Development of food bioactives through the valorization of olive oil production wastes

Abstract

Olive oil production is one of the most important industries in the countries of the Mediterranean region. Olive pomace is the residue generated from these industries that use a two-phase centrifugation system. These residues have harmful effects in the environment due to their high organic content and phytotoxicity. Thus, their management and disposal have raised increasing concern by the environmentalists, researchers and society in general. On the other hand, these residues present high content of bioactive compounds with putative health benefits and their recovery has aroused considerable interest for food industries in developing functional foods, due to the demonstrated antioxidant, antimicrobial, antiviral, anti-inflammatory and anticancer activities of these valuable compounds. Ionizing radiation is a simple, modern and environment friendly technology capable to decompose complex molecules into small ones, as well as to promote an improvement in the beneficial bioactive properties of different matrices such as industrial and municipal wastewaters, agro-industrial residues, fresh fruits and aromatic and medicinal plants. In the present work, the feasibility of using gamma radiation for improving the extractability of valuable compounds present in olive pomace samples was evaluated. Furthermore, the extraction conditions to recover the main compounds from the irradiated olive pomace were also optimized in order to find out the most promising extract to incorporate as a food preservative in fresh-cut apples and to extend their shelf life. Two types of olive pomace, Crude Olive Pomace (COP) and Extracted Olive Pomace (EOP), and different absorbed doses (5, 10, 16 and 22 kGy) were initially assayed to determine the best conditions to improve the extractability of the natural bioactives. Hydroxytyrosol was the most abundant compound (25 ± 1 mg/g extract in EOP and 23.9 ± 0.3 mg/g extract in COP) present in the studied olive pomace extracts, followed by hydroxytyrosol-1-ß-glucoside (9.8 ± 0.4 mg/g extract in EOP and 9.94 ± 0.08 mg/g extract in COP), tyrosol (5.9 ± 0.2 mg/g extract in EOP and 5.9 ± 0.4 mg/g extract in COP), luteolin-7-O-rutinoside (3.1 ± 0.1 mg/g extract in EOP and 4.1 ± 0.2 mg/g extract in COP), oleuropein aglycon (3.0 ± 0.3 mg/g extract in EOP and 3.1 ± 0.1 mg/g extract in COP) and verbascoside (2.29 ± 0.03 mg/g extract in EOP y 3.4 ± 0.2 mg/g extract in COP). Regarding the effect of gamma radiation, an increasing of at least 2-fold in polyphenols extractability from EOP extracts was achieved using an absorbed dose of 5 kGy, while improving the antioxidant activity of the extracts by the inhibition of lipid peroxidation and antihemolytic activity, maintaining the latter in values similar to those obtained for the reference antioxidant, Trolox. Both extracts of COP and EOP samples presented antimicrobial activity against different studied bacterial (MIC = 20-40 mg/mL) and fungi (MIC = 40 mg/mL) strains, but EOP extracts were more effective against Escherichia coli and Pseudomonas fluorescens (MIC = 20 mg/mL). Moreover, no toxicity in non-tumor hepatic cells (PLP2) was observed for any of the extracts and gamma radiation treatment did not cause variation in the antimicrobial activity and hepatotoxicity of the extracts. With the achieved results, EOP sample irradiated at 5 kGy was selected to optimize the extraction conditions of the main bioactive compounds, especially hydroxytyrosol and tyrosol, present in the extracts using heat-assisted extraction (HAE) and ultrasound-assisted extraction (UAE). Response Surface Methodology (RSM) was employed to explore the possible relations between different extraction variables (i.e., time - t, solvent concentration - S, temperature - T, and Power - P). In the optimal conditions obtained, UAE reduced the extraction time and the solvent consumption when compared to HAE (t = 28 min, P = 490 W and S = 7.3% ethanol for UAE; t = 120 min, T = 85 ºC and S = 76% ethanol for HAE) and led to higher extraction yields (UAE yield = 30%; HAE yield = 13.7%). Nevertheless, when comparing the bioactivities of the extracts obtained by the two processes, HAE extracts presented higher antioxidant capacity, requiring less concentration of extract to inhibit TBARS formation and oxidative hemolysis, as well as higher antibacterial and anti-inflammatory activities. Other promising results for the HAE extracts were obtained by the increased antidiabetic activity, evaluated by the inhibition in alpha-amylase and alpha-glucosidase activities, and cytotoxic effect against breast adenocarcinoma (MCF-7) cells compared to UAE extracts. In fact, the alpha-glucosidase inhibition of HAE extracts (IC50 value of 14 ± 1 mg/mL) did not differ significantly from that of the standard positive control acarbose (IC50 value of 11 ± 1 mg/mL). In order to maintain the consumers' interest in healthier foods, food industries are always seeking for innovative approaches to develop new functional products, and a great attention has been given to natural additives. In this sense, extracts of irradiated olive pomace recovered using HAE optimal conditions were tested as potential substitutes to ascorbic acid in delaying the oxidation of fresh-cut apples. Besides the antioxidant capacity, the potential inhibition of microbial growth and changes produced in physicochemical characteristics (texture, weight loss and colour) were evaluated during 12 days of refrigerated storage. Furthermore, two different films, biodegradable polylactic acid (PLA) and commercial oriented polypropylene (OPP) films, were explored for packaging the fruit slices in order to assess the most adequate one to ensure the desired quality of the fruit. Fresh-cut apples with natural olive pomace extracts presented higher growth inhibition of mesophilic bacteria and filamentous fungi for 5 days of storage at 4 ºC, and no detection of coliforms was verified along the 12 days of storage, contrary to what was observed for the samples treated with ascorbic acid. In general, the olive pomace extracts preserved or improved the phenolic content and antioxidant potential of the fruits packaged in PLA bags, when compared to ascorbic acid and to OPP bags, without significant changes in their texture. On the other hand, olive pomace extracts showed lower microbial diversity than ascorbic acid samples, supporting their potential to be used as a natural antimicrobial preservative in ready-to-eat foods. A storage of 5 days in refrigerated conditions could be proposed to produce these snacks using olive pomace extracts with similar or higher quality than the ones using the commercial antioxidant. The overall results obtained in this Thesis can be useful to fulfil the needs of the food industries in producing new ingredients for the development of functional food products (Sustainable Development Goals - SDG 2 and 3). The reuse of agro-industrial wastes to recover new compounds as natural alternatives to the synthetic preservatives contributes for the sustainability of both agro-industrial sector and environment, also promoting the circular economy concepts and the awareness of 'zero waste' strategy (SDG 9).