Characterization of laccase_catalyzed oxidation of phenolic compounds from apple | 漆酶对苹果酚类物质的催化特性

Chinese. 研究白腐菌漆酶对苹果多酚类物质的催化特征，为开发新型具有生物活性的色素物质提供理论依据。采用单因素和正交组合试验确定白腐菌漆酶对苹果多酚酶促氧化的最佳条件，并对氧化产物的稳定性进行分析；然后对酚类氧化产物进行HPLC分析，初步确定产物组分，最后分析了苹果多酚及其氧化产物对•OH和DPPH•的清除作用。①漆酶对苹果多酚的最佳酶促氧化条件为：反应温度55 ℃，pH 5.5，苹果多酚质量浓度2.5 mg/mL，漆酶浓度 3.5 U/mL。②氧化产物在363 nm处有最大吸收峰，对温度和光照不敏感，随着pH增大颜色加深。③HPLC分析表明，模式底物儿茶酚、表儿茶素、儿茶素分别能产生2，2，4种氧化产物。④抗氧化活性研究表明，质量浓度较高时，苹果多酚氧化产物对•OH和DPPH• 的清除作用强于苹果多酚，且在一定质量浓度范围内呈现量效关系。在氧化产物质量浓度为500 μg/mL时，对•OH的清除率最高，达85.42%；在氧化产物1 000 μg/mL、反应时间为60 min时，对DPPH•的清除率最高，达94.32%。优化了漆酶催化苹果多酚氧化的工艺条件，苹果酚类物质经漆酶催化氧化后形成了新的可溶性、光热稳定性的有色产物，高质量浓度下产物的抗氧化活性较原提取物增强。

English. In order to develop novel and bio-active possessed pigment,the catalytic characteristics of apple phenolic compounds catalyzed by Trametes versicolor laccase were investigated. The process parameters of apple phenolic compounds catalyzed by T.versicolor laccase were optimized through both single_factor test and orthogonal experiment and the stability of oxidation products was analyzed; the composition of the end products was determined through HPLC and its properties were measured by scavenging free radical of •OH as well as DPPH•. ①The optimal parameters for the process of laccase catalyzed oxidation were as follows:reaction temperature of 55 ℃, substrate concentration of 2.5 mg/mL, pH 5.5, enzyme dosage of 3.5 U/mL. ②The oxidative products presented maximum absorption peak at 363 nm,the color of which darkened with the increase of pH, and less sensitive to heating and light. ③HPLC analysis showed that substrates of catechol, epicatechin and catechin produced 2, 2 and 4 oxidized products, respectively. ④Each product revealed higher activity to scavenge free radical of •OH as well as DPPH•at their high mass concentration than the substrate, which showed dosage_effect effect within certain mass concentration scope, and a maximum scavenging activity to •OH of 85.42% was reached at mass concentration of 500 μg/mL, while that to DPPH• was 94.32% at 1 000 μg/mL. The conditions of apple phenolic compounds catalyzed by T.versicolor laccase were optimized, and new colorful and soluble product formed from apple polyphenol catalyzed by laccase. The product was stable to light and heating, and showed increased antioxidant activity than the original extract in high mass concentrations.