Study On Preparation Of Chickpea Polypeptides And Its Biological Activities

Chickpea has three characteristics:balanced composition of essential amino acids,low anti-nutritional factors and high bioavailability.It is a high-quality plant protein source.The objective of this study is to screen the optimum protease for chickpea protein isolate hydrolysis,to investigate the effect of hydrolysis conditions on the hydrolysis degree and yield of chickpea polypeptide,to evaluate the physicochemical properties and functional properties of chickpea polypeptide with different hydrolysis degrees,and to evaluate the antioxidant and anti-fatigue effects of the optimized enzymatic hydrolysates with different hydrolysis degrees through in vitro antioxidant model and animal experiments.The target peptide fraction was separated and purified.Finally,the amino acid sequence of the target peptide was analyzed and predicted by reversed-phase high performance liquid chromatography(RP-HPLC)and on-line Q Exactive mass spectrometer to develop the function of chickpea.Peptide products provide a theoretical basis.The main results are as follows::(1)The effects of four factors on the protein extraction rate of Xinjiang mulei base chickpea were studied by using alkali soluble acid precipitation method to extract the protein of chickpea and to investigate the ratio of material to liquid,alkali soluble pH,extraction time and extraction temperature.On the basis of single factor experiment,the optimum extraction conditions of chickpea protein were optimized by response surface methodology(RSM).SDS-PAGE gel electrophoresis was applied to chickpea protein extraction.The subunit composition of chickpea protein was analyzed.The results showed that,the extraction rate of chickpea protein was 83.29%when the ratio of material to liquid was 1:10(g/mL),alkali-soluble pH was 10.0,the extraction time was 82 minutes,and the extraction temperature was 30?.Under these conditions,the subunits of chickpea protein were mainly distributed in the seven regions of 157,75,52,48,37,28 and 18 KDa.(2)The conditions of composite enzymatic hydrolysis of chickpea protein were established.the influencing factors on the chickpea protein hydrolysate of Alcalase enzyme further hydrolyzed by Neutrase and Flavorzyme enzyme were investigated on the basis of the hydrolysis of chickpea protein with Alcalase,The regression model for the relationship between the impact factors and the TCA-NSI was established.The results showed that the optimal processing conditions for hydrolyzing chickpea protein hydrolysate of Alcalase with Neutrase and Flavorzyme are as follows:pH 7.0,temperature 55?,time 216 min and the composite enzyme concentration 5678 U/g.Under these conditions,the TCA-NSI is 63.79%,Compared with Alcalase hydrolyzing alone,the TCA-NSI increased by 8.64%,DH was 26.74%.(3)The impact of enzymatic hydrolysis by complex enzymatic on the conformational and functional properties of chickpea protein isolate(CPI)was investigated.The physicochemical,functioal characteristics of CPI and their hydrolysates(CPH)according to the degree of hydrolysis(DH)were also determined.These parameters were then related to the changes in the emulsification activity(EAI)and stability(ESI).The enzymatic hydrolysis was found to improve protein solubility,especially at the pH near the isoelectric point of chickpea protein isolate;improve foaming and Foam stability;the secondary structure of chickpea protein isolate(CPI)changed significantly,the ?-helix and ?-turn content were significantly reduced,and the ?-sheet and disorder structure were significantly increased.(4)The functional properties of chickpea polypeptides were evaluated by different in vitro antioxidant models and animal experiments.The in vitro antioxidant activity and anti-fatigue ability of chickpea polypeptides with different degrees of hydrolysis were studied.The results showed that the chickpea polypeptides with different degrees of hydrolysis can scavenge free radicals and have certain reducing ability and metal chelation,especially when the concentration is increased.The chickpea polypeptide with a degree of hydrolysis of 16.5%has better antioxidant capacity than other chickpea polypeptides with other degrees of hydrolysis.The results of anti-fatigue test showed that compared with the control group,the weight-bearing swimming time of the mice in each dose group was significantly prolonged(P<0.05),which increased the thymus index and spleen index(P<0.05),which could increase the liver glycogen.It can significantly reduce the levels of lactic acid,malondialdehyde(MDA)and urea nitrogen(BUN)(P<0.05),and has certain anti-fatigue effects.(5)The hydrolyzate was separated into four components by ultrafiltration.The CPH-? component(<3 kDa)has the strongest free radical scavenging capacity.Then,the component CPH-? was separated into three components(F-?,F-?,F-?)by gel chromatography(Sephadex G-25),The F-? components with the highest free radical scavenging activity were separated by RP-HPLC,and 12 antioxidant components were obtained.The components of P8 with strong antioxidant activity were screened with the free radical capture rate as the index,and identified as single peak by RP-HPLC.The amino acid sequence of antioxidant peptides was identified by Q Exactive mass spectrometry as DGRYNDPFNDGAGSQ with molecular weight of 1432.67.