牛乳粉中乳蛋白糖基化分析及其水解程度研究
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摘要
探讨牛乳粉中酪蛋白和乳清蛋白的糖基化程度及其水解特性。采用不同方法提取牛乳粉中的酪蛋白和乳清蛋白,SDS3PAGE电泳分析其组成;高碘酸-希夫碱染色法及莫氏试验检测糖基化的酪蛋白和乳清蛋白;木瓜蛋白酶水解牛乳和乳粉中的酪蛋白和乳清蛋白。鞣酸提取酪蛋白的质量分数46.13%显著高于其他方法(p<0.05),乳酸发酵法是提取乳清蛋白的有效方法;电泳结果表明,酪蛋白有分子质量为24、34和60 ku的3条带,乳清蛋白有分子质量为14和18 ku的2条带;高碘酸-希夫碱糖蛋白染色结果表明,分子质量为24和34 ku的酪蛋白和分子质量为14 ku的乳清蛋白均是糖基化蛋白;莫氏试验结果表明,乳粉中存在糖基化的酪蛋白和乳清蛋白;乳蛋白木瓜蛋白酶水解结果表明,乳粉酪蛋白和乳清蛋白水解游离氨基酸含量均显著低于鲜乳(p<0.05)。牛乳粉中存在糖基化的酪蛋白和乳清蛋白,前者分子质量分别为24和34 ku,后者为14 ku;乳粉中乳蛋白水解程度较鲜乳低。
The glycosylation of casein and whey protein in milk powder and its enzymatic hydrolysis property were studied. The casein and whey protein in milk powder were extracted by different methods. The compositions of casein and whey protein were analyzed by SDS-PAGE electrophoresis,and glycosylated casein and whey protein were identified by periodate-schiff alkali dyeing and Morse experiment. Amino acid content was determined after papain hydrolysis of casein and whey protein in milk powder and fresh milk. The results showed that the percentage of casein extracted by tannic acid was 46. 13%,which was significantly higher than that of other groups( p < 0. 05). The method of lactic acid fermentation was the most efficient method to extract whey protein. The results of SDS-PAGE showed that the molecular weights of the casein and whey protein were 24,34,and 60 ku,and 14 and 18 ku,respectively. The casein of 24 and 34 ku,and the whey protein of 14 ku were both glycoproteins by periodate-schiff alkali dyeing. The results of Morse experiment of casein and whey protein showed that milk powder exist glycoprotein. The results of milk protein hydrolysis by papain showed that the content of free amino acid in casein dehydrated from milk powder was significantly lower than that from fresh milk( p < 0. 05),and the same results to that of whey protein. Milk powder contained glycosylated casein,they were 24 and 34 ku of casein and 14 ku whey protein. The degree of milk protein hydrolysis from milk powder is lower than that from fresh milk.
The glycosylation of casein and whey protein in milk powder and its enzymatic hydrolysis property were studied. The casein and whey protein in milk powder were extracted by different methods. The compositions of casein and whey protein were analyzed by SDS-PAGE electrophoresis,and glycosylated casein and whey protein were identified by periodate-schiff alkali dyeing and Morse experiment. Amino acid content was determined after papain hydrolysis of casein and whey protein in milk powder and fresh milk. The results showed that the percentage of casein extracted by tannic acid was 46. 13%,which was significantly higher than that of other groups( p < 0. 05). The method of lactic acid fermentation was the most efficient method to extract whey protein. The results of SDS-PAGE showed that the molecular weights of the casein and whey protein were 24,34,and 60 ku,and 14 and 18 ku,respectively. The casein of 24 and 34 ku,and the whey protein of 14 ku were both glycoproteins by periodate-schiff alkali dyeing. The results of Morse experiment of casein and whey protein showed that milk powder exist glycoprotein. The results of milk protein hydrolysis by papain showed that the content of free amino acid in casein dehydrated from milk powder was significantly lower than that from fresh milk( p < 0. 05),and the same results to that of whey protein. Milk powder contained glycosylated casein,they were 24 and 34 ku of casein and 14 ku whey protein. The degree of milk protein hydrolysis from milk powder is lower than that from fresh milk.
引文
[1]刘丽波,孙迪,李春,等.糖基化修饰牛乳清蛋白过敏原性研究进展[J].食品科学,2012,33(13):334-338.
[2]朱科学,周惠明,郭晓娜,等.植物来源糖蛋白的结构与功能[J].食品与发酵工业,2002,28(12):57-61.
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[4]齐晓彦,苏米亚.婴幼儿水解配方奶粉的研究进展[J].中国乳品工业,2016,44(3):46-50.
[5]MONTRCUIL J,VLIEGENTHARL J.Glycoproteins[M].Amsterdam:Elsevier Science Pubishing Company,INC,1995.
[6]仇娟.糖肽/糖蛋白富集新技术的研究[D].上海:复旦大学,2009:1-4.
[7]王承健,姜宽,邢伟博,等.鸡蛋中两种食品过敏原糖链决定簇结构分析研究[C]//中国食品科学技术学会年会.哈尔滨:东北农业大学,2011:26-32.
[8]杨剑婷.白果过敏蛋白及其致敏机理的研究[D].南京:南京林业大学,2010:82-84.
[9]应有成.牛乳主要过敏原α-乳白蛋白和β-乳球蛋白标准物质的制备[D].南昌:南昌大学,2013:3-4.
[10]李冰,龙再浩.牛乳蛋白过敏及其改性研究[J].农产品加工·学刊,2012(6):41-44.
[11]白振宇.牛奶中主要过敏原的消除及检测技术的研究[D].天津:天津商业大学,2007:1-5.
[12]罗永康,沈小琴,李朝慧,等.牛乳蛋白过敏原改性的研究[J].中国乳品工业,2005,33(10):4-8.
[13]刘丽波,孙迪,李春,等.美拉德反应影响α-乳白蛋白和β-乳球蛋白过敏原性研究[C]//中国食品科学技术学会儿童食品分会学术年会.哈尔滨:东北农业大学,2011:26-32.
[14]刘媛,王健,李研东,等.不同沉淀方法对牛乳中蛋白质质量分数测定的影响[J].中国乳品工业,2014,42(1):59-61.
[15]朱晓莹,覃靖娟,朱日生,等.等电沉淀法提取酪蛋白的实验方法改进[J].卫生职业教育,2013,31(9):93-94.
[16]陈惠,谢彩军,叶广辉,等.几种儿童牛奶酪蛋白含量的测定与比较[J].食品研究与开发,2014,35(20):101-103.
[17]LEJAR A,NOGRE M,TELLIER M,et al.Method for the extraction of one or several proteins present in milk:EP,WO/2007/138198[P].2007-02-25.
[18]李林强,朱莉莉,万威,等.牛羊乳热处理蛋白质变性程度比较及机理分析[J].西北农林科技大学学报:自然科学版,2016,44(11):149-154.
[19]宋宏新,刘立新,柏红梅,等.牛乳中蛋白质的电泳分析技术研究[J].食品与机械,2010,26(6):51-53.
[20]管方方.牛乳中掺假大豆蛋白的SDS-PAGE方法研究[D].上海:上海应用技术学院,2015:28-46.
[21]萧能赓.生物化学实验原理和方法[M].北京:北京大学出版社,2005:145-151.
[22]陈钧辉.生物化学实验[M].北京:科学出版社,2003:1-2.
[23]胡京枝,董小海,余大杰,等.牛奶及其制品中游离氨基酸含量的测定方法研究[J].河南畜牧兽医:综合版,2007,28(10S):29-31.
[24]邵金良,黎其万,董宝生,等.茚三酮比色法测定茶叶中游离氨基酸总量[J].中国食品添加剂,2008(2):162-165.
[25]向天勇,黄健,沈卫红,等.缫丝废水中丝胶蛋白的回收工艺[J].浙江农业科学,2016,57(8):1 335-1 337.
[26]胡鹏刚,刘强.高纯食品鞣酸在啤酒中的应用[J].酿酒科技,2004(3):61-62.
[27]杨勇,徐志霞,黄循吟,等.等电点法提取酪蛋白的方法改进[J].海南师范大学学报:自然科学版,2016(1):109-111.
[28]孙雯.乳蛋白与大豆蛋白组分分离及鉴别方法的研究[D].西安:陕西科技大学,2015:23-36.
[29]宋宏新,张歌,李红心,等.牛羊乳酪蛋白制备及电泳分析比较[J].中国酿造,2012,31(10):65-67.
[30]薛建龙.牛羊乳中蛋白质和脂肪的差异比较研究[D].西安:陕西科技大学,2015:26-29.
[31]张永忠,赵新淮,于国萍,等.乳品化学[M].北京:科学出版社,2007:88-119.
[32]张和平,张佳程.乳品工艺学[M].北京:中国轻工业出版社,2007:12-26.
[33]陈静廷,卜登攀,马露,等.不同等电点沉淀法和超速离心法提取牛奶乳清蛋白的双向电泳分析[J].食品科学,2014,35(20):180-184.
[34]阳晖,刘燕.不同方法提取乳清浓缩蛋白的研究[J].安徽农业科学,2011,39(2):657-659.
[35]刘翠芳,蒋继志.高碘酸-硝酸银染色法鉴定糖蛋白[J].生物技术通报,2006(S1):534-535.
[36]陈小萍,吴钦穗.PAS组织化学染色的几点体会[J].福建医药杂志,2006,28(6):178-179.
[37]赵新淮,徐红华,姜毓君.食品蛋白质:结构,性质与功能[M].北京:科学出版社,2009:183-194.
[38]许亚杰.非酶糖基化对部分血液中蛋白质结构和功能的影响[D].北京:中国科学院生物物理研究所,2004.
[39]O'RIORDAN,NOELLE.Targeting the glycome of major milk proteins[J].Febs Journal,2016,278(19):3 769-3 781.
[40]白振宇.牛奶中主要过敏原的消除及检测技术的研究[D].天津:天津商业大学,2007:69-71.
[2]朱科学,周惠明,郭晓娜,等.植物来源糖蛋白的结构与功能[J].食品与发酵工业,2002,28(12):57-61.
[3]吴芝岳,杨娜,孙俊,等.部分水解蛋白配方乳粉的研究进展及应用[J].食品安全导刊,2017(1):73-75.
[4]齐晓彦,苏米亚.婴幼儿水解配方奶粉的研究进展[J].中国乳品工业,2016,44(3):46-50.
[5]MONTRCUIL J,VLIEGENTHARL J.Glycoproteins[M].Amsterdam:Elsevier Science Pubishing Company,INC,1995.
[6]仇娟.糖肽/糖蛋白富集新技术的研究[D].上海:复旦大学,2009:1-4.
[7]王承健,姜宽,邢伟博,等.鸡蛋中两种食品过敏原糖链决定簇结构分析研究[C]//中国食品科学技术学会年会.哈尔滨:东北农业大学,2011:26-32.
[8]杨剑婷.白果过敏蛋白及其致敏机理的研究[D].南京:南京林业大学,2010:82-84.
[9]应有成.牛乳主要过敏原α-乳白蛋白和β-乳球蛋白标准物质的制备[D].南昌:南昌大学,2013:3-4.
[10]李冰,龙再浩.牛乳蛋白过敏及其改性研究[J].农产品加工·学刊,2012(6):41-44.
[11]白振宇.牛奶中主要过敏原的消除及检测技术的研究[D].天津:天津商业大学,2007:1-5.
[12]罗永康,沈小琴,李朝慧,等.牛乳蛋白过敏原改性的研究[J].中国乳品工业,2005,33(10):4-8.
[13]刘丽波,孙迪,李春,等.美拉德反应影响α-乳白蛋白和β-乳球蛋白过敏原性研究[C]//中国食品科学技术学会儿童食品分会学术年会.哈尔滨:东北农业大学,2011:26-32.
[14]刘媛,王健,李研东,等.不同沉淀方法对牛乳中蛋白质质量分数测定的影响[J].中国乳品工业,2014,42(1):59-61.
[15]朱晓莹,覃靖娟,朱日生,等.等电沉淀法提取酪蛋白的实验方法改进[J].卫生职业教育,2013,31(9):93-94.
[16]陈惠,谢彩军,叶广辉,等.几种儿童牛奶酪蛋白含量的测定与比较[J].食品研究与开发,2014,35(20):101-103.
[17]LEJAR A,NOGRE M,TELLIER M,et al.Method for the extraction of one or several proteins present in milk:EP,WO/2007/138198[P].2007-02-25.
[18]李林强,朱莉莉,万威,等.牛羊乳热处理蛋白质变性程度比较及机理分析[J].西北农林科技大学学报:自然科学版,2016,44(11):149-154.
[19]宋宏新,刘立新,柏红梅,等.牛乳中蛋白质的电泳分析技术研究[J].食品与机械,2010,26(6):51-53.
[20]管方方.牛乳中掺假大豆蛋白的SDS-PAGE方法研究[D].上海:上海应用技术学院,2015:28-46.
[21]萧能赓.生物化学实验原理和方法[M].北京:北京大学出版社,2005:145-151.
[22]陈钧辉.生物化学实验[M].北京:科学出版社,2003:1-2.
[23]胡京枝,董小海,余大杰,等.牛奶及其制品中游离氨基酸含量的测定方法研究[J].河南畜牧兽医:综合版,2007,28(10S):29-31.
[24]邵金良,黎其万,董宝生,等.茚三酮比色法测定茶叶中游离氨基酸总量[J].中国食品添加剂,2008(2):162-165.
[25]向天勇,黄健,沈卫红,等.缫丝废水中丝胶蛋白的回收工艺[J].浙江农业科学,2016,57(8):1 335-1 337.
[26]胡鹏刚,刘强.高纯食品鞣酸在啤酒中的应用[J].酿酒科技,2004(3):61-62.
[27]杨勇,徐志霞,黄循吟,等.等电点法提取酪蛋白的方法改进[J].海南师范大学学报:自然科学版,2016(1):109-111.
[28]孙雯.乳蛋白与大豆蛋白组分分离及鉴别方法的研究[D].西安:陕西科技大学,2015:23-36.
[29]宋宏新,张歌,李红心,等.牛羊乳酪蛋白制备及电泳分析比较[J].中国酿造,2012,31(10):65-67.
[30]薛建龙.牛羊乳中蛋白质和脂肪的差异比较研究[D].西安:陕西科技大学,2015:26-29.
[31]张永忠,赵新淮,于国萍,等.乳品化学[M].北京:科学出版社,2007:88-119.
[32]张和平,张佳程.乳品工艺学[M].北京:中国轻工业出版社,2007:12-26.
[33]陈静廷,卜登攀,马露,等.不同等电点沉淀法和超速离心法提取牛奶乳清蛋白的双向电泳分析[J].食品科学,2014,35(20):180-184.
[34]阳晖,刘燕.不同方法提取乳清浓缩蛋白的研究[J].安徽农业科学,2011,39(2):657-659.
[35]刘翠芳,蒋继志.高碘酸-硝酸银染色法鉴定糖蛋白[J].生物技术通报,2006(S1):534-535.
[36]陈小萍,吴钦穗.PAS组织化学染色的几点体会[J].福建医药杂志,2006,28(6):178-179.
[37]赵新淮,徐红华,姜毓君.食品蛋白质:结构,性质与功能[M].北京:科学出版社,2009:183-194.
[38]许亚杰.非酶糖基化对部分血液中蛋白质结构和功能的影响[D].北京:中国科学院生物物理研究所,2004.
[39]O'RIORDAN,NOELLE.Targeting the glycome of major milk proteins[J].Febs Journal,2016,278(19):3 769-3 781.
[40]白振宇.牛奶中主要过敏原的消除及检测技术的研究[D].天津:天津商业大学,2007:69-71.