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 Esters with 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 /. Membr.Sci. 99, 21-30 Permeation Processes

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

Box 1clases of proteasas The hydrolysis of proteins is catalyzed by peptide-bond-splitting enzymes. These are classified by the Enzyme Commission of the International Union of Biochemistry, according to the peptide bond hydrolysis reaction they catalyze, as proteases,the hydrolase subclass EC 3.4.X.X. Those proteases catalyzing the splitting of peptide bonds within the protein are coded in the hydrolase sub-subclass as EC 3.4.21-24.X, proteinases or endopeptidases. Those proteases catalyzing the splitting of peptide bonds at either the amino or carboxyl ends of the protein are coded in the hydrolase subsubclassas EC 3.4.1 l-l 7.X, peptidases or exopeptidases.The term protease is used only when the speciFicity and mechanism of hydrolysis of an enzyme are unknown.

Box 2. Proteaseinhibitors An enzyme inhibitor is any substance that reduces the rate of an enzyme-catalyzed reaction . Protease inhibitors mimic the protein substrate by binding to the active site of the protease. In general, protease inhibitors are specific for the active site of a specific class of protease; the exception is the mammalian plasma protease inhibitor 01-2 macrogIobulin, which is non-specific for the enzyme or the class of protease. Chelators that remove cations from metaldependent proteases, and denaturants that alter catalytic sites are known as inactivators rather than inhibitors. Irreversible inhibitors are generally low molecular weight sitedirected compounds. Most of the known irreversible inhibitors are synthetic substances that are used to determine the class of a proteinase. For further information on this topic, see Refs 2-4. Reversibleinhibitors are, in general, naturally occurring proteins. They usually have measurable kinetic binding constants; however, these are orders of magnitude lower than those of the enzymesubstrate complex. Because protease inhibitors generally bind the protease very tightly, it is difficult to evaluate their binding kinetics using the Michaelis-Menten equation. For further kinetic considerations, see Ref. 4.

plants protecting themselves against insect predation. However, when a plant or animal tissue is intended for use in food production, proteolysis inhibition can be an unwanted process. The presence of proteinase inhibitors in foods decreases the apparent nutritional quality of protein in the diet, by affecting the ability of the body s digestive enzymes to degrade dietary protein, and thus limiting the intake of amino acids needed to construct new proteins. For example, when Atlantic salmon were fed diets containing soybean trypsin inhibitor, their ability to digest protein and fat, their ability to gain weight, and trypsin activity were all reduced in a dose-dependent manneI5. The fish were able to counteract low concentrations of the inhibitor by increasing trypsin secretion, but high levels of the inhibitor exhausted the ability of the fish s pancreas to synthesize the digestive enzyme5. In rodents, exposure to diets containing 0.1% soybean or potato trypsin inhibitor produced pancreatic pathology; the effect was dependent on the length of exposure6. In this study, the co-administration of proteinase inhibitors together with dietary protein reduced the rodents ability to degrade the protein. In humans, consumption of raw
98

soybeans reduced both chymotrypsin and trypsin activity in the digestive tract, presumably as a result of the presence of the dietary proteinase inhibitors. Ten minutes after the challenge, a subsequent increase in both trypsin and chymotrypsin activity was found. Because this increase in proteinase activity was not preceded by an elevation in the plasma cholecystokinin concentration, it appeared that a neural regulation system was involved in mediating the inhibitor-stimulated increase in enzyme secretion7. This illustrates the potential adverse effects of proteinase inhibitors in the diet. However, in certain situations the effects of inhibitors on protein digestion might be advantageous, for example by improving the intact absorption of some therapeutic proteins such as orally delivered insulin*. Proteinaceous proteinase inhibitors are sensitive to physical treatment, and can be denatured by heat. Heat treatment can thus improve the nutritional value of foods by reducing proteinase inhibition, thus increasing the availability of lysine and other amino acids (however, care must be taken to avoid using excessive heat, as it can impair the nutritional value of dietary protein by causing crosslinking reactions or amino acid racemization). Soybeans intended as feed for animals are first treated with hexane to extract the oil; the remaining hexane is then removed by heating. In this process, most of the proteinase inhibitors are destroyed. An alternative method of treatment is the extrusion, at 104-120C for 30-60 s, of diets containing inhibitors. When this treatment method was used to prepare food for chicks, animal growth rates were greater and pancreas weights were lowerg. The thermostability of plant proteinase inhibitors is species specific. Seed meals made from six different legumes (intended for use as shrimp feed) that inhibited mammalian trypsin, chymotrypsin and papain, as well as shrimp hepatopancreas proteinases, were each affected differently by heat treatment. When these seed meals were heated for 60min at 85C the degree of hydrolysis of the seed-meal protein by shrimp hepatopancreas extract increased by between l.l-fold and 3.7-fold, depending on the type of legume. The protein of untreated seed meals was hydrolyzed by the hepatopancreas extract to some extent, yielding a low degree of hydrolysis of only 6-32%. This explains why animals fed with raw seed meal can survive, but have a poor growth performance.

Inhibitors in food processing

Besides affecting the enzymes involved in protein digestion and thus reducing the nutritive quality of dietary protein, the presence of inhibitors in food ingredients can adversely affect intended enzymatic modifications of ingredients during food processing. Water-holding capacity, foaming, gel-forming and whipping abilities, and texture are examples of attributes that can be improved by using enzymatic modification to change the functional properties of food proteins, giving added value to commercially processed foods. On the other hand, sometimes, both in research and food technology, proteases are pervasive, perplexing,
Trends in Food Science & Technology June 1996 [Vol. 71

persistent and pernicious for protein, but with proper precautions, preventable (the 9~s rule) - and in these cases, inhibitors of proteases can be desirable. In surimi production, proteinase inhibitors are deliberately added as a means of circumventing the problem of reduced gel-forming ability that occurs as a result of the action of endogenous myosin-degrading proteinases. Unfortunately, enzymatic protein hydrolysis is also a problem in day-to-day laboratory work: autolysis during protein studies, including protein purification and characterization work, is an annoying problem. Methods for its control are needed; some suggestions of how to solve this problem are given by Baynon O.

Fish processing
Postmortem muscle softening in fish, mollusks and crustaceans, and blackspot development in crustaceans are serious problems in seafood storage and processing. This softening or mushiness is mainly caused by muscle proteinases, mostly cathepsins. Some fish proteinases are activated during cooking, causing myosin degradation and subsequent textural destruction. Most of the fish proteinases involved in textural deterioration are activated at 6OC, although some squid enzymes are activated at temperatures as low as 35C (Ref. 11); this is why some fish harvests are not intended for direct human consumption an6 are used only in commercial processing, where the proteolytic activity can be controlled. An example of the deleterious effects of endogenous proteinases can be seen during surimi production. During the water-minced flesh wash, -60% of the muscle proteinase responsible for softening can be eliminated *; however, the remaining activity is sufficient to degrade the myosin. It has been suggested that meat softening is also associated with the presence of myxosporean parasites in the flesh13. However, this relation has yet to be established, because the available information is not conclusive. The enzymes responsible for fish muscle mushiness are proteinases that are located in the muscle tissue, and are activated at 60C and pH5.5. According to the pattern of inhibition by class-specific inhibitors14, the enzymes are cysteine proteinases that resemble cathepsin L. The pH profiles for maximum activity indicate the presence of several minor enzymes. On comparing enzymes from Pacific whiting (Merluccius productus), An et al. found cathepsin B to be the most active proteinase in fish fillets; cathepsin L was the most active in surimi14. In this study, cathepsins B and H, but not cathepsin L, were shown to be removed by washing during surimi production. The myosin molecule was the primary target of the enzyme. The pH of the fish muscle is 6.8-6.9, which is above the optimum for the enzymatic activity, but close enough to allow considerable activity. It is possible to reduce enzyme activity during surimi production by adding proteinase inhibitors at certain steps of the process; generally these are mixed in with the cryoprotectants that are added before freezing. Inhibitors from potato, bovine plasma and egg white are currently used in the production of surimi from several
Trends in Food Science & Technology June 1996 [Vol. 71

fish species l5. Potato and egg white contain specific competitive inhibitors for the active sites of the fish muscle proteinases. Mammalian plasma contains a high molecular weight protein, an o-2 macroglobulin, that non-specifically traps the proteinase(s). At least two proteinase inhibitors, namely cystatin and an ovomucoid, are present in egg white. Cystatins are a family of inhibitors of cysteine proteinases. It seems that the NH,terminal segment of the inhibitor cystatin is a conserved region in the family, and is responsible for the competitive interaction with the active site of the enzyme16. Ovomucoids are glycoproteins with a molecular mass of -28 000 Da, containing -25% carbohydrate, no tryptophan, but a high proportion of cysteine. In general, ovomucoids have three complex-forming domains, which react with trypsin-like and chymotrypsin-like enzymes independently. The main limitations of the potato, egg white and bovine plasma inhibitors are that they impart off-flavors and off-colors to the surimi. They are also relatively expensive. Alternative food-grade inhibitors are needed. Inhibitors from rice (oryzacystatin)18, whey protein concentrate19 and legume seed meals15are under study.

Underusedmarine resources
Squid from the Atlantic and Pacific Oceans are underused marine resources, partly because of their low gelforming ability, which limits their use for surimi production. Proteinases are responsible for this effect. A cathepsin C from Atlantic squid (Mex illecebrosus) has been characterized, showing the transpeptidase activity that is characteristic of this enzyme. The enzyme is dependent on the presence of Cl- and sulphydryl activators*O. The spear squid (Loligo bleekeri) has several muscle proteinases. One of them, a trypsin-like enzyme, degraded myosin to a great extent, following activation at 35C. The enzyme was inhibited by serine proteinase inhibitors.

Crustaceans
Some fresh-water and ocean crustaceans, such as krill (mostly Euphausia superba) from the Antarctic Ocean, langostilla (Pleuroncodes planipes) from the Pacific shoreline of the Baja California peninsula, and crayfish (Pacifastacus astacus) from European and North American rivers, represent extensive resources. Wild and cultivated crustaceans for human consumption, such as shrimp, prawn, crayfish and American lobster, are becoming important because of their mass and value. Crustaceans possess the highly active proteinases trypsin, chymotrypsin and collagenase3~21*22, which are released from the hepatopancreas by postmortem processes autolysing the meat. Krill has long been considered a source for protein and enzyme production. Several proteinases from krill have been reported, and a trypsin-like proteinase was shown to be the most important enzyme involved in autolysis at tissue pHz3. Thus, krill may be a useful commercial source of proteinase enzymes, although the endogenous enzyme activity may limit the direct use of krill as a food source. In crayfish,
199

endogenous proteinase activity is significantly reduced Several research groups have been enthusiastic about the potential of proteinase inhibitors for the control of following the 7 min boiling step commonly used to facilitate the peeling of the crayfish. However, in langostilla, insect pests. One approach to provide defense against elimination of enzyme activity has not been that simple, herbivorous insects is the incorporation of the gene showing that there are important differences in the pro- encoding the proteinase inhibitor into the genome of teinases among species *l . Controlled proteolysis can in the plant. This strategy has proven to be successful. some circumstances be used to advantage; a protein Biotechnology is taking advantage of the co-evolution hydrolysate derived from the cephalothorax of crayfish over millions of years of insects and flowering plants (which is normally considered as waste, once the tail that has generated insect resistance genes30. A number has been removed for human consumption) has been re- of plants possess two wound-induced small multigene ported to have potential applications as a flavoring in- families that code for proteinase inhibitors; these ingredient for seafood%. hibitors are now targets for studies on gene transfer into . Blackspot development in crustaceans is dependent food crops3 on the proteolytic activation of a phenolase, which Transgenic plants expressing a proteinase inhibitor transforms tyrosine into the dark pigment known as have enhanced levels of insect resistance. The gene melanin. Sulphite is currently used to reduce post- encoding the cysteine proteinase inhibitor oryzacystatin mortem blackspot development. However, there is some has been successfully introduced into tobacco plants32. potential hazard associated with this practice, and alter- The transgenic inhibitor was present at levels of up to native methods of control need to be developed. Three OS-0.6% of the total soluble protein in the leaves and proteinases have been isolated from Norway lobster roots. The use of the bacterium Agrobucterium tume(Nephrops nowegicus). Two of them are thiol (cysteine) fuciens as a vector for the introduction and expression proteinases and the third is a metal-dependent serine of foreign genes has also proven to be successful in proteinase25. Inhibition of the serine proteinase reduced other plants, including in cocoa leaf cells, even though phenolase activation, and therefore blackspot develop- A. tumefuciensis not a natural pathogen for the cocoa ment. Inhibition of the thiol proteinases, however, had pla&. negligible effect on blackspot development* j. An alternative approach for pest control is the production of proteinase inhibitors on a large scale in a bacinhibitors in pest control: transgenic plants terial system for subsequent purification and use as a Plants protect themselves against predation by pro- crop spray. The expression of the genes for oryzacysducing insecticides and proteinase inhibitors of the pest tatin I and II in Escherichiu coli using a glutathione digestive system, impairing predator physiology. Phyto- S-transferase gene fusion system has yielded active phagous insect pests have digestive proteinases belong- forms of the inhibitors34. Because of the specificity of ing to the serine and cysteine proteinase classes. Serine the fusion products, these inhibitors might be useful in proteinases predominate in Lepidoptera, and cysteine pro- the control of predators that digest dietary protein by teinases in Coleoptera27. Hines et uZ.*~ showed that feed- using cysteine proteinases. Plants respond naturally to biological attack. One ing the insect Acunthoscelidesabtectus with an artificial bean-seed system containing the cysteine proteinase in- mechanism of defense is the production of woundhibitor E-64 delayed the development and increased the induced proteinase inhibitors. In the gray alder (Alnus mortality of the larvae. These effects were a direct func- incunu), an increase of trypsin inhibitor after defoliation tion of the concentration of the proteinase inhibitor in was concomitant with a decrease of soluble plant prothe feed. The cysteine proteinase activity in extracts of the tein. Leaves from attacked trees, which contain an inlarvae was severely affected. The addition of free amino creased ratio of trypsin inhibitor to soluble protein, have acids to the diet did not prevent the reduction in the en- antifeeding properties, causing retarded growth, delayed zyme activity, but improved larval development and re- pupation, reduced egg production and low survival of duced mortality. In another study, the growth of the black the predator beetle (Guferucellu lineolu)35. The toxic effield cricket (Teleogrylluscommodus) was evaluated when fect can be reversed if the insect s diet is rich in essenrearing it on diets containing different levels of protein tial amino acids. A similar effect could explain the flucand inhibitors for trypsin and elastase, the major cricket tuating dynamics of a herbivore population, for example . In this study, all of the proteinase in- the possible effects of grazing-induced proteinase ingut proteinases 29 hibitors that inhibited trypsin or elastase in vitro reduced hibitors on lemming population cycles36. When the ratio the cricket s growth. Inhibitors that reacted with both of trypsin inhibitor to soluble protein in plant extracts enzymes were more effective. Diets containing potato was increased, a decrease in lemming densities ocmulticystatin caused a dose-dependent reduction in the curred. The animals from the declining populations growth of Western and Southern corn rootworm larvae showed the pancreatic hypertrophy that is correlated (Diubrotica virgiferu virgij2ru)27. Multicystatin from with a prolonged intake of proteinase inhibitors. potato tubers is a cysteine proteinase inhibitor belonging to the cystatin family. The inhibitor is a protein consist- Proteinaseinhibitors and cancer prevention ing of eight tandem 10.8 kDa cystatin domains linked by Cancers are a complex group of diseases. Their causes trypsin-sensitive bonds. These studies demonstrate the are multiple. Several pathological conditions associated effects of proteinase inhibitors in foods on predators. with carcinogenesis are related to changes in proteases
200 Trends in Food Science & Technology June 1996 [Vol. 71

responsible for tumor promotion and neoplastic transformation. The inhibition of such activities explains the effectiveness of protease inhibitors in the prevention of some types of cancers; for comprehensive reviews, see DeClerck and Imren3 , and Das and Mukhopadhyay3*. The need for agents to treat or to prevent cancers has driven a search for molecules with anticancer activities, and many different ones have been found and identified. Some of these are proteinase inhibitors, both naturally occurring inhibitors found in plants and synthetic inhibitors. Legume seeds are known to contain proteinase inhibitors. Soybean seeds, for example, contain at least three different compounds with anti-proteolytic activity 39 . During the past 20 years, information has been gained about the protective effects against cancers of diets containing legume proteinase inhibitors. Such an effect has been shown in the case of mouse skin tumors induced by nitroquinoline oxide and phorbol myristate acetate, as well as for rat breast tumors and mouse liver cancer induced by ionizing radiation38. Several serine proteinase inhibitors from soybean and potato suppressed oral carcinogenesis in hamsters that was induced by 7,12-dimethylbenz[a]anthracene. The inhibitors, applied topically to the cheek pouch, were effective in reversing carcinogenesis at concentrations as low as O.Ol%, even when used 45 d after the beginning of exposure to the carcinogen40. Details about the mechanisms of action of the preventive effects of proteinase inhibitors on cancers remain to be elucidated. One problem that needs to be solved is how the therapeutic form of the proteinase inhibitor should be administered. It appears that inhibitors could complement cytotoxic therapies. Steinmetz and Potter4 , in their extensive review, concluded that consumption of higher levels of vegetables and fruits is associated consistently, although not universally, with a reduced risk of cancer at most sites, and particularly with epithelial cancers of the alimentary and respiratory tracts . The authors postulate a mechanism to explain how plant proteinase inhibitors affect enzymes produced by neoplastic cells, reducing damage to the extracellular matrix caused by tumor proteinases, and thus limiting tissue invasion. They also point out that humans are adapted for a high intake of plant foods. Some food components that are not thought to be essential nutrients may be crucial for the prevention of cancers. If some cancers are the result of ingesting too low a level of metabolically necessary foods, they may be a disease of maladaptation.

enzymes. The purification of enzymes using affinity chromatography has been the subject of extensive investigation, and is beyond the scope of this review; for further information, see the comprehensive chapter by Wilchek et a1.43 The purification of proteinases by affinity chromatography is more complicated than that of other enzymes. This is because of the unique substrate specificity of proteinases for an amino acid residue forming the carboxylic half of a target peptide bond, rather than for a particular protein. Furthermore, the possibility that the proteinase being purified will hydrolyze the immobilized protein substrate makes it infeasible to use proteins as ligands. Synthetic substrates mimicking the target peptide bond [e.g. tryptophan methyl ester, for chymotrypsin; (Ala),, for elastase; and aminobenzoyl-Arg, for carboxypeptidase] have instead been used. Proteinaceous proteinase inhibitors, which resist hydrolysis, have also been used. Affinity chromatography using immobilized soybean trypsin inhibitor produced a peptidase-enriched fraction from each of two decapod (langostilla and crayfish) hepatopancreas samples that contained both proteinases and peptidases44. In each case, only the proteinases bound to the agarose - soybean inhibitor column because they belong to the serine proteinase class. The peptidases were enriched up to 15-fold, whereas the proteinases were reduced to negligible levels. The peptidases are intended for future use in accelerating the ripening of Cheddar cheese. Evaluating proteinase activity and proteinase inhibitors Many methods have been reported for evaluating the activity of proteinases and proteinase inhibitors. A great number of proteins have been proposed for use as the substrate; casein or its derivatives such as azocasein are the most commonly used. A complex of casein and an azo dye permits more sensitive spectroscopic determination, as the molar light absorbance (at 366nm) of the azocasein complex is higher than that of the commonly used aromatic amino acids at 280nm. A simple method for evaluating the proteinase activity of an enzyme preparation is outlined in Table 1. Casein-based substrates are useful at neutral and basic pH values. For assays at acid pH values, hemoglobin is used. A problem in evaluating the kinetic properties of proteinases is that the concentration of a proteinaceous substrate increases rather than decreases with time, as a protein can have several peptide bonds available for attack: when the enzyme hydrolyzes a bond to create two peptides, the molar concentration of the substrate increases. Furthermore, the cleavage of only one peptide bond does not always produce peptides that are soluble in the trichloroacetic acid (TCA) solution used to stop the enzymatic reaction and to precipitate the undigested protein substrate, allowing spectrophotometric determination of the TCA-soluble peptides. Thus, non-protein substrates are needed. Synthetic substrates mimicking the natural substrates are therefore used; these are also useful to help obtain information about the specificity and the class of an enzyme.
20

Proteinaseinhibitors in the laboratory

Proteinase purification by affinity chromatography Thirty years after its introduction, affinity chromatography is still the best purification technique. It is based on specific recognition between the compound to be purified and a ligand immobilized on an affinity column, and renders the maximum possible purification factor, which averages loo-fold (Ref. 42). It is commonly used to purify biologically active molecules such as antigens, antibodies, cell receptors, hormones, nucleic acids and
Trends in Food Science & Technology June 1996 [Vol. 71

Table 1. Assay for proteinase activity and inhibition

suitability of using proteinase inhibitor genes to transform plants or bacteria for large-scale production and to enhance 7-9 10-12 Tube numbers l-3 4-6 insect resistance has been proven, and 500 bl 500 p,l 500 /.Ll Bufferd 500 pl represents an alternative to the conventional breeding of resistant plants. S-20 pl 5-20 ~1 Enzyme extract S-20 j,d S-20 ~1 Food processing is already benefiting Inhibitor solution 1Opl from the use of inhibitors to reduce 1Opl Inhibitor solvent unwanted protein hydrolysis, as in the -----------------10-60minat25-650C -- ~~~~~~~~____ case of surimi production. Alternative lncubatione inhibitors that do not alter food quality 0.5 ml TCA and improve the profit of the process 0.5 ml 0.5 ml 0.5 ml Substrate 0.5 ml will always be welcome. The discovery of proteinase inhibitors -----------------10-60minat25-65C -------_____ ---IncubatiorF with particular kinetic properties might 0.5 ml 0.5 ml 0.5 ml TCA prove useful as biochemical tools in _-----------------6500gforsmin ---~~~~~~~~___---Centrifugation affinity chromatography. The purification of proteinases by bioaffinity ----------------------Record--------------------Absorbance (366 nm) chromatography should provide cheaper and more specific proteolytic enzymes Adapted from Ref. 2 for industrial purposes. bControl for the enzyme extract (somecrude extracts might absorb at 366 nm becauseof the presenceof Proteinase inhibitors in foods can be carotenoids or other yellow substances) CControlfor the inhibitor solvent (some inhibitor solvents might modify the activity of the enzyme) either good or bad : good when they d Usually 50 IIIM Tris/HCI, pH 7.8 are used to avoid unwanted proteolysis, The temperatureand time of the incubation depends on the thermostabilityand the time required to obtain a to control pests, to prevent certain dislinear reaction; for thermophilic organisms,the temperaturecould be as high as 65C eases, or to improve gel strength in 1.5% azocasein in 50m~ Tris/HCl, pH 7.8 food technology; bad when they reTCA, Trichloroacetic acid (20%, w/v, in water) duce the digestibility of food or feed. Improved knowledge of how inhibitors The easiest way to test for the presence of proteinase interact with their target enzyme(s), their mechanism of inhibitors in a biological sample is to challenge it with inhibition, their specificity, and their bioavailability will commercial proteinases belonging to different classes. allow better use and control of these biochemical agents. The sample is mixed with the proteinase for some time, taking into account the fact that some inhibitors may Future trends Biotechnology and the food sciences can profit from react slowly with the enzyme. Then, the inhibitorenzyme mixture is mixed with the substrate at zero time the use of proteinase inhibitors. Further research on new of the reaction. The time course of the reaction in the inhibitors, their mechanism of action and kinetic propercontrol assay without added inhibitor must be linear to ties will expand the possibilities of the use of the inhibiguarantee that any reduction in enzyme activity is due to tion process in practical applications. Most of the information about proteinase inhibitors the inhibitor and not because of a shortage of the substrate, product inactivation or denaturation of the en- has been derived from terrestrial organisms. Of the zyme. Table 1 shows a general assay protocol for study- -400 papers reviewed, only a few concerned marine organisms. The search for unique inhibitors from organing the activity of proteinases and proteinase inhibitors. isms from both marine and extreme environments is An exquisite method for evaluating the composition and molecular mass of proteinases and proteinaceous expected to provide molecules that function at high proteinase inhibitors is the substrate - gel electrophoresis temperatures or low temperatures, with the possibility of technique45. This method takes advantage of the fact using heat or low ion concentrations to inactivate them. The control of insect pests and herbivores should be that proteinases are proteins that are composed of just one polypeptide chain and are therefore resistant to de- possible by using transgenic plants or by producing naturation following sodium dodecyl sulphate treatment. large amounts of ecologically benign, easy-to-inactivate inhibitors that provide protection during plant growth, Figure 1 shows the protocol followed in this technique. harvest or storage. Methods of evaluating proteinases and proteinase inConclusions Proteinase inhibitors are ubiquitous molecules. They hibitors are needed. The use of rational selection and artificial neural network methods are needed to select suitare present in all organisms as a means of regulating physiological proteolysis and predation. Huge quantities of able inhibitors when analyzing several sources. Methods crops are lost, during culture, harvest and storage, owing such as surface response methodology will help to optito fungal and viral diseases or insect pests and vertebrate mize the variables of evaluation. While there is growing emphasis on the applications herbivores. Most predators have sensitive enzymatic systems, some related to the digestion of protein. The of molecular biology techniques, further studies are
202 Trends in Food Science & Technology June 1996 [Vol. 71

Inhibition assay

Activity assay

Extract controlb

Solvent controlc

Fig. 1 Substrate - sodium dodecyl sulphate (SDS) - polyacrylamide gel electrophoresis (PAGE) protocols for determining proteinase composition, proteinase activity and the presence of proteinaceous proteinase inhibitors. Left, To determine the proteinase activity of electrophoretically twin gel is soaked for 30-60min separated samples, one of the two identical SDS-PAGE gels is stained with Coomassie (Brilliant) Blue for protein, showing the protein composition of the sample. The in a solution of the substrate, 2% casein, in buffer (50 mM Tris/HCI, pH 7.8 is recommended for serine proteinases) then washed with distilled water and stained for protein, showing the proteinase composition by negative staining. In those places where a proteinase was located, the digestion of the casein produces a clear band in a blue background of the undigested and stained substrate. Right, To determine the presence of proteinase inhibitors in electrophoretically separated samples, one of two identical SDS-PAGE gels is stained with Coomassie Blue for protein, showing the protein composition of the sample. The twin gel is soaked for 30min in a solution containing the target enzyme that is to be inhibited, washed with distilled water, and soaked in a solution of the substrate as at left. The target enzyme will digest the substrate except in those places where the inhibitor restrained the enzyme. For additional information on this technique, see Ref. 45.

needed on the protein biochemistry, mechanism of action and reaction kinetics of enzymes and enzyme inhibitors. Studies on nucleic acids are important because they are responsible for storing information about the physiological processes. Nucleic acids are the software of life. However, no-one goes to the theater to read the script of a play. We want to see the actors perform. In physiology, pathology and food technology, the actors are the enzymes that dictate the route and speed of important biotransformations. Enzymes are the hardware of life. Acknowledgements Grant 3589-N by CONACyT, which partially supported this work, is appreciated. Thanks are given to Paty Hernandez for her help in the preparation of electrophoresis slides, Drs Gloria Yepiz at CIAD, Hermosillo and Norman Haard at UC Davis for their suggestions on the early draft, and Ellis Glazier for editorial work on the manuscript. References
1 Whitaker, JR., ed. (1994) Principles of Enzymology for the Food Sciences (2nd edn), Marcel Dekker 2 Garcia-Carreiio, F.L. (1992) Protease Inhibition in Theory and Practice in Biotechnol. Educ. 3, 145-l 50 3 Garcia-CarreRo, F.L. and Haard, N. (1993) Characterization of Proteinase Classes in Langostilla (Pleuroncodes planipes) and Crayfish (Pacific astacus) Extracts in 1. Food Biochem. 17, 97-l 13 4 Salvesen, C. and Nagase, H. (1989) Inhibition of Proteolytic Enzymes in Proteolytic Enzymes (Beynon, R. and Bond, J., eds), pp. 83-104,

IRL Press at Oxford University Press 5 OIIi, J., Hjelmeland, K. and Krodahl, A. (1994) Soybean Trypsin Inhibitor in Diets for Atlantic Salmon (Salmo salar, L): Effects on Nutrients Digestibilities and Trypsin in Pyloric Caeca Homogenate and Intestinal Content in Camp. Biochem. Physiol. lOgA, 923-928 6 Cummann, M., Dugan, G., Spangler, W., Baker, E. and Rackis, J. (1989) Pancreatic Response in Rats and Mice to Trypsin Inhibitors from Soy and Potato AtIer Short- and Long-term Dietary Exposure in /. Nutr. 119, 1598-l 609 7 Holm, H., Reseland, J., Thorsen, L., Flatmark, A. and Hanssen, L. (1992) Raw Soybean Stimulate Human Pancreatic Proteinase Secretion in 1. Nutr. 122,1407-1416 8 Yamamoto, A., Taniguchi, T., Rikyuu, K., Tsuji, T., Fujita, T., Murakami, M. and Muranishi, S. (1994) Effects of Various Protease Inhibitors on the Intestinal Absorption and Degradation of Insulin in Ratsin Pharm. Res. 11, 1496-l 500 9 Zhang, Y., Parsons, C., Weingartner, K. and Wijeratne, W. (1993) Effects of Extrusion and Expelling on the Nutritional Quality of Conventional and Kunitz Trypsin-free Soybean in Poultry Sci. 72, 2299-2308 10 Baynon, R. (1988) Prevention of Unwanted Proteolysis in New Protein Technology (Walker, J., ed.), pp. l-24, Humana Press, Clifton, NJ, USA 11 Ebina, H., Nagashima, Y., Ishizaki, S. and Taguchi, T. (1995) Myosin Heavy Chain-degrading Proteinase from Spear Squid Muscle in Food Res. ht. 28,31-36 12 Chang-Lee, M., Pacheco-Aguilar, R., Crawford, L. and Lampila, L. (1989) Proteolytic Activity of Surimi from Pacific Whiting (Meduccius productus) and Heat-set Gel Texture in j. Food Sci. 54, 1116-l 119, 1124 13 Kabata, Z. and Whitaker, D. (1981) Two Species of Kudos (Myxosporea: Multivalda) Parasite in the Flesh of Meduccius productus (Ayres, 1855) (Pisces: Teleostei) in the Canadian Pacific in Can. 1. Zool. 59, 2085-2091 14 An, H., Weerasinghe, V., Seymour, T. and Morrissey, M. (1994) Cathepsin Degradation of Pacific Whiting Surimi Proteins in I. food Sci. 59, 1013-1017 15 Garcia-CarreAo, F.L., Navarrete del Toro, M.A., Diaz-Lopez, M., HernandezCartes, M.P. and Ezquerra, J.M. (1996) Proteinase Inhibition of Fish Muscle Enzymes Using Legume Seed Extracts in 1. Food Protect. 58, 1-8 16 Takeda, A., Iwasawa, A., Nakamura, Y., Omata, K. and Nakaya, K. (1994)

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

203

17

18

19

20

21

22 23

24 25

26

27

28

29

30

Monoclonal Antibodies as Probes to Detect Conformational Changes in the Rat Cysteine Proteinase Inhibitor Cystatin A in 1. Immunol. Methods 168,69-78 Feeney, R. and Osuga, D. (1988) Egg-white and Blood-serum Proteins Functioning by Noncovalent Interactions: Studies by Chemical Modification and Comparative Biochemistry in 1. Protein Chem. 7, 667-687 Izquierdo, M., Haard, T., Hung, J. and Haard, N. (1994) Oryzacystatin and Other Proteinase Inhibitors in Rice Grain: Potential Use as Fish Processing Aid in 1. Agric. Food Chem. 42,616-622 Weerasinghe, V., Morrissey, M., Chung, Y. and An, H. (1996) Whey Protein Concentrate as a Proteinase Inhibitor in Pacific Whiting Surimi in /. FoodSci. 61(2), 367-371 Hameed, K. and Haard, N. (1985) Isolation and Characterization of Cathepsin C from Atlantic Short Finned Squid ///ex illecebrosus in Comp. Biochem. f hysiol. 828,241-246 Garcia-Carretio, F.L., Hernandez-Cartes, M.P. and Haard, N.F. (1994) Enzymes with Peptidase and Proteinase Activity from Digestive System of Fresh Water and Marine Decapods in 1. Agric. Food Chem. 42, 1442-1456 Osnes, K. and Mohr, V. (1985) Peptide Hydrolases of Antarctic Krill, Euphasia superba in Camp. Biochem. Physiof. B2B, 599-606 Kawamura, Y., Nishimura, K., Matoba, T. and Yonezawa, D. (1984) Effects of Protease Inhibitors on the Autolysis and Protease Activities of Antarctic Krill in Agric. Biol. Chem. 48, 923-930 Baek, H. and Cadwallader, K. (1995) Enzymatic Hydrolysis of Crayfish By-products in /. FoodSci. 60,929-935 Wang, Z., Taylor, A. and Yan, X. (1992) Studies on the Protease Activities in Norway Lobster (Nephrops norvegicus) and their Role in the Phenolase Activation Processin Food Chem. 45,ll l-l 16 Wang, Z., Taylor, A. and Yan, X. (1994) Further Studies on the Role of Proteases in the Activation of Phenolase from Norway Lobster (Nephrops norvegicus) in Food Chem. 51,99-l 03 Orr, C., Strickland, J. and Walsh, T. (1994) Inhibition of Diabrotica Larval Growth by a Multicystatin from Potato Tubers in 1. insect Physiol. 40, 893400 Hines, M., Nielsen, S., Shade, R. and Pomeroy, M. (1990) The Effect of Two Proteinase Inhibitors, E-64 and the Bowman-Birk Inhibitor, on the Developmental Time and Mortality of Acanthoscelides obtectus in Entomol. Exp. Appl. 57,201-207 Burgess, E., Stevens, P., Keen, G., Laing, W. and Christeller, J. (1991) Effects of Protease Inhibitors and Dietary Protein Level on the Black Field Cricket Telegryllus commodus in fntomol. Exp. Appl. 61, 123-l 30 Boulter, D., Gatehouse, M. and Hilder, V. (1989) Use of Cowpea Trypsin

31

32

33

34

35

36

37 38 39 40

41 42 43

44

45

Inhibitor (Cpti) to Protect Plants Against Insect Predation in Biotechnol. Adv. 7,489-497 Ryan, C. (1984) Defense Response of Plantsin Plant Gene Research: Genes involved in Microbe P/ant Interaction (Verma, D. and Hihn, T., eds), pp. 375-386, Springer-Verlag Masoud, S., Johnson, L., White, F. and Reeck, G. (1993) Expression of a Cysteine Proteinase Inhibitor (Oryzacystatin I) in Transgenic Tobacco Plantsin P/antMo/. Biol. 21, 655-663 Sain, S., Oduro, K. and Furter, D. (1994) Genetic Transformation of Cocoa Leaf Cells Using Agrobacterium tumefaciens in Plant Cell, Tissue Organ Cult. 37,243-251 Michaud, D., Nguyen-Quoc, B. and Yelle, S. (1994) Production of Oryzacystatin I and II in Escherichia co/i Using the Clutathione Stransferase Gene Fusion Systemin Biotechnol. Prog. 10, 155-l 59 Seldal, T., Dybwad, E., Andersen, K. and Hogstedt, G. (1994) Woundinduced Proteinase Inhibitor in Grey Alder (Alnus incana): A Defense Mechanism Against Attacking Insects in OlKOS 71,239-245 Seldal, T., Andersen, K. and Hogstedt, G. (1994) Grazing-induced Proteinaje Inhibitors: A Possible Cause for Lemming Population Cycles in OIKOS 70, 3-l 1 DeClerck, Y. and Imren, S. (1994) Protease Inhibitors: Role and Potential Therapeutic Use in Human Cancer in Eur. I. Cancer 30A, 2170-2180 Das, S. and Mukhopadhyay, P. (1994) Protease Inhibitors in Chemoprevention of Cancer in Acta Oncol. 33,859-865 Laskowski, M. and Laskowski, M., Jr (1954) Naturally Occurring Trypsin Inhibitors in Adv. Protein Chem. IX, 203-242 Kennedy, A., Billings, P., Maki, A. and Newberne, P. (1993) Effect of Various Preparations of Dietary Protease Inhibitors on Oral Carcinogenesis in Hamster Induced by DMBA in Nutr. Cancer 19,191-200 Steinmetz, K. and Potter, D. (1991) Vegetables, Fruit and Cancer. II. Mechanisms in Cancer Causes Control 2,427-442 Bonnerjea, J. and Oh, S. (1986) Protein Purification: The Right Step at the Right Time in Biotechnology4,954-958 Wilchek, M., Miron, T. and Kohn, J. (1984) Affinity Chromatography in Methods in Enzymology (Vol. 104) Uakoby, W., ed.), pp. 3-55, Academic Press Garcia-CarreAo, F.L. and Haard, N. (1994) Preparation of an Exopeptidase Enriched Fraction from the Hepatopancreas of Decapods in Process Biochem. 29,663-670 Garcia-Carreno, F.L., Dimes, N. and Haard, N. (1993) Substrate-gel Electrophoresis for Composition and Molecular Weight of Proteinases or Proteinaceous Proteinase Inhibitors in Anal. Biochem. 214(l), 65-69

letter

BSE on the World W ide Web

The April issue of TIFS (p. 131) lists a number of World Wide Web sites carrying links to other sites where information about bovine spongiform encephalopathy (BSE) is available. If one accesses a professional institute Web site such as the BSE page of the UK Institute of Food Science & Technology (IFST) http;//www.easynet.co,uWifstihottop5.htm or the new BSE backgrounder page of the US Institute of Food Technologists G=T) http;//www.ift.orglsc/stat-tes/G-055.htmI one can rely on the information having gone through a rigorous

to ill-informed opinionated garbage (sadly, some of it put out by people who should know better) at the other. A large volume of speculation is peer-review process. Similarly, presented as though it were research reports on sites such as CAB established fact, with a great deal of INTERNATIONAL quoting of second-hand media http$www.cabi.orglwhatsnew/bs$seindex.htm stories that purport to give accounts of, but grossly misrepresent, papers in are mostly abstracts of papers in peer-reviewed journals. research journals. However, when it comes to Web Those seeking information about sites that carry links to other Web sites, BSE on the Internet need, therefore, to for the most part the compilers be aware of this state of affairs and to exercise no quality control read everything with a critical eye. whatsoever over the contents of the linked sites. Indeed, one such J. Ralph Blanchfield compiler has stated that to do so Food Science, Food Technology & would be unwarranted censorship. Food Law Consultant; The fact is that BSE material on the Chair, IFST Member Relations & Services Internet ranges from peer-reviewed Committee; Web Editor, IFST scientific information at one extreme (Web address: http;//www.easynet.co.ui$fst/).

204

01996, Elsevier Science Ltd PII:so924-2244(96)2m3-2

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