Study On Preparation Of Chickpea Protein Hydrolysates And Its Antioxidant Peptides

Chickpea (Cicer arietinum L.), an annual herbage plant, is grown mostly in arid and temperate regions of the world. In 1980s, hundreds of chickpea species were imported from the International Center for Agricultural Research in the Dry Areas (ICARDA) and the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT) and have been planted in Gansu, Qinghai and Xinjiang of China.The content of protein isolates from different chickpea cultivars ranged between 15-30%. Chickpea proteins have been considered a suitable source of dietary protein due to their good balance in essential amino acids composition, high bioavailability, and low level of antinutritional factors. In this study, our objective was to investigate hydrolysis parameter of chickpea protein, antioxidant activities of hydrolysates, and the separation and analysis of antioxidant peptides using enzyme hydrolysis, antioxidant activity assay, gel filtration chromatography, RP-HPLC and MALDI-TOF-TOF MS. Using high hydrostatic pressure to promote the release of antioxidant peptides was also studied. Our results laid a theoretical foundation for the development and utilization of chickpea protein antioxidant peptides.The degree of hydrolysis (DH) and superoxide radical scavenging activity of CPHs obtained by 6 types of proteases were studied and the results showed that Alcalase hydrolysates presented stronger scavenging activity against superoxide radical. On the basis of single factor test, optimum conditions to achieve the maximum antioxidant ability were determined by response surface methodology: [E]/[S], 2.72%; Enzymolysis Temperature, 52°C; Enzymolysis time, 31 min. Under these conditions, reducibility and superoxide anion radical capturing rate of the enzymolysis product reached 0.667 and 61.55% respectivelyn and hydrolysis degree is 15.13%.In this dissertation, the antioxidant properties of CPHs at various DH are evaluated using different antioxidant assays in vitro. CPH at DH 15 has the most powerful antioxidative activeties and radical scavenging activites, such as reducing power, hydroxyl radical scavenging activity, scavenging of superoxide anion, DPPH radical scavenging activity. The molecular weight distribution profile of CPHs at various DH indicated the molecular weight decreased at higher DH and the peptide with molecular weight (Mw) between 300 and 1,500Da dominated in hydrolysate at DH15. The antioxidant testing in vivo showed that high and middle dose of CPH could significantly (P<0.01) keep the activities of SOD and GSH-PX stable and decreased MDA contents in the serum, heart and liver of aged mice induced by D-galactose.Chickpea protein hydrolysate (CPH) was fractionated by gel filtration on Sephadex G-25. The antioxidant and free radical-scavenging activities of four CPH fractions (Fra.I, Fra.II, Fra.III, and Fra.IV) were measured using reducing power, inhibition of linoleic acid autoxidation, and DPPH/superoxide/hydroxyl radical scavenging assay. The antioxidant activity of Fra.IV (81.13%) was closer to that ofα-tocopherol (83.66%) but lower than that of BHT (99.71%) in the linoleic acid oxidation system. Amino acid analyses showed that Fra.IV with the strongest antioxidant activity also had the highest total hydrophobic amino acids content (38.94% THAA) and hydrophobicity (125.62 kcal/mol amino acid residue) compared with the other three fractions. The molecular weight distribution of Fra.IV indicated that this fraction was composed of low molecular weight peptides whose major peaks were located at near 1286 (49.21%) and 652 (40.63%).The purified peptide purified by using consecutive chromatographic methods had a molecular mass of 717.37 Da and amino acid sequence was identified as Asn-Arg-Tyr-His-Glu by amino acid analysis and MALDI-TOF-TOF MS/MS. The antioxidant activity of this peptide efficiently quenched different sources of free radical: 1,1-diphenyl-2-pycryl-hydrazyl (DPPH) (45.33%), hydroxyl (79.81%), and superoxide radicals (60.09%). This peptide possessed strong Fe2+ and Cu2+ chelating effect (76.92% and 63.08%, respectively), and it exhibits 88.81% inhibition of peroxidation in linoleiacid system at 50μg/L higher than that of VE.The effect of high-pressure (HP) treatment on the hydrolysis of CPI by Alcalase was analysed. Isostatic pressure (100-600 MPa) was applied to the protein substrate prior to its enzymatic hydrolysis. The degree of hydrolysis was measured by the pH-stat and TCA method, and the antioxidant activity was assessed using reducing power and superoxide anion radical scavenging. The results showed a significant increase of DH and antioxidant activity when CPI was digested after the HP treatment at 300 MPa and 400 MPa for 10 min. Digestion was also conducted at atmospheric pressure (0.1 MPa) and high pressure. The hydrolysis and antioxidant activity were also enhanced significantly (P<0.01) when digestion took place under high pressure. The highest DH were found at 200 MPa for 30 min, while the highest reducibility and superoxide anion radical scavenging acitivity of hydrolysates reached 0.406±0.006 and 66.26%±1.67 respectively when hydrolysis was conducted at 200 MPa for 20 min. The peptide profile analysed by SE-HPLC showed that high-pressure treatment can improve the content of small peptides with the molecular distribution ranging from 500 to 1000 which mainly contribute to the antioxidant activity.