Pickering emulsion stabilized by full-component egg white protein-EGCG (epigallocatechin gallate) as well as preparation method and application of Pickering emulsion

Abstract

The invention discloses a Pickering emulsion stabilized by full-component egg white protein-EGCG (epigallocatechin gallate) as well as a preparation method and application of the Pickering emulsion, and belongs to the field of functional carrier emulsion preparation. The full-component egg white protein-EGCG composite stabilizer is formed by compounding a cooked egg white protein microparticle solution and an EGCG solution through pH regulation and control, the full-component egg white protein-EGCG composite stabilizer can be used for well stabilizing a Pickering emulsion and loading bioactive substances; the prepared Pickering emulsion with the stable astaxanthin-loaded full-component egg white protein-EGCG compound has relatively strong self-supporting property, relatively high encapsulation efficiency, excellent rheological property, moisture distribution property and biologically active substance slow release property, can be used as a 3D printing material, and has good 3D printing stability and accuracy.

Description

Technical Field

[0001] This invention relates to a Pickering emulsion stabilized by whole-component egg white protein-EGCG, its preparation method and application, belonging to the field of functional carrier emulsion preparation. Background Technology

[0002] Astaxanthin possesses potent antioxidant activity and can effectively scavenge free radicals. As a chain-breaking antioxidant, it can effectively neutralize reactive oxygen species and nitrogenous substances, protecting against oxidative stress damage. However, astaxanthin is extremely sensitive to temperature, light, and oxygen, leading to degradation and loss during processing, and even the development of undesirable flavors. Furthermore, its high lipophilicity and hydrophobicity result in naturally low solubility in the gastrointestinal tract, significantly limiting its application in water-based dispersion systems commonly found in food. Therefore, developing suitable delivery systems to enhance the stability and solubility of astaxanthin is of practical significance.

[0003] Pickering emulsions are a novel type of emulsion that uses solid particles to stabilize emulsion systems instead of traditional organic surfactants. Compared to conventional surfactant-stabilized emulsions, Pickering emulsions reduce the use of surfactants while avoiding toxicity issues and regulatory restrictions associated with food systems. Pickering emulsions are widely considered one of the most suitable methods for encapsulating lipophilic bioactive compounds. These particle-stabilized colloids exhibit enhanced physicochemical stability and biocompatibility, and hold promise as a versatile delivery platform for nutritional supplements and bioactive compounds.

[0004] In addition, Pickering emulsions have good applicability to 3D printing. It is worth considering that the application of Pickering emulsions in 3D printed food systems often requires the use of edible emulsifiers to ensure the stability of the 3D structure.

[0005] For example, Chinese patent CN119699575A discloses a method for preparing a Pickering emulsion stably loaded with lutein using a Chlamydomonas reinhardtii protein-xanthan gum complex and its application in 3D printing. The materials used are prepared by the following method: 1) Chlamydomonas reinhardtii algal powder is dissolved in water and subjected to ultrasonic treatment. After adjusting the pH to 9-11, the supernatant is collected by centrifugation. The pH of the supernatant is then adjusted to 3-5, and the precipitate is collected by centrifugation to obtain Chlamydomonas reinhardtii protein; 2) Xanthan gum is dissolved in water and heated to obtain a xanthan gum solution; 3) The Chlamydomonas reinhardtii protein solution and xanthan gum solution are compounded in a certain proportion and then mixed with edible oil containing lutein, followed by high-speed shearing to obtain a Pickering emulsion loaded with lutein. However, this method for extracting Chlamydomonas reinhardtii protein from Sargassum fusiforme is extremely complex, with issues such as complex raw material sources, waste and unusable byproducts, limiting its practical industrial production and utilization. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a Pickering emulsion stabilized by whole-component egg white protein-EGCG, its preparation method, and its application. This whole-component egg white protein (cooked egg white protein)-EGCG composite stabilizer is composed of cooked egg white and EGCG. The cooked egg white is obtained by peeling fresh poultry eggs after cooking, making it readily available and inexpensive. Furthermore, the strong electrostatic interaction between cooked egg white protein and EGCG results in a highly stable composite. When used in the preparation of Pickering emulsions, it exhibits high thermal and storage stability. In addition, when used as a carrier to load astaxanthin, the loading rate reaches as high as 98.58%, demonstrating a high encapsulation rate and stability. When used in 3D printing, it can print complete patterns with clearly visible holes and clear and complete printing paths, displaying high precision and a clear printed structure, exhibiting good 3D printing stability and accuracy.

[0007] To achieve the above objectives, the following technical solution is provided:

[0008] The first objective of this invention is to provide a whole-component egg white protein (cooked egg white protein)-EGCG complex stabilizer, wherein the cooked egg white protein-EGCG complex stabilizer is composed of a cooked egg white protein microparticle solution and an EGCG solution through pH adjustment.

[0009] In one embodiment, the pH value is 7 to 9.

[0010] In one embodiment, the cooked egg white protein microparticle solution refers to the solution obtained by separating the cooked egg white from the yolk after cooking poultry eggs, mixing the cooked egg white with water, and crushing it in a high-speed blender.

[0011] In one embodiment, the poultry eggs include one or more of chicken eggs, duck eggs, and goose eggs.

[0012] In one embodiment, the heating temperature during the cooking process is 80–100°C, and the time is 10–30 minutes.

[0013] In one embodiment, the parameters for crushing in the blender are a crushing rate of 20,000 to 40,000 rpm and a crushing time of 2 to 6 minutes.

[0014] In one embodiment, the concentration of the cooked egg white protein microparticle solution is 20-60%, w / v, g / mL; preferably 20-40%, w / v, g / mL.

[0015] In one embodiment, the concentration of the EGCG solution is 0.5–2%, w / v, g / mL.

[0016] In one embodiment, the volume ratio of the cooked egg white protein microparticle solution to the EGCG solution is 1:1.

[0017] A second objective of this invention is to provide a method for preparing Pickering emulsion based on the aforementioned full-component egg white protein-EGCG complex stabilizer, the method comprising the following:

[0018] The whole-component egg white protein-EGCG complex stabilizer is mixed with edible oil and sheared to obtain a Pickering emulsion stabilized by the whole-component egg white protein-EGCG complex.

[0019] In one embodiment, the edible oil includes one or more of corn oil, soybean oil, peanut oil, and rapeseed oil.

[0020] In one embodiment, the proportion of the oil phase in the Pickering emulsion system is 50% to 80%.

[0021] In one embodiment, the shearing speed is 6000-13000 rpm and the time is 1-4 min.

[0022] In one embodiment, the concentration of EGCG in the whole-component egg white protein-EGCG complex stabilizer is 0.25-1%; 0.75-1%.

[0023] A third objective of this invention is to provide a Pickering emulsion prepared by the method described above.

[0024] A fourth object of the present invention is to provide an application of the Pickering emulsion described above as a functional carrier.

[0025] A fifth object of the present invention is to provide a method for improving the thermal stability and storage stability of Pickering emulsions, the method comprising:

[0026] The whole-component egg white protein-EGCG complex stabilizer was mixed with edible oil and sheared to obtain a Pickering emulsion stabilized by the whole-component egg white protein-EGCG complex; wherein the concentration of EGCG in the whole-component egg white protein-EGCG complex stabilizer was 0.75% to 1%, w / v, g / mL.

[0027] The sixth object of the present invention is to provide a method for preparing a Pickering emulsion loaded with astaxanthin, the method comprising the following:

[0028] The whole-component egg white protein-EGCG complex stabilizer was mixed with edible oil containing astaxanthin and then sheared to obtain the astaxanthin-loaded Pickering emulsion.

[0029] In one embodiment, the astaxanthin concentration in the edible oil containing astaxanthin is 0.05–5%, w / v, mg / mL.

[0030] In one embodiment, the edible oil includes one or more of corn oil, soybean oil, peanut oil, and rapeseed oil.

[0031] In one embodiment, the proportion of the oil phase in the astaxanthin-loaded Pickering emulsion system is 50% to 80%.

[0032] In one embodiment, the shearing speed is 6000-13000 rpm and the time is 1-4 min.

[0033] A seventh object of the present invention is to provide a Pickering emulsion loaded with astaxanthin prepared by the method described above.

[0034] The eighth objective of this invention is to provide a 3D printed food product, which is made by 3D printing based on the astaxanthin-loaded Pickering emulsion described above as a raw material, wherein the EGCG concentration of the whole-component egg white protein-EGCG complex stabilizer in the astaxanthin-loaded Pickering emulsion is 1%, w / v, mg / mL.

[0035] Beneficial effects:

[0036] (1) This invention uses cooked egg white as raw material and combines it with EGCG to form a composite stabilizer. This composite stabilizer can effectively stabilize Pickering emulsion and load bioactive substances. The cooked egg white is derived from eggs. The protein content in egg white is about 12%, and there is no fat content. After heating, the egg white protein denatures. The cooked egg white protein microparticles have strong emulsifying ability and thermal stability. Furthermore, the protein is not extracted separately, which greatly simplifies the cost.

[0037] (2) The present invention uses cooked egg white protein-EGCG complex as a stabilizer to prepare Pickering emulsion. The emulsion has good stability. When heated at 90°C for 60 min, stored at room temperature for 14 days, or centrifuged at 10,000 rpm for 10 min, the cooked egg white protein-EGCG stabilized Pickering emulsion remains stable without phase separation or flow.

[0038] (3) The Pickering emulsion with cooked egg white protein-EGCG complex loaded with astaxanthin prepared in this invention has strong self-support, high encapsulation rate, excellent rheological properties, water distribution properties and slow release properties of bioactive substances, and can be used as a 3D printing material to load bioactive substances; specifically, the Pickering emulsion with cooked egg white protein-EGCG complex loaded with astaxanthin does not flow after being inverted and still has self-support, indicating that it has good stability and has the potential to be used as a 3D printing material.

[0039] (4) The cooked egg white protein-EGCG complex-stabilized Pickering emulsion prepared in this invention achieves an encapsulation rate of up to 98.58% for astaxanthin, effectively realizing the function of encapsulating hydrophobic bioactive substances. In rheological experiments, the storage modulus is higher than the loss modulus, indicating typical emulsion characteristics. In vitro simulated gastrointestinal digestion experiments show that the astaxanthin-loaded cooked egg white protein-EGCG complex-stabilized Pickering emulsion exhibits controlled-release characteristics, slowly releasing astaxanthin in gastric juice and rapidly releasing it in intestinal juice, thereby optimizing bioavailability under physiological conditions. Notably, the astaxanthin-loaded cooked egg white protein-EGCG complex-stabilized Pickering emulsion shows great potential in 3D printing applications, especially when the formulation of the stabilized Pickering emulsion at an EGCG concentration of 1% (w / v) is used as an edible ink, demonstrating precise pattern deposition and structural integrity. Attached Figure Description

[0040] Figure 1 Zeta potential data for different concentrations of cooked egg white protein-EGCG complexes prepared in Example 1;

[0041] Figure 2 The appearance of the cooked egg white protein-EGCG stabilized Pickering emulsion prepared in Example 2 after heating at 90°C for 0, 30 and 60 min;

[0042] Figure 3 The images show the appearance of the cooked egg white protein-EGCG stabilized Pickering emulsion prepared in Example 2 after storage at room temperature for 0, 7, and 14 days.

[0043] Figure 4 The appearance of the cooked egg white protein-EGCG stabilized Pickering emulsion prepared in Example 2 after centrifugation at 10,000 rpm for 10 min at 25°C.

[0044] Figure 5The inverted experiment results of the astaxanthin-loaded cooked egg white protein-EGCG stabilized Pickering emulsion, the astaxanthin-loaded undenatured egg white protein-EGCG stabilized Pickering emulsion, and the astaxanthin-loaded denatured isolated whey protein-EGCG stabilized Pickering emulsion prepared in Example 3 are shown in the figure.

[0045] Figure 6 Cryo-scanning electron microscopy images of the cooked egg white protein-EGCG stabilized Pickering emulsion loaded with astaxanthin prepared in Example 3, and optical microscopy images of the undenatured egg white protein-EGCG stabilized Pickering emulsion loaded with astaxanthin and the denatured isolated whey protein-EGCG stabilized Pickering emulsion loaded with astaxanthin.

[0046] Figure 7 The encapsulation efficiency data of the astaxanthin-loaded cooked egg white protein-EGCG stabilized Pickering emulsion prepared in Example 3 is shown in the figure.

[0047] Figure 8 The transverse relaxation time (T2) distribution of the astaxanthin-loaded cooked egg white protein-EGCG stabilized Pickering emulsion prepared in Example 3;

[0048] Figure 9 Strain scanning data of the astaxanthin-loaded cooked egg white protein-EGCG stabilized Pickering emulsion prepared in Example 3;

[0049] Figure 10 Viscosity data of the astaxanthin-loaded cooked egg white protein-EGCG stabilized Pickering emulsion prepared in Example 3;

[0050] Figure 11 The oscillation frequency scan data of the astaxanthin-loaded cooked egg white protein-EGCG stabilized Pickering emulsion prepared in Example 3;

[0051] Figure 12 Dynamic temperature scan data of the astaxanthin-loaded cooked egg white protein-EGCG stabilized Pickering emulsion prepared in Example 3;

[0052] Figure 13 Astaxanthin release curves for in vitro simulated digestion of cooked egg white protein-EGCG-stabilized Pickering emulsion loaded with astaxanthin.

[0053] Figure 14 Front and side views of the 3D printed Chinese knot pattern of the astaxanthin-loaded cooked egg white protein-EGCG stabilized Pickering emulsion prepared in Example 3. Detailed Implementation

[0054] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. The specific embodiments described below further illustrate the present invention.

[0055] The testing method involved in this invention:

[0056] 1. Astaxanthin encapsulation rate

[0057] Specific steps: 4 mL dichloromethane: Add methanol (2:1, v / v) mixed with organic reagents to 1 mL of astaxanthin-loaded cooked egg white protein-EGCG stabilized Pickering emulsion, then measure the absorbance of the lower liquid at 480 nm, and calculate the astaxanthin concentration using the astaxanthin standard curve; the standard regression curve of astaxanthin is y = 192.67x - 0.0119, R2 = 0.9998 (y represents the absorbance value, x represents the astaxanthin concentration, and R2 is the correlation retention coefficient);

[0058] Astaxanthin encapsulation efficiency (%) = (concentration of encapsulated astaxanthin / initial concentration of astaxanthin) × 100%.

[0059] 2. The transverse relaxation (T2) curve of the Pickering emulsion stabilized with cooked egg white protein-EGCG loaded with astaxanthin was determined by low-field nuclear magnetic resonance.

[0060] Specifically, the sample is placed in a sample vial, and the parameters are set as follows: 90° pulse duration is 31μs, 180° pulse duration is 62μs, π value is 1500ms, repetition time is 1600ms, and echo time is 50ms.

[0061] 3. The rheological properties of the astaxanthin-loaded cooked egg white protein-EGCG-stabilized Pickering emulsion were characterized using a rheometer system.

[0062] Thixotropic recovery properties and apparent viscosity from 0.1 to 100% were evaluated by sequentially applying strains of 0.1% to 100% at a fixed frequency of 1 Hz at 25 °C. -1 The changes at different shear rates.

[0063] All tests were conducted at 25°C with a test gap of 1 mm. The rheological behavior of the samples was tested in frequency scanning mode (0.1–10 Hz, constant stress 1 Pa). In addition, temperature scanning tests were conducted from 20 to 80°C with a fixed strain of 1.0% and an angular frequency of 10.0 rad·s. -1The heating rate is 5℃ / min.

[0064] 4. The in vitro simulated digestion experiment is divided into three stages: salivary digestion, gastric juice digestion, and intestinal juice digestion.

[0065] All steps were performed at 37°C. Before the experiment, the digestive fluid was preheated for 2 hours and the emulsion was diluted 15 times. In the salivary digestion stage, an equal volume of astaxanthin-loaded cooked egg white protein-EGCG stabilized Pickering emulsion was added to the artificial simulated saliva, the pH was adjusted to 6.8, and then the mixture was placed in a shaker for 10 minutes. In the gastric digestion stage, the above saliva mixture was added to an equal volume of gastric juice, the pH was adjusted to 2, and the mixture was placed in a shaker for 110 minutes. In the intestinal digestion stage, the intestinal juice was mixed with an equal volume of the digestive fluid mixture from the previous stage, the pH was adjusted to 7, and the mixture was placed in a shaker for 120 minutes.

[0066] To determine the astaxanthin release rate, 1 mL of the mixture was removed from the digestion system every 30 min. The sample was mixed with 9 mL of dichloromethane-methanol solution (2:1, v / v), vortexed for 2 min, centrifuged at 8000 rpm for 10 min, and the absorbance of the lower layer was measured at 480 nm. The astaxanthin concentration was calculated using a standard curve. The astaxanthin release rate (%) = (astaxanthin concentration in the digestion solution / initial astaxanthin concentration) * 100%.

[0067] Example 1

[0068] The preparation method of cooked egg white protein-EGCG complex includes the following steps:

[0069] (1) Heat the eggs at 100°C for 20 minutes, then separate the cooked egg whites from the yolks; then mix 120g of cooked egg whites with 300mL of deionized water and stir with a blender at 35,000rpm / min for 3 minutes to prepare a 40% (w / v, g / mL) cooked egg white protein microparticle solution.

[0070] (2) Dissolve 1 g of EGCG in 50 mL of deionized water, stir at room temperature for 1 h, and concentrate to a 2% (w / v, g / mL) EGCG solution; use a stepwise gradient method to continuously dilute the 2% (w / v, g / mL) EGCG solution to 1.5% (w / v, g / mL), 1% (w / v, g / mL) and 0.5% (w / v, g / mL);

[0071] (3) The cooked egg white protein microparticle solution obtained in step (1) is mixed with the EGCG solution obtained in step (2) at a ratio of 1:1 (v / v), and the pH value is adjusted to 8 with 0.1M NaOH solution to obtain 0%, 0.25%, 0.5%, 0.75% and 1% EWP-EGCG complexes.

[0072] The zeta potential of the cooked egg white protein-EGCG complex is as follows: Figure 1 As shown. Surface potential is the main determinant of charge-related interaction mechanisms. For example... Figure 1 As shown, at low EGCG concentrations (0.25% and 0.5%), the Zeta potential of the cooked egg white protein-EGCG complex did not show significant changes. However, the Zeta potentials of 0.75% and 1% EWP-EGCG increased to -36.95 mV and -35.66 mV, respectively. The increase in the absolute value of the Zeta potential indicates that higher EGCG concentrations can enhance the stability of the cooked egg white protein-EGCG complex, confirming that the stronger electrostatic interaction between cooked egg white protein and EGCG is beneficial to emulsion stability.

[0073] Example 2

[0074] A method for preparing a Pickering emulsion stabilized by a cooked egg white protein-EGCG complex includes the following steps:

[0075] Different concentrations of cooked egg white protein-EGCG complex prepared in Example 1 were mixed with corn oil at a ratio of 2:3 (v / v) and then sheared at a high speed of 12,000 rpm for 2.5 min to obtain a stable Pickering emulsion of cooked egg white protein-EGCG complex.

[0076] Performance testing:

[0077] In this embodiment, the appearance of the Pickering emulsion stabilized by the cooked egg white protein-EGCG complex after heating for 60 minutes is shown in the figure below. Figure 2 As shown, after heat treatment in a 90℃ water bath for 30 and 60 minutes, compared with the unheated state, varying degrees of collapse and water separation were observed in the cooked egg white protein-EGCG-stabilized Pickering emulsion, and the color changed from milky white to light brown. This is because the EGCG in the emulsion oxidizes after heating, causing the color to darken. However, when no EGCG was added, the emulsion exhibited phase separation after heating, with some oil precipitating from the upper layer. The high-concentration EGCG-stabilized Pickering emulsion remained relatively stable after heating, without phase separation.

[0078] In this embodiment, the appearance of the Pickering emulsion stabilized by the cooked egg white protein-EGCG complex after 14 days of storage at room temperature is shown in the image below. Figure 3 As shown, the Pickering emulsion without EGCG became thinner after 7 days of storage, and this thinning became more pronounced after 14 days. However, the Pickering emulsion with a high concentration of EGCG remained stable and showed no significant change in appearance after 14 days of storage.

[0079] In this embodiment, the appearance of the Pickering emulsion stabilized by the cooked egg white protein-EGCG complex after centrifugation at 10,000 rpm for 10 min at 25°C is shown in the figure below. Figure 4 As shown in the figure, the degree of water-oil separation in a cooked egg white protein-EGCG-stabilized Pickering emulsion under centrifugal force is one of the important indicators for evaluating emulsion stability. It can be observed that the volume of the aqueous phase separated from the Pickering emulsion without added EGCG after centrifugation is significantly higher than that of the Pickering emulsion with high concentrations of EGCG.

[0080] Example 3

[0081] A method for preparing a cooked egg white protein-EGCG complex-stabilized astaxanthin-loaded Pickering emulsion includes the following steps:

[0082] Different concentrations of cooked egg white protein-EGCG complex prepared in Example 1 were mixed with corn oil containing 1 mg / mL astaxanthin at a ratio of 2:3 (v / v), and then sheared at 12,000 rpm for 2.5 min to obtain Pickering emulsions loaded with astaxanthin stabilized by different concentrations of cooked egg white protein-EGCG complex.

[0083] Comparative Example 1

[0084] A method for preparing an astaxanthin-loaded Pickering emulsion stable by an undenatured protein-EGCG complex includes the following steps:

[0085] (1) Separate fresh egg whites from egg yolks. Then, mix 20g of undenatured egg whites with 50mL of deionized water and stir magnetically for 2h at room temperature to prepare a 40% (w / v, g / mL) undenatured egg white solution.

[0086] (2) Take the undenatured egg white solution and mix it with 0.02 g / mL EGCG aqueous solution at a ratio of 1:1 (v / v), and adjust the pH value to 8 with 0.1 M NaOH solution to obtain the undenatured egg white-EGCG complex;

[0087] (3) Take the undenatured egg white-EGCG complex and mix it with corn oil containing 1 mg / mL astaxanthin at a ratio of 2:3 (v / v). Then, shear it at 12,000 rpm for 2.5 min to obtain a stable Pickering emulsion of undenatured egg white-EGCG complex loaded with astaxanthin.

[0088] Comparative Example 2

[0089] A method for preparing a stable astaxanthin-loaded Pickering emulsion for isolating whey protein-EGCG complex includes the following steps:

[0090] (1) Take 20g and 2.2g of isolated whey protein and mix them with 50mL of deionized water respectively. Then, heat the mixture in a water bath at 100℃ for 20min and vortex for 2min to prepare a 40% (w / v, g / mL) denatured isolated whey protein solution.

[0091] (2) Take the denatured whey protein solution and mix it with 0.02 g / mL EGCG aqueous solution at a ratio of 1:1 (v / v), and adjust the pH value to 8 with 0.1 M NaOH solution to obtain denatured whey protein-EGCG complex.

[0092] (3) Take the denatured whey protein-EGCG complex and mix it with corn oil containing 1 mg / mL astaxanthin at a ratio of 2:3 (v / v). Then, shear the mixture at 12,000 rpm for 2.5 min to obtain a stable Pickering emulsion loaded with astaxanthin from the denatured whey protein-EGCG complex.

[0093] Results Analysis

[0094] The appearance of Pickering emulsions stably loaded with astaxanthin from cooked egg white protein-EGCG complexes prepared with different EGCG concentrations is shown in the figure. Figure 5 As shown. Figure 5 As can be seen, the emulsions were generally stable, without stratification or bubbles. The brown color of the emulsions was due to the loading of astaxanthin; the concentration of EGCG did not significantly change the sample color. During turnover experiments, it was observed that the emulsions without EGCG and the stable Pickering emulsions at low EGCG concentrations exhibited strong flowability, indicating an unstable state. Conversely, as the EGCG concentration gradually increased, the prepared emulsions remained non-flowing, demonstrating good structural enhancement. This can be attributed to the interfacial adsorption of the cooked egg white protein-EGCG complex, which increases the emulsion stability. It is noteworthy that from... Figure 5As can be seen, the Pickering emulsion stabilized by undenatured egg white-EGCG exhibits oil droplet separation within a short storage period. During the inversion experiment, the emulsion exhibits extremely high fluidity, indicating its instability. While the Pickering emulsion stabilized by denatured whey protein isolate-EGCG shows relatively uniform texture and no phase separation within a short time, the inversion experiment also reveals its extremely high fluidity. Although the emulsion becomes more uniform when the concentration of whey protein isolate increases from 2.2% to 20%, its fluidity remains high. This suggests that neither the astaxanthin-loaded Pickering emulsions stabilized by undenatured egg white-EGCG nor those stabilized by denatured whey protein isolate-EGCG are suitable for 3D printing food.

[0095] Cryo-SEM images of Pickering emulsions stabilized by different concentrations of cooked egg white protein-EGCG complex are shown below. Figure 6 As shown, with increasing EGCG concentration in the cooked egg white protein-EGCG complex, the resulting emulsion droplets became smaller and the droplet size distribution more concentrated, indicating that EGCG enhanced the emulsifying ability of the cooked egg white protein-EGCG complex. Under higher magnification cryo-scanning electron microscopy images, the emulsion droplets showed some degree of adhesion, most notably in the emulsion prepared with a 1% (w / v) EGCG concentration. These more compact structures effectively inhibited relative movement between droplets, effectively improving emulsion stability. Specifically, Figure 6 These are optical microscopic images of undenatured egg white-EGCG-stabilized Pickering emulsions and denatured whey protein-EGCG-stabilized Pickering emulsions loaded with astaxanthin. The optical microscopic images show that the Pickering emulsion stabilized by undenatured egg white-EGCG has a large and relatively non-uniform particle size, which is detrimental to emulsion stability. In contrast, the microscopic images of the Pickering emulsion stabilized by denatured whey protein-EGCG show that while the particle size remains large at a whey protein concentration of 2.2%, it becomes very small and more uniform at a protein concentration of 20%. However, the emulsion still exhibits high fluidity and poor stability.

[0096] like Figure 7The image shows the determination of the encapsulation efficiency of astaxanthin in Pickering emulsions by the combination of egg white protein microparticles and EGCG. Pickering emulsions with EGCG concentrations of 0% (w / v), 0.25% (w / v), 0.5% (w / v), 0.75% (w / v), and 1% (w / v) showed encapsulation efficiencies of 88.40%, 92.02%, 95.21%, 96.79%, and 98.58% for astaxanthin, respectively. It can be seen that all Pickering emulsions exhibited relatively high encapsulation efficiencies, with the emulsion at a EGCG concentration of 1% (w / v) showing an even higher encapsulation efficiency. The encapsulation efficiency of astaxanthin increased with increasing EGCG concentration. This phenomenon may be attributed to the formation of a more stable cooked egg white protein-EGCG complex at higher EGCG concentrations, which can increase the interaction at the oil-water interface.

[0097] Figure 8 The diagram shows the transverse relaxation time (T2) distribution of cooked egg white protein-EGCG complex stably loaded with astaxanthin. Figure 8 As shown, the Pickering emulsion T stabilized by the astaxanthin-loaded egg white protein-EGCG complex. 22 (Fixed water) and T 23 The free water content decreases with increasing EGCG concentration, indicating a reduction in the degree of freedom of oil and free water. Therefore, higher concentrations of EGCG restrict the flowability of oil, fixed water, and free water in the emulsion, thereby enhancing the emulsion's stability.

[0098] The storage modulus and loss modulus of pickering emulsions as a function of strain are as follows: Figure 9 As shown. Figure 9 In the strain scan (range from 0.01% to 100%), the storage modulus increased significantly with increasing EGCG concentration, indicating enhanced viscoelasticity of the emulsion. This suggests that the emulsion structure's ability to resist mechanical forces is enhanced, exhibiting strong self-supporting properties. Compared to Pickering emulsions with other concentrations, the storage modulus of the emulsion with a 1% (w / v) EGCG concentration increased rapidly, indicating that a 1% (w / v) concentration of EGCG can more effectively affect the viscoelasticity of Pickering emulsions than other EGCG concentrations (0%–0.75%).

[0099] The viscosity of the pickering emulsion changes with shear rate as follows: Figure 10 As shown. Figure 10 The viscosity and shear rate (0-100 s) of the Pickering emulsion are shown. -1The relationship between the shear rate and the viscosity of the Pickering emulsion is discussed. Increased shear rate reduces the viscosity of the emulsion, which can be attributed to shear-thinning behavior induced by the increased shear rate. As the shear rate gradually increases, the droplet network of the emulsion is disrupted by shear forces, leading to a decrease in viscosity. Furthermore, the viscosity of the emulsion increases with increasing EGCG concentration, indicating improved emulsion stability. This is likely because interactions between particles and between particles and the liquid are enhanced at higher EGCG concentrations, resulting in a denser network.

[0100] The storage modulus and loss modulus of pickering emulsions as a function of frequency are as follows: Figure 11 As shown, the storage modulus and loss modulus of all samples were frequency-dependent, and across the entire frequency range from 0.01 to 10 Hz, the storage modulus of all emulsions was greater than the loss modulus, indicating that the reversible deformation of the Pickering emulsion was greater than the irreversible deformation, resulting in an elastic network structure. With increasing EGCG concentration in the cooked egg white protein-EGCG complex, the values ​​of both storage modulus and loss modulus increased accordingly, indicating that the network structure of the emulsion was strengthened.

[0101] The storage modulus and loss modulus of pickering emulsions change with temperature as follows: Figure 12 As shown. Figure 12 As shown, under dynamic temperature scanning (20℃-80℃) testing, the storage modulus and loss modulus of stable Pickering emulsions at EGCG concentrations of 0.75% and 1% (w / v) remained relatively stable during heating, indicating that the network structure of the emulsion was not destroyed under heat treatment. Furthermore, the thermal stability of the Pickering emulsion increased with increasing EGCG concentration. The excellent thermal stability of Pickering emulsions gives them unique advantages in food processing.

[0102] Astaxanthin release rates of Pickering emulsion loaded with astaxanthin at different stages of digestion in the gastrointestinal tract, as shown in the figure. Figure 13As shown, the astaxanthin pickering emulsion with 1% (w / v) EGCG showed a low release rate of 22.17% when the sample was in the gastric fluid stage, but the release rate increased significantly to 79.24% when it was in the intestinal fluid stage. Similarly, similar results were found in the astaxanthin pickering emulsion without EGCG, with a release rate of 33.07% in gastric fluid and 58.90% in intestinal fluid. Notably, the astaxanthin release rate of the astaxanthin pickering emulsion without EGCG increased by only 25.83% in intestinal fluid, far lower than the 57.07% of the astaxanthin pickering emulsion with 1% (w / v) EGCG. During gastric digestion, the low release rate of astaxanthin in a 1% (w / v) astaxanthin pickering emulsion can be attributed to the better protection of astaxanthin at the oil-water interface provided by the cooked egg white protein-EGCG complex stabilized emulsion prepared with high-concentration EGCG, effectively reducing the release rate. Therefore, a pickering emulsion stabilized by cooked egg white protein particles and EGCG can serve as an effective delivery system to achieve a sustained release of astaxanthin in the gastrointestinal tract.

[0103] Pickering emulsion loaded with astaxanthin was 3D printed. The emulsion was loaded into the 3D printer and printed at a speed of 1 mm. 3 ·s -1 The material is extruded at a constant rate through a nozzle with a diameter of 1 mm to print a "Chinese knot" pattern. For example... Figure 14 As shown, 3D printing was observed from the front and side to evaluate the potential of pickering emulsions stabilized with cooked egg white protein-EGCG complex and loaded with astaxanthin in 3D printing. The results showed that the "Chinese knot" shape printed by the pickering emulsion stabilized by cooked egg white protein alone still exhibited strong fluidity, with relatively blurry patterns and relatively poor support. With the addition and increase of EGCG concentration, the pattern became increasingly complete. When the EGCG concentration was 1% (w / v), the support and integrity of the pickering emulsion were significantly improved, and the outline was clear. In particular, the 1% (w / v) EGCG concentration produced the most complete pattern, with clearly visible holes and a clear and complete printing path, demonstrating high precision and forming a clear printed structure with good 3D printing stability and accuracy.

[0104] Based on the above rheological and stability analyses, the concentration of EGCG in the cooked egg white protein-EGCG complex plays a crucial role in the Pickering emulsion. Therefore, the astaxanthin-loaded Pickering emulsion prepared at an EGCG concentration of 1% (w / v) can be used for 3D printing food, and various structures can be designed and produced to meet consumer needs in 3D printing, demonstrating broad application potential and value.

[0105] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. A full component egg white protein-EGCG complex stabilizer, characterized by, The cooked egg white protein-EGCG complex stabilizer is formed by pH regulation complexing of a cooked egg white protein microparticle solution and an EGCG solution; wherein the cooked egg white protein microparticle solution is obtained by mixing cooked egg white with water in a broken wall machine after separating the cooked egg white from the yolk of a boiled egg.

2. The all-component egg white protein-EGCG complex stabilizer according to claim 1, characterized by, The concentration of the EGCG solution is 0.5-2%, w / v, g / mL.

3. The all-component egg white protein-EGCG complex stabilizer according to claim 1, characterized by, The volume ratio of the cooked egg white protein microparticle solution and the EGCG solution is 1:

1.

4. A method for preparing a Pickering emulsion based on the all-component egg white protein-EGCG complex stabilizer according to any one of claims 1 to 3, characterized by, The method comprises the following steps: The whole-component egg white protein-EGCG complex stabilizer is mixed with edible oil and sheared to obtain a Pickering emulsion stabilized by the whole-component egg white protein-EGCG complex.

5. The Pickering emulsion prepared by the method of claim 4.

6. The Pickering emulsion of claim 5 for use as a functional carrier.

7. A method of improving the thermal stability and storage stability of a Pickering emulsion, characterized in that, The method comprises the following steps: The whole-component egg white protein-EGCG complex stabilizer of any one of claims 1-3 is mixed with edible oil and sheared to obtain a Pickering emulsion stabilized by the whole-component egg white protein-EGCG complex; wherein the concentration of EGCG in the whole-component egg white protein-EGCG complex stabilizer is 0.75%-1%, w / v, g / mL.

8. A method for the preparation of a Pickering emulsion loaded with astaxanthin, characterized in that, The method comprises the following steps: The whole-component egg white protein-EGCG complex stabilizer of any one of claims 1-3 is mixed with edible oil containing astaxanthin and sheared to obtain a Pickering emulsion loaded with astaxanthin.

9. The Pickering emulsion loaded with astaxanthin prepared by the method of claim 8.

10. A 3D printed food product, characterized in that, The 3D-printed food is based on the Pickering emulsion loaded with astaxanthin of claim 9 as raw material and is formed by 3D printing, wherein the concentration of EGCG in the whole-component egg white protein-EGCG complex stabilizer in the Pickering emulsion loaded with astaxanthin is 1%, w / v, mg / mL.

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