| The objective of this research is to study the stabilization of Tea polyphenols(TP) on two sulfhydryl proteases(bromelain and papain) of comprehensive application and inactivation of two trypsin inhibitors(KSTI and BBI),which exist in most legumes. The main research conclusions were as follows:The effects of TP concentration, temperature, pH, action time, buffer type and concentration on bromelain and papain recovery were discussed when they were complexing with TP. The results showed that the maximum recovery of bromelain activity was 81.97%and the protein recovery was 79.56%under the conditions of 0.03mol.L-1 citric acid -Na2HPO4 buffer , 0.8% TP concentration at 30℃and pH 5 for 30 min; When 0.7% TP was added to papain dissolved in 0.1mol.L-1 citric acid-Na2HPO4 buffer at 0~4℃and pH 4.75 for 45 min, the maximum papain recovery was 91.24% and protein recovery was 92.45%, respectively.The catalyzing properties and stabilization of bromelain and papain before and after complexing with TP were compared. Complexing with TP didn't change the optimum temperature of bromelain(55℃) and papain(65℃);It was showed that the optimum pH of the free and the complexed bromelain didn't change(7.0) ,but papain changed from 7 to 7.5. Comparing with free bromelain and papain, the thermal stabilities of the complexed were greatly improved: after warming in 80℃for 2h, residue ratio of bromelain activity was improved from 20% to 57.34%,whereas papain activity from 48.07% to 76.47%; when stored under normal temperature(25℃) , the half-time(time to lose half of the activity) of bromelain was increased from 8d to 20d, papain was increased from 5d to more than 35d; when stored under low temperature(0~4℃) for 30 days, residue ratio of bromelain activity was changed from 15.1% to 77.89%, papain from 49.84% to 84.86%. In addition, the results showed that the Km values for the complexed was higher than the free: bromelain from 0.6mg.mL-1 to 1.4mg.mL-1, papain from 3.07 mg.mL-1 to 5.71 mg.mL-1, which indicated that complexing with TP lowers the affinity of the enzyme for the casein substrate. These results indicated that the complexed had enhanced their utilization by improving their stabilities obviously, but not changed reaction characteristic of bromelain and papain.The inactivation of TP on trypsin inhibitors of soybean milk was studied.Optimum inactivation ratio was 52.3% when the rate of TP to soybean milk was 1:2.The inactivation effects of several chemical compounds on KSTI and BBI were compared.It was showed that to KSTI:GSH>TP>Na2SO3>Lys>Na2S2O3, and to BBI: Na2SO3>TP>Na2S2O3>Lys>GSH. The results indicated TP was a prospective biology inactivator.Optimal reaction conditions for TP to inactive KSTI and BBI were also studied. Optimal inactivation ratio was 83.15% under the conditions: the quantity ratio of TP to KSTI (mg: mg) was 25 at pH 4.5 and temperature 45℃for 1.5h; maximum inactivation ratio of BBI was 76.58% when the quantity ratio of TP to BBI is 20 at pH 4.5 and temperature 40℃for 1h.In order to summarize the action mechanisms, the antioxidant activities, sulfhydryl contents, UV and fluorescence spectrum of these several sulfhydryl proteins before and after complexing with TP were determined. The inhibitory activity to pyrogallol and Feton of papain and bromelain were greatly improved after complexing, reducing trend of sulfhydryl content with storing time of bromelain and papain was slower than that of free enzyme, and the sulfhydryl group content of KSTI and BBI complex were fewer than of non-complexing KSTI and BBI. UV and fluorescence spectrum showed that covalent bonds structure of these sulfhydryl protein hadn't been destroyed after complexing with TP, but polarity of Trp in microenvironment decreased. |
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