Protein glycosylation modification method based on assistance of static magnetic field

Abstract

The invention relates to the technical field of protein glycosylation modification, in particular to a method for assisting protein glycosylation modification based on a static magnetic field. Comprising the following steps: preparing a protein solution with the concentration of 3-8%, and adjusting the pH value of the protein solution to 8-12; placing the protein solution in a 2.5-10mT static magnetic field to assist the protein in hydration reaction; after hydration is completed, carbohydrates are added and fully mixed for 20-40 min under the condition of magnetic stirring, and a protein-carbohydrate mixed solution is prepared; adjusting the pH of the protein-saccharide mixed solution to be the same as that in the first step; carrying out constant-temperature magnetic stirring on the protein-saccharide mixed solution under the conditions that the temperature is 75-85 DEG C and the rotating speed is 150-250 rpm for reaction, and keeping the pH value constant in the reaction process; and after the reaction is finished, cooling in an ice bath and freeze-drying. The method has the advantages that the grafting rate of the Maillard product is remarkably increased, the browning process can be inhibited, and the color deterioration of the glycosylation product is avoided.

Description

Technical Field

[0001] The invention relates to the technical field of protein glycosylation modification, and in particular to a method for protein glycosylation modification based on static magnetic field assistance. Background Art

[0002] Protein is an amphiphilic macromolecule. Due to its excellent functional properties and biocompatibility, it is widely used in food, medicine and other fields. However, natural protein is easily affected by factors such as pH and temperature, and has the disadvantage of low solubility, which limits its application value. Maillard reaction, also known as glycosylation reaction, refers to the carbonyl-amine reaction between the free amino group of the amino acid side chain in the protein molecule and the carbonyl group at the reducing end of the polysaccharide molecule. The reaction process can be controlled by regulating temperature and pH value, and the solubility, thermal stability, foaming and other physical and chemical and functional properties of the protein can be effectively improved. It is an ideal means to improve the properties of protein. Common glycosylation methods are divided into dry heat method and wet heat method. However, the dry heat method has a long reaction cycle, high temperature, and is not suitable for industrial production. The wet heat method has incomplete reaction, low grafting rate, difficult to control the reaction process, and easy to generate undesirable late end products (AGEs). Therefore, improving the glycosylation reaction, increasing the grafting rate and reducing the generation of AGEs are key issues in achieving industrial applications.

[0003] At present, the grafting rate of Maillard reaction is improved by using ultrasound-assisted, pulsed electric field-assisted, high-voltage-assisted, microwave-assisted and other methods, which effectively improves the functional properties of proteins. Static magnetic field (SMF) is an energy field with multiple biological effects. Studies have shown that static magnetic field can act on the secondary bonds of proteins and change the secondary structure of proteins.

[0004] Traditional glycosylation methods include dry heat method and wet heat method. The dry heat method has a long reaction cycle, high temperature, and is not suitable for industrial production. The wet heat method has an incomplete reaction, low grafting rate, difficult to control the reaction process, and is prone to generate undesirable late end products (AGEs). Therefore, improving the glycosylation reaction, increasing the grafting rate, and reducing the generation of AGEs are the key to realizing the industrial application of glycosylated modified proteins. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a method for protein glycosylation modification based on static magnetic field assistance.

[0006] The present invention provides a method for protein glycosylation modification based on static magnetic field assistance, which specifically comprises the following steps: S1. Prepare a protein solution with a concentration of 3-8%, and adjust the pH value of the protein solution to 8-12; S2. Under shaking culture conditions of 5-12°C and 150-250 rpm, the protein solution is placed in a static magnetic field with an induction intensity of 2.5-10 mT to assist the protein in hydration reaction; S3. After hydration is completed, add sugars and mix thoroughly for 20 to 40 minutes under magnetic stirring to prepare a protein-sugar mixed solution; the mass ratio of protein to sugar is 1:1 to 2; S4. Adjust the pH of the protein-carbohydrate mixed solution to the same as step S1; S5. The protein-sugar mixed solution is placed in a constant temperature magnetic stirring state at 75-85°C and a rotation speed of 150-250 rpm for reaction, and the pH value is maintained constant during the reaction; S6. After the reaction is completed, the temperature is quickly cooled in an ice bath, and the glycosylated protein product is obtained after freeze-drying.

[0007] Preferably, the hydration reaction time in step S2 is 10-20 hours, and the reaction temperature is 10°C.

[0008] Preferably, the hydration reaction time in step S2 is 12 hours, and the induction intensity of the static magnetic field is 10 mT.

[0009] Preferably, the protein is zein, soy protein isolate, whey protein isolate or casein.

[0010] Preferably, the carbohydrate substance is an oligosaccharide or a polysaccharide.

[0011] Preferably, the oligosaccharide is xylooligosaccharide, and the mass ratio of protein to xylooligosaccharide is 1:2.

[0012] Preferably, the polysaccharide is pullulan, and the mass ratio of protein to pullulan is 1:1.

[0013] Preferably, the reaction time in step S5 is 1.5-2.5 h, and the reaction temperature is 80° C.

[0014] Preferably, the protein solution in step S1 is prepared by dissolving the protein in a phosphate buffer, stirring it thoroughly, and leaving it for more than 8 hours to fully hydrate it.

[0015] Preferably, the concentration of the protein solution in step S1 is 5%.

[0016] Compared with the prior art, the present invention can achieve the following beneficial effects: The present invention provides a method for protein glycosylation modification based on static magnetic field assistance, which uses static magnetic field to assist protein hydration to change the secondary structure of protein, thereby improving the degree of glycosylation reaction. Compared with the traditional wet method for Maillard reaction, the use of static magnetic field to assist glycosylation modification of protein can significantly increase the grafting rate of Maillard product, while inhibiting the browning process and avoiding the color deterioration of glycosylation product. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the measurement result of the grafting rate of the glycosylated protein product and the amount of Maillard intermediates generated according to Example 1 of the present invention.

[0018] Figure 2 This is the measurement result of the grafting rate of the glycosylated protein product and the amount of Maillard intermediates generated according to Example 2 of the present invention.

[0019] Figure 3 This is the measurement result of the grafting rate of the glycosylated protein product and the amount of Maillard intermediates generated according to Example 3 of the present invention.

[0020] Figure 4 This is the measurement result of the grafting rate of the glycosylated protein product and the amount of Maillard intermediates generated according to Example 4 of the present invention.

[0021] Figure 5 This is the measurement result of the grafting rate of the glycosylated protein product and the amount of Maillard intermediates generated according to Example 5 of the present invention.

[0022] Figure 6 This is the measurement result of the grafting rate of the glycosylated protein product and the amount of Maillard intermediates generated according to Example 6 of the present invention.

[0023] Figure 7 This is the measurement result of the grafting rate of the glycosylated protein product and the amount of Maillard intermediates generated according to Example 7 of the present invention.

[0024] Figure 8 This is the measurement result of the grafting rate of the glycosylated protein product and the amount of Maillard intermediates generated according to Example 8 of the present invention. DETAILED DESCRIPTION

[0025] Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings. In the following description, the same modules are represented by the same reference numerals. In the case of the same reference numerals, their names and functions are also the same. Therefore, the detailed description thereof will not be repeated.

[0026] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and do not constitute a limitation of the present invention.

[0027] The present invention provides a method for protein glycosylation modification based on static magnetic field assistance, which specifically comprises the following steps: S1. Prepare a protein solution with a concentration of 3-8%, and adjust the pH value of the protein solution to 8-12; in a specific embodiment, the concentration of the protein solution is 5%.

[0028] Preferably, the protein is zein, soy protein isolate, whey protein isolate or casein; the optimal pH values ​​of the protein solution are: zein pH=12, soy protein isolate pH=11, whey protein isolate pH=9, casein pH=8; Preferably, the protein solution is prepared by dissolving the protein in a phosphate buffer, stirring it thoroughly, and leaving it for more than 8 hours (overnight) to fully hydrate it.

[0029] S2. Under shaking culture conditions of 5-12°C and 150-250 rpm, the protein solution is placed in a static magnetic field with an induction intensity of 2.5-10 mT to assist the protein in hydration reaction; Preferably, the hydration reaction time is 10-20 hours; in a specific embodiment, the hydration reaction time is 12 hours, the reaction temperature is 10° C., and the induction intensity of the static magnetic field is 10 mT.

[0030] S3. After hydration is completed, add sugars and mix thoroughly for 20 to 40 minutes under magnetic stirring to prepare a protein-sugar mixed solution; the mass ratio of protein to sugar is 1:1 to 2; Preferably, the carbohydrate substance is an oligosaccharide or a polysaccharide; Preferably, the oligosaccharide is xylooligosaccharide; the mass ratio of protein to xylooligosaccharide is 1:2; Preferably, the polysaccharide is pullulan, and the mass ratio of protein to pullulan is 1:1.

[0031] S4. Adjust the pH of the protein-carbohydrate mixed solution to the same as step S1 (optimal reaction temperature).

[0032] S5. The protein-sugar mixed solution is placed in a constant temperature magnetic stirring state at 75-85°C and a rotation speed of 150-250 rpm for reaction, and the pH value is maintained constant during the reaction; Preferably, the reaction time in step S5 is 1.5-2.5 h, and the reaction temperature is 80° C.

[0033] S6. After the reaction is completed, the temperature is quickly cooled in an ice bath, and the glycosylated protein product is obtained after freeze-drying.

[0034] Example 1 A method for protein glycosylation modification based on static magnetic field assistance specifically comprises the following steps: S1. Prepare a 5% zein solution, and adjust the pH value of the zein solution to 12; S2. Under shaking culture conditions at 10°C and 200 rpm, the zein solution was placed in static magnetic fields with induction strengths of SMF 0mT (Control), SMF 2.5mT, SMF 5mT, and SMF 10mT to assist the protein in hydration reaction for 12 hours. S3. After hydration, add xylooligosaccharides (XOS) and mix thoroughly for 30 minutes under magnetic stirring to prepare a protein-xylooligosaccharide mixed solution; the mass ratio of protein to xylooligosaccharides (XOS) is 1:2; S4. Adjust the pH of the protein-xylo-oligosaccharide mixed solution to 12; S5. The protein-xylo-oligosaccharide mixed solution was placed at 80°C and 200 rpm for 2 h under constant temperature magnetic stirring, and the pH value was maintained constant during the reaction; S6. After the reaction is completed, the temperature is quickly cooled in an ice bath and freeze-dried to obtain the glycosylated protein product (ZEIN-XOS).

[0035] The grafting rate and the amount of Maillard intermediates produced were measured on the prepared products. Figure 1 The results showed that compared with the control group, SMF 2.5mT, SMF 5mT and SMF 10mT treatments significantly increased the grafting rate. The SMF5mT and SMF 10mT treatments increased the grafting rate by more than 50%, with the highest reaching 55.63%. In addition, the generation of Maillard intermediates (the size of the A294 value represents the amount of Maillard intermediates generated, which is proportional to the amount of Maillard intermediates generated) was promoted. The SMF 10mT treatment increased the content of Maillard intermediates by 5.59%, and at the same time inhibited the degree of Maillard browning (the size of the A420 value represents the degree of Maillard browning, which is proportional to the amount of Maillard browning). Under the optimal conditions, the browning degree only increased by 10.37%.

[0036] Example 2 A method for protein glycosylation modification based on static magnetic field assistance specifically comprises the following steps: S1. Prepare a 5% soy protein isolate (SPI) solution, and adjust the pH value of the soy protein isolate (SPI) solution to 11; S2. Under shaking culture conditions at 10°C and 200 rpm, the soy protein isolate (SPI) solution was placed in a static magnetic field with an induction strength of SMF 0mT (Control), SMF 2.5mT, SMF 5mT, and SMF 10mT to assist the protein in hydration reaction for 12 hours; S3. After hydration, add xylooligosaccharides (XOS) and mix thoroughly for 30 minutes under magnetic stirring to prepare a protein-xylooligosaccharide mixed solution; the mass ratio of protein to xylooligosaccharides (XOS) is 1:2; S4. Adjust the pH of the protein-xylo-oligosaccharide mixed solution to 11; S5. The protein-xylo-oligosaccharide mixed solution was placed at 80°C and 200 rpm for 2 h under constant temperature magnetic stirring, and the pH value was maintained constant during the reaction; S6. After the reaction is completed, the temperature is quickly cooled in an ice bath and freeze-dried to obtain the glycosylated protein product (SPI-XOS).

[0037] The grafting rate and the amount of Maillard intermediates produced were measured on the prepared products. Figure 2 The results showed that compared with the control group, SMF 2.5mT and SMF 5mT treatments increased the grafting rate by 50.86% and 74.14%, respectively, and the grafting rate increased to 2.2 times that of the control group under SMF 10mT treatment. In addition, SMF 10mT increased the amount of Maillard intermediates by 36.42%, while inhibiting the Maillard browning process, with the browning degree only increasing by 10.83%.

[0038] Example 3 A method for protein glycosylation modification based on static magnetic field assistance specifically comprises the following steps: S1. Prepare a whey protein isolate (WPI) solution with a concentration of 5%, and adjust the pH value of the whey protein isolate (WPI) solution to 9; S2. Under shaking culture conditions at 10°C and 200 rpm, whey protein isolate (WPI) was placed in static magnetic fields with induction strengths of SMF0mT (Control), SMF 2.5mT, SMF 5mT, and SMF 10mT to assist the protein in hydration reaction for 12 hours; S3. After hydration, add xylooligosaccharides (XOS) and mix thoroughly for 30 minutes under magnetic stirring to prepare a protein-xylooligosaccharide mixed solution; the mass ratio of protein to xylooligosaccharides (XOS) is 1:2; S4. Adjust the pH of the protein-xylo-oligosaccharide mixed solution to 9; S5. The protein-xylo-oligosaccharide mixed solution was placed at 80°C and 200 rpm for 2 h under constant temperature magnetic stirring, and the pH value was maintained constant during the reaction; S6. After the reaction is completed, the temperature is quickly cooled in an ice bath and freeze-dried to obtain the glycosylated protein product (WPI-XOS).

[0039] The grafting rate and the amount of Maillard intermediates produced were measured on the prepared products. Figure 3 The results showed that compared with the control group, SMF 5mT and SMF 10mT treatments increased the grafting rate by 83.51% and 90.67%, respectively, and increased the amount of Maillard intermediates (6.6%-9.14%), effectively inhibiting the increase in the degree of Maillard browning. Under the condition that the grafting rate was nearly doubled, the browning degree only increased by 10%-11%.

[0040] Example 4 A method for protein glycosylation modification based on static magnetic field assistance specifically comprises the following steps: S1. Prepare a 5% casein (CS) solution, and adjust the pH value of the casein (CS) solution to 8; S2. Under shaking culture conditions at 10°C and 200 rpm, the casein (CS) solution was placed in a static magnetic field with an induction strength of SMF 0mT (Control), SMF 2.5mT, SMF 5mT, and SMF 10mT to assist the protein in hydration reaction for 12 hours; S3. After hydration, add xylooligosaccharides (XOS) and mix thoroughly for 30 minutes under magnetic stirring to prepare a protein-xylooligosaccharide mixed solution; the mass ratio of protein to xylooligosaccharides (XOS) is 1:2; S4. Adjusting the pH of the protein-xylo-oligosaccharide mixed solution to 8; S5. The protein-xylo-oligosaccharide mixed solution was placed at 80°C and 200 rpm for 2 h under constant temperature magnetic stirring, and the pH value was maintained constant during the reaction; S6. After the reaction is completed, the temperature is quickly cooled in an ice bath and freeze-dried to obtain the glycosylated protein product (CS-XOS).

[0041] The grafting rate and the amount of Maillard intermediates produced were measured on the prepared products. Figure 4The results showed that compared with the control group, SMF 2.5mT, SMF 5mT, and SMF 10mT treatments increased the grafting rates by 18.48%, 38.52%, and 40.66%, respectively. SMF treatment increased the production of Maillard intermediates by 2.94%, and significantly inhibited the Maillard browning process. Compared with the control group, SMF 5mT and SMF 10mT significantly reduced the degree of Maillard browning.

[0042] Example 5 A method for protein glycosylation modification based on static magnetic field assistance specifically comprises the following steps: S1. Prepare a 5% zein solution, and adjust the pH value of the zein solution to 12; S2. Under shaking culture conditions at 10°C and 200 rpm, the zein solution was placed in static magnetic fields with induction strengths of SMF 0mT (Control), SMF 2.5mT, SMF 5mT, and SMF 10mT to assist the protein in hydration reaction for 12 hours. S3. After hydration is completed, pullulan is added and mixed thoroughly for 30 minutes under magnetic stirring to prepare a protein-pullulan mixed solution; the mass ratio of protein to pullulan is 1:1; S4. Adjusting the pH of the protein-pullulan mixed solution to 12; S5. The protein-pullulan mixed solution was placed at 80°C and 200 rpm under constant temperature magnetic stirring for 2 h, and the pH value was maintained constant during the reaction; S6. After the reaction is completed, the temperature is quickly cooled in an ice bath and freeze-dried to obtain the glycosylated protein product (ZEIN-Pullulan).

[0043] The grafting rate and the amount of Maillard intermediates produced were measured on the prepared products. Figure 5 The results showed that compared with the control group, SMF 2.5mT, SMF 5mT, and SMF 10mT treatments increased the grafting rate by 65.81%, 88.89%, and 128.21%, significantly increased the production of Maillard intermediates (20.7%-43.4%), and significantly reduced the degree of Maillard browning (-6.05%--13.95%).

[0044] Example 6 A method for protein glycosylation modification based on static magnetic field assistance specifically comprises the following steps: S1. Prepare a 5% soy protein isolate (SPI) solution, and adjust the pH value of the soy protein isolate (SPI) solution to 11; S2. Under shaking culture conditions at 10°C and 200 rpm, the soy protein isolate (SPI) solution was placed in a static magnetic field with an induction strength of SMF 0mT (Control), SMF 2.5mT, SMF 5mT, and SMF 10mT to assist the protein in hydration reaction for 12 hours; S3. After hydration is completed, pullulan is added and mixed thoroughly for 30 minutes under magnetic stirring to prepare a protein-pullulan mixed solution; the mass ratio of protein to pullulan is 1:1; S4. Adjusting the pH of the protein-pullulan mixed solution to 11; S5. The protein-pullulan mixed solution was placed at 80°C and 200 rpm under constant temperature magnetic stirring for 2 h, and the pH value was maintained constant during the reaction; S6. After the reaction is completed, the temperature is quickly cooled in an ice bath and freeze-dried to obtain the glycosylated protein product (SPI-Pullulan).

[0045] The grafting rate and the amount of Maillard intermediates produced were measured on the prepared products. Figure 6 The results showed that compared with the control group, SMF 5mT and SMF 10mT treatments increased the grafting rate by 43.81% and 39.69%, and SMF 10mT increased the production of Maillard intermediates by 13.27%.

[0046] Example 7 A method for protein glycosylation modification based on static magnetic field assistance specifically comprises the following steps: S1. Prepare a whey protein isolate (WPI) solution with a concentration of 5%, and adjust the pH value of the whey protein isolate (WPI) solution to 9; S2. Under shaking culture conditions at 10°C and 200 rpm, whey protein isolate (WPI) was placed in static magnetic fields with induction strengths of SMF0mT (Control), SMF 2.5mT, SMF 5mT, and SMF 10mT to assist the protein in hydration reaction for 12 hours; S3. After hydration is completed, pullulan is added and mixed thoroughly for 30 minutes under magnetic stirring to prepare a protein-pullulan mixed solution; the mass ratio of protein to pullulan is 1:1; S4. Adjusting the pH of the protein-pullulan mixed solution to 9; S5. The protein-pullulan mixed solution was placed at 80°C and 200 rpm under constant temperature magnetic stirring for 2 h, and the pH value was maintained constant during the reaction; S6. After the reaction is completed, the temperature is quickly cooled in an ice bath and freeze-dried to obtain the glycosylated protein product (WPI-Pullulan).

[0047] The grafting rate and the amount of Maillard intermediates produced were measured on the prepared products. Figure 7 The results showed that compared with the control group, SMF 10mT treatment significantly increased the grafting rate (22.43%) and the amount of Maillard intermediates generated (13.42%), and significantly reduced the degree of Maillard browning (-16.49%).

[0048] Example 8 A method for protein glycosylation modification based on static magnetic field assistance specifically comprises the following steps: S1. Prepare a 5% casein (CS) solution, and adjust the pH value of the casein (CS) solution to 8; S2. Under shaking culture conditions at 10°C and 200 rpm, the casein (CS) solution was placed in a static magnetic field with an induction strength of SMF 0mT (Control), SMF 2.5mT, SMF 5mT, and SMF 10mT to assist the protein in hydration reaction for 12 hours; S3. After hydration is completed, pullulan is added and mixed thoroughly for 30 minutes under magnetic stirring to prepare a protein-pullulan mixed solution; the mass ratio of protein to pullulan is 1:1; S4. Adjusting the pH of the protein-pullulan mixed solution to 8; S5. The protein-pullulan mixed solution was placed at 80°C and 200 rpm under constant temperature magnetic stirring for 2 h, and the pH value was maintained constant during the reaction; S6. After the reaction is completed, the temperature is quickly cooled in an ice bath and freeze-dried to obtain the glycosylated protein product (CS-Pullulan).

[0049] The grafting rate and the amount of Maillard intermediates produced were measured on the prepared products. Figure 8 The results showed that compared with the control group, SMF 2.5mT, SMF 5mT, and SMF 10mT treatments significantly increased the grafting rate by 20.48%, 29.19%, and 41.18%, respectively; SMF 10mT treatment increased the amount of Maillard intermediates by 6.25%; and reduced the increase in the degree of Maillard browning, which only increased by 9.25% compared with the control group.

[0050] It should be understood that the various forms of processes shown above can be used to reorder, add or delete steps. For example, the steps described in the disclosure of the present invention can be performed in parallel, sequentially or in different orders, as long as the desired results of the technical solution disclosed in the present invention can be achieved, and this document does not limit this.

[0051] The above specific implementations do not constitute a limitation on the protection scope of the present invention. It should be understood by those skilled in the art that various modifications, combinations, sub-combinations and substitutions can be made according to design requirements and other factors. Any modification, equivalent substitution and improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for protein glycosylation modification based on static magnetic field, characterized in that: The specific steps include: S1. Prepare a protein solution with a concentration of 3-8%, and adjust the pH value of the protein solution to 8-12; S2. Under shaking culture conditions of 5-12°C and 150-250 rpm, the protein solution is placed in a static magnetic field with an induction intensity of 2.5-10 mT to assist the protein in hydration reaction; S3. After hydration is completed, add sugars and mix thoroughly for 20 to 40 minutes under magnetic stirring to prepare a protein-sugar mixed solution; the mass ratio of protein to sugar is 1:1 to 2; S4. Adjust the pH of the protein-carbohydrate mixed solution to the same as step S1; S5. The protein-sugar mixed solution is placed in a constant temperature magnetic stirring state at 75-85°C and a rotation speed of 150-250 rpm for reaction, and the pH value is maintained constant during the reaction; S6. After the reaction is completed, the temperature is quickly cooled in an ice bath, and the glycosylated protein product is obtained after freeze-drying.

2. The method for protein glycosylation modification based on static magnetic field assistance according to claim 1, characterized in that: The hydration reaction time in step S2 is 10-20 hours, and the reaction temperature is 10°C.

3. The method for protein glycosylation modification based on static magnetic field assistance according to claim 2, characterized in that: The hydration reaction time in step S2 is 12 hours, and the induction intensity of the static magnetic field is 10 mT.

4. The method for protein glycosylation modification based on static magnetic field assistance according to claim 1, characterized in that: The protein is zein, soy protein isolate, whey protein isolate or casein.

5. The method for protein glycosylation modification based on static magnetic field assistance according to claim 4, characterized in that: The carbohydrate substance is oligosaccharide or polysaccharide.

6. The method for protein glycosylation modification based on static magnetic field assistance according to claim 5, characterized in that: The oligosaccharide is xylooligosaccharide, and the mass ratio of protein to xylooligosaccharide is 1:

2.

7. The method for protein glycosylation modification based on static magnetic field assistance according to claim 5, characterized in that: The polysaccharide is pullulan, and the mass ratio of protein to pullulan is 1:

1.

8. A method for protein glycosylation modification based on static magnetic field assistance according to any one of claims 1 to 7, characterized in that: The reaction time in step S5 is 1.5-2.5 hours, and the reaction temperature is 80°C.

9. The method for protein glycosylation modification based on static magnetic field assistance according to claim 8, characterized in that: The protein solution in step S1 is prepared by dissolving the protein in a phosphate buffer, stirring it thoroughly, and leaving it for more than 8 hours to fully hydrate it.

10. The method for protein glycosylation modification based on static magnetic field assistance according to claim 9, characterized in that: The concentration of the protein solution in step S1 is 5%.

Images