**4. Enzymatic modification**

Enzymatic reactions are based on the selective biotransformation of determined compounds in order to modify their chemical or physical properties. Hence, the utilization of enzymes, for instance, makes easier the separation of tocopherols from SODD by converting sterols to steryl esters, acylglycerols to free fatty acids and free fatty acids to fatty acid methyl or ethyl esters (FAMEs or FAEEs). Then, it is easier to separate the new product mixture by distillation or supercritical fluid extraction. From published literature, it can be point out that the main difficulties of the enzymatic processes are the numerous parameters involved such as moisture content, enzyme concentration, time, temperature, ratio of the reactants, stability, recovery and reutilization of the enzyme preparation, among others (Ramamurthi *et al.*, 1991), (Ramamurthi & McCurdy, 1993).

The conversion of FFAs to FAMEs or FAEEs is an important step in the concentration and purification of tocopherols. If this step is omitted, the separation of FFA and tocopherols by distillation cannot be achieved due to their similar boiling points (Shimada, 2000). Furthermore, if methanol is used for the biotransformation of FFA to FAMEs, concomitant sterol esterification with fatty acids is inhibited. To avoid this problem, a lipase can be used in a two stages procedure: first to carry out hydrolysis of acylglycerols and then to promote the esterification of sterols with free fatty acids. The different components are then successfully separated by short path distillation or supercritical fluid extraction since their boiling points are now sufficiently different.

In the literature, many enzymatic procedures for the preparation of sterol esters are described, but most of them require organic solvents, water and molecular sieves or other drying agents (Haraldsson, 1992), (Shimada *et al.*, 1999), (Jonzo *et al.*, 1997), (Hedström *et al.*, 1992). Although these strategies gave good conversion rates for the formation of sterol esters, the use of such multiphasic systems may complicate the final purification of the products in the case of larger scale productions. However, the enzymatic preparation of fatty acid esters of sterols, stanols and steroids in high yield by esterification and transesterification of fatty acids and other carboxylic acid esters, in vacuum at moderate

Extraction and Enzymatic Modification of

solvent fractionation.

**5. Molecular distillation** 

and environmental problems.

molecules such as tocopherols.

others.

when subjected to high temperatures.

Functional Lipids from Soybean Oil Deodorizer Distillate 457

esters (FAEEs) from SODD. Firstly, SODD was mixed with oleic acid to reduce its melting point and to enhance the free phytosterols esterification. The first enzymatic step (using *Candida rugosa* lipase) allowed the efficient conversion of more than 90% free phytosterols within 5 h. The second one (using Novozym 435) converted more than 95% FFAs in less than 3 h. The final product obtained was used as starting material to purify FASEs,

Weber and coworkers (Weber *et al.*, 2002) have also reported the use of lipases for the conversion of sterols into steryl esters leading to a higher degree of purity (90%), however the methodology is more complex and involves deacidification, flash chromatography and

Another methodology was developed to focus more specifically on the conversion of FFAs into fatty acid butyl esters (FABEs) (Nagesha *et al.*, 2004). Nagesha and coworkers (Nagesha, 2004) used immobilized *Mucor miehei* lipase in supercritical carbon dioxide at high pressure

Most of the substances that are present in soybean deodorizer distillate are molecules of high molecular weight and thermally sensitive. These properties hinder the separation or purification of these compounds through traditional methods, because they are decomposed

An alternative separation/purification procedure of such products is the use of molecular or short-path distillation. It consists of transferring molecules from the surface of an evaporating liquid to the cooled surface of a condenser through a short path, which is on the order of 2-5 cm. In this process, distillation of heat-sensitive materials is accompanied by only negligible thermal decomposition (Lutisan *et al.*, 2002) because materials, by using high vacuum, are submitted to relatively reduced temperatures, and short residence times (Lutisan, 2002) inside the equipment. Furthermore, this process has advantages over other techniques that use toxic or flammable solvents as the separating agent, avoiding toxicity

The combination of a small distance between the evaporator and the condenser of only a few centimetres and a high vacuum in the distillation gap, results in a specific mass transfer mechanism with evaporation outputs as high as 20–40 gm−2 s−1 (Cvengros *et al.*, 2000). Under these conditions (e.g., short residence time and low temperature), distillation of heatsensitive materials is accomplished without or only negligible thermal decomposition. Therefore, molecular distillation shows potential in the separation, purification and/or concentration of natural products, usually constituted by complex and thermally sensitive

In lipid chemistry, it has been used for the purification of monoacylglycerols (Szelag & Zwierzykowski, 1983), recovery of carotenoids from palm oil (Batistella & Wolf Maciel, 1998), fractionation of polyunsatured fatty acids from fish oils (Breivik *et al.*, 1997), recovery of squalene (Sun *et al.*, 1997), and recovery of tocopherols (Batistella *et al.*, 2002), among

Normally, SODD have a high content of FFA and acylglycerol. To increase the separation efficiency of the compounds of interest, esterification and/or transesterification reactions are usually carried out prior to molecular distillation. Free fatty acids and acylglycerols are converted to fatty acid methyl esters, which are more easily removed by vacuum distillation

and obtained a maximum recovery of 88% and a FABE purity of 95% from SODD.

tocopherols, and FAEEs via supercritical CO2 extraction (Torres, 2009).

temperature using immobilized lipases have been also reported (Weber *et al.*, 2001). In this case neither organic solvent, nor water or any drying reagent such as molecular sieves, are used. This and others studies (Shimada, 2000) showed that in the process of esterification of sterols with free fatty acids, the best results are obtained with *Candida rugosa* lipase and *Pseudomonas* sp. However, enzymatic conversion of FFAs to FAMEs or FAEEs is carry out frequently in the presence of *Candida antactica* lipase or *Alcalygenes* sp. (Torres, 2007), (Nagao *et al.*, 2005).

In the following paragraphs some examples of methodologies using enzymes in the pretreatment of SODD are described. Most of them will be further developed in following sections:

Shimada and coworkers (Shimada, 2000) converted sterols from SODD to fatty acid sterol esters and completely hydrolyzed acylglycerols by applying lipase reactions (*Candida rugosa*  or *Pseudomonas* sp., at 35 ºC for 24 h) to the purification of tocopherols and sterols, resulting in an efficient fractionation of tocopherols and sterols as fatty acids steryl esters (FASEs) by short-path distillation. This process included the drawback that FFA and tocopherols were not efficiently fractionated because the boiling points of the two substances were close. This problem could be solved by conversion of the FFA to their corresponding methyl esters. An attempt to develop a reaction system in which the methyl esterification of FFA proceeded simultaneously with the conversion of sterols to FASEs and the hydrolysis of acylglycerols has been also reported (Watanabe *et al.*, 2004).

Nagao and coworkers (Nagao, 2005) and Watanabe and coworkers (Watanabe, 2004) have applied a procedure based on using a lipase to promote the simultaneous esterification of sterols with free fatty acids and hydrolysis of acylglycerols before the esterification of the free fatty acids with methanol. These authors use *Candida rugosa* lipase for the purification of tocopherol in SODD. Watanabe and coworkers reported 80% conversion of the initial sterols to FASEs, complete hydrolysis of the acylglycerols, and a 78% decrease in the initial FFA content by methyl esterification in 40 h. Tocopherols did not change throughout the process. Distillation of the reaction mixture purified tocopherols to 76.4% (recovery, 89.6%) and sterols to 97.2% as FASEs (recovery, 86.3%). Nagao and coworkers reported a more effective sterols esterification, with a degree of esterification reached 95%. The second-step reaction was then conducted at 30 ºC for 20 h with *Alcaligenes* sp. lipase. 95% FFAs were converted to FAME, and steryl esters synthesized by the first-step reaction were not reconverted to free sterols. Finally, tocopherols and steryl esters were purified from the reaction mixture by short-path distillation. Tocopherols were purified to 72% (yield, 88%) and steryl esters were purified to 97% (yield, 97%). One of the main disadvantages of this method is that the remaining free fatty acids are not completely separated from the tocopherols.

Lipase-catalyzed esterification of sterols and ethyl esterification simultaneously, are governed by the concentration of water present. The degree to which esterification of sterols occurs relative to ethyl esterification requires to attain a balance not always easy to achieve because the presence of an excess of water favours hydrolysis, whereas esterification predominates when a very limited amount of water is present (Marangoni & Rousseau, 1995). By appropriate choice of reaction conditions, however, it is possible to separate the sterol esterification and ethyl esterification in time or space. It is then possible to optimize each of these reactions independently, thereby minimizing costs or improving the yield of the desired final reaction products.

This is precisely the procedure carried out by Torres and coworkers (Torres, 2007), who proposed a two-step enzymatic procedure to obtain FASEs, tocopherols, and fatty acid ethyl esters (FAEEs) from SODD. Firstly, SODD was mixed with oleic acid to reduce its melting point and to enhance the free phytosterols esterification. The first enzymatic step (using *Candida rugosa* lipase) allowed the efficient conversion of more than 90% free phytosterols within 5 h. The second one (using Novozym 435) converted more than 95% FFAs in less than 3 h. The final product obtained was used as starting material to purify FASEs, tocopherols, and FAEEs via supercritical CO2 extraction (Torres, 2009).

Weber and coworkers (Weber *et al.*, 2002) have also reported the use of lipases for the conversion of sterols into steryl esters leading to a higher degree of purity (90%), however the methodology is more complex and involves deacidification, flash chromatography and solvent fractionation.

Another methodology was developed to focus more specifically on the conversion of FFAs into fatty acid butyl esters (FABEs) (Nagesha *et al.*, 2004). Nagesha and coworkers (Nagesha, 2004) used immobilized *Mucor miehei* lipase in supercritical carbon dioxide at high pressure and obtained a maximum recovery of 88% and a FABE purity of 95% from SODD.
