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

Abstract:

 

In this study, walnut protein/Cistanche deserticola polysaccharide (WP/CDPS) composite nanoparticles were constructed and employed as a stabilizer to prepare a Pickering emulsion. The nanoparticles and the Pickering emulsion were evaluated in terms of particle size, polydispersity index and zeta potential. The effect of WP/CDPS mass ratio on the interfacial tension, storage stability, thermal stability, encapsulation efficiency, microstructure and oxidation stability of the Pickering emulsion was investigated. The results showed that with the increase in the proportion of CDPS, the zeta potential of the WP/CDPS nanoparticles gradually decreased from −22 to −37 mV. The Pickering emulsion (C1W1R) with a mass ratio of WP to CDPS of 1:1 had the smallest average particle size (5.927 μm), the lowest interfacial tension (11.88 mN/m), good storage stability and thermal stability. After 480 h of storage, the proportion of the emulsified layer was 95.6%. The particle size of C1W1R had the smallest change with temperature. The results of confocal laser scanning microscopy (CLSM) showed that the WP/CDPS-stabilized emulsion could effectively encapsulate resveratrol (RT) with an encapsulation efficiency of more than 85%, which was higher than that of WP-stabilized emulsion, and the encapsulation efficiency reached 92.9% after 35 days of storage. Pickering emulsions stabilized by WP/CDPS offer a promising alternative carrier for steady-state delivery of resveratrol in the functional food industry and other related industries.

Keywords: walnut protein; Cistanche deserticola polysaccharide; resveratrol; Pickering emulsion; stability

CISTANCHE HERAL SUPPLEMENTS WITH HIGH ECHINACOSIDE AND ACTEOSIDE

 

 

 

 

Resveratrol (RT) is a non-flavonoid natural polyphenol organic compound extracted from plants. It is widely used for its various pharmacological activities such as anti-inflammatory, antioxidant, anti-tumor, neuroprotection, and improvement of ischemic damage. widespread concern[1-2]. In recent years, RT has been widely used in food, pharmaceutical, cosmetics and other industries. Among various functional factors, the trans isomer structure of RT contains functional groups such as aromatic rings, phenolic hydroxyl groups and double bonds, which has higher biological activity [3-4]. However, it is reported that the isomerization phenomenon and poor water solubility of RT limit its development and utilization in food processing, drug preparation, and active membrane preparation. Encapsulation in delivery systems such as nanoparticles and emulsions can significantly improve its stability and water solubility, and can effectively control the slow and sustained release of RT in a specific environment of the gastrointestinal tract, thus improving the bioavailability of RT [5-6].
Plant protein has good biocompatibility and surface activity and is used in the food field [7]. Currently, more and more studies have found that water-insoluble protein nanoparticles are excellent carrier materials for preparing Pickering emulsions [8]. Walnut protein (WP) can be made into walnut protein nanoparticles (WPN) through self-assembly. WPN has good biocompatibility and bioadhesion and is an ideal carrier material [9]. However, due to the poor water solubility of WPN, WPN-stabilized Pickering emulsions are often unstable. Composite nanoparticles are prepared through protein-polysaccharide combination to adjust the water solubility of WPN, such as stevia, nanocellulose, chitosan, etc. coupled with protein Coupling has been proven to be an effective method to improve the interfacial properties of Pickering emulsions [10-12]. In view of the high stability and high safety characteristics of the food-grade solid particle-stabilized Pickering emulsion prepared from plant protein, it can be used to protect and deliver RT, broadening the application scope of RT in the food field.
Cistanche deserticola polysaccharide (CDPS) is the main component of Cistanche deserticola. It is an acidic heteropolysaccharide composed of glucose, galactose, rhamnose, arabinose, fructose and other monosaccharides. It has the functions of protecting nerves and improving intestinal function. Tract flora, regulating immunity
It has functions such as preventing epidemics and improving memory, and can be used as raw materials for health products or medicines [13]. ˆ
The combination of CDPS and proteoglycan gives it a certain emulsification stability and can be used in food processing instead of some emulsifiers. Therefore, taking advantage of the strong negative charge, enhanced hydrophilicity and better dispersibility of CDPS, one can try to prepare composite particles by compounding with positively charged WP to stabilize ickering emulsions. However, few studies have attempted to prepare WP/CDPS composite nanoparticles with different mass ratios of WP and CDPS to stabilize Pickering emulsions.
Based on this, this study prepared WP/CDPS composite nanoparticles by changing the mass ratio of WP and CDPS and used them to stabilize RT. Study the physical properties of different WP/CDPS composite nanoparticles and study the effects of different WP/CDPS composite nanoparticles on the performance of Pickering emulsion, in order to provide a theoretical basis for improving the emulsification performance and emulsion stability of WP/CDPS particles, and for the utilization of WP/CDPS Provides reference as nuclear material for protection and delivery of RT.

CISTANCHE HERAL SUPPLEMENTS WITH HIGH ECHINACOSIDE AND ACTEOSIDE

 

1 Materials and methods

1.1 Materials and reagents

WP powder (purity 90%) Peptide Love Biotechnology (Xi'an) Co., Ltd.; NaOH, HCI, anhydrous ethanol, RT (purity 99%), sodium chloride (analytical grade) Xinjiang Hongdao Instrument Co., Ltd.; RT standard (HPLC ≥ 98%, molecular weight 228.24 Da) Chengdu Dester Biotechnology Co., Ltd.; CDPS (powder after 80-120 mesh sieve, purity 98%, main components: phenylethyl glycosides, echinacoside, verbascoside, eugenol glycoside, cistancheside A, etc., molecular weight 488.44 Da) Shanghai Yuanye Biotechnology Co., Ltd.; Nile red dye Sigma-Aldrich (Shanghai) Trading Co., Ltd.; fluorescein isothiocyanate ester (FITC) Beijing Solebow Technology Co., Ltd.; non-esterified fatty acid kit Suzhou Keming Biotechnology Co., Ltd.; methanol, n-hexane, dichloromethane (chromatographic grade) Sinopharm Chemical Reagent Co., Ltd.

 

1.2 Instruments and equipment

CIENTZ-30ND freeze dryer, JY92-IINe ultrasonic cell crusher Ningbo Xinzhi Biotechnology Co., Ltd.; DF-101S magnetic stirrer Shanghai Lichen Bangxi Instrument Technology Co., Ltd.; PHS-3Ce acidity meter Shanghai Yidian Scientific Instrument Co., Ltd.; Winner2005e laser particle size analyzer Jinan Micro-Nano Particle Instrument Co., Ltd.; JS94H Zeta potential meter Shanghai Zhongchen Digital Technology Equipment Co., Ltd.; AXRP confocal laser scanning microscopy (CLSM) Nikon Corporation, Japan.

 

1.3 Methods

1.3.1 Preparation of WPN

WPN was prepared by anti-solvent precipitation method [14]. 2 g WP was dissolved in 100 mL 0.5 mol/L NaCl solution and stirred at 75 °C and 300 r/min for 12 to 24 h until it was completely dissolved. Then centrifuge at 3 000 r/min for 10 min to remove large particles and other insoluble substances. Finally, the pH value of the obtained supernatant was adjusted to 12.0 with 0.1 mol/L HCl or NaOH solution, and the dispersion was pre-frozen in a refrigerator at -80 ℃ for 12 h, and then vacuum freeze-dried at -50 ℃ for 72 h to obtain WPN.

 

1.3.2 Preparation of WP/CDPS composite nanoparticles

1.0 g CDPS and WP were dispersed in 100 mL distilled water to prepare 1% CDPS suspension and WP suspension. Then, the WP suspension was added to the CDPS suspension at different mass ratios of CDPS to WP (4:1, 3:2, 1:1, 2:3 and 1:4), and a mixture of WP and CDPS was obtained under stirring. Excess water was evaporated by rotary evaporation under vacuum (-0.1 MPa) at 40 ℃. The nanoparticle dispersion was pre-frozen in a refrigerator at -80 °C for 12 h, and then vacuum freeze-dried at -50 °C for 72 h to obtain WP/CDPS composite nanoparticles. The WP/CDPS composite nanoparticles with a CDPS to WP mass ratio of 4:1, 3:2, 1:1, 2:3 and 1:4 were named C4W1, C3W2, C1W1, C2W3 and C1W4, respectively.

 

1.3.3 Particle size, polydispersity index (particle size, PDI) and Zeta potential determination

 

According to the literature [15] with slight modifications, the particle size of WPN and WP/CDPS composite nanoparticles was measured using a laser particle size analyzer wet method. PDI was determined using a dynamic light scattering instrument. The Zeta potential of the Pickering emulsion was measured using a Zeta potential analyzer. Before analysis, the sample was diluted 100 times with ultrapure water to avoid multiple scattering effects.

 

1.3.4 Preparation of Pickering emulsions

100 mL suspensions of WPN and different WP/CDPS composite nanoparticles (mass fraction 1%) were prepared. RT (volume fraction 10%) was mixed with each suspension. Ultrasonic cell disruption was performed at 250 W for 4 min. According to the different WP/CDPS composite nanoparticles, the Pickering emulsions were named C4W1R, C3W2R, C1W1R, C2W3R and C1W4R. The same method was used to prepare the Pickering emulsion stabilized by WPN and named WPR.

 

1.3.5 Determination of interfacial tension of Pickering emulsions

The interfacial tension of Pickering emulsions stabilized by WP/CDPS composite nanoparticles was determined according to the method of reference [16] with some modifications.
20 μL of Pickering emulsion was added to the syringe and the dynamic interfacial tension was measured at 25 °C. The interfacial tension of each sample was measured 3 times.

 

1.3.6 Determination of RT embedding rate

The embedding rate of RT was determined by ultraviolet spectrophotometry using the method of Mei Yuqi et al. [17]. The diluted emulsion (volume fraction 1%) was centrifuged at 9,000 r/min for 10 min at 25 °C. The supernatant was collected and the absorbance was measured at 306 nm. After RT was fully dissolved, it was appropriately diluted and the content of RT was calculated according to its standard curve (y = 0.124 2x + 0.035 4, R2 = 0.997 7). The embedding rate was calculated using formula (1):