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Supercritical Fluid Technology for Extraction of Bioactive Components


John Shi and Lamin S. Kassama
Agriculture and Agri-Food Canada

Yukio Kakuda
University of Guelph

CONTENTS
Introduction ......................................................................................................4 Process Concept Schemes and Systems ..........................................................6 1.2.1 Process Scheme and System................................................................7 1.2.1.1 Single-Stage Extraction Process...........................................7 1.2.1.2 Multistage Extraction Process ..............................................9 1.2.2 Physicochemical Properties of Supercritical (CO2) Fluids ...............10 1.2.2.1 Phase Diagram ....................................................................10 1.2.2.2 Physical Properties..............................................................10 1.3 Applications in the Food Industry .................................................................12 1.3.1 Extraction of Bioactive Compounds..................................................12 1.3.2 Fractionation of Flavors and Fragrances ...........................................18 1.3.3 Cholesterol-Free Food Products ........................................................20 1.3.4 Separation of Spices and Essential Oils............................................21 1.3.5 Decaffeination of Coffee and Tea......................................................23 1.3.6 Fish Oil Concentration.......................................................................26 1.4. Factors Affecting Extraction Yield ................................................................27 1.4.1 Pressure ..............................................................................................27 1.4.2 Temperature........................................................................................28 1.4.3 Moisture Content of Raw Materials ..................................................29 1.4.4 Cosolvent............................................................................................30 1.1 1.2

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1.4.5 Particle Size........................................................................................32 1.4.6 Flow Rate ...........................................................................................32 1.4.7 Effect of Time on Yield .....................................................................34 1.5 Summary ........................................................................................................36 References................................................................................................................37

1.1 INTRODUCTION
Some chemicals are toxic and if consumed can lead to cancer and other ailments of public concern. As a result, the conventional solvent extraction methods are viewed with suspicion with regards to their role in the manufacturing of functional foods. Extracts from natural sources are key elements in the manufacturing of functional foods. These are any food fortied with extracts from natural sources or nutraceutical. A new paradigm, supercritical extraction technology, was developed to extract bioactive components for use as supplements for functional foods. This technology uses supercritical uids as the extracting solvent which has a signicant attribute paramount to the food and pharmaceutical industry because it leaves no residue in the extract and is gaseous at ambient temperature. Extraction with organic solvents is a well-established technique for selective separation of specic constituents from plant products. Organic solvents with low boiling point such as ethyl acetate, methanol, dichloromethane, and so forth, have been successfully used to isolate bioactive components from hops, spices, oil seeds, and other plant products. The procedure is used to decaffeinate coffee and tea, and to remove nicotine from tobacco. The solvents used for this purpose must meet the legal requirements put in place to ensure food quality and safety, and these regulations vary from country to country. These requirements are: high degree of purity, chemical stability, inert (no reaction with food constituent), low boiling point, and no toxic effects. Criteria for these regulations are set by national and international bodies such as the US Food and Drug Administration (FDA); European Economic Commission (EEC) Codex committee; the Canadian Food Inspection Agency (CFIA); and FAO/WHO Codex Alimentarius Commission. Most countries have regulations stating which extraction solvents are generally regarded as safe (GRAS). For example, the Canadian food additive regulation (FDAR: B.10.045) for food chemical codex specication and the EEC directive (88/344/EEC) state the regulations regarding the use of extraction solvents in the production of foodstuffs and food ingredients within Canada and the EEC. Although only mandatory within Canada and the EEC, in general they are comparable to those in other countries. Public health, environmental, and safety issues are the major concerns in the use of organic solvents in food processing. The possibility of solvent residues remaining in the nal product has been a growing concern to consumers, thus warranting stringent environmental regulations. The demand for ultrapure and highadded-value products is redirecting the focus of the food and pharmaceutical industries into seeking the development of new and clean technologies for their products. The supercritical uid extraction (SFE) technology has provided an excellent alternative to the conventional organic solvent extraction methods. Although the

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Supercritical Fluid Technology for Extraction of Bioactive Components

technology was known for more than 100 years, its application in the food and pharmaceutical industry began only three decades ago.1,2 Since that time, over 100 plants of different capacities have been built globally for extraction of desired solutes from solid materials.3 Supercritical extraction is a novel separation technique that utilizes the solvent properties of uids near their thermodynamic critical points.4 A variety of processes involving extractions with supercritical uids (SCF) have been developed and are regarded as a viable extraction technology that meets the food quality and safety requirements. The physicochemical properties of supercritical uids, such as the density, diffusivity, viscosity, and dielectric constant can be controlled by varying the operating conditions of pressure and temperature or both in combination.1,3,5 Many supercritical uids (carbon dioxide, ethane, propane, butane, pentane, ethylene, ammonia, sulfur dioxide, water, chlorodiuoromethane, etc.) are used in supercritical extraction processes. Sihvonen et al.,1 Brunner,3 and Rozzi and Singh6 recommended carbon dioxide (CO2) in their reports because of its favorable properties and the ease of changing selectivity by the addition of agents such as ethanol and other polar solvents. Supercritical carbon dioxide (SC-CO2) is the most desirable supercritical uid for extracting natural products for foods and medicinal uses. Its characteristic traits are inert, nonammable, noncorrosive, inexpensive, availability, odorless, tasteless, environmentally friendly, and GRAS status.7 Its near-ambient critical temperature makes it ideal for thermolabile natural products.8,9 Supercritical CO2 has the following advantageous attributes over other solvents: (a) it has a solvating power similar to organic liquid solvents and higher diffusivities, lower surface tension, and viscosity; (b) separation can be affected by simply changing the operating pressure or temperature to alter the solvating power of the solvent; and (c) modifying CO2 with a cosolvent can signicantly augment the selective and separation power and in some cases extend its solvating powers to polar components. Supercritical carbon dioxide is being given a great deal more attention as an alternative to industrial solvents as a result of (a) increased governmental scrutiny and new regulations restricting the use of common industrial solvents such as chlorinated hydrocarbons; (b) its nontoxic and environmentally friendly attributes, given that it leaves no traces of solvent residue in food; (c) sharp increase in energy cost, which increased the cost of traditional energy-intensive separation technique, such as distillation; (d) carbon dioxide is cheap, safe to use, recyclable, and has minimum disposal cost required; (e) stringent pollution-control legislation prompting industries to seek alternative means of waste treatment and utilization; and (f) increased performance demands on materials, which traditional processing techniques cannot meet. It must, however, be stated that commercial applications of the SFE technology remain limited to a few high-value products due to high capital investment,10,11 its novelty, and complex operating system. Adoption of the technology is on the rise as a result of advances in processing, equipment, and the realization of producing high-value products with high protability.1,10 Supercritical uid extraction is one of the most promising technologies being adopted in the chemical, food, pharmaceutical, and neutraceutical industries. Analytical techniques are superior to the conventional organic solvents when applied for qualitative and quantitative extraction

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of natural thermolabile products.12,13 Research efforts have proven that the SFE technologies could be successfully employed in the extraction and fractionation of fats and oils; purication of solid matrix; separation of tocopherols and antioxidants; removal of pesticide residue from herbs, medicines, and food products; detoxication of shellsh; and concentration of fermented broth, fruit juices, essential oils, spices, and coffee; and separation of caffeine, and so forth.3,1416

1.2 PROCESS CONCEPT SCHEMES AND SYSTEMS


The supercritical uid extraction technology was conceptualized on the basis of obtaining pure extract without solvent residues which could be detrimental to consumers of food and pharmaceutical products. Extraction is an analytical process used to separate and isolate a targeted component from other substances. The success of the process is dependent on the distribution of the analyte between two phases, the separation and stationary phases.17 In the phase equilibrium between liquid and gas, the partition of the liquid phase increases with increasing pressure and decreases with increasing temperature. If the temperature and pressure are simultaneously increased, the transport properties of both liquid and gas increases and thus convergence occurs. When used under practical pressure and temperature conditions of 5 to 50 MPa and ambient to 300C, respectively, the solubility properties of the supercritical uid are greatly inuenced by its density, diffusivity, and viscosity. The SC-CO2 is liquid-like and has a higher extraction ux than those obtained with organic liquids solvents. King et al.17 stated that at high CO2 densities, its solvent properties were similar to organic solvents like chloroform and acetone, and if intermediate compression were applied, it behaved like a nonpolar hydrocarbon such as n-pentane or diethyl ether. The separation phase occurs during the dynamic extraction period, while the stationary phase is the sample material loaded as a xed-bed in the extraction column. Supercritical extraction (SCE) involves the use of compressed gases at or above their critical temperature (Tc) and pressure (Pc). It utilizes the ability of these special uids to become excellent solvents to solvate certain solutes (bioactive components) from a solid matrix.6 The solute extraction stream from the sample matrix is directly proportional to the product of solubility and diffusivity in the supercritical medium. Hence the solutes solubility increases with pressure, while its corresponding diffusivity is expected to decrease by two orders of magnitude. The solvent capacity is mainly the function of density and can be improved with the addition of a cosolvent, which modies the density and polarity of the supercritical uid, thus signicantly increasing the yield. This technology has been successfully applied in the extraction of bioactive components (antioxidants, avonoids, lycopene, essential oils, lectins, carotenoids, etc.) from a variety of biological materials such as hops, spices, tomato skins, and other raw or waste agricultural materials. The process requires intimate contact between the packed bed formed by a ground solid substratum (xed-bed of extractable material) with a supercritical uid.18 During the supercritical extraction process, the solid phase comprised of the solute and the insoluble residuum (matrix) is brought

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Supercritical Fluid Technology for Extraction of Bioactive Components

CO2 Pump Extractor column Heat exchanger

Back pressure valve CO2 outlet Separator Extracts outlet

Cooling bath

Heat exchanger

Pressure valve CO2 Source

FIGURE 1.1 Schematic diagram of a typical single-stage supercritical uid extraction system with CO2.

into contact with the uid phase which is the solution of the solute in the supercritical uid (solvent). The extracted material is then conveyed to a separation unit. The power of supercritical uid extraction is linked to the solubility and phase equilibrium of substances in the compressed gas. The targeted bioactive component being extracted must be soluble in the supercritical uid. Controlling the pressure and temperature of SCF varies the solubility and phase equilibria. The extraction of pure and high-value extract is accomplished without risk of environmental pollution or residual solvent contamination in the nal product.

1.2.1 PROCESS SCHEME

AND

SYSTEM

The extraction with supercritical uids is comparable to liquid-liquid solvent extraction even though, with supercritical extraction, compressed gas is used instead of organic solvents and the applied pressure is crucial. The supercritical uid extraction process is governed by four key steps: extraction, expansion, separation, and solvent conditioning. The steps are accompanied by four generic primary components: extractor column (high-pressure vessel), pressure control valves, separator column, and pressure intensier (pump) (Figure 1.1) for the recyclable solvent.3,5,17,19,20 The system has other built-in accessories, such as heat exchangers for providing a source of heating; condensers for condensing supercritical uids to liquid; storage vessels; and a supercritical uid source. Raw materials are usually ground and charged into a temperature-controlled extractor column forming a xed bed, which is usually the case for batch and single-stage mode. 1.2.1.1 Single-Stage Extraction Process The supercritical uid is fed at high pressure by means of a pump, which pressurizes the extraction tank and also circulates the supercritical medium throughout the system. Figure 1.2 shows an example of a typical single-stage supercritical extraction system. Once the SCF and the feed reach equilibrium in the extraction vessel, through the manipulation of pressure and temperature to achieve the ideal operating conditions, the extraction process proceeds. The mobile phase consisting of the SCCO2 uid and the solubilized components are transferred to the separator where the

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Dry test meter

Pressure regulator

Three stage separator columns

Extractor column

Ice water trap

CO2 pump Mixer

Co-solvent inlet

CO-solvent pump

FIGURE 1.2 Schematic diagram of supercritical uid extraction system used to fractionate bioactive components from plant matrix using supercritical carbon dioxide.

solvating power of the uid is decreased by increasing the temperature or decreasing the pressure of the system. The extract precipitates in the separator while the supercritical uid is either released or recycled back to the extractor. In the case where highly volatile components are being extracted, a multistage conguration may have to be employed as shown in Figure 1.2. As the solutions leave the extractor and ow to the rst separation vessel via the pressure regulator, the pasty oleoresins settle to the bottom as they separate and can be collected, while the remaining solution goes to the second-stage separator where the fractionation of the volatile components occurs. For more sensitive products, the third stage of separation would be required for the complete isolation of pure volatile components. Saltzman et al.21 presented a design (Figure 1.2) where the solution ows through a heated valve and precipitates into a preweight U-tube in an ice-water bath. The glass wool at the U-tube exit traps the entrained solutes in the gas, while the gas ows through a dry-test meter which monitors the ow rate. Oszagyan et al.22 used a similar system as illustrated in Figure 1.2 to extract essential oil from Lavandula intermedia Emeric ex aloisel and herb of Thymus vulgaris L., and further fractionated volatile components (-Cymene, -Terpinene, Thymol, and Carvacrol) while Ozcan23 used it to fractionate volatile components from Turkish herbal tea (Salvia aucheri Bentham Var. canaescen Boiss and Heldr.). Duquesnoy et al.24 and Boutekedjiret et al.25 extracted and fractionated volatile compounds from plant materials using SCE with a similar multistage fractionation method. The processes described above are semibatch continuous, where the SCF ows in a continuous mode while the extractable solid feed is charged into the extraction vessel in batches. In a commercial processing plant, multiple extraction vessels are sequentially used to enhance process performance and output. Although the system is interrupted at the end of the extraction period, when the process is switched to a

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Extracts

CO2 inlet

Supercritical Fluid Technology for Extraction of Bioactive Components

2 Stage separation vessels 3 Stage extraction columns

CO2 inlet Pu mps

Co-solvent inlet

FIGURE 1.3 Schematic diagram of commercial scale multistage supercritical uid extraction system used to fractionate bioactive components.

prepared vessel for extraction, the unloading and/or loading of the spent vessels can be carried out while extraction is in progress, reducing the downtime and improving the production efciency. 1.2.1.2 Multistage Extraction Process A semicontinuous approach on a commercial scale uses a multiple-stage extraction process which involves running the system concurrently by harnessing a series of extraction vessels in tandem as shown in Figure 1.3. In this system the process is not interrupted at the end of extraction period for each vessel, because the process is switched to the next prepared vessel by control valves for extraction while unloading or loading the spent vessels; although imperfect, continuity is attained. The primary extraction stages operate in a similar mode to the ones depicted in Figure 1.2 and Figure 1.3. The rafnate from the premier stage enters the rst separation vessel while separation and fractionation of different compounds occurs based on their relative solubility. The options of cosolvent are available to enhance the solvent power of separation of specic components. This is effective for cases where more than one targeted component is to be extracted, giving the exibility to vary the extraction parameters such as pressure and temperature to achieve different solubilities for different components being extracted at each stage of operation. Gamse26 suggested that highly soluble substances could be extracted at the initial stages at low SCF density, and by increasing the density in the subsequent stages remove the less soluble substances. The supercritical pressure, temperature, and ow rate at each stage could be controlled independently.

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Storage tank

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OF

1.2.2 PHYSICOCHEMICAL PROPERTIES FLUIDS

SUPERCRITICAL (CO2)

The physicochemical properties of the supercritical uids are crucial to the understanding of the process design calculation and modeling of the extraction process. Therefore, selectivity of solvents to discriminate solutes is a key property for the process engineer. Physical characteristics such as density and interfacial tension are important for separation to proceed; the density of the extract phase must be different from that of the rafnate phase and the interfacial properties inuence coalescence, a step that must occur if the extract and rafnate phase are to separate. 1.2.2.1 Phase Diagram Supercritical state of uid is inuenced by temperature and pressure above the critical point. The critical point is the end of the vapor-liquid coexistence curve as shown on the pressure-temperature curve in Figure 1.4 where a single gaseous phase is generated. When pressure and temperature are further increased beyond this critical point, the uid enters a supercritical state. At this state no phase transition will occur regardless of any increase in pressure or temperature nor will it transit to a liquid phase. Hence, diffusion and mass transfer during supercritical extraction are about two orders of magnitude greater than in the liquid state. 1.2.2.2 Physical Properties Substances that have similar polarities will be soluble in each other, but increasing deviation in polarity will make solubility increasingly difcult. Intermolecular polarities exist as a result of van der Waals forces, and, although solubility behaviors

Supercritical region Pressure (MPa) Liquid phase PC Solid phase

Triple point

Gas phase Tc = 31.1C Pc = 7.38 MPa TC

Temperature (C)

FIGURE 1.4 Supercritical pressure-temperature diagram for carbon dioxide.

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Supercritical Fluid Technology for Extraction of Bioactive Components

11

TABLE 1.1 Comparison of Physical Properties of Supercritical CO2 at 20 MPa and 55C with Some Selected Liquid Solvent at 25C
Properties Density (g/mL) Kinematic viscosity (m2/s) Diffusivity (m2/s) Cohesive energy density ((cal/cm)) Source: Modied from King et al.17 CO2 0.75 1.0 6.0 109 10.8 n-Hexane 0.66 4.45 4.0 109 7.24 Methylene Chloride 1.33 3.09 2.9 109 9.93 Methanol 0.79 6.91 1.8 109 14.28

depend on the degree of intermolecular attraction between molecules, the discrimination between different types of polarities is also important. Substances dissolve in each other if their intermolecular forces are similar, or if the composite forces are made up in the same way. Properties such as the density, diffusivity, dielectrical constant, viscosity, and solubility are paramount to supercritical extraction process design. The dissolving power of SCF depends on its density and the mass transfer characteristic, and is superior due to its high diffusivity, low viscosity, and interfacial tension to liquid solvents. Although many different types of supercritical uids are in existence and have many industrial applications, CO2 is the most desired for SCE of bioactive components. Table 1.1 shows some physical properties of compressed (20 MPa) supercritical CO2 at 55C compared to condensed liquids commonly used as extraction solvents at 25C. It should be noticed that supercritical CO2 exhibited similar density as those of the liquid solvents, while less viscous and highly diffusive. This uidlike attribute of CO2 coupled with its ideal transport properties and other quality attributes outlined above make it a better choice over other solvents. The specic heat capacity (Cp) of CO2 rapidly increases as the critical point (31.1C temperature, 7.37 MPa pressure, and 467.7 g/L ow rate) is approached. Like enthalpy and entropy, the heat capacity is a function of temperature, pressure, and density.5 Under constant temperature both the enthalpy and entropy of supercritical CO2 decreases with increased pressure and increases with temperature at constant pressure. The change in the specic heat as a result of varying the pressure and temperature is also dependent on density. For example, under constant temperature, specic heat increases with increased density up to a certain critical level. Above this critical level, any further increase of density decreases the specic heat. Sample matrix is an important parameter that inuences solubility and mass transfer process during SCE. Properties such as particle shape and size distribution, porosity and pore size distributions, surface area, and moisture content inuence solubility and mass transfer. The presence of water (moisture content) in the sample matrix during supercritical extraction also has an effect on the extraction outcome.

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1.3 APPLICATIONS IN THE FOOD INDUSTRY


One of the most important trends in the food industry today is the demand for allnatural food ingredients that are free of chemical additives. Natural food antioxidants are derivatives of plant by-products. A quantum leap in supercritical uid extraction technology was made by its applications in decaffeinating coffee, tea, and other bioactive (essential oils from spices) components used as ingredients in foods. Likewise, SCF extraction is used to extract avor and fragrance components, highvalue compounds used in the food, pharmaceuticals, and neutraceutical industries. With this technology, extracts of natural nutrients could be utilized without the fear of organic solvent residues. A compendium of process parameters used for different product applications is listed in Table 1.2. Various processes of extracting bioactive components from agricultural materials have been used commercially. The major hurdles to overcome in SCE are to obtain extracted compounds with similar bioactivity as the synthetic compounds and also to maintain the required avor, odor, and color components that may be detectable in the treated food product. Many extraction processes employ the following methods to extract bioactive components from plants (Labiatae family): solvent (polar and nonpolar) extraction and aqueous alkaline58; extraction of vegetable oils and mono/diglycerides59 by steam distillation and molecular distillation.23 These processes are limited by numerous disadvantages, because the solvents used are not very selective for the bioactive components. Consequently, the resulting extracts are not as pure as synthetic chemical compounds. The solvents used include hydrocarbons such as hexane, acetone, and methyl chloride which leave unwanted residues in the food products, which are prohibited in some instances by regulatory bodies for use in food as discussed earlier. Many researchers applied SCE technology for the extraction of bioactive components.3,56,6062

1.3.1 EXTRACTION

OF

BIOACTIVE COMPOUNDS

Some bioactive components are phenolic or polyphenolic compounds of plant origins which interfere with the formation of free radicals, thus preventing the formation of hydroperoxides. However, during food processing, especially conventional thermal processing of food products, these bioactive components are lost. To restore these components necessitates fortication. Some of the more common bioactive compounds include lycopene, avonoids, tocopherol, lecithin, ascorbic acid, citric acid, polyphenols, and so forth. Most of the separation procedures involve physical and chemical processes such as centrifugation, ltration, membrane separation, precipitation, chromatography, solvent extraction, crystallization, evaporation, molecular distillation, and SC-CO2. To overcome the separation problems encountered when producing soluble materials via such processing procedures requires conditions that may have detrimental effects on the nature of the extraction technique or the product stability. In order to achieve extract labile compounds, a rapid separation process may be needed to avoid any signicant product loss. Most bioactive components used as food additives are in a concentrated form. Appropriate extraction procedures are consequently required

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TABLE 1.2 Supercritical Extraction Process Parameters for Some Selected Bioactive Components from Agricultural Material by SC-CO2
Product Extracted Sunower oil Soybean oil Corm germ oil Seed oil Raw Material Sunower seed Soybean Corn germ Avocado Cottonseed Paprika Peanuts Peanut hearts Rice bran Sorghum bran Sorghum germ Soybean Wheat bran Wheat germ Raw Material Pretreatment Ground Flaked, 20.1 0.385.1mm Dried, milled 23.4 Dried, ground (< 0.25 mm) Component Concentration (%) Temp (C) Pressure (MPa) CO2 Flow Rate Time (L/h) (h) 2.5 9001080 9.8 3.5 3.4 8.6 5 9.0 2.8 6.3 6.1 9.4 11.4 11.4 10.1 Ethanol 50 58.2 30.8 7.2 48.0 42.6 19.2 5.0 16.8 19.4 4.0 7.0 MC (%) Recovery (%) 36

Cosolvent Ethanol

Source Cocero & Calvo27 Friedich et al.28 Ronyai et al.29 Friedich & Pryde30

4050 3235 50 50 70 50 50 7075 50 70 78 70 50 50 50 7.588.27 55.2 75.8 55.2 55.2 68.9 68.9 65.5 66.2 68.9 55.2 55.2 55.2

1140

24 8 7 8 11 6 11 7 8 5 6

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TABLE 1.2 (CONTINUED) Supercritical Extraction Process Parameters for Some Selected Bioactive Components from Agricultural Material by SC-CO2
Product Extracted (Essential oil) Limonene Carvone Anethole Eugenol and Caryophyllene Yaleranone Wheat germ oil Raw Material Raw Material Pretreatment Component Concentration (%) Temp (C) Pressure (MPa) CO2 Flow Rate Time (L/h) (h) 60 MC (%) 6.7 Recovery (%) 60

Cosolvent

Source Sovava et al.31

Licochalcone A Licochalcone B Defatted mustard Canola oil

Bergaptene

Sweet Dried ground orange Sieved (1, 0.7, Caraway 0.4, 0.2, 0.08 seed mm) Anise seed Cloves Spikenard Wheat germ Milled, 10.2 powder (20 m diameter) Licorile Cut root Mustard Dried, fully 16 seed pressed (1mm) Canola seed Flaking (0.20.5 mm) cooking (90C) Bergamot Peel dried 0.15 Citrus ground

2340 910

3550 1341

0.12

4.311.5

98.7

Ge et al.32

35 40

12.7 30.4

360 300400

0.5 3

5% Ethanol 2530 80 Taniguchi et al.33

4570 4162

180

Ethanol

44

Dunford and Temelli34

4060 810

2.6

1719

85

Poiana et al.35

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Carotene lutein Defatted & decholesterol of muscle Oil Bixin pigment Defatted & decholesterol beef Bixin pigment

Leaf protein concentration Dehydrated Spray dried beef powder + air dried chunk Antarctic Freeze-dried 16.2 krill meal & ground Annatto Seed, 3.92 seed mm Fresh 19 frozen Ground beef Annatto Whole seed 1.3 seed (11.6 oil) Evening primrose seed Cardamom seeds Crude beef tallow Sweet potato Woolgrease Dried, ground, 0.355 mm Freshly ground seeds, mesh 21.9

Alfalfa

40

30 70

300360

90 70 1.43.2 87

Favati et al.36

4555 233927.6 300600

Wehling et al.37

80 50 60 35 50

24.5 34.5 31 31. 29.6

51.4 117.2 144.8 117.2 144.8 1.2

34 12 48

7.83

99

Yamaguchi et al.38 Chao et al.39 Chao et al.40

56-lipid 26-cholesterol Soybean oil 20% v

Degnan et al.41

Evening primrose oil Cardamom oil

60

70

64.8

1.75 < 8

97

Favati et al.42

4060

40

10

10

8595

Gopalakrishnan & Narayanan43 Chao et al.44 Spanos et al.45

Decholesterol beef tallow -carotene

0.150.2 Freeze dried ground ( 0.25 mm) 94

40 48

34.5 41.4

226.7250 8401080

46

6070 80

Lanolin

80

38

3962880

15.5

CygnarowiczProvost et al.46

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TABLE 1.2 (CONTINUED) Supercritical Extraction Process Parameters for Some Selected Bioactive Components from Agricultural Material by SC-CO2
Product Extracted Phospholipid mixture Raw Material Soybean akes Raw Material Pretreatment Component Concentration (%) Temp (C) 80 Pressure (MPa) 68.2 CO2 Flow Rate Time (L/h) (h) 43.6109 MC (%) 10.8 Recovery (%)

Cosolvent Ethanol (5% v)

Source

Ground, 5.134 mg/g thinly akes, (0.10.25 mm)

Tocopherol enrichment Carotene Essential oil

Soybean akes Rice bran Carrot Star anise fruit

80

25

300

Protein Essential oil Piperine Essential oil Essential oils gingeroles Triglycerides carotenoid

Spring mackerel Black pepper Eucalyptus leaves Ginger rhizomes Palm oil ber

Frozen puree (0.93mm) Dried, ground, particle size (0.57 mm) Freeze-dried, chopped Dried ground Air drying Dried ground Oven dried

500 ppm 89

55 26.5

20.7 6.5

90 5.4 1.5

81 12.7

Ethanol 10%

60-Soybean King et al.47 70-Rice bran 80-Wheat germ Vega et al.48 97

35 1.5 5.7 40 50 30 40 45 55

34.5 915 9 30 2030

1080 2.55 1.1 138 1.45

15

1.6 9

87 18 2.4 8.4 7 Perakis et al.49 Porta et al.50 Catchpole et al.51 Franca and Meirele52

2.5 2 2.25

9.5 8.8 5

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Lycopene -Carotene Essential oil Menthol Essential oil vanillin Essential oil Capsaicine alkaloids Essential oil Antioxidants

Tomatoes Peppermint & Spearmint Vanilla pod Chili pepper Coriander

Dried skin ground Cut leaves

65 35

4080 1728 2443 618

30 3.42

0.5 49 1482

Chloroform 76

Cadoni et al.53 Barton et al.54

Dried ground Dried ground

36 2

36 40

11 30 2 8.8

97 Catchpole et al.51

Essential oil

Dried grounds 96 (0.4 mm) Sweet Thai Dried ground Tamarinds (mesh 4070) Grape seed Wash dry 80 ground

50

15

3 0.3

3 5 Ethanol

0.61

Anitescu et al.55 Luengthanaphol et al.56 Lee et al.57

3580 1030

3575 2047

0.75

10

2% Ethanol 77

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when preparing them from their original matrices. Some compounds in the concentrated form are thermolabile, volatile, and prone to degradation when subject to intensive heat. Supercritical extraction with CO2 is the most viable method for food applications. Baysal et al.63 extracted lycopene and -carotene from tomato using SC-CO2. The processing conditions used were extraction pressure of 20, 25, and 30 MPa; temperatures of 35C, 45C, 55C, and 65C; resident time of 1, 2, and 3 h; and CO2 ow rate of 2, 4, and 8 kg/h. The best conditions for lycopene extraction were 2 h at a ow rate of 4 kg/h, pressure of 30 MPa, temperature of 55C, and with the addition of 5% cosolvent (ethanol). They noted that if too much ethanol is used, it decreased the homogeneity of the extraction mixture, and reduced the separation efciency. Brunner and Peter64 and Vega et al.48 corroborated this nding. Luengthanaphol et al.56 compared the SC-CO2 extraction to other methods and their effects on bioactivity. Their results compared to Tsuda et al.65 indicated the superiority of SC-CO2 coupled with cosolvent as shown in Table 1.3. Both studies show the superiority of antioxidant extraction with SC-CO2 and modiers, although some disparity occurred which could have been caused by the variety used. Macias-Sanchez et al.61 extracted carotenoids and chlorophyll from Nannochloropsis gaditana and achieved the highest yield at 20 MPa and 60C, the optimal pressure and temperature, respectively. Wang et al.66 also reported that the antioxidant activity of Bupleurum kaoi Liu fractionated with SC-CO2 gave the highest yield of phenol and the strongest antioxidant capacities.

1.3.2 FRACTIONATION

OF

FLAVORS

AND

FRAGRANCES

The extraction of compounds, for avor and fragrances, by supercritical CO2 is paramount in the food industry. Mother Nature is a splendid synthesizer of avors and fragrances in natural products. The cleaner and safer attributes make supercritical

TABLE 1.3 Yields and Activities of Antioxidants Extracted by Different Methods from Sweet Thai Tamarind Seed. SC-CO2 Extraction at 30 MPa and 80C; Organic Solvent Extraction at Room Temperature
(-) Epicatechin Yield (mg/100 g dry weight ) Extraction SC-CO2 SC-CO2 + Cosolvent (10% Ethanol) Ethanol -tocopherol Luengthanaphol et al.56 Tsuda et al.65 0.022 13 0.336 26 PV after 24 h (meq/kg dry weight) Luengthanaphol et al.56 231 Tsuda et al.65 26

25

32

157

25

Source: Modied from Luengthanaphol et al.56 and Tsuda et al.65

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TABLE 1.4 The Results of Gas Chromatography and Mass Spectrophotometer Analysis of Flavor Compounds Extracted by the Likens-Nickerson Extraction and the Supercritical CO2 Extraction Methods of Basmati Rice
Likens-Nickerson Extraction Peak No. 2 3 5 8 13 14 19 21 22 24 28 30 31 34 35 37 Retention Time (min) 7.51 8.11 9.51 14.52 21.98 22.68 27.71 28.53 30.45 32.14 35.14 36.12 37.34 40.22 42.77 43.82 Peak No. SC-CO2 Retention Time (min)

Compound Hexamethyl disiloxane* Silicate anion tetramer* Dibutyl phthalate* Hexane cyclotrisiloxane* Pentanal Butan-2-one-3-Me Heptanol 2-Heptanone Octanol Octanal 2-Octanone Nonanal Decanol 2-Decenal Undecane Benzoic acid 2,5-bis (trimethylsiloxy) benzene*

Compound

12 13 19 20 21 23 29 30 31 32 34 37 40 44

22.11 22.56 28.32 29.27 30.92 32.26 35.19 35.44 36.13 37.42 40.31 42.52 45.11 45.24

Pentanal Butan-2-one-3-Me Heptanol 2-Heptanone Octanol Octanal 2-Octenal 2-Octanone Nonanal Decanol 2-Decenal Undecane Dodecane Tetradecane

39

46.28

Compounds identied as artifacts. Source: Modied from Bhattacharjee et al.62

technology an ideal candidate for extracting such valuable and heat-sensitive products when compared to toxic organic solvents. The high-value-added natural products are good for use in soft drinks. An example is ginger extract which gives the pungency and avor in ginger ale drinks. Bhattacharjee et al.62 compared the Likens-Nickerson extraction and the SCCO2 extraction methods on Basmati rice. The results showed that the SCE technique was superior and extracted the most avor components that bore the closest resemblance to the original Basmati avor (Table 1.4). The SC-CO2 technique extracted more avor compounds and produced purer (resembles original basmati avor) extracts than its counterpart (Liken-Nickerson method). Desired fragrances are isolated from concentrates extracted from owers. The process consists of initial solvent extraction, usually with hexane, which yields an intermediate product called concrete. This product contains fragrances and other components like parafn, fatty acids, fatty acids methyl ester, di-, and tri-terpenic

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compounds, pigments, and so forth. Postprocessing of the concrete can be done using SC-CO2 extraction. Reverchon67 used single-step SCE at a pressure of 8 MPa and temperature of 40C followed by a two-stage separation procedure at a pressure of 8 MPa and temperature of 16C at the rst stage and a pressure of 1.5 MPa and a temperature of 0C at the second stage. Under these optimum conditions the extracted volatile rose oil contains 50% 2-phenylethanol. When a cosolvent (ethanol) is mixed with SC-CO2, a yield of 50% to 60% was observed.68 Jasmine fragrance extracted at 12 MPa and 40C gave results superior to those of other solvents. Sastry and Mukhopadhyay68 experienced increased yield of 45% to 53% with the use of cosolvents. Similarly, SC-CO2 has been used effectively to extract fragrances from orange, marigold, sandalwood, vetiver, and so forth.

1.3.3 CHOLESTEROL- FREE FOOD PRODUCTS


Cholesterol is an unavoidable substance we require for the daily maintenance of our body. Lipoproteins are vehicles that transport cholesterol to various body tissues to be used, stored, or excreted, but if cholesterol is in excess it can lead to coronary heart problems (atherosclerosis). Low-density lipoprotein (LDL) termed bad cholesterol causes fat buildup in the arteries increasing the risk of heart disease. Highdensity lipoprotein (HDL) termed good cholesterol transports cholesterol back to the liver, where endogenous metabolism prevents cholesterol buildup and reduces the risk of heart disease. The indiscriminate consumption of saturated fats in our diet may raise the total LDL (> 100 mg/dL) level and decrease HDL (< 35 mg/dL) level, thus increasing the risk of heart disease.69 The recommended daily intake of cholesterol is about 300 mg.70 The correlation between serum cholesterol level and mortality rate of cardiovascular disease has been reported in many studies.69,71 Pork has cholesterol content of 30 to 450 mg/100 g, poultry 70 mg, sh 35 to 70 mg/100 g, and beef 65 to 331mg. One common source of cholesterol is from the consumption of fried fast-food products. The fast-food industry uses hydrogenated fats for their deep-fat frying processes because of its stability and high economic turnover. The hydrogenated fat is the potential source of trans fatty acids which are taken up by the fried foods during cooking (French fries, onion rings, chicken nuggets, etc.) and ultimately ingested by the consumer. Trans fats have been shown to increase LDL cholesterol levels and reduce HDL cholesterol levels, thus raising the risk of heart disease. Public health initiatives such as the National Cholesterol Education programs have raised consumer awareness, resulting in the advocation for healthy foods with low cholesterol. Thus, the food industry is under tremendous pressure to address this consumer concern. Supercritical uid extraction is an emerging technology with great potential to revolutionize the oil/fat industry. Many researchers (Dunford and Temelli34; Temelli et al.34,72) reported the feasibility of supercritical uid extraction of lipids from food without compromising their organoleptic quality. Chao et al.44 used a similar extractor conguration (Figure 1.3) as discussed earlier with three stage separations, to remove cholesterol. By decreasing the pressure at each stage sequentially from 17 to 11 to 4 MPa, they were able to achieve higher selectivity for cholesterol at the lower pressures. The results also showed that the fraction collected from the third

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TABLE 1.5 Solubility of Cholesterol in Supercritical CO2 under Different Operating Conditions
Run No. 1 2 3 4 5 6 7 8 Pressure (MPa) 10 10 10 10 13 13 13 13 Temperature (C) 40 45 50 55 40 45 50 55 Density of CO2 (g/L) 602.4 494.4 408.1 342.3 723.4 655.8 588.5 527.7 Solubility of Cholesterol (mg/L) 146.9 82.6 47.2 28.5 289.2 234.8 182.7 141.1

Source: Modied from Yeh et al.74

separator at 4 MPa contained concentrated cholesterol ranging from 272 to 433 mg/100 g lipid. Chao et al.40 used SC-CO2 at an operating pressure of 10 to 30 MPa and temperature range of 30C to 50C to reduce the cholesterol level in ground beef. Similarly, Hardardottir and Kinsella73 explored the removal of lipids and cholesterol from sh muscle with SC-CO2. They removed over 80% to 99% cholesterol using pressures of 14 to 35 MPa and temperatures of 40C to 50C. Although the authors noted limited effect on lipid/cholesterol yield with increased extraction pressure and temperature, increased extraction time from 3 to 9 h signicantly increased the yield. Yeh et al.74 used eight operating conditions shown in Table 1.5 to optimize their process and observed that at 10.3 MPa and 55C operating pressure and temperature, respectively, cholesterol level was reduced from 2867 mg/100 g to 14.1 mg/100 g. Supercritical-CO2 technology was used to fractionate milk fat, which is an excellent raw material with specic functionalities used in many products.75 Extracting cholesterol from anhydrous milk fat with SC-CO2 used in conjunction with adsorbents (silica gel) to maximize yield was demonstrated by Huber et al.76

1.3.4 SEPARATION

OF

SPICES

AND

ESSENTIAL OILS

Spices have strongly avored or aromatic components that can be used in small quantities in food as a preservative or avoring ingredient. Chili (capsicium species), ginger (Zingiber ofcinalis), and pepper (piper nigrum L) are classic pungent avorines while ginger and chili have additional nutraceutical values. These products have high economic value in their concentrated form. The extraction of spices is usually carried out in two stages: stage one separates pungent oleoresins and the second stage the essential oil fractions. Essential oils are typically volatile terpenes and esters. Essential oils are concentrated pure plant extracts that have long been revered for their therapeutic applications and are derivatives from owers, leaves,

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stems, berries, rinds, resins, or roots of plants. These are very important ingredients and food additives of high value. Catchpole et al.51 performed a detailed study on the extraction of spices and essential oils using SC-CO2, propane, and dimethyl ether. They reported ginger to be the easiest of all spices in terms of optimized yield relative to pressure and temperature, while capsaicin in chili could be extracted at moderate pressure and temperature, especially with the use of modiers. Chili oil fraction contains fatty oil and carotenoids, and is speculated that the fatty oil acts as modier for the capsaicins.77 The author extracted black pepper with much duress, because of its viscous characteristics, thus requiring higher pressure and moderate to high temperatures. The use of supercritical propane for extracting spices was reported by Illes et al.78 They found propane to adequately extract fatty oils, tocopherols, and carotenoids, but inadequate for capsaicins, while CO2 was adequate for capsaicinoids, fatty oils, and tocopherols, but not for carotenoids. Figure 1.5 shows the supercritical extraction of ginger with three extraction uids (CO2, propane, and dimethyl ether).51 Propane gave the lowest yields while dimethyl ether gave the highest yield. They reported dimethyl ether to have mutual solubility with water. Ginger contains high amounts of volatiles, and CO2 extraction offers the advantage of dividing the extract into oleoresins and essential oil fractions by using a two-stage separation procedure with sequential pressure reduction. Similarly, if propane or dimethyl ether is used, considerable heating is required which ultimately results in thermal degradation, and a larger energy requirement in the form of cooling, depressurization, and boiling to recover the essential oils. In the case of ginger, the oxygenated fraction was much greater than the steam distilled oils and the gingerols in the oleoresin were extracted without decomposition. Oleoresins and piperine from pepper were extracted with insignicant losses although a longer processing time was required. Similar trends were observed for chili and pepper (Figure 1.6 and Figure 1.7). The extracts contained carotenoid pigments, and those obtained with SC-CO2 were bright red with pink residues, while those from propane and dimethyl ether were dark red. The extract obtained from chili with SCCO2 corroborated the results of other researchers5,79 in being viscous, pastry yellow, and semisolid, while those extracted with dimethyl ether were yellow/black and liquid at room temperature with a high quantity of water resulting in the dilution of the essential oil and piperine content. Nguyen et al.80 described the extraction of antioxidants from Labiatae herbs (rosemary, sage, oregano, and thyme) with SCCO2 at pressures in the vicinity of 50 MPa and temperatures ranging from 80C to 100C. The extracted oleoresin was precipitated into two fractions at various pressures and temperatures. The rst fraction consisted of a green-brown, oil-soluble, heat-stable, resin containing less than 2% essential oil and exhibiting remarkable antioxidant properties. The second fraction was the essential oil containing more than 95 mL steam distilled oil/100 g. The use of SC-CO2 for the production of essential oils or oleoresins from spices is possible by selecting suitable pressures and temperatures. The oils extracted with supercritical technology were found to be superior in terms of their chemical composition and higher percentage of sesquiterpene compounds. Supercritical extraction with CO2 and hydrodistillation extraction methods were used to extract essential oil

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160 120 Yield (g/kg) 80 40 0 0 5 CO2, 313 K 10 15 Solvent (kg/kg ginger ) Propane, 323 K 20 Dimethylether, 313 K 25

FIGURE 1.5 Supercritical extraction yield of ginger using CO2, propane, and dimethyl ether (modied from Catchpole et al.51).

160 120 Yield (g/kg) 80 40 0 0 5 CO2, 313 K 10 Propane, 323 K 15 20 25 Solvent (kg/kg chili) Dimethylether, 313 K

FIGURE 1.6 Supercritical extraction yield of chili using CO2, propane, and dimethyl ether (modied from Catchpole et al.51).

(Juniperus communis L.)9 (Table 1.6). Oils obtained by SC-CO2 and hydrodistillation showed signicant differences; the former was more selective and particularly efcient for the isolation of -thoujone and limonene. Anitescu et al.55 did a comparative analysis of coriander oil with supercritical CO2 and stream distillation (Table 1.7). They concluded that oils obtained by supercritical extraction gave a superior aroma compared to both the commercial and hydrodistillation extracted oils.

1.3.5 DECAFFEINATION

OF

COFFEE

AND

TEA

Caffeine (1,3,7-trimethylxanthine) is a bioactive plant component commonly found (since 1820s) in popular beverages such as teas (Camellia sinensis), coffees (Coffee Arabica, canephora, liberica), and soft drinks.81 Caffeine is a secondary metabolite, the product of nucleic acid catabolism, and belongs to the group of compounds

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200 150 Yiled (g/kg) 100 50 0 0 5 CO2, 313 K 10 15 20 25 Solvent (kg/kg pepper) Propane, 323 K 30 35

Dimethylether, 313 K

FIGURE 1.7 Supercritical extraction yield of pepper using CO2, propane, and dimethyl ether (modied from Catchpole et al.51).

TABLE 1.6 Essential Oil (Juniperus communis) Oil Extracted by Comparing Supercritical Extraction and Hydrodistillation (HD) Methods
Pressure Temperature Dynamic time Modier No. Compound 1 -Thoujone 2 -Pinene 3 Sabinene 4 Myrcene 5 3-Carene 6 Limonene 7 Terpinolene (MPa) (C) (min) (L) RI* 928 943 972 987 1007 1020 1071 20 45 20 1 25.1 1.3 2.3 2.8 35.0 22.0 2.6 20 45 30 2 26.2 1.4 2.1 3.8 36.8 24.2 20 55 30 3 26.9 1.4 2.3 3.8 37.1 22.9 2.6 35 45 30 4 17.0 1.8 2.1 1.3 19.9 9.0 0.8 35 55 30 5 13.0 1.8 1.2 17.1 8.6 2.1 35. 55 30 80 6 22.0 1.9 1.9 2.4 29.1 15.6 2.7 35 55 30 400 7 4.0 22.4 34.6 4.2 2.6 6.1 2.7 HD

24.5 0.4 3.4 39.4 3.1

* Retention Index; Source: Modied from Pourmortazavi et al.9

known as purine alkaloids. Excessive ingestion of caffeine may cause certain health problems such as palpitations, gastrointestinal disturbance, anxiety, tremor, increased blood pressure, dizziness, and insomnia.8285 The aroma and avor coupled with the stimulant effects comes from caffeine. Coffee beans have about 2% to 3% while tea leaves have about 5% caffeine,82 depending on the variety and species. Decaffeinated coffee must contain less than 0.1% caffeine on a dry weight basis, as specied by EEC regulations. Therefore, decaffeination of coffee and tea poses signicant challenges to both the producers and processors. The demand for decaffeinated coffee is high on the world market. It accounts for more than 20% of all coffee sales in the USA, with a 50% growing demand among the adult population.82 Research in

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TABLE 1.7 Results of Comparative Analysis of Essential (Coriander) Oil between Supercritical CO2 and Steam Distillation Processes
Kovats RI 928 936 951 975 980 990 1006 1030 1033 1103 1147 1174 1184 1186 1192 1226 1241 1245 1287 1299 1363 1382 1428 1463 1526 1594 Comm Oil Trace (Tr) 3.3 0.6 0.1 1.0 1.2 1.1 2.4 Tr 63.8 5.5 Tr Tr Tr 1.0 0.1 0.1 0.5 0.7 Tr 0.1 1.0 Tr Tr Tr 3.8 Steam Distillation Oil Tr 2.3 0.4 0.3 0.3 0.8 0.3 2.3 0.1 62.8 5.6 0.1 0.1 0.1 0.9 0.3 0.1 1.0 0.4 0.1 0.1 1.8 2.1 0.3 Tr Tr 2.9 SC-CO2 Oil 0.1 2.8 1.5 0.9 0.9 1.0 0.3 2.7 0.1 61.9 5.6 0.1 0.3 0.2 0.6 0.2 0.2 1.0 0.4 0.2 0.2 2.4 0.8 0.2 0.2 0.2 1.7

No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

Compounds -Thujene -Pinene Camphene Sabinene -Pinene -Myrcene 3-Carene Limonene 1,8-Cineole Linalol Camphor Menthol -Cymen-8-ol Cis-Hex-3-enyl butyrate -Terpineol -Citronellol Neral Carvone Anethole Carvacrol Neryl acetate Geranyl acetate -Caryophylene -Humulene Eugenyl acetate -Caryophylene Unidentied Compounds

Source: From Anitescu et al.55

genetics engineering to produce transgenic tea and coffee plants decient in caffeine is in progress.82,83,86 However, the consumption of genetically modied products is still contentious globally, and SCE technology gives the best option in combating these critical issues. The decaffeination of coffee and tea using SCE technology is among the rst known commercial operation in the food industry. In the past, methylene chloride was used for decaffeination of coffee with one cycle of production lasting from 24 to 36 h and the end products usually contained toxic residues, thus posing more harm than the caffeine. Due to its suspected carcinogenic effect, the FDA placed regulations against methylene chloride use. However, decaffeination process with SC-CO2 uid can be accomplished on green

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coffee or roasted coffee beans or tea leaves without deleterious effects on the avor even after 10 h of processing, and many patents exist for such processes. The process requires charging the extraction vessel containing the coffee beans with CO2 at a pressure of 7 to 22 MPa and temperature of 31C. The caffeine is dissolved in the SC-CO2 stream, which subsequently enters a washing tower or alternatively activated carbon scrubbers, distillation, recrystallization, or reverse osmosis are used in some instances to entrain caffeine. The method can strip coffee of its caffeine content (0.7% to 3%) by 71% to 97%.87 The caffeine recovered is sold for medicinal purposes and or for use in soft drinks. Peker et al.88 reported that soaking raw coffee beans in water prior to processing enhances the rate of decaffeination.

1.3.6 FISH OIL CONCENTRATION


Fish oils are characterized by a high percentage of unsaturated straight-chain fatty acids ranging from C14 to C22 with one to six double bonds. They contain essential fatty acids (EFA) and polyunsaturated acids, grouped into omega-6 and -3 EFAs. The main sources of omega-3 (-3) are axseed, walnut, and marine plankton and sh. This review would focus on -3 oils derived from sh. Eicosapentaenoic acid (EPA) and decosahexaenoic acid (DHA) are predominant in sh oil, and have been reported to contribute to the prevention of atherosclerosis, heart attack, depression, and cancer if consumed in sufcient quantities.89 Fish oil derivatives in the form of -3 oils are in high demands as food additives. For example, Ocean Nutritions ME3TM Omega-3 powder is currently used in several breads. In the US, Wegmans Food Markets, Rochester, NY, launched breads fortied with MEG omega-3 fats, two slices of which offers 80 to 90 mg of omega-3s.90 Encapsulated omega-3 fatty acids forms are available for fortied bakery products. Fish oils are processed as fatty acids or as methyl or ethyl esters which are more stable than the free acids form.91 Fatty acids are highly soluble in CO2 and as a result SC-CO2 extraction is a preferred method of fractionation. With this technology, it is possible to separate heat-sensitive compounds (-3 fatty acids) and avoid toxic solvent residues in the nal product. The isolation and fractionation of -3 PUFA (polyunsaturated fatty acid) from sh, sh oil, and esters using SC-CO2 have been studied by several researchers.72,92,93 Eisenbach93 fractionated the ethyl esters from cold sh oil using SC-CO2 at a pressure of 15 MPa and an extracting temperature of 50C. Alkio et al.94 produced EPA and DHA with 50% and 90% purity, respectively, from trans-esteried tuna oil using carbon dioxide. Temelli et al.72 obtained the highest yield of -3 fatty acids at 35 MPa and 35C without denaturing the protein during SCE. They also compared solvent (hexane) extraction to SC-CO2 as shown in Table 1.8. A higher concentration of -3 was achieved with SC-CO2 (Table 1.8). At 25 MPa pressure and temperature from 40C to 80C, no signicant effect on yield was observed in oil extraction from krill.38 Hardardotti and Kinsella73 did not see any yield change on the recovery of rainbow trout at operating pressure ranging from 13 to 35 MPa and temperature range of 40C to 50C.

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TABLE 1.8 Fatty Acid Composition of Fall Atlantic Mackerel Oil Concentrate Extracted with Hexane and SC-CO2 at Pressure 34.5 MPa and Temperature of 35C Fractionated by Gas Chromatograph
Fatty Acid C16:0 C16:1 C18:0 C18:1 C18:3 C20:0 C20:1 C20:5 (EPA,-3) C22:5 C20:5 (DHA,-3) EPA+DHA (-3) GC Retention Time (min) 8.07 8.53 12.49 13.09 15.58 18.05 18.14 22.79 30.08 31.43 Hexane Extract 15.86 6.65 3.53 3.59 0.88 11.22 1.20 6.07 1.34 8.58 14.65 SC-CO2 Extract 16.66 7.94 3.16 3.16 1.04 7.84 1.20 8.76 1.38 8.97 17.73

Source: Modied from Tamelli et al.72

1.4. FACTORS AFFECTING EXTRACTION YIELD


Several bioactive components were extracted successfully by the SC-CO2 extraction method as outlined in the preceding sections. Optimization of yield is a function of various independent parameters. Process parameters such as solvent ow rate, resident time, moisture content, particle sizes, and particle size distribution in conjunction with supercritical pressures and temperatures are key parameters for achieving optimum results. Most of these parameters can have individual or combined effects on the extraction rate of a process; for example, the resident time can have an immense inuence on the composition of the extracted compound.

1.4.1 PRESSURE
Figure 1.8 is a typical extraction rate curve, and it is apparent that pressure significantly inuences the rate of extraction, and likewise the extraction time. Extrapolating the normalized yield at the point where the yield curve become asymptotic gives signicant different normalized yields of 15, 11, and 4% for pressures of 10, 9, and 8 MPa, respectively.8 Macias-Sanchez et al.61 observed similar trends in the SC-CO2 extraction of carotenoids and chlorophyll a from Nannochloropsis gaditana, although as pressure increased beyond a critical point the yields dropped as a result of increased density. Higher density causes a double effect: increases the solvation power and reduces the interaction between the uids and matrix, thus decreasing the diffusion coefcient. Excessive pressure also increases the compactness of the sample matrix, thus reducing the pore sizes and apparently reducing the mass transport, which eventually diminishes the yield.95

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16 12 Yield (%) 8 4 0 0 100 200 8 MPa 300 400 Time (min) 9 MPa 500 10 MPa 600 700

FIGURE 1.8 The effect of pressure change on bioactive compound yield during supercritical extraction of essential oil from Juniperus oxycedrus on extraction rate at ow rate 1.5 kg/h and temperature 50C (modied from Marongiu et al.8).

The selectivity of solute extraction is a function of extraction pressure, and if it increases, different solutes are extracted. DAndrea et al.96 also found that with a working pressure of 25 MPa and temperature of 55C, they attained optimum yield for the extraction of azadirachtin and 3-tigloylazadirachtol from neem seeds. Similarly, Tonthubthimthong et al.95 reported optimum yield at a pressure of 23 MPa and a temperature of 55C.

1.4.2 TEMPERATURE
Temperature is a parameter with signicant inuence on SFE; therefore manipulating it could have adverse implication on the process and yield. Figure 1.9 shows the general trend of increased extraction yield as temperature increases relative to the pressure. Tonthubthimthong et al.95 reported similar trends for extracting nimbin from neem seeds at 20 MPa and a CO2 ow rate of 0.62 mL/min, and found 35C the optimum temperature for their process. Ge et al.32 indicated a temperature of 35C gave the highest yield in the rst 45 min of extraction, but during prolonged extraction from 45 to 120 min, the highest temperature condition was shown to produce the highest yield (Figure 1.9). Although many literature reports correlated increased temperature to yield,45 many others showed no particular trends as far as temperature was concerned.97 Some researchers reported yield was inversely proportional to temperature under 15 MPa. The combined effect of pressure and temperature on cholesterol extraction was studied by Chao et al.44 At a pressure-temperature setting of 34 MPa and 50C, cholesterol yield of 160 mg/100 g was realized compared to 430 mg/100 g when the temperature drops to 40C and 2.5 kg of CO2 was used. As the mass of CO2 increased, the yields decreased, but the lowest temperature still maintains the highest yield as shown in Figure 1.10. Also, under constant temperature, increases were achieved when pressure decreased. The results demonstrated that higher selectivity is possible at lower pressures and higher temperatures. Froning et al.98 corroborated this fact based on their experiment with lipid and cholesterol extraction from

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2600 Yield (mg/100 g)

2200

1800

1400 10 15 35C 20 25 Pressure (MPa) 40C 30 35 50C 40

45C

FIGURE 1.9 The effect of temperature change on bioactive compound yield during supercritical extraction of wheat germ on extraction, time 120 min, rate at ow rate 2.0 mL/min and sample size 5g (modied from Ge et al.32).

500 Cholesterol (mg/100 g) 400 300 200 100 0 0 5 10 15 20 25 Carbon dioxide (kg)

34.5 MPa/50C 34.5 MPa/40C

13.8 MPa/50C 24.1 MPa/40C

FIGURE 1.10 Supercritical carbon dioxide extraction of cholesterol in beef tallow, different pressures and temperatures (modied from Chao et al.44).

dehydrated chicken meat. The combination of pressure and temperature of 38.6 MPa and 55C, respectively, yielded lipid (89%) and cholesterol (90%), while a pressure and temperature combination of 30.3 MPa and 45C, respectively, produced much lower yield.

1.4.3 MOISTURE CONTENT

OF

RAW MATERIALS

Moisture content is a factor that inuences extraction yield of bioactive compounds as shown in Table 1.9. A maxima yield of 1678 mg/100 g was achieved at 5% moisture content, and any further increase or decrease in moisture reduced that yield. Therefore, it is important to establish this magic number in order to maximize yield in the SCE process. The effect of sample pretreatment is crucial in attaining this

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TABLE 1.9 The Effect of Moisture Variation on Bioactive Compound Yield during Supercritical CO2 Extraction of Wheat Germ
Water Content (% wet basis) 4 5 8 12 Yield (mg/100 g) 1470 1678 1352 1290

Source: Modied from Ge et al.32

objective. Water removal in most cases frees the internal pores and thus increases the mass transport intensity, because water in the sample matrix would inhibit the ow of SCF by changing surface tension and contact angles as a result of phase interaction between the three components (water, sample matrix, and SCF). For example, the higher the moisture content, the higher the probability for the formation of a thin lm of water between the sample matrix and the SCF phase. Water has a small but nite solubility in SC-CO2, and as a result it can also be extracted with the targeted components, and its separation can then only be done at the end of the process.

1.4.4 COSOLVENT
The use of cosolvent (entrainers) during SCF extraction is key to enhancing the extraction efciency and cost effectiveness of the processes. Joslin et al.99 indicated two signicant attributes of cosolvents: the interaction between the cosolvent and the solute (direct effect) and the cosolvent-solvent interactions (indirect effect). Cosolvents used in small doses (1% to 5% mol) in SCF can change the overall characteristics of the extraction uid such as polarity, solvent strength, and specic interactions. These changes in turn can signicantly alter the density and compressibility of the original SCF.5 Additionally, they can improve selectivity for desired components and facilitate selective fractional separations. Table 1.10 summarizes the results of different cosolvents (ethanol, methylene chloride, and methanol). Ethanol was found to have the greatest enhancement factor while methanol had the lowest.100 The solubility enhancement is the result of complex interaction between the -carotene, SC-CO2 , and cosolvent as indicated by Joslin et al.99 Temelli et al.101 observed an enhancement factor of 64, 63, and 29 by using ethanol as cosolvent to extract palmitic acid, stearic and behenic fatty acids, respectively. Baysal et al.63 used ethanol at different concentrations (5%, 10%, and 15%) to recover -carotene and lycopene from tomato paste. Although they observed that, with a high ethanol

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TABLE 1.10 The Effect of Fractionation of -Carotene in Cosolvent in Supercritical Carbon Dioxide Mixtures at the Temperature of 70C. Enhancement Factors were Determined Based on Density (17 mol1)
Pressure (MPa) CO2 Density (mol1) -Carotene CO2 15.71 16.73 17.65 18.43 18.91 19.49 19.95 Yields ( 107)

21.2 24.9 28.7 32.8 35.8 40.0 43.9

1.95 3.33 6.23 10.00 12.50 19.10 25.4

-Carotene CO2 + 1 wt % Ethanol (Enhancement factor = 4.7) 22.3 15.92 9.6 24.9 16.73 19.5 31.6 18.22 25.2 37.4 19.14 37.5 -Carotene CO2 + 1 wt % methylene chloride (Enhancement factor = 3.5) 23.4 16.28 12.8 24.7 16.67 13.3 31.2 18.16 21.7 37.0 19.08 27.7 -Carotene CO2 + 1 wt % Methanol (Enhancement factor = 2.1) 18.0 13.92 3.98 26.8 17.26 9.62 33.0 18.47 15.60 37.3 19.12 30.60 Source: Modied from Cygnarowicz et al.100

concentration, the extraction was hindered due to a decrease in the homogeneity of the extraction mixture, no statistically signicant differences were found between the 10% and 15% concentrations. Quancheng et al.102 also investigated the effect of cosolvents on the extraction of tocopherols. They extracted tocopherols from rapeseed deodorizer distillate at 2%, 4%, and 6% with methanol, ethanol, and mixed

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ethanol as cosolvents, under operating conditions of 12 MPa and 60C. The use of 4% cosolvent gave the best tocopherol yield followed by 2% and 6%, and the 2% and 6% methanol concentration gave the best yield of the three and the mixed ethanol the worst.

1.4.5 PARTICLE SIZE


Particle size may have a signicant impact on the ow behavior of SCF in the sample matrix. The mechanism of sample pretreatment, for example the methods of drying (air, oven, vacuum, or freeze drying), would inuence particle sizes when subjected to attrition or size reduction. The sizes of particles, shapes, and their random layout (size distribution) would determine what goes through the medium and how fast. The layout would inuence the type of pore, either open or blind pores, or their degree of interconnectedness. Process parameters such as pressure inuence particle size distributions. Pressure tends to create compactedness,95 thus decreases the intergranular porosity resulting in increased solid density. The smaller the particle sizes, the larger is the surface area, and as a result bioactive components are released easily. However, Coelho et al.103 observed no signicant effect of particle sizes on the extraction yield as a function of extraction time at a xed ow rate. The oxygenated compounds increased from 81% to 85% as particle size decreased, as shown in Table 1.11. However, the ndings of Ge et al.32 were contrary to Coelho et al.103 in their study on the effect of particle sizes on wheat germ (Table 1.12). Papamichail et al.104 extracted essential oil from celery with SC-CO2. They experienced increased yield (more oil released) as the particle sizes of the seed decreased and attributed that to the pretreatment milling and sieving. Maximum yield of 1838 mg/100 g was obtained with optimum particle size of 0.505 mm. Also, they observed that very ne and big particle sizes have low extraction yield probably due to higher resistances to mass transfer because of the compact tendency reecting reduced pore sizes in ner particle sizes while less interactions with the supercritical uids in the case for the latter. Likewise larger particles contain undamaged cell walls rendering then impervious,60 although some believed that higher SC-CO2 ow rate is capable of degrading protein structure to release the targeted bioactive components.105 Nelson106 revealed the need to break the lipoprotein matrix in order to release the embedded lipids in sh during SCE. Papamichail et al.104 reported that it was possible to extract more essential oil per kg of CO2 at a lower ow rate due to the intraparticle diffusion resistance. Therefore, a threshold level for each product has to be experimentally determined in pursuance to pilot plant or industrial processing.

1.4.6 FLOW RATE


Coelho et al.103 presented three scenarios of 2.3, 1.5, and 0.85 kg/h ow rates as a function of extraction time and concluded the highest ow rate of 2.3 kg/h gave the higher rate of extraction of Foeniculum vulgare volatile oil with SC-CO2 at 9 MPa and 40C. A similar trend was observed by Ge et al.32 who reported a ow rate of 3 mL/min yielded 1927 mg/100 g of bioactive component from wheat germ. Some

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TABLE 1.11 Comparative Supercritical CO2 and Hydrodistillation Extraction of Volatile Components from Fennel (Foeniculum vulgare) Fruits of Different Particle Sizes
HD (%) Volatile Components Canfene Sebinene Myrcene -Phellandrene Limonene -Terpinene Terpinolene Fenchone Estragol (E)-Anethole Piperitenone oxide Unknowns Waxes Stalks 0.2 0.1 1.2 2.0 2.1 Tr 0.5 15.8 18.9 42.5 0.2 4.8 Fruits (0.5 mm) 0.2 0.2 1.4 2.2 3.6 0.1 0.6 16.8 20.9 42.2 0.2 3.4 SCE CO2 (%) Fruits (0.55 mm) 0.2 0.2 1.4 2.2 3.5 Tr 0.6 16.2 21.0 42.5 0.3 5.5 0.6 Fruits (0.35 mm) 0.1 0.2 1.3 1.9 3.1 Tr 0.6 17.1 21.9 44.6 0.3 0.3

Tr (Trace < 0.05)). Source: Modied from Coelho et al.103

TABLE 1.12 The Effect of Particle Sizes on Bioactive Compound Yield during Supercritical CO2 Extraction of Wheat Germ
Sieving (mesh) No grinding 20 30 40 60 80 100 Particle Size (mm) 2.1 0.86 0.51 0.40 0.22 0.18 0.13 Yield (mg/100 g) 1610 1710 1838 1550 1070 890 742

Source: Modied from Ge et al.32

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0.18 Yield (kg/kg feed)

0.12

0.06

0 0 50 100 150 Time (min) 1.1 kg/h 3 kg/h 200 250

FIGURE 1.11 The effect of ow rate on supercritical extraction of essential oil from celery at pressure 15 MPa and temperature 45C (modied from Baysal et al.63).

of these facts were corroborated by Peker et al.88 in their experimental study on extraction rates of coffee beans with SC-CO2. They indicated the need for long extraction time in conditions where low ow rate is used. Figure 1.11 shows an apparent yield at high ow rate for extracting celery oil104 and Juniperus oxycedrus essential oil.8 Summarized in Table 1.13 are the results of Baysal et al.63 where a ow rate of 4 kg/h was identied as the optimum condition for attaining the highest yield. Similar trend was observed by Ferreira et al.18 for extracting essential oil from black pepper. They observed larger yield at 30 MPa using the upper-level ow rate (10.54 kg/s). When maximum solubility is attained, the highest CO2 ow rate would offer the highest recovery in extracting lipids from sh.72

1.4.7 EFFECT

OF

TIME

ON

YIELD

Several factors have direct or indirect implication on yield during SCE. Resident time is an important factor that inuences yield and the economic viability of the process. Other factors such as temperature and pressure could have individual or

TABLE 1.13 Effect of Flow Rate on the Supercritical Extraction of Lycopene and -Carotene from Tomato Paste
Flow Rate (kg/h) 2 4 8 Extraction Time (h) 4 2 1 Lycopene (%) 14 22 20 -carotene (%) 30 43 34

Source: Baysal et al.63

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60 50 Content (%) 40 30 20 10 0 0 50 100 HM 150 200 Time (min) OM (a) HS 250 OS 300 350

Cumulative quantity (g)

1.5

0.5

0 0 50 HM 100 OM 150 HS (b) 200 OS 250 300 Overall 350 Time (min)

FIGURE 1.12 Families of avor compounds extracted from Santolina Insularis at different supercritical extraction times. HM: hydrocarbon monoterpenes; OM: Oxygenated monoterpenes; HS: Hydrocarbon sesquiterpenes; and OS: Oxygenated sesquiterpenes (modied from Cherchi et al.107).

combined effects. Cherchi et al.107 performed detailed analysis of avor compounds in essential oil extracted from Santolina insularis by SC-CO2 extraction. They reported the change in concentration exhibited by the monoterpenes from 50% in the fraction collected after 30 min to 10% in the fraction collected after 240 min (Figure 1.12a) under optimum conditions of 9 MPa and 50C and a two-stage separation. The rst-stage separation was accomplished under 9 MPa and 12C, while the nal stage used 2 MPa and 15C. The yield becomes asymptotic at 1.75% with increased extraction time while the rate decreases (Figure 1.12b). Hawthorne et al.108 studied the extraction rate of basil conducted at 30 MPa and 45C for 10 min and identied 1,8-cineole, estragole, eugenol, and selinene. Yield in most cases

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was dependent on time. Tamelli et al.72 considered 3 to 4 h sufcient time to extract all extractable lipids from freeze-dried krill and 6 h for rainbow trout regardless of the conditions.

1.5 SUMMARY
Supercritical carbon dioxide extraction has been shown to be a viable alternative to the conventional solvent extraction technique to extract bioactive components from agricultural materials. It offers a unique advantage of adding value to agricultural waste by extracting antioxidants and avonoids (lycopene from tomato skin, essential oils, and avor and fragrances, etc.), which are then used for the fortication of foods and other applications. Its drawbacks are the difculties in extracting polar compounds and its susceptibility of extracting compounds from a complex matrix where the phase interaction with the intrinsic properties of the product inhibits its effectiveness. Many of these drawbacks can be ameliorated by using cosolvents. However, much investigation is required to understand the solvation effects on targeted bioactive components being extracted. Supercritical uid extraction technology can be utilized to provide healthy snack foods, a problem that faces the snack food industry, with regard to fat/oil contents, which is becoming a greater public health concern. A lot of cost is incurred reformulating products such as French fries, onion rings, and other snack foods to eliminate trans fats and high cholesterol content. Defatting and decholesterol with SCF has been demonstrated as applicable to food products. Although most of the tests were conducted on dehydrated products, research has shown successful application of SC-CO2 on high moisture products where extraction could be accomplished without compromising the organoleptic characteristics. The SCF technology is available in the form of single-stage batch and could be upgraded to multistage semicontinuous batch coupled with a multiseparation process. Although much work was accomplished, it would not warrant complacency, since batch modes render the SCF technology cumbersome for certain industrial applications, and has been the drawback for broader commercial adaptation. However, the needs to improve the design into continuous modes are growing. One possibility is to integrate membrane technology into a supercritical process. Study on membrane and SC-CO2 has been attempted and needs an aggressive pursuance. This concept would make feasible the extraction of bioactive components from aqueous, less viscous, and pumpable substances in a continuous mode. Supercritical carbon dioxide extraction could only be cost effective under largescale production, which made it ideal for decaffeination of coffee, tea, and hops. Although it is expensive, it is much more economical when compared to conventional solvent extraction at high production scales. With improved processing conditions and reduced cost, SCE will become even more economical at low throughput.

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