脯氨酸的检测方法

1、脯氨酸的检测方法Coca-cola standardization office ZZ5AB-ZZSYT-ZZ2C-ZZ682T-ZZT18脯氨酸的检测方法1原理样品用前三酮处理，样品中的脯氨酸反应生成橙色物质，在520nm检测吸光度，并作空白对照实验。2 .应用范围本方法适用于蜂蜜、果汁和浓缩汁中的脯氨酸的检测。本方法在其它方面的应 用必须经过个别论证。3 .试剂及仪器脯氨酸3%苛三酮2-甲氧基乙醇溶液（即三酮溶解于10ml 2-甲氧基乙醇，每天保持 新鲜，即现用现配）2-甲氧基乙醇蚁酸（96%）,即甲酸异丙醇（2-丙醇）水溶液：水：丙醇二1： 115ml旋盖玻璃离心管（确保离心管的边缘没有

2、缺口，将有缺口的离心管丢 掉）分光光度计，检测吸光度设定在520nm下1cm玻璃比色皿4 .样品准备苹果，梨和大部分浆果的果汁用单一浓度（即原果汁浓度），橙汁用1： 20的水进行稀 释。其它的果汁应进行适当的稀释，以保证稀释后的样品中的肺氨酸的含量低于 50mg/'L（见稀释表）。浓缩果汁用水稀释到单一浓度，然后再进一步进行适当的稀释。 蜂蜜的稀释，采用蜂蜜加50ml水。将样品在分析之前离心5min左右，去除悬浮物，取上清液进行分析。原果汁稀释表稀释比例期望值样品类型颜色空白Apple苹果1:1yesApricot 杏21:1yesAronia 野樱鞋1:1yesBanana香蕉1:1

3、yesBlackberry 黑莓1:1yesCherry樱桃1:1yesCranberry 蔓越莓1:1yesGrape葡萄16:1yesGrapefruit 柚子21:1noGuava番石榴3:1yesKiwi泥猴桃1:1yesLemon柠檬21:1noLime酸橙21:1noMandarin中国柑桔21:1noMango芒果1:1yesNectarine 油桃21:1noOrange柑桔21:1noPapaya番木瓜1:1yesPassion Fruit 西番莲1:1yesPeach 桃6:1yesPineapple 菠萝1:1yesPlum李子7:1yesPrune 西梅16:1yesR

4、aspberry悬钩子（树5:1yes梅）Strawberry 草莓1:1yesTangerine 橘子21:1noTomato番茄1:1yes5 .标准溶液的制备脯氨酸贮备液（500mg/L）：脯氨酸加水定容至100ml。此贮备溶液冷藏存 放，每月重新配置一次。50mlg/L准工作溶液：每天配置一次，取5nli脯氨酸贮备溶液，用水定容 至50ml。用该溶液作为分析用的标准溶液。6 .结果判定将离心管编号，并设计样品编号与离心管标号的对照表格。按下表，向离心 管内移取相应溶液：*颜色水样品空白标准一门的三酮溶液mL mL mL2-甲氧基乙醇 mL 标准溶液 mL 样品 mLmL 水 mL蚁酸m

5、L mL mLmL深颜色的果汁或者稀释度较低的果汁中的色素物质，将会对检测结果的判定 产生影响。为纠正因此产生的结果偏差，使用颜色空白对照实验。对苹 果汁、梨汁、浆果汁和蜂蜜采用颜色空白对照实验。50mg/l标准溶液的水空白对照实验用于柑橘汁及其它高稀释倍数的果汁 检测。盖紧离心管，充分摇匀（处理苛三酮时要戴乳胶手套）。同时将所有离心 管放入沸水浴中加热15分钟。将离心管放在室温水浴中冷却5分钟。向每个离 心管中加入5ml异丙醇水溶液（1： 1） °充分混匀后，用1cm比色皿，在 520nm下测量每管溶液的吸光度（A520）（对于有絮状物的样品，如芒果，将样 品离心后，把上清液用移液

6、管小心转移至比色肌中，直接川离心管倾倒会将絮 状物混到上清液中）。每次测量之前，要先用待测溶液将比色皿润洗一下，倒 掉润洗液，然后再进行测量。在35分钟的冷却时间，内将所有待测溶液的 A520检测完毕。应戴乳胶手套，以避免药三酮溶液滴溅到皮肤上，药三酮溶液 滴溅到皮肤上，会产生深紫色的色斑。7 .计算如果50 mg/L标准溶液的附值 ,结果需经实验室主管的核准，才能上报结 果，否则样品可能需要进行复测脯氨酸（mg/L）二（AA样品/AA标准溶液）x CC是标准溶液的浓度（通常为50 mg/L）对于脯氨酸含量的水果来说，结果必须要乘上稀释倍数（稀释后体积/原体 积）对于浓缩果汁和蜂蜜来说，结果必

7、须乘以稀释倍数（稀释后体积ml/预 案重量g）结果用mg/kg表示&参考资料A0AC官方分析方法出版时间 15th Ed. （1990）方法编号工作表脯氨酸样品编号壮 Date.样品类型 Analyst样品标准稀释度(w/v)稀释度(v/v) 离心管编号#空白管号A520吸光度A520空白吸光度AA520如果50 mg/L标准溶液的口儿值 ,结果需经实验室主管的核准,样品 脯氨酸mg./L or mg./kg mg/L or mg/kg mg/L or mg/kg mg/L or mg/kg mg/L or mg/kg mg/L or mg/kg mg/L or mg/kgPROLIN

8、EPrincipleThe prepared sample is treated with ninhydrin. Proline in the sample reacts to form an orange derivative. Absorbance is measured at 520 nm versus appropriate reagent blank.ApplicabilityThis method is applicable to the determination of proline in honey, fruit juices and concentrates. Other

9、applications of this method must be individually validated.Reagents and EquipmentProline脯氨酸3% ninhydrin in 2-methoxy ethanol g of ninhydrin dissolved in 10 mL 2-methoxyethano1 is satisfactory; make up fresh daily)3%的荀三酮在2-甲氧基乙醇(g荀三酮溶解在10 mL 2-中氧基乙醇；每天补 充新的) 2-methoxy ethanol2-甲氧基乙醇formic acid (96%)蚁

10、酸(96%)(甲酸)aqueous isopropanol(2- Propanol ) - diluted 1+1水：异丙醇1: 115 mL screw cap glass centrifuge tubes (be sure that there are no chips on the rim of the tube; dispose of chipped tubes)15 mL的带螺帽的玻璃离心管(确定管子的边没有破损；除掉有破损的瓶子) spectrophotometer set to measure absorbance at 520 nm520 nm测定吸光度1 cm glass c

11、uvettes 1 cm的玻璃小管Sample PreparationApple, pear and most berry juices are run single strength.苹果,梨和 多数浆果类的汁液是直接做。Citrus juices are diluted 1+20 with water. 柑橘类的汁液要用水稀释 1： 20 o Other fruit juices should be diluted appropriately such that the proline concentration in the diluted sample is less than 50

12、mg/L (see dilution table). 其它水果汁液也 应该适当的稀释使脯氨酸的浓度在稀释后达到小于50 mg/L o Fruit juice concentrates are diluted to single strength with water, and then further diluted appropriately.浓缩果汁用水稀释到原来的浓度，然后再适当的稀释。Honey is diluted g to 50 mL with water.蜂蜜取 g 用 50 mL 的水稀释。Centifuge turbid samples for approximately 5

13、 minutes prior to analysis to remove suspended material; use clear supernatant for analysis.浑浊的样品，在分析前5分钟离心，移去悬浮物，用清光的浮 在表面的去测。Dilution Table for Single Strength JuiceDilutionColorExpectedSample TypefromBlankValueSingleStrengthApple1:1yesApricot21:1yesAronia1:1yesBanana1:1yesBlackberry1:1yesCherry1:

14、1yesCranberry1:1yesGrape16:1yesGrapefruit21:1noGuava3:1yesKiwi1:1yesLemon21:1noLime21:1noMandarin21:1noMango1:1yesNectarine21:1noOrange21:1noPapaya1:1yesPassion Fruit1:1yesPeach6:1yesPineapple1:1yesPlum7:1yesPrune16:1yesRaspberry5:1yesStrawberry1:1yesTangerine21:1noTomato1:1yesStandard PreparationPr

15、epare a proline stock solution (500 mg/L) by dissolving g proline in water and making up to 100 mL. This solution should be stored refrigerated and prepared fresh monthly.准备脯氨酸标准储备溶液(500 mg/L)：用水溶解g肺氨酸至100 mL,溶液保存 到冰箱，每月配制新的标品。Prepare fresh daily a 50 mg/L working standard solution by diluting 5 mL

16、proline stock solution to 50 mL with water. Use this standard for the analysis.每天准备新的脯氨酸标准溶液(50 mg/L),将5 mL脯氨酸标准储备溶液(500 mg/L)用水稀释到50 mLo用这个浓度做分析。DeterminationNumber a series of screw cap centrifuge tubes, and prepare a table correlating sample numbers to tube numbers. Pipet the following into tubes

17、:给一系列的带有螺帽的离心管编号，准备相关于管子编号的表，将下面的物质 用移液管移到管子里：color*water*sample blank stdblankninhydrin solutionmL mL mL2-me thoxye thano 1 mL standard solution mLprepared sample mLmL water mLformic acidmLmLmLmL* Intensly colored fruit juices or juices with very little dilution may contain pigments that will inter

18、fere with the determination. To correct for this, use a color blank. Run color blanks for apple, pear, berry juices and honey颜色深的果汁如果不稀释可能含有色素会干扰检测。校正使用颜色空白，用于 苹果、梨等浆果类和蜂蜜。The water blank for 50 mg/L standard will be suficient to correct for the background in citrus and other highly diluted fruit ju

19、ices. 水空白为50 mg/L的标准溶液用于校正柑橘类等高稀释度的果汁。Cap tubes tightly and shake well (wear latex gloves while handling ninhydrin solutions).盖紧盖子充分振摇(当操作荀三酮溶液时戴上乳胶手 套)。Simultaneously place all tubes in a boiling water bath for exactly 15 minutes.同时放置所有的管子到沸水浴中15分钟。Cool tubes for 5 minutes in a room temperature wat

20、er bath. 在室温的水浴中冷却管:5 分钟。 Add 5 mL aqueous isopropanol (1+1) into each tube.力口 5 mL 水：异丙醇(1： 1)溶液到每个管子中。Mix well and measure theabsorbance at 520 nm (A520) of the solution in each tube in a 1 cmglass cuvette充分混合后在520 nm处测量吸光度。(for samples that have some floc, such as mango, centrifuge the tube and c

21、arefully transfer the clear layer into the cuvette with a disposable pipet, tipping the tube may resuspend the floc).(有絮状物的样品，例如芒 果，离心此管子，小心的用移液管将清液移到比色皿中，倾倒管子可能重新悬 浮絮状物) Rinse the cuvette with the solution once and discard before taking each measurement.用溶液漂洗比色皿一次并且在测量前丢 弃这些漂洗液。Measure A52O of all

22、of the solutions within 35 minutesof cooling.在35分钟之内将所有溶液测量520 nm处吸光度。Avoid spills of ninhydrin solution on the skin by wearing latex gloves; a deep purple discoloration will result from exposure of the skin to ninhydrin solution.戴上乳胶手套避免给皮肤上溅上的三酮溶液，如果给皮肤 上溅上司三酮溶液，皮肤上会有深紫色的污点。CalculationsDA* = A52O

23、- A52O blank*if the DA value for a 50 mg/L standard is < , the results should be approved by a supervisor before they are distributed to the active folder, samples may need to be rerun如果DA (50 mg/L的标准溶液)< ,在分发前应该经过主管审核，样品可能需 要复检。Proline (mg/L) = (DAsample /DAstandard) x CWhere C is the concentration of the standard (typically 50 mg/