Pickering emulsion with stable nano-selenium-chitin nanocrystalline composite particles as well as preparation method and application of Pickering emulsion

Abstract

The invention belongs to the technical field of functional foods and biological medicines, and particularly relates to a Pickering emulsion with stable nano-selenium-chitin nanocrystalline composite particles as well as a preparation method and application of the Pickering emulsion. The nano-selenium-chitin nanocrystalline composite particles are mixed with an oil phase, and the nano-selenium Pickering emulsion which is uniform, free of layering, high in grease embedding rate (larger than or equal to 80%) and high in oxidation resistance is obtained after sufficient emulsification. Compared with a traditional chemical emulsifier, the nano-selenium-chitin nanocrystalline composite particle has the advantages of renewability and environmental friendliness; moreover, the nano-selenium Pickering emulsion obtained by taking the nano-selenium-chitin nanocrystalline composite particles as a stabilizer not only can be used as an oral selenium source, but also can be used as a conveying carrier of hydrophobic active substances, so that the problem of poor compatibility of nano-selenium and oil-soluble components is effectively solved, and the application range of nano-selenium is widened; wide application values are realized in the fields of functional foods and biological medicines.

Description

Technical Field

[0001] The present invention belongs to the field of functional food and biomedicine technology, and more specifically relates to a Pickering emulsion stabilized by nano-selenium-chitin nanocrystal composite particles, and a preparation method and application thereof. Background Art

[0002] Pickering emulsions, as emulsion systems stabilized by nano- / micron-sized solid particles, offer significant advantages in stability and biosafety over traditional surfactant-stabilized emulsions, and therefore hold great promise for applications in biomedicine, food, and cosmetics. However, commonly used inorganic particle stabilizers (such as silica and titanium dioxide) suffer from poor biocompatibility and difficulty in degradation, severely restricting their application in bio-related fields. To address this, researchers have begun exploring soft microparticles based on biopolymers (such as proteins and polysaccharides) as novel Pickering emulsion stabilizers.

[0003] Selenium nanoparticles (SeNPs) have shown great potential in the application of functional ingredients, due to their excellent biocompatibility, low toxicity, and significant antioxidant and anti-tumor bioactivities, in health supplements, functional foods, and pharmaceuticals. However, existing research on SeNPs has primarily focused on solution systems, while their application in emulsion systems is still in its infancy. More importantly, the current SeNP-based Pickering emulsion construction technology still has several key limitations.

[0004] First, existing technologies often rely on chemically synthesized surfactants to modify SeNPs. For example, Chinese patent application CN112544982A uses Tween 80 to modify SeNPs to prepare Pickering emulsions. While achieving good biological activity and stability, Tween 80, as a chemically synthesized surfactant, not only poses potential safety risks at high doses, but its synthesis process also fails to adhere to green chemistry principles, severely limiting the practical application value of this technology. Second, existing SeNP-stabilized emulsion systems exhibit poor interfacial stability. The zein-modified SeNPs system reported by Wang et al. exhibits this prominent problem: when the oil phase ratio exceeds 60%, its oil encapsulation efficiency drops sharply to less than 40%, indicating a significant decrease in the emulsion's interfacial stability. This poor interfacial stability not only leads to low oil encapsulation efficiency but also makes it difficult to effectively protect the active ingredients encapsulated in the oil phase, severely restricting the practical application of this technology in areas such as functional foods and drug delivery.

[0005] In summary, developing a SeNPs-based Pickering emulsion construction method that is completely independent of chemically synthesized surfactants and can maintain excellent interfacial stability under high oil phase conditions has become a key scientific issue that needs to be urgently addressed in this field. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of the existing SeNPs-based Pickering emulsion construction that relies on chemically synthesized surfactants and the poor performance of the SeNPs-stabilized emulsion system constructed by the existing technology in terms of interface stability, and to provide a Pickering emulsion stabilized by nano-selenium-chitin nanocrystal composite nanoparticles.

[0007] Another object of the present invention is to provide an application of the Pickering emulsion stabilized by the nano-selenium-chitin nanocrystal composite nanoparticles.

[0008] The above-mentioned purpose of the present invention is achieved through the following technical solutions: The present invention protects a Pickering emulsion stabilized by nano-selenium-chitin nano-crystal composite nano-particles, which is obtained by uniformly mixing the nano-selenium-chitin nano-crystal composite nano-particles and an oil phase and fully emulsifying them; The nano-selenium-chitin nanocrystal composite nanoparticles include elemental selenium and chitin nanocrystals composited therewith.

[0009] Furthermore, the nano-selenium-chitin nanocrystal composite nanoparticles are prepared by the following steps: S1. The chitosan was dispersed in an acidic reagent solution, fully hydrolyzed and centrifuged, the precipitate was dispersed in water and homogenized to obtain a chitosan microcrystal suspension; S2. Mixing the selenium source with the chitosan microcrystal suspension obtained in step S1, adding a reducing agent, and allowing the mixture to react sufficiently to obtain nano-selenium-chitosan nanocrystal composite nanoparticles.

[0010] Preferably, the concentration of the chitosan microcrystal suspension is 0.25 wt% to 1 wt%, more preferably 0.5 to 1 wt%, and most preferably 1 wt%.

[0011] Furthermore, the mass volume ratio of the selenium source to the chitin microcrystal suspension is (0.5~2) mg:1 mL.

[0012] Preferably, the molar ratio of the selenium source to the reducing agent is 1:(3-5).

[0013] Furthermore, the selenium source includes one or more of sodium selenite, sodium hydrogen selenite, potassium selenite, sodium selenate, and selenium dioxide.

[0014] Furthermore, the pH of the chitosan microcrystal suspension is 6.8-7.2, preferably 7.0.

[0015] Furthermore, the reducing agent includes one or more of ascorbic acid, sodium citrate, and glutathione.

[0016] Preferably, the acidic agent is hydrochloric acid or sulfuric acid, preferably hydrochloric acid.

[0017] Preferably, the concentration of the acidic reagent solution is 2-4 mol / L, preferably 3 mol / L.

[0018] Preferably, the sufficient hydrolysis is carried out under boiling water bath conditions.

[0019] Furthermore, the sufficient hydrolysis time is 1 to 3 hours, preferably 2 hours.

[0020] Furthermore, the centrifugal conditions are 15-25 min, 5000-7000 r / min, preferably 20 min, 6000 r / min.

[0021] Furthermore, the centrifugation further includes water washing, and the number of water washings is 1 to 3, preferably 2 times.

[0022] Furthermore, in step S1, the homogenization conditions are: homogenization pressure is 200-400 bar (preferably 350 bar), and time is 3-6 min (preferably 5 min).

[0023] Specifically, the chitosan microcrystal suspension is prepared by dispersing weighed chitosan in a hydrochloric acid solution, hydrolyzing it in a boiling water bath for 1 to 3 hours, then centrifuging it at 5000 to 7000 r / min for 20 minutes, washing it with water for 1 to 3 minutes, centrifuging it again, dispersing the precipitate in water, and homogenizing it under high pressure at 200 to 400 bar for 3 to 6 minutes to obtain a chitosan microcrystal suspension.

[0024] Furthermore, the volume content of the oil phase in the Pickering emulsion is 20% to 75%.

[0025] Furthermore, the oil phase includes one or more of soybean oil, medium chain triglycerides, and corn oil.

[0026] Preferably, the conditions for sufficient emulsification are: homogenization at 10k~20k rpm for 1~3 min, preferably homogenization at 14k rpm for 2 min.

[0027] The present invention also protects the use of the Pickering emulsion in preparing functional foods or medicines.

[0028] Compared with the prior art, the present invention has the following beneficial effects: (1) Chitosan nanocrystals are derived from natural renewable materials and are renewable and environmentally friendly. Compared with traditional chemical emulsifiers, their production process is greener and has less impact on the environment. (2) Using nano-selenium modified with chitin nanocrystals as an emulsifier, a nano-selenium Pickering emulsion with uniformity, no stratification, high oil embedding rate (≥80%), and high antioxidant properties was prepared; (3) The nano-selenium Pickering emulsion can not only be used as an oral selenium source, but also as a delivery carrier for hydrophobic active substances, effectively solving the problem of poor compatibility between nano-selenium and oil-soluble components and broadening the application range of nano-selenium. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 1 is an appearance diagram of the Pickering emulsions prepared in Examples 1, 13 and 14.

[0030] Figure 2 These are the appearance pictures of the Pickering emulsions prepared in Example 1 (right) and Comparative Example 1 (left).

[0031] Figure 3 1 and 2 are appearance diagrams of the Pickering emulsions prepared in Example 1 and Comparative Examples 5-6.

[0032] Figure 4 This is a graph showing the test results of the particle size of the composite nanoparticles prepared in Example 1.

[0033] Figure 5 This is a transmission electron microscope observation image of the composite nanoparticles prepared in Example 1.

[0034] Figure 6 These are optical microscope morphologies of the Pickering emulsions prepared in Examples 9 to 12.

[0035] Figure 7 This is a statistical diagram of the oil embedding efficiency of the Pickering emulsion prepared in Examples 1 to 12.

[0036] Figure 8 The graph is a statistical diagram of the peroxide content of the Pickering emulsions obtained in Examples 3, 7, 11 and Comparative Examples 2 to 4. DETAILED DESCRIPTION

[0037] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0038] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0039] Chitosan: Deacetylation degree: <50%; Manufacturer: Zhejiang Aoxing Biological Co., Ltd. Chitosan: Deacetylation degree: ≥95%; Manufacturer: Shanghai McLean Biochemical Technology Co., Ltd.

[0040] Example 1 Preparation of Nano-Selenium-Chitosan Nanocrystal Composite Nanoparticle Pickering Emulsion (1) Preparation of chitin nanocrystals Weigh 8 g of chitin and disperse it in 3 M hydrochloric acid, stirring thoroughly. Hydrolyze the mixture in a boiling water bath at 100°C for 2 hours, stirring with a rotor to ensure uniform hydrolysis. After cooling, remove the supernatant, add deionized water, and centrifuge at 6000 rpm for 20 minutes. Repeat the process twice, rinse with water, filter, and centrifuge. Add an appropriate amount of deionized water to 400 mL, and homogenize at 350 bar for 5 minutes to obtain a 2 wt% chitin nanocrystal suspension.

[0041] (2) Preparation of nano-selenium-chitin nanocrystal composite nanoparticles S1. Take 10 mL of the above chitosan nanocrystal suspension and dilute it to 80 mL with deionized water to obtain a 0.25 wt% chitosan nanocrystal suspension (adjust the pH to 7.0).

[0042] S2. Weigh 40 mg of sodium selenite powder and pour it into 80 mL of the 0.25 wt% chitosan nanocrystal suspension obtained in step S1. Stir for 10 min.

[0043] S3. According to the molar ratio of sodium selenite to ascorbic acid of 1:4, ascorbic acid was added to the system of step S2, and magnetic stirring was performed at room temperature for 1 h to obtain low-concentration nano-selenium-chitin nanocrystal composite nanoparticles (CNC-SeNPs L).

[0044] (3) Preparation of Pickering emulsion of nanoselenium-chitin nanocrystal composite nanoparticles 12 mL of CNC-SeNPs L obtained in step S3 was mixed with 8 mL of soybean oil and homogenized at 14 k rpm for 2 min to obtain a CNC-SeNPs L-stabilized Pickering emulsion (the oil phase content was 40%, and the obtained emulsion was referred to as CNC-SeNPs L40%).

[0045] Examples 2-14 Preparation of Nano-Selenium-Chitosan Nanocrystal Composite Nanoparticle Pickering Emulsion The differences between Examples 2 to 14 and Example 1 are shown in Table 1. Other steps and parameters were the same as in Example 1. It should be noted that the volume of the chitin nanocrystal suspension to which sodium selenite powder was added was 80 mL; the total volume of the composite nanoparticles and the oil phase in the Pickering emulsion system was 20 mL; and chitin nanocrystal suspensions of varying concentrations (0.25 wt % to 1 wt %) were prepared by diluting the 2 wt % chitin nanocrystal suspension obtained in Example 1 with water.

[0046] Table 1 Preparation conditions of Examples 1 to 14

[0047] Note: MCT stands for medium chain triglycerides.

[0048] Comparative Example 1 Preparation of Chitosan Nanocrystal Pickering Emulsion The difference from Example 1 is that no selenium source is added. The specific preparation process is as follows: (1) Preparation of chitin nanocrystals Weigh 8 g of chitin and disperse it in 3 M hydrochloric acid, stirring thoroughly. Hydrolyze the mixture in a boiling water bath at 100°C for 2 hours, stirring with a rotor to ensure uniform hydrolysis. After cooling, remove the supernatant, add deionized water, and centrifuge at 6000 rpm for 20 minutes. Repeat the process twice, rinse with water, filter, and centrifuge. Add an appropriate amount of deionized water to 400 mL, and homogenize at 350 bar for 5 minutes to obtain a 2 wt% chitin nanocrystal suspension.

[0049] Take 10 mL of the above 2 wt% chitosan nanocrystal suspension and dilute it to 80 mL with deionized water to obtain a 0.25 wt% chitosan nanocrystal suspension (adjust the pH to 7.0).

[0050] (2) Preparation of Chitosan Nanocrystal Pickering Emulsion 12 mL of 0.25 wt% chitosan nanocrystal suspension was mixed with 8 mL of soybean oil and homogenized at 14 krpm for 2 min to obtain a chitosan nanocrystal Pickering emulsion (the oil phase content was 40%, and the obtained emulsion was referred to as CNC L 40%).

[0051] Comparative Examples 2-4 Preparation of Chitosan Nanocrystal Composite Nanoparticle Pickering Emulsion The differences between Comparative Examples 2 to 4 and Comparative Example 1 are shown in Table 2. Other steps and parameters were the same as those in Comparative Example 1. It should be noted that the volume of the 2 wt % chitosan nanocrystal suspension after dilution with deionized water was 80 mL; the total volume of the composite nanoparticles and oil phase in the Pickering emulsion system was 20 mL; and chitosan nanocrystal suspensions of varying concentrations (0.25 wt % to 1 wt %) were prepared by diluting the 2 wt % chitosan nanocrystal suspension obtained in Comparative Example 1 with water.

[0052] Table 2 Preparation conditions of Examples 1 to 14

[0053] Comparative Example 5 Preparation of Nano-Selenium-Chitin Composite Nanoparticle Pickering Emulsion The difference from Example 1 is that the 0.25 wt % chitosan microcrystal suspension is replaced with a 0.25 wt % chitosan suspension to prepare the composite nanoparticles and Pickering emulsion. The other steps and parameters are the same as those in Example 1.

[0054] Preparation of 0.25 wt% chitosan suspension: Weigh 0.25 g chitosan and dissolve it in 100 mL of 1% dilute acetic acid solution.

[0055] Comparative Example 6 Preparation of Pickering Emulsion of Nano-Selenium-Chitosan Composite Nanoparticles The difference from Example 1 is that the 0.25 wt % chitin microcrystal suspension is replaced with a 0.25 wt % chitosan suspension to prepare the composite nanoparticles and Pickering emulsion. The other steps and parameters are the same as those in Example 1.

[0056] Preparation of 0.25 wt% chitosan solution: Weigh 0.25 g chitosan and dissolve it in 100 mL of 1 vol% dilute acetic acid solution.

[0057] Experimental Example 1: Measurement of Appearance The appearance of the Pickering emulsions obtained in Examples 1, 13 and 14 was taken, and the results were as follows: Figure 1 As shown in the figure, different oils (soybean oil, MCT, corn oil) can be successfully used to prepare stable nano-selenium-chitin nanocrystal composite nanoparticle Pickering emulsions.

[0058] The appearance of the Pickering emulsions obtained in Example 1 and Comparative Example 1 was taken, and the results were as follows: Figure 2 As shown in the figure, under the same oil phase volume conditions, the Pickering emulsion stabilized by chitosan nanocrystals alone has a small amount of water precipitation, while the Pickering emulsion prepared by nano-selenium-chitosan nanocrystal composite nanoparticles has no obvious stratification phenomenon and no oil precipitation, showing a stable state, indicating that the presence of selenium is conducive to the stability of the Pickering emulsion.

[0059] The appearance of the Pickering emulsions obtained in Example 1 and Comparative Examples 5 to 6 was photographed. The results are as follows Figure 3 As shown, the Pickering emulsion based on nanoselenium and chitin nanoparticles exhibited significant demulsification. The Pickering emulsion based on nanoselenium and chitosan nanoparticles also exhibited demulsification, with the lower liquid layer being distinctly red, indicating that a significant amount of selenium remained in the aqueous phase and did not reach the oil-water interface, leading to emulsion instability and demulsification. These results indicate that neither chitin nor chitosan can be used to prepare stable nanoselenium particle-stabilized Pickering emulsions.

[0060] Experimental Example 2: Determination of the particle size of composite nanoparticles The particle size of the composite nanoparticles prepared in Example 1 was measured using a Malvern Nanozetasizer ZS90. The test results are shown in FIG. Figure 4 .

[0061] Depend on Figure 4 It can be seen that the composite particles have a uniform particle size distribution, with an average particle size of 293 nm, which belongs to the nanometer range.

[0062] Experimental Example 3 Determination of the morphology of composite nanoparticles The morphology of the composite nanoparticles prepared in Example 1 was observed using a Japanese JEOL JEM-F200 transmission electron microscope. Figure 5 shown.

[0063] Depend on Figure 5 It can be seen that the prepared nano-selenium has a spherical structure and is uniform in size.

[0064] Experimental Example 4 Determination of Pickering Emulsion Particle Size The particle sizes of the Pickering emulsions prepared in Examples 1, 5, and 9 were observed using an MS2000 laser particle size analyzer. The results are shown in Table 3.

[0065] Table 3 Pickering emulsion particle size

[0066] As can be seen from Table 3, the D 3,2 Particle size is 7~19μm, D 4,3 The particle size ranged from 20 to 32 μm, and the overall emulsion particle size was small, among which the ratio of CNC-SeNPs H 40% was the smallest, indicating that the particle size distribution of this emulsion was the most concentrated and uniform.

[0067] Experimental Example 5 Determination of the optical microscopic morphology of Pickering emulsion The morphology of the Pickering emulsions prepared in Examples 9 to 12 was observed using an MD50 optical microscope. Figure 6As shown in the figure, under different oil phase volume conditions, the emulsion oil droplets remain round and relatively uniform in size. As the oil phase content increases, the oil droplet size gradually increases.

[0068] Experimental Example 6 Determination of the oil embedding rate of Pickering emulsion Take a 2 mL centrifuge tube and label it, weigh the centrifuge tube and record it. Take 1 g of freshly prepared emulsion sample (Examples 1 to 12) and add it to the marked centrifuge tube, and then add 1 mL of n-hexane to extract the unentrapped oil. Then shake it evenly, centrifuge it at 5000 g (6000 rpm) for 5 minutes using a high-speed centrifuge, and then carefully extract the organic phase with a syringe. Then wash it with distilled water, that is, add 1 mL of distilled water, centrifuge it at 5000 g for 5 minutes, and then remove the lower aqueous phase with a syringe, and repeat three times. Record the weight, open the centrifuge tube lid, and place it in a 70°C oven to dry to constant weight. The formula for calculating the oil entrapment rate is as follows:

[0069] M W : Refers to the weight after continuous drying to constant weight, that is, the weight of the sample after removing the unembedded soybean oil and water (g) M D : Refers to the weight of the fresh emulsion sample after drying to remove moisture (g).

[0070] All reagents were prepared and used immediately, the experiment was repeated three times, and the results were the average of the three experiments.

[0071] The results are as follows Figure 7 As shown in the figure, overall, with the increase of the oil phase content, the oil embedding efficiency of the Pickering emulsions obtained in Examples 1 to 12 did not differ significantly. When the oil phase content was as high as 75%, the oil embedding efficiency of the Pickering emulsions stabilized by the three composite particles with different concentrations was still above 80%. In particular, the oil embedding efficiency of the Pickering emulsion stabilized by the high-concentration composite particles (CNC-SeNPs H) was above 90%, showing extremely excellent oil embedding stability.

[0072] Experimental Example 6 Determination of hydrogen peroxide content after accelerated oxidation of Pickering emulsion 2 mL of freshly prepared Pickering emulsion (Examples 3, 7, and 11, Comparative Examples 2-4) was added to a centrifuge tube and stored in a 37°C incubator for 7 days, followed by 2 days at 65°C to accelerate oxidation. A 0.6 mL emulsion sample was added to 3.0 mL of a 3:1 isooctane-isopropanol mixture, mixed thoroughly, and centrifuged at 3000 g for 6 min. 400 μL of the supernatant (organic phase) was added to 5.6 mL of a 2:1 methanol-n-butanol mixture, 30 μL of a 3.94 M ammonium thiocyanate solution, and 30 μL of a ferrous ion solution consisting of equal volumes of 0.144 M ferrous sulfate and 0.132 M barium chloride. After reacting at 25°C for 20 minutes, the absorbance of the sample was measured at 510 nm using a UV spectrophotometer (UV-2600, SHIMADZU, Japan). All reagents were prepared freshly before use, and the experiment was repeated three times, with the average of the three experiments being used as the mean.

[0073] The results of the accelerated oxidation treatment of Pickering emulsion are as follows Figure 8 As shown in the figure, under the same storage conditions, the hydrogen peroxide concentration of the nano-selenium-chitin nanocrystal composite nanoparticle Pickering emulsion was significantly lower than that of the chitin nanocrystal composite nanoparticle Pickering emulsion without the addition of selenium source, which indicates that nano-selenium and chitin nanocrystals synergistically enhance the stability of the emulsion, making the nano-selenium-chitin nanocrystal composite nanoparticle stabilized Pickering emulsion exhibit excellent antioxidant properties and can better avoid lipid oxidation in the emulsion.

[0074] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A Pickering emulsion stabilized by nano-selenium-chitin nanocrystal composite nanoparticles, characterized in that: The nano-selenium-chitin nanocrystal composite nanoparticles and the oil phase are mixed and fully emulsified to obtain; The nano-selenium-chitin nanocrystal composite nanoparticles include elemental selenium and chitin nanocrystals composited therewith.

2. The Pickering emulsion according to claim 1, wherein The nano-selenium-chitin nanocrystal composite nanoparticles are prepared by the following steps: S1. The chitosan was dispersed in an acidic reagent solution, fully hydrolyzed and centrifuged, the precipitate was dispersed in water and homogenized to obtain a chitosan microcrystal suspension; S2. Mixing the selenium source with the chitosan microcrystal suspension obtained in step S1, adding a reducing agent, and allowing the mixture to react sufficiently to obtain nano-selenium-chitosan nanocrystal composite nanoparticles.

3. The Pickering emulsion according to claim 2, wherein The concentration of the chitosan microcrystal suspension is 0.25 wt % to 1 wt %.

4. The Pickering emulsion according to claim 2, wherein The mass volume ratio of the selenium source to the chitin microcrystal suspension is (0.5-2) mg:1 mL.

5. The Pickering emulsion according to claim 2, wherein The molar ratio of the selenium source to the reducing agent is 1:(3-5).

6. The Pickering emulsion according to claim 2, characterized in that The selenium source includes one or more of sodium selenite, sodium hydrogen selenite, potassium selenite, sodium selenate, and selenium dioxide.

7. The Pickering emulsion according to claim 2, wherein The reducing agent includes one or more of ascorbic acid, sodium citrate, and glutathione.

8. The Pickering emulsion according to any one of claims 1 to 7, characterized in that The volume content of the oil phase in the Pickering emulsion is 20% to 75%.

9. The Pickering emulsion according to claim 8, characterized in that The oil phase includes one or more of soybean oil, medium chain triglycerides, and corn oil.

10. Use of the Pickering emulsion according to any one of claims 1 to 9 in the preparation of functional foods or medicines.

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