(Phaseolus Vulgaris L and Vicia Faba L) 7S and 11S Globulins in the Sm

Journal of the Science of Food and Agriculture J Sci Food Agric 85:65–72 (2005) DOI: 10.1002/jsfa.1940

Heat-induced denaturation impairs digestibility of legume (Phaseolus vulgaris LandVicia faba L) 7S and 11S globulins in the small intestine of rat M Carbonaro,1∗ GGrant2 and M Cappelloni1 1Istituto Nazionale di Ricerca per gli Alimenti e la Nutrizione, V Ardeatina 546, 00178 Roma, Italy 2Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen AB21 9SB, Scotland, UK

Abstract: 7S globulin from common bean (Phaseolus vulgaris L) and 11S globulin from faba bean (Vicia faba L) were isolated to over 90% purity and the digestibility of the proteins, either in native or denatured (120 ◦C, 20 min, 1 atm) state, was tested in the small intestine of growing rats in acute (1 h) experiments. Native globulins were well digested (92 and 95% for 7S and 11S proteins, respectively). However, after thermal denaturation, protein digestibility of 7S globulin was reduced to 88%, while that of 11S globulin to only 79%. SDS-PAGE revealed that high amounts of the intermediate proteolytic products of phaseolin (MW 22 000–27 000 Da) were present in the small intestine of rats after 1 h digestion of the denatured 7S globulin, while protein material in the high MW range (>55 000 Da) were recovered from the 11S globulin. The overall negative charge of unavailable proteins from the 7S globulin was found by anion exchange–FPLC separation to be higher than that of products from the 11S globulin. MALDI-MS analysis of proteins in the small intestine conﬁrmed the presence of half-size phaseolin subunits (MW 23 700 Da) as breakdown products from the denatured 7S globulin, and of highly hydrophobic basic subunits (MW 20 000 Da) from the 11S globulin.  2004 Society of Chemical Industry

Keywords: common bean; faba bean; 7S/11S globulins; small intestinal digestibility; thermal denaturation

ABBREVIATIONS for the structure and functional properties of 7S and FPLC fast performance liquid chromatography 11S protein families. However, much remains to be HMW high molecular weight known about the structural properties of concern for LMW low molecular weight protein quality so as to achieve the goal of improving MALDI-MS matrix-assisted laser desorption ioniza- nutritional potential through genetically modified tion mass spectrometry globulins. Indeed, little information is available so PBS phosphate-buffered saline far about molecular features affecting the protein SDS-PAGE sodium dodecyl sulfate–polyacrylamide digestibility behaviour in monogastric animals before gel electrophoresis and after processing of legumes. In vitro studies on digestibility of native storage proteins initially suggested they were resistant to INTRODUCTION proteolysis because of structural properties, such as Legume seed storage proteins, legumin-type 11S and compact structure and stability conferred by S–S vicilin-type 7S globulins, are oligomeric proteins con- bonds and by the carbohydrate moiety.4,5 Later, sisting of six subunit pairs (MW 300 000–400 000 Da) in vivo studies on isolated globulin preparations and three subunits (MW 140 000–200 000 Da), indicated they can in fact be readily digested in respectively, held together by non-covalent links.1 the small intestine.6–8 This led us to reconsider the Progress in the knowledge of protein properties generally accepted view that low biological value of at a molecular level, notably the acquirement of X- legume proteins is at least in part determined by their ray three-dimensional structure (2.2 A resolution) of poor intestinal digestibility. Phaseolus vulgaris L7Sprotein2 and of Glycine max As far as the effect of heating on digestibility of L 11S globulin3 has been achieved. This allowed a legume proteins is concerned, results are still scarce suggestion to be made for a unified canonical model and conflicting. While in vitro studies indicated that

∗ Correspondence to: M Carbonaro, Istituto Nazionale di Ricerca per gli Alimenti e la Nutrizione, V Ardeatina 546, 00178 Roma, Italy E-mail: [email protected] (Received 9 May 2003; revised version received 10 May 2004; accepted 15 July 2004) Published online 9 September 2004  2004 Society of Chemical Industry. J Sci Food Agric 0022–5142/2004/$30.00 65 M Carbonaro, G Grant, M Cappelloni isolated heated phaseolin could be digested readily EXPERIMENTAL by various proteases, improvement in digestibility of Purification of 7S and 11S globulins legume proteins in whole seed upon thermal treatment Commercial Italian varieties of white common bean was not always observed.9 Increasing evidence from (Phaseolus vulgaris L) and faba bean (Vicia faba L) were in vivo studies suggests that the digestibility of heated purchased from a local supermarket (Rome, Italy). legume proteins is quite low, even if inactivation or Raw seeds were ground in a Cyclotec 1093 Tecator removal of most of the antinutritional compounds (50 µm). is recognized to occur during processing of legume 7S globulin (vicilin) was purified from raw common seeds.10– 12 bean seed flour according to Pusztai and Stewart24 Legume globulins undergo heat-induced dissoci- and 11S globulin (legumin) was purified from raw ation of subunits with formation of novel macro- faba bean as described by Schwenke et al.25 complexes, whose structure and digestibility have not Briefly, 7S globulin was extracted from raw bean been thoroughly investigated.12,13– 15 Inthecaseof flour homogenate in 0.2 M Tris-HCl buffer, pH 8.0 glycinin (11S) soy protein, two forms of aggregates (1:10, w/v) by stirring at room temperature overnight. are formed: a soluble complex, built of acidic polypep- After centrifugation (35 000 × g, 45 min) the super- tides, and an insoluble aggregate, composed of basic natant was adjusted to pH 4.5 and centrifuged again polypeptides.16 at 12 000 × g for 30 min. The precipitate (globulin) Analysis of far-UV CD spectra of heated (100 ◦C, was redissolved in distilled water. Globulin pro- 10 min) legumin from broad bean revealed a high teins other than 7S protein (glycoprotein II) were content of secondary structure, with the typical α- removed from solution by precipitation with ammo- helix pattern of native legume proteins, thus indicating nium sulfate at 60% saturation. After centrifugation only slight alterations of the legumin molecule (18 000 × g, 10 min) the supernatant was recovered, upon thermal aggregation.17 However, even though extensively dialyzed against distilled water and freeze- complete unfolding of vicilins (7S proteins) from dried. According to Pusztai and Stewart,24 7S protein both pea (Pisum sativum L) and common bean is obtained in over 90% purity. (Phaseolus vulgaris L) was not observed upon heating, For 11S globulin preparation, protein was extracted structural rearrangement of the pea protein was in water at pH 8.0 (1:10, w/v) for 1 h. After adjusting responsible for a decreased susceptibility to proteolysis the supernatant to 0.5 M NaCl and the pH at by trypsin.14 4.8, a globulin fraction was precipitated at a salt Mechanisms for heat-induced aggregation of concentration of 0.3 M. The precipitate was dissolved legume proteins, that may account for the digestibil- in 0.5 M NaCl, pH 7.5 and reprecipitated at pH ity behaviour, have been proposed on the basis of 4.8 (NaCl 0.3 M) to obtain purified legumin.25 The in vitro14,15,17,18 and in vivo evidence.12,19 precipitate was resuspended in phosphate-buffered Recommendations for quantifying digestibility of saline (PBS). dietary proteins have questioned the use of classical Protein content (N × 6.25) of globulin preparations nitrogen balance studies as an accurate indicator of was determined by the Kjeldahl method.26 protein availability, especially in the case of plant Globulins were denatured by autoclaving a 3% (w/v) ◦ foods: in this case, their strong effect on microbial protein solution in PBS for 20 min at 120 C(1atm) metabolism in the large intestine may result in a in a laboratory autoclave (Priorclave 55 Autoclave, substantial modification of the pattern of nitrogen Priorclave Ltd, London, UK). excretion.20 Moreover, currently used methods for protein digestibility determinations do not allow us to Amino acid analysis follow the events that take place during gastrointestinal Amino acid composition of 7S and 11S globulins was digestion (ie interaction with other meal components, determined after hydrolysis under vacuum with 6 M size of protein and peptides). Alternative in vivo HCl at 110 ◦C for 24 and 72 h, respectively. Amino approaches for the evaluation of the nutritional value acids were analysed with a Beckman 118BL amino of proteins through the assessment of ileal than acid analyzer (Beckman Instruments, Fullerton, CA, faecal digestibility, with the possibility of monitoring USA) and quantified after reaction with ninhydrin.27 whole protein digestive pattern, have recently been Cysteine and methionine were determined as cysteic developed.12,21– 23 acid and methionine sulfone, respectively, after In the present study, to gain knowledge about qual- oxidation with performic acid.28 Tryptophan was ity of heat-processed legume proteins, the digestibility determined after alkaline hydrolysis.29 of common bean 7S (Phaseolus vulgaris L) and faba bean 11S (Vicia faba L) purified globulins, in either In vivo trials native or denatured state, was tested in the small intes- In vivo digestibility was determined in acute (1 h) tine of rats in short-term (1 h) experiments. Charac- experiments with growing rats. Five rats were used terization of protein components in the small intestinal for each treatment. Male rats of the Hooded Lister content was carried out in order to derive mechanisms strain (40 days of age), weaned at 19 days of age, were responsible for bioavailability of storage globulins from adapted to experimental conditions by feeding them a legumes. semi-synthetic lactalbumin-control diet for 7 days.30

66 J Sci Food Agric 85:65–72 (2005) Heat-induced denaturation impairs digestibility of legume globulins

Rats (140 ± 1 g) were then housed individually in Anion exchange-FPLC polypropylene and stainless-steel cages and fasted Characterization of proteins components in the overnight before the experiment. small intestinal content of rats was carried out Rats were intubated with 100 mg of protein by anion exchange-FPLC separation on a Merck (N × 6.25) of the various samples suspended in PBS. Superformance 150-10 column (Merck, Darmstadt, Control rats were given PBS in the same amount Germany). Peptides were separated by a gradient that was given to the corresponding treated rats. One system (A: 0.05 M Na-phosphate buffer + 0.05 M hour after gavage, the rats were killed by anaesthetic NaCl, pH 7.4; B: 0.05 M Na-phosphate buffer + 0.5 M (halothane) overdose. A longitudinal incision along the NaCl, pH 7.4), at a flow rate of 1 ml min−1.The midline of the abdomen was made and the stomach eluate was monitored at 240 nm with a Waters 490E and small intestine were taken out separately. Their multiwavelength detector (Waters Chromatography contents (digesta) were washed out with 10 ml of ice- Division, Milford, MA, USA). cold water containing 0.1mgml−1 Aprotinin (Sigma Chemical Co, St Louis, MO, USA), and centrifuged Matrix-assisted laser desorption ionisation mass at 4000 × g for 45 min. Protein recovery after the spectrometry (MALDI-MS) centrifugation step was ≥95%. The protein content Samples were prepared by mixing 3 µlofsample of the supernatants was determined by the method − with 3 µl of matrix (sinnapinic acid 10 mg ml 1 in of Lowry et al.31 Endogenous proteins in the stomach acetonitrile:water 70:30+0.1% trifluoracetic acid). and intestinal contents were estimated in rats given One microlitre of this mixture was then deposited PBS only. These values were subtracted from the onto a sample slide and allowed to air dry. Once dry, stomach and intestinal protein contents determined for the sample was admitted into the mass spectrometer. the test samples. Using the corrected protein content All analyses were carried out on a Perceptive values, protein digestibility (%) was calculated by the Biosystems Voyager-DE (Warrington, UK) mass ratio between protein absorbed in the small intestine spectrometer with a 1.5-metre linear flight tube in and protein ingested, after subtraction of protein in the delayed extraction mode. Ions are produced by the stomach: firing a VSL-337 nm nitrogen laser focused onto the P − P − P sample spot. The ions are extracted through a series of PD (%) = ing st int × 100 Ping − Pst grid lenses before acceleration into the analyser with a voltage of 25 kV. The ions were detected with an  where PD = protein digestibility; Ping = mg of protein electron multiplier and data collected on Millenia ingested; Pst = mg of protein in the stomach; Pint = mg Lxe computer (Micron Electronics, Idaho, USA). of protein in the intestine. With each sample multiple scans were recorded and averaged using the system software. SDS-PAGE SDS-PAGE was carried out according to Laemmli32 Statistical analysis on a slab gel of 13% polyacrylamide in the The results were subjected to analysis of variance. presence of β-mercaptoethanol. Pooled intestinal The significance of the differences between means was contents of rats in each treatment were ana- estimated by a two-sided Student t-test. lyzed. Samples and standard proteins were dissolved in sample buffer (0.05 M Tris-HCl, pH 6.8, 3% SDS, 12% glycerol, 2% β-mercaptoethanol, 0.01% bromophenol blue (BioRad Laboratories, Her- RESULTS AND DISCUSSION cules, CA, USA) and heated at 100 ◦C for 5 min Protein content of globulin samples from common immediately prior to electrophoresis. High molec- bean and faba bean was 93 and 95%, respectively. ular weight (HMW) and low molecular weight Absence of gross contamination by non-protein (LMW) reference proteins were provided from Sigma. seed compounds in the globulin preparations was HMW protein markers were: myosin (205 000 Da), denoted by good correspondence of values of protein β-galactosidase (116 000 Da), phosphorylase b determination by the Kjeldahl (N × 6.25) and Lowry (97 000 Da), fructose-6-P-kinase (84 000 Da), bovine methods after freeze-drying of the samples. Degree of serum albumin (66 000 Da), glutamic dehydrogenase purity of the 7S and 11S preparations was checked by (55 000 Da), ovalbumin (45 000 Da), glyceraldehyde- amino acid analysis (Table 1) and SDS-PAGE (Figs 1 3-P-dehydrogenase (36 000 Da). LMW protein mark- and 2). ers were: bovine serum albumin (66 000 Da), ovalbu- Amino acid compositions of the purified globulins min (45 000 Da), glyceraldehyde-3-P-dehydrogenase corresponded well with those previously provided for (36 000 Da), carbonic anhydrase (29 000 Da), the 7S protein from common bean and from the trypsinogen (24 000 Da), trypsin inhibitor (20 000 11S protein from faba bean.1 Both proteins presented Da), α-lactalbumin (14 200 Da), aprotinin (6500 Da). a high content of glutamic acid/glutamine, aspartic For each sample, 200 µg of protein was loaded on the acid/asparagine, lysine and arginine, as is typical of gel. After the electrophoretic run (20 mA, 4 h), the gel reserve proteins (Table 1). The presence of low, but was stained with Coomassie Brilliant Blue R-250. detectable, amounts of cysteine and methionine was

J Sci Food Agric 85:65–72 (2005) 67 M Carbonaro, G Grant, M Cappelloni

Table 1. Amino acid composition of puriﬁed common bean and faba bean globulins (g per 16g N)a

Amino acid Common bean globulin Faba bean globulin

Lysine 7.01 ± 0.13 6.42 ± 0.01 Histidine 3.10 ± 0.10 2.51 ± 0.05 Arginine 5.22 ± 0.08 9.45 ± 0.21 Aspartic acid 13.47 ± 0.27 11.40 ± 0.38 Threonine 3.14 ± 0.44 3.70 ± 0.02 Serine 6.86 ± 0.51 5.95 ± 0.17 Glutamic acid 19.45 ± 0.33 17.66 ± 0.26 Proline 2.99 ± 0.47 4.94 ± 0.39 Glycine 3.94 ± 0.01 3.96 ± 0.01 Alanine 3.58 ± 0.13 4.24 ± 0.11 Half cystine 0.26 ± 0.04 0.75 ± 0.04 Figure 2. ± ± SDS-PAGE of in vivo gastrointestinal digestion of 11S Valine 5.44 0.37 5.66 0.12 globulin from faba bean (1) Endogenous protein from stomach of ± ± Methionine 0.48 0.15 0.80 0.13 overnight fasted rats; (2) endogenous protein from small intestine of Isoleucine 5.15 ± 0.33 5.06 ± 0.09 overnight fasted rats; (3) HMW protein markers; (4) LMW protein Leucine 9.49 ± 0.15 8.53 ± 0.01 markers; (5) native 11S globulin; (6) stomach content from 11S Tyrosine 3.43 ± 0.51 3.29 ± 0.19 globulin; (7) intestinal content from 11S globulin; (8) denatured 11S Phenylalanine 7.01 ± 0.15 4.48 ± 0.09 globulin; (9) stomach content from denatured 11S globulin; Tryptophan 0.81 ± 0.01 0.76 ± 0.02 (10) intestinal content from denatured 11S globulin. a The values represent the means and standard deviations of four 24 determinations. that correspond to the four subunits of phaseolin. Bands at MW lower than 43 000 Da were also present. Because this preparation, unlike crude globulin extract from the same common bean variety,12 was found totally devoided of lectin (G2 globulin, MW 30 000–32 000 Da) activity, these bands probably represent minor subunits of phaseolin, whose presence in globulin preparations has been described.1 Major components of faba bean globulin were the 34 000 Da legumin α-chain and the 18 000–26 000 Da β-chains (Fig 2, lane 5). Even though removal of vicilin (7S protein) from faba bean 11S preparations has been shown to be a difficult task, the procedure of Schwenke et al25 employed in this study was able to provide, in most cases, a very low amount of contam- inating 7S protein. Bands at MW 44 000–53 000 Da, typical of vicilin, were present in small amounts in the SDS-PAGE pattern of purified faba bean globulin, as it was also found in some faba bean varieties by Schwenke et al.25 Therefore, our globulin preparations were consid- Figure 1. SDS-PAGE of in vivo gastrointestinal digestion of 7S globulin from common bean. (1) HMW protein markers; (2) LMW ered suitable to be assayed in in vivo digestibility trials. protein markers; (3) native 7S globulin; (4) stomach content from 7S Protein digestibility in the small intestine of native globulin; (5) intestinal content from 7S globulin; (6) denatured 7S and thermally treated purified globulins from common globulin; (7) stomach content from denatured 7S globulin; bean and faba bean is reported in Table 2. In (8) intestinal content from denatured 7S globulin. Bracket indicates agreement with previous claims,6 native globulins were the 22–27 kDa bands. well digested (92 and 95% for 7S and 11S proteins, respectively). However, after thermal denaturation, confirmed, in agreement with the results of Derbyshire protein digestibility of both globulins was reduced, et al.1 the reduction being highly significant for faba Because common bean seed storage globulins are bean globulin. The values obtained for heat-treated predominantly 7S proteins, whereas those of faba bean globulins, which comprise the bulk of the proteins, are mainly 11S proteins,1 our preparations contained were close to those measured for whole cooked seeds major protein components of the two legume seeds. (89 and 79% for cooked common bean and faba bean, SDS-PAGE analysis of the storage proteins puri- respectively).12,19 It is worth considering that, in the fied from common bean and faba bean seed flours same experimental conditions, digestibility of animal is presented in Figs 1 and 2, respectively. Elec- proteins, casein and BSA, was found to be 98%.33 trophoretic pattern of common bean globulin (Fig 1, The proteolytic pattern of common bean globulin lane 3) showed bands at MW 43 000–53 000 Da during gastrointestinal digestion (Fig 1) indicated no

68 J Sci Food Agric 85:65–72 (2005) Heat-induced denaturation impairs digestibility of legume globulins

Table 2. Effect of thermal treatment (120 ◦C, 20 min, 1 atm) on the amount of total protein (mg) in stomach and small intestine and on small intestinal digestibility (%) of common bean (PvulgarisL) and faba bean (VfabaL) puriﬁed globulinsa

Common bean globulin Faba bean globulin

Sample Native Thermally treated Native Thermally treated

Protein in: Stomach 3.04 ± 1.11 5.33 ± 1.43 7.51 ± 1.73 6.91 ± 1.05 Small intestine 7.74 ± 0.62∗ 10.78 ± 1.71 4.55 ± 1.81∗∗ 19.72 ± 6.24 Intestinal digestibility 92.02 ± 0.76∗ 88.61 ± 1.91 95.08 ± 3.04∗∗ 78.79 ± 6.82

Rats were killed after 1 h of intubation with 100 mg of proteins. a Values are means and standard deviations of five replicates. Native vs thermally treated: significantly different, ∗ P < 0.05; ∗∗ P < 0.01. degradation in the stomach (Fig 1, lane 4), confirming 0.70 in vitro data showing marked resistance of the 7S 0.70 protein to pepsin hydrolysis.5 In the small intestine, (A) (B) 0.60 the major proteolytic breakdown products of phaseolin 0.60 (MW 22 000–27 000 Da) were clearly detectable after 0.50 0.50 1 h of digestion (Fig 1, lane 5). Similarly to the results of in vitro proteolysis of the protein,5 in our digestion 0.40 0.40 conditions phaseolin native molecule appears to be 0.30 cleaved to give digestion products of approximately 0.30 half the molecular weight of the original subunits. O D 240 nm O D 240 nm 0.20 After thermal treatment, the SDS profile was very 0.20 similar (Fig 1, lanes 6–8). However, the appearance 0.10 of bands at MW lower than 44 000 Da indicated a 0.10 small extent of degradation of the heated protein in 0 the stomach (Fig 1, lane 7). Notwithstanding, in the 0 small intestine, the 22 000–27 000 Da bands appeared 36 32 28 24 20 16 12 8 4 0 36 32 28 24 20 16 12 8 4 0 in a higher concentration in the denatured than in the Time (mins) Time (mins) native 7S globulin, on the basis of visual observation Figure 3. Anion exchange–FPLC separation of proteins in small of the gel, thus implying a lower extent of digestion intestinal contents of rats 1 h after feeding with faba bean native (A) or (Fig 1, lane 8). This result is in contrast with the denatured (B) 11S globulins. evidence provided by in vitro studies on phaseolin, all showing the heated protein could be readily hydrolyzed 0.50 0.50 by trypsin.5 (A) (B) Unlike common bean protein, faba bean globulin 0.40 0.40 showed very little protein staining bands in the stomach, indicating degradation to very low MW 0.30 0.30 peptides (Fig 2, lane 6). Only a small amount of proteins, mostly in the high MW range (>55 000 Da), 0.20 0.20 O D 240 nm O D 240 nm could be detected in the small intestinal contents (lane

7). In this case, the amount of residual proteins in the 0.10 0.10 small intestine was higher in the heated than in the unheated protein (lanes 10 and 7, respectively). A very 0 0 signiﬁcant trend towards aggregation of the faba bean 4036 32 28 24 20 16 12 8 4 0 4036 32 28 24 20 16 12 8 4 0 Time (mins) globulin upon heating was indicated by the presence of Time (mins) protein material that did not enter the stacking gel and Figure 4. Anion exchange–FPLC separation of proteins in small by precipitation during the electrophoretic run (Fig 2). intestinal contents of rats 1 h after feeding with common bean native Because the charge density of legume proteins was (A) or denatured (B) 7S globulins. previously found to affect the protein digestibility behaviour,9,12 anion exchange–FPLC analysis of indicating that the overall negative charge of peptides proteinaceous material in the small intestinal content and undigested proteins recovered in the small was carried out (Figs 3 and 4). Although the amount intestine after 1 h of digestion was decreased. of protein in the gut was increased greatly when In contrast, in the case of common bean, heat heat-treated globulins were given (Table 2), the treatment markedly increased the net negative charge overall peptide proﬁles obtained with native and of the peptides (Fig 4B). This is likely to be the denatured faba bean globulins were very similar reason for a lower extent of aggregation, as a (Fig 3). However, protein material at tR>16 min was consequence of the effect of repulsive charges, of markedly reduced in the heated globulin (Fig 3B), peptides from common bean than of those from

J Sci Food Agric 85:65–72 (2005) 69 M Carbonaro, G Grant, M Cappelloni faba bean globulins. In the latter case, strong The present results add further support to the hydrophobic interactions are a predominant factor hypothesis that the low digestibility of heat-treated 11S in the stabilization (and subsequent precipitation) of protein is mainly related to the high hydrophobicity of peptide complexes. basic polypeptides. These polypeptides were shown to MALDI-MS analysis of protein species in the give rise to macrocomplexes of very high MW upon small intestinal digests of heat-treated globulin from heating, probably stabilized by SS linkage formation common bean (Fig 5) conﬁrmed the presence of only in hydrophobic regions.13,17 As far as the 7S protein partially digested 7S protein with MW 23 700 Da, is concerned, trapping of lysine residues inside high- that is very close to half the MW of the β- MW aggregates, formed from subunits of denatured phaseolin subunit (MW 47 500 Da).1,34 Two other proteins, has recently been demonstrated by electron major peptide peaks of MWs 8290 and 13 740 Da spin resonance studies for PvulgarisLproteins.15 in the low region of the spectrum (m/z <14 000) Therefore, similar events are likely to be responsible were resolved. In the MALDI-mass spectrum of faba for impaired digestibility of legume 7S and 11S heated bean globulin (Fig 6), only peaks between MWs globulins in the small intestine. 7580 and 9160 or of 20 057 Da were found. The latter represents undigested basic subunits of the 11S globulin, whose MW has been reported to be CONCLUSIONS 20 000–20 100.1,25 Heat-induced denaturation of 7S and 11S stor- The 11S basic chain has been reported to be age globulins is responsible for a decrease in similar in sequence to the C-terminal region of the digestibility, faba bean 11S globulin being particularly 7S protein.35 Basic polypeptides from legumin (11S affected. protein) are characterized by a highly ordered structure The electrical charge of peptides that are coming and high hydrophobicity of their β-polypeptide.36 In a from in vivo digestion of globulins affects the overall widely accepted 6-subunit pair model of 11S globulin, aggregation process and, consequently, the extent basic polypeptides are forming the core of the subunits of digestibility: the less negative the protein, the (and of the globulin) and are therefore protected from lower the digestibility. Highly hydrophobic basic the attack of proteases.16 Indeed, a high concentration subunits of MW 20 000 Da were found in undigested of hydrophobic amino acids, such as alanine, valine, material recovered in the small intestine of rats given methionine, isoleucine, phenylalanine, in undigested denatured 11S protein, while a half-size β-phaseolin proteins recovered in the small intestine of rats fed on subunit was found in that from rats assayed for 7S faba bean meal has been previously found.12 protein.

Figure 5. MALDI-mass spectrum of proteins in small intestinal content of rat 1 h after feeding with denatured 7S globulin.

70 J Sci Food Agric 85:65–72 (2005) Heat-induced denaturation impairs digestibility of legume globulins

Figure 6. MALDI-mass spectrum of proteins in small intestinal content of rat 1 h after feeding with denatured 11S globulin.

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