Double emulsion formulation of W1 / O / W2 type embedding IgY and method for its preparation

Abstract

The application provides a W1 / O / W2 double emulsion preparation embedding IgY and a preparation method thereof, and the preparation method comprises the following steps: providing an IgY extract; dissolving an ion-responsive gel matrix in the IgY extract, stirring to obtain an inner water phase W1; dissolving a small molecule emulsifier and a first substrate in edible oil, adding lipase, incubating to obtain an oil phase O; mixing the inner water phase W1 with the oil phase O, adding gluconolactone, stirring, cooling to room temperature, and then emulsifying to obtain a water-in-oil primary emulsion W1 / O; dissolving a water activity regulator and a second substrate in water to obtain an outer water phase W2; adding the water-in-oil primary emulsion W1 / O to the outer water phase W2, emulsifying, and then incubating, deactivating the enzyme to obtain the W1 / O / W2 double emulsion preparation embedding IgY. The application can significantly improve the structural stability of the double emulsion and improve the oral availability of IgY.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a W1 / O / W2 type dual emulsion formulation encapsulated with IgY and its preparation method. Background Technology

[0002] Egg yolk immunoglobulin (IgY) is the main immunoglobulin in avian serum. It reacts with most antigens, is produced upon stimulation by specific antigens, and is selectively transferred into the yolk via specific receptors on the surface of the yolk membrane through blood circulation. IgY has advantages such as high accuracy in immunodetection, strong responsiveness, good water solubility, and good temperature stability, and has significant application potential in areas such as infection prevention, immune regulation, and antibiotic alternatives. IgY consists of two heavy chains and two light chains, forming a Y-shaped structure with a total molecular weight of approximately 180 kDa. It is divided into a Fab moiety that binds to antigens and antibodies and an Fc moiety that interacts with cell surface receptors (Fc) and activates the immune system. The site for specific antigen recognition and binding is located at the end of the light chain. However, the highly acidic conditions in the gastric environment during digestion and the presence of pepsin can severely impair the activity of IgY, resulting in poor bioavailability for oral administration. The pH conditions in the industrial production environment of products also pose a significant threat to IgY activity, seriously hindering its application.

[0003] Given the unique environmental dependence of IgY, various encapsulation strategies have been developed to improve its stability and protect its immune activity. Among them, water-in-oil-in-water (W1 / O / W2) double emulsions, with their bilayer structure, can encapsulate highly water-soluble IgY within the inner aqueous phase, thereby increasing its loading capacity and providing a protective effect during gastric digestion, achieving targeted release into the intestine. However, while the multiple structures of water-in-oil-in-water (W1 / O / W2) double emulsions offer promising applications, they also introduce problems such as poor stability during storage, leading to a more complex mechanism for emulsion system instability. Summary of the Invention

[0004] In view of this, the present invention provides a W1 / O / W2 type dual emulsion formulation encapsulated with IgY and a method for preparing the same, which can significantly improve the structural stability of the dual emulsion and improve the oral accessibility of IgY.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a method for preparing a W1 / O / W2 type dual emulsion formulation encapsulated with IgY, comprising the following steps: S1. Provide IgY extraction solution; S2. Dissolve the ion-responsive gel matrix in the IgY extract and stir to obtain the internal aqueous phase W1; wherein the ion-responsive gel matrix includes polysaccharides and calcium ion source; S3. Dissolve the small molecule emulsifier and the first substrate in edible oil, add lipase, and incubate to obtain oil phase O; wherein, the first substrate includes at least one of polyglycerol-10 laurate, polyglycerol-10 oleate, ethyl laurate, and glyceryl monolaurate. S4. Mix the aqueous phase W1 with the oil phase O, add gluconolactone, stir, cool to room temperature, and then emulsify to obtain a water-in-oil primary emulsion W1 / O. S5. Dissolve the water activity regulator and the second substrate in water to obtain an external aqueous phase W2; wherein the second substrate includes at least one of a natural macromolecular protein and its hydrolyzed peptides; S6. Add the water-in-oil primary emulsion W1 / O to the external aqueous phase W2, emulsify, then incubate to inactivate the enzyme, and obtain a W1 / O / W2 type dual emulsion formulation encapsulating IgY.

[0006] Preferably, step S1 includes: S11. Separate the yolk from the egg, wash with water, break the yolk membrane, filter, and obtain yolk liquid; S12. Mix the egg yolk liquid with water, freeze, thaw overnight, centrifuge to obtain IgY extract.

[0007] Preferably, in step S2: The polysaccharide includes at least one of sodium alginate and low-methoxyl pectin; the calcium ion source includes calcium carbonate; and / or, The proportion of the ion-responsive gel matrix in the internal aqueous phase W1 is 1~5 wt%.

[0008] Preferably, in step S3: The small molecule emulsifier includes at least one of polyglycerol ricinoleate, Span derivatives, polyol fatty acid esters, and phospholipids; and / or, The lipase includes at least one of the following: Candida antarcticis lipase B (CALB) and its lipase-like enzymes, Ustilago maydis lipase B and its lipase-like enzymes, and bacterial lipases with structural similarity to Candida antarcticis lipase B (CALB); and / or, The edible oil includes at least one of sunflower seed oil, soybean oil, and rapeseed oil.

[0009] Preferably, in step S3: The proportion of the small molecule emulsifier in the oil phase O is 1~10 wt%; and / or, The proportion of the first substrate in the oil phase O is 1~5 wt%; and / or, The proportion of the lipase in the oil phase O is 0.5~5 wt%.

[0010] Preferably, in step S4: The mass ratio of the internal aqueous phase W1 to the oil phase O is (1~5):1; and / or, The mass-to-volume ratio of the gluconolactone to the internal aqueous phase W1 is (1~2) g: 8 mL.

[0011] Preferably, in step S5: The water activity regulator includes at least one of small molecule sugars, sugar alcohols, polyols, and small molecule salts; and / or, The natural macromolecular protein includes at least one of sodium caseinate, whey protein concentrate, soy protein isolate, pea protein, and yeast protein.

[0012] Preferably, in step S5: The water activity regulator in the external aqueous phase W2 accounts for 8-15 wt%; and / or, The proportion of the second substrate in the external aqueous phase W2 is 0.1~10 wt%.

[0013] Preferably, in step S6, the mass ratio of the external aqueous phase W2 to the water-in-oil primary emulsion W1 / O is (1~5):1.

[0014] In a second aspect, the present invention also provides a W1 / O / W2 type dual emulsion formulation encapsulating IgY, which is prepared by the preparation method described in the first aspect.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The preparation method of the present invention yields a W1 / O / W2 type dual emulsion formulation with encapsulated IgY based on the dual stabilization mechanism of interfacial condensation and internal aqueous phase gelation. The dual stabilization mechanism of interfacial condensation and internal aqueous phase gelation can control each phase in the dual emulsion layer by layer, significantly improving the structural stability of the dual emulsion and maintaining its multilayer structure under storage, processing, gastric digestion and other conditions. At the same time, encapsulating IgY in the internal aqueous phase of the dual emulsion, the oil film and interfacial film can keep IgY active during processing and gastric digestion, reduce the influence of the external environment on IgY, and improve the oral accessibility of IgY.

[0016] (2) The preparation method of the present invention is simple and can effectively reduce costs. It has important application value for enhancing the bioaccessibility of oral IgY products and ensuring the active function of oral IgY health products. The prepared IgY-encapsulated W1 / O / W2 type double emulsion formulation can be used in food, cosmetics, oral care products and animal health products such as ice cream, popsicles, yogurt, cheese, milk powder and beverages, with broad application prospects. Attached Figure Description

[0017] Figure 1Laser microscope image of the dual emulsion formulation of Example 1 provided by the present invention; Figure 2 The graph shows the change in IgY encapsulation efficiency during storage of the double emulsion formulations of Examples 1-5 and Control Groups 2-4 provided by this invention; Figure 3 The graph shows the change of the solid-liquid balance point (SLB) index of the double emulsion formulations of Examples 1-5 and Control Groups 2-4 provided by the present invention during storage. Figure 4 The graph shows the change in elasticity factor (EI) index of the double emulsion formulations of Examples 1-5 and Control Groups 2-4 provided by the present invention during storage. Figure 5 The graph shows the changes in the macroscopic viscosity factor (MVI) index of the dual emulsion formulations of Examples 1-5 and Control Groups 2-4 provided by the present invention during storage. Figure 6 The graph shows the change of the flow factor (FI) index of the double emulsion formulations of Examples 1-5 and Control Groups 2-4 provided by the present invention during storage. Figure 7 The graph shows the changes in IgY activity retention rate of the double emulsions of Examples 1-5, Control Groups 2-4, and Control Group 1 under different pH conditions provided by the present invention. Figure 8 The graph shows the changes in IgY activity retention rate after different in vitro simulated digestion stages of the double emulsions of Examples 1-5, Control Groups 2-4, and Control Group 1 provided by the present invention. Detailed Implementation

[0018] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can more clearly understand the present invention.

[0019] In a first aspect, the present invention provides a method for preparing a W1 / O / W2 type dual emulsion formulation encapsulated with IgY, comprising the following steps: S1. Provide IgY extraction solution; S2. Dissolve the ion-responsive gel matrix in the IgY extract and stir to obtain the internal aqueous phase W1; wherein the ion-responsive gel matrix includes polysaccharides and calcium ion source; S3. Dissolve the small molecule emulsifier and the first substrate in edible oil, add lipase, and incubate to obtain oil phase O; wherein, the first substrate includes at least one of polyglycerol-10 laurate (PG-10L), polyglycerol-10 oleate, ethyl laurate, and glyceryl monolaurate (GML). S4. Mix the aqueous phase W1 with the oil phase O, add gluconolactone, stir, cool to room temperature, and then emulsify to obtain a water-in-oil primary emulsion W1 / O. S5. Dissolve the water activity regulator and the second substrate in water to obtain an external aqueous phase W2; wherein the second substrate includes at least one of a natural macromolecular protein and its hydrolyzed peptides; S6. Add the water-in-oil primary emulsion W1 / O to the external aqueous phase W2, emulsify, then incubate to inactivate the enzyme, and obtain a W1 / O / W2 type dual emulsion formulation encapsulating IgY.

[0020] In this invention, ion crosslinking allows the inner aqueous phase loaded with IgY to form a three-dimensional network via calcium ion bridges, causing a gelation reaction. A primary emulsion of water-in-oil is then formed through initial emulsification, followed by secondary emulsification with the outer aqueous phase to form a dual emulsion. An interfacial condensation reaction, utilizing the first substrate in the oil phase and the second substrate in the outer aqueous phase, catalyzed by lipase, forms a polyamide interfacial film at the oil-water interface. This yields a W1 / O / W2 type dual emulsion formulation encapsulated with IgY, based on a dual stabilization mechanism of interfacial condensation and inner aqueous phase gelation. This dual stabilization mechanism of interfacial condensation and inner aqueous phase gelation allows for layer-by-layer control of each phase in the dual emulsion, significantly improving its structural stability and maintaining its multilayer structure under storage, processing, and gastric digestion conditions. Simultaneously, encapsulating IgY within the inner aqueous phase of the dual emulsion, along with the oil and interfacial films, ensures IgY activity during processing and gastric digestion, reducing the impact of the external environment on IgY and improving its oral accessibility.

[0021] It should be noted that internal aqueous phase gelation is a method of immobilization. Through calcium ion bridge crosslinking, the internal aqueous phase is transformed from a liquid state to a gel or gel-like state, reducing the fluidity of the internal aqueous phase and enabling it to be uniformly dispersed in the oil phase in a stable manner for a long time. This avoids problems such as aggregation, seepage, migration, and Ostwald ripening of the internal aqueous phase. Interfacial condensation is the process at the oil-water interface between the external aqueous phase and the water-in-oil primary emulsion. Under the catalysis of lipase, amino and ester groups undergo an interfacial condensation reaction to form a continuous and robust interfacial film, which can stabilize the oil phase and prevent problems such as aggregation, precipitation, and rupture. The order of steps (S1~S2) for preparing the internal aqueous phase W1, step (S3) for preparing the oil phase O, and step (S5) for preparing the external aqueous phase W2 is not specifically limited. In step S3, the first substrate is preferably polyglycerol-10 laurate.

[0022] In some embodiments, step S1 includes: S11. Separate the yolk from the egg, wash with water, break the yolk membrane, filter, and obtain yolk liquid; S12. Mix the egg yolk liquid with water, freeze, thaw overnight, centrifuge to obtain IgY extract.

[0023] In some embodiments, in step S12, the volume ratio of the yolk fluid to water is 1:(3~10). Further, the volume ratio of the yolk fluid to water is preferably 1:5.

[0024] In some embodiments, the freezing time in step S12 is 0.5 to 8 hours. Further, the freezing time is preferably 1 to 2 hours.

[0025] In some embodiments, in step S12, the overnight thawing temperature is 0~4 ℃.

[0026] In some embodiments, in step S12, the centrifugation temperature is 0~4 ℃, the rotation speed is 8000~12000 r / min, and the time is 15~45 min.

[0027] In some embodiments, in step S2, the polysaccharide includes at least one of sodium alginate and low-methoxyl pectin, and the calcium ion source includes calcium carbonate (CaCO3). Further, the ion-responsive gel matrix is ​​preferably composed of sodium alginate and calcium carbonate, wherein in the internal aqueous phase W1, the proportion of sodium alginate is 0.5~2.5 wt%, and the proportion of calcium carbonate is 0.5~2.5 wt%.

[0028] In some embodiments, in step S2, the proportion of the ion-responsive gel matrix in the inner aqueous phase W1 is 1~5 wt%.

[0029] In some embodiments, in step S3, the small molecule emulsifier includes at least one of polyglycerol ricinoleate (PGPR), Span derivatives, polyol fatty acid esters, and phospholipids. It should be noted that the Span derivative can be Span 80, the polyol fatty acid ester can be glyceryl monostearate, and the phospholipid can be lecithin; furthermore, the small molecule emulsifier is preferably polyglycerol ricinoleate.

[0030] In some embodiments, in step S3, the lipase includes at least one of *Candida antarcticis* lipase B (CALB) and its lipase-like enzymes, *Ustilago maydis* lipase B and its lipase-like enzymes, and bacterial lipases with structural similarity to *Candida antarcticis* lipase B (CALB). It should be noted that bacterial lipases with structural similarity to *Candida antarcticis* lipase B (CALB) may include at least one of *Bacillus subtilis* lipase, *Streptococcus thermophilus* lipase, *Pseudomonas* lipase, *Streptomyces* lipase, and *Enterobacter* lipase.

[0031] In some embodiments, in step S3, the edible oil includes at least one of sunflower seed oil, soybean oil, and rapeseed oil.

[0032] In some embodiments, in step S3, the proportion of the small molecule emulsifier in the oil phase O is 1~10 wt%.

[0033] In some embodiments, in step S3, the proportion of the first substrate in the oil phase O is 1~5 wt%.

[0034] In some embodiments, in step S3, the proportion of the lipase in the oil phase O is 0.5~5 wt%.

[0035] In some embodiments, in step S3, the method of dissolving the small molecule emulsifier and the first substrate in edible oil is to add the small molecule emulsifier and the first substrate to the edible oil at 30~50 °C and stir for 20~45 min.

[0036] In some embodiments, in step S3, lipase is added and incubated by adding lipase at 40-50 °C and in the dark, and stirring at 300-400 r / min for 20-40 min to ensure that the lipase is fully dissolved and suitable for catalyzing the interfacial condensation reaction.

[0037] In some embodiments, in step S4, the mass ratio of the internal aqueous phase W1 to the oil phase O is (1~5):1.

[0038] In some embodiments, in step S4, the mass-to-volume ratio of the gluconolactone (GDL) to the internal aqueous phase W1 is (1~2) g: 8 mL.

[0039] In some embodiments, in step S4, the method for mixing the internal aqueous phase W1 with the oil phase O is to add the internal aqueous phase W1 to the oil phase O while stirring at 40~50°C, and then continue stirring for 2~5 minutes.

[0040] In some embodiments, in step S4, after adding gluconolactone (GDL) at 40-50 °C, the mixture is stirred for 1-3 min and then cooled to room temperature.

[0041] In some embodiments, in step S4, the emulsification is performed using a low-energy emulsification method or a high-energy emulsification method. The low-energy emulsification method includes phase transition method, spontaneous emulsification method or membrane emulsification method, and the high-energy emulsification method includes high-speed shearing, high-pressure homogenization emulsification or microfluidic emulsification.

[0042] In some embodiments, in step S4, the emulsification is carried out using a high-energy emulsification method, wherein the high-energy emulsification method is shearing at a rotation speed of 10000~15000 r / min for 8~15 min.

[0043] In some embodiments, in step S5, the water activity regulator includes at least one of small molecule sugars, sugar alcohols, polyols, and small molecule salts. It should be noted that the small molecule sugar can be trehalose, the sugar alcohol can be sorbitol, the polyol can be glycerol, and the small molecule salt can be sodium chloride (NaCl); furthermore, the water activity regulator is preferably trehalose.

[0044] In some embodiments, in step S5, the natural macromolecular protein includes at least one selected from sodium caseinate, whey protein concentrate, soy protein isolate (SPI), pea protein, and yeast protein. Further, the second substrate is preferably sodium caseinate.

[0045] In some embodiments, in step S5, the proportion of the water activity regulator in the external aqueous phase W2 is 8~15wt%.

[0046] In some embodiments, in step S5, the proportion of the second substrate in the external aqueous phase W2 is 0.1~10 wt%.

[0047] In some embodiments, after the water activity regulator and the second substrate are dissolved in water, they are allowed to stand at 0-4 °C for 30-60 min to achieve full hydration.

[0048] In some embodiments, in step S6, the mass ratio of the external aqueous phase W2 to the water-in-oil primary emulsion W1 / O is (1~5):1.

[0049] In some embodiments, in step S6, the emulsification is performed using a low-energy emulsification method or a high-energy emulsification method. The low-energy emulsification method includes phase transition method, spontaneous emulsification method, or membrane emulsification method. The high-energy emulsification method includes high-speed shearing, high-pressure homogenization emulsification, or microfluidic emulsification.

[0050] In some embodiments, in step S6, the emulsification is performed using a high-energy emulsification method, wherein the high-energy emulsification method is shearing at a rotation speed of 4000~6000 r / min for 3~5 min.

[0051] In some embodiments, in step S6, the incubation temperature is 35~45 ℃ and the time is 2~4 h.

[0052] In some embodiments, in step S6, the enzyme inactivation method is to inactivate the enzyme by heating at 90~98 °C for 0.5~2 min.

[0053] Secondly, the present invention also provides a W1 / O / W2 type dual emulsion formulation encapsulating IgY, which is prepared by the preparation method described in the first aspect.

[0054] Thirdly, the present invention also provides the application of the W1 / O / W2 type dual emulsion formulation for encapsulating IgY as described in the second aspect in IgY health products.

[0055] In some embodiments, the IgY health products include ice cream, popsicles, yogurt, cheese, milk powder, beverages, cosmetics, oral care products, or animal health products.

[0056] Example 1 This embodiment provides a method for preparing a W1 / O / W2 type dual emulsion formulation encapsulated with egg yolk immunoglobulin (IgY) based on a dual stabilization mechanism of interfacial condensation and internal aqueous phase gelation, comprising the following steps: (1) Collection of yolk fluid: Take a fresh egg, break it and place it on a separator to separate the yolk. Wash the yolk in distilled water to remove the residual clear liquid. Then, puncture the yolk membrane and put it into a filter bag to filter and collect the filtrate. (2) Water dilution-freeze-thaw method for IgY extraction: Egg yolk liquid was diluted with 5 times the volume of distilled water and mixed well. It was frozen in a -18 ℃ refrigerator for 1 h, thawed overnight at 4 ℃, and centrifuged at 4 ℃ and 10000 r / min for 30 min. The supernatant was collected to obtain IgY extract. (3) Preparation of the inner aqueous phase W1: The ion-responsive gel matrix (sodium alginate and calcium carbonate (CaCO3)) was dissolved in the IgY extract and stirred to disperse it evenly to form the inner aqueous phase W1; in the inner aqueous phase W1, the proportion of sodium alginate was 1 wt% and the proportion of calcium carbonate was 1 wt%; (4) Preparation of oil phase O: The small molecule emulsifier (polyglycerol ricinoleate (PGPR)) and the first substrate (polyglycerol-10 laurate (PG-10L)) were added to liquid edible oil (sunflower seed oil) and dissolved completely. Then, Candida antarcticis lipase B (CAL-B) was slowly added. The mixture was heated at 300 r / min for 45 min under light-protected conditions at 40 ℃ to allow it to fully incubate and form oil phase O. In oil phase O, the proportion of small molecule emulsifier was 4 wt%, the proportion of first substrate was 2 wt%, and the proportion of Candida antarcticis lipase B was 0.5 wt%. (5) Gelation of the inner aqueous phase: Under the heating of a water bath at 40 °C, the inner aqueous phase W1 was added dropwise to the oil phase O while stirring for initial mixing. The mass ratio of oil phase O to inner aqueous phase W1 was 1.5:1. Then, gluconolactone (GDL) was added and stirred for 1 min. Then, it was cooled to room temperature. The amount of gluconolactone (GDL) added was 1 g per 8 mL of inner aqueous phase W1. (6) Preparation of water-in-oil primary emulsion W1 / O: The mixture cooled to room temperature was emulsified by high-energy emulsification (shearing at 13000 r / min for 10 min) to obtain water-in-oil primary emulsion W1 / O; (7) Preparation of external aqueous phase W2: The second substrate (sodium caseinate) and water activity regulator (trehalose) were added to deionized water and allowed to dissolve completely. The solution was then allowed to stand at 4 °C for 30 min to fully hydrate, thus obtaining external aqueous phase W2. In external aqueous phase W2, the proportion of the second substrate was 5 wt% and the proportion of the water activity regulator was 10 wt%. (8) Preparation of W1 / O / W2 type double emulsion: Add water-in-oil primary emulsion W1 / O to the external aqueous phase W2. The mass ratio of external aqueous phase W2 to water-in-oil primary emulsion W1 / O is 1.5:1. Emulsify using high-energy emulsification method (shearing at 6000 r / min for 5 min) to obtain W1 / O / W2 type double emulsion.

[0057] (9) Catalysis of interfacial condensation reaction: After incubation at 40 °C for 3 h, the double emulsion was heated at 95 °C for 1 min to inactivate the enzyme, and a W1 / O / W2 type double emulsion formulation with encapsulated IgY based on the dual stabilization mechanism of interfacial condensation and internal aqueous phase gelation was obtained.

[0058] Example 2 This embodiment provides a method for preparing a W1 / O / W2 type dual emulsion formulation encapsulated with egg yolk immunoglobulin (IgY) based on a dual stabilization mechanism of interfacial condensation and internal aqueous phase gelation. The difference from Example 1 is that: In step (3), sodium alginate is replaced with low-methoxyl pectin; In step (4), polyglycerol ricinoleate (PGPR) is replaced with Span 80, and polyglycerol-10 lauryl ester (PG-10L) is replaced with polyglycerol-10 oleate; In step (7), sodium caseinate is replaced with whey protein concentrate and trehalose is replaced with sorbitol; The rest is the same as in Example 1.

[0059] Example 3 This embodiment provides a method for preparing a W1 / O / W2 type dual emulsion formulation encapsulated with egg yolk immunoglobulin (IgY) based on a dual stabilization mechanism of interfacial condensation and internal aqueous phase gelation. The difference from Example 1 is that: In step (4), polyglycerol ricinoleate (PGPR) is replaced with glyceryl monostearate, and polyglycerol-10 laurate (PG-10L) is replaced with ethyl laurate; In step (7), sodium caseinate is replaced with soy protein isolate (SPI) and trehalose is replaced with glycerol; The rest is the same as in Example 1.

[0060] Example 4 This embodiment provides a method for preparing a W1 / O / W2 type dual emulsion formulation encapsulated with egg yolk immunoglobulin (IgY) based on a dual stabilization mechanism of interfacial condensation and internal aqueous phase gelation. The difference from Example 1 is that: In step (4), polyglycerol ricinoleate (PGPR) is replaced with polyglycerol ricinoleate (PGPR) and lecithin in a mass ratio of 1:1, and polyglycerol-10 laurate (PG-10L) is replaced with glyceryl monolaurate (GML). In step (7), sodium caseinate is replaced with pea protein and trehalose is replaced with sodium chloride (NaCl). The rest is the same as in Example 1.

[0061] Example 5 This embodiment provides a method for preparing a W1 / O / W2 type dual emulsion formulation encapsulated with egg yolk immunoglobulin (IgY) based on a dual stabilization mechanism of interfacial condensation and internal aqueous phase gelation. The difference from Example 1 is that: In step (7), sodium caseinate is replaced with yeast protein; The rest is the same as in Example 1.

[0062] Compare with Example 1 This comparative example provides an IgY extract that is not encapsulated in an emulsion system and is prepared using steps (1) and (2) in Example 1, without performing steps (3) to (9) in Example 1.

[0063] Compare with Example 2 This comparative example provides a method for preparing a W1 / O / W2 type dual emulsion formulation containing encapsulated egg yolk immunoglobulin (IgY) without interfacial condensation and internal aqueous phase gelation stabilization, which differs from Example 1 in that: Step (3) is not performed. In step (4), the first substrate is not added, and the process of "slowly adding Candida antarctica lipase B (CAL-B) and continuing to stir at 300 r / min for 45 min under the conditions of heating at 40 ℃ in the dark to allow it to be fully incubated" is not performed. In step (5), the internal aqueous phase W1 is the IgY extract obtained in step (2), without the "addition of gluconolactone (GDL) and stirring for 1 min, followed by cooling to room temperature"; In step (7), no second substrate is added; The rest is the same as in Example 1.

[0064] Compare with Example 3 This comparative example provides a method for preparing a W1 / O / W2 type dual emulsion formulation containing encapsulated egg yolk immunoglobulin (IgY) without internal aqueous phase gelation stabilization, which differs from Example 1 in that: In step (5), the process of "adding gluconolactone (GDL) and stirring for 1 min, followed by cooling to room temperature" is not performed. The rest is the same as in Example 1.

[0065] Compare with Example 4 This comparative example provides a method for preparing a W1 / O / W2 type dual emulsion formulation containing encapsulated egg yolk immunoglobulin (IgY) without interfacial condensation stabilization, which differs from Example 1 in that: In step (4), the following steps are not performed: “slowly add Candida antarcticis lipase B (CAL-B) and continue stirring at 300 r / min for 45 min under 40 ℃ dark heating conditions to allow it to fully incubate”. The rest is the same as in Example 1.

[0066] Example 6 Structural and storage evaluation of W1 / O / W2 type dual emulsion formulations encapsulating egg yolk immunoglobulin (IgY) based on the dual stabilization mechanism of interfacial condensation and internal aqueous phase gelation. 1. Microscopic structural morphology observation The structural morphology of the dual emulsion formulation prepared in Example 1 was observed using a laser confocal microscope (CLSM). The dye was prepared and the emulsion was made and digested under light-protected conditions. Nile red fluoresces at an excitation wavelength of 530 nm. 1 mg of Nile red was dissolved in 1 mL of anhydrous ethanol as the oil phase dye. 500 μL of the dual emulsion was mixed thoroughly with 10 μL of Nile red dye, and then another 10 μL of the mixture was placed on a glass slide. The microstructure was observed using a 20x objective lens. Commonly observed emulsion structures were photographed and recorded. The results are shown in [Figure number missing]. Figure 1 .

[0067] like Figure 1 As shown, the dual emulsion formulation prepared in Example 1 exhibits a clear and stable water-in-oil-in-water structure. This result not only confirms the realization of the dual emulsion structure, but also demonstrates that the dual stabilization mechanism based on interfacial condensation and internal aqueous phase gelation has a good effect on stabilizing the dual emulsion structure.

[0068] 2. Measurement of embedding efficiency The IgY activity of the double emulsion formulations prepared in Examples 1-5 and Control Examples 2-4 was detected by indirect ELISA at 0, 7, and 15 days of storage, and compared with the IgY activity of the IgY extract of Control Example 1 to determine the encapsulation efficiency of IgY. Samples were diluted to the same concentration with distilled water, and then diluted 5000 times with sodium bicarbonate buffer (0.05 mol / L, pH 9.6). A 3% BSA solution was used as a blank control, and the samples were incubated overnight at 4 °C. The ELISA plates containing the samples were removed from the refrigerator and brought to room temperature. They were washed five times with PBS buffer containing 0.05% Tween 20 (PBS-Tw), and then blocked at 37 °C for 1 h with 3% BSA. After rinsing five times with PBS-Tw, HRP-conjugated rabbit anti-chicken IgY (1:5000 dilution) was added, and the plates were incubated at 37 °C for 30 min. The plates were then washed five times with PBS-Tw. Add tetramethylbenzidine (TMB) substrate solution and react at room temperature for 15 min. Then, add a colorimetric stop solution to stop the reaction. Measure the developing color at 450 nm and record the absorbance value as OD450 (subtracting the blank value). The embedding efficiency (%) is expressed as the ratio of the emulsion sample to the IgY extract. Results are shown below. Figure 2 .

[0069] like Figure 2 As shown, the dual emulsion formulations of Examples 1-5 all exhibited excellent encapsulation efficiency, ranging from 93.2% to 97.4%, with a slight decrease in encapsulation efficiency over extended storage time. However, the dual emulsion formulations of Control Examples 2-4 showed poor encapsulation efficiency, only about 30% to 36%, and the encapsulation efficiency decreased significantly over extended storage time. This indicates that the W1 / O / W2 type dual emulsion formulation, based on a dual stabilization mechanism of interfacial condensation and internal aqueous phase gelation, possesses excellent encapsulation and loading effects.

[0070] 3. Microrheological measurement The microrheological properties of the dual emulsion formulations prepared in Examples 1-5 and Control Examples 2-4 were analyzed using an optical microrheometer. Samples were added to the instrument analysis vials, ensuring all liquid levels were at the same height. Monitoring was performed at 0, 7, and 15 days of sample storage. Instrument parameters were set as follows: temperature 25 °C, Full Characterization mode, and each measurement lasted 2 hours. Changes in the root mean square displacement (MSD) of the solid-liquid equilibrium point (SLB), elasticity factor (EI), macroviscosity factor (MVI), and fluidity factor (FI) were recorded. The results are shown in the table below. Figures 3-6 .

[0071] The SLB value represents the state of the sample; the closer the value is to 0, the more solid-like the sample behaves, while the closer it is to 1, the more liquid-like the sample behaves. For example... Figure 3 As shown, the SLB values ​​of the dual emulsion formulations in Examples 1-5 were all below 0.3, and decreased slightly during storage, indicating that the internal network structure of the dual emulsion structure with dual stabilization mechanism was stable and no demulsification occurred. However, the SLB values ​​of the dual emulsion formulations in Control Examples 2-4 were higher and gradually increased during storage, indicating that the dual emulsion structure without dual stabilization mechanism was unstable. Among them, the dual emulsion formulation in Control Example 2, which lacked both stabilization mechanisms, had the highest SLB value and exhibited a liquid-like state.

[0072] The EI value reflects the elastic characteristics of the sample. For example... Figure 4 As shown, compared with Comparative Examples 2-4, the dual emulsion formulations of Examples 1-5 have higher EI values, indicating that a robust network structure is formed inside the dual emulsion under the participation of the dual stabilization mechanism, thereby exhibiting excellent emulsion stabilization ability.

[0073] The MVI value reflects the viscosity characteristics of a sample. For example... Figure 5 As shown, in Examples 1-5 and Comparative Examples 2-4, except for Comparative Example 2, all other dual emulsion formulations contained a water activity modifier and an ion-responsive gel matrix. Although the dual emulsion formulation of Comparative Example 3 did not undergo calcium ion bridging activated by GDL solution, the viscosity of the dual emulsion was still increased. This indicates that the contribution of viscosity change to the stability of the dual emulsion is far less than the contribution of the polyamide film formed by the interfacial condensation reaction in synergistic with the internal aqueous phase gel.

[0074] The FI value refers to the fluidity of a sample. For example... Figure 6 As shown, the trend of FI value change is opposite to that of EI value. Compared with control groups 2-4, the FI values ​​of the double emulsion formulations in Examples 1-5 are lower and further decrease with the extension of storage time, which is consistent with the results of EI value.

[0075] The results above indicate that the dual stabilization mechanism based on interfacial condensation and internal aqueous phase gelation is the most important factor in maintaining the stability of the dual emulsion structure, and the W1 / O / W2 type dual emulsion formulation prepared based on this dual stabilization mechanism has excellent stability.

[0076] Example 7 pH stability assay of W1 / O / W2 type dual emulsion formulations encapsulated with egg yolk immunoglobulin (IgY) based on the dual stabilization mechanism of interfacial condensation and internal aqueous phase gelation. 1. Preparation of samples under different pH conditions For the double emulsion formulations prepared in Examples 1-5 and Control Examples 2-4, the sample preparation method was as follows: the freshly prepared double emulsion formulation was added to an equal volume of PBS buffer, and the pH value of the mixture was adjusted to 2, 3, 4, 5, and 6 (the original pH value of the emulsion) with 2 mol / L hydrochloric acid, 15 mL for each group; then the mixture was stirred on a magnetic stirrer for 1 h to obtain samples under different pH conditions.

[0077] For the IgY extract of Control Example 1, the sample preparation method was as follows: 15 mL of IgY extract was placed in a glass bottle, and the pH value of the extract was adjusted to 2, 3, 4, 5 and 6 with a 2 mol / L hydrochloric acid solution to obtain test samples under different pH conditions.

[0078] 2. Determination of IgY activity retention rate The IgY activity of samples prepared with the double emulsion formulations of Examples 1-5, Control Examples 2-4, and Control Example 1 IgY extract at different pH values ​​was detected using an indirect ELISA method. The IgY activity was compared with that of Control Example 1 IgY extract to determine the IgY activity retention rate of the W1 / O / W2 double emulsion formulation and IgY extract encapsulating egg yolk immunoglobulin (IgY) under different pH conditions. Results are shown in [Figure number missing]. Figure 7 .

[0079] like Figure 7 As shown, in the IgY extract of Control Example 1, IgY was not encapsulated and protected, and its activity decreased rapidly, especially when the pH dropped below 3, where more than 87% of the IgY activity was destroyed. In the dual emulsion formulations of Examples 1-5, the IgY activity retention rate was only slightly affected at lower pH levels; when the pH dropped to 2, 81.5% of the IgY activity was still retained. The dual emulsion formulations of Control Examples 2-4, due to their poor structural stability, exhibited weaker IgY loading and protection capabilities. This demonstrates that the W1 / O / W2 dual emulsion formulation, based on a dual stabilization mechanism of interfacial condensation and internal aqueous phase gelation, exhibits excellent IgY protection through its complex structure and strong stabilization mechanism, making it suitable for the industrial development of formulations and their applications.

[0080] Example 8 In vitro digestive stability assay of W1 / O / W2 type dual emulsion formulations encapsulated with egg yolk immunoglobulin (IgY) based on the dual stabilization mechanism of interfacial condensation and internal aqueous phase gelation. 1. In vitro simulated digestion For the double emulsion formulations prepared in Examples 1-5 and Comparative Examples 2-4, in vitro simulated digestion includes the following steps; (1) Preparation of oral digestive fluid: Mix 15 mL of PBS and 20 mL of distilled water, add 5 g of double emulsion preparation to prepare oral digestive fluid; (2) Add 0.1 g mucin (dissolved in 1 mL PBS) to the oral digestion fluid, adjust the pH to 6.8, and place it in a 37℃ water bath to simulate oral digestion for 10 min; (3) Preparation of gastric digestion mother fluid: Mix 2 g sodium chloride and 7 mL hydrochloric acid, add water to make up to 1 L, and prepare gastric digestion mother fluid; (4) Preparation of gastric digestive juice: Take 20 mL of the sample after the completion of simulated oral digestion and mix it with 20 mL of gastric digestive mother juice to prepare gastric digestive juice; (5) Adjust the pH of the solution to 2.0, add 0.064 g of pepsin (dissolved in 1 mL PBS), and place it in a 37 ℃ water bath to simulate gastric digestion for 1 h.

[0081] (6) Preparation of small intestinal digestive juice: Take 30 mL of gastric digestive juice, add 0.2 g of bile salt (dissolved in 4 mL of PBS), 0.064 g of pancreatic enzyme (dissolved in 1 mL of PBS), 0.022 g of calcium chloride and 0.3564 g of sodium chloride (dissolved in 5 mL of PBS) to prepare small intestinal digestive juice; (7) Adjust the pH of the solution to 7 and place it in a 37°C water bath to simulate intestinal digestion for 2 hours.

[0082] For the IgY extract of Control Example 1, the difference between the in vitro simulated digestion and the in vitro simulated digestion of the above-mentioned double emulsion preparation is as follows: (1) Preparation of oral digestion solution: Take 20 mL of IgY extract and mix it with 20 mL of PBS solution to prepare oral digestion solution. The rest is the same as the in vitro simulated digestion of the above-mentioned double emulsion preparation.

[0083] 2. Determination of IgY activity retention rate The IgY activity of the double emulsion formulations of Examples 1-5, the double emulsion formulations of Control Examples 2-4, and the IgY extract of Control Example 1 after completing different in vitro simulated digestion stages (initial, oral, gastric, and small intestine) was detected using an indirect ELISA method. The IgY activity was compared with that of the IgY extract of Control Example 1 to determine the IgY activity retention rate of the W1 / O / W2 double emulsion formulations and IgY extracts encapsulating egg yolk immunoglobulins (IgY) after completing different in vitro simulated digestion stages. Results are shown in [Figure number missing]. Figure 8 .

[0084] like Figure 8As shown, in the IgY extract of Control Example 1, IgY was not encapsulated for protection, and its activity decreased rapidly, especially after gastric digestion, where the IgY activity retention rate was only 0.96%. In the dual emulsion formulations of Examples 1-5, the IgY activity retention rate remained above 80% after gastric digestion, with significant activity loss only occurring after intestinal digestion. The dual emulsion formulations of Control Examples 2-4 showed significantly weaker resistance to gastric environmental damage compared to those of Examples 2-4, with a substantial decrease in IgY activity retention rate after simulated gastric digestion. This indicates that during the gastric digestion stage, when IgY activity is most vulnerable to degradation, the W1 / O / W2 dual emulsion formulation, based on a dual stabilization mechanism of interfacial condensation and internal aqueous phase gelation, exhibits excellent IgY protection through its complex structure and strong stabilization mechanism. Subsequently, it is effectively released during intestinal digestion, which improves bioavailability and human digestion and absorption, confirming that the dual emulsion formulation based on this dual stabilization mechanism is suitable for the development of oral IgY formulations and their applications.

[0085] Unless otherwise specified, all raw materials used in this invention are existing substances that can be purchased directly from the market.

[0086] The above are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing a W1 / O / W2 type dual emulsion formulation encapsulating IgY, characterized in that, Includes the following steps: S1. Provide IgY extraction solution; S2. Dissolve the ion-responsive gel matrix in the IgY extract and stir to obtain the internal aqueous phase W1; wherein the ion-responsive gel matrix includes polysaccharides and calcium ion source; S3. Dissolve the small molecule emulsifier and the first substrate in edible oil, add lipase, and incubate to obtain oil phase O; wherein, the first substrate includes at least one of polyglycerol-10 laurate, polyglycerol-10 oleate, ethyl laurate, and glyceryl monolaurate. S4. Mix the aqueous phase W1 with the oil phase O, add gluconolactone, stir, cool to room temperature, and then emulsify to obtain a water-in-oil primary emulsion W1 / O. S5. Dissolve the water activity regulator and the second substrate in water to obtain an external aqueous phase W2; wherein the second substrate includes at least one of a natural macromolecular protein and its hydrolyzed peptides; S6. Add the water-in-oil primary emulsion W1 / O to the external aqueous phase W2, emulsify, then incubate to inactivate the enzyme, and obtain a W1 / O / W2 type dual emulsion formulation encapsulating IgY.

2. The method for preparing the W1 / O / W2 type dual emulsion formulation encapsulating IgY according to claim 1, characterized in that, Step S1 includes: S11. Separate the yolk from the egg, wash with water, break the yolk membrane, filter, and obtain yolk liquid; S12. Mix the egg yolk liquid with water, freeze, thaw overnight, centrifuge to obtain IgY extract.

3. The method for preparing the W1 / O / W2 type dual emulsion formulation encapsulating IgY according to claim 1, characterized in that, In step S2: The polysaccharide includes at least one of sodium alginate and low-methoxyl pectin; the calcium ion source includes calcium carbonate; and / or, The proportion of the ion-responsive gel matrix in the internal aqueous phase W1 is 1~5 wt%.

4. The method for preparing the W1 / O / W2 type dual emulsion formulation encapsulating IgY according to claim 1, characterized in that, In step S3: The small molecule emulsifier includes at least one of polyglycerol ricinoleate, Span derivatives, polyol fatty acid esters, and phospholipids; and / or, The lipase includes at least one of Candida antarcticis lipase B and its lipase-like enzymes, Ustilago esculenta lipase B and its lipase-like enzymes, and bacterial lipases with structural similarity to Candida antarcticis lipase B; and / or The edible oil includes at least one of sunflower seed oil, soybean oil, and rapeseed oil.

5. The method for preparing the W1 / O / W2 type dual emulsion formulation encapsulating IgY according to claim 1, characterized in that, In step S3: The proportion of the small molecule emulsifier in the oil phase O is 1~10 wt%; and / or, The proportion of the first substrate in the oil phase O is 1~5 wt%; and / or, The proportion of the lipase in the oil phase O is 0.5~5 wt%.

6. The method for preparing the W1 / O / W2 type dual emulsion formulation encapsulating IgY according to claim 1, characterized in that, In step S4: The mass ratio of the internal aqueous phase W1 to the oil phase O is (1~5):1; and / or, The mass-to-volume ratio of the gluconolactone to the internal aqueous phase W1 is (1~2) g: 8 mL.

7. The method for preparing the W1 / O / W2 type dual emulsion formulation encapsulating IgY according to claim 1, characterized in that, In step S5: The water activity regulator includes at least one of small molecule sugars, sugar alcohols, polyols, and small molecule salts; and / or, The natural macromolecular protein includes at least one of sodium caseinate, whey protein concentrate, soy protein isolate, pea protein, and yeast protein.

8. The method for preparing the W1 / O / W2 type dual emulsion formulation encapsulating IgY according to claim 1, characterized in that, In step S5: The water activity regulator in the external aqueous phase W2 accounts for 8-15 wt%; and / or, The proportion of the second substrate in the external aqueous phase W2 is 0.1~10 wt%.

9. The method for preparing the W1 / O / W2 type dual emulsion formulation encapsulating IgY according to claim 1, characterized in that, In step S6, the mass ratio of the external aqueous phase W2 to the water-in-oil primary emulsion W1 / O is (1~5):

1.

10. A W1 / O / W2 type dual emulsion formulation encapsulating IgY, characterized in that, It is prepared by the preparation method according to any one of claims 1 to 9.

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