**2.2 Deacidification**

294 Olive Oil – Constituents, Quality, Health Properties and Bioconversions

pressure and temperature. Selectivity can also be changed by the addition of a co-solvent such as ethanol, methanol, hexane, acetone, chloroform and water to increase or decrease the polarity. Ethanol is the most preferred co-solvent because it is non-toxic and meets green

SC-CO2 processing adds value because products obtained may be considered as natural. Although SFT is used for extraction of plants and vegetables of different sources (Table 1), applications in the olive oil industry have been limited. SFT can be used in olive oil processing for extraction and deacidification, as well as separation, purification or

SC-CO2 has been used to replace hexane in the olive oil industry and meets the growing demand for natural products (Temelli, 2009). The most common applications are extraction of total lipids from olive husk or minor lipid components from olive oil. Extraction of high value minor components without degradation led industry and researchers to focus on SC-CO2 extraction. Fig. 1 represents a typical lab scale SC-CO2 system used for extraction of lipids.

Fig. 1. Schematic diagram of a lab scale SC-CO2 extraction system: 1, CO2 tank; 2,4,10, shutoff valves; 3, pressure gauge; 5, filter; 6, compressor; 7, back pressure regulator; 8, extraction vessel; 9, thermocouple; 11, micrometering valve; 12; sample collector; 13, oil and moisture

technology criteria (GRAS status) (Dunford, 2004).

Sample Analyte Reference Carrot Carotenes Vega et al. (1996) Tomato skin Lycopene Ollanketo et al. (2001) Mushrooms Oleoresins del Valle & Aguilera (1989) Tea Caffeine Calabuig Aracil (1998) Grape skin Anthocyanins Blasco et al. (1999) Cottonseed Lipids Bhattacharjee et al. (2007) Hops Humulone, lupulone and essential oils Langezaal et al. (1990) Rosemary Oil Bensebia et al. (2009)

Table 1. Supercritical fluid extraction of different plants and vegetables.

concentration of minor components.

trap; 14, flowmeter; 15, gas meter.

**2.1 Extraction** 

Crude olive oil contains free fatty acids (FFA) and other impurities which must be removed, yielding a triacylglycerol (TAG) rich fraction. A high FFA content decreases the oxidative stability of the oil and leads to rancidity. A reduction in FFA content in virgin olive oil results in an increase in commercial value (Vázquez et al., 2009).

Supercritical fluid extraction has been proposed as an alternative technology for deacidification of oils and has been used for deacidification of olive pomace oil, an important by-product of olive oil industry. Crude olive pomace oil is often very acidic, darkly colored and highly oxidized. Intensive refining is thus required to make it suitable for human consumption. Neutralization is currently applied, but there are drawbacks to this process. Product yield is very low and neutralization increases the cost per unit. Therefore, it is necessary to reduce the FFA content before refining (Fadiloglu et al., 2003). Supercritical deacidification is actually a selective supercritical fluid extraction process. During the process, FFAs preferentially extracted with minimum neutral oil (TAGs, tocopherols, phytosterols) loss (Vázquez et al., 2009). A schematic diagram of a supercritical fluid extraction system for deacidification of oils is shown in Fig. 2. The oil is fed to the extraction column by a pump. The extraction column consists of two sections: an enriching (above of the oil feeding point) section, and a stripping section (below the oil feeding point). Raffinate is first separated from the extract and sent to the stripping section. Then, in the stripping section, the extract is separated from raffinate and transported to the enriching section. Extract rich in minor lipid compounds and CO2 is separated in the separator. A specified amount of the extract is transferred to the top of the column as reflux (Brunner, 2009). CO2 can be purified and recycled into the system. Raffinate is collected at the bottom of the column.

Deacidification of different oil sources using supercritical fluids have been performed at laboratory scale by several researchers. Turkay et al. (1996) achieved a selective and quantitative (90%) FFA extraction for deacidification of high acidic black cumin seed oil using SC-CO2 at relatively low pressure (15 MPa) and relatively high (60 °C) temperature. Ooi et al. (1996) decreased the FFA content of palm oil to 0.1% in a continuous SC-CO2 extractor.

Brunetti et al. (1989) obtained deacidification of high acidic olive oil with SC-CO2 at pressures of 20 and 30 MPa, and temperatures of 40 and 60 °C. They reported that the selectivity for FFAs was highest at 20 MPa and 60 °C. Bondioli et al. (1992) studied the supercritical fluid deacidication of olive oil in the pressure range of 9–15 MPa and 40–50 °C. The acidity was reduced from 6.3% to values less than 1% at 40 °C and 13 MPa. In another application, Vázquez et al. (2009) used SC-CO2 as an extraction solvent to remove FFAs from cold-pressed olive oil in a packed column. The acidity was reduced from 4 to 1.43% at 25 MPa and 40 °C.

Potential Applications of Green Technologies in Olive Oil Industry 297

transport of substrates to the catalyst and, in the case of enzyme within the pores of enzyme support, this results in an easier access to the enzyme sites leading to higher reaction rates. In addition to the previously mentioned advantages of supercritical fluids, the finding that enzymes can retain their biocatalytic activity at high pressures has also encouraged the use

In general, expansion of the substrates in CO2 seems to be the main advantage of enzymatic lipid reactions in SC-CO2. Expanded substrates have better diffusivity, low surface tension and low viscosity. In addition, a lesser amount of substrate available per unit amount of enzyme per unit time will increase the reaction rate (Ciftci & Temelli, 2011). However, at very high pressures, mass transfer properties of the substrates may be affected negatively. High CO2 densities at high pressures lead to a decrease in enzymatic conversions. It has been reported that diffusion coefficients of fatty acids, fatty acid esters and glycerides in SC-CO2 may also decrease at high pressures due to increase in the density of CO2 (Rezaei & Temelli, 2000). Therefore, optimization of the process in terms of pressure and temperatures is crucial. Esmelindro et al. (2008) produced MAGs from olive oil in compressed propane. Their results showed that lipase-catalyzed glycerolysis in compressed propane might be a potential replacement for conventional methods, as high contents of reaction products, MAG and diacylglycerol (DAG), were achieved at mild temperature and pressure conditions (30 °C and 3 MPa) with a low solvent to substrates mass ratio (4:1) in short-reaction times (1 h). Lee et al. (2009) produced biodiesel from various oils, namely, olive, soybean, rapeseed, sunflower and palm oil, using lipase in SC-CO2. The highest yield (65.18%) was obtained from olive oil at 13

Membrane technology is becoming increasingly important as a green processing and separation method in food processing and waste water treatment Membranes are used as filters in separation processes and have a wide variety of applications Membrane technology is now competitive compared to conventional techniques such as adsorption, ion exchangers

The main advantage of membrane processing is that it avoids the use of any chemicals that have to be discharged. It works with relatively high efficiency and low energy consumption (Mulder, 1996). It also has the advantage of operating at ambient temperature, resulting in preservation of heat-sensitive components and nutritional value of food products

Membrane separation processes differ greatly in the type of membranes and driving forces used for separation, the process design, and the area of application. There are many different membrane processes, including reverse osmosis, micro-, ultra- and nanofiltration, dialysis, electrodialysis, Donnan dialysis, pervaporation, gas seperation, membrane contactors, membrane distillation, membrane based solvent extraction, membrane reactors, etc. Among them, the innovative methods preferred by the food industry are pressure driven separation processes such as reverse osmosis, nanofiltration, ultrafiltration and microfiltration. These preferred methods facilitate the separation of components with a large range of particle sizes. The obtained products are generally of high quality and less post-

of enzymes under supercritical condition (Rezaei et al., 2007b; Rezaei et al., 2007a).

MPa, 45 °C and 20% of lipase concentration (based on weight of oil).

**3. Membrane technology** 

and sand filters.

(Dewettinck & Le, 2011).

treatment procedures are required (Baker, 2004).

Fig. 2. Schematic diagram of supercritical fluid extraction pilot plant used for deacidification, separation, concentration and purification of oils.

#### **2.3 Separation, concentration and purification of minor lipid compounds**

Extraction of high value minor components from natural products is of great interest to food industry. SFT has been applied for purification, separation or concentration of several compounds from vegetable oils, essential oils and deodorizer distillates. These applications include purification of monoacylglycerols (MAGs) and lecithin, removal of cholesterol and limonene, and separation of squalene, tocopherols and fatty acid esters (Brunner, 2009).

Products of the olive oil industry are important sources of high value components such as tocopherols, phytosterols, squalene and fatty acids. The system used for separation of minor lipid compounds is the same as shown in Fig. 1. Fornari et al. (2008) purified squalene from a by-product obtained after distillation, esterication and transesterication of olive oil deodorizer distillates. They obtained 89.4% squalene purity and 64.2% yield at 70 °C and 18 MPa, and obtained a raffinate concentrated in TAGs and sterol compounds.

Dauksas et al. (2002) extracted tocopherols from olive tree leaves using SC-CO2. They obtained a high value extract of 97.1% (w/w) tocopherol at 25 MPa and 40 °C after 1 h of extraction, and 74.48 % at the same pressure and temperature after 2 h. Le Floch et al. (1998) used supercritical fluid extraction for isolation of phenols from olive leave samples using SC-CO2 modified with 10% methanol at 33.4 MPa and 100 °C.

#### **2.4 Use of supercritical fluids as reaction media for enzymatic modification of lipids**

Enzymatic interesterification in organic solvents leads to very important modifications of lipids. However, the use of organic solvents in these reactions is a disadvantage. Therefore, biosynthesis in supercritical uids is attracting much attention. Replacement of organic solvents by supercritical fluids makes the process green and eliminates the need of solvent separation. The lower viscosity and the higher diffusivity of supercritical fluids allow easier

Fig. 2. Schematic diagram of supercritical fluid extraction pilot plant used for

**2.3 Separation, concentration and purification of minor lipid compounds** 

MPa, and obtained a raffinate concentrated in TAGs and sterol compounds.

SC-CO2 modified with 10% methanol at 33.4 MPa and 100 °C.

Extraction of high value minor components from natural products is of great interest to food industry. SFT has been applied for purification, separation or concentration of several compounds from vegetable oils, essential oils and deodorizer distillates. These applications include purification of monoacylglycerols (MAGs) and lecithin, removal of cholesterol and limonene, and separation of squalene, tocopherols and fatty acid esters (Brunner, 2009).

Products of the olive oil industry are important sources of high value components such as tocopherols, phytosterols, squalene and fatty acids. The system used for separation of minor lipid compounds is the same as shown in Fig. 1. Fornari et al. (2008) purified squalene from a by-product obtained after distillation, esterication and transesterication of olive oil deodorizer distillates. They obtained 89.4% squalene purity and 64.2% yield at 70 °C and 18

Dauksas et al. (2002) extracted tocopherols from olive tree leaves using SC-CO2. They obtained a high value extract of 97.1% (w/w) tocopherol at 25 MPa and 40 °C after 1 h of extraction, and 74.48 % at the same pressure and temperature after 2 h. Le Floch et al. (1998) used supercritical fluid extraction for isolation of phenols from olive leave samples using

**2.4 Use of supercritical fluids as reaction media for enzymatic modification of lipids**  Enzymatic interesterification in organic solvents leads to very important modifications of lipids. However, the use of organic solvents in these reactions is a disadvantage. Therefore, biosynthesis in supercritical uids is attracting much attention. Replacement of organic solvents by supercritical fluids makes the process green and eliminates the need of solvent separation. The lower viscosity and the higher diffusivity of supercritical fluids allow easier

deacidification, separation, concentration and purification of oils.

transport of substrates to the catalyst and, in the case of enzyme within the pores of enzyme support, this results in an easier access to the enzyme sites leading to higher reaction rates. In addition to the previously mentioned advantages of supercritical fluids, the finding that enzymes can retain their biocatalytic activity at high pressures has also encouraged the use of enzymes under supercritical condition (Rezaei et al., 2007b; Rezaei et al., 2007a).

In general, expansion of the substrates in CO2 seems to be the main advantage of enzymatic lipid reactions in SC-CO2. Expanded substrates have better diffusivity, low surface tension and low viscosity. In addition, a lesser amount of substrate available per unit amount of enzyme per unit time will increase the reaction rate (Ciftci & Temelli, 2011). However, at very high pressures, mass transfer properties of the substrates may be affected negatively. High CO2 densities at high pressures lead to a decrease in enzymatic conversions. It has been reported that diffusion coefficients of fatty acids, fatty acid esters and glycerides in SC-CO2 may also decrease at high pressures due to increase in the density of CO2 (Rezaei & Temelli, 2000). Therefore, optimization of the process in terms of pressure and temperatures is crucial. Esmelindro et al. (2008) produced MAGs from olive oil in compressed propane. Their results showed that lipase-catalyzed glycerolysis in compressed propane might be a potential replacement for conventional methods, as high contents of reaction products, MAG and diacylglycerol (DAG), were achieved at mild temperature and pressure conditions (30 °C and 3 MPa) with a low solvent to substrates mass ratio (4:1) in short-reaction times (1 h). Lee et al. (2009) produced biodiesel from various oils, namely, olive, soybean, rapeseed, sunflower and palm oil, using lipase in SC-CO2. The highest yield (65.18%) was obtained from olive oil at 13 MPa, 45 °C and 20% of lipase concentration (based on weight of oil).
