[目的] 优化福林酚法测定牛奶中蛋白酶活力的试验条件,解决检测结果重复性差、检测效率不高等问题。[方法] 用酶标仪测定牛奶中蛋白酶活力,同步对比各试验条件对测定结果的影响。[结果] 酶标仪测定时,显色保温时间15 min以上、取样350 μL、福林试剂浓度0.33~0.5 mol/L时,牛奶中蛋白酶活力测定结果重复性好,检测效率高。[结论] 酶标仪测定牛奶中蛋白酶活力是一种检测效率高、结果重复性好的方法,更适合乳品企业大批量筛查牛奶中蛋白酶活力。
[Objective] To optimize the conditions for the determination of protease activity in milk using the national standard method - Folin-phenol method. [Method] By measuring the protease activity in milk,the influence of various experimental parameters on the results was compared and analyzed. [Result] When using an enzyme-linked immunosorbent assay（ELISA） reader for measurement,the color retention time should be more than 15 minutes,and a sample of 350 μL.When the concentration of Folin reagent is 0.33~0.5 mol/L,the absorbance constant K value of the standard curve meets the requirements. [Conclusion] When detecting protease activity in milk,it is necessary to control the color retention time,sampling volume,and clarity of the sample solution.After the color reaction is fully completed,the system of the standard solution and sample solution has stabilized,and there is no significant change in the absorbance value within 1 hour of further insulation or room temperature storage,which has no significant impact on the results.
[1] 王春燕,任彩霞,任向东,等.影响超高温灭菌乳货架期品质的相关蛋白酶类研究进展[J].现代食品,2020(18):40-47.
[2] 吕加平,张维清,逄晓阳,等.一种高通量检测液态乳制品中脂肪酶酶活性的方法及其试剂盒[P].北京:CN105651715B,2019-04-09.
[3] 王传宝,张艳凌,陆剑锋,等.高钙奶苦味原因分析[J].中国食品添加剂,2020,31(4):56-61.
[4] 付建平,靳烨,马玉玲.乳中蛋白酶与UHT乳贮存中的胶凝现象[J].中国乳品工业,2004(7):22-25.
[5] 吴正钧,花陈剑,吴昊.嗜冷菌耐热性胞外蛋白酶对液态奶的影响[J].乳业科学与技术,2001(1):9-12.
[6] 贺凯茹,刘至立,薛瑞霞,等.货架期内品质劣变UHT乳的理化特性及风味品质特性研究[J].中国乳品工业,2023,51(1):19-25.
[7] Yang X,Wang Z,Zhang C,et al.Assessment of the production of Bacillus cereus protease and its effect on the quality of ultra-high temperature-sterilized whole milk[J].Journal of Dairy Science,2021,104(6):6577-6587.
[8] 郭荣,剧柠,卞永霞,等.乳及乳制品中的酶及对其关键品质的影响[J].中国乳品工业,2023,51(11):40-46.
[9] 王学标,何云侠,刘强.福林酚试剂法与缩二脲试剂法测定饲用蛋白酶活力的比较分析[J].畜牧与兽医,2019,51(3):23-28.
[10] 杨旭,屈小玄,郭庆贺,等.牛奶中蛋白酶活力测定的方法比较[J].中国乳品工业,2014,42(6):48-51.
[11] 杨帆,李淑梅,杨晓,等.分光光度法与酶标仪微量法测量菌液浓度的比较[J].光谱实验室,2011,28(4):1752-1754.
[12] 贾瑞波,韩春晖,贺建华,等.市售不同来源福林试剂对多肽含量测定结果的影响[J].中国药师,2016,19(12):2378-2380.
[13] 王贞,张英超,徐鹏,等.2709碱性蛋白酶酶活测定的影响因素研究[J].食品与药品,2015,17(5):323-327.
[14] 柴林山,李剑梅,孙翠焕,等.一种测定蛹虫草中虫草酸含量的酶标仪微量反应方法[J].微生物学杂志,2022,42(4):80-85.
[15] 马跃龙,张益萍,褚佳玥.基于水-甲醇体系的ONPG法测定调制乳粉中乳糖酶活力研究[J].中国乳品工业,2023,51(9):47-51.
[16] 郑东影,陈玮,张卫卫,等.福林酚法测定酿酒白曲酸性蛋白酶活力的条件试验[J].酿酒,2020,47(2):63-66.
[17] Ganado Z M,Bordagaray A,Ezenarro J,et al.Adaptation of the Folin-Ciocalteu and Fast Blue BB spectrophotometric methods to digital image analysis for the determination of total phenolic content:Reduction of reaction time,interferences and sample analysis[J].LWT,2024,193115756.
