Application of response surface methodology for the optimization of supercritical CO2 extraction of oil from olive paste: yield, content of bioactive molecules and biological effects in vivo

The modern two-phase technology of the Leopard series for olive oil industrial extraction developed by the Pieralisi Group (Pieralisi Maip S.p.A., Jesi, AN, Italy) generates large quantities of a novel by-product (olive paste - OP) constituted of the partially defatted wet drupe pulp without any traces of the kernel. OP is rich in lipophilic and hydrophilic molecules able to exert beneficial effects on human health, including triterpenic acids, phytosterols, tocopherols, carotenoids, polyphenols, minerals and fibers [1]. In the perspective of by-product valorization through a modern biorefinery approach, it is worth noting that dried OP contains more than 10% oil characterized by a well-balanced lipid profile, rich in mono and polyunsaturated fatty acids, and a very good oxidative stability, due to the high concentration of fat-soluble antioxidants. This makes OP oil particularly suitable as functional ingredient for food/feed industry, as well as for the formulation of nutraceutical, cosmeceutical and pharmaceutical products. Supercritical carbon dioxide (SC-CO2) is a green and environmentally friendly technology for the effective extraction of high-value natural molecules. It is gaining a foothold in industrial production of solvent-free vegetable oils and has been also applied to concentrate oil products of lipophilic micronutrients (e.g. vitamin E in soybean and olive oils) [2]. Nevertheless, the industrial application of SC-CO2 technology requires a careful optimization of the operative parameters to make the process efficient from both an economic and a productive point of view. In this work, a response surface methodology approach, based on the Box-Behnken Design, was used to determine the optimal parameters of pressure, temperature and time to simultaneously maximize oil production from the freeze-dried OP of cultivar Leccino and concentrate the most abundant lipophilic bioactives. The experimental data were fitted to a second-order polynomial equation by multiple regression analysis, and examined using appropriate statistical methods. The 3-D response surface plots derived from the mathematical models were applied to determine the optimal extraction parameters, which resulted: temperature 70 °C, pressure 35.5 MPa and time 62 min. Under these conditions, the oil experimental yield was 14.0±0.7%, in close agreement with the predicted value. Compared to the freeze-dried OP, SC-CO2 extracted oil was much concentrated in phytosterols (~13 fold), tocopherols (~6 fold) and squalene (~8 fold); total carotenoid concentration remained almost unchanged, while triterpenic acids, being polar, were substantially reduced (- 79.5%). However, their total content was much higher than in extra virgin olive oil. The biological effects produced by OP oil intake were examined on 3 groups of healthy BALB/c mice (n=5/group) orally administered (for 4 weeks) with 20 or 40 mg/die of OP oil. Untreated animals were used as control. OP oil supplementation had no significant effects neither on body weight, nor on the activity of most liver cytoprotective enzymes (glutathione-disulfide reductase, glutathione S-transferase and glucose-6-phosphate dehydrogenase) with the exception of NAD(P)H:Quinone Oxidoreductase 1, whose activity was significantly increased (P<0.05; 30%) as compared to controls, by the 40 mg treatment. A significant decrease in the amount of liver triglycerides was also detected in the mice fed with 20 mg oil. The investigation of the molecular mechanisms of oil action revealed a significant increase in the activity of carnitine palmitoyl-transferase I, suggesting a concomitant stimulation of hepatic fatty acid oxidation. A parallel increase in the activities of mitochondrial respiratory complexes was also observed in freshly isolated mice liver mitochondria. Interesting, the stimulation of respiratory chain activity retained an efficient mitochondrial oxidative phosphorylation, as suggested by the respiratory control ratio values. Keywords: antioxidant defenses; by-products; mathematical modelling; olive oil; respiratory chain; supercritical fluids References [1] Padalino et al., 2018. Antioxidants, 7(4), 59. [2] Fine et al., 2016. Eur. J. Lipid Sci. Technol., 118, 680-697.