Biochemical studies on some bioactive compounds produced from fermented olive cake as antioxidant and anticancer

Abstract

Natural bioactive compounds have received much attention as possible alternatives for synthetic ones. In this context, olive oil cake (OC) which is considered as one of the worst agro-industrial wastes in Mediterranean countries, was tested for production of bioactive compoundsunder solid state fermentation technique. Tannase enzyme was successively produced by the yeast Kluyveromyces marxianus NRRL Y-8281 using OC as a solid support. Optimization of the production process resulted in 1.4fold enhanced tannase yield (1714.7 U/gds) as compared to un-optimized conditions (1226.6 U/gds) by growing the yeast at 45C with initial pH 6.0, 35% moisture level and 20% inoculum size for 48 hours without additive nitrogen or carbon sources. The enzyme was purified using ammonium sulfate fractional precipitation followed by Sephadex G-200 gel filtration chromatographic column resulting in 64.6% enzyme yield with 1026.12 U/mg specific activity and 24.21 purification fold. The pure tannase had molecular weight of 65 KDa and 66.62KDa by SDS-PAGE and gel filtration column chromatography, respectively. Pure tannase showed a maximal activity at 35C having two different pH optima, one of which is acidic (4.5) and the other one is alkaline (8.5). The enzyme was stable in the acidic range of pH (4.0–5.5) for 30 minutes, and thermostable within the temperature range 30-70C. Using tannic acid as a substrate, the enzyme had a Km value of 0.77 mM and Vmax of 263.16 µmole min-1 ml-1. The effect of different metal ions on enzymatic activity was evaluated. HPLC analysis data indicated that the purified enzyme could carry out 24.65% tannic acid conversion with 5.25 folds increase in gallic acid concentration within 30 minutes only.Having potent biological activities, olive cake phenolic compounds from both unfermented (UFOC) and fermented (FOC) formswere extracted. The highest total phenolic recovery (160.08 and 149.00 mgGAE/gds) and antioxidant activity (96% and 97%) for UFOC and FOC, respectively, were obtained using methanol at a sample to solvent ratio of 1:10 for 2 hours at 50°C using a sample concentration of 4 mg/ml. Furthermore, HPLC analysis data revealed that K. marxianus mediated fermentation of OC resulted in degradation of96.75% of OC tannin content accompanied with 2.8 times increase in gallic acid.In vitro anticancer activity of both extractswas assessed against different human cancer cell lines. The results revealed that although both extracts did not show any effect against lung A549, cervix Hela cancer cell lines or normal HFB4 cells, they exerted anticancer effect close to the value of the doxorubicin drug against liver HepG2 and breast MCF-7. Moreover, both extracts showed moderate activity against prostate PC3 and colon HCT116 cell lines. However, the fermented extract was more potent than the unfermented one. GC/MS analysis was carried out to determine the compounds responsible for antioxidant and anticancer activities.SSF of OC by K. marxianus is a new eco-friendly valorization technique that enables the production of valuable bioactive compounds and their associated enzyme (tannase).

Key words: Bioactive compounds - Fermentation - Olive cake – Tannase – Antioxidant - Anticancer.