Study On Preparation Of Chickpea Protein Isolates And Their Functional Properties

The purpose of this investigation was to evaluate protein properties of chickpea produced in Xinjiang. First, the chickpea protein isolate (CPI) preparation art, alkaline extraction and acid precipitation was optimized. Second, based on purification, the structure and functional properties of CPI and its major fractions were investigated. Futher, the detail study of solubility, emulsifying and gelation properties of CPI and their relations to the structure were conducted to expand the use of CPI in food industries. Finally, CPI modification methods were explained.Chickpea used in this paper was deduced to be Kabuli type according to its appearance. Using the lowest protein content in the supernatants after precipitation at various pHs as the index, the isoelectric point (pI) was determined to be pH 5.0. Then, the extraction parameters were optimized by the means of response surface methodology, including extraction temperature, pH, solid to water ratio and time. Under the optimal conditions, protein extraction yield and protein content of the product were 84.5% and 91.53%, respectively.Following above, CPI was separated with Sephacryl S-200 and DEAE-Sepharose CL-6B successively. Two major fractions were obtained, accounting for 60.4% and 17.7% of the total proteins, of which molecular weights were 170 kDa (signed as B) and 110 kDa (signed as D), respectively. The subunit molecular weights of 45, 38.6, 35, 20.1 andl4.4 kDa for B and 60, 55, 51, 38.6 and 35 kDa for D were obtained by SDS-PAGE. The two major fractions B and D were determined to be 11S and 7S, respectively.Amino acid analysis indicated that CPI contained all essential amino acids. The average hydrophobicity of CPI, 11S and 7S were 4.4, 4.05 and 4.65 KJ/mol amino acid residues. Hydrophobicity probe (ANS) and differential scanning calorimetry (DSC) were used to determine the surface hydrophobicity (So) and thermal denaturation temperature (Td) of CPI, 11S and 7S. S₀ were 94.3, 144 and 77.7, while Td were 95.7℃, 88.8 ℃ and 55.4 ℃, respectively. The free sulphydryl (Sf), total sulphydryl (S_T) and disulfide bond (SS) estimated by DTNB were 12.01, 33.50 and 10.75 μmol/g protein for CPI, 13.14, 35.9 and 11.38 μmol/g protein for 11S, and 21.37, 33.83 or 6.23 μmol/g protein for 7S.The CPI solubility was diverse at different temperature, pH, ionic strength and salt (MgSO₄, NaCl, Na₂HPO₄). It was the highest at 45 ℃ compared to that of other temperatures, hit 80%. The solubility increased on both sides of pI (pH 5.0). The effects of ionic strengths on the solubility showed that it was the lowest at ionic strength 0.1, and increased on each side of 0.1. The types of salt affected the solubility little when the ionic strength is below 1. Whereas the effects of salt types on solubility were in accordance with Hofmerister order when the ionic strength is above 1.The effects of protein concentration, oil volume and environment factors (such as pH, ionic strength) on CPI emulsifying properties were investigated. The emulsifying properties included emulsifying activity (EA), emulsion stability (ES) and oil diameter (d3,2). With the protein content increasing, EA increased, and d3,2 decreased. At the same protein content, with the increasing of oil volume, both EA and d3>2 increased. The results also showed that EA was the lowest, while ES and d3,2 the highest at pH 5.0. EA increased and ES kept at about 1.0 on both sides of pH 5.0. Under the ionic strength 0.1, EA was the lowest, while both ES and d3,2 were the highest.In order to understand the relationships between the emulsification and hydrophobicity of CPI, the effects of pH and ionic strength on its So were studied. The results showed that emulsifying activity at each pH tested was not positively dependant on So, while it was positively relevant to So at various ionic strengths.The least gelation concentration endpoints (LGE) of CPI gelation were 14% and 18% for samples prepared with water at pH 7.0 or pH 3.0, respectively. However, it reduced to 10% for the samples with 0.1 mol/L NaCl at pH 3.0. The viscoelastic properties affected by pH, ionic strength, the amount and types of the assayed salts were tested in oscillatory mode with the CPI concentration 15%. The results of frequency sweep (0.1-10 Hz) showed that the samples behaved as diluted macromolecular or semidiluted solutions under the conditions of pH 3.0 with water or 0.1 mol/L NaCl, while at high ionic strengths (0.5-1.0 mol/L NaCl) or 0.1-0.3 mol/L CaCb, they were more elastic with a gel-like behavior, the same as they were at pH 7.0 with water or 0.1 mol/L NaCl. Additionally, at high ionic strength (0.5-1.0 mol/L NaCl) and pH 7.0, semidiluted solutions were observed and G' intersected G" at a frequency of 1 Hz. The effects of CaC^ concentration on gelation properties at pH 7.0 showed that sample behaved as gel-like at 0.1 mol/L CaC^, while a semidiluted solution was observed at 0.3 mol/L CaCh, G' and G" intersecting at 1 Hz. Time sweep mode was used to study the gelation kinetics under different conditions. The kinetics of sample prepared at pH 7.0 with water was similar to that observed at pH 3.0 with 0.5 or 1.0 mol/L NaCl. Tested by puncture method, gels made with CaCU were harder than those with NaCl under the same ionic strength and pH.The structure changes of CPI after gelation were studied by Raman spectroscopy. The P-sheet and random coil increased at the expense of a-helix after gelation. The intensity ratio hso/h3O increased showed a tyrosine residue in a strong hydrogen bond as a proton acceptor. Exposure of buried tryptophan residues in proteins was observed by the decrease of the peak intensity near 760 cm"1, which indicated that tryptophan may play a role in hydrophobic interaction in the gels. The bond 525 cm"1 could be assigned as disulfide bond in the gauche-gauche-trans conformation. This peak disappeared or intensity weakened showed that the conformation changed after gelation.The enzyme modifications by alcalase and transglutaminase (TGase) were performed toovercome the low solubility and emulsifying activity of CPI under low ionic strength. After CPI was hydrolyzed by alcalase for 60 min (DH 5.91%), the solubility of CPI were no longer affected by low ionic strength. When hydrolyzed for 30 min (DH 5.80%), the emulsifying activity was 1.22 or 2.78 fold those of unmodified samples with water or 0.1 mol/L NaCl, respectively. TGase showed no good effect on the solubility and emulsifying activity of CPI at 0.1 mol/L NaCl.