CN116004019B - Preparation and application of gelatin carrier structure homopolymer formed by emulsifying gelatin and gamma protein film - Google Patents
Preparation and application of gelatin carrier structure homopolymer formed by emulsifying gelatin and gamma protein film Download PDFInfo
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- Peptides Or Proteins (AREA)
- Medicinal Preparation (AREA)
Abstract
The application relates to preparation and application of a gelatin carrier structure homopolymer formed by emulsifying gelatin and gamma protein films, which comprises the following steps: s1, preparing a modifier, modifying S2 gelatin, and crosslinking S3. In the application, the gelatin is modified, the basic amino group is blocked, and the isoelectric point of the gelatin is reduced, so that the gelatin can not disintegrate and swell under the strong acid condition, but can swell under the neutral bias condition, a carrier with pH response is formed, and the gelatin can be protected from being decomposed by acid liquid after being compounded with gamma protein, so that the activity of the gelatin is reduced. When the modified gelatin is compounded with gamma protein, the modified gelatin is emulsified and crosslinked in a manner of dripping polyethylene glycol and stearic acid aqueous solution, and the sedimentation of gamma protein and the speed of emulsifying sizing material can be controlled, so that gamma protein crosslinking and coating can be just realized, and the encapsulation rate is improved.
Description
Technical Field
The application relates to the technical field of biological products, in particular to preparation and application of a gelatin carrier structure homopolymer formed by emulsifying gelatin and gamma protein films.
Background
Egg yolk antibody (IgY) refers to the main serum antibody of birds, amphibians and reptiles, and at present, chicken IgY is the most widely used IgY, which is mainly produced by injecting egg-laying chicken through immunity, namely, the corresponding antibody can be extracted from egg yolk produced by the chicken IgY, and can be used for preventing and treating corresponding diseases, and the preparation is called egg yolk antibody. The egg yolk antibody gradually enters the chick embryo blood in the chick embryo hatching process, provides passive immune protection for the chick just coming out of the shell, and plays an important role in chick disease prevention. The main components in the yolk are protein and fat, and the ratio of the protein to the fat is 1:2. Most proteins are lipoproteins, present in egg yolk particles, insoluble in water, only vitellins (α, β, γ) are water soluble, and IgY is gamma vitellin (γ protein).
The IgY has the advantages of stable chemical property, strong specificity, simple preparation, high yield, low cost, green safety and small interference in immunodetection, and is widely applied to the aspects of food, medicine, biological products, biological detection and the like. IgY has been used in infant foods, middle-aged and elderly health products for improving the resistance of the human body to a certain disease, for example, in the food field; igY has been used in the pharmaceutical field for the control of gastrointestinal infectious diseases in humans and animals.
Egg yolk antibody IgY, although having many advantages, is also subject to a number of limitations in its use. IgY trypsin and chymotrypsin are resistant to a certain degree, but are very sensitive to acidic environments, especially acidic environments containing pepsin, are easy to deactivate gradually in acidic environments (pH less than or equal to 4), and are rapidly deactivated in conditions where pH less than or equal to 4.5 and pepsin is contained; the digestive absorption of proteins is mainly in the intestinal tract, and premature gastric inactivation can seriously affect IgY.
In order to improve the activity of IgY, the IgY is generally embedded or loaded by a carrier, so that the stability of the IgY is improved; currently, the carriers for IgY mainly include liposomes, polymers, etc.; however, liposome carriers tend to have a low entrapment rate and a poor effect on pepsin resistance; the polymer carrier has better embedding rate and protective effect on IgY activity, but has potential degradability and harm of being absorbed or not, so that the polymer carrier is difficult to be applied to the fields of foods and medicines.
Disclosure of Invention
In order to improve the activity and the utilization rate of IgY, the application provides the preparation and the application of a gelatin carrier structure homopolymer formed by emulsifying gelatin and gamma protein films.
In a first aspect, the present application provides a preparation method of a homopolymer of a gelatin carrier structure formed by emulsifying gelatin and a gamma protein film, which adopts the following technical scheme:
the preparation of the gelatin carrier structure homopolymer formed by emulsifying gelatin and gamma protein film comprises the following steps:
s1, preparing a modifier: adding salicylic acid into the solvent 1, then dropwise adding acetic anhydride for reflux reaction, and distilling the solvent 1 after the reaction is finished to obtain an intermediate 1; mixing the intermediate 1 with a solvent 2, adding thionyl chloride for reaction, distilling to remove the solvent 2 after the reaction is finished, purifying and drying to obtain a modifier;
the specific synthetic route pattern is as follows:
s2: gelatin modification: adding gelatin into water for swelling, adding NaOH to adjust the pH value to 8-9, adding the modifier in the step S1, then stirring for reaction, adding hydrochloric acid for sedimentation and washing after the reaction is finished, then adopting distilled water for swelling, adding NaOH to adjust the pH value to 6-7, washing and freeze-drying to obtain modified gelatin powder;
the specific synthetic route is as follows:
s3, crosslinking: adding the modified gelatin powder and the IgY in the step S2 into PBS buffer solution to prepare mixed solution; and (3) dripping the aqueous solution of polyethylene glycol and stearic acid into the mixed solution under the stirring condition to cause the aqueous solution to perform emulsification and crosslinking, adding hydrochloric acid after the reaction is finished to enable the aqueous solution to settle, centrifugally washing, and freeze-drying to obtain the gelatin carrier structure homopolymer formed by emulsifying gelatin and gamma protein membranes.
By adopting the technical scheme, the modifier containing the acyl chloride is firstly prepared, and the acyl chloride group reacts with an amino group on gelatin, so that the free amino group in the modifier is reduced, the amino group is blocked, the isoelectric point of the gelatin is reduced by pH 1.5-2.5, and thus, coagulation can occur under an acidic condition, and the decomposition and swelling of the gelatin under a strong acidic condition are reduced. In the application, after the modified gelatin is mixed with the IgY, polyethylene glycol and stearic acid are added to enable the modified gelatin and the IgY to be emulsified and crosslinked, the IgY is emulsified and crosslinked and embedded in the modified gelatin, and then acid is added to enable the modified gelatin to be coagulated, so that a gelatin carrier structure homopolymer formed by emulsifying gelatin and gamma protein films is formed. Gamma protein (IgY) in the homopolymer can be embedded in modified gelatin gel, and is converged and precipitated in gastric juice environment, so that the effect of protecting IgY is achieved, and the IgY can be released after swelling occurs in the biased neutral condition, so that the pH response release process can be realized in the coating process, the effect of protecting IgY can be achieved, and the utilization rate of IgY can be improved.
Preferably, in the step S1, the molar ratio of salicylic acid to acetic anhydride is 1 (0.75-1.5), the solvent 1 is one of acetone and tetrahydrofuran, and the solvent is added to 1/2-2/3 of the volume of the reaction vessel; the reflux reaction temperature is 60-80 ℃ and the reaction time is 2-4 h.
Through adopting above-mentioned technical scheme, through proportion and the reaction condition of control salicylic acid and acetic anhydride in this application, can make the hydroxy on the salicylic acid can be better take place the esterification with the anhydride, can promote its sedimentation performance under acidic condition.
Preferably, in the step S1, the mol ratio of the intermediate 1 to the thionyl chloride is 1 (1-1.5), the solvent 2 is one of chloroform and tetrahydrofuran, and the solvent is added to 1/2-2/3 of the volume of the reaction vessel; the reaction temperature is 65-75 ℃ and the reaction time is 3-6 h.
Through adopting above-mentioned technical scheme, through the proportion and the reaction condition of control intermediate 1 and thionyl chloride in this application, make the carboxyl on the benzene ring change into acyl chloride bond, reduce the content of impurity, promote its purity, consequently can be better take place the modification reaction with gelatin.
Preferably, in the step S2, the concentration of the gelatin in the water is 0.2-0.5 g/mL, the mass ratio of the gelatin to the modifier is 5 (2-3), the stirring reaction temperature is 40-50 ℃, and the reaction time is 30-60 min.
By adopting the technical scheme, the acyl chloride bond on the modifier reacts with the amino in the gelatin by controlling the reaction condition, so that the content of the residual amino in the gelatin is reduced, the isoelectric point of the gelatin is reduced, and the benzene ring and the ester bond of the modifier are easy to precipitate under the acidic condition, so that the gelatin is not swelled and disintegrated; under the condition of being in neutral, the residual carboxyl makes the carboxyl easy to swell, so that the pH targeted release of the carboxyl is realized.
Preferably, in the step S2, hydrochloric acid is added to adjust the pH to 3 to 4 for sedimentation, the supernatant is removed, and then the solution is continuously dissolved in water and adjusted to be neutral.
Through adopting above-mentioned technical scheme, adjust pH through hydrochloric acid in this application and make it take place to subside to get rid of the supernatant, can effectively get rid of impurity and modified incomplete gelatin, improve the pH effect of gelatin, follow-up continue to adjust with pure water and sodium hydroxide, make it be neutral, avoid in the dissolution, the gelatin acid value is too high, influences subsequent cladding process.
Preferably, in the step S3, the mass concentration of gelatin in the mixed solution is 2-6%, and the concentration of IgY is 0.04-0.08%; the concentration of the polyethylene glycol and the stearic acid aqueous solution is 0.5-0.8 g/mL and 0.4-0.6 g/mL respectively; the volume ratio of the mixed solution to the polyethylene glycol and the stearic acid aqueous solution is 1 (0.05-0.1).
Through adopting above-mentioned technical scheme, through mixing IgY and gelatin together, then drip polyethylene glycol and stearic acid into it in this application, polyethylene glycol can make IgY subside, and stearic acid can make the gelatin and IgY take place partial emulsification cross-linking, therefore make IgY crosslinked into the gelatin structure, through stirring effect, form microcapsule structure. In the prior emulsification process, stearic acid emulsifying crosslinking substances are generally directly added, but in the application, aqueous solution is dripped under the stirring condition, mainly for controlling the settling speed and emulsifying crosslinking speed of the IgY, so that the IgY is embedded in a gelatin structure as much as possible, and the encapsulation efficiency of the IgY is improved.
Preferably, in the step S3, the emulsification crosslinking temperature is 40 to 50 ℃, the stirring speed is 1000 to 2000rpm, and the emulsification crosslinking time is 15 to 25 minutes.
By adopting the technical scheme, the emulsion crosslinking temperature is controlled, so that the crosslinking effect can be ensured while the activity of the IgY is ensured; controlling the stirring speed helps to promote the embedding and crosslinking effects.
Preferably, in the step S3, hydrochloric acid is added to adjust the pH of the solution to 3 to 4 to precipitate the modified gelatin.
By adopting the technical scheme, the gelatin is settled by hydrochloric acid, and the IgY is already encapsulated in the modified gelatin, so that the activity of the IgY is not influenced; the solution can be better separated by coagulation.
In a second aspect, a homopolymer of gelatin carrier structure formed by emulsifying gelatin and gamma protein film is prepared according to the preparation method.
In a third aspect, the gelatin and gamma protein film emulsified gelatin carrier structure homopolymer is used as food and medicine field.
In summary, the present application includes at least one of the following beneficial technical effects:
1. in the application, the gelatin is modified, the basic amino group is blocked, and the isoelectric point of the gelatin is reduced, so that the gelatin can not disintegrate and swell under the strong acid condition, but can swell under the neutral bias condition, a carrier with pH response is formed, and the gelatin can be protected from being decomposed by acid liquid after being compounded with gamma protein, so that the activity of the gelatin is reduced.
2. When the modified gelatin is compounded with gamma protein, the modified gelatin is emulsified and crosslinked in a manner of dripping polyethylene glycol and stearic acid aqueous solution, and the sedimentation of gamma protein and the speed of emulsifying sizing material can be controlled, so that gamma protein crosslinking and coating can be just realized, and the encapsulation rate is improved.
3. In the application, the modified gelatin is adopted to crosslink, emulsify and embed the gamma protein, and the modified gelatin is combined with the gamma protein mainly through emulsification and crosslinking, so that the encapsulation rate of the modified gelatin is higher, the encapsulated homopolymer can protect and improve the stability of the gamma protein, and the targeted release and dilution of the modified gelatin in intestinal tracts are realized, so that the effect of the modified gelatin can be improved.
Drawings
FIG. 1 is a synthetic scheme for the modifier of the present application.
FIG. 2 is a reaction scheme of gelatin and modifier in the present application.
FIG. 3 the release profile of the homopolymers of example 1 and comparative examples 1 to 3 in simulated gastric fluid.
FIG. 4 Release curves of homopolymers from example 1 and comparative examples 1 to 3 in sodium hydroxide solution
Detailed Description
The egg yolk antibody used in the present application was anti-E.coli CMCC44102 egg yolk antibody purchased from Beijing millibiosciences, inc.
Example 1
S1, preparing a modifier: 30g of salicylic acid was added to a 250mL round-bottomed flask, followed by 150mL of acetone, the temperature was raised to 70℃and 20g of acetic anhydride was added dropwise thereto, after the completion of the dropwise addition, the reflux reaction was continued for 3 hours, and after the completion of the reaction, distillation was performed at 85℃to obtain intermediate 1. Adding 35g of intermediate 1 into 200mL of chloroform, stirring and mixing, adding 25.45g of thionyl chloride into the mixture, heating to 70 ℃, carrying out reflux reaction for 4 hours, absorbing waste in the reaction process by sodium hydroxide, distilling at 85 ℃ to remove a solvent after the reaction is finished, purifying and drying to obtain a modifier; the reaction scheme is shown in FIG. 1.
S2: adding 30g of gelatin powder into 100mL of aqueous solution, stirring and swelling for 30min, adding 2mol/L sodium hydroxide solution into the aqueous solution to adjust the pH to 8-9, adding 15g of modifier in the step S1, adding the aqueous solution to 45 ℃ for modification reaction for 45min, adding hydrochloric acid to adjust the pH of reaction solution to 3-4 after the reaction is finished, at the moment, gelatin can be subjected to coagulation, removing supernatant on the surface, adding pure water into the aqueous solution, adjusting the pH to 6-7 by using 2mol/L sodium hydroxide solution, centrifuging for multiple times, removing upper solution, adding water for 2 times of washing and centrifuging operation, and finally freeze-drying to obtain modified gelatin powder. The reaction process is shown in figure 2.
S3: 10g of modified gelatin powder is dissolved in 250mL of PBS (pH=7.4) buffer solution, then 0.15g of egg yolk antibody IgY is added into the solution, the solution is heated to 40 ℃ under the stirring speed of 1500rpm, 25mL of polyethylene glycol-400 and stearic acid aqueous solution (the concentration of polyethylene glycol-400 is 0.06g/mL and the concentration of stearic acid is 0.05 g/mL) are dropwise added into the solution, the reaction is continued for 20min after the dropwise addition, hydrochloric acid is added into the solution to adjust the pH to 3-4 after the reaction is completed, sedimentation is carried out on the solution, centrifugation and washing are carried out on the solution, and freeze-drying is carried out on the solution, thus obtaining the gelatin carrier structure homopolymer formed by emulsifying gelatin and gamma protein film.
Comparative example 1
The preparation method comprises the following steps:
10g of gelatin powder is dissolved in 250mL of PBS (pH=7.4) buffer solution, then 0.15g of egg yolk antibody IgY is added into the solution, the solution is heated to 40 ℃ under the stirring speed of 1500rpm, 25mL of polyethylene glycol-400 and stearic acid aqueous solution (the concentration of polyethylene glycol-400 is 0.06g/mL and the concentration of stearic acid is 0.05 g/mL) are dropwise added into the solution, the reaction is continued for 20min after the dropwise addition, the solution is centrifuged after the reaction is finished, part of supernatant is removed, and freeze-drying is directly carried out, so that a gelatin carrier structural homopolymer formed by emulsifying gelatin and gamma protein films is obtained.
Comparative example 2
Substantially the same as in example 1, except that the following differences exist in step S3:
s3: dissolving 10g of modified gelatin powder in 250mL of PBS (pH=7.4) buffer solution, adding 0.15g of egg yolk antibody IgY, heating to 40 ℃ at a stirring speed of 1500rpm, directly adding 25mL of polyethylene glycol-400 and stearic acid aqueous solution (the concentration of polyethylene glycol-400 is 0.06g/mL and the concentration of stearic acid is 0.05 g/mL), continuing to react for 20min after the addition, adding hydrochloric acid to adjust the pH to 3-4 after the reaction is finished, settling, centrifuging and washing, and freeze-drying to obtain a gelatin carrier structural homopolymer formed by emulsifying gelatin and gamma protein films.
Comparative example 3
Substantially identical to example 1, the difference is that step S1 is the following:
s1, preparing a modifier: adding 35g of salicylic acid into 200mL of chloroform, stirring and mixing, adding 25.45g of thionyl chloride into the mixture, heating the mixture to 70 ℃, carrying out reflux reaction for 4 hours, absorbing waste in the reaction process by sodium hydroxide, distilling the mixture at 85 ℃ to remove the solvent after the reaction is finished, purifying and drying the mixture to obtain the modifier.
Performance test:
encapsulation efficiency test:
adding 1g of prepared gelatin and gelatin carrier structure homopolymer formed by emulsifying a gamma protein membrane into a PBS buffer solution with pH of 6.8, swelling and releasing for 24 hours, centrifuging, removing supernatant, measuring the content of gamma protein (egg yolk antibody) in the solution by using a Folin-phenol method, and calculating the encapsulation efficiency according to an encapsulation efficiency formula. Encapsulation efficiency (%) = actual mass of egg yolk antibody in microcapsule/theoretical mass of egg yolk antibody in microcapsule x 100%.
pH responsive release:
2g of prepared gelatin and a gelatin carrier structure homopolymer formed by emulsifying a gamma protein membrane are added into 100mL of simulated gastric fluid (16.4 mL of diluted hydrochloric acid, 800mL of water and 10g of pepsin are added, shaking is carried out uniformly, then water is added for dilution into 1000mL, and the mixture is soaked for 4h, and the content of gamma protein (egg yolk antibody) in 0.5h,1h,2h,3h and 4h of the solution is tested.
2g of prepared gelatin and a gelatin carrier structure homopolymer formed by emulsifying a gamma protein membrane are added into 100mL of PBS solution with pH=6.8 for soaking and release, and the content of gamma protein (egg yolk antibody) in the solutions of 0.5h,1h,2h,3h and 4h is tested by the test solutions.
Activity test:
soaking 2g of prepared gelatin and gelatin carrier structural homopolymer formed by emulsifying gamma protein membrane in 20mL of simulated gastric fluid for 4h, centrifuging, removing gastric fluid, washing with pure water, adding 20mL of PBS buffer solution with pH of 6.8, swelling and releasing for 4h, testing the activity of egg yolk antibody in the solution, and taking the activity of egg yolk antibody with theoretical concentration (content actually measured in encapsulation efficiency) as the counterRatio. Activity retention% = test solution OD450nm Contrast solution OD450nm ×100％。
The activity test method is as follows:
the activity of IgY is determined by an indirect ELISA method, and the operation steps are as follows: adding 120 mu L of diluted antigen (the antigen is escherichia coli thallus broken liquid) into each well of a 96-well ELISA plate, and incubating at 4 ℃ overnight; each well was blocked by adding 250. Mu.L of BSA solution and incubated for 2h at 37 ℃; sequentially adding a gradient diluted sample (IgY initial concentration is 1mg/m L, gradient dilution is 1:1) and a blank control, and incubating for 2 hours at 37 ℃ in 100 mu L of each well; mu.L of rabbit anti-chicken Ig G-HRP (1:5000 dilution) was added to each well, incubated at 37℃for 40min, the liquid in the wells was dried at the end of each of the preceding steps, washed again with 300. Mu.L of PBST solution (PBS-0.05% Tween, adjusted p H to 7.4 with 1mol/L Na OH), repeated 5 times for 1min each, and patted dry on absorbent paper. The TMB substrate solution is prepared, 100 mu L of the TMB substrate solution is added into each hole for color development, and the mixture is placed in a 37 ℃ incubator for 15min for incubation, and the timing is accurate. After the reaction was completed, 50. Mu.L of 10% sulfuric acid was added to each well by a row gun to terminate the reaction, and the absorbance OD450nm was measured by an ELISA reader.
The encapsulation efficiency and activity test data in example 1 and comparative examples 1 to 3 are shown in table 1, and the response release data are shown in fig. 2 and 3:
TABLE 1
| Encapsulation efficiency (%) | Activity ratio (%) | |
| Example 1 | 85.1 | 71.18 |
| Comparative example 1 | 50.7 | 19.44 |
| Comparative example 2 | 64.5 | 61.44 |
| Comparative example 3 | 80.4 | 56.04 |
As can be seen from the data in table 1, the encapsulation efficiency and activity rate are highest in example 1, and the encapsulation efficiency and activity rate of the unmodified gelatin used in comparative example 1 are lower, and mainly the unmodified gelatin may not have the effect of coagulation shrinkage embedding, so that the encapsulation efficiency is lower; in comparative example 2, the polyethylene glycol-400 and the aqueous solution of stearic acid were not added dropwise, and the encapsulation efficiency and the activity rate were also lowered to some extent, probably because the egg yolk antibody was too fast in sedimentation rate and crosslinking rate, resulting in poor embedding and acid-resistant effects. In comparative example 3, the modification agent was mainly free from blocking hydroxyl groups, so that the embedding rate was slightly lowered, but the activity was more remarkably lowered, probably because the-OH group was not changed, so that the acid and pepsin resistance was deteriorated, and the activity rate was lowered to some extent.
As can be seen from the data in fig. 2 and 3, in the simulated gastric fluid, the release rate of the egg yolk antibody in example 1 was the lowest, and the release rate in comparative example 2 was the highest, reaching more than 60%, and comparative examples 2 and 3 were slightly improved compared with example 1; in fig. 3, the difference between the release of the neutral solution is not great in example 1 and comparative examples 1 to 3, and it can be seen from fig. 2 and 3 that the unmodified gelatin (comparative example 2) does not have a pH-responsive effect, whereas the pH-responsive effect is provided in both example 1 and comparative examples 2 and 3.
Example 2
S1, preparing a modifier: to a 250mL round-bottomed flask was added 30g of salicylic acid, followed by 150mL of tetrahydrofuran, and then heated to 65℃to which 30g of acetic anhydride was added dropwise, and after the completion of the dropwise addition, the reflux reaction was continued for 2 hours, and after the completion of the reaction, distillation was performed at 85℃to obtain intermediate 1. 35g of intermediate 1 is added into 200mL of chloroform, stirred and mixed, 27.53g of thionyl chloride is added into the mixture, the temperature is raised to 75 ℃, the reflux reaction is carried out for 3 hours, waste in the reaction process is absorbed by sodium hydroxide, after the reaction is finished, the solvent is distilled and removed at 85 ℃, and the modifier is obtained after purification and drying.
S2: adding 30g of gelatin powder into 120mL of aqueous solution, stirring and swelling for 40min, adding 2mol/L sodium hydroxide solution into the aqueous solution to adjust the pH to 8-9, adding 12g of modifier in the step S1, adding the aqueous solution to 50 ℃ for modification reaction for 30min, adding hydrochloric acid to adjust the pH of reaction solution to 3-4 after the reaction is finished, at the moment, gelatin can be subjected to coagulation, removing supernatant on the surface, adding pure water into the aqueous solution, adjusting the pH to 6-7 by using 2mol/L sodium hydroxide solution, centrifuging for multiple times, removing upper solution, adding water for 2 times of washing and centrifuging operation, and finally freeze-drying to obtain modified gelatin powder.
S3: 10g of modified gelatin powder is dissolved in 400mL of PBS (pH=7.4) buffer solution, then 0.32g of egg yolk antibody IgY is added into the solution, the solution is heated to 45 ℃ under the stirring speed of 1500rpm, 30mL of polyethylene glycol-400 and stearic acid aqueous solution (the concentration of polyethylene glycol-400 is 0.05g/mL and the concentration of stearic acid is 0.04 g/mL) are dropwise added into the solution, the reaction is continued for 15min after the dropwise addition, hydrochloric acid is added into the solution to adjust the pH to 3-4 after the reaction is finished, sedimentation is carried out on the solution, centrifugation and washing are carried out on the solution, and freeze-drying is carried out on the solution, thus obtaining the gelatin carrier structure homopolymer formed by emulsifying gelatin and gamma protein film.
Example 3
S1, preparing a modifier: 30g of salicylic acid was added to a 250mL round-bottomed flask, followed by 150mL of acetone, the temperature was raised to 75 ℃, 26g of acetic anhydride was added dropwise thereto, and after the completion of the dropwise addition, the reflux reaction was continued for 4 hours, and after the completion of the reaction, distillation was performed at 85℃to obtain intermediate 1. 35g of intermediate 1 is added into 200mL of chloroform, stirred and mixed, 29.82g of thionyl chloride is added into the mixture, the temperature is raised to 65 ℃, the reflux reaction is carried out for 5h, waste in the reaction process is absorbed by sodium hydroxide, after the reaction is finished, the solvent is distilled and removed at 85 ℃, and the modifier is obtained after purification and drying.
S2: adding 30g of gelatin powder into 80mL of aqueous solution, stirring and swelling for 60min, adding 2mol/L sodium hydroxide solution into the aqueous solution to adjust the pH to 8-9, adding 18g of modifier in the step S1, adding the aqueous solution to 50 ℃ for modification reaction for 50min, adding hydrochloric acid to adjust the pH of reaction solution to 3-4 after the reaction is finished, at the moment, gelatin can be subjected to coagulation, removing supernatant on the surface, adding pure water into the aqueous solution, adjusting the pH to 6-7 by using 2mol/L sodium hydroxide solution, centrifuging for multiple times, removing upper solution, adding water for 2 times of washing and centrifuging operation, and finally freeze-drying to obtain modified gelatin powder.
S3: 10g of modified gelatin powder is dissolved in 300mL of PBS (pH=7.4) buffer solution, then 0.24g of egg yolk antibody IgY is added into the solution, the solution is heated to 45 ℃ under the stirring speed of 2000rpm, 28mL of polyethylene glycol-400 and stearic acid aqueous solution (the concentration of polyethylene glycol-400 is 0.08g/mL and the concentration of stearic acid is 0.06 g/mL) are dropwise added into the solution, the reaction is continued for 25min after the dropwise addition, hydrochloric acid is added into the solution to adjust the pH to 3-4 after the reaction is completed, sedimentation is carried out on the solution, centrifugation and washing are carried out on the solution, and freeze-drying is carried out on the solution, thus obtaining the gelatin carrier structure homopolymer formed by emulsifying gelatin and gamma protein film.
The encapsulation efficiency and activity of homopolymers of gelatin carrier structures formed by emulsifying gelatin with gamma protein film in examples 2 and 3 were tested, and the results are shown in table 2.
TABLE 2
| Encapsulation efficiency (%) | Activity ratio (%) | |
| Example 2 | 84.7 | 67.2 |
| Example 3 | 81.5 | 74.9 |
As can be seen from the data in Table 2, the performance of examples 2 and 3, which varied from the process parameters, was somewhat floating, but remained essentially in a relatively stable range from the data point of view, and the egg yolk antibody was protected in simulated gastric fluid to maintain a relatively stable activity.
The foregoing are all preferred embodiments of the present application, and are not intended to limit the scope of the present application in any way, therefore: all equivalent changes in structure, shape and principle of this application should be covered in the protection scope of this application.
Claims (8)
1. The preparation method of the gelatin carrier structure homopolymer formed by emulsifying gelatin and gamma protein film is characterized by comprising the following steps:
s1, preparing a modifier: adding salicylic acid into the solvent 1, then dropwise adding acetic anhydride for reflux reaction, and distilling the solvent 1 after the reaction is finished to obtain an intermediate 1; mixing the intermediate 1 with a solvent 2, adding thionyl chloride for reaction, distilling to remove the solvent 2 after the reaction is finished, purifying and drying to obtain a modifier;
the specific synthetic route pattern is as follows:
s2: gelatin modification: adding gelatin into water for swelling, adding NaOH to adjust the pH value to 8-9, adding the modifier in the step S1, then stirring for reaction, adding hydrochloric acid for sedimentation and washing after the reaction is finished, then adopting distilled water for swelling, adding NaOH to adjust the pH value to 6-7, washing and freeze-drying to obtain modified gelatin powder;
the specific synthetic route is as follows:
s3, crosslinking: adding the modified gelatin powder and the IgY in the step S2 into PBS buffer solution to prepare mixed solution; dripping the aqueous solution of polyethylene glycol and stearic acid into the mixed solution under the stirring condition to cause the aqueous solution to have the emulsification and crosslinking effects, adding hydrochloric acid after the reaction is finished to enable the aqueous solution to be settled, centrifugally washing, and freeze-drying to obtain a gelatin carrier structure homopolymer formed by emulsifying gelatin and gamma protein films;
in the step S3, the mass concentration of gelatin in the mixed solution is 2-6%, and the concentration of IgY is 0.04-0.08%; the concentration of the polyethylene glycol and the stearic acid aqueous solution is 0.5-0.8 g/mL and 0.4-0.6 g/mL respectively; the volume ratio of the mixed solution to the polyethylene glycol and the stearic acid aqueous solution is 1 (0.05-0.1);
in the step S3, the emulsifying and crosslinking temperature is 40-50 ℃, the stirring speed is 1000-2000 rpm, and the emulsifying and crosslinking time is 15-25 min.
2. The method for preparing a homopolymer of a gelatin carrier structure formed by emulsifying gelatin and gamma protein film according to claim 1, wherein in the step S1, the molar ratio of salicylic acid to acetic anhydride is 1 (0.75-1.5), the solvent 1 is one of acetone and tetrahydrofuran, and the solvent is added to 1/2-2/3 of the volume of the reaction vessel; the reflux reaction temperature is 60-80 ℃ and the reaction time is 2-4 h.
3. The method for preparing a homopolymer of a gelatin carrier structure formed by emulsifying gelatin and gamma protein films according to claim 1, wherein in the step S1, the molar ratio of the intermediate 1 to thionyl chloride is 1 (1-1.5), the solvent 2 is one of chloroform and tetrahydrofuran, and the solvent is added to 1/2-2/3 of the volume of a reaction container; the reaction temperature is 65-75 ℃ and the reaction time is 3-6 h.
4. The method for preparing a homopolymer of a gelatin carrier structure formed by emulsifying gelatin and gamma protein film according to claim 1, wherein in the step S2, the concentration of gelatin in water is 0.2-0.5 g/mL, the mass ratio of gelatin to modifier is 5 (2-3), the temperature of stirring reaction is 40-50 ℃, and the reaction time is 30-60 min.
5. The method for preparing a homopolymer of gelatin carrier structure formed by emulsifying gelatin and gamma protein film according to claim 1, wherein in the step S2, hydrochloric acid is added to adjust the pH to 3-4 for sedimentation, the supernatant is removed, and then the solution is continuously dissolved in water and adjusted to be neutral.
6. The method for preparing a homopolymer of gelatin carrier structure formed by emulsifying gelatin and gamma protein film according to claim 1, wherein in the step S3, hydrochloric acid is added to adjust the pH of the solution to 3-4 so that the modified gelatin is settled.
7. A homopolymer of gelatin carrier structure formed by emulsifying gelatin and gamma protein film prepared by the preparation method according to any one of claims 1 to 6.
8. Use of a homopolymer of gelatin carrier structure formed by emulsification of gelatin and gamma protein film as claimed in claim 7 in the manufacture of food and pharmaceutical products.
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