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Angiotensin I-converting enzyme inhibitory properties and SDS-PAGE of red lentil protein hydrolysates
LWT - Food Science and Technology 43 (2010) 987–991
Contents lists available at ScienceDirect
LWT - Food Science and Technology journal homepage: www.elsevier.com/locate/lwt
Research Note
Angiotensin I-converting enzyme inhibitory properties and SDS-PAGE of red lentil protein hydrolysates Joyce Irene Boye a, *, Samira Roufik b, Noemie Pesta c, Chockry Barbana a a
Food Research and Development Centre, Agriculture and Agri-Food Canada, Casavant Blvd. West, St Hyacinthe, QC J2S 8E3, Canada Health Canada, Ottawa, Canada c Universite´ de Reims Champagne-Ardenne, 9 Boulevard de la Paix, Reims, Champagne-Ardenne, France b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 May 2009 Received in revised form 14 January 2010 Accepted 15 January 2010
Several research studies have shown that protein hydrolysates from milk and soy contain peptides that possess angiotensin I converting enzyme (ACE) inhibitory properties and may help to prevent hypertension. To date, no studies have been conducted to determine if red lentil (Lens culinaris) proteins contain peptides with ACE-inhibitory properties. The objective of the present work was to characterize the proteins present in red lentils and determine if tryptic hydrolysis could liberate peptides with ACEinhibitory properties. Red lentil protein extracts were prepared and fractionated to obtain enriched albumin, legumin and vicilin fractions. Protein/peptide profiles were studied by electrophoresis and ACEinhibitory activity was measured using the HPLC hippuryl-His-Leu (HHL) substrate method. Our results revealed that red lentil protein hydrolysates posses ACE-inhibitory properties. Furthermore, we demonstrated that the ACE-inhibitory property of the hydrolysates varied as a function of the protein fraction with the total lentil protein hydrolysate having the lowest half maximal inhibitory concentration (IC50) (111 1 mmol/L) (i.e., highest ACE-inhibitory activity), followed by the enriched legumin (119 0.5 mmol/L), albumin (127 2 mmol/L) and vicilin (135 2 mmol/L) fractions, respectively. Crown Copyright Ó 2010 Published by Elsevier Ltd. All rights reserved.
Keywords: ACE-inhibitory activity Bioactivity Protein hydrolysate Pulse Red lentils
1. Introduction A major risk factor for developing cardiovascular disease, one of the leading causes of death in North America, is elevated blood pressure. Angiotensin II, a potent vasoconstrictor, is a major contributor to high blood pressure. Vasoconstriction occurs when rennin, an enzyme liberated by the kidneys, proteolytically acts on circulating angiotensinogen and converts it to angiotensin I (a decapeptide). In the presence of angiotensin converting enzyme (ACE), angiotensin I is cleaved to the octapeptide, angiotensin II resulting in arterial constriction and blood pressure elevation. ACE also breaks down bradykinin, a vasodilator, further contributing to the elevation in blood pressure. Inhibition of ACE is, therefore, important for the lowering of blood pressure as this results in a decrease in the concentration of angiotensin II and an increase in the levels of bradykinin (Erdos, 1975; Yang, Erdos, & Levin, 1970). ACE is a dipeptidyl carboxypeptidase (EC3.4.15.1) and many studies in recent years have focused on identifying compounds that can inhibit its activity (Quiros et al., 2007; Tsai, Chen, Pan, Gong, &
* Corresponding author. Tel.: þ1 450 768 3232; fax: þ 1 450 773 8461. E-mail address: [email protected] (J.I. Boye).
Chung, 2008; Vermeirssen, Van Camp, & Verstraete, 2002; Wu & Ding, 2002). Various high blood pressure medications available on the market today are targeted towards ACE inhibition. Examples of such drugs include Captopril, Enalapril, Ramipril, and Quinapril (Accupril). Due to potential side effects of pharmaceutical drugs, there is increased interest to identify foods that naturally contain peptides with hypotensive properties. Several research studies have shown that protein hydrolysates from certain plant and animal sources contain peptides that demonstrate ACE-inhibitory properties and may help to prevent hypertension (Hartmann & Meisel, 2007; Wu & Muir, 2008; Yang, Yang, Chen, & Chen, 2008). Recently, some studies reported novel antihypertensive peptides in fermented milk products (Chen, Tsai, & Pan, 2007; Quiros et al., 2007; Tsai et al., 2008). In other studies, ACE-inhibitory activity after hydrolysis of different milk protein products with a variety of enzymes has been reported (Otte, Shalaby, Zakora, Pripp, & ElShabrawy, 2007). Plant proteins such as soy, chickpea and pea, have also been shown to contain ACE-inhibitory peptides (Aluko, 2008; Chiang, Tsou, Tsai, & Tsai, 2006; Kuba, Tanaka, Tawata, Takeda, & Yasuda, 2003; Wu & Ding, 2002). To date, however, no studies have been conducted to determine if red lentil proteins contain peptides with ACE-inhibitory properties. IC50 values of 0.78–0.83, 0.15–0.69 and 0.008–0.89 mg protein/mL for common
0023-6438/$ – see front matter Crown Copyright Ó 2010 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2010.01.014
988
J.I. Boye et al. / LWT - Food Science and Technology 43 (2010) 987–991
Fig. 1. Schematic representation of the process used for the fractionation of the albumin, vicilin and legumin fractions from red lentil protein isolate. (Based on the method described by Gupta and Dhillon (1993)).
beans, pinto beans and green lentils, respectively, after in vitro gastrointestinal digestion have been very recently reported (Akıllıog˘lu & Karakaya, 2009). The major proteins found in lentils are albumins and globulins (Bhatty, 1988; Gupta & Dhillon, 1993). Legumins (molecular weight (MW) 350–400 kDa) and vicilins (MW 150–200 kDa) are the two main globulins. Other minor proteins present in lentils are prolamins and glutelins. Hydrolysis of lentil proteins could potentially liberate peptide sequences with ACEinhibitory properties. The aim of the proposed work, therefore, was to extract the proteins present in red lentils and determine if tryptic hydrolysis could liberate peptides with ACE-inhibitory properties. Additionally, the polypeptide profiles of the different red lentil protein fractions and their tryptic hydrolysates were characterized by SDS-PAGE.
temperature at 12,000 g for 30 min. The supernatant was subjected to ultrafiltration using a 50 kDa MW Cut-Off membrane and a volume concentration ratio of 5 followed by diafiltration using a volume dilution factor of 4. The retentate was neutralized to pH 7.0 using 1 mol equiv/L HCl, frozen and then freeze-dried (red lentil protein isolate). The protein isolate was further fractionated to the respective albumin, globulin and glutelin fractions using a combination of salt extraction, isoelectric precipitation and membrane separation techniques as described previously (Gupta & Dhillon, 1993) (Fig. 1). The pH of solutions was adjusted using 1 mol equiv/L NaOH or HCl. The enriched fractions were hydrolyzed as described below and freeze-dried for ACE-inhibitory studies.
2. Materials and methods
Protein content of the red lentil protein isolate and the enriched fractions were analysed by Leco (Leco FP-428, Leco Corp., St. Joseph, Mich., U.S.A.), using the combustion method (AOAC, 1995) and a nitrogen conversion factor of 6.25.
2.1. Materials The red lentil used for this study was the Common Blaze certified variety which was provided by Simpson Seeds Inc. (Saskatchewan, Canada). All other chemicals used were of analytical grade. 2.2. Protein extraction and fractionation Protein isolates were prepared from red lentils using alkaline extraction followed by ultrafiltration. Briefly, whole lentil seeds were frozen in liquid nitrogen and ground using a Brinkmann centrifugal mill (Brinkmann Instruments Canada, Mississauga, ON, Canada) equipped with a 5-mesh sieve with 1.5 mm pore size. Ground seeds (10 g) were suspended in water (100 g/L) and the pH was adjusted to 9 using 1 mol equiv/L NaOH. The dispersions were stirred for 60 min at 25 1 C to facilitate protein solubilization, while maintaining the pH at 9, and then centrifuged at room
2.3. Protein analysis
2.4. Protein hydrolysis Trypsin was used to hydrolyze the red lentil protein fractions into peptide fragments under saturated substrate conditions. The tryptic digestion (E/S: 1/25) was conducted for 24 h at 37 C and pH 6.5. The reaction was stopped by heating at 90 C for 10 min, and all samples were centrifuged at 12,000 g for 20 min at 4 C, and the supernatant was filtered and lyophilized. 2.5. SDS-polyacrylamide gel electrophoresis SDS-PAGE analysis of the lentil protein isolate, enriched fractions and their tryptic digests was performed on precast 10–20% gradient polyacrylamide Tris/HCl and Tris/Tricine gels using the
J.I. Boye et al. / LWT - Food Science and Technology 43 (2010) 987–991
Bio-Rad Criterion cell (St. Louis, Mo., U.S.A.). For experiments under reducing conditions, 5 mL/100 mL b-mercaptoethanol (2-ME) was added to the protein/peptide solutions and the samples were heated at 100 C for 5 min prior to the electrophoresis run. The gels were stained with Bio-Rad Coomassie Blue R-250 in the case of protein fractions and with Bio-Rad Coomassie Blue G-250 in the case of tryptic protein digests. Amersham Pharmacia Biotech (Uppsala, Sweden) Low Molecular Weight Calibration Kit (MW 14.1–97 kDa) and Bio-Rad Polypeptide SDS-PAGE Molecular Weight Standards (MW 1.423–26.625 kDa) were used as molecular markers for SDS-Tris/HCl and SDS Tris/Tricine-PAGE, respectively. 2.6. ACE-inhibitory properties ACE-inhibitory activity was determined in triplicate using the HPLC hippuryl-His-Leu (HHL) substrate method described previously (Shalaby, Zakora, & Otte, 2006). The method involves the calculation of the amount of hippuric acid liberated from hippurylHis-Leu (HHL) during 30 min of incubation at 37 C with the angiotensin I-converting enzyme in the presence (treatment sample) and absence (control sample) of the lentil protein hydrolysates. Inhibition (%) was calculated as follows:
Inhibitory activityð%Þ ¼ ½ðAc As Þ=ðAc Ab Þ 100 where Ac is the absorbance of the control, As is the absorbance of the sample, and Ab is the absorbance of the blank. The IC50 was calculated as the concentration of the hydrolysates that provided 50% inhibition of ACE activity.
989
The electrophoretic pattern of the legumin-rich fraction shows the acidic subunits (30–45 kDa) and basic subunit (between 20 and 25 kDa) (Shewry et al., 2004). The four minor bands with molecular weights between 50 and 95 kDa as well as the two low molecular weight polypeptides (MW <20 kDa) are very likely to be contaminants from the vicilin and albumin fractions. SDS-PAGE of the tryptic hydrolysates of the fractions were also analysed under reducing and non-reducing conditions (Fig. 2b). Electrophoretic patterns observed for trypsin hydrolysis showed the presence of various peptide bands confirming the effectiveness of enzymatic cleavage of lentil proteins. Moreover, all fraction digests showed the presence of major bands with molecular weights estimated at around 4 kDa. In addition larger peptides with molecular weight of w6, 15, and 20 kDa, were also observed in the electrophoregrams of all the fraction digests. However, some differences could be observed in the intensity of the bands for the different digests. In the case of the hydrolyzed albumin-rich fraction, major bands with molecular weight of approximately 15 and 27 kDa polypeptide could be observed after hydrolysis (Fig. 2b). These polypeptides could correspond to trypsin inhibitors which were resistant to hydrolysis or to aggregates linked by disulfide bridges (Macedo, Garcia, Freire, & Richardson, 2007). To ascertain this, studies are currently ongoing to evaluate the level of trypsin inhibitors present in the hydrolysates and to characterize these proteins. In the presence of 2-ME, a different peptide profile was observed characterized by a disappearance of the major bands
2.7. Statistical analysis The data obtained was subjected to analysis of variance (ANOVA). Statistical differences were calculated using the Fisher test analysis (P < 0.01) and Statistica (version 5) software (StatSoft Inc., Oklahoma, USA). 3. Results and discussion The composition of the red lentil protein isolate and fractions used in this study were studied. The purity (mass of protein in isolate (g)/total mass of isolate (g)) of the red lentil protein isolate was 86.35 0.31 g/100 g, while the enriched albumin, vicilin and legumin fractions contained 74.99 1.06, 77.65 0.72 and 90.47 0.57 g/100 g protein, respectively. 3.1. SDS-PAGE of the different lentil protein fractions before and after hydrolysis Electrophoretic profiles of the different fractions and their tryptic hydrolysates are presented in Fig. 2. The figure shows distinct differences in the electrophoretic profiles of the 3 enriched fractions. The albumin-rich fraction mostly contained smaller molecular weight proteins ranging in size from 14 to 28 kDa (Fig. 2a). Minor bands were observed with higher molecular weights which are likely to be contaminants from vicilin and legumin proteins. Electrophoretic profile of the vicilin-rich fraction shows the extract was composed of at least 9 polypeptides ranging from 14 to 66 kDa. Two of the most intense bands had molecular weights corresponding to w50 and 60 kDa which probably represent the subunits of vicilin (48 kDa) and convicilin (63 kDa), respectively (Shewry, Jenkins, Beaudoin, & Clare Mills, 2004). The three intense lower molecular weight bands (14–20 kDa) may be gamma-vicilin storage proteins or polypeptides from the posttranslational cleavage of the storage proteins (Shewry et al., 2004).
Fig. 2. (a) SDS-PAGE of red lentil protein fractions: MW markers (lane 1), Albumin-rich fraction (lane 2), Vicilin-rich fraction (lane 3) and Legumin-rich fraction (lane 4). (b) SDS-PAGE in Tris/Tricine of tryptic digests: MW markers (lane 1), in non-reducing conditions (lane 2: Albumin digest; lane 3: Vicilin digest and lane 4: Legumin digest) and in reducing conditions with 2-ME (lane 5: Albumin digest; lane 6: Vicilin digest and lane 7: Legumin digest).
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J.I. Boye et al. / LWT - Food Science and Technology 43 (2010) 987–991 100%
a
ACE inhibition (%)
90% 80% 70% 60% 50% 40% 30% 20% 10% 0% 0
0.5
1
1.5
2
2.5
Digest concentration (mg/ml) 560 540
b
IC50 ( g/ml)
520 500 480
sequences contained in the legumin hydrolysate or to the higher protein content of the legumin-rich fraction compared to the vicilin and albumin fractions (i.e., 90.47, 77.65, 74.99 g protein/100 g, respectively). Since the purity of the albumin and vicilin fractions were almost the same, the differences observed in ACE-inhibitory properties are likely due to differences in the inhibitory properties of the specific peptide sequences contained in these hydrolysates. Further studies are needed to purify the protein fractions and compare the ACE-inhibitory properties of the pure fractions. The ACE-inhibitory activity of lentil protein hydrolysates observed in this work is high and promising especially when compared to activities reported for other seed protein hydrolysates and tryptic whey digest; IC50 values of 1 mg/mL have been reported for whey and pea tryptic digests (Marczak et al., 2003; Pihlanto-Leppala, Koskinen, Piilola, Tupasela, & Korhonen, 2000; Vermeirssen et al., 2002). Recently, Akilliog˘lu and Karakaya (2009) have also reported ACEinhibitory activity of in vitro gastrointestinal digests of ‘‘Sultani’’ green lentil, with IC50 values ranging between 0.008 and 0.89 mg/mL depending on the heat treatment applied prior to enzymatic hydrolysis.
4. Conclusions
460 440 420 400 Lentil Protein digest
Legumin digest
Albumin digest
Vicilin digest
Fig. 3. (a) ACE-inhibitory activity of red lentil protein isolate digest (C) and enriched fraction digests of: legumin (A), albumin (-) and vicilin (:) (b) IC50 (mg/mL) values for digests obtained from lentil protein isolate and lentil protein fractions (mean SD). Each IC50 value represents the mean value of three experiments.
described above suggesting that these polypeptides were linked by disulfide bridges. For the digested legumin and vicilin fractions, additional bands were also observed under reducing conditions suggesting the presence of intra disulfide bonds within the structure of some hydrolysate polypeptides. 3.2. ACE-inhibitory activity of the different tryptic digests The bioactivity of the different tryptic digests obtained from the lentil fractions was studied. All red lentil protein fractions displayed ACE-inhibitory activities. Fig. 3a shows that ACE-inhibitory activity increased with increasing tryptic digest concentrations of lentil protein isolate, enriched albumin, legumin and vicilin fractions. IC50 values were calculated as 440 4, 476 2, 509 8 and 539 9 mg/ mL for lentil protein isolate, legumin, albumin, and vicilin hydrolysates, respectively (Fig. 3b). Assuming an average molecular weight of 4 kDa for the ACE-inhibitory peptides based on the SDS-PAGE results (Fig. 2b), IC50 values could be expressed as 111 1, 119 0.5, 127 2 and 135 2 mmol/L for lentil protein, legumin, albumin, and vicilin hydrolysates, respectively. The differences in the IC50 values were found to be highly significant (P < 0.01) suggesting marked differences in the capacity of the total lentil protein isolate hydrolysate and the hydrolysates of the different fraction to inhibit ACE. The highest inhibition effect on ACE was found for the crude lentil protein isolate hydrolysate which may be due to the combined effect of the activity from the different fractions. The higher ACE-inhibitory activity of the legumin fraction compared to the albumin and vicilin fractions could be due either to the nature of the specific peptide
Our results reveal that red lentil protein hydrolysates posses ACE-inhibitory properties. Furthermore, we demonstrated that the IC50 values of the hydrolysates varied significantly as a function of the protein fraction with the total lentil protein isolate having the lowest IC50, followed by the legumin-rich, albumin-rich, and vicilin-rich fractions, respectively. The ACE-inhibitory activity of the total red lentil protein hydrolysate obtained in this study (IC50 of 440 4 mg/mL or 111 mmol/L) is particularly promising considering that no fractionation was performed. Our results further suggests that while each of the enriched protein fraction demonstrated ACEinhibitory activity, it is likely that lentil varieties having higher amounts of legumin and albumin proteins may have higher ACEinhibitory properties. Further studies are, therefore, ongoing to compare the ACE-inhibitory activity of different lentil varieties and to determine the mechanisms of inhibition.
Acknowledgment The authors would like to thank Mr. Denis Belanger and Ms Sabine Ribe´reau for their valuable technical assistance.
References Akilliog˘lu, 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. AOAC. (1995). Official method 992.15: Crude protein in meat and meat products including pet foods. Washington, DC: Association of Official Analytical Chemists. Bhatty, R. S. (1988). Composition and quality of lentil (Lens culinaris Medik): a review. Canadian Institute of Food Science and Technology, 21, 144–160. Chen, G. W., Tsai, J. S., & Pan, B. S. (2007). Purification of angiotensin I-converting enzyme inhibitory peptides and antihypertensive effect of milk produced by protease-facilitated lactic fermentation. International Dairy Journal, 17, 641–647. Chiang, W. D., Tsou, M. J., Tsai, Z. Y., & Tsai, T. C. (2006). Angiotensin I-converting enzyme inhibitor derived from soy protein hydrolysate and produced by using membrane reactor. Food Chemistry, 98, 725–732. Erdos, E. G. (1975). Angiotensin I-converting enzyme. Circulation Research, 36, 247–255. Gupta, R., & Dhillon, S. (1993). Characterization of seed storage proteins of Lentil (Lens culinaris M.). Annals of Biology, 9, 71–78. Hartmann, R., & Meisel, H. (2007). Food-derived peptides with biological activity: from research to food applications. Current Opinion in Biotechnology, 18, 1–7.
J.I. Boye et al. / LWT - Food Science and Technology 43 (2010) 987–991 Kuba, M., Tanaka, K., Tawata, S., Takeda, Y., & Yasuda, M. (2003). Angiotensin Iconverting enzyme inhibitory peptides isolated from tufuyo fermented soybean food. Bioscience Biotechnology and Biochemistry, 67, 1278–1283. Macedo, M. L. R., Garcia, V. A., Freire, M. G. M., & Richardson, M. (2007). Characterization of a Kunitz trypsin inhibitor with a single disulfide bridge from seeds of Inga laurina (SW.) willd. Phytochemistry, 68, 1104–1111. Marczak, E. D., Usui, H., Fujita, H., Yang, Y., Yokoo, M., Lipkowski, A. W., et al. (2003). New antihypertensive peptides isolated from rapeseed. Peptides, 24, 791–798. Otte, J., Shalaby, S. M., Zakora, M., Pripp, A. H., & El-Shabrawy, S. A. (2007). Angiotensin-converting enzyme inhibitory activity of milk protein hydrolysates: effect of substrate, enzyme and time of hydrolysis. International Dairy Journal, 17, 488–503. Pihlanto-Leppala, A., Koskinen, P., Piilola, K., Tupasela, T., & Korhonen, H. (2000). Angiotensin I-converting enzyme inhibitory properties of whey protein digests: concentration and characterization of active peptides. Journal of Dairy Research, 67, 53–64. Quiros, A., Ramos, M., Muguerza, B., Delgado, M. A., Miguel, A., Aleixandre, A., et al. (2007). Identification of novel antihypertensive peptides in milk fermented with Enterococcus faecalis. International Dairy Journal, 17, 33–41. Shalaby, S. M., Zakora, M., & Otte, J. (2006). Performance of two commonly used angiotensin-converting enzyme inhibition assays using FA-PGG and HHL as substrates. Journal of Dairy Research, 73, 178–186.
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Shewry, P. R., Jenkins, J. A., Beaudoin, F., & Clare Mills, E. N. (2004). The classification, functions and evolutionary relationships of plant proteins in relation to food allergies. In E. N. Clare Mills, & P. R. Shewry (Eds.), Plant food allergens (pp. 24– 41). Oxford, UK: Blackwell Science. Tsai, J. S., Chen, T. J., Pan, B. S., Gong, S. D., & Chung, M. Y. (2008). Antihypertensive effect of bioactive peptides produced by protease-facilitated lactic acid fermentation of milk. Food Chemistry, 106, 552–558. Vermeirssen, V., Van Camp, J., & Verstraete, W. (2002). Optimisation and validation of an angiotensin-converting enzyme inhibition assay for the screening of bioactive peptides. Journal of Biochemical and Biophysical Methods, 51, 75–87. Wu, J., & Ding, X. (2002). Characterization of inhibition and stability of soy-proteinderived angiotensin I-converting enzyme inhibitory peptides. Food Research International, 35, 367–375. Wu, J., & Muir, A. D. (2008). Comparative structural, emulsifying and biological properties of 2 major canola proteins, cruceferin and napin. Journal of Food Science, 73, C210–C216. Yang, H. Y. T., Erdos, E. G., & Levin, Y. A. (1970). A dipeptidyl carboxypeptidase that converts angiotensin I and inactivates bradykinin. Biochimica et Biophysica Acta, 214, 374–376. Yang, H. Y., Yang, S. C., Chen, S. T., & Chen, J. R. (2008). Soy protein hydrolysate ameliorates cardiovascular remodeling in rats with L-NAME-induced hypertension. Journal of Nutritional Biochemistry, 19, 833–839.
Contents lists available at ScienceDirect
LWT - Food Science and Technology journal homepage: www.elsevier.com/locate/lwt
Research Note
Angiotensin I-converting enzyme inhibitory properties and SDS-PAGE of red lentil protein hydrolysates Joyce Irene Boye a, *, Samira Roufik b, Noemie Pesta c, Chockry Barbana a a
Food Research and Development Centre, Agriculture and Agri-Food Canada, Casavant Blvd. West, St Hyacinthe, QC J2S 8E3, Canada Health Canada, Ottawa, Canada c Universite´ de Reims Champagne-Ardenne, 9 Boulevard de la Paix, Reims, Champagne-Ardenne, France b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 May 2009 Received in revised form 14 January 2010 Accepted 15 January 2010
Several research studies have shown that protein hydrolysates from milk and soy contain peptides that possess angiotensin I converting enzyme (ACE) inhibitory properties and may help to prevent hypertension. To date, no studies have been conducted to determine if red lentil (Lens culinaris) proteins contain peptides with ACE-inhibitory properties. The objective of the present work was to characterize the proteins present in red lentils and determine if tryptic hydrolysis could liberate peptides with ACEinhibitory properties. Red lentil protein extracts were prepared and fractionated to obtain enriched albumin, legumin and vicilin fractions. Protein/peptide profiles were studied by electrophoresis and ACEinhibitory activity was measured using the HPLC hippuryl-His-Leu (HHL) substrate method. Our results revealed that red lentil protein hydrolysates posses ACE-inhibitory properties. Furthermore, we demonstrated that the ACE-inhibitory property of the hydrolysates varied as a function of the protein fraction with the total lentil protein hydrolysate having the lowest half maximal inhibitory concentration (IC50) (111 1 mmol/L) (i.e., highest ACE-inhibitory activity), followed by the enriched legumin (119 0.5 mmol/L), albumin (127 2 mmol/L) and vicilin (135 2 mmol/L) fractions, respectively. Crown Copyright Ó 2010 Published by Elsevier Ltd. All rights reserved.
Keywords: ACE-inhibitory activity Bioactivity Protein hydrolysate Pulse Red lentils
1. Introduction A major risk factor for developing cardiovascular disease, one of the leading causes of death in North America, is elevated blood pressure. Angiotensin II, a potent vasoconstrictor, is a major contributor to high blood pressure. Vasoconstriction occurs when rennin, an enzyme liberated by the kidneys, proteolytically acts on circulating angiotensinogen and converts it to angiotensin I (a decapeptide). In the presence of angiotensin converting enzyme (ACE), angiotensin I is cleaved to the octapeptide, angiotensin II resulting in arterial constriction and blood pressure elevation. ACE also breaks down bradykinin, a vasodilator, further contributing to the elevation in blood pressure. Inhibition of ACE is, therefore, important for the lowering of blood pressure as this results in a decrease in the concentration of angiotensin II and an increase in the levels of bradykinin (Erdos, 1975; Yang, Erdos, & Levin, 1970). ACE is a dipeptidyl carboxypeptidase (EC3.4.15.1) and many studies in recent years have focused on identifying compounds that can inhibit its activity (Quiros et al., 2007; Tsai, Chen, Pan, Gong, &
* Corresponding author. Tel.: þ1 450 768 3232; fax: þ 1 450 773 8461. E-mail address: [email protected] (J.I. Boye).
Chung, 2008; Vermeirssen, Van Camp, & Verstraete, 2002; Wu & Ding, 2002). Various high blood pressure medications available on the market today are targeted towards ACE inhibition. Examples of such drugs include Captopril, Enalapril, Ramipril, and Quinapril (Accupril). Due to potential side effects of pharmaceutical drugs, there is increased interest to identify foods that naturally contain peptides with hypotensive properties. Several research studies have shown that protein hydrolysates from certain plant and animal sources contain peptides that demonstrate ACE-inhibitory properties and may help to prevent hypertension (Hartmann & Meisel, 2007; Wu & Muir, 2008; Yang, Yang, Chen, & Chen, 2008). Recently, some studies reported novel antihypertensive peptides in fermented milk products (Chen, Tsai, & Pan, 2007; Quiros et al., 2007; Tsai et al., 2008). In other studies, ACE-inhibitory activity after hydrolysis of different milk protein products with a variety of enzymes has been reported (Otte, Shalaby, Zakora, Pripp, & ElShabrawy, 2007). Plant proteins such as soy, chickpea and pea, have also been shown to contain ACE-inhibitory peptides (Aluko, 2008; Chiang, Tsou, Tsai, & Tsai, 2006; Kuba, Tanaka, Tawata, Takeda, & Yasuda, 2003; Wu & Ding, 2002). To date, however, no studies have been conducted to determine if red lentil proteins contain peptides with ACE-inhibitory properties. IC50 values of 0.78–0.83, 0.15–0.69 and 0.008–0.89 mg protein/mL for common
0023-6438/$ – see front matter Crown Copyright Ó 2010 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2010.01.014
988
J.I. Boye et al. / LWT - Food Science and Technology 43 (2010) 987–991
Fig. 1. Schematic representation of the process used for the fractionation of the albumin, vicilin and legumin fractions from red lentil protein isolate. (Based on the method described by Gupta and Dhillon (1993)).
beans, pinto beans and green lentils, respectively, after in vitro gastrointestinal digestion have been very recently reported (Akıllıog˘lu & Karakaya, 2009). The major proteins found in lentils are albumins and globulins (Bhatty, 1988; Gupta & Dhillon, 1993). Legumins (molecular weight (MW) 350–400 kDa) and vicilins (MW 150–200 kDa) are the two main globulins. Other minor proteins present in lentils are prolamins and glutelins. Hydrolysis of lentil proteins could potentially liberate peptide sequences with ACEinhibitory properties. The aim of the proposed work, therefore, was to extract the proteins present in red lentils and determine if tryptic hydrolysis could liberate peptides with ACE-inhibitory properties. Additionally, the polypeptide profiles of the different red lentil protein fractions and their tryptic hydrolysates were characterized by SDS-PAGE.
temperature at 12,000 g for 30 min. The supernatant was subjected to ultrafiltration using a 50 kDa MW Cut-Off membrane and a volume concentration ratio of 5 followed by diafiltration using a volume dilution factor of 4. The retentate was neutralized to pH 7.0 using 1 mol equiv/L HCl, frozen and then freeze-dried (red lentil protein isolate). The protein isolate was further fractionated to the respective albumin, globulin and glutelin fractions using a combination of salt extraction, isoelectric precipitation and membrane separation techniques as described previously (Gupta & Dhillon, 1993) (Fig. 1). The pH of solutions was adjusted using 1 mol equiv/L NaOH or HCl. The enriched fractions were hydrolyzed as described below and freeze-dried for ACE-inhibitory studies.
2. Materials and methods
Protein content of the red lentil protein isolate and the enriched fractions were analysed by Leco (Leco FP-428, Leco Corp., St. Joseph, Mich., U.S.A.), using the combustion method (AOAC, 1995) and a nitrogen conversion factor of 6.25.
2.1. Materials The red lentil used for this study was the Common Blaze certified variety which was provided by Simpson Seeds Inc. (Saskatchewan, Canada). All other chemicals used were of analytical grade. 2.2. Protein extraction and fractionation Protein isolates were prepared from red lentils using alkaline extraction followed by ultrafiltration. Briefly, whole lentil seeds were frozen in liquid nitrogen and ground using a Brinkmann centrifugal mill (Brinkmann Instruments Canada, Mississauga, ON, Canada) equipped with a 5-mesh sieve with 1.5 mm pore size. Ground seeds (10 g) were suspended in water (100 g/L) and the pH was adjusted to 9 using 1 mol equiv/L NaOH. The dispersions were stirred for 60 min at 25 1 C to facilitate protein solubilization, while maintaining the pH at 9, and then centrifuged at room
2.3. Protein analysis
2.4. Protein hydrolysis Trypsin was used to hydrolyze the red lentil protein fractions into peptide fragments under saturated substrate conditions. The tryptic digestion (E/S: 1/25) was conducted for 24 h at 37 C and pH 6.5. The reaction was stopped by heating at 90 C for 10 min, and all samples were centrifuged at 12,000 g for 20 min at 4 C, and the supernatant was filtered and lyophilized. 2.5. SDS-polyacrylamide gel electrophoresis SDS-PAGE analysis of the lentil protein isolate, enriched fractions and their tryptic digests was performed on precast 10–20% gradient polyacrylamide Tris/HCl and Tris/Tricine gels using the
J.I. Boye et al. / LWT - Food Science and Technology 43 (2010) 987–991
Bio-Rad Criterion cell (St. Louis, Mo., U.S.A.). For experiments under reducing conditions, 5 mL/100 mL b-mercaptoethanol (2-ME) was added to the protein/peptide solutions and the samples were heated at 100 C for 5 min prior to the electrophoresis run. The gels were stained with Bio-Rad Coomassie Blue R-250 in the case of protein fractions and with Bio-Rad Coomassie Blue G-250 in the case of tryptic protein digests. Amersham Pharmacia Biotech (Uppsala, Sweden) Low Molecular Weight Calibration Kit (MW 14.1–97 kDa) and Bio-Rad Polypeptide SDS-PAGE Molecular Weight Standards (MW 1.423–26.625 kDa) were used as molecular markers for SDS-Tris/HCl and SDS Tris/Tricine-PAGE, respectively. 2.6. ACE-inhibitory properties ACE-inhibitory activity was determined in triplicate using the HPLC hippuryl-His-Leu (HHL) substrate method described previously (Shalaby, Zakora, & Otte, 2006). The method involves the calculation of the amount of hippuric acid liberated from hippurylHis-Leu (HHL) during 30 min of incubation at 37 C with the angiotensin I-converting enzyme in the presence (treatment sample) and absence (control sample) of the lentil protein hydrolysates. Inhibition (%) was calculated as follows:
Inhibitory activityð%Þ ¼ ½ðAc As Þ=ðAc Ab Þ 100 where Ac is the absorbance of the control, As is the absorbance of the sample, and Ab is the absorbance of the blank. The IC50 was calculated as the concentration of the hydrolysates that provided 50% inhibition of ACE activity.
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The electrophoretic pattern of the legumin-rich fraction shows the acidic subunits (30–45 kDa) and basic subunit (between 20 and 25 kDa) (Shewry et al., 2004). The four minor bands with molecular weights between 50 and 95 kDa as well as the two low molecular weight polypeptides (MW <20 kDa) are very likely to be contaminants from the vicilin and albumin fractions. SDS-PAGE of the tryptic hydrolysates of the fractions were also analysed under reducing and non-reducing conditions (Fig. 2b). Electrophoretic patterns observed for trypsin hydrolysis showed the presence of various peptide bands confirming the effectiveness of enzymatic cleavage of lentil proteins. Moreover, all fraction digests showed the presence of major bands with molecular weights estimated at around 4 kDa. In addition larger peptides with molecular weight of w6, 15, and 20 kDa, were also observed in the electrophoregrams of all the fraction digests. However, some differences could be observed in the intensity of the bands for the different digests. In the case of the hydrolyzed albumin-rich fraction, major bands with molecular weight of approximately 15 and 27 kDa polypeptide could be observed after hydrolysis (Fig. 2b). These polypeptides could correspond to trypsin inhibitors which were resistant to hydrolysis or to aggregates linked by disulfide bridges (Macedo, Garcia, Freire, & Richardson, 2007). To ascertain this, studies are currently ongoing to evaluate the level of trypsin inhibitors present in the hydrolysates and to characterize these proteins. In the presence of 2-ME, a different peptide profile was observed characterized by a disappearance of the major bands
2.7. Statistical analysis The data obtained was subjected to analysis of variance (ANOVA). Statistical differences were calculated using the Fisher test analysis (P < 0.01) and Statistica (version 5) software (StatSoft Inc., Oklahoma, USA). 3. Results and discussion The composition of the red lentil protein isolate and fractions used in this study were studied. The purity (mass of protein in isolate (g)/total mass of isolate (g)) of the red lentil protein isolate was 86.35 0.31 g/100 g, while the enriched albumin, vicilin and legumin fractions contained 74.99 1.06, 77.65 0.72 and 90.47 0.57 g/100 g protein, respectively. 3.1. SDS-PAGE of the different lentil protein fractions before and after hydrolysis Electrophoretic profiles of the different fractions and their tryptic hydrolysates are presented in Fig. 2. The figure shows distinct differences in the electrophoretic profiles of the 3 enriched fractions. The albumin-rich fraction mostly contained smaller molecular weight proteins ranging in size from 14 to 28 kDa (Fig. 2a). Minor bands were observed with higher molecular weights which are likely to be contaminants from vicilin and legumin proteins. Electrophoretic profile of the vicilin-rich fraction shows the extract was composed of at least 9 polypeptides ranging from 14 to 66 kDa. Two of the most intense bands had molecular weights corresponding to w50 and 60 kDa which probably represent the subunits of vicilin (48 kDa) and convicilin (63 kDa), respectively (Shewry, Jenkins, Beaudoin, & Clare Mills, 2004). The three intense lower molecular weight bands (14–20 kDa) may be gamma-vicilin storage proteins or polypeptides from the posttranslational cleavage of the storage proteins (Shewry et al., 2004).
Fig. 2. (a) SDS-PAGE of red lentil protein fractions: MW markers (lane 1), Albumin-rich fraction (lane 2), Vicilin-rich fraction (lane 3) and Legumin-rich fraction (lane 4). (b) SDS-PAGE in Tris/Tricine of tryptic digests: MW markers (lane 1), in non-reducing conditions (lane 2: Albumin digest; lane 3: Vicilin digest and lane 4: Legumin digest) and in reducing conditions with 2-ME (lane 5: Albumin digest; lane 6: Vicilin digest and lane 7: Legumin digest).
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J.I. Boye et al. / LWT - Food Science and Technology 43 (2010) 987–991 100%
a
ACE inhibition (%)
90% 80% 70% 60% 50% 40% 30% 20% 10% 0% 0
0.5
1
1.5
2
2.5
Digest concentration (mg/ml) 560 540
b
IC50 ( g/ml)
520 500 480
sequences contained in the legumin hydrolysate or to the higher protein content of the legumin-rich fraction compared to the vicilin and albumin fractions (i.e., 90.47, 77.65, 74.99 g protein/100 g, respectively). Since the purity of the albumin and vicilin fractions were almost the same, the differences observed in ACE-inhibitory properties are likely due to differences in the inhibitory properties of the specific peptide sequences contained in these hydrolysates. Further studies are needed to purify the protein fractions and compare the ACE-inhibitory properties of the pure fractions. The ACE-inhibitory activity of lentil protein hydrolysates observed in this work is high and promising especially when compared to activities reported for other seed protein hydrolysates and tryptic whey digest; IC50 values of 1 mg/mL have been reported for whey and pea tryptic digests (Marczak et al., 2003; Pihlanto-Leppala, Koskinen, Piilola, Tupasela, & Korhonen, 2000; Vermeirssen et al., 2002). Recently, Akilliog˘lu and Karakaya (2009) have also reported ACEinhibitory activity of in vitro gastrointestinal digests of ‘‘Sultani’’ green lentil, with IC50 values ranging between 0.008 and 0.89 mg/mL depending on the heat treatment applied prior to enzymatic hydrolysis.
4. Conclusions
460 440 420 400 Lentil Protein digest
Legumin digest
Albumin digest
Vicilin digest
Fig. 3. (a) ACE-inhibitory activity of red lentil protein isolate digest (C) and enriched fraction digests of: legumin (A), albumin (-) and vicilin (:) (b) IC50 (mg/mL) values for digests obtained from lentil protein isolate and lentil protein fractions (mean SD). Each IC50 value represents the mean value of three experiments.
described above suggesting that these polypeptides were linked by disulfide bridges. For the digested legumin and vicilin fractions, additional bands were also observed under reducing conditions suggesting the presence of intra disulfide bonds within the structure of some hydrolysate polypeptides. 3.2. ACE-inhibitory activity of the different tryptic digests The bioactivity of the different tryptic digests obtained from the lentil fractions was studied. All red lentil protein fractions displayed ACE-inhibitory activities. Fig. 3a shows that ACE-inhibitory activity increased with increasing tryptic digest concentrations of lentil protein isolate, enriched albumin, legumin and vicilin fractions. IC50 values were calculated as 440 4, 476 2, 509 8 and 539 9 mg/ mL for lentil protein isolate, legumin, albumin, and vicilin hydrolysates, respectively (Fig. 3b). Assuming an average molecular weight of 4 kDa for the ACE-inhibitory peptides based on the SDS-PAGE results (Fig. 2b), IC50 values could be expressed as 111 1, 119 0.5, 127 2 and 135 2 mmol/L for lentil protein, legumin, albumin, and vicilin hydrolysates, respectively. The differences in the IC50 values were found to be highly significant (P < 0.01) suggesting marked differences in the capacity of the total lentil protein isolate hydrolysate and the hydrolysates of the different fraction to inhibit ACE. The highest inhibition effect on ACE was found for the crude lentil protein isolate hydrolysate which may be due to the combined effect of the activity from the different fractions. The higher ACE-inhibitory activity of the legumin fraction compared to the albumin and vicilin fractions could be due either to the nature of the specific peptide
Our results reveal that red lentil protein hydrolysates posses ACE-inhibitory properties. Furthermore, we demonstrated that the IC50 values of the hydrolysates varied significantly as a function of the protein fraction with the total lentil protein isolate having the lowest IC50, followed by the legumin-rich, albumin-rich, and vicilin-rich fractions, respectively. The ACE-inhibitory activity of the total red lentil protein hydrolysate obtained in this study (IC50 of 440 4 mg/mL or 111 mmol/L) is particularly promising considering that no fractionation was performed. Our results further suggests that while each of the enriched protein fraction demonstrated ACEinhibitory activity, it is likely that lentil varieties having higher amounts of legumin and albumin proteins may have higher ACEinhibitory properties. Further studies are, therefore, ongoing to compare the ACE-inhibitory activity of different lentil varieties and to determine the mechanisms of inhibition.
Acknowledgment The authors would like to thank Mr. Denis Belanger and Ms Sabine Ribe´reau for their valuable technical assistance.
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