pores of the network topology structure is 18-25 nm. A network topological structure with holes of 18-25 nm is formed in the hydrogel complexing agent, and the network topological structure is matched with a three-dimensional structure of the royal jelly major protein, so that the hydrogel complexing agent can be used as a royal jelly major protein trap; the network topology structure is convenient for the royal jelly main protein to enter the holes, and the chemical groups in the holes can form supramolecular interaction with the royal jelly main protein, so that the capture of the hydrogel complexing agent on the royal jelly main protein is realized, and a simple, low-cost and high-yield method is provided for separating the main protein from the royal jelly.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a hydrogel complexing agent and application thereof in separating royal jelly main protein. Background Art

[0002] Royal jelly is a milky mixture secreted by worker bees that are 5 to 15 days old. In fact, the components of royal jelly mainly come from the royal gland and mandibular gland of the worker bees. The royal gland is located in the head of the worker bee, and the mandibular gland is located in the mouth of the worker bee. When the secretions of the royal gland (the main source of protein in royal jelly) pass through the duct to reach the mouth, the mandibular gland will also produce a secretion (the main source of fatty acids in royal jelly), and royal jelly is a mixture of the secretions of these two glands.

[0003] Royal jelly is a complex bee product, its chemical composition varying with bee species, age, season, and pollen source. Generally speaking, its composition is: water 64.5-69.5%, crude protein 11-14.5%, carbohydrates 13-15%, lipids 6.0%, minerals 0.4-2%, and unidentified substances 2.84-3.0%.

[0004] The nutritional value of royal jelly is widely known, but many people suspect that its consumption may affect estrogen levels and increase the risk of certain gynecological diseases. While there is no clear evidence that royal jelly contains estrogen or hormone disruptors, it does contain some components with unknown functions. Therefore, compared to consuming royal jelly or freeze-dried royal jelly powder, consuming royal jelly by precisely isolating its key nutrients ensures that they have no negative effects on the human body.

[0005] As we all know, the main nutrients in royal jelly include royal jelly acid and royal jelly main protein. However, royal jelly acid only accounts for 1.4~2.0% of the royal jelly content, and royal jelly main protein only accounts for 5%~10% of the royal jelly content. These two characteristic nutrients are low in content and difficult to separate. Traditional methods for separating and purifying royal jelly main protein mainly include dialysis, superposition and combination of multiple chromatographic separation methods, etc., which are not only time-consuming and labor-intensive, but also high in cost and low in yield, making it difficult to meet the needs of large-scale production. Summary of the Invention

[0006] The invention aims to provide a hydrogel complexing agent and its application in separating the main protein of royal jelly, so as to provide a simple, low-cost and high-yield method for separating the main protein from royal jelly.

[0007] In order to achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows: The present invention first provides a hydrogel complexing agent, which includes a poly (N-isopropylacrylamide) hydrogel matrix. A network topology structure is formed in the poly (N-isopropylacrylamide) hydrogel, and the pore diameter of the network topology structure is 18-25 nm.

[0008] The hydrogel complexing agent of the present invention forms a network topology with pores of 18-25 nm. This network topology matches the three-dimensional structure of the royal jelly main protein, so the hydrogel complexing agent can be used as a royal jelly main protein trap; that is, this network topology not only facilitates the royal jelly main protein to enter the pores, but the chemical groups in the pores can also form supramolecular interactions with the royal jelly main protein, thereby achieving the capture of the royal jelly main protein by the hydrogel complexing agent, thereby quickly and effectively separating the main protein from other substances in the royal jelly.

[0009] Based on this, the present invention also provides the use of the above-mentioned hydrogel complexing agent in separating the main protein of royal jelly.

[0010] Specifically, the application includes: S1: under neutral or weakly acidic conditions, allowing royal jelly and the hydrogel complexing agent to undergo supramolecular reaction in water to obtain a hydrogel complexing agent loaded with royal jelly main protein; S2: soaking the hydrogel complexing agent loaded with the royal jelly main protein in an alkaline solution to dissociate the royal jelly main protein from the hydrogel complexing agent, thereby obtaining an alkaline hydrogel complexing agent and a royal jelly main protein solution respectively; S3: acidifying and regenerating the alkaline hydrogel complexing agent to obtain the hydrogel complexing agent; and adjusting the royal jelly main protein solution to a neutral pH value and then freeze-drying to obtain the royal jelly main protein.

[0011] The hydrogel complexing agent of the present invention is used to separate the main royal jelly protein. The separation method is a spontaneous process based on pH response, which does not require high-energy-consuming equipment such as a pump group, and significantly reduces energy consumption and cost. The separation process is a supramolecular process and does not change the structure and activity of the main royal jelly protein. The total yield of the main royal jelly protein can reach 5% to 10%, and the extraction rate can reach 90% to 100%. The acidified regenerated hydrogel complexing agent can also be continuously used for recycling in step S1.

[0012] Moreover, in step S1, the hydrogel complexing agent loaded with the main royal jelly protein can be completely separated from the remaining royal jelly components. The remaining royal jelly components can continue to be used to extract other effective ingredients such as fatty acids and amino acids, and have secondary utilization value. The hydrogel complexing agent will not cause pollution to the extraction of these components.

[0013] Preferably, in the above-mentioned application step S1, the mass ratio of the hydrogel complexing agent, royal jelly and water is 2: (1-2): 1; The supramolecular reaction was carried out at 20-45°C under a disturbed state (such as stirring, nitrogen bubbling, etc.) for 1-2 h.

[0014] As a further preference, the mass ratio of the hydrogel complexing agent, royal jelly and water is 2:1:1; and the supramolecular reaction is carried out at 30°C with stirring for 2 h.

[0015] Preferably, in step S2 of the above application, the hydrogel complexing agent loaded with royal jelly major protein is immersed in an alkaline solution of equal mass and pH 8 for 3-6 hours.

[0016] Preferably, in step S2 of the above application, the hydrogel complexing agent loaded with royal jelly major protein is immersed in an alkaline solution (such as sodium carbonate solution) of equal mass with a pH of 8 for 3-6 h (more preferably, for 4 h).

[0017] Preferably, in the above-mentioned step S2, the alkaline hydrogel complexing agent is immersed in an acidic solution (such as a hydrochloric acid solution) with a pH of 5-6 for acidification for 4 hours.

[0018] Preferably, the hydrogel complexing agent is prepared by the following process: (1) Preparing a hydrogel prepolymer solution containing magnetic particles; (2) causing the hydrogel prepolymer to undergo a polymerization reaction to obtain a hydrogel containing magnetic particles; (3) soaking the hydrogel in an alkaline solution and using a magnet to release magnetic particles to obtain a complexing agent precursor; (4) Acidifying the complexing agent precursor to obtain the hydrogel complexing agent.

[0019] The present invention forms a special network topology in the hydrogel complexing agent by first preparing a hydrogel containing magnetic particles and then removing the magnetic particles. By controlling the size of the magnetic particles, the pore diameter of the network topology can be controlled between 18-25nm.

[0020] Preferably, in step (1) of the preparation process of the hydrogel complexing agent, the magnetic particles include Fe3O4@C magnetic particles; The preparation method of Fe3O4@C magnetic particles is as follows: Ferrocene was dissolved in butanone, and 30% hydrogen peroxide was added dropwise at a mass volume ratio of 1 g: (2-4) mL. After stirring for 3-5 hours, the mixture was subjected to hydrothermal reaction at 150-200°C for 72 hours. The mixture was subjected to magnetic separation at least once to obtain Fe3O4@C magnetic particles.

[0021] As a further preferred method, the preparation method of Fe3O4@C magnetic particles is: Ferrocene was dissolved in butanone, and 30% hydrogen peroxide was added dropwise at a mass-to-volume ratio of 1 g:3 mL. After stirring for 4 hours, the mixture was subjected to a hydrothermal reaction at 180°C for 72 hours. The magnetic material was magnetically separated, dispersed in acetone, and then magnetically separated at least once to obtain Fe3O4@C magnetic particles.

[0022] Preferably, in step (1) of the process for preparing the hydrogel complexing agent, the method for preparing the hydrogel prepolymer solution containing magnetic particles is: In a light-proof environment, poly (N-isopropylacrylamide), acrylic acid, a crosslinking agent, and a solvent are first mixed, and then a photoinitiator is added and mixed, and finally magnetic particles are added and mixed to obtain a hydrogel prepolymer solution containing magnetic particles; The mass volume ratio of poly (N-isopropylacrylamide), acrylic acid, crosslinker, solvent, photoinitiator and magnetic particles is 1 g:1.5 mL:0.03 g:1.5 mL:0.02 mL:0.002 g.

[0023] Preferably, the crosslinking agent includes BIS (N,N'-methylenebisacrylamide), the photoinitiator includes HMPP, and the mass volume ratio of poly (N-isopropylacrylamide) to the photoinitiator is 1g: The solvent includes at least one of ethanol and ethylene glycol.

[0024] For the above-mentioned hydrogel complexing agent, preferably, in step (2) of the preparation process of the hydrogel complexing agent, a hydrogel prepolymer solution containing magnetic particles is first injected into a mold, and a magnet is used to induce the magnetic particles to arrange in order; then, ultraviolet polymerization is carried out at 356 nm for 1-2 minutes, and the hydrogel containing magnetic particles is obtained after demoulding and expansion.

[0025] Preferably, in step (3) of the process for preparing the hydrogel complexing agent, the hydrogel containing the magnetic particles is immersed in an alkaline solution with a pH of 8.

[0026] Preferably, in step (4) of the preparation process of the hydrogel complexing agent, the complexing agent precursor is placed in an acidic solution with a pH of 5-6 and acidified for 3-5 h.

[0027] Compared with the prior art, the technical effects of the present invention are embodied in: (1) The hydrogel complexing agent of the present invention forms a network topology with pores of 18-25 nm. This network topology matches the three-dimensional structure of the royal jelly protein, so the hydrogel complexing agent can be used as a royal jelly protein trap; that is, this network topology not only facilitates the royal jelly protein to enter the pores, but also the chemical groups in the pores can form supramolecular interactions with the royal jelly protein, thereby achieving the capture of the royal jelly protein by the hydrogel complexing agent, thereby quickly and effectively separating the protein from other substances in the royal jelly.

[0028] (2) The present invention forms a special network topology in the hydrogel complexing agent by first preparing a hydrogel containing magnetic particles and then removing the magnetic particles. By controlling the size of the magnetic particles, the pore diameter of the network topology can be controlled between 18-25 nm.

[0029] (3) The hydrogel complexing agent of the present invention is used to separate the main protein of royal jelly. The separation method is a spontaneous process based on pH response, which does not require high-energy-consuming equipment such as pumps, and significantly reduces energy consumption and costs. The separation process is a supramolecular process and will not change the structure and activity of the main protein of royal jelly. The total yield of the main protein of royal jelly can reach 5% to 10%, and the extraction rate can reach 90% to 100%. The acidified regenerated hydrogel complexing agent can also be used for recycling in step S1.

[0030] (4) In the present invention, after the hydrogel complexing agent completes the capture of the royal jelly main protein, the hydrogel complexing agent loaded with the royal jelly main protein can be completely separated from the remaining royal jelly components. The remaining royal jelly components can continue to be used to extract other effective ingredients such as fatty acids and amino acids, and have secondary utilization value. The hydrogel complexing agent will not cause pollution to the extraction of these components. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is an electron microscope observation image of the hydrogel complexing agent of the present invention (200 μm); Figure 2 This is an electron microscope observation image of the hydrogel complexing agent of the present invention (50 μm); Figure 3 This is an electron microscope observation image of the hydrogel complexing agent of the present invention (50 μm); Figure 4 This is an electron microscope observation image (30 μm) of the hydrogel complexing agent of the present invention. DETAILED DESCRIPTION

[0032] The technical solution of the present invention is further described in detail below with reference to the accompanying drawings and embodiments.

[0033] Example 1 This embodiment provides a hydrogel complexing agent, and its preparation process includes the following steps: (1) Preparing a hydrogel prepolymer solution containing magnetic particles; Specifically, Fe3O4@C magnetic particles were first prepared: 1 g of ferrocene was added to 100 mL of butanone and ultrasonically treated for 25 minutes to promote dissolution; then, 3 mL of 30% hydrogen peroxide was added dropwise and stirred for 4 hours; the mixture was then subjected to hydrothermal reaction at 180°C for 72 hours; the magnetic material was separated using an electromagnet, dispersed with 100 mL of acetone, and magnetically separated again to obtain Fe3O4@C magnetic particles; the Fe3O4@C magnetic particles were mixed with ethylene glycol at a ratio of 10 g / L and ultrasonically dispersed to obtain a Fe3O4@C magnetic particle suspension for later use; Then, a hydrogel prepolymer solution containing magnetic particles was prepared: 2 g PNIPAM (poly (N-isopropylacrylamide),) 3 mL AAc (acrylic acid), 0.06 g BIS (N,N'-methylenebisacrylamide), 1.5 mL ethanol, and 1.5 mL ethylene glycol were added to a light-proof brown bottle and mixed evenly by ultrasonication. 0.04 mL HMPP was added and 0.2 mL Fe3O4@C magnetic particle suspension was added under ultrasonication or oscillation and mixed evenly to obtain a hydrogel prepolymer solution containing magnetic particles. (2) causing the hydrogel prepolymer to undergo polymerization reaction to obtain a hydrogel containing magnetic particles; Specifically, a hydrogel prepolymer solution containing magnetic particles was injected into a 2mm glass plywood mold, which was then placed under a 0.25T magnet and induced to align for 30 minutes. The solution was then polymerized under 356nm UV light for 1 minute, demolded, and the resulting hydrogel was expanded three times with PBS to obtain a hydrogel containing magnetic particles. (3) Soaking the hydrogel in an alkaline solution and using a magnet to free the magnetic particles to obtain a complexing agent precursor; Specifically, the hydrogel containing magnetic particles was immersed in a sodium carbonate solution with a pH of 8, and a 0.25T magnet was used to induce magnetic attraction to release Fe3O4@C magnetic particles, thus obtaining a complexing agent precursor. (4) Acidifying the complexing agent precursor to obtain a hydrogel complexing agent; Specifically, the complexing agent precursor was immersed in a hydrochloric acid solution with a pH of 5 and acidified for 4 h to obtain the hydrogel complexing agent of this embodiment, which was named BR003.

[0034] The obtained hydrogel complexing agent BR003 (hereinafter referred to as acid BR003) was analyzed by electron microscopy. The results are as follows: Figure 1 、 Figure 2 、 Figure 3 and Figure 4 shown.

[0035] from Figure 3 and Figure 4 It can be clearly seen that a network topology is formed in the acid BR003. The pore diameter is measured to be between 18-25 nm.

[0036] Example 2 The present embodiment provides a method for isolating royal jelly main protein, comprising the following steps: S1: Under neutral or weakly acidic conditions, royal jelly and the hydrogel complexing agent undergo supramolecular reaction in water to obtain a hydrogel complexing agent loaded with royal jelly main protein; Specifically, 20 g of acid BR003, 10 g of royal jelly, and 10 g of water were added to the reactor and stirred at 30°C for 2 h; then the acid BR003 loaded with royal jelly main protein was taken out; S2: soaking the hydrogel complexing agent loaded with the royal jelly main protein in an alkaline solution to dissociate the royal jelly main protein from the hydrogel complexing agent, thereby obtaining an alkaline hydrogel complexing agent and a royal jelly main protein solution respectively; Specifically, the acidic BR003 loaded with royal jelly protein was placed in 20g of sodium carbonate solution with a pH of 8 and soaked for 4 hours. During this process, the royal jelly protein dissociated from the acidic BR003 and released into the solution, obtaining a royal jelly protein solution. The acidic BR003 was then converted into the basic BR003 and removed. S3: acidifying and regenerating the alkaline hydrogel complexing agent to obtain a hydrogel complexing agent; and adjusting the royal jelly main protein solution to a neutral pH and then freeze-drying to obtain the royal jelly main protein; Specifically, on the one hand, the alkaline BR003 was placed in 20g of hydrochloric acid solution with a pH of 5 and soaked for 4 hours to acidify it and regenerate it into acid BR003; on the other hand, the royal jelly main protein solution was adjusted to a neutral pH and then freeze-dried to obtain 1.0g of royal jelly main protein.

[0037] In this embodiment, the total yield of the royal jelly main protein (the proportion of the royal jelly) is 10%, and the extraction efficiency (the proportion of the total protein in the royal jelly) is 100%.

[0038] Example 3 The present embodiment provides a method for isolating royal jelly main protein, comprising the following steps: S1: Under neutral or weakly acidic conditions, royal jelly and the hydrogel complexing agent undergo supramolecular reaction in water to obtain a hydrogel complexing agent loaded with royal jelly main protein; Specifically, 20 g of acid BR003, 20 g of royal jelly, and 10 g of water were added to the reactor and stirred at 45°C for 1 h; then the acid BR003 loaded with royal jelly main protein was taken out; S2: soaking the hydrogel complexing agent loaded with the royal jelly main protein in an alkaline solution to dissociate the royal jelly main protein from the hydrogel complexing agent, thereby obtaining an alkaline hydrogel complexing agent and a royal jelly main protein solution respectively; Specifically, the acidic BR003 loaded with royal jelly protein was placed in 20g of sodium carbonate solution with a pH of 8 and soaked for 4 hours. During this process, the royal jelly protein dissociated from the acidic BR003 and released into the solution, obtaining a royal jelly protein solution. The acidic BR003 was then converted into the basic BR003 and removed. S3: acidifying and regenerating the alkaline hydrogel complexing agent to obtain a hydrogel complexing agent; and adjusting the royal jelly main protein solution to a neutral pH and then freeze-drying to obtain the royal jelly main protein; Specifically, on the one hand, the alkaline BR003 was placed in 20g of hydrochloric acid solution with a pH of 5 and soaked for 4 hours to acidify it and regenerate it into acid BR003; on the other hand, the royal jelly main protein solution was adjusted to a neutral pH and then freeze-dried to obtain 1.8g of royal jelly main protein.

[0039] In this embodiment, the total yield of the royal jelly main protein (the proportion of the royal jelly) is 9%, and the extraction efficiency (the proportion of the total protein in the royal jelly) is 90%.

[0040] Example 4 The present embodiment provides a method for isolating royal jelly main protein, comprising the following steps: S1: Under neutral or weakly acidic conditions, royal jelly and the hydrogel complexing agent undergo supramolecular reaction in water to obtain a hydrogel complexing agent loaded with royal jelly main protein; Specifically, 20 g of acid BR003, 15 g of royal jelly, and 10 g of water were added to the reactor and stirred at 20°C for 2 h; then the acid BR003 loaded with royal jelly main protein was taken out; S2: soaking the hydrogel complexing agent loaded with the royal jelly main protein in an alkaline solution to dissociate the royal jelly main protein from the hydrogel complexing agent, thereby obtaining an alkaline hydrogel complexing agent and a royal jelly main protein solution respectively; Specifically, the acidic BR003 loaded with royal jelly protein was placed in 20g of sodium carbonate solution with a pH of 8 and soaked for 4 hours. During this process, the royal jelly protein dissociated from the acidic BR003 and released into the solution, obtaining a royal jelly protein solution. The acidic BR003 was then converted into the basic BR003 and removed. S3: acidifying and regenerating the alkaline hydrogel complexing agent to obtain a hydrogel complexing agent; and adjusting the royal jelly main protein solution to a neutral pH and then freeze-drying to obtain the royal jelly main protein; Specifically, on the one hand, the alkaline BR003 was placed in 20g of hydrochloric acid solution with a pH of 5 and soaked for 4 hours to acidify it and regenerate it into acid BR003; on the other hand, the royal jelly main protein solution was adjusted to a neutral pH and then freeze-dried to obtain 1.45g of royal jelly main protein.

[0041] In this embodiment, the total yield of the royal jelly main protein (the proportion of the royal jelly) is 9.7%, and the extraction efficiency (the proportion of the total protein in the royal jelly) is 97%.

[0042] Example 5 The present embodiment provides a method for isolating royal jelly main protein, comprising the following steps: S1: Under neutral or weakly acidic conditions, royal jelly and the hydrogel complexing agent undergo supramolecular reaction in water to obtain a hydrogel complexing agent loaded with royal jelly main protein; Specifically, 20 g of acid BR003, 12 g of royal jelly, and 10 g of water were added to the reactor and stirred at 35°C for 1.5 h; then the acid BR003 loaded with royal jelly main protein was taken out; S2: soaking the hydrogel complexing agent loaded with the royal jelly main protein in an alkaline solution to dissociate the royal jelly main protein from the hydrogel complexing agent, thereby obtaining an alkaline hydrogel complexing agent and a royal jelly main protein solution respectively; Specifically, the acidic BR003 loaded with royal jelly protein was placed in 20g of sodium carbonate solution with a pH of 8 and soaked for 4 hours. During this process, the royal jelly protein dissociated from the acidic BR003 and released into the solution, obtaining a royal jelly protein solution. The acidic BR003 was then converted into the basic BR003 and removed. S3: acidifying and regenerating the alkaline hydrogel complexing agent to obtain a hydrogel complexing agent; and adjusting the royal jelly main protein solution to a neutral pH and then freeze-drying to obtain the royal jelly main protein; Specifically, on the one hand, the alkaline BR003 was placed in 20g of hydrochloric acid solution with a pH of 5 and soaked for 4 hours to acidify it and regenerate it into acid BR003; on the other hand, the royal jelly main protein solution was adjusted to a neutral pH and then freeze-dried to obtain 1.1g of royal jelly main protein.

[0043] In this embodiment, the total yield of the royal jelly main protein (the proportion of the royal jelly) is 5.5%, and the extraction efficiency (the proportion of the total protein in the royal jelly) is 92%.

[0044] Example 6 The present embodiment provides a method for isolating royal jelly main protein, comprising the following steps: S1: Under neutral or weakly acidic conditions, royal jelly and the hydrogel complexing agent undergo supramolecular reaction in water to obtain a hydrogel complexing agent loaded with royal jelly main protein; Specifically, 20 g of acid BR003, 18 g of royal jelly, and 10 g of water were added to the reactor and stirred at 25°C for 2 h; then the acid BR003 loaded with royal jelly main protein was taken out; S2: soaking the hydrogel complexing agent loaded with the royal jelly main protein in an alkaline solution to dissociate the royal jelly main protein from the hydrogel complexing agent, thereby obtaining an alkaline hydrogel complexing agent and a royal jelly main protein solution respectively; Specifically, the acidic BR003 loaded with royal jelly protein was placed in 20g of sodium carbonate solution with a pH of 8 and soaked for 4 hours. During this process, the royal jelly protein dissociated from the acidic BR003 and released into the solution, obtaining a royal jelly protein solution. The acidic BR003 was then converted into the basic BR003 and removed. S3: acidifying and regenerating the alkaline hydrogel complexing agent to obtain a hydrogel complexing agent; and adjusting the royal jelly main protein solution to a neutral pH and then freeze-drying to obtain the royal jelly main protein; Specifically, on the one hand, the alkaline BR003 was placed in 20g of hydrochloric acid solution with a pH of 5 and soaked for 4 hours to acidify it and regenerate it into acid BR003; on the other hand, the royal jelly main protein solution was adjusted to a neutral pH and then freeze-dried to obtain 1.74g of royal jelly main protein.

[0045] In this embodiment, the total yield of the royal jelly main protein (the proportion of the royal jelly) is 9.7%, and the extraction efficiency (the proportion of the total protein in the royal jelly) is 96%.

[0046] Example 7 The present embodiment provides a method for isolating royal jelly main protein, comprising the following steps: S1: Under neutral or weakly acidic conditions, royal jelly and the hydrogel complexing agent undergo supramolecular reaction in water to obtain a hydrogel complexing agent loaded with royal jelly main protein; Specifically, 20 g of acid BR003, 10 g of royal jelly, and 10 g of water were added to the reactor and stirred at 20°C for 2 h; then the acid BR003 loaded with royal jelly main protein was taken out; S2: soaking the hydrogel complexing agent loaded with the royal jelly main protein in an alkaline solution to dissociate the royal jelly main protein from the hydrogel complexing agent, thereby obtaining an alkaline hydrogel complexing agent and a royal jelly main protein solution respectively; Specifically, the acidic BR003 loaded with royal jelly protein was placed in 20g of sodium carbonate solution with a pH of 8 and soaked for 4 hours. During this process, the royal jelly protein dissociated from the acidic BR003 and released into the solution, obtaining a royal jelly protein solution. The acidic BR003 was then converted into the basic BR003 and removed. S3: acidifying and regenerating the alkaline hydrogel complexing agent to obtain a hydrogel complexing agent; and adjusting the royal jelly main protein solution to a neutral pH and then freeze-drying to obtain the royal jelly main protein; Specifically, on the one hand, the alkaline BR003 was placed in 20g of hydrochloric acid solution with a pH of 5 and soaked for 4 hours to acidify it and regenerate it into acid BR003; on the other hand, the royal jelly main protein solution was adjusted to a neutral pH and then freeze-dried to obtain 0.99g of royal jelly main protein.

[0047] In this embodiment, the total yield of the royal jelly main protein (the proportion of the royal jelly) is 9.9%, and the extraction efficiency (the proportion of the total protein in the royal jelly) is 99%.

[0048] Example 8 The present embodiment provides a method for isolating royal jelly main protein, comprising the following steps: S1: Under neutral or weakly acidic conditions, royal jelly and the hydrogel complexing agent undergo supramolecular reaction in water to obtain a hydrogel complexing agent loaded with royal jelly main protein; Specifically, 20 g of acid BR003, 16 g of royal jelly, and 10 g of water were added to the reactor and stirred at 40°C for 1.2 h; then the acid BR003 loaded with royal jelly main protein was taken out; S2: soaking the hydrogel complexing agent loaded with the royal jelly main protein in an alkaline solution to dissociate the royal jelly main protein from the hydrogel complexing agent, thereby obtaining an alkaline hydrogel complexing agent and a royal jelly main protein solution respectively; Specifically, the acidic BR003 loaded with royal jelly protein was placed in 20g of sodium carbonate solution with a pH of 8 and soaked for 4 hours. During this process, the royal jelly protein dissociated from the acidic BR003 and released into the solution, obtaining a royal jelly protein solution. The acidic BR003 was then converted into the basic BR003 and removed. S3: acidifying and regenerating the alkaline hydrogel complexing agent to obtain a hydrogel complexing agent; and adjusting the royal jelly main protein solution to a neutral pH and then freeze-drying to obtain the royal jelly main protein; Specifically, on the one hand, the alkaline BR003 was placed in 20g of hydrochloric acid solution with a pH of 6 and soaked for 4 hours to acidify it and regenerate it into acid BR003; on the other hand, the royal jelly main protein solution was adjusted to a neutral pH and then freeze-dried to obtain 1.45g of royal jelly main protein.

[0049] In this embodiment, the total yield of the royal jelly main protein (the proportion of the main protein in the royal jelly) is 9%, and the extraction efficiency (the proportion of the main protein in the royal jelly) is 91%.

Claims

1. A hydrogel complexing agent, characterized in that The invention comprises a poly N-isopropylacrylamide hydrogel, wherein a network topology structure is formed in the poly N-isopropylacrylamide hydrogel, and the pore diameter of the network topology structure is 18-25 nm.

2. The hydrogel complexing agent according to claim 1, wherein Prepared by the following process: (1) Preparing a hydrogel prepolymer solution containing magnetic particles; (2) causing the hydrogel prepolymer to undergo a polymerization reaction to obtain a hydrogel containing magnetic particles; (3) soaking the hydrogel in an alkaline solution and using a magnet to release magnetic particles to obtain a complexing agent precursor; (4) Acidifying the complexing agent precursor to obtain the hydrogel complexing agent.

3. The hydrogel complexing agent according to claim 1, wherein In step (1), the magnetic particles include Fe3O4@C magnetic particles; The preparation method of Fe3O4@C magnetic particles is as follows: Ferrocene was dissolved in butanone, and 30% hydrogen peroxide was added dropwise at a mass volume ratio of 1 g: (2-4) mL. After stirring for 3-5 hours, the mixture was subjected to hydrothermal reaction at 150-200°C for 72 hours. The mixture was subjected to magnetic separation at least once to obtain Fe3O4@C magnetic particles.

4. The hydrogel complexing agent according to claim 1, wherein In step (1), the preparation method of the hydrogel prepolymer solution containing magnetic particles is: In a light-proof environment, poly (N-isopropylacrylamide), acrylic acid, a crosslinking agent, and a solvent are first mixed, and then a photoinitiator is added and mixed, and finally magnetic particles are added and mixed to obtain a hydrogel prepolymer solution containing magnetic particles; The mass volume ratio of poly (N-isopropylacrylamide), acrylic acid, crosslinker, solvent, photoinitiator and magnetic particles is 1 g:1.5 mL:0.03 g:1.5 mL:0.02 mL:0.002 g.

5. The hydrogel complexing agent according to claim 1, wherein In step (2), the hydrogel prepolymer solution containing magnetic particles is first injected into the mold, and the magnetic particles are induced to arrange in order using a magnet; then ultraviolet polymerization is carried out at 356 nm for 1-2 minutes, and the hydrogel containing magnetic particles is obtained after demoulding and expansion.

6. The hydrogel complexing agent according to claim 1, wherein In step (3), the hydrogel containing magnetic particles is immersed in an alkaline solution with a pH of 8; In step (4), the complexing agent precursor is placed in an acidic solution with a pH of 5-6 and acidified for 3-5 hours.

7. Use of the hydrogel complexing agent according to any one of claims 1 to 6 in separating the main protein of royal jelly.

8. The use according to claim 7, characterized in that include: S1: Under neutral or weakly acidic conditions, royal jelly and the hydrogel complexing agent undergo supramolecular reaction in water to obtain a hydrogel complexing agent loaded with royal jelly main protein; S2: soaking the hydrogel complexing agent loaded with the royal jelly main protein in an alkaline solution to dissociate the royal jelly main protein from the hydrogel complexing agent, thereby obtaining an alkaline hydrogel complexing agent and a royal jelly main protein solution respectively; S3: acidifying and regenerating the alkaline hydrogel complexing agent to obtain a hydrogel complexing agent; The royal jelly main protein solution is adjusted to a neutral pH and then freeze-dried to obtain the royal jelly main protein.

9. The use according to claim 8, characterized in that In step S1, the mass ratio of the hydrogel complexing agent, royal jelly and water is 2: (1-2): 1; The supramolecular reaction was carried out at 20-45°C under agitation for 1-2 h.

10. The use according to claim 8, characterized in that In step S2, the hydrogel complexing agent loaded with the royal jelly main protein is immersed in an alkaline solution of equal mass and pH 8 for 3-6 hours.

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