Quality Improvement Of Tea Beverage With Tannase Tahm From Aspergillus Niger

Tannase(EC 3.1.1.20)belongs to tannin acyl hydrolase that specifically hydrolyzes the ester and dephenolic acid bonds in the gallotannins.As a specific enzyme for the re-solution of "tea cheese",tannase plays an important role in tea beverage industry.Currently,commercially tannase products are mixture leading to unclear reaction pattern and side-reaction due to the others enzyme activities existed in the products.In this thesis,an Aspergillus niger tannase,TahA,was molecularly cloned and expressed.Its enzymatic properties and hydrolytic activity were investigated.The role in improving the quality of tea beverage was further explored.The main results are as follows:(1)Cloning and expression of A.niger tahA in Pichia pastorisA.niger F0510 tahA was cloned using regular molecular cloning techniques.Its coding region was 1689 bp and deduced 562 amino acid residues.The recombinant yeast,An-tahA,was developed by constructing the expression plasmid in pPIC9K and then transformed into Pichia pastoris GS115.The recombinant expressed 1.04 U/mL of tannase on the shake flask level.(2)Purification and enzymatic properties of recombinant tannaseThe crude enzyme was prepared by fermentation.The enzyme was purified by ammonium sulfate precipitation and then by gel filtration chromatography.The specific activity of purified TahA was 11.5 U/mg with purification folds and yield of 13.9 and 21.4%,respectively.Its maximum activity was at 30? and pH 4.5.The enzyme had good stability when the temperature was lower than 50? or pH 3.0-6.0.The activity was stimulated by Cu2+,Mn2+and K+ and slightly inhibited by Co2+,Fe3+,Mg2+,Zn2+and EDTA,stongly inhibited by Ca2+,Sn2+,while completely inactivated by SDS.The Km and Vmax values were 5.143 mmol·L-10.436 ?mol-mL-1·min-1 on methyl gallate.The enzyme was able to hydrolyze esterified catechins,including epigallocatechin gallate(EGCG),epicatechin gallate(ECG)and galloocetesin gallate(GCG)with hydrolyse strength ECG>EGCG>GCG.(3)The role of recombinant tannase in improving the quality of tea beverages.The condition of the tannase on tea extracts with the turbidity reduced and the brightness increased were experimentally investigated with optimal condition of 0.1 U/mL for 60 min.The enzyme was able to reduce 26.7%of the turbidity of green tea from 48.7 NTU to 35.7 NTU and reduce 20.46%of the turbidity of black tea from 68.23 NTU to 52.47 NTU.The brightness both of tea extracts were increased and the total antioxidant capacity of the green tea and black tea extracts increased by 62.8%and 49.82%after enzyme treatment.The radial scavenging rate of tea extracts was examined by using 1,1-diphenyl-2-picrylhydrazyl(DPPH)scavenging method.26.3 mg/L of the enzyme-treated green tea extract was inquired,which was 77.58%of that green tea extract without enzyme treatment(33.9 mg/L),in addition,52.63 mg/L of the enzyme-treated black tea extract was inquired,which was 90.12%of that black tea extract without enzyme treatment(58.4 mg/L)?...