特殊医学用途配方乳粉中色氨酸测定酶解条件优化

Optimization of enzymatic hydrolysis conditions for determining tryptophan in special medical purpose formula milk powder

中文关键词:  色氨酸  氨基酸分析仪  酶解  特殊医学用途配方乳粉  响应曲面

英文关键词:tryptophan  amino acid analyzer  enzymatic hydrolysis  special medical purpose formula milk powder  response surface methodology

中文摘要:

      目的 基于氨基酸分析仪建立酶解法测定特殊医学用途配方乳粉中色氨酸的方法。方法 在单因素试验基础上, 运用Box-Behnken响应面设计, 将酶加入量、酶解温度和酶解时间作为影响因子, 以色氨酸回收率为响应值, 确定酶解最佳条件, 同时对比不同净化方式对结果的影响。结果 酶解最佳条件为: 酶添加量0.74 mL、酶解温度45.2 °C, 酶解时间12 h; 通过实验比对确定12%三氯乙酸为最优沉淀剂。对以上优化条件进行方法学验证, 色氨酸回收率为95.5%~101.0%, 相对标准偏差(relative standard deviations, RSDs, n=7)为0.9%~1.6%, 检出限(limit of detection, LOD)和定量限(limit of quantitation, LOQ)分别为0.3 mg/100 g和1.0 mg/100 g。结论 该方法具有操作简便、准确度高、精密度好等优点, 适用于特殊医学用途配方乳粉中色氨酸的定量检测。

英文摘要:

      Objective To establish a method for determining tryptophan in special medical purpose formula milk powder by enzymatic hydrolysis based on amino acid analyzer. Methods Based on single-factor experiments, the Box-Behnken response surface methodology was employed, with enzyme addition amount, enzymatic hydrolysis temperature and enzymatic hydrolysis time as influencing factors, and tryptophan recovery rate as the response value. The optimal enzymatic hydrolysis conditions were determined, and the effects of different purification methods on the results were also compared. Results The optimal enzymatic hydrolysis conditions were: Enzyme addition amount 0.74 mL, enzymatic hydrolysis temperature 45.2 ℃ and enzymatic hydrolysis time 12 h. Through experimental comparison, 12% trichloroacetic acid was identified as the optimal precipitant. Under the optimized conditions: Methodological validation showed a tryptophan recovery rate of 95.5%–101.0%, with relative standard deviations (RSDs, n=7) ranging from 0.9% to 1.6%, and limit of detection (LOD) andlimit of quantitation (LOQ) of 0.3 mg/100 g and 1.0 mg/100 g, respectively. Conclusion This method offers ease of operation, high accuracy and excellent precision, making it suitable for the quantitative determination of tryptophan in special medical purpose formula milk powder.