Measurement of β-carotene stability
The physicochemical stability of β-carotene encapsulated in the nanoemulsions and in the filled hydrogel particles was tested: 4 mL of nanoemulsions (5% oil) or 4 mL filled hydrogel beads (5% oil) were transferred into glass tubes and stored in the dark at 55 °C for 12 days (pH 7). The β-carotene was isolated from the samples using a solvent extraction method: each sample was extracted by chloroform and alcohol (volume fraction 1:1) solution at least twice. For the filled hydrogel beads, saturated ethylenediaminetetraacetic acid (EDTA) solution was used to dissolve the beads and release the encapsulated β-carotene. The transparent lower organic phase containing the β-carotene was collected and transferred to a cuvette, and then its absorbance was measured at 450 nm using a UV–visible spectrometer. A pure chloroform and alcohol solution was used as a blank. A nanoemulsion containing no added β-carotene was also analyzed as a control. The β-carotene content was determined using a standard curve created from solutions with varying amounts of known β-carotene. All measurements were repeated three times.