CN112142839A - A method suitable for industrial large-scale extraction of egg yolk antibodies - Google Patents

Abstract

The invention discloses a method for extracting a yolk antibody in an industrialized large scale, which is characterized by comprising the following steps: (1) washing and sterilizing high-immunity egg white, separating to obtain egg yolk, and stirring to obtain egg yolk liquid; (2) adding a nano carbon dot into an acetic acid-sodium acetate buffer solution with the pH value of 5.0-5.2 to prepare a yolk antibody extraction buffer solution containing the carbon dot; (3) the yolk liquid is added into the yolk antibody extraction buffer solution, the mixture is stirred evenly, the supernatant is obtained by standing and precipitating at room temperature, then the supernatant is obtained by centrifuging the precipitate, and the supernatant obtained by combining the two times is the yolk antibody extracting solution.

Description

A method suitable for industrial large-scale extraction of egg yolk antibodies

technical field

The invention relates to a method for extracting egg yolk antibodies, in particular to a method suitable for industrial large-scale extraction of egg yolk antibodies.

Background technique

Egg yolk antibody, also known as egg yolk immunoglobulin (egg yolk immunoglobulin, IgY), is a specific antibody formed by the transfer of chicken serum IgG into egg yolk, and has the same immunological activity as mammalian IgG, but compared with mammalian IgG, IgY has more It has the advantages of good chemical stability, high yield, simple preparation, low cost, green safety, etc., and has been widely used in food, medicine, biological products, etc.

Chicken egg yolk contains about 48% water, about 17% protein and about 30% fat. Fat is easy to combine with protein to form lipoprotein, and egg yolk antibody belongs to soluble egg yolk protein. At present, there are many methods for extracting egg yolk antibody, and the common methods are: 1. Polyethylene glycol (PEG) precipitation method, which is the most commonly used method for large-scale extraction of egg yolk antibody. The advantage is that PEG is an organic solvent for non-ionic water-soluble polymers, easily soluble in water, low in heat dissipation, and short in time to form protein precipitation. However, PEG is an organic solvent. At present, there is no way to remove PEG in the extract. Whether the residual PEG is harmful to farmed animals is still inconclusive. At the same time, the high price of PEG and the large amount of PEG will increase a lot of costs. 2. Ammonium sulfate precipitation method. The advantages of this method are that it has low requirements on equipment and conditions, low price of ammonium sulfate, and mild properties. The disadvantage is that the amount of ammonium sulfate is too large, and the buffering capacity is poor, which has an impact on the activity of the yolk antibody, and the waste liquid of a large amount of ammonium sulfate causes environmental pollution. 3. Organic solvent extraction method such as octanoic acid or chloroform. The organic solvent in this method not only affects the activity of the antibody, but also is toxic and pollutes the environment. 4. The acidified water extraction method, the principle is that in the acidic pH and low-concentration ion environment, the lipoproteins in the egg yolk aggregate and separate out, so as to achieve the purpose of separating and extracting the egg yolk antibody. This method has a large loss of yolk antibody. In addition, the acidified water stability system is poor, and the shelf life of the extracted egg yolk antibody is short. Therefore, it is a technical difficulty that those skilled in the art need to break through to explore a method for purifying egg yolk antibody with simple process, good degreasing effect, low cost, stable antibody and environmental friendliness.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is to provide a method suitable for industrialized large-scale extraction of egg yolk antibody with high extraction rate, good stability, no pollution and high-efficiency bacteriostatic effect.

The technical scheme adopted by the present invention to solve the above-mentioned technical problems is: a method suitable for industrialized large-scale extraction of egg yolk antibody, which is characterized by comprising the following steps:

(1) separating the egg yolk after cleaning and sterilizing the high-free egg, stirring evenly to obtain egg yolk liquid;

(2) adding a kind of nano carbon dots to an acetic acid-sodium acetate buffer solution with a pH value of 5.0-5.2 to prepare a carbon dot-containing egg yolk antibody extraction buffer;

(3) Add the egg yolk solution to the egg yolk antibody extraction buffer, stir evenly, let the precipitation stand at room temperature to get the supernatant, then centrifuge the precipitation to obtain the supernatant, and combine the supernatants obtained twice to obtain the egg yolk antibody extract .

Further, in step (1), the stirring speed is 50-500 r/min, and the time is 10-20 min.

Further, in step (2), the concentration of nano carbon dots in the acetic acid-sodium acetate buffer solution is 5-25 μg/ml.

Further, the carbon nanodots are zero-dimensional carbon-based nanomaterials with a size below 50 nm obtained by preparing vitamin C by a one-step electrochemical method.

Further, the concentration of the acetic acid-sodium acetate buffer solution described in step (2) is 0.1-0.5M, and the pH value is 5.0-5.2.

Further, the mass-volume ratio of the egg yolk solution described in step (3) and the egg-yolk antibody extraction buffer is 1: (5-10).

Further, the specific process of step (3) is as follows: adding the egg yolk solution to the egg yolk antibody extraction buffer at a ratio of 1: (5-10) by mass, stirring at 50-500 r/min for 10-20 min, and leaving it to settle at room temperature for 8 ~15h; after aspirating the supernatant, centrifuge the precipitate at 4000-12000 r/min for 10-30 min to take the supernatant, and combine the supernatants obtained twice to obtain the egg yolk antibody extract.

Compared with the prior art, the advantages of the present invention are:

1) Compared with the traditional acidified water extraction method, the present invention adds decomposable and residue-free carbon dots prepared with vitamin C in the buffer system, further improving the yield of IgY, and the obtained IgY has good stability. The problems of high cost and large loss of antibodies in the method for extracting egg yolk antibodies in the prior art are solved.

2) Due to the strong antibacterial effect of carbon dots, the extraction process of egg yolk antibody can be carried out at room temperature. Therefore, compared with the traditional extraction method, which requires overnight precipitation at 4° C., the device of the present invention has simple requirements and is easy to operate. At the same time, because carbon dots can be completely degraded into CO ₂ , CO and H ₂ O under visible light in the air without toxicity, the sediment can also be developed into food or feed raw materials to improve the utilization rate and achieve zero pollution.

3) Compared with the traditional acidified water extraction method, the present invention adopts "0.1M-0.5M acetic acid-sodium acetate buffer solution", so that the activity of IgY is stable in the buffer system.

4) After the carbon dot extraction process adopted in the present invention is completed, it is completely degraded into CO ₂ , CO and H ₂ O under natural light for about 20 days, with no residue, and is green and safe. The technical problem of potential risks to animals or humans caused by residual organic matter and high-dose preservatives in the extraction solution in the conventional method is solved.

To sum up, the present invention provides a method suitable for industrial large-scale extraction of egg yolk antibody, the method has simple steps, low cost and high efficiency; the added nano-carbon dots have high bacteriostatic effect, which can improve the yield of egg yolk antibody. At the same time, it avoids the disadvantage of overnight precipitation at a low temperature of 4°C when extracting egg yolk antibodies with traditional acidified water. No or less preservatives are added, and the product is green and pollution-free.

Description of drawings

Fig. 1 is the result of circular dichroism analysis of the effect of different concentrations of carbon dots on the structure of IgY;

Figure 2 is the preliminary detection of IgY yields by SDS-PAGE electrophoresis of egg yolk antibodies extracted from Cdots of different concentrations and buffers of different proportions after precipitation with 19wt% ammonium sulfate solution. Among them: M is the protein marker, lanes 1 to 3 are the carbon dots concentration of 0 μg/mL, lanes 4 to 6 are the carbon dots concentration of 6.25 μg/mL, respectively, lanes 7 to 9 are the carbon dots concentration of 12.5 μg/mL , lanes 10 to 12 respectively indicate that the concentration of carbon dots is 25 μg/mL; at the same time, lanes 1, 4, 7, and 10 also indicate that the mass-volume ratio of egg yolk and buffer is 1:5, and lanes 2, 5, 8, and 11 also indicate egg yolk The mass-volume ratio to buffer is 1:7, and lanes 3, 6, 9, and 12 also indicate that the mass-volume ratio of egg yolk to buffer is 1:9;

Fig. 3 is the result of measuring antibody titer in anti-Turbot IL-1β egg yolk antibody extract by ELISA method;

Fig. 4 is the result that ELISA method measures the antibody titer in anti-crucian carp gill hemorrhage virus (CyHV-2) yolk antibody extract;

Figure 5 shows that the mass-to-volume ratio of yolk against crucian carp gill hemorrhage virus (CyHV-2) to the buffer is 1:9, and the yolk antibodies extracted from carbon dots with different concentrations were left for 15h at 4°C and room temperature. total bacterial count.

Detailed ways

The present invention will be further described in detail below with reference to the embodiments of the accompanying drawings.

1. Specific Examples

Example 1

Extraction of Anti-Turbot IL-1β Egg Yolk Antibody

1. Anti-Turbot IL-1β high-immunity eggs were washed with tap water at room temperature for 1 min, and the washed eggs were disinfected with 0.25mg/L chlorine dioxide aqueous solution for 1 min; then spray the sterilized eggs with tap water for 2 min, remove the disinfectant, The washed eggs are sent to the egg white and yolk separator, the egg shell, egg white and egg yolk are separated, the egg yolk is collected, weighed, and stirred at 100 r/min for 10 minutes to obtain egg yolk liquid, which is ready for use;

2. Add the new zero-dimensional carbon-based nanomaterial carbon dots into the acetic acid-sodium acetate buffer solution with a concentration of 0.1M and a pH value of 5.1 to prepare a yolk antibody extraction buffer containing carbon dots with a concentration of 6.25 μg/mL;

3. Add egg yolk antibody extraction buffer to the egg yolk solution at a ratio of 1:5 by mass to volume, stir at 100 r/min

Stir for 20 min, and settle for 8 h at room temperature; suck the supernatant into a clean container with a vacuum pump, and then centrifuge the supernatant at 12,000 r/min for 20 min to take the supernatant, and combine the supernatant to obtain the egg yolk antibody extract.

Example 2

Extraction of Anti-Turbot IL-1β Egg Yolk Antibody

1. Anti-Turbot IL-1β high-immunity eggs were washed with tap water at room temperature for 1 min, and the washed eggs were disinfected with 0.25mg/L chlorine dioxide aqueous solution for 1 min; then spray the sterilized eggs with tap water for 2 min, remove the disinfectant, The washed eggs are sent to the egg white and yolk separator to separate the egg shell, egg white and egg yolk, collect the yolk, weigh, stir at 200 r/min for 10 min to obtain egg yolk liquid, which is ready for use;

2. Add the new zero-dimensional carbon-based nanomaterial carbon dots into the acetic acid-sodium acetate buffer solution with a concentration of 0.2M and a pH value of 5.1 to prepare a yolk antibody extraction buffer containing carbon dots with a concentration of 6.25 μg/mL;

3. Add egg yolk antibody extraction buffer to the egg yolk solution at a ratio of 1:7 by mass to volume, stir at 200 r/min for 20 minutes, and let it stand for 12 hours at room temperature for precipitation; use a vacuum pump to suck the supernatant into a clean container, and then precipitate at 8000 r/min. Centrifuge for 30 min to take the supernatant, and combine the supernatant to obtain the egg yolk antibody extract.

Example 3

Extraction of Anti-Turbot IL-1β Egg Yolk Antibody

1. Anti-Turbot IL-1β high-immunity eggs were washed with tap water at room temperature for 1 min, and the washed eggs were disinfected with 0.25mg/L chlorine dioxide aqueous solution for 1 min; then spray the sterilized eggs with tap water for 2 min, remove the disinfectant, The washed eggs are sent to the egg white and yolk separator, the egg shell, egg white and egg yolk are separated, the egg yolk is collected, weighed, and stirred at 300 r/min for 10 minutes to obtain egg yolk liquid, which is ready for use;

2. Add the new zero-dimensional carbon-based nanomaterial carbon dots into an acetic acid-sodium acetate buffer solution with a concentration of 0.3M and a pH value of 5.1 to prepare a yolk antibody extraction buffer containing carbon dots with a concentration of 6.25 μg/mL;

3. Add egg yolk antibody extraction buffer to the egg yolk solution at a ratio of 1:9 by mass to volume, stir at 500 r/min for 10 minutes, and let it stand for 15 hours at room temperature for precipitation; use a vacuum pump to suck the supernatant into a clean container, and then precipitate at 5000 r/min. Centrifuge for 30 min to take the supernatant, and combine the supernatant to obtain the egg yolk antibody extract.

Example 4

Extraction of Anti-Turbot IL-1β Egg Yolk Antibody

1. Anti-Turbot IL-1β high-immunity eggs were washed with tap water at room temperature for 1 min, and the washed eggs were disinfected with 0.25mg/L chlorine dioxide aqueous solution for 1 min; then spray the sterilized eggs with tap water for 2 min, remove the disinfectant, The washed eggs are sent to the egg white and yolk separator, the egg shell, egg white and egg yolk are separated, the egg yolk is collected, weighed, and stirred at 300 r/min for 10 minutes to obtain egg yolk liquid, which is ready for use;

2. Add the new zero-dimensional carbon-based nanomaterial carbon dots into the acetic acid-sodium acetate buffer solution with a concentration of 0.3M and a pH value of 5.1 to prepare a yolk antibody extraction buffer containing carbon dots with a concentration of 12.5 μg/mL;

3. Add egg yolk antibody extraction buffer to the egg yolk solution at a ratio of 1:5 by mass to volume, stir at 400 r/min for 10 minutes, and let stand for 9 hours at room temperature for precipitation; use a vacuum pump to suck the supernatant into a clean container, and then precipitate at 12,000 r/min. Centrifuge for 20 min to take the supernatant, and combine the supernatant to obtain the egg yolk antibody extract.

Example 5

Extraction of Anti-Turbot IL-1β Egg Yolk Antibody

1. The high-immunity eggs against turbot IL-1β were washed with tap water at room temperature for 1 min, and the washed eggs were disinfected with 0.25mg/L chlorine dioxide aqueous solution for 2 min; then the sterilized eggs were sprayed with tap water for 2 min to remove the disinfectant, The washed eggs are sent to the egg white and yolk separator, the egg shell, egg white and egg yolk are separated, the egg yolk is collected, weighed, and stirred at 400 r/min for 10 minutes to obtain egg yolk liquid, which is ready for use;

2. Add the new zero-dimensional carbon-based nanomaterial carbon dots into an acetic acid-sodium acetate buffer solution with a concentration of 0.2M and a pH value of 5.1 to prepare a yolk antibody extraction buffer containing carbon dots with a concentration of 12.5 μg/mL;

3. Add egg yolk antibody extraction buffer to egg yolk solution at a ratio of 1:7 by mass to volume, stir at 300 r/min for 10 min, and let stand for 10 h at room temperature; suck the supernatant into a clean container with a vacuum pump, and precipitate at 9000 r/min. Centrifuge for 30 min to take the supernatant, and combine the supernatant to obtain the egg yolk antibody extract.

Example 6

Extraction of Anti-Turbot IL-1β Egg Yolk Antibody

1. The high-immunity eggs against turbot IL-1β were washed with tap water at room temperature for 1 min, and the washed eggs were disinfected with 0.25mg/L chlorine dioxide aqueous solution for 2 min; then the sterilized eggs were sprayed with tap water for 2 min to remove the disinfectant, The washed eggs are sent to the egg white and yolk separator, the egg shell, egg white and egg yolk are separated, the egg yolk is collected, weighed, and stirred at 400 r/min for 10 minutes to obtain egg yolk liquid, which is ready for use;

2. Add the new zero-dimensional carbon-based nanomaterial carbon dots into the acetic acid-sodium acetate buffer solution with a concentration of 0.3M and a pH value of 5.1 to prepare a yolk antibody extraction buffer containing carbon dots with a concentration of 12.5 μg/mL;

3. Add egg yolk antibody extraction buffer to the egg yolk solution at a ratio of 1:9 by mass to volume, stir at 200 r/min for 30 minutes, and let it stand for 15 hours at room temperature for precipitation; suck the supernatant into a clean container with a vacuum pump, and then precipitate at 4000 r/min. Centrifuge for 30 min to take the supernatant, and combine the supernatant to obtain the egg yolk antibody extract.

Example 7

Extraction of Anti-Turbot IL-1β Egg Yolk Antibody

1. The high-immunity eggs against turbot IL-1β were washed with tap water at room temperature for 1 min, and the washed eggs were disinfected with 0.25mg/L chlorine dioxide aqueous solution for 2 min; then the sterilized eggs were sprayed with tap water for 2 min to remove the disinfectant, The washed eggs are sent to the egg white and yolk separator, the egg shell, egg white and egg yolk are separated, the egg yolk is collected, weighed, and stirred at 200 r/min for 15 minutes to obtain egg yolk liquid, which is ready for use;

2. Add the new zero-dimensional carbon-based nanomaterial carbon dots into an acetic acid-sodium acetate buffer solution with a concentration of 0.1 M and a pH value of 5.1 to prepare a yolk antibody extraction buffer containing carbon dots with a concentration of 25 μg/mL;

3. Add egg yolk antibody extraction buffer to egg yolk solution at a ratio of 1:5 by mass to volume, stir at 400 r/min for 10 min, and let stand for 12 h at room temperature for precipitation; suck the supernatant into a clean container with a vacuum pump, and precipitate at 12000 r/min. Centrifuge for 20 min to take the supernatant, and combine the supernatant to obtain the egg yolk antibody extract.

Example 8

Extraction of Anti-Turbot IL-1β Egg Yolk Antibody

1. Anti-Turbot IL-1β high-immunity eggs were washed with tap water at room temperature for 1 min, and the washed eggs were disinfected with 0.25mg/L chlorine dioxide aqueous solution for 1 min; then spray the sterilized eggs with tap water for 2 min, remove the disinfectant, The washed eggs are sent to the egg white and yolk separator, the egg shell, egg white and egg yolk are separated, the egg yolk is collected, weighed, and stirred at 100 r/min for 20 minutes to obtain egg yolk liquid, which is ready for use;

2. Add the new zero-dimensional carbon-based nanomaterial carbon dots into the acetic acid-sodium acetate buffer solution with a concentration of 0.2M and a pH value of 5.1 to prepare a yolk antibody extraction buffer containing carbon dots with a concentration of 25 μg/mL;

3. Add egg yolk antibody extraction buffer to egg yolk solution at a ratio of 1:7 by mass to volume, stir at 300 r/min for 30 minutes, and let stand for 10 hours at room temperature for precipitation; suck the supernatant into a clean container with a vacuum pump, and precipitate at 7000 r/min. Centrifuge for 30 min to take the supernatant, and combine the supernatant to obtain the egg yolk antibody extract.

Example 9

Extraction of Anti-Turbot IL-1β Egg Yolk Antibody

1. Anti-Turbot IL-1β high-immunity eggs were washed with tap water at room temperature for 1 min, and the washed eggs were disinfected with 0.25mg/L chlorine dioxide aqueous solution for 1 min; then spray the sterilized eggs with tap water for 2 min, remove the disinfectant, The washed eggs are sent to the egg white and yolk separator, the egg shell, egg white and egg yolk are separated, the egg yolk is collected, weighed, and stirred at 300 r/min for 10 minutes to obtain egg yolk liquid, which is ready for use;

2. Add the new zero-dimensional carbon-based nanomaterial carbon dots into the acetic acid-sodium acetate buffer solution with a concentration of 0.3M and a pH value of 5.1 to prepare a yolk antibody extraction buffer containing carbon dots with a concentration of 25 μg/mL;

3. Add egg yolk antibody extraction buffer to the egg yolk solution at a ratio of 1:9 by mass to volume, stir at 300 r/min for 15 minutes, and let it stand for 15 hours at room temperature for precipitation; suck the supernatant into a clean container with a vacuum pump, and precipitate at 6000 r/min. Centrifuge for 20 min to take the supernatant, and combine the supernatant to obtain the egg yolk antibody extract.

Example 10

Extraction of yolk antibody against crucian carp gill hemorrhage virus (CyHV-2)

1. The high-immunity eggs against crucian carp gill hemorrhage virus (CyHV-2) were washed with tap water at room temperature for 1 min, and the washed eggs were sterilized with 0.25mg/L chlorine dioxide aqueous solution for 2 min; and then sprayed with tap water for 2 min. Remove the disinfectant, send the washed eggs to the egg white and yolk separator, separate the egg shell, egg white and egg yolk, collect the yolk, weigh, stir at 200 r/min for 15 minutes, and obtain egg yolk liquid, which is ready for use;

2. Add the new zero-dimensional carbon-based nanomaterial carbon dots into the acetic acid-sodium acetate buffer solution with a concentration of 0.1M and a pH value of 5.1 to prepare a yolk antibody extraction buffer containing carbon dots with a concentration of 6.25 μg/mL;

3. Add egg yolk antibody extraction buffer to the egg yolk solution at a ratio of 1:9 by mass to volume, stir at 400 r/min for 10 minutes, and let stand for 12 hours at room temperature for precipitation; suck the supernatant into a clean container with a vacuum pump, and precipitate at 5000 r/min. Centrifuge for 30 min to take the supernatant, and combine the supernatant to obtain the egg yolk antibody extract.

Example 11

Extraction of yolk antibody against crucian carp gill hemorrhage virus (CyHV-2)

1. The high-immunity eggs against crucian carp gill hemorrhage virus (CyHV-2) were washed with tap water at room temperature for 1 min, and the washed eggs were disinfected with 0.25mg/L chlorine dioxide aqueous solution for 1 min; and then sprayed with tap water for 2 min. Remove the disinfectant, send the washed eggs to the egg white and yolk separator, separate the egg shell, egg white and egg yolk, collect the yolk, weigh, stir at 100 r/min for 20 minutes, and obtain egg yolk liquid, which is ready for use;

2. Add the new zero-dimensional carbon-based nanomaterial carbon dots into the acetic acid-sodium acetate buffer solution with a concentration of 0.1M and a pH value of 5.1 to prepare a yolk antibody extraction buffer containing carbon dots with a concentration of 12.5 μg/mL;

3. Add egg yolk antibody extraction buffer to the egg yolk solution at a ratio of 1:9 by mass to volume, stir at 300 r/min for 30 minutes, and let stand for 12 hours at room temperature for precipitation; suck the supernatant into a clean container with a vacuum pump, and precipitate at 6000 r/min. Centrifuge for 30 min to take the supernatant, and combine the supernatant to obtain the egg yolk antibody extract.

Example 12

Extraction of yolk antibody against crucian carp gill hemorrhage virus (CyHV-2)

1. The high-immunity eggs against crucian carp gill hemorrhage virus (CyHV-2) were washed with tap water at room temperature for 1 min, and the washed eggs were sterilized with 0.25mg/L chlorine dioxide aqueous solution for 2 min; and then sprayed with tap water for 2 min. Remove the disinfectant, send the washed eggs into the egg white and yolk separator, separate the egg shell, egg white and egg yolk, collect the yolk, weigh, stir at 300 r/min for 10 minutes, and obtain egg yolk liquid, which is ready for use;

2. Add the new zero-dimensional carbon-based nanomaterial carbon dots into an acetic acid-sodium acetate buffer solution with a concentration of 0.1 M and a pH value of 5.1 to prepare a yolk antibody extraction buffer containing carbon dots with a concentration of 25 μg/mL;

3. Add egg yolk antibody extraction buffer to the egg yolk solution at a ratio of 1:9 by mass to volume, stir at 300 r/min for 15 minutes, and let it stand for 15 hours at room temperature for precipitation; suck the supernatant into a clean container with a vacuum pump, and then precipitate at 7000 r/min. Centrifuge for 25 min to take the supernatant, and combine the supernatant to obtain the egg yolk antibody extract.

In the above embodiments, the carbon dots are prepared as follows: a zero-dimensional carbon-based nanomaterial with a size of less than 50 nm is prepared from vitamin C by a one-step electrochemical method. Different concentrations of zero-dimensional carbon-based nano-carbon dots were added to the purified IgY protein solution, and the effect of different concentrations of carbon dots on the structure of IgY protein was analyzed by circular dichroism. When the concentration of carbon dots reaches 50 μg/mL, the peak height between 190-210 nm decreases slightly, and the stability of the secondary structure of IgY is slightly lower (see Figure 1). Therefore, low concentrations of carbon nanodots below 25 μg/mL can be used to extract egg yolk antibodies.

2. Analysis of the results

1. Determination of protein content in crude extract of anti-Turbot IL-1β yolk antibody. The BCA protein concentration assay kit was used to measure the water-soluble protein content in the egg yolk antibody extract (purchased from Beijing Solaibao Biotechnology Co., Ltd.), and the results are shown in Table 1.

Table 1 Efficiency (mg/g egg yolk) of extracting water-soluble protein from egg yolk with different concentrations of carbon dots and different proportions of buffers

Note: All data with different shoulder letters indicate significant difference (P<0.05).

It can be seen from Table 1 that the concentration of water-soluble protein in the yolk antibody extract of each test group increased with the increase of the concentration of carbon dots. Comprehensive comparison, when the concentration of carbon dots was 25μg/mL, the egg yolk antibody extraction effect was the best in the buffer ratio of 1:9 group, with a very significant difference (P<0.01), followed by the buffer ratio of 1:7 group (P<0.05); when the carbon dot concentration was 12.5 μg/mL and the buffer ratio was 1:9, the protein extraction efficiency also reached a significant level (P<0.05). It shows that the addition of carbon dots is helpful for the extraction of water-soluble proteins.

2. Visual determination of the amount of IgY in the crude extract of anti-Turbot IL-1β yolk antibody. Because there is a certain amount of impurity protein in the egg yolk antibody extract, in order to further visually detect the extraction effect of IgY in the anti-Turbot IL-1β egg yolk antibody extract, the present invention uses the egg yolk antibody extract with a mass-volume ratio of 19% sulfuric acid. After precipitation with ammonium solution (Sinopharm Group, AR grade, CAS: 7783-20-2), resuspend with the same volume of PBS (0.1M, pH 7.2) buffer of egg yolk antibody extract, and then SDS-PAGE electrophoresis to identify the antibody The extraction effect, the results are shown in Figure 2. Wherein M: Protein Marker, lanes 1-3: the concentration of carbon dots is 0 μg/mL, lanes 4-6 (Example 1-3): the concentration of carbon dots is 6.25 μg/mL, respectively, lanes 7-9 (implementation Example 4-6): the concentration of carbon dots is 12.5 μg/mL, respectively, lane 10-12 (Example 7-9): the concentration of carbon dots is 25 μg/mL; the yolk and The mass-volume ratio of buffer solution is 1:5, the mass-volume ratio of egg yolk to buffer solution in lanes 2, 5, 8, and 11 is 1:7, and the mass-volume ratio of egg yolk and buffer solution in lanes 3, 6, 9, and 12 is 1: 9.

It can be seen from Figure 2 that the yolk antibody SDS-PAGE electrophoresis can observe the IgY heavy chain of about 66kDa, the IgY light chain of about 25kDa, and two heteroprotein bands above and below 40kDa. From the analysis of antibody purity (i.e., the gray scale of the bands), the antibody extraction effect was the best in the same buffer solution ratio group when the carbon dot concentration was 6.25 μg/mL, followed by the 12.5 μg/mL concentration group, and the difference between the other groups was not obvious. This result is inconsistent with the results of water-soluble protein content in egg yolk antibody extract, indicating that high concentration of carbon dots can improve the water-soluble impurity protein of some non-yolk antibody while extracting egg yolk antibody, and low concentration of carbon dots is more conducive to the extraction of egg yolk antibody IgY .

3. Determination of IgY titer in crude extract of anti-Turbot IL-1β yolk antibody and anti-Crucian carp gill hemorrhage virus (CyHV-2) yolk antibody. The antibody titer in the egg yolk antibody extract was determined by indirect ELISA, namely, the recombinant protein of turbot IL-1β, the tandem gene of carp gill hemorrhage virus CyHV-2 (ORF72, ORF66, ORF81, ORF82, namely CyHV-2-D4ORF) The recombinant protein of ) is the coating antigen, and the egg yolk antibody extract of anti-Turbot IL-1β and anti-CyHV-2-D4ORF of different dilutions is used as the primary antibody, and the HRP goat anti-chicken IgY diluted at 1:2000 is used as the primary antibody. The secondary antibody was used to detect the antibody titer. The negative control was egg yolk antibody extracted from common eggs under the same conditions, and the blank control was PBS. The judgment standard is: P/N=(OD value to be measured - OD value of blank control)/(OD value of negative control - OD value of blank control), when P/N≥2.1, the maximum dilution of the antibody is the antibody titer. The results are shown in Figure 3 and Figure 4, respectively.

It can be seen from the ELISA results in Figure 3 that when the ratio of egg yolk to buffer solution is 1:5, the antibody titers in the egg yolk antibody extracts of each group are increased by 1:16000 of the control group after adding carbon dots to the acetic acid-sodium acetate buffer solution. When the ratio of yolk and buffer solution was 1:7, the antibody titer of the Cdot concentration 6.25μg/mL and 12.5μg/mL groups increased from 1:16000 to 1:32000, but the Cdot concentration was 25μg/mL. The antibody titer of the mL group was not different from that of the non-carbon dots group; when the ratio of egg yolk to buffer solution was 1:9, the antibody titer of the control group was 1:8000, and the antibody titer of the carbon dot concentration 25μg/mL group was 1 : 16000, and the antibody titer of the C-dot concentration 6.25μg/mL and 12.5μg/mL groups reached 1:32000. On the one hand, this result shows that the addition of carbon dots is helpful for the extraction of IgY, and on the other hand, it shows that the effect of low concentration carbon dots to extract IgY is better.

Antibody titers in the yolk antibody extract against crucian carp gill hemorrhage virus (CyHV-2-D4ORF) were determined by ELISA, and the results are shown in Figure 4. When the ratio of egg yolk to buffer was 1:9, the antibody titer of the control group was 1:8000, and the antibody titers of the 6.25μg/mL, 12.5μg/mL and 25μg/mL groups were 1:16000, 1:16000 and 1:8000. This result further verifies that low-concentration carbon dots have the best extraction effect of IgY, and the titer is significantly improved.

4. Determination of bacterial content in crude extract of yolk antibody against crucian carp gill hemorrhage virus (CyHV-2). Coat with LB solid plate, and measure the total bacterial content in the yolk antibody extract of anti-Crucian carp gill hemorrhage virus (CyHV-2) treated with different concentrations of carbon dots (Examples 10-12). After mixing the egg yolk antibody extract with the original fat precipitate in the egg yolk, 100 μL were taken and coated, and the number of colonies was counted after culturing for 24 hours.

LB solid plate coating method was used to determine the effect of different concentrations of carbon dots on the content of total bacteria in the yolk antibody extract against crucian carp gill hemorrhage virus (CyHV-2-D4ORF), and the treatment parameters were the same at room temperature and 4 ℃. The difference of bacterial content in 15h precipitation. The results are shown in Figure 5. The results showed that when other parameters were the same, the bacterial content in the antibody solution extracted at room temperature was higher than that in 4 ℃; when the Cdots were added to the buffer, the bacterial content in the egg yolk antibody extraction solution decreased significantly (P<0.05), and the number of colonies increased with the Cdots. The concentration increases and decreases until bacterial growth is completely inhibited. It is indicated that under the condition of adding a certain concentration of carbon dots, egg yolk antibody can be extracted at room temperature, and the carbon dots will be degraded and non-toxic, so that the by-products after extraction of egg yolk antibody can be further developed and utilized. Therefore, the process parameters can be changed from 4°C to room temperature after adding nanomaterials, which is simple and convenient. increase profit.

Conclusion: After comprehensively evaluating the effects of different concentrations of carbon dots and buffer solutions of different proportions on the extraction effect of egg yolk antibody, the present invention finally determines that when the concentration of carbon dots is 6.25 μg/mL ~ 12.5 μg/mL, the ratio of egg yolk solution to buffer solution is 1: 5～1:9 can achieve better extraction effect. Further, when a high concentration of antibody is required, the antibody can be further concentrated by other technical methods.

The above description does not limit the present invention, and the present invention is not limited to the above examples. Changes, modifications, additions or substitutions made by those skilled in the art within the essential scope of the present invention should also belong to the protection scope of the present invention.

Claims (7)

1. a method that is applicable to industrialized large-scale extraction of egg yolk antibody, is characterized in that comprising the following steps: (1) separating the egg yolk after cleaning and sterilizing the high-free egg, stirring evenly to obtain egg yolk liquid; (2) adding a kind of nano carbon dots to an acetic acid-sodium acetate buffer solution with a pH value of 5.0-5.2 to prepare a carbon dot-containing egg yolk antibody extraction buffer; (3) Add the egg yolk solution to the egg yolk antibody extraction buffer, stir evenly, let the precipitation stand at room temperature to get the supernatant, then centrifuge the precipitation to obtain the supernatant, and combine the supernatants obtained twice to obtain the egg yolk antibody extract . 2. A method suitable for industrialized large-scale extraction of egg yolk antibody according to claim 1, characterized in that: in step (1), the stirring speed is 50～500r/min, and the time is 10～20min. 3. A method suitable for industrialized large-scale extraction of egg yolk antibody according to claim 1, characterized in that: in step (2), the concentration of nano-sized carbon dots in the acetic acid-sodium acetate buffer solution is 5-25 μg/ml. 4. a kind of method suitable for industrialized large-scale extraction of egg yolk antibody according to claim 1, is characterized in that: described nanometer carbon dots is that the size of vitamin C prepared by one-step electrochemical method is zero below 50nm. Carbon-based nanomaterials. 5. a kind of method suitable for industrialized large-scale extraction of egg yolk antibody according to claim 1, is characterized in that: the concentration of acetic acid-sodium acetate buffer solution described in step (2) is 0.1～0.5M, pH value is 5.0 to 5.2. 6. a kind of method that is applicable to industrialized large-scale extraction of egg yolk antibody according to claim 1, is characterized in that: the mass volume ratio of egg yolk liquid described in step (3) and described egg yolk antibody extraction buffer is 1: (5 to 10). 7. a kind of method suitable for industrialized large-scale extraction of egg yolk antibody according to claim 1 is characterized in that the concrete process of step (3) is: egg yolk liquid is added in the ratio of mass volume ratio 1:(5～10) Add to egg yolk antibody extraction buffer, stir at 50-500 r/min for 10-20 min, and settle at room temperature for 8-15 h; after aspirating the supernatant, centrifuge the precipitate at 4000-12000 r/min for 10-30 min to take the supernatant and combine them The supernatant obtained twice is the egg yolk antibody extract.

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