Preparation method of double Pickering emulsion loaded with anthocyanin

Abstract

The invention discloses a preparation method for constructing a dual Pickering emulsion on the basis of a phosphorylated purple perilla isolated protein-pectin compound (PPSPI-PEC), realizes application of the dual Pickering emulsion in anthocyanin protection and delivery functions, and belongs to the technical field of food. Degreased purple perilla seed meal is used as a raw material, purple perilla protein isolate (PSPI) is prepared through isoelectric precipitation, then PPSPI is obtained through phosphorylation treatment in a system added with sodium tripolyphosphate, PPSPI dispersion and pectin dispersion with the same volume are mixed and subjected to heat treatment to form a PPSPI-PEC compound, the proportion of PPSPI to PEC is adjusted, and the optimal compound ratio is determined to be 1: 1. And finally, dissolving the anthocyanin solution and PGPR in the perilla seed oil, adjusting the content of PGPR to 5%, the concentration of PPSPI to 2.0 wt% and (W1 / O = 3: 7): W2 = 3: 7, and homogenizing by a high-pressure homogenizer to prepare the stable dual Pickering emulsion. The application of the perilla isolated protein in the field of food science is expanded, and a technical support is provided for developing a novel anthocyanin delivery system.

Description

Technical Field

[0001] The present invention belongs to the technical field of food, and particularly relates to a phosphorylated perilla seed isolate-pectin complex and a preparation method of a double Pickering emulsion thereof. Background Art

[0002] Double emulsion is a multiple emulsion with a unique two-membrane three-phase structure. The most common double emulsion is the water-in-oil-in-water (W / O / W) emulsion. Due to the partitioning of its internal structure, the double emulsion is an ideal delivery system for encapsulating different polar hydrophilic and hydrophobic bioactive substances. It can load hydrophilic and lipophilic substances separately or simultaneously. While each phase achieves a high utilization rate, it also provides effective protection and controlled release for functional substances that are unstable and easily degraded and inactivated. In the field of food processing, double emulsions are widely regarded as an effective method for reducing the oil content of emulsified foods while maintaining the taste characteristics of the foods. In addition, this technology is also used to encapsulate and protect water-soluble components in foods that are extremely sensitive to environmental changes (such as pressure, temperature, light, etc.), including nutritional components, flavor substances, natural pigments, etc. Therefore, double emulsions have received extensive attention due to their wide application potential in multiple fields such as the food industry, cosmetics, pharmaceuticals, and materials science.

[0003] Phosphorylated perilla seed isolate (PPSI) is a polypeptide obtained by phosphorylation modification. PPSI is rich in hydrophobic amino acids, especially proline, which makes it excellent in aspects such as water solubility, antioxidant property, anti-aging property, and blood pressure lowering. The phosphorylation treatment binds to PPSI through the C-O-P bond, increasing the exposure of its hydrophobic groups, enhancing the solubility and emulsifying property of PPSI, and also improving its thermal stability and oil-holding capacity, thereby enhancing its application potential in the food industry, especially as an emulsifier and stabilizer.

[0004] Pectin (PEC) is a polysaccharide with a complex structure and is mainly present in the cell walls of higher plants. Due to its characteristic of forming a stable oil-in-water (O / W) emulsion, it is widely used as an emulsifier, stabilizer, gelling agent, and thickening agent. Since pectin is an anionic polysaccharide, it cannot be digested by enzymes in the human body. Instead, it can electrostatically bind to other biomolecules (such as positively charged proteins or polysaccharides) to form a stable barrier to prevent enzymatic destruction. These characteristics give pectin potential advantages compared with other types of emulsifiers (such as modified starch and proteins).

[0005] Anthocyanin is a water-soluble natural pigment widely present in various plants in nature. It belongs to the flavonoid compound and is commonly used as an additive in food, medicine, and health products due to its rich resources, high safety, non-toxicity, and strong antioxidant ability. The high sensitivity of anthocyanin to environmental conditions leads to its low stability during food processing and storage, making it difficult to be fully applied. The stability of anthocyanin can be improved in various ways, including using appropriate emulsifiers and stabilizers, adjusting the pH value, controlling temperature and light, etc.

[0006] Pickering particles are often used as stabilizers to improve the stability of multiple emulsion systems. Pickering emulsions use solid particles instead of surfactants to inhibit droplet aggregation and achieve system stability by the adsorption of particles on the surface of oil droplets. By increasing the steric hindrance and changing the properties or rheology of the continuous phase interface, the stability of the sensitive active substances encapsulated in the inner phase and their sustained-release ability in the gastrointestinal tract are improved.

[0007] The present invention constructs a complex using perilla seed protein isolate and pectin as raw materials and prepares a double emulsion therefrom. While characterizing the double emulsion, the protective effect and delivery function on anthocyanin are achieved. Summary of the Invention

[0008] Object of the present invention: Emulsions rich in anthocyanin prepared by traditional methods are vulnerable to environmental factors such as heat, oxygen, and pH value during application, resulting in the loss of their functional activities. The present invention constructs a novel protein-polysaccharide complex by utilizing the interaction between phosphorylated perilla seed protein isolate and pectin to form a stable double Pickering emulsion. This emulsion can effectively encapsulate anthocyanin, improve its stability under adverse environmental conditions, and enhance the bioavailability of anthocyanin.

[0009] Technical solution: A method for preparing a double Pickering emulsion loaded with anthocyanin using perilla seed protein isolate and pectin as raw materials, comprising the following steps: (1) Grind defatted perilla seeds to prepare defatted perilla seed powder, disperse the defatted perilla seed powder in deionized water, and adjust the pH to 9.0 with NaOH; after stirring at a constant temperature of 45 °C for 2 h, centrifuge at 6000×g for 15 min. Add HCl to the collected supernatant to change the pH to 4.5; centrifuge and separate the protein precipitate, and then wash and neutralize it with deionized water to obtain perilla seed protein isolate (PSPI); (2) Disperse PSPI in deionized water and stir for 4 h to form a dispersion; after overnight hydration, add sodium tripolyphosphate and adjust the pH of the mixed system to 9.0; incubate at 45 °C for 2 h, dialyze, and freeze-dry to obtain phosphorylated perilla seed protein isolate (PPSPI); (3) Disperse PPSPI in deionized water and stir for 4 h to obtain the protein stock solution. After storing overnight at 4 °C, centrifuge at 6000×g for 10 min to remove insoluble substances. Dissolve pectin in deionized water and stir for 60 min to prepare the pectin stock solution, and store overnight at 4 °C. After heating the obtained protein dispersion at 90 °C for 20 min, immediately cool it to room temperature. Mix the same volume of PPSPI dispersion and pectin dispersion to prepare the protein-pectin dispersion. After stirring for 1 h, change the pH of the complex to 4.0 with 1.0 M HCl, and continue stirring for 1 h. The mixing system is 2.0 wt%, and the phosphorylated perilla seed protein isolate-pectin (PPSPI-PEC) complex is prepared. (4) Prepare the W / O emulsion with the anthocyanin solution as W1. Dissolve PGPR in perilla seed oil. After magnetic stirring for 1 h, add W1. Add the aqueous phase to the oil phase and subject it to high-speed shearing by a high-speed homogenizer to prepare the W1 / O emulsion. (5) Use the PPSPI-PEC complex dispersion as the continuous phase (W2) and the emulsion prepared in step (4) as the dispersed phase. Add the W1 / O emulsion to the PPSPI-PEC complex dispersion, homogenize, and then homogenize it through a high-pressure homogenizer to obtain the W1 / O / W2 emulsion.

[0010] Determination of encapsulation efficiency (EE) and encapsulation stability (ES): Take 1 mL of fresh or stored emulsions for a certain time, add 2 mL of deionized water into a centrifuge tube. Shake and disperse the emulsion. W2 has a good dispersion effect in water. Centrifuge at 4000 r / min for 10 min, then carefully recover the aqueous phase from the lower supernatant with a syringe and filter it through a 0.45 μm polyethersulfone (PES) filter. The pH difference method is used to detect the concentration of anthocyanin in the supernatant.

[0011] Confocal laser scanning microscopy (CLSM): Use a Leica TCS SP2 CLSM to observe the double Pickering emulsion. The oil phase is pre-stained with Nile red (0.1%, w / v), and the protein is pre-stained with Nile blue (0.1%, w / v) to observe the microstructure of the emulsion.

[0012] In vitro simulated digestion: Mix 20 g of the double emulsion with 20 g of simulated gastric juice. Adjust the pH value of the mixture to 2.0 and stir at 37 °C for 1 h to simulate gastric digestion. Take 20 g of gastric chyme, adjust the pH to 7.0, and add 20 g of simulated intestinal juice to the gastric chyme, including sodium chloride (120 mM / L), calcium chloride (10 mM / L), bile salts (5 mg / mL), lipase (3.2 mg / mL), and trypsin (2.4 mg / mL). Maintain the pH of the mixture at 7.0 and stir at 37 °C for 2 h to simulate intestinal digestion.

[0013] Anthocyanin content during digestion: The digested mixture was centrifuged at 12,000 rpm for 30 min at 4°C. The upper layer of the mixture was undigested oil, the middle layer was the micelle layer containing anthocyanins, and the lower layer was the indigestible or undigested solid. The micelle layer was filtered through a 0.45 μm PES filter, and the anthocyanin content was determined by the pH difference method.

[0014] Compared with the prior art, the present invention has the following advantages and beneficial effects: (1) The present invention discloses for the first time the use of phosphatidyl perilla seed protein isolate - pectin (PPSPI - PEC) composite particles as stabilizers for double Pickering emulsions. By precisely controlling the ratio of PPSPI to PEC to 1:1, the stability of the emulsion was optimized. (2) The emulsion prepared using PPSPI - PEC composite particles exhibits excellent viscoelasticity and stability, which is mainly attributed to the strong film layer formed by the composite particles at the oil - water interface, effectively resisting the influence of external environmental changes on the emulsion stability. (3) The emulsion obtained in the present invention has a unique "hierarchical" double structure (W1 / O / W2), which provides more superior conditions for the encapsulation and controlled release of active ingredients, enhancing the protective effect of the emulsion during the simulated gastrointestinal digestion process. (4) The composite emulsion obtained in the present invention can enable anthocyanins to effectively resist degradation during the gastric digestion stage (the free anthocyanin content in the gastric digestion stage is 21.4%), effectively improving the stability and bioavailability of anthocyanins, which provides new possibilities for the development of new and efficient active ingredient delivery systems. Description of the Drawings

[0015] Figure 1 Turbidity diagrams of PPSPI - PEC complexes at different protein - pectin ratios (4:1, 2:1, 1.5:1, 1:1, and 1:1.5).

[0016] Figure 2 Fourier transform infrared spectra of PPSPI - PEC complexes at different protein - pectin ratios (4:1, 2:1, 1.5:1, 1:1, and 1:1.5).

[0017] Figure 3 X - ray diffraction spectra of PPSPI - PEC complexes at different protein - pectin ratios (4:1, 2:1, 1.5:1, 1:1, and 1:1.5).

[0018] Figure 4 Encapsulation efficiency of W1 / O / W2 emulsions prepared at different (W1 / O) to W2 volume ratios (2:8, 3:7, 4:6, 5:5, and 6:4).

[0019] Figure 5 Confocal laser scanning microscopy images of fresh double emulsions loaded with anthocyanins and double emulsions stored for 7 days prepared under the optimal conditions (containing 5% PGPR, PPSPI concentration of 2.0 wt%, (W1 / O = 3:7):W2 = 3:7). (Nile red fluorescence images at 488 nm and Nile blue fluorescence images at 633 nm).

[0020] Figure 6 The encapsulation stability of double Pickering emulsions loaded with anthocyanins prepared under the optimal conditions (containing 5% PGPR, PPSPI concentration of 2.0 wt%, (W1 / O = 3:7):W2 = 3:7).

[0021] Figure 7 For the free fatty acid release curve (G) during simulated intestinal digestion.

[0022] Figure 8 For the change in the content of free anthocyanins in total anthocyanins during simulated intestinal digestion. Detailed implementation manners

[0023] The present invention will be further clarified below in conjunction with specific embodiments. The specific implementation manners are carried out on the premise of the technical solution of the present invention. It should be understood that these manners are only used to illustrate the present invention and not to limit the scope of the present invention. Specific embodiment 1: (1) Extraction of perilla seed protein isolate: First, 200 g of defatted perilla seed powder was soaked in 500 mL of deionized water, and the pH was adjusted to 9.0 with 1 mol / L NaOH. After stirring at a constant temperature of 45 °C for 3 h, it was centrifuged at 6000×g for 20 min. The supernatant was adjusted to pH 4.5 with 1 mol / L HCl, the protein precipitate was collected, washed with deionized water and neutralized. (2) Preparation of phosphorylated perilla seed protein isolate (PPSPI): The perilla seed protein isolate (PSPI) was dispersed in deionized water at a concentration of 6% (w / v). After stirring for 4 h, sodium tripolyphosphate (8%, based on the mass of PSPI) was added, the pH was adjusted to 9.0, incubated at 45 °C for 3 h, dialyzed and freeze-dried to obtain PPSPI. (3) Preparation of phosphorylated perilla seed protein isolate - pectin (PPSPI-PEC) complex: PPSPI was dispersed in deionized water at a concentration of 4% (w / v). After stirring for 4 h, it was centrifuged at 6000×g for 10 min to remove insoluble substances. Pectin was dissolved in deionized water at a concentration of 4% (w / v). After stirring for 1 h, the PPSPI and PEC solutions were mixed, the pH was adjusted to 4.0, and after stirring for 1 h, the PPSPI-PEC complex was prepared. (4) Preparation of double Pickering emulsion: Using 1% (w / v) anthocyanin solution as W1, dissolve PGPR in perilla seed oil at a ratio of 5% (v / v). After stirring for 1 h, add W1, add the aqueous phase to the oil phase, and use a high-speed homogenizer to shear at 15000 rpm for 5 min to prepare the W1 / O emulsion. Then, using 2% (w / v) PPSPI-PEC complex as the continuous phase (W2), add the W1 / O emulsion to W2 and homogenize using a high-pressure homogenizer at 50 bar pressure to prepare the W1 / O / W2 emulsion. Specific Example 2: (1) Extraction of perilla seed protein isolate: First, soak 200 g of defatted perilla seed powder in 500 mL of deionized water, adjust the pH to 9.0 with 1 mol / L NaOH, stir at a constant temperature of 45 °C for 3 h, and then centrifuge at 6000×g for 20 min. Adjust the pH of the supernatant to 4.5 with 1 mol / L HCl, collect the protein precipitate, and wash and neutralize it with deionized water. (2) Preparation of phosphorylated perilla seed protein isolate (PPSPI): Disperse perilla seed protein isolate (PSPI) in deionized water at a concentration of 6% (w / v). After stirring for 4 h, add sodium tripolyphosphate (8%, based on the mass of PSPI), adjust the pH to 9.0, incubate at 45 °C for 3 h, dialyze and lyophilize to obtain PPSPI. (3) Preparation of phosphorylated perilla seed protein isolate - pectin (PPSPI-PEC) complex: Disperse PPSPI in deionized water at a concentration of 4% (w / v). After stirring for 4 h, centrifuge at 6000×g for 10 min to remove insoluble matter. Dissolve pectin in deionized water at a concentration of 4% (w / v). After stirring for 1 h, mix the PPSPI and PEC solutions, adjust the pH to 4.0, and stir for 1 h to obtain the PPSPI-PEC complex. (4) Preparation of double Pickering emulsion: Using 1% (w / v) anthocyanin solution as W1, dissolve PGPR in perilla seed oil at a ratio of 5% (v / v). After stirring for 1 h, add W1, add the aqueous phase to the oil phase, and use a high-speed homogenizer to shear at 15000 rpm for 5 min to prepare the W1 / O emulsion. Then, using 2.5% (w / v) PPSPI-PEC complex as the continuous phase (W2), add the W1 / O emulsion to W2 and homogenize using a high-pressure homogenizer at 50 bar pressure to prepare the W1 / O / W2 emulsion. Specific Example 3: (1) Extraction of perilla seed protein isolate: First, soak 200 g of defatted perilla seed powder in 500 mL of deionized water, adjust the pH to 9.0 with 1 mol / L NaOH, stir at a constant temperature of 45 °C for 3 h, and then centrifuge at 6000×g for 20 min. Adjust the pH of the supernatant to 4.5 with 1 mol / L HCl, collect the protein precipitate, and wash and neutralize it with deionized water. (2) Preparation of phosphorylated perilla seed protein isolate (PPSPI): Dispersed perilla seed protein isolate (PSPI) in deionized water at a concentration of 6% (w / v). After stirring for 4 h, sodium tripolyphosphate (8%, based on the mass of PSPI) was added, the pH was adjusted to 9.0, incubated at 45 °C for 3 h, dialyzed and freeze-dried to obtain PPSPI. (3) Preparation of phosphorylated perilla seed protein isolate - pectin (PPSPI-PEC) complex: Dispersed PPSPI in deionized water at a concentration of 4% (w / v). After stirring for 4 h, centrifuged at 6000×g for 10 min to remove insoluble matter. Pectin was dissolved in deionized water at a concentration of 4% (w / v). After stirring for 1 h, the PPSPI and PEC solutions were mixed, the pH was adjusted to 4.0, and stirred for 1 h to obtain the PPSPI-PEC complex. (4) Preparation of double Pickering emulsion: Using 1% (w / v) anthocyanin solution as W1, dissolved PGPR in perilla seed oil at a ratio of 5% (v / v). After stirring for 1 h, added W1, added the aqueous phase to the oil phase, and sheared at 15000 rpm for 5 min using a high-speed homogenizer to prepare the W1 / O emulsion. Then, using 3% (w / v) PPSPI-PEC complex as the continuous phase (W2), added the W1 / O emulsion to W2, and homogenized at 50 bar pressure using a high-pressure homogenizer to prepare the W1 / O / W2 emulsion.

[0027] The above-described embodiments are only examples for easy understanding. It should be noted that for those skilled in the art of this technology, without departing from the principle of the present invention, several improvements can be made according to the technical solution of the present invention and the description of its embodiments, and these improvements should also fall within the protection scope of the present invention.

Claims

1. A preparation method of double Pickering emulsion loaded with anthocyanins. Compared with double emulsions prepared using other stabilizers, the double Pickering emulsion prepared using perilla seed protein-pectin complex as a stabilizer effectively entraps anthocyanins, realizes the sustained release of blue honeysuckle anthocyanins. After being stored at room temperature for one week, the emulsion can still maintain the W1 / O / W2 structure, showing good stability and improving the bioavailability of anthocyanins in the simulated gastrointestinal tract.

2. A preparation method of double Pickering emulsion loaded with anthocyanins, comprising the following steps: (1) Grind defatted perilla seeds to prepare defatted perilla seed powder. Disperse the defatted perilla seed powder in deionized water and adjust the pH to 9.0 with NaOH. Stir at a constant temperature of 45 °C for 2 h, then centrifuge at 6000×g for 15 min. Add HCl to the collected supernatant to change the pH to 4.

5. Centrifuge and separate the protein precipitate, and then wash and neutralize it with deionized water to obtain perilla seed protein isolate (PSPI); (2) Disperse PSPI in deionized water and stir for 4 h to form a dispersion. After overnight hydration, add sodium tripolyphosphate and adjust the pH of the mixed system to 9.

0. Incubate at 45 °C for 2 h, dialyze and freeze-dry to obtain phosphorylated perilla seed protein (PPSPI); (3) Disperse PPSPI in deionized water and stir for 4 h to obtain a protein stock solution; After storing overnight at 4 °C, centrifuge at 6000×g for 10 min to remove insoluble substances; Dissolve pectin in deionized water and stir for 60 min to obtain a pectin stock solution, and store it overnight at 4 °C. Heat the obtained protein dispersion at 90 °C for 20 min and then immediately cool it to room temperature. Mix the same volume of PPSPI dispersion and pectin dispersion to form a protein-pectin dispersion. After stirring for 1 h, change the pH of the complex to 4.0 with 1.0 M HCl and continue to stir for 1 h. The mixed system is 2.0 wt%, and phosphorylated perilla seed protein-pectin (PPSPI-PEC) complex is obtained; (4) Prepare a W / O emulsion with an anthocyanin solution as W1, and dissolve PGPR in perilla oil. After magnetic stirring for 1 h, add W 1； Add the aqueous phase to the oil phase and subject it to high-speed shearing by a high-speed homogenizer to prepare a W1 / O emulsion; (5) Use the PPSPI-PEC complex dispersion as the continuous phase (W2), and the emulsion prepared in step (4) as the dispersed phase. Add the W1 / O emulsion to the PPSPI-PEC complex dispersion, homogenize, and then homogenize it through a high-pressure homogenizer to obtain a W1 / O / W2 emulsion.

3. The preparation method of a double Pickering emulsion loaded with anthocyanins according to claim 2, characterized in that, The concentration of the defatted perilla seed powder described in step (1) is 1:15, w / v.

4. The preparation method of a double Pickering emulsion loaded with anthocyanins according to claim 2, characterized in that, The concentration of the dispersion described in step (2) is 6% (w / v).

5. The preparation method of a double Pickering emulsion loaded with anthocyanins according to claim 2, characterized in that, The content of the protein stock solution obtained in step (3) is 4 wt%, and the protein-pectin ratio is 1:

1.

6. The preparation method of a double Pickering emulsion loaded with anthocyanins according to claim 2, characterized in that, The water-oil volume ratio of the W1 / O emulsion used in step (5) is 3:7.

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