阅读说明：本技术 一种制备卵黄免疫球蛋白的方法 (Method for preparing yolk immunoglobulin ) 是由 顾璐萍 王旭婷 杨严俊 苏宇杰 常翠华 李俊华 于 2020-06-05 设计创作，主要内容包括：本发明公开了一种制备卵黄免疫球蛋白的方法,属于食品技术领域。所述方法是先采用超声结合冷冻对蛋黄进行预处理,再结合絮凝剂和超滤浓缩进行纯化得到卵黄免疫球蛋白。本发明先对蛋黄进行超声处理,破坏卵黄球体结构,再对其进行冷冻处理,使得脂蛋白和油脂形成凝胶,这有利于水溶性部分与非水溶性部分实现快速分离,进一步采用低倍数的水进行稀释,使得免疫球蛋白溶解在水溶液中,再进行后续的纯化工序,该工艺具有处理量小、耗时短、杂质少、絮凝剂用量少、产物得率和纯度较高等优点,最终免疫球蛋白的得率为1.0％～4.0％,纯度为30％～70％；同时,大幅度降低后续纯化工序的负荷,提高膜的使用效率,适合工业化大规模生产。(The invention discloses a method for preparing yolk immunoglobulin, belonging to the technical field of food. The method comprises the steps of firstly adopting ultrasound and freezing to pretreat egg yolk, and then combining flocculating agent and ultrafiltration concentration to purify to obtain egg yolk immunoglobulin. The invention firstly carries out ultrasonic treatment on the yolk to destroy the yolk sphere structure, and then carries out freezing treatment on the yolk to lead lipoprotein and grease to form gel, which is beneficial to realizing rapid separation of a water-soluble part and a non-water-soluble part, and further adopts low-multiple water to dilute so as to lead the immunoglobulin to be dissolved in a water solution, and then carries out subsequent purification procedures, the process has the advantages of small treatment amount, short time consumption, less impurities, less flocculant consumption, higher product yield and purity and the like, and finally the yield of the immunoglobulin is 1.0-4.0 percent, and the purity is 30-70 percent; meanwhile, the load of the subsequent purification process is greatly reduced, the use efficiency of the membrane is improved, and the method is suitable for industrial large-scale production.)

1. A method for preparing yolk immunoglobulin is characterized in that yolk is pretreated by ultrasonic and freezing, and then purified by flocculating agent and ultrafiltration concentration to obtain the yolk immunoglobulin.

2. The method of claim 1, wherein the ultrasonic pre-treatment conditions are: the ultrasonic power is 40-600W, and the ultrasonic time is 2-30 min.

3. The method according to claim 1 or 2, wherein the freezing pretreatment is to freeze the egg yolk at-20 to-15 ℃ for 12 to 24 hours, and then to unfreeze the egg yolk at 0 to 8 ℃ for 12 to 24 hours after being taken out.

4. A method according to any of claims 1-3, characterized in that the method comprises the steps of:

(1) raw material treatment: washing fresh egg white, sterilizing, separating egg white and yolk, and stirring yolk uniformly;

(2) ultrasonic treatment: carrying out ultrasonic treatment on the yolk;

(3) freezing and thawing yolk: freezing the yolk obtained in the step (1) and then unfreezing;

(4) fat removal: adding water into the yolk gel obtained in the step (2), adjusting the pH value by using a food acidity regulator, stirring, standing for precipitation, and centrifuging to obtain an immunoglobulin extracting solution;

(5) flocculation purification: adding a flocculating agent into the immunoglobulin supernatant in the step (3), and centrifuging or filtering by a plate frame to obtain a high-purity immunoglobulin extracting solution;

(6) and (3) ultrafiltration concentration and purification: separating and purifying the immunoglobulin extracting solution obtained in the step (4) by using a low-temperature ultrafiltration membrane to obtain a concentrated extracting solution;

(7) and (3) microfiltration sterilization: sterilizing the concentrated solution obtained in the step (5) by a low-temperature microfiltration membrane;

(8) and (3) freeze drying: and (4) freeze-drying the microfiltration permeating liquid obtained in the step (6) to obtain the product, namely the egg yolk immunoglobulin.

5. The method according to claim 4, wherein the mass ratio of the egg yolk to the water in the step (4) is 1: 4-1: 9, the pH is 4.0-7.0, the food acidity regulator can be acetic acid, lactic acid, citric acid, tartaric acid, sodium carbonate, sodium citrate, potassium citrate and the like, the stirring speed is 600-1000 rpm, the stirring time is 10-30 min, the centrifugation is carried out at 3000-10000 rpm for 3-15 min, and the precipitation is carried out for 1-5 h.

6. The method according to claim 4 or 5, wherein the flocculating agent in the step (5) is one or more selected from sodium alginate, xanthan gum, chitosan, pectin, carrageenan, konjac gum, guar gum and sodium carboxymethylcellulose, the concentration is 0.2-10%, and the addition amount is 0-0.8% of the volume of the supernatant.

7. The method according to any one of claims 4 to 6, wherein the ultrafiltration temperature in step (6) is 4 to 10 ℃ and the molecular weight cut-off is 20000 to 150000.

8. A yolk immunoglobulin prepared by the method of any one of claims 1 to 7.

9. Use of the yolk immunoglobulin of claim 8 for the preparation of a medicament, food, health product and daily product.

10. A pretreatment method for extracting yolk immunoglobulin is characterized in that the method adopts ultrasonic and freezing to pretreat yolk; the ultrasonic power is 40-600W, and the ultrasonic time is 2-30 min; the freezing pretreatment is to freeze the yolk at a temperature of between 20 ℃ below zero and 15 ℃ below zero for 12 to 24 hours, and unfreeze the yolk at a temperature of between 0 and 8 ℃ for 12 to 24 hours after the yolk is taken out.

Technical Field

The invention relates to a method for preparing yolk immunoglobulin, belonging to the technical field of food.

Background

Yolk immunoglobulin (IgY) refers to the antibody protein with specific activity produced by chicken group through passive immunization. The IgY has the characteristics of high purity, strong specificity, high safety, strong heat stability, no complement activation and the like, and has wide application prospect in the fields of food, medicine, daily chemical industry and the like.

The composition of yolk comprises 50% of water, 30% of fat and 20% of protein, most of the protein is combined with fat and exists in the form of lipoprotein, while immunoglobulin is a water-soluble protein in yolk, therefore, the technical difficulty of extracting the yolk immunoglobulin is to remove the water-insoluble components in the yolk. The existing method for extracting the yolk immunoglobulin mainly comprises an inorganic matter precipitation method, an organic matter polymerization method, a water dilution method and the like. Wherein, the inorganic substance precipitation method, namely the salting-out method, is a very classical method for extracting protein, but because the egg yolk has more protein types, the product prepared by the method has lower purity; the organic precipitation method and the organic polymerization method have good purification effect, but have the problems of organic residue and the like; the water dilution method is economical, simple to operate and suitable for large-scale industrial production, but has the problems of high water dilution ratio requirement, long process time consumption, large process treatment capacity, more impurities in crude extracts, heavy load of subsequent purification processes, low product extraction rate, low purity and the like.

Disclosure of Invention

In order to solve the problems, the invention firstly carries out ultrasonic treatment on yolk to destroy a yolk sphere structure, and then carries out freezing treatment on the yolk to enable lipoprotein and grease to form gel, which is beneficial to realizing rapid separation of a water-soluble part and a non-water-soluble part, and further adopts low-multiple water to dilute so as to enable immunoglobulin to be dissolved in aqueous solution, and then carries out subsequent purification procedures.

The first purpose of the invention is to provide a pretreatment method for extracting yolk immunoglobulin, which adopts the pretreatment of yolk by combining ultrasound with freezing.

The second purpose of the invention is to provide a method for preparing the yolk immunoglobulin, which is to firstly pretreat the yolk by combining ultrasound with freezing, and then combine a flocculating agent and ultrafiltration concentration for purification to obtain the yolk immunoglobulin.

In one embodiment of the present invention, the ultrasonic pretreatment conditions are: the ultrasonic power is 40-600W, and the ultrasonic time is 2-30 min.

In one embodiment of the invention, the freezing pretreatment is to freeze the egg yolk at-20 to-15 ℃ for 12 to 24 hours, and then unfreeze the egg yolk at 0 to 8 ℃ for 12 to 24 hours after being taken out.

In one embodiment of the invention, the method comprises the steps of:

(1) raw material treatment: washing fresh egg white, sterilizing, separating egg white and yolk, and stirring yolk uniformly;

(2) ultrasonic treatment: putting the yolk in an ultrasonic instrument for ultrasonic treatment;

(3) freezing and thawing yolk: freezing the yolk obtained in the step (1) and then unfreezing;

(4) fat removal: adding water into the yolk gel obtained in the step (2), adjusting the pH value by using a food acidity regulator, stirring, standing for precipitation, and centrifuging to obtain an immunoglobulin extracting solution;

(5) flocculation purification: adding a flocculating agent into the immunoglobulin supernatant in the step (3), and centrifuging or filtering by a plate frame to obtain a high-purity immunoglobulin extracting solution;

(6) and (3) ultrafiltration concentration and purification: separating and purifying the immunoglobulin extracting solution obtained in the step (4) by using a low-temperature ultrafiltration membrane to obtain a concentrated extracting solution;

(7) and (3) microfiltration sterilization: sterilizing the concentrated solution obtained in the step (5) by a low-temperature microfiltration membrane;

(8) and (3) freeze drying: and (4) freeze-drying the microfiltration permeating liquid obtained in the step (6) to obtain the product, namely the egg yolk immunoglobulin.

In one embodiment of the invention, the eggs in the step (1) are common eggs or fresh eggs laid by immunized laying hens, antigens are common human pathogenic bacteria or viruses, are cultured and purified to reach a certain order of magnitude, are inactivated as the antigens, and are added with antigen adjuvants to carry out 3-6 times of immunization injection on healthy laying hens of 15-20 weeks old;

in one embodiment of the invention, the specific conditions in the step (2) are that the ultrasonic power is 40-600W, and the ultrasonic time is 2-30 min. The step has the effects of destroying the yolk sphere structure, promoting the release of immunoglobulin and being beneficial to improving the yield of the immunoglobulin.

In one embodiment of the invention, the specific conditions in the step (3) are that the yolk obtained in the step (3) is frozen at-18 +/-2 ℃ for 12-24 h, and then taken out and thawed at 0-8 ℃ for 12-24 h. The key step has the effects that most fat-soluble proteins in the egg yolk are subjected to structural change in the low-temperature process to form irreversible gel which is easy to separate from water-soluble immunoglobulin, so that the operation difficulty and the working strength of subsequent extraction, flocculation, membrane separation and other processes are reduced, the energy consumption is reduced, and the efficiency is improved. Compared with other crude extraction methods, the process has the advantages of simple operation, low energy consumption, short time consumption, high efficiency, high product yield, high purity and the like.

In one embodiment of the present invention, in the step (4), the yolk gel obtained in the step (3) is added into deionized water, pH is adjusted, stirred, stood for precipitation, and centrifuged to obtain an immunoglobulin extract, wherein the specific technical conditions are that the yolk: deionized water is 1: 4-1: 9(w/w), a food acidity regulator is used for regulating the pH value to 4.0-7.0, the food acidity regulator can be acetic acid, lactic acid, citric acid, tartaric acid, sodium carbonate, sodium citrate, potassium citrate and the like, the stirring speed is 600-1000 rpm, the stirring time is 10-30 min, a centrifuge is used for separating at 3000-8000 rpm for 3-15 min, and the precipitation is carried out for 1-5 h. The step is used for separating immunoglobulin and other water insoluble substances to obtain relatively pure immunoglobulin extractive solution. Compared with the prior water dilution method, the method has the advantages that the water consumption is greatly reduced, the required water is only deionized water instead of ultrapure water, the production cost is reduced, the stirring and precipitating time is shortened, in addition, the obtained upper clear and transparent liquid is removed, most of water-insoluble substances are removed, and the workload of subsequent flocculation and ultrafiltration is reduced.

In one embodiment of the present invention, in the step (5), a flocculant is further added to the immunoglobulin extract obtained in the step (4), the flocculant is an aqueous solution of one or more than two natural polysaccharides such as sodium alginate, xanthan gum, chitosan, pectin, carrageenan, konjac gum, guar gum, sodium carboxymethylcellulose, the concentration of the flocculant is 0.8 to 1.5%, and the addition amount of the flocculant is 0 to 0.8% of the volume of the supernatant, and then the immunoglobulin clear extract is obtained by centrifugation or plate-and-frame filtration. The step has the effects of purifying the immunoglobulin extracting solution, removing impurity protein, reducing the working pressure of subsequent ultrafiltration membranes and microfiltration membranes and prolonging the service cycle of the membranes. The method has the advantages that harmless natural polysaccharide is used, the method is environment-friendly and healthy, the using amount is small, and the clarification degree of the extracting solution obtained in the previous step is added as appropriate or even not added.

In one embodiment of the present invention, step (6) is to perform low-temperature ultrafiltration membrane separation on the immunoglobulin extract obtained in step (5) to obtain an immunoglobulin concentrated retentate. The specific technical conditions are that the temperature is 4-10 ℃, and the molecular weight cutoff is 20000-150000. The function of this step is to concentrate and purify the immunoglobulin extract and further remove small molecular hetero-proteins.

In one embodiment of the present invention, step (7) is to cold sterilize the immunoglobulin concentrated retentate obtained in step (6) to obtain a microfiltration permeate. The specific technical conditions of the step are that the aperture of the micro-filtration membrane is 0.22 mu m, and the microbial index of the main product is that the total number of bacteria is less than or equal to 3000 CFU. The method has the advantages of good degerming effect and no damage to the biological activity of the product.

In one embodiment of the invention, step (8) is to freeze-dry the microfiltration permeate obtained in step (7) at-30 to-50 ℃ to obtain a high-purity immunoglobulin powder product, wherein the yield is 1.0 to 4.0 percent and the purity is 30 to 70 percent, and the obtained product can be applied to preparation of medicines, foods, health products, daily necessities and the like; egg white obtained by separating raw materials is used for preparing protein powder, and yolk precipitate obtained by centrifuging in the step (4) is used for preparing products such as yolk powder, lecithin and the like.

The third purpose of the invention is to provide the yolk immunoglobulin prepared by the method.

The fourth purpose of the invention is to provide the application of the yolk immunoglobulin in the aspects of food, medicine and daily chemical industry.

The invention has the beneficial effects that:

the invention has the advantages of high product purity, less product impurities and purity up to 70 percent; the extraction process is short in time consumption, and the subsequent operation can be carried out only after the stirring time is 10-30 min through ultrasonic-freezing pretreatment; the addition amount of the flocculating agent is 0-0.8%, and the dosage is small; the addition of water is reduced in the degreasing process, so that the treatment capacity of concentration processes such as subsequent plate frames and ultrafiltration is reduced, and the recycling efficiency of the filtering membrane is improved. The invention not only improves the extraction efficiency and the extraction purity of the immunoglobulin, but also reduces the production cost, and is a feasible industrialized large-scale production method.

Drawings

FIG. 1 is a flow chart of the method for producing yolk immunoglobulin by ultrasound assistance.

Detailed Description

The following description of the preferred embodiments of the present invention is provided for the purpose of better illustrating the invention and is not intended to limit the invention thereto.

1. Determination of protein yield

The protein concentration of the supernatant is measured by using a Bradford method, the protein content of the supernatant can be obtained, and then the protein content is divided by the mass of the yolk to obtain the protein mass yield.

The calculation formula is as follows:

2. determination of immunoglobulin purity

Performing polyacrylamide gel electrophoresis on the protein with activity maintained by using a non-denaturing polyacrylamide gel electrophoresis method (Native-PAGE) without adding denaturants such as SDS mercaptoethanol and the like to obtain a plurality of protein bands, and scanning according to the content of the bands to obtain the proportion of the immunoglobulin bands, namely the purity of the immunoglobulin.

3. The eggs mentioned in the embodiment are common eggs or fresh eggs laid by immunized laying hens, and the immunization of the laying hens is carried out by inactivating common human pathogenic bacteria or viruses as antigens and adding antigen adjuvants for 3-6 times of immunization injection on healthy laying hens aged 15-20 weeks.