阅读说明：本技术 从鸡蛋清中高效提取卵类黏蛋白的方法 (Method for efficiently extracting ovomucoid from egg white ) 是由 付星 黄茜 梁怡鑫 黄孝懿 蔡朝霞 金永国 靳国峰 盛龙 邱宁 马美湖 于 2019-08-01 设计创作，主要内容包括：本发明涉及一种从鸡蛋清中高效提取卵类黏蛋白的方法,该方法包括鸡蛋清溶液的样品制备、利用乙醇预处理蛋清溶液除杂蛋白、微波辅助硫酸铵第一步沉淀、微波辅助硫酸铵第二步沉淀四个步骤；所述的卵类黏蛋白提取工艺是采用微波功率140W、微波时间20s的参数来辅助硫酸铵沉淀过程。本发明解决了卵类黏蛋白分离纯化操作复杂、周期长、纯度及得率低等问题,利用微波技术诱导蛋白分子极化,及促进传质的效应,建立了一种新兴、简单、快速和高效的卵类黏蛋白提取方法,该方法大大缩短了普通盐析过程中的静置时间,简化操作过程,降低提取成本,提高分离效率,适用于大范围生产,为微波技术在禽蛋类蛋白提纯中的应用提供支持。(The invention relates to a method for efficiently extracting ovomucoid from egg white, which comprises four steps of sample preparation of an egg white solution, pretreatment of the egg white solution by using ethanol to remove impurity proteins, microwave-assisted first-step precipitation of ammonium sulfate and microwave-assisted second-step precipitation of ammonium sulfate; the ovomucoid extraction process is assisted by ammonium sulfate precipitation process by adopting parameters of microwave power of 140W and microwave time of 20 s. The invention solves the problems of complex operation, long period, low purity and yield of separation and purification of ovomucoid, utilizes the microwave technology to induce protein molecule polarization and promote mass transfer effect, establishes a novel, simple, rapid and efficient ovomucoid extraction method, greatly shortens standing time in the common salting-out process, simplifies the operation process, reduces the extraction cost, improves the separation efficiency, is suitable for large-scale production, and provides support for the application of the microwave technology in purification of avian egg proteins.)

1. A method for efficiently extracting ovomucoid from egg white is characterized by comprising the following steps: the method comprises the following steps:

(1) Sample preparation

Breaking fresh eggs, separating egg white, adding water with the same volume to the egg white, and homogenizing for 15-30min to obtain egg white solution;

(2) Pretreatment of ethanol solution

Slowly adding anhydrous ethanol into egg white solution to make the final concentration of ethanol in the solution reach 20-25%, mixing uniformly to obtain mixed solution, adjusting the pH value of the mixed solution to 4.5-5, magnetically stirring for 15-30min, centrifuging, and ultrafiltering and freeze-drying the obtained supernatant to obtain crude ovomucoid powder;

(3) Microwave-assisted first-step precipitation of ammonium sulfate

adding distilled water with one volume into crude ovomucoid powder to obtain crude protein solution, adding ammonium sulfate until the saturation of ammonium sulfate reaches 30-70%, placing in a microwave oven, extracting under sealed condition of microwave power of 0-280W for 0-40s to obtain extractive solution, and centrifuging to obtain supernatant;

(4) Microwave-assisted second-step precipitation of ammonium sulfate

And continuously adding ammonium sulfate into the supernatant until the saturation of the ammonium sulfate reaches 70-90%, placing the obtained solution in a microwave oven, extracting for 0-40s under the sealing condition of the microwave power of 0-280W, centrifuging, removing the supernatant, taking the precipitate, re-dissolving the precipitate in water, dialyzing, and freeze-drying to prepare the ovomucoid, wherein the extraction rate of the ovomucoid is 63.1-72.7%, and the purity of the ovomucoid is more than 95%.

2. The method for efficiently extracting ovomucoid from egg white according to claim 1, wherein the method comprises the following steps: the centrifugation conditions were: centrifuging at 4 deg.C and 5000g for 10 min.

3. The method for efficiently extracting ovomucoid from egg white according to claim 1, wherein the method comprises the following steps: in the step 3), the saturation degree of ammonium sulfate reaches 50%.

4. The method for efficiently extracting ovomucoid from egg white according to claim 3, wherein the method comprises the following steps: in the step 4), the saturation degree of the ammonium sulfate is 80%.

5. The method for efficiently extracting ovomucoid from egg white according to claim 4, wherein the method comprises the following steps: in the steps 3) and 4), microwave extraction parameters are as follows: the power was 140W for 20 s.

6. the method for efficiently extracting ovomucoid from egg white according to claim 5, wherein the method comprises the following steps: the extraction rate of the ovomucoid is 72.7%, and the purity is more than 95%.

Technical Field

The invention relates to a high-efficiency extraction method of ovomucoid, in particular to a method for efficiently extracting ovomucoid from egg white.

Background

The egg white has wide source and low price, and the protein content in dry matter exceeds 80 percent, which is one of the most ideal protein resources. Ovomucoid (ovmou, abbreviated as OVM) accounts for about 11% of the total amount of egg white protein. The protein has compact structure, heat resistance, acid and alkali resistance, trypsin digestion resistance and other characteristics, and can cause immune reaction. The ovomucoid has important immunological effects of inhibiting tumor, resisting cancer and the like, can be used as a marker in food detection, and can also be used for predicting the occurrence of egg allergy in children. Therefore, the purification to obtain the ovomucoid has very important significance for the fields of food and medicine.

although there are many methods for protein isolation and purification, no simple, economical and widely applicable method has been found to extract ovomucoid from complex egg components. In the early days, ovomucoid was extracted by organic solvent precipitation, such as trichloroacetic acid and acetone, but organic solvents resulted in varying degrees of denaturation of ovomucoid. At present, most of the methods for separating and purifying ovomucoid include aqueous two-phase extraction, electrophoresis, chromatography, precipitation and the like. The double aqueous phase extraction method and the electrophoresis method have small treatment capacity and no practical application value; the purity of the target protein obtained by the chromatography is high, but the treatment amount is small, the cost is high, and the technology is very complicated; at present, most of precipitation technologies for separating and purifying ovomucoid are precipitation by using a salting-out method, the method is simple and easy to operate, but the problems exist in that the precipitation method needs longer standing time and extremely low efficiency, and the purity of the ovomucoid obtained by separation is generally lower than 90%. In foreign studies, in 2009, Anupong Tankrathok purified ovomucoid using a combination of two-step ion exchange chromatography and precipitation (Tankrathok A, Daduang S, Patramanon R, et al purification processes for the Preparation and characterization of Hen Egg White Ovalbumin, Lysozyme, Ovotransferin, and Ovomucoid [ J ]. PREPARATIVE BIOCHEMISTRY & BITECHNOLOGY, 2009,39(4):380-399.), but the recovery efficiency was low (purity 80%, recovery 21%) and only applicable to laboratory-level preparations. The acetone precipitation method used by xiaxia in 2012 (xiaxia egg white ovomucoid separation and purification, structural characterization and research on its allergenicity [ D ] university of agriculture in china, 2012) extracted ovomucoid with a purity of 95.3%, but it was purified again by using cellulose ion exchange after precipitation treatment with cold acetone, which was complicated in steps, long in time, and only recovered to 23.8%. E.d.n.s.aberrathne isolated ovomucoid from ethanol extraction supernatant in 2014 using high concentration ethanol in combination with citric acid (aberrathne E D N S, Lee H Y, Ahn D u.separation of ovomucor and ovomucoid from chicken egg white [ J ] Poult Sci 2014,93(4):1010-1017.) although the protein recovery rate reached 95%, the process was cumbersome, multiple steps required standing overnight, the waiting time was too long, not conducive to large scale preparation; in domestic research, in 2014, in king commander (Wuzi Jian, Queensu Ying, etc., chicken egg mucoprotein [ J ] is purified by a three-step precipitation method, food science 2014,35(24): 74-80), the chicken egg mucoprotein is purified by a three-step salting-out precipitation method, so that high purity can be obtained, but the extraction rate is only 27.05%, and the salting-out reaction takes longer time (about 12 hours) in the process. Therefore, the development of a separation and purification method of ovomucoid with simple operation, short time consumption, high purity and high recovery rate has important application value.

The microwave is different from the traditional heating mode, the microwave is heated inside and outside simultaneously through two modes of dipole rotation and ion conduction, and the temperature gradient is avoided, so that the temperature rise is rapid and uniform, the reaction time is greatly shortened, and the reaction rate is improved. The application of microwave in biotechnology is a relatively new research field. The microwave-assisted salting-out effect is utilized to promote the mass transfer process, accelerate the position replacement of electrolyte ions and hydration layers around the protein, and quickly replace water molecules by the ions, so that the hydration layers of the protein are replaced by the electrolyte ions, and part of charges carried on the surface of the protein are neutralized. As the microwave field induces protein molecules to generate a polarization phenomenon, non-covalent bonds (hydrophobic interaction, disulfide bond and electrostatic interaction) maintaining the spatial structure of the protein are destroyed, the protein molecules are partially unfolded, the flexibility of the molecules is improved, more protein molecules are dissolved in a water phase, and the solubility of the protein is enhanced; when the microwave treatment time is further prolonged, polarized protein molecules interact with each other, and molecular aggregates are formed again through hydrophobic interaction, disulfide bonds, electrostatic interaction, hydrogen bonds and the like, so that the flexibility of the molecules is reduced, and the solubility of the protein with weaker stability is reduced, and precipitation is generated. Therefore, the salting-out system after the microwave action does not need to be kept still, and the salting-out separation speed of the protein is greatly improved. In patent CN101962403A (Nianxian' e, Lirijun, Huang Yongchun, etc., a method for extracting silkworm pupa protein by using microwave-assisted salt solution, CN101962403A [ P ] 2011) uses microwave-assisted separation of silkworm pupa protein, and proves that microwave can effectively accelerate extraction efficiency.

however, no reports about the application of microwave technology to separation and purification of poultry egg proteins exist at present.

disclosure of Invention

The invention aims to overcome the defects of long time consumption, complex steps and the like of a chromatography or electrophoresis method in a purification process, and provides a simple, quick and efficient method for extracting ovomucoid from egg white.

in order to achieve the purpose, the invention designs a method for efficiently extracting ovomucoid from egg white, which comprises the following steps:

(1) Sample preparation

breaking fresh eggs, separating egg white, adding water with the same volume to the egg white, and homogenizing for 15-30min to obtain egg white solution;

(2) pretreatment of ethanol solution

Slowly adding anhydrous ethanol into the egg white solution to enable the final concentration of the ethanol in the solution to reach 20-25% (v/v), uniformly mixing to obtain a mixed solution, adjusting the pH value of the mixed solution to 4.5-5, magnetically stirring for 15-30min, centrifuging, and performing ultrafiltration and freeze-drying on the obtained supernatant to obtain crude ovomucoid powder;

(3) Microwave-assisted first-step precipitation of ammonium sulfate

Adding distilled water with one volume into crude ovomucoid powder to obtain crude protein solution, adding ammonium sulfate until the saturation of ammonium sulfate reaches 30-70%, placing in a microwave oven, extracting under sealed condition of microwave power of 0-280W for 0-40s to obtain extractive solution, and centrifuging to obtain supernatant;

(4) microwave-assisted second-step precipitation of ammonium sulfate

And continuously adding ammonium sulfate into the supernatant until the saturation of the ammonium sulfate reaches 70-90%, placing the obtained solution in a microwave oven, extracting for 0-40s under the sealed condition of the microwave power of 0-280W, centrifuging, removing the supernatant, taking the precipitate, re-dissolving the precipitate in water, dialyzing, and freeze-drying to prepare the ovomucoid, wherein the extraction rate of the ovomucoid is 63.1-72.7%, and the purity of the ovomucoid is more than 95% (electrophoresis grade purity).

further, the centrifugation conditions are: centrifuging at 4 deg.C and 5000g for 10 min.

Still further, in the step 3), the saturation degree of ammonium sulfate reaches 50%.

Still further, in the step 4), the saturation degree of ammonium sulfate is 80%.

Still further, in the steps 3) and 4), microwave extraction parameters are as follows: the power was 140W for 20 s.

Still further, the extraction rate of the ovomucoid is 72.7%, and the purity is more than 95% (electrophoresis grade purity).

The invention has the beneficial effects that:

(1) By using a novel microwave technology and utilizing the advantage of promoting the mass transfer process, the non-covalent bond of the space structure of the protein is accelerated to be destroyed, the solubility of the protein is greatly reduced, the standing time is shortened on the basis of the traditional salting-out precipitation method, the purification efficiency is improved, and the aim of quickly and efficiently extracting the protein is fulfilled;

(2) The influence of different parameters on the experimental result is considered, and the scheme for extracting the protein by microwave is optimized. Through analysis, the optimal process conditions for extracting the ovomucoid in the egg white by microwave-assisted salting-out are microwave power 140W and microwave time 20s, and the protein extraction rate is 72.7 percent obtained by three times of parallel experiments. The method has the advantages of thorough extraction of protein in egg white, high yield, high purity, simple operation, environmental protection and certain reference value for improving the added value of egg products.

Compared with the prior method, the method saves the reaction time of 12h, has obvious experimental effect and can be used for large-scale preparation of the OVM protein. In conclusion, the microwave-assisted salting-out technology provided by the invention is economic and environment-friendly, and has feasibility, innovation and good application prospect in the field of protein purification.

Drawings

FIG. 1 is a flow chart of the separation and purification technique of ovomucoid;

FIG. 2 is the effect of different microwave powers on OVM protein extraction rate;

FIG. 3 is the effect of different microwave times on the extraction rate of OVM protein;

FIG. 4 is a graph of the effect of different combinations of ammonium sulfate saturation on OVM protein extraction in a two-step precipitation process;

FIG. 5 is a SDS-PAGE gel electrophoresis of samples extracted at each step;

Detailed Description

The present invention is described in further detail below with reference to specific examples so as to be understood by those skilled in the art.

The method for efficiently extracting ovomucoid from egg white comprises the following steps:

(1) Sample preparation

Breaking fresh eggs, separating egg white, adding water with the same volume to the egg white, and homogenizing for 15-30min to obtain egg white solution;

(2) Pretreatment of ethanol solution

Slowly adding anhydrous ethanol into the egg white solution to enable the final concentration of the ethanol in the solution to reach 20-25% (v/v), uniformly mixing to obtain a mixed solution, adjusting the pH value of the mixed solution to 4.5-5, magnetically stirring for 15-30min, centrifuging, and performing ultrafiltration and freeze-drying on the obtained supernatant to obtain crude ovomucoid powder;

(3) Microwave-assisted first-step precipitation of ammonium sulfate

Adding distilled water with one volume into crude ovomucoid powder to obtain crude protein solution, adding ammonium sulfate until the saturation of ammonium sulfate reaches 30-70%, placing in a microwave oven, extracting under sealed condition of microwave power of 0-280W for 0-40s to obtain extractive solution, and centrifuging to obtain supernatant;

(4) Microwave-assisted second-step precipitation of ammonium sulfate

Adding ammonium sulfate into the supernatant until the saturation of ammonium sulfate reaches 70-90%, placing the obtained solution in a microwave oven, extracting under sealed condition of microwave power of 0-280W for 0-40s, centrifuging, removing supernatant, collecting precipitate, dissolving the precipitate in water, dialyzing, and lyophilizing to obtain ovomucoid.

The preparation method has the following theoretical basis of parameter selection:

1. extraction rate of ovomucoid OVM under different microwave power

using the same preparation method steps, when the microwave time is set to 20s, ovomucoid OVM is extracted under different microwave powers (0, 70, 140, 210, 280W), and the extraction rate of ovomucoid OVM under different microwave powers is calculated as follows:

as shown in fig. 2: the protein extraction rate is highest when the microwave power is 140W;

2. extraction rate of ovomucoid OVM under different microwave time

Using the same preparation method steps as above, when the microwave power is set to 140W, ovomucoid OVM is extracted at different microwave times (0, 10, 20, 30, 40s), and the extraction rate of ovomucoid OVM at different microwave powers is calculated by the above method:

as shown in fig. 3: the protein extraction rate is highest when the microwave time is 20 s.

3. extraction rate of ovomucoid OVM under different ammonium sulfate saturation combinations

according to the steps of the same preparation method, when the optimal parameters of microwave power and time are selected, the saturation degrees (30, 40, 50, 60 and 70 percent) of ammonium sulfate precipitated in the first step and the saturation degrees (70, 75, 80, 85 and 90 percent) of ammonium sulfate precipitated in the second step are adjusted to extract ovomucoid OVM, and the extraction rates of the ovomucoid OVM under different combinations of the saturation degrees of the ammonium sulfate are calculated by the method:

Combination A: the first step of ammonium sulfate saturation is 30 percent and the second step of ammonium sulfate saturation is 70 percent

combination B: the first step of ammonium sulfate saturation is 40% and the second step of ammonium sulfate saturation is 75%

And (3) combination C: the first step of ammonium sulfate saturation is 50% and the second step of ammonium sulfate saturation is 80%

combination D: the saturation of the first step of ammonium sulfate is 60 percent and the saturation of the second step of ammonium sulfate is 85 percent

Combination E: the first step of ammonium sulfate saturation is 70 percent and the second step of ammonium sulfate saturation is 90 percent

As shown in fig. 4: and the combination C, namely the saturation of the ammonium sulfate precipitated in the first step is 50 percent, and the saturation of the ammonium sulfate precipitated in the second step is 80 percent, the protein extraction rate is highest.

4. SDS-PAGE detection of samples obtained in four of the above steps

Egg white is obtained by the four steps of the method in sequence, a large amount of foreign proteins are removed from an egg white solution and an ethanol solution, the first step of precipitation is carried out on a 50% ammonium sulfate solution, and the second step of precipitation is carried out on an 80% ammonium sulfate solution to obtain protein supernatant or precipitate; the four products were examined by SDS-PAGE:

SDS-PAGE protein sample conditions:

separating gel with mass fraction of 12%, concentrating gel with mass fraction of 5%, loading 10 μ L, performing 80V constant voltage electrophoresis for 0.5h, and performing 120V constant voltage electrophoresis for 1h

b. As a result:

After electrophoresis is finished, fixing for 0.5h by using a fixing solution, dyeing for 0.5h by using a dyeing solution, and taking a picture after the decoloring solution is eluted for multiple times; comparative display of electrophoretic bands: the extraction process of the invention can obtain relatively pure ovomucoid.