Study on Preparation and Antioxidative Activity of Tea Residues Polypeptide
|School||Nanjing Agricultural College|
|Keywords||tea residues protein protease hydrolyze peptide antioxidative activity|
Peptide is a product constructed by amino acids, and despite having the property of protein is quiet different from protein. Antioxidative peptide is one kind of activity peptide which has substantial antioxidative activities. In contrast to protein, activity peptide has good physico-chemical property and hydrability; As a foundational nutrient substance, the biology availability of peptide is high, is much easier to accept than protein, and it has the efficiency of regulting human body physiology functions. These properties lack in the original protein and amino acids. In this study, crude protein was extracted from tea residues, and activity peptide was prepared using the enzymic method. The primary findings are listed as follows:(1) The tea residues which were used in this experiment contained 10.7%,23.055% and 0.1% tea polyphenol, protein and caffeine. In order to further increase the extraction yield of protein from wast tea leaves, the residual polyphenol was removed through pretreatment. And according to single factor experiments, the rerults showed that the optimum process was:liquid to solid ratio of 40,70℃temperature,15 min, and 1 times. The condition gave a polyphenol extraction rate of 67.3%.(2) The effect of ion strenth agent-NaCl on the yield of protein was analyzed on the basis of the comparison of alcohol method and base digestion. According to orthogonal experiments, the results showed that the optimum condition of alcohol method was as follows:ethanol concentration of 40%, pH 12,70℃, liquid to solid ratio of 40,60 min. The yield of protein reached 19.784% under this condition. And the optimum condition of base digestion was as follows:pH 12,80℃, liquid to solid ratio of 50,60 min, where the protein extraction rate was 33.496% under this condition that was 13.712% higher than that of the alcohol method. In base digestion experiment, ion strenth agent-NaCl inhibited the leaching of protein with the inhibition rate increasing with the adding of NaCl concentration. The product extracted by base digestion had 41.0% of protein.(3) Based on the research of extraction methods of tea leaf protein, the scavenging capacity to hydroxyl radical was the index to compare the hydrolysis effect with four kinds of proteases including Protease M "Amano" G, Protease A "Amano" 2 G, Protex 6 L and ProteAX to hydrolyze tea residues protein to peptide, separately. Where a method to determine the scavenging capacity to hydroxyl radical was optimized as follows:salicylic acid-alcohol solution concentration of 10 mmol/L, adding amount of 0.5 mL; FeSO4·7H2O concentration of 10 mmol/L, adding amount of 0.5 mL; sample solution concentration of 3.0 mg/mL, adding amount of 0.5 mL(pH 7.0); adding 8 mL distilled water; then increased 0.5 mL 10 mmol/L H2O2to start Fenton reaction; shaked, then standing at 25℃for 20 min, distilled water as the comparison, rejected salicylic acid for baseline control; determine the absorbance at 510 nm lastly.a. Protease M"Amano" G, Protease A "Amano" 2 G and ProteAX were compared under their optimal conditions and the same amount of 1000 U/g. The experiment showed that: ProteAX had the best capacity of 16.9%; the capacity of Protease M"Amano" G was 12.1%; Protease A "Amano" 2 G had the poorest activity of 9.9%.b. According to single factor experiments, both ProteAX and Protex 6L had the better hydrolysis effect, thier capacity to scavenging hydroxyl radical reached 27.3% and 25.3% with their optimum conditions being crude protein concentration of 0.5%, enzyme dosage of 600 U/g,55℃, pH 8.0, hydrolysis time of 5 h and crude protein concentration of 0.5%, enzyme dosage of 400 DU/g, pH 8.5,50℃, hydrolysis time of 1 h individully. Although the effect had no significant differences, the reaction time was reduced 4 h when used Protex 6L.c. After hydrolyzed by Protex 6L, the crude polypeptide contained 28.4%,11.0% and 1.5 % protein, peptide and free amino acids, respectively. And the capacity to semi-scavenge hydroxyl radical was 8.432 mg/mL, the Vc was 0.897 mg/mL under the same condition.