Preparation And Stability Of Resveratrol-Loaded Pickering Emulsion Stabilized By Walnut Protein/ Cistanche Deserticola Polysaccharide Composite Nanoparticles

2 Results and analysis

2.1 Characterization of WP/CDPS composite nanoparticles

Figure 1A shows the average particle size of WPN and all WP/CDPS composite nanoparticles. The PDI of all nanoparticles is lower than 0.2, indicating that the nanoparticles are uniformly distributed [21]. Figure 1B shows the droplet size distribution of WP/CDPS composite nanoparticles and WPN. All nanoparticles show a unimodal distribution. The droplet size of WPN is about 17.20 μm, and the droplet size of WP/CDPS composite nanoparticles is significantly smaller than that of WPN, indicating that CDPS changes the surface and shape of WP, among which C4W1 has the largest droplet size (8.891 μm). The sizes of C1W4, C3W2, and C2W3 are 7.063, 7.868, and 8.631 μm respectively. The WP/CDPS composite nanoparticles formed by combining CDPS and WP have a more compact structure. Polysaccharides and proteins are complexed to form hybrid particles. The particles can be irreversibly adsorbed on the oil-water surface to form a strong and orderly interface structure. Polysaccharides and proteins are complexed. The interactions between particles, between emulsified oil droplets, and between emulsified oil droplets and particles can significantly stabilize the system [22].

When the WP content is greater than CDPS (C1W4, C2W3) or less than CDPS (C3W2, C4W1), the droplet size of WP/CDPS shows an increasing trend with the decrease of WP. The C1W1 nanoparticle droplet size is the smallest at 5.927 μm, which is significantly smaller than other WP/CDPS composite nanoparticles. WP and CDPS particles form the best combination to cover the droplet surface and form a stable structure around the droplet, which can effectively Prevent droplet aggregation and further enhance stability. Therefore, the optimal mass ratio of WP/CDPS composite nanoparticles is 1:1.
The zeta potential of different nanoparticle dispersions is shown in Figure 1C. WPN suspension shows positive charge, while CDPS suspension shows negative charge. This indicates that WP and CDPS form WP/CDPS core-shell composite nanoparticles mainly through electrostatic interaction. As the ratio of CDPS increases, WP/CDPS complex
The zeta potential of the combined nanoparticles gradually decreased from -22 mV to -37 mV.
The low incorporation amount of CDPS can neutralize the positive charges on the WP and weaken the electrostatic repulsion of the particles. As the CDPS content increases, the zeta potential of WP/CDPS composite nanoparticles decreases, which can provide sufficient electrostatic repulsion between particles to prevent their aggregation.

Cistanche Herbal Extract With High Active Ingredient

2.2 Interfacial tension of Pickering emulsion

Interfacial tension, as an indicator of surface properties, plays a crucial role in the characteristics of emulsions [23]. Figure 2 shows the interfacial tension changes with time for different Pickering emulsions. Over time, the interfacial tension of all Pickering emulsions gradually decreased and then stabilized. Comparing the interfacial tension under stable conditions, the interfacial tension of these Pickering emulsions showed a trend of first decreasing and then increasing with the increase of WP content in the composite nanoparticles.
C1W1R has the lowest interfacial tension and the highest surface activity. The higher surface activity improves the adsorption efficiency on the interface, resulting in the lowest interfacial tension at adsorption equilibrium (11.88 mN/m). In summary, under an appropriate WP/CDPS mass ratio, WP/CDPS composite nanoparticles with low interfacial tension can be formed. This trend is consistent with the research results of the interfacial tension of zein/carboxylated cellulose composite nanoparticles by Qin Weili et al. [8]. Zein/carboxylated cellulose nanocrystal composite nanoparticles stabilized zein zein Emulsions with a higher content of protein or carboxylated cellulose nanocrystals had higher interfacial tension, indicating lower adsorption efficiency of their nanoparticles.

Fig. 2 Interfacial tension of Pickering emulsion stabilized by WP/ CDPS composite nanoparticles

2.3 Embedding efficiency of Pickering emulsion

As shown in Figure 3, the embedding efficiency of RT in WPR after storage for 35 days was 65.6%, which indicates that single WP as an emulsifier also has a certain protective effect on RT. The embedding efficiency of the Pickering emulsion stabilized by WP/CDPS composite nanoparticles on RT exceeded 85%, which was higher than that of WPR. When the WP/CDPS ratio was 4:1 and 3:2, the embedding efficiency of RT after storage for 35 days was 89.2% and 88.6%, and when the WP/CDPS ratio was 1:4 and 2:3, the embedding efficiency after storage for 35 days was 86.2% and 87.6%, respectively. Compared with other WP/CDPS ratios, the embedding efficiency of 1:1 was the highest at 92.9% after storage for 35 days. Zhang Yali et al. [24] used zein/chitosan particle stabilized Pickering emulsion to embed RT. The experiment proved that the stability and sustained release of RT in this emulsion system were better than those of RT embedded in nanoemulsion in the same study. In this experiment, when the addition ratio of WP/CDPS was 1:1, RT was encapsulated in Pickering emulsion, which had better free radical scavenging activity, loading capacity and stability. It can be considered that when the addition ratio of WP/CDPS was 1:1, it was an effective delivery system for bioactive compounds.

Fig. 3 Effect of storage time on the encapsulation efficiency of RT in the emulsion

2.4 Storage stability of Pickering emulsions

Figure 4 shows the changes in the storage stability of WPR and WP/CDPS composite nanoparticle-stabilized Pickering emulsions within 20 days. The fresh emulsions stabilized with different nanoparticles were uniform and milky white. A small amount of water layer appeared in WPR after 12 h, which gradually increased with the extension of storage time. Finally, equilibrium was reached at 240 h, and the proportion of the emulsion layer was 63.8%.

This is mainly due to the fact that WP contains a large amount of non-polar amino acids, which causes WP to aggregate in the emulsion [25]. For WP/CDPS composite nanoparticle-stabilized emulsions, C1W1R showed the best storage stability at 480 h, with an emulsion layer proportion of 95.6%. This is consistent with the interfacial tension results observed in Pickering emulsions. The lower the interfacial tension, the higher the surface activity of the composite nanoparticles. The enhancement of surface activity plays a key role in stabilizing the emulsion. The composite nanoparticles are effectively adsorbed at the oil-water interface, forming a protective layer, reducing aggregation, and improving the stability of the Pickering emulsion [26-27]. When the CDPS content in the composite nanoparticles is high, the stability of the emulsion decreases. At 240 h, the emulsion layer of C3W2R reached a stable level of 72.5%. At 300 h, the emulsion layer of C4W1R reached a stable level of 75.7%. This is mainly due to the high hydrophobicity of the composite nanoparticles containing higher CDPS. There is a strong electrostatic repulsion in the emulsion, and the structural stability of the composite nanoparticles is poor, resulting in the instability of the emulsion. As time goes by, the composite nanoparticles flocculate and sink under the action of gravity. The storage stability of C1W4R and C2W3R is better than that of C4W1R and C3W2R, and both remain in a uniform and stable state before 96 h. They reach a stable state at 300 h and 252 h, respectively, and the final emulsion layer ratio is about 92.6%. Therefore, a more stable emulsion can be formed in C1W1R. Wu Bi et al. [28] prepared a new starch-based Pickering emulsion by modifying starch with octenyl succinate starch anhydride for encapsulating RT. This system can have a good sustained-release effect on RT and can remain stable for 60 days at 4°C and 25°C, with good centrifugation and dilution stability.

Fig. 4 Appearances (A) and proportion of emulsified layer (B) of Pickering emulsion stabilized by WP/CDPS composite nanoparticles at 25 ℃

2.5 Thermal stability of Pickering emulsion

The thermal stability of Pickering emulsions stabilized by WPR and WP/CDPS composite nanoparticles is shown in Figure 5. The initial particle size of WPR is 45.6 μm.
The initial particle size of the Pickering emulsion stabilized by WP/CDPS composite nanoparticles increased slightly. C1W1R has the smallest particle size (53.8 μm). When the WP or CDPS content increases, the initial particle size of the emulsion increases, which may be related to their corresponding composite nanoparticles. During the heat treatment process at different temperatures, when the WP/CDPS mass ratio is 1:1, polysaccharides and proteins complex to form hybrid particles, and the particles are irreversibly adsorbed on the oil-water surface to form a strong and ordered interface structure. WP The interaction between /CDPS forms a significantly stable system. The particle size of C1W1R changes the smallest with temperature, indicating that it has the best thermal stability. It shows that the C1W1R composite particles form the strongest wrapping structure outside the oil droplets. The higher the WP content, the particle size of C1W4R and C2W3R also increases with the increase of temperature. The main reason may be that heat treatment causes protein denaturation, exposes hydrophobic groups, promotes aggregation between nanoparticles, and induces droplet agglomeration. [29]. After heat treatment, different Pickering emulsions show different degrees of flocculation, and the degree of flocculation increases with the increase in heating temperature. Among the stable emulsions of different composite nanoparticles, C1W1R has the smallest apparent change and the best thermal stability.

Consistent with these results, Lu Siyi et al. [30] used pectin-walnut protein complex emulsion to study its stability and found that after 14 days of storage, the particle size of curcumin-encapsulated pectin-walnut protein complex emulsion (D4, 3) There was a slight increase without phase separation. The curcumin-encapsulated walnut albumin complex emulsion showed good stability to NaCl treatment (up to 300 mmol/L) and heat treatment (up to 90 ℃), indicating that the complex It is an effective delivery system for bioactive compounds and can be widely used in functional foods.

Fig. 5 Particle size change of Pickering emulsion stabilized by WP/ CDPS composite nanoparticles

2.6 Lipid oxidation stability of Pickering emulsion

The primary products of lipid oxidation are one of the indicators for determining the oxidative stability of emulsion [31]. As shown in Figure 6, with the extension of storage time, the POV of Pickering emulsions stabilized by WP/CDPS composite nanoparticles in different proportions gradually increased, which was caused by the auto-oxidation of oils.
As the proportion of CDPS increases, the POV of the emulsion decreases significantly at first, indicating that CDPS has the effect of delaying oil oxidation. However, as the proportion of CDPS continues to increase, the POV of the emulsion gradually increases; when the WP/CDPS ratio is 1:1 (C1W1R), The production of POV is lowest in emulsions. Wang Ran [32] used tea polyphenols-starch nanoparticles to stabilize Pickering emulsions. The study found that tea polyphenols can significantly reduce the POV in the emulsion and effectively delay oil oxidation, which is consistent with the conclusion of this study.

Fig. 6 POV of Pickering emulsions stabilized by different WP/CDPS composite nanoparticles as a function of storage time up to 30 days

2.7 Microstructure of Pickering emulsion

The microstructure of the Pickering emulsion prepared with WP/CDPS at a mass ratio of 1:1 and the distribution of RT in the emulsion system were observed by CLSM. Figure 7A is a stacking state image under simultaneous excitation at 488 nm and 633 nm, which is displayed in yellow (superimposed colors of green and red). The network structure of WP/CDPS around the wrapped oil droplets is observed, which may be due to the strong electrostatic interaction between WP/CDPS. Figure 7B shows a green image of WPN stained with FITC protein dye under 488 nm wavelength laser excitation, indicating that there is a WPN wrapping layer outside the oil droplets. Figure 7C shows a red image of RT stained with Nile red under 633 nm laser excitation.
In the CLSM image, it can be observed that the spherical oil droplets of WP/CDPS Pickering emulsion are dispersed. Overlapping fluorescence micrographs show that RT (red part) is densely encapsulated by WP/CDPS (green part), indicating that WP/CDPS with a mass ratio of 1:1 can be adsorbed on the oil-water interface to effectively stabilize the Pickering emulsion. Yang Tang et al. [15] investigated a new type of edible high internal phase emulsion gel that was effectively stabilized by unique polysaccharide-protein hybrid nanoparticles, and observed spherical dispersion similar to the emulsion in the study.

Fig. 7 Confocal micrographs of Pickering emulsion stabilized by C1W1 composite nanoparticles

3 Conclusion

WP/CDPS composite nanoparticles were constructed using WP and CDPS, and Pickering emulsions were prepared with them as stabilizers, and their stability and RT encapsulation rate were further investigated. The droplet size of WP/CDPS composite nanoparticles was significantly smaller than that of WPN. CDPS changed the surface and shape of WP, and combined with WP to form a more compact structure of WP/CDPS composite nanoparticles, which can effectively prevent droplet aggregation and further enhance stability. Among them, when the mass ratio of WP to CDPS was 1:1, the average particle size of the Pickering emulsion was the smallest (5.927 μm), the interfacial tension was the lowest (11.88 mN/m), and it had good storage stability and thermal stability. After 480 h of storage, the emulsion stratification rate was 95.6%. During the heat treatment at different temperatures, the emulsion particle size of C1W1R changed the least with temperature. CLSM test results show that WP/CDPS can effectively encapsulate RT. The RT embedding efficiency of Pickering emulsion stabilized by WP/CDPS composite nanoparticles exceeded 85%, which was higher than that of WPR emulsion. The RT embedding efficiency was as high as 92.9% after storage for 35 days.

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