Disclosure of Invention
The invention aims to solve the technical problem of providing a method for extracting the yolk antibody in an industrialized large scale, which has high extraction rate, good stability, no pollution and high-efficiency bacteriostatic action.
The technical scheme adopted by the invention for solving the technical problems is as follows: a method for extracting yolk antibodies in an industrialized large scale 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) adding the yolk solution into the yolk antibody extraction buffer solution, stirring uniformly, standing at room temperature for precipitation to obtain a supernatant, centrifuging the precipitate to obtain a supernatant, and combining the supernatants to obtain the yolk antibody extract.
Further, in the step (1), the stirring speed is 50-500 r/min, and the time is 10-20 min.
Further, in the step (2), the concentration of the nano carbon dots in the acetic acid-sodium acetate buffer solution is 5-25 mu g/ml.
Further, the nano carbon dots are zero-dimensional carbon-based nano materials with the size below 50nm, which are prepared by a one-step electrochemical method of vitamin C.
Further, the concentration of the acetic acid-sodium acetate buffer solution in the step (2) is 0.1-0.5M, and the pH value is 5.0-5.2.
Further, the mass-to-volume ratio of the yolk solution to the yolk antibody extraction buffer solution in the step (3) is 1: (5-10).
Further, the specific process of the step (3) is as follows: mixing yolk liquid according to the mass volume ratio of 1: (5-10), adding the yolk antibody into the yolk antibody extraction buffer solution in a ratio of 50-500 r/min, stirring for 10-20 min, and standing and precipitating for 8-15 h at room temperature; and sucking the supernatant, centrifuging the precipitate at 4000-12000 r/min for 10-30 min, taking the supernatant, and combining the supernatants to obtain the egg yolk antibody extracting solution.
Compared with the prior art, the invention has the advantages that:
1) compared with the traditional acidified water extraction method, the invention adds decomposable residue-free carbon dots prepared by vitamin C into the buffer system, further improves the yield of IgY and ensures that the obtained IgY has good stability. Solves the problems of high cost and high antibody loss in the method for extracting the yolk antibody in the prior art.
2) The yolk antibody can be extracted at room temperature due to the strong bacteriostatic action of the carbon dots. Therefore, compared with the traditional extraction method which needs to be precipitated at 4 ℃ overnight, the method has the advantages of simple equipment requirement and simple and convenient operation. Meanwhile, the carbon dots can be completely degraded into CO under visible light in the air2CO and H2O is non-toxic, so the precipitate can be developed into food or feed raw materials, the utilization rate is improved, and zero pollution is realized.
3) Compared with the traditional acidified water extraction method, the invention adopts 0.1-0.5M acetic acid-sodium acetate buffer solution to ensure that the activity of IgY is stable in a buffer system.
4) After the carbon point extraction process adopted by the invention is finished, the carbon point is completely degraded into CO within about 20 days under natural light2CO and H2And O, no residue, green and safe. Solves the technical problem that residual organic matters and high-dose preservatives in the extracting solution have potential risks to animals or human bodies in the conventional method.
In conclusion, the invention provides a method for extracting the yolk antibody in an industrialized large scale, which has simple steps, low cost and high efficiency; the added nano carbon dots have high-efficiency antibacterial action, improve the yield of the yolk antibody, avoid the defect that the yolk antibody needs to be precipitated at low temperature of 4 ℃ overnight when being extracted by traditional acidified water, and are green and pollution-free due to no or little addition of a preservative.
Detailed Description
The invention is described in further detail below with reference to the accompanying examples.
Detailed description of the preferred embodiments
Example 1
Extraction of anti-turbot IL-1 beta yolk antibody
1. Washing anti-turbot IL-1 beta hyperimmune egg with normal temperature tap water for 1min, and disinfecting the washed egg with 0.25mg/L chlorine dioxide water solution for 1 min; spraying sterilized egg with tap water for 2min, removing disinfectant, separating egg shell, egg white and yolk in an egg white and yolk separator, collecting yolk, weighing, and stirring at 100r/min for 10min to obtain yolk liquid;
2. adding the carbon dots of the novel zero-dimensional carbon-based nano material into an acetic acid-sodium acetate buffer solution with the concentration of 0.1M and the pH value of 5.1 to prepare a yolk antibody extraction buffer solution with the carbon dot concentration of 6.25 mug/mL;
3. adding yolk antibody extraction buffer solution into yolk solution at a mass-to-volume ratio of 1:5, stirring at 100r/min
Stirring for 20min, standing at room temperature for precipitation for 8 h; sucking the supernatant with vacuum pump, centrifuging at 12000r/min for 20min, collecting supernatant, and mixing the supernatants to obtain yolk antibody extractive solution.
Example 2
Extraction of anti-turbot IL-1 beta yolk antibody
1. Washing anti-turbot IL-1 beta hyperimmune egg with normal temperature tap water for 1min, and disinfecting the washed egg with 0.25mg/L chlorine dioxide water solution for 1 min; spraying the sterilized egg with tap water for 2min, removing disinfectant, separating egg shell, egg white and yolk in an egg white and yolk separator, collecting yolk, weighing, and stirring at 200r/min for 10min to obtain yolk liquid;
2. adding the carbon dots of the novel zero-dimensional carbon-based nano material into an acetic acid-sodium acetate buffer solution with the concentration of 0.2M and the pH value of 5.1 to prepare a yolk antibody extraction buffer solution with the carbon dot concentration of 6.25 mug/mL;
3. adding a yolk antibody extraction buffer solution into the yolk solution according to the mass-volume ratio of 1:7, stirring at 200r/min for 20min, and standing and precipitating at room temperature for 12 h; sucking the supernatant with vacuum pump, centrifuging at 8000r/min for 30min, collecting supernatant, and mixing the supernatants to obtain yolk antibody extractive solution.
Example 3
Extraction of anti-turbot IL-1 beta yolk antibody
1. Washing anti-turbot IL-1 beta hyperimmune egg with normal temperature tap water for 1min, and disinfecting the washed egg with 0.25mg/L chlorine dioxide water solution for 1 min; spraying the sterilized egg with tap water for 2min, removing disinfectant, separating egg shell, egg white and yolk in an egg white and yolk separator, collecting yolk, weighing, and stirring at 300r/min for 10min to obtain yolk liquid;
2. adding the carbon dots of the novel zero-dimensional carbon-based nano material into an acetic acid-sodium acetate buffer solution with the concentration of 0.3M and the pH value of 5.1 to prepare a yolk antibody extraction buffer solution with the carbon dot concentration of 6.25 mug/mL;
3. adding a yolk antibody extraction buffer solution into the yolk solution according to the mass-volume ratio of 1:9, stirring at 500r/min for 10min, and standing and precipitating at room temperature for 15 h; sucking the supernatant with vacuum pump, centrifuging the precipitate at 5000r/min for 30min, collecting supernatant, and mixing the supernatants to obtain yolk antibody extractive solution.
Example 4
Extraction of anti-turbot IL-1 beta yolk antibody
1. Washing anti-turbot IL-1 beta hyperimmune egg with normal temperature tap water for 1min, and disinfecting the washed egg with 0.25mg/L chlorine dioxide water solution for 1 min; spraying the sterilized egg with tap water for 2min, removing disinfectant, separating egg shell, egg white and yolk in an egg white and yolk separator, collecting yolk, weighing, and stirring at 300r/min for 10min to obtain yolk liquid;
2. adding the carbon dots of the novel zero-dimensional carbon-based nano material into an acetic acid-sodium acetate buffer solution with the concentration of 0.3M and the pH value of 5.1 to prepare a yolk antibody extraction buffer solution with the carbon dot concentration of 12.5 mug/mL;
3. adding a yolk antibody extraction buffer solution into the yolk solution according to the mass-volume ratio of 1:5, stirring at 400r/min for 10min, and standing and precipitating at room temperature for 9 h; sucking the supernatant with vacuum pump, centrifuging at 12000r/min for 20min, collecting supernatant, and mixing the supernatants to obtain yolk antibody extractive solution.
Example 5
Extraction of anti-turbot IL-1 beta yolk antibody
1. Washing anti-turbot IL-1 beta hyperimmune egg with normal temperature tap water for 1min, and disinfecting the washed egg with 0.25mg/L chlorine dioxide water solution for 2 min; spraying the sterilized egg with tap water for 2min, removing disinfectant, separating egg shell, egg white and yolk in an egg white and yolk separator, collecting yolk, weighing, and stirring at 400r/min for 10min to obtain yolk liquid;
2. adding the carbon dots of the novel zero-dimensional carbon-based nano material into an acetic acid-sodium acetate buffer solution with the concentration of 0.2M and the pH value of 5.1 to prepare a yolk antibody extraction buffer solution with the carbon dot concentration of 12.5 mug/mL;
3. adding a yolk antibody extraction buffer solution into the yolk solution according to the mass-volume ratio of 1:7, stirring at 300r/min for 10min, and standing and precipitating at room temperature for 10 h; sucking the supernatant with vacuum pump, centrifuging at 9000r/min for 30min, collecting supernatant, and mixing the supernatants to obtain yolk antibody extractive solution.
Example 6
Extraction of anti-turbot IL-1 beta yolk antibody
1. Washing anti-turbot IL-1 beta hyperimmune egg with normal temperature tap water for 1min, and disinfecting the washed egg with 0.25mg/L chlorine dioxide water solution for 2 min; spraying the sterilized egg with tap water for 2min, removing disinfectant, separating egg shell, egg white and yolk in an egg white and yolk separator, collecting yolk, weighing, and stirring at 400r/min for 10min to obtain yolk liquid;
2. adding the carbon dots of the novel zero-dimensional carbon-based nano material into an acetic acid-sodium acetate buffer solution with the concentration of 0.3M and the pH value of 5.1 to prepare a yolk antibody extraction buffer solution with the carbon dot concentration of 12.5 mug/mL;
3. adding a yolk antibody extraction buffer solution into the yolk solution according to the mass-to-volume ratio of 1:9, stirring at 200r/min for 30min, and standing and precipitating at room temperature for 15 h; sucking the supernatant with vacuum pump, centrifuging the precipitate at 4000r/min for 30min, collecting supernatant, and mixing the supernatants to obtain yolk antibody extractive solution.
Example 7
Extraction of anti-turbot IL-1 beta yolk antibody
1. Washing anti-turbot IL-1 beta hyperimmune egg with normal temperature tap water for 1min, and disinfecting the washed egg with 0.25mg/L chlorine dioxide water solution for 2 min; spraying the sterilized egg with tap water for 2min, removing disinfectant, separating egg shell, egg white and yolk in an egg white and yolk separator, collecting yolk, weighing, and stirring at 200r/min for 15min to obtain yolk liquid;
2. adding the carbon dots of the novel zero-dimensional carbon-based nano material into an acetic acid-sodium acetate buffer solution with the concentration of 0.1M and the pH value of 5.1 to prepare a yolk antibody extraction buffer solution with the carbon dot concentration of 25 mug/mL;
3. adding a yolk antibody extraction buffer solution into the yolk solution according to the mass-volume ratio of 1:5, stirring at 400r/min for 10min, and standing and precipitating at room temperature for 12 h; sucking the supernatant with vacuum pump, centrifuging at 12000r/min for 20min, collecting supernatant, and mixing the supernatants to obtain yolk antibody extractive solution.
Example 8
Extraction of anti-turbot IL-1 beta yolk antibody
1. Washing anti-turbot IL-1 beta hyperimmune egg with normal temperature tap water for 1min, and disinfecting the washed egg with 0.25mg/L chlorine dioxide water solution for 1 min; spraying sterilized egg with tap water for 2min, removing disinfectant, separating egg shell, egg white and yolk in an egg white and yolk separator, collecting yolk, weighing, and stirring at 100r/min for 20min to obtain yolk liquid;
2. adding the carbon dots of the novel zero-dimensional carbon-based nano material into an acetic acid-sodium acetate buffer solution with the concentration of 0.2M and the pH value of 5.1 to prepare a yolk antibody extraction buffer solution with the carbon dot concentration of 25 mug/mL;
3. adding a yolk antibody extraction buffer solution into the yolk solution according to the mass-to-volume ratio of 1:7, stirring at 300r/min for 30min, and standing and precipitating at room temperature for 10 h; sucking the supernatant with vacuum pump, centrifuging at 7000r/min for 30min to obtain supernatant, and mixing the supernatants to obtain yolk antibody extractive solution.
Example 9
Extraction of anti-turbot IL-1 beta yolk antibody
1. Washing anti-turbot IL-1 beta hyperimmune egg with normal temperature tap water for 1min, and disinfecting the washed egg with 0.25mg/L chlorine dioxide water solution for 1 min; spraying the sterilized egg with tap water for 2min, removing disinfectant, separating egg shell, egg white and yolk in an egg white and yolk separator, collecting yolk, weighing, and stirring at 300r/min for 10min to obtain yolk liquid;
2. adding the carbon dots of the novel zero-dimensional carbon-based nano material into an acetic acid-sodium acetate buffer solution with the concentration of 0.3M and the pH value of 5.1 to prepare a yolk antibody extraction buffer solution with the carbon dot concentration of 25 mug/mL;
3. adding a yolk antibody extraction buffer solution into the yolk solution according to the mass-to-volume ratio of 1:9, stirring at 300r/min for 15min, and standing and precipitating at room temperature for 15 h; sucking the supernatant with vacuum pump, precipitating, centrifuging at 6000r/min for 20min, collecting supernatant, and mixing the supernatants to obtain yolk antibody extractive solution.
Example 10
Extraction of yolk antibody against crucian gill hemorrhage virus (CyHV-2)
1. Washing high-immunity eggs for resisting crucian gill hemorrhage virus (CyHV-2) with normal temperature tap water for 1min, and disinfecting and cleaning eggs with 0.25mg/L chlorine dioxide water solution for 2 min; spraying the sterilized egg with tap water for 2min, removing disinfectant, separating egg shell, egg white and yolk in an egg white and yolk separator, collecting yolk, weighing, and stirring at 200r/min for 15min to obtain yolk liquid;
2. adding the carbon dots of the novel zero-dimensional carbon-based nano material into an acetic acid-sodium acetate buffer solution with the concentration of 0.1M and the pH value of 5.1 to prepare a yolk antibody extraction buffer solution with the carbon dot concentration of 6.25 mug/mL;
3. adding a yolk antibody extraction buffer solution into the yolk solution according to the mass-volume ratio of 1:9, stirring at 400r/min for 10min, and standing and precipitating at room temperature for 12 h; sucking the supernatant with vacuum pump, centrifuging the precipitate at 5000r/min for 30min, collecting supernatant, and mixing the supernatants to obtain yolk antibody extractive solution.
Example 11
Extraction of yolk antibody against crucian gill hemorrhage virus (CyHV-2)
1. Washing high-immunity eggs for resisting crucian gill hemorrhage virus (CyHV-2) with normal temperature tap water for 1min, and disinfecting and cleaning eggs with 0.25mg/L chlorine dioxide water solution for 1 min; spraying sterilized egg with tap water for 2min, removing disinfectant, separating egg shell, egg white and yolk in an egg white and yolk separator, collecting yolk, weighing, and stirring at 100r/min for 20min to obtain yolk liquid;
2. adding the carbon dots of the novel zero-dimensional carbon-based nano material into an acetic acid-sodium acetate buffer solution with the concentration of 0.1M and the pH value of 5.1 to prepare a yolk antibody extraction buffer solution with the carbon dot concentration of 12.5 mug/mL;
3. adding a yolk antibody extraction buffer solution into the yolk solution according to the mass-to-volume ratio of 1:9, stirring at 300r/min for 30min, and standing and precipitating at room temperature for 12 h; sucking the supernatant with vacuum pump, precipitating, centrifuging at 6000r/min for 30min, collecting supernatant, and mixing the supernatants to obtain yolk antibody extractive solution.
Example 12
Extraction of yolk antibody against crucian gill hemorrhage virus (CyHV-2)
1. Washing high-immunity eggs for resisting crucian gill hemorrhage virus (CyHV-2) with normal temperature tap water for 1min, and disinfecting and cleaning eggs with 0.25mg/L chlorine dioxide water solution for 2 min; spraying the sterilized egg with tap water for 2min, removing disinfectant, separating egg shell, egg white and yolk in an egg white and yolk separator, collecting yolk, weighing, and stirring at 300r/min for 10min to obtain yolk liquid;
2. adding the carbon dots of the novel zero-dimensional carbon-based nano material into an acetic acid-sodium acetate buffer solution with the concentration of 0.1M and the pH value of 5.1 to prepare a yolk antibody extraction buffer solution with the carbon dot concentration of 25 mug/mL;
3. adding a yolk antibody extraction buffer solution into the yolk solution according to the mass-to-volume ratio of 1:9, stirring at 300r/min for 15min, and standing and precipitating at room temperature for 15 h; sucking the supernatant with vacuum pump, precipitating, centrifuging at 7000r/min for 25min, collecting supernatant, and mixing the supernatants to obtain yolk antibody extractive solution.
In the above embodiments, the carbon dot preparation method comprises: the zero-dimensional carbon-based nano material with the size below 50nm is prepared from vitamin C by a one-step electrochemical method. The zero-dimensional carbon-based nano carbon dots with different concentrations are added into the purified IgY protein solution, and the influence of the carbon dots with different concentrations on the structure of the IgY protein is analyzed through circular dichroism chromatography, so that the protein structure and the stability of the IgY are basically not influenced when the concentration of the carbon dots is lower than 25 mu g/mL, the peak height between 190 and 210nm is slightly reduced when the concentration of the carbon dots reaches 50 mu g/mL, and the stability of the secondary structure of the IgY is slightly lower (see figure 1). Therefore, the nano carbon dots with low concentration below 25 mug/mL can be used for extracting the yolk antibody.
Second, result analysis
1. And (3) determining the protein content of the crude yolk antibody against the turbot IL-1 beta. The content of water-soluble protein in the yolk antibody extract was measured by using BCA protein concentration assay kit (purchased from Beijing Solebao Biotech Co., Ltd.), and the results are shown in Table 1.
TABLE 1 efficiency of extracting water-soluble protein from yolk (mg/g yolk) with different concentrations of carbon dots and different ratios of buffers
Note: all data with different shoulder letters indicated significant differences (P < 0.05).
As is clear from Table 1, the concentration of the water-soluble protein in the yolk antibody extract of each test group increased as the concentration of the carbon dots increased. Comprehensively comparing, when the carbon dot concentration is 25 mug/mL, the yolk antibody extraction effect of the group with the buffer ratio of 1:9 is the best, the extremely significant difference (P < 0.01) is achieved, and then the group with the buffer ratio of 1:7 (P <0.05) is achieved; protein extraction efficiency also reached significant levels (P <0.05) when the carbon spot concentration was 12.5. mu.g/mL and the buffer ratio was 1: 9. It is shown that the addition of carbon dots aids in the extraction of water-soluble proteins.
2. And (3) visually measuring the amount of IgY in the crude extract of the anti-turbot IL-1 beta yolk antibody. Because a certain amount of impure protein exists in the egg yolk antibody extracting solution, in order to further visually detect the extraction effect of IgY in the anti-turbot IL-1 beta egg yolk antibody extracting solution, the egg yolk antibody extracting solution is precipitated by using an ammonium sulfate solution (national drug group, AR level, CAS: 7783-20-2) with the mass-volume ratio of 19%, then the egg yolk antibody extracting solution is re-suspended by using PBS (0.1M, pH 7.2) buffer solution with the same volume as the egg yolk antibody extracting solution, and then SDS-PAGE electrophoresis is carried out to identify the antibody extraction effect, and the result is shown in figure 2. Wherein M: protein Marker, lanes 1-3: carbon spot concentration of 0. mu.g/mL, lanes 4-6 (examples 1-3): carbon spot concentration of 6.25. mu.g/mL, lanes 7-9 (examples 4-6), respectively: carbon spot concentrations of 12.5. mu.g/mL, lanes 10-12 (examples 7-9), respectively: respectively, the carbon dot concentration is 25 mug/mL; the yolk to buffer mass/volume ratio in lanes 1, 4, 7, 10 was 1:5, the yolk to buffer mass/volume ratio in lanes 2, 5, 8, 11 was 1:7, and the yolk to buffer mass/volume ratio in lanes 3, 6, 9, 12 was 1: 9.
As shown in FIG. 2, the SDS-PAGE electrophoresis of the yolk antibody shows the heavy IgY chain of about 66kDa, the light IgY chain of about 25kDa, and two hetero-protein bands of about 40 kDa. From the analysis of antibody purity (i.e., band gray scale), the antibody extraction effect was the best when the carbon spot concentration was 6.25. mu.g/mL in the same buffer solution ratio group, and the difference was not significant in the other groups, which were the second group with a concentration of 12.5. mu.g/mL. The result is inconsistent with the result of the content of the water-soluble protein in the yolk antibody extracting solution, which shows that the water-soluble hybrid protein of part of non-yolk antibodies is improved when the yolk antibody is extracted by the high-concentration carbon points, and the extraction of the yolk antibody IgY is more facilitated by the low-concentration carbon points.
3. And (3) measuring the titer of the IgY in the crude extract of the anti-turbot IL-1 beta yolk antibody and the anti-crucian gill hemorrhagic disease virus (CyHV-2) yolk antibody. The antibody titer in the yolk antibody extracting solution is determined by adopting an indirect ELISA method, namely recombinant proteins of turbot IL-1 beta and crucian gill hemorrhage virus CyHV-2(ORF72, ORF66, ORF81 and ORF82 tandem genes, namely CyHV-2-D4ORF) are respectively used as coating antigens, yolk antibody extracting solutions of anti-turbot IL-1 beta and anti-CyHV-2-D4 ORF with different dilutions are respectively used as primary antibodies, and the antibody titer in the yolk antibody extracting solution is determined by adopting a 1: and detecting the antibody titer by using HRP goat anti-chicken IgY diluted by 2000 as a secondary antibody, wherein a negative control is a yolk antibody extracted from a common egg under the same condition, and a blank control is PBS. The judgment standard is as follows: and (2) P/N (OD value to be measured-blank control OD value)/(negative control OD value-blank control OD value), and when P/N is more than or equal to 2.1, the maximum dilution of the antibody is the antibody titer. The results are shown in FIGS. 3 and 4, respectively.
As can be seen from the ELISA results in FIG. 3, when the ratio of yolk to the buffer solution was 1:5, the antibody titer in the yolk antibody extract of each group was increased from 1:16000 to 1:32000 in the control group by adding carbon dots to the acetic acid-sodium acetate buffer solution; when the ratio of the yolk to the buffer solution is 1:7, the antibody titer of the groups with the carbon spot concentration of 6.25 mug/mL and 12.5 mug/mL is improved from 1:16000 to 1:32000, but the antibody titer of the group with the carbon spot concentration of 25 mug/mL is not different from that of the group without the carbon spot; when the ratio of yolk to the buffer solution was 1:9, the antibody titer of the control group was 1:8000, the antibody titer of the group with a carbon spot concentration of 25. mu.g/mL was 1:16000, and the antibody titers of the groups with carbon spot concentrations of 6.25. mu.g/mL and 12.5. mu.g/mL reached 1: 32000. this result indicates that the addition of carbon dots contributes to the extraction of IgY, and that the extraction of IgY from low-concentration carbon dots is more effective.
The antibody titer in the egg yolk antibody extract against crucian gill hemorrhage virus (CyHV-2-D4ORF) was determined by ELISA method, and the results are shown in FIG. 4. When the ratio of yolk to buffer was 1:9, the antibody titers of the control group were 1:8000, and those of the groups with a carbon spot concentration of 6.25. mu.g/mL, 12.5. mu.g/mL and 25. mu.g/mL were 1:16000, 1:16000 and 1:8000, respectively. The results further verify that the effect of extracting IgY from the low-concentration carbon spots is best and the titer is obviously improved.
4. And (3) measuring the bacterial content in the crude egg yolk antibody extract for resisting crucian gill hemorrhage virus (CyHV-2). The total bacterial content of the carbon-point-treated egg yolk antibody extract against crucian gill hemorrhage virus (CyHV-2) was determined by plating with LB solid plates (example 10-12). Mixing yolk antibody extractive solution and yolk original fat precipitate, coating 100 μ L, culturing for 24 hr, and counting colony number.
The influence of carbon points with different concentrations on the total bacteria content in the egg yolk antibody extracting solution for resisting crucian gill hemorrhage virus (CyHV-2-D4ORF) is measured by an LB solid plate coating method, and the difference of the bacteria content of 15h of standing precipitation at room temperature and 4 ℃ when the processing parameters are the same. The results are shown in FIG. 5. The results show that: when other parameters are the same, the bacteria content of the antibody liquid extracted at room temperature is higher than that of the antibody liquid extracted at 4 ℃; after the carbon dots are added into the buffer solution, the content of bacteria in the yolk antibody extracting solution is obviously reduced (P is less than 0.05), and the colony number is reduced along with the increase of the concentration of the carbon dots until the growth of the bacteria is completely inhibited. The method can realize the extraction of the yolk antibody at room temperature under the condition of adding carbon points with certain concentration, and the carbon points can be degraded to cause no toxicity, so that byproducts after the yolk antibody is extracted can be further developed and utilized. Therefore, the technological parameters can be changed from 4 ℃ to room temperature after the nano material is added, and the method is simple and convenient. The utilization rate is improved.
And (4) conclusion: comprehensively evaluating the influence of carbon dots with different concentrations and buffer solutions with different proportions on the yolk antibody extraction effect, the invention finally determines that when the carbon dot concentration is 6.25-12.5 mug/mL, the ratio of the yolk solution to the buffer solution is 1: 5-1: 9, the yolk antibody can achieve better extraction effect. Further, when high concentrations of antibody are desired, the antibody can be further concentrated by other technical means.
The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Those skilled in the art should also realize that changes, modifications, additions and substitutions can be made without departing from the true spirit and scope of the invention.