碱解法测定动物水解蛋白中L-羟脯氨酸的含量

Determination of L-hydroxyproline in hydrolyzed animal protein based on alkaline ydrolysis

目的建立了使用聚四氟乙烯带盖水解管,以NaOH溶液为水解液的碱解法测定动物水解蛋白中L-羟脯氨酸(L-Hydroxyproline,L-Hyp)含量。方法将样品置于10 ml水解管中,加入6 ml 2.5 mol/L NaOH溶液在110℃烘箱中加热2 h,水解出的L-Hyp经氯胺T氧化后与对二甲氨基苯甲醛反应生成红色络合物,在(558±2)nm处测定其吸光度。结果在优化实验条件下,该方法的线性范围0～10μg/ml(r=0.999 3),检出限1.35μg/g,样品测定的RSD在1.0%～2.3%,加标回收率为88.7%～96%(加标量为30 mg/kg)。结论相对酸解法,碱解法的水解效率和酸解法基本一致,样品前处理操作简单,缩短了测定时间,灵敏度、重复性和稳定性良好。

Objective To establish a method based on alkaline hydrolysis for determination of L-hydroxyproline（L-Hyp） in hydrolyzed animal protein,in which NaOH solution and polytetrafluoroethylene（PTEF） hydrolysis tube with cover were used.Method The sample was hydrolysed by 6 ml 2.5 mol/L NaOH soultion in 10 ml hydrolysis tube and heated in the oven at 110 ℃ for 2 h.The hydrolysate of L-Hyp was oxidized by chloramine-T and then reacted with paradime thylaminobenzaldehyde to generate red complex.The absorbency was determined at（558±2） nm.Results Under the optimal conditions,the calibration curve was linear in the range of 0-10 μg/ml with detection limit of 1.35 μg/g,and the relative standard deviation（RSD） was 1.0%-2.3%.It was effective in the determination of L-Hyp in milk and dairy products with recoveries of 88.7%-96%.Conclusion Compared with acidolysis,the alkaline hydrolysis had more simple experimental procedure,fewer measurement time,highly sensitive and reproducible with consistency.