Optimization of the Enzymatic Hydrolysis of Lupin (Lupinus) Proteins

Article pubs.acs.org/JAFC

Optimization of the Enzymatic Hydrolysis of Lupin (Lupinus) Proteins for Producing ACE-Inhibitory Peptides Giovanna Boschin,*,† Graziana Maria Scigliuolo,‡ Donatella Resta,‡ and Anna Arnoldi†,‡ †

Department of Pharmaceutical Sciences, University of Milan, via Mangiagalli 25, 20133 Milano, Italy HPF Nutraceutics s.r.l., via Balzaretti 9, 20133 Milano, Italy

‡

ABSTRACT: Recently, the enzymatic hydrolysis of Lupinus albus and Lupinus angustifolius proteins with pepsin was showed to produce peptides able to inhibit the angiotensin-converting enzyme (ACE). The objective of the present work was to test different hydrolytic enzymes and to investigate three lupin species (L. albus, L. angustifolius, Lupinus luteus) with the final goal of selecting the best enzyme/species combination for an efficient production of ACE-inhibitory peptide mixtures. Pepsin gave peptides with the best IC50 values (mean value on three species 186 ± 10 μg/mL), followed by pepsin + trypsin (198 ± 16 μg/ mL), chymotrypsin (213 ± 83 μg/mL), trypsin (405 ± 54 μg/mL), corolase PP (497 ± 32 μg/mL), umamizyme (865 ± 230 μg/mL), and flavourzyme (922 ± 91 μg/mL). The three species showed similar activity scales, but after pepsin + trypsin and chymotrypsin treatments, L. luteus peptide mixtures resulted to be significantly the most active. This investigation indicates that lupin proteins may be a valuable source of ACE-inhibitory peptides, which may explain the activity observed in experimental and clinical studies and foresee the application of lupin proteins into functional foods or dietary supplements. KEYWORDS: enzymatic hydrolysis, functional foods, hypertension, lupin, nutraceutics

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INTRODUCTION Lupin seed represents a promising source of innovative food ingredients because it has a good protein content (34−43% of dry matter, competitive with soybean), an acceptable composition of essential amino acids,1 and an interesting content of some other relevant nutrients such as tocopherols and unsaturated fatty acids.2,3 Moreover, the former toxicological issue of lupin seeds, i.e., the presence of quinolizidine alkaloids (QAs), has been solved by the selection of lowalkaloid cultivars.4 Recent literature indicates that lupin consumption may provide some useful health benefits such as the reduction of systemic hypertension. The hypotensive effect of lupin protein has been assessed in vivo in Goto−Kakizaki rats, which develop hypertension when fed with a high-salt diet.5 The rats were fed with a 6% NaCl diet containing lupin protein isolates (20% weight/weight) for two weeks. At the end of the study, the systolic blood pressure (SBP) was 18.6 mmHg lower than in the control group.5 In an open study on subjects with moderate hypertension, the daily consumption of 35 g of lupin protein in a model beverage reduced the SBP by 9.5 mmHg (from 137.1 to 127.6 mmHg) after 1 month and 9.0 mmHg (from 137.1 to 128.1 mmHg) after 3 months, whereas it lowered the diastolic blood pressure by 3.0 mmHg (from 81.5 to 78.52 mmHg) and by 4.4 mmHg (from 81.50 to 77.14 mmHg), respectively.6 Finally, two long-term clinical studies showed that the consumption of bread enriched with lupin flour produced very small, but statistically significant, decreases of blood pressure with respect to the control bread in normotensive overweight or obese subjects.7,8 Hypertension is usually treated with drugs, for example, with angiotensin I converting enzyme (ACE; EC 3.4.15.1) inhibitors.9 They inhibit ACE, an enzyme that plays an important role in regulating blood pressure in the renin− © 2014 American Chemical Society

angiotensin system, because it catalyzes the conversion of the biologically inactive angiotensin I to the potent vasoconstrictor angiotensin II and inactivates the potent vasodilator bradykinin.10 Nonpharmacologic measures, such as changes in lifestyle and use of dietary supplements and functional foods, are encouraged in subjects with mild hypertension to avoid or delay the use of synthetic drugs that may produce side effects. Specific dietary ingredients are the ACE-inhibitory peptides produced by hydrolyzing the proteins from different animal or plant foods, in particular, milk, soy, pea, and other legumes.11−15 In a previous paper, we have compared the ACE-inhibitory activity of samples obtained by hydrolyzing the proteins from different legume seeds with pepsin and have shown that lupin is the most active, together with soybean.16 The generic term lupin actually indicates the main domesticated and cultivated species: Lupinus albus (white lupin), Lupinus angustifolius (narrow-leaf lupin), and Lupinus luteus (yellow lupin). The objectives of the present investigation were to test different enzymes for preparing peptide mixtures and to investigate three lupin species, L. albus, L. angustifolius, and L. luteus, as substrates. The final goal is selecting the best species/ enzyme combinations for an efficient production of ACEinhibitory peptide mixtures, which may be possibly used in innovative functional foods or dietary supplements. Although numerous papers have reported the ACE-inhibitory activity of peptides from different foods, lupin is still an unexplored Received: Revised: Accepted: Published: 1846

September 3, 2013 January 23, 2014 January 31, 2014 January 31, 2014 dx.doi.org/10.1021/jf4039056 | J. Agric. Food Chem. 2014, 62, 1846−1851

Journal of Agricultural and Food Chemistry

Article

(containing 0.6 M sodium citrate, 0.9 M sodium carbonate, and 0.07 M copper sulfate, 2.4 M NaOH, pH 10.6) was prepared. The reaction mixture was carefully mixed, incubated for 15 min at 20 °C, and then the absorbance was measured at 330 nm. A sterile solution of peptone from casein at 10 mg/mL in water was used as standard for the calibration curve; the assay is linear in the range 100−1000 μg of peptides in cuvette. ACE-Inhibitory Activity Assay. After hydrolysis and filtration, all samples were tested for determining their ACE-inhibitory activity, as recently reported.16 Briefly, 100 μL of 2.5 mM hippuryl-histidylleucine (HHL) in buffer 1 (100 mM Tris-HCOOH, 300 mM NaCl pH 8.3) was mixed with 30 μL of sample in buffer 1 at different concentrations. Usually, six concentrations were used for each sample, and each solution was tested twice. Samples were preincubated at 37 °C for 15 min, then 15 μL of ACE solution, corresponding to 3 mU of enzyme in buffer 2 (100 mM Tris-HCOOH, 300 nM NaCl, 10 μM ZnCl2, pH 8.3), were added; samples were incubated for 60 min at 37 °C. The reaction was stopped with 125 μL of 0.1 M HCl. The aqueous solution was extracted twice with 600 μL of ethyl acetate; the solvent was evaporated at 95 °C and the residue was dissolved in 500 μL of buffer 1 and then analyzed by HPLC. 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 mm × 250 mm, 5 μm; Grace, Italy) using water and acetonitrile as solvent and following the gradient: 0 min 5% acetonitrile, 10 min 60% acetonitrile, 12 min 60% acetonitrile, 15 min 5% acetonitrile. Injection volume was 10 μL, wavelength 228 nm, flow 0.5 mL/min. The evaluation of ACE inhibition was based on the comparison between the concentrations of HA (hippuric acid) in the presence or absence of an estimated inhibitor. The phenomenon of autolysis of HHL to give HA was evaluated by a reaction blank, i.e., a sample with the higher evaluated inhibitor concentration and without the enzyme. The percentage of ACE inhibition was computed considering the area of HA peak with the following formula:

substrate, also considering the clinical and experimental data reported above.

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MATERIALS AND METHODS

Chemicals. The commercial sources of proteases were as follows: pepsin (from porcine gastric mucosa; EC 3.4.23.1; commercial code P6887), trypsin (from bovine pancreas; EC 3.4.21.4; commercial code T1426), chymotrypsin (from bovine pancreas; EC 3.4.21.1; commercial code C4129), and flavourzyme (from Aspergillus oryzae; EC 232.752.2; commercial code P6110) from Sigma-Aldrich (St. Louis, MO, USA), corolase PP (from porcine pancreas; EC 3.4.21.4; commercial code 89082) from AB Enzymes (Darmstadt, Germany), and umamizyme (from Aspergillus oryzae; EC not specified; declared activity of 74.3 U/g) from Amano Enzyme Inc. (Nagoya, Japan). Amicon Ultra-0.5 filters (centrifuge tubes) were bought from Millipore (Billerica, MA, USA). HPLC-grade water was prepared with a Milli-Q purification system (Millipore). Bio-Safe Coomassie and Precision Plus Protein Standards Dual Color marker for SDS-PAGE were from Biorad (Biorad Laboratories Inc., Hercules, CA). All other chemicals (reagents and solvents) were from Sigma-Aldrich. Sampling. Lupin seeds of the species L. albus (cultivar Ares) were provided by Terrena (Matrignè-Ferchaud, France), L. angustifolius (cultivar Boregine) by Fraunhofer institute IVV (Freising, Germany), and L. luteus (cultivar Mister) by Prof. Biagina Chiofalo (University of Messina, Messina, Italy). TPE Preparation. The total protein extracts (TPEs) of the lupin seeds were obtained as previously reported17 and stored at −20 °C. Briefly, proteins were extracted from defatted flour with 100 mM TrisHCl/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, using bovine serum albumin (BSA) as standard.18 Enzymatic Hydrolysis. TPEs were dissolved in Tris-HCl buffer 100 mM, pH 8. The pH value were adjusted to the optimal hydrolysis conditions for each enzyme with the addition of 1 M NaOH or 1 M HCl, if necessary. The enzymes were dissolved in appropriate solutions: 30 mM NaCl for pepsin, and 1 mM HCl for trypsin, chymotrypsin, and corolase PP. Umamizyme was added as a powder to the TPEs, whereas flavourzyme was a ready-to-use solution. Both enzyme solutions and the umamizyme powder were directly added to the TPE solutions. Reaction mixtures were incubated at a fixed temperature for different reaction times. For the two-stage digestion with pepsin + trypsin, TPEs were initially incubated with pepsin at pH 2 for 4 h, the pH was adjusted to 8 with 1 M NaOH, and trypsin was added to the reaction mixture. Finally, pepsin was inactivated by adjusting the pH to 7 with 1 M NaOH, whereas all other enzymes were inactivated by heating the solutions at 100 °C for 10 min. Then the solutions were cooled and stored at −20 °C. SDS-PAGE. Drawings were taken at various intervals and analyzed by SDS-PAGE. They were separated on 15% SDS-polyacrylamide gel using Mini Protean 3 Cell (Biorad Laboratories Inc., Hercules, CA). 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 analyzed with Quantity One 4.6.8 Software (Biorad). Ultrafiltration. After the hydrolysis, all low-molecular-weight peptides were separated from intact enzymes, proteins, and highmolecular-weight polypeptides by ultrafiltration through 3000 Da cutoff centrifuge filters (Amicon Ultra-0.5, Millipore, Billerica, MA, USA) at 12000g for 30 min at 4 °C. The permeates were then stored at −20 °C. Determination of Peptide Concentration. The peptide concentration in the hydrolyzed samples was measured according to literature methods19,20 based on chelating the peptide bonds by Cu(II) in alkaline media and monitoring the change of absorbance at 330 nm. In brief, a solution of X μL peptide mixture, (500 − X) μL of water, 500 μL of 6% (w/w) NaOH in water, and 50 μL of active reagent

⎡ (A − A ) ⎤ N % ACE inhibition = ⎢ IB ⎥ × 100 ⎣ (AIB − ARB) ⎦ where AIB is the area of HA in inhibitor blank (IB) sample (i.e., sample with enzyme but without any estimated inhibitor), AN is the area of HA in the n samples containing different amounts of the estimated inhibitor (in our case the hydrolyzate), and ARB is the area of HA in the reaction blank (RB) sample (i.e., sample without enzyme and with the estimated inhibitor at the highest concentration). The percentages of ACE inhibition were plotted vs the peptide concentrations. When the maximum ACE-inhibition concentration was over 50%, the percentages of ACE-inhibition were plotted vs the log10 peptide concentrations obtaining a sigmoid curve. This curve permitted to calculate the inhibitory concentration 50% (IC50), i.e., the concentration needed to observe a 50% inhibition of ACE activity. The IC50 values were obtained testing independently each sample three times and are expressed as mean value ± standard deviation. The most active peptides have the lowest IC50 values. Statistical Analysis. Statistical analyses were performed with Statgraphics Plus (version 2.1 for Windows). The data were evaluated using one-way analysis of variance followed by Fisher’s least significant difference (LSD) procedure; values with different letters are significantly different for p < 0.05.

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RESULTS AND DISCUSSION

In a preliminary part of the present investigation, we tested the total protein extract from the seeds of L. albus before hydrolysis. As expected, it resulted to be inactive as ACEinhibitor, according to literature.13,21 In fact, owing to the dimension of the active site of the enzyme, the interaction may take place only when bioactive peptides are released from the parent protein by enzymatic or hydrolytic reactions, such as digestion, or industrial processing, such as fermentation. 1847

dx.doi.org/10.1021/jf4039056 | J. Agric. Food Chem. 2014, 62, 1846−1851


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Preparation of Enzymatic Hydrolyzates. The first part of the experimental work was devoted to establish the best reaction conditions for each proteolytic enzyme. Drawings were taken at various intervals and analyzed by SDS-PAGE in order to evaluate the hydrolysis efficiency, changing the following parameters: enzyme/substrate (E/S) ratio, pH, temperature, and reaction time. In the case of pepsin, the best pH was 2, the temperature 37 °C, the E/S ratio 1/100, and 18 hours were necessary to complete the reaction. Figure 1a shows the SDS-

Figure 2. Total protein extract of Lupinus albus seed digested with pepsin: comparison between the ACE inhibition curves obtained filtering with 10000 and 3000 Da cutoff membranes.

Table 1. Optimized Parameters for the Hydrolysis of the Lupin Total Protein Extract: Enzyme, pH, Temperature (°C), Enzyme/Substrate Ratio (E/S ratio), and Time (h) enzyme

pH

temperature (°C)

E/S ratio (w/w)

time (h)

pepsin trypsin chymotrypsin corolase PP umamizyme flavourzyme

2 8 8 8 7 7

37 37 37 37 45 60

1/100 1/100 1/100 1/50 1/20 1/20

18 18 18 18 24 24

After the digestion, the samples were ultrafiltered in order to separate low-molecular-weight peptides from high-molecular ones and intact enzymes. To select the best filtration conditions for maximizing the ACE-inhibitory activity, the digested mixture obtained by hydrolyzing L. albus protein with pepsin was ultrafiltered either with 10000 or 3000 Da cutoff membranes. Figure 2 shows two ACE inhibition curves obtained by plotting the peptide concentration vs the percentage ACE-inhibition for the samples with molecular weight (MW) lower than 3000 or 10000 Da, respectively. Whereas the latter sample did not reach the 50% ACE-inhibition even at the highest tested concentration (1110 μg/mL), the former, having molecular weight less than 3000 Da, was much more active, showing a 87% ACE-inhibition at the concentration of 893 μg/mL. A possible explanation is that the sample having MW lower than 10000 Da contains numerous inactive peptides (with MW in the range 3000 and 10000 Da), which decrease the concentration of the active peptides. Low MW peptides, usually under 3000 Da, are known as ACE-inhibitors in vivo because they can be absorbed at gut level.21 After these results, all subsequent experimentation was performed on hydrolyzed mixtures filtered through a 3000 Da cutoff membrane. Evaluation of ACE-Inhibitory Activity. The ACEinhibitory activity assays were performed after having equalized the peptide concentrations of all samples. The peptide concentration was determined by using a literature colorimetric assay optimized for peptides.19,20 In our opinion, the evaluation of the peptide concentrations and their equalization before performing the ACE-inhibitory test are of outmost importance in order to get a reliable comparison of their activities. The results of the ACE inhibition assays (maximum percentage of ACE inhibition and IC50 value) of the peptide mixtures from L. albus, L. angustifolius, and L. luteus are reported in Tables 2, 3, and 4, respectively.

Figure 1. SDS-PAGE obtained after digestion of: a) Lupinus albus (A) and Lupinus luteus (L) proteins with pepsin, at 37 °C with enzyme/ substrate (E/S) ratio 1/100; drawings collected immediately after enzyme addition (A0 and L0), after 1 h (A1h and L1h), and after 18 h (A18h and L18h). b) Lupinus angustifolius proteins digested with pepsin + trypsin drawings collected immediately after pepsin addition (P0), after 1 h (P1h), after 4 h (P4h), immediately after trypsin addition (Tro), after 1 h (Tr1h), after 24 h (overnight, Tro/n). M is the marker.

PAGEs of the drawings of the hydrolysis of L. albus (A) and L. luteus (L) proteins collected immediately after the enzyme addition (A0 and L0), after 1 h (A1h and L1h), and after 18 h (A18h and L18h). In general, the optimal conditions for using trypsin are 37 °C and pH 7−8; in our experience, the best pH value for hydrolyzing lupin proteins is 8. With an E/S ratio of 1/100, the digestion was complete after 18 h. The same conditions were used also for chymotrypsin. The double digestion with the sequence pepsin + trypsin was performed treating with pepsin at pH 2 for 4 h and then, after changing the pH value to 8, adding trypsin and incubating for 24 h. Figure 2b shows the SDS-PAGE of the two-step digestion. For corolase PP digestion, better results were obtained by changing the E/S ratio to 1/50. For flavourzyme and umamizyme, several changes were performed; the reaction time was extended to 24 h and the E/S ratio was changed to 1/ 20 because both 1/50 and 1/100 ratios were ineffective. In addition, the incubation temperatures were increased to 45 and 60 °C, respectively. Table 1 reports the final conditions used for producing the samples used in the following experimentations. 1848



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umamizyme and flavourzyme give the least active samples (Tables 2, 3, 4). Pepsin gives always peptide mixtures with the highest ACEinhibitory activity, with a mean IC50 value among the three species of 186 ± 10 μg/mL. When a trypsin treatment follows the pepsin one, for mimicking the physiological digestion, the ACE inhibition does not improve, but instead slightly decreases (mean value 198 ±16 μg/mL), even if no significant differences are revealed towards pepsin digestion (same letter in the statistical analysis for each species in Tables 2, 3, 4). Also chymotrypsin gives samples with good ACE-inhibitory activities with a mean value of 213 ± 83 μg/mL. Lower activities were observed treating the proteins with trypsin and corolase PP, with mean IC50 values of 405 ± 54 μg/mL and 497 ± 32 μg/ mL, respectively. The other two enzymes gave even less satisfactory results: umamizyme gave a mean IC50 value of 865 ± 230 μg/mL and flavourzyme of 922 ± 91 μg/mL. Figure 3 enables a direct comparison of the three species. The statistical analysis indicates that the treatment of the proteins of the three lupin species with pepsin and corolase PP produces peptide mixtures whose activities do not depend on the species, whereas by treating with trypsin, pepsin + trypsin as well as with chymotrypsin, significant differences are observed, the peptides from L. luteus being the most active. Also with umamyzime, the peptides from L. luteus were significantly the most active, whereas the peptides from L. angustifolius were the best after proteolysis with flavourzyme. The comparison of data from different papers is a complex task because the differences in the ACE-inhibitory activity may be related to several different causes such as the protein extraction procedure, the enzyme selected for the proteolysis, the parameters of the digestion process (substrate/enzyme ratio, pH, time, temperature), or the different analytical method used for the determination of the ACE-inhibitory activity.22 As already indicated in the introduction, very few data are available on ACE-inhibitory activity of lupin proteins. In a previous paper by our group, L. albus and L. angustifolius proteins were digested with pepsin showing an ACE-inhibitory activity similar to soybean and higher than other legumes (pea, common bean, lentils, chickpea).16 Bioactive peptides are usually formed by sequences of 3−20 amino acids encrypted within the primary sequence of proteins. During food processing, fermentation, and/or gastrointestinal digestion, they are released through proteolysis of the parent protein. Although the complexity of the tested hydrolyzates impairs their direct comparison, it is clear that the observed activities reflect the different specificities of the tested enzymes. Pepsin and chymotrypsin are specific endopeptidases, which mainly break the peptidic bonds involving hydrophobic and aromatic amino acids; for this reason they produce peptides having a hydrophobic and/or aromatic amino acid as terminal group, preferably, proline, tryptophan, phenylalanine, or tyrosine. These features are particularly suitable for interacting with the ACE catalytic site, thus permitting the inhibition.21 Trypsin, instead, is a specific endopeptidase that cleaves mainly peptidic bonds involving basic amino acids.23 Combining pepsin + trypsin in a two-stage process resulted in a small overall reduction of ACE-inhibitory activity. Possibly, the active peptides formed during the pepsin digestion are in part degraded by trypsin. The same explanation was proposed for other sequential enzymatic treatments impairing the ACEinhibitory activity: in particular, the two-step digestion with

Table 2. Results of ACE Inhibition Assay for Lupinus albus Proteins: Digestion Enzyme, Highest Inhibitor Concentration (μg/mL), Maximum ACE Inhibition (%), and IC50 Value (μg/mL)a enzyme pepsin trypsin pepsin + trypsin chymotrypsin corolase PP umamizyme flavourzyme

highest inhibitor concentration (μg/mL)

max ACE inhibition (%)

IC50 (μg/mL)

856 939 908

76 ± 6.3 67 ± 4.3 84 ± 2.5

197 ± 1.6a 427 ± 16.8c 205 ± 4.5a

989 1039 1066 1037

73 60 58 51

± ± ± ±

3.0 4.7 2.6 0.6

301 488 1053 930

± ± ± ±

4.8b 42.8c 77.8e 3.71d

IC50 values are reported as mean value ± standard deviation of three independent experiments; values with different letters are significantly different (p < 0.05).

a

Table 3. Results of ACE Inhibition Assay for Lupinus angustifolius Proteins: Digestion Enzyme, Highest Inhibitor Concentration (μg/mL), Maximum ACE Inhibition (%), and IC50 Value (μg/mL)a enzyme pepsin trypsin pepsin + trypsin chymotrypsin corolase PP umamizyme flavourzyme



IC50 (μg/mL)

723 614 1047

80 ± 3.9 58 ± 3.2 82 ± 3.2

185 ± 13.3a 446 ± 17.5b 210 ± 1.9a

894 953 990 1151

79 67 53 55

± ± ± ±

4.6 2.3 1.5 2.8

203 470 933 827

± ± ± ±

5.9a 27.5b 0.7d 41.9c


a

Table 4. Results of ACE Inhibition Assay for Lupinus luteus Proteins: Digestion Enzyme, Highest Inhibitor Concentration (μg/mL), Maximum ACE Inhibition (%), and IC50 Value (μg/mL)a enzyme pepsin trypsin pepsin + trypsin chymotrypsin corolase PP umamizyme flavourzyme



IC50 (μg/mL)

1014 999 999

84 ± 3.7 72 ± 3.5 83 ± 4.0

176 ± 2.3a 343 ± 28.1b 181 ± 2.4a

1014 896 1232 1081

87 66 67 54

± ± ± ±

4.3 2.6 3.9 3.8

136 533 608 1009

± ± ± ±

4.5a 5.7c 64.1d 33.0e


a

IC50 values range from 136 μg/mL in the case of the L. luteus sample treated with chymotrypsin to 1053 μg/mL in the case of the L. albus sample treated with umamizyme. Comparing the three species, generally, the effects of the enzymes on the three lupin species are comparable: pepsin, followed by pepsin + trypsin, and chymotrypsin samples give the lowest IC50 values, i.e., the highest ACE-inhibitory activities, whereas corolase PP gives intermediate IC50 values and 1849



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Figure 3. Comparison of mean values of ACE-inhibition IC50 values (expressed in μg/mL) of peptides mixtures obtained from Lupinus albus, Lupinus angustifolius, and Lupinus luteus proteins digested with pepsin, trypsin, pepsin + trypsin, chymotrypsin, corolase PP, umamizyme, and flavourzyme. Values with different letters are significantly different (p < 0.05).

Of course, this is only a first step in the study of ACEinhibitory activity of lupin peptides because other studies are necessary to determine their stability and bioavailability. In fact, an open question is their capability to survive to the gastrointestinal digestion and to be absorbed in order to reach the bloodstream in their active form. Indeed, the literature shows that peptides from other food sources (for example milk) are systemically distributed.21,30 The problem could be overcome also by using new emerging technologies, such as micro- or nanoencapsulation, that may offer feasible solutions for improving the stability of peptides in various food products and during digestion.21,30 The bioavailability of ACEinhibitory peptides may also be increased by cross-linking the target peptide to protein transduction domains or by means of specific peptide carriers able to deliver biologically active peptides into cells. Moreover, peptide permeation may be achieved by chemical enhancers and surfactant-like agents.31 Finally, further studies are needed to determine the composition of active peptide mixtures, to isolate the peptide(s) responsibles for ACE-inhibitory activity, and to confirm their activity through in vitro and in vivo studies.

alcalase and flavourzyme performed on common bean protein and chicken protein isolates.24,25 Corolase PP, a patented enzymatic complex used to simulate the in vitro protein digestion in the intestinal tract, consists of a mixture of proteolytic enzymes from pig pancreas, containing trypsin and chymotrypsin, together with other aminopeptidases and carboxypeptidases. Its moderate ACE-inhibitory activity may be related to the absence of pepsin that seems to be the best enzyme to produce lupin peptides with good ACEinhibitory activity. Obviously, this enzyme may give good results with other proteins; for example, satisfactory results were obtained on sea cucumber proteins digested with pepsin and subsequently with corolase PP.26 Flavourzyme and umamizyme are “generally recognized as safe” (GRAS) enzymes used in the food industry. They are food-grade fungal protease/peptidase complexes produced by submerged fermentation of a strain of Aspergillus oryzae, exhibiting both endoprotease and exopeptidase activities. They were tested with the final aim of a large-scale production of hydrolyzates to be used as food supplements production but did not provided useful results. In the literature, flavourzyme digestion was applied to cowpea proteins, giving peptides with IC50 values in the large range 0.04−170.6 μg/mL and to rapeseed protein producing peptides with an ACE-inhibitory activity of about 50%.27,28 Moreover a double digestion with alcalase/flavourzyme on two different chickpea varieties gave peptide mixtures with IC50 values of 316 and 228 μg/mL, while on yellow pea proteins gave an IC50 value of 412 μg/mL.22 Umamizyme was used to digest canola meal proteins, giving peptide mixtures with low ACE-inhibitory activity.29 Unfortunately, with lupin proteins, they give lower ACE-inhibitory activity than the other tested enzymes. In conclusion, this investigation confirms that lupin seed may be a valuable source of ACE-inhibitory peptides, possibly explaining the activity observed in experimental and clinical studies.5−8 This may foresee their future applications into either functional foods or dietary supplements.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +39-02-50318210. Fax: +39-02-50318202. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to Mariantonietta Loredana Mesce for her precious help in experimental work, Biagina Chiofalo for L. luteus seeds, the Fraunhofer Institute IVV for L. angustifolius seeds, Terrena for L. albus seeds, AB enzymes for the gift of corolase PP, Giovanna Speranza for the gift of umamizyme and 1850



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flavourzyme, and Andrea Meregaglia for enzymatic digestions advices.

(18) Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein−dye binding. Anal. Biochem. 1976, 72, 248−254. (19) Levashov, P. A.; Sutherland, D. S.; Besenbacher, F.; Shipovskov, S. A robust method of determination of high concentrations of peptides and proteins. Anal. Biochem. 2009, 395, 111−112. (20) Goa, J. A micro biuret method for protein determination. Determination of total protein in cerebrospinal fluid. Scand. J. Clin. Lab. Invest. 1953, 5, 218−222. (21) Vermeirssen, V.; van Camp, J.; Verstraete, W. Bioavailability of angiotensin I converting enzyme inhibitory peptides. Br. J. Nutr. 2004, 92, 357−366. (22) Roberts, P. R.; Burney, J. D.; Black, K. W.; Zaloga, G. P. Effect of chain length on absorption of biologically active peptides from the gastrointestinal tract. Digestion 1999, 60, 332−337. (23) Rui, X.; Boye, J. I.; Simpson, B. K.; Prasher, S. O. Angiotensin Iconverting enzyme inhibitory properties of Phaseolus vulgaris bean hydrolysates: effects of different thermal and enzymatic digestion treatments. Food Res. Int. 2012, 49, 739−746. (24) Pedroche, J.; Yust, M. M.; Giron-Calle, J.; Alaiz, M.; Millan, F.; Vioque, J. Utilisation of chickpea protein isolates for production of peptides with angiotensin I-converting enzyme (ACE)-inhibitory activity. J. Sci. Food Agric. 2002, 82, 960−965. (25) Barbana, C.; Boye, J. I. Angiotensin I-converting enzyme inhibitory activity of chickpea and pea protein hydrolysates. Food Res. Int. 2010, 43, 1642−1649. (26) Pérez-Vega, J. A.; Olivera-Castillo, L.; Gómez-Ruiz, J. Á .; Hernández-Ledesma, B. Release of multifunctional peptides by gastrointestinal digestion of sea cucumber (Isostichopus badionotus). J. Funct. Foods 2013, 5, 869−877. (27) Segura Campos, M. R.; Chel Guerrero, L. A.; Betancur Ancona, D. A. Angiotensin-I converting enzyme inhibitory and antioxidant activities of peptide fractions extracted by ultrafiltration of cowpea Vigna unguiculata hydrolysates. J. Sci. Food Agric. 2010, 90, 2512− 2518. (28) He, R.; Alashi, A.; Malomo, S. A.; Girgih, A. T.; Chao, D.; Ju, X.; Aluko, R. E. Antihypertensive and free radical scavenging properties of enzymatic rapeseed protein hydrolysates. Food Chem. 2013, 141, 153− 159. (29) Wu, J.; Aluko, R. E.; Muir, A. D. Production of angiotensin Iconverting enzyme inhibitory peptides from defatted canola meal. Biores. Technol. 2009, 100, 5283−5287. (30) Korhonen, H. Milk-derived bioactive peptides: from science to applications. J. Funct. Foods 2009, 1, 177−187. (31) Hong, F.; Ming, L.; Yi, S.; Zhanxia, L.; Yongquan, W.; Chi, L. The antihypertensive effect of peptides: a novel alternative to drugs? Peptides 2008, 29, 1062−1071.

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ABBREVIATIONS USED ACE, angiotensin-converting enzyme; BSA, bovine serum albumin; E/S, enzyme/substrate ratio; HA, hippuric Acid; HHL, hippuryl-histidyl-leucine; HL, histidyl-leucine; IC50, inhibitory concentration 50%; MW, molecular weight; PP, from porcine pancreas; SDS-PAGE, sodium dodecyl sulfate− polyacrylamide gel electrophoresis; TPE, total protein extract

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REFERENCES

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Optimization of the Enzymatic Hydrolysis of Lupin (Lupinus) Proteins

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