**Extraction and Enzymatic Modification of Functional Lipids from Soybean Oil Deodorizer Distillate**

Carlos F. Torres, Guzmán Torrelo and Guillermo Reglero *Departamento de Producción y Caracterización de Nuevos Alimentos Instituto de Investigación en Ciencias de la Alimentación (CIAL), CSIC-UAM Madrid Spain* 

### **1. Introduction**

446 Recent Trends for Enhancing the Diversity and Quality of Soybean Products

Zeeman, S.C.; Smith, S.M. & Smith, A.M. (2007). The diurnal metabolism of leaf starch.

Zeeman, S.C.; Kossmann, J. & Smith, A.M. (2010). Starch:its metabolism, evolution and biotechnological modification in plants. Annual Review Plant Biology 61:209-234. Zenobi, R.; & Knochenmuss, R. (1998). Ion formation in MALDI mass spectrometry. Mass

Zhu, S.G; Long, S.P. & Ort, D.R. (2010). Improving photosynthetic efficiency for greater

*Biochemical Journal* 401:13-28.

Spectrom. Rev. 17:337-366.

yield. Annual Review Plant Biol. 61:235-261.

Crude vegetable oils contain triacylglycerols as major component and various minor components such as diacylglycerols, monoacylglycerols, free fatty acids, phospholipids, tocopherols, sterols, squalene, color pigments, waxes, aldehydes, ketones, triterpene alcohols and metals that may affect the quality of the final product. The minor components are removed partially or entirely by either physical or chemical refining in order to make the vegetable oils suitable for human consumption. Deodorization is the last major processing step in the refining of edible oils. It has the responsibility for removing both the undesirable ingredients occurring in natural fats and oils and those which may be imparted by prior unit processes such as caustic refining, bleaching, hydrogenation, or even storage conditions. It is this unit process that finally establishes the oil characteristics of "flavor and odor," which are those most readily recognized by the consumer (Gavin, 1978).

Deodorizer distillate is a by-product of deodorization, which is the last major step in vegetable oil refining process. It is a complex mixture of free fatty acids, mono-, di- and triacyglycerols, sterols and their esters, tocopherols, hydrocarbons, pesticides, and breakdown products of fatty acids, aldehydes, ketones and acylglycerol species (Ramamurthi & McCurdy, 1993). Deodorizer distillate is an excellent source of valuable compounds such as phytosterols, tocopherols and squalene, which can be recovered and further used as food additives, in pharmaceutical industry and cosmetics. Their commercial value however, is mainly dependent on their tocopherol content (Fernandes & Cabral, 2007). Although in recent years, efforts from industry resulted in a significant number of reports, describing better and improved methods for phytosterol recovery and purification. Such surge is closely related to growing market for phytosterols, particularly given the widespread dissemination of functional foods (Fernandes & Cabral, 2007).

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 three generic categories: crystallization and precipitation, chemical and enzymatic modification, and extraction and fractionation.

One important bioactive compound concentrated in intermediate byproducts and waste streams during the refining of soybean oil is squalene. Recently, steroidal hydrocarbons and

Extraction and Enzymatic Modification of

from the oil and recycled.

centrifugation.

dehulling.

Functional Lipids from Soybean Oil Deodorizer Distillate 449

Soybean preparation generally includes the steps of cleaning, drying, cracking, and

Oil extraction basically consists of separating the oil from the remainder of the soybean, known as soybean meal. The great majority of commercial soybean extraction processes use a solvent to separate the oil from the meal. In the solvent extraction process, the beans are flaked to provide a large surface area. A solvent, commonly hexane, is then pumped through the soybean flakes, dissolving the oil in the hexane. The hexane is then separated

The crude oil resulting from the extraction process must then be subjected to additional treatments, collectively called "refining", to remove various materials in order for the oil to be suitable for consumption. These materials include hydratable and non-hydratable

Crude soybean oil contains phosphorous compounds called hydratable phospholipids, and small amounts of calcium and magnesium that complex with a portion of the phospholipids to form non-hydratable phospholipids. Hydratable phospholipids are normally removed by a process known as "degumming", in which the oil is agitated or otherwise intimately combined with water to precipitate gums from the oil. The gums are then removed by

These precipitated gums can be used as a feed additive, or evaporated to remove moisture, the end product is called lecithin. Lecithin has various end uses such as food emulsifier. The degummed oil is dried under vacuum to remove any water. Removal of non-hydratable phospholipids is considerably more difficult and expensive, requiring further chemical treatment, typically chemical refining, to break the chemical bonds between the calcium or

In most processes, free fatty acids are then removed from the oil by a process known as caustic refining, also called chemical or alkali refining, in which the oil is mixed with a caustic material, such as sodium or potassium hydroxide, which undergoes a saponification reaction with the acids, forming soaps that are then removed by centrifugation. In this case, the non-hydratable phosphotide are removed along with the free fatty acids. In addition, a

Free fatty acid removal by a process known as physical refining has been used for oils that are low in non-hydratable phospholipids, such as lauric oils, particularly palm oil. In physical refining, the oil is vacuum distilled at high temperatures, e.g., from about 230 ºC to about 260 ºC to separate more volatile components from the oil. This process is used to remove various flavor components, and will also remove free fatty acids. However, the process has not been viable for removing free fatty acids from oils such as soybean oil, which contains higher levels of non-hydratable phospholipids (more than 20 ppm based of phosphorous content). The high temperatures required for physical refining tend to break down the non-hydratable phospholipids that are present in the soybean oil, producing

Conventional refining processes also involve some bleaching of the soybean oil to remove color pigments (i. e., carotenoids, chlorophyll) that adversely affect the color of the oil.

Finally, deodorization is the last process step used to improve the taste, odor, color and stability of the oil by means of removing undesirable substances. The goal of deodorization is to obtain a final product, finished oil that has a bland flavor, a maximum FFA content of 0.05% and a zero peroxide value. All commercial deodorization, whether in continuous,

magnesium ions and the phospholipids, followed with extensive bleaching of the oil.

significant quantity of the oil is captured by the soaps, adversely affecting oil yield.

chemical compounds that cause an unacceptable flavor and color.

Bleaching process employs the use of adsorbents such as acid-activated clays.

phospholipids, free fatty acids, and various color and flavor components.

squalene in soybean oil deodorizer distillate have been isolated and identified. Separation, purification, and chemical characterization of hydrocarbons fraction in soybean oil deodorizer distillate will help researchers finding a better utilization of these byproduct (Gunawan *et al.*, 2008b, Kasim *et al.*, 2009). Both squalene and steroidal hydrocarbons have been purified from soybean oil deodorizer distillate by means of modified soxhlet extractions with hexane to obtain two main fractions: one fraction rich in fatty acid steryl esters and squalene and a second fraction rich in tocopherols, free phytosterols, free fatty acids (FFAs) and acylglycerols. Then hydrocarbons are isolated via silica gel column chromatography. Similarly, other procedures described in the literature for isolation of sterols and tocopherols in soybean oil deodorizer distillate are based on the utilization of organic solvents (Lin *et al.*, 2004).

Recently greener technologies for isolation, purification and fractionation of bioactive compounds from soybean oil deodorizer distillate (SODD) have been developed. These methodologies can work in combination or independently to improve the fractionation of soybean oil deodorizer distillate. Hence, lipase-catalyzed methyl or ethyl esterification of SODD to transform free fatty acids into their corresponding fatty acid methyl or ethyl esters coupled to molecular distillation and/or supercritical fluid extraction has been described as a strategy to improve the separation between tocopherols, sterols and free fatty acids (Torres *et al.*, 2009). Alternatively, enzymatic esterification of the sterols with the fatty acids already present in the deodorizer sludge makes the separation of tocopherols and sterols simpler using short-path distillation or supercritical fluid extraction (Shimada *et al.*, 2000). However, short path distillation (Ito *et al.*, 2006), supercritical fluid extraction (Chang *et al.*, 2000), and enzymatic modifications (Torres *et al.*, 2007) have been also utilized independently on soybean oil deodorizer distillate. Therefore, these three technologies will be analyzed and discussed on the present chapter to evaluate the feasibility of these procedures for the valorization of side-stream products obtained during refining of soybean oil.

In addition, oxidation of sterols during refining steps such as heating, degumming, neutralization, bleaching, and deodorization, and during storage and handling should be also considered (Verleyen *et al.*, 2002b). However, limited information is available on the levels of sterols in the by-product fractions collected from chemical and physical refining processes (Verleyen *et al.*, 2001c) and the biological effects of phytosterol oxidation products on animal and human health need more investigation, as feed quality is crucial for animal health and welfare, and ultimately human health. 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 *et al.*, 2006). Positive correlations between total sterols and total phytosterol oxidation products in the by-products collected from refining processes have been found (Ubhayasekera & Dutta, 2009). Therefore, other aspects concerning soybean oil deodorizer distillate such as nonfood applications, direct consumption, and oxidative quality will be also analyzed and discussed in the present chapter.

#### **2. Soybean oil deodorizer distillate production and characterization**

The vegetable oils correspond about 70% of demand of natural oils and fatty acids consumed in the world. The soybean oil corresponds from 20 to 30% of the vegetable oils world market (Bockisch, 1998) and its production involves several steps that are necessary to render the soybean oil suitable for human consumption. These production steps have been broadly characterized as 1) soybean preparation, 2) oil extraction, and 3) oil refining.

squalene in soybean oil deodorizer distillate have been isolated and identified. Separation, purification, and chemical characterization of hydrocarbons fraction in soybean oil deodorizer distillate will help researchers finding a better utilization of these byproduct (Gunawan *et al.*, 2008b, Kasim *et al.*, 2009). Both squalene and steroidal hydrocarbons have been purified from soybean oil deodorizer distillate by means of modified soxhlet extractions with hexane to obtain two main fractions: one fraction rich in fatty acid steryl esters and squalene and a second fraction rich in tocopherols, free phytosterols, free fatty acids (FFAs) and acylglycerols. Then hydrocarbons are isolated via silica gel column chromatography. Similarly, other procedures described in the literature for isolation of sterols and tocopherols in soybean oil deodorizer distillate are based on the utilization of

Recently greener technologies for isolation, purification and fractionation of bioactive compounds from soybean oil deodorizer distillate (SODD) have been developed. These methodologies can work in combination or independently to improve the fractionation of soybean oil deodorizer distillate. Hence, lipase-catalyzed methyl or ethyl esterification of SODD to transform free fatty acids into their corresponding fatty acid methyl or ethyl esters coupled to molecular distillation and/or supercritical fluid extraction has been described as a strategy to improve the separation between tocopherols, sterols and free fatty acids (Torres *et al.*, 2009). Alternatively, enzymatic esterification of the sterols with the fatty acids already present in the deodorizer sludge makes the separation of tocopherols and sterols simpler using short-path distillation or supercritical fluid extraction (Shimada *et al.*, 2000). However, short path distillation (Ito *et al.*, 2006), supercritical fluid extraction (Chang *et al.*, 2000), and enzymatic modifications (Torres *et al.*, 2007) have been also utilized independently on soybean oil deodorizer distillate. Therefore, these three technologies will be analyzed and discussed on the present chapter to evaluate the feasibility of these procedures for the

valorization of side-stream products obtained during refining of soybean oil.

quality will be also analyzed and discussed in the present chapter.

**2. Soybean oil deodorizer distillate production and characterization** 

The vegetable oils correspond about 70% of demand of natural oils and fatty acids consumed in the world. The soybean oil corresponds from 20 to 30% of the vegetable oils world market (Bockisch, 1998) and its production involves several steps that are necessary to render the soybean oil suitable for human consumption. These production steps have been broadly characterized as 1) soybean preparation, 2) oil extraction, and 3) oil refining.

In addition, oxidation of sterols during refining steps such as heating, degumming, neutralization, bleaching, and deodorization, and during storage and handling should be also considered (Verleyen *et al.*, 2002b). However, limited information is available on the levels of sterols in the by-product fractions collected from chemical and physical refining processes (Verleyen *et al.*, 2001c) and the biological effects of phytosterol oxidation products on animal and human health need more investigation, as feed quality is crucial for animal health and welfare, and ultimately human health. 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 *et al.*, 2006). Positive correlations between total sterols and total phytosterol oxidation products in the by-products collected from refining processes have been found (Ubhayasekera & Dutta, 2009). Therefore, other aspects concerning soybean oil deodorizer distillate such as nonfood applications, direct consumption, and oxidative

organic solvents (Lin *et al.*, 2004).

Soybean preparation generally includes the steps of cleaning, drying, cracking, and dehulling.

Oil extraction basically consists of separating the oil from the remainder of the soybean, known as soybean meal. The great majority of commercial soybean extraction processes use a solvent to separate the oil from the meal. In the solvent extraction process, the beans are flaked to provide a large surface area. A solvent, commonly hexane, is then pumped through the soybean flakes, dissolving the oil in the hexane. The hexane is then separated from the oil and recycled.

The crude oil resulting from the extraction process must then be subjected to additional treatments, collectively called "refining", to remove various materials in order for the oil to be suitable for consumption. These materials include hydratable and non-hydratable phospholipids, free fatty acids, and various color and flavor components.

Crude soybean oil contains phosphorous compounds called hydratable phospholipids, and small amounts of calcium and magnesium that complex with a portion of the phospholipids to form non-hydratable phospholipids. Hydratable phospholipids are normally removed by a process known as "degumming", in which the oil is agitated or otherwise intimately combined with water to precipitate gums from the oil. The gums are then removed by centrifugation.

These precipitated gums can be used as a feed additive, or evaporated to remove moisture, the end product is called lecithin. Lecithin has various end uses such as food emulsifier. The degummed oil is dried under vacuum to remove any water. Removal of non-hydratable phospholipids is considerably more difficult and expensive, requiring further chemical treatment, typically chemical refining, to break the chemical bonds between the calcium or magnesium ions and the phospholipids, followed with extensive bleaching of the oil.

In most processes, free fatty acids are then removed from the oil by a process known as caustic refining, also called chemical or alkali refining, in which the oil is mixed with a caustic material, such as sodium or potassium hydroxide, which undergoes a saponification reaction with the acids, forming soaps that are then removed by centrifugation. In this case, the non-hydratable phosphotide are removed along with the free fatty acids. In addition, a significant quantity of the oil is captured by the soaps, adversely affecting oil yield.

Free fatty acid removal by a process known as physical refining has been used for oils that are low in non-hydratable phospholipids, such as lauric oils, particularly palm oil. In physical refining, the oil is vacuum distilled at high temperatures, e.g., from about 230 ºC to about 260 ºC to separate more volatile components from the oil. This process is used to remove various flavor components, and will also remove free fatty acids. However, the process has not been viable for removing free fatty acids from oils such as soybean oil, which contains higher levels of non-hydratable phospholipids (more than 20 ppm based of phosphorous content). The high temperatures required for physical refining tend to break down the non-hydratable phospholipids that are present in the soybean oil, producing chemical compounds that cause an unacceptable flavor and color.

Conventional refining processes also involve some bleaching of the soybean oil to remove color pigments (i. e., carotenoids, chlorophyll) that adversely affect the color of the oil. Bleaching process employs the use of adsorbents such as acid-activated clays.

Finally, deodorization is the last process step used to improve the taste, odor, color and stability of the oil by means of removing undesirable substances. The goal of deodorization is to obtain a final product, finished oil that has a bland flavor, a maximum FFA content of 0.05% and a zero peroxide value. All commercial deodorization, whether in continuous,

Extraction and Enzymatic Modification of

(Svensson, 1976).

2007).

2009).

Min, 1990, Warner, 2005).

in the functional food industry.

Fernandes & Cabral, 2007).

Functional Lipids from Soybean Oil Deodorizer Distillate 451

molecular synthesis schemes such as the production of dibasic acids of different chain lengths (Gangopadhyay *et al.*, 2007). Alternatively, deodorizer distillate have non-food applications, such as biodiesel or can be used mixed with the fuel oil to fire the steam boilers

Tocopherols (vitamin E) are natural antioxidants found in vegetable oils and contribute significantly to their oxidative stability. Due to the high tocopherol content of crude oils, they can be stored long periods of time if they are protected from air, moisture and high temperature (Norris, 1979) without any significant deterioration. However, the concentration of natural tocopherols in soybean oil is too high for optimum oxidative stability and flavor, because they can act as pro-oxidants by peroxide formation (Jung &

Moreover, tocopherols exert several beneficial activities, such as protective role of vitamin A, β-carotene and essential fatty acids (Ferrari *et al.*, 1996). Tocopherols also prevent diseases like cancer (Kline *et al.*, 2007), cardiovascular and cataracts (Block & Langseth, 1994, Munteanu & Zingg, 2007, Rimm *et al.*, 1993). They are used in food, cosmetics and pharmaceutical industries (Chu, 2002) and a mixture of α, β, γ and δ isomers containing 60

On other hand, in recent years a significant number of reports, patents, and scientific publications describing improved methods for phytosterol recovery and purification have been developed. This phenomenon is closely related to the growing market for phytosterols, particularly given the widespread dissemination of functional foods (Fernandes & Cabral,

Phytosterols are useful hypocholesterolemic agents since a daily intake of 2-3 g lowers LDL cholesterol concentrations by 10-15 % as found in various populations (Kritchevsky & Chen, 2005, Quílez *et al.*, 2003). The proposed mechanism is that plant sterols reduce the micellar solubility of cholesterol and consequently lower intestinal absorption of both exogenous and endogenous cholesterol (de Jong *et al.*, 2003, Trautwein *et al.*, 2003), but also experimental investigations suggest that sterols may act modulating lipid and protein metabolism (Mulligan *et al.*, 2003, Plat & Mensink, 2005). In addition to their cholesterol lowering effect, plant sterols may possess anti-cancer (Awad *et al.*, 2003), antiatherosclerosis (Moghadasian *et al.*, 1999, Moghadasian *et al.*, 1997), anti-inflammation (Bouic, 2001) and antioxidation activities (van Rensburg *et al.*, 2000). Phytosterol compounds exhibit virtually no side effects and they have shown no evidence of *in vitro* mutagenic activity or subchronic toxicity in animals (Rozner, 2006). These compounds have been extensively used as a food ingredient

Moreover, phytosterols are valuable precursors in the production of hormones (Donova, 2007). They are used in manufacturing progesterone, corticoids, estrogens, contraceptives, diuretics, male hormones and vitamin D. They are, also, used in cosmetics (Balazs, 1987,

Another important bioactive compound concentrated in intermediate byproducts and waste streams during the refining of soybean oil, is squalene, a hydrocarbon that has been used in applications such as natural moisturizer in cosmetics and biochemical precursor in the synthesis of steroids. Recently, steroidal hydrocarbons and squalene in soybean oil deodorizer distillate have been isolated and identified. Separation, purification, and chemical characterization of hydrocarbons fraction in soybean oil deodorizer distillate will help researchers finding a better utilization of these byproduct (Gunawan, 2008b, Kasim,

wt% tocopherols is widely used as additive to many kinds of foods (Shimada, 2000).

semicontinuous or batch units, is essentially a form of physical distilling by steam, in which the oil is subjected to high temperatures (210 ºC - 280 ºC) under a high vacuum (1 – 6 mm Hg) for a short period of time, which is sufficient to remove FFA and other volatile flavorcausing compounds. During the process, peroxide decomposition products, color bodies and their decomposition products are eliminated and the content of sterols, sterol esters and tocopherols is also reduced.

The modern commercial deodorizers are equipped with a packed column that has three sections: vapour scrubbing section, stripping section and heat bleaching section.

Bleached oil is pre-heated by outgoing deodorized oil and sprayed into the Deaerator where dissolved air and moisture are reduced to a minimum. The oil is then heated to full temperature by hot deodorized oil in the Deodorizing Economizer and high pressure steam in the Final Heater. A portion of the free fatty acids in the oil will be flashed off as the oil temperature increases.

The hot oil enters the Packed Column, which is filled with special structured packing so that the oil is distributed into a thin film and is evenly agitated by stripping steam flowing counter currently from the bottom of the column. As a result, free fatty acids and other remaining volatile impurities in the oil are evaporated and removed with the steam. The residence time in the column is only a few minutes.

Next, the stripped oil enters the heat bleaching section where it flows through the channels of a series of vertically stacked compartments (trays) while agitated by stripping steam. The prolonged thermal action breaks down color bodies (carotenes) and other heat sensitive compounds are volatilized and removed, or rendered inactive, resulting in a lighter oil color. Also, the amount of remaining free fatty acids in the oil is reduced to an absolute minimum.

The deodorized oil is pre-cooled by deaerated oil and then sprayed into the Post Deodorizer where the final "off-flavor" compounds are removed.

Fatty acids and other materials, evaporated from the oil, are condensed by contact with recycled and cooled distillate in the Vapor Scrubbing section. The soybean oil deodorizer distillate is circulated by the Distillate Pump via the Distillate Cooler where it is cooled by cooling water. Accumulated distillate is discharged from the Scrubber to storage.

SODD is a by-product of deodorization and is a complex mixture of free fatty acids, mono-, di- and triacyglycerols, sterols and their esters, tocopherols, hydrocarbons, pesticides, and breakdown products of fatty acids, aldehydes, ketones and acylglycerol species (Ramamurthi & McCurdy, 1993, Verleyen, 2001c). SODD corresponds between 0.1 and 0.4% of crude soybean oil. Deodorizer distillate is an excellent source of valuable compounds such as phytosterols and tocopherols, corresponding approximately 10% and 20% respectively (Czuppon *et al.*, 2003), which can be recovered and further used as food additives, in pharmaceutical industry and cosmetics (Lin & Koseoglu, 2003). Their commercial value however, is mainly dependent on their tocopherol content, depending on the market demand for this ingredient (Dumont & Narine, 2007).

SODD contains high levels of free fatty acids and acylglycerols (Chu *et al.*, 2002). Fatty acids represent 25-75% and acylglycerols about 3-56% of Vegetal Oil Deodorizer Distillates (VODD), depending on the raw material being refined and on the type and conditions of the refining process (Ramamurthi & McCurdy, 1993). The free fatty acids from deodorizer distillate are mostly used as additives for animal food, fluidizing agents for lecithin or as medium-grade soaps. Such fatty acids also can be used as precursors in a wide variety of

semicontinuous or batch units, is essentially a form of physical distilling by steam, in which the oil is subjected to high temperatures (210 ºC - 280 ºC) under a high vacuum (1 – 6 mm Hg) for a short period of time, which is sufficient to remove FFA and other volatile flavorcausing compounds. During the process, peroxide decomposition products, color bodies and their decomposition products are eliminated and the content of sterols, sterol esters and

The modern commercial deodorizers are equipped with a packed column that has three

Bleached oil is pre-heated by outgoing deodorized oil and sprayed into the Deaerator where dissolved air and moisture are reduced to a minimum. The oil is then heated to full temperature by hot deodorized oil in the Deodorizing Economizer and high pressure steam in the Final Heater. A portion of the free fatty acids in the oil will be flashed off as the oil

The hot oil enters the Packed Column, which is filled with special structured packing so that the oil is distributed into a thin film and is evenly agitated by stripping steam flowing counter currently from the bottom of the column. As a result, free fatty acids and other remaining volatile impurities in the oil are evaporated and removed with the steam. The

Next, the stripped oil enters the heat bleaching section where it flows through the channels of a series of vertically stacked compartments (trays) while agitated by stripping steam. The prolonged thermal action breaks down color bodies (carotenes) and other heat sensitive compounds are volatilized and removed, or rendered inactive, resulting in a lighter oil color. Also, the amount of remaining free fatty acids in the oil is reduced to an absolute

The deodorized oil is pre-cooled by deaerated oil and then sprayed into the Post Deodorizer

Fatty acids and other materials, evaporated from the oil, are condensed by contact with recycled and cooled distillate in the Vapor Scrubbing section. The soybean oil deodorizer distillate is circulated by the Distillate Pump via the Distillate Cooler where it is cooled by

SODD is a by-product of deodorization and is a complex mixture of free fatty acids, mono-, di- and triacyglycerols, sterols and their esters, tocopherols, hydrocarbons, pesticides, and breakdown products of fatty acids, aldehydes, ketones and acylglycerol species (Ramamurthi & McCurdy, 1993, Verleyen, 2001c). SODD corresponds between 0.1 and 0.4% of crude soybean oil. Deodorizer distillate is an excellent source of valuable compounds such as phytosterols and tocopherols, corresponding approximately 10% and 20% respectively (Czuppon *et al.*, 2003), which can be recovered and further used as food additives, in pharmaceutical industry and cosmetics (Lin & Koseoglu, 2003). Their commercial value however, is mainly dependent on their tocopherol content, depending on

SODD contains high levels of free fatty acids and acylglycerols (Chu *et al.*, 2002). Fatty acids represent 25-75% and acylglycerols about 3-56% of Vegetal Oil Deodorizer Distillates (VODD), depending on the raw material being refined and on the type and conditions of the refining process (Ramamurthi & McCurdy, 1993). The free fatty acids from deodorizer distillate are mostly used as additives for animal food, fluidizing agents for lecithin or as medium-grade soaps. Such fatty acids also can be used as precursors in a wide variety of

cooling water. Accumulated distillate is discharged from the Scrubber to storage.

the market demand for this ingredient (Dumont & Narine, 2007).

sections: vapour scrubbing section, stripping section and heat bleaching section.

tocopherols is also reduced.

temperature increases.

minimum.

residence time in the column is only a few minutes.

where the final "off-flavor" compounds are removed.

molecular synthesis schemes such as the production of dibasic acids of different chain lengths (Gangopadhyay *et al.*, 2007). Alternatively, deodorizer distillate have non-food applications, such as biodiesel or can be used mixed with the fuel oil to fire the steam boilers (Svensson, 1976).

Tocopherols (vitamin E) are natural antioxidants found in vegetable oils and contribute significantly to their oxidative stability. Due to the high tocopherol content of crude oils, they can be stored long periods of time if they are protected from air, moisture and high temperature (Norris, 1979) without any significant deterioration. However, the concentration of natural tocopherols in soybean oil is too high for optimum oxidative stability and flavor, because they can act as pro-oxidants by peroxide formation (Jung & Min, 1990, Warner, 2005).

Moreover, tocopherols exert several beneficial activities, such as protective role of vitamin A, β-carotene and essential fatty acids (Ferrari *et al.*, 1996). Tocopherols also prevent diseases like cancer (Kline *et al.*, 2007), cardiovascular and cataracts (Block & Langseth, 1994, Munteanu & Zingg, 2007, Rimm *et al.*, 1993). They are used in food, cosmetics and pharmaceutical industries (Chu, 2002) and a mixture of α, β, γ and δ isomers containing 60 wt% tocopherols is widely used as additive to many kinds of foods (Shimada, 2000).

On other hand, in recent years a significant number of reports, patents, and scientific publications describing improved methods for phytosterol recovery and purification have been developed. This phenomenon is closely related to the growing market for phytosterols, particularly given the widespread dissemination of functional foods (Fernandes & Cabral, 2007).

Phytosterols are useful hypocholesterolemic agents since a daily intake of 2-3 g lowers LDL cholesterol concentrations by 10-15 % as found in various populations (Kritchevsky & Chen, 2005, Quílez *et al.*, 2003). The proposed mechanism is that plant sterols reduce the micellar solubility of cholesterol and consequently lower intestinal absorption of both exogenous and endogenous cholesterol (de Jong *et al.*, 2003, Trautwein *et al.*, 2003), but also experimental investigations suggest that sterols may act modulating lipid and protein metabolism (Mulligan *et al.*, 2003, Plat & Mensink, 2005). In addition to their cholesterol lowering effect, plant sterols may possess anti-cancer (Awad *et al.*, 2003), antiatherosclerosis (Moghadasian *et al.*, 1999, Moghadasian *et al.*, 1997), anti-inflammation (Bouic, 2001) and antioxidation activities (van Rensburg *et al.*, 2000). Phytosterol compounds exhibit virtually no side effects and they have shown no evidence of *in vitro* mutagenic activity or subchronic toxicity in animals (Rozner, 2006). These compounds have been extensively used as a food ingredient in the functional food industry.

Moreover, phytosterols are valuable precursors in the production of hormones (Donova, 2007). They are used in manufacturing progesterone, corticoids, estrogens, contraceptives, diuretics, male hormones and vitamin D. They are, also, used in cosmetics (Balazs, 1987, Fernandes & Cabral, 2007).

Another important bioactive compound concentrated in intermediate byproducts and waste streams during the refining of soybean oil, is squalene, a hydrocarbon that has been used in applications such as natural moisturizer in cosmetics and biochemical precursor in the synthesis of steroids. Recently, steroidal hydrocarbons and squalene in soybean oil deodorizer distillate have been isolated and identified. Separation, purification, and chemical characterization of hydrocarbons fraction in soybean oil deodorizer distillate will help researchers finding a better utilization of these byproduct (Gunawan, 2008b, Kasim, 2009).

Extraction and Enzymatic Modification of

of sterols was not provided in these publications.

mixture (ratio of 4:1, v/v) at -5 ºC.

and hydrocarbons.

Functional Lipids from Soybean Oil Deodorizer Distillate 453

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

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

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,

The refining process induces changes in the structure and concentration of tocopherols, sterols (free and bound) and squalene.

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 oils were constant during processing.

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 in unchanged form.

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 enzymatic modification.
