Proteinase inhibitors-Review Garcia-Carreño

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33 Nguyen, U., Evans, D.A., Berger, D.J. and Calderon, LA. (1992) Process forthe Supercritical Extraction and Fractionation of Spi ces, US Patent 5 120 558

34 Saito, N., tkushima, Y., Hatakeda, K., Ito, S. and Coto, T. (1991) FractionalExtraction of Rice Bran Oil and Its Esterswith Supercritical Carbon Dioxidein 1. Agric. Gem. Sot. Ipn 65, 153-l 61

35 Bondioli, P. etal. (1992) Lampante Olive Oil Refining with SupercriticalCarbon Dioxide in 1. Am. Oil Chem. Sot. 69,477-480

36 Li, S. and Hartland, S. (1992) Influence of Co-solvents on Solubility andSelectivity in Extraction of Xanthines and Cocoa Butter from Cocoa Beanswith Supercritical CO, in 1. Supercrit. Fluids 5, 7-l 2

37 Karlsson, H.O.E. and Tragdrdh, G. (1993) Pervaporation of Dil uteOrganic-Water Mixtures. A Literature Review on Modelling Studies and

Applications to Aroma Compound Recovery in 1. Membr. Sci. 76,121-14638 Bengtsson, E., Trlgardh, C. and Hallstrom, B. (1993) Concentration

Polarization During the Enrichment of Aroma Compounds from a WaterSolution by Pervaporation i n 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 MembraneTechnology t o Food Processing in Trends FoodSci. Technol. 4,277-282

41 Pare, J. (1992) Microwave Extraction of Volatile Oils and ApparatusTherefore, 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 ofAroma Compounds in Thermal/y Generated F/avows. Maillard, Microwaveand Extrusion Processes ACS Symposium Series 543) (Parli ment, T.H.,Morello, M.J. and McCorrin, R. J., eds), pp. 403-413, ACS Press, Washington,DC, USA

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46 Pettersen, T. and Lien, K.M. (1995) Design of Hybrid Distillation and VaporPermeation Processesn /. Membr.Sci. 99, 21-30

Review

1 Yroteinase inhibitorsEnzymatic protein hydrolysis plays a major role in variousphysiological processes, including digestion, and is regulated

by proteinase inhibitors. Inhibitors in foods and food ingredi -

ents can reduce the absorption of free amino acids, and can

impair protein hydrol ysis in industrial processes. However,

inhibitors can be useful tools in pest control, in the preven-

tion 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 protei nase classes and specificities. This

article discusses how proteinase inhibition is involved insome processes of current interest to food scientists and

technologists.

Enzymatic protein hydrolysis is a major concern for bio-logical scientists. The hydrolysis of proteins is catalyzedby peptide-bond-splitting enzymes (Box 1). Proteinasesand peptidases are involved in the hydrolysis of proteinduring digestion, and have important roles in physiologyand pathology. Enzymatic protein hydrolysis is con-trolled in several ways, including by the use of specificinhibitors (Box 2). Proteinase inhibition is a commonprocess in nature. Proteinase-inhibitor interactions areinvolved in protein digestion, various physiologicalprocesses (e.g. blood coagulation, fibrinolysis, com-plement activation and phagocytosis), pathologicalprocesses (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 01996, Elsevier Science Ltd

I ernando his Garcia-Carreiio(e.g. with AIDS or invasive parasites). Another naturalmethod of controlling proteinase activity is the synthesisof an inactive form of the enzyme, the zymogen.Zymogens are activated, usually by the action of an-other proteinase, in the digestive system and also duringregulatory physiological processes. When an enzyme isin its active form, proteinase inhibition is an exquisitemeans of enzyme control in physiological processes,which is achieved by highly specific inhibitors. The im-portance of the control of proteolytic activity by in-hibitors in physiological processes is demonstrated bythe fact that inhibitor molecules exceed 10% of the totalprotein in human plasma.

The fact that the control of proteolysis by inhibitors isso specific makes it a valuable tool in medicine, agri-culture and food technology. The human immune de-ficiency virus proteinase, the digestive systems of croppests, and fish muscle proteases are some examples oftargets for study. Most organisms produce proteinaseinhibitors as a means to control proteolytic processes.Some organisms store huge amounts of inhibitors, forexample legume seeds and some leaves. This seems tobe an evolutionary response to predation.

Inhibitors for digestive proteinases in food and feedSome food ingredients contain so-called antinutritive

factors: lectins, phenols, and other factors, includingcertain proteins that inhibit proteinases. The presence ofproteinase inhibitors in living tissues seems to be a natu-ral regulatory process, well represented by the case of

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endogenous proteinase activity is significantly reducedfollowing the 7 min boil ing step commonly used to facili-tate the peeling of the crayfish. However, in langostilla,elimination of enzyme activity has not been that simple,showing that there are important differences in the pro-teinases among species *l Controlled proteolysis can insome circumstances be used to advantage; a proteinhydrolysate derived from the cephalothorax of crayfish(which is normally considered as waste, once the tailhas been removed for human consumption) has been re-ported to have potential applications as a flavoring in-gredient for seafood%.

Blackspot development in crustaceans is dependenton the proteolytic activation of a phenolase, whichtransforms tyrosine into the dark pigment known asmelanin. Sulphite is currently used to reduce post-mortem blackspot development. However, there is somepotential hazard associated with this practice, and alter-native methods of control need to be developed. Threeproteinases have been isolated from Norway lobster(Nephrops nowegicus). Two of them are thiol (cysteine)proteinases and the third is a metal-dependent serineproteinase25. Inhibition of the serine proteinase reducedphenolase activation, and therefore blackspot develop-ment. Inhibition of the thiol proteinases, however, hadnegligible effect on blackspot development*j.

inhibitors in pest control: transgenic plantsPlants protect themselves against predation by pro-

ducing insecticides and proteinase inhibitors of the pestdigestive system, impairing predator physiology. Phyto-phagous insect pests have digestive proteinases belong-ing to the serine and cysteine proteinase classes. Serineproteinases predominate in Lepidoptera, and cysteine pro-teinases in Coleoptera27. Hines et uZ.*~howed that feed-ing the insect Acunthoscelidesabtectuswith an artificialbean-seed system containing the cysteine proteinase in-hibitor E-64 delayed the development and increased themortality of the larvae. These effects were a direct func-tion of the concentration of the proteinase inhibitor inthe feed. The cysteine proteinase activity in extracts of thelarvae was severely affected. The addition of free aminoacids to the diet did not prevent the reduction in the en-zyme activity, but improved larval development and re-duced mortality. In another study, the growth of the blackfield cricket(Teleogryllus ommodus)wasevaluated whenrearing it on diets containing different levels of proteinand inhibi tors for trypsin and elastase, the major cricketgut proteinases 29 In this study, al l of the proteinase in-hibitors that inhibi ted trypsin or elastase invitro reducedthe crickets growth. Inhibitors that reacted with bothenzymes were more effective. Diets containi ng potatomulticystatin caused a dose-dependent reduction in thegrowth of Western and Southern corn rootworm larvae(Diubrotica virgiferu virgij2ru)27.Multicystatin frompotato tubers is a cysteine proteinase inhibitor belongingto the cystatin family. The inhibitor is a protein consist-ing of eight tandem 10.8 kDa cystatin domains l inked bytrypsin-sensitive bonds. These studies demonstrate theeffects of proteinase inhibitors in foods on predators.

Several research groups have been enthusiastic aboutthe potential of proteinase inhibitors for the control ofinsect pests. One approach to provide defense againstherbivorous insects is the incorporation of the geneencoding the proteinase inhibitor into the genome ofthe plant. This strategy has proven to be successful.Biotechnology is taking advantage of the co-evolutionover millions of years of insects and flowering plantsthat has generated insect resistance genes30. A number

of plants possess two wound-induced small multigenefamilies that code for proteinase inhibitors; these in-hibitors are now targets for studi es on gene transfer intofood crops3.

Transgenic plants expressing a proteinase inhibitorhave enhanced levels of insect resistance. The geneencoding the cysteine proteinase inhibi tor oryzacystatinhas been successfully introduced into tobacco plants32.The transgenic inhibitor was present at levels of up toOS-0.6% of the total soluble protein in the leaves androots. The use of the bacterium Agrobucterium tume-fuciens as a vector for the introduction and expressionof foreign genes has also proven to be successful i nother plants, including in cocoa leaf cells, even thoughA. tumefucienss not a natural pathogen for the cocoapla&.

An al ternative approach for pest control is the produc-tion of proteinase inhibitors on a large scale in a bac-terial system for subsequent purification and use as acrop spray. The expression of the genes for oryzacys-tatin I and II in Escherichiu coli using a glutathioneS-transferase gene fusion system has yielded activeforms of the inhibi tors34. Because of the specificity ofthe fusion products, these inhibi tors might be useful inthe control of predators that digest dietary protein byusing cysteine proteinases.

Plants respond naturally to biological attack. Onemechanism of defense is the production of wound-induced proteinase inhibitors. In the gray alder(Alnusincunu),an increase of trypsin inhibi tor after defoliati onwas concomitant with a decrease of soluble pl ant pro-tein. Leaves from attacked trees, which contain an in-creased ratio of trypsin inhibi tor to soluble protein, haveantifeeding properties, causing retarded growth, delayedpupation, reduced egg production and low survival ofthe predator beetle (Guferucellu ineolu)35. hetoxic ef-fect can be reversed if the insects diet is rich in essen-tial amino acids. A similar effect could explain the fluc-tuating dynamics of a herbivore population, for examplethe possible effects of grazing-induced proteinase in-hibitors on lemming population cycles36. When the ratioof trypsin inhibi tor to soluble protein in plant extractswas increased, a decrease in l emming densities oc-curred. The animals from the declining populationsshowed the pancreatic hypert rophy that is correlatedwith a prolonged intake of proteinase inhibitors.

Proteinase nhibitors and cancer preventionCancers are a complex group of diseases. Their causes

are multiple. Several pathological conditions associatedwith carcinogenesis are related to changes in proteases

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responsible for tumor promotion and neoplastic trans-formation. The inhibi tion of such activities explains theeffectiveness of protease inhibi tors in the prevention ofsome types of cancers; for comprehensive reviews, seeDeClerck and Imren3, and Das and Mukhopadhyay3*.The need for agents to treat or to prevent cancers hasdriven 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 inhibi tors found in plants and syntheticinhibitors.Legume seeds are known to contain proteinase in-

hibitors. Soybean seeds, for example, contain at leastthree different compounds with anti-proteolytic activ-ity .9 During the past 20 years, information has beengained about the protective effects against cancers ofdiets containing legume proteinase inhibitors. Such aneffect has been shown in the case of mouse skin tumorsinduced by nitroquinoline oxide and phorbol myristateacetate, as well as for rat breast tumors and mouse livercancer induced by i onizing radiation38. Several serineproteinase inhibitors from soybean and potato sup-pressed oral carcinogenesis in hamsters that was inducedby 7,12-dimethylbenz[a]anthracene. The inhibitors, ap-plied topically to the cheek pouch, were effective inreversing carcinogenesis at concentrations as low asO.Ol%, even when used 45 d after the beginning of ex-posure to the carcinogen40.

Details about the mechanisms of action of the preven-tive effects of proteinase inhibitors on cancers remain tobe elucidated. One problem that needs to be solved ishow the therapeutic form of the proteinase inhibitorshould be administered. It appears that inhibitors couldcomplement cytotoxic therapies. Steinmetz and Potter4,in their extensive review, concluded that consumptionof higher levels of vegetables and fruits is associatedconsistently, although not universally, with a reducedrisk of cancer at most sites, and particularly with epi-thelial cancers of the alimentary and respiratory tracts.The authors postulate a mechanism to explain how plantproteinase inhibitors affect enzymes produced by neo-plastic cells, reducing damage to the extracellular matrixcaused by tumor proteinases, and thus limiting tissue in-vasion. They also point out that humans are adapted fora high intake of plant foods. Some food components thatare not thought to be essential nutrients may be crucialfor the prevention of cancers. If some cancers are the re-sult of ingesting too low a level of metabolically necess-ary foods, they may be a disease of maladaptation.

Proteinase nhibitors in the laboratoryProteinase purification by affinity chromatography

Thirty years after its introduction, affinity chroma-tography is still the best purification technique. It is basedon specific recognition between the compound to be puri-fied and a ligand i mmobili zed on an affinity column,and renders the maximum possible purification factor,which averages loo-fold (Ref. 42). It is commonly usedto purify biologically active molecules such as antigens,antibodies, cell receptors, hormones, nucleic acids and

enzymes. The purification of enzymes using affinitychromatography has been the subject of extensive investi-gation, and is beyond the scope of this review; forfurther information, see the comprehensive chapter byWilchek et a1.43

The purification of proteinases by affinity chroma-tography is more complicated than that of other enzymes.This i s because of the unique substrate specificity ofproteinases for an amino acid residue forming the car-

boxylic half of a target peptide bond, rather than for aparticular protein. Furthermore, the possibility that theproteinase being purified will hydrolyze the immobi-lized protein substrate makes it infeasible to use proteinsas ligands. Synthetic substrates mimicking the targetpeptide bond [e.g. tryptophan methyl ester, for chymo-trypsin; (Ala),, for elastase; and aminobenzoyl -Arg, forcarboxypeptidase] have instead been used. Protein-aceous proteinase inhibi tors, which resist hydrolysis,have also been used. Affinity chromatography usingimmobilized soybean trypsin inhibitor produced apeptidase-enriched fraction from each of two decapod(langostill a and crayfish) hepatopancreas samples thatcontained both proteinases and peptidases44. In eachcase, only the proteinases bound to the agarose - soy-bean inhibitor column because they belong to the serineproteinase class. The peptidases were enriched up to15-fold, whereas the proteinases were reduced to negli-gible levels. The peptidases are intended for future usein accelerating the ripening of Cheddar cheese.

Evaluating proteinase activity and proteinase inhibitorsMany methods have been reported for evaluating the

activity of proteinases and proteinase inhibi tors. A greatnumber of proteins have been proposed for use as thesubstrate; casein or its derivatives such as azocasein arethe most commonly used. A complex of casein and anazo dye permits more sensitive spectroscopic determi-nation, as the molar light absorbance (at 366nm) of theazocasein complex is higher than that of the commonlyused aromatic amino acids at 280nm. A simple methodfor evaluating the proteinase activity of an enzymepreparation is outlined in Table 1. Casein-based sub-strates are useful at neutral and basic pH values. For as-says at acid pH values, hemoglobin is used. A problemin evaluating the kinetic properties of proteinases isthat the concentration of a proteinaceous substrate in-creases rather than decreases with time, as a protein canhave several peptide bonds available for attack: whenthe enzyme hydrolyzes a bond to create two pepti des,the molar concentration of the substrate increases.Furthermore, the cleavage of only one peptide bonddoes not always produce peptides that are soluble in thetrichloroacetic acid (TCA) solution used to stop the en-zymatic reaction and to precipitate the undigested pro-tein substrate, allowing spectrophotometric determi-nation of the TCA-soluble peptides. Thus, non-proteinsubstrates are needed. Synthetic substrates mimickingthe natural substrates are therefore used; these are alsouseful to help obtain i nformation about the specificityand the class of an enzyme.

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Table 1. Assay or proteinase activity and inhibition

Inhibition Activity Extract Solventassay assay controlb controlc

Tube numbers l-3 4-6 7-9 10-12

Bufferd 500 pl 500 bl 500 p,l 500 /.Ll

Enzymeextract S-20 j,d S-20 ~1 S-20 pl 5-20 ~1

Inhibitor solution 1OplInhibitor solvent 1Opl

lncubatione -----------------10-60minat25-650C -- ~~~~ ~~~~ ____ -

TCA 0.5 ml

Substrate 0.5 ml 0.5 ml 0.5 ml 0.5 ml

IncubatiorF -----------------10-60minat25-65C -------_____ ----

TCA 0.5 ml 0.5 ml 0.5 ml

Centrifugation _------------------ 6500gforsmin ---~~~~~~~~___----

Absorbance (366 nm) ----------------------Record---------------------

Adapted from Ref. 2bControl or the enzyme extract (somecrude extracts might absorb at 366 n m becauseof the presenceofcarotenoidsor other yellow substances)CControl or the inhibitor solvent some nhibitor solventsmight modify the activity of the enzyme)d Usually 50IIIM Tris/HCI, pH 7.8The temperatureand time of the incubation dependson the thermostabilityand the time required o obtain alinear reaction; or t hermophili c organisms, he temperaturecould be as high as 65C1.5% azocasein n 50m~ Tris/HCl, pH 7.8TCA, Trichloroacetic acid (20%, w/v, in water)

The easiest way to test for the presence of proteinaseinhibitors in a biological sample is to challenge it withcommercial proteinases belonging to different classes.The sample is mixed with the proteinase for some time,taking into account the fact that some inhibitors mayreact slowly with the enzyme. Then, the inhibitor-enzyme mixture is mixed with the substrate at zero timeof the reaction. The time course of the reaction in thecontrol assay without added inhibitor must be linear toguarantee that any reduction in enzyme activity is due tothe inhibitor and not because of a shortage of the sub-strate, product inactivati on or denaturation of the en-zyme. Table 1 shows a general assay protocol for study-ing the activity of proteinases and proteinase inhibitors.

interact with their target enzyme(s), their mechanism ofinhibition, their specificity, and their bioavailability willallow better use and control of these biochemical agents.

Future rendsBiotechnology and the food sciences can profit from

the use of proteinase inhibitors. Further research on newinhibitors, their mechanism of action and kinetic proper-ties will expand the possibiliti es of the use of the inhibi-tion process in practical applications.

An exquisite method for evaluating the compositionand molecular mass of proteinases and proteinaceousproteinase inhibitors is the substrate - gel electrophoresistechnique45. This method takes advantage of the factthat proteinases are proteins that are composed of justone polypeptide chain and are therefore resistant to de-naturation following sodium dodecyl sulphate treatment.Figure 1 shows the protocol followed in this technique.

Most of the information about proteinase inhibitorshas been derived from terrestrial organisms. Of the-400 papers reviewed, only a few concerned marine or-ganisms. The search for unique inhibitors from organ-isms from both marine and extreme environments isexpected to provide molecules that function at hightemperatures or low temperatures, with the possibility ofusing heat or low ion concentrations to inactivate them.

The control of insect pests and herbivores should bepossible by using transgenic plants or by producinglarge amounts of ecologically benign, easy-to-inactivateinhibitors that provide protection during plant growth,harvest or storage.

ConclusionsProteinase inhibitors are ubiquitous molecules. They

are present in all organisms as a means of regulatingphysiological proteolysis and predation. Huge quantities ofcrops are lost, during culture, harvest and storage, owingto fungal and viral diseases or insect pests and vertebrateherbivores. Most predators have sensitive enzymatic

systems, some related to the digestion of protein. The

suitability of using proteinase inhibitorgenes to transform plants or bacteria forlarge-scale production and to enhanceinsect resistance has been proven, andrepresents an alternative to the con-ventional breeding of resistant plants.

Food processing is already benefitingfrom the use of inhibitors to reduceunwanted protein hydrolysis, as in thecase of surimi production. Alternativeinhibitors that do not alter food qualityand improve the profit of the processwill always be welcome.

The discovery of proteinase inhibitorswith particular kinetic properties mightprove useful as biochemical tools inaffinity chromatography. The purifi-cation of proteinases by bioaffinitychromatography should provide cheaperand more specific proteolytic enzymesfor industrial purposes.

Proteinase inhibitors in foods can beeither good or bad: good when theyare used to avoid unwanted proteolysis,to control pests, to prevent certain dis-eases, or to improve gel strength infood technology; bad when they re-duce the digestibility of food or feed.Improved knowledge of how inhibitors

Methods of evaluating proteinases and proteinase in-hibitors are needed. The use of rational selection and arti-ficial neural network methods are needed to select suit-able inhibitors when analyzing several sources. Methodssuch as surface response methodology will help to opti-mize the variables of evaluation.

While there is growing emphasis on the applications

of molecular biology techniques, further studies are

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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 separated samples,one of the two identical SDS-PA GE gels is stained with Coomassi e (Brilliant) Blue for protein, showing the protein composition of the sample. The

twin gel is soaked for 30-60min 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-PA GE 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 ac-tion and reaction kinetics of enzymes and enzyme in-hibitors. Studies on nucleic acids are important becausethey are responsible for storing information about thephysiological processes. Nucleic acids are the softwareof life. However, no-one goes to the theater to read thescript of a play. We want to see the actors perform. Inphysiology, pathology and food technology, the actorsare the enzymes that dictate the route and speed ofimportant biotransformations. Enzymes are the hard-ware of life.

AcknowledgementsGrant 3589-N by CONACyT, which partially sup-

ported this work, is appreciated. Thanks are given toPaty Hernandez for her help in the preparation ofelectrophoresis slides, Drs Gloria Yepiz at CIAD,Hermosillo and Norman Haard at UC Davis for theirsuggestions on the early draft, and Ellis Glazier foreditorial work on the manuscript.

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32 Masoud, S., Johnson, L., White, F. and Reeck, G. (1993) Expression of aCysteine Proteinase Inhibitor (Oryzacystatin I) in Transgenic Tobacco Plants inP/antMo/. Biol. 21, 655-663

33 Sain, S., Oduro, K. and Furter, D. (1994) Genetic Transformation of CocoaLeaf Cells Using Agrobacterium tumefaciens in Pl ant Cell, Tissue Organ Cult.37,243-251

34 Mi chaud, D., Nguyen-Quoc, B. and Yelle, S. (1994) Production of

Oryzacystatin I and II in Escherichia co/i Using the Clutathione StransferaseGene Fusion System in Biotechnol. Prog. 10, 155-l 59

35 Seldal, T., Dybwad, E., Andersen, K. and Hogstedt, G. (1994) Wound-induced Proteinase Inhibitor in Grey Alder (Alnus incana): A DefenseMechanism Against Attacking Insects in OlKOS 71,239-245

36 Seldal, T., Andersen, K. and Hogstedt, G. (1994) Grazing-induced ProteinajeInhibitors: A Possibl e Cause for Lemming Population Cycles i n OIKOS 70,3-l 1

37 DeClerck, Y. and Imren, S. (1994) Protease Inhibitors: Role and PotentialTherapeutic Use in Human Cancer i n Eur. . Cancer 30A, 2170-2180

38 Das, S. and Mukhopadhyay, P. (1994) Protease Inhibitors i n Chemopreventionof Cancer in Acta Oncol. 33,859-865

39 Laskowski, M. and Laskowski, M., Jr (1954) Naturally Occurring TrypsinInhibitors in Adv. Protein Chem. IX, 203-242

40 Kennedy, A., Billi ngs, P., Maki, A. and Newberne, P. (1993) Effect of VariousPreparations of Dietary Protease Inhibitors on Oral Carcinogenesis in HamsterInduced by DMBA in Nutr. Cancer 19,191-200

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BSE on the World Wide WebThe April issue of TIFS (p. 131) l ists anumber of World Wide Web sitescarrying links to other sites whereinformation about bovine spongiformencephalopathy (BSE) is available.

If one accesses a professionalinstitute Web site such as the BSEpage of the UK Institute of FoodScience & Technology (IFST)

http;//www.easynet.co,uWifstihottop5.htm

or the new BSE backgrounder page ofthe US Institute of Food TechnologistsG=T)

http;//www.ift.orglsc/stat-tes/G-055.htmI

one can rely on the informationhaving gone through a rigorous

peer-review process. Similarly,research reports on sites such as CABINTERNATIONAL

http www.cabi.orglwhatsnew/bs seindex.htm

are mostly abstracts of papers inpeer-reviewed journals.

However, when it comes to Websites that carry links to other Web sites,for the most part the compilersexercise no quality controlwhatsoever over the contents of thelinked sites. Indeed, one suchcompiler has stated that to do sowould be unwarranted censorship.

The fact is that BSE material on theInternet ranges from peer-reviewedscientific information at one extreme

to ill -informed opinionated garbage(sadly, some of it put out by peoplewho should know better) at the other.A large volume of speculation ispresented as though it wereestablished fact, with a great deal ofquoting of second-hand mediastories that purport to give accountsof, but grossly misrepresent, papers inresearch journals.

Those seeking information aboutBSE on the Internet need, therefore, tobe aware of this state of affairs and toread everything with a critical eye.

J. Ralph BlanchfieldFood Science, Food Technology &

Food Law Consultant;Chair, IFST Member Relations & Services

Committee; Web Editor, IFST(Web address: http;//www.easynet.co.ui fst/).

20401996, Elsevier Science Ltd

PII:so924-2244(96)2m3-2 Trends n Food Science & Technology June 1996 [Vol. 71

Proteinase inhibitors-Review Garcia-Carreño

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