Preliminary Study On The Enzyme And Substrates Of Black Pigment From Vaccinium Bracteatum Thunb Leaf

Black pigment of Vaccinium Bracteatum Thunb leaf is a kind of natural black pigmentfrom plant, which is fairly safe and used widely in the Chinese folk for many years. Recentlyit has attracted more and more attention of researchers. Many researches have been done onblack pigment of Vaccinium Bracteatum Thunb leaf during recent years, but the composition,formation mechanism and detection method about black pigment of Vaccinium BracteatumThunb leaf are still unkown, which have hindered its further development. In order to offer atheory basis for the deep process of Vaccinium Bracteatum Thunb leaf, in the present essay,we investigated the formation mechanism of black pigment.Firstly, the major components of black pigment of Vaccinium Bracteatum Thunb leafwere analyzed. Then, a primary research on the formation mechanism of black pigment wasmade, and from the research we could find that the enzyme catalyzing the formation of blackpigment was contained in the fresh Vaccinium Bracteatum Thunb leaf. The enzyme showedno catalytic activity in the native state, however, it could separate from tissue and be activatedby homogenate. The enzyme by homogenate could catalyze the formation of pigment, whichmight form stable black pigment by heating.Then, black pigment enzyme was purified through a series of meanings, including buffersolution method with PVPP,20%ammonium sulphate precipitation and Sephadex G-150. Thepurified enzyme showed a specific activity of207.89U/mg, which exhibited a purificationfold of4.07and a enzymatic activity recovery rate of1.2%. Then the enzymaticcharacterizations were analyzed. The optimal pH and temperature were6.5and45oCrespectively. The effect of metal ions was also tested, indicating that K+and Na+showed noinfluence on enzymatic catalysis, while catalytic efficiency could be significantly enhancedby Ca2+, Zn2+, Mg2+and Cu2+. By comparison, Fe2+showed an inhibition on the enzymaticactivity. The black pigment enzyme showed a high affinity with hydroxyl phenolics(pyrocatechol, L-DOPA, pyrogallic acid, catechinic acid and chlorogenic acid), however, itshowed a poor affinity with single hydroxyl phenolics (L-tyrosine and guaiacol). At the sametime, the optimal substrate was catechol.After that, we made simulation process of the formation of black pigment by usingLC-MS. The experiment of LC-MS indicated that the content of two substances wasdecreased. It was speculated that they were the main precursor of black pigment withmolecular weight354and536respectively, and the substance with molecular weight354wasScopoletin glycoside.Final, we made coloring experiments on a variety of food raw material with freshVaccinium Bracteatum Thunb leaf extraction. The experiment of coloring experiments indicated that the coloring effect of Vaccinium Bracteatum Thunb leaf extraction on thepowdery food material is fast，the coloring effect of Vaccinium Bracteatum Thunb leafextraction on the high starch food is good, the coloring effect of Vaccinium Bracteatum Thunbleaf black pigment on the high protein food is generally satisfactory.