**4.1 Enzymatic conversions**

Interesterification reactions are widely studied to produce margarines and shortenings with zero-*trans* fatty acids, cocoa butter equivalents, structured lipids with specific nutritional properties, partial glycerides and biodiesel. Chemical interesterification uses metal

Potential Applications of Green Technologies in Olive Oil Industry 301

1,3-selective *Rhizopus oryzae* heterologous lipase (rROL) immobilized in Eupergit C and modified sepiolite. These structured lipids are low caloric and and have dietetic properties for controlling obesity and malabsorption. They showed that rROL immobilized in Eupergit C was able to catalyze the incorporation of 21.6 and 34.8 mol% of caprylic or capric acid into virgin olive oil, after 24 h at 40 °C in solvent-free media. Fumoso and Akoh (2002) also used lipase-catalyzed acidolysis of olive oil and caprylic acid to produce structured lipids. They used a *sn*-1,3-specific lipase from *Rhizomucor miehei* in a bench-scale packed bed bioreactor. They studied the effect of solvent, temperature, substrate mol ratio, and flow rate/residence time. The optimal solvent-free production of structured lipid was obtained at a substrate flow rate of 1 ml/min, a residence time 2.7 h, 60 °C, and a mol ratio 1:5 (olive oil/caprylic acid). The structured lipid produced at optimal conditions had 7.2% caprylic acid, 69.6% oleic acid, 21.7% linoleic acid and 1.5% palmitic acid at the *sn*-2 position. Another structured lipid used as a constituent of infant formulas, consisting mainly of UPU triglycerides (U=unsaturated acyl chains, P=palmitic acyl group), can be prepared by lipase catalyzed reactions of fractionated

Biodiesel can be obtained from vegetable oils, animal fats, recycled grease, or algae and can be produced by the reaction of TAGs with methanol (methanolysis). Lipase-catalyzed methanolysis is more attractive than conventional base-catalyzed method since the glycerol produced as a by-product can easily be recovered and the purification process for fatty acid methyl esters (FAMEs) is relatively simple. In the oil and fat industry, conversion of waste edible oil and soapstock (a by-product generated in alkali refining of vegetable oils) to biodiesel has attracted a great deal of attention (Azócar et al., 2010; Safieddin Ardebili et al., 2011; Singaram, 2009). Unlike the conventional chemical routes for synthesis of diesel fuels, biocatalytic routes permit one to carry out the interesterification of a wide variety of oil

Olive pomace oil was used by Yucel (2011) for enzymatic production of biodiesel. Yucel (2011) immobilized microbial lipase from *Thermomyces lanuginosus* on olive pomace by covalent binding, and then used this immobilized lipase for the methanolysis of olive pomace oil. Under the optimized conditions for solvent-free reaction, the maximum yield was reported to be 93% at 25 °C after 24 h. Sanchez and Vasudevan (2006) produced biodiesel by transesterification of olive oil triolein with methanol using lipase. They studied the effects of the molar ratio of methanol to triolein, semibatch (stepwise addition of methanol) vs batch operation, enzyme activity, and reaction temperature on overall conversion. Because of the inactivation of the enzyme by insoluble methanol, stepwise methanolysis with a 3:1 methanol

One method to reduce the FFA content in fats and oils is to convert the FFAs to TAGs. This

A reported application of enzymatic deacidification of olive pomace oil is the enzymatic glycerolysis of highly acidic (32%) olive pomace oil (Fadiloglu et al., 2003). FFAs of olive pomace oil were esterified with glycerol using a nonspecific immobilized lipase, reducing

palm oil, rich in tripalmitin, and oleic acid from olive oil (Schmid et al., 1998).

to triolein molar ratio and an overall ratio of 8:1 gave the best results.

is carried out by direct esterification of fatty acids with glycerol.

**4.1.2 Biodiesel** 

feedstocks in the presence of excess FFAs.

**4.2 Enzymatic deacidification** 

alcoholate catalysts to incorporate fatty acids randomly. This reaction produces a complete positional randomization of acyl groups in TAGs. In enzymatic interesterification the final structure of TAGs is controlled and a desired acyl group can be guided into TAGs using nonspecific, regiospecific (*sn*-1,3- or 2- specific) and fatty acid specific lipases as catalysts. This results in products with predictable composition. Enzymatic interesterification is becoming a more attractive method to convert cheap oils such as olive pomace oil, soya bean oil, rape seed oil, lard, tallow, etc. to high-value-added products and modified fats (An et al., 2007; Liua et al., 1997; Macrae, 1983; Miller et al., 1991; Pomier et al., 2007; Xu, 2003). Furthermore, enzymatic interesterification has milder reaction conditions and produces less waste than the chemical alternative. In addition, the same immobilized enzyme can be used many times (Akoh et al., 1998; Marangoni & Rousseau, 1995; Willis et al., 1998; Willis & Marangoni, 2002). Therefore, intensive research has aimed at replacing chemical interesterification with enzymatic interesterification.

There are three types of interesterification reactions: acidolysis, which is the reaction between an ester and a fatty acid, alcoholysis, the reaction between an ester and an alcohol, and transesterification, the reaction of an ester with another ester, also called ester-ester exchange (Macrae, 1983; Xu, 2003). Production of structured lipids and biodiesel has been the major topics of enzymatic interesterification studies.

#### **4.1.1 Structured lipids**

Structured lipids are novel modified TAGs produced by the incorporation of desirable fatty acids at specific positions or by changing the position of the fatty acids on the glycerol backbone. These processes allow for specific characteristics to be obtained such as melting behavior, functionality, and metabolism. Lipases, especially those which are *sn-*1,3 specific, are used for this purpose because these enzymes can make changes at *sn*-1 and *sn*-3 positions by keeping *sn*-2 ester group position unchanged.

Cocoa butter (CB) has a narrow melting range due to its unique TAG composition. This melting behavior is critical. The steepness of the melting profile (% solid fat as a function of time) has an impact on flavor release and crystallization. The high price of cocoa butter has prompted the industry search for CB alternatives. CB equivalents (CBEs) can be produced from palm oil and exotic fats by means of fractionation. Enzymatic synthesis of CBEs from cheap oils and fats using *sn*-1,3 specific lipases is also an alternative method. CB-like fats could be produced which have even more desirable properties than natural CB. Ciftci et al. (2010) used olive pomace oil for the production of CB-like fat using *sn*-1,3 specific lipase. They interesterified refined olive pomace oil, palmitic acid and stearic acid at a molar ratio of 1:2:6, respectively, at 45°C using a pack bed reactor filled with *sn*-1,3 specific lipase. They reported that the CB-like fat could replace CB up to 30% without significantly changing the physical and chemical properties of the product. Chang et al. (1990) also produced CB-like fat by enzymatic interesterification of fully hydrogenated cotton seed and olive oils. The melting point of their CB-like fat was 39°C; close to 36°C, the melting point of CB.

Any lipid containing medium-chain and long-chain unsaturated fatty acids might be useful for certain applications and functionalities. Nunes et al. (2011) produced structured lipids containing medium-chain fatty acids at *sn*-1,3 position and long-chain unsaturated fatty acids at the *sn*-2 position by acidolysis of virgin olive oil and caprylic or capric acids using 1,3-selective *Rhizopus oryzae* heterologous lipase (rROL) immobilized in Eupergit C and modified sepiolite. These structured lipids are low caloric and and have dietetic properties for controlling obesity and malabsorption. They showed that rROL immobilized in Eupergit C was able to catalyze the incorporation of 21.6 and 34.8 mol% of caprylic or capric acid into virgin olive oil, after 24 h at 40 °C in solvent-free media. Fumoso and Akoh (2002) also used lipase-catalyzed acidolysis of olive oil and caprylic acid to produce structured lipids. They used a *sn*-1,3-specific lipase from *Rhizomucor miehei* in a bench-scale packed bed bioreactor. They studied the effect of solvent, temperature, substrate mol ratio, and flow rate/residence time. The optimal solvent-free production of structured lipid was obtained at a substrate flow rate of 1 ml/min, a residence time 2.7 h, 60 °C, and a mol ratio 1:5 (olive oil/caprylic acid). The structured lipid produced at optimal conditions had 7.2% caprylic acid, 69.6% oleic acid, 21.7% linoleic acid and 1.5% palmitic acid at the *sn*-2 position. Another structured lipid used as a constituent of infant formulas, consisting mainly of UPU triglycerides (U=unsaturated acyl chains, P=palmitic acyl group), can be prepared by lipase catalyzed reactions of fractionated palm oil, rich in tripalmitin, and oleic acid from olive oil (Schmid et al., 1998).
