Enzymatic Preparation Of Acetylated EGCG And Its Antioxidant Properties In Edible Oils

Epigallocatechin gallate (EGCG) is one of the main active compounds in tea polyphenols. It possesses a wide spectrum of biological activities:scavenging free radicals, anti-oxidation, radiation resistance, lowering blood pressure and blood glucose and blood lipid, sterilization, prevention of cardiovascular diseases and cancer, and other biological activities. However, the use of EGCG in the biological, pharmacological, and medicinal industries, especially in edible oil, is limited by its low stability and solubility in the fatty and aqueous phases. By chemical or enzymatic method, it is feasible to change the structure of tea polyphenols to increase its liposolubility. The enzymatic method has many advantages over the chemical method in reaction steps, reaction time, solvent consumption and efficiency. The domestic study on enzymatic modification of catechin EGCG monomer is not reported. Therefore, in this paper, enzymatic acylation of EGCG was carried out to increase the lipid solubility of EGCG. The conditions of the enzymatic acylation were optimized and the acylated products were separated by high-speed countercurrent chromatography (HSCCC). Moreover, the solubility and oxidation resistance of acylated EGCG in edible oil were studied. Main researched contents include:By single factor and orthogonal experiments, the conditions of the enzymatic acylation of EGCG were optimized as follows. The kind of enzyme was the lipase TL IM LIPOZYME, reaction solvent system was made up of acetonitrile and isopropanol at a ratio of5to5, reaction temperature was45℃, reaction time was11hours, molar ratio of substrates vinyl acetate and EGCG was0.4:1, enzyme dosage was5g per100g substrate. Under those conditions, EGCG acetylated conversion rate reached96.73%.The determination of the acylated product by liquid chromatography tandem mass spectrometry (LC-MS) and infrared spectrometry. comparing the infrared spectrum of EGCG and acetylated EGCG, showed that the reaction product has obvious carbonyl (C=O) absorption peak, means acetylation reaction was successful.LC-MS showed that the enzymatic acylation of EGCG was successful and the acylated products could be classified into three groups, one acetyl-replaced EGCG, two acetyls-replaced EGCG and three acetyls-replaced EGCG. According to the different replace position, each type of substitution product was divided into different monomers.The efficient preparative HSCCC separation and purification of the acylated EGCG was developed at a flow rate of1.5mL, using a two-phase solvent system composed of n-hexane: ethyl acetate:methanol:wate (1.5:5:1.5:5, v/v/v) which was selected according to the measurement of partition coefficient (K). The structure was identified by TOF MS/MS,1H NMR and13C. The results showed that the Peak2was5"-O-acetylation EGCG.Through the solubility and light transmittance experiments showed that the acetylation EGCG has good solubility and high transparency in edible oil, solubility in30℃was0.50g/kg. Chromatic aberration experiments showed that when the content is200mg/kg, acetylated EGCG has little effect on color and brightness for soybean oil. Using peroxide value (POV) method to examine and compare the antioxidant properties of different antioxidants on edible oil. Results showed that when the adding quantity was200mg/kg, antioxidant activity of acetylated EGCG was higher than EGCG and BHT, slightly lower than TBHQ. For the different acetyl number replaced EGCG, the antioxidant activity in order to:EGCG replaced by one acetyl product> mixture of EGCG replaced by one acetyl product and EGCG replaced by two acetyls product> acetylated EGCG> EGCG. DPPH free radical scavenging rate was:VC> EGCG> acetylated EGCG> TBHQ.