Optimization Of Fermentation Conditions And Studys Of The Characteristics And Application Of Tannase From Microorganisms

Tannase,the tannin acyl hydrolase?EC 3.1.1.20?,was named for its ability to hydrolyze the ester bonds and depside bonds in tannins.The hydrolysable tannins,complex tannins,and esters of gallic acid and alcohol were catalyzed by tannase.And it was widely used in the food,pharmaceutical,and animal husbandry industries.The enzyme was obtained mainly from Penicillium,Aspergillus,and also was found in yeast,bacteria,actinomycetes and some plants.In this paper,the tannase-producing strain Aspergillus niger YY was screened from the strains preserved in the laboratory.And then the liquid fermentation of tannase production was optimized.The optimal medium formulation was?100 mL?:3 g tannic acid,1 g sucrose,0.2 g NaNO₃,0.05 g MgSO₄,0.1 g K₂HPO₄.Optimal culture conditions were as follows as below 30?,160 rpm,inoculum size 1%,5 d.After optimization,the enzyme activity was increased from the initial 0.782 U/L to 2.686 U/L,which was 3.43folds.The tannase was preliminarily purified by ammonium sulfate fractional precipitation method.The catalytic properties of tannase were investigated.The first step,the protein was removed by precipitation with 50%ammonium sulfate,and the tannase was precipitated with 90%ammonium sulfate for the second step.The purification ratio was2.03,and the yield was 41.36%.The enzyme activity was the highest with pH 5 and 70°C.The enzyme was stable below 60°C.After storage for 15 d and 30 d at room temperature,the enzyme activity was decreased by 6.60%and 27.77%,respectively.After storage for15 d and 30 d at 4?,the enzyme activity was decreased by 2.11%and 14.91%,respectively.The enzyme activity was increased by Ca²⁺and Mg²⁺,inhibited by Zn²⁺and Mn²⁺,and no significant effect by Cu²⁺,Ba²⁺,Al³⁺,Fe²⁺and Fe³⁺.The enzyme was significantly inhibited by 9.5%ethanol and 0.25%?w/v?SDS.The tannase was used to extract polyphenols from pomegranate leaves.The content of total phenolic and gallic acid and antioxidant activity of the extracts were found to be higher than those without the addition of tannase.After adding 0.8 U of tannase,the total phenolic of pomegranate leaf extract was increased 0.48 folds,the gallic acid was increased 2.15 folds,and the DPPH free radical scavenging ability was improved 21%.It is preliminarily proved that tannase can be used for biotransformation of pomegranate leaf polyphenols.Resource utilization of pomegranate leaves can be improved.