Sardine protein / konjac glucomannan compound stable Pickering emulsion gel and application

Abstract

The invention discloses Pickering emulsion gel with stable sardine protein / konjac glucomannan compound and application of the Pickering emulsion gel, and belongs to the technical field of Pickering emulsion. According to the preparation method, sardine protein is taken as a raw material, konjac glucomannan is added to form a compound, the Pickering emulsion is stabilized based on the gel characteristic of the compound, and the stable Pickering emulsion gel can be prepared only through a one-step emulsification method. A good physical barrier can be provided for active ingredients, and the retention rate of astaxanthin under the conditions of storage, heating and ultraviolet irradiation is increased. Meanwhile, the emulsion prepared by the method disclosed by the invention has an intestinal flora probiotic effect. The emulsion gel is easy to prepare, raw materials are green and safe, and the emulsion gel can be used for delivering active ingredients and can be applied to the field of food.

Description

Technical Field

[0001] The invention relates to a Pickering emulsion gel stabilized by a sardine protein / konjac glucomannan complex and application thereof, belonging to the technical field of Pickering emulsions. Background Art

[0002] Astaxanthin (AXT) is a ketone-like, fat-soluble carotenoid found primarily in algae, yeast, shrimp, and crustacean shells. Natural AXT molecules possess a unique carbon-carbon double bond structure that effectively reduces reactive oxygen species and scavenges free radicals, resulting in excellent antioxidant and anti-inflammatory activities and potential use as a nutritional supplement in the food industry. However, astaxanthin's low water solubility and instability result in low bioavailability, limiting its practical application.

[0003] Pickering emulsions, stabilized by solid particles rather than surfactants, have attracted considerable attention due to their resistance to aggregation, Ostwald solidification, and high stability. Currently, Pickering emulsions are widely used to load functional substances with low water solubility. Common food-grade stabilizers include proteins, polysaccharides, and protein-polysaccharide complexes, which stabilize emulsions by reducing interfacial tension or increasing electrostatic repulsion between droplets. Protein-stabilized Pickering emulsions are prone to aggregation and flocculation during prolonged storage. Therefore, forming complexes with other substances, such as polysaccharides and polyphenols, is an effective means of improving the emulsification stability of protein particles. Polysaccharides can improve the interfacial adsorption state of particles by modifying the secondary structure and wettability of proteins. Furthermore, polysaccharides can prevent flocculation and demixing by increasing the thickness of the oil-water interface, thereby directly reducing the aggregation / accumulation of oil droplets and providing a good physical barrier to the active substance, minimizing its loss. Therefore, protein-polysaccharide complexes are ideal stabilizers for the preparation of Pickering emulsions for the loading and delivery of astaxanthin. Currently, there are existing technologies for preparing Pickering emulsions using protein-polysaccharide complexes as stabilizers. For example, patent CN 116172186A discloses a method for preparing a Pickering emulsion using myofibrillar protein / konjac glucomannan as a stabilizer; patent CN112370419 A discloses a method for preparing a Pickering emulsion using zein-konjac glucomannan as a stabilizer. However, the Pickering emulsions prepared using the above-mentioned protein-polysaccharide complexes as stabilizers still suffer from uneven oil droplet distribution, which easily leads to droplet aggregation and phase separation. Therefore, it is necessary to explore a method for improving the storage stability, thermal stability, and centrifugal stability of Pickering emulsions. Summary of the Invention

[0004] [Technical Issues]

[0005] The technical problem to be solved by the present invention is that the existing technology of using protein-polysaccharide complexes as stabilizers to prepare Pickering emulsions suffers from internal droplet coalescence and flocculation, which easily leads to phase separation.

[0006] [Technical solution]

[0007] In order to solve the above problems, the present invention provides a Pickering emulsion gel stabilized by a sardine protein / konjac glucomannan complex. The present invention uses sardine protein as a raw material, forms a complex by adding konjac glucomannan (KGM), stabilizes the Pickering emulsion based on the gel properties of the complex, and can prepare a stable Pickering emulsion gel by only one-step emulsification method. The present invention can provide a good physical barrier for the active ingredients and improve the retention rate of astaxanthin under storage, heating and ultraviolet light conditions. At the same time, the emulsion prepared by the method of the present invention has a probiotic effect on intestinal flora. The emulsion gel of the present invention is simple to prepare, the raw materials are green and safe, can be used for the delivery of active ingredients, and can be applied to the food field.

[0008] The first object of the present invention is to provide a Pickering emulsion gel stabilized by a sardine protein / konjac glucomannan complex. The Pickering emulsion gel is obtained by mixing a sardine protein aqueous solution and a konjac glucomannan aqueous solution to obtain a mixture solution as an aqueous phase and oil as an oil phase, and then subjecting the mixture to high-speed shearing.

[0009] In one embodiment of the present invention, the preparation method of the sardine protein is as follows: chopped sardines are mixed with deionized water in a ratio of 1:10 (g / mL), and the pH value of the mixture is adjusted to 10.5 with a 2mol / L NaOH solution. After stirring for 2 hours, the mixture is centrifuged at 9000 rpm for 30 minutes at 4°C, and the pH value of the obtained supernatant is adjusted to 4.5 with a 2mol / L HCl solution. Then, the suspension is placed at room temperature for 30 minutes, centrifuged at 8000 rpm for 15 minutes at 4°C, and the obtained protein precipitate is suspended in deionized water, the pH value is adjusted to 7.0, stirred at room temperature for 12 hours, and centrifuged at 8000 rpm for 10 minutes at 4°C. The obtained supernatant is freeze-dried to obtain sardine protein.

[0010] In one embodiment of the present invention, the sardine protein aqueous solution and the konjac glucomannan aqueous solution are mixed in equal volumes to allow them to fully interact with each other.

[0011] In one embodiment of the present invention, the concentration of the sardine protein aqueous solution in the aqueous phase is 1-2 wt%.

[0012] In one embodiment of the present invention, the concentration of the konjac glucomannan aqueous solution in the aqueous phase is 0.4-2 wt%.

[0013] In one embodiment of the present invention, the oil is one or more of DHA algae oil, fish oil, γ-linolenic acid oil, krill oil, chia seed oil, corn oil, and soybean oil.

[0014] In one embodiment of the present invention, high-speed shearing is shearing at 8000-12000 rpm for 1-3 minutes.

[0015] In one embodiment of the present invention, the volume ratio of the water phase to the oil phase is 60:40 to 30:70, more preferably 45:55 to 35:65.

[0016] The second object of the present invention is to provide a Pickering emulsion gel loaded with active ingredients, wherein the Pickering emulsion gel loaded with active ingredients is prepared from the Pickering emulsion gel stabilized by the sardine protein / konjac glucomannan complex described above.

[0017] In one embodiment of the present invention, the active ingredient is astaxanthin.

[0018] In one embodiment of the present invention, a method for preparing a Pickering emulsion gel loaded with active ingredients comprises: mixing the Pickering emulsion gel stabilized by the sardine protein / konjac glucomannan complex and corn oil in which astaxanthin is dissolved in proportion, and homogenizing the mixture.

[0019] The third object of the present invention is to provide the use of the above-mentioned Pickering emulsion gel loaded with active ingredients in protecting nutrients and promoting the probiotic growth of intestinal flora.

[0020] The fourth object of the present invention is to provide the use of the above-mentioned Pickering emulsion gel loaded with active ingredients in the preparation of foods, medicines and health products.

[0021] [Beneficial Effects]

[0022] (1) The present invention stabilizes the Pickering emulsion gel based on the sardine protein / konjac glucomannan complex, and has good storage stability, thermal stability, centrifugal stability, pH stability and salt ion stability.

[0023] (2) The preparation process of this method is simple and easy to operate, and the amount of the complex used is small, and it has good biosafety and environmental compatibility.

[0024] (3) Pickering emulsion gel can reduce the degree of damage to the active substances loaded therein by gastric acid and maintain good integrity. At the same time, the smaller size can increase the contact between the droplets and lipase, promote the release of active ingredients in the intestine, and thus better protect the balance of intestinal flora. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 : Particle sizes of SDP and SKCs.

[0026] Figure 2 : Intrinsic fluorescence spectra of SDP and SKCs.

[0027] Figure 3 : Circular dichroism spectra of SDP and SKCs.

[0028] Figure 4 : Secondary structures of SDP and SKCs.

[0029] Figure 5 : Infrared spectra of SDP and SKCs.

[0030] Figure 6 : Appearance (A) and microstructure (B, C) of SPEs and SKEGs.

[0031] Figure 7 : Particle size of SPEs and SKEGs.

[0032] Figure 8 : Storage stability (A), thermal stability (B), and centrifugation stability (C) of SPEs and SKEGs.

[0033] Figure 9 : Storage stability (A, B), thermal stability (C), and UV stability (D) of AXT-SPEs and AXT-SKEGs.

[0034] Figure 10 : Effects of AXT-SKEGs on gut microbial abundance at the phylum level.

[0035] Figure 11 : Effects of AXT-SKEGs on gut microbial abundance at the genus level. DETAILED DESCRIPTION

[0036] Example 1

[0037] This embodiment provides a Pickering emulsion gel stabilized by a sardine protein / konjac glucomannan complex, the preparation method of which includes:

[0038] (1) Preparation of sardine protein (SDP): minced sardines were mixed with deionized water at a ratio of 1:10 (w / v, g / mL), and the pH of the mixture was adjusted to 10.5 with 2 mol / L NaOH solution. After stirring for 2 h, the mixture was centrifuged at 9000 rpm at 4°C for 30 min. The pH of the supernatant was adjusted to 4.5 with 2 mol / L HCl solution. Then, the suspension was allowed to stand at room temperature (25°C) for 30 min and then centrifuged at 8000 rpm at 4°C for 15 min. The resulting protein precipitate was suspended in deionized water and the pH was adjusted to 7.0. After stirring at room temperature for 12 h, the mixture was centrifuged at 8000 rpm at 4°C for 10 min. The supernatant was freeze-dried to obtain sardine protein.

[0039] (2) An aqueous solution of sardine protein and an aqueous solution of konjac glucomannan (KGM) were mixed in equal volumes to obtain a complex solution with a sardine protein concentration of 1% (w / v, g / mL) and a konjac glucomannan concentration of 1.2% (w / v, g / mL) as the aqueous phase and corn oil as the oil phase (the volume ratio of the aqueous phase to the oil phase was 40:60). The mixture was homogenized at 10,000 rpm for 2 minutes using a high-speed disperser to obtain a Pickering emulsion gel, named SKEG (1.2%).

[0040] Example 2

[0041] The preparation method of Pickering emulsion gel was the same as that in Example 1, except that the concentration of the konjac glucomannan aqueous solution was 0.4%, and was named SKEG (0.4%).

[0042] Example 3

[0043] The preparation method of Pickering emulsion gel was the same as that in Example 1, except that the concentration of the konjac glucomannan aqueous solution was 0.8%, and was named SKEG (0.8%).

[0044] Example 4

[0045] The preparation method of Pickering emulsion gel was the same as that in Example 1, except that the concentration of the konjac glucomannan aqueous solution was 1.6%, and was named SKEG (1.6%).

[0046] Example 5

[0047] The preparation method of Pickering emulsion gel was the same as that in Example 1, except that the concentration of the konjac glucomannan aqueous solution was 2%, and was named SKEG (2%).

[0048] Comparative Example 1

[0049] The preparation method of Pickering emulsion gel was the same as that in Example 1, except that the concentration of the konjac glucomannan aqueous solution was 0%, and was named SPE (0%).

[0050] Comparative Example 2

[0051] The preparation steps of the sardine protein-konjac glucomannan (SKCs) complex solution are as follows: an aqueous sardine protein solution and an aqueous konjac glucomannan solution are mixed in equal volumes to obtain a complex solution having a sardine protein concentration of 1% (w / v, g / mL) and a konjac glucomannan concentration of 1.2% (w / v, g / mL), which is named SKCs.

[0052] The particle size of SDP and SKCs was determined by using a particle size analyzer (Zetasizer 3000HSA). Figure 1 As shown in the figure, the particle size of SDP is 256.70±6.13μm and the average particle size of SKCs is 375.6±13.61nm.

[0053] The endogenous fluorescence of SDP and SKCs was measured by using a fluorescence spectrophotometer (F-2700). Figure 2 As can be seen, when the excitation wavelength is 280 nm, the maximum emission peak of SDP is at 302 nm. Furthermore, the fluorescence of SDP decreases with the addition of KGM. This is likely due to the fact that the introduction of KGM promotes the unfolding of the SDP protein structure and the exposure of hydrophobic groups. This structural change alters the polarity of the microenvironment of the chromogenic amino acids in the protein, resulting in a decrease in intrinsic fluorescence intensity.

[0054] The circular dichroism spectra of SDP and SKCs were measured using a CD spectropolarimeter fluorescence spectrophotometer (J-1500). Figure 3 and Figure 4 Four secondary structures can be observed in SDP, including α-helix (21.5%), β-sheet (40.7%), β-turn (8.9%), and random coil (28.9%). The results indicate that the α-helix content increases with the addition of KGM, which is attributed to the formation of hydrogen bonds between SDP and KGM. These results suggest that the secondary structure of SDP can be altered by hydrogen bonding during the formation of colloidal particles.

[0055] The infrared spectra of SDP and SKCs were determined by using a Fourier transform infrared spectrometer (Frontier). Figure 5As the KGM concentration increases, the blue shift of the OH stretching band confirms the formation of hydrogen bonds between SDP and KGM. Furthermore, the amide II band of SKCs shifts to higher wavenumbers, indicating a hydrophobic interaction between SDP and KGM. The amide I band, on the other hand, remains unchanged, indicating that electrostatic interactions are not the primary force between SDP and KGM.

[0056] The structure and morphology of the oil droplets were measured using a fluorescence inverted microscope (Ti-S, Nikon) and a cryogenic scanning electron microscope (SU-8010, Hitachi). Figure 6 The size distribution of oil droplets was analyzed using Image J software. A total of 200 oil droplets were counted. The results are shown in Figure 7 .from Figure 6 As can be seen from A, when the sample bottle is inverted, the SPEs show a flowing state, while all SKEGs show a solid appearance, indicating that a stable gel structure has been formed. Figure 6 B shows that with the increase of KGM concentration, the oil droplets in SKEGs are denser and more uniform than those in SPEs. This is probably due to the formation of a gel network structure that prevents the merging and flow of droplets. Excessive addition of KGM (>1.2%) leads to an increase in droplet size because excessive addition of KGM hinders the adsorption of SDP at the oil-water interface, causing SDP to accumulate in the continuous phase, further affecting the stability of SKEGs. In addition, Figure 6 As can be seen from Figure C, a gel network structure is observed between the oil droplets, which effectively inhibits the relative motion between the droplets and enhances the stability of the Pickering emulsion. Figure 7 It can be seen that the average particle size of oil droplets in SPEs is 15.26±0.84μm, and the average particle size of oil droplets in SKEGs is related to the KGM concentration. The oil droplet size of SKEGs (1.2%) is the smallest, with an average particle size of 7.12±0.14μm.

[0057] SKEG was placed in a glass sample bottle and stored at room temperature for 14 days. The appearance changes were recorded using a camera and the microstructural changes were observed using a fluorescence inverted microscope (Ti-S, Nikon). Figure 8 A. SKEGs were placed in glass sample bottles and heated at 95°C for 30 min. The appearance changes were recorded using a camera and the microstructural changes were observed using a fluorescence inverted microscope (Ti-S, Nikon). Figure 8 B. From Figure 8As shown in A and B, after storage for 14 days and heating at 95°C for 30 minutes, the emulsion stabilized by SDP alone showed obvious phase separation and oil leakage, while the appearance of the Pickering emulsion stabilized by SKCs remained better. It can also be observed that the size of the oil droplets increased during storage, and the degree of increase was affected by the KGM concentration. With the increase of KGM concentration, the increase in oil droplet size decreased significantly, and when the KGM concentration was 1.2%, the increase in oil droplet size was the smallest, with better stability. The results show that the oil droplets are fixed in the gel network structure, and the adsorption of the composite particles at the oil-water interface effectively enhances the interfacial barrier, so that the movement of the oil droplets is inhibited, thereby preventing the merging of adjacent oil droplets and avoiding the occurrence of phase separation. It can be seen that SKEGs (1.2%) have better storage and thermal stability. SKEGs were placed in glass sample bottles and centrifuged at 9000g for 10 minutes. The appearance changes were recorded using a camera, and the microstructural changes were observed using a fluorescence inverted microscope (Ti-S, Nikon). The results are shown in Figure 8 C. After centrifugation, a small amount of aqueous phase appeared at the bottom of emulsions containing lower KGM concentrations (0.4%-0.8%), while emulsions containing higher KGM concentrations (1.2%-2%) showed no significant change in appearance, indicating that the appropriate addition of KGM can promote the formation of a network structure and prevent phase separation caused by centrifugation. From a microstructural perspective, due to the high centrifugal force, the oil droplets in all samples squeezed against each other after centrifugation, forming a denser microstructure.

[0058] Comparative Example 3

[0059] AXT was dissolved in corn oil (1 mg / mL) and stirred in the dark for 24 h until it was completely dissolved. The solution was then centrifuged at 8000g for 10 min to remove insoluble matter and named Free AXT.

[0060] Comparative Example 4

[0061] The SDP prepared in Example 1 was mixed with the AXT-dissolved corn oil prepared in Comparative Example 3 (the volume ratio of the aqueous phase to the oil phase was 40:60). The mixture was homogenized at 10,000 rpm for 2 min using a high-speed disperser to obtain an AXT-loaded Pickering emulsion, which was named AXT-SPEs (0%).

[0062] Example 6

[0063] The SKEG (1.2%) prepared in Example 1 was mixed with the AXT-dissolved corn oil prepared in Comparative Example 3 in a proportion (the volume ratio of the aqueous phase to the oil phase was 40:60). The mixture was homogenized at 10,000 rpm for 2 min using a high-speed disperser to obtain an AXT-loaded Pickering emulsion gel, named AXT-SKEGs (1.2%).

[0064] Stability of the samples of Comparative Examples 3, 4 and Example 6 and their protective effects on the balance of intestinal flora in IBD mice:

[0065] (1) Storage stability of AXT-SKEG

[0066] The samples of Comparative Examples 3, 4 and Example 6 were stored at room temperature for 14 days, and samples were taken every 2 days to measure the AXT retention rate. The results are shown in Table 1. Figure 9 A, B.

[0067] (2) Thermal stability of AXT-SKEG

[0068] The samples of Comparative Examples 3, 4 and Example 6 were heated in a water bath at 90°C for 14 h in the dark, and samples were taken every 2 h to determine the AXT retention rate. The results are shown in Table 1. Figure 9 C.

[0069] (3) UV stability of AXT-SKEG

[0070] All samples were exposed to 6W ultraviolet light for 48 hours, and samples were taken every 2 hours to determine the AXT retention rate. The results are shown in Figure 9 D.

[0071] from Figure 9 It can be seen that after 14 days of storage, the retention rates of AXT in corn oil, Pickering emulsion stabilized by SDP, and Pickering emulsion gel stabilized by SKCs were 28.32 ± 1.64%, 42.31 ± 0.64%, and 61.23 ± 0.76%, respectively. Figure 3Figure 2B shows a semi-logarithmic plot of AXT retention. The AXT in corn oil exhibited the highest decay rate, primarily due to the susceptibility of unprotected AXT to photolysis during storage. The decay rate of AXT in the Pickering emulsion gel stabilized by SKCs was significantly lower than that in the Pickering emulsion stabilized by SDP. After heating at 90°C for 14 h, the AXT retention in the SKCs-stabilized Pickering emulsion gel (77.27 ± 1.67%) was significantly higher than that in the SDP-stabilized Pickering emulsion (60.12 ± 1.58%), while only 37.60 ± 1.90% of AXT remained in the corn oil. After 14 h of UV irradiation, the AXT content in the corn oil dropped to 67.69 ± 1.69%, demonstrating its photostability. In contrast, the AXT retention in the Pickering emulsion stabilized by SDP reached 74.73 ± 1.52%, and in the Pickering emulsion gel stabilized by SKCs reached 82.19 ± 1.52%, both higher than that of unencapsulated AXT. The interfacial barrier and compact gel network provided by KGM enhanced the stability of AXT and reduced its loss during storage, heat, and UV exposure.

[0072] (5) Protective effect of AXT-SKEGs on intestinal flora balance in IBD mice

[0073] Mice were randomly divided into five groups: control, DSS, free AXT, AXT-SPEs, and AXT-SKEGs. After full acclimation, mice in the control and DSS groups received daily gavage with water. Mice in the free AXT, AXT-SPE, and AXT-SKEGs groups received daily gavage with free AXT, AXT-SPE, or AXT-SKEGs, respectively, from days 0 to 21. AXT dose of 10 mg / kg / day was administered. Except for the control group, all other groups received 3.5% DSS dissolved in their drinking water from days 15 to 21. Feces were collected on day 22, and the intestinal microbial flora in the feces was analyzed by 16S rRNA.

[0074] from Figure 10As can be seen, Bacteroidetes and Firmicutes were the most dominant bacterial phyla in all samples, followed by Proteobacteria, Campilobacteriota, and Actinobacteria. Notably, compared with the control group, the abundance of harmful bacteria associated with colitis (Bacteroidetes and Proteobacteria) increased in the DSS group, while the abundance of beneficial bacteria (Firmicutes) decreased. After AXT intervention, the changes in species composition caused by DSS were restored. In particular, the AXT-SKEGs group showed a stronger regulatory effect compared with the AXT-SPEs group, and its overall intestinal microbiota composition was close to that of the control group. Figure 11 The species composition and abundance of each group at the genus level were presented in a ternary plot. Under DSS treatment, the expression level of Bacteroidetes was associated with increased inflammation. Compared with the DSS group, the Bacteroidetes content decreased in the free AXT, AXT-SPEs, and AXT-SKEGs groups, and the Bacteroidetes abundance in the AXT-SKEGs group was lower than that in the free AXT and AXT-SPEs groups. Furthermore, high expression of Lactococcus has a positive effect on the alleviation of IBD. Compared with the DSS group, the content of Lactococcus increased in the free AXT, AXT-SPEs, and AXT-SKEGs groups, and the abundance of Lactococcus was higher in the AXT-SPEs and AXT-SKEGs groups than in the free AXT group. In summary, AXT-SPEGs can maintain the balance of the intestinal flora by increasing the number of beneficial bacteria and reducing the number of harmful bacteria.

[0075] The embodiments provided above are not intended to limit the scope of the present invention, nor are the steps described to limit their execution order. Any obvious improvements to the present invention made by those skilled in the art in combination with existing common knowledge shall fall within the scope of protection defined by the claims of the present invention.

Claims

1. A Pickering emulsion gel stabilized by a sardine protein / konjac glucomannan complex, characterized in that: The sardine protein aqueous solution and the konjac glucomannan aqueous solution are mixed, the obtained mixture solution is used as the water phase, and the fat is used as the oil phase, and the mixture is subjected to high-speed shearing to obtain the Pickering emulsion gel.

2. The Pickering emulsion gel stabilized by the sardine protein / konjac glucomannan complex according to claim 1, characterized in that The sardine protein aqueous solution and the konjac glucomannan aqueous solution were mixed in equal volumes.

3. The Pickering emulsion gel stabilized by the sardine protein / konjac glucomannan complex according to claim 1, characterized in that In the aqueous phase, the concentration of the sardine protein aqueous solution is 1-2 wt %; the concentration of the konjac glucomannan aqueous solution is 0.4-2 wt %.

4. The Pickering emulsion gel stabilized by the sardine protein / konjac glucomannan complex according to claim 1, characterized in that The oil is one or more of DHA algae oil, fish oil, γ-linolenic acid oil, krill oil, chia seed oil, corn oil, and soybean oil.

5. The Pickering emulsion gel stabilized by the sardine protein / konjac glucomannan complex according to claim 1, characterized in that High-speed shearing is 8000-12000 rpm shearing for 1-3 minutes.

6. The Pickering emulsion gel stabilized by the sardine protein / konjac glucomannan complex according to claim 1, characterized in that The volume ratio of the water phase to the oil phase is 60:40 to 30:

70.

7. The Pickering emulsion gel stabilized by the sardine protein / konjac glucomannan complex according to claim 1, characterized in that The preparation method of sardine protein is as follows: chopped sardines are mixed with deionized water in a ratio of 1:10, g / mL, and the pH value of the mixture is adjusted to 10.5 with a 2mol / L NaOH solution. After stirring for 2 hours, the mixture is centrifuged at 9000 rpm for 30 minutes at 4°C, and the pH value of the obtained supernatant is adjusted to 4.5 with a 2mol / L HCl solution. Then, the suspension is allowed to stand for 30 minutes, and then centrifuged at 8000 rpm for 15 minutes at 4°C. The obtained protein precipitate is suspended in deionized water, and the pH value is adjusted to 7.

0. After stirring for 12 hours, the mixture is centrifuged at 8000 rpm for 10 minutes at 4°C, and the obtained supernatant is freeze-dried to obtain sardine protein.

8. A Pickering emulsion gel loaded with active ingredients, characterized in that: The Pickering emulsion gel loaded with active ingredients is prepared from the Pickering emulsion gel stabilized by the sardine protein / konjac glucomannan complex according to any one of claims 1 to 7.

9. The Pickering emulsion gel loaded with active ingredients according to claim 8, characterized in that: The active ingredient is astaxanthin.

10. Use of the active ingredient-loaded Pickering emulsion gel according to claim 9 in the preparation of foods, medicines and health products.

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