Optimization Of The Synthesis Conditions Of Theaflavins By Enzymatic Oxidation

Theaflavins, first found by Roberts E.A.H found, refer to the soluble in ethyl acetate orange-yellow substances derived from tea polyphenols and their derivatives oxidative polymerization. Theaflavins have a close relationship to black tea quality and are responsible for bright-yellow color of black tea liquor and the appearance of the golden ring of black tea. Therefore, the content of theaflavins in black tea determined the price of black tea. Theaflavins are not only the important quality indicator of black tea, but also the natural products of tea owning broad development prospects because of their pharmacological functions, such as anti-oxidation, anti-tumor, anti-inflammatory anti-bacterial, anti-viral and other activity. All of the factors promote theaflavins become the hotspot of research before and in the foreseeable future. The traditional method for obtaining theaflavins is extracting them from black tea with different kinds of solvent. But low content of theaflavins in black tea and the difficulties in extracting and isolation of theaflavins make the traditional extraction method inefficient and high-cost, which seriously hinder the utilization and development of theaflavins as natural products and functional food additives. The aim of this study is to synthesize theaflavins from tea catechins mixture using enzymatic oxidation methods. It has important theoretical and practical significance.1. Single-phase testThis study based on the previous research explored the parameters of enzymatic oxidation method for TFs synthesis. Using single phase reaction to probe the optimum enzyme oxidation conditions from catechins to theaflavins on pH, temperature, enzyme concentration, substrate concentration et al. Single factor test results showed:the theaflavins generate the largest amount on the condition of pH5.0, temperature30℃, substrate concentration1.75g/200mL, enzyme concentration of50g/200mL. By quadratic regression orthogonal rotation combination tests on the reaction factors, including pH, temperature, substrate concentration and enzyme concentration, the optimal condition of pH4.96, temperature30.5℃, substrate concentration of1.78g/200mL, enzyme concentration50.55g/200mL was reached. Under the optimum condition the rate of conversion from catechins to theaflavins can reach19.44%. Based on the basis of the optimal condition,50mins was selected as best reaction time. Results showed introducing oxygen can significantly improve oxidation conversion rate from catechins to theaflavins, especially for frozen fresh tea leaves. It was also showed that when introducing enzyme can also increase the amount of produced theaflavins. Theaflavins production significantly increased when adding Polygonum cuspidatum enzyme. 2. Two-phase testOn the basis of the single experiment results, it was found that adding ethyl acetate in the reaction system improved the conversion rate of catechins. The experiment results showed that when the ratio of buffer solution and ethyl acetate was1:1, theaflavins purity can be up to43.38%and the conversion rate can reach28.75%, far higher than the single phase experiments. After selected the best rate of buffer solution and ethyl acetate, the other parameters such as pH, temperature, substrate concentration and esters were optimized by quadratic regression orthogonal rotation combination tests. The obtained best condition was pH5.06, temperature31.23℃, the substrate concentration1.71g/200mL, the rate of buffer and ethyl acetate1:1.2and under the best condition, yield rate of theaflavins can reach28.90%.