拟目乌贼肌肉多糖酶法脱蛋白工艺优化及体外清除自由基能力

Optimization of Enzymatic Deproteinization of Polysaccharides from Cuttlefish Muscle(Sepia lycidas) and Its in Vitro Free Radical Scavenging Potential

以加酶量、酶解温度、pH值、酶解时间为影响因素,以蛋白脱除率和多糖损失率为评价指标,通过正交试验优化拟目乌贼肌肉多糖酶法脱蛋白工艺,并与三氯乙酸法、等电点法脱蛋白效果进行比较。同时对脱蛋白多糖的体外清除自由基能力进行研究。结果表明,拟目乌贼肌肉多糖脱蛋白最佳工艺条件为:加酶量3.0 g/100 m L、酶解温度53℃、p H 8.2、酶解时间1.5 h。此条件下蛋白脱除率为89.43%,多糖损失率为1.78%,优于三氯乙酸法和等电点法。清除自由基结果显示,多糖质量浓度为10 mg/m L时,酶法、三氯乙酸法和等电点法得到的脱蛋白多糖对羟自由基清除率分别为45.72%、38.72%和25.18%,相应的IC_(50)值分别为12.44、16.37、34.64 mg/m L;对1,1-二苯基-2-三硝基苯肼自由基的清除率分别为68.00%、30.08%和23.10%,相应的IC50值分别为7.62、16.71、28.96 mg/m L。3种脱蛋白方法所得拟目乌贼肌肉多糖清除自由基效果依次为:酶法>三氯乙酸法>等电点法。

The enzymatic deproteinization of polysaccharides from cuttlefish muscle(Sepia lycidas)was optimized using an orthogonal array design.Trypsin was determined to be the optimal enzyme to hydrolyze the protein in the crude polysaccharide extract.Enzyme dosage,hydrolysis temperature,pH and hydrolysis time were selected as independent variables for optimization,and the response variables were deproteinization efficiency and polysaccharide loss.The enzymatic method was compared with trichloroacetic acid(TCA)and isoelectric point(IP)methods.Meanwhile,the free radical scavenging capacity of the deproteinated polysaccharides was also studied.The results showed that the optimum deproteinization conditions were obtained as follows:trypsin dosage,3.0g/100mL;hydrolysis temperature,53℃;pH,8.2;and hydrolysis time,1.5h,yielding a deproteinization rate as high as89.43%and a polysaccharide loss of only1.78%.In terms of both dependent variables,the enzymatic method was better than TCA and IP methods.The percentage scavenging of hydroxyl radical by the deproteinated polysaccharides obtained by the enzymatic,TCA and IP methods were45.72%,38.72%and25.18%,with IC50values of12.44,16.37and34.64mg/mL,respectively,and the percentage scavenging of1,1-diphenyl-2-picrylhydrazyl(DPPH)radical by these purified polysaccharides were68.00%,30.08%and23.10%,with IC50values of7.62,16.71and28.96mg/mL,respectively.The free radical scavenging activities of the deproteinated polysaccharides from S.lycidas muscle were ranked as follows:enzymatic method>TCA method>IP method.