Melanin and humic acid-like polymer complex from olive mill waste waters. Part I. Isolation and characterization
Introduction
Development of natural food additives has been of growing interest in recent years, mainly due to the strong consumer demand for food containing natural rather than synthetic additives and ingredients. Food colorants, emulsifiers, flavor enhancers, antioxidants and antimicrobials from natural sources have become increasingly popular, since synthetic ones are perceived as harmful and unhealthy (Brewer, 2011, Chen et al., 2008, Negi, 2012, Wang et al., 2006). Apart from consumers’ preference for clean labeling, the claim for sustainable sources and environmentally friendly production is forcing the food industry to move in the direction of a greener chemistry.
Oil mill waste water (OMWW) is a by-product of olive oil production and for a long time it has been the most polluting waste material in the Mediterranean countries. Producing huge amounts of OMWW represents a great environmental problem, especially on soil and water quality. The organic fraction of this effluent involves sugars, phenolics, proteins, lipids and other components (Mulinacci et al., 2001).
OMWW represents a natural and inexpensive source of bioactive compounds and many studies have focused on phenolic fractions of OMWW (Lafka, Lazou, Sinanoglou, & Lazos, 2011). Re-examination of organic compounds of OMWW with new fractionation processes will continue to provide new biomaterial. In previous works, the isolation and characterization of a humic acid-like biomaterial named polymerin, composed of carbohydrates, melanin and proteins, by applying a precipitation scheme using methanol, a denaturing organic solvent was reported (Capasso et al., 2002, Iorio et al., 2010).
Until now, no reports have focused on the isolation of a protein fraction of OMWW using non-denaturing agents. Thus, the choice of a suitable method of protein fractionation is crucial to guide isolation of new biopolymers from OMWW containing biologically active proteins, which can be of interest in the food industry for their potential use as natural additives. In this respect, we have already applied ammonium sulfate precipitation for the fractionation of proteins from olive oil and olive fruits water extracts as an initial step for the isolation and characterization of oxidizing enzyme activities (Georgalaki et al., 1998, Tzika et al., 2009). The main aim of the present investigation was the isolation of new biomaterial from OMWW using a classical method of protein fractionation. Biochemical, physicochemical and antioxidant properties of the isolated and purified protein complex were characterized using a variety of techniques and compared with those of humic acid and melanin polymers.
Section snippets
OMWW samples and chemicals
Fresh oil mill waste waters were procured in the harvest season (December 2013) from olives (Olea europea, Chetoui cultivars) using a three phase olive mill located in the North of Tunisia. Samples were stored at −18 °C until use. Sephacryl S-300 and Blue Dextran 2000 were purchased from Pharmacia Biotech (Uppsala, Sweden), caffeic acid, TEMPOL free radical, bovine serum albumin and all materials used for sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE) were purchased from
Protein, polyphenol and carbohydrate content
OMWW proteins were precipitated by 80% ammonium sulfate. After dialysis, a clear and dark solution was obtained with a dry biomass content of 25.90 ± 0.20 g/l. Total dry matter recovered from 1 l of OMWW filtrate was 1.15 ± 0.01 g. It has a protein content of 8.05 ± 0.18 g/l (31.08%), which is much higher than that of polyphenols (0.42 ± 0.02 g/l (1.62%)) and sugars (1.04 ± 0.27 g/l (4.02%)) (Table 1). The low total content of these organic components (36.72%) related to dry biomass (100%) can be mainly due to
Conclusion
Ammonium sulfate fractionation of OMWW lead to a dark product mainly composed of proteins, polyphenols and carbohydrates linked in supramolecular assemblies by either covalent or non-covalent bonds. OMWW–ASP complex was proved to be formed by polydispersed aggregates similar to melanin and humic substances. Moreover, OMWW–ASP complex is an efficient free radical scavenger. The biochemical and physicochemical characterization of OMWW–ASP suggest that this water soluble humic acid- and
Acknowledgements
This work was supported by grants from the Ministry of Higher Education and Scientific Research, Tunisia. Dr. S. Avramiotis from the Institute of Biology, Medicinal Chemistry & Biotechnology, NHRF, is acknowledged for his support in EPR simulations. The authors would like to express their gratitude to Dr. A. Xenakis from the Institute of Biology, Medicinal Chemistry & Biotechnology, NHRF, for valuable assistance and helpful discussion.
The authors have declared no conflict of interest.
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