高效液相色谱法测定婴幼儿配方奶粉中酪蛋白磷酸肽含量
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摘要
目的建立高效液相色谱法(highperformanceliquidchromatography,HPLC)测定婴幼儿配方奶粉中酪蛋白磷酸肽(casein phosphopeptide, CPP)的含量的方法。方法对样品静置温度、静置时间、沉淀分离方式进行优化,用高效液相色谱法对婴幼儿配方奶粉中CPP的含量进行测定。结果最优的前处理条件为样品溶解后调整pH至4.6,于4℃条件下静置1 h,过滤后上清液经0.22μm滤膜过滤,以Ultimate AQ-C_(18)色谱柱(250mm×4.6mm,5μm)分离。经钼蓝比色法鉴定,保留时间为8.387min的组分为CPP。该方法在10～1000mg/L范围内线性关系良好,相关系数为0.9993,定量限为4.40 mg/L,婴幼儿奶粉中添加量为500、1000、5000 mg/kg时, CPP的回收率为96.63%%～101.42%。结论该方法样品前处理简单,稳定性和可靠性良好,检测结果符合定性定量要求,可用于婴幼儿配方奶粉中CPP含量的测定。
Objective To establish a method of determination of casein phosphopeptides(CPP) in infant formula milk powder by high performance liquid chromatography(HPLC). Methods The sample standing temperature, standing time and precipitation separation method were optimized, and the content of CPP in infant formula was determined by HPLC. Result The optimal pretreatment conditions were as follows: the sample was dissolved and adjusted to pH 4.6, and allowed to stand at 4 ℃ for 1 h. After filtration, the supernatant was filtered through a 0.22 μm filter and separated by an Ultimate AQ-C_(18) column(250 mm×4.6 mm, 5 μm). It was identified by molybdenum blue colorimetric method, and the component with retention time of 8.387 min was CPP. The method had a good linearity in the range of 10-1000 mg/L, the correlation coefficient was 0.9993, and the limit of quantification was 4.40 mg/L. When the dosages of infant milk powder were 500, 1000, and 5000 mg/kg, the recovery rates of CPP were 96.63%%-101.42%. Conclusion Themethod is simple, stable and reliable, and the results complied with the qualitative and quantitative requirements, which can be used to detect CPP content in infant milk powder.
Objective To establish a method of determination of casein phosphopeptides(CPP) in infant formula milk powder by high performance liquid chromatography(HPLC). Methods The sample standing temperature, standing time and precipitation separation method were optimized, and the content of CPP in infant formula was determined by HPLC. Result The optimal pretreatment conditions were as follows: the sample was dissolved and adjusted to pH 4.6, and allowed to stand at 4 ℃ for 1 h. After filtration, the supernatant was filtered through a 0.22 μm filter and separated by an Ultimate AQ-C_(18) column(250 mm×4.6 mm, 5 μm). It was identified by molybdenum blue colorimetric method, and the component with retention time of 8.387 min was CPP. The method had a good linearity in the range of 10-1000 mg/L, the correlation coefficient was 0.9993, and the limit of quantification was 4.40 mg/L. When the dosages of infant milk powder were 500, 1000, and 5000 mg/kg, the recovery rates of CPP were 96.63%%-101.42%. Conclusion Themethod is simple, stable and reliable, and the results complied with the qualitative and quantitative requirements, which can be used to detect CPP content in infant milk powder.
引文
[1]冯凤琴,王博诚,倪莉,等.酪蛋白磷酸肽(CPP)分离纯化及分子结构的鉴定[J].无锡轻工大学学报,1999,18(4):33-37.Feng F,Wang BC,Ni L,et al.Isolation,purification and molecular structure identification of casein phosphopeptide(CPP)[J].J Wuxi Univ Light Ind,1999,18(4):33-37.
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[7]Miquel E,FarréR.Effects and future trends of casein phosphopeptides on zinc bioavailability[J].Trend Food Sci Technol,2007,18(3):139-143.
[8]Donida BM,Mrak E,Gravaghi C,et al.Casein phosphopeptides promote calcium uptake and modulate the differentiation pathway in human primary osteoblast-like cells[J].Peptides,2009,30(12):2233-2241.
[9]Perego S,Zabeo A,Marasco E,et al.Casein phosphopeptides modulate calcium uptake and apoptosis in Caco2 cells through their interaction with the TRPV6 calcium channel[J].J Funct Food,2013,5(2):847-857.
[10]Zhou C,Zhang DL,Bai YX,et al.Casein phosphopeptide-amorphous calcium phosphate remineralization of primary teeth early enamel lesions[J].J Dent,2014,24(1):21-29.
[11]Park OJ,Allen JC.Antigenicity of casein phosphopeptides prepared with immobilized glutamic acid-specific endopeptidase or trypsin[J].Nutr Res,2000,20(3):359-370.
[12]唐婷,马力.酪蛋白磷酸肽的营养作用及其应用[J].中国食物与营养,2008,2(3):28-29.Tang T,Ma L.Nutritional function and application of casein phosphopeptides[J].Food Nutr Chin,2008,2(3):28-29.
[13]郭长慧,张娜,刘丽洁.酪蛋白磷酸肽制备工艺参数的优化研究[J].中国乳品工业,2016,(1):4-8.Guo CH,Zhang N,Liu LJ.Study on the optimization of technological parameters for the preparation of casein phosphopeptides[J].Chin Dair Ind,2016,(1):4-8.
[14]庞广昌,朱文欣,陈庆森.酪蛋白磷酸肽(CPPs)测定方法的研究[J].食品科学,2001,22(5):49-52.Pang GC,Zhu WX,Chen QS.Study on the determination of casein phosphopeptide(CPPs)[J].Food Sci,2001,22(5):49-52.
[15]眸光庆,张丽萍,王立民,等.毛细管区带电泳测定酪蛋白磷酸肽方法的研究[J].中国食品学报,2002,21(2):21-24.Mou GQ,Zhang LP,Wang LM,et al.Determination of casein phosphopeptide by capillary zone electrophoresis[J].Chin J Food,2002,21(2):21-24.
[16]Adamson N,Riley PF,Reynolds EC.The analysis of multiple phosphoseryl-containing casein peptides using capillary zone electrophoresis[J].J Chromatogr A,1993,646(2):391-396.
[17]高启奇,黄思棋,肖光辉,等.高效液相色谱法测定乳粉中酪蛋白磷酸肽含量[J].食品科技,2016,41(7):292-296.Gao QQ,Huang SQ,Xiao GH,et al.Determination of casein phosphopeptide in milk powder by high performance liquid chromatography[J].Food Sci Technol,2016,41(7):292-296.
[18]刘玉娟.高效液相色谱法测定婴幼儿配方乳粉中酪蛋白磷酸肽的含量[J].乳业科学与技术,2016,39(4):26-28.Liu YJ.Determination of casein phosphopeptide in infant formula milk powder by high performance liquid chromatography[J].Dairy Sci Technol,2016,39(4):26-28.
[19]张帅,刘飞,罗珍,等.高效液相色谱法测定钙片中酪蛋白磷酸肽的含量[J].现代食品科技,2014,30(7):258-262.Zhang S,Liu F,Luo Z,et al.Determination of casein phosphopeptide in calcium tablets by high performance liquid chromatography[J].Mod Food Sci Technol,2014,30(7):258-262.
[20]李则均.高效液相色谱法测定婴幼儿配方羊奶粉中酪蛋白磷酸肽含量[J].中国乳业,2017,(2):66-70.Li ZJ.Determination of casein phosphopeptide in infant formula sheep milk powder by high performance liquid chromatography[J].Dairy Ind Chin,2017,(2):66-70.
[21]梁艳,石丹,王艳萍.高效液相色谱法检测乳制品的酪蛋白磷酸肽[J].中国乳品工业,2016,44(9):56-57.Liang Y,Shi D,Wang YP.Determination of casein phosphopeptide in dairy products by high performance liquid chromatography[J].Chin Dairy Ind,2016,44(9):56-57.
[22]董浩,于洋,晏嘉泽,等.利用高效液相色谱-串联质谱追踪酸奶发酵过程中的酪蛋白磷酸肽[J].色谱,2017,35(6):587-593.Dong H,Yu Y,Yang JZ,et al.Casein phosphopeptides in yoghurt fermentation were tracked by high performance liquid chromatography-tandem mass spectrometry[J].Chromatography,2017,35(6):587-593.
[23]胡蓓,张京顺,柯星,等.高效液相色谱串联质谱法测定婴幼儿配方奶粉中酪蛋白磷酸肽含量[J].食品科学,2017,4(39):26-30.Hu B,Zhang JS,Ke X,et al.Determination of casein phosphopeptide in infant formula milk powder by high performance liquid chromatography-tandem mass spectrometry[J].Food Sci,2017,4(39):26-30.
[24]Olumee-Shabon Z,Boehmer JL.Detection of casein phosphopeptides in goat milk[J].J Prot Res,2013,12(6):3034-3041.
[25]冯凤琴,陈闽军,杜永盛,等.高效液相色谱法测定奶粉中酪蛋白磷酸肽(CPP)含量[J].中国食品学报,2006,6(3):128-132.Feng FQ,Chen MJ,Du YS,et al.Determination of casein phosphopeptide(CPP)in milk powder by high performance liquid chromatography[J].Chin J Food,2006,6(3):128-132.
[26]Morrison WR.A fast,simple and reliable method for the microdetermination of phosphorus in biological materials[J].Anal Biochem,1964,7(2):218-224.
[27]陈雪香,罗珍,刘飞,等.酪蛋白磷酸肽的质量评价方法研究[J].食品工业科技,2012,33(8):75-77.Chen XX,Luo Z,Liu F,et al.Study on the quality evaluation method of casein phosphopeptides[J].Food Ind Sci Technol,2012,33(8):75-77.
[2]Zhao W,Xu G,Yang R,et al.Preparation of casein phosphopeptides using a novel continuous process of combining an enzymatic membrane reactor with anion-exchange chromatography[J].J Food Eng,2013,117(1):105-112.
[3]Laparra JM,Alegria A,Barbera R,et al.Antioxidant effect of casein phosphopeptides compared with fruit beverages supplemented with skimmed milk against H2O2-induced oxidative stress in Caco-2 cells[J].Food Res Int,2008,41(7):773-779.
[4]徐曼.酪蛋白磷酸肽的制备及其持钙能力的研究[D].武汉:武汉工业学院,2011.XU M.Preparation of casein phosphopeptide and its calcium holding capacity[D].Wuhan:Wuhan Inst Technol,2011.
[5]Cross KJ,Huq NL,Palamara JE,et al.Physicochemical characterization of casein phosphopeptide-amorphous calcium phosphate nanocomplexes[J].J Biol Chem,2005,280(15):15362-15369.
[6]Miquel E,Alegría A,BarberáR,et al.Speciation analysis of calcium,iron,and zinc in casein phosphopeptide fractions from toddler milk-based formula by anion exchange and reversed-phase high-performance liquid chromatography-mass spectrometry/flame atomic-absorption spectroscopy[J].Anal Bioanal Chem,2005,381(5):1082-1088.
[7]Miquel E,FarréR.Effects and future trends of casein phosphopeptides on zinc bioavailability[J].Trend Food Sci Technol,2007,18(3):139-143.
[8]Donida BM,Mrak E,Gravaghi C,et al.Casein phosphopeptides promote calcium uptake and modulate the differentiation pathway in human primary osteoblast-like cells[J].Peptides,2009,30(12):2233-2241.
[9]Perego S,Zabeo A,Marasco E,et al.Casein phosphopeptides modulate calcium uptake and apoptosis in Caco2 cells through their interaction with the TRPV6 calcium channel[J].J Funct Food,2013,5(2):847-857.
[10]Zhou C,Zhang DL,Bai YX,et al.Casein phosphopeptide-amorphous calcium phosphate remineralization of primary teeth early enamel lesions[J].J Dent,2014,24(1):21-29.
[11]Park OJ,Allen JC.Antigenicity of casein phosphopeptides prepared with immobilized glutamic acid-specific endopeptidase or trypsin[J].Nutr Res,2000,20(3):359-370.
[12]唐婷,马力.酪蛋白磷酸肽的营养作用及其应用[J].中国食物与营养,2008,2(3):28-29.Tang T,Ma L.Nutritional function and application of casein phosphopeptides[J].Food Nutr Chin,2008,2(3):28-29.
[13]郭长慧,张娜,刘丽洁.酪蛋白磷酸肽制备工艺参数的优化研究[J].中国乳品工业,2016,(1):4-8.Guo CH,Zhang N,Liu LJ.Study on the optimization of technological parameters for the preparation of casein phosphopeptides[J].Chin Dair Ind,2016,(1):4-8.
[14]庞广昌,朱文欣,陈庆森.酪蛋白磷酸肽(CPPs)测定方法的研究[J].食品科学,2001,22(5):49-52.Pang GC,Zhu WX,Chen QS.Study on the determination of casein phosphopeptide(CPPs)[J].Food Sci,2001,22(5):49-52.
[15]眸光庆,张丽萍,王立民,等.毛细管区带电泳测定酪蛋白磷酸肽方法的研究[J].中国食品学报,2002,21(2):21-24.Mou GQ,Zhang LP,Wang LM,et al.Determination of casein phosphopeptide by capillary zone electrophoresis[J].Chin J Food,2002,21(2):21-24.
[16]Adamson N,Riley PF,Reynolds EC.The analysis of multiple phosphoseryl-containing casein peptides using capillary zone electrophoresis[J].J Chromatogr A,1993,646(2):391-396.
[17]高启奇,黄思棋,肖光辉,等.高效液相色谱法测定乳粉中酪蛋白磷酸肽含量[J].食品科技,2016,41(7):292-296.Gao QQ,Huang SQ,Xiao GH,et al.Determination of casein phosphopeptide in milk powder by high performance liquid chromatography[J].Food Sci Technol,2016,41(7):292-296.
[18]刘玉娟.高效液相色谱法测定婴幼儿配方乳粉中酪蛋白磷酸肽的含量[J].乳业科学与技术,2016,39(4):26-28.Liu YJ.Determination of casein phosphopeptide in infant formula milk powder by high performance liquid chromatography[J].Dairy Sci Technol,2016,39(4):26-28.
[19]张帅,刘飞,罗珍,等.高效液相色谱法测定钙片中酪蛋白磷酸肽的含量[J].现代食品科技,2014,30(7):258-262.Zhang S,Liu F,Luo Z,et al.Determination of casein phosphopeptide in calcium tablets by high performance liquid chromatography[J].Mod Food Sci Technol,2014,30(7):258-262.
[20]李则均.高效液相色谱法测定婴幼儿配方羊奶粉中酪蛋白磷酸肽含量[J].中国乳业,2017,(2):66-70.Li ZJ.Determination of casein phosphopeptide in infant formula sheep milk powder by high performance liquid chromatography[J].Dairy Ind Chin,2017,(2):66-70.
[21]梁艳,石丹,王艳萍.高效液相色谱法检测乳制品的酪蛋白磷酸肽[J].中国乳品工业,2016,44(9):56-57.Liang Y,Shi D,Wang YP.Determination of casein phosphopeptide in dairy products by high performance liquid chromatography[J].Chin Dairy Ind,2016,44(9):56-57.
[22]董浩,于洋,晏嘉泽,等.利用高效液相色谱-串联质谱追踪酸奶发酵过程中的酪蛋白磷酸肽[J].色谱,2017,35(6):587-593.Dong H,Yu Y,Yang JZ,et al.Casein phosphopeptides in yoghurt fermentation were tracked by high performance liquid chromatography-tandem mass spectrometry[J].Chromatography,2017,35(6):587-593.
[23]胡蓓,张京顺,柯星,等.高效液相色谱串联质谱法测定婴幼儿配方奶粉中酪蛋白磷酸肽含量[J].食品科学,2017,4(39):26-30.Hu B,Zhang JS,Ke X,et al.Determination of casein phosphopeptide in infant formula milk powder by high performance liquid chromatography-tandem mass spectrometry[J].Food Sci,2017,4(39):26-30.
[24]Olumee-Shabon Z,Boehmer JL.Detection of casein phosphopeptides in goat milk[J].J Prot Res,2013,12(6):3034-3041.
[25]冯凤琴,陈闽军,杜永盛,等.高效液相色谱法测定奶粉中酪蛋白磷酸肽(CPP)含量[J].中国食品学报,2006,6(3):128-132.Feng FQ,Chen MJ,Du YS,et al.Determination of casein phosphopeptide(CPP)in milk powder by high performance liquid chromatography[J].Chin J Food,2006,6(3):128-132.
[26]Morrison WR.A fast,simple and reliable method for the microdetermination of phosphorus in biological materials[J].Anal Biochem,1964,7(2):218-224.
[27]陈雪香,罗珍,刘飞,等.酪蛋白磷酸肽的质量评价方法研究[J].食品工业科技,2012,33(8):75-77.Chen XX,Luo Z,Liu F,et al.Study on the quality evaluation method of casein phosphopeptides[J].Food Ind Sci Technol,2012,33(8):75-77.
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