油橄榄叶橄榄苦苷酶解制备羟基酪醇的研究

Preparation of Hydroxytyrosol by Enzymatic Hydrolysis of Oleuropein from Olea europaea L. Leaf Extract

以1 g油橄榄叶提取物为原料,采用高效液相色谱法测定油橄榄叶橄榄苦苷(OE)和羟基酪醇(HT)的含量,筛选得到适合用于OE酶解制备HT的酶,通过单因素和正交试验优化酶解工艺,并考察了HT、OE和油橄榄叶提取物清除二苯基苦基肼(DPPH)自由基的抗氧化活性。结果表明,在相同酶活力条件下,半纤维素酶对OE的降解和HT的制备都有着很好的效果,其次为纤维素酶和β-葡萄糖苷酶。半纤维素酶降解OE的最佳工艺为温度60℃、p H值5.5、酶量40 mg、时间6 h,得到HT质量分数为6.07%,OE降解率为85.28%。酸解、碱解和酶解方法的比较结果表明,盐酸水解后HT质量分数可达7.41%,效果略优于酶水解;但Na OH水解后HT质量分数仅为4.09%。DPPH自由基清除实验结果表明,DPPH自由基清除能力为HT>维生素C>OE>2,6-二叔丁基-4-甲基苯酚>油橄榄叶提取物,HT的半数抑制质量浓度(IC50)值可达1.07 mg/L。

The hydroxytyrosol （HT） was prepared from oleuropein （OE） in the olive leaves by enzymatic hydrolysis. The qualitative and quantitative determination of OE and HT from the olive leaf extract were analysed by HPLC, and the best enzyme to hydrolyze OE for HT from olive leaf extrct was selected. Moreover, the optimal technology of enzymatic process was obtained by single factor test and orthogonal test. The DPPH radical scavenging effects of HT, OE and olive leaf extract were examined, too. The results showed that hemicellulase was the best enzyme to hydrolyze OE for HT in the same enzymatic activity from 10 enzymes, and the cellulase and β-glucosidase were second. The optimal conditions were temperature 60℃, pH value 5.5, enzyme quantity 40 mg and time 6 h. The degradation rate of OE was 85.28 %, and HT content was 6.07 % by the best enzymatic process. Furthermore, HT was obtained by acid- and base-treatment, respectively. Acid hydrolysis performance （7.41% ） was better than enzymatic hydrolysis, and base-treatment result（4.09 % ） was the worst. By comparison of the two methods, it was found that enzymatic condition was mild and could reduce the catalyst use, and it is a new hydrolysis method to further develop and utilize. The IC50 of HT was 1.07 mg/L, and the radical scavenging effect was in the order of HT 〉 Vc 〉 OE 〉 BHT 〉 olive leaf extract. Therefore, HT has a good ability of anti-oxidation compound.