Extraction of secondary metabolites from plant material: A review

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 sepa-

metabolites from plant material: A review

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

ration processes, a few general conclusions about the applicability of the different types of extraction techniques are drawn.

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.

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.

Solvent extraction

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: [email protected]). ganic solvents to a prescribed maximum residual level

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

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

01996.

19

Elsevier Science Ltd PII: SO924-2244(96110020-O

Steamdistillation to obtain essential oils from plant material has been a research subject for many years, and Function Source/compound Application many famousresearchershave given it their attention. As early as 1910, von Colorant Beetrootjuice Ice cream Rechenberg’ l explained the phenomSpearmint 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, becauseoxygen-rich Carvone Sprout suppressant Potato storage compoundswill dissolve more easily Palm oil Food additive, cosmetics Food industry, skin softener in the watery phasepenetratingthe plant material. For instance, when caraway (i.e. 5-30mg hexaneper kg of product) if the product is seedsare extracted,the releaseof carvone (T,., = 71°C 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,, = 60-61°C insoluble in water). evaporationprocesses.In addition, the extra emissionof 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 examplesof steamdistillation are the recovery of antioxidants from the waterNew developments Not only is the recovery of the solvent from the ex- dispersed ‘concrete’that is obtained after evaporation 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 superheatedsolvent. 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 higher than in a liquid phase. Thus, although the solu- vapour streamare easily separatedfrom it by decreasing 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 olites using an extraction procedure with a gaseous heat-exchangeunit. The resulting liquid m ixture is led 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 Qm to cope with renewed (i.e. decreased) maximum emission standards.AddiResidue Extract tionally, the loss of valuable volatile compounds should be restricted for Fig*l economicreasons.The total amountof 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 )

/

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.

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

phase

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

l

l

l

l

Supercritical extraction

Organic

phase

It has a critical temperature of 31’C; this means that 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.

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

Gaseous

Supercritical

Liquid

Diffusion coefficient (cm2/s)

0.1

1O-3

5 x 10-6

Density (g/cm3)

10”

0.3

1

Viscosity (g/cm.s)

10-h

1o-4

10-2

Extractor

Separator

Condenser

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

A

. . . . .t.. ::::.:::. l . ..-•..

separatorvessels,each operatedat different temperature and pressurecombinations” . This results in the fractionation of the organic compoundsaccordingto their solubility in the gaseousphaseat particular conditions. New developmentsin supercritical extraction Removal of undesirable constituents

::::,:~.,.

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 can be selectively extracted with supercriticalcarbondioxide. Fig. 3 The common awarenessthat saturatedfatty acids and A simplified representationof a supercritical extraction processsetup. cholesterolplay a crucial role in the developmentof cardiovasculardiseaseshas triggeredresearchon de-fatting 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, thermostaticallycontrolledextractor,vesselA. The super- malt23,peanuts”and other foodstuffs25. critical fluid is circulatedthroughthe extractor,dissolving organiccompoundsfrom the materialinsidethe container. Aroma extraction from seeds and other foods Terpene hydrocarbonsfound in the essential oils of Subsequently,the pressureis partly releasedin the separator, 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 separatedfrom the gas, which is then liquefied in the and floral scents.In addition to their usagein perfumes condenser,C, by decreasingthe temperature.Finally, the and perfumedproducts,thesehydrocarbonscan be used liquefied gas is re-pressurizedto supercriticalconditions to flavour foods and beverages,underlining their value 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-inducedchanges. I I Current developmentsin the extraction of terpene ,---+A hydrocarbons concernthe isolations28and enantiospecific I .J= I characterization2gof limonene derivatives and higher I I terpenessuch as sesquiterpenes30 and triterpenes31.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 have also receivedspecialinterest. .g$,$ :$--

t

Fractionation of low vapour pressure oils Temperature

-

Fig. 4 A pressure-temperaturediagram of carbon dioxide. 94

Usually, essential oils isolated from plant material containa m ixture of interestingcompounds.The essential 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.

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

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

1 Table 3. Characteristics of conventional extraction processes

Method

Total costs (capital + operating)

Mode of operation

Liquid extraction

$4

Steam extraction

Supercritical extraction

Pros

Cons

Contitiuous

Low processing costs, easy operation

Toxic solvents, expensive recovery, explosion hazard

$ +$$

Batch

No toxic solvents

High temperatures, high energy needs

$34 •t $$$

Batch

High quality, no toxic solvents, rapid extraction

High-pressure precautions, high capital costs

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

invention of new proceduresfor the isolation of valuable compoundsfrom such resources. Promising techniquesinclude the isolation of volatile compounds using a gas stream led over m icrowaveheated plant materia145. As well as resulting in the invention 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 Sage’in 1. Food Sci. 42, 1102-1106 4 Hartmann, V.E., Racine, P., Garnero, R.J. and d’Audiffret, Y.T. (1980) ‘Les Extraits de Romarin (Rosmarinus oficinalis Linnaeus) Antioxygenes Naturels Utilisables dans la Protection des Huiles Essentielles’in 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 Processes’in 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

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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

I

Fernando his Garcia-Carreiio

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

is involved in

some processes of current interest to food scientists and technologists.

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

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: [email protected]).

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

(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,

197

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