飼料中非蛋白氮20090516

1、飼料中非蛋白氮的摻假及其檢測(cè) 北京 2009-5-16國(guó)家飼料質(zhì)量監(jiān)督檢驗(yàn)中心1Institute of quality standards and testing technology for agri-food, CAAS.IQSTAP2為什么用非蛋白氮摻假？現(xiàn)有蛋白測(cè)定方法的缺陷：重要假設(shè)為，所有氮來(lái)自于蛋白質(zhì)，蛋白質(zhì)含氮量固定。3The sample is digested to convert the protein nitrogen into ammonium sulfate, which is treated with an alkali, thus liberating amm

2、onia. The ammonia is received by an acid and quantified through titrationquantified titration.456Ureaammonium sulfateBiuret Melaminemelamine formaledehyde resin, Urea-farmaldehyde resinMixed compounds Next ?曾經(jīng)用于摻假的物質(zhì):7適宜于摻假的化合物特性：價(jià)低 高含氮量沒(méi)有刺激性氣味 對(duì)動(dòng)物無(wú)毒/或?qū)嶋H無(wú)毒不溶于水/酸/堿8無(wú)機(jī)氮化合物 磷酸氫銨，硫酸銨，碳酸銨等其N(xiāo): 17.7%，20.6%

3、，21.2%。價(jià)格：300-1000元/噸問(wèn)題：已被檢測(cè)9尿素N%：46.7,白色固體，無(wú)臭，但有刺激性氣味。 熔點(diǎn):132.7135 C由無(wú)機(jī)物合成的第一個(gè)有機(jī)物。10尿素加熱所生成的物質(zhì)ureaheatingtrichloroisocyanuric acid Cyanuric acid meliminebiuretpolymertriuret112分子尿素在加熱至其熔點(diǎn)溫度時(shí)，釋放出1個(gè)分子氨氣，生成1分子縮二脲： 2 CO(NH2)2 H2N-CO-NH-CO-NH2 + NH3 白色固體，溶于熱水， 在 186189 C時(shí)降解.液態(tài)位藍(lán)色，遇蛋白或多肽時(shí)變成紫色。12三聚氰酸首次合成于

4、1829年，通過(guò)尿素和氰酸熱解重組。目前工業(yè)合成路線(xiàn)為3個(gè)分子尿素?zé)峤庵亟M，釋放3個(gè)分子氨，生成1個(gè)分子三聚氰酸。反應(yīng)溫度約為 175 ：3 H2N-CO-NH2 C(O)NH3 + 3 NH3 在三聚氰胺粗品結(jié)晶過(guò)程中，通過(guò)水解產(chǎn)生三聚氰酸。三聚氰酸一旦產(chǎn)生將與三聚氰胺結(jié)合，產(chǎn)生沉淀。 13中間產(chǎn)物與雜質(zhì)：在脫水的過(guò)程中將產(chǎn)生氰酸、縮二脲、縮三脲：H2N-CO-NH2 HNCO + NH3 H2N-CO-NH2 + HNCO H2N-CO-NH-CO-NH2 H2N-CO-NH-CO-NH2 + HNCO H2N-CO-NH-CO-NH-CO-NH2 雜質(zhì)之一為三聚氰酸一酰胺，判斷這是由縮二

5、脲降解重組而形成。當(dāng)反應(yīng)溫度超過(guò)190 時(shí)該雜質(zhì)顯著增加：3 H2N-CO-NH-CO-NH2 C(O)2(CNH2)(NH)2N + 2 NH3 + H2O 當(dāng)溫度在325 和 350 時(shí)，產(chǎn)生少量三聚氰胺。14三聚氰胺目前，工業(yè)合成的主要工藝為尿素的熱反應(yīng)：6 (NH2)2CO C3H6N6 + 6 NH3 + 3 CO2 反應(yīng)過(guò)程分兩個(gè)步驟.首先，尿素在吸熱反應(yīng)中降解為氰酸和氨:(NH2)2CO HCNO + NH3 接著，氰酸聚合成三聚氰胺，并放出二氧化碳:6 HCNO C3H6N6 + 3 CO2 15尿素土法加熱處理：為了避免尿素的刺激性氣味和容易檢測(cè)的弊病，摻假者土法對(duì)其進(jìn)行加熱

6、處理后刺激性氣味明顯減少，經(jīng)檢測(cè)：真蛋白氮2.56%，總氮41.32%，尿素28.8%，三聚氰酸55%，三聚氰胺4%，未知物？16添加物經(jīng)常變化 添加混合物難以通過(guò)感官來(lái)辨別摻假者也不知道添加的物質(zhì)檢測(cè)所要面臨的問(wèn)題：17如何檢測(cè)這些摻假 目前飼料中非蛋白氮檢測(cè)有諸多方法，歸納有三類(lèi): 目標(biāo)物檢測(cè)方法扣除分析法蛋白質(zhì)檢測(cè)方法18目標(biāo)物檢測(cè): 準(zhǔn)確、精確、靈敏、適應(yīng)性強(qiáng)，分析費(fèi)用昂貴（儀器/耗材），明確摻假物后，才能建立方法。以下以三聚氰胺檢測(cè)為例說(shuō)明。 19202122231 ppb的三聚氰胺、三聚氰酸和其內(nèi)標(biāo)物13C3標(biāo)記的三聚氰胺和三聚氰酸的色譜圖（由上至下依次為：三聚氰酸、13C3標(biāo)記的

7、三聚氰酸、三聚氰胺、13C3標(biāo)記的三聚氰胺）2413C3標(biāo)記的三聚氰胺Q(chēng)ED-MS/MS譜圖，右圖為三聚氰胺Q(chēng)ED-MS/MS譜圖25誰(shuí)最高興？26直接測(cè)定蛋白法: 不受摻假物影響，方法相對(duì)簡(jiǎn)便、無(wú)需大型精密設(shè)備，背景影響加大。27Estimation of quantities of protein using the dye eosin YA. A. Waheed, and P. D. Gupta (1996)a method for estimating submicrogram quantities of proteins using the dye eosin Y. The incr

8、ease in sensitivity of this assay is approximately two fold under optimal assay condition. 0.01 and 0.05% The proteindye complex formation is completed within 2 min and its absorbance is stable up to 60 min with a variation of 4.0%. The interference due to sugars, reducing agents, glycerol and some

9、neutral detergents like Triton X-100, NP-40 and Tween-20 is less than 12% whereas Brij-35, ethanol, acetone and chelators like EGTA and EDTA suppress the absorbance by about 1218%. However, basic buffers like Tris, urea, CHAPS and NaN3 interfere with the formation of the proteindye complex. 28扣除分析法首

10、先將蛋白氮與非蛋白氮分開(kāi)，然后測(cè)定蛋白氮，則得到非蛋白氮；或測(cè)定非蛋白氮，得到蛋白氮。關(guān)鍵：將蛋白氮與非蛋白氮分離。問(wèn)題：動(dòng)植物組織存在非蛋白氮，且含量不定。 29分離區(qū)分蛋白氮與尿素及無(wú)機(jī)氮化合物三氯乙酸、鎢酸鈉、酸性硫酸銅等溶液用于沉淀蛋白?？梢院芎玫貙⑵渑c蛋白氮分離，測(cè)定。尿素及無(wú)機(jī)氮化合物水溶液中完全溶解。30分離區(qū)分蛋白氮與三聚氰酸/三聚氰胺堿性硫酸銅溶液可以沉淀蛋白，將其與三聚氰胺、三聚氰酸、尿素、無(wú)機(jī)氮化合物很好分離。三聚氰胺、三聚氰酸不溶于水、酸，但溶于堿溶液。31雙匯公司的研究結(jié)果32Separation by copper sulfate in alkaline solut

11、ion0.459200018.50.45210.03210.0241018.00.47150.03380.02230.021118.40.469000.02100.025417.900.024600.021418.10.46610.031000.034117.633A critical study of methods for the determination of nonprotein nitrogen Pamela M. BellSome twenty methods for the removal of protein from biological materials were us

12、ed to prepare the nonprotein nitrogen fraction from skim milk, serum, a water extract of flour, and a water extract of bran. The results obtained by different methods on one solution or the same methods on different solutions were not entirely concordant and showed that NPN fractions obtained in these ways cannot validly be compared. Dialysis or related techniques appeared to achieve separations most closely related to NPN as theoretically defined. The binding of amino acids to protein was demonstrated using isotopically labeled amino acids in flour-protein solutions