摘要
采用双向电泳技术对牛羊乳品的蛋白质指纹进行分析。首先通过全乳图谱分析牛羊乳的差异蛋白,然后针对婴幼儿配方粉、乳清粉等开展方法特异性研究,通过婴幼儿配方粉添加实验开展方法灵敏度研究,最后对市售配方粉、液态乳、酸乳等进行检测。总体上,双向电泳技术信息丰度高,信息直观,重复性良好,能准确分辨牛羊乳及乳清,灵敏度达到5%。通过检测,8份市售样品中有1份酸羊乳实际为牛乳,图谱蛋白指纹信息清晰,直观反映产品真实性。
Two-dimensional?gel?electrophoresis(2 DGE)?was?applied?in?protein?fingerprinting?of?cow?and?goat?milk?products.?First?of?all,?the?protein?profiles?of?cow?and?goat?milk?were?compared?to?find?out?the?differential?proteins.?Afterwards,?the?specificity?of?this?method?was?tested?by?applying?it?to?infant?formula?and?whey?protein?powder?from?cow?and?goat?milk?and the sensitivity was evaluated by using different proportion mixtures of cow milk infant formula and goat milk infant formula.?Finally,?commercialized?infant?formula,?liquid?milk?and?yoghurt?samples?were?tested.?2 DGE?method?was?confirmed?to?be?highly?informative?and?highly?repeatable?and?it?could?visually?present?protein?profiles.?As?proved?by?our?data,?liquid?milk, whey protein and infant formula could be distinguished clearly by 2 DGE and the detection limit reached 5%. Of 8 commercialized?samples,?one?labelled?as?fermented?goat?milk?was?identified?as?cow?milk?with?a?clear?protein?fingerprint?which?could?visually?reflect?the?authenticity?of?the?product
引文
[1]解庆刚,赵鑫,董长春,等.山羊乳作为婴幼儿配方粉乳原料的营养优势评估[J].中国乳品工业, 2015, 43(9):26-29. DOI:10.3969/j.issn.1001-2230.2015.09.007.
[2]?ROMERO?C,?PEREZ-ANDúJAR?O,?OLMEDO?A,?et?al.?Detection?of?cow’s?milk?in?ewe’s?or?goat’s?milk?by?HPLC[J].?Chromatographia,?1996, 42(3/4):181-184. DOI:10.1007/BF02269650.
[3]HUANGXQ,ZHONGXJ,ZHANGDM,etal.Studyonthesterilizationconditionsforthecorngoatmilk[J].ModernFoodScience&Technology,2011,27(12):1487-1489.DOI:10.1016/j.tifs.2014.06.009.
[4]张歌.牛乳和羊乳蛋白质差异比较及检测方法的研究[D].西安:陕西科技大学, 2013.
[5]王坤,叶兴乾,关荣发.羊乳及其奶酪中掺入牛乳检测的研究[J].食品与发酵工业, 2005, 31(10):136-138. DOI:10.3321/j.issn:0253-990X.2005.10.035.
[6]?YURCHENKO?S,?SATS?A,?POIKALAINEN?V,?et?al.?Method?fordeterminationoffattyacidsinbovinecolostrumusingGC-FID[J].FoodChemistry,2016,212:117-122.DOI:10.1016/j.foodchem.2016.05.103.
[7]黎颖,巫坚,林波,等.羊乳及其制品中掺入牛属牛乳和水牛乳的检测[J].基因组学与应用生物学, 2015, 34(5):977-981. DOI:10.13417/j.gab.034.000977.
[8]?DALMASSO?A,?SACCHI?P,?BOTTERO?M?T.?Development?of?a?real-time PCR assay for the detection of cow and donkey milk[J]. EuropeanFoodResearch&Technology,2012,235(1):47-52.DOI:10.1007/s00217-012-1732-9.
[9]PESIC M, BARAC M, VRVIC M, et al. Qualitative and quantitativeanalysisofbovinemilkadulterationincaprineandovinemilksusingnative-PAGE[J].FoodChemistry,2011,125(4):1443-1449.DOI:10.1016/j.foodchem.2010.10.045.
[10]?CALVANO?C?D,?DE?CEGLIE?C,?ARESTA?A,?et?al.?MALDI-TOF?mass spectrometric determination of intact phospholipids as markers ofillegal bovine milk adulteration of high-quality milk[J]. Analytical&BioanalyticalChemistry,2013,405(5):1641-1649.DOI:10.1007/s00216-012-6597-z.
[11]石燕,姜金斗,刘宁.利用毛细管电泳测定牛乳和山羊乳混合乳的蛋白质[J].东北农业大学学报, 2010, 41(11):119-124. DOI:10.3969/j.issn.1005-9369.2010.11.022.
[12]BRAMANTIE,SORTINOC,ONORM,etal.Separationanddetermination of denaturedαS1-,αS2-,β-andκ-caseins by hydrophobicinteractionchromatographyincows’,ewes’andgoats’milk,milkmixturesandcheeses[J].JournalofChromatography A,2003,994(1/2):59-74. DOI:10.1016/S0021-9673(03)00483-7.
[13]张世伟,王士峰,赖心田,等.水牛乳和羊乳中掺伪牛乳高特异性酶联免疫检测方法[J].食品工业, 2015(5):191-194.
[14]王帅星.羊乳中牛乳掺假的胶体金免疫层析试纸条检测研究[D].杨凌:西北农林科技大学, 2016.
[15]BANERJEE D, GOHAINA M, SINGH K P. Simplex and duplex PCRassaysforspeciesspecificidentificationofcattleandbuffalomilkandcheese[J].FoodControl,2011,22(5):690-696.DOI:10.1016/j.foodcont.2010.09.026.
[16]?CHEN?R?K,?CHANG?L?W,?CHUNG?Y?Y,?et?al.?Quantification?of?cowmilkadulterationingoatmilkusinghigh-performanceliquidchromatographywithelectrosprayionizationmassspectrometry[J].RapidCommunicationsinMassSpectrometry,2010,18(10):1167-1171. DOI:10.1002/rcm.1460.
[17]CHEN Q, KE X, ZHANG J S, et al. Proteomics method to quantifythepercentageofcow,goat,andsheepmilksinrawmaterialsfordairy products[J]. Journal of Dairy Science, 2016, 99(12):9483-9492.DOI:10.3168/jds.2015-10739.
[18]?ANGUITA?G,?MARTIN?R,?GARCIA?T,?et?al.?Indirect?ELISA?for?detection of cow’s milk in ewes’ and goats’ milks using a monoclonalantibodyagainstbovinebeta-casein[J].JournalofDairyResearch,1995, 62(4):655-659. DOI:10.1016/j.scienta.2016.02.003.
[19]王宁.乳糖含量对乳清分离蛋白储藏过程老化及影响机制的研究[D].哈尔滨:哈尔滨工业大学, 2016.
[20]TSUGITA A,KAWAKAMI T,UCHIDA T,etal.Proteomeanalysisofmousebrain:two-dimensionalelectrophoresisprofilesoftissueproteins during the course of aging[J]. Electrophoresis, 2000, 21(9):1853-1871. DOI:10.1002/(SICI)1522-2683.
[21]?CHANG?C?I,?CHEN?L?H,?HU?Y?F,?et?al.?Determining?the?cleavage?site?for the mature antimicrobial peptide of Nile tilapiaβ-defensin using2D electrophoresis, western blot, and mass spectrometry analysis[J].Fish&ShellfishImmunology,2017,62:41-46.DOI:10.1016/j.fsi.2017.01.010.
[22]李倩,于振,江帆,等.双向电泳技术在蛋白质组学中的应用[J].实验室科学,2009(2):81-83.DOI:10.3969/j.issn.1672-4305.2009.02.032.
[23]毛雪飞,任敏.双向电泳技术新进展[J].农学学报,2007(9):87-89.DOI:10.3969/j.issn.1007-7774.2007.09.042.
[24]熊伟.蛋白质组双向电泳技术在生物医学研究中的应用进展[J].生命科学仪器,2010,8(1):7-10.DOI:10.3969/j.issn.1671-7929.2010.01.002.
[25]?BRUNEEL?A,?HABAROU?F,?STOJKOVIC?T,?et?al.?Two-dimensional?electrophoresishighlightshaptoglobinbetachainasanadditionalbiomarker of congenital disorders of glycosylation[J]. Clinica ChimicaActa, 2017, 470:70-74. DOI:10.1016/j.cca.2017.04.022.
[26]王静.龙眼子叶胚发育过程中差异表达蛋白的研究[D].福州:福建农林大学, 2009.
[27]郭丽丽.表征属性识别技术在燕窝真伪鉴别中的应用研究[D].北京:中国农业大学, 2014.
[28]赵方圆.蜂王浆蛋白质组学研究及新鲜度指示蛋白的应用初探[D].北京:中国农业大学, 2013.
[29]汪家政,范明.蛋白质技术手册[M].北京:科学出版社, 2000:77-100.
[30]?MAURICIO?G?M,?CONSTANZA?T?M,?R?THLISBERGER?S,?etal.Preprocessingof2-dimensionalgelelectrophoresisimagesappliedtoproteomicanalysis:areview[J].GenomicsProteomics&Bioinformatics, 2018, 16:63-72. DOI:10.1016/j.gpb.2017.10.001.
[31]李则均.婴幼儿配方羊奶粉中乳铁蛋白含量测定[J].中国乳业,2017(4):71-75. DOI:10.16172/j.cnki.114768.2017.04.015.
[32]程妮.牛羊乳蛋白质的双向电泳分析及免疫检测技术研究[D].西安:陕西科技大学, 2017.
