active bowman–birk inhibitors survive gastrointestinal digestion at the terminal ileum of pigs fed...

Journal of the Science of Food and Agriculture J Sci Food Agric 88:513–521 (2008)

Active Bowman–Birk inhibitors survivegastrointestinal digestion at the terminalileum of pigs fed chickpea-based dietsAlfonso Clemente,∗ Elisabeth Jimenez, M Carmen Marin-Manzano and Luis A RubioDepartment of Physiology and Biochemistry of Animal Nutrition, Estacion Experimental del Zaidın (CSIC), Professor Albareda 1, 18008Granada, Spain

Abstract

BACKGROUND: Protease inhibitors of the Bowman–Birk family have been demonstrated to be naturallyoccurring chemopreventive agents in a wide range of in vitro and in vivo models. In vitro and in vivo experimentshave reported that Bowman–Birk inhibitors (BBIs) may exert colorectal chemopreventive effects. To exert sucheffects, these proteins have to survive, at least to some extent, the digestive process within the gastrointestinaltract.

RESULTS: In order to determine the survival rates of functional BBI proteins in vivo, five castrated male pigs(100 ± 2 kg body weight) fitted with T-shaped cannulas at the terminal ileum were fed a chickpea-based diet.Pigs fed hydrolysed casein as the only protein source were used as an experimental control diet. The survivalrates of BBI proteins from chickpea-based diets at the terminal ileum, expressed in terms of trypsin (TIA) andchymotrypsin inhibitory activity (CIA), were 7.3 and 4.4%, respectively. The presence of BBI proteins in ilealsamples from pigs fed chickpea-based diet was confirmed by SDS-PAGE and peptide mass fingerprinting.

CONCLUSION: It is concluded that significant amounts of active BBI proteins reach the large intestine of thepig. Further pharmacological studies are necessary in order to determine the potential of dietary BBI proteins ascolorectal chemopreventive agents in humans. 2007 Society of Chemical Industry

Keywords: Bowman–Birk inhibitors; digestive proteases; gastrointestinal survival; protease inhibitors; chickpea;swine

INTRODUCTIONBowman–Birk inhibitors (BBI) are plant proteinsof low molecular weight (6–9 kDa) with the abilityto inhibit serine proteases through competition withsubstrates for access to the active site of the enzyme.Protease inhibitors of the Bowman–Birk family arewidely distributed in the plant kingdom, beingparticularly abundant in legume seeds compared withtheir concentrations in other legume organs or inother plant families. BBI proteins have two inhibitorydomains and form stable stoichiometric complexeswith the digestive enzymes, trypsin (EC 3.4.21.4)and chymotrypsin (EC 3.4.21.1). They can interactsimultaneously and independently with two targetproteases without any substantial conformationalchange. The resulting non-covalent complex rendersthe proteases inactive. The significance of each of thetwo inhibitory sites, in terms of their action as pseudo-substrate of target proteases, is likely to differ with

respect to biological relevance. The trypsin inhibitorysite of soybean BBI has been associated with negativeeffects on bioavailability of dietary proteins1 whereasthe chymotrypsin inhibitory site of BBI proteins hasbeen implicated in cancer chemopreventive effects.2,3

BBI proteins have been demonstrated to be effectiveat preventing or suppressing radiation-induced andchemical carcinogen-induced transformation, in awide variety of in vitro assays, and carcinogenesisin in vivo model systems, as has been reviewedelsewhere.4–7 Additionally, BBI-like proteins arebeing currently investigated for the developmentof innovative therapies against neurodegenerativediseases and inflammatory processes.7

In order to exert a biological effect, either localor systemic, BBI proteins from feed or foodstuffshave to resist and survive, at least to some extent,the digestive process within the gastrointestinal tract(GIT). It seems clear that the exploitation of these

∗ Correspondence to: Alfonso Clemente, Department of Physiology and Biochemistry of Animal Nutrition, Estacion Experimental del Zaidın (CSIC), ProfesorAlbareda 1, 18008 Granada, SpainE-mail: [email protected]/grant sponsor: Spanish CICYT; contract/grant number: AGL2004-03260ALIContract/grant sponsor: Junta de Andalucıa; contract/grant number: AGR2006-00706Contract/grant sponsor: Ramon and Cajal contract(Received 4 May 2007; revised version received 28 August 2007; accepted 29 August 2007)Published online 8 November 2007; DOI: 10.1002/jsfa.3115

2007 Society of Chemical Industry. J Sci Food Agric 0022–5142/2007/$30.00

A Clemente et al.

proteins in human health-promotion programmeswill depend largely upon their relative survival ratesafter digestion and their further distribution to targetorgans. BBI proteins from different plant sources arehighly resistant to heat treatment, under either neutralor acidic conditions;8–11 it has been also establishedthat most of the trypsin inhibitory activity (TIA)present in processed legumes is due to heat-stable BBIproteins. Furthermore, BBI proteins have been shownto be highly resistant to both the acidic conditions andthe action of proteolytic enzymes of the upper GITin vitro.12 The structure of BBI proteins linked to thenumber and distribution of disulphide bridges seemsto be a major contributor to such high stability.13

The resistance of BBI proteins to harsh conditionshas made these proteins attractive for evaluationas chemopreventive agents, through modulating cellviability and tumour progression, within the GIT.Under acidic conditions, BBI proteins from commonbeans (Phaseolus vulgaris) have been shown tobe biologically active in suppressing benzopyrene-induced forestomach carcinogenesis in mice, followingoral treatment.14 Purified soybean (Glycine max)BBI and a soybean BBI concentrate exerted aprotective effect in dimethylhydrazine (DMH)-treatedanimals, reducing the incidence and frequency ofcolon tumours in mice15,16 and rats,17 withoutany adverse side effects documented for animalgrowth or organ physiology. Although such studieshave demonstrated potential health benefits of BBIproteins within the GIT, there have been very fewattempts to quantify the intestinal recovery of BBIproteins and results are controversial mainly due tomethodological difficulties.18,19 The presence of highlevels of trypsin- and chymotrypsin-like proteases inileal flows makes it particularly difficult to accuratelyevaluate the inhibitory activity of these samples byenzymatic methods; additionally, BBI proteins have astrong affinity for the digestive enzymes, trypsin andchymotrypsin and, under neutral conditions, are likelyto be complexed with the proteases they inhibit.

Although most of nutritional studies have beencarried out with soybean BBI, BBI-like proteins fromother sources, including chickpea (Cicer arietinum),common beans, lentil (Lens culinaris) and pea (Pisumsativum), have been documented to have nutritionalproperties equivalent or superior to those documentedfor soybean BBI.3,6 In this study, we have exploitedchickpea seeds as a source of BBI-like proteins in orderto (1) develop a reliable and reproducible method todetect active BBI in ileal samples, and (2) quantify theinhibitory activity derived from BBI proteins foundat the terminal ileum of animals fed a chickpea-based diet. In this work, we have detected significantamounts of functional BBI at the terminal ileum ofcannulated pigs fed chickpea-based diets that couldpotentially exert a chemopreventive function in thelarge intestine. These data further our knowledge onthe gastrointestinal survival of potentially therapeutic

proteins and could assist in determining suitable BBIdoses for pre-clinical trials.

MATERIALS AND METHODSMaterialsHydrolysed casein, Bowman–Birk inhibitor from soy-bean, trypsin (type III) and α-chymotrypsin (typeVII) from bovine pancreas, phenylmethylsulpho-nyl fluoride (PMSF), N-α-benzoyl-DL-arginine-p-nitroanilide (BAPNA) and N-benzoyl-L-tyrosine ethylester (BTEE) were obtained from Sigma (Alcobendas,Madrid, Spain). All other chemicals were of analyticalgrade.

AnimalsMale castrated pigs [100 ± 2 kg mean live body weight(b.w.), n = 5] were purchased from Sanchez RomeroCarvajal S.A. (Huelva, Spain) and housed individuallyin 4 m2 pens. Pigs were fitted with T-shaped ilealcannulas, surgically implanted about 15 cm anterior tothe ileocaecal junction20 and allowed to recover fromsurgery. Water was freely available from low-pressuredrinking nipples. Cannulated pigs remained in goodcondition throughout the whole experimental periodand digesta flowed through the cannulas withoutany blockage. All management and experimentalprocedures were implemented, in strict accordancewith the guidelines of good practice of laboratoryanimals of the Spanish Ministry of Agriculture (ActNo. 1201/2005, October 10, 2005), by staff trained tocarry out such procedures.

Diets and feeding regimePrior to the experimental period, pigs were feda commercial cereal-based diet. Experimental dietswere based on defatted chickpea (Cicer arietinum cv.Athenas) seed meal (731 g kg−1) or hydrolysed casein(122 g kg−1) as the only protein sources (Table 1).Both diets, containing similar levels of digestibleenergy (14.2–15.1 kJ g−1), were fortified with vitaminsand minerals to meet requirements.21 Chromiumoxide (10 g kg−1 diet) was used as an indigestiblemarker and added to the diets mixed with maize starch.Feed was given at about twice their calculated energymaintenance needs [maintenance energy requirement(MEm) = 458 kJ per kg b.w.0.75] and offered in twomeals (at 9:00 h and 18:00 h) of 1 kg each. Theanimals were adapted to the experimental diets fora 7-day period prior to the collection day, so thatany carry-over effect from the previous diet wouldbe abolished. Samples from cannulas were taken inplastic bags attached to them. On day 8, the entireileal contents from 9:00 h to 18:00 h were collected ina time interval of less than 30 min. The bags containingileal digesta were immediately frozen in liquid nitrogenand stored at −20 ◦C until use. After thawing, samplesfrom the same animal were mixed, freeze dried, finelyground and stored at 4 ◦C for further analysis.

514 J Sci Food Agric 88:513–521 (2008)DOI: 10.1002/jsfa

Survival of active BBI in ileum

Table 1. Composition (g kg−1) of the experimental diets

Hydrolysed casein Chickpea

Dieta

Maize starch 630 204.9Sunflower oil 30 –Vitamins + mineralsb 3 3Ca carbonate 5 7.1Ca diphosphate 25 3.7Cr2O3/maize starch (1:5) 50 50Hydrolysed casein 122Chickpea seed meal – 731.3

CompositionDigestible energy (MJ kg−1) 15.1 14.2Proteinc 73 108

Protease inhibitor activityd

TIU ND 8.6CIU ND 8.9

a For details see materials and methods.b As in reference 21.c N was determined by Kjeldahl. Protein content in diets was calculatedas N × 5.5 and N × 6.25 in chickpea- and hydrolysed casein-baseddiets, respectively.23

d Trypsin and chymotrypsin inhibitory activity are expressed as TIUand CIU per mg of dry weight, respectively.ND, not detected.

Chemical analysisExperimental diets and freeze-dried ileal samples wereanalysed for chromium oxide by a micromethodinvolving dry ashing and alkaline fusion mixture.22

Using the Kjeldahl method, crude protein contentof feed was determined as N × 6.25 for hydrolysedcasein and N × 5.5 for chickpea proteins.23 Otherchemical determinations were carried out as previouslydescribed.19

Measurement of protease activitiesFinely ground freeze-dried ileal samples (100 mg) wereextracted with 1.5 mL of 50 mmol L−1 HCl (acidicconditions) or 50 mmol L−1 Tris, pH 8.0, 0.5 MNaCl (buffered conditions) at 4 ◦C for 2 h and theextracts centrifuged at 12 000 × g for 10 min. Super-natants were assayed for hydrolysis of BAPNA andBTEE, as described below (see Section on Measure-ment of protease activites), except that the exogenousenzymes trypsin and chymotrypsin were omitted fromthe assay. The amount of ileal sample necessary togive a linear relationship between ileal content andsubstrate hydrolysis was established. Units of pro-tease were determined from the quantitative assays (asgiven for inhibitor measurements, except that trypsinand chymotrypsin were omitted), where one unit oftrypsin (TU) and chymotrypsin (CU) as defined asthat amount giving an increase of 0.01 absorbanceunits at 410 and 256 nm, respectively, in a 10 mLof sample volume. The effect of heat treatment (70,80 or 90 ◦C for 10 min) on protease activities of ilealsamples, extracted under acidic conditions, was alsoinvestigated.

Measurement of protease inhibitory activitiesChickpea seed meal and experimental diets wereassessed for trypsin (TIA) and chymotrypsin inhibitoryactivity (CIA). Finely ground samples (25 mg) wereextracted with 1.5 mL of 50 mmol L−1 HCl at 4 ◦Cfor 2 h and centrifuged at 12 000 × g for 10 min atroom temperature. TIA was measured using a mod-ified small-scale quantitative assay, with BAPNA asspecific substrate, using 50 mmol L−1 Tris, pH 7.5,instead of 0.01 mol L−1 NaOH. One trypsin inhibitorunit (TIU) was defined as that which gives a reductionin absorbance at 410 nm of 0.01, relative to trypsincontrol reactions, in a define assay volume of 10 mL.24

CIA was measured using BTEE as specific substrate.One chymotrypsin inhibitor unit (CIU) was defined asthat which gives a reduction in absorbance at 256 nmof 0.01, relative to chymotrypsin control reactions,in a defined assay volume of 10 mL, as previouslydescribed.25

The high content of trypsin- and chymotrypsin-like protease activities in the ileal samples, evenunder acidic extraction conditions, made it extremelydifficult to measure protease inhibitory activitiesdirectly. To determine the survival of functional BBIproteins at the terminal ileum, a two-step procedurewas carried out. This included acidic extractionof freeze-dried ileal samples (100 mg in 1.5 mL50 mmol L−1 HCl, 2 h at 4 ◦C) to maximise thetotal amount of free BBI proteins, and further heattreatment (70, 80 or 90 ◦C for 10 min) of the acid-soluble fraction in order to selectively inactivate theproteolytic enzymes, but not the protease inhibitors.To confirm that protease inhibitors were unaffectedby such heat treatment, TIA and CIA of pure soybeanBBI in solution was investigated. In addition, theability of exogenous soybean BBI and the non-specific serine protease inhibitor PMSF to inhibittrypsin- and/or chymotrypsin-like activities of ilealsamples from pigs fed hydrolysed casein-based dietswas determined. PMSF was dissolved in anhydrousethanol to 100 mmol L−1 and incubated at a rangeof concentrations with ileal samples for some of theprotease inhibition experiments. The concentrationof soybean BBI or PMSF required to achieve 50%of inhibition was calculated by the non-linear fit ofinhibition data obtained for digestive proteases, usingthe GraFit software (GraFit Version 5, Erithacussoftware, Horley, UK).

In order to measure the recovery of BBI proteinsfrom ileal samples, an experimental model systembased on commercially available soybean BBI andileal samples from pigs fed hydrolysed casein-baseddiet was developed. Different amounts of exogenoussoybean BBI (80–400 µg) were incubated in 1 mL ofileal samples (100 mg in 1 mL of 50 mmol L−1 HCl)from pigs fed the hydrolysed casein-based diet. Themixture was incubated at 4 ◦C for 2 h and centrifugedat 12 000 × g for 10 min at room temperature.Supernatants were heated at 80 ◦C for 10 min andtheir inhibitory activities (TIA and CIA) compared

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with those exogenously added to ileal samples. Suchexperimental conditions were subsequently used fordetermining the protease inhibitory activities of ilealsamples from pigs fed the chickpea-based diet.

Ileal apparent digestibilityThe ileal apparent digestibility (%) of BBI pro-teins, based on TIA and CIA, was calcu-lated according to the expression: [(IAf/Cr2O3f ) −(IAi/Cr2O3i)]/(IAf/Cr2O3f ), where IAf and IAi repre-sent TIA or CIA (units per 100 mg of ileal sample) infeed and ileal samples, respectively, and Cr2O3f andCr2O3i are chromium oxide concentrations (g 100 g−1)in feed and ileal samples.

Cation exchange chromatography andSDS-PAGE analyses of ileal samplesFreeze-dried ileal samples (50 mg) from pigs fedchickpea or hydrolysed casein-based diets wereextracted under acidic conditions (50 mmol L−1

sodium citrate, pH 2.4) at 4 ◦C for 1 h and the extractscentrifuged at 12 000 × g for 10 min. Supernatantswere heat-treated at 80 ◦C for 10 min and centrifugedas above. Protein extracts were fractionated ona MonoS 5/50 GL cation exchange column (GEHealthcare, Uppsala, Sweden), connected to anAKTA FPLC system (GE Healthcare), using a lineargradient of 0–0.5 mol L−1 NaCl in 50 mmol L−1

sodium citrate buffer, pH 2.4, at a flow rate of 0.25 mLmin−1. The elution was monitored at 280 nm and0.5 mL fractions were collected. TIA measurementswere carried out in flat-bottom microtitre platesand scanned at 405 nm;24 CIA was monitoredas described above. Column fractions containinginhibitory activity were pooled. For SDS-PAGE,300 µL of pooled fractions containing inhibitoryactivity were precipitated with 1200 µL of cold acetoneand resuspended in NuPAGE LDS sample buffer(Invitrogen, Paisley, UK). Samples were analysed on4–12% Bis-Tris precast gels using NuPAGE MES asrunning buffer (Invitrogen). Prior to loading, proteinsamples were reduced and alkylated with DTT andiodoacetamide, respectively.25 Proteins were visualisedby Colloidal Blue staining (Invitrogen).

Peptide mass fingerprintingElectrophoretic bands corresponding to pooled chro-matographic fractions containing inhibitory activitywere excised from Colloidal Blue-stained gels andsubjected to trypsin digestion. Peptide fragments fromdigested proteins were desalted and concentratedby using C-18 ZipTip columns (Millipore, Madrid,Spain) and directly loaded onto the MALDI plate,using α-cyano hydroxycinnamic acid as the matrixfor MALDI-MS analysis. MS spectra were obtainedautomatically in a 4700 Proteomics Analyser (AppliedBiosystems, Cheshire, UK) operating in reflectronmode with delayed extraction. Peptide mass data weresearched for protein identification against the MSDBsequence database. Additionally, a further attempt to

identify the peptide mass data against the theoreticalmass of peptides derived from an N-terminal sequenceof a major BBI from chickpea seeds,26 not included indatabases, was carried out.

Statistical analysisThe data are presented as means ± SD. Statisticalevaluation of data was performed by one-way analysisof variance (ANOVA) and the comparison of meanvalues between groups was carried out by Tukey’sHSD test (P < 0.05), using the Minitab statisticalsoftware (State College, PA).

RESULTSDietsSeeds from the chickpea cultivar (cv.) Athenas wereused as a protein source to feed cannulated pigsin order to evaluate the survival of functional BBIproteins at the terminal ileum. Seeds from thecv. Athenas showed relatively high specific TIA(11.81 ± 0.66 TIU mg−1 of dry weight) and CIA(12.88 ± 0.67 CIU mg−1 of dry weight) in comparisonwith other chickpea genotypes previously screenedfrom the Center for Plant Genetic Resources (CRF-INIA, Spain) (data not shown). In agreement with thepercentage of chickpea meal used in the preparationof the feed (73%), the chickpea-based diet showedspecific TIA and CIA of 8.63 ± 1.20 TIU mg−1 ofdry weight and 8.86 ± 0.99 CIU mg−1 of dry weight,respectively. A diet containing hydrolysed casein wasused as a control due to its lack of measurableTIA and CIA. Neither of the diets showed trypsin-like or chymotrypsin-like protease activity underthe experimental conditions used in this study (notshown).

Proteolytic activities of ileal samplesIn order to evaluate trypsin- and chymotrypsin-likeactivities of ileal samples from pigs fed chickpea-and hydrolysed casein-based diets, buffered (50 mmolL−1 Tris, pH 8.0, 0.5 mol L−1 NaCl) and acidic(50 mmol L−1 HCl) extraction conditions were tested;statistically significant (P < 0.05) higher levels oftrypsin- and chymotrypsin-like activity were foundin ileal samples extracted under buffered conditions(Fig. 1). The relative levels of proteolytic activitiesobtained under buffered conditions, in comparisonwith those found under acidic conditions, were likelyto be significantly affected by the presence (chickpea-based diet) or absence (hydrolysed casein-based diet)of BBI proteins. Accordingly, the ability of soybeanBBI to inhibit trypsin- and chymotrypsin-like activitiesin buffered extracts of ileal samples from pigs fedhydrolysed casein-based diets was investigated. Adose-dependent inhibition of exogenous soybean BBIagainst trypsin-like activity of ileal samples is shownin Fig. 2. Trypsin-like activity of ileal samples wascompletely inhibited by exogenous soybean BBI; 79 ngof soybean BBI was sufficient to achieve a 50%



reduction in activity of the trypsin-like activity in 1 mgdry weight of ileal sample. In contrast, inhibition ofchymotrypsin-like activity did not exceed a 48% of thetotal activity, even when the amount of soybean BBIadded was in vast excess of that required to completelyinhibit trypsin-like activity of ileal samples (Fig. 2).These results support the idea that, under bufferedconditions, BBI proteins are more likely to take part ofcomplexes with digestive proteases; protease inhibitoryactivities of BBI proteins could be responsible for thelower levels of trypsin- and chymotrypsin-like activitiesobserved in ileal samples from pigs fed chickpea-based diets when compared to those levels from pigsfed hydrolysed casein-based diets (Fig. 1). Althoughonly partial inhibition of chymotrypsin-like activityof ileal samples from pigs fed hydrolysed casein-based diets was found, it was further noted thatsuch activity was completely inhibited by PMSF whenadded to the reaction buffer; 920 ng of PMSF wassufficient to inhibit 50% of the total chymotrypsin-likeactivity in 1 mg of ileal sample. These data suggestthat, under our experimental conditions, most of thechymotrypsin-like activity from ileal samples (∼50%)derives from a serine protease that share some commoncharacteristics with chymotrypsin but is not inhibitedby soybean BBI.

The presence of endogenous trypsin- and chymo-trypsin-like activities in ileal samples can mask theinhibitory activity of BBI proteins against the exoge-nous bovine trypsin and chymotypsin, commonly usedin inhibition assays, impairing their enzymatic evalu-ation. Therefore, thermal treatment was exploited toinactivate free enzymes present in the acid-solublefraction of ileal samples. Preliminary assays demon-strated the resistance to thermal inactivation (10 minat 80 ◦C) of pure soybean BBI in solution, with TIAand CIA totally unaffected (data not shown). The

Figure 1. Trypsin (T) and chymotrypsin activities (C) of ileal samples,extracted under acidic (50 mmol L−1 HCl) and buffered (50 mmol L−1

Tris, pH 8.0, 0.5 mol L−1 NaCl) conditions, from pigs fed chickpea-and hydrolysed casein-based diets. Values are means of five and twoanimals fed chickpea- and hydrolysed casein-based diets,respectively. Bars represent standard deviations (n = 10).

Figure 2. Inhibition curves of trypsin- and chymotrypsin-like activityof ileal samples from pigs fed hydrolysed casein-based diets by puresoybean BBI. Protease activities were determined for extractsobtained from 1 mg of ileal sample extracted under bufferedconditions (50 mmol L−1 Tris, pH 8.0, 0.5 mol L−1 NaCl).

effect of heat treatment on trypsin- and chymotrypsin-like activity of the acid-soluble fraction of ileal samplesfrom pigs fed chickpea-based diets is shown in Fig. 3.Under acidic conditions, significant differences in thethermal resistance of the two enzymatic activities wereobserved, with trypsin-like activity being less suscepti-ble to inactivation at high temperature when comparedwith chymotrypsin-like activity. A decrease of morethan 50% of trypsin-like activity was observed whenan acidic extract was heated at 70 ◦C for 10 min; suchtreatment reduced the chymotrypsin-like activity ofileal samples to less than 10% of the initial inhibitoryactivity. Heat treatment at 80 ◦C for 10 min reducedmore than 90% of the trypsin-like activity in ileal sam-ples, with chymotrypsin-like activity almost completelyabolished; such a reduction in protease activities wasconsidered sufficient for further evaluation of proteaseinhibitor activities of ileal samples.

Survival rates of functional BBI proteins at theterminal ileum in cannulated pigsPrior to investigating the survival rates of activeBBI proteins at the terminal ileum of cannulatedpigs fed chickpea-based diets, an experimental model

Figure 3. Effect of heat treatment on trypsin- and chymotrypsin-likeactivities of ileal samples, extracted under acidic conditions (50 mmolL−1 HCl), from pigs fed chickpea-based diets. Values are means offive animals; bars represent standard deviations (n = 10).


A Clemente et al.

Figure 4. Recovery rates of soybean BBI exogenously added to ilealsamples of pigs fed hydrolysed casein-based diets. BBI amounts inthe range of 80–400 µg were added to 100 mg of ileal sample underacidic conditions (50 mmol L−1 HCl). Values are means of threeindependent determinations (n = 6); bars represent standarddeviations.

system based on exogenous soybean BBI and ilealsamples from pigs fed hydrolysed casein-based dietwas developed. Using this experimental model, weevaluated the recovery of functional BBI exogenouslyadded to ileal samples (80–400 µg of inhibitor per100 mg of ileal sample from hydrolysed casein-fedpigs). Samples were subjected to acidic extraction(50 mmol L−1 HCl, 2 h at 4 ◦C) and further heat-treatment (80 ◦C for 10 min). The values determinedfor soybean BBI activity were in the range of 53–71%and 27–33% of that added, when based on TIAand CIA, respectively (Fig. 4). The relatively lowrecovery rates of soybean BBI obtained are likelyto be due to the strong protein–protein interaction

Table 2. Trypsin (TIA) and chymotrypsin inhibitor activity (CIA) in the

diet and in ileal contents (units 100 mg−1) and ileal apparent

digestibility of activity (% of activity in the diet) in pigs fed

chickpea-based dietsa

TIA CIA

Diet 863 ± 120 886 ± 99Ileal contents 222 ± 7 120 ± 13Apparent digestibility 92.7 ± 0.2 95.6 ± 1.3

a Data are the mean ± SD of ileal samples from five animals.Measurements of inhibitory activities of ileal samples were carriedout on the acid-soluble fraction after heat treatment 10 min at 80 ◦C.For details of the determination of trypsin and chymotrypsin inhibitoractivity, see Materials and Methods.

between digestive proteases and BBI proteins, evenwhen harsh conditions were used.13 Under theseexperimental conditions, the survival rates determinedfor BBI proteins from chickpea-based diets at theterminal ileum of cannulated pigs, expressed in termsof TIA and CIA, were 7.3 and 4.4%, respectively(Table 2). Differences found in survival rates of BBIproteins, based on inhibitory activities (TIA and CIA),are likely to be linked to the variation in recovery ratesof BBI proteins (Fig. 4); such variation might be due todifferences in the affinity of BBI proteins against targetproteases from ileal samples. Further studies will benecessary to characterise protein–protein interactionsbetween BBI proteins and trypsin- and chymotrypsin-like proteases from ileal samples. In order toconfirm the presence of active BBI proteins in ilealsamples from pigs fed chickpea-based diets, samplefractionation by cation exchange chromatography andfurther enzymatic and electrophoretic analyses wascarried out. Figure 5(A) shows the elution profile

(A)

(C)

(B)

Figure 5. (A) Elution profile of proteins of ileal samples from pigs fed chickpea-based diets, on a MonoS 5/50 GL cation exchange column.Absorbance at 280 nm and the linear gradient of NaCl (0–0.5 mol L−1) are shown (solid and dotted lines, respectively). Using BAPNA and BTEE asspecific substrates, the trypsin (�) and chymotrypsin (�) inhibitory activity, measured on every single fraction is shown; (B) SDS-PAGE of totalextract (lane 1) and partially purified BBI (lane 2) from ileal samples of pigs fed chickpea-based diets. Molecular weight markers are in lane 3;(C) Identification by mass peptide fingerprinting of chickpea BBI from ileal samples of cannulated pigs fed chickpea-based diets. An experimentalpeptide sequence (in bold) was identified when its peptide mass (857.4 Da) was matched up to a previously reported N-terminal sequence of amajor BBI isoform from chickpea seeds.26



of ileal proteins from cannulated pigs fed chickpea-based diets on a cation exchange MonoS column atpH 4.4. Ileal proteins showing TIA were bound andeluted in a single peak in the range 60–130 mmol L−1

NaCl; further enzymatic studies demonstrated thatprotein fractions containing TIA were also able toinhibit bovine chymotrypsin. Protein fractions havingonly TIA or CIA were not detected. Protein fractionscontaining protease inhibitory activity were pooledand analysed by SDS-PAGE following reduction andalkylation of cysteine residues (see section ‘Cationexchange chromatography’). An electrophoretic bandof approximately 7 kDa, which was absent in ilealsamples from pigs fed hydrolysed casein-based diets(not shown), was detected by Colloidal Blue staining(Fig. 5B). These data suggest that only BBI proteinshaving both active inhibitory domains were present inileal samples.

In order to verify the identity of BBI in ileal samplesfrom cannulated pigs fed chickpea-based diets, in-geltrypsin digestion of an isolated electrophoretic band(∼7 kDa) followed by separation of generated peptidesand further mass spectrometric-based analysis was car-ried out. The search of peptide mass data for proteinidentification against the MSDB sequence databasewas unsuccessful; however, the experimental peptidemass value of 857.4 Da was in perfect agreement withthe theoretical mass computed for the peptide SIP-PQCR derived from an available N-terminal sequence(ACCDSCVCTKSIPPQCRCNDM), not included indatabases, of a major BBI isoform from chickpeaseeds26 (Fig. 5C).

DISCUSSIONIn this work, we have demonstrated that significantamounts of BBI proteins from chickpea-based dietsare able to reach the terminal ileum of cannulatedpigs in active form. The survival rates of functionalBBI proteins were 7.3 and 4.4%, in terms of TIAand CIA, respectively (Table 2). These values arelikely to be under-represented due to the fact thatrelatively low recovery values were obtained whensoybean BBI was exogenously added to ileal samplesfrom pigs fed hydrolysed casein-based diets (Fig. 4).BBI proteins had been previously reported to behighly resistant to both the acidic conditions andthe action of proteolytic enzymes of the upper GITin vitro.12 In vivo studies have recently demonstratedthe presence of BBI proteins in the small intestine.Using an ELISA method, Hajos et al.18 reported thesurvival (4.8% of the total ingested) of soybean BBI inimmunologically intact form in the stomach and thesmall intestine of rats. Similar survival rates were foundfor cowpea (Vigna unguiculata) BBI-like proteins(CpTI) in rat feeding trials.27 It was suggested by theseauthors that some of the protease inhibitors couldmaintain their biological activities during passagethrough the GIT; however, no quantitative data forthe functional properties of BBI proteins present

in the GIT were reported. In this study we haveclearly demonstrated the resistance of functional BBIproteins from chickpea seeds to the harsh conditionsof GIT in vivo. Chromatographic, electrophoretic andfunctional data obtained from ileal samples suggestthat most of the BBI activity is derived from aprotein core containing both trypsin and chymotrypsininhibitory domains. Although processing at both N-and C-terminal ends of BBI proteins can occur,the presence of disulfide bridges seems to exert aprotective effect, precluding further protein hydrolysis.The rigid structure of BBI proteins consisting of a well-conserved skeleton of cysteine residues, which formseven disulfide bridges, is thought to play a major rolein maintaining the structural stability and inhibitoryactivity of this protein class.13,28 The presence ofdisulfide bonds might prevent structural changesinduced by harsh conditions (i.e. heat treatment,acidic conditions or the action of proteolytic enzymes),making it possible that BBI proteins reach the terminalsmall intestinal sections in intact form.17

There are few quantitative data for the gastroin-testinal survival of BBI proteins presumably due tothe complexity of the biological samples and limita-tions in methodology. Although immunoassays offerthe specificity and sensitivity necessary to recogniseBBI proteins in complex samples,18,29 they havesome limitations in comparison with the enzymaticmethods. Antibody-based enzyme-linked immunoab-sorbent assays (ELISA) can be misleading, particularlyin assays of samples from the GIT where some proteindigestion is expected, and immunoreactive BBI formsmay be detected that are not functional.18,19 In addi-tion, unusual patterns of temperature-dependent bind-ing displayed by monoclonal antibodies towards soy-bean BBI have been reported; even mild thermal treat-ment of samples seems to significantly affect the affinityof soybean BBI and specific antibodies.30 In the caseof enzymatic inhibition, only functional BBI proteinswith the ability to form complexes with digestive pro-teases, trypsin and/or chymotrypsin, can be evaluated.In this study, we have reported that soybean BBI iscapable of inhibiting, to different extents, trypsin- andchymotrypsin-like activities of ileal samples from can-nulated pigs (Fig. 2). The presence of proteolytic activ-ities at high levels and the fact that substantial amountsof the BBI proteins are likely to be complexed withdigestive enzymes makes the detection of BBI proteinsby enzymatic assays difficult. In order to determine theinhibitory activities of ileal samples, it was necessaryto establish suitable extraction conditions to maximisethe amounts of unbound BBI and, subsequently, toinactivate free digestive enzymes by heat treatment(Fig. 3), without affecting the inhibitory activity ofBBI proteins. Acidic extraction conditions prior toheat treatment (10 min at 80 ◦C) was established asnear-optimal conditions to evaluate the highest lev-els of active BBI proteins in ileal samples from pigsfed chickpea-based diets. Even under such conditions,substantial losses of BBI can occur, as shown by the


A Clemente et al.

recovery values obtained from exogenous soybean BBIincubated with ileal samples from pigs fed hydrolysedcasein-based diets (Fig. 4). As soybean BBI demon-strated substantial resistance to acidic conditions andheat treatment, these losses may be attributed mainlyto the ability of these proteins to take part in tight-binding complexes with digestive proteases.

Colon cancer is a complex disease that combinesboth lifestyle factors and multi-step genetic alterations.It has become one of the major causes of morbidity andmortality in western countries, being the second mostcommon cause of death from cancer within the EU,which makes the digestive tract an obvious strategictarget for health research. One of the most effectivemeans of preventing or reducing colon cancer riskseems to be through appropriate diet and/or nutritionalmanipulation. In this paper, we have demonstratedthat naturally occurring protease inhibitors of theBBI class reach the colon in active form. The anti-carcinogenic properties of BBI proteins have beenspecifically attributed to the presence of an activechymotrypsin inhibitory domain.2,3 Because of theapparent association of the chymotrypsin inhibitorybinding site with anti-carcinogenic properties, it hasbeen hypothesised that chymotrypsin-like proteasesare likely to be involved in carcinogenesis.31 Therefore,it is predicted that survival of significant amounts ofBBI proteins that are active against chymotrypsin-likeproteases at the terminal ileum could exert a cancerchemo-preventive role in the colon, a main targetin cancer research programmes. In agreement withthis, in vivo studies have demonstrated that soybeanBBI can prevent or suppress cancer developmentin many different animal models, including DMH-induced colon and anal gland tumours in mice.15,16

Soybean BBI has been reported to be effective whenused at concentrations as low as 10 mg 100 g−1

of diet, using the dimethylhydrazine (DMH) ratmodel, in reducing the incidence or frequency ofcolorectal tumours when compared with animalstreated with DMH alone.17 BBI-like proteins frompeas at concentrations as low as 20 µmol L−1 wereshown to have a significant suppressive effect onthe growth of human colon adenocarcinoma HT29cells in vitro.3 It has also been shown that colonicepithelial cells in culture internalise soybean BBI in atime-dependent manner.32 Strikingly, the internalisedinhibitor was able to bind to a chymotrypsin affinitycolumn, indicating that the inhibitor still possessedprotease inhibitor activity.

In summary, we have reported that significantamounts of BBI proteins are functionally resistantto gastrointestinal digestion in the pig, which is gener-ally held to be a suitable model for human diges-tive physiology.33 The presence of functional BBIproteins at the terminal ileum together with theirwell-recognised ability in preventing or suppressingcarcinogen-induced transformation in vitro and car-cinogenesis in a wide variety of in vivo model systemsoffers novel opportunities for therapeutic intervention.

Pharmacological and pre-clinical studies are necessaryin order for these proteins to be exploited fully inhuman health-promotion programmes. Such studieswill provide important knowledge and tools for thera-peutic or preventive medicine.

ACKNOWLEDGEMENTThis work was supported by grants AGL2004-03260/ALI and AGR2006-00706 from the SpanishCICYT and Junta de Andalucıa, respectively. A.C.acknowledges receipt of a Ramon and Cajal contract.We are grateful to Dr C. Domoney (John InnesCentre, UK) for her constructive comments on thispaper. We are very grateful to Dr Samuel Ogueta fromthe Proteomics Facility of University of Cordoba forcarrying out peptide mass fingerprinting.

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active bowman–birk inhibitors survive gastrointestinal digestion at the terminal ileum of pigs fed...

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