【作者】 朱秀高；

【导师】 王捍东；

【作者基本信息】 扬州大学 ， 临床兽医学， 2010， 硕士

【摘要】 目前,人们对乳制品的需求量越来越大,而作为乳制品基本来源的原料乳供应却呈明显短缺态势。在此情况下,一些不法者为了自身利益而非法向原料乳中加入其他外源物质,获取不正当利益。当前牛奶的掺假主要针对乳蛋白成分,使用的掺假物质主要包括缩二脲、三聚氰胺等化工原料、尿素、硫酸铵等农用化肥。引起这种掺假现象蔓延的原因主要在于现有蛋白检测技术的缺陷以及掺假物质检测方法的局限性。本研究针对目前乳品业发展中存在的上述状况,分别对乳真蛋白(TP)和尿素氮与铵态氮检测方法及其检测体系进行了研究。其中乳TP的检测通过建立微量化Lowry反应体系进行,尿素氮和铵态氮的检测通过建立酶解-Berthelot反应体系进行。研究中对每种反应体系建立过程中所涉及到的主要问题进行了实验摸索,并依据方法学要求,对建立的每种反应体系从各主要方面进行了详细评价。实验结果表明,两种反应体系分别可以准确、高效、灵敏的对乳TP和尿素氮与铵态氮总量进行测定,酶解-Berthelot体系还能分别定量尿素氮与铵态氮浓度。各检测体系的主要特点如下：微量化Lowry反应体系在自动分析应用中,其TP测定结果准确性高,与凯氏定氮法(不添加无机含氮物质、有机非蛋白质含氮物质时)间无显著性差异；反应体系中样品液蛋白浓度为10～100μg/mL线性良好,最低检测浓度为5μg；可以将乳样做500倍稀释后直接测定,乳蛋白含量在0.5～5.0g/100g时可准确定量。方法在不同样品、不同测定时间内的重复性良好；乳中可能存在或人为添加的叠氮化钠、碳酸氢钠、硫酸钠、尿素、三聚氰胺、硫酸铵、盐酸羟胺、硫脲、亚硝酸盐、蔗糖和氯化钠等不会对测定结果产生干扰,重铬酸钾只有在浓度超过10mg/mL时,才对反应产生影响,其能使反应结果升高。酶解-Berthelot法测定乳中尿素氮与铵态氮,在0.5～400mg N/100g范围内可准确定量(即尿素1.073mg/100g～0.858g/100g或硫酸铵2.36mg/100g～1.888g/100g,相当于3.125 mg/100g～2.5g/100g蛋白质的含氮量),添加回收率高(>96%)、结果准确,能够准确定量尿素氮与铵态氮总量及分别浓度,既能对人为添加的尿素、硫酸铵等进行测定,也可以检测牛奶中含有的内源尿素氮(MUN)。反应中牛奶样品的前处理采用TCA沉淀法,该过程在脲酶处理之后进行。方法在不同样品、不同测定时间、不同操作人员间的重复性好,乳中可能同时存在的叠氮化钠、碳酸氢钠、硫酸钠、蔗糖、葡萄糖和氯化钠等不会对测定结果产生干扰,超过一定浓度的重铬酸钾(>10mg/mL)、过氧化氢(>0.6%)、硫酸铜(>10mg/mL)、盐酸羟胺(>10mg/mL)、硫脲(>10mg/mL)、亚硝酸盐(>10mg/mL)会对反应产生影响,且均表现为对反应的抑制作用。更多还原

【Abstract】 The requirement amounts for diary product are increasing, but there is an obvious lack of the raw milk which the origin for it. In this case, some badmen mix external substances into it for the interest of themselves. The phenomenon fastens on milk protein, and its reasons contain lack of determination methods for it and localization of detection methods for adulteration.In the face of above-mentioned complexion, we research the detection methods and systems for milk true protein (TP) and urea nitrogen together with ammonium nitrogen separately. The TP’s determination employ the Micro-Lowry method that set up by experimentation, and for urea nitrogen with ammonium nitrogen are used Urease-Berthelot method. In the study, we researched the major issues relating to each reaction system that was in the process of establishing, and evaluate the systems based on methodology.The result showed that two reaction systems are accuracy, efficiency and sensitive, not only detect the total amount of TP and urea nitrogen with ammonium nitrogen, but distinguish urea nitrogen from ammonium nitrogen. The principal characteristics of every system are following.In the auto-analyze system, the Micro-Lowry reaction’s result is accurate, and no marked difference with Kjeldahl method (when no inorganic nitrogen and organic nonprotein nitrogen substances in sample). The linear is well when the protein concentration in the range of 10～100μg/mL in reaction solution, and it’s least detection limit is 5μg. The sample can direct determination by dilution 500 times. The milk protein can accurate quantitation while its concentration in the range of 0.5～ 5.0g/100g. The method’s reproducibility is well between distinct sample and assay time. Sodium azide, sodium bicarbonate, sodium sulphate, urea, ammonium sulphate, hydroxylamine hydrochloride, thiourea, nitrite, sucrose and sodium chloride in milk don’t influence result. Potassium dichromate increase reaction result in the concentration above 10mg/mL.The recovery rate are high (>96%) and data are exact, when the Urease-Berthelot method used for determination outside urea nitrogen and ammonium nitrogen in milk. The linear is well when the nitrogen concentration in the range of 1～40μg N in reaction solution, and it’s least detection limit is 0.09μg N. The urea nitrogen and ammonium nitrogen in milk can can accurate quantitation while its concentration in the range of 0.5～400mg/100g (correspond with urea 1.073mg/100g～0.858g/100g or ammonium sulfate 2.36mg/100g～1.888g/100g, equivalent to nitrogen concentration in protein quantity 3.125 mg/100g～2.5g/100g), because of sample dilution to 1～50 time after by urease hydrolysis. The reproducibility is well among different sample, assay time and operator. Sodium azide, sodium bicarbonate, sodium sulphate, sucrose, glucose and sodium chloride in milk don’t influence result. Potassium dichromate (>10mg/mL), hydrogen peroxide (>0.6%), copper sulfate (>10mg/mL),hydroxylamine hydrochloride (>10mg/mL), thiourea(>50mg/mL) and nitrite(>10mg/mL) decrease reaction result in the above concentration.更多还原