Lipids from natural products

Prospective applications for the SFE of lipids or oils, apart from common vegetable oils (soy oils, corn oil, rice bran oil, sunflower oil, olive oil, etc.), also include animal fats, fish oil, oil from seaweeds and oil from microorganisms like fungi (Walker et al., 1999; Mukhopadhyay, 2000; Shen et al., 1996). Major components of lipids include monoglycerides, diglycerides, triglycerides and free fatty acids (FFAs); minor constituents include sterols, toco-pherols, gums, alkaloids, flavonoids, wax and volatile compounds, which provide taste and odor. Most studies concerning SFE of lipids are focused on the optimization of extraction conditions to increase the yield of extract-able materials (Hu, 1995). Several components of lipids have significant health and nutritional implications for the food and pharmaceutical industries. PUFAs have important therapeutic value (Shahidi and Wanasundara, 1998; Galli and Butrum, 1991). Unsaturated fatty acids and saturated fatty acids have different health effects. Sterols, antioxidants, wax and volatile compounds are also significantly important for health. Major SFE applications include separation of FFAs from vegetable oils, separation of PUFA from animal fats, refining and deodorization of vegetable oils, fractionation of glycerides, recovery of oil from biological materials, de-oiling of lecithin, and de-cholesterolization and de-lipidation of food products (Mukhopadhyay, 2000).

King and List (1991) reviewed the SFE of fat and observed that the solubility of fats in supercritical CO2 generally increases with pressure and temperature. At very low pressures, the solubility of fats is slightly higher at lower temperatures. However, they found that SFE of fat worked best at high temperatures above 80 °C and pressure above 55.16 MPa or 8000 psi. Smaller particles yielded more oil as did dryer material. They described SFE

Instrumentation

Valves

A

CO2 cylinder

1

CO2 cylinder supply (from A)

B

CO2 syringe pump

2

CO2 pump supply

C

Water bath

3

CO2 syringe pump isolation

D

Preheating coil for CO2

4

Vessel isolation

E

Explosion/extraction vessel

5a

CO2 flow control

F

Hexane reservoir

5b

Hexane rinse control

G

Vessel pressure transducer

6

CO2 micrometering

H

Gas flow meter

7

Explosion high pressure release

I

Hexane cold trap

8a

CO2 by-pass

J

Flow control section heating tape 8b

Hexane reservoir outlet

K

CO2 feed ice bath

9

CO2 pressure release

Fig. 10.3 Schematic of the supercritical CO2 extraction apparatus.

Fig. 10.3 Schematic of the supercritical CO2 extraction apparatus.

as a promising technology for the extraction of small-scale, high-value products from fat-bearing materials. Bjergegaard et al. (1999) compared SFE and conventional solvent extraction for the extraction of volatiles and hydrophilic compounds from rapeseed, sunflower and soybean. SFE was also useful for analytical purposes to ascertain lipid content. Montanari et al. (1996) used supercritical CO2 and a co-solvent (ethanol) for the selective extraction of phospholipids from soybean flakes. Taylor and King (2000) used analytical-scale SFE and supercritical fluid chromatography (SFC) for the optimization and fractionation of corn bran oil to achieve a high concentration of ferulate phytosterol esters (FPE). They extracted a maximum 1.25% FPE from corn bran over the different combinations of temperatures (40, 60 and 80 °C) and pressures (13.8, 34.5 and 69 MPa) tried during the experiments.

Eggers and Sievers (1989) studied the SFE of rapeseed with different pretreatments and observed that flaked rapeseed cake and higher pressures were beneficial. Fattori et al. (1988) studied supercritical extraction of canola seed oil (25-90 °C, 10-36 MPa) and found that oil solubility in supercritical CO2 was strongly dependent on pressure, but was not significantly dependent on temperature. The total oil recovery was also significantly dependent on the pretreatment of the seed (flaking, cooking, pressure rupture, chopping, crushing). Greater amounts of oil were recovered from flaked and cooked seed compared with whole seed.

Brown seaweed extraction with supercritical CO2 (24.1-37.9 MPa, 40-50 °C) was compared with Soxhlet extraction using chloroform : methanol (2 : 1, v/v) (Cheung et al., 1998). Oil yields of SFE at 37.9 MPa (40-50 °C) were comparable to Soxhlet extraction, but w-3-fatty acid concentrations were higher (31.4%) in supercritical extract compared with Soxhlet extraction (23.5%). For constant pressure (24.1 MPa), SFE yielded more lipids at 40 °C than at 50 °C. The concentration of total PUFAs in the oil decreased significantly, and that of total saturated fatty acids increased significantly with increased pressure and solvent density.

Studies on the extraction of spearmint oil (essential oil of Mentha spicata) from Turkish mint plant leaves with supercritical CO2 indicated that the concentration of the monoterpenes fraction in the oil and oil yields were inversely related. Compared with conventional methods of hydrodistillation (HD), SFE produced lower concentrations of the monoterpenes in the oil at a low temperature that was safe for heat-sensitive essential oils (Ozer et al., 1996). Lavender essential oil and wax extraction with supercritical CO2 resulted in higher linalyl acetate content in the oil (34.7%) compared with conventional hydro-distillation (12.1%) (Reverchon and Porta, 1995).

Other recent lipid extraction studies using SFE include canola oil (Bulley and Fattori, 1984; Temelli, 1992), citrus oil (Sato et al., 1998), menhaden oil (Rizvi et al., 1988; Nilsson et al., 1988), rapeseed oil (Eggers and Sievers, 1989), evening primrose oil (Favati et al., 1991), soybean oil (List et al., 1993), soya, canola and corn germ oils (Taylor et al., 1993), peppermint oil (Motonobu et al., 1993), caraway essential oil (Sovova et al., 1994a), soybean oil (Reverchon and Osseo, 1994), ginger oil (Roy et al., 1996), cloudberry seed oil (Manninen et al., 1997), sunflower oil (Perrut et al., 1997), pistachio nut lipids (Palazoglu and Balaban, 1998), almond oil (Marrone et al., 1998), lavender essential oils and waxes (Akgun et al., 2000), grape seed oil (Lee et al., 2000; Murga et al., 2002), hiprose seed oil (Reverchon et al., 2000) and Romanian mentha hybrids oil (Eugenia and Danielle, 2001).

King et al. (1996) used combined SFE (25 MPa and 80 °C) and SFC (1.7 cm diameter and 20 cm long columns charged with 60-200 mesh silica gel, 16 g, in a preparative mode) to fractionate and enrich tocopherol components of the oil from soybean flakes and rice bran. Total tocopherol recovery and enrichments were observed as a function of the mass ratio of CO2/seed charge. Also, tocopherol recovery differed from one seed type to another. Garcia et al. (1996) found that at 28 MPa and 70 °C (highest allowable pressure and temperature in their system) they obtained 16-60% of solvent-extractable oil yield from rice bran. Oil obtained by SFE was lighter in color, high in waxes and had greater long-chain fatty acids (C20-C34) compared with hexane-extracted oil.

Kuk and Dowd (1998) carried out SFE of rice bran (6% moisture, below 0.297 mm particle size) at 48.26 and 62.05 MPa for 1.5 h and reported 19.2-20.4% rice bran oil yield, compared with 20.5% extraction yield using hexane in 4 h. They also found increases in rice bran oil yield with increasing temperatures at constant pressure. Sterol extraction was found to increase with increasing pressure and temperature. Kim et al. (1999) compared essential fatty acids (EFAs) in rice bran oil extracted under different conditions (40, 50 and 70 °C; 20.68, 27.58, 34.47 and 41.37 MPa). They found yields to be dependent on reduced density of supercritical CO2. Up to 70-80% of rice bran oil may be extracted in 4 h. Xu and Godber (2000) compared solvent extraction (50% hexane and 50% isopropanol v/v) of rice bran with supercritical CO2 extraction at 50 °C and 68.9 MPa pressure for extraction of y-oryzanol, an important antioxidant component (Xu and Godber, 1999; Xu et al., 2001). Their studies indicated that SFE extraction may extract up to four times more y-oryzanol (5.39 mg/g of rice bran) in less time compared with solvent extraction. Table 10.2 shows the major sterols co-extracted with various natural oils. These sterols have great potential as precursors to pharmaceutical compounds.

Lipids from fungal and algal biomass

The beneficial health effects of consuming PUFAs, which include (C20:5; rn-3) eicosapentaenoic acid (EPA) and (C22:6; rn-3) docosahexaenoic acid (DHA), have been well documented over the years (Gilli and Valivety, 1997; Li et al., 2003). These fatty acids have been linked to visual and mental health as well as regulating critical biological functions (Simopoulos et al., 1991; Bajpai and Bajpai, 1993; Barclay, 1997). PUFAs are associated with the prevention and treatment of coronary heart disease and abnormal cholesterol levels, in addition to alleviating inflammatory conditions (Babcock

Table 10.2 Comparison of sterols and triterpenes in different oils (% in oil) (from Rukmini and Raghuram, 1991)

Oil

Campesterol

Stigmasterol

P-Sito-sterol

Cycloartanol

Cycloartenol

24-Methylene cyloartanol

Rice bran

0.506

0.271

0.885

0.106

0.482

0.494

Safflower

0.045

0.031

0.181

0.001

0.034

0.007

Corn

0.410

0.110

1.180

0.004

0.008

0.011

Sunflower

0.031

0.031

0.235

-

0.029

0.016

Cottonseed

0.017

0.004

0.400

-

0.010

0.017

Sesame

0.117

0.062

0.382

0.004

0.062

0.107

Soybean

0.072

0.072

0.191

-

0.168

0.008

Groundnut

0.036

0.021

0.153

0.001

0.011

0.016

Source: Rukmani and Raghuram (1991)

Source: Rukmani and Raghuram (1991)

et al., 2000) and even retarding growth of tumor cells (Youdim et al., 2000; Tapiero et al., 2002; Voet and Voet, 2004). Filamentous fungi, like Pythium irregulare (Cheng et al., 1999; Singh and Ward, 1997; Stredansky et al., 2000), and algae like Crypthecodinium cohnii (DeSwaaf et al., 2002), are identified as microorganisms that can produce health beneficial, valuable PUFA rich oils (Walker et al., 1999) and can produce EPA and DHA at 10-50% of the total intracellular lipids (De Swaaf et al., 2002). The downstream recovery and purification of this PUFA-rich oil impose certain restraints since these compounds are thermally labile, fragile and easily oxidized. SFE techniques have been applied to the extraction of said oils; however, the intracellular oils can be difficult to extract from the biomass since most of the cells remain intact during the extraction process.

Fish oil supplements dominate the current PUFA market; however, fish oil possesses objectionable tastes and odors along with cholesterol and small amounts of pollutants that may include mercury (Simopoulos, 2004). Microorganisms are promising producers of PUFA-rich oils that can serve as an alternative to the oils obtained from agricultural and animal sources. The technology to produce EPA and DHA from microalgae and fungi has proven to be commercially feasible (Kyle, 2001); these organisms are capable of year-round oil production on a variety of cheap substrates (Bajpai and Bajpai, 1993; Nettleton, 1995; Kyle, 1996; Uauy et al., 2001; Simopoulos, 2004; Voet and Voet, 2004; Ward and Singh, 2005). The filamentous fungi Pythium irregulare is a well-researched organism capable of producing EPA and other long-chain fatty acids such as linoleic (C18:2 rn-6) and arachidonic (C20:4 rn-6) acids (Certik and Shimizu, 1999; Belarbi Medina et al., 2000; Robles Medina et al., 1998; Wen and Chen, 2003).

Badal (2002) studied the effects of particle size (16-48 mesh and >48 mesh) and biotreatment with Pythium irregulare fungi, on the yield and the quality of rice bran oil extracted with supercritical CO2 (40 °C, 27.57 MPa, 200 standard cm3 per min). The extraction yield was approximately 50% of the total ether Soxhlet extractable oil (in 2 h) from the smaller-particle rice bran. Eicosapentaenoic acid and arachidonic acid produced during the treatment by Pythium irregulare were extracted by SFE.

Enzyme reactions in supercritical fluids

Enzymes show significant stability when immobilized in CO2. The stability is partially attributed to the absence of oxidation reactions (Shishikura et al., 1994). Fungal enzymes are typically more stable in non-aqueous environments than bacteria-derived enzymes due to the functionality of fungi at lower water activities (Svensson et al., 1994). Fungal lipases are particularly well suited to catalyze reactions in non-aqueous environ ments. This is not only due to this enzyme's stability, but the reacting substrates and products are usually soluble in the non-aqueous phase. Therefore, the equilibrium of the enzyme-catalyzed reaction may be improved by the facilitated removal of byproducts with CO2 (Shishikura et al., 1994).

An important group of lipases include the 1,3 regiospecific enzymes (e.g. from Mucor miehei and Rhizopus arrhizus) that produce diacyl-glycerols, 2-monoacylglycerols and FFAs. This leads to a number of important reactions that include transesterification and interesterification of lipids. For instance, Shishikura et al. (1994) found that lipase-catalyzed interesterification - using the immobilized Mucor miehei lipase, lipozyme -between medium-chain length triglycerides and free long-chain fatty acids may be accomplished, while the byproducts of the reaction are simultaneously extracted with CO2.

Enzymes, however, require an aqueous phase directly adjacent to the enzyme to remain active. Therefore, water activity is an important parameter for enzyme functionality in non-aqueous environments and should be controlled to optimize the reaction kinetics (Svensson et al., 1994). Aqueous phases are required for enzyme reactions, but also cause unwanted hydrolysis reactions during interesterification with the lipase enzyme (Shishikura et al., 1994). Therefore it is advantageous to bring the reaction mixture into contact with CO2 to extract fatty acid moieties produced during the interesterification to improve equilibrium conditions.

Mucor miehei lipase has been shown to be very effective in the inter-esterification reaction that incorporates long-chain fatty acids into triglycerides to produce a product of greater value (Mukherjee and Kiewitt, 1991; Shishikura et al., 1994; Nagesha et al., 2004). Shishikura et al. (1994) noted that glycerol was required at 1-2% to carry out the incorporation of long-chain fatty acids successfully. They noted that 1,3 diglycerides are also formed with glycerol addition. The goal is to improve enzymatic catalysis by increasing the contact of supercritical CO2 with lipases attached to the silica adsorption phase (from rice ash) for simultaneous reaction and byproduct extraction in the presence of small amounts of water contained in humidified CO2. Blattner et al. (2006) first attempted encapsulation of lipases from Mucor miehei and Candida antarctica in stable lecithin-based water-in-oil microemulsion organogels for immobilized-enzyme supercritical-CO2 technology to apply sustainable 'green' chemistry to commercial processes.

Supercritical fluid fractionation of lipids

Concentration and fractionation of the PUFAs and important minor constituents - including antioxidants e.g. oryzanols - may be accomplished during the extraction process (Nilsson et al., 1988; Xu and Godber, 1999;

Xu and Godber, 2000). Friedrich and Pryde (1984) applied SFE to soybean, cotton seed, corn germ, wheat germ and bran, and observed that supercritical extracted oil was light colored compared with hexane-extracted oil. Moreover, they observed some fractionation during the extraction where more polar and higher-molecular-weight compounds were found to increase during later stages of the extraction process. They noted that more polar and high-molecular-weight compounds tended to appear at higher concentrations in the later fractions. Zosel (1978) separated up to 50 fractions of triglycerides from cod liver oil based on increasing molecular weight and degree of unsaturation. McHugh and Krukonis (1994) and Eisenbach (1984) noted that by first transesterifying the triglycerides to EPA ethyl esters, fractionation of individual fatty acids would be possible during the SFE process. Transesterification also increases volatility which would correspond to an increase in solubility of the fatty acids in supercritical CO2 (Harrison et al., 1994; Smith et al., 1998; Fleck et al., 1998; Riha and Brunner, 2000).

Polar lipid fractions may be significantly solubilized in CO2 with the addition of small amounts of polar entrainers such as ethanol (Temelli, 1992). Hardardottir and Kinsella (1988) reported that SFE could remove up to 97% of fish lipids with the addition of 10% ethanol compared with 78% of lipids without ethanol. Also cholesterol removal was about 99.5%. Mendes et al. (1994) extracted hydrocarbons from a slightly crushed, freeze-dried alga and discovered that the polar phospholipids were not extracted (verified by thin-layer chromatography). Cygnarowicz-Provost et al. (1992), however, noted that supercritical CO2 extraction with 10% ethanol gave a recovery of 89% lipids from the filamentous fungi Saprolegnia parasitica, which contains 25% polar lipids. This compared with only 49% lipids extracted with pure CO2.

Dunford and King (2000) studied extraction of rice bran oil by supercritical CO2 fractionation for reducing FFAs and minimizing losses of phyto-sterols. From their experiments at pressures of 20.5-32.0 MPa and temperatures ranging from 45 to 80 °C, they found that lower pressures and higher temperatures reduced the loss of triglycerides and phytosterols during removal of FFAs from crude rice bran oil. Rice bran oil containing less than 1% FFA, up to 95% triglycerides, 0.35% free sterols and 1.8% oryzanol, may be obtained by SFE extraction. During olive oil de-acidification with supercritical CO2 at different pressures (20 and 30 MPa) and temperatures (35-60 °C), CO2 extracted fatty acids more selectively than triglycerides (at 60 °C and 20 MPa). Moreover the physical state of the solute significantly affected solubility trends as a function of temperature and pressure. Supercritical fluid de-acidification of olive oil was found suitable, especially for oils with relatively high FFA content (<10%) due to a higher selectivity factor for FFA (Brunetti et al., 1989).

Taylor and King (2000) used SFE (13.8, 34.5 and 69 MPa; 40, 60 and 80 °C) to extract high-value ferulate phytosterol esters from corn bran; highest yields (1.25%) were obtained in the extract at 69 MPa and 80 °C as well as at 34.5 MPa and 40 °C. Furthermore, SFE (34.5 MPa, 40 °C) extracted corn bran oil with subsequent fractionation with SFC (amino propyl sorbent, commenced at 69 MPa at 80 °C and subsequently lowered to 34.5 MPa at 40 °C with addition of ethanol modifier at the lower pressure) that produced up to a 14.5% FPE enrichment level. Dunford et al. (2003) used continuous counter-current supercritical fluid processing (CO2 flow rate of 2 l/min and oil flow rate of 0.7 l/min) for de-acidification of rice bran oil at isobaric and isothermal conditions (at a pressure range of 13.8-27.5 MPa and a temperature range of 45-80 °C) and observed that fractionation at 13.8 MPa and 80 °C was effective in de-acidification without loss of oryzanol.

In the past few years, attempts have been made to extract lipids from stabilized rice bran using SFE. Kim et al. (1999) extracted and separated rice bran oil rich in essential fatty acids (EFAs) using the SFE process. Zhao et al. (1987) conducted the fractional extraction of rice bran oil with SFE at pressures of 14.7-34.3 MPa, and at a fixed temperature of 40 °C. They found differences in oil yield (18.6-22.0%) extracted at different pressures. Qualitative differences indicated that the fractions obtained at high pressures contained less FFA and waxes or unsaponifiables in the oil. Grinding of bran was also found to be effective in reducing the required CO2 and extraction time. Ramsay et al. (1991) compared different rice bran oil extraction processes including solvent extraction (hexane), SFE, and SFE with 5% ethanol co-solvent. The oil yield was 20.2% for solvent extraction, 18.0% for SFE extraction and 18.2% for SFE with modifier. For SFE and SFE co-solvent extractions, they used 35 °C for 5 h at a flow rate of 20.5 g/min in a 1 liter vessel at 30.0 MPa. They also compared concentrations of sterol components in the extracts: 9.4, 7.3 and 8.3 mg of sterol per gram of rice bran oil for hexane, SFE and SFE co-solvent extractions, respectively. Entrainers (ethanol and chloroform) and separation columns were used by Saito et al. (1993) for SFE of rice bran oil with CO2 at 40-100 °C and at 8.2-19.8 MPa. A separation column (silica gel-supported nitric acid column) was effective in the fractionation of fatty acids whereas the ethanol entrainer increased extraction efficiency up to 1.6 times. There was not much difference in FFA composition with or without entrainers. For example, C16:0, C18:1, C18:2 were 18.6, 42.5 and 35.1% of total FFA for SFE extraction, respectively whereas their concentrations in SFE with ethanol extraction were 18.2, 43.1 and 35.4%, respectively. Higher temperatures increased the fractionation of fatty acid esters.

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