Study On The Extraction Process Of Tea Leaves With Compound Enzymes

The function of hydrolysis of exogenous protease,pectinase,cellulase and their complex enzyme on tea leaf were studied in this paper.After that we detected the change of reducing sugar,amino acids physicochemical index in the tea soup by the method of capillary electrophoresis(CE)and high performance liquid chromatography(HPLC),in order to obtain a kind of complex enzyme with high hydrolysis efficiency to tea leaf and improve the quality of the tea soup.The results showed as follows:1.Nine reducing carbohydrates which may be existed in the green tea enzymatic hydrolysate were derivatized with 1-phenyl-3-methyl-5-pyrazolone(PMP),and baseline separated by CZE under the condition of 200 mmol/L borate buffer pH 11.0 and running voltage 25 kv at temperature 25 ?.The standard curves of main reducing carbohydrate derivative in the green tea including maltose,lactose,xylose,arabinose,glucose,rhamnose,galactose,mannose and galacturonic acid showed good linearity in the range of 0.5?10 mmol/L and the R2 value equivalent to 0.9939?0.9994.The minimum limit point of determination ranged from 0.09 to 0.23 ?mol/mL and quantitative recoveries of the nine reducing carbohydrates ranged from 94.04%to 101.9%.The optimized CZE method was found to be feasible to examine the compositions and content of reducing carbohydrates in the green tea extracts with enzyme treatment.The result showed that the enzymatic solution mainly contain five kinds of reducing carbohydrates including xylose,arabinose,glucose,galactose and galacturonic acid.2.The HPLC method for determination of amino acids:Using phenylisothiocyanate(PITC)as derivating agent,the Hypersil BDS C18 column(200mm×4.6mm,5?m)was used.Under the condition of gradient elution,the flow rate was 1.0 mL/min,the column temperature was 40 ?and the UV detection wavelength was 254 nm.The contents of 17 kinds of hydrolyzed amino acids in green tea determined by HPLC can be separated well.While the corelation coefficient in equation of linear regression were 0.99821?0.99977,which can be meet the linear requirement of substance detection well.By this method,we detected the contents of amino acids in green tea soup and the result showed 13 kinds of amino acids were detected.3.The determination of amino acids in tea soup by HPLC method,we selected better hydrolytic protease green protein C from six protease.After that,C protease digestion conditions were optimized on single factor and orthogonal in order to obtain the best protease C processing conditions.The result showed the best processing conditions were that protease dosage 10 mg/g,hydrolysis sampling time 8 h,hydrolysis pH value of 5.0,reaction temperature 50 ?.Total amino acids showed 2.58 times over the control under the best enzyme treatment condition through detection of acid analyzer,which greatly improve the amino acid content in tea soup.4.We screened and recomposed pectinase and cellulase existed in our lab,obtained a new pectinase and complex plant hydrolase.The new pectinase consist of pectinase D and pectinase E and the proportion of them was 2:3.The complex plant hydrolase composed by the cellulase 0.76 IU/g and pectinase 5.06 U/g.A complex protease was recomposed with protease complex 10 mg/g,new pectinase 5.06 U/g and cellulase 0.76 IU/g.5.The sense of tea soup was evaluated after hydrolyzed by the complex protease.The result indicated that the color,aroma and taste has greatly improved.And applied it to seven other commercially available tea including Green tea,Longjing tea,white tea,Tieguanyin,Oolong tea,Keemun black tea,Pu'er tea,we found reducing sugar,total sugar,amino acid,the leaching rate and other physical and chemical indicators in enzymatic hydrolysate of unfermented and mild fermented tea were significantly better than the effect of fermented tea and Semi-fermented.Meanwhile the former were increased by several times compared with the control,which proved the complex protease has great application prospect.