Optimized Enzymatic Hydrolysis of Olive Pomace Proteins Using Response Surface Methodology

Background and Objective: Olive pomace is a by-product of olive oil extraction used in livestock and poultry feeds. Nevertheless, it can be a valuable source for the production of bioactive peptides, which include positive effects on the natural functions and health of the body. The objective of this study was to optimize enzymatic hydrolysis conditions of olive pomace protein for the production of hydrolysates with the highest degrees of hydrolysis and investigation of bioactive characteristics of the treatments under optimal conditions. Material and Methods: After extraction of proteins from olive pomace, response surface method based on the Box-Behnken design was used to optimize the enzymatic hydrolysis of olive pomace protein by trypsin. Degrees of the hydrolysis were considered as response. Antioxidant activity in the hydrolysates was assessed using 2,-2-diphenyl-1-picryl-hydroxyl radical scavenging and iron (II) chelating activity. Antiproliferative activity was assessed on human breast cancer cells (MCF-7) using 3-(4,-5-dimethylthiazol-2-yl)-2,-5-diphenyl tetrazolium bromide assay. Microscopic structure and amino acid content were studied using scanning electron microscopy and reverse phase-high performance liquid chromatography, respectively. Results and Conclusion: The optimal hydrolysis conditions were as follows: time of 5 h, pH 8.5 and the temperature of 39 °C. Under these conditions, the maximum degree of hydrolysis was achieved as 70.62%. Furthermore, 2,-2-dipheny1-1-picryl-hydroxyl radical scavenging activity and iron (II) chelating activity of the olive pomace protein hydrolysate under optimal conditions respectively were 42.26% ± 2.46 and 30.91% ± 0.26 at a concentration of 3.5 mg ml-1. Although, antioxidant activity of the protein hydrolysate was significantly lower than that of ascorbic acid and ethylene-diaminetetraacetic acid. The highest antiproliferative activity of the olive pomace protein hydrolysate on MCF-7 breast cancer cells was observed at a concentration of 80 mg ml-1 and the proportion of cell viability was achieved at 24 and 48 h at 15.06% ±0.40 and 9.71% ±1.32, respectively. Microscopic analysis of the hydrolysate verifies that the enzyme trypsin was able to hydrolyze large protein molecules, converting them into small fragments. The amino acid analysis showed increases in hydrophobic and aromatic amino acids in the hydrolyzed protein. As a conclusion, protein hydrolysates from olive pomace with antioxidant and antiproliferative potentials can be used as functional additive foods in livestock.