Preparation method of enteric-targeted slow-release antarctic krill oil pickering emulsion

Abstract

This invention discloses a method for preparing an intestinal-targeted sustained-release Antarctic krill oil Pickering emulsion: Zein / κ-CA binary composite nanoparticles are prepared by dissolving zein in an ethanol aqueous solution and rapidly adding it to a κ-carrageenan solution via antisolvent precipitation. These binary composite nanoparticles are then added to a sodium alginate aqueous solution to construct a Zein / κ-CA / SA ternary composite nanoparticle through layer-by-layer adsorption. Zein / κ-CA / SA are used as stabilizers to organically combine with Antarctic krill oil, and a Pickering emulsion is constructed through high-speed shearing. Finally, K... + and Ca 2+ The solution was ionically cross-linked to construct an Antarctic krill oil Pickering emulsion with a dual-network interpenetrating structure at the oil-water interface. The emulsion exhibited good structural stability and good intestinal-targeted sustained-release properties based on the directional hydrolysis of κ-CA and SA by intestinal β-mannanase.

Description

Technical Field

[0001] This invention belongs to the field of Pickering emulsion oil-water interface modification technology, specifically involving a protein / polysaccharide composite nanoparticle forming a network interpenetrating oil-water interface stabilizer through ionic double cross-linking, and developing an intestinal-targeted sustained-release Antarctic krill oil Pickering emulsion based on the directional hydrolysis of intestinal β-mannanase. Background Technology

[0002] Antarctic krill oil is a red, mixed lipid extracted from Antarctic krill, comprising approximately one-sixth of the krill's body weight. It is rich not only in ω-3 polyunsaturated fatty acids such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), but also in bioactive components such as flavonoids, phospholipids, and fat-soluble vitamins. The synergistic effects of DHA, EPA, phospholipids, and astaxanthin give Antarctic krill oil various metabolic regulatory functions in the human body, improving health, preventing cardiovascular disease and premenstrual syndrome, inhibiting inflammation, improving memory, and inhibiting tumors. However, the phospholipid-type ω-3 polyunsaturated fatty acids and other active components in Antarctic krill oil are easily degraded in the stomach due to pepsin, low acidity, and ionic strength, making them difficult to digest and absorb in the small intestine, resulting in low oral bioavailability. Constructing an Antarctic krill oil Pickering emulsion system is a potential way to improve these problems, with oil-in-water (O / W) emulsions showing the greatest potential as a delivery system for Antarctic krill oil.

[0003] Pickering emulsions are systems stabilized by micron- or even nano-sized solid particles. They are thermodynamically and kinetically stable systems that prevent droplet aggregation through interparticle interactions, reduce the total free energy of the system, and create a spatial physical barrier. Relying on the hydrophobicity of the particle surface, this process is considered irreversible adsorption. Compared to traditional emulsions, picking emulsions offer significant advantages: lower emulsifier dosage, reducing raw material costs; and environmental friendliness, with minimal impact from factors such as temperature and ionic strength. Based on considerations of safety, sustainability, biocompatibility, and biodegradability, research on food-grade solid particle-stabilized picking emulsions is increasing. These can be categorized into three types: first, picking emulsions using proteins as emulsifiers, such as soy protein and zein; second, picking emulsions using polysaccharides as emulsifiers, such as modified starch and cellulose nanocrystals; and third, picking emulsions using binary or higher complexes as emulsifiers, such as the zein-pectin binary complex system.

[0004] Sodium alginate (SA) is a sodium salt of a polyanionic polysaccharide extracted from natural brown algae. It is characterized by its green and safe nature, biocompatibility, and biodegradability. When it encounters cations such as calcium ions, it forms a gel layer, thus achieving a sustained-release effect on drugs. κ-carrageenan (κ-CA) is also a polyanionic polysaccharide, and its disaccharide units contain sulfate groups, allowing it to cross-link with specific ions such as calcium ions. Furthermore, due to its shear-thinning properties, it has adjustable viscosity and can be compounded with SA. This can alter its viscosity, gelling properties, and swelling properties, which is beneficial for the long-term retention of active substances, achieving a sustained-release effect. Both can be hydrolyzed by β-mannanase, resulting in targeted release.

[0005] Omega-3 polyunsaturated fatty acids can only be absorbed and utilized by the human body after interacting with lipases in the small intestine. During digestion, omega-3 polyunsaturated fatty acids undergo structural and property changes due to the action of other enzymes and oxygen, reducing their nutritional value. Therefore, reducing the release of omega-3 polyunsaturated fatty acids before they reach the small intestine digestion stage and increasing their release during the small intestine digestion stage can improve their bioavailability. The Antarctic krill oil Pickering emulsion prepared by this invention, before the small intestine digestion stage, can reduce the emulsion particle size due to the strong electrostatic and ionic cross-linking effects of the composite nanoparticles. It can also aggregate on the droplet surface to form a physical barrier, effectively reducing the release of ω-3 polyunsaturated fatty acids even in the low pH environment of the stomach. During the small intestine digestion stage, the directional hydrolysis of α-1,4 glycosidic bonds by β-mannanase in the intestine enhances the targeted release of ω-3 polyunsaturated fatty acids in the intestine. At the same time, the double network interpenetrating structure of the composite nanoparticles competitively adsorbs with lipase at the oil-water interface, improving the slow release effect of ω-3 polyunsaturated fatty acids, and finally improving its oral bioavailability over a long period of time. Summary of the Invention

[0006] This invention is based on the directed hydrolysis of intestinal β-mannanase, using Zein / κ-CA / SA ternary nanoparticles as a stabilizer and organically combining them with Antarctic krill oil to prepare a network-interpenetrating intestinal-targeted sustained-release Antarctic krill oil Pickering emulsion. The emulsion contains K... + Cross-linked κ-CA forms the first polymeric network, with Ca... 2+ The cross-linked SA forms the second polymer network. (The text abruptly ends here, likely due to an incomplete sentence or missing information.) + Solution, Ca 2+ Solution cross-linking can form an interpenetrating network structure, which is convenient to operate and has a simple process. Dual ionic cross-linking based on β-mannanase-directed hydrolysis endows Antarctic krill oil Pickering emulsion with good intestinal targeting and sustained release effects.

[0007] The technical solution of the present invention is as follows:

[0008] A method for preparing an intestinal-targeted sustained-release Antarctic krill oil Pickering emulsion includes:

[0009] Zein (zein protein) was dissolved in an ethanol-water solution. The resulting Zein solution was added to a κ-CA (κ-carrageenan) solution, and Zein / κ-CA binary composite nanoparticles were prepared by antisolvent precipitation. Subsequently, the Zein / κ-CA binary composite nanoparticles were added to an SA (sodium alginate) aqueous solution to construct Zein / κ-CA / SA ternary composite nanoparticles through layer-by-layer adsorption. Using the Zein / κ-CA / SA ternary composite nanoparticles as a stabilizer, they were organically combined with Antarctic krill oil, and a Pickering emulsion was constructed by high-speed shearing. Finally, K... + Solution, Ca 2+ The solution was subjected to ionic double cross-linking to construct an Antarctic krill oil Pickering emulsion with an oil-water interface double network interpenetrating structure.

[0010] The Antarctic krill oil Pickering emulsion prepared by this invention has good structural stability, and based on the targeted hydrolysis of κ-CA and SA by intestinal β-mannanase, the emulsion has good intestinal targeted sustained-release properties.

[0011] Furthermore, the method described in this invention includes the following steps:

[0012] (1) Preparation of Zein solution: Add Zein to an aqueous ethanol solution, seal and stir to dissolve, and the Zein solution is obtained.

[0013] Preferably, the volume fraction of ethanol in the aqueous ethanol solution is 80%.

[0014] The preferred Zein solution has a Zein concentration of 10 mg / mL.

[0015] (2) Preparation of SA solution: Add SA to water, stir, and let stand to allow it to fully hydrate, thus obtaining SA solution;

[0016] The preferred mass concentration ratio of Zein solution to SA solution is 20:1 to 1:2;

[0017] (3) Preparation of κ-CA solution: Add κ-CA to water, heat in an 80℃ water bath for 30 minutes, stir, and let stand to allow it to fully hydrate, thus obtaining κ-CA solution;

[0018] The preferred mass concentration ratio of Zein solution to κ-CA solution is 1:1;

[0019] (4) Preparation of Zein / κ-CA binary composite nanoparticles: The Zein solution obtained in step (1) is added to the κ-CA solution obtained in step (3), and stirred under sealed conditions to obtain a Zein / κ-CA binary composite nanoparticle solution.

[0020] The preferred volume ratio of Zein solution to κ-CA solution is 1:1;

[0021] The preferred stirring speed is 600–800 rpm, and the stirring time is 10–30 min;

[0022] (5) Preparation of Zein / κ-CA / SA ternary composite nanoparticles: The Zein / κ-CA binary composite nanoparticle solution obtained in step (4) is added to the SA solution obtained in step (2), stirred under sealed conditions, and the ethanol is removed by rotary evaporation to obtain the Zein / κ-CA / SA ternary composite nanoparticle solution.

[0023] The preferred volume ratio of Zein / κ-CA binary composite nanoparticle solution to SA solution is 1:3;

[0024] The preferred stirring speed is 600–800 rpm, and the stirring time is 10–30 min;

[0025] (6) Preparation of Antarctic krill oil dilution: Disperse Antarctic krill oil in corn germ oil, seal and stir well in the dark to obtain Antarctic krill oil dilution;

[0026] The preferred composition of Antarctic krill oil in corn germ oil is 10%–30% by mass;

[0027] (7) Homogenization emulsification: The Antarctic krill oil dilution obtained in step (6) and the Zein / κ-CA / SA ternary composite nanoparticle solution obtained in step (5) are mixed and sheared in an ice bath using a high-speed shearing machine to obtain an emulsion;

[0028] The preferred volume ratio of Antarctic krill oil dilution to Zein / κ-CA / SA ternary composite nanoparticle solution is 9:1 to 1:9;

[0029] The preferred shearing conditions are 18000 r / min for 3 min;

[0030] (8)K + Crosslinking: Add KCl solution to the emulsion obtained in step (7) and crosslink at 33-35℃ for 1-4 hours to obtain a crosslinked emulsion;

[0031] The preferred concentration of KCl solution is 1 mol / L, and the preferred amount of KCl solution added is 1% to 2% (volume fraction).

[0032] (9)Ca 2+Crosslinking: Add CaCl2 solution to the crosslinked emulsion obtained in step (8) and crosslink at 44-55℃ for 12-24h to obtain Antarctic krill oil Pickering emulsion with oil-water interface double network interpenetrating structure.

[0033] The preferred concentration of the CaCl2 solution is 1 mol / L, and the preferred amount of CaCl2 solution added is 0.2% to 2% (volume fraction).

[0034] The beneficial effects of this invention are as follows:

[0035] The Zein, SA, and κ-CA required in the preparation method of this invention are widely available, inexpensive, and readily available, exhibiting good biocompatibility. The preparation method used in this invention is simple, low-cost, and easily applicable to large-scale enterprise production. The Antarctic krill oil Pickering emulsion obtained by this invention can maintain the stability of the gastric digestive structure and has good intestinal targeting and sustained-release effects, showing great application potential in the fields of food, medicine, and materials. Attached Figure Description

[0036] Figure 1 This is a flow chart of the Pickering emulsion process for Antarctic krill oil.

[0037] Figure 2 The figures show a comparison of the average particle size and polydispersity index (PDI) of different nanoparticles in Example 1 and Comparative Example 2 (A) and a comparison of the zeta potential values ​​(B).

[0038] Figure 3 Comparison chart (A) shows the average particle size, polydispersity index (PDI), and zeta potential value of Antarctic krill oil Pickering emulsions from Examples 1-4, Comparative Example 1, and Comparative Example 2.

[0039] Figure 4 The chart shows the trend of emulsification index of Antarctic krill oil Pickering emulsion in Examples 1-4, Comparative Example 1, and Comparative Example 2.

[0040] Figure 5 The graphs (A) and (B) show the changes in FFA release rate and emulsion particle size after external digestion of Antarctic krill oil Pickering emulsion in Example 1 and Comparative Example 1.

[0041] Figure 6 The graph shows the changes in DHA (A) and EPA (B) concentrations in serum after internal digestion of Antarctic krill oil Pickering emulsion in Example 1 and Comparative Example 1. Detailed Implementation

[0042] The present invention will be further described below with reference to specific embodiments. The following are only specific embodiments of the present invention, but the scope of protection of the present invention is not limited thereto.

[0043] Example 1

[0044] A pickering emulsion of Antarctic krill oil with intestinal targeting and sustained release, and its preparation method, comprising the following steps:

[0045] (1) Preparation of Zein solution

[0046] Add 1g of Zein to 100mL of 80% ethanol aqueous solution, seal and stir for 30min at room temperature to obtain a Zein solution.

[0047] (2) Preparation of SA solution

[0048] Dissolve SA in water, stir thoroughly, and let stand overnight to allow it to fully hydrate. The mass concentration ratio of Zein solution to SA solution is 5:1.

[0049] (3) Preparation of κ-CA solution

[0050] Dissolve κ-CA in water, incubate at 80°C for 30 minutes with thorough stirring, and let stand overnight to allow for complete hydration. The mass concentration ratio of Zein solution to κ-CA solution is 1:1.

[0051] (4) Preparation of Zein / κ-CA binary composite particles

[0052] Add the Zein solution from step (1) to the κ-CA solution from step (3) at a volume ratio of 1:1, and stir at 700 rpm for 30 min under sealed conditions to obtain a composite nanoparticle solution.

[0053] (5) Preparation of Zein / κ-CA / SA ternary composite nanoparticles

[0054] The Zein / κ-CA binary composite nanoparticle solution from step (4) was added to the SA solution from step (2) at a volume ratio of 1:3. Under sealed conditions, the solution was stirred at 700 rpm for 30 min to obtain a ternary composite nanoparticle solution. Finally, the ethanol was removed by rotary evaporation.

[0055] (6) Prepare Antarctic krill oil dilution

[0056] Antarctic krill oil was dispersed in corn germ oil at a mass fraction of 15%, and the mixture was stirred in a sealed container away from light to obtain a diluted Antarctic krill oil solution.

[0057] (7) Homogenization and emulsification

[0058] The Antarctic krill oil dilution in step (6) and the composite nanoparticle solution in step (5) were mixed at a ratio of 7:3 (v / v), and the mixture was sheared for 3 minutes at 18000 r / min in an ice bath using a high-speed shearing machine to obtain an emulsion.

[0059] (8)K + Crosslinking: Add 1% 1mol / L KCl solution to the emulsion obtained in step (7) and crosslink at 40℃ for 4h to obtain a crosslinked emulsion.

[0060] (9)Ca 2+ Crosslinking: Add 0.5% 1mol / L CaCl2 solution to the emulsion obtained in step (8) and crosslink at 40℃ for 24h to obtain Antarctic krill oil Pickering emulsion with an oil-water interface double network interpenetrating structure.

[0061] Example 2

[0062] A pickering emulsion of Antarctic krill oil with intestinal targeting and sustained release, and its preparation method, comprising the following steps:

[0063] (1) Preparation of Zein solution

[0064] Add 1g of Zein to 100mL of 80% ethanol aqueous solution, seal and stir for 30min at room temperature to obtain a Zein solution.

[0065] (2) Preparation of SA solution

[0066] Dissolve SA in water, stir thoroughly, and let stand overnight to allow it to fully hydrate. The mass concentration ratio of Zein solution to SA solution is 5:1.

[0067] (3) Preparation of κ-CA solution

[0068] Dissolve κ-CA in water, incubate at 80°C for 30 minutes with thorough stirring, and let stand overnight to allow for complete hydration. The mass concentration ratio of Zein solution to κ-CA solution is 1:1.

[0069] (4) Preparation of Zein / κ-CA binary composite particles

[0070] The Zein solution from step (1) was added to the κ-CA solution from step (3) at a volume ratio of 3:1, and the mixture was stirred at 700 rpm for 30 min under sealed conditions to obtain a composite nanoparticle solution.

[0071] (5) Preparation of Zein / κ-CA / SA ternary composite nanoparticles

[0072] The Zein / κ-CA binary composite nanoparticle solution from step (4) was added to the SA solution from step (2) at a volume ratio of 1:3. Under sealed conditions, the solution was stirred at 700 rpm for 30 min to obtain a ternary composite nanoparticle solution. Finally, the ethanol was removed by rotary evaporation.

[0073] (6) Prepare Antarctic krill oil dilution

[0074] Antarctic krill oil was dispersed in corn germ oil at a mass fraction of 15%, and the mixture was stirred in a sealed container away from light to obtain a diluted Antarctic krill oil solution.

[0075] (7) Homogenization and emulsification

[0076] The Antarctic krill oil dilution in step (6) and the composite nanoparticle solution in step (5) were mixed at a ratio of 7:3 (v / v), and the mixture was sheared for 3 minutes at 18000 r / min in an ice bath using a high-speed shearing machine to obtain an emulsion.

[0077] (8)K + Crosslinking: Add 1% 1mol / L KCl solution to the emulsion obtained in step (7), and crosslink at 40°C for 4 hours to obtain K + Cross-linked Antarctic krill oil Pickering emulsion.

[0078] Example 3

[0079] A pickering emulsion of Antarctic krill oil with intestinal targeting and sustained release, and its preparation method, comprising the following steps:

[0080] (1) Preparation of Zein solution

[0081] Add 1g of Zein to 100mL of 80% ethanol aqueous solution, seal and stir for 30min at room temperature to obtain a Zein solution.

[0082] (2) Preparation of SA solution

[0083] Dissolve SA in water, stir thoroughly, and let stand overnight to allow it to fully hydrate. The mass concentration ratio of Zein solution to SA solution is 5:1.

[0084] (3) Preparation of κ-CA solution

[0085] Dissolve κ-CA in water, incubate at 80°C for 30 minutes with thorough stirring, and let stand overnight to allow for complete hydration. The mass concentration ratio of Zein solution to κ-CA solution is 1:1.

[0086] (4) Preparation of Zein / κ-CA binary composite particles

[0087] Add the Zein solution from step (1) to the κ-CA solution from step (3) at a volume ratio of 1:1, and stir at 700 rpm for 30 min under sealed conditions to obtain a composite nanoparticle solution.

[0088] (5) Preparation of Zein / κ-CA / SA ternary composite nanoparticles

[0089] The Zein / κ-CA binary composite nanoparticle solution from step (4) was added to the SA solution from step (2) at a volume ratio of 1:3. Under sealed conditions, the solution was stirred at 700 rpm for 30 min to obtain a ternary composite nanoparticle solution. Finally, the ethanol was removed by rotary evaporation.

[0090] (6) Prepare Antarctic krill oil dilution

[0091] Antarctic krill oil was dispersed in corn germ oil at a mass fraction of 15%, and the mixture was stirred in a sealed container away from light to obtain a diluted Antarctic krill oil solution.

[0092] (7) Homogenization and emulsification

[0093] The Antarctic krill oil dilution in step (6) and the composite nanoparticle solution in step (5) were mixed at a ratio of 7:3 (v / v), and the mixture was sheared for 3 minutes at 18000 r / min in an ice bath using a high-speed shearing machine to obtain an emulsion.

[0094] (8)Ca 2+ Crosslinking: Add 0.5% 1 mol / L CaCl2 solution to the emulsion obtained in step (8), and crosslink at 40℃ for 24 h to obtain Ca 2+ Cross-linked Antarctic krill oil Pickering emulsion.

[0095] Example 4

[0096] A pickering emulsion of Antarctic krill oil with intestinal targeting and sustained release, and its preparation method, comprising the following steps:

[0097] (1) Preparation of Zein solution

[0098] Add 1g of Zein to 100mL of 80% ethanol aqueous solution, seal and stir for 30min at room temperature to obtain a Zein solution.

[0099] (2) Preparation of SA solution

[0100] Dissolve SA in water, stir thoroughly, and let stand overnight to allow it to fully hydrate. The mass concentration ratio of Zein solution to SA solution is 5:1.

[0101] (3) Preparation of κ-CA solution

[0102] Dissolve κ-CA in water, incubate at 80°C for 30 minutes with thorough stirring, and let stand overnight to allow for complete hydration. The mass concentration ratio of Zein solution to κ-CA solution is 1:1.

[0103] (4) Preparation of Zein / κ-CA binary composite particles

[0104] Add the Zein solution from step (1) to the κ-CA solution from step (3) at a volume ratio of 1:1, and stir at 700 rpm for 30 min under sealed conditions to obtain a composite nanoparticle solution.

[0105] (5) Preparation of Zein / κ-CA / SA ternary composite nanoparticles

[0106] The Zein / κ-CA binary composite nanoparticle solution from step (4) was added to the SA solution from step (2) at a volume ratio of 1:3, and stirred at 700 rpm for 30 min under sealed conditions to obtain a ternary composite nanoparticle solution.

[0107] (6) Prepare Antarctic krill oil dilution

[0108] Antarctic krill oil was dispersed in corn germ oil at a mass fraction of 15%, and the mixture was stirred in a sealed container away from light to obtain a diluted Antarctic krill oil solution.

[0109] (7) Homogenization and emulsification

[0110] The Antarctic krill oil dilution in step (6) and the composite nanoparticle solution in step (5) were mixed at a ratio of 7:3 (v / v), and the mixture was sheared for 3 minutes at 18000 r / min in an ice bath using a high-speed shearing machine to obtain an uncrosslinked Antarctic krill oil Pickering emulsion.

[0111] Comparative Example 1

[0112] (1) Preparation of Zein solution

[0113] Add 1g of Zein to 100mL of 80% ethanol aqueous solution, seal and stir for 30min at room temperature to obtain a Zein solution.

[0114] (2) Preparation of Zein particles

[0115] The Zein solution from step (1) was added to deionized water at a volume ratio of 1:3, and stirred at 700 rpm for 30 min under sealed conditions to obtain Zein particles. Finally, the ethanol was removed by rotary evaporation.

[0116] (3) Preparation of Antarctic krill oil dilution

[0117] Antarctic krill oil was dispersed in corn germ oil at a mass fraction of 15%, and the mixture was stirred in a sealed container away from light to obtain a diluted Antarctic krill oil solution.

[0118] (4) Homogenization and emulsification

[0119] The Antarctic krill oil dilution in step (3) and the Zein nanoparticle solution in step (2) were mixed at a ratio of 7:3 (v / v), and the mixture was sheared for 3 minutes at 18000 r / min in an ice bath using a high-speed shearing machine to obtain the Antarctic krill oil Pickering emulsion.

[0120] Comparative Example 2

[0121] (1) Preparation of Zein solution

[0122] Add 1g of Zein to 100mL of 80% ethanol aqueous solution, seal and stir for 30min at room temperature to obtain a Zein solution.

[0123] (2) Preparation of SA solution

[0124] Dissolve SA in water, stir thoroughly, and let stand overnight to allow it to fully hydrate. The mass concentration ratio of Zein solution to SA solution is 5:1.

[0125] (3) Preparation of CMC solution

[0126] Dissolve CMC in water, stir thoroughly, and let stand overnight to allow it to fully hydrate. The mass concentration ratio of Zein solution to CMC solution is 1:1.

[0127] (4) Preparation of Zein / CMC binary composite particles

[0128] Add the Zein solution from step (1) to the CMC solution from step (3) at a volume ratio of 1:1, and stir at 700 rpm for 30 min under sealed conditions to obtain a composite particle solution.

[0129] (5) Preparation of Zein / CMC / SA ternary composite particles

[0130] Add the Zein / CMC binary composite particle solution from step (4) to the CMC solution from step (2) at a volume ratio of 1:3, and stir at 700 rpm for 30 min under sealed conditions to obtain a ternary composite particle solution. Finally, remove the ethanol by rotary evaporation.

[0131] (6) Prepare Antarctic krill oil dilution

[0132] Antarctic krill oil was dispersed in corn germ oil at a mass fraction of 15%, and the mixture was stirred in a sealed container away from light to obtain a diluted Antarctic krill oil solution.

[0133] (7) Homogenization and emulsification

[0134] The Antarctic krill oil dilution in step (6) and the composite nanoparticle solution in step (5) were mixed at a ratio of 7:3 (v / v), and the mixture was sheared for 3 minutes at 18000 r / min in an ice bath using a high-speed shearing machine to obtain an emulsion.

[0135] (8)K + Crosslinking: Add 1% 1mol / L KCl solution to the emulsion obtained in step (7) and crosslink at 40℃ for 4h to obtain a crosslinked emulsion.

[0136] (9)Ca 2+ Crosslinking: Add 0.5% 1mol / L CaCl2 solution to the emulsion obtained in step (8) and crosslink at 40℃ for 24h to obtain Antarctic krill oil Pickering emulsion.

[0137] Nanoparticle characterization

[0138] The different nanoparticles prepared in Example 1 and Comparative Example 2 were diluted 5 times, and their average particle size, zeta potential and PDI value were measured three times for each data point.

[0139] Result: As Figure 2 As shown in (A), in Example 1, the particle size and PDI of the Zein / κ-CA / SA ternary composite nanoparticles did not increase significantly, while the absolute value of the ζ potential was above 30mV and the largest, indicating that it was more stable than Zein and Zein / κ-CA. In contrast, in Comparative Example 2, the particle size and PDI of the Zein / CMC / SA ternary composite particles were much larger than those of the Zein / κ-CA / SA ternary composite nanoparticles, while the absolute value of the ζ potential was smaller.

[0140] Emulsion characterization

[0141] Measurement of average particle size, PDI value, and zeta potential

[0142] Pickering emulsions prepared in Examples 1-4, Comparative Example 1, and Comparative Example 2 were diluted 1000 times, and their average particle size, zeta potential, and PDI value were measured three times for each data point.

[0143] Liquefaction Index Measurement

[0144] Take 5 mL of each of the Pickering emulsions prepared in Examples 1-4, Comparative Example 1, and Comparative Example 2 and place them in sealed test tubes. Let them stand at 25°C. After the emulsions stabilize, they separate into two layers: an oil layer on top and an emulsion separation layer on the bottom. Record the height change of the emulsion separation interface from 1 to 30 days. CI is the ratio of the height of the lower emulsion separation layer (Hs) to the height of the overall emulsion (Ht), i.e., the emulsion separation index.

[0145] Result: As Figure 3 You can see that K is being performed.+ and Ca 2+ The crosslinked emulsion of Example 1 had the smallest particle size, the smallest PDI value, and the largest absolute value of ζ-potential, making it the most stable; while only K... + Crosslinked Example 2 and Ca 2+ The crosslinked emulsion of Example 3 had a slightly larger emulsion particle size and PDI value, and a slightly smaller zeta potential value, but it was more stable than Example 4, which did not undergo crosslinking. In contrast, Comparative Example 1, which was stabilized with Zein, and Comparative Example 2, which was stabilized with Zein / CMC / SA, had larger emulsion particle sizes and PDI values, and smaller absolute values ​​of zeta potential values, making them relatively unstable.

[0146] like Figure 4 As can be seen, the trend of the stratification index is consistent with the above. Example 1 is the most stable, with no stratification observed for 30 days, and only K... + Crosslinked Example 2 and Ca 2+ Crosslinked Example 3 showed delamination on day 22 and day 24, but was more stable than Example 4 which was not crosslinked. Comparative Examples 1 and 2 showed delamination earliest and were less stable.

[0147] In vitro digestion simulation experiment

[0148] Oral digestion: Prepare 90 mL of simulated saliva (SSF) using the following ingredients: 80.64 mg KCl, 79.92 mg NaH2PO4, 18 mg KSCN, 26.82 mg NaCl, 51.3 mg Na2SO4, 98.46 mg NaHCO3, 18 mg urea, 1.35 mg uric acid, and 54 mg α-amylase. Preheat 15 mL of Pickering emulsion and 90 mL of SSF to 37°C for 5 min, respectively. Place the dialysis bag into the 90 mL SSF. Quickly adjust the pH of the system to 6.8 with 1 mol / L HCl, and digest for 10 min in a constant temperature shaking water bath at 37°C (100 rpm). At each time point (0, 2, 4, 6, 8, and 10 min), remove 5 mL of release medium from the incubation bath.

[0149] Gastric digestion: Prepare simulated gastric juice (SGF) using 180 mg NaCl, 630 mg HCl, and 288 mg pepsin. Preheat 90 mL of SGF to a 37°C water bath for 5 min. Place the dialysis bag containing the oral digested sample into the SGF. Quickly adjust the pH of the mixture to 2.5 with 1 mol / L NaOH. Digest for 2 h in a 37°C shaking water bath (100 rpm). At each time point (10, 30, 60, 90, and 120 min), remove 5 mL of release medium from the incubation bath.

[0150] Intestinal digestion: A simulated intestinal fluid (SIF) was prepared using 550.5 mg CaCl2·2H2O, 75 mg bile salts, and 67.5 mg pancreatic enzymes. After gastric digestion, the pH of the resulting system was rapidly adjusted to 7.0 with 0.25 mol / L NaOH. Simultaneously, the SIF was preheated in a 37°C water bath for 5 min. The dialysis bag, after gastric digestion, was placed into the SIF, and the pH of the mixture was adjusted to 7.0 with 1 mol / L NaOH. Digestion was carried out in a 37°C shaking water bath (100 r / min) for 2 h. During this time, the pH of the mixture was continuously adjusted with NaOH to maintain it at 7.0. At each time point (10, 30, 60, 90, and 120 min), 5 mL of release medium was removed from the incubation bath. The cumulative release rate of fatty acids was evaluated based on the amount of NaOH used in the titration at different digestion stages, and the emulsion particle size was measured at different time points.

[0151] Results: This embodiment conducted an in vitro digestion simulation experiment on the Antarctic krill oil Pickering emulsion from Example 1 and Comparative Example 1. Figure 5 As shown in Figure (A), during in vitro simulated digestion, it was found that fatty acids are mainly absorbed in the small intestine, especially ω-polyunsaturated fatty acids, which can enter the bloodstream to exert physiological functions. Antarctic krill oil Pickering emulsion was stabilized using Zein / κ-CA / SA ternary composite nanoparticles and subjected to K... + Ca 2+ Cross-linking is crucial for enhancing the targeted and sustained release of ω-polyunsaturated fatty acids in vivo. During simulated gastrointestinal digestion, pepsin and pancreatic enzymes may hydrolyze Zein in Comparative Example 1, thereby disrupting the nanoparticle structure and causing the release of fatty acids from the stable Pickering emulsion into SGF and SIF. The Pickering emulsion prepared in Example 1 exhibits better pH stability, ensuring the stability of fatty acids within the Pickering emulsion in SGF and facilitating targeted release in SIF. Meanwhile, as... Figure 5 As shown in (B), during in vitro digestion, the increase in emulsion particle size also shows the same trend as the cumulative release rate of fatty acids.

[0152] In vivo ω-3 unsaturated fatty acid metabolism assay (using EPA and DHA as examples)

[0153] Two hundred male rats (weighing 220±6g) were selected. They were fed the same diet under controlled conditions (3±50℃; relative humidity, 5±12%) with a 1-hour light / dark cycle. After 2 weeks of acclimatization, the rats were randomly divided into two groups (Example 1 group and Comparative Example 1 group) and orally administered either the emulsion of Example 1 or Comparative Example 1. Venous blood samples were collected at 2, 4, 6, 8, 12, 24, and 48 hours later. Serum was stored at -80℃ until analysis was required. Whole blood concentrations of DHA and EPA were measured by LC-MS / MS within 2 weeks after sampling, and the maximum plasma concentration C was recorded. max and reaching C max Time T max .

[0154] Result: As Figure 6 Example 1 shows the C of DHA and EPA. max All were greater than those of Comparative Example 1, but reached C. max Time T max The results are identical. Therefore, under the same timeframe, the DHA and EPA in Example 1 are absorbed and metabolized faster, and a greater amount of DHA and EPA reaches the intestines. This demonstrates that Antarctic krill oil Pickering emulsion with an oil-water interface dual-network interpenetrating structure can enhance the targeted release of ω-3 polyunsaturated fatty acids and promote gastrointestinal absorption.

Claims

1. A method for preparing an intestinal-targeted sustained-release Antarctic krill oil Pickering emulsion, characterized in that, Includes the following steps: (1) Preparation of Zein solution: Add Zein to an aqueous ethanol solution, seal and stir to dissolve, and the Zein solution is obtained; (2) Preparation of SA solution: Add SA to water, stir, and let stand to allow it to fully hydrate, thus obtaining SA solution; (3) Preparation of κ-CA solution: Add κ-CA to water to prepare κ-CA solution; (4) Preparation of Zein / κ-CA binary composite nanoparticles: The Zein solution obtained in step (1) is added to the κ-CA solution obtained in step (3), and stirred under sealed conditions to obtain a Zein / κ-CA binary composite nanoparticle solution; (5) Preparation of Zein / κ-CA / SA ternary composite nanoparticles: The Zein / κ-CA binary composite nanoparticle solution obtained in step (4) is added to the SA solution obtained in step (2), stirred under sealed conditions, and the ethanol is removed by rotary evaporation to obtain the Zein / κ-CA / SA ternary composite nanoparticle solution. (6) Preparation of Antarctic krill oil dilution: Disperse Antarctic krill oil in corn germ oil, seal and stir well in the dark to obtain Antarctic krill oil dilution; (7) Homogenization emulsification: The Antarctic krill oil dilution obtained in step (6) and the Zein / κ-CA / SA ternary composite nanoparticle solution obtained in step (5) are mixed and sheared in an ice bath using a high-speed shearing machine to obtain an emulsion; (8) K + Crosslinking: Add KCl solution to the emulsion obtained in step (7) and crosslink at 33~35℃ for 1~4h to obtain a crosslinked emulsion; (9) Ca 2+ Crosslinking: Add CaCl2 solution to the crosslinked emulsion obtained in step (8) and crosslink at 44~55℃ for 12~24 h to obtain Antarctic krill oil Pickering emulsion with oil-water interface double network interpenetrating structure. In this context, Zein represents zein, κ-CA represents κ-carrageenan, and SA represents sodium alginate.

2. The preparation method according to claim 1, characterized in that, In step (1), the volume fraction of ethanol in the aqueous ethanol solution is 80%.

3. The preparation method according to claim 1, characterized in that, The mass concentration of Zein in the Zein solution is 10 mg / mL; the mass concentration ratio of Zein solution to SA solution is 20:1 to 1:2; the mass concentration ratio of Zein solution to κ-CA solution is 1:

1.

4. The preparation method according to claim 1, characterized in that, Antarctic krill oil accounts for 10-30% of corn germ oil by mass.

5. The preparation method according to claim 1, characterized in that, The volume ratio of Zein solution to κ-CA solution is 1:1, the volume ratio of Zein / κ-CA binary composite nanoparticle solution to SA solution is 1:3, and the volume ratio of Antarctic krill oil dilution to Zein / κ-CA / SA ternary composite nanoparticle solution is 9:1 to 1:

9.

6. The preparation method according to claim 1, characterized in that, In step (4) or step (5), the stirring rate is 600~800 rpm and the stirring time is 10~30 min.

7. The preparation method according to claim 1, characterized in that, In step (7), the shearing conditions are 18000 r / min for 3 min.

8. The preparation method according to claim 1, characterized in that, In step (8), the concentration of KCl solution is 1 mol / L, and the amount of KCl solution added is 1~2%.

9. The preparation method according to claim 1, characterized in that, In step (9), the concentration of CaCl2 solution is 1 mol / L, and the amount of CaCl2 solution added is 0.2~2%.

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