**8.1 Influence of refining on phytosterols**

During refining of edible fats and oils, the content of total sterols decreases due to degradation and formation of products through isomerization (D5 to D7-sterol), dehydration, polymerization, and formation of hydrocarbons or sterenes and sterol oxidation products (Dutta, 2006). These qualitative and quantitative changes in sterols can be traced in the refined oil and in by-products such as soapstocks and distillate fractions collected after chemical and physical refining processes (Dowd, 1998).

Acid hydrolysis of steryl esters may occur upon bleaching with an acid activated bleaching earth. The slight reduction of the total sterol content is due to the formation of steradienes and disteryl ethers. A gradual reduction in the total sterol content is observed at increasing deodorization temperature due to distillation and steradiene formation. Increasing the temperature from 220 °C to 260 °C resulted in a gradual reduction of the total sterol recovery from 90.4% to 67.7% in physical refining and from 93% to 62.7% in chemical refining. However in physical refining, an increase of 40% in the steryl ester fraction is observed due to an esterification reaction, promoted by high temperature between a sterol and a fatty acid. Due to the absence of free fatty acids in the chemical refining their esterification did not occur (Verleyen *et al.*, 2001b). The influence of refining on free and esterified sterols has been studied by (Verleyen, 2002b).

Phytosterols are progressively lost during refining while continuously altering the ratio of free and esterified sterols (Kochhar, 1983). During chemical neutralization, the free sterol content is significantly reduced especially upon addition of weak caustic solution due to the loss in the soapstock (Gutfinger & Letan, 1974).

Bleaching effects on phytosterols are generally minor and mainly limited to the formation of some nonpolar dehydration products (Ferrari, 1996) and partial hydrolysis of sterol esters (Homberg & Bielefeld, 1982). Steradienes and disteryl ether dehydration products (Figure 1) are formed during bleaching step by the bleaching temperature and the degree of acid activation of the bleaching earth, while during the deodorization, the degree of sterol dehydration is mainly influenced by deodorization temperature giving rise to a concentration of the steradienes in the distillate (Verleyen, 2002b, Verleyen, 2001c). The presence of steradienes can also be used as a marker for the presence of refined oils (Grob *et al.*, 1994).

Whenever applied, hydrogenation has a tremendous effect on sterol structures, including hydrogenation of double bonds, opening of cyclopropane rings, and positional isomerization of side chain unsaturation (Strocchi & Marascio, 1993).

A part of a multinational EU research project (FOOD-CT2004-007020) was to carry out qualitative and quantitative assessment of sterols and sterol oxidation products in samples of by-products from chemical and physical refining of edible fats and oils collected from various locations in Europe. To the best of our knowledge, this is the first report on the contents of oxidized sterols in soapstock and distillate fractions from edible oil refining processes. The levels of sterol oxidation products were higher in acid oil obtained from

Extraction and Enzymatic Modification of

parent sterol.

in humans.

chemical origin.

**8.2 Influence of refining on tocopherols** 

compared to oxycholesterol has not been elucidated yet.

and therefore, the potential to modulate human metabolism.

Functional Lipids from Soybean Oil Deodorizer Distillate 467

The biological effects of oxycholesterol have been extensively studied (Bjorkhem *et al.*, 2002); however, the amount of biological research on oxyphytosterols is rather scarce, mostly dated and has never been extensively reviewed before (Francesc, 2004) and (Dieter, 2004). Most reports available so far on oxyphytosterols cover the methodological aspects of their measurement in foods. The usual perception about oxyphytosterols is that these components present a concern in terms of food quality and health. This perception originates from the parallel that is made between oxycholesterol and oxyphytosterols. Whether oxyphytosterols may indeed play similar and/or different biological roles

A review (Hovenkamp *et al.*, 2008) summarise the current knowledge on the possible biological effects of oxyphytosterols and to identify future research needs, which will help in clarifying the possible impact of oxyphytosterols on human health. The review focuses on the more common oxyphytosterols which differ only in a few structural changes from the

Over the last thirty years a diversity of potential biological effects, including modulation of cholesterol homeostasis, anti-inflammatory and anti-tumour activities, as well as lipidlowering and anti-diabetic properties, have been attributed to specific oxyphytosterols. Although these studies were not all carried out with oxyphytosterols also identified in the human body, these results suggest that oxyphytosterols may have systemic effects in vivo

Despite some putative desirable effects, oxyphytosterols may be perceived as presenting a concern in terms of food quality and health. Indeed, oxyphytosterols have been reported to exert, in vitro, cytotoxic effects comparable to those attributed to oxycholesterol. However, high, non-physiological concentrations of oxyphytosterols were needed to exert adverse effects. In addition, data from one animal study do not support a role of oxyphytosterols in atherosclerosis promotion. However, this aspect deserves more attention in future research. Altogether, the currently available observations do not suggest that oxyphytosterols, in relatively low concentrations such as those reported in human plasma, may exert in vivo deleterious effects similar to those attributed to oxycholesterol. In addition, although probably different in structure than the potentially deleterious ones, some oxyphytosterols may also have the ability to activate transcription factors involved in cholesterol metabolism. Nevertheless, more detailed investigations are needed to evaluate the biological impact of long-term exposure to physiologically relevant concentrations of oxyphytosterols

During deodorization, all tocopherols present in the bleached oil will be partitioned either in the deodorized oil or in the deodorizer distillate. A significant loss in the tocopherol mass balance in the range of 25%-35% was observed originating from technological and/or

The loss of tocopherols can be caused either by a thermal breakdown at temperatures higher than 240 °C, by oxidation reaction or by chemical reaction such as the formation of tocopheryl esters (Verleyen *et al.*, 2001a). Extensive analysis of vegetable oils by HPLC and comparison with synthesized tocopheryl esters did not show any adsorption in the elution region of tocopheryl esters, indicating that esters of tocopherols with fatty acids are not present in crude oils (Verleyen, 2001c). Therefore the stability of tocopherols during

chemical refining (AOCHE) samples than in acid oil obtained from physical refining (AOPHY) samples, with ranges 0.02–17.0 and 0.01–1.5 mg/100 g, respectively. The lower content of sterol oxidation products in AOPHY samples may be due to the high temperature applied during vacuum distillation accelerating the breakdown and transformation of the sterol oxidation products into other unidentified degradation products. Further formation of sterol oxidation products has been prevented by the high amounts of natural antioxidants in AOPHY distillate (Verleyen, 2001c). Some sterols appeared to be more liable to breakdown than others, e.g. there was a higher content of oxybrassicasterols than the other sterol oxidation products in this study, although the content of brasicasterol in the sample was lower than other sterols. Similar results have been reported previously (Dutta, 2006). This may be due to the structural arrangement in the brassicasterol molecule rendering it more easily oxidized than other sterols. However, systematic studies are required to clarify this phenomenon. Although stigmasterol has a double bond in the side-chain, similar to brassicasterol, the quantities of phytosterol oxidation products or oxyphytosterols observed in this study were quite different. Stigmasterol has an ethyl group at position C24 while brasicasterol has a methyl group, and this difference may affect in the relative rate of formation of their oxidation products (Dutta, 2006). Further studies are needed on this point.

Fig. 1. Reaction products of sterols during refining.

It has been reported that the formation of sterol oxidation products is affected not only by the chemical nature of the sterols but also by their quantity (Dutta, 2006). There were positive correlations between total sterols and total phytosterol oxidation products in the by-products collected from both refining processes.

chemical refining (AOCHE) samples than in acid oil obtained from physical refining (AOPHY) samples, with ranges 0.02–17.0 and 0.01–1.5 mg/100 g, respectively. The lower content of sterol oxidation products in AOPHY samples may be due to the high temperature applied during vacuum distillation accelerating the breakdown and transformation of the sterol oxidation products into other unidentified degradation products. Further formation of sterol oxidation products has been prevented by the high amounts of natural antioxidants in AOPHY distillate (Verleyen, 2001c). Some sterols appeared to be more liable to breakdown than others, e.g. there was a higher content of oxybrassicasterols than the other sterol oxidation products in this study, although the content of brasicasterol in the sample was lower than other sterols. Similar results have been reported previously (Dutta, 2006). This may be due to the structural arrangement in the brassicasterol molecule rendering it more easily oxidized than other sterols. However, systematic studies are required to clarify this phenomenon. Although stigmasterol has a double bond in the side-chain, similar to brassicasterol, the quantities of phytosterol oxidation products or oxyphytosterols observed in this study were quite different. Stigmasterol has an ethyl group at position C24 while brasicasterol has a methyl group, and this difference may affect in the relative rate of formation of their oxidation products (Dutta, 2006). Further studies are needed on this point.

Fig. 1. Reaction products of sterols during refining.

by-products collected from both refining processes.

It has been reported that the formation of sterol oxidation products is affected not only by the chemical nature of the sterols but also by their quantity (Dutta, 2006). There were positive correlations between total sterols and total phytosterol oxidation products in the The biological effects of oxycholesterol have been extensively studied (Bjorkhem *et al.*, 2002); however, the amount of biological research on oxyphytosterols is rather scarce, mostly dated and has never been extensively reviewed before (Francesc, 2004) and (Dieter, 2004).

Most reports available so far on oxyphytosterols cover the methodological aspects of their measurement in foods. The usual perception about oxyphytosterols is that these components present a concern in terms of food quality and health. This perception originates from the parallel that is made between oxycholesterol and oxyphytosterols. Whether oxyphytosterols may indeed play similar and/or different biological roles compared to oxycholesterol has not been elucidated yet.

A review (Hovenkamp *et al.*, 2008) summarise the current knowledge on the possible biological effects of oxyphytosterols and to identify future research needs, which will help in clarifying the possible impact of oxyphytosterols on human health. The review focuses on the more common oxyphytosterols which differ only in a few structural changes from the parent sterol.

Over the last thirty years a diversity of potential biological effects, including modulation of cholesterol homeostasis, anti-inflammatory and anti-tumour activities, as well as lipidlowering and anti-diabetic properties, have been attributed to specific oxyphytosterols. Although these studies were not all carried out with oxyphytosterols also identified in the human body, these results suggest that oxyphytosterols may have systemic effects in vivo and therefore, the potential to modulate human metabolism.

Despite some putative desirable effects, oxyphytosterols may be perceived as presenting a concern in terms of food quality and health. Indeed, oxyphytosterols have been reported to exert, in vitro, cytotoxic effects comparable to those attributed to oxycholesterol. However, high, non-physiological concentrations of oxyphytosterols were needed to exert adverse effects. In addition, data from one animal study do not support a role of oxyphytosterols in atherosclerosis promotion. However, this aspect deserves more attention in future research. Altogether, the currently available observations do not suggest that oxyphytosterols, in relatively low concentrations such as those reported in human plasma, may exert in vivo deleterious effects similar to those attributed to oxycholesterol. In addition, although probably different in structure than the potentially deleterious ones, some oxyphytosterols may also have the ability to activate transcription factors involved in cholesterol metabolism. Nevertheless, more detailed investigations are needed to evaluate the biological impact of long-term exposure to physiologically relevant concentrations of oxyphytosterols in humans.

#### **8.2 Influence of refining on tocopherols**

During deodorization, all tocopherols present in the bleached oil will be partitioned either in the deodorized oil or in the deodorizer distillate. A significant loss in the tocopherol mass balance in the range of 25%-35% was observed originating from technological and/or chemical origin.

The loss of tocopherols can be caused either by a thermal breakdown at temperatures higher than 240 °C, by oxidation reaction or by chemical reaction such as the formation of tocopheryl esters (Verleyen *et al.*, 2001a). Extensive analysis of vegetable oils by HPLC and comparison with synthesized tocopheryl esters did not show any adsorption in the elution region of tocopheryl esters, indicating that esters of tocopherols with fatty acids are not present in crude oils (Verleyen, 2001c). Therefore the stability of tocopherols during

Extraction and Enzymatic Modification of

**BIODIESEL**

acylglycerols route (B).

best conversion.

lipid:methanol:sulphuric acid.

Distillation

**FAME**, acylglycerols, sterols, tocopherols

One step or Multiple step esterification

**DEODORIZER DISTILLATES**  (FFA, sterols, tocopherols, acylglycerols, etc.)

**A B**

**STEROLS,** 

**9.1 Production of biodiesel by direct conversion** 

**9.1.1 Chemically catalyzed process** 

the recovery of sterols and tocopherols.

**TOCOPHEROLS BIODIESEL**

Transesterification

Hydrolysis

**FAME**, sterols, tocopherols

Functional Lipids from Soybean Oil Deodorizer Distillate 469

Fig. 2. Production of biodiesel from deodorizer distillates by direct conversion (A) and *via* 

Verhé and coworkers (Verhé *et al.*, 2008) reported a process of converting the deodorizer distillates to biodiesel using methanol in a weight ratio 1:1 and 5 % w/w sulphuric acid as catalyst, at 75 °C for 5 h. Under the mentioned conditions, the FFA have undergone esterification while MAG reacted *via* transesterification, resulting in methyl esters. The crude biodiesel was further washed with 20 % water for 15 min, dried and distilled in order to increase the quality of the methyl esters. The distillation pitch was further processed for

Facioli and Arellano (Facioli & Barrera-Arellano, 2002) described a process to obtain ethyl esters from SODD. SODD contained 47.5 % FFA C18:1, 26.2 % acylglycerols and 26.2 % unsaponifiable matter using concentrated sulphuric acid as catalyst. The optimum conditions found in this study were for EtOH:FFA between 6.4:1 to 11.2:1, H2SO4 from 0.9- 1.5 % and reaction time from 1.3 h to 2.6 h. Under the described conditions a conversion of 94 % of the fatty acids to ethyl esters was achieved. Tocopherols losses were below 5.5 %. A molar excess of ethanol in relation to SODD:FFA was found to be necessary to obtain the

Hammond and coworkers (Hammond & Tong, 2005) described a three-stage acid catalyzed esterification using a molar ratio acid oil:methanol:sulphuric acid of 1:1.3:0.03 for the first stage (25 h). The reaction mixture was centrifuged, the supernatant lipid phase was separated from the sludge (glycerol, water, acid and methanol), and further reacted with methanol and acid, keeping the previous mentioned ratios of unreacted

Acylglycerols

Esterification

**DEODORIZER DISTILLATES**  (FFA, sterols, tocopherols, acylglycerols, etc.)

Transesterification

**FAME**

**BIOFUEL**

**Glycerol, FFA**

**BIODIESEL**

Distillation

**GLYCEROL**

deodorization has been studied under various process conditions. The presence of oxidation products has no influence on the loss of tocopherols during deodorization based on the fact that two successive deodorization steps yielded identical loss of tocopherols.

Experiments using spiked triolein with 2000 ppm of α-tocopherol showed that the addition of tertbutylhydroquinone (TBHQ) as a strong antioxidant reduces the loss of tocopherols with more than 50% in comparison with the reference procedure. α- Tocopherol (2000 ppm) was dissolved in triolein and heated to 254 °C, 5-6 mbar, for 80 min, with no steam injection. 9% of tocopherol loss was observed in the control sample and 3% for the sample with 1500 ppm TBHQ. The more active TBHQ will compete with tocopherols to scavenge radicals and consequently the tocopherol loss in the mass balance is reduced as more natural tocopherols stay in the oil or in the distillate (Verleyen *et al.*, 2002a, Verleyen *et al.*, 2003).

In vegetable oils, the addition of TBHQ from 0 to 1500 ppm establishes a gradual reduction in tocopherol loss from 26.7% to 17.6% while the concentration of tocopherols in the distillate rises from 1.85% to 2.35%. Performing deodorization with nitrogen as stripping agent showed an important reduction in the tocopherol loss (Verleyen, 2002a). In the model study with triolein no reduction of α-tocopherol was observed while using corn oil a reduction of 30%-50% was observed. The highest reduction was detected at severe deodorization conditions (260 °C, 3 mbar) (Verleyen, 2002a). These experiments show that tocopherols are thermally stable compounds and probably the loss of tocopherols is due to oxidation reactions, which leads to compounds such as α- tocopherol dimer quinone, 4α, 5 epoxytocopherolquinone, 7, 8-epoxy tocopherol quinone, tocopherol dimer quinone, tocopherol spirotrimer and ditocopherol ethers (Verleyen, 2001a). These compounds can be found in the finished oil and in the distillate.

In a model experiment using 3500 ppm α- tocopherol in triolein and heating at 240 °C for 90 min at a reduced pressure of 6-7 mbar 4α, 5-epoxytocopherolquinone, 7, 8-epoxy tocopherolquinone and α-tocopherol quinone were identified as oxidation products supporting that the tocopherol loss during deodorization is mainly due to oxidative degradation (Verleyen, 2002a).
