| Peanut, as an important resource of oil plant protein,contains eight kinds of essential amino acids that is well balanced of the human body, is high quality edible protein source which is ideally suited for human consumption. In this dissertation, peanut protein was gained with dried peanut, the anticoagulant activity of different kinds of protein isolates was studied and compared. High quality peanut protein was selected for futher isolate preparation of peanut peptides,which is prepared by the method of enzymatic, via testing the anticoagulant activity of different hydrolysates, the suitable proteases on further hydrolysis of peanut protein isolate is chosed. The anticoagulant activitypeanut of peptide is higher than that of peanut protein; After the peanut peptides is freeze dryed, the high anticoagulant activity of peanut peptides was prepared by preparative liquid chromatography, semi-preparative RP-HPLC and SEC-HPLC,then explore and analysis the relationship between the anticoagulant activity and molecular structure via HPLC-MS.Different degree of hydrolysis, different anticoagulation activity,though the degree of anticoagulant activity of enzymatic hydrolysate is high,the hydrolysis may be low, here comes the conclusion that the hydrolyzed degree of enzymatic hydrolysate and its anticoagulant activity are not necessarily linked. Via the enzyme screening, according to the comparison of the overall enzymatic hydrolysate anticoagulant activity, we determined to hydrolyze peanut protein with the Alcalase 2.4L enzyme.The best hydrolysis process conditions were determined according to the response surface experimental design: substrate concentration 5.0 g/100 m L, enzyme dosage is 5000 U/(G protein), reaction temperature 55 C, p H 9, hydrolysis time 2h. When the density was 60mg/ml,The inhibition rate of thrombin under this condition reached 86.62%, while the degree of peanut enzyme hydrolys was 19.1%.Preparation of peanut peptides is separated by liquid chromatograph system, the optimal separation condition is determined through single factor experiment: acetonitrile concentration : 25%, the concentration of sample :20mg/m L, sample volume: 1m L, flow rate of 5m L/min,Under which condition the peanut peptide separation purity and yield the best: 3 main components were seperated, the anticoagulant activity of component G2 is best, At the concentration of 40mg/m L, the inhibition rate on thrombin reached 94.2%.The G2 component was seperated by semi preparative RP-HPLC, according to optimizing separation conditions of single factor test, the best conditions for the elution gradient elution is determined:A flow rate of 1.5 m L/min, the mobile phase with gradient elution: 0 ~ 5min and l00 ~ 85%A; 5 ~ 20 min, 85 ~ 60% A; 20 ~ 26 min, 60 ~ 0%A ~ 28 min; 26 0% A; 28 ~ 30 min, 0 ~ 100% A and 30 ~ 40 min; 100% A(A:100% water, 0.1% TFA; B:100% pure acetonitrile, containing 0.1% TFA), feed concentration for the 1.5 mg/m L;in the same way,when the eluent was at a certain proportion, we determined the best condition: proportion of acetonitrile: water =15:85(v:v), flow rate of 1.5m L/min, inlet concentration of 7mg/m L, the injection volume 80 μl.Through SEC-HPLC the component of G2-R3,G2-R5 was analysised under this condition:the eluate ratio of acetonitrile: water =15:85(v:v), flow rate of 1.5m L/min,inlet concentration of 7mg/m L, the injection volume 80 μl, Both of them were notpure. The anticoagulant effect of G2-R3 was the best, and the inhibition rate was 100.07 at 0.41mg/m L, the anticoagulant effect of G2-R5 was second, and the anticoa gulant activity was 100% at 0.40mg/m L. The structural of G2-R3, G2-R5 was analy sised by MALDI SYNAPT Q-TOF MS, we got four active peptides. Gly-Asn-His-Gl u-Ala-Gly-Glu(M:712.19)and Cys-Phe-Asn-Glu-Tyr-Glu(M:803.23)were from G2-R3, Pro-Ala-Ser-Gln-Gly-Glu(M:587.24)and Asn-Ala-Gly-Leu-Asn-Asp-Pro-Met-AspHis-Glu(M:1211.47)were from G2-R5, in which Asp(aspartic acid) was a main co mponent of cotyledons of peanut protein. |
PDF Full Text Request