Refining should be carried out so as to minimize costs, including reduced equipment and minimal energy, as well as minimal losses of neutral oil (Rodrigues, Pessôa Filho, & Meirelles, 2004). The common chemical RBO refining process includes degumming, neutralisation, bleaching, dewaxing and deodorisation (Pestana et al., 2008)

(Fig. 1). Degumming removes phospholipids and lipoproteins, through hydration, by adding water and either citric or phosphoric acid, followed by centrifugation (Baruffaldi and de Oliveira, 1998 and Zambiazi, 1997). During neutralisation, free fatty acids are removed by precipitation with a sodium hydroxide solution (Araújo, 1999), and the sodium salts of the free fatty acids (soaps) are separated by centrifugation (Baruffaldi & de Oliveira, 1998). The pigments naturally present in the crude oil (including Ipilimumab chemical structure 17-AAG chlorophylls and carotenoids) are removed by adsorption on bleaching earth (Ferrari, 2001 and Weiss, 1983). During dewaxing, the oil is maintained at low temperatures to provoke wax crystallisation; then solidified waxes are removed by filtration or centrifugation (Zambiazi, 1997). Finally, during deodorising, volatile substances that are responsible for undesirable odours are removed; for this purpose, the oil is heated to 200–250 °C at low pressures (3–5 mm Hg) (Kao & Luh, 1991; Baruffaldi

& de Oliveira, 1998). On the other hand, precipitated soap is further processed for fatty acid recovering. As illustrated in Fig. 2, acid hydrolysis is initially carried out; the resulting raw fatty acids are separated from a hydrosoluble fraction, mainly containing HCl and NaCl. Finally, the raw fatty acids are distilled at low pressure to recover a 99.9% Amylase pure fraction. Therefore, during fatty acid recovering, raw fatty acids (or hydrolysed soap as an intermediate product), purified fatty acids (final product), and two residues (hydrosoluble fraction from hydrolysis and distillation residue) are produced. In a previous work we have investigated the variations of the

concentrations of several phytochemicals, including γ-oryzanol and tocopherols, during the steps of industrial RBO refining (Pestana et al., 2008). These two compound classes are important antioxidants, being also of interest from a nutritional viewpoint (Ferrari, 2001 and Pestana et al., 2008). During RBO refining, the concentration of γ-oryzanol is largely reduced. Therefore, the concentration of γ-oryzanol in refined RBO is merely 2% of its initial value in crude RBO (Pestana et al., 2008). On the other hand, the concentration of tocopherols in refined RBO is similar to or slightly lower than that in crude RBO; thus, taking into account that refined RBO represents less initial mass of crude RBO, it can be deduced that an important fraction of the tocopherols present in crude RBO is lost during refining.