4.7.2. Western Blotting

To confirm the data of quantitative PCR, we evaluated gene expressions in protein level. Briefly, total protein was extracted from cells before and after treatment. Equal amounts of extracted proteins (40 mg/sample) were separated by SDS polyacrylamide gel electrophoresis and transferred to 0.45-mm polyvinylidene difluoride membrane. The membrane was blocked with 5% (*w/v*) non-fat dried milk (Difco/Becton Dickinson, Franklin Lakes, NJ, USA) and incubated for 2 h at room temperature with primary antibody [1:200, BCL-2 (sc-492), BAX (sc-7480), and β-actin (sc-47778)]. Mouse anti-rabbit IgG-HRP in 5% defatted dry milk-TBS-0.1% Tween (Santa Cruz Biotechnology, Dallas, TX, United States) were employed to visualize proteins. All steps were performed at room temperature. All

signals were visualized using enhanced Western Blotting Luminol Reagent (Santa Cruz Inc., Dallas, TX, USA).


**Beach Nourishment: A 21st Century Review**

Editors

**Juan J. Munoz-Perez Luis Moreno**

MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade • Manchester • Tokyo • Cluj • Tianjin

*Editors* Juan J. Munoz-Perez Applied Physics University of Cadiz Puerto Real Spain

Luis Moreno Hydraulics, Energy and Environmental Engineering Politecnica de Madrid Madrid Spain

*Editorial Office* MDPI St. Alban-Anlage 66 4052 Basel, Switzerland

This is a reprint of articles from the Special Issue published online in the open access journal *Journal of Marine Science and Engineering* (ISSN 2077-1312) (available at: www.mdpi.com/journal/ jmse/special issues/beach nourishment).

For citation purposes, cite each article independently as indicated on the article page online and as indicated below:

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Sustainable Management of Phosphorus in Agriculture for Environmental Conservation

*Tahsina Sharmin Hoque, Deepranjan Sarkar, Rahul Datta, Mohammad Golam Kibria, Rafi Ullah, Nazeer Ahmed, Mohammad Anwar Hossain, Asim Masood and Naser A. Anjum*


**3. Acid mine drainage**

Acid Mine Drainage (AMD) is the problem of acid drainage, traditionally referred in Australia and North America as Acid Rock Drainage (ARD). It seems to be a significant environmental impact of mining activities especially in opencast mines. It may damage long after the operation has ended because process and reaction have taken time. Runoff passing through the sourcing area can then give rise to severe threat. Moreover, AMD potentially dissolves and leaches out some toxic metals from the heap, mining waste dump and even natural soil and rock prior to contamination of surface water and groundwater. AMD is usually generated by the oxidation of sulfides in mining wastes; consequently, water supply from the area would be sulfide-rich drainage with acidic leaching property that may lead to mobilization of metals. Sulfides bound up in the waste rocks and tailings usually have various forms. Mineral sulfides are crystalline substances that contain sulfur combined with metal or semi-metal without oxygen. The most general form is "pyrite" (FeS2), moreover, other forms also include Fe1-xSx, Fe3S4. FeS, CuFeS4, ZnS, PbS, HgS, CoAsS etc. After these sulfide minerals are exposed to the air and water, the sulfide ions are oxidized into soluble sulfates as well as toxic metal ions and hydrogen ions may in turn be released into the environment. Initial factors for acid generation are: 1) sulfide minerals in the solid wastes (e.g., rocks and tailings); 2) water or a humid atmosphere; 3) an oxidant (usually oxygen in the form of O2). Therefore, processes of acid generation and metal release would be taken place together during the formation of AMD which are closely related to oxidation of pyrite and precipitation of Fe hydroxides. There are four common chemical reactions represent AMD formed from pyrite. The first equation shows that an important oxidant of pyrite is oxygen. Ferrous iron is released and sulfur is oxidized and changed to sulfate. This equation shown 2 moles of acidity generated from each mole of pyrite. The second equation is the conversion of ferrous iron to ferric iron. It consumes one mole of acidity. The third equation is a hydrolysis reaction which splits the water molecule; consequently, moles of acidity are generated as by-product. The fourth reaction is the oxidation of additional pyrite by ferric iron. The ferric irons generated in reaction steps 1 and 2 are cycle and propagation of the overall reaction. They take place very rapidly and continue until either the ferric iron or pyrite are depleted. In this reaction, iron is the main oxidizing agent instead of oxygen.

$$\begin{array}{ccccccccc}2\text{FeS}\_2 & + & 7\text{O}\_2 & + & 2H\_2\text{O} & \rightarrow & 2Fe^{2+} & + & 2SO\_4^{2-} & +4H^+\\ \text{pyruvate} & & \text{oxygen} & \text{water} & \text{fermions iron} & \text{sulfate} & \text{acidity}\end{array} \tag{1}$$

$$\begin{array}{ccccc} 4\text{Fe}^{2+} & + & O\_2 & + & 4H^+ & \rightarrow & 4Fe^{3+} & + & 2H\_2O \\ \text{fermous iron} & & \text{oxygen acidity} & \text{fercic iron} & & & \text{water} \end{array} \tag{2}$$

$$\begin{array}{rcl} 4Fe^{3+} + & 12H\_2O \rightarrow & 4Fe(OH)\_3^- & + & 12H^+ \\ \text{ferric iron} & \text{Water} & \text{ferrichydrooxide} & \text{acidity} \end{array} \tag{3}$$

$$\begin{array}{ccccccccc}\text{FeS}\_2 & + & 14\text{Fe}^{3+} & + & 8\text{H}\_2\text{O} & \rightarrow & 15\text{Fe}^{2+} & + & 2\text{SO}\_4^{2-} & + & 16\text{H}^+\\\text{pyruvate} & \text{ferric iron} & & \text{water} & \text{ferrous iron} & & \text{sulfate} & & \text{acidity}\end{array} \tag{4}$$

Many procedures have been developed to assess the acid forming characteristics of mine waste materials. The most widely used methods are Acid-Base Accounting (ABA) test and the Net Acid Generation (NAG) test. These procedures are described below.

#### **3.1 Acid-base accounting**

94 Environmental Monitoring

Fig. 2. Tailing pond (left photo) for disposal of slurry-like waste produced from gold dressing and sample collection (right photo), a routine monitoring program which

Due to tailings comprise both solid wastes mixing with water during the operation period of mine and they will become drying after the mining end, redox reaction would be taking place and may in turn change stability of some elements which can be leached out to the environment by accidence. Moreover, their property may also cause AMD. Therefore, routine monitoring plans for both water and tailing must be designed and continuously followed up. Monitoring data should be used for protection at the end and may be very

Acid Mine Drainage (AMD) is the problem of acid drainage, traditionally referred in Australia and North America as Acid Rock Drainage (ARD). It seems to be a significant environmental impact of mining activities especially in opencast mines. It may damage long after the operation has ended because process and reaction have taken time. Runoff passing through the sourcing area can then give rise to severe threat. Moreover, AMD potentially dissolves and leaches out some toxic metals from the heap, mining waste dump and even natural soil and rock prior to contamination of surface water and groundwater. AMD is usually generated by the oxidation of sulfides in mining wastes; consequently, water supply from the area would be sulfide-rich drainage with acidic leaching property that may lead to mobilization of metals. Sulfides bound up in the waste rocks and tailings usually have various forms. Mineral sulfides are crystalline substances that contain sulfur combined with metal or semi-metal without oxygen. The most general form is "pyrite" (FeS2), moreover, other forms also include Fe1-xSx, Fe3S4. FeS, CuFeS4, ZnS, PbS, HgS, CoAsS etc. After these sulfide minerals are exposed to the air and water, the sulfide ions are oxidized into soluble sulfates as well as toxic metal ions and hydrogen ions may in turn be released into the environment. Initial factors for acid generation are: 1) sulfide minerals in the solid wastes (e.g., rocks and tailings); 2) water or a humid atmosphere; 3) an oxidant (usually oxygen in the form of O2). Therefore, processes of acid generation and metal release would be taken place together during the formation of AMD which are closely related to oxidation of pyrite and precipitation of Fe hydroxides. There are four common chemical reactions represent AMD formed from pyrite. The first equation shows that an important oxidant of pyrite is

has to be carried out regularly

**3. Acid mine drainage**

useful for development of the mineral processing.

Characterization of rock types and geologic setting in the mine should be initially concerned prior to determination of capacity acid drainage generation of these rocks (Environment Australia, 1997). Acid-Base Accounting (ABA) is the most commonly-used static procedure that has been used for estimation/qualification of the acid generation potential of mine wastes (Furguson & Erickson, 1988). This procedure was developed at West Virginia University in late 1960s. ABA tests are designed to measure the balance between potentially acid-generating potential, particularly oxidation of sulfide materials and acid neutralizing potential in sample such as dissolution of alkaline, carbonates, displacement of exchangeable bases and weathering of silicate. The values arising from ABA are referred to the Maximum Potential Acidic (MPA) and the Acid Neutralizing Capacity (ANC), respectively. After MPA and NAC have been determined for a sample, both values are compared with set criteria. Two methods of combination commonly used are: 1) The difference in value between MPA and ANC or Net Acid Producing Potential (NAPP) where NAPP = MPA-ANC; 2) The ratio of ANC to MPA (ANC/MPA). NAPP is a theoretical calculation commonly used to indicate where a waste material has potential to produce acidic drainage. NAPP values represent balance between capacity of acid generation and capacity of acid neutralization. Unit of NAPP is also expressed as kg H2SO4/t in MPA and ANC. In addition, ANC/MPA ratio is also considered for assessment of acid generation from mine waste material. The main purpose of ANC/MPA ratio is to indicate relatively

Geochemical Application for Environmental Monitoring and Metal Mining Management 97

represents the inherent acid neutralizing capacity of the sample. Calculation will be carried

Net Acid Generation (NAG) test was developed as an assessment tool for acid producing potential of sample for longer than 20 years ago. The NAG test is usually used in association with NAPP. It is direct method to measure ability of sample to produce acid via sulfide oxidation. Hydrogen peroxide (H2O2) is used to activate and complete oxidation process of the sulfide minerals contained in the sample. H2O2 added during the NAG test leads to simultaneous reactions of acid generation and acid neutralization. Then pH measurement of solution has to be carried out after the completion of reaction. The acidity of solution under the NAG is a direct measurement of net acid generation of sample. Shu et al. (2001) studied the effect of lead/zinc mine acidity on heavy metal mobility using both NAG test and ABA method. They concluded, based on their results that NAG test, direct measurements of ANC from acid produced from oxidized sulfide, yields more accurate than that of ABA method. This is because prediction of acid forming potential from the total pyritic sulfur content as done for ABA method may overestimate amount of acid generation due to uncompleted

However, classifications of waste rock have generally used NAPP estimation based on ABA method in combination of NAG pH testing. Schematic classification is present in Fig. 4. Three types of west rocks from mining activity can be grouped as No Net Acid Forming (NAF), Potentially Net Acid Forming (PAF), and Uncertainly Net Acid Forming (UC).

Fig. 4. NAG pH plot against NAPP for classification potential of net acid formation of waste

**No Net Acid Forming (NAF):** either there is minimal or no sulfides present or the neutralization potential exceeds the acid potential. This type of waste rock gives a negative

out and expressed in terms of kg H2SO4/t.

**3.2 Net acid generation**

acidification of pyritic sulfur.

rock

Definitions of these groups are given below.

NAPP and NAG pH greater than or equal to 4.5.

safety margin of material. Safe values for prevention of acid generation are reported with different ANC/MPA values ranging from 1 to 3. The higher ANC/MPA value indicates high probability of the material that may remain circum-neutral in pH and should not be problematic by acid rock drainage. Both NAPP value and ANC/MPA ratio are usually used together for placement planning of rock waste and other overburdens (Skousen et al., 1987). Sulfur and ANC data are often used in combination with ANC/MPA ratio as presented in Fig. 3.

Fig. 3. Plots of all parameters considered in Acid-Base Accounting (ABA)

*Maximum Potential Acidic:* MPA is the maximum amount of acid that can be produced from the oxidation of sulfur-containing minerals in the rock material. It can be measured and calculated from the sulfur content. Total sulfur content of a sample is commonly determined by the LECO high temperature combustion method or other appropriate methods. For instant, it is assumed that all sulfurs occur as iron-sulfide (or pyrite; FeS2) and this iron-sulfide reacts under oxidizing condition to generate acid according to the following reaction:

$$\text{FeS}\_2 + 15/4 \,\text{O}\_2 + 7/2 \,\text{H}\_2\text{O} \quad \Delta \quad \text{Fe(OH)}\_3 \quad + 2 \,\text{H}\_2\text{SO}\_4$$

According to the stoichiometry, the maximum amount of acid that could be produced by a sample containing 1%S as pyrite would be 30.6 kilograms of H2SO4 per ton of material. The MPA is calculated from the total sulfur content as:

$$\text{MPA (kg H}\text{\u3}\text{\u3}\text{\u4/t)} \quad = \text{ (Total \u3}\text{\u3}\text{\u4}\text{\u4)}$$

*Acid Neutralizing Capacity:* ANC is calculated from the amount of acid neutralizer in the sample and it is expressed in metric tons/1000 metric tons of material. Acid generated from pyrite oxidation will be partly reacted by acid neutralizing minerals contained within the sample. This inherent acid buffering is resulted in term of the ANC. Most of the minerals which contribute the acid neutralizing capacity usually are carbonates such as calcite and dolomite. The modified Sobek method is the most common method used to determine ANC. This method is determined experimentally by reaction of a known amount of standardized acid (hydrochloric acid, HCL) with a known amount of sample and then the mixed solution sample is back-titrated by sodium hydroxide (NaOH). The amount of acid consumed represents the inherent acid neutralizing capacity of the sample. Calculation will be carried out and expressed in terms of kg H2SO4/t.

#### **3.2 Net acid generation**

96 Environmental Monitoring

safety margin of material. Safe values for prevention of acid generation are reported with different ANC/MPA values ranging from 1 to 3. The higher ANC/MPA value indicates high probability of the material that may remain circum-neutral in pH and should not be problematic by acid rock drainage. Both NAPP value and ANC/MPA ratio are usually used together for placement planning of rock waste and other overburdens (Skousen et al., 1987). Sulfur and ANC data are often used in combination with ANC/MPA ratio as presented

Fig. 3. Plots of all parameters considered in Acid-Base Accounting (ABA)

under oxidizing condition to generate acid according to the following reaction:

MPA is calculated from the total sulfur content as:

*Maximum Potential Acidic:* MPA is the maximum amount of acid that can be produced from the oxidation of sulfur-containing minerals in the rock material. It can be measured and calculated from the sulfur content. Total sulfur content of a sample is commonly determined by the LECO high temperature combustion method or other appropriate methods. For instant, it is assumed that all sulfurs occur as iron-sulfide (or pyrite; FeS2) and this iron-sulfide reacts

FeS2 + 15/4 O2 + 7/2 H2O Fe(OH)3 + 2 H2SO4 According to the stoichiometry, the maximum amount of acid that could be produced by a sample containing 1%S as pyrite would be 30.6 kilograms of H2SO4 per ton of material. The

MPA (kg H2SO4/t) = (Total %S) X 30.6 *Acid Neutralizing Capacity:* ANC is calculated from the amount of acid neutralizer in the sample and it is expressed in metric tons/1000 metric tons of material. Acid generated from pyrite oxidation will be partly reacted by acid neutralizing minerals contained within the sample. This inherent acid buffering is resulted in term of the ANC. Most of the minerals which contribute the acid neutralizing capacity usually are carbonates such as calcite and dolomite. The modified Sobek method is the most common method used to determine ANC. This method is determined experimentally by reaction of a known amount of standardized acid (hydrochloric acid, HCL) with a known amount of sample and then the mixed solution sample is back-titrated by sodium hydroxide (NaOH). The amount of acid consumed

in Fig. 3.

Net Acid Generation (NAG) test was developed as an assessment tool for acid producing potential of sample for longer than 20 years ago. The NAG test is usually used in association with NAPP. It is direct method to measure ability of sample to produce acid via sulfide oxidation. Hydrogen peroxide (H2O2) is used to activate and complete oxidation process of the sulfide minerals contained in the sample. H2O2 added during the NAG test leads to simultaneous reactions of acid generation and acid neutralization. Then pH measurement of solution has to be carried out after the completion of reaction. The acidity of solution under the NAG is a direct measurement of net acid generation of sample. Shu et al. (2001) studied the effect of lead/zinc mine acidity on heavy metal mobility using both NAG test and ABA method. They concluded, based on their results that NAG test, direct measurements of ANC from acid produced from oxidized sulfide, yields more accurate than that of ABA method. This is because prediction of acid forming potential from the total pyritic sulfur content as done for ABA method may overestimate amount of acid generation due to uncompleted acidification of pyritic sulfur.

However, classifications of waste rock have generally used NAPP estimation based on ABA method in combination of NAG pH testing. Schematic classification is present in Fig. 4. Three types of west rocks from mining activity can be grouped as No Net Acid Forming (NAF), Potentially Net Acid Forming (PAF), and Uncertainly Net Acid Forming (UC). Definitions of these groups are given below.

Fig. 4. NAG pH plot against NAPP for classification potential of net acid formation of waste rock

**No Net Acid Forming (NAF):** either there is minimal or no sulfides present or the neutralization potential exceeds the acid potential. This type of waste rock gives a negative NAPP and NAG pH greater than or equal to 4.5.

Geochemical Application for Environmental Monitoring and Metal Mining Management 99

geological classification as well as mining operation. Placement and disposal may be designed based on this classification in cooperation with other testing methods. Rock powdering using appropriate crusher and miller must be done prior to further analyses. Subsequently, the powdered rock samples may be fused to glass beads or pressed as pellet for X-ray Fluorescence (XRF) analyses of 9 major oxides (i.e., SiO2, TiO2, FeOt, MnO, MgO, CaO, Na2O, K2O and P2O5) and perhaps some trace elements (e.g., Ba, Zn, Sr, Rb, Zr, Co, Cr, Ni, Y and V). Rock standards should be used for calibration at the same analytical condition. Moreover, loss on ignition (LOI) should also be measured by weighting rock powders before and after ignition at 900º C for 3 hrs in an electric furnace. Trace and rare earth elements may be additionally analyzed using advanced instruments such as Inductively Coupled Plasma (ICP) Spectrometer, Atomic Absorption Spectrometer (AAS) and other spectrometric techniques. Rock samples have to be digested totally without remaining of rock powders. About 0.1000 g (±0.0001 g) of powdered samples are weighted and then dissolved in a concentrate HF-HNO3-HClO4 acid mixture in sealed Teflon beakers. The digested samples were diluted immediately and added mixed standard solution to all samples. Proportion of these concentrate acids is usually adapted in laboratory as well as time of digestion. Hotplate has been engaged traditionally but it may take long time. Alternatively, microwave has been applied to shorten the digestion time. This method is total digestion which most elements including toxic elements and non toxic ones are dissolved for analyses. However, these contents do not clearly reflect environmental impact. Microwave-assisted acid solubilization has been proved to be the most suitable method for the digestion of complex matrices such as sediments and soil. This method shortens the digestion time, reduces the risk of external contamination and uses smaller quantities of acid (Wang et al., 2004). However, there are different procedures required for appropriate sample types. Some standard digestion techniques are usually used for soil, sediment and

sludge; for example, EPA 3052, EPA 3050B and EPA 3051 are described below.

should be obtained from EPA (1996).

*EPA 3052:* This method is an acid digestion of siliceous matrices, and organic matrices and other complex matrices (e.g., ashes, biological tissues, oils, oil contaminated soils, sediments, sludges and soils) which they may be totally decomposed for analysis. Powdered sample of up to 0.5 g is added into 9 ml of concentrated nitric acid and usually 3 ml hydrofluoric acid for 15 minutes using microwave. Several additional alternative acids and reagents have been applied for the digestion. These reagents include hydrochloric acid and hydrogen peroxide. A maximum sample of 1.0 g can be prepared by this method. Mixed acids and sample are placed in an inert polymeric microwave vessel then sealed prior to heating in the microwave system. Temperature may be set for specific reactions and incorporates reaching 180 ± 5 ºC in approximately shorter than 5.5 minutes and remaining at 180 ± 5 ºC for 9.5 minutes to complete specific reactions. Solution may be filtered before appropriate volume is made by dilution. Finally, the solution is now ready for analyses (e.g., AAS or ICP). More details

**EPA 3050:** Two separate procedures have been proposed for digestion of sediment, sludge and soil etc. The first procedure is preparation for analysis of Flame Atomic Absorption Spectrometry (FLAA) or Inductively Coupled Plasma-Atomic Emission Spectrometry (ICP-AES) whereas the other is for Graphite Furnace AA (GFAA) or Inductively Coupled Plasma Mass Spectrometry (ICP-MS). Appropriate elements and their detection limits must be concerned and designed for selection of both methods (EPA, 2009). Alternative determination techniques may also be modified as far as scientific validity is proven. This method can also be applied to other elements and matrices but performance need to be

**Potentially Net Acid Forming (PAF):** the acid potential exceeds the neutralization potential. These rocks are described as potentially acid forming. They may generate AMD if they are exposed to sufficient oxygen to allow sulfide oxidations. Geochemical tests usually yield positive NAPP and NAG pH below 4.5.

**Uuncertain Net Acid Forming (UC):** uncertain classification is obtained when there is an apparent conflict between the NAPP result and NAG pH; for example, NAPP is negative but NAG pH lower than 4.5 or NAPP is positive but NAG pH higher than 4.5. However, further testing work would be performed for such rock types to determine proportion between NAF and PAF if they occur.

Recently, this classification has been using widely for geochemical study of waste rock and assessment of acid forming potential. Tran et al. (2003), for an example, also used NAG together with NAPP tests to figure out key criteria for construction design of waste rock dumps to avoid AMD. They collected samples from 2 sites in which have different temperatures. NAG and NAPP tests were applied to classify PAF, NAF and UC materials prior to placement control of waste rocks within the dumps. They succeeded to have reduced AMD load that may be generated from both dumps.


**5. Molecular distillation**

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

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

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

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

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

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

*et al.*, 2005).

sections:

has been also reported (Watanabe *et al.*, 2004).

the desired final reaction products.

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 when subjected to high temperatures.

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 and environmental problems.

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 molecules such as tocopherols.

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 others.

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

Extraction and Enzymatic Modification of

FASEs (86.3% recovery).

(fourth) residue.

Functional Lipids from Soybean Oil Deodorizer Distillate 459

tocopherols to 76.4 wt% purity (89.6% recovery) and free phytosterols to 97.2 wt% purity as

Nagao and coworkers (Nagao, 2005) carried out similar steps of isolation of tocopherols and free phytosterols as FASEs from SODDTSC. SODDTSC were first treated with *Candida rugosa* lipase (250 U/g activity) to convert free phytosterols to FASEs at 40 ºC for 24 h, achieving about 95% conversion. Unreacted FFAs contained in the reaction mixture was then converted to FAMEs by *Alcaligenes* sp. lipase at 30 ºC for 20 h, achieving 95% conversion. Reaction mixture was then subjected to a four-stage molecular distillation (160 ºC and 0.2 mm Hg, 175 ºC and 0.2 mm Hg, 230 ºC and 0.02 mm Hg, and 240 ºC and 0.02 mm Hg, respectively) to isolate tocopherols (72.3 wt% purity, 87.6% recovery) in the third distillate fraction and free phytosterols as FASEs (97 wt% purity, 97% recovery) in the last

Jacobs (Jacobs, 2005) proposed a method for recovering tocotrienols from fatty acid distillate FAD, which initially contained 1 wt% tocopherols and 0.3 wt% free phytosterols, by stripping FFAs (condition: 0.5–1.5 mm Hg, 180–240 ºC, and 0.5–1.5 min) to form a first stripped product. Short path distillation of the first stripped product gave a first distillate. Saponifying the second stripped product resulted in a saponified product with all FFA converted to FAMEs. A second short path distillation of the saponified product generated a second distillate without FAMEs. Solvent wintering (via filtration) of the second distillate gave a stripped filtrate. The stripped filtrate from the previous step is subjected to a third short path distillation at a temperature of 180° and an absolute pressure of 0.01 mm Hg. The resulting final tocotrienol product, about 1% of the original feed, contains from about 50% tocotrienols, 1% sterols and 49% other unsaponifiables and unknowns. Additionally, the

Fizet (Fizet, 1996) esterified free phytosterols from deodorizer distillate with FFAs at either 180 ºC for 2.5 h or 250 ºC for 1.5 h. Esterification product was then distilled at 120–150 ºC and 0.08 mm Hg to obtain a residue containing mostly tocopherols and FASEs and a distillate containing mostly fatty acids. The residue was then distilled again at 200–220 ºC and 0.1 mbar to obtain a distillate containing mostly tocopherols and a residue containing mostly FASEs. Tocopherols enriched in distillate were then subjected to an ion exchange chromatography and FASEs enriched in residue were then subjected to an acid-catalyzed

Even so, some authors (Martins *et al.*, 2006) have been trying to achieve an efficient FFA separation from SODD with the lowest loss of tocopherols by molecular distillation, without preliminary steps. This separation is difficult to achieve although is technologically viable at least at lab scale, due to the differences between molecular weights and vapour pressures of FFA (MW 180 g·mol-1, VP at 200 ºC = 4 mm Hg) and tocopherols (MW 415 g·mol-1, VP at 200 ºC = 0.15 mm Hg). Martins and coworkers (Martins, 2006) employed molecular distillation at 160 ºC, 7.5×10-4 mm Hg, and 10.4 g/min feed flow rate to remove FFAs into the distillate fraction and obtain a residue fraction, which contained 6.4 wt% FFAs and 18.3 wt% tocopherols from a SODD feed which contained 57.8 wt% FFAs and 8.97 wt% tocopherols. They succeeded in removing 96.16% FFAs and recovering 81.23% tocopherols. Martins and coworkers (Martins *et al.*, 2005) reported the isolation of tocopherols by first converting acylglycerols in SODD into FFAs through saponification at 65 ºC followed by acidulation, and then submitting the unsaponifiable product to five stages of molecular distillation. They succeeded in enriching tocopherols (34.14% purity) by 5.8 times. The major disadvantage in

final product contains from about 15% to about 30% tocopherols.

transesterification with methanol to produce free phytosterols.

these cases is the residual free fatty acids in the tocopherol mixture.

due to their higher vapor pressure than those of the corresponding free fatty acids and acylglycerols. However, this step made the whole process more complicated and laborintensive when compared with the saponification process. Other problem is that the separation of tocopherols from phytosterols is difficult because they have similar molecular weights, boiling points and vapor pressure, and, consequently, they are distillated together (Ghosh & Bhattacharyya, 1996).

Different processes have been proposed in the literature to eliminate FFA by molecular distillation and purify tocopherols and phytosterols. Most of them include a preliminary chemical or enzymatic treatment step.

Ramamurthi and McCurdy (Ramamurthi & McCurdy, 1993) studied the pretreatment of deodorizer distillate using a lipase-catalyzed esterification reaction to convert FFA into methyl esters, followed by vacuum distillation (1-2 mm Hg) to remove them and concentrate tocopherols and sterols (recoveries were over 90%).

Hirota and coworkers (Hirota *et al.*, 2003) isolated naturally occurring Fatty Acid Steryl Esters (FASEs) from SODD. SODD was firstly subjected to molecular distillation at 250 ºC and 0.02 mm Hg to obtain a residue which was rich in DAGs and TAGs, and steryl esters. Enzymatic lipolysis was then conducted to specifically hydrolyze DAGs and TAGs at 35 ºC for 24 h, resulting in a mixture from which fatty acid steryl esters were later purified using a two-stage molecular distillation (180 ºC and 0.2 mm Hg, and 250 ºC and 0.02 mm Hg). The recovery and purity of FASEs were about 87.7% and 97.3 wt%, respectively.

Purification of tocopherols from SODD was carried out by Shimada and coworkers (Shimada, 2000). SODD was distilled using molecular distillation at 250 ºC and 0.02 mm Hg and the resulting distillate was used as a starting material. Sterols in SODD were converted to FA sterol esters and acylglycerols were completely hydrolyzed by applying lipase reactions. FASEs were recovered as residue from the reaction mixture via molecular distillation at 250 ºC and 0.2 mm Hg. However, the last stage of molecular distillation failed to separate FFAs and tocopherols. A second esterification of free phytosterols was applied at 35 ºC for 24 h, followed by another four-stage molecular distillation (160 ºC and 0.2 mm Hg, 200 ºC and 0.2 mm Hg, 230 ºC and 0.04 mm Hg, and 255 ºC and 0.03 mm Hg) which yielded tocopherols with purity and recovery of about 65.3 wt% and 54.6%, respectively.

Watanabe and coworkers (Watanabe, 2004) isolated tocopherols and free phytosterols as their esters from SODD tocopherol/sterol concentrate (SODDTSC). SODDTSC was obtained via molecular distillation at 240 ºC and 0.02 mm Hg, resulting in a distillate rich in tocopherols and free phytosterols (SODDTSC), and a residue rich in FASEs, DAGs, and TAGs. SODDTSC, which contained also MAGs, DAGs, FFAs, and unidentified hydrocarbons, were then subjected to a two-step in situ enzymatic reaction. SODDTSC were treated with *Candida rugosa* lipase (200 U/g activity) to convert free phytosterols to FASEs, acylglycerols (MAGs and DAGs) to FFAs, and FFAs to FAMEs at 30 ºC for 40 h, achieving 80% conversion of the initial sterols to FA steryl esters, complete hydrolysis of the acylglycerols, and a 78% decrease in the initial FFA content by methyl esterification. Tocopherols did not change throughout the process. To enhance degree of steryl and methyl esterification, FASEs and FAMEs enriched in the reaction product were then removed by a two-step molecular distillation. In the first step, FAMEs was removed in the distillate (160 ºC and 0.2 mm Hg). In the second step (240 ºC, 0.2 mm Hg), FASEs was isolated in the residue and the distillate containing tocopherols, free phytosterols, and FFAs were treated again with lipase. A three-step molecular distillation of the reaction mixture purified

due to their higher vapor pressure than those of the corresponding free fatty acids and acylglycerols. However, this step made the whole process more complicated and laborintensive when compared with the saponification process. Other problem is that the separation of tocopherols from phytosterols is difficult because they have similar molecular weights, boiling points and vapor pressure, and, consequently, they are distillated together

Different processes have been proposed in the literature to eliminate FFA by molecular distillation and purify tocopherols and phytosterols. Most of them include a preliminary

Ramamurthi and McCurdy (Ramamurthi & McCurdy, 1993) studied the pretreatment of deodorizer distillate using a lipase-catalyzed esterification reaction to convert FFA into methyl esters, followed by vacuum distillation (1-2 mm Hg) to remove them and

Hirota and coworkers (Hirota *et al.*, 2003) isolated naturally occurring Fatty Acid Steryl Esters (FASEs) from SODD. SODD was firstly subjected to molecular distillation at 250 ºC and 0.02 mm Hg to obtain a residue which was rich in DAGs and TAGs, and steryl esters. Enzymatic lipolysis was then conducted to specifically hydrolyze DAGs and TAGs at 35 ºC for 24 h, resulting in a mixture from which fatty acid steryl esters were later purified using a two-stage molecular distillation (180 ºC and 0.2 mm Hg, and 250 ºC and 0.02 mm Hg). The

Purification of tocopherols from SODD was carried out by Shimada and coworkers (Shimada, 2000). SODD was distilled using molecular distillation at 250 ºC and 0.02 mm Hg and the resulting distillate was used as a starting material. Sterols in SODD were converted to FA sterol esters and acylglycerols were completely hydrolyzed by applying lipase reactions. FASEs were recovered as residue from the reaction mixture via molecular distillation at 250 ºC and 0.2 mm Hg. However, the last stage of molecular distillation failed to separate FFAs and tocopherols. A second esterification of free phytosterols was applied at 35 ºC for 24 h, followed by another four-stage molecular distillation (160 ºC and 0.2 mm Hg, 200 ºC and 0.2 mm Hg, 230 ºC and 0.04 mm Hg, and 255 ºC and 0.03 mm Hg) which yielded

Watanabe and coworkers (Watanabe, 2004) isolated tocopherols and free phytosterols as their esters from SODD tocopherol/sterol concentrate (SODDTSC). SODDTSC was obtained via molecular distillation at 240 ºC and 0.02 mm Hg, resulting in a distillate rich in tocopherols and free phytosterols (SODDTSC), and a residue rich in FASEs, DAGs, and TAGs. SODDTSC, which contained also MAGs, DAGs, FFAs, and unidentified hydrocarbons, were then subjected to a two-step in situ enzymatic reaction. SODDTSC were treated with *Candida rugosa* lipase (200 U/g activity) to convert free phytosterols to FASEs, acylglycerols (MAGs and DAGs) to FFAs, and FFAs to FAMEs at 30 ºC for 40 h, achieving 80% conversion of the initial sterols to FA steryl esters, complete hydrolysis of the acylglycerols, and a 78% decrease in the initial FFA content by methyl esterification. Tocopherols did not change throughout the process. To enhance degree of steryl and methyl esterification, FASEs and FAMEs enriched in the reaction product were then removed by a two-step molecular distillation. In the first step, FAMEs was removed in the distillate (160 ºC and 0.2 mm Hg). In the second step (240 ºC, 0.2 mm Hg), FASEs was isolated in the residue and the distillate containing tocopherols, free phytosterols, and FFAs were treated again with lipase. A three-step molecular distillation of the reaction mixture purified

(Ghosh & Bhattacharyya, 1996).

chemical or enzymatic treatment step.

concentrate tocopherols and sterols (recoveries were over 90%).

recovery and purity of FASEs were about 87.7% and 97.3 wt%, respectively.

tocopherols with purity and recovery of about 65.3 wt% and 54.6%, respectively.

tocopherols to 76.4 wt% purity (89.6% recovery) and free phytosterols to 97.2 wt% purity as FASEs (86.3% recovery).

Nagao and coworkers (Nagao, 2005) carried out similar steps of isolation of tocopherols and free phytosterols as FASEs from SODDTSC. SODDTSC were first treated with *Candida rugosa* lipase (250 U/g activity) to convert free phytosterols to FASEs at 40 ºC for 24 h, achieving about 95% conversion. Unreacted FFAs contained in the reaction mixture was then converted to FAMEs by *Alcaligenes* sp. lipase at 30 ºC for 20 h, achieving 95% conversion. Reaction mixture was then subjected to a four-stage molecular distillation (160 ºC and 0.2 mm Hg, 175 ºC and 0.2 mm Hg, 230 ºC and 0.02 mm Hg, and 240 ºC and 0.02 mm Hg, respectively) to isolate tocopherols (72.3 wt% purity, 87.6% recovery) in the third distillate fraction and free phytosterols as FASEs (97 wt% purity, 97% recovery) in the last (fourth) residue.

Jacobs (Jacobs, 2005) proposed a method for recovering tocotrienols from fatty acid distillate FAD, which initially contained 1 wt% tocopherols and 0.3 wt% free phytosterols, by stripping FFAs (condition: 0.5–1.5 mm Hg, 180–240 ºC, and 0.5–1.5 min) to form a first stripped product. Short path distillation of the first stripped product gave a first distillate. Saponifying the second stripped product resulted in a saponified product with all FFA converted to FAMEs. A second short path distillation of the saponified product generated a second distillate without FAMEs. Solvent wintering (via filtration) of the second distillate gave a stripped filtrate. The stripped filtrate from the previous step is subjected to a third short path distillation at a temperature of 180° and an absolute pressure of 0.01 mm Hg. The resulting final tocotrienol product, about 1% of the original feed, contains from about 50% tocotrienols, 1% sterols and 49% other unsaponifiables and unknowns. Additionally, the final product contains from about 15% to about 30% tocopherols.

Fizet (Fizet, 1996) esterified free phytosterols from deodorizer distillate with FFAs at either 180 ºC for 2.5 h or 250 ºC for 1.5 h. Esterification product was then distilled at 120–150 ºC and 0.08 mm Hg to obtain a residue containing mostly tocopherols and FASEs and a distillate containing mostly fatty acids. The residue was then distilled again at 200–220 ºC and 0.1 mbar to obtain a distillate containing mostly tocopherols and a residue containing mostly FASEs. Tocopherols enriched in distillate were then subjected to an ion exchange chromatography and FASEs enriched in residue were then subjected to an acid-catalyzed transesterification with methanol to produce free phytosterols.

Even so, some authors (Martins *et al.*, 2006) have been trying to achieve an efficient FFA separation from SODD with the lowest loss of tocopherols by molecular distillation, without preliminary steps. This separation is difficult to achieve although is technologically viable at least at lab scale, due to the differences between molecular weights and vapour pressures of FFA (MW 180 g·mol-1, VP at 200 ºC = 4 mm Hg) and tocopherols (MW 415 g·mol-1, VP at 200 ºC = 0.15 mm Hg). Martins and coworkers (Martins, 2006) employed molecular distillation at 160 ºC, 7.5×10-4 mm Hg, and 10.4 g/min feed flow rate to remove FFAs into the distillate fraction and obtain a residue fraction, which contained 6.4 wt% FFAs and 18.3 wt% tocopherols from a SODD feed which contained 57.8 wt% FFAs and 8.97 wt% tocopherols. They succeeded in removing 96.16% FFAs and recovering 81.23% tocopherols. Martins and coworkers (Martins *et al.*, 2005) reported the isolation of tocopherols by first converting acylglycerols in SODD into FFAs through saponification at 65 ºC followed by acidulation, and then submitting the unsaponifiable product to five stages of molecular distillation. They succeeded in enriching tocopherols (34.14% purity) by 5.8 times. The major disadvantage in these cases is the residual free fatty acids in the tocopherol mixture.

Extraction and Enzymatic Modification of

2002).

column.

Functional Lipids from Soybean Oil Deodorizer Distillate 461

recycling the solvent does not endanger the viability of the process. The value of the rate of return on investment and time of return on investment for the process that does not recycle the carbon dioxide is higher than those of recycling the solvent. This is due to the compression cost that represents more than 59% of the total cost of the production (Mendes,

 SODD as such will not be feasible to work with SC-CO2 for the tocopherol enrichment, owing to its poor SC-CO2 solubility. So, to concentrate tocopherols from SODD, pretreatment of the raw material, including the esterification of free fatty acids and the removal of sterols with alcohol recrystallization, is needed to obtain the primary tocopherols concentrate with improve solubility in SC-CO2 (Shishikura *et al.*, 1988). For that, triglycerides and FFAs which constitute a major component in SODD have to be chemically

Several researchers have tried to concentrate tocopherols from SODD by supercritical CO2 (Lee, 1991), (Brunner *et al.*, 1991), (Brunner, 1994b), (Zhao *et al.*, 2000), (Nagesha, G. K. *et al.*, 2003), (Fang *et al.*, 2007), but the operation parameters, especially pressure, differ from author to author. Moreover, in all the cases, tocopherols content of the extract depended on

The interest in the tocopherol concentration using supercritical fluid extraction started with Lee and coworkers (Lee, 1991) followed by Brunner and coworkers (Brunner, 1991) and Brunner (Brunner, 1994b). The operational conditions used varied from 35 to 90 °C and from 200 to 400 bar using extractors or countercurrent columns. Lee and coworkers (Lee, 1991) attempted to modify soybean sludge chemically, to improve the solubility in SC-CO2. A simple batch process was utilized to recover tocopherols at 40% concentration at a pressure of 400 bar from the esterified soybean sludge which initially contained 13-14% tocopherols. The solubility of the esterified soybean sludge in supercritical carbon dioxide was more than 4 to 6 times higher than that of the sterols. Brunner and coworkers (Brunner, 1991) obtained a higher enrichment of tocopherols from SODD using supercritical carbon dioxide as a solvent compared to results obtained by Lee and coworkers This group recovered tocopherols from a model mixture of squalene, tocopherols, and sterols using two continuous countercurrent fractionation columns. Squalene was separated from the model mixture in the first column. Sterols were removed from the bottom of the second column, resulting in 85-95% concentration of tocopherol being obtained at the top of the second

These works concluded that the fatty acids are extracted initially and the tocopherols are enriched inside the extractor. The results also indicated that the solubility of the tocopherols

Chang and coworkers (Chang, 2000) worked on the separation of several SODD components. Their supercritical fluid extraction apparatus had a separation and an extraction unit. Free fatty acids, squalene and tocopherols were recovered in the extract and the sterols were recovered in the raffinates. The average tocopherol concentration factor was 1.38, which means that the mixture in the extract did not separate. However, the author mentioned that with the increase of CO2 volume, the separation factor can reach 1.7, but the poor increase in the concentration factor does not justify the raise in gas volume. The following research groups focused on the separation of the problematic pairs by supercritical fluids using synthetic mixtures: (Mendes, 2005), (Mendes *et al.*, 2000), (Wang *et*

is intermediary when compared to the solubilities of squalene and stigmasterol.

modified to obtain free fatty acids and then their methyl esters by esterification.

the composition and properties of the natural matrix.

*al.*, 2004), (Nagesha, 2004) and (Nagesha, 2003).

Among the great variety of processes that have been patented for the purification of the compounds of the SODD, only the processes of esterification of fatty acids and acylglycerols with methanol or ethanol followed by high vacuum distillation, have been developed on a commercial scale for the concentration of tocopherols (Takagi & Kai, 1984), (Su-Min *et al.*, 1992), (Yong-Bo *et al.*, 1994), (Rohr & Trujillo-Quijano, 2002). These processes are the most time efficient and economical methods, however high purity of sterols or tocopherols cannot be achieved due to the similar boiling points of these two substances.

In the case of separation by distillation of unsaponifiable valuable products of SODD subjected to saponification, the difference between the boiling point of volatile products, such as unsaponifiable components, and the boiling point of the sodium and potassium organic acid soaps is so great that separation is theoretically possible at a high level of efficiency. However, a problem related to this separation technique is that the soaps have a very high melting point, close to the decomposition temperature of the sodium or potassium soaps (i.e. the sodium or potassium salts of fatty acids, rosin acids etc), and, when melted, these soaps produce an extremely viscous liquids. These two factors combined make industrial handling difficult. Furthermore, while at the high temperature necessary to maintain their flow, these soaps are in permanent decomposition, compromising the separation output and the quality of the final product, as many of the unsaponifiable valuable products are heat sensitive.

#### **6. Supercritical fluid extraction (SFE)**

Although the conventional methods, vacuum and molecular distillation, have been applied to commercial production of tocopherols from SODD, there are some drawbacks such as residual solvents, high temperature, large amounts of energy consumption, high production costs and the unreliable quality of the products that require further developments. Since thermal degradation of tocopherols is commonly caused by processing at high temperatures (de Lucas *et al.*, 2002), new alternative isolation techniques are desired.

Supercritical carbon dioxide extraction is a process where carbon dioxide passes through a mixture of interest at a certain temperature and pressure until it reaches an extractor. This process is used because supercritical carbon dioxide has a low viscosity, a high diffusivity and a low surface tension that provides selective extraction, fractionation and purification, allowing its penetration in micro- and macro-porous materials. Carbon dioxide is the most desirable supercritical fluid solvent for the separation of natural products used in foods and medicines because of its inertness, nontoxicity, low cost, and high volatility. The major advantage of this method is the easy post-reaction separation of the components by depressurization, so resultant extract does not contain solvent residue and hence naturalquality extracts can be obtained. Another advantage is the low temperatures used for the majority of the experimentations because carbon dioxide has a near-ambient critical temperature (31.1 °C), so is suitable for thermolabile natural products.

However, the use of high pressure conditions to concentrate tocopherols makes the system energetically expensive, but the industrial process can be economically viable using conditions of approximately 90 atm and 40 °C (Mendes *et al.*, 2002). At these specific conditions, only fatty acids are separated from tocopherol (Mendes *et al.*, 2005). An increase in pressure and temperature increases the oil extraction and tocopherol recovery, although different pressure–temperature systems need to be used in order to separate the different components (sterols, tocopherols, fatty acids and squalene). It is important to know that

Among the great variety of processes that have been patented for the purification of the compounds of the SODD, only the processes of esterification of fatty acids and acylglycerols with methanol or ethanol followed by high vacuum distillation, have been developed on a commercial scale for the concentration of tocopherols (Takagi & Kai, 1984), (Su-Min *et al.*, 1992), (Yong-Bo *et al.*, 1994), (Rohr & Trujillo-Quijano, 2002). These processes are the most time efficient and economical methods, however high purity of sterols or tocopherols cannot

In the case of separation by distillation of unsaponifiable valuable products of SODD subjected to saponification, the difference between the boiling point of volatile products, such as unsaponifiable components, and the boiling point of the sodium and potassium organic acid soaps is so great that separation is theoretically possible at a high level of efficiency. However, a problem related to this separation technique is that the soaps have a very high melting point, close to the decomposition temperature of the sodium or potassium soaps (i.e. the sodium or potassium salts of fatty acids, rosin acids etc), and, when melted, these soaps produce an extremely viscous liquids. These two factors combined make industrial handling difficult. Furthermore, while at the high temperature necessary to maintain their flow, these soaps are in permanent decomposition, compromising the separation output and the quality of the final product, as many of the unsaponifiable

Although the conventional methods, vacuum and molecular distillation, have been applied to commercial production of tocopherols from SODD, there are some drawbacks such as residual solvents, high temperature, large amounts of energy consumption, high production costs and the unreliable quality of the products that require further developments. Since thermal degradation of tocopherols is commonly caused by processing at high temperatures

Supercritical carbon dioxide extraction is a process where carbon dioxide passes through a mixture of interest at a certain temperature and pressure until it reaches an extractor. This process is used because supercritical carbon dioxide has a low viscosity, a high diffusivity and a low surface tension that provides selective extraction, fractionation and purification, allowing its penetration in micro- and macro-porous materials. Carbon dioxide is the most desirable supercritical fluid solvent for the separation of natural products used in foods and medicines because of its inertness, nontoxicity, low cost, and high volatility. The major advantage of this method is the easy post-reaction separation of the components by depressurization, so resultant extract does not contain solvent residue and hence naturalquality extracts can be obtained. Another advantage is the low temperatures used for the majority of the experimentations because carbon dioxide has a near-ambient critical

However, the use of high pressure conditions to concentrate tocopherols makes the system energetically expensive, but the industrial process can be economically viable using conditions of approximately 90 atm and 40 °C (Mendes *et al.*, 2002). At these specific conditions, only fatty acids are separated from tocopherol (Mendes *et al.*, 2005). An increase in pressure and temperature increases the oil extraction and tocopherol recovery, although different pressure–temperature systems need to be used in order to separate the different components (sterols, tocopherols, fatty acids and squalene). It is important to know that

be achieved due to the similar boiling points of these two substances.

(de Lucas *et al.*, 2002), new alternative isolation techniques are desired.

temperature (31.1 °C), so is suitable for thermolabile natural products.

valuable products are heat sensitive.

**6. Supercritical fluid extraction (SFE)**

recycling the solvent does not endanger the viability of the process. The value of the rate of return on investment and time of return on investment for the process that does not recycle the carbon dioxide is higher than those of recycling the solvent. This is due to the compression cost that represents more than 59% of the total cost of the production (Mendes, 2002).

 SODD as such will not be feasible to work with SC-CO2 for the tocopherol enrichment, owing to its poor SC-CO2 solubility. So, to concentrate tocopherols from SODD, pretreatment of the raw material, including the esterification of free fatty acids and the removal of sterols with alcohol recrystallization, is needed to obtain the primary tocopherols concentrate with improve solubility in SC-CO2 (Shishikura *et al.*, 1988). For that, triglycerides and FFAs which constitute a major component in SODD have to be chemically modified to obtain free fatty acids and then their methyl esters by esterification.

Several researchers have tried to concentrate tocopherols from SODD by supercritical CO2 (Lee, 1991), (Brunner *et al.*, 1991), (Brunner, 1994b), (Zhao *et al.*, 2000), (Nagesha, G. K. *et al.*, 2003), (Fang *et al.*, 2007), but the operation parameters, especially pressure, differ from author to author. Moreover, in all the cases, tocopherols content of the extract depended on the composition and properties of the natural matrix.

The interest in the tocopherol concentration using supercritical fluid extraction started with Lee and coworkers (Lee, 1991) followed by Brunner and coworkers (Brunner, 1991) and Brunner (Brunner, 1994b). The operational conditions used varied from 35 to 90 °C and from 200 to 400 bar using extractors or countercurrent columns. Lee and coworkers (Lee, 1991) attempted to modify soybean sludge chemically, to improve the solubility in SC-CO2. A simple batch process was utilized to recover tocopherols at 40% concentration at a pressure of 400 bar from the esterified soybean sludge which initially contained 13-14% tocopherols. The solubility of the esterified soybean sludge in supercritical carbon dioxide was more than 4 to 6 times higher than that of the sterols. Brunner and coworkers (Brunner, 1991) obtained a higher enrichment of tocopherols from SODD using supercritical carbon dioxide as a solvent compared to results obtained by Lee and coworkers This group recovered tocopherols from a model mixture of squalene, tocopherols, and sterols using two continuous countercurrent fractionation columns. Squalene was separated from the model mixture in the first column. Sterols were removed from the bottom of the second column, resulting in 85-95% concentration of tocopherol being obtained at the top of the second column.

These works concluded that the fatty acids are extracted initially and the tocopherols are enriched inside the extractor. The results also indicated that the solubility of the tocopherols is intermediary when compared to the solubilities of squalene and stigmasterol.

Chang and coworkers (Chang, 2000) worked on the separation of several SODD components. Their supercritical fluid extraction apparatus had a separation and an extraction unit. Free fatty acids, squalene and tocopherols were recovered in the extract and the sterols were recovered in the raffinates. The average tocopherol concentration factor was 1.38, which means that the mixture in the extract did not separate. However, the author mentioned that with the increase of CO2 volume, the separation factor can reach 1.7, but the poor increase in the concentration factor does not justify the raise in gas volume. The following research groups focused on the separation of the problematic pairs by supercritical fluids using synthetic mixtures: (Mendes, 2005), (Mendes *et al.*, 2000), (Wang *et al.*, 2004), (Nagesha, 2004) and (Nagesha, 2003).

Extraction and Enzymatic Modification of

tocopherol recovery (about 80%).

Functional Lipids from Soybean Oil Deodorizer Distillate 463

initial pressure, feed location, temperature gradient, and ratio of CO2 to ME-DOD were optimized for separating FAMEs. For the following tocopherol concentration step, a final pressure of 200 bar resulted in the greatest average tocopherol content (>50%) and

The important step in concentrating natural tocopherols from these systems is to remove the FAMEs. FAMEs are important chemical materials in biofuel, metal-cutting oil, and cleaning agent production, as well as in the synthesis of other fatty acid products (Swern, 1986). Some works on phase equilibrium for the realistic system of modified esterification SODD/supercritical CO2 (Fang *et al.*, 2005) established that the separation factor 1 between tocopherols and FAMEs was always smaller than unity in the range investigated. This indicates that when supercritical CO2 is used as the separation solvent, tocopherols, unlike FAMEs, tend to enrich in the liquid phase. In particular, the separation factors at pressures lower than 200 bar were relatively small. At 40 ºC, for instance, the separation factor remained lower than 0.2 for all pressures lower than 150 bar. As pressure increased, the separation factor increased greatly, reaching 0.35 at 200 bar. The influence of temperature was contrary to that of pressure, with an increase in temperature leading to a decrease in separation factor. Low pressure and high temperature result in high selectivity, indicated by a low separation factor, which is advantageous in the separation of FAMEs from tocopherols with supercritical CO2. King and coworkers (King *et al.*, 1996) combined supercritical fluid extraction (SFE) with supercritical fluid chromatography (SFC) for concentrating tocopherols and the optimized conditions were 250 bar/80 ºC for SFE and 250 bar/40 ºC for SFC. Approximately 60% of the available tocopherols in soyflakes can be recovered in the SFE step, yielding enrichment factors of 1.83-4.33 for the four tocopherol species found in soybean oil. Additional enrichment of tocopherol species can be realized in the SFC stage, with enrichment factors 2

Starting with a feed containing 48.3 wt% tocopherols, Gast and coworkers (Gast *et al.*, 2005) were able to obtain tocopherols with a purity of 94.4 wt% in bottom phase by supercritical CO2 extraction at 230 bar and 80 ºC, with a solvent-to-feed ratio of 110 and a reflux ratio of

Torres and coworkers (Torres, 2007) proposed a two-step enzymatic procedure to obtain FASEs, tocopherols, and fatty acid ethyl esters (FAEEs) from SODD, together with minor amounts of squalene, free fatty acids, free sterols and triacylglycerols. The final product obtained was used as starting material to purify FASEs, tocopherols, and FAEEs via supercritical CO2 extraction The phytosterol esters were then purified from this mixture using supercritical carbon dioxide (Torres, 2009). Experimental extractions were carried out in an isothermal countercurrent column (without reflux), with pressures ranging from 200 to 280 bar, temperatures of 45-55 °C and solvent-to-feed ratios from 15 to 35 kg/kg. Using these extraction conditions, the fatty acid esters were completely extracted and, thus, the fractionation of tocopherols and phytosterol esters was studied. At 250 bar, 55 °C and a

1 The separation factor represents the process selectivity for separating methyl oleate from tocopherol. In detail, a lower value indicates higher selectivity, whereas a higher value indicates that it is more difficult to separate the two compounds under certain conditions. Furthermore, when the separation factor equals unity, the composition in the gas phase is similar to that in the liquid, and the supercritical

2 The enrichment factors were the ratio of individual tocopherols in extracts versus the same tocopherol

ranging from 30.8 for delta-tocopherol to 2.41 for beta-tocopherol.

4.6. Squalene was completely recovered in top phase.

CO2 process cannot separate methyl oleate from tocopherol.

content initially found in the soyflakes.

There are some interesting relationships and conclusions that can be deduced from the results obtained by Mendes (Mendes, 2005, Mendes, 2000) and Chang (Chang, 2000) regarding the yield and concentration factor of pairs of compounds at different conditions of pressure and temperature. The binary mixture of tocopherol and squalene cannot be separated at low pressure conditions. An acceptable separation needs a raise in pressure to almost 203 bar ((Mendes, 2005) and (Mendes, 2000)). However, a recovery of 90% and a purity of 60% of α-tocopherol has been achieved using a pressure swing adsorption (PSA) device, that is a widely used process in the separation of gas mixtures for air-drying, oxygen and nitrogen separation of air, hydrogen purification, and various other separations. The PSA process is based on the regeneration of adsorber by the difference in adsorbed amounts of gas solute as a function of pressure. In the case of a two-bed process, one bed is in the adsorption step, while the other is simultaneously in the desorption step. The adsorption and desorption steps had pressure conditions of 160 and300 bar, respectively (Wang, 2004).

The ternary mixture of tocopherol, fatty acids and squalene behaved differently from the tocopherol–fatty acid binary mixture. For the same conditions of pressure (160 and 300 bar), the binary mixture had a total separation while the ternary mixture did not achieve any separation. Squalene and stigmasterol mixtures are also very difficult to separate. At low pressure conditions, the yield is less than 10% but at higher pressure, the yield is 76%. It is important to note that low temperatures were used in these studies.

Results of these authors suggest that the supercritical-CO2 process could be used for the separation of squalene, fatty acids and tocopherols. In order to enhance the squalene, fatty acids and tocopherols separation, deodorizer distillate mixtures should be processed several times in supercritical-CO2 at different temperature and pressure conditions. However, there is no data reported regarding the total extraction time of the mixture and this makes it impossible to estimate the operational cost. The major advantage of this method is the total removal of free fatty acids from the mixture.

Zhao and coworkers (Zhao, 2000) concentrated tocopherols up to 75% at 120 bar using a fractionation column with a gradient of temperature from 30–80 °C in a pilot plant scale.

Nagesha and coworkers (Nagesha, 2003) performed chemical modification of SODD containing about 2.9 wt% of tocopherols, as well as triglycerides (56 wt%), free fatty acids (25.3 wt%), sterols (7.8 wt%), hydrocarbons (0.6 wt%), and unsaponifiables (6.4 wt%) apart from tocopherols. Chemical modification of SODD included saponification and esterification steps to result in fatty acid methyl esters from free fatty acids, so as to improve the solubility of SODD in SC-CO2 extraction. Reactions were conducted in dark with continuous flushing of N2 and 1.0 wt% of pyrogallol was added to prevent the oxidation of tocopherols. After chemical modification, esterified SODD contained about 3.7 wt% of tocopherols. Tocopherols concentrates of about 36% was obtained by SC-CO2 extraction at the pressure 180 bar and temperature 60 ºC.

Fang and coworkers (Fang, 2007) carried out a pretreatment of methyl esterification and methanolysis reactions, which converted most of free fatty acids and glycerides to fatty acid methyl esters (FAMEs), respectively, to simplify the composition of SODD and improve his solubility in supercritical CO2 extraction. The mixture was held at 3 ºC in a refrigerator for 12 h, as a result most of sterols were crystallized and removed by filtering under a reduced pressure. Supercritical CO2 fractionation was employed to concentrate tocopherols from Methyl Ester Oil Deodorizer Distillate (ME-DOD) product, mainly contained FAMEs (65– 80 wt.%), tocopherols (10–15 wt.%), and impurities (such as residual sterols, glycerides, squalene, pigments, and long chain paraffins, comprising in total about 10–15 wt.%). The

There are some interesting relationships and conclusions that can be deduced from the results obtained by Mendes (Mendes, 2005, Mendes, 2000) and Chang (Chang, 2000) regarding the yield and concentration factor of pairs of compounds at different conditions of pressure and temperature. The binary mixture of tocopherol and squalene cannot be separated at low pressure conditions. An acceptable separation needs a raise in pressure to almost 203 bar ((Mendes, 2005) and (Mendes, 2000)). However, a recovery of 90% and a purity of 60% of α-tocopherol has been achieved using a pressure swing adsorption (PSA) device, that is a widely used process in the separation of gas mixtures for air-drying, oxygen and nitrogen separation of air, hydrogen purification, and various other separations. The PSA process is based on the regeneration of adsorber by the difference in adsorbed amounts of gas solute as a function of pressure. In the case of a two-bed process, one bed is in the adsorption step, while the other is simultaneously in the desorption step. The adsorption and desorption steps had pressure conditions of 160 and300 bar, respectively (Wang, 2004). The ternary mixture of tocopherol, fatty acids and squalene behaved differently from the tocopherol–fatty acid binary mixture. For the same conditions of pressure (160 and 300 bar), the binary mixture had a total separation while the ternary mixture did not achieve any separation. Squalene and stigmasterol mixtures are also very difficult to separate. At low pressure conditions, the yield is less than 10% but at higher pressure, the yield is 76%. It is

Results of these authors suggest that the supercritical-CO2 process could be used for the separation of squalene, fatty acids and tocopherols. In order to enhance the squalene, fatty acids and tocopherols separation, deodorizer distillate mixtures should be processed several times in supercritical-CO2 at different temperature and pressure conditions. However, there is no data reported regarding the total extraction time of the mixture and this makes it impossible to estimate the operational cost. The major advantage of this method is the total

Zhao and coworkers (Zhao, 2000) concentrated tocopherols up to 75% at 120 bar using a fractionation column with a gradient of temperature from 30–80 °C in a pilot plant scale. Nagesha and coworkers (Nagesha, 2003) performed chemical modification of SODD containing about 2.9 wt% of tocopherols, as well as triglycerides (56 wt%), free fatty acids (25.3 wt%), sterols (7.8 wt%), hydrocarbons (0.6 wt%), and unsaponifiables (6.4 wt%) apart from tocopherols. Chemical modification of SODD included saponification and esterification steps to result in fatty acid methyl esters from free fatty acids, so as to improve the solubility of SODD in SC-CO2 extraction. Reactions were conducted in dark with continuous flushing of N2 and 1.0 wt% of pyrogallol was added to prevent the oxidation of tocopherols. After chemical modification, esterified SODD contained about 3.7 wt% of tocopherols. Tocopherols concentrates of about 36% was obtained by SC-CO2 extraction at

Fang and coworkers (Fang, 2007) carried out a pretreatment of methyl esterification and methanolysis reactions, which converted most of free fatty acids and glycerides to fatty acid methyl esters (FAMEs), respectively, to simplify the composition of SODD and improve his solubility in supercritical CO2 extraction. The mixture was held at 3 ºC in a refrigerator for 12 h, as a result most of sterols were crystallized and removed by filtering under a reduced pressure. Supercritical CO2 fractionation was employed to concentrate tocopherols from Methyl Ester Oil Deodorizer Distillate (ME-DOD) product, mainly contained FAMEs (65– 80 wt.%), tocopherols (10–15 wt.%), and impurities (such as residual sterols, glycerides, squalene, pigments, and long chain paraffins, comprising in total about 10–15 wt.%). The

important to note that low temperatures were used in these studies.

removal of free fatty acids from the mixture.

the pressure 180 bar and temperature 60 ºC.

initial pressure, feed location, temperature gradient, and ratio of CO2 to ME-DOD were optimized for separating FAMEs. For the following tocopherol concentration step, a final pressure of 200 bar resulted in the greatest average tocopherol content (>50%) and tocopherol recovery (about 80%).

The important step in concentrating natural tocopherols from these systems is to remove the FAMEs. FAMEs are important chemical materials in biofuel, metal-cutting oil, and cleaning agent production, as well as in the synthesis of other fatty acid products (Swern, 1986).

Some works on phase equilibrium for the realistic system of modified esterification SODD/supercritical CO2 (Fang *et al.*, 2005) established that the separation factor 1 between tocopherols and FAMEs was always smaller than unity in the range investigated. This indicates that when supercritical CO2 is used as the separation solvent, tocopherols, unlike FAMEs, tend to enrich in the liquid phase. In particular, the separation factors at pressures lower than 200 bar were relatively small. At 40 ºC, for instance, the separation factor remained lower than 0.2 for all pressures lower than 150 bar. As pressure increased, the separation factor increased greatly, reaching 0.35 at 200 bar. The influence of temperature was contrary to that of pressure, with an increase in temperature leading to a decrease in separation factor. Low pressure and high temperature result in high selectivity, indicated by a low separation factor, which is advantageous in the separation of FAMEs from tocopherols with supercritical CO2.

King and coworkers (King *et al.*, 1996) combined supercritical fluid extraction (SFE) with supercritical fluid chromatography (SFC) for concentrating tocopherols and the optimized conditions were 250 bar/80 ºC for SFE and 250 bar/40 ºC for SFC. Approximately 60% of the available tocopherols in soyflakes can be recovered in the SFE step, yielding enrichment factors of 1.83-4.33 for the four tocopherol species found in soybean oil. Additional enrichment of tocopherol species can be realized in the SFC stage, with enrichment factors 2 ranging from 30.8 for delta-tocopherol to 2.41 for beta-tocopherol.

Starting with a feed containing 48.3 wt% tocopherols, Gast and coworkers (Gast *et al.*, 2005) were able to obtain tocopherols with a purity of 94.4 wt% in bottom phase by supercritical CO2 extraction at 230 bar and 80 ºC, with a solvent-to-feed ratio of 110 and a reflux ratio of 4.6. Squalene was completely recovered in top phase.

Torres and coworkers (Torres, 2007) proposed a two-step enzymatic procedure to obtain FASEs, tocopherols, and fatty acid ethyl esters (FAEEs) from SODD, together with minor amounts of squalene, free fatty acids, free sterols and triacylglycerols. The final product obtained was used as starting material to purify FASEs, tocopherols, and FAEEs via supercritical CO2 extraction The phytosterol esters were then purified from this mixture using supercritical carbon dioxide (Torres, 2009). Experimental extractions were carried out in an isothermal countercurrent column (without reflux), with pressures ranging from 200 to 280 bar, temperatures of 45-55 °C and solvent-to-feed ratios from 15 to 35 kg/kg. Using these extraction conditions, the fatty acid esters were completely extracted and, thus, the fractionation of tocopherols and phytosterol esters was studied. At 250 bar, 55 °C and a

 1 The separation factor represents the process selectivity for separating methyl oleate from tocopherol. In detail, a lower value indicates higher selectivity, whereas a higher value indicates that it is more difficult to separate the two compounds under certain conditions. Furthermore, when the separation factor equals unity, the composition in the gas phase is similar to that in the liquid, and the supercritical CO2 process cannot separate methyl oleate from tocopherol.

<sup>2</sup> The enrichment factors were the ratio of individual tocopherols in extracts versus the same tocopherol content initially found in the soyflakes.

Extraction and Enzymatic Modification of

or complement other methods at industrial scale.

**8.1 Influence of refining on phytosterols**

**8. Degradation and oxidation of functional lipids from SODD**

collected after chemical and physical refining processes (Dowd, 1998).

esterified sterols has been studied by (Verleyen, 2002b).

loss in the soapstock (Gutfinger & Letan, 1974).

*al.*, 1994).

Functional Lipids from Soybean Oil Deodorizer Distillate 465

These methods do not present a significant advance regarding the most frequently utilized methods and probably their application to production scale would be little profitable. However, they can be used to isolate tocopherols and sterols from SODD at laboratory scale

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

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

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

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*

Whenever applied, hydrogenation has a tremendous effect on sterol structures, including hydrogenation of double bonds, opening of cyclopropane rings, and positional

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

isomerization of side chain unsaturation (Strocchi & Marascio, 1993).

solvent-to-feed ratio of 35, the phytosterol esters were concentrated in the raffinate up to 82.4 wt-% with satisfactory yield (72%).

Other supercritical fluids have been explored but unsuccessfully for the separation of different pairs of components. An attempt at using similar methodology to (Mendes, 2000) and (Mendes, 2005) but using liquid gas petroleum instead of carbon dioxide did not change the poor concentration factor between the critical pairs of components (Buczenko *et al.*, 2003). Buczenko and coworkers (Buczenko, 2003) performed the saponification of the raw material and the extraction of unsaponifiable matter as pre-treatment of VODD.

As discussed above, there are a lot of experimental studies proving the efficiency of the supercritical extraction to concentrate the vitamin E from different raw material or in some cases, from synthetic mixtures representing the deodorizer distillate, but the extraction of sterols using supercritical fluid from the deodorizer distillate was not described in the literature.

On the other hand, due to the low content of squalene in SODD, specific extraction processes of squalene using supercritical fluid from SODD was not described in the literature. Existing studies are models of fractionation of artificial mixtures such as those mentioned above (Chang, 2000). For example, Brunner (Brunner, 1994a) studied the phase equilibrium for recovering α-tocopherol from a mixture of squalene, tocopherol, and campesterol. He concluded that the separation factor for squalene/α-tocopherol varied between a value of 4 at low squalene concentrations (0.5 wt %), to a value of 1 at high squalene concentrations (85 wt %), at pressures ranging from 200 to 300 bar and temperatures ranging from 70 to 100 ºC.

Bondioli and coworkers (Bondioli *et al.*, 1993) esterified FFAs into their corresponding glycerides and then applied a supercritical carbon dioxide extraction to produce a squaleneenriched fraction (purity 90.0%, yield 91.1%), but from olive oil deodorizer distillates.