Food Chemistry 145 (2014) 34–40

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ACE-inhibitory activity of enzymatic protein hydrolysates from lupin and other legumes Giovanna Boschin a,⇑, Graziana Maria Scigliuolo b, Donatella Resta b, Anna Arnoldi a,b a b

Laboratory of Food Chemistry and Mass Spectrometry, Department of Pharmaceutical Sciences, Università degli Studi di Milano, via Mangiagalli 25, 20133 Milano, Italy HPF Nutraceutics s.r.l., via Balzaretti 9, 20133 Milano, Italy

a r t i c l e

i n f o

Article history: Received 14 February 2013 Received in revised form 3 June 2013 Accepted 17 July 2013 Available online 24 July 2013 Keywords: Angiotensin converting enzyme (ACE) inhibitors Enzymatic protein hydrolysates Hypertension Hippuric acid Legumes Lupin Pepsin Protein isolates

a b s t r a c t The objective of this investigation was to compare the angiotensin converting enzyme (ACE)-inhibitory activity of the hydrolysates obtained by pepsin digestion of proteins of some legumes, such as chickpea, common bean, lentil, lupin, pea, and soybean, by using the same experimental procedure. The ACE-inhibitory activity was measured by using the tripeptide hippuryl-histidyl-leucine (HHL), as model peptide, and HPLC-DAD, as analytical method. The peptide mixtures of all legumes were active, with soybean and lupin the most efficient, with IC50 values of 224 and 226 lg/ml, respectively. Considering the promising results obtained with lupin, and aiming to identify the protein(s) that release(s) the peptides responsible for the activity, the peptides obtained from the pepsin digestion of some industrial lupin protein isolates and purified protein fractions were tested. The most active mixture, showing an IC50 value of 138 lg/ml, was obtained hydrolysing a mixture of lupin a + b conglutin. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Cardiovascular diseases, such as atherosclerosis, coronary heart disease, stroke, and heart failure, are a major health concern, because they are one of the leading causes of death in most industrialised countries. One of the main risk factors is hypertension, defined by the World Health Organisation (WHO) as the exceeding of 90 mmHg for the diastolic arterial pressure, and 140 mmHg for the systolic pressure. Hypertension is commonly treated with blood pressure lowering drugs, in particular with the inhibitors of the angiotensin I converting enzyme (ACE; EC 3.4.15.1), which plays an important role in regulating blood pressure in the renin-angiotensin system. This enzyme catalyses the conversion of the biologically inactive angiotensin I to the potent vasoconstrictor angiotensin II, and inactivates the potent vasodilator bradykinin (Skeggs, Kahn, & Shumway, 1956). Inhibitors bind tightly to the ACE active site, competing

Abbreviations: ACE, angiotensinconverting enzyme; BSA, bovin serum albumin; E/S, enzyme/substrate; HA, hippuric acid; HHL, hippuryl-histidyl-leucine; HL, histidyl-leucine; LPI, lupin protein isolate; SDS–PAGE, sodium dodecyl sulphate– polyacrylamide gel electrophoresis; TPE, total protein extract. ⇑ Corresponding author. Tel.: +39 02 50318210; fax: +39 02 50318204. E-mail address: [email protected] (G. Boschin). 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.07.076

with angiotensin I for occupancy; as a consequence, ACE cannot convert angiotensin I to angiotensin II (Skeggs et al., 1956). Considering that synthetic ACE inhibitors, such as captopril, lisinopril and enalapril, may produce side effects, such as coughing, taste disturbances and skin rashes, there is interest in natural inhibitors, and numerous studies are focused on the production and isolation of ACE-inhibitory peptides from different food proteins. Food derived ACE-inhibitory peptides can be introduced into functional foods or dietary supplements. An alternative solution may be the incorporation into functional foods of specific proteins that can release ACE-inhibitory peptides during digestion. In both cases, their integrity, absorption and bioavailability are relevant issues (Roberts, Burney, Black, & Zaloga, 1999; Vermeirssen, van Camp, & Verstraete, 2004). The first active peptides were obtained from milk proteins (Nakamura et al., 1995), but some have been isolated from plant seeds, such as soybean (Chen, Yang, Suetsuna, & Chao, 2004; Wu & Ding, 2002), pea (Aluko, 2008; Barbana & Boye, 2010; Roy, Boye, & Simpson, 2010), rice, sunflower, and wheat (Guang & Phillips, 2009). The present study aims to compare the ACE-inhibitory activity of peptides derived from the enzymatic digestion of lupin and other grain legume proteins using the same experimental protocol. In addition to lupin, peptides derived from some purified protein

G. Boschin et al. / Food Chemistry 145 (2014) 34–40

fractions and industrial protein isolates were investigated, since a clinical trial has demonstrated that consuming bread containing lupin flour may positively influence the blood pressure of overweight individuals (Lee et al., 2009). Different methods for the measurement of ACE-inhibitory activity have been reported in literature using various substrates, media, and analytical techniques (Cushman & Cheung, 1971; Lam et al., 2007; Vermeirssen, Van Camp, & Verstraete, 2002; Wu, Aluko, & Muir, 2002). Numerous papers still use the method developed by Cushman and Cheung in 1971, based on the determination of the concentration of hippuric acid (HA), by spectrophotometry, at 228 nm, after ethyl acetate extraction, which is formed from HHL (hippuryl-histidyl-leucine) by the action of ACE. Although this assay is simple and economic, it has some limitations when it is applied to the complex peptide mixtures derived from the hydrolysis of plant proteins, because of the presence of interfering molecules. For this reason, a chromatographic method based on HPLC coupled with a DAD detector was preferred following published procedures (Lam et al., 2007; Wu et al., 2002), with some modifications.

2. Materials and methods 2.1. Materials Amicon Ultra-0.5 filters were bought from Millipore. HPLCgrade water was prepared with a Milli-Q purification system (Millipore, Billerica, MA, USA). Bio-Safe Coomassie and Precision Plus Protein Standards Dual Colour marker for SDS–PAGE were from Biorad. All other chemicals (reagents and solvents) were from Sigma–Aldrich (St. Louis, MO, USA).

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2.4. Preparation of total protein extracts (TPEs) The total protein extracts (TPEs) from seeds of different legumes and from lupin isolates (LPI, LPI-E, and LPI-F) were obtained as previously reported (Brambilla, Resta, Isak, Zanotti, & Arnoldi, 2009). Briefly, proteins were extracted from defatted flour with 100 mM Tris–HCl/0.5 M NaCl buffer (pH 8.2) for 2 h at 4 °C. The solid residue was eliminated by centrifugation at 6500g, for 20 min at 4 °C, and the supernatant was dialyzed against 100 mM Tris–HCl buffer (pH 8.2) for 24 h at 4 °C. The protein content was assessed according to Bradford (Bradford, 1976), using BSA as standard. Dialyzed solutions were stored at 20 °C. 2.5. Extraction of a, b, c, and d conglutin from lupin seed The main lupin proteins, a, b, c, and d conglutin, were extracted with a literature procedure (Sironi et al., 2005). Briefly, defatted lupin flour was suspended in water (1/20 w/v) and brought to pH 8.0–8.5 with a diluted NaOH solution under magnetic stirring for 3 h at room temperature. The slurry was centrifuged at 10,000g for 60 min at 18 °C. The supernatant was precipitated by acidifying to pH 4.5 with dilute HCl. Upon a further centrifugation at 10,000g for 30 min, a pellet containing a, b and d conglutin, and a supernatant containing c conglutin were obtained. In order to separate d conglutin from a + b conglutin, the pellet was resuspended in 0.1 M CH3COONa (pH 4), containing 0.4 M NaCl and 40% ethanol, and stirred for 1 h at room temperature. Since this treatment enables to dissolve d conglutin, by a further centrifugation (as described above) a pellet containing a + b conglutin and a supernatant containing d conglutin were obtained. The latter was recovered in a solid form by cold precipitation at 20 °C overnight (18 h) and subsequent centrifugation at 0 °C at 10,000g for 30 min. 2.6. Chromatographic separation of c conglutin from lupin TPE

2.2. Sampling Lupins (L. albus, cv ares) were kindly provided by HPF Nutraceutics s.r.l.; all other legumes, pea (Pisum sativum), lentil (Lens culinaria), chickpea (Cicer arietinum), common bean (Phaseolus vulgaris), and soybean (Glycine max), were purchased in a local supermarket, and no indication about cultivar was available. Three lupin protein industrial isolates from L. albus, LPI (lupin protein isolate), LPI-E (lupin protein isolate E), and LPI-F (lupin protein isolate F), were provided by the Fraunhofer Institute IVV (Freising, Germany). LPI is a total protein isolate containing mostly globulins, LPI-E was separated by alkaline extraction and isoelectric precipitation and comprises mostly a and b conglutin, whereas LPI-F, recovered by acid extraction and ultrafiltration, is enriched in c conglutin (D’Agostina et al., 2006). Samples of purified globulins (a mixture of a + b conglutin, c conglutin and d conglutin) were obtained from the total protein extract using a fractionation procedure described in the literature (Sironi, Sessa, & Duranti, 2005). Additionally, a sample of c conglutin was obtained as reported by us (Resta, Brambilla, & Arnoldi, 2012).

2.3. SDS–page Protein hydrolysates were separated on 15% SDS–polyacrylamide gel using Mini Protean 3 Cell (Biorad). Electrophoresis was performed at a constant voltage of 80 V for stacking, and 120 V for separation. Gels were stained with Bio-Safe Coomassie (Biorad), scanned with Versa Doc 3000 (Biorad) and analysed with Quantity One 4.6.8 Software (Biorad).

c Conglutin was isolated as previously reported (Resta et al., 2012). Briefly, isolation was performed with two chromatographic steps, i.e. anion-exchange chromatography and gel filtration chromatography. The TPE was injected onto a DEAE-FF column (1.6  2.5 cm, 15–70 mm bead size, 5 ml column volume; GE Healthcare Bio-Sciences AB, Uppsala, Sweden) and fractions were eluted from the column with a linear salt gradient (0–100% NaCl over 16 column volumes). The c conglutin enriched fraction, eluted at the beginning of the gradient, was then loaded onto a gel-filtration column (Superdex 200, 1  30 cm, 24 ml column volume; GE Healthcare Bio-Sciences AB) for a second purification step. Purified c conglutin was dialysed against 30 mM Tris–HCl buffer (pH 8.2) and stored at 20 °C. 2.7. Preparation of protein hydrolysates The hydrolysis reaction was optimised, testing different experimental conditions, such as the time and the enzyme/substrate (E/ S) ratio using lupin TPE (data not shown). The TPEs from different legumes, from lupin industrial protein isolates as well as the isolated lupin proteins were dissolved in Tris–HCl buffer 100 mM, pH 8, the protein solutions were then acidified at pH 2 with HCl, and pepsin, dissolved in 30 mM NaCl, was added at a 1:100 ratio of enzyme to substrate (w/w). The reaction mixture was incubated at 37 °C for 18 h, and few microliters of each sample were withdrawn at various intervals. The enzyme was inactivated by adjusting the pH to 7 with NaOH and then the solutions were cooled. The efficiency of the hydrolysis was evaluated by SDS–PAGE considering S0, sample collected immediately after enzyme addition (time 0), S1, sample collected after 1 h of incubation and So/n, collected after the night of incubation (18 h). A 15% glycine gel,

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G. Boschin et al. / Food Chemistry 145 (2014) 34–40

Table 1 Values of highest inhibitor concentration (lg/ml), maximum percentage of ACEinhibition and IC50 (lg/ml) for hydrolysates obtained with pepsin digestion of total protein extracts from grain legumes. Values are reported as mean value ± standard deviation of three independent experiments; values with different letters are significantly different (p < 0.05). Sample

Highest inhibitor concentration (lg/ml)

Max ACE inhibition (%)

IC50 (lg/ml)

Chickpea Common bean Lentil Lupin 1 (L. albus) Lupin 2 (L. angustifolius) Pea Soybean

802 944 829 902 828 861 983

86 77 79 80 89 71 88

673 ± 15.3e 633 ± 13.0d 606 ± 10.4c 268 ± 11.9b 226 ± 15.8a 595 ± 11.4c 224 ± 13.1a

instead of a 16% tricine gel, was used to verify the effectiveness of the hydrolysis reaction in order to point out differences among high molecular weight proteins, which sometimes are still present, in small amount, at the end of the hydrolysis. The low-molecular weight peptides obtained after 18 h of hydrolysis were separated from the intact enzymes, proteins and high-molecular weight peptides by ultrafiltration through a 3000 Da cut-off membrane (Amicon Ultra-0.5, Millipore, Billerica, MA, USA) at 12,000 g for 30 min at 4 °C. The permeate, containing only peptides with a molecular weight lower than 3 kDa, was freezed and stored at 20 °C. For common beans, the procedure was slightly modified, taking advantage of some suggestions from (Venkatachalam & Sathe, 2003). Briefly, the TPE from common beans was dissolved in Tris HCl buffer 100 mM pH 8 as usual and then boiled for 30 min. After cooling at room temperature, it was acidified by HCl to pH 2 and then the enzyme was added at 1:50 ratio of enzyme to substrate (w/w). Each reaction mixture was incubated for 18 h at 37 °C, the hydrolysate was taken at various intervals and the enzyme was inactivated by adjusting the pH to 7 with NaOH. 2.8. Determination of the peptide concentration The peptide concentration in the hydrolysates was measured according to the method of(Levashov, Sutherland, Besenbacher, & Shipovskov, 2009), which is based on chelating the peptide bonds by Cu (II) in alkaline media and monitoring the change of absorbance at 330 nm, following an experimental method proposed by Goa (Goa, 1953). In brief, a solution of X ll peptide mixture, (500  X) ll water, 500 ll 6% (w/w) NaOH in water, and 50 ll of active reagent (containing 0.6 M sodium citrate, 0.9 M sodium

carbonate, and 0.07 M copper sulphate, 2.4 M NaOH, pH 10.6) was prepared. The reaction mixture was mixed, incubated for 15 min at 20 °C and then the absorbance was measured at 330 nm. As standard for the calibration curve, a sterile solution of peptone from casein at 10 mg/ml in water was used; the assay is linear in the range 100–1000 lg of peptides in the cuvette.

2.9. ACE-inhibitory activity assay The assay was performed by using HHL as substrate (Cushman & Cheung, 1971) and HPLC-DAD for the detection of HA (Wu et al., 2002). Some modifications were introduced, as described below. A volume of 100 ll of 2.5 mM HHL in buffer 1 (100 mM Tris–HCOOH, 300 mM NaCl pH 8.3) was mixed with 30 ll of ‘inhibitor’ in buffer 1 at different concentrations. The ‘‘inhibitor’’ is a peptide mixture obtained from the digestion of proteins, i.e. TPEs, industrial isolates, or laboratory purified proteins. Usually, at least 6 concentrations were used for each inhibitor, and each solution was tested twice: the highest concentrations used in the assays, expressed in lg of peptides/ml, are reported in Tables 1 and 2; serially dilutions were performed to obtain the other concentrations. HHL solution was daily prepared because, in line with our experience and literature data (Shalaby, Zakora, & Otte, 2006), it is not very stable. Samples were pre-incubated at 37 °C for 15 min, then 15 ll of ACE solution, corresponding to 3 mU of enzyme in buffer 2 (100 mM Tris–HCOOH, 300 nM NaCl, 10 lM ZnCl2, pH 8.3), were added; samples were then incubated for 60 min at 37 °C. The reaction was stopped with 125 ll of 0.1 M HCl. The aqueous solution was extracted twice with 600 ll of ethyl acetate, and the solvent was evaporated at 95 °C, the residue was then dissolved in 500 ll of buffer 1 and analysed by HPLC. In our experiments, the direct injection in HPLC did not give optimal results regarding peak shape and baseline for a reliable and accurate quantification. For this reason, we decided to perform the extraction step, using ethyl acetate and a double step of extraction followed by the evaporation of solvent in glass vials. The evaporation conditions greatly influenced the results of the assay: a trace of residual solvent results in abnormally high absorbance values (Lam et al., 2007). Obviously the sample preparation requires one more step, but the extraction assures good reproducibility in the quantification of the HA chromatographic peak and the absence of interfering peaks due to peptides. Sigmoid curves (Fig. 2) fit very well the data points representing the ACE inhibition in function of inhibitor concentration, as pointed out by the correlation coefficient (R2). HPLC analyses were performed with a HPLC 1200 Series equipped with an autosampler (Agilent Technologies, Santa Clara, US) with a Lichrospher 100, C18 column (4.6  250 mm, 5 lm; Grace, Italy). Water and acetonitrile were used as solvents with

Table 2 The values of highest inhibitor concentration (lg/ml), maximum percentage of ACE-inhibition and IC50 (lg/ml) of hydrolysates obtained by pepsin digestion of purified protein fractions or protein isolates from L. albus cv. ares. Values are reported as mean value ± standard deviation of three independent experiments; values with different letters are significantly different (p < 0.05). Sample Laboratory samples

Industrial samples

a + b conglutin c conglutin 1 c conglutin 2 d conglutin LPI LPI-E LPI-F

Highest inhibitor concentration (lg/ml)

Max ACE inhibition (%)

IC50 (lg/ml)

749 777 946 1082 1104 1185 1109

93 48 44 28 91 89 81

138 ± 4.7a n.d. n.d. n.d. 165 ± 3.0a 142 ± 4.2a 768 ± 35.0b

LPI: lupin protein isolate. c conglutin 1: obtained by chemical separation procedure (Sironi et al., 2005). c conglutin 2: obtained by chromatographic separation method (Resta et al., 2012). n.d.: not determined, since the maximum percentage of ACE-inhibition did not reach 50%.

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3 .8 1 4

DAD1 B, Sig=228,4 Ref=360,100 (ACIDOIPP\HA0025.D) mAU

(A)

250

200

150

100

50

0 0

2

4

6

8

10

12

14

min

4 .0 5 4

DAD1 B, Sig=228,4 Ref=360,100 (28072011\A11.D) mAU

(B)

250

200

150

100

50

0

0

2

4

6

8

10

12

14

min

Fig. 1. Chromatogram at 228 nm of (A) a standard solution of hippuric acid, (B) a representative sample of an assay with peptidic mixture from pea protein, showing the peak of HA.

the following gradient: 0 min 5% acetonitrile, 10 min 60% acetonitrile, 12 min 60% acetonitrile, 15 min 5% acetonitrile. Injection volume was 10 ll, wavelength 228 nm, flow 0.5 ml/min. The retention time of hippuric acid (HA) was 4.2 min. An exemplary HPLC chromatogram is reported in Fig. 1: (A) is a standard solution of HA, (B) is a representative chromatogram of an assay with peptidic mixture from pea protein. In these chromatographic conditions HHL eluted at 11 min. The detector response for standard HA was linear in the range 1–100 lg/ml. The relative standard deviation for determination of the 10 lg/ml HA solution was 2.5% (n = 8), very similar to already published data (Wu et al., 2002).

where AIB is the area of HA in Inhibitor Blank (IB) sample (i.e. sample with enzyme but without inhibitor), AN is the area of HA in the samples containing different inhibitor amounts and ARB is the area of HA in the reaction blank (RB) sample (i.e. sample without enzyme and with inhibitor in the highest concentration). The percentages of ACE inhibition were plotted vs. log10 inhibitor concentrations, obtaining a sigmoid curve; IC50 was defined as the inhibitor concentration needed to observe a 50% inhibition of the ACE activity. Only when the ACE-inhibitory activity was more than 50%, the IC50 value was calculated. The IC50 values were obtained independently testing each inhibitor three times.

2.10. ACE inhibition measurement

2.11. Statistical analyses

The evaluation of ACE inhibition was based on the comparison between the concentrations of HA in the presence or absence of an inhibitor (inhibitor blank). The phenomenon of autolysis of HHL to give HA was evaluated by a reaction blank, i.e. a sample with the higher inhibitor concentration and without the enzyme. The percentage of ACE inhibition was computed considering the area of HA peak with the following formula:

Statistical analyses were performed with Statgraphics Plus (ver. 2.1 for Windows). IC50 values labelled with different letters are significantly different (p < 0.05).

% ACE inhibition ¼





ðAIB  AN Þ  10 ðAIB  ARB Þ

3. Results and discussion 3.1. ACE-inhibitory activity of legume hydrolysates The principal aim of this work was the comparison of the ACEinhibitory activity of protein hydrolysates of some grain legumes,

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G. Boschin et al. / Food Chemistry 145 (2014) 34–40

Fig. 2. Exemplary diagrams reporting % ACE inhibition vs. log10 concentrations of pepsin hydrolysates of (A) soybean TPE, (B) lupin TPE, and (C) LPI-E (TPE, total protein extract, LPI, lupin protein isolate).

i.e. chickpea, common bean, lentil, lupin, pea, and soybean, using the same experimental protocol for each sample. The protein extraction was performed from defatted flour obtained by milling the different seeds, using a method already optimised by us for lupin kernel (Brambilla et al., 2009). In general, legume seed proteins are mainly globulins and their extraction is performed at alkaline pH values because they have higher solubility at pH > 7. The enzymatic digestion was performed with pepsin, which has been used by other Authors (Akillioglu & Karakaya, 2009; Aluko, 2008) to prepare peptide mixtures with good ACE-inhibitory activity. The digestion was optimised by changing different parameters, such as the time and the enzyme/substrate (E/S) ratio. Preliminary tests (data not shown) indicated that the digestion had to be performed over-night (18 h) and at an E/S ratio of 1:100 to obtain the best results for all TPEs. Unfortunately these conditions were not suitable for common bean proteins, because SDS–PAGEs (data not shown) showed that no digestion took place. In fact phaseolin, the major protein fraction, is known for its resistance to both in vitro and in vivo proteolysis by digestive enzymes (Jivotovskaya, Senyuk, Rotari, Horstmann, & Vaintraub, 1996). For this reason, we were compelled to modify the original procedure, taking advantage of the suggestions of (Venkatachalam & Sathe, 2003), i.e. a pretreatment of boiling.

Samples were withdrawn at the beginning of proteolysis, after one hour, and at the end of the overnight incubation: the SDS– PAGEs confirmed the digestion by showing the disappearance of the coloured gel bands. Since ACE-inhibitory peptides are generally small, the hydrolysates were filtered through a 3000 Da cut-off membrane. The intact protein was tested only once in the case of lupin and showed no activity, as expected (Aluko, 2008). In order to compare the activities of the different hydrolysates, in our view it is necessary to know the concentration of the peptides formed by enzymatic hydrolysis, although the majority of available literature references do not give indications on this parameter. In this work a colorimetric assay optimised for peptides was used (Goa, 1953; Levashov et al., 2009). The ACE-inhibitory activity assay was performed after having equalised the peptide concentration of all samples in order to get a reliable comparison of their activities. Fig. 2 shows two exemplary charts obtained plotting the % ACE inhibition vs. log10 inhibitor concentrations for soybean TPE (Fig. 2A), and lupin TPE (Fig. 2B). Very good fits with sigmoid curves and reproducible IC50 values were obtained. The maximum percentage of ACE inhibition and the IC50 values are reported in Table 1. The results indicate that there are significant differences (p < 0.05) in ACE-inhibitory activity of the pepsin hydrolysates of TPE from different legumes. The hydrolysates from soybean and both lupin species (L. albus and L. angustifolius) are the most efficient ACE inhibitors having the lowest IC50. In detail, L. angustifolius is equivalent to soybean, whereas L. albus is slightly less active; lentil and pea have intermediate activities, with IC50 values of 595 and 606 lg/ml, respectively; whereas common bean and chickpea are the least active. Previous investigations on pulses were primarily focussed on soybeans (Wu & Ding, 2002), but recent papers have dealt also with peas, chickpeas, lentils, and common beans (Akillioglu & Karakaya, 2009; Aluko, 2008; Roy et al., 2010). The comparison of our data with those obtained by other authors is a complex task, since the observed differences in the ACE-inhibitory activity may be related to a variety of causes, such as the sample and the protein extraction procedure, as well as the differences in the peptide mixture composition, related to changes in the parameters of the digestion process (enzyme, pH, time, temperature, substrate/enzyme ratio, etc.) (Barbana & Boye, 2010), or the different analytical method used for the determination of the ACE-inhibitory activity. For example, the IC50 value of chickpeas (673 lg/ml) is much higher than those reported in previous studies, in which the digestion of two varieties of chickpea had been performed with alcalase/ flavourzyme, papain, and pepsin/trypsin/a-chymotrypsin (Barbana & Boye, 2010; Pedroche et al., 2002; Yust et al., 2003). On the contrary, the IC50 value for common beans (633 lg/ml) is in agreement with the values reported in a previous study (Akillioglu & Karakaya, 2009), in which common bean proteins had been hydrolyzed with pepsin: 780–830 lg/ml. The literature lentil IC50 values vary in a wide range: 8–890 lg/ml, depending on the sample (green or red lentils), enzyme, and any thermal treatment applied before the digestion (Akillioglu & Karakaya, 2009; Boye, Roufik, Pesta, & Barbana, 2010). Our pea protein hydrolysate showed lower activity than that observed in previous studies in which pea proteins had been digested with the same or other enzymes (Aluko, 2008; Boye et al., 2010; Roy et al., 2010). Literature data on ACE-inhibitory activity of soybean hydrolysates are numerous and our experimental data are in good agreement with the majority of them (Chen et al., 2004; Guang & Phillips, 2009; Wu & Ding, 2002). On the contrary, data on lupin are very poor: the only available IC50 value, related to a pepsin/pancreatin hydrolysate, is 290 lg/ml, in agreement with our data (Yoshie-Stark, Bez, Wada, & Waesche, 2004).

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3.2. ACE-inhibitory activity of peptides from lupin protein isolates Lupin storage proteins consist of two major globulins, a and b conglutin, and two minor globulins, c and d conglutin (Sironi et al., 2005). Considering the promising results obtained with lupin peptides and aiming to identify the protein(s) that had released the peptides responsible for the hypotensive activity, it was decided to purify these globulins from the total protein extract using a fractionation procedure described in the literature, based on extraction and precipitation stages at different pH values (Sironi et al., 2005). This enabled us to obtain three samples: a mixture of a + b conglutin, c conglutin (named as c conglutin 1 in Table 2), and d conglutin. Additionally, a chromatographic separation method recently optimised by our group (Resta et al., 2012) enabled us to obtain another sample of c conglutin (named as c conglutin 2 in Table 2). The ACE-inhibitory activity parameters (maximum ACE inhibition percentage, and IC50 values) of peptide mixtures obtained from these samples are reported in Table 2. Clearly, the active component was the a + b conglutin mixture, which was more active than the total lupin protein extract (Table 1), whereas both c conglutin samples and d conglutin were inactive (IC50 values could not be calculated because the 50% of ACE inhibition was not reached even at the highest concentration). Further investigations were performed on the peptides obtained from some industrial isolates from L. albus, i.e. a total protein isolate (LPI) and two lupin protein isolates called E (LPI-E), able to stabilise emulsions, and F (LPI-F), useful to stabilize foams, whose production and physico-chemical characteristics were described in a previous paper (D’Agostina et al., 2006). LPI contains mostly globulins, LPI-E, separated by alkaline extraction and isoelectric precipitation, comprises mostly a and b conglutin, whereas LPI-F, recovered by acid extraction and ultrafiltration, is enriched in c conglutin. Fig. 2C shows the chart obtained plotting the% ACE inhibition vs. log10 inhibitor concentrations for the peptides derived from LPI-E, showing the good fit obtained with the sigmoid curve. The ACE-inhibitory activities of peptides from the industrial isolates were reported in Table 2. Surprisingly, the activity of peptides from the industrial lupin total protein isolate LPI was higher than that of the peptides obtained from the TPE prepared in laboratory (Lupin 1 in Table 1). Possibly the spray-drying treatment may have transformed the proteins (perhaps with an unfolding process), enabling pepsin to digest them in a different way, producing more active peptides. The experimental result that the spray-drying procedure has not damaged the ACE-inhibitory activity may be useful for practical applications. LPI-E, containing a mixture of a and b conglutin, is statistically equivalent to the laboratory a + b conglutin sample. On the contrary, a big difference was observed comparing the activities of the protein isolate LPI-F and the corresponding laboratory samples c conglutins 1 and 2. This is in agreement with SDS– PAGE analysis (data not shown) that revealed that LPI-F contains also relevant amounts of a and b conglutin, which may explain its higher activity in respect to highly purified samples of c conglutin (Resta et al., 2012). As already indicated, only incomplete data are available in the literature for lupin. The same group that has provided us these industrial samples, some years ago has independently published some preliminary data on the ACE-inhibitory activity of samples similar to LPI, LPI-E and LPI-F (Yoshie-Stark et al., 2004). In that case, the peptide mixtures were obtained using pepsin or pepsin and pancreatin and the ACE-inhibitory activity was determined at a single concentration by spectrophotometry. The results were expressed as the % ACE-inhibition activity achieved at 1 mg/ml sample concentration and the IC50 values were calculated, as indicated by the authors, from the linear increase of inhibition with

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increased amount of sample (Yoshie-Stark et al., 2004). Although a direct comparison with our data is impaired by the completely different methodological approaches, also in that case the ACEinhibition activity of LPI-E was significantly higher than that of LPI-F. 4. Conclusions The direct comparison of the pepsin hydrolysates from numerous legume seeds has shown that those from soybean and lupin are the most promising. In addition our work has demonstrated that a and b conglutin are the main sources of ACE-inhibitory peptides in lupin seed. Unfortunately, a direct comparison of our results with published data is very difficult, since each research group uses a very different experimental protocol. Up to now, only milk peptides have found practical applications as hypotensive food ingredients (Vermeirssen et al., 2004). Our work has demonstrated that also some plant proteins may become a valuable source of ACE-inhibitory peptides, which in the future may be used for the formulation of functional foods or dietary supplements for the prevention or treatment of hypertension. Acknowledgements The authors gratefully acknowledge Valentina Locaso for her precious experimental work, HPF Nutraceutics s.r.l. and Fraunhofer Institute IVV for the lupin samples. This research was supported by the project ‘‘Nutraceutici innovativi per la prevenzione delle malattie cardiovascolari’’ Art. 11 DM 593 (08/08/2000). References Akillioglu, H. G., & Karakaya, S. (2009). Effects of heat treatment and in vitro digestion on the angiotensin converting enzyme inhibitory activity of some legume species. European Food Research and Technology, 229, 915–921. Aluko, R. E. (2008). Determination of nutritional and bioactive properties of peptides in enzymatic pea, chickpea, and mung bean protein hydrolysates. Journal of AOAC International, 91, 947–956. Barbana, C., & Boye, J. I. (2010). Angiotensin I-converting enzyme inhibitory activity of chickpea and pea protein hydrolysates. Food Research International, 43, 1642–1649. Boye, J. I., Roufik, S., Pesta, N., & Barbana, C. (2010). Angiotensin I-converting enzyme inhibitory properties and SDS-PAGE of red lentil protein hydrolysates. LWT–Food Science and Technology, 43, 987–991. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 248–254. Brambilla, F., Resta, D., Isak, I., Zanotti, M., & Arnoldi, A. (2009). A label-free internal standard method for the differential analysis of bioactive lupin proteins using nano HPLC-chip coupled with ion trap mass spectrometry. Proteomics, 9, 272–286. Chen, J.-R., Yang, S.-C., Suetsuna, K., & Chao, J. C. J. (2004). Soybean protein-derived hydrolysate affects blood pressure in spontaneously hypertensive rats. Journal of Food Biochemistry, 28(1), 61–73. Cushman, D. W., & Cheung, H. S. (1971). Spectrophotometric assay and properties of the angiotensin-converting enzyme of rabbit lung. Biochemical Pharmacology, 20, 1637–1648. D’Agostina, A., Antonioni, C., Resta, D., Arnoldi, A., Bez, J., Knauf, U., et al. (2006). Optimization of a pilot-scale process for producing lupin protein isolates with valuable technological properties and minimum thermal damage. Journal of Agriculture and Food Chemistry, 54(1), 92–98. Goa, J. (1953). A micro biuret method for protein determination. Determination of total protein in cerebrospinal fluid. Scandinavian Journal of Clinical and Laboratory Investigation, 5, 218–222. Guang, C., & Phillips, R. D. (2009). Plant food-derived angiotensin I converting enzyme inhibitory peptides. Journal of Agriculture and Food Chemistry, 57(12), 5113–5120. Jivotovskaya, A. V., Senyuk, V. I., Rotari, V. I., Horstmann, C., & Vaintraub, I. A. (1996). Proteolysis of phaseolin in relation to its structure. Journal of Agriculture and Food Chemistry, 44, 3768–3772. Lam, L. H., Shimamura, T., Sakaguchi, K., Noguchi, K., Ishiyama, M., Fujimura, Y., et al. (2007). Assay of angiotensin I-converting enzyme-inhibiting activity based on the detection of 3-hydroxybutyric acid. Analytical Biochemistry, 364, 104–111. Lee, Y. P., Mori, T. A., Puddey, I. B., Sipsas, S., Ackland, T. R., Beilin, L. J., et al. (2009). Effects of lupin kernel flour-enriched bread on blood pressure: A controlled intervention study. American Journal of Clinical Nutrition, 89(3), 766–772.

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ACE-inhibitory activity of enzymatic protein hydrolysates from lupin and other legumes.

The objective of this investigation was to compare the angiotensin converting enzyme (ACE)-inhibitory activity of the hydrolysates obtained by pepsin ...
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