**3. Classic methods to obtain functional lipids from SODD**

In the past, recovering tocopherols and sterols from deodorizer distillates and related mixtures has been proved to be complicated and expensive. One difficulty associated with isolating one or more distillate fractions enriched in fatty acids, tocopherols, and/or sterols from deodorizer distillates is that the molecular weights and volatilities of sterols are similar to those of tocopherols (Ghosh & Bhattacharyya, 1996). For this reason, it is difficult to recover concentrates of tocopherols and phytosterols with good yield and high quality (Lin, 2002). In addition, in order to separate the squalene present in the distillate, the main challenge is to isolate them from each other, especially in the case of the following pairs of components: tocopherol–squalene, tocopherol–fatty acids, tocopherol–sterol and sterol– squalene.

Another difficulty is that deodorizer distillate can undergo thermal degradation if it is processed for extended periods at the temperatures at which sterols and tocopherols vaporize, such temperature conditions which can cause fatty acids to convert into undesirable trans isomeric forms and may cause the degradation of tocopherols (Chu, 2002).

Classical methods for recovering tocopherols and sterols include solvent extraction, chemical treatment, crystallization, complexation, and molecular distillation (Rohr & Trujillo-Quijano, 2005). The separation process involves a series of chemical and physical techniques which are used alone or in combination. In general, most processes are designed to remove either fatty acids or sterols in the initial step, followed by tocopherol concentration by other methods.

The refining process induces changes in the structure and concentration of tocopherols,

Of these various components, most attention is given to the tocopherols. Jung and coworkers (Jung *et al.*, 1989) and Ferrari (Ferrari, 1996) have studied the tocopherol content at all stages of processing for all isomers in the finished oil. The tocopherol content decreases during each step of processing and may be markedly reduced during deodorization, as the tocopherols are volatile under these conditions. The processing removed between 30-60% of tocopherols in crude soybean oil. Even though total tocopherol content decreased during processing, the relative compositions of tocopherols in soybean

Sterol content present in soybean oil also tend to be diminished in processing and the magnitude of such decrease is about the same as the tocopherols (Ferrari, 1996). It has been shown that the absorption of sterols is increased extensively with increased amounts of bleaching clay. The lipid extract from the bleaching clay had high concentrations of sterols

Squalene content also decreases during processing (Nergiz & Çelikkale, 2010), but not drastically until deodorization, when it is partially volatilized. Total losses during all the stages of refining were found to be 31 % as compared to its content in crude soybean oil. Numerous procedures have been described to isolate bioactive compounds from soybean oil deodorizer distillate to improve the value and the quality of this by-product. All these procedures can be grouped in four generic categories: classic method such as crystallization and precipitation, molecular distillation, supercritical fluid extraction and chemical and

In the past, recovering tocopherols and sterols from deodorizer distillates and related mixtures has been proved to be complicated and expensive. One difficulty associated with isolating one or more distillate fractions enriched in fatty acids, tocopherols, and/or sterols from deodorizer distillates is that the molecular weights and volatilities of sterols are similar to those of tocopherols (Ghosh & Bhattacharyya, 1996). For this reason, it is difficult to recover concentrates of tocopherols and phytosterols with good yield and high quality (Lin, 2002). In addition, in order to separate the squalene present in the distillate, the main challenge is to isolate them from each other, especially in the case of the following pairs of components: tocopherol–squalene, tocopherol–fatty acids, tocopherol–sterol and sterol–

Another difficulty is that deodorizer distillate can undergo thermal degradation if it is processed for extended periods at the temperatures at which sterols and tocopherols vaporize, such temperature conditions which can cause fatty acids to convert into undesirable trans isomeric forms and may cause the degradation of tocopherols (Chu,

Classical methods for recovering tocopherols and sterols include solvent extraction, chemical treatment, crystallization, complexation, and molecular distillation (Rohr & Trujillo-Quijano, 2005). The separation process involves a series of chemical and physical techniques which are used alone or in combination. In general, most processes are designed to remove either fatty acids or sterols in the initial step, followed by tocopherol

**3. Classic methods to obtain functional lipids from SODD** 

sterols (free and bound) and squalene.

oils were constant during processing.

in unchanged form.

enzymatic modification.

squalene.

2002).

concentration by other methods.

Crystallization has frequently been used to purify sterols from SODD, either following or preceding other separation methods. Brown (Brown & Smith, 1964) reported a phytosterols product prepared by a continuous two-stage liquid-liquid extraction (LLE) with a solvent pair of methanol and hexane, and then followed by crystallization using acetone as a solvent at 4 ºC for 24 h. By this approach, 73% sterol concentrate was obtained from SODD containing 6.5% sterol. Sheabar and Neeman (Sheabar & Neeman, 1987) have shown the preparation of a tocopherol concentrate through removal of sterol from SODD by a twostage crystallization at -20 ºC with hexane and acetone as crystallization solvents. Attempts have been made to isolate tocopherols from SODD by supercritical fluid extraction technology with crystallization as pretreatment to first remove sterols (Lee *et al.*, 1991). SODD was esterified with methanol using HCl as catalyst, then a solvent pair of hexanemethanol was used to obtain tocopherols-sterols concentrate from which sterols were recovered by crystallization at -20 ºC with acetone as a solvent (Brown & Smith, 1964). The results were similar to those mentioned above. Nevertheless, the information of total yield of sterols was not provided in these publications.

Crystallization seems successful as a simple and efficient process to remove and concentrate sterols and tocopherols from SODD. This process has the advantage of not causing tocopherol oxidation, because the low temperature utilized, and it does not use high pressure. While there is much information in the literature on the recovery of sterols from SODD by crystallization, little attempt is made pertaining to its optimal conditions such as solvent type, crystallization temperature and time. Lin and Koseoglu (Lin & Koseoglu, 2003) have shown crystallization of sterols from SODD without any pretreatment is practical. The best results were achieved by crystallization at -20 ºC for 24 h using a solvent mixture of acetone-methanol (4:1, v/v) at a solvent-to-SODD ratio of 3:1 (v/w), followed by centrifugation, filtration, and twice washing of the wet cake. Over 90% of the original tocopherols and squalene, were retained in the filtrate fraction, while 80% of the original sterols were crystallized in the cake fraction. Khatoon and coworkers (Khatoon *et al.*, 2010) developed a method for the preparation of phytosterols from SODD by crystallisation using hexane and water. Direct crystallisation yielded a phytosterol fraction with lower recovery of 13.2–17.8% while treatment with alkali to remove FFA and the glycerides followed by organic solvent extraction yielded unsaponifiable matter containing phytosterols with a recovery of 74.6%. Later the unsaponifiable matter was purified by double crystallisation into a mixture of phytosterols of 87% purity. Moreira and Baltanás (Moreira & Baltanás, 2004) studied the impact of the principal process variables (solvents and cosolvents, cooling rate, crystallization temperature, and ripening time) on the quality and yield of the recovered phytosterols, but in this case by using a sunflower oil deodorizer distillate "enriched" (i.e., preconcentrated). In this study, a sterols recovery as high as 84% (with 36% purity) was achieved by using a single-stage batch crystallization of hexane/ethanol mixture (ratio of 4:1, v/v) at -5 ºC.

On the other hand, a modified industrial process was developed by Xu and coworkers (Xu *et al.*, 2005) to recovery and purify valuable compounds from SODD. In this process, tocopherols and fatty acids methyl esters (FAMEs) was obtained from SODD after a process with methyl esterification by sulfuric acid catalyst, transesterification by alkaline catalyst, crystallization of sterols and molecular distillation. The waste residue of SODD was obtained after the molecular distillation and it mainly contains steryl esters, acylglycerols, and hydrocarbons.

Extraction and Enzymatic Modification of

content of the soap mass is too high.

final product is also expensive.

**4. Enzymatic modification** 

*et al.*, 1991), (Ramamurthi & McCurdy, 1993).

boiling points are now sufficiently different.

Functional Lipids from Soybean Oil Deodorizer Distillate 455

facilitate the separation of the soluble tocopherols and sterols from the insoluble soap mass. Unfortunately, this process requires a large amount of powdering agent which remains in the soap mass, and the effectiveness of the powdering agent is diminished if the moisture

Although saponification is effective to remove free fatty acids and acylglycerols, it involves the use of a large amount of alkali which is harmful to tocopherols, thus leading to low yields. Recently, molecular distillation combined with crystallization was more attractive to separately concentrate tocopherols and sterols (Gapor *et al.*, 1989, Hunt *et al.*, 1997, Kijima, 1964, Kim & Rhee, 1982, Smith Frank, 1967). To increase the separation efficiency, esterification and/or transesterification are usually carried out prior to molecular distillation. Free fatty acids and acylglycerol are converted to fatty acid methyl esters, which are more easily removed by vacuum distillation due to their higher vapor pressure than those of the corresponding free fatty acids and acylglycerol. However, this step made the whole process more complicated and labor-intensive when compared with the saponification process. Another drawback of molecular distillation is that it is energy consuming to maintain high vacuum all of the time during operation. Consequently, the

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

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

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

In turn, Yang and coworkers (Yang *et al.*, 2009) developed a catalytic and crystallization process to recover phytosterols from waste residue of SODD (WRSODD). A catalyst was employed to decompose WRSODD so as to transform steryl esters into phytosterols. The mixed solvent that generated the best crystallization results was acetone and ethanol (4:1, v/v). The yield and the purity of recovered phytosterols were 22.95 wt. % and 92-97 %, respectively.

Nevertheless, crystallization has the disadvantage of the solvents available at present are not sufficiently selective to obtain, through the current processes, a reasonable separation between the unsaponifiable components and free fatty acids. Due to this, it is often necessary to use more than one solvent, which in turn complicates and increases tremendously the cost of recovery and recycling of these solvent mixtures. Furthermore, solvents or solvent mixtures are used in very large proportions, when compared to the quantity of the material submitted for extraction, and the solvents need additional processes for their removal and/or recycling in the extraction and pre-concentration process of the valuable products. The foregoing reasons make solvent based-processes, expensive, unattractive and less environmentally friendly, resulting in a scarce and expensive final product.

Saponification is also a common practice to concentrate tocopherols and sterols since it produces alkali metal soap which, due to its insolubility in the solvent used in the process, can be separated from the dissolved tocopherols, thereby permitting recovery of the tocopherols in a form relative free from fatty acids and glycerides. The processes themselves are costly, however, and tocopherols are produced in low yield. The sterols are then isolated from the resulting concentrate mixture by crystallization (Brown & Meag, 1963, Kijima *et al.*, 1964, Kim & Rhee, 1982).

Of the saponification processes, the lime saponification process is the most widely used. Hickman, U.S. Patent No. 2.349.270 (Hickman, 1944), discloses that deodorizer distillate can be treated with calcium hydroxide, traditionally called slaked lime, to saponify the fatty acids, followed by extraction of the unsaponifiable fraction (tocopherols and sterols) with acetone, in which the saponification products are insoluble. The extract is then washed and concentrated, as for example by solvent distillation, and then cooled to crystallize sterols which are removed by filtration, leaving a high purity tocopherol fraction. The fatty acid soaps formed by the process can be acidulated and converted into free fatty acids. Andrews, U.S. Patent No. 2.263.550 (Andrews, 1941), discloses saponification of deodorizer distillates with sodium hydroxide, followed by metathesis (a molecular process involving the exchange of bonds between the two reacting chemical species, in this case a ion exchange) with calcium chloride to convert the sodium soaps to calcium soaps (not water soluble), from which the tocopherols and other unsaponifiable matter are then extracted with acetone.

The disadvantage of each of these processes is that the calcium soap is formed in a wide particle size distribution, ranging from fine particles to lumps. The result is a soap mass which is lumpy in form and from which the unsaponificable matter is difficult to extract. To permit the extraction to take place, the soap mass must be ground into particulate form, a process which entails a substantial capital investment. Even then, solvent consumption is high and the recovery of tocopoherols and other useful unsaponificable matter such as sterols is low.

Grinding is avoided in the process disclosed by Brown and coworkers (Brown & Meag, 1963), which uses calcium silicate as a powdering agent in combination with acetone to

In turn, Yang and coworkers (Yang *et al.*, 2009) developed a catalytic and crystallization process to recover phytosterols from waste residue of SODD (WRSODD). A catalyst was employed to decompose WRSODD so as to transform steryl esters into phytosterols. The mixed solvent that generated the best crystallization results was acetone and ethanol (4:1, v/v). The yield and the purity of recovered phytosterols were 22.95 wt. % and 92-97 %,

Nevertheless, crystallization has the disadvantage of the solvents available at present are not sufficiently selective to obtain, through the current processes, a reasonable separation between the unsaponifiable components and free fatty acids. Due to this, it is often necessary to use more than one solvent, which in turn complicates and increases tremendously the cost of recovery and recycling of these solvent mixtures. Furthermore, solvents or solvent mixtures are used in very large proportions, when compared to the quantity of the material submitted for extraction, and the solvents need additional processes for their removal and/or recycling in the extraction and pre-concentration process of the valuable products. The foregoing reasons make solvent based-processes, expensive, unattractive and less environmentally friendly, resulting in a scarce and expensive final

Saponification is also a common practice to concentrate tocopherols and sterols since it produces alkali metal soap which, due to its insolubility in the solvent used in the process, can be separated from the dissolved tocopherols, thereby permitting recovery of the tocopherols in a form relative free from fatty acids and glycerides. The processes themselves are costly, however, and tocopherols are produced in low yield. The sterols are then isolated from the resulting concentrate mixture by crystallization (Brown & Meag, 1963, Kijima *et al.*,

Of the saponification processes, the lime saponification process is the most widely used. Hickman, U.S. Patent No. 2.349.270 (Hickman, 1944), discloses that deodorizer distillate can be treated with calcium hydroxide, traditionally called slaked lime, to saponify the fatty acids, followed by extraction of the unsaponifiable fraction (tocopherols and sterols) with acetone, in which the saponification products are insoluble. The extract is then washed and concentrated, as for example by solvent distillation, and then cooled to crystallize sterols which are removed by filtration, leaving a high purity tocopherol fraction. The fatty acid soaps formed by the process can be acidulated and converted into free fatty acids. Andrews, U.S. Patent No. 2.263.550 (Andrews, 1941), discloses saponification of deodorizer distillates with sodium hydroxide, followed by metathesis (a molecular process involving the exchange of bonds between the two reacting chemical species, in this case a ion exchange) with calcium chloride to convert the sodium soaps to calcium soaps (not water soluble), from which the tocopherols and other unsaponifiable matter are then extracted with

The disadvantage of each of these processes is that the calcium soap is formed in a wide particle size distribution, ranging from fine particles to lumps. The result is a soap mass which is lumpy in form and from which the unsaponificable matter is difficult to extract. To permit the extraction to take place, the soap mass must be ground into particulate form, a process which entails a substantial capital investment. Even then, solvent consumption is high and the recovery of tocopoherols and other useful unsaponificable matter such as

Grinding is avoided in the process disclosed by Brown and coworkers (Brown & Meag, 1963), which uses calcium silicate as a powdering agent in combination with acetone to

respectively.

product.

acetone.

sterols is low.

1964, Kim & Rhee, 1982).

facilitate the separation of the soluble tocopherols and sterols from the insoluble soap mass. Unfortunately, this process requires a large amount of powdering agent which remains in the soap mass, and the effectiveness of the powdering agent is diminished if the moisture content of the soap mass is too high.

Although saponification is effective to remove free fatty acids and acylglycerols, it involves the use of a large amount of alkali which is harmful to tocopherols, thus leading to low yields. Recently, molecular distillation combined with crystallization was more attractive to separately concentrate tocopherols and sterols (Gapor *et al.*, 1989, Hunt *et al.*, 1997, Kijima, 1964, Kim & Rhee, 1982, Smith Frank, 1967). To increase the separation efficiency, esterification and/or transesterification are usually carried out prior to molecular distillation. Free fatty acids and acylglycerol are converted to fatty acid methyl esters, which are more easily removed by vacuum distillation due to their higher vapor pressure than those of the corresponding free fatty acids and acylglycerol. However, this step made the whole process more complicated and labor-intensive when compared with the saponification process. Another drawback of molecular distillation is that it is energy consuming to maintain high vacuum all of the time during operation. Consequently, the final product is also expensive.
