阅读说明：本技术 一种包载卵黄免疫球蛋白的壳聚糖脂质体及其制备方法和用途 (Chitosan liposome for encapsulating yolk immunoglobulin and preparation method and application thereof ) 是由 蔡朝霞 陈晨 盛龙 黄茜 李小萌 夏民权 柳聪 董宛宜 于 2021-08-30 设计创作，主要内容包括：本发明涉及一种包载卵黄免疫球蛋白的壳聚糖脂质体及其制备方法和用途,所述脂质体包含以下组份:卵磷脂、胆固醇、IGY以及壳聚糖；其中,所述脂质体以卵磷脂和胆固醇形成的磷脂双分子层为脂质体骨架材料,所述IGY包埋于所述脂质体的内水相,所述壳聚糖包覆于所述脂质体中磷脂双分子层的内层表面和外层表面,所述卵磷脂选自蛋黄卵磷脂、大豆卵磷脂、磷脂酰胆碱、磷脂酰乙醇胺中的一种。基于本发明的脂质体稳定性好且包封率高。(The invention relates to a chitosan liposome for encapsulating yolk immunoglobulin, a preparation method and application thereof, wherein the liposome comprises the following components of lecithin, cholesterol, IGY and chitosan; the liposome takes a phospholipid bilayer formed by lecithin and cholesterol as a liposome skeleton material, the IGY is embedded in an internal water phase of the liposome, the chitosan is coated on the surface of an inner layer and the surface of an outer layer of the phospholipid bilayer in the liposome, and the lecithin is selected from one of egg yolk lecithin, soybean lecithin, phosphatidylcholine and phosphatidylethanolamine. The liposome based on the invention has good stability and high entrapment rate.)

1. A chitosan liposome entrapping yolk immunoglobulin is characterized in that the liposome comprises lecithin, cholesterol, yolk immunoglobulin and chitosan; the liposome takes a phospholipid bilayer formed by lecithin and cholesterol as a liposome skeleton material, the IGY is embedded in an internal water phase of the liposome, and the chitosan is coated on the surface of an inner layer and the surface of an outer layer of the phospholipid bilayer in the liposome; the lecithin is selected from one of egg yolk lecithin, soybean lecithin, phosphatidylcholine and phosphatidylethanolamine.

2. The yolk immunoglobulin-encapsulating chitosan liposome of claim 1, wherein the mass ratio of lecithin, cholesterol, IGY and chitosan is (18-22): (4-6): (1-1.5): (18-22).

3. The yolk immunoglobulin-encapsulating chitosan liposome of claim 1 or 2, wherein the lecithin is yolk lecithin.

4. A preparation method of chitosan liposome entrapping yolk immunoglobulin is characterized by comprising the following steps:

s1, dispersing lecithin and cholesterol in a buffer solution with the pH value of 7-8 to obtain a suspension A, dissolving IgY in the suspension A, and then dispersing a chitosan solution in the suspension A to obtain a suspension B; the concentration of lecithin in the suspension B is 5-10 mg/mL, and the content of chitosan is 0.5% -1.4%;

s2, pressurizing the suspension B to 5-25MPa at the temperature of 40-60 ℃, stirring for 0.5-2 h, incubating for 5-20 min, and decompressing to obtain the yolk immunoglobulin-encapsulated chitosan liposome suspension.

5. The method for preparing yolk immunoglobulin-encapsulating chitosan liposome of claim 4, wherein the buffer solution is phosphate buffered saline solution, and the pH value is 7.2-7.6.

6. The method for preparing yolk immunoglobulin-entrapped chitosan liposome according to claim 4, wherein the chitosan solution is prepared by dissolving chitosan in 1-2% acetic acid solution.

7. The method for preparing yolk immunoglobulin-entrapped chitosan liposome according to claim 4, wherein the process of dispersing chitosan solution in the suspension A is as follows: and adding the chitosan solution into the suspension A, continuously stirring for 0.5-2 h at 1000-1500 r/min, and performing ultrasonic treatment for 5-20 min to prepare a suspension B.

8. The method for preparing yolk immunoglobulin-encapsulating chitosan liposome of claim 4, wherein the stirring speed in S2 is 100-300 r/min.

9. The method for preparing yolk immunoglobulin-encapsulating chitosan liposome of claim 4, wherein the pressure release rate in S2 is 0.2-2 Mpa/min.

10. Use of yolk immunoglobulin-entrapped chitosan liposomes according to any of claims 1 to 3 as antibody food or food additive.

Technical Field

The invention belongs to the field of oral products, and particularly relates to a chitosan liposome entrapping yolk immunoglobulin, and a preparation method and application thereof.

Background

The application of IgY in food industry is diverse, mainly including development of functional foods, bacteriostasis and preservation, and immune rapid detection of pathogenic bacteria and functional components. A large number of researches show that the IgY in the eggs is protein with stronger immune function, and the IgY is used as a food additive or directly used as a raw material of antibody food to prepare functional food, which plays an immeasurable role in the aspects of preventing and treating diseases, regulating and enhancing the immunity of human bodies. IgY is a protein immune active substance, is quite stable to digestion of intestinal protease, is stable when the temperature reaches 65 ℃ at the pH value of 4-9, only part of IgY is inactivated when the pH value is less than 4, and both antitrypsin and chymotrypsin have stronger decomposition capacity. The encapsulation of IgY is embedded by physical, chemical or combined processes, etc. to prevent it from being decomposed by pepsin, and to improve the immunological activity when reaching the small intestine.

Chitosan is usually produced by chemical or enzymatic deacetylation of chitin, one of the most abundant natural polymers present in arthropod cuticles, making it very cheap. Chitosan acts as a penetration enhancer, regulating the intestinal barrier, and it opens the tight junctions between epithelial cells instantaneously, while acting only on mucosal surfaces for later clearance, thus providing a very promising material for effective oral delivery.

The traditional liposome preparation technology comprises a thin film hydration method, a reverse evaporation method and the like. The thin film hydration method firstly uses a round-bottom flask to evaporate an organic solvent and a lipid mixture to form a lipid thin film, then rehydration is carried out to form a liposome, if the rehydration is carried out by violent shaking, multi-layer vesicles with uneven size distribution can be formed, if the rehydration is carried out by mild hydration, macro-unilamellar vesicles are generated, if uniform vesicles are generated, subsequent operations such as probe ultrasound, water bath ultrasound or multiple extrusion through a polycarbonate thin film are required, and the method has the defects that: the entrapment rate of the hydrophilic material is low, organic solvent remains, pollution of subsequent operation and damage to liposome vesicles are caused; the reverse evaporation method comprises the steps of adding a lipid mixture into a flask, evaporating to obtain a lipid film, adding an organic phase (ethanol or isopropyl ether) to dissolve to form an inverted gel, adding an aqueous phase to form a water-in-oil two-phase system, performing ultrasonic dispersion, and finally evaporating an organic solvent under low pressure to form a liposome suspension.

SCCO₂Is a preferred solvent in the food and other industries because it is non-toxic, low cost, low processing temperature, non-flammable and easily separable from the extracted compounds by SCCO₂The liposome prepared by the auxiliary technology can solve the problem of solvent residue in conventional liposome microencapsulation. Otake et al by SCCO₂The chitosan-coated L-R-dipalmitoyl phosphatidylcholine cationic liposome is prepared by the technology, chitosan, lipid materials and water phase containing glucose are simultaneously put into a reaction kettle under the condition of not using acid or organic solvent (including ethanol), and then the mixture is converted into the liposome from the supercritical state under reduced pressure. Although the stable liposome is prepared, the maximum encapsulation rate of the prepared liposome is only 17 percent, the encapsulation rate is low, and the advantages of no organic solvent residue and the like exist, but the encapsulation rate and SCCO are comprehensively considered₂The requirement for high-voltage equipment, compared to conventional methods, uses SCCO₂The application of techniques for preparing liposomes is relatively limited.

Disclosure of Invention

The technical problem solved by the invention is as follows: provides a chitosan liposome entrapping yolk immunoglobulin, a preparation method and application thereof, which aim to solve the problems of wide size distribution, low entrapment rate, poor stability and organic solvent residue in the traditional liposome preparation method.

The specific solution provided by the invention comprises the following steps:

the invention provides a chitosan liposome (IgY-CS-LP) encapsulating yolk immunoglobulin, which comprises the following components of lecithin, Cholesterol (CHOL), yolk Immunoglobulin (IGY) and Chitosan (CS); the liposome takes a phospholipid bilayer formed by lecithin and cholesterol as a liposome skeleton material, the IGY is embedded in an internal water phase of the liposome, the chitosan is coated on the surface of an inner layer and the surface of an outer layer of the phospholipid bilayer in the liposome, and the lecithin is selected from one of egg yolk lecithin (EPC), soybean lecithin (SPC), phosphatidylcholine and phosphatidylethanolamine.

On the basis of the technical scheme, the invention can be further improved as follows:

further, the mass ratio of the lecithin to the cholesterol to the IGY to the chitosan is (18-22): (4-6): (1-1.5): (18-22).

Further, the lecithin is egg yolk lecithin.

The chitosan liposome entrapped with the yolk immunoglobulin has the following beneficial effects:

(1) the chitosan improves the membrane stability and mechanical strength of liposome IgY-CS-LP, has better embedding effect on an IgY structure, improves the stomach stability of the IgY, delays the release of the IgY and improves the activity of an antibody reaching the small intestine. According to the IgY-entrapped chitosan liposome, chitosan can exist in an inner liposome layer and an outer liposome layer, and due to the electrostatic interaction between the inner surface and the outer surface of the lipid bilayer membrane, the chitosan on the surface of the inner layer and the outer layer of the bilayer membrane has a fixing effect on phospholipid in the liposome membrane, so that the transverse movement of the phospholipid can be hindered, and the rigidity and the mechanical stability of the lipid membrane can be improved; the addition of the chitosan enables the surface of the liposome to have higher charges, improves the stability of the liposome, and the chitosan shell has certain viscosity, reduces the fluidity of the liposome, thereby further enhancing the stability of the liposome; the electrostatic action and the tackifying mechanism of the chitosan enable the liposome to have physical stability and enhance the slow release effect.

The invention also provides a preparation method of the chitosan liposome entrapping the yolk immunoglobulin, which comprises the following steps:

s1, dispersing lecithin and cholesterol in a buffer solution with the pH value of 7-8 to obtain a suspension A, dissolving IgY in the suspension A, and then dispersing a chitosan solution in the suspension A to obtain a suspension B; the concentration of lecithin in the suspension B is 5-10 mg/mL, and the content of chitosan is 0.5% -1.4%;

s2, pressurizing the suspension B to 5-25MPa at the temperature of 40-60 ℃, stirring for 0.5-2 h, incubating for 5-20 min, and decompressing to obtain the yolk immunoglobulin-encapsulated chitosan liposome (IgY-CS-LP) suspension.

Further, the buffer solution is phosphate buffer salt solution, and the pH value is 7.2-7.6.

Further, the chitosan solution is prepared by dissolving chitosan in 1% -2% acetic acid solution.

Further, the process of dispersing the chitosan solution in the suspension a is as follows: and adding the chitosan solution into the suspension A, continuously stirring for 0.5-2 h at 1000-1500 r/min, and performing ultrasonic treatment for 5-20 min to prepare a suspension B.

Further, the pressure relief rate in S2 is 0.2-2 Mpa/min.

The preparation method of the chitosan liposome entrapping the yolk immunoglobulin has the following beneficial effects:

(1) by using supercritical CO₂Auxiliary method for preparing IgY-entrapped chitosan liposome IgY-CS-LP, CO₂The liposome has the advantages of no toxicity, incombustibility, chemical stability, no residue and mild supercritical condition, and can be used as an encapsulation solvent of an unstable active substance IGY liposome without causing organic solvent residue.

(3) Given the risk that the lipid bilayer membrane of oral liposomes may leak in the gastrointestinal tract, the reinforcement of the lipid bilayer by the addition of a chitosan coating allows for more release of IgY in the intestinal environment, and the chitosan has a pH response, thereby enhancing the protective effect of IgY in the gastric environment.

(3)SCCO₂The solvent is used for dissolving phospholipid and uniformly dispersing the phospholipid in a system, the phospholipid spontaneously forms a lipid bilayer after pressure is released, and due to the electrostatic interaction between chitosan and lipid, positively charged chitosan is adsorbed with negatively charged phospholipid polar head in the liposome forming process, and adsorption layers are formed inside and outside the lipid bilayer to form a liposome with positive charges, so that the mechanical stability of the liposome is improved, the Zeta potential of the liposome in the finally obtained liposome suspension is higher than 40mV, and the stability of the liposome suspension is high.

(4) The chitosan liposome with high stability and high encapsulation rate is prepared by a phospholipid, cholesterol and chitosan one-pot method, and the method has the advantages of simple process and high production efficiency. In a buffer solution with the pH value of 7-8, IGY is dissolved in the buffer solution, the surface of the buffer solution is negatively charged (-about 17.3 mV), a chitosan solution mostly exists in the form of an aggregate, phospholipid molecules mainly exist in an aqueous medium in a double-layer or curvature sheet form, and cholesterol aggregates are randomly doped into the phospholipid double-layer; after pressurization, CO₂Rapidly dissolved in the aqueous phase with CO₂Molecule, HCO₃-, and H₂CO₃CO in the form of unhydrated₂The molecules will be partially trapped between the hydrophobic fatty acyl chains of the phospholipid bilayer and between the cholesterol molecules, resulting in an expansion of the length and thickness of the bilayer membrane, while CO is present₂The pH value of the buffer solution is slightly reduced by dissolution, the solubility of the chitosan aggregate is increased, and the dissolved chitosan ions and the surfaces of the negatively charged IGY and phospholipid bilayer in the buffer solution have electrostatic interaction respectively; after depressurization, CO₂The molecules will be released rapidly from the phospholipid bilayer, temporarily breaking the phospholipid bilayer into highly dispersed phospholipids and the cholesterol aggregates, chitosan aggregates will also break down into highly dispersed monomers, and this strong dispersion will lead to the formation of transient IgY solutions of discrete phospholipids, chitosan, cholesterol, with CO₂The pH value in the water phase is converted into an original state to form an instant solution; after the formation of the transient solution, the phospholipids, chitosan and cholesterol, which are temporarily separated, will rapidly recombine due to electrostatic and van der Waals forces, eventually assembling into lipidsIn the liposome of the IgY-CS-LP and IgY-CS-LP, chitosan exists an inner liposome layer and an outer liposome layer, on one hand, chitosan is coated on the surface of the outer liposome layer due to the electrostatic interaction with phospholipid in a large amount, the chitosan induces larger self-assembly aggregates on the outer surface of the liposome to form a chitosan coating, on the other hand, the chitosan electrostatically combined with the IGY enters a liposome vesicle along with an IGY solution and is then gradually adsorbed on the surface of the inner liposome layer, the higher chitosan concentration induces the larger self-assembly aggregates on the surface of the liposome to form a bonding layer with a certain thickness, wherein the chitosan is adsorbed on a polar head of a phospholipid bilayer to form the liposome with high electropositivity, and the chitosan on the surface of the outer liposome layer in the bilayer has a fixing effect on the phospholipid in the liposome membrane, so that the rigidity and the mechanical stability of the liposome membrane are obviously increased, the stability of the liposome is improved, so that the liposome with uniform particle size and high encapsulation efficiency is obtained.

The invention also provides the application of the chitosan liposome entrapping the yolk immunoglobulin, which is used as antibody food or food additive.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Drawings

FIG. 1 is a CLSM map of blank shell liposomes prepared from different phospholipids and of liposomes IgY-CS-LP.

FIG. 2 is a Zeta potential diagram of IgY, blank liposome EPC-CS and liposome EPC-CS-IGY.

FIG. 3 shows the sedimentation height of IgY-CS-LP of liposomes prepared at different chitosan concentrations over time.

FIG. 4 shows Zeta potentials of IgY-CS-LP liposomes prepared at different chitosan concentrations.

FIG. 5 shows the encapsulation efficiency of liposome IgY-CS-LP prepared at different chitosan concentrations.

FIG. 6 is a different SCCO₂DSC thermogram of liposome IgY-CS-LP prepared under pressure.

FIG. 7 is a different SCCO₂Infrared of liposomes IgY-CS-LP prepared under pressureA spectrogram.

Detailed Description

The following detailed description of embodiments of the invention is intended to be illustrative, and is not to be construed as limiting the invention.

The invention is described in detail below with reference to the figures and specific embodiments.

Example 1

The embodiment provides a preparation method of a chitosan liposome carrying yolk immunoglobulin, which specifically comprises the following steps:

(1) extracting IgY from yolk by water dilution and salting-out, freeze-drying to obtain IgY pure product, and freezing and storing in-20 deg.C refrigerator for use.

(2) Dispersing egg yolk lecithin (EPC) and cholesterol in 40mL of PBS (phosphate buffer solution) with the pH value of 7.4, slowly adding 10mL of IgY solution, finally adding into 50mL of chitosan solution, continuously stirring for 1h at 1200r/min by using a magnetic stirrer, and adsorbing chitosan on the surface of a phospholipid bilayer to obtain a suspension B, wherein the concentration of the chitosan in the suspension B is 1.1%, the concentration of the EPC is 6.28mg/mL, and the mass ratio of the phospholipid, the cholesterol and the IgY is 20:5: 1.2; wherein the chitosan solution is prepared by adding chitosan into 50ml of 1.0% acetic acid, heating to 50 ℃, sequentially stirring for 1h, and performing ultrasonic treatment for 10 min.

(3) Sealing 100mL of the suspension B in the step (2) in a high-pressure reaction kettle, controlling the temperature of the high-pressure reaction kettle to be 50 ℃ by using a water bath kettle, and then using CO₂Pressurizing to 10MPa, stirring at 200r/min for 1h, incubating for 10min, opening a valve of the high-pressure reaction kettle, and rapidly relieving pressure (0.5MPa/min) to obtain liposome IgY-CS-LP suspension.

Example 2

(1) Extracting IgY from yolk by water dilution and salting-out, freeze-drying to obtain IgY pure product, and freezing and storing in-20 deg.C refrigerator for use.

(2) Dispersing soybean lecithin (SPC) and cholesterol in 40mL of PBS (phosphate buffer solution) with the pH value of 7.4, slowly adding 10mL of IgY solution, finally adding into 50mL of chitosan solution, continuously stirring for 1h at 1200r/min by using a magnetic stirrer, adsorbing chitosan on the surface of a phospholipid bilayer layer to obtain a suspension B, wherein the concentration of the chitosan in the suspension B is 1.1%, the concentration of the SPC is 6.28mg/mL, and the mass ratio of the SPC, the cholesterol and the IgY is 20:5: 1.2; wherein the chitosan solution is prepared by adding chitosan into 50ml of 1.0% acetic acid, heating to 50 ℃, sequentially stirring for 1h, and performing ultrasonic treatment for 10 min.

(3) Sealing 100ml of the suspension B in the step (2) in a high-pressure reaction kettle, controlling the temperature of the high-pressure reaction kettle to be 50 ℃ by using a water bath kettle, and then using CO₂Pressurizing to 10MPa, stirring at 200r/min for 1h, incubating for 10min, opening a valve of the high-pressure reaction kettle, and rapidly relieving pressure (0.5MPa/min) to obtain liposome IgY-CS-LP suspension.

Example 3

The embodiment provides a preparation method of a chitosan liposome carrying yolk immunoglobulin, which specifically comprises the following steps:

(1) extracting IgY from yolk by water dilution and salting-out, freeze-drying to obtain IgY pure product, and freezing and storing in-20 deg.C refrigerator for use.

(2) Dispersing phosphatidylcholine and cholesterol in 40mL of PBS (phosphate buffer solution) with the pH value of 7.6, slowly adding 10mL of IgY solution, finally adding into 50mL of chitosan solution, continuously stirring for 0.5h at 1200r/min by using a magnetic stirrer, adsorbing chitosan on the surface of a phospholipid bilayer to obtain a suspension B, wherein the concentration of chitosan in the suspension B is 1.4%, the concentration of phosphatidylcholine is 10mg/mL, and the mass ratio of phosphatidylcholine to cholesterol to IgY is 22: 4: 1; wherein the chitosan solution is prepared by adding chitosan into 50ml of 1.0% acetic acid, heating to 50 ℃, sequentially stirring for 1h, and performing ultrasonic treatment for 10 min.

(3) Sealing the suspension B in the step (2) in a high-pressure reaction kettle, controlling the temperature of the high-pressure reaction kettle to be 40 ℃ by using a water bath kettle, and then using CO₂Pressurizing to 5MPa, stirring at 100r/min for 1h, incubating for 20min, opening a valve of the high-pressure reaction kettle, and rapidly relieving pressure (0.5MPa/min) to obtain liposome IgY-CS-LP suspension.

Example 4

The embodiment provides a preparation method of a chitosan liposome carrying yolk immunoglobulin, which specifically comprises the following steps:

(1) extracting IgY from yolk by water dilution and salting-out, freeze-drying to obtain IgY pure product, and freezing and storing in-20 deg.C refrigerator for use.

(2) Dispersing phosphatidylethanolamine and cholesterol in 40mL of PBS (phosphate buffer solution) with the pH value of 7.2, slowly adding 10mL of IgY solution, finally adding into 50mL of chitosan solution, continuously stirring for 2h at 1200r/min by using a magnetic stirrer, and adsorbing chitosan on the surface of a phospholipid bilayer to obtain a suspension B, wherein the concentration of the chitosan in the suspension B is 0.5 percent, the concentration of the phosphatidylethanolamine is 5mg/mL, and the mass ratio of the phosphatidylethanolamine, the cholesterol and the IgY is 18:6: 1.5; wherein the chitosan solution is prepared by adding chitosan into 50ml of 1.0% acetic acid, heating to 50 ℃, sequentially stirring for 1h, and performing ultrasonic treatment for 10 min.

(3) Sealing the suspension B in the step (2) in a high-pressure reaction kettle, controlling the temperature of the high-pressure reaction kettle to be 60 ℃ by using a water bath kettle, and then using CO₂Pressurizing to 25MPa, stirring for 1h at 300r/min, incubating for 5min, opening a valve of the high-pressure reaction kettle, and rapidly relieving pressure (0.5MPa/min) to obtain liposome IgY-CS-LP suspension.

Comparative example 1

The comparative example provides a chitosan liposome entrapping IgY, which specifically comprises the following steps:

(1) extracting IgY from yolk by water dilution and salting-out, freeze-drying to obtain IgY pure product, and freezing and storing in-20 deg.C refrigerator for use.

(2) Egg yolk lecithin (EPC) and cholesterol were dispersed in 40mL of a PBS solution with pH 7.4, 10mL of the IgY solution was slowly added, and the mixture was continuously stirred at 1200r/min for 1 hour using a magnetic stirrer to obtain a suspension having an EPC concentration of 6.28mg/mL and a mass ratio of phospholipids, cholesterol and IgY of 20:5: 1.2.

(3) Sealing the suspension in the step (2) in a high-pressure reaction kettle, controlling the temperature of the high-pressure reaction kettle to be 50 ℃ by using a water bath kettle, and then using CO₂Pressurizing to 20MPa, stirring at 200r/min for 1h, incubating for 10min, opening valve, and rapidly relieving pressure (0.5MPa/min) to obtain liposome suspension.

(4) Finally adding the chitosan into 50ml of chitosan solution, and continuously stirring for 1h at 1200r/min by using a magnetic stirrer, wherein the concentration of chitosan in the mixed solution is 1.1%; wherein the chitosan solution is prepared by adding chitosan into 50ml of 1.0% acetic acid, heating to 50 ℃, stirring for 1h and performing ultrasonic treatment for 10 min.

Comparative example 2

The comparative example provides a method for preparing a chitosan liposome entrapping yolk immunoglobulin based on a thin film dispersion method, which specifically comprises the following steps:

(1) extracting IgY from yolk by water dilution and salting-out, freeze-drying to obtain IgY pure product, and freezing and storing in-20 deg.C refrigerator for use.

(2) The preparation method comprises the following steps of preparing liposome by using egg yolk lecithin (EPC), cholesterol and IgY as raw materials and adopting a thin film dispersion method, wherein the mass ratio of the egg yolk lecithin (EPC), the cholesterol and the IgY is 20:5:1.2, and the preparation method comprises the following specific steps: dissolving phospholipid and cholesterol in anhydrous ethanol, removing ethanol at 45 deg.C with rotary evaporator after completely dissolving, adding PBS solution containing IgY, ultrasonic treating for 1min for convenient hydration, and hydrating for 30min to obtain liposome suspension.

Comparative example 3

The same as example 1, except that pure water was used in place of PBS buffer.

The test method comprises the following steps: measuring the particle size, particle size distribution and Zeta potential of the liposome by using a nano-particle size and Zeta potential analyzer; determining the content of IgY by adopting a Coomassie brilliant blue method to determine the entrapment rate; the phase transition temperature of the liposomes was tested using Differential Scanning Calorimetry (DSC); observing the micro morphology of the liposome by a confocal laser scanning electron microscope (CLSM); structural characterization of the starting material and liposomes was performed using fourier transform infrared spectroscopy (FT-IR).

Comparative example 4:

the same as example 1, except that the IgY concentration was 0%.

Comparative example 5

The same as example 2, except that the IgY concentration was 0%.

1. The liposome prepared in examples 1-2 and comparative examples 1-3 were tested for particle size, particle size distribution, Zeta potential and encapsulation efficiency, and the results are shown in table 1,

TABLE 1 test data Table for liposomes prepared in examples 1-2 and comparative examples 1-3

(1) As can be seen from Table 1, in example 1, the particle size increased from 2659.32 to 3629, the PDI increased from 0.48 to 0.21, the potential increased from 29.83mV to 44.63mV, and the encapsulation efficiency increased from 58.87% to 76.85% as compared to comparative example 1, indicating that the addition of chitosan followed by SCCO was performed first₂The chitosan is treated to better act on the inner layer surface and the outer surface of the liposome, so that the stability of the liposome is obviously improved, and the entrapment rate and the Zeta potential of the liposome are improved; as can be seen from the comparison of the data in example 1 and comparative example 2, the particle size of the liposome prepared by the membrane dispersion method is significantly smaller than SCCO₂The liposome prepared by the aid has poor liposome uniformity and obviously lower encapsulation effect than SCCO₂Auxiliary prepared liposome; compared with the PBS buffer solution, the liposome prepared under the pure water condition has smaller particle size, relatively poorer dispersibility and lower entrapment rate compared with the PBS buffer solution in example 1, because carbon dioxide is rapidly dissolved in the water phase after pressurization under the pure water condition, the pH of the solution is significantly reduced, chitosan is rapidly and massively dissolved to form highly dispersed monomers, the chitosan monomers are more likely to be adsorbed on the surface of phospholipid bimolecular layer compared with IGY (isoelectric point is 5.5) during stirring, the concentration of chitosan in the main body of the IGY solution is lower, and CO is reduced after depressurization₂The molecules are released from the phospholipid bilayer rapidly, and the highly dispersed monomer phospholipid, chitosan and cholesterol are recombined rapidly due to the action of electrostatic force and van der waals force to finally assemble the IgY-CS-LP liposomeLiposome IgY-CS-LP, liposome prepared in pure water, has unstable vesicular structure and uneven texture, resulting in large PDI and low encapsulation efficiency.

(2) As can be seen from the data of examples 1 and 2 in table 1, compared to the liposome prepared with SPC as the membrane material, the liposome prepared with EPC as the membrane material has larger particle size and better dispersibility and homogeneity, and EPC is a better IGY encapsulating material. Shown in FIG. 1, are confocal laser scanning electron micrographs of liposomes prepared in example 1(EPC-CS), example 2(SPC-CS), comparative example 4(EPC-CS-IGY) and comparative example 5(SPC-CS-IGY), respectively, it can be seen that the blank liposome EPC-CS and liposome EPC-CS-IGY prepared by using EPC as membrane material have larger particle size, and has better dispersibility and uniformity, probably because EPC belongs to animal lecithin, is mainly saturated phospholipid and can form a closely-arranged phospholipid membrane structure, SPC is a plant lecithin, most of which is unsaturated phospholipid, and cannot form a compact arrangement structure, therefore, the inner phospholipid layer of the lipid is likely to have larger curvature, a smaller inner water core is formed, and the particle size is smaller, and the larger particle size of the liposome prepared by the EPC is likely to be one of the reasons for higher encapsulation efficiency.

(3) As can be seen from Table 1, the phase transition temperature of the liposomes prepared in examples 1-2 is significantly higher than that of comparative examples 1-3, and the thermal stability of the liposomes is higher.

2. The Zeta potential test was performed on the liposomes prepared in IgY, example 1 and comparative example 4, respectively, and the results are shown in FIG. 2, the Zeta potential of IgY is-17.3 mV in PBS buffer, and there is no significant difference in Zeta potential between the vacant shell liposomes and the IgY-CS-LP liposomes, indicating that IgY is mainly encapsulated in the lipid bilayer membrane and thus has little effect on the surface charge of the liposomes.

3. The influence of the addition amount of the chitosan on the stability of the IgY-CS-LP liposome is explored

The preparation method of the IgY-CS-LP liposome is the same as that of example 1, except that the concentrations of chitosan are respectively controlled to be 0%, 0.5%, 0.8%, 1.1% and 1.4%.

The results of the sedimentation velocity test, the Zeta potential test and the encapsulation efficiency test of a series of IgY-CS-LP liposomes obtained by preparation are shown in figures 3-5. As shown in fig. 3, the sedimentation rate of the liposome without chitosan is the fastest, the sedimentation rate is slowed down along with the increase of the concentration of chitosan, the sedimentation rate of the liposome with the concentration of chitosan of 1.4% is the slowest, and the chitosan is beneficial to the stability of the liposome solution; as shown in figure 4, the Zeta potential of the IgY liposome after chitosan is added is obviously increased, the surface charge of the IgY-CS-LP is changed from negative charge to positive charge, and the surface charge is increased from-8.54 mV to 44.63 mV; the encapsulation efficiency results are shown in figure 5. The result shows that the chitosan has good stability, encapsulation efficiency and stability within the range of 0.5-1.4%.

4. Exploring different SCCOs₂Thermal stability and structural properties of IgY-CS-LP liposomes prepared under pressure.

The thermal stability of IGY, EPC and IgY-CS-LP liposomes prepared under different pressures was tested and the results are shown in FIG. 6. After the IgY is subjected to liposome encapsulation under the pressure of 5-25MPa, the phase transition temperature of the IgY is increased to a certain extent compared with that of the IgY (75.3 ℃), namely 114.7 ℃, 120.7 ℃, 138.9 ℃, 126.7 ℃ and 100.5 ℃, respectively, so that the liposome encapsulation has the effect of improving the thermal stability of the IgY, wherein the maximum phase transition temperature of the liposome is 138.9 ℃ under the pressure of 15MPa, the peak shape is narrow, and the liposome prepared under the pressure has relatively compact lipid accumulation and optimal thermal stability.

Exploration of different SCCOs by FT-IR₂The structural characteristics of IgY-CS-LP under pressure, the results are shown in FIG. 7, which is an infrared spectrum of IgY, Cholesterol (CHOL), Chitosan (CS), EPC and liposomes IgY-CS-LP prepared under different pressures, the IgY is at 3276cm^-1The strong absorption peak represents the stretching vibration of N-H and is 1632cm^-1、1529cm^-1The strong absorption peak at position (b) is a characteristic peak of the protein in the amide I, II band, and represents the stretching vibration of C ═ O and the bending deformation vibration of N — O, respectively; the EPC has a characteristic region comprising a polar hydrophilic head region, an interfacial region and a nonpolar hydrophobic tail region, and representative groups thereof are PO₂ ^-、C＝O、CH₂And CH₃The corresponding absorption peaks are 1241cm respectively^-1、1735cm^-1、2921cm^-1、2955cm^-1(ii) a ChitosanAt 3355cm^-1The left and right sides have a broad peak, which is formed by overlapping the stretching vibration of O-H and the stretching vibration absorption peak of N-H, and the width is 2872cm^-1The absorption peak represents the C-H stretching vibration absorption peak of methyl or methine; in addition, there is 1647cm^-1、1590cm^-1And 1320cm^-1The three characteristic absorption peaks respectively represent amide I, II and III bands; suspension B at 1559cm^-1、1408cm^-1The strong absorption peak is the characteristic peak of the protein in the amide II and III bands and respectively represents the bending vibration of C-H and the stretching vibration of C-H, which shows that the protein in the suspension B is not coated although the protein is changed; EPC at 1241cm^-1The absorption peak of (a) corresponds to the lecithin phosphate group, the phosphate group shifts and is significantly weakened after the suspension B is formed due to the electrostatic interaction between the phospholipid and the chitosan, and 1735cm of the carbonyl vibration of the fatty acid ester after the chitosan is added^-1The absorption band is significantly reduced, also indicating that there is a strong interaction between chitosan and lipids; carrying out SCCO₂After treatment, the positions of the characteristic peaks under the condition of 5-25MPa are basically consistent, and the characteristic peaks of chitosan and EPC are found to be at 3250-3275cm^-1A wider absorption peak indicates that hydroxyl groups form associations of intermolecular hydrogen bonds; and the characteristic peaks of the protein in the amide I, II and III bands are red-shifted and are obviously weakened compared with the suspension B, which can indicate that the suspension B is processed by SCCO in the method₂After hydration treatment, not only is the IgY physically mixed with other components simply, but the IgY is not completely encapsulated in a free state in a vesicular structure formed by lipids and chitosan, and a new phase is formed, which is also an important reason for the high encapsulation efficiency and stability of IgY.

Although embodiments of the present invention have been described in detail above, those of ordinary skill in the art will understand that: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.