Method for preparing high-purity lysozyme and ovotransferrin by adopting combined ion exchange chromatography

Abstract

The invention discloses a method for preparing high-purity lysozyme and ovotransferrin through combined ion exchange chromatography, and belongs to the technical field of protein separation and purification and functional food. The lysozyme with the purity of 95.57% and the ovotransferrin with the purity of 97.89% are obtained through separation by combining cation exchange resin with anion exchange filler. Wherein the ovotransferrin has good oxidation resistance, has a relatively high inhibition rate on the growth of pseudomonas fluorescens, and does not lose the biological function in an acid environment, so that a basis is provided for improving the additional value of the egg white and developing antibacterial and anti-inflammatory functional foods.

Description

Technical Field

[0001] The invention belongs to the technical field of protein separation and purification and functional food, and specifically relates to a method for preparing high-purity lysozyme and ovotransferrin by using combined ion exchange chromatography. Background Art

[0002] Egg white protein is the most ideal high-quality protein in food, mainly composed of ovalbumin, ovotransferrin, ovomucin and lysozyme. Among them, ovotransferrin accounts for about 12%-13% of the total protein content of egg white. It has antioxidant, antibacterial, antiviral, anti-inflammatory and immunomodulatory functions. It is a biologically active protein that has attracted much attention from scientific researchers and has great application potential in the fields of functional foods and food preservation. Lysozyme is recognized as a natural substance with bactericidal effects. It not only has antibacterial and antiviral effects, but also can improve the body's immune function. It is a functional food with great application prospects.

[0003] At present, the main methods for preparing ovotransferrin include ethanol extraction, ammonium sulfate precipitation, ultrafiltration and nanofiltration extraction, etc. Among them, the use of ethanol and ammonium sulfate for separation will denature ovotransferrin to varying degrees, resulting in a significant decrease in the activity of ovotransferrin; the ovotransferrin obtained by ultrafiltration and nanofiltration extraction has a low purity.

[0004] At present, publication number CN118515751A discloses a method for quickly and efficiently preparing highly active ovotransferrin, but the purity of the ovotransferrin obtained is less than 95%; publication number CN110066778B discloses a method for the combined separation of lysozyme and ovotransferrin in egg white, but the metal ions introduced therein are likely to cause environmental pollution.

[0005] Based on this, the present invention is proposed. Summary of the invention

[0006] In view of the above problems, the present invention provides a method for preparing high-purity and high-activity ovotransferrin by combined ion exchange chromatography, comprising the following steps: (1) Egg white pretreatment: Wash fresh eggs, separate the yolk and egg white, take the egg white liquid, shear it at high speed for 3-5 minutes, and then filter it with a filter to remove insoluble impurities; (2) Removing ovomucin: adjusting the pH of the egg white solution treated in step (1) with a citric acid solution, and centrifuging at 4°C to remove the ovomucin precipitate after magnetic stirring; (3) Separating lysozyme: Using cation exchange chromatography, the pH of the supernatant obtained in step (2) is adjusted and passed through a cation exchange resin, and then eluted with a NaCl solution to obtain lysozyme, and the permeate containing ovalbumin and ovotransferrin is collected; (4) Separating ovotransferrin: Using anion exchange chromatography, the pH of the flowthrough obtained in step (3) is adjusted and passed into an anion exchange column Q Sepharose Fast Flow, and then the ovalbumin is eluted with a NaCl solution, and the flowthrough containing ovotransferrin is collected; (5) Concentration and drying of lysozyme and ovotransferrin: The permeate obtained in step (4) is filtered through an ultrafiltration membrane using a membrane concentration method, and then vacuum freeze-concentrated to obtain high-purity and high-activity ovotransferrin and lysozyme.

[0007] As a preferred embodiment of the present invention, in step (2), the pH of the egg white solution is adjusted to 4-6, the magnetic stirring speed is 500-2000 rpm, and the stirring time is 1-3 h.

[0008] As a preferred embodiment of the present invention, in the step (3), the adsorption time of the egg white supernatant on the cation exchange resin is 1-3 h, the adsorption pH is 7.5-9.0, the mass fraction of the eluent NaCl solution is 3-9%, and the elution time is 30-60 min.

[0009] As a preferred embodiment of the present invention, in the step (4), the penetration time of the permeate containing ovalbumin and ovotransferrin in the anion exchange column is 1 to 3 h, the adsorption pH is 5.0 to 6.0, the adsorption flow rate is 1 to 5 mL / min, the NaCl concentration of the eluent is 2 to 8%, and the elution flow rate is 4 to 8 mL / min; As a preferred embodiment of the present invention, in step (5), the pore size of the ultrafiltration membrane is 20-50 kDa, the pressure is 0.5-1.0 MPa, the flow rate is 10-20 Hz, and the ultrafiltration times are 1-3 times.

[0010] Beneficial effects of the present invention: (1) Combined protein separation: The present invention innovatively utilizes cation exchange resin in combination with anion exchange filler to simultaneously separate and prepare two kinds of egg white active proteins. Most of the reagent solutions used in the entire preparation process can be recycled and reused. The purity of the obtained lysozyme and ovotransferrin is higher than 95%.

[0011] (2) Maintaining biological activity: The protein extraction and purification process of the present invention does not involve heat treatment, and can maintain the good antioxidant and antibacterial biological activities of ovotransferrin.

[0012] (3) Enriching resource utilization dimensions: The innovative method of the present invention simultaneously extracts two kinds of egg white active proteins, which not only enriches the utilization scope of egg white resources, but also increases the added value of egg white, making it have broad market prospects in the fields of functional foods and food preservation. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The appearance of the egg white solution obtained in Examples 1-5 of the present invention after being adjusted to different pH values ​​and centrifuged and layered; Figure 2 for Figure 1 Appearance of the supernatant after centrifugation of the egg white solution; Figure 3 for Figure 1 Appearance of the precipitate after centrifugation of the egg white solution; Figure 4 The SDS-PAGE electrophoresis diagram of the egg white solution obtained in Examples 1-5 of the present invention.

[0014] Figure 5 This is the SDS-PAGE electrophoresis diagram of the egg white permeate after cation exchange chromatography obtained in Examples 6-14 of the present invention.

[0015] Figure 6 The SDS-PAGE electrophoresis diagram of the lysozyme obtained in Example 10 of the present invention.

[0016] Figure 7 This is the SDS-PAGE electrophoresis diagram of the egg white eluate after anion exchange chromatography obtained in Examples 15-19 of the present invention.

[0017] Figure 8 This is the SDS-PAGE electrophoresis diagram of ovotransferrin obtained in Example 17 of the present invention.

[0018] Fig. 9 This is a scanning electron micrograph of the ovotransferrin obtained in Example 17 of the present invention.

[0019] Fig.10 This is a graph showing the inhibitory effect of different protein concentrations (0.03125-16 mg / mL) on the growth of Pseudomonas fluorescens in a 96-well plate in an antibacterial activity test of ovotransferrin against Pseudomonas fluorescens at different pH values ​​(3, 5, 7, 9) obtained in Example 17 of the present invention; Fig.11 for Fig.10 In the experiment, 1 mg / mL ovotransferrin inhibited the growth of Pseudomonas fluorescens on the plate.

[0020] Fig.12 The UV spectra of ovotransferrin at different pH values ​​(3, 5, 7, 9) obtained in Example 17 of the present invention are shown.

[0021] Fig.13 : This is the fluorescence spectra of ovotransferrin at different pH values ​​(3, 5, 7, 9) obtained in Example 17 of the present invention. DETAILED DESCRIPTION

[0022] The technical solutions described in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0023] Example 1 (1) Egg white pretreatment: Wash fresh eggs, separate the yolk and egg white, take the egg white liquid, shear it at high speed for 5 minutes, and then filter it with a filter to remove insoluble impurities; (2) Removal of ovomucin: The pH of the egg white solution treated in step (1) was adjusted to 6.0 with citric acid solution, and the mixture was stirred magnetically at 1000 rpm for 2 h, and then centrifuged at 4°C to remove the ovomucin precipitate to obtain a supernatant.

[0024] Example 2 (1) Egg white pretreatment: Wash fresh eggs, separate the yolk and egg white, take the egg white liquid, shear it at high speed for 5 minutes, and then filter it with a filter to remove insoluble impurities; (2) Removal of ovomucin: The pH of the egg white solution treated in step (1) was adjusted to 5.5 with citric acid solution, and the mixture was stirred magnetically at 1000 rpm for 2 h, and then centrifuged at 4°C to remove the ovomucin precipitate to obtain a supernatant.

[0025] Example 3 (1) Egg white pretreatment: Wash fresh eggs, separate the yolk and egg white, take the egg white liquid, shear it at high speed for 5 minutes, and then filter it with a filter to remove insoluble impurities; (2) Removal of ovomucin: The pH of the egg white solution treated in step (1) was adjusted to 5.0 with citric acid solution, and the mixture was stirred magnetically at 1000 rpm for 2 h, and then centrifuged at 4°C to remove the ovomucin precipitate to obtain a supernatant.

[0026] Example 4 (1) Egg white pretreatment: Wash fresh eggs, separate the yolk and egg white, take the egg white liquid, shear it at high speed for 5 minutes, and then filter it with a filter to remove insoluble impurities; (2) Removal of ovomucin: The pH of the egg white solution treated in step (1) was adjusted to 4.5 with citric acid solution, and the mixture was stirred magnetically at 1000 rpm for 2 h, and then centrifuged at 4°C to remove the ovomucin precipitate to obtain a supernatant.

[0027] Example 5 (1) Egg white pretreatment: Wash fresh eggs, separate the yolk and egg white, take the egg white liquid, shear it at high speed for 5 minutes, and then filter it with a filter to remove insoluble impurities; (2) Removal of ovomucin: The pH of the egg white solution treated in step (1) was adjusted to 4.0 with citric acid solution, and the mixture was stirred magnetically at 1000 rpm for 2 h, and then centrifuged at 4°C to remove the ovomucin precipitate to obtain a supernatant.

[0028] Referring to Examples 1-5, observe the centrifugal stratification of egg white solution and the molecular weight of supernatant protein after different pH treatments distributed: 1) Appearance evaluation method: After centrifugation at 4°C and 5000 rpm for 10 min, take photos of the layers, and filter out the lower precipitate and the upper clear liquid and take photos for observation.

[0029] Results: Figure 1 As shown in Figure 2, the centrifugal separation of egg white solution after adjusting pH to 6-4 was quite different; the turbidity of the supernatant increased first and then decreased from pH = 6 to pH = 4, reaching the maximum turbidity at pH = 5.5 (see Figure 2 );also, Figure 3 The precipitation exhibited increased with decreasing pH value, which was due to the excessive precipitation of OVT and OVA.

[0030] 2) SDS-PAGE method: Electrophoresis was performed using a separation gel containing 12% sodium dodecyl sulfate (SDS) and a 5% stacking gel. The sample was prepared using a sample buffer (10 mM Tris-HCl, 10 wt% glycerol, 0.02 wt% bromophenol blue, 2 wt% SDS, 5% [v / v] β-mercaptoethanol, pH 8.0). Then, the mixed solution was boiled for 7 min and cooled. After electrophoresis, the sample was stained with Coomassie Brilliant Blue for 2 h and then destained.

[0031] Results: Figure 4 As shown, Figure 2 The obtained supernatant samples were run on electrophoresis, and it can be seen that as the pH decreases from 6 to 4, the color of the OVT and OVA bands becomes lighter, indicating that OVT and OVA are simultaneously lost and precipitated as ovomucin is separated. OVT and OVA at pH 6 and pH 5.5 can still be retained in the egg white solution, so in summary, pH 5.5 was selected as the most optimal pH for separating ovomucin.

[0032] Example 6 (1) Egg white pretreatment: Wash fresh eggs, separate the yolk and egg white, take the egg white liquid, shear it at high speed for 5 minutes, and then filter it with a filter to remove insoluble impurities; (2) Removing ovomucin: adjusting the pH of the egg white solution treated in step (1) to 5.5 with a citric acid solution, stirring with a magnetic stirrer at 1000 rpm for 2 h, and then centrifuging at 4°C to remove the ovomucin precipitate to obtain a supernatant; (3) Isolation of lysozyme: The supernatant obtained in step (2) was adjusted to pH 8.5 by cation exchange chromatography and passed into cation exchange resin 1 (D152 macroporous cation exchange resin, Shanghai Yuanye Biotechnology Co., Ltd.) for adsorption for 1 h. Lysozyme was then eluted with a 6% by mass NaCl solution for 45 min, and the permeate containing ovalbumin and ovotransferrin was collected.

[0033] Example 7 (1) Egg white pretreatment: Wash fresh eggs, separate the yolk and egg white, take the egg white liquid, shear it at high speed for 5 minutes, and then filter it with a filter to remove insoluble impurities; (2) Removing ovomucin: adjusting the pH of the egg white solution treated in step (1) to 5.5 with a citric acid solution, stirring with a magnetic stirrer at 1000 rpm for 2 h, and then centrifuging at 4°C to remove the ovomucin precipitate to obtain a supernatant; (3) Isolation of lysozyme: Using cation exchange chromatography, the supernatant obtained in step (2) was adjusted to pH 8.5 and passed into cation exchange resin 1 for adsorption for 2 h. Lysozyme was then eluted with a 6% by mass NaCl solution for 45 min, and the permeate containing ovalbumin and ovotransferrin was collected.

[0034] Example 8 (1) Egg white pretreatment: Wash fresh eggs, separate the yolk and egg white, take the egg white liquid, shear it at high speed for 5 minutes, and then filter it with a filter to remove insoluble impurities; (2) Removing ovomucin: adjusting the pH of the egg white solution treated in step (1) to 5.5 with a citric acid solution, stirring with a magnetic stirrer at 1000 rpm for 2 h, and then centrifuging at 4°C to remove the ovomucin precipitate to obtain a supernatant; (3) Isolation of lysozyme: Using cation exchange chromatography, the supernatant obtained in step (2) was adjusted to pH 8.5 and passed into cation exchange resin 1 for adsorption for 3 h. Lysozyme was then eluted with a 6% by mass NaCl solution for 45 min, and the permeate containing ovalbumin and ovotransferrin was collected.

[0035] Example 9 (1) Egg white pretreatment: Wash fresh eggs, separate the yolk and egg white, take the egg white liquid, shear it at high speed for 5 minutes, and then filter it with a filter to remove insoluble impurities; (2) Removing ovomucin: adjusting the pH of the egg white solution treated in step (1) to 5.5 with a citric acid solution, stirring with a magnetic stirrer at 1000 rpm for 2 h, and then centrifuging at 4°C to remove the ovomucin precipitate to obtain a supernatant; (3) Isolation of lysozyme: The supernatant obtained in step (2) was adjusted to pH 8.5 by cation exchange chromatography and passed into cation exchange resin 2 (SA780N cation exchange resin, Xi'an Lanxiao Technology New Materials Co., Ltd.) for adsorption for 1 h. Lysozyme was then eluted with a 6% by mass NaCl solution for 45 min, and the permeate containing ovalbumin and ovotransferrin was collected.

[0036] Example 10 (1) Egg white pretreatment: Wash fresh eggs, separate the yolk and egg white, take the egg white liquid, shear it at high speed for 5 minutes, and then filter it with a filter to remove insoluble impurities; (2) Removing ovomucin: adjusting the pH of the egg white solution treated in step (1) to 5.5 with a citric acid solution, stirring with a magnetic stirrer at 1000 rpm for 2 h, and then centrifuging at 4°C to remove the ovomucin precipitate to obtain a supernatant; (3) Isolation of lysozyme: Using cation exchange chromatography, the supernatant obtained in step (2) was adjusted to pH 8.5 and passed into cation exchange resin 2 for adsorption for 2 h. Lysozyme was then eluted with a 6% by mass NaCl solution for 45 min, and the permeate containing ovalbumin and ovotransferrin was collected.

[0037] Embodiment 11 (1) Egg white pretreatment: Wash fresh eggs, separate the yolk and egg white, take the egg white liquid, shear it at high speed for 5 minutes, and then filter it with a filter to remove insoluble impurities; (2) Removing ovomucin: adjusting the pH of the egg white solution treated in step (1) to 5.5 with a citric acid solution, stirring with a magnetic stirrer at 1000 rpm for 2 h, and then centrifuging at 4°C to remove the ovomucin precipitate to obtain a supernatant; (3) Isolation of lysozyme: Using cation exchange chromatography, the supernatant obtained in step (2) was adjusted to pH 8.5 and passed into cation exchange resin 2 for adsorption for 3 h. Lysozyme was then eluted with a 6% by mass NaCl solution for 45 min, and the permeate containing ovalbumin and ovotransferrin was collected.

[0038] Example 12 (1) Egg white pretreatment: Wash fresh eggs, separate the yolk and egg white, take the egg white liquid, shear it at high speed for 5 minutes, and then filter it with a filter to remove insoluble impurities; (2) Removing ovomucin: adjusting the pH of the egg white solution treated in step (1) to 5.5 with a citric acid solution, stirring with a magnetic stirrer at 1000 rpm for 2 h, and then centrifuging at 4°C to remove the ovomucin precipitate to obtain a supernatant; (3) Isolation of lysozyme: The supernatant obtained in step (2) was adjusted to pH 8.5 by cation exchange chromatography and passed into cation exchange resin 3 (ZGC180 cation exchange resin, Shenzhen Hongsen Environmental Protection Technology Co., Ltd.) for adsorption for 1 h. Lysozyme was then eluted with 6% NaCl solution for 45 min, and the permeate containing ovalbumin and ovotransferrin was collected.

[0039] Example 13 (1) Egg white pretreatment: Wash fresh eggs, separate the yolk and egg white, take the egg white liquid, shear it at high speed for 5 minutes, and then filter it with a filter to remove insoluble impurities; (2) Removing ovomucin: adjusting the pH of the egg white solution treated in step (1) to 5.5 with a citric acid solution, stirring with a magnetic stirrer at 1000 rpm for 2 h, and then centrifuging at 4°C to remove the ovomucin precipitate to obtain a supernatant; (3) Isolation of lysozyme: Using cation exchange chromatography, the supernatant obtained in step (2) was adjusted to pH 8.5 and passed into cation exchange resin 3 for adsorption for 2 h. Lysozyme was then eluted with a 6% by mass NaCl solution for 45 min, and the permeate containing ovalbumin and ovotransferrin was collected.

[0040] Embodiment 14 (1) Egg white pretreatment: Wash fresh eggs, separate the yolk and egg white, take the egg white liquid, shear it at high speed for 5 minutes, and then filter it with a filter to remove insoluble impurities; (2) Removing ovomucin: adjusting the pH of the egg white solution treated in step (1) to 5.5 with a citric acid solution, stirring with a magnetic stirrer at 1000 rpm for 2 h, and then centrifuging at 4°C to remove the ovomucin precipitate to obtain a supernatant; (3) Isolation of lysozyme: Using cation exchange chromatography, the supernatant obtained in step (2) was adjusted to pH 8.5 and passed into cation exchange resin 3 for adsorption for 3 h. Lysozyme was then eluted with a 6% by mass NaCl solution for 45 min, and the permeate containing ovalbumin and ovotransferrin was collected.

[0041] Referring to Examples 6-14, the effects of different cation exchange resins and different adsorption times on lysozyme in egg white were determined. Influence of adsorption effect: SDS-PAGE method: Electrophoresis was performed using a separating gel containing 12% sodium dodecyl sulfate (SDS) and a 5% stacking gel. The sample was prepared using a sample buffer (10 mM Tris-HCl, 10 wt% glycerol, 0.02 wt% bromophenol blue, 2 wt% SDS, 5% [v / v] β-mercaptoethanol, pH 8.0). Then, the mixed solution was boiled for 7 min and cooled. After electrophoresis, the sample was stained with Coomassie Brilliant Blue for 2 h and then destained.

[0042] Results: Figure 5 As shown, after 1 h of adsorption, the lysozyme bands in the penetration of cation exchange resin 1 (Example 6) and cation exchange resin 2 (Example 7) were lighter than those in cation exchange resin 3 (Example 8); after 2 h of adsorption, the lysozyme bands in the penetration of cation exchange resin 2 (Example 10) almost disappeared; and after 3 h of adsorption, the lysozyme bands in the penetration of all three resins (Examples 12-14) completely disappeared. Therefore, based on the comprehensive results, cation exchange resin 2 was selected as the most preferred resin for separating lysozyme.

[0043] Referring to Example 10, the purity of the obtained lysozyme was determined: Purity determination method: Electrophoresis was performed using a separation gel containing 12% sodium dodecyl sulfate (SDS) and a 5% stacking gel. The sample was prepared using a sample buffer (10 mM Tris-HCl, 10 wt% glycerol, 0.02 wt% bromophenol blue, 2 wt% SDS, 5% [v / v] β-mercaptoethanol, pH 8.0). Then, the mixed solution was boiled for 7 min and cooled. After electrophoresis, the sample was stained with Coomassie brilliant blue for 2 h and then destained. Image J was used to calculate the protein purity based on the grayscale of the electrophoresis bands.

[0044] Results: Figure 6 As shown in the figure, the lysozyme band at about 14 kDa is very obvious, and no obvious bands of other molecular weights appear, indicating that the obtained lysozyme has a high purity; in addition, according to Image J calculation, the purity of lysozyme is about 95.57%.

[0045] Embodiment 15 (1) Egg white pretreatment: Wash fresh eggs, separate the yolk and egg white, take the egg white liquid, shear it at high speed for 5 minutes, and then filter it with a filter to remove insoluble impurities; (2) Removing ovomucin: adjusting the pH of the egg white solution treated in step (1) to 5.5 with a citric acid solution, stirring with a magnetic stirrer at 1000 rpm for 2 h, and then centrifuging at 4°C to remove the ovomucin precipitate to obtain a supernatant; (3) Isolation of lysozyme: Using cation exchange chromatography, the supernatant obtained in step (2) was adjusted to pH 8.5 and passed into cation exchange resin 2 for adsorption for 2 h, and then eluted with 6% NaCl solution for 45 min to obtain lysozyme, and the permeate containing ovalbumin and ovotransferrin was collected; (4) Separation of ovotransferrin: Using anion exchange chromatography, the pH of the flowthrough obtained in step (3) was adjusted to 6.0 and passed into an anion exchange column Q Sepharose Fast Flow, adsorbed at an adsorption flow rate of 4 mL / min for 1 h, and then eluted with 6% NaCl solution at an elution flow rate of 8 mL / min to remove ovalbumin, and the flowthrough containing ovotransferrin was collected; (5) Concentration and drying of ovotransferrin: The permeate obtained in step (4) was filtered three times through a 50 kDa ultrafiltration membrane using a membrane concentration method at a pressure of 1.0 MPa and a flow rate of 10 Hz, and then vacuum freeze-concentrated to obtain high-purity and high-activity ovotransferrin.

[0046] Example 16 (1) Egg white pretreatment: Wash fresh eggs, separate the yolk and egg white, take the egg white liquid, shear it at high speed for 5 minutes, and then filter it with a filter to remove insoluble impurities; (2) Removing ovomucin: adjusting the pH of the egg white solution treated in step (1) to 5.5 with a citric acid solution, stirring with a magnetic stirrer at 1000 rpm for 2 h, and then centrifuging at 4°C to remove the ovomucin precipitate to obtain a supernatant; (3) Isolation of lysozyme: Using cation exchange chromatography, the supernatant obtained in step (2) was adjusted to pH 8.5 and passed into cation exchange resin 2 for adsorption for 2 h, and then eluted with 6% NaCl solution for 45 min to obtain lysozyme, and the permeate containing ovalbumin and ovotransferrin was collected; (4) Separation of ovotransferrin: Using anion exchange chromatography, the pH of the flowthrough obtained in step (3) was adjusted to 6.0 and passed into an anion exchange column Q Sepharose Fast Flow, adsorbed at an adsorption flow rate of 4 mL / min for 1.5 h, and then eluted with 6% NaCl solution at an elution flow rate of 8 mL / min to remove ovalbumin, and the flowthrough containing ovotransferrin was collected; (5) Concentration and drying of ovotransferrin: The permeate obtained in step (4) was filtered three times through a 50 kDa ultrafiltration membrane using a membrane concentration method at a pressure of 1.0 MPa and a flow rate of 10 Hz, and then vacuum freeze-concentrated to obtain high-purity and high-activity ovotransferrin.

[0047] Embodiment 17 (1) Egg white pretreatment: Wash fresh eggs, separate the yolk and egg white, take the egg white liquid, shear it at high speed for 5 minutes, and then filter it with a filter to remove insoluble impurities; (2) Removing ovomucin: adjusting the pH of the egg white solution treated in step (1) to 5.5 with a citric acid solution, stirring with a magnetic stirrer at 1000 rpm for 2 h, and then centrifuging at 4°C to remove the ovomucin precipitate to obtain a supernatant; (3) Isolation of lysozyme: Using cation exchange chromatography, the supernatant obtained in step (2) was adjusted to pH 8.5 and passed into cation exchange resin 2 for adsorption for 2 h, and then eluted with 6% NaCl solution for 45 min to obtain lysozyme, and the permeate containing ovalbumin and ovotransferrin was collected; (4) Separation of ovotransferrin: Using anion exchange chromatography, the pH of the flowthrough obtained in step (3) was adjusted to 6.0 and passed into an anion exchange column Q Sepharose Fast Flow, adsorbed at an adsorption flow rate of 4 mL / min for 2.0 h, and then eluted with 6% NaCl solution at an elution flow rate of 8 mL / min to remove ovalbumin, and the flowthrough containing ovotransferrin was collected; (5) Concentration and drying of ovotransferrin: The permeate obtained in step (4) was filtered three times through a 50 kDa ultrafiltration membrane using a membrane concentration method at a pressure of 1.0 MPa and a flow rate of 10 Hz, and then vacuum freeze-concentrated to obtain high-purity and high-activity ovotransferrin.

[0048] Embodiment 18 (1) Egg white pretreatment: Wash fresh eggs, separate the yolk and egg white, take the egg white liquid, shear it at high speed for 5 minutes, and then filter it with a filter to remove insoluble impurities; (2) Removing ovomucin: adjusting the pH of the egg white solution treated in step (1) to 5.5 with a citric acid solution, stirring with a magnetic stirrer at 1000 rpm for 2 h, and then centrifuging at 4°C to remove the ovomucin precipitate to obtain a supernatant; (3) Isolation of lysozyme: Using cation exchange chromatography, the supernatant obtained in step (2) was adjusted to pH 8.5 and passed into cation exchange resin 2 for adsorption for 2 h, and then eluted with 6% NaCl solution for 45 min to obtain lysozyme, and the permeate containing ovalbumin and ovotransferrin was collected; (4) Separation of ovotransferrin: Using anion exchange chromatography, the pH of the flowthrough obtained in step (3) was adjusted to 6.0 and passed into an anion exchange column Q Sepharose Fast Flow, adsorbed at an adsorption flow rate of 4 mL / min for 2.5 h, and then eluted with 6% NaCl solution at an elution flow rate of 8 mL / min to remove ovalbumin, and the flowthrough containing ovotransferrin was collected; (5) Concentration and drying of ovotransferrin: The permeate obtained in step (4) was filtered three times through a 50 kDa ultrafiltration membrane using a membrane concentration method at a pressure of 1.0 MPa and a flow rate of 10 Hz, and then vacuum freeze-concentrated to obtain high-purity and high-activity ovotransferrin.

[0049] Embodiment 19 (1) Egg white pretreatment: Wash fresh eggs, separate the yolk and egg white, take the egg white liquid, shear it at high speed for 5 minutes, and then filter it with a filter to remove insoluble impurities; (2) Removing ovomucin: adjusting the pH of the egg white solution treated in step (1) to 5.5 with a citric acid solution, stirring with a magnetic stirrer at 1000 rpm for 2 h, and then centrifuging at 4°C to remove the ovomucin precipitate to obtain a supernatant; (3) Isolation of lysozyme: Using cation exchange chromatography, the supernatant obtained in step (2) was adjusted to pH 8.5 and passed into cation exchange resin 2 for adsorption for 2 h, and then eluted with 6% NaCl solution for 45 min to obtain lysozyme, and the permeate containing ovalbumin and ovotransferrin was collected; (4) Separation of ovotransferrin: Using anion exchange chromatography, the pH of the flowthrough obtained in step (3) was adjusted to 6.0 and passed into an anion exchange column Q Sepharose Fast Flow, adsorbed at an adsorption flow rate of 4 mL / min for 3.0 h, and then eluted with 6% NaCl solution at an elution flow rate of 8 mL / min to remove ovalbumin, and the flowthrough containing ovotransferrin was collected; (5) Concentration and drying of ovotransferrin: The permeate obtained in step (4) was filtered three times through a 50 kDa ultrafiltration membrane using a membrane concentration method at a pressure of 1.0 MPa and a flow rate of 10 Hz, and then vacuum freeze-concentrated to obtain high-purity and high-activity ovotransferrin.

[0050] Referring to Examples 15-19, the effects of different adsorption times on the adsorption of ovalbumin in egg white on the adsorption efficiency of anion exchange columns were determined. The impact of the fruit: SDS-PAGE method: Electrophoresis was performed using a separating gel containing 10% sodium dodecyl sulfate (SDS) and a 5% stacking gel. The sample was prepared using a sample buffer (10 mM Tris-HCl, 10 wt% glycerol, 0.02 wt% bromophenol blue, 2 wt% SDS, 5% [v / v] β-mercaptoethanol, pH 8.0). Then, the mixed solution was boiled for 7 min and cooled. After electrophoresis, the sample was stained with Coomassie Brilliant Blue for 2 h and then destained.

[0051] Results: Figure 7 As shown in the figure, the band morphology of ovalbumin in the eluate after 1-3 h of adsorption is similar, so the adsorption effect of ovalbumin at different times is similar; however, according to Examples 15-16, the band of ovotransferrin is thicker after 1-1.5 h of adsorption, indicating that too short an adsorption time will cause ovotransferrin and ovalbumin to be adsorbed together, resulting in the loss of ovotransferrin. Therefore, in order to save time and reduce loss, 2 h (Example 17) is selected as the most preferred adsorption time for adsorbing ovalbumin.

[0052] Referring to Example 17, the purity of the obtained ovotransferrin was determined, the scanning electron micrographs, and the results of the experiments under different pH conditions. Functional properties and structural characterization of: 1) Purity determination method: Electrophoresis was performed using a separation gel containing 12% sodium dodecyl sulfate (SDS) and a 5% stacking gel. The sample was prepared using a sample buffer (10 mM Tris-HCl, 10 wt% glycerol, 0.02 wt% bromophenol blue, 2 wt% SDS, 5% [v / v] β-mercaptoethanol, pH 8.0). Then, the mixed solution was boiled for 7 min and cooled. After electrophoresis, the sample was stained with Coomassie Brilliant Blue for 2 h and then destained. Image J was used to calculate the protein purity based on the grayscale of the electrophoresis bands.

[0053] Results: Figure 8 As shown, the electrophoresis diagram showed that ovotransferrin appeared at 77-80 kDa, and no other obvious molecular weight bands appeared; according to Image J, the purity of ovotransferrin was calculated by the grayscale of the electrophoresis bands to be about 97.89%, indicating that this method can extract high-purity (>95%) protein.

[0054] 2) Scanning electron microscopy method: The microstructure of ovotransferrin was observed using a scanning electron microscope. First, the sample was glued to the sample stage of the scanning electron microscope, and then gold was sprayed on the surface of the material using a gold sprayer.

[0055] Results: Fig. 9 As shown, according to the scanning electron micrographs at three magnification ratios, we found that ovotransferrin was in the form of tiny particles with relatively uniform particle size and wrinkled surface.

[0056] 3) Solubility determination method: Use 0.5 M NaOH and 0.5 M hydrochloric acid to adjust the pH of the protein sample solution (10 mg / mL) to 3, 5, 7, and 9, stir the solution at 25°C to mix thoroughly, then centrifuge at 5000 × g for 15 min, take the supernatant and measure the protein concentration using the Coomassie Brilliant Blue method. Calculate the solubility according to the formula: Solubility (%) = (measured protein concentration / original protein concentration) × 100%.

[0057] Results: As shown in Table 1, as the solution system environment increases from acidic pH 3 to alkaline pH 9, the solubility of ovotransferrin first decreases and then increases, reaching the lowest around pH 5 and pH 7 (isoelectric point around 6-6.5).

[0058] Table 1 Solubility, antioxidant activity (DPPH free radical scavenging rate) and antibacterial rate of ovotransferrin under different pH environments

[0059] 4) Antioxidant activity determination method: 2 mL of sample solution was added with 2 mL of DPPH ethanol solution (0.1 mM), vortexed and immediately placed in the dark for incubation for 30 min, then centrifuged and the absorbance was measured at 517 nm. Anhydrous ethanol was used as a blank control, and the scavenging rate of DPPH free radicals was calculated according to the following formula: DPPH free radical scavenging rate (%) = (1-A / A 0 )×100%, where A is the absorbance of the sample at 517 nm. 0 is the absorbance of the blank sample at 517 nm.

[0060] Results: As shown in Table 1, with the increase of pH, the DPPH free radical scavenging rate decreased from 81.96% to 20.64%, indicating that ovotransferrin can exert a greater antioxidant effect in an acidic environment.

[0061] 5) Method for determination of antibacterial rate: Sterile MH broth medium was added to each sample well in a 96-well plate, and then ovotransferrin was diluted twice under different pH conditions and added to the sample wells to obtain sample wells with different concentrations of antibacterial agent. Diluted bacterial suspension (fluorescing Pseudomonas) was added to each sample well, and 1% red tetrazolium was added after incubation and continued to incubate for 30 min. The absorbance value of each well at 600 nm was measured using an ELISA instrument, and the antibacterial rate was obtained by comparing the bacterial turbidity in the antibacterial wells with the negative / positive blank wells of different concentrations. The culture solution of the wells with the same antibacterial concentration was applied to the solid culture medium, and the bacterial growth was observed after constant temperature incubation.

[0062] Results: As shown in Table 1, the inhibition rate of 1 mg / mL ovotransferrin against Pseudomonas fluorescens was 62.83-99.87%. As the pH gradually increased, the inhibition rate showed a trend of first decreasing and then increasing, and the inhibition rate reached the lowest in an environment of pH 7. Most microorganisms could not grow in an environment of pH = 3, while the inhibition rate of ovotransferrin reached 88.49% under pH = 5, indicating that ovotransferrin can exert the greatest antibacterial effect in a weakly acidic environment.

[0063] The principle of red tetrazolium staining bacteria is based on the fact that the dehydrogenase produced during bacterial growth reduces red tetrazolium to a red formazan compound, thereby indicating the vitality or presence of bacteria. Fig.10As shown in the figure, the inhibitory effect of ovotransferrin on Pseudomonas fluorescens at different concentrations and different pH values ​​(3-9) was determined. According to the red tetrazolium staining results, no bacteria grew in the antibacterial wells at pH=3, while the antibacterial wells at pH=5 began to turn pink when the ovotransferrin concentration was less than 0.5 mg / mL, indicating that the minimum inhibitory concentration was 0.5 mg / mL; at pH=7, all antibacterial wells were pink, and the pink color became darker as the inhibitory concentration decreased from 1 mg / mL, indicating that ovotransferrin had only partial antibacterial effect but no bactericidal effect under this environmental condition; and the color change of the antibacterial wells at pH=9 was similar to that at pH=5, but the pink color of the antibacterial wells was darker.

[0064] Plate the 1 mg / mL inhibition wells at different pH values, such as Fig.11 As shown, no bacteria grew at pH=3, the number of bacteria on the plate at pH=5 was within the countable range (30-300), the number of bacteria at pH=7 was too many to be counted, and the number of bacteria at pH=9 was between pH=5 and pH=9.

[0065] In summary, pH 3-5 is the optimal antibacterial pH for ovotransferrin.

[0066] 6) UV spectrometry method: UV spectrophotometer was used to collect UV absorption spectra of ovotransferrin at different pH values. The protein sample was dissolved in PBS solution to a concentration of 1 mg / mL, the measurement speed was medium, the resolution was 0.5 nm, and the measurement wavelength range was 240-400 nm.

[0067] Results: Fig.12 As shown in the figure, with the increase of environmental pH, the ultraviolet maximum absorption wavelength of ovotransferrin gradually red-shifted from 284.2 nm to 286.4 nm, indicating that the amino acid environment shifted to a more hydrophobic environment. This is because the conformation of the protein system changed, and the alkaline environment made the protein structure more compact.

[0068] 7) Fluorescence spectroscopy method: The fluorescence emission spectra of OVA and SOVA were measured using a fluorescence spectrophotometer. The sample was diluted to 1 mg / mL with PBS, the excitation wavelength was set to 290 nm, the emission wavelength scanning range was 300-450 nm, the excitation and emission slit widths were both 10 nm, and PBS was used as a blank control.

[0069] Results: The changes in the tertiary structure of proteins were evaluated based on the intrinsic fluorescence emission spectrum reflecting the polarity changes of the tryptophan microenvironment. Fig.10As shown in the figure, as the environmental pH increases from 3 to 9, the maximum emission wavelength blue-shifts from 336.2 nm to 329.8 nm. As the pH increases from 5 to 9, the maximum fluorescence intensity of the protein gradually decreases from 6867.3 to 5373.0, which is due to the tryptophan residues deeply buried in the protein and the hydrophobic interactions within the protein molecules.

[0070] In summary, the present invention uses a cation exchange resin in combination with an anion exchange filler to simultaneously separate and prepare lysozyme and ovotransferrin, and the obtained lysozyme and ovotransferrin are both high-purity proteins (purity>95%); in addition, ovotransferrin exhibits a higher solubility in an alkaline environment and exhibits better antioxidant and antibacterial properties in an acidic environment.

[0071] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for preparing high-purity lysozyme and ovotransferrin by combined ion exchange chromatography, characterized in that: The following steps are involved: Step 1: Egg white pretreatment: Wash fresh eggs, separate the yolk and egg white, take the egg white liquid, and filter to remove insoluble impurities after high-speed shearing; Step 2: Remove ovomucin: The egg white solution treated in step 1 is adjusted to pH with a citric acid solution, stirred, and centrifuged at 4°C to remove ovomucin precipitate to obtain a refined egg white solution; Step 3, separation of lysozyme: The purified egg white solution is adjusted to pH and passed through a cation exchange resin by cation exchange chromatography, and then lysozyme is obtained by elution with a NaCl solution, and the permeate containing ovalbumin and ovotransferrin is collected; Step 4: Isolation of ovotransferrin: Using anion exchange chromatography, adjusting the pH of the flow-through obtained in step 3 and passing it into an anion exchange column, then eluting with a NaCl solution to remove the ovalbumin, and collecting the flow-through containing ovotransferrin; Step 5, concentration and drying of lysozyme and ovotransferrin: The permeate obtained in step 4 is filtered through an ultrafiltration membrane using a membrane concentration method, and then vacuum frozen and concentrated to obtain high-purity and high-activity ovotransferrin and lysozyme.

2. The preparation method according to claim 1, characterized in that: In step 2, the pH of the egg white solution treated in step 1 is adjusted to 4-6 with a citric acid solution; the stirring speed is 500-2000 rpm, and the stirring time is 1-3 h.

3. The preparation method according to claim 1, characterized in that: In step 3, the adsorption time of the refined egg white solution on the cation exchange resin is 1-3 h, the adsorption pH is 7.5-9.0, the mass fraction of the NaCl solution is 3-9%, and the elution time is 30-60 min.

4. The preparation method according to claim 1, characterized in that: In step 4, the penetration time of the flowthrough containing ovalbumin and ovotransferrin in the anion exchange column is 1-3 h, the adsorption pH is 5.0-6.0, the adsorption flow rate is 1-5 mL / min, the mass fraction of the NaCl solution is 2-8%, and the elution flow rate is 4-8 mL / min.

5. The preparation method according to claim 1, characterized in that: In step 5, the pore size of the ultrafiltration membrane is 20-50 kDa, the pressure during ultrafiltration is 0.5-1.0 MPa, the flow rate is 10-20 Hz, and the ultrafiltration times are 1-3 times.

6. A high-purity ovotransferrin, characterized in that: The invention is prepared by the preparation method according to any one of claims 1 to 5.

7. Use of the high-purity ovotransferrin as claimed in claim 6 in the field of functional food and food preservation.

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