Impact of hydrolysis on functional properties, antioxidant, ACE-I inhibitory and antiproliferative activity of Cicer arietinum and Cicer reticulatum hydrolysates
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Chickpea is an important food legume of the world offering valuable nutrients. Employing the degree of hydrolysis (DH) by the enzyme alcalase and flavourzyme, the functional properties of two chickpea species i.e. Cicer arietinum and Cicer reticulatum was investigated. This includes antioxidant, physicochemical properties and angiotensin-converting enzyme (ACE-I) inhibitory activities.
Hydrolysis reaction was performed in different time intervals including 0, 20, 40, 60, 80, and 100 min by adding 2% (w/v) alcalase (pH 8) and flavourzyme (pH 7) at 50 °C temperature and pH was kept constant during hydrolysis time.
DH improved through solubility of chickpea isolate in the pH range of 5–10. Alcalase produced a maximum degree of hydrolysis at 60 min for both the Cicer species. Flavourzyme demonstrated the highest degree hydrolysis for C. arietinum at 80 min and C. reticulatum at 100 min. The Cicer protein hydrolysates showed the highest antioxidant, ACE-I inhibitory and antiproliferative activity.
As a prerequisite to food industry, present study revealed that chickpea protein hydrolysate (CPH) can be appraised as a source of functional food.
KeywordsChickpea, Cicer arietinum Cicer reticulatum ACE-I inhibitors, Enzymatic hydrolysis
Plant proteins besides providing adequate nutrition conferred multiple health benefits as functional foods. These plant proteins exhibit regulatory functions in human body and release bioactive peptides under in vivo and in vitro conditions . Such peptides offer different health benefits and remain inactive within the sequence of parent protein . The DH is a measure of protein hydrolysis and constitutes an important parameter in analyzing functional properties of protein hydrolysate [3, 4]. DH affects the size and taste of protein hydrolysate generating peptide of bioactive features. The DH correspondingly demonstrates variation in biological activity due to changes in amino acid composition that in turn modulates the biological performance of the peptides formed during hydrolysis [3, 4]. Published reports describe that hydrolysis of proteins by means of various enzyme activates chemical, functional and nutritional properties . Hydrolysis enhance the functional properties of dietary proteins without affecting its nutritive value by converting it into peptides of desired size, charge, and surface properties . Such hydrolyzed bioactive peptides are short chain peptides with 2–15 amino acid residues . The bioactive peptides isolated include animal (chicken and fish muscle) and plant (wheat gluten, soy, sunflower, and mung bean, etc.) sources .
ACE, a dipeptidyl carboxypeptidase (EC 188.8.131.52), is a zinc-metallopeptidase formed in the lung and involved in peripheral hypertension as well as overall cardiovascular functioning. Various effective synthetic ACE inhibitors such as captopril, enalapril, lisinopril and ramipril are broadly prescribed in the clinical treatment of hypertension-associated cardiac failures. These synthetic ACE inhibitors, however, have contrary side effects such as cough, taste disturbances, rashes and angioedema . Therefore, the quest for searching natural plant product acting as a principal constituent in the management of hypertension is essential .
Chickpea of the Leguminosae family is a major food legume crop of India and alleged to be a preferred source of protein after milk. Chickpea seed protein contains essential amino acid needed by the human body. Earlier studies reported health-promoting functions of chickpea seeds [10, 11, 12]. However, hydrolysis of chickpea proteins ensuing functional properties in relation to antioxidant, ACE-I inhibitory activities and antiproliferative effects are scanty. The present study was therefore undertaken to evaluate the properties of chickpea seed protein and its hydrolysates to provide insights in developing nutraceuticals.
Materials and methods
Chickpea seeds of two species, i.e., Cicer arietinum and Cicer reticulatum were obtained from Indian Institute of Pulses Research (IIPR), Kanpur, India. Chemicals used for the assays including 1,1-diphenyl-2-picrylhydrazyl (DPPH), butylated hydroxyanisole (BHA), β-carotene, ethylene diamine tetra acetic acid (EDTA), linoleic acid, N-Hippuryl-His-Leu hydrate (HHL), alcalase and flavourzyme were purchased from Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals and reagents used were of analytical grade.
Protein concentrate preparation
Ground flour of chickpea seeds was defatted with hexane (1:10 w/v) employing constant stirring for 8 h at 4 °C. Isoelectric precipitation was carried out for protein extraction. Briefly, protein extraction using flour to solvent ratio of 1:10 (w/v) was performed . This flour suspension (25 g flour in 250 ml of water at pH 8) was stirred for 1 h and subsequently centrifuged at 10,000×g for 15 min. The supernatant obtained was vacuum filtered using 100–160 m pore size filter funnel, freeze-dried and stored at − 20 °C till further use and named as chickpea protein (CP).
To ascertain protein concentrate hydrolysis, individual enzyme viz. alcalase and flavourzyme was employed following the method of Pedroche et al. . Hydrolysis reaction was performed for different time intervals including 0, 20, 40, 60, 80 and 100 min by adding 2% (w/v) alcalase (pH 8) and flavourzyme (pH 7) at 50 °C temperature and pH was kept constant during hydrolysis time. Subsequently, the reaction was stopped and filtered through 0.45-nm filters to remove residual matter. The filtrates were lyophilized, maintained at − 80 °C and named as alcalase-generated chickpea protein hydrolysate (ACPH) and flavourzyme-generated chickpea protein hydrolysate (FCPH) respectively.
Degree of hydrolysis (DH)
The protocol of De Castro and Sato  was followed to determine DH with slight modifications. Briefly, to the hydrolysates (1 ml), 12% trichloroacetic acid (1 ml) was added and centrifuged at 10000×g for 20 min. The DH was calculated as the ratio of TCA-soluble protein to total protein in the hydrolysate.
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)
The protein hydrolysates were subjected to SDS-PAGE according to the method of Laemmli . The electrophoresis was carried out in 11% gel at 60 V for 4 h and the gel was stained using Coomassie Brilliant Blue R-250 stain (0.05%) for 2 h, destained and subsequently documented. The molecular weights of the subunits were estimated using protein molecular weight markers.
Assays for the physicochemical properties
The method of Pearce and Kinsella  was employed to calculate the emulsifying activity index (EAI). Protein solutions and soya bean oil were stirred and the pH was adjusted to 2.0, 4.0, 6.0, 8.0, and 10.0 and centrifuged at 10000×g for 15 min. Hundred microliters of emulsion was taken from the bottom of the emulsion and diluted to 10 ml with 0.1:100 (w/v) sodium dodecyl sulfate. The absorbance was read at 500 nm and used to calculate the EAI (m2 g−1):
DPPH radical scavenging assay
Where A blank is the absorbance of the control reaction (containing all reagents except the sample) and A sample is the absorbance of CPHs (with the DPPH solution).
Antioxidant assay using the ß-carotene bleaching method
The ability of the chickpea hydrolysate to prevent the bleaching of ß-carotene was determined following the method of Koleva et al. . Briefly, ß-carotene (0.5 mg) in chloroform (1 ml) was mixed with Tween-40 (200 μl) and linoleic acid (25 μl). The samples were kept in an incubator (45 °C) to evaporate chloroform completely and milli-Q water (100 ml) was added and stirred vigorously. Before each experiment, the emulsion was freshly prepared. The ß-carotene-linoleic acid emulsion aliquot (2.5 ml) was transferred to 0.5 ml of tubes containing the sample, incubated at 50 °C for 2 h and at 470 nm, the absorbance was recorded. A blank of 0.5 ml of milli-Q water without sample and BHA was used as a positive control.
Extraction of ACE from rabbit lungs
Assay for ACE-inhibitory activity
Determination of the ACE inhibitory activities of the digests was performed according to the method of Cushman and Cheung  with minor modifications. CP, ACPH and FCPH were added to 500 μl mixture containing 100 μl of 100 mM phosphate buffer (pH 8.3), 100 μl of 300 mM NaCl, 200 μl of 5 mM HHL and 100 μl of ACE. The mixture was incubated at 37 °C for 30 min on incubator shaker and the reaction was stopped by adding 500 μl of 1 N HCl. To this reaction mixture was added with 3.0 ml of ethyl acetate and vortexed for 15 s. Ethyl acetate layer was obtained and allowed to evaporate. The residue was redissolved in 1.0 ml of distilled water and the absorbance of the resulting solution at 228 nm was recorded. For the blank, no peptide sample was added. The negative control was devoid of peptide sample. Pulverized captopril served as a positive control.
Cell lines and culture conditions
Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 mg/ml streptomycin was used for growth and maintenance of the cell lines in a constant humidified incubator at 37 °C in 5% CO2. Human endometrial adenocarcinoma cell line (Ishikawa) and human breast cancer cell line (MCF-7) were used in the present study. Control cells were maintained in parallel and subjected to identical culture conditions.
Cell viability assay
As and Ac are the absorbance of sample and control respectively.
Morphological observation under inverted microscope
Morphological observation of cells treated with Cicer hydrolysates was done to determine the changes induced. After 48 h of treatment, the morphology of cells was observed under inverted microscope and images were captured. The control cells were well adhered displaying the normal morphology of cells. In contrast, majority of cells treated with Cicer hydrolysates showed changes such as cells became round, shrunken, membrane blebbing and formation of apoptotic bodies were observed in predicting the apoptotic mechanism for cell death. Cells could not be affixed to the walls and floating in the medium.
DNA ladder assay
Cancer cells were treated with Cicer hydrolysates for 24 h to observe DNA fragmentation. For this objective, genomic DNA was isolated using an apoptotic DNA ladder kit (Roche, USA). Integrity of DNA was analyzed using electrophoresis on 1% (w/v) agarose gel.
Statistical analysis of the data was done using the Graph pad prism (version 5) software. The differences in mean were calculated using the Duncan multiple-range tests for means with 95% confidence limit (P < 0.05).
Results and discussion
The degree of hydrolysis differed (P < 0.05) depending on the use of enzyme types viz. alcalase, flavourzyme and the reaction time. At 60 min, alcalase was catalyzed to produce highest degree of hydrolysis in both the Cicer species, i.e., arietinum and reticulatum. The C. arietinum resulted DH 43.95% while C. reticulatum produced 39.51%. With flavourzyme, the degree of hydrolysis was increased over time from 14.34% (20 min) to 30.26% (100 min) in C. reticulatum. The C. arietinum hydrolysate producing DH due to alcalase revealed similar trend like that of Vigna unguiculata L. . Earlier reports on chickpea hydrolysate produced by alcalase and flavourzyme exhibited 65% DH at 150 min , while hydrolysis of mung bean protein isolate with alcalase and neutrase produced 22% and 12% DH at 10 h .
To produce the protein hydrolysates with better functional and nutritional characteristics than the original protein, the preferred alkaline protease is alcalase. However, it also generated bioactive peptides with ACE-I inhibitory activity from other protein sources like bovine skin gelatin , plasma , and sardine muscle . Alcalase from bacterial origin (Bacillus licheniformis) exhibits endopeptidase activity due to subtilisin-Carlsberg serine group in its active site. This enzyme hydrolyzes peptides with a wide specificity, releasing hydrophobic amino acids such as Phe, Tyr, Trp, Leu, Ile, Val, and Met . Hydrophobic amino acid (aromatic or branched lateral chain) residues in each of the C-terminal positions are accessible to ACE-I as a substrate or competitive inhibitor . Both species of C. arietinum and C. reticulatum are therefore suitable for producing peptides with ACE-I inhibitory activity using alcalase.
Foam formation is an important parameter in food industry applications such as beverages, cakes and whipped toppings. The foaming properties of C. arietinum and C. reticulatum isolates and hydrolysates showed a pH-dependent tendency (Figs. 4b and 5b). C. arietinum exhibited lowest foaming capacity at pH 2 corresponding to CP (34.79%), ACPH (44.79%), and FCPH (35.79%), while at pH 10.0 produced highest foaming capacity of 74.21%, 84.71%, and 78.71% respectively. Similarly, C. reticulatum hydrolysates showed the highest foaming capacity at pH 10.0 and lowest at pH 2.0. Hydrolysis of the protein furthermore releases peptides with altered polarity or hydrophobicity, which could also affect properties such as foaming and solubility. The smaller peptides are able to incorporate more air into the solution than larger peptides and increase the foaming capacity and do not have enough strength to give stable foam . Chickpea protein hydrolysate (CPH) exhibited stronger foaming capacity in a neutral environment, while CP exhibited the strongest foaming capacity in an alkaline environment. The basis of this might be due to the different protein confirmations of CP and CPHs.
Chickpea being a rich source of proteins, its functional property in terms of emulsifying capacity is mostly warranted. The capacity of protein as an emulsifying agent was measured in terms of emulsifying activity index (EAI). In the present study, C. arietinum depict highest EAI of CP, ACPH, and FCPH at pH 13.0 (4.21 m2 g−1, 4.81 m2 g−1, and 4.46 m2 g−1, respectively), while the lowest at pH 2.0 (2.16 m2 g−1, 2.59 m2 g−1, and 2.64 m2 g−1, respectively) (Fig. 4c). Similarly, C. reticulatum hydrolysates showed the highest emulsifying capacity at pH 10.0 and lowest at pH 2.0 (Fig. 5c). Significant (P < 0.05) difference was observed between the EAI of ACPH and FCPH for all the pH values tested. At pH 2, lowest EAI of CP and CPH observed might be due to the protein being close to the isoelectric point. At the isoelectric point, protein is neutral which lacks electrostatic reprography repulsive force between the molecules. Emulsifying capacity depends on the number of carboxyl groups (–COO). The number of –COO under acidic and neutral conditions is less than that under alkaline conditions, which is not conducive to the formation of micelles, therefore emulsification is lesser .
Hygroscopic capacity is one of the accurate measures of hydrophilicity of CP and CPH of both chickpea species. Figure 4d and 5d depict increased hygroscopic capacity of CP and CPH between 0 to 4 days. After 4 days, the changes in hygroscopic capacity curve were smooth. The hygroscopic capacity of C. arietinum CP changed from 4.29 to 6.52% which was less than that of ACPH (5.05 to 10.43%) and FCPH (6.08 to 10.21%). Similarly, C. reticulatum showed highest capacity from 3.1 to 9.0% by alcalase hydrolysate. There were significant differences between the hygroscopic capacities of CP, ACPH, and FCPH (P < 0.05). CPH showed higher hygroscopic capacity than CP. This might be because CPH has low molecular weight and increased exposure of amino acids. High hygroscopic capacity could help to reduce moisture loss in packaged bakery goods. Moreover, water-holding is indispensably required to maintain freshness and a moist mouth-feel of baked foods. Accordingly, CPH might prove to be a good candidate for bakery products.
Free radical scavenging and β-carotene inhibition activity
Lipid oxidation products react with proteins causing oxidation. In this study, lipid peroxidation inhibition activity of chickpea protein and its hydrolysate was determined to assess their ability to inhibit oxidation of linoleic acid in an emulsified model system. Lipid oxidation of both species obtained at different time intervals viz., C. arietinum with alcalase at 60 min, C. arietinum with flavourzyme at 80 min, C. reticulatum with alcalase at 60 min, and C. arietinum with flavourzyme at 100 min. Among the enzymatic treatments degree of hydrolysis at 80 min, C. arietinum flavourzyme hydrolysate (67.4 ± 4.4%) had the highest β-carotene inhibition activity. On the other hand, C. reticulatum flavourzyme hydrolysate exhibited lowest β-carotene inhibition activity. C. arietinum and C. reticulatum alcalase hydrolysate showed the β-carotene inhibition activity at 60 min degree of hydrolysis (Fig. 6). The antioxidant activity index of peptides/proteins in the free radical-mediated lipid peroxidation system is influenced by molecular size, chemical properties, and electron transferring ability of amino acid residues in the sequence .
ACE-I inhibitory activity
SRB assay is widely used to determine the cell density based on the measurement of cellular protein content. SRB having features to bind stoichiometrically with proteins under mildly acidic conditions and can be extracted using basic conditions; bound dye can be used as a proxy for cell mass, which can then be inferred to measure cell proliferation . Endometrial cancer (cancer of the inner lining of the uterus) is the second most common type and fourth most common cancer in women from developed countries. On the other hand, breast cancer is one of the most common malignancies and heterogeneous cancer among the females worldwide, with an estimated 1.7 million new cases (25.2%) and 0.5 million cancer deaths (14.7%) in 2012 .
DNA ladder assay
Based on our findings, chickpea seed protein hydrolysate could be a promising source for the manufacture of bioactive peptides in developing functional foods for blood pressure and chemoprevention. Further testing of chickpea protein hydrolysate in animal system is essential to understand their role in in vivo especially with regard to the antihypertensive and cytotoxic effects.
The authors are grateful to Prof. Sangeeta Shukla the Hon’ble Vice-Chancellor, Jiwaji University, Gwalior (M.P.), India for providing research grants and encouragements.
SSB supervised the present research work and gave the required tips and scientific instructions. NG did the experiments and obtained the data. All the achieved data were analyzed by both authors. The article was critically checked by SSB. Both authors read and approved the final manuscript.
Compliance with ethical standards
The authors declare that they have no competing interests.
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