3.6. Characterization of Astaxanthin Nanoparticles

Z-average (mean diameter), zeta-potential (charge) and polydispersity index (PDI) of NPs were analyzed by dynamic light scattering (DLS) principles using a Malvern Zetasizer (Nano-ZS; Malvern Instruments, Worcestershire, UK) at 25 °C. Prior to the analysis, the samples were diluted 80 times to avoid multiple scattering effects. PDI values ranging from 0 to 1 indicated the distributions of the particle sizes, with a value close to 0 indicating a uniform population of particles and a value close to 1 indicating a wide variety of dimensions among the particle size distribution. Zeta-potential gives important information about particle stability, and for values closer to 0, the system is considered not stable due to the absence of a net charge that can contrast the aggregation process of NPs.

NPs were treated enzymatically with trypsin (2 mg/mL final concentration) in phosphate buffer pH 7.0 for 4 h at 37 °C in a thermoshaker (Biosan, Riga, Latvia). The enzymatically digested solution was mixed with a double volume of ethyl acetate and placed on a rotating shaker for 60 min. The solution was centrifuged at 12,000 × g for 5 min to allow the separation of the two immiscible phases. ASX was recovered from the upper phase, diluted opportunely, and quantified by spectrophotometry as described above. The efficiency of encapsulation (EE%) was estimated by the following formula:

where ASXi represents the initial amount of ASX loaded in the NPs and ASXf refers to the amount of ASX extracted from NPs after the enzymatic degradation of the protein shell.

Surface ASX (ASXs) was calculated as follows: 0.5 mL of NPs was mixed with 1 mL of ethyl acetate for 5 min. After centrifugation at 12,000 × g for 5 min, the supernatant was analyzed by spectrophotometry to measure ASXs by the following formula:

For the ABTS assay, the procedure proposed by Thaipong et al. [59] with some modifications was followed. Two stock solutions of 7.4 mM ABTS and 2.6 mM potassium persulfate were prepared. The working solution was then obtained by mixing the two stock solutions in equal quantities and allowing them to react for 12 h at room temperature in the dark. The solution was then diluted opportunely with methanol (for the H.p. oleoresin), or PBS (for ASX NPs) to obtain an absorbance of 0.75 units at 734 nm. Fresh ABTS solution was prepared for each assay. Samples (20 μL) were loaded in a 96-well plate and left to react with 180 μL of the ABTS solution for 2 h in the dark. Then the absorbance was taken at 734 nm using a microplate reader (Bio-Tek, Winooski, VT, USA). Results were presented as % scavenging activity following the equation:

where A blank is the absorbance given by the solvent at 734 nm while A sample is the absorbance given by the sample.