Review

Extraction of secondary
This review article intends to give an overview of the developments in the extraction technology of secondary metabolites from plant material. There are three types of conventional extraction techniques. In order of increasing technological difficulty, these involve the use of solvents, steam or supercritical fluids. Each of these types of extraction methods is described in detail with respect to typical processing parameters and recent developments. Following the discussion of some technical and economic aspects of conventional and novel separation processes, a few general conclusions about the applicability of the different types of extraction techniques are drawn.

metabolites from plant material: A review

I Dick A.J. Starmans and Herry H. Nijhuis

tea. Basically, pretreated plant material is put into contact with hot water, which takes up the flavour compounds and colouring agents. After filtering off the solid residue, the extract is ready for consumption. In the case of the isolation of certain compounds from plant material by means of liquid extraction, some technological problems have to be overcome. First, the plant material has to be pretreated in order to obtain reasonable extraction yields. Convenient methods are maceration and cutting, followed by drying and breaking. A somewhat newer method is cryogenic milling, which results in a product with a large surface area and minor losses of volatile organic compounds owing to the low temperature used. Therefore, pretreatment using cryogenic milling can lead to a more efficient extraction. Another problem is the need for special (i.e. toxic organic) solvents to be used in the extraction procedure. Extraction has been performed using edible oils and , some common organic solvents (e.g. hexane*, ether, chloroform3, benzene, ethano14). With these solvents, antioxidants from well-known spices such as rosemary3, sage5, thyme and marjoram6 have been isolated as natural replacements for the synthetic antioxidants butylated hydroxyanisole and butylated hydroxytoluene. More recently, attention has broadened from the isolation of specific antioxidants towards the extraction of other valuable organic compounds that can be used in the food industry. Of particular interest is the isolation of aromas and fragrances from plant essential oils and ,* fruit9.

Although chemistry has provided humankind with a large number of different materials to make life easier, we still cannot make some compounds as efficiently as mother nature does. Plant cells synthesize a vast supply of natural compounds that are not strictly necessary for growth or reproduction, but whose presence can be demonstrated genetically, physiologically or biochemically. The natural function of these so-called secondary metabolites is often closely related to their function when used in a pure form. Thus, natural sprout suppressants, insecticides, fungicides, as well as aromas, oils and fragrances originate from plant material (see Table 1). To exploit plant material resources, extraction techniques have been developed to obtain such secondary metabolites commercially. In addition to the well-known extraction processes, some new techniques for the separation of compounds from plant material have received a considerable amount of attention. These techniques are largely focused on finding technological solutions to diminish or even prevent the use of organic solvents in extraction processes, to obtain more highly purified products containing fewer additional toxins, rendering them useful in a wider range of applications. Furthermore, the use of plant material as the source of such products allows consumer demands for the replacement of synthetic compounds with natural substitutes to be met.

Solvent extraction

In the 18th century, scientific research regarding the isolation of valuable compounds from plant material focused on the isolation of odours from flowers. In a technique called enfleurage, fat was brought into direct contact with flowers, thus extracting the odours from the plant matrix. Nowadays, in practically all households worldwide, solvent extraction is practised while making coffee or

Processing Solvent extraction on an industrial scale is often performed using an inclined diffusor (Fig. 1). In this type of apparatus, a pair of intermeshed helical screws transports the material through a heated solvent phase. The pulp is pressed and the press liquid is heated before it is returned to the diffusor. The extraction liquid is then drawn off at the bottom of the trough. The total volume of solvent in the diffusor is kept constant by feeding fresh solvent into it at the upper end. Following extraction, simple evaporation of the solDick A.1. Starmans and Herry H. Nijhuis are at the Agrotechnological vent is often sufficient to obtain the final product in its ResearchInstitute (ATO-DLO), PO Box 17, NL-6700 AA Wageningen, The pure form, but legislation requires the removal of orNetherlands(fax: +31-317-412260; e-mail: d.starmans@ato.dlo.nI). ganic solvents to a prescribed maximum residual level
01996. Elsevier Science Ltd PII: SO924-2244(96110020-O

Trends in Food Science & Technology June 1996 [Vol. 71

19

Steamdistillation to obtain essential oils from plant material has been a research subject for many years, and Function Source/compound Application many famousresearchers have given it their attention. As early as 1910, von Colorant Beetrootjuice Ice cream Rechenberg explained the phenoml Spearmint oil Aroma Toothpaste,chewing gum enon that oxygen-richcompoundswith Carsonic acid Antioxidant French fries a high boiling point will be isolated before oxygen-poorcompoundswith a Antioxidant Rosemaryoil Preventionof fat oxidation low boiling point, because oxygen-rich Carvone Sprout suppressant Potato storage compoundswill dissolve more easily Palm oil Food additive, cosmetics Food industry, skin softener in the watery phasepenetrating plant the material. For instance, when caraway (i.e. 5-30mg hexaneper kg of product) if the product is seedsare extracted,the releaseof carvone (T,., = 71C to be used in food applications. Also, the thermal in- solubility = 1.32g/l) is promoted despiteits high boiling stability of the compoundsof interest may lim it the use point, becauseit dissolves in water, unlike limonene of elevated temperatures during the extraction and (T,, = 60-61C insoluble in water). evaporationprocesses. addition, the extra emissionof In Vapour-phaseextraction can also be performed on toxic chemicals into the environment and the fact that liquid samples. For instance, steam distillation of the higher-boiling impurities are retained in the final prod- volatile componentsof black tea was used to separate uct have brought about serious interest in alternative aroma m ixtures into basic and neutral components12, thus allowing the further characterizationof the whole liquid-solid and liquid-liquid separationprocesses. aromaof black tea. Another two examples steamdistilof lation are the recovery of antioxidants from the waterNew developments concretethat is obtained after evaporation Not only is the recovery of the solvent from the ex- dispersed tract phaseimportant, but it is also important to recover of the solvent used in the extraction of herb spices6, the solventpresentin the residuephase.A new technique and the isolation of essentialoils that have antioxidant which frees residuesof a solvent activity from caraway,clove, cumin, rosemary,sageand is flash desolventizinglO, from a solid phaseby evaporationat low temperaturein thyme13. a high-velocity streamof superheated solvent. Processing Steamdistillation is the most commontype of vapourSteamextraction Besides liquid extraction procedures,there are also phaseextraction. In this extraction technique,a packed vapour-phaseextraction procedures.A vapour can pen- bed of plant material, which is placed on a porous supetrate coarsely pretreated plant material better than a port, is continuouslyflushedwith steam(Fig. 2). Volatile liquid can becauseof its low viscosity, and thereforere- organic compounds present in the plant material are sults in a larger effective contact area. Furthermore,the taken up by the vapour phasebecauseof their low pardiffusion of small compoundsin a vapour phaseis much tial vapour pressure. Thus, compoundscarried by the from it by decreasing higher than in a liquid phase. Thus, although the solu- vapour streamare easily separated bilizing capacity of a vapour phaseis far less than that the solubility. This is achievedby lowering the temperaof a liquid phase,the net recovery of secondarymetab- ture of the vapour stream by forced condensationin a unit. The resulting liquid m ixture is led olites using an extraction procedure with a gaseous heat-exchange phasecan surpassthat of one that usesa liquid phases. into a separator,where static separationof the oil phase from the water phaseoccurs. The isolated compoundswill redistribute bePress liquid tween both the oil and watery phase, according to their respective partition ter coefficients. Thus, any water-soluble componentswill reside to someextent in the water phase( hydrolate ). Legislationregardingindustrialwaste water is rapidly increasing. This im0 0 plies that ways will have to be found m Q to cope with renewed (i.e. decreased) maximum emission standards.AddiResidue Extract tionally, the loss of valuable volatile compounds should be restricted for Fig* economicreasons.The total amountof l
Table 1, Examplesof applications of secondary metabolites from plant material A schematic representationof a diffusor (shown in cross-sectionat left) for continuous solvent extraction of a solid sample. 192 waste cm h decreased by p&d

con-

densationof the vapour phase. When

Trends in Food Science & Technology June 1996 [Vol. 71

this is sufficient to separate the oil phase from the vapour phase, the vapour phase can be reheated and repressurized before it is re-circulated. Thus, the oil phase is in equilibrium with only a fraction of the total amount of the aqueous phase, leading to a decrease in the total amount of waste. Furthermore, by applying re-circulation, the process is much more energy efficient. Another possibility for decreasing the total amount of waste water generated is the application of new membrane-based processes such as pervaporation. This technique is still gaining ground in processes that require the removal of volatile organic compounds from liquid systems; consequently, it will be described in more detail in a later section.
/

Condenser

Sample Divider tray

Steam generator

Separator Watery ) Organic phase phase

New developments Although the use of vapours and gases for the isolation of organic compounds is already well established, innovations in this area are still being reported in the literature. For instance, much research has been carried out on instant coffee. By changing process parameters such as temperature, pressure and residence time of the coffee beans in the extractor, the total efficiency of the extraction can be altered. Futhermore, by adjusting these parameters, the unwanted extraction of typical offflavours can be counteracted to a moderate extent, albeit at the cost of the total yield of instant coffee. A new multistage method of steam extraction of ground coffee to obtain instant coffee with a natural taste has therefore been patented14. Not only have the process conditions been optimized, but also the hardware used in extraction processes has been subjected to modernization. Recovery of organic contaminants from dairy products proved possible up to the parts-per-trillion range by using a simultaneous steam distillation and solvent extraction technique15. Although this technique has not yet been used for the isolation of secondary metabolites from plant material, further improvements to increase the capacity of this laboratory-scale system might be worth looking into.

Fig. 2
A schematic representation of a steam distillation setup.

and subsequently recycled. There are five motives for choosing supercritical carbon dioxide as the extraction medium:
l

It has a critical temperature of 31 this means that C; extractions can be conducted at temperatures that are low enough not to harm the physicochemical properties of the extract. It is inert in nature; thus, there is no risk of side reactions such as oxidation. It is nontoxic; carbon dioxide is a harmless material that is often used in beverages. It has been accepted by most European food and drugs acts as an extraction medium for the isolation of food-related compounds. It has a low polarity; the polarity of carbon dioxide is close to that of pentane and hexane, which are solvents commonly used in liquid extraction procedures. Thus, a similar range of compounds can be extracted using both techniques. It allows fractionated separation; by simply choosing different temperature and pressure conditions for a number of sequential separator vessels, a fractionated separation of the organic compounds can be achieved.

Supercritical extraction
Because of the low solubilizing power of vapours and gases, research has been carried out on gas-phase extractions performed at elevated pressures and temperatures. When a gas is compressed and heated, it can reach a state of aggregation at which no distinction between the gas and liquid can be observed. Such a so-called supercritical gas has improved solubilizing properties that are roughly comparable to those of liquids, yet has an extremely high diffusion coefficient, resembling that of a natural gas. A supercritical gas also has a viscosity similar to that of a gaseous phase, which means that the contacting area between the plant material and a supercritical fluid is also similar. The orders of magnitude of physical data for extraction agents in various phases are given in Table 2 (Ref. 16). When the pressure of a supercritical gas is decreased, the extracted organic compounds will easily separate from the gaseous phase. The gas can be re-pressurized
Trends in Food Science & Technology June 1996 [Vol. 71

Processing Extraction with supercritical fluids is rather similar to normal liquid-extraction methods. A simplified
Table 2. Orders of magnitude of physical data for extraction agents in various phases Phaseof the extraction agent Property
Diffusion coefficient (cm2/s) Density (g/cm3) Viscosity (g/cm.s)

Gaseous
0.1 10 10-h

Supercritical
1O-3 0.3 1o-4

Liquid
5 x 10-6 1 10-2

Processdesign and material handling are often easier when dealing with small material streamsthan with bulk quantities. Therefore, the selective removal of small amountsof undesirableconstituentspresentin an otherExtract wise desirableproduct is preferred to the selective removal of the desirable product itself. Some foodstuffs contain compounds that have to be removed before consumption.In certain corn-derivedproducts,naturally present glucosinolates undesiredflavourslg,and even *, toxins such as pesticideszOJ1 be selectively extracted can with supercriticalcarbondioxide. Fig. 3 The common awareness saturatedfatty acids and that A simplified representationof a supercritical extraction processsetup. cholesterolplay a crucial role in the developmentof cardiovasculardiseases triggeredresearchon de-fatting has representationof a possible process is given in Fig. 3. procedures.Becauseof the low polarity of supercritical The materialthat is to be extractedis put into a cylindrical carbon dioxide, fatty products can be extracted with container that has porous ends, which is placed inside a ease,leading to products such as reduced-fatrice bran22, peanutsand other foodstuffs25. thermostatically controlledextractor,vesselA. The super- malt23, critical fluid is circulatedthroughthe extractor,dissolving organiccompounds from the materialinsidethe container. Aroma extraction from seeds and other foods Terpene hydrocarbonsfound in the essential oils of Subsequently, pressureis partly releasedin the sepathe rator, vesselB, which convertsthe supercriticalfluid into plants are among the most important flavour and frathe gaseousphase. The dissolved organic compounds grancecompounds,and offer a wide variety of pleasant are separated from the gas, which is then liquefied in the and floral scents.In addition to their usagein perfumes condenser, by decreasing temperature. C, the Finally, the and perfumedproducts,thesehydrocarbonscan be used liquefied gas is re-pressurized supercriticalconditions to flavour foods and beverages,underlining their value to in modern-daychemistry. with a pump, before it is re-circulatedto the extractor. Flowers, fruit and herbs are typical sources of fraThe temperatureand pressureof the extraction medium in the extractor, separatorand condenserare indi- grance and aroma compounds. When aroma components are incorporated into processedfoods, by using catedby points A, B and C on Fig. 4, respectively. The separationof organic compoundscan also be ac- hydrodistilled essential oils or by adding solvent-excomplished in more than one step by choosing several tracted oleoresins,they do not fully resemblethe natural aromas. This may be the result of heat- and waterinduced changes taking place during the distillation processor the presenceof solvent residue in the oleoI I resin. However, by using extraction with supercritical Liquid Supercritical ,olid 1 I fluids, at moderatetemperatures, fragrancesand aromas can be isolatedwithout any process-induced changes. I I Current developmentsin the extraction of terpene ,---+A hydrocarbons concernthe isolations28and enantiospecific I .J= I characterization2g limonene derivatives and higher of I I terpenessuch as sesquiterpenes30 triterpenes31. and The I enantiospecificcharacterizationof terpenescan be apI I ;/ Critical Ipoint i plied as an analytical tool to distinguish betweennatural and nature-identical flavours and fragrances.Essential oils from spices32 also receivedspecialinterest. have
.g$,$ :$-t

.t::x R
r-+-l-

Extractor

Separator

Condenser

separatorvessels,each operatedat different temperature and pressurecombinations . This results in the fraction ation of the organic compoundsaccordingto their solubility in the gaseous phaseat particular conditions. New developmentsin supercritical extraction
Removal of undesirable constituents

. . . . .t.. ::::.:::. l . ..-..

::::,:~.,.

Fractionation of low vapour pressure oils

Temperature -

Fig. 4 A pressure-temperature diagram of carbon dioxide. 94

Usually, essential oils isolated from plant material containa m ixture of interestingcompounds. essential The oils of plant materialsuchas ginger,cinnamonand pepper contain non-volatile, pungentcompoundsthat determine to an importantextentthe overall flavourprofile. However,
Trends in Food Science & Technology June 1996 [Vol. 71

in the essential oils of other plant material such as the members of the Umbelliferae family (e.g. celery, fennel, caraway, anise, dill, coriander), high levels of odourless triacylglycerol oils are present. The valuable organic compounds in essential oils can be separated from such contaminants by passing the supercritical carbon dioxide extract through two or more individually controlled separator vessels, each operated at a particular pressure and temperature. The resulting stage-wise decrease in solubility of the multicomponent extract thus leads to the separation of pure secondary metabolites at low temperatures33. Other authors have reported the enhanced isolation of palmitate by fractionation of rice-bran oiP4, and the elaborated countercurrent purification of the otherwise inedible lampante olive oiP5. New supercritical solvents The drawback of using supercritical carbon dioxide for the extraction of organic compounds from plant material is the fact that extraction performance is decreased considerably when the material contains water. In addition, only compounds that have a moderate polarity and contain nonpolar components are extracted well. To circumvent these problems, polar solvents such as ethanol (?!=516K, Pc=6.2 MPa) are often mixed with supercnttcal carbon dioxidez8. For example, the isolation of theobromine from cocoa beans with supercritical carbon dioxide is enhanced 30-fold by the addition of 30% (w/w) ethano136. Also, supercritical fluid extraction is not limited to the use of carbon dioxide. Small gases such as methane, ethylene and chlorotrifluoromethane also display supercritical behaviour, even below the supercritical temperature and pressure of carbon dioxide (304 K, 7.38 MPa), although the toxicity of chlorotrifluoromethane may limit its practical use. Higher alkanes such as ethane and propane have increasingly higher supercritical temperatures (305 K and 370 K, respectively), which restricts their usage when temperature-sensitive organic compounds are involved.

Microwave heating Treatment of plant material with microwave irradiation before and/or during an extraction procedure can result in an enhanced recovery of secondary metabolites and aroma compounds. The forced heating of water in the core of the material can cause the steam-induced opening of the outer layers of the plant material. Such opening (or puffing) of the matrix material effectively shortens the path of diffusion of the secondary metabolites, and therefore promotes a more successful extraction of the material. The extracted compounds are taken up by a suitable surrounding medium to facilitate the separation from the remaining plant material. This surrounding medium can be either a liquid4 or a gas42.In the case of a liquid, a somewhat more elaborate separation step is necessary to obtain the pure compounds, whereas in the case of a gas, only a simple condensation step is sufficient. The final choice depends largely on the ease of volatilization of the desired compounds43.

Conclusions
Investigations concerning the extraction of valuable organic compounds from plant material have received a great deal of attention in the literature. A synopsis of the characteristics of the conventional extraction processes is presented in Table 3. On comparing the different extraction methods listed in this table, it is clear that each method has some distinct advantages. The total costs are closely related to the level of technology necessary to reach safely the temperature and pressure values required for the specific extraction method: In this respect, liquid extraction is favoured over both steam extraction and supercritical fluid extraction. Another important aspect on comparing the different extraction methods is the quality of the resultant extracts. This is especially important if thermolabile components are to be extracted. Thus, a high-temperature process such as steam distillation for the isolation of fragrances and/or aromas from plant material can result in the formation of off-notes due to unwanted decomposition of the desired product. Furthermore, such temperature conditions can even cause the generation of additional (unwanted) compounds from precursors present in the plant material. In general, low-temperature extraction and processing of plant material with inert solvents results in extracts with aroma and/or flavour compositions close to those of the plant material they were derived from. Therefore, the low-temperature extraction of plant material using supercritical carbon dioxide results in extracts whose chemical composition more closely resembles that of the original material than is possible with the hightemperature extracts obtained by steam distillation44. Future perspectives A large part of the aforementioned work has been motivated by consumer demands for high end-usage of natural resources, which in turn can lead to the improvement of existing preparation techniques and the
195

Other new developments

Pervaporation Pervaporation is currently being developed as a technique for the recovery of organic compounds from liquid media. In pervaporation, non-porous membranes are used, which have a high affinity for such compounds. With hydrophilic membranes, trace amounts of water can be removed from an organic phase. Analogously, by using hydrophobic membranes, organic compounds can be removed from an aqueous phase37.This has opened up possibilities for the isolation of aroma compounds from apple juice38 and grape juice9 before they undergo industrial processing. Nowadays, special membranes are developed for the separation of mixtures of organic compounds39. The ongoing developments in membrane technology will undoubtedly result in the fabrication of highly (enantio) selective membranes for separations at the molecular leve140.
Trends in Food Science & Technology June 1996 [Vol. 71

1 Table 3. Characteristics of conventional extraction processes Total costs (capital + operating)

$4

Method
Liquid extraction

Mode of operation
Contitiuous

Pros
Low processing costs, easy operation No toxic solvents

Cons
Toxic solvents, expensive recovery, explosion hazard High temperatures, high energy needs High-pressure precautions, high capital costs

Steam extraction

$ +$$

Batch

Supercritical extraction

$34 t $$$

Batch

High quality, no toxic solvents, rapid extraction

Given as approximaterelative orders of magnitude:$ = low; $$$ = high

invention of new procedures the isolation of valuable for compoundsfrom such resources. Promising techniquesinclude the isolation of volatile compounds using a gas stream led over m icrowaveheated plant materia145. well as resulting in the inAs vention of new techniques,researchhas led to increased knowledge about existing extraction techniques. The numerical simulation of binary m ixtures in a vapourpermeation system allows, for instance, the numerical solution of complex hybrid distillation processeP. In general, the art of separation is still improving, with new methods and new proceduresrapidly following each other. But not only the proceduresare changing. The ongoing quest for new materials, new natural sources,is also ever increasing. References
1 Bracco,U., Liiliger,J. and Viret,J-L.(1981) Productionand Use of Natural Antioxidants in 1. Am. Oil Chem. Sot. 58, 686-690 2 Inatani, R., Nakatani, N. and Fuwa, H. (1983) Antioxidative Effect of the Constituents of Rosemary (Rosmarinus officinalis L.) and Their Derivatives in Agric. Biol. Chem. 47,521-528 3 Chang, S.S., Ostric-Matijasevic, B., Hsieh, O.A.L. and Huang, C-L. (1977) Natural Antioxidants from Rosemary and Sagein 1. Food Sci. 42, 1102-1106 4 Hartmann, V.E., Racine, P., Garnero, R.J. and d Audiffret, Y.T. (1980) Extraits de Romarin (Rosmarinus oficinalis Linnaeus) Antioxygenes Les Naturels Utilisables dans la Protection des Huiles Essentiellesin Parfums, CosmBt., ArGmes 36, 33-40 5 Kimura, Y. and Kanamori, T. (1982) Method of Frying Foods in the Presence of a Spice Antioxidant US Patent 4 363 823 , 6 Yajima, M. (1985) Method for Prevention of Oxidation of Oils and Fats and Soft Capsules Containing the Treated Oils and Fats US Patent 4 525 306 , 7 Brueske, C.D. (1993) Oil/Meal Separation Processesin World Conference on Oilseed Technology and Utilization (Applewhite, T.H., ed.), pp. 126-l 36, AOCS Press, Champaign, IL, USA 8 Cu, J.Q., Perineau, F., Delmas, M. and Gaset, A. (1989) Comparison of the Chemical Composition of Carrot Seed Essential Oil Extracted by Different Solvents in Flavour Fragrancel. 4,22S-231 9 Rajagopalan, N. and Cheryan, M. (1995) Pervaporation of Grape Juice Aroma in 1. Membr. Sci. 104,243-250 10 Vavlitis, A. and Milligan, E.D. (1993) Flash Desolventizing in World Conference on Oilseed Technology and Utilization (Applewhite, T.H., ed.), pp. 286-289, AOCS Press, Champaign, IL, USA 11 von Rechenberg, C. (1910) Theorie der Gewinnung und Trennung der Atherischen Ole, Selbstverlag von Schimmel & Co. 12 Vitzthum, O.G., Werkhoff, P. and Hubert, P. (1975) New Volatile Constituents of Black Tea Aroma in I. Agric. Food Chem. 23, 999-1003 13 Farag, R.S., Badei, A.Z.M.A., Hewedi, F.M. and El-Baroty, C.S.A. (1989) Antioxidant Activity of Spice Essential Oils on Linoleic Acid Oxidation in Aqueous Media in 1. Am. Oil Chem. Sot. 66,792-799

14 Vitzthum, 0. and Koch, K.D. (1992) Verfahren zur Herstellung von Laslichem Kaffee German Patent 40 38 526 Al , 15 Seidel, V. and Lindner, W. (1993) Universal Sample Enrichment Technique for Organochlorine Pesticides in Environmental and Biological Samples Using a Redesigned Simultaneous Steam Distillation-solvent Extraction Apparatus in Anal. Chem. 65,3677-3683 16 Paulaitis, M.E., Krukonis, V.J., Kurnik, R.T. and Reid, R.C. (1983) Supercritical Fluid Extraction in Reviews in Chem. Eng. l(2), 180-249 17 Reverchon, E., Ambruosi, A. and Senatore, F. (1994) Isolation of Peppermint Oil Using Supercritical CO, Extraction in Flavour Fragrance). 9, 19-23 18 Diosady, L.L. and Rubin, L.J. (1993) New Approach to Canola and A Rapeseed Processingin World Conference on Oilseed Technology and Utilization (Applewhite, T.H., ed.), pp. 498-504, AOCS Press, Champaign, IL, USA 19 Wu, Y.V., King, J.W. and Warner, K. (1994) Evaluation of Corn Gluten Meal Extracted with Supercritical Carbon Dioxide and Other Solvents: Flavor and Composition in Cereal Chem. 71,217-219 20 Thomson, CA. and Chesney, D.J. (1992) Supercritical Carbon Dioxide Extraction of 2,4-Dichlorophenol from Food Crop Tissuesin Anal. Chem. 64,848-853 21 King, J.W., Hopper, M.L., Luchtefeld, R.C., Taylor, S.L. and Orton, W.L. (1993) Optimization of Experimental Conditions for the Supercritical Carbon Dioxide Extraction of Pesticide Residues from Grains in 1. AOAC Int. 76, 857-864 22 Ramsay, M.E., Hsu, J.T., Novak, R.A. and Reightler, W.J. (1991) Processing Rice Bran by Supercritical Fluid Extraction in Food Technol. 45,98-104 23 Ono, M., Arakawa, K., Furukubo, S. and Hamatani, K. (1992) Lipid-removed Malt for Brewing, Beer Using the Said Malt and Method of Preparing the Said Beer European Patent 0 509 432 A2 , 24 Santerre, C.R., Coodrum, J.W. and Kee, J.M. (1994) Roasted Peanuts and Peanut Butter Quality are Affected by Supercritical Fluid Extraction in ). Foo&Sci. 59,382-386 25 Forster, A. (1991) High-pressure Extraction with CO, in the Food Industry in Weihenstephaner 59,147-l 51 26 Ayano, S. etal. (1992) Extraction of Limonoids and Limonoid Glucosides with Supercritical Carbon Dioxide in 1. Ipn. Sot. Food Sci. Technol. 39,684-689 27 Shin, D.H., Shimoda, M., Kawano, T. and Osajima, Y. (1992) Supercritical Carbon Dioxide Extraction of Terpene Hydrocarbons from Citrus Peel Oil in 1. jpn. Sot. FoodSci. Technol. 39, 377-382 28 Shimoda, M., Shin, D.H., Kawano, T. and Osajima, Y. (1992) Effects of Ethanol as an Entrainer in Preparation of Terpeneless Oil by Supercritical Carbon Dioxide Extraction in 1. jpn. Sot. Food Sci. Technol. 39,339-343 29 Weinreich, B. and Nitz, S. (1992) Influences of Processing on the Enantiomeric Distribution of Chiral Flavour Compounds. Part A. Linalyl Acetate and Terpene Alcohols in Chem. Mikrobiol. Technol. lebensm. 14,117-124 30 Castaneda-Acosta, J., Cain, A.W., Fischer, N.H. and Knopf, F.C. (1995) Extraction of Bioactive Sesquiterpene Lactones from Magnolia grandir7ora Using Supercritical Carbon Dioxide and Near-critical Propane in ). Agric. Food Chem. 43,63-68 31 Sewram, V., Nair, J.J., Mulholland, D.A. and Raynor, M.W. (1995) Open Tubular Supercritical Fluid Chromatography of Triterpene Acids from Dysoxylum pettigrewianum (Meliaceae) in ). High Resolut. Chromatog. 18, 363-366 32 Nitz, S., Kollmannsberger, H. and Punkert, M. (1992) CO,Hochdruckextraktion von Cewiirzen in Chem. Mikmbiol. Technol. lebensm. 14,108-116

96

Trends in Food Science & Technology June 1996 [Vol. 71

33 Nguyen, U., Evans, D.A., Berger, D.J. and Calderon, LA. (1992) Process for the Supercritical Extraction and Fractionation of Spices US Patent 5 120 558 , 34 Saito, N., tkushima, Y., Hatakeda, K., Ito, S. and Coto, T. (1991) Fractional Extraction of Rice Bran Oil and Its Esterswith Supercritical Carbon Dioxide in 1. Agric. Gem. Sot. Ipn 65, 153-l 61 35 Bondioli, P. etal. (1992) Lampante Olive Oil Refining with Supercritical Carbon Dioxide in 1. Am. Oil Chem. Sot. 69,477-480 36 Li, S. and Hartland, S. (1992) Influence of Co-solvents on Solubility and Selectivity in Extraction of Xanthines and Cocoa Butter from Cocoa Beans with Supercritical CO, in 1. Supercrit. Fluids 5, 7-l 2 37 Karlsson, H.O.E. and Tragdrdh, G. (1993) Pervaporation of Dilute Organic-Water Mixtures. A Literature Review on Modelling Studies and Applications to Aroma Compound Recovery in 1. Membr. Sci. 76,121-146 38 Bengtsson, E., Trlgardh, C. and Hallstrom, B. (1993) Concentration Polarization During the Enrichment of Aroma Compounds from a Water Solution by Pervaporation in 1. Food Eng. 19, 399407 39 Park, H.C., Ramaker, N.E., Mulder, M.H.V. and Smolders, C.A. (1995) Separation of MTBE-Methanol Mixtures by Pervaporation in Sep. Sci. Technol. 30,41 g-l33

40 Cuperus, F.P. and Nijhuis, H.H. (1993) Applications of Membrane Technology to Food Processingin Trends FoodSci. Technol. 4,277-282 41 Pare, J. (1992) Microwave Extraction of Volatile Oils and Apparatus Therefore European Patent 0 485 668 Al , 42 Craveiro, A.A., Matos, F.J.A., Alencar, J.W. and Plumef, M.M. (1989) Microwave Oven Extraction of an Essential Oil in Flavour Fragrancel. 4,43-44 43 Lindstrom, T.R. and Parliment, T.H. (1994) Microwave Volatilization of Aroma Compounds in Thermal/y Generated F/avows. Maillard, Microwave and Extrusion Processes(ACS Symposium Series 543) (Parliment, T.H., Morello, M.J. and McCorrin, R. J., eds), pp. 403-413, ACS Press, Washington, DC, USA 44 Moyler, D.A. (1993) Extraction of Essential Oils with Carbon Dioxide in Flavour Fragrance J 8,235-247 45 Craveiro, A.A., Matos, F&A., Alencar, J.W. and Plumel, M.M. (1989) Microwave Oven Extraction of an Essential Oil in FlavourFragrance/. 4,43-44 46 Pettersen, T. and Lien, K.M. (1995) Design of Hybrid Distillation and Vapor in /. Permeation Processes Membr.Sci. 99, 21-30

Review

1 Yroteinase inhibitors
Enzymatic protein hydrolysis plays a major role in various physiological processes, including digestion, and is regulated by proteinase inhibitors. Inhibitors in foods and food ingredients can reduce the absorption of free amino acids, and can impair protein hydrolysis in industrial processes. However, inhibitors can be useful tools in pest control, in the prevention and treatment of diseases such as cancers and AIDS, and in the elimination of unwanted proteinase activity in food processes. Proteinase inhibitors are also useful biochemical tools for studying proteinase classes and specificities. This article discusses how proteinase inhibition technologists. is involved in some processes of current interest to food scientists and

Fernando his Garcia-Carreiio

Enzymatic protein hydrolysis is a major concern for biological scientists. The hydrolysis of proteins is catalyzed by peptide-bond-splitting enzymes (Box 1). Proteinases and peptidases are involved in the hydrolysis of protein during digestion, and have important roles in physiology and pathology. Enzymatic protein hydrolysis is controlled in several ways, including by the use of specific inhibitors (Box 2). Proteinase inhibition is a common process in nature. Proteinase-inhibitor interactions are involved in protein digestion, various physiological processes (e.g. blood coagulation, fibrinolysis, complement activation and phagocytosis), pathological processes (e.g. cancers and hypertension) and infection

(e.g. with AIDS or invasive parasites). Another natural method of controlling proteinase activity is the synthesis of an inactive form of the enzyme, the zymogen. Zymogens are activated, usually by the action of another proteinase, in the digestive system and also during regulatory physiological processes. When an enzyme is in its active form, proteinase inhibition is an exquisite means of enzyme control in physiological processes, which is achieved by highly specific inhibitors. The importance of the control of proteolytic activity by inhibitors in physiological processes is demonstrated by the fact that inhibitor molecules exceed 10% of the total protein in human plasma. The fact that the control of proteolysis by inhibitors is so specific makes it a valuable tool in medicine, agriculture and food technology. The human immune deficiency virus proteinase, the digestive systems of crop pests, and fish muscle proteases are some examples of targets for study. Most organisms produce proteinase inhibitors as a means to control proteolytic processes. Some organisms store huge amounts of inhibitors, for example legume seeds and some leaves. This seems to be an evolutionary response to predation. Inhibitors for digestive proteinases in food and feed Some food ingredients contain so-called antinutritive factors: lectins, phenols, and other factors, including certain proteins that inhibit proteinases. The presence of proteinase inhibitors in living tissues seems to be a natural regulatory process, well represented by the case of
01996, Elsevier Science Ltd PII: SO924-2244(96110023-6

Fernando his Carcia-CarreRo

is at Centro de lnvestigaciones Biologicas del

Noroeste, PO Box 128, La Paz, BCS, 23000, Mexico (fax: t52-112-5-4710;

e-mail: fgarcia@cibnor.conacyt.mex).

Trends in Food Science & Technology June 1996 [Vol. 71

197