Extraction of β-carotene from carrot pomace using microemulsions and pulsed electric fields

Classical extraction methods, such as low pressure solvent extraction (LPSE) are widely used to obtain hydrophobic bioactive compounds from plant sources. Although LPSE is a simple method, it has limited application in food processing due to most organic solvents being banned for food products extraction, and they are costly, environmentally hazardous, and require expensive disposal procedures. Thus, the use of inexpensive and efficient alternate extraction systems might be suitable to replace the use of LPSE techniques. The objective of this research was to develop a new strategy to extract hydrophobic bioactive compounds from plant materials. The effect of electric field strength and frequency during pulsed electric field (PEF) processing on the extractability of carotenoids in carrots was examined. This study attempted to evaluate the effectiveness of oil-in-water (O/W) microemulsions as a potential alternative to replace the use of organic solvents for extracting β-carotene from carrot pomace. The carotenoids extractability of carrot pomace was improved using PEF processing at different electric field strengths (0.1-1 kV cm-1) and frequencies (5-75 Hz). Electroporation due to PEF treatment at moderate field strengths up to 1 kV cm-1 at 5 Hz improved the extractability of carotenoids in carrot pomace when organic solvents and vegetable oils were used as extraction media. Increasing PEF frequency above 10 Hz at an electric field strength of 1 kV cm-1 did not improve the amount of extracted material. The impact of oil type and fatty acid chain length on the development of food-grade microemulsions for the entrapment of β-carotene was investigated. The microemulsion region of a ternary phase diagram containing short chain monoglycerides was larger than for di- and triglycerides when Tween 80 was used as surfactant. Due to the higher solubilization of monoglycerides compared to other types of lipids studied, β-carotene-loaded O/W microemulsions were developed using medium chain monoglycerides (Capmul MCM), non-ionic surfactant (Tween 80), phosphate buffer, and synthetic β-carotene as the main constituents. Incorporation of β-carotene into the microemulsion affected the structure in terms of size and polydispersity index (PDI), whereas no significant differences in the zeta potential, pH and refractive index values between the blank and β-carotene-loaded microemulsions were observed. Amongst the formulations, the microemulsion contained 30% Capmul MCM oil, 20% Tween 80 and 50% buffer had the highest β-carotene loading with a particle size less than 100 nm. The feasibility of using an O/W microemulsion as the medium to extract natural β-carotene from PEF-treated carrot pomace was further investigated. The optimum carotenoids extraction conditions by O/W microemulsion were determined by means of three levels Box-Behnken experimental design combined with response surface modelling (RSM) and the extraction efficiency was compared to other extraction medium (e.g. organic solvent and oil). Similar to organic solvents and vegetable oils, the β-carotene extracted from PEF-treated carrot pomace using O/W microemulsion was higher than for untreated carrot pomace. The extraction efficiency of β-carotene using microemulsions was higher compared to 100% hexane or 100% glycerol monocaprylocaprate oil, which shows the advantage of using an O/W microemulsion for extraction of hydrophobic compounds compared to other solvents. The mathematical model predicted that an extraction time of 49.4 min, temperature of 52.2°C and carrot/microemulsion ratio of 1:70 (w/w) would result in microemulsions with β-carotene loading of 19.6 μg g-1, PDI of 0.27 and particle size of 74 nm. The cytotoxicity of microemulsions loaded with natural β-carotene was determined on human intestinal epithelial (Caco-2) cells using three assays including lactate dehydrogenase (LDH), nitric oxide, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in the presence and absence of H2O2. Cytotoxicity of the formulations significantly decreased when lowering the microemulsion concentration. Increasing the cytotoxicity of microemulsions at concentrations higher than 0.0313% (Tween 80 concentration of 0.006%) might be the result of Tween 80 precipitation on the cell culture monolayer and β-carotene loaded in the microemulsion did not appear to protect cells from Tween 80 associated damage. However, a β-carotene loaded microemulsion concentration of 0.0313% gave bioprotection to the cells suffering oxidative damage caused by H2O2. These results of this study showed the potential of using microemulsions as a media for extraction of hydrophobic bioactive compound (e.g. β-carotene), from plant materials (e.g. carrot pomace). The natural β-carotene microemulsions prepared in this study could be used as a delivery system at a safe level of Tween 80 for application in pharmaceutical and food sciences.