A method for preparing low-concentration bovine serum albumin synergized by pulsed electric field

Abstract

The present invention discloses a method for preparing low-concentration bovine serum albumin with pulse electric field synergistic glycosylation. In the case of 0.8wt%-3wt% low-concentration bovine serum albumin, the temperature is raised to 85-95°C to coordinate pulse electric field treatment, which greatly improves the reaction efficiency of the glycosylation reaction, achieves a higher grafting degree, improves the emulsification and emulsifying properties of BSA, and reduces unnecessary color browning, so that it has a broader application prospect.

Description

Technical field:

[0001] The invention relates to the field of protein modification, and in particular to a method for preparing pulse electric field-coordinated glycosylated low-concentration bovine serum albumin. Background technology:

[0002] Glycosylation modification of proteins is a common protein modification method, which involves reversible or irreversible chemical recombination reactions such as oxidation, cyclization and polymerization between the hemiacetal group of reducing sugars and the free amino acids of proteins. Studies have shown that glycosylation modification can improve the functional properties of proteins such as gelling, foaming and emulsification, and also has a certain effect on antioxidant activity and antibacterial properties. Compared with other protein modification methods, glycosylation modification does not require the addition of additional chemical reagents that may be harmful to health, and is more environmentally friendly and safe.

[0003] Due to the compact structure of proteins and fewer reaction sites, especially in scenarios with low protein concentrations, the protein has even fewer reaction sites. Glycosylation reactions generally require higher temperatures (>110°C) and longer reaction times (generally several hours or even days) to achieve a higher degree of reaction, and the browning degree of the reaction is very deep, which may have a negative impact on the quality and appearance of the food, and may also generate some potential substances that are harmful to human health. In addition, long-term high-temperature reactions also cause a waste of energy and time. Therefore, more and more emerging physical processing technologies are being used to assist glycosylation reactions to improve their reaction efficiency. Among them, pulsed electric fields, as a new type of green physical non-thermal processing technology, cause less temperature rise during the treatment process and almost no energy waste. At present, this technology is mainly used in the field of sterilization, and there is relatively little research on the application and principle of pulsed electric fields in glycosylation modification. Chinese invention patent CN114246247A discloses a method for preparing an antioxidant camellia seed peptide, which discloses a high-voltage pulse electric field treatment to unfold the protein structure, and then sequentially hydrolyzes the protein with neutral protease, alkaline protease, and trypsin to obtain the camellia seed peptide. Chinese invention patent CN 101584421B, a previous research result of our research group, discloses a method for treating the Maillard reaction at low temperature with a pulse electric field, which uses a pulse electric field to strengthen the Maillard reaction between reducing sugars or oligosaccharides or polysaccharides that are decomposed into reducing sugars under alkaline conditions and proteins, polypeptides, or amino acids to prepare a food antioxidant that can improve the color of food. The pulse electric field conditions are as follows: (1) pulse electric field strength: 15 kV / cm~30 kV / cm; (2) pulse frequency: 100 Hz~700 Hz; (3) pulse width: 10 μs~100 μs; (4) reaction temperature: below 35°C. It has the advantages of no pollution, low energy consumption, short preparation time, low processing temperature, high reaction efficiency, maximum preservation of product nutrition and flavor components, improved food quality, increased product color value and food safety. However, this method mainly introduces the promotion effect of high pulse electric field strength (15-30kV / cm) on the glycosylation reaction of high-concentration protein (the concentration of protein aqueous solution is 9-25wt%), and does not involve the effect of pulse electric field on protein glycosylation reaction in low-concentration protein scenario. Summary of the invention:

[0004] The purpose of the present invention is to provide a method for preparing low-concentration bovine serum albumin with pulsed electric field synergistic glycosylation. In the case of low-concentration bovine serum albumin of 0.8wt%-3wt%, the temperature is raised to 85-95°C to coordinate pulsed electric field treatment, which greatly improves the reaction efficiency of the glycosylation reaction, achieves a higher grafting degree, improves the emulsification and emulsification properties of BSA, and reduces unnecessary color browning, so that it has a broader application prospect.

[0005] The present invention is achieved through the following technical solutions:

[0006] A method for preparing pulsed electric field-coordinated glycosylated low-concentration bovine serum albumin, the method comprising the following steps:

[0007] S1: dissolving bovine serum albumin in water to prepare a bovine serum albumin aqueous solution with a concentration of 0.8 wt% to 3 wt%, preferably 1 wt% to 3 wt%, and most preferably 1 wt%;

[0008] S2: adding glucose to the bovine serum albumin aqueous solution to make the mass ratio of bovine serum albumin to glucose be 1:0.25-1:2, preferably 1:0.5-1:1.5, and stirring to prepare a bovine serum albumin / glucose mixed solution;

[0009] S3: adjusting the pH value of the mixture in step S2 to 6.7-10.7, preferably 8.7-10.7, with a base;

[0010] S4: raising the temperature of the mixed solution to 85-95°C, preferably 90°C; then pumping it into the pulse electric field treatment chamber for pulse electric field treatment; pulse electric field treatment conditions: pulse electric field intensity of 4-14kV / cm, preferably 8-12kV / cm; pulse frequency of 100-1000Hz, preferably 700-1000Hz; pulse width of 20-60μs, preferably 20-40μs; pulse treatment time of 15-45min, preferably 25-35min; the temperature remains unchanged during the reaction;

[0011] S5: The solution after the reaction is rapidly cooled to room temperature, and then dialyzed and freeze-dried to obtain the final product.

[0012] The inventors unexpectedly discovered that in a 0.8wt%-3wt% protein aqueous solution, heating to 85-95°C and coordinating with a pulsed electric field treatment can greatly improve the reaction efficiency of the glycosylation reaction.

[0013] In the pulse electric field strength range of 4-14kV / cm, the grafting degree of the protein first increases and then decreases with the increase of the pulse electric field strength. This is because after being treated with a low-intensity pulse electric field, the protein structure unfolds, exposing more reactive groups, increasing the contact probability between sugar and protein, and thus increasing the grafting degree of the reaction; however, continuously increasing the electric field strength will cause protein aggregation and the reactive groups will be embedded in the protein, which is not conducive to the reaction.

[0014] Moreover, the grafting degree of the protein first increases and then decreases with the increase of protein concentration. This is because when the protein concentration is too low, the probability of collision with glucose in the solution is small, so the grafting degree is low; when the protein concentration is high, under the pulse electric field and 85-95℃ water bath treatment, the protein is prone to aggregation, which leads to a decrease in the grafting degree.

[0015] The beneficial effects of the present invention are as follows: The present invention discloses a method for preparing low-concentration bovine serum albumin with pulsed electric field synergistic glycosylation. In the case of low-concentration bovine serum albumin of 0.8wt%-3wt%, the temperature is raised to 85-95°C and the synergistic pulsed electric field treatment greatly improves the reaction efficiency of the glycosylation reaction, achieves a higher grafting degree, improves the emulsification and emulsification properties of BSA, and reduces unnecessary color browning, so that it has a broader application prospect. Description of the drawings:

[0016] Figure 1 It is an infrared spectrum (from top to bottom, they are raw material bovine serum albumin (BSA), the product obtained in Comparative Example 9 (P-BSA), the product obtained in Comparative Example 11 (W-BSA), the product obtained in Example 6 (PW), and the product obtained in Comparative Example 10 (W)).

[0017] Figure 2 This is the zeta potential diagram of bovine serum albumin.

[0018] Figure 3 Circular dichroism characterization of bovine serum albumin. Specific implementation method:

[0019] The following is a further description of the present invention, rather than a limitation of the present invention.

[0020] Embodiment 1:

[0021] S1: dissolving bovine serum albumin in distilled water to a protein concentration of 1 wt%, and mixing and stirring for 0.5 h to obtain a uniform solution;

[0022] S2: adding glucose to the protein solution to make the protein / sugar mass ratio 1:1, mixing and stirring for 0.5 h to obtain a bovine serum albumin / glucose mixed solution;

[0023] S3: Adjust the pH of the mixed solution to about 10.7;

[0024] S4: The solution treated with S3 was heated to 90°C and pumped into the pulse electric field treatment chamber for electric field treatment. The pulse field strength was controlled to be 10 kV / cm and the pulse time was 30 min. After the reaction, it was quickly cooled to room temperature.

[0025] S5: Use a dialysis bag to dialyze the product in S4 until the conductivity is stable, and freeze-dry the dialysate to obtain a bovine serum albumin / glucose complex.

[0026] Product indicators: The obtained product was prepared into a 3 mg / mL protein aqueous solution, and the free amino acid content of the protein was determined by the OPA method to reflect its grafting degree. The grafting degree was 20.03%.

[0027] Comparative Example 1

[0028] Refer to Example 1, except that the S4 pulse electric field treatment condition is 20 kV / cm. The free amino group content of the protein is measured by OPA method, and the protein grafting degree is 16.64%.

[0029] Comparative Example 2

[0030] Refer to Example 1, except that the S4 treatment temperature is 70° C. The free amino group content of the protein is measured by OPA method, and the protein grafting degree is 15.94%.

[0031] Comparative Example 3

[0032] Refer to Example 1, except that the S4 treatment temperature is 85° C. The free amino group content of the protein is measured by OPA method, and the protein grafting degree is 17.87%.

[0033] Comparative Example 4

[0034] Refer to Example 1, except that the S4 treatment temperature is 95° C. The protein free amino group content is measured by OPA method, and the protein grafting degree is 21.47%.

[0035] Comparative Example 5

[0036] Refer to Example 1, except that the S4 treatment temperature is 100° C. The free amino group content of the protein is measured by OPA method, and the protein grafting degree is 23.16%.

[0037] Comparative Example 6

[0038] Referring to Example 1, the difference is that the protein concentration at S1 is 0.5 wt %. The free amino group content of the protein is measured by OPA method, and the protein grafting degree is 14.43%.

[0039] Comparative Example 7

[0040] Referring to Example 1, the difference is that the protein concentration at S1 is 0.8 wt %. The free amino group content of the protein is measured by OPA method, and the protein grafting degree is 16.10%.

[0041] Comparative Example 8

[0042] Referring to Example 1, the difference is that the protein concentration at S1 is 5 wt %. The free amino group content of the protein is measured by OPA method, and the protein grafting degree is 13.72%.

[0043] Example 2

[0044] S1: dissolving bovine serum albumin in distilled water to a protein concentration of 1 wt%, and mixing and stirring for 0.5 h to obtain a uniform solution;

[0045] S2: adding glucose to the protein solution to make the protein / sugar mass ratio 1:1, mixing and stirring for 0.5 h to obtain a bovine serum albumin / glucose mixed solution;

[0046] S3: Adjust the pH of the mixed solution to about 10.7;

[0047] S4: The solution treated with S3 was heated to 90°C and pumped into the pulse electric field treatment chamber for electric field treatment. The pulse field strength was controlled to be 5 kV / cm and the pulse time was 30 min. After the reaction, it was quickly cooled to room temperature.

[0048] S5: Use a dialysis bag to dialyze the product in S4 until the conductivity is stable, and freeze-dry the dialysate to obtain a bovine serum albumin / glucose complex.

[0049] Product indicators: The obtained product was prepared into a 3 mg / mL protein aqueous solution, and the free amino acid content of the protein was determined by the OPA method to reflect its grafting degree. The grafting degree was 17.81%.

[0050] Example 3

[0051] S1: Dissolve bovine serum albumin in distilled water to a protein concentration of 3 wt %, and mix and stir for 0.5 h to obtain a uniform solution;

[0052] S2: adding glucose to the protein solution to make the protein / sugar mass ratio 1:1, mixing and stirring for 0.5 h to obtain a bovine serum albumin / glucose mixed solution;

[0053] S3: Adjust the pH of the mixed solution to about 10.7;

[0054] S4: The solution treated with S3 was heated to 90°C and pumped into the pulse electric field treatment chamber for electric field treatment. The pulse field strength was controlled to be 10 kV / cm and the pulse time was 30 min. After the reaction, it was quickly cooled to room temperature.

[0055] S5: Use a dialysis bag to dialyze the product in S4 until the conductivity is stable, and freeze-dry the dialysate to obtain a bovine serum albumin / glucose complex.

[0056] Product indicators: The obtained product was prepared into a 3 mg / mL protein aqueous solution, and the free amino acid content of the protein was determined by the OPA method to reflect its grafting degree. The grafting degree was 17.37%.

[0057] Example 4

[0058] S1: dissolving bovine serum albumin in distilled water to a protein concentration of 1 wt%, and mixing and stirring for 0.5 h to obtain a uniform solution;

[0059] S2: adding glucose to the protein solution to make the protein / sugar mass ratio 1:1, mixing and stirring for 0.5 h to obtain a bovine serum albumin / glucose mixed solution;

[0060] S3: Adjust the pH of the mixed solution to about 10.7;

[0061] S4: The solution treated with S3 was heated to 90°C and pumped into the pulse electric field treatment chamber for high voltage electric field treatment. The pulse field strength was controlled to be 10 kV / cm and the pulse time was 25 min. After the reaction, it was quickly cooled to room temperature.

[0062] S5: Use a dialysis bag to dialyze the product in S4 until the conductivity is stable, and freeze-dry the dialysate to obtain a bovine serum albumin / glucose complex.

[0063] Product indicators: The obtained product was prepared into a 3 mg / mL protein aqueous solution, and the free amino acid content of the protein was determined by the OPA method to reflect its grafting degree. At this time, the grafting degree was 20.61%.

[0064] Example 5

[0065] S1: dissolving bovine serum albumin in distilled water to a protein concentration of 1 wt%, and mixing and stirring for 0.5 h to obtain a uniform solution;

[0066] S2: adding glucose to the protein solution to make the protein / sugar mass ratio 1:1, mixing and stirring for 0.5 h to obtain a bovine serum albumin / glucose mixed solution;

[0067] S3: Adjust the pH of the mixed solution to about 9.7;

[0068] S4: The solution treated with S3 was heated to 90°C and pumped into the pulse electric field treatment chamber for high voltage electric field treatment. The pulse field strength was controlled to be 10 kV / cm and the pulse time was 30 min. After the reaction, it was quickly cooled to room temperature.

[0069] S5: Use a dialysis bag to dialyze the product in S4 until the conductivity is stable, and freeze-dry the dialysate to obtain a bovine serum albumin / glucose complex.

[0070] Product indicators: The obtained product was prepared into a 3 mg / mL protein aqueous solution, and the free amino acid content of the protein was determined by the OPA method to reflect its grafting degree. At this time, the grafting degree was 15.36%.

[0071] Example 6

[0072] S1: dissolving bovine serum albumin in distilled water to a protein concentration of 1 wt%, and mixing and stirring for 0.5 h to obtain a uniform solution;

[0073] S2: adding glucose to the protein solution to make the protein / sugar mass ratio be 1:1.5, and mixing and stirring for 0.5 h to obtain a bovine serum albumin / glucose mixed solution;

[0074] S3: Adjust the pH of the mixed solution to about 10.7;

[0075] S4: The solution treated with S3 was heated to 90°C and pumped into the pulse electric field treatment chamber for high voltage electric field treatment. The pulse field strength was controlled to be 12 kV / cm and the pulse time was 28 min. After the reaction, it was quickly cooled to room temperature.

[0076] S5: Use a dialysis bag to dialyze the product in S4 until the conductivity is stable, and freeze-dry the dialysate to obtain a bovine serum albumin / glucose complex, which is recorded as product PW. Its infrared spectrum can be found in Figure 1 .

[0077] Product indicators: The obtained product was prepared into a 3 mg / mL protein aqueous solution, and the free amino acid content of the protein was determined by the OPA method to reflect its grafting degree. At this time, the grafting degree was 21.71%.

[0078] Comparative Example 9

[0079] Refer to Example 6, except that glucose is not added in step S2, the temperature is not raised to 90°C in step S4, and the electric field treatment is directly performed, and after the treatment, the temperature is quickly cooled to room temperature, dialyzed and freeze-dried to obtain the product P-BSA.

[0080] Comparative Example 10

[0081] Reference Example 6, except that, in S4, the solution treated with S3 was heated to 90°C and then not pumped into the pulse electric field treatment chamber for electric field treatment, and was rapidly cooled to room temperature after treatment, dialyzed and freeze-dried to obtain product W. At this time, the protein grafting degree was measured to be 12.09%. The absorbance at 420nm was 0.185.

[0082] Comparative Example 11

[0083] Refer to Example 6, except that glucose is not added in step S2, and in step S4 the protein is heated to 90° C. in a water bath without pulse electric field treatment, and then rapidly cooled to room temperature, dialyzed and freeze-dried to obtain the product W-BSA.

[0084] Example 7

[0085] S1: dissolving bovine serum albumin in distilled water to a protein concentration of 1 wt%, and mixing and stirring for 0.5 h to obtain a uniform solution;

[0086] S2: adding glucose to the protein solution to make the protein / sugar mass ratio 1:1, mixing and stirring for 0.5 h to obtain a bovine serum albumin / glucose mixed solution;

[0087] S3: Adjust the pH of the mixed solution to about 10.7;

[0088] S4: The solution treated with S3 was heated to 90°C and pumped into the pulse electric field treatment chamber for high voltage electric field treatment. The pulse field strength was controlled to be 8 kV / cm and the pulse time was 30 min. After the reaction, it was quickly cooled to room temperature.

[0089] S5: Use a dialysis bag to dialyze the product in S4 until the conductivity is stable, and freeze-dry the dialysate to obtain a bovine serum albumin / glucose complex.

[0090] Product indicators: The obtained product was prepared into a 3 mg / mL protein aqueous solution, and the free amino acid content of the protein was determined by the OPA method to reflect its grafting degree. At this time, the grafting degree was 18.92%.

[0091] Example 8

[0092] S1: dissolving bovine serum albumin in distilled water to a protein concentration of 1 wt%, and mixing and stirring for 0.5 h to obtain a uniform solution;

[0093] S2: adding glucose to the protein solution to make the protein / sugar mass ratio be 1:0.5, and mixing and stirring for 0.5 h to obtain a bovine serum albumin / glucose mixed solution;

[0094] S3: Adjust the pH of the mixed solution to about 10.7;

[0095] S4: The solution treated with S3 was heated to 90°C and pumped into the pulse electric field treatment chamber for high voltage electric field treatment. The pulse field strength was controlled to be 10 kV / cm and the pulse time was 30 min. After the reaction, it was quickly cooled to room temperature.

[0096] S5: Use a dialysis bag to dialyze the product in S4 until the conductivity is stable, and freeze-dry the dialysate to obtain a bovine serum albumin / glucose complex.

[0097] Product indicators: The obtained product was prepared into a 3 mg / mL protein aqueous solution, and the free amino acid content of the protein was determined by the OPA method to reflect its grafting degree. At this time, the grafting degree was 16.62%.

[0098] Detection method:

[0099] ①Determination of grafting degree

[0100] The degree of grafting was determined by the OPA reagent method. 80 mg of OPA was dissolved in 2 mL of anhydrous ethanol, mixed with 50 mL of 0.1 mol / L sodium tetraborate buffer and 5 mL of 20% SDS solution, and then 0.2 mL of β-mercaptoethanol was added. After mixing, the volume was adjusted to 100 mL with distilled water to prepare the OPA reagent. 200 μL of the sample solution (3 mg / mL) was mixed with 4 mL of the OPA reagent and reacted accurately at 35 ° C for 2 minutes, and then the ultraviolet absorption intensity at a wavelength of 340 nm was immediately measured. Water was used as the blank control, and lysine was used instead of the sample to make the standard curve. The standard curve equation was: y = 8.83143x + 0.0941, where: y is the absorbance and x is the free amino content.

[0101]

[0102] Where: C0——free amino content when not reacted; C t ——Free amino content after reaction.

[0103] It can be found from Example 1 and Comparative Example 1 that the grafting degree of the protein of the present invention is better. This may be because increasing the electric field strength to 20 kV / cm will cause protein aggregation, and the reactive groups are buried inside the protein, which is not conducive to the reaction.

[0104] Through Example 1 and Comparative Examples 2-5, it can be seen that the grafting degree of the glycosylation reaction also shows an increasing trend with the increase of temperature. However, in actual laboratory operations, when the temperature is ≥100°C, the reaction conditions are too intense and the reaction process is difficult to control. Therefore, the temperature of this experimental condition is preferably 85-95°C, and most preferably 90°C.

[0105] It can be found from Examples 1, 3 and Comparative Examples 6-8 that the grafting degree of the protein first increases and then decreases with the increase of the protein concentration. The effect is better when the protein concentration is 0.8wt-3wt%, preferably 1wt-3wt%, and most preferably 1wt%. This is because when the protein concentration is too low (less than 0.8wt%), the probability of collision with glucose in the solution is small, so the grafting degree is low; when the protein concentration is high (greater than 3wt%), under the pulse electric field and 90°C water bath treatment, the protein is prone to aggregation, and the reactive groups are embedded in the protein, resulting in a decrease in the grafting degree.

[0106] The grafting degree results of Example 6 and Comparative Example 10 show that the reaction efficiency of the glycosylation reaction can be greatly improved by the synergistic treatment of pulsed electric field and temperature increase, and the color of the solution of Comparative Example 10 is darker and the degree of browning is higher, indicating that the browning of the glycosylation reaction can be reduced by pulsed electric field treatment, which is beneficial to the retention of product color.

[0107] ②Determination of browning degree

[0108] 1.5 mL of the glycoprotein solution after the glycosylation reaction was taken out, diluted to 5 mL with distilled water, and the absorbance of the diluted solution was measured at 420 nm using an ultraviolet spectrometer. The absorbance of the product of Example 6 at 420 nm was 0.081.

[0109] ③ Particle size and ζ-potential determination

[0110] The raw material bovine serum albumin, the lyophilized bovine serum albumin obtained in Example 6 and Comparative Examples 9-11 were dissolved in distilled water to prepare a 0.4 mg / mL protein solution. After sufficient dissolution, the particle size and ζ-potential were measured by Malvern nanoparticle size and Zeta potential analyzer at an equilibrium time of 120 s and 25° C. The measurements were repeated three times in parallel. The results are shown in Tables 1 and Figure 2 .

[0111] Table 1 Particle size and ζ-potential measurement results

[0112]

[0113]

[0114] It can be seen from Table 1 that the average particle size of Example 6 and Comparative Example 10 increases, which may be due to the fact that under the pulsed electric field treatment, the protein structure unfolds, the rigid structure weakens, the flexibility increases, and the molecular radius slightly increases; at the same time, compared with the original protein, the absolute value of the ζ-potential of the treated protein, especially Example 6, increases, which may be because the protein structure is more unfolded, thereby exposing more negatively charged charges on the surface, which indicates that the pulsed electric field synergistic glycosylation treatment may change the structure of the protein and make the solution system more stable.

[0115] ④Circular dichroism measurement

[0116] Natural bovine serum albumin, and the freeze-dried bovine serum albumin obtained in Example 6 and Comparative Examples 9-11 were dissolved in distilled water to prepare a 0.2 mg / mL protein solution. After sufficient dissolution, the protein secondary structure was measured by Chirascan circular dichroism spectrometer at a wavelength range of 190 nm to 260 nm.

[0117] Table 2 Circular dichroism measurement results

[0118]

[0119] As shown in Table 2, the pulse electric field and water bath heating treatment of the protein can reduce the α-helical structure content of the protein and increase the random coil content. It is known that the α-helical structure of the protein is the most stable, indicating that the structure of the protein is stretched after treatment to expose more reactive groups. In the presence of sugar, the α-helical structure content of the protein in Example 6 decreases and the random coil content increases more significantly, indicating that pulse-assisted glycosylation can make the protein structure more unfolded, which explains the reason for the increase in the glycosylation reaction rate.

Claims

1. A method for preparing low-concentration bovine serum albumin synergistically glycosylated by pulsed electric field, characterized in that: The method comprises the following steps: S1: dissolving bovine serum albumin in water to prepare a 1 wt%-3 wt% bovine serum albumin aqueous solution; S2: adding glucose to the bovine serum albumin aqueous solution to make the mass ratio of bovine serum albumin to glucose be 1:0.25-1:2, and stirring to prepare a bovine serum albumin / glucose mixed solution; S3: adjusting the pH value of the mixture in step S2 to 9.7-10.7 with alkali; S4: The mixed solution is heated to 85-95 °C and then pumped into the pulse electric field treatment chamber for pulse electric field treatment; the pulse electric field treatment conditions are: pulse electric field intensity of 4-14 kV / cm; pulse frequency of 100-1000 Hz; pulse width of 20-60μs, pulse treatment time of 15-45 min; the temperature remains unchanged during the reaction process; S5: The solution after the reaction is rapidly cooled to room temperature, and then dialyzed and freeze-dried to obtain the final product.

2. The preparation method according to claim 1, characterized in that: In step S1, the concentration of the bovine serum albumin aqueous solution is 1 wt %.

3. The preparation method according to claim 1, characterized in that: In step S2, the mass ratio of bovine serum albumin to glucose is 1:0.5-1:1.

5.

4. The preparation method according to claim 1, characterized in that: The pH value in step S3 is 10.

7.

5. The preparation method according to claim 1, characterized in that: The temperature of step S4 is 90°C.

6. The preparation method according to claim 1, characterized in that: In step S4, the pulse electric field strength is 8-12 kV / cm; the pulse frequency is 700-1000 Hz; and the pulse width is 20-40 μs.

7. The preparation method according to claim 1, characterized in that: In step S4, the pulse treatment time is 25-35 minutes.

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