Food Chemistry xxx (2011) xxx–xxx

Contents lists available at SciVerse ScienceDirect

Food Chemistry

journal homepage: www.elsevier .com/locate / foodchem

Effect of instant controlled pressure drop on the oligosaccharides, inositolphosphates, trypsin inhibitors and lectins contents of different legumes

Mercedes M. Pedrosa a, Carmen Cuadrado a, Carmen Burbano a, Karim Allaf b, Joseph Haddad b,Eva Gelencsér c, Krisztina Takács c, Eva Guillamón d, Mercedes Muzquiz a,⇑a Departamento de Tecnología de Alimentos, SGIT-INIA, Ctra Coruña Km 7.5, 28040 Madrid, Spainb Laboratoire Maîtrise des Technologies Agro-Industrielles (LMTAI), Université de La Rochelle, Avenue Michel Crepeau, 17049 La Rochelle cedex 01, Francec Department of Food Safety, Central Food Research Institute (CFRI), Herman Ottó út 15, H-1022 Budapest, Hungaryd Centro para la Calidad de los Alimentos, INIA, José Tudela prox. 20, Campus Duques de Soria, 42004 Soria, Spain

a r t i c l e i n f o a b s t r a c t

Article history:Received 28 January 2011Received in revised form 13 July 2011Accepted 19 September 2011Available online xxxx

Keywords:Thermal processingDICNutritionally active factorsElectrophoretic analysisHPLCLegumes

0308-8146/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.foodchem.2011.09.061

⇑ Corresponding author.E-mail address: muzquiz@inia.es (M. Muzquiz).

Please cite this article in press as: Pedrosa, M.inhibitors and lectins contents of different legu

Well-controlled technologies for seed treatment have become a necessity for the food industry. Instantcontrolled pressure drop treatment (DIC�) is a new and highly controlled process that combines steampressure (up to 8 bar) with heat (up to 170 �C) for a short time (up to 3 min). The end-product is a wholeseed with a porous texture. The aim of this work was to evaluate the effect of this new (DIC) process onthe contents of nutritionally active factors (NAFs) in soybean, lupin, lentil, chickpea and roasted peanut.Unprocessed (control) and processed (DIC treatment under different pressure and time conditions)ground samples were analysed for oligosaccharides, inositol phosphates, trypsin inhibitors and lectins.The effect of DIC treatment on NAFs in legume seeds has shown that this process considerably reducesmost of these components; the optimum condition for DIC treatment in all the seeds was DIC-3 (6 bar,1 min). The main advantages of DIC are its short processing time and the possibility of treating wholeseeds for industrial applications.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Legumes and oilseeds are of great global importance due totheir significance in human and animal nutrition. The presence ofboth protein and starch in adequate proportions, along with fibre,vitamins and microelements, has made the legumes a focus of jus-tified nutritional interest (Leterme, 2002). However, their use is of-ten limited by the presence of a series of compounds, generallyknown as antinutritional factors or nutritionally active factors(NAFs), that impede the digestion and absorption of some of theirmost interesting components (e.g. proteins, vitamins), or, in somecases, they are simply toxic (alkaloids) or cause undesirable phys-iological side effects (e.g. flatulence) (Muzquiz & Wood, 2007). Onthe other hand, recent research has shown potential beneficial ef-fects of some of these compounds, e.g. oligosaccharides, inositolphosphates, protease inhibitors, lectins (Gelencsér, 2009; Lajolo,Genovese, Pryme, & Dale, 2004).

Although early studies on NAFs were rather simplistic andmechanistic in nature, they have still provided useful qualitative,and sometimes quantitative indications about these harmful

ll rights reserved.

M., et al. Effect of instant contmes. Food Chemistry (2011), do

effects. Several methods have been developed and tested innutritional practise for the reduction, or possible elimination, ofthe negative effects of antinutrients. Heating is the most com-monly used treatment for the elimination of NAFs although otherprocesses are also used, such as germination and fermentation(Cuadrado et al., 1996; Sanchez et al., 2005). High temperatureheating denatures almost all proteins, for this reason, the mostconvenient and frequently used methods for the elimination ofthe harmful effects of protein antinutritional factors are based onvarious forms of heat treatment (microwave, boiling, extrusion-cooking, autoclaving). Severe heating can limit protein digestionand amino acid availability. Previous studies have demonstratedthat lupin allergenic proteins are relatively heat-stable, and a com-bination of heat and pressure is required to eliminate their aller-genic potency (Frias, Vidal-Valverde, Sotomayor, Diaz Pollan, &Urbano, 2000; Guillamón et al., 2008a; Shimelis & Rakshit, 2007).

Therefore a new, simple and well-controlled technology forseed treatment has become a necessity for the food industry.Instant controlled pressure drop (DIC�; patent F2708419, 1995)treatment is a new and highly controlled process used in foodtechnology that combines steam pressure (up to 8 bar) with heat(up to 170 �C) for a short time (up to 3 min). DIC treatment of someNAFs in lupin and soybean seeds considerably reduced thosecomponents without affecting the total protein or lipid contents

rolled pressure drop on the oligosaccharides, inositol phosphates, trypsini:10.1016/j.foodchem.2011.09.061

2 M.M. Pedrosa et al. / Food Chemistry xxx (2011) xxx–xxx

(Haddad & Allaf, 2007; Haddad, Greiner, & Allaf, 2007; Haddad,Muzquiz, & Allaf, 2006). To our knowledge little is known aboutthe effect of this treatment on the main NAFs of legume seeds forhuman consumption (e.g. lentils, chickpeas and peanuts).

This work is aimed to study the effect of different conditions ofpressure and time applied during DIC treatment of soybean, lupin,lentil, chickpea and roasted peanut seeds to reduce their NAFs

contents.

2. Materials and methods

2.1. Plant material

Lupin seeds (Lupinus albus var. Multolupa) were supplied fromCentro de Investigación Agraria Finca la Orden, Badajoz, Spain.Soybean (Glycine max var. Ostrumi) and chickpea seeds (Cicerarietinum var. Athenas) were obtained from the Instituto de Forma-ción e Investigación Agraria y Pesquera (IFAPA, Córdoba, Spain).Lentil seeds (Lens culinaris var. Magda) were provided by the Insti-tuto Técnico Agronómico Provincial (ITAP, Albacete, Spain) androasted peanuts (Arachis hypogaea var. Virginia) were purchasedfrom Aperitivos Medina (Madrid, Spain).

2.2. DIC treatment

DIC treatment was carried out as previously described (Guilla-món et al., 2008b). Briefly, the moistened product is placed in aprocessing chamber and exposed to steam pressure (up to 8 bar)at high temperature (up to 170 �C), over a relatively short time(few seconds to some minutes). This high-temperature-short timestage is followed by an instant pressure drop towards a vacuum atabout 50 mbar. This abrupt pressure drop, at a rate DP/Dt higherthan 5 bar s�1, simultaneously provokes an auto-vapourization ofpart of the water in the product, and an instantaneous coolingof the products, which stops thermal degradation. Whole-mealsof the selected legumes (lupin, soybean, lentil, chickpea, peanut)were treated with DIC at two pressures for two periods of time:DIC-1 (3 bar, 1 min), DIC-2 (3 bar, 3 min), DIC-3 (6 bar, 1 min)and DIC-4 (6 bar, 3 min).

2.3. Chemical analysis

2.3.1. GeneralControl and DIC processed seeds were ground to pass through a

1 mm sieve (Tecator, Cyclotec 1093), and the flours were defattedwith n-hexane (34 ml g�1 of flour) for 4 h, shaken and air-driedafter filtration and used for the determination of the followingcompounds:

2.3.2. Soluble sugarsThe concentration of soluble sugars in seed fractions was deter-

mined by HPLC, using a modification of the Muzquiz, Rey, Cuadra-do, and Fenwick (1992) method. A sample (0.1 g) washomogenised in aqueous ethanol (50% v/v, 5 ml) for 1 min usingan Ultraturrax homogenizer. The mixture was centrifuged for5 min at 12,100g. The supernatant was decanted and the procedurerepeated twice. The combined supernatants were passed throughSep-Pak C18 cartridges (500 mg, Waters, Milford, MA, USA) andthe column was washed with 3 ml of aqueous ethanol (50% v/v).The combined extracts and washings were collected and evapo-rated to dryness. The residue was redissolved in 1 ml of doubledeionized water, and centrifuged for 8 min at 12,100g. Beforeinjection samples were filtered through a 0.45 lm Milliporemembrane.

Please cite this article in press as: Pedrosa, M. M., et al. Effect of instant continhibitors and lectins contents of different legumes. Food Chemistry (2011), do

Samples were analysed in duplicate. Aliquots of 20 ll were in-jected into an HPLC system (Beckman System Gold Instrument,Los Angeles, CA, USA) equipped with a refractive index detector.A Spherisorb-5-NH2 column (250 � 4.6 mm i.d., Waters, Milford,MA, USA), equilibrated with acetonitrile/water 60:40 (v/v), wasused, with a flow rate of 1 ml min�1.

Individual sugars were quantified by comparison with externalstandards of pure sucrose, raffinose, ciceritol and stachyose, (Sig-ma, St. Louis, MO, USA). Verbascose was purified and kindly sup-plied by Dr. A. I. Piotrowicz-Cieslak (Olsztyn-Kortowo, Poland).Calibration curves were constructed for all standard sugar solu-tions. A linear response was evident in the range (0–5 mg ml�1),with a correlation coefficient of 0.99.

2.3.3. Inositol phosphatesIndividual inositol phosphates (IP3–IP6) were extracted accord-

ing to Burbano, Muzquiz, Osagie, Ayet, and Cuadrado (1995) withsome modifications and determined by the Lehrfeld method (Lehr-feld, 1994). A 0.5 g sample was extracted with 5 ml of 0.5 M HCl for1 min, using an Ultraturrax homogenizer. The extract (2.5 ml) wasdiluted with 25 ml of deionized water and placed onto a SAX col-umn (Varian, Lake, Forest, California, USA). The column waswashed with 2 ml of deionized water, and the inositol phosphateseluted with 2 ml of 2 M HCl. The eluate was evaporated to drynessand the residue dissolved in a buffer solution. The solution wascentrifuged at 12,100g for 6 min to remove any suspended materialprior to injection into the HPLC (Beckman System Gold Instrument,Los Angeles, CA, USA). The column consisted of a macroporouspolymer PRP-1 (150 � 4.1 mm i.d., 5 lm, Hamilton, Reno, Nevada,USA) which was maintained at 45 �C.

2.3.4. Trypsin inhibitor activitiesQuantitative trypsin inhibitor measurements were performed

and trypsin inhibitor units (TIU) were defined according to the as-say described by Welham and Domoney (2000). Trypsin inhibitorwas determined, using a-N-benzoyl-DL-arginine-p-nitroanilidehy-drochloride (BAPNA) as the trypsin substrate. Trypsin inhibitorunits (TIUmg flour�1) calculated from the absorbance read at410 nm against a reagent blank. One unit of TIU was defined asthat, which gave a reduction in A410nm of 0.01, relative to trypsincontrol reactions, using a 10 ml assay volume (Welham, O’Neill,Johnson, Wang, & Domoney, 1998). All assays were performed intriplicate.

For electrophoretic analysis on native gels, 1 g of flour fromeach sample was extracted in 10 ml of 50 mM HCl for 2 h with con-tinuous stirring. All steps were carried out at 4 �C. Following centri-fuging at 12,100g for 10 min, the residue was re-extracted with2 ml of 50 mM HCl and centrifuged again. Pooled supernatantswere dialysed against distiled water overnight. The supernatantswere then centrifuged at 12,100g for 20 min and freeze-dried.Samples were prepared in Tris–glycine native sample buffer (NO-VEX) and analysed on native 4–16% Zymogram gradient gels (NO-VEX Zymogram (blue Casein) 4–16%) using the buffers suppliedand a soybean trypsin inhibitor (BBI) as a standard (Type I-S, SigmaT9003). Following electrophoresis, the Zymogram gels were incu-bated in renaturing buffer with gentle agitation for 30 min at roomtemperature (Muzquiz et al., 2004).

The renaturing buffer was replaced with Zymogram developingbuffer and incubated for 30 min at room temperature with gentleagitation. The developing buffer was then replaced with a freshsolution of trypsin in the same buffer (20 mg trypsin per 100 ml)and incubated at 37 �C, with gentle agitation, for 90 min. The gelswere treated with 5% acetic acid and placed in distiled water. Areasof the gels that remained blue indicated where trypsin had beeninhibited (Guillamón et al., 2008b).

rolled pressure drop on the oligosaccharides, inositol phosphates, trypsini:10.1016/j.foodchem.2011.09.061

M.M. Pedrosa et al. / Food Chemistry xxx (2011) xxx–xxx 3

2.3.5. LectinsUnprocessed (control) and processed (DIC treated) ground sam-

ples were extracted with 0.1 M PBS (pH 7.4) according to the pro-cedure of Trugo et al. (1999). Haemagglutinating activity in the PBSextracts was estimated by a serial dilution procedure, using trypsintreated rat blood cells (Grant, 1991). The amount of material (mg)causing 50% agglutination of erythrocytes was defined as that,which contained 1 haemagglutinating unit (HU). For comparison,values were expressed as HU g�1 seed meal. The assays werereproducible to ± 1 dilution and the final values were the meanof four separate measurements. Phaseolus vulgaris cvs. Processorand Pinto were included in each assay as positive and negativecontrols, respectively. Pure lentil lectin (LCA), previously obtained(Cuadrado et al., 2002) and soybean lectin (SBA), from Sigma di-luted in PBS (0.01 M PBS, pH 7.4), were used as standards.

2.4. Statistical analysis

All analyses were done in duplicate. A one way ANOVA analysiswas applied to the obtained analytical data, as well as Duncan’smultiple range test, in order to establish the statistical significance(p < 0.05). A Statgraphics Plus 4.1 computer package was used forthis purpose.

3. Results and discussion

3.1. Soluble sugars

The results of the effect of DIC treatments on the soluble sugarscontent of legume seeds are presented in Table 1.

All control samples contained different amounts of sucrose, raf-finose, ciceritol and stachyose. Lupin and lentil also contained ver-bascose, but ciceritol was not detected in soybean. The highestconcentration of total a-galactosides was present in lupin seeds.The a-galactosides (raffinose, stachyose and verbascose) are wellknown as antinutritional factors for causing flatulence. Flatulenceoccurs because mammals (including humans) have no a-galactosi-dase present in their intestinal mucosa.

Table 1The effect DIC treatment on soluble sugars content (mg g�1d.m.) of different legume seed

Sample Sucrose Raffinose Cicerit

Lupin control 28.8 ± 1.20 6.35 ± 0.34 5.97 ±DIC-1 28.8 ± 1.83 6.95 ± 0.47 9.22 ±DIC-2 27.7 ± 0.78 7.08 ± 0.26 9.30 ±DIC-3 26.0 ± 0.70 6.98 ± 0.29 8.68 ±DIC-4 21.2 ± 0.57 5.88 ± 0.36 8.29 ±

Soybean control 40.8 ± 0.33 9.30 ± 0.18DIC-1 42.0 ± 0.30 9.75 ± 0.11DIC-2 40.6 ± 0.24 8.79 ± 0.12DIC-3 41.0 ± 1.07 9.30 ± 0.42DIC-4 42.6 ± 1.93 8.90 ± 0.34

Lentil control 10.4 ± 1.29 2.92 ± 0.39 12.8 ±DIC-1 13.1 ± 1.03 3.08 ± 0.94 15.1 ±DIC-2 12.6 ± 0.06 2.87 ± 0.10 16.2 ±DIC-3 13.0 ± 0.60 2.61 ± 0.16 15.3 ±DIC-4 11.0 ± 0.30 2.21 ± 0.04 16.8 ±

Chickpea control 21.5 ± 1.17 6.53 ± 0.87 38.3 ±DIC-1 26.1 ± 0.24 5.37 ± 0.20 42.8 ±DIC-2 25.4 ± 0.81 6.41 ± 0.17 42.2 ±DIC-3 23.8 ± 0.94 5.89 ± 0.56 42.1 ±DIC-4 23.7 ± 0.25 4.97 ± 0.10 39.2 ±

Roasted peanut control 77.6 ± 2.87 4.68 ± 0.31 0.70 ±DIC-1 75.1 ± 0.37 4.40 ± 0.05 0.67 ±DIC-2 80.6 ± 6.99 4.25 ± 0.30 0.71 ±DIC-3 80.1 ± 0.38 4.91 ± 0.04 0.73 ±DIC-4 73.1 ± 3.06 4.51 ± 0.20 0.89 ±

a–c Figures (means ± SE; n = 4) followed by different superscripts in the same column ar

Please cite this article in press as: Pedrosa, M. M., et al. Effect of instant continhibitors and lectins contents of different legumes. Food Chemistry (2011), do

Chickpea and roasted peanut showed a slight reduction in thea-galactosides content (15% and 7%, respectively) at DIC-4 (6 bar3 min). In general, DIC treatment increased the soluble sugar con-tent, probably due to a mechanical-structure modification (higherporosity, rupture of the cell walls), which increased the specificsurface area, improved the diffusion of solvent inside such seedsand increased the availability of these oligosaccharides. However,the different analysed sugars were not affected to the same extentby the treatment and there were differences between seeds: raffi-nose content at DIC-1 (3 bar, 1 min) showed a 5% increase in soy-bean although a 18% reduction was observed in chickpea. Theamount of stachyose decreased by 12% in lupin, but increased inlentil 18% at DIC-4 (6 bar, 3 min). The a-galactosides convey manybenefits, to humans and monogastric animals alike. They can act asprebiotics, increasing the colonic population of bifidobacteria,reducing both constipation and diarrhoea, stimulating the immunesystem and increasing resistance to infection (Lajolo et al., 2004).

Amor, Lamy, Andre, and Allaf (2008) studied the effect of DICtreatments on the extraction of ciceritol and stachyose in the caseof Tephrosia purpurea seeds and noticed that there was a significantand positive impact of the pressure, which means that, the higherthe steam pressure, the greater is the extraction yield.

Shimelis and Rakshit (2007) observed that a-galactoside con-tent was only partly affected by the processing methods, e.g.hydration, cooking, autoclaving and their combinations, due totheir heat-stable nature. This is in agreement with the present re-sults obtained by DIC treatment.

The increase of the availability of the soluble sugar observedafter DIC treatment could be an advantage for obtaining a-galacto-sides extracts which can be used as prebiotic ingredients in foodproducts of high added value, in accordance with Martinez-Villalu-enga, Frias, and Vidal-Valverde (2006) and Martinez-Villaluengaand Gomez (2007).

3.2. Inositol phosphates

The effects of different conditions of DIC treatment on theinositol phosphates content are presented in Table 2. Control

s.

ol Stachyose Verbascose Total a-galactosides

0.23 61.3 ± 0.93 11.7 ± 1.81 79.3 ± 1.40a

0.59 62.8 ± 0.22 12.8 ± 0.26 70.0 ± 1.92b

0.14 66.6 ± 1.10 12.1 ± 0.61 85.8 ± 1.47c

0.43 61.5 ± 0.41 19.7 ± 0.95 88.2 ± 1.63d

0.31 53.6 ± 1.39 10.5 ± 0.87 82.5 ± 0.81e

40.7 ± 0.89 50.0 ± 1.04a

43.0 ± 0.39 52.8 ± 0.37b,c

41.9 ± 0.86 51.5 ± 1.37a

42.2 ± 0.96 50.7 ± 0.97a,b

45.0 ± 2.10 53.9 ± 2.36c

0.96 22.6 ± 0.99 21.9 ± 0.77 47.5 ± 1.87a

0.22 25.4 ± 1.58 26.5 ± 1.18 55.1 ± 3.17b

1.01 25.7 ± 0.34 24.0 ± 0.51 52.6 ± 1.14c

0.49 25.8 ± 0.50 23.6 ± 0.91 52.0 ± 0.99b,c

0.62 26.6 ± 0.94 24.9 ± 0.72 53.7 ± 1.44b,c

1.84 22.9 ± 0.92 29.4 ± 2.30a

0.51 20.8 ± 0.33 26.2 ± 0.83b,c

0.71 20.9 ± 0.33 27.4 ± 1.72a,b

1.45 22.0 ± 0.63 27.9 ± 0.99a,b

0.31 19.8 ± 0.47 24.8 ± 0.85c

0.47 6.69 ± 0.09 11.4 ± 0.35a

0.02 6.31 ± 0.25 10.5 ± 0.34a,b

0.07 6.54 ± 0.80 10.8 ± 1.10a,b

0.03 5.98 ± 0.09 10.7 ± 0.28a,b

0.06 6.01 ± 0.15 10.9 ± 0.12c

e significantly different (p < 0.05) compared by Duncan test.

rolled pressure drop on the oligosaccharides, inositol phosphates, trypsini:10.1016/j.foodchem.2011.09.061

Table 2The effect of DIC treatment on inositol phosphates content (mg g�1 d.m.) of different legume seeds.

Sample IP3 IP4 IP5 IP6 IP total

Lupin control 0.52 ± 0.00 1.07 ± 0.05 7.54 ± 0.15 8.87 ± 0.12a

DIC-1 0.81 ± 0.02 5.10 ± 0.10 5.91 ± 0.09b

DIC-2 0.53 ± 0.02 1.07 ± 0.04 3.77 ± 0.04 5.24 ± 0.32c

DIC-3 0.56 ± 0.03 1.11 ± 0.02 3.67 ± 0.13 5.21 ± 0.20c

DIC-4 0.07 ± 0.04 0.18 ± 0.03 0.63 ± 0.12 0.87 ± 0.13d

Soybean control 0.49 ± 0.01 0.51 ± 0.02 1.53 ± 0.04 17.6 ± 0.08 20.1 ± 0.15a

DIC-1 0.45 ± 0.00 0.52 ± 0.01 1.76 ± 0.09 15.0 ± 0.42 17.8 ± 0.48b

DIC-2 0.44 ± 0.00 0.59 ± 0.02 2.48 ± 0.05 14.1 ± 0.05 17.6 ± 0.07b

DIC-3 0.44 ± 0.01 0.73 ± 0.05 2.78 ± 0.01 14.2 ± 0.13 18.2 ± 0.14c

DIC-4 0.43 ± 0.03 1.26 ± 0.01 3.64 ± 0.03 11.6 ± 0.09 16.9 ± 0.09d

Lentil control 0.62 ± 0.02 1.17 ± 0.06 2.81 ± 0.13 7.63 ± 0.04 12.2 ± 0.22a

DIC-1 0.58 ± 0.02 1.04 ± 0.06 2.03 ± 0.03 5.02 ± 0.05 8.68 ± 0.09b

DIC-2 0.59 ± 0.01 0.91 ± 0.07 2.05 ± 0.15 4.13 ± 0.05 7.68 ± 0.19c

DIC-3 0.65 ± 0.01 1.05 ± 0.02 2.11 ± 0.04 3.71 ± 0.06 7.53 ± 0.03c

DIC-4 0.64 ± 0.01 0.99 ± 0.04 1.91 ± 0.03 2.92 ± 0.05 6.46 ± 0.07d

Chickpea control 0.57 ± 0.02 1.44 ± 0.06 4.01 ± 0.02 6.02 ± 0.07a

DIC-1 0.49 ± 0.00 1.02 ± 0.15 3.76 ± 0.59 5.03 ± 0.73b

DIC-2 0.50 ± 0.04 1.00 ± 0.07 3.16 ± 0.11 4.41 ± 0.29c

DIC-3 0.54 ± 0.02 1.21 ± 0.02 3.58 ± 0.02 5.20 ± 0.28b

DIC-4 0.56 ± 0.03 1.11 ± 0.03 2.21 ± 0.05 3.89 ± 0.03c

Roasted peanut control 0.46 ± 0.01 0.96 ± 0.02 3.11 ± 0.03 15.6 ± 0.64 20.1 ± 0.64a

DIC-1 0.45 ± 0.01 0.99 ± 0.02 3.53 ± 0.04 14.0 ± 0.04 18.9 ± 0.06b

DIC-2 0.46 ± 0.01 1.12 ± 0.09 3.64 ± 0.09 12.6 ± 0.03 17.8 ± 0.18c

DIC-3 0.46 ± 0.01 1.23 ± 0.02 4.12 ± 0.03 12.0 ± 0.06 17.8 ± 0.05c

DIC-4 0.64 ± 0.01 2.04 ± 0.05 5.11 ± 0.14 10.3 ± 0.16 18.1 ± 0.23c

a–d Figures (means ± SE; n = 4) followed by different superscripts in the same column are significantly different (p < 0.05) compared by Duncan test.

4 M.M. Pedrosa et al. / Food Chemistry xxx (2011) xxx–xxx

samples contained mainly IP6 (phytic acid) and different amountsof IP3, IP4 and IP5, except that lupin and chickpea that did not con-tain IP3.

DIC treatment induced a reduction in inositol phosphates con-centration but did not affect all seeds to the same extent. AfterDIC-2 (3 bar, 3 min), total phytate content decreased by 41% in lu-pin, 13% in soybean, 37% in lentil, 27% in chickpea and 12% in pea-nut. Following DIC-4 treatment (6 bar for 3 min), the reduction washigher, 90% in lupin, 16% in soybean, 47% in lentil, 35% in chickpeaand only 10% in peanut. Under the harsh condition, soybean and

Table 3The effect of DIC treatment on trypsin inhibitor (TIU mg�1d.m.) and lectin content(HU (g mg�1 d.m.)) in control and treated legume seeds.

Sample Trypsin inhibitor Lectin

Soybean control 86.1 ± 0.65a 40.0 ± 0.00a

DIC-1 0.48 ± 0.01b 10.2 ± 0.00b

DIC-2 0.22 ± 0.02b 10.2 ± 0.00c

DIC-3 0.24 ± 0.01b 5.13 ± 0.00b

DIC-4 0.35 ± 0.03b 20.4 ± 0.00d

Lentil control 4.88 ± 0.14a 5000 ± 167a

DIC-1 0.16 ± 0.02b 2.56 ± 0.00b

DIC-2 0.20 ± 0.02b,c 2.56 ± 0.00b

DIC-3 0.25 ± 0.02b 2.56 ± 0.00b

DIC-4 0.20 ± 0.03b,c 7.67 ± 2.54b

Chickpea control 11.7 ± 0.62a n.d.DIC-1 0.24 ± 0.02b n.d.DIC-2 0.23 ± 0.01b n.d.DIC-3 0.20 ± 0.01b n.dDIC-4 0.30 ± 0.01b n.d.

Roasted peanut control 1.28 ± 0.03a 5.13 ± 0.00a

DIC-1 0.72 ± 0.01b 0.64 ± 0.00b

DIC-2 0.68 ± 0.03b 1.28 ± 0.00c

DIC-3 0.57 ± 0.01c 1.28 ± 0.00c

DIC-4 0.29 ± 0.01d 1.28 ± 0.00c

n.d. Not detected.a–d Figures (means ± SE; n = 4) followed by different superscripts in the samecolumn are significantly different (p < 0.05) compared by Duncan test.

Please cite this article in press as: Pedrosa, M. M., et al. Effect of instant continhibitors and lectins contents of different legumes. Food Chemistry (2011), do

roasted peanut, showed increases of the amounts of IP4 and IP5,probably due to the IP6 dephosphorylation; this is in agreementwith the transformation of phytic acid reported by other authors(Greiner, 2001; Vidal-Valverde, Frias, Lambein, & Kuo, 2001). How-ever, lupin, lentil and chickpea showed a reduction of all inositolphosphate forms. While IP6 is considered to be harmful (it bindswith other nutrients making them inaccessible to digestion), thelower forms have pharmaceutical and medical properties (Champ,2001; Greiner, 2001; Lajolo et al., 2004).

Haddad et al. (2007) obtained a 16% decrease of phytate contentin L. albus and 19% in Lupinus mutabilis after DIC treatment at 7 barfor 1 min and up to 55% (L. albus) and 60% (L. mutabilis) after DICtreatment for 7 min.

The results of this work are consistent with previous workswhich showed that heat treatment only partly affected the phyticacid content (Shimelis & Rakshit, 2007).

3.3. Trypsin inhibitors

Table 3 summarises the effect of different DIC treatments ontrypsin inhibitor amounts in the legume seeds. The data clearlyshow that trypsin inhibitor units in control samples ranged fromnegligible, as in lupin seeds (data not shown) and roasted peanut(1.3 TIU mg�1), to very high in soybean (86.1 TIU mg�1). Gallardo,Araya, Pak, and Tagle (1974) showed, as in the present study, a lackof trypsin inhibitors in lupin seeds. However, there are large differ-ences among the values of TIU reported by some authors in otherlegume seeds (Berger, Siddique, & Loss, 1999; Guillamón et al.,2008b).

After DIC treatment, the reduction ratio varied with the sam-ples. All DIC treatments almost abolished trypsin inhibitor activity(>99.5%) in soybean samples, and led to 96% reduction in lentil andchickpeas. The initial TIU content of roasted peanut was 76% re-duced for the treatment carried out at DIC-4 (6 bar, 3 min) havingvalues (around 0.30 TIU) similar to those of the other legumesunder the same conditions. The fall in trypsin inhibitor contentconfirms their heat-sensitive nature. Haddad and Allaf (2007)

rolled pressure drop on the oligosaccharides, inositol phosphates, trypsini:10.1016/j.foodchem.2011.09.061

Soybean Chickpea Peanut

1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6

Fig. 1. Zymograms of trypsin inhibitor isoform patterns of BBI standard (lane 1), control samples (lane 2), DIC-1 (lane 3), DIC-2 (lane 4), DIC-3 (lane 5) and DIC-4 (lane 6).

M.M. Pedrosa et al. / Food Chemistry xxx (2011) xxx–xxx 5

obtained a reduction in trypsin inhibitor content in soybean ofabout 94% with 7 bar pressure and a processing time which doesnot exceed 1 min. Several reports suggest that 70–87% reductionin trypsin inhibitor activity is satisfactory (Leontowicz, Kostyra,Leontowicz, & Kulasek, 1998). All the values obtained with DICtreatment, under any condition of pressure and time, were lowerthan the most restrictive values established as safe for human con-sumption (1–1.5 TIU mg�1) (Pipa, 1988). Protease inhibitors havebeen linked, over the past two decades, to health-promoting prop-erties. The natural bioactive substances Bowman–Birk inhibitors(BBI) have been shown to be effective in preventing or suppressingcarcinogen-induced effects (Clemente, Mackenzie, Johnson, &Domoney, 2004).

Although most compounds with protease inhibitor activity areheat-labile, it has been found that the thermostability of trypsininhibitors in legumes varies, not only with legume source, but alsowith the different conditions used during processing (pH, humid-ity, time temperature, pressure) (Frias et al., 2000; Urbano et al.,1995). Several authors have reported that cooking and autoclavingproduce larger trypsin inhibitor activity (TIA) reduction than doesdry heating (Griffiths, 1984; Khalil & Mansour, 1995). Borowska,Kozlowska, and Scheneider (1989) and Kozlowska, Borowska, For-nal, Scheneider, and Schamndke (1989) found total inactivation oftrypsin inhibitors isolated from faba bean seeds after autoclaving.Jourdan, Noreña, and Brandelli (2007) found that after 15 min ofautoclaving (121 �C at 1.4 bar), the inactivation of TIA was 100%,irrespective of the variety tested. It has been reported that theinactivation of trypsin inhibitor occurs mostly in the first minutesof thermal treatment.

The qualitative variation of trypsin inhibitors isoform patternsof soybean, lentil, chickpea and peanut, using Zymogram gels, isshown in Fig. 1. In relation to control samples, at least six isoformswere clearly detected in soybean, two in lentil and in chickpea andthree in peanut. Practically all inhibitor isoforms appeared in zonesof higher electronegativity than the BBI used as standard. After DICtreatment, no bands could be detected in soybean or lentil. The re-sults confirm the lack of activity in the DIC-treated seeds. However,chickpea and roasted peanut showed some isoforms after bothtreatments at 3 bar (DIC-1 and -2) but only peanut showed bandsunder the DIC-3 (6 bar, 1 min) condition. This is probably due tothe fact that chickpea and peanut isoforms are more resistant toDIC treatment than are other legume isoforms (Frias et al., 2000;Urbano et al., 1995).

3.4. Lectins

Table 3 shows the effect of DIC treatment on lectin content (HU(g mg�1 d.m.)) in control and treated legume seeds. Lupin andchickpea did not present haemagglutinating activity. Lentil control

Please cite this article in press as: Pedrosa, M. M., et al. Effect of instant continhibitors and lectins contents of different legumes. Food Chemistry (2011), do

showed the highest amount of lectin (500 HU mg�1) and roastedpeanut had 100-fold lower content than had lentil (5.13 HU mg�1).Lectins can be toxic, although, a small amount may be beneficial bystimulating gut function, limiting tumour growth and amelioratingobesity (Lam & Ng, 2010; Pusztai, Bardocz, & Martin-Cabrejas,2004).

There was a meaningful decrease in the haemagglutinatingactivity of all DIC-treated legumes in comparison with the controlones. A negligible value was found in lentil under all DIC condi-tions, while a significant value was obtained in soybean (from13% to 51% of residual activity) and roasted peanut (from 13% to25%). This may be due to the presence of different isolectins, asfor peanut agglutinin (PNA) (Pueppke, 1981), and/or because ofits variable thermostability in legumes as has been observed fortrypsin inhibitors.

Soybean showed a greater amount of lectin (SBA) at 6 bar, 3 min(DIC-4) in comparison with the other DIC conditions. According toMa and Wang (2010), after heat treatment alone (boiling and auto-claving), or followed by enzymatic hydrolysis, SBA still had 44–62%residual activity. Native SBA is a tetramer, each of whose subunitsare capable of sugar binding (Sharmistha & Avadhesha, 2005). Thisprotein shows a very high degree of stability during food process-ing, compared to the other lectins, due to a high degree of subunitinteractions (Ghosh & Mandal, 2001; Umezawa, Sato, Naganuma,Ogawa, & Muramoto, 1999).

PNA is a homotetrameric non-glycosylated legume lectin with avery unusual quaternary structure, described as an ‘open’ quater-nary structure. Earlier unfolding studies on PNA led to the discov-ery of a partially folded intermediate that had retained acarbohydrate binding property (Sagarika, Nirmala, Sharmistha, &Avadhesha, 2006). This would be related to the remaining haemag-glutinating activity, noticed even under the harsh conditionsassayed.

According to the present results, lectin activity of soybean, lentiland roasted peanut is clearly sensitive to heat treatment such asthat applied in the DIC treatment (170 �C). Leontowicz, Leontowicz,Kostyra, Gralak, and Kulasek (1999) concluded that extrusion at150 �C is adequate for elimination of lectin in pea and faba beanbut in the case of soybean lectin, 170 �C is required. Boiling for20 min was not effective to destroy SBA. However, Alonso, Rubio,Muzquiz, and Marzo (2001) showed that extrusion at 148 �C re-duced pea and kidney bean lectin activities by 98%.

4. Conclusion

The DIC treatment increases the availability of soluble sugars;the level of the most antinutritional phytate compound, IP6, de-creased as processing conditions became harsher; lectin contentwas strongly reduced and trypsin inhibitor activity was almost

rolled pressure drop on the oligosaccharides, inositol phosphates, trypsini:10.1016/j.foodchem.2011.09.061

6 M.M. Pedrosa et al. / Food Chemistry xxx (2011) xxx–xxx

abolished. Considering the processing conditions attempted in thepresent study and, since, PNA, SBA and lentil lectin showed a high-er amount of lectin at DIC-4, the DIC-3 treatment (6 bar, 1 min) ap-pears to be the most effective for reducing levels of all theinvestigated NAFs in all the seeds studied.

One of the major advantages of the DIC is its short processingtime. Treatments of a few minutes are sufficient with regard tothe safe limit of heat-labile compounds such as trypsin inhibitorsand lectins. The possibility of processing whole seeds is anotheradvantage of this technique for industrial applications. Therefore,the DIC treatment opens the way to new perspectives, especiallya more effective use of legumes as a plant protein source and withbetter industrial exploitation.

Acknowledgments

This work was financially supported by the Acción Integrada(HH2006-0039) from the Foreign Affairs Ministry.

References

Alonso, R., Rubio, L. A., Muzquiz, M., & Marzo, F. (2001). The effect of extrusion-cooking on mineral bioavailability in pea and kidney bean seed meals. AnimalFeed Science and Technology, 94, 1–13.

Amor, B. B., Lamy, C., Andre & Allaf, K. (2008). Effect of instant controlled pressuredrop treatments on the oligosaccharides extractability and microstructure ofTephrosia purpurea seeds. Journal of Chromatography A, 1213, 118–124.

Berger, J. D., Siddique, K. H. M., & Loss, S. P. (1999). Cool season grain legumes forMediterranean environments: species x environment interaction in seedsquality traits and anti-nutritional factors in the genus Vicia. Australian Journalof Agricultural Research, 50, 389–401.

Borowska, J., Kozlowska, H., & Scheneider, Ch. (1989). Preparation of faba bean(Vicia faba, L. minor) products. Part V. Effect of hydrothermal treatment of fababean and peas on the quality of protein isolates. Acta Alimentria Polonica, 15,171–177.

Burbano, C., Muzquiz, M., Osagie, A., Ayet, G., & Cuadrado, C. (1995). Determinationof phytate and lower inositol phosphates in Spanish legumes by HPLCmethodology. Food Chemistry, 52, 321–325.

Champ, M. (2001). Non-nutrient bioactives substances of pulses. British Journal ofNutrition, 88, S307–319.

Clemente, A., Mackenzie, D. A., Johnson, I. T., & Domoney, C. (2004). Investigation oflegume seed protease inhibitors as potential anticarcinogenic proteins. In M.Muzquiz, G. D. Hill, C. Cuadrado, M. M. Pedrosa, & C. Burbano (Eds.), Recentadvances of research in antinutritional factors in legume seeds and oilseeds(pp. 137–142). Toledo, Spain: EAAP publication.

Cuadrado, C., Ayet, G., Robredo, L. M., Tabera, J., Villa, R., Pedrosa, M. M., et al. (1996).Effect of natural fermentation on the content of inositol phosphates in lentils.Zeitschrift Lebensmittel Untersuchung und Forschung, 203, 268–271.

Cuadrado, C., Grant, G., Rubio, L. A., Muzquiz, M., Bardocz, S., & Pusztai, A. (2002).Nutritional utilization by the rat of diets based in lentil (Lens culinaris) seedmeal or its fractions. Journal of Agricultural and Food Chemistry, 50, 4371–4376.

Frias, J., Vidal-Valverde, C., Sotomayor, C., Diaz Pollan, C., & Urbano, G. (2000).Influence of processing on available carbohydrate content and antinutritionalfactors of chickpeas. European Food Research and Technology, 210, 340–345.

Gallardo, F., Araya, H., Pak, N., & Tagle, M. A. (1974). Factores tóxicos de leguminosascultivadas en Chile. II. Inhibidores de tripsina. Archivos Latinoamericanos deNutrición, 24, 183–189.

Gelencsér, É. (2009). Novel research strategy for safe use of legume proteins inhuman nutrition. Acta Alimentaria, 38 (Suppl.), pp. 61–70, doi: 10, 1556/AAlim.38. Suppl.4.

Ghosh, M., & Mandal, D. K. (2001). Analysis of equilibrium dissociation andunfolding in denaturants of soybean agglutinin and two of derivatives.International Journal of Biological Macromolecules, 29, 273–280.

Grant, G. (1991). Legumes. In J. P. F. D’Mello, C. M. Duffus, & J. H. Duffus (Eds.), Toxicsubstances in crop plants (pp. 49–67). Cambridge: Royal Society of Chemistry.

Greiner, R. (2001). Properties of phytate-degrading enzymes from germinatedlupine seeds (Lupinus albus var Amiga). In A. EP (Ed.), Towards the sustainableproduction of healthy food, feed and novel products (pp. 398–399). Poland:Cracow.

Griffiths, D. W. (1984). The trypsin and chymotrypsin inhibitor activities of variouspea (Pisum spp.) and field bean (Vicia faba) cultivars. Journal of the Science of Foodand Agriculture, 35, 481–486.

Guillamón, E., Burbano, C., Cuadrado, C., Muzquiz, M., Pedrosa, M. M., Sánchez, M.,et al. (2008a). Effect of instantaneous controlled pressure drop on in vitro lupinallergenicity to lupins (Lupinus albus var Multolupa). International Archives ofAllergy Immunology, 145, 9–14.

Guillamón, E., Pedrosa, M. M., Burbano, C., Cuadrado, C., Sánchez, M. C., & Muzquiz,M. (2008b). The trypsin inhibitors present in seed of different grain legumespecies and cultivar. Food Chemistry, 107, 68–74.

Please cite this article in press as: Pedrosa, M. M., et al. Effect of instant continhibitors and lectins contents of different legumes. Food Chemistry (2011), do

Haddad, J., Muzquiz, M., & Allaf, K. (2006). Treatment of lupin seed using theinstantaneous controlled pressure drop technology to reduce alkaloid content.Food Science and Technology International, 12, 365–370.

Haddad, J., & Allaf, K. (2007). A study of the impact of instantaneous controlledpressure drop on the trypsin inhibitors of soybean. Journal of Food Engineering,79, 353–357.

Haddad, J., Greiner, R., & Allaf, K. (2007). Effect of instantaneous controlled pressuredrop on the phytate content of lupin. Swiss Society of Food Science andTechnology, 40, 448–453.

Jourdan, G. A., Noreña, C. P. Z., & Brandelli, A. (2007). Inactivation of trypsin inhibitoractivity from brazilian varieties of beans (Phaseolus vulgaris L.). Food Science andTechnology International, 13, 195–198.

Khalil, H. A., & Mansour, E. H. (1995). The effect of cooking, autoclaving andgermination on the nutritional quality of faba beans. Food Chemistry, 54,177–182.

Kozlowska, H., Borowska, J., Fornal, J., Scheneider, Ch., & Schamndke, H. (1989).Preparation of faba bean (Vicia faba, L. minor) products. Part IV. Effect ofhydrothermal treatment of faba bean on the quality of flour. Acta AlimentriaPolonica, 15, 161–169.

Lajolo, F. M., Genovese, M. I., Pryme & Dale, T. M. (2004). Beneficial(antiproliferative) effects of different substances. In M. Muzquiz, G. D. Hill, C.Cuadrado, M. M. Pedrosa, & C. Burbano (Eds.), Recent advances of research inantinutritional factors in legume seeds and oilseeds (pp. 123–137). Toledo, Spain:EAAP publication.

Lam, S.K. & Ng, T.B. (2010). Lectins: production and practical applications. AppliedMicrobiology Biotechnology. doi: 10.1007/s00253-010-2892-9. (on line 3October 2010).

Lehrfeld, J. (1994). HPLC Separation and quantification of phytic acid and someinositol phosphates in foods: problems and solutions. Journal of Agricultural andFood Chemistry, 42, 2726–2731.

Leontowicz, H., Kostyra, H., Leontowicz, M., & Kulasek, G. W. (1998). Theinactivation of legume seeds haemmagglutinin and trypsin inhibitors byboiling. In A. J. M. Jasman, G. D. Hill, & A. F. B. Van der Poel (Eds.), Recentadvances of research in antinutritional factors in legume seeds and rapeseeds. (pp429–432). The Netherlands: Wageningen Press.

Leontowicz, H., Leontowicz, M., Kostyra, H., Gralak, M. A., & Kulasek, G. W. (1999).The influence of extrusion or boiling on trypsin inhibitor and lectin activity inleguminous seeds and protein digestibility in rats. Polish Journal of Food andNutrition Sciences, 8, 77–87.

Leterme, P. (2002). Recommendations by health organizations for pulseconsumption. British Journal of Nutrition, 88, S239–S242.

Ma, Y., & Wang, T. (2010). Deactivation of soybean agglutinin by enzymatic andother physical treatments. Journal of Agricultural Food Chemistry, 58,11413–11419.

Martinez-Villaluenga, C., Frias, J., & Vidal-Valverde, C. (2006). Lupinus albus L. andLupinus luteus L. after extraction of a-galactosides. Food Chemistry, 98, 291–299.

Martinez-Villaluenga, C., & Gomez, R. (2007). Characterization of bifidobacteria asstarters in fermented milk containing raffinose family of oligosaccharides fromlupin as prebiotic. International Dairy Journal, 17, 116–122.

Muzquiz, M., Rey, C., Cuadrado, C., & Fenwick, R. (1992). Effect of germination onoligosaccharide content of lupin species. Journal of Chromatogaphy, 607,349–352.

Muzquiz, M., Welham, T., Altares, P., Goyoaga, C., Cuadrado, C., Romero, R., et al.(2004). The presence of trypsin inhibitors in Vicia faba and Cicer arietinumduring germination. Journal of the Science of Food and Agriculture, 84, 556–560.

Muzquiz, M., & Wood, J. A. (2007). Antinutritional Factors. In S. S. Yadav, R. Redden,W. Chen, & B. Sharma (Eds.), Chickpea Breeding & Management (pp. 143–167).Trowbridge, UK: Cromwell Press.

Pipa, F. (1988). Traitement hydro-thermique des matieres premieres. In Les traitementshydro-thermiques des matieres premieres. TECALIMAN-Symposium 1988. (pp 59–69). France: Nantes.

Pueppke, S. G. (1981). Multiple molecular forms of peanut lectin: Classification ofisolectins and isolectin distribution among genotypes of the genus Arachis.Archives of Biochemistry and Biophysics, 212, 254–261.

Pusztai, A., Bardocz, S., & Martin-Cabrejas, M.A. (2004). The mode of action of ANFson the gastrointestinal tract and its microflora. In: M. Muzquiz, G.D. Hill, C.Cuadrado, M.M. Pedrosa, & C. Burbano (Eds.). Recent advances of research inantinutritional factors in legume seeds and oilseeds (pp 87–100). Toledo, Spain:EAAP publication.

Sagarika, D., Nirmala, D. K., Sharmistha, S., & Avadhesha, S. (2006). ThermodynamicAnalysis of Three State Denaturation of Peanut Agglutinin. Life, 58, 549–555.

Sanchez, M. C., Altares, P., Pedrosa, M. M., Burbano, C., Cuadrado, C., Goyoaga, C.,Muzquiz, M., Jimenez-Martinez, C., & Dávila-Ortiz, G. (2005). Alkaloid variationduring germination in different lupin species. Food Chemistry, 90, 347–355.

Sharmistha, S., & Avadhesha, S. (2005). Oligomerization Endows Enormous Stabilityto Soybean Agglutinin: A Comparison of the Stability of Monomer and Tetramerof Soybean Agglutinin. Biophysical Journal, 88, 4243–4251.

Shimelis, E. A., & Rakshit, S. K. (2007). Effect of processing on antinutrients andin vitro protein digestibility of kidney bean (Phaseolus vulgaris L.) varietiesgrown in East Africa. Food Chemistry, 103, 161–172.

Trugo, L., Muzquiz, M., Pedrosa, M. M., Ayet, G., Burbano, C., & Cuadrado, C. (1999).Influence of malting on the composition of non-nutrient components ofdifferent seeds. Food Chemistry, 65, 85–90.

Umezawa, Y., Sato, Y., Naganuma, T., Ogawa, T., & Muramoto, K. (1999). Stability ofSoybean Lectin during Food Processing and in Digestive Tract. Soy ProteinResearch, 2, 59–64.

rolled pressure drop on the oligosaccharides, inositol phosphates, trypsini:10.1016/j.foodchem.2011.09.061

M.M. Pedrosa et al. / Food Chemistry xxx (2011) xxx–xxx 7

Urbano, G., Lopez-Jurado, M., Hernandez, J., Moreu, M. C., Frias, J., Diaz-Pollan, C.,et al. (1995). Ca and P bioavailability of processed lentils as affected by dietaryfiber and phytic acid content. Nutrition Research, 19, 49–64.

Vidal-Valverde, C., Frias, J., Lambein, F., & Kuo, Y. H. (2001). Increasing thefunctionality of legumes by germination. In A. EP (Ed.), Towards the sustainableproduction of healthy food, feed and novel products. (pp 422). Poland: Cracow.

Please cite this article in press as: Pedrosa, M. M., et al. Effect of instant continhibitors and lectins contents of different legumes. Food Chemistry (2011), do

Welham, T., & Domoney, C. (2000). Temporal and spatial activity of a promoter froma pea enzyme inhibitor gene and its exploitation for seed quality improvement.Plant Science, 159, 289–299.

Welham, T., O’Neill, M., Johnson, S., Wang, T. L., & Domoney, C. (1998). Expressionpatterns of genes encoding seed trypsin inhibitors in Pisum sativum. PlantScience, 131, 13–24.

rolled pressure drop on the oligosaccharides, inositol phosphates, trypsini:10.1016/j.foodchem.2011.09.061