CN116941754A - Preparation method of intestinal targeted slow-release euphausia superba oil Pickering emulsion - Google Patents

Abstract

The invention discloses a preparation method of intestinal targeted slow-release euphausia superba oil Pickering emulsion, which comprises the following steps: zein is dissolved in ethanol water solution, is quickly added into kappa-carrageenan solution, and is prepared into Zein/kappa-CA binary composite nano particles by an anti-solvent precipitation method, the binary composite nano particles are added into sodium alginate water solution, zein/kappa-CA/SA ternary composite nano particles are constructed by layer-by-layer adsorption, zein/kappa-CA/SA is taken as a stabilizer to be organically combined with antarctic krill oil, pickering emulsion is constructed by high-speed shearing, and finally K is sequentially added ⁺ And Ca ²⁺ Carrying out ionic crosslinking on the solution to construct antarctic krill oil Pickering emulsion with an oil-water interface double-network interpenetrating structure; the emulsion has good structural stability, and has good intestinal targeting slow release characteristic based on the directional hydrolysis of the intestinal beta-mannase on kappa-CA and SA.

Description

Preparation method of intestinal-targeted sustained-release Antarctic krill oil Pickering emulsion

Technical field

The invention belongs to the technical field of Pickering emulsion oil-water interface modification. Specifically, it relates to a protein/polysaccharide composite nanoparticle that forms a network interpenetrating oil-water interface stabilizer through ion double cross-linking. It is developed based on intestinal β-mannanase directional hydrolysis. Targeted sustained-release Antarctic krill oil Pickering emulsion.

Background technique

Antarctic krill oil refers to a red mixed lipid extracted from Antarctic krill. The oil content accounts for about one-sixth of the shrimp's body weight. It is not only rich in docosahexaenoic acid (DHA) and Omega-3 polyunsaturated fatty acids such as eicosapentaenoic acid (EPA), as well as bioactive ingredients such as flavonoids, phospholipids, and fat-soluble vitamins. It is precisely because of the synergistic effect of various active substances such as DHA, EPA, phospholipids and astaxanthin that Antarctic krill oil has various metabolic effects in the human body, improves human health, and can prevent cardiovascular diseases. , premenstrual syndrome, inhibit inflammation, improve memory and inhibit tumors, etc. However, the phospholipid-type omega-3 polyunsaturated fatty acids and other active ingredients in Antarctic krill oil are easily degraded in the gastric digestion stage due to the influence of pepsin, low acidity and ionic strength, and are difficult to enter the small intestine for digestion and absorption, resulting in oral bioavailability. Not high. Constructing an Antarctic krill oil Pickering emulsion system can be a potential way to improve the above problems. Among them, oil-in-water (O/W) emulsion has the most potential application as an Antarctic krill oil delivery system.

Pickering emulsion refers to a system that can be stabilized by micron-scale or even nano-scale solid particles. It is a system that can achieve thermodynamic and kinetic stability. It can prevent emulsion droplets from coalescing through particle interface effects and reduce the total freedom of the system. It can produce a spatial physical barrier, which depends on the surface hydrophobicity of the particles and is considered to be irreversible adsorption. Compared with traditional emulsions, Pickering emulsions have great advantages: the amount of emulsifier added is small, saving raw material costs; they are environmentally friendly, and factors such as temperature and ionic strength have little impact on the emulsions. Based on factors such as safety, greenness, biocompatibility, and biodegradability, research on food-grade solid particle-stabilized Pickering emulsions is increasing, which can be divided into the following three types: First, Pickering emulsions made from proteins as emulsifiers , such as soy protein, zein (Zein), etc.; the second is Pickering emulsions in which polysaccharides are used as emulsifiers, such as modified starch and cellulose nanocrystals; the third is binary or higher complexes as emulsifiers, such as zein. Protein-pectin binary complex system, etc.

Sodium alginate (SA) is a sodium salt of polyanionic polysaccharide extracted from natural brown algae. It has the characteristics of green safety, biocompatibility and biodegradability. When it encounters cations such as calcium ions, it produces a gel layer, which plays a role in Sustained drug release. κ-carrageenan (κ-CA) is also a polyanionic polysaccharide and contains a sulfate group in its disaccharide unit, which can be cross-linked with specific ions such as calcium ions. At the same time, because of its shear thinning properties, it has The viscosity is adjustable and can be compounded with SA to change its viscosity, gelling, swelling and other characteristics, which is beneficial to retaining active substances for a long time and achieving a sustained release effect. At the same time, both can be hydrolyzed by β-mannanase. , achieving the effect of targeted release.

Omega-3 polyunsaturated fatty acids can only be absorbed and utilized by the body after interacting with lipase in the small intestine. During the digestion process, omega-3 polyunsaturated fatty acids will change their structure and properties due to the action of other enzymes and oxygen, reducing the Its nutritional value, therefore reducing the release of omega-3 polyunsaturated fatty acids before reaching the intestinal digestive stage and increasing its release during the small intestinal digestive stage can improve its bioavailability. The Antarctic krill oil Pickering emulsion prepared by the present invention can not only reduce the particle size of the emulsion before the small intestinal digestion stage because the composite nanoparticles have strong electrostatic and ionic cross-linking effects, but can also gather on the surface of the droplets to form a A layer of physical barrier can effectively reduce the release of ω-3 polyunsaturated fatty acids even in the low pH environment of the stomach; during the small intestinal digestion stage, the orientation of α-1,4 glycosidic bonds by β-mannanase in the intestine Hydrolysis improves the targeted release of omega-3 polyunsaturated fatty acids in the intestine. At the same time, the double network interpenetrating structure of the composite nanoparticles forms competitive adsorption with lipase at the oil-water interface, improving the release of omega-3 polyunsaturated fatty acids. The slow release effect of fatty acids ultimately improves their oral bioavailability over a long period of time.

Contents of the invention

The present invention is based on the directional hydrolysis of intestinal β-mannanase, using Zein/κ-CA/SA ternary nanoparticles as stabilizers and organic combination with Antarctic krill oil to prepare network interpenetrating intestinal targeted sustained-release Antarctic krill. Oil Pickering emulsion, in which κ-CA cross-linked with K ⁺ is the first polymer network, and SA cross-linked with Ca ²⁺ is the second polymer network. An interpenetrating network structure can be formed by cross-linking with K ⁺ solution and Ca ²⁺ solution, with convenient operation and simple process. Double-ion cross-linking based on directional hydrolysis of β-mannanase gives Antarctic krill oil Pickering emulsion good intestinal targeting and sustained release effects.

The technical solution of the present invention is as follows:

A preparation method for intestinal-targeted sustained-release Antarctic krill oil Pickering emulsion, including:

Zein (zein) is dissolved in an ethanol aqueous solution, and the resulting Zein solution is added to a κ-CA (κ-carrageenan) solution. Zein/κ-CA binary composite nanoparticles are prepared by an antisolvent precipitation method, and then Zein/ κ-CA binary composite nanoparticles are added to SA (sodium alginate) aqueous solution, and Zein/κ-CA/SA ternary composite nanoparticles are constructed through layer-by-layer adsorption; Zein/κ-CA/SA ternary composite nanoparticles are constructed In order to organically combine the stabilizer with Antarctic krill oil, high-speed shearing is used to construct a Pickering emulsion. Finally, K ⁺ solution and Ca ²⁺ solution are added in sequence for ion double cross-linking to construct an Antarctic krill oil Pickering with a double network interpenetrating structure at the oil-water interface. Lotion.

The Antarctic krill oil Pickering emulsion prepared by the present invention has good structural stability, and is based on the directional hydrolysis of κ-CA and SA by intestinal β-mannanase. The emulsion has good intestinal targeted sustained release characteristics. .

Further, the method of the present invention includes the following steps:

(1) Prepare Zein solution: Add Zein to ethanol aqueous solution, seal and stir to dissolve, to obtain Zein solution;

Preferably, the volume fraction of ethanol in the ethanol aqueous solution is 80%;

Preferably, the mass concentration of Zein in the Zein solution is 10 mg/mL;

(2) Prepare SA solution: Add SA to water, stir, and let it stand to fully hydrate to obtain SA solution;

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

(3) Prepare κ-CA solution: Add κ-CA to water, bathe in 80°C water for 30 minutes, stir, and let stand to fully hydrate to obtain κ-CA solution;

Preferably, the mass concentration ratio of Zein solution and κ-CA solution is 1:1;

(4) Prepare Zein/κ-CA binary composite nanoparticles: Add the Zein solution obtained in step (1) to the κ-CA solution obtained in step (3), and stir under sealed conditions to obtain Zein/κ-CA binary composite nanoparticles. granular solution;

The preferred volume ratio of Zein solution and κ-CA solution is 1:1;

The preferred stirring rate is 600~800rpm, and the stirring time is 10~30min;

(5) Prepare Zein/κ-CA/SA ternary composite nanoparticles: Add the Zein/κ-CA binary composite nanoparticle solution obtained in step (4) to the SA solution obtained in step (2), and stir under sealed conditions. The ethanol is removed by rotary evaporation to obtain a Zein/κ-CA/SA ternary composite nanoparticle solution;

Preferably, the volume ratio of Zein/κ-CA binary composite nanoparticle solution and SA solution is 1:3;

The preferred stirring rate is 600~800rpm, and the stirring time is 10~30min;

(6) Preparing Antarctic krill oil diluent: Disperse Antarctic krill oil in corn germ oil, seal in a dark place, stir and mix, and obtain Antarctic krill oil diluent;

The preferred mass fraction of Antarctic krill oil in corn germ oil is 10% to 30%;

(7) Homogeneous emulsification: Mix the Antarctic krill oil diluent obtained in step (6) and the Zein/κ-CA/SA ternary composite nanoparticle solution obtained in step (5), and cut them with a high-speed shear in an ice bath. Cut to get emulsion;

The preferred volume ratio of the Antarctic krill oil diluent and the Zein/κ-CA/SA ternary composite nanoparticle solution is 9:1 to 1:9;

The preferred shearing conditions are 18000r/min, 3min;

(8) K ⁺ cross-linking: Add KCl solution to the emulsion obtained in step (7), and cross-link at 33-35°C for 1-4 hours to obtain a cross-linked emulsion;

The preferred KCl solution concentration is 1 mol/L, and the preferred KCl solution addition amount is 1% to 2% (volume fraction);

(9) Ca ²⁺ cross-linking: Add CaCl ₂ solution to the cross-linked emulsion obtained in step (8), and cross-link at 44-55°C for 12-24 hours to obtain Antarctic krill with a double network interpenetrating structure at the oil-water interface. Oil Pickering Lotion;

The preferred concentration of the CaCl ₂ solution is 1 mol/L, and the preferred addition amount of the CaCl ₂ solution is 0.2% to 2% (volume fraction).

The beneficial effects of the present invention are:

Zein, SA and κ-CA required in the preparation method of the present invention are widely sourced, cheap and easy to obtain, and have good biocompatibility. The preparation method used in the present invention has simple process, low cost, and is easy to be popularized and applied in the production of large enterprises. The Antarctic krill oil Pickering emulsion prepared by the invention can maintain the stability of the digestive structure of the stomach and has good intestinal targeting and sustained-release effects, and has huge application potential in the fields of food, medicine, materials and other fields.

Description of the drawings

Figure 1 is the process flow chart of Antarctic krill oil Pickering emulsion.

Figure 2 is a comparison chart (A) of the average particle size and polydispersity index (PDI) of different nanoparticles in Example 1 and Comparative Example 2, and a comparison chart of zeta potential values (B).

Figure 3 is a comparison chart (A) of the average particle size, polydispersity index (PDI), and zeta potential value (B) of the Antarctic krill oil Pickering emulsion of Examples 1 to 4, Comparative Example 1, and Comparative Example 2.

Figure 4 is a comparison chart of the emulsion index trend of the Antarctic krill oil Pickering emulsion of Examples 1 to 4, Comparative Example 1, and Comparative Example 2.

Figure 5 is a graph showing changes in FFA release rate (A) and changes in emulsion particle size (B) of external digestion of Antarctic krill oil Pickering emulsion in Example 1 and Comparative Example 1.

Figure 6 is a graph showing the concentration changes of DHA (A) and EPA (B) in the serum of internal digestion of Antarctic krill oil Pickering emulsion in Example 1 and Comparative Example 1.

Detailed ways

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

Example 1

An intestinal-targeted sustained-release Antarctic krill oil Pickering emulsion and a preparation method thereof, including the following steps:

(1) Prepare Zein solution

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

(2) Prepare SA solution

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

(3) Prepare κ-CA solution

Dissolve κ-CA in water, place in a water bath at 80°C for 30 minutes, stir thoroughly, and let it stand overnight to fully hydrate. The mass concentration ratio of Zein solution and κ-CA solution is 1:1.

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

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

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

Add the Zein/κ-CA binary composite nanoparticle solution in step (4) to the SA solution in step (2) with a volume ratio of 1:3, and stir at 700 rpm for 30 minutes under sealed conditions to obtain the ternary composite nanoparticles. particle solution, and finally, the ethanol is removed by rotary evaporation.

(6) Preparation of Antarctic krill oil diluent

Antarctic krill oil is dispersed in corn germ oil at a mass fraction of 15%, sealed and stirred away from light to obtain an Antarctic krill oil dilution.

(7) Homogeneous emulsification

Mix the Antarctic krill oil diluent in step (6) and the composite nanoparticle solution in step (5) at a ratio of 7:3 (v/v), and shear with a high-speed shear at 18000r/min in an ice bath. Cut for 3 minutes to get the emulsion.

(8) K ⁺ cross-linking: Add 1% 1 mol/L KCl solution to the emulsion obtained in step (7), and cross-link at 40°C for 4 hours to obtain a cross-linked emulsion.

(9) Ca ²⁺ cross-linking: Add 0.5% 1 mol/L CaCl ₂ solution to the emulsion obtained in step (8), and cross-link at 40°C for 24 hours to obtain Antarctic krill oil with a double network interpenetrating structure at the oil-water interface. Pickering lotion.

Example 2

An intestinal-targeted sustained-release Antarctic krill oil Pickering emulsion and a preparation method thereof, including the following steps:

(1) Prepare Zein solution

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

(2) Prepare SA solution

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

(3) Prepare κ-CA solution

Dissolve κ-CA in water, place in a water bath at 80°C for 30 minutes, stir thoroughly, and let it stand overnight to fully hydrate. The mass concentration ratio of Zein solution and κ-CA solution is 1:1.

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

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

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

Add the Zein/κ-CA binary composite nanoparticle solution in step (4) to the SA solution in step (2) with a volume ratio of 1:3, and stir at 700 rpm for 30 minutes under sealed conditions to obtain the ternary composite nanoparticles. particle solution, and finally, the ethanol is removed by rotary evaporation.

(6) Preparation of Antarctic krill oil diluent

Antarctic krill oil is dispersed in corn germ oil at a mass fraction of 15%, sealed and stirred away from light to obtain an Antarctic krill oil dilution.

(7) Homogeneous emulsification

Mix the Antarctic krill oil diluent in step (6) and the composite nanoparticle solution in step (5) at a ratio of 7:3 (v/v), and shear with a high-speed shear at 18000r/min in an ice bath. Cut for 3 minutes to get the emulsion.

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

Example 3

An intestinal-targeted sustained-release Antarctic krill oil Pickering emulsion and a preparation method thereof, including the following steps:

(1) Prepare Zein solution

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

(2) Prepare SA solution

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

(3) Prepare κ-CA solution

Dissolve κ-CA in water, place in a water bath at 80°C for 30 minutes, stir thoroughly, and let it stand overnight to fully hydrate. The mass concentration ratio of Zein solution and κ-CA solution is 1:1.

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

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

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

Add the Zein/κ-CA binary composite nanoparticle solution in step (4) to the SA solution in step (2) with a volume ratio of 1:3, and stir at 700 rpm for 30 minutes under sealed conditions to obtain the ternary composite nanoparticles. particle solution, and finally, the ethanol is removed by rotary evaporation.

(6) Preparation of Antarctic krill oil diluent

Antarctic krill oil is dispersed in corn germ oil at a mass fraction of 15%, sealed and stirred away from light to obtain an Antarctic krill oil dilution.

(7) Homogeneous emulsification

Mix the Antarctic krill oil diluent in step (6) and the composite nanoparticle solution in step (5) at a ratio of 7:3 (v/v), and shear with a high-speed shear at 18000r/min in an ice bath. Cut for 3 minutes to get the emulsion.

(8) Ca ²⁺ cross-linking: Add 0.5% 1 mol/L CaCl ₂ solution to the emulsion obtained in step (8), and cross-link at 40°C for 24 hours to obtain Ca ²⁺ cross-linked Antarctic krill oil Pickering emulsion.

Example 4

An intestinal-targeted sustained-release Antarctic krill oil Pickering emulsion and a preparation method thereof, including the following steps:

(1) Prepare Zein solution

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

(2) Prepare SA solution

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

(3) Prepare κ-CA solution

Dissolve κ-CA in water, place in a water bath at 80°C for 30 minutes, stir thoroughly, and let it stand overnight to fully hydrate. The mass concentration ratio of Zein solution and κ-CA solution is 1:1.

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

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

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

Add the Zein/κ-CA binary composite nanoparticle solution in step (4) to the SA solution in step (2) with a volume ratio of 1:3, and stir at 700 rpm for 30 minutes under sealed conditions to obtain the ternary composite nanoparticles. granular solution.

(6) Preparation of Antarctic krill oil diluent

Antarctic krill oil is dispersed in corn germ oil at a mass fraction of 15%, sealed and stirred away from light to obtain an Antarctic krill oil dilution.

(7) Homogeneous emulsification

Mix the Antarctic krill oil diluent in step (6) and the composite nanoparticle solution in step (5) at a ratio of 7:3 (v/v), and shear with a high-speed shear at 18000r/min in an ice bath. Cut for 3 minutes to obtain uncrosslinked Antarctic krill oil Pickering emulsion.

Comparative example 1

(1) Prepare Zein solution

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

(2) Preparation of Zein particles

Add the Zein solution in step (1) to deionized water at a volume ratio of 1:3, and stir at 700 rpm for 30 minutes under sealed conditions to obtain Zein particles. Finally, ethanol is removed by rotary evaporation.

(3) Preparation of Antarctic krill oil diluent

Antarctic krill oil is dispersed in corn germ oil at a mass fraction of 15%, sealed and stirred away from light to obtain an Antarctic krill oil dilution.

(4) Homogeneous emulsification

Mix the Antarctic krill oil diluent in step (3) and the Zein nanoparticle solution in step (2) according to 7:3 (v/v), and shear with a high-speed shear at 18000r/min in an ice bath. Cut for 3 minutes to get the Antarctic Krill Oil Pickering Emulsion.

Comparative example 2

(1) Prepare Zein solution

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

(2) Prepare SA solution

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

(3) Prepare CMC solution

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

(4) Preparation of Zein/CMC binary composite particles

Add the Zein solution in step (1) to the CMC solution in step (3) with a volume ratio of 1:1, and stir at 700 rpm for 30 minutes under sealed conditions to obtain a composite particle solution.

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

Add the Zein/CMC binary composite particle solution in step (4) to the CMC solution in step (2) with a volume ratio of 1:3, and stir at 700 rpm for 30 minutes under sealed conditions to obtain a ternary composite particle solution. , rotary evaporate to remove ethanol.

(6) Preparation of Antarctic krill oil diluent

Antarctic krill oil is dispersed in corn germ oil at a mass fraction of 15%, sealed and stirred away from light to obtain an Antarctic krill oil dilution.

(7) Homogeneous emulsification

Mix the Antarctic krill oil diluent in step (6) and the composite nanoparticle solution in step (5) at a ratio of 7:3 (v/v), and shear with a high-speed shear at 18000r/min in an ice bath. Cut for 3 minutes to get the emulsion.

(8) K ⁺ cross-linking: Add 1% 1 mol/L KCl solution to the emulsion obtained in step (7), and cross-link at 40°C for 4 hours to obtain a cross-linked emulsion.

(9) Ca ²⁺ cross-linking: Add 0.5% 1 mol/L CaCl ₂ solution to the emulsion obtained in step (8), and cross-link at 40°C for 24 hours to obtain Antarctic krill oil Pickering emulsion.

Nanoparticle characterization

Different nanoparticles prepared in Example 1 and Comparative Example 2 were diluted 5 times respectively, and their average particle size, zeta potential and PDI value were measured. Each data was measured three times.

Results: As shown in Figure 2 (A), in Example 1, the relative increase in particle size and PDI of the Zein/κ-CA/SA ternary composite nanoparticles was not very large, and at the same time, the absolute value of the ζ potential value was above 30mV. And the absolute value is the largest, which is more stable than Zein and Zein/κ-CA. In Comparative Example 2, the particle size and PDI value of Zein/CMC/SA ternary composite particles are much larger than those of Zein/κ-CA/SA ternary composite nanoparticles. particles, and the absolute value of the ζ potential value is small.

Emulsion characterization

Determination of average particle size, PDI value, and zeta potential value

The Pickering emulsion prepared in Examples 1 to 4, Comparative Example 1, and Comparative Example 2 was diluted 1000 times, and its average particle size, zeta potential, and PDI value were measured. Each data was measured three times.

Lactation index determination

Take 5 mL of the Pickering emulsions prepared in Examples 1 to 4, Comparative Example 1, and Comparative Example 2 respectively and place them in sealed test tubes, and let them stand at 25°C. After the emulsions are left to stand and stabilize, they are divided into two layers. The upper layer of the emulsion is the oil layer. The lower layer is the emulsion layer, and the height change of the emulsion interface is recorded from 1 to 30 days. CI is the ratio of the height of the lower emulsion layer (Hs) to the height of the overall emulsion (Ht), that is, the emulsion index.

Results: As shown in Figure 3, it can be seen that the emulsion of Example 1 with K ⁺ and Ca ²⁺ cross-linking has the smallest particle size, the smallest PDI value, the largest absolute value of ζ potential value, and is the most stable; while only K ⁺ cross-linking is performed The emulsion particle size and PDI value of Example 2 cross-linked with Ca ²⁺ and Example 3 cross-linked with Ca 2+ are slightly larger, and the zeta potential value is slightly smaller, but they are more stable than Example 4 without cross-linking. In contrast, Comparative Example 1 stabilized by Zein and Comparative Example 2 stabilized by Zein/CMC/SA have larger emulsion particle sizes, PDI values, smaller absolute values of zeta potential values, and are relatively unstable.

As shown in Figure 4, it can be seen that the trend of the lactation index is also consistent with the above. Example 1 is the most stable, and there is no delamination phenomenon for 30 days. Example 2 only performs K ⁺ cross-linking and Ca ²⁺ cross-linking. Example 3 showed delamination on the 22nd and 24th days respectively, but was more stable than Example 4 without cross-linking. Comparative Examples 1 and 2 showed delamination at the earliest and were more unstable.

In vitro digestion simulation experiment

Oral digestion _{: Prepare simulated saliva (} SSF)90mL. Take 15 mL Pickering emulsion and 90 mL SSF respectively in the dialysis bag, preheat them at 37°C for 5 minutes, and put the dialysis bag into 90 mL SSF. Quickly adjust the pH of the system to 6.8 with 1 mol/L HCl, and digest in a 37°C constant-temperature shaker water bath (100 r/min) for 10 minutes. At each time point of 0, 2, 4, 6, 8, and 10 min, remove 5 mL of release medium from the incubation bath.

Gastric digestion: Take 180 mg of NaCl, 630 mg of HCl, and 288 mg of pepsin to prepare simulated gastric juice (SGF). Take 90mL SGF and preheat it in a constant temperature water bath at 37°C for 5 minutes. Place the above oral digestion dialysis bag into the SGF. Use 1mol/L NaOH to quickly adjust the pH of the mixed system to 2.5 and place it in a 37°C constant temperature shaker water bath (100r/min). Digest for 2 hours. At each time point of 10, 30, 60, 90, and 120 min, remove 5 mL of release medium from the incubation bath.

Intestinal digestion: Take 550.5 mg of CaCl ₂ ·2H ₂ O, 75 mg of bile salts, and 67.5 mg of pancreatic enzyme to prepare simulated intestinal fluid (SIF). After gastric digestion, the pH of the resulting system was quickly adjusted to 7.0 with 0.25 mol/L NaOH. At the same time, preheat the SIF in a constant temperature water bath at 37°C for 5 minutes, place the gastric-digested dialysis bag into the SIF, and adjust the pH of the mixed system to 7.0 with 1 mol/L NaOH. Digest in 37℃ constant temperature shaker water bath (100r/min) for 2h. During this period, NaOH was continuously used to adjust the pH of the mixed system at 7.0. At each time point of 10, 30, 60, 90, and 120 min, remove 5 mL of release medium from the incubation bath. The cumulative release rate of fatty acids was evaluated according to the amount of NaOH used in the titration at different stages of digestion, and the emulsion particle size at different time points was determined.

Results: This example conducted an in vitro digestion simulation experiment on the Antarctic krill oil Pickering emulsion in Example 1 and Comparative Example 1. As shown in Figure 5(A), during the simulated digestion process in vitro, it can be found that since fatty acids are mainly absorbed in the small intestine, especially ω-polyunsaturated fatty acids can enter the blood to exert physiological functions, using Zein/κ-CA/SA triple Metacomposite nanoparticles stabilize Antarctic krill oil Pickering emulsion and perform K ⁺ and Ca ²⁺ cross-linking, which is of great significance to improve the targeted and sustained release of ω-polyunsaturated fatty acids in the body. During the simulated gastrointestinal digestion process, pepsin and pancreatin may hydrolyze Zein in Comparative Example 1, thereby destroying the structure of the nanoparticles and causing the release of fatty acids in its stable Pickering emulsion in SGF and SIF. The Pickering emulsion prepared in Example 1 has good pH stability and can stabilize the fatty acids in the Pickering emulsion in SGF and perform targeted release in SIF. At the same time, as shown in Figure 5 (B), during the in vitro digestion process, the increase in emulsion particle size also showed the same trend as the cumulative release rate of fatty acids.

Measurement of omega-3 unsaturated fatty acid metabolism in vivo (taking EPA and DHA as examples)

200 male rats (weight 220±6g) were selected. Animals were raised on the same diet source under controlled conditions (3±50°C; relative humidity, 5±12%) on a 1 hour light/dark cycle. After adapting for 2 weeks, the rats were randomly divided into two groups (Example 1 group and Comparative Example 1), and the emulsion of Example 1 or Comparative Example 1 was administered orally. Venous blood samples were collected after 2, 4, 6, 8, 12, 24, and 48 h, and serum was stored in a -80°C refrigerator until required for analysis. The whole blood concentrations of DHA and EPA were measured within 2 weeks after LC-MS/MS sampling, and the maximum plasma concentration C _max and the time to reach C _max T _max were recorded simultaneously.

Result: As shown in Figure 6, the C _max of DHA and EPA in Example 1 are both greater than that of Comparative Example 1, but the time T _max to reach C _max is the same. It can be seen that at the same time, the DHA and EPA of Example 1 are absorbed and metabolized faster, and the amount of DHA and EPA reaching the intestine is greater. It can be seen that the Antarctic krill oil Pickering emulsion has a double network interpenetrating structure at the oil-water interface. It can improve the targeted release of omega-3 polyunsaturated fatty acids and promote gastrointestinal absorption.

Claims (10)

1. The preparation method of the intestinal targeting slow-release euphausia superba oil Pickering emulsion is characterized by comprising the following steps:

dissolving Zein in ethanol water solution, adding the obtained Zein solution into kappa-CA solution, preparing Zein/kappa-CA binary composite nano particles by an anti-solvent precipitation method, then adding the Zein/kappa-CA binary composite nano particles into SA water solution, and constructing the Zein/kappa-CA/SA ternary composite nano particles by layer-by-layer adsorption; zein/kappa-CA/SA ternary composite nano particles are taken as a stabilizer to be organically combined with antarctic krill oil, pickering emulsion is constructed by high-speed shearing, and finally K is sequentially added ⁺ Solution, ca ²⁺ Carrying out ion double cross-linking on the solution to construct antarctic krill oil Pickering emulsion with an oil-water interface double-network interpenetrating structure;

wherein Zein represents Zein, kappa-CA represents kappa-carrageenan, and SA represents sodium alginate.

2. The method for preparing the intestinal targeted slow-release type antarctic krill oil Pickering emulsion according to claim 1, which is characterized by comprising the following steps:

(1) Preparing Zein solution: adding Zein into ethanol water solution, sealing, stirring and dissolving to obtain Zein solution;

(2) Preparing SA solution: adding SA into water, stirring, standing to enable the SA to be fully hydrated, and obtaining SA solution;

(3) Preparing a kappa-CA solution: adding kappa-CA into water, water-bathing at 80deg.C for 30min, stirring, and standing to allow full hydration to obtain kappa-CA solution;

(4) Preparing Zein/kappa-CA binary composite nano particles: adding the Zein solution obtained in the step (1) into the kappa-CA solution obtained in the step (3), and stirring under a sealing condition to obtain a Zein/kappa-CA binary composite nanoparticle solution;

(5) Preparation of Zein/kappa-CA/SA ternary composite nanoparticles: adding the Zein/kappa-CA binary composite nanoparticle solution obtained in the step (4) into the SA solution obtained in the step (2), stirring under a sealing condition, and removing ethanol by rotary evaporation to obtain the Zein/kappa-CA/SA ternary composite nanoparticle solution;

(6) Preparing antarctic krill oil diluent: dispersing euphausia superba oil in corn germ oil, sealing in a dark place, stirring and uniformly mixing to obtain euphausia superba oil diluent;

(7) Homogenizing and emulsifying: mixing the antarctic krill oil diluent obtained in the step (6) and the Zein/kappa-CA/SA ternary composite nanoparticle solution obtained in the step (5), and shearing in an ice bath by using a high-speed shearing machine to obtain emulsion;

(8)K ⁺ crosslinking: adding KCl solution into the emulsion obtained in the step (7), and crosslinking for 1-4 hours at 33-35 ℃ to obtain crosslinked emulsion;

(9)Ca ²⁺ crosslinking: adding CaCl to the crosslinked emulsion obtained in the step (8) ₂ And (3) crosslinking the solution at 44-55 ℃ for 12-24 hours to obtain the antarctic krill oil Pickering emulsion with the oil-water interface double-network interpenetrating structure.

3. The method of claim 2, wherein the ethanol in the aqueous ethanol solution in step (1) has a volume fraction of 80%.

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

5. The preparation method as claimed in claim 2, wherein the antarctic krill oil is 10-30% by mass of the maize germ oil.

6. The method of claim 2, wherein the Zein solution and the kappa-CA solution are in a volume ratio of 1:1, the Zein/kappa-CA binary composite nanoparticle solution and the SA solution are in a volume ratio of 1:3, and the euphausia superba oil diluent and the Zein/kappa-CA/SA ternary composite nanoparticle solution are in a volume ratio of 9:1-1:9.

7. The process according to claim 2, wherein in the step (4) or the step (5), the stirring rate is 600 to 800rpm and the stirring time is 10 to 30 minutes.

8. The method of claim 2, wherein in step (7), the shearing condition is 18000r/min for 3min.

9. The method according to claim 2, wherein in the step (8), the concentration of the KCl solution is 1mol/L and the addition amount of the KCl solution is 1 to 2%.

10. The method of claim 2, wherein in step (9), caCl is added to the mixture ₂ The concentration of the solution is 1mol/L, caCl ₂ The addition amount of the solution is 0.2-2%.