脯氨酸的檢測(cè)方法

1、脯氨酸的檢測(cè)方法Coca-cola standardization office ZZ5AB-ZZSYT-ZZ2C-ZZ682T-ZZT18脯氨酸的檢測(cè)方法1原理樣品用前三酮處理，樣品中的脯氨酸反應(yīng)生成橙色物質(zhì)，在520nm檢測(cè)吸光度，并作空白對(duì)照實(shí)驗(yàn)。2 .應(yīng)用范圍本方法適用于蜂蜜、果汁和濃縮汁中的脯氨酸的檢測(cè)。本方法在其它方面的應(yīng) 用必須經(jīng)過(guò)個(gè)別論證。3 .試劑及儀器脯氨酸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 新鮮，即現(xiàn)用現(xiàn)配）2-甲氧基乙醇蟻酸（96%）,即甲酸異丙醇（2-丙醇）水溶液：水：丙醇二1： 115ml旋蓋玻璃離心管（確保離心管的邊緣沒(méi)有

2、缺口，將有缺口的離心管丟 掉）分光光度計(jì)，檢測(cè)吸光度設(shè)定在520nm下1cm玻璃比色皿4 .樣品準(zhǔn)備蘋果，梨和大部分漿果的果汁用單一濃度（即原果汁濃度），橙汁用1： 20的水進(jìn)行稀 釋。其它的果汁應(yīng)進(jìn)行適當(dāng)?shù)南♂?，以保證稀釋后的樣品中的肺氨酸的含量低于 50mg/'L（見稀釋表）。濃縮果汁用水稀釋到單一濃度，然后再進(jìn)一步進(jìn)行適當(dāng)?shù)南♂尅?蜂蜜的稀釋，采用蜂蜜加50ml水。將樣品在分析之前離心5min左右，去除懸浮物，取上清液進(jìn)行分析。原果汁稀釋表稀釋比例期望值樣品類型顏色空白Apple蘋果1:1yesApricot 杏21:1yesAronia 野櫻鞋1:1yesBanana香蕉1:1

3、yesBlackberry 黑莓1:1yesCherry櫻桃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檸檬21:1noLime酸橙21:1noMandarin中國(guó)柑桔21:1noMango芒果1:1yesNectarine 油桃21:1noOrange柑桔21:1noPapaya番木瓜1:1yesPassion Fruit 西番蓮1:1yesPeach 桃6:1yesPineapple 菠蘿1:1yesPlum李子7:1yesPrune 西梅16:1yesR

4、aspberry懸鉤子（樹5:1yes梅）Strawberry 草莓1:1yesTangerine 橘子21:1noTomato番茄1:1yes5 .標(biāo)準(zhǔn)溶液的制備脯氨酸貯備液（500mg/L）：脯氨酸加水定容至100ml。此貯備溶液冷藏存 放，每月重新配置一次。50mlg/L準(zhǔn)工作溶液：每天配置一次，取5nli脯氨酸貯備溶液，用水定容 至50ml。用該溶液作為分析用的標(biāo)準(zhǔn)溶液。6 .結(jié)果判定將離心管編號(hào)，并設(shè)計(jì)樣品編號(hào)與離心管標(biāo)號(hào)的對(duì)照表格。按下表，向離心 管內(nèi)移取相應(yīng)溶液：*顏色水樣品空白標(biāo)準(zhǔn)一門的三酮溶液mL mL mL2-甲氧基乙醇 mL 標(biāo)準(zhǔn)溶液 mL 樣品 mLmL 水 mL蟻酸m

5、L mL mLmL深顏色的果汁或者稀釋度較低的果汁中的色素物質(zhì)，將會(huì)對(duì)檢測(cè)結(jié)果的判定 產(chǎn)生影響。為糾正因此產(chǎn)生的結(jié)果偏差，使用顏色空白對(duì)照實(shí)驗(yàn)。對(duì)蘋 果汁、梨汁、漿果汁和蜂蜜采用顏色空白對(duì)照實(shí)驗(yàn)。50mg/l標(biāo)準(zhǔn)溶液的水空白對(duì)照實(shí)驗(yàn)用于柑橘汁及其它高稀釋倍數(shù)的果汁 檢測(cè)。蓋緊離心管，充分搖勻（處理苛三酮時(shí)要戴乳膠手套）。同時(shí)將所有離心 管放入沸水浴中加熱15分鐘。將離心管放在室溫水浴中冷卻5分鐘。向每個(gè)離 心管中加入5ml異丙醇水溶液（1： 1） °充分混勻后，用1cm比色皿，在 520nm下測(cè)量每管溶液的吸光度（A520）（對(duì)于有絮狀物的樣品，如芒果，將樣 品離心后，把上清液用移液

6、管小心轉(zhuǎn)移至比色肌中，直接川離心管傾倒會(huì)將絮 狀物混到上清液中）。每次測(cè)量之前，要先用待測(cè)溶液將比色皿潤(rùn)洗一下，倒 掉潤(rùn)洗液，然后再進(jìn)行測(cè)量。在35分鐘的冷卻時(shí)間，內(nèi)將所有待測(cè)溶液的 A520檢測(cè)完畢。應(yīng)戴乳膠手套，以避免藥三酮溶液滴濺到皮膚上，藥三酮溶液 滴濺到皮膚上，會(huì)產(chǎn)生深紫色的色斑。7 .計(jì)算如果50 mg/L標(biāo)準(zhǔn)溶液的附值 ,結(jié)果需經(jīng)實(shí)驗(yàn)室主管的核準(zhǔn)，才能上報(bào)結(jié) 果，否則樣品可能需要進(jìn)行復(fù)測(cè)脯氨酸（mg/L）二（AA樣品/AA標(biāo)準(zhǔn)溶液）x CC是標(biāo)準(zhǔn)溶液的濃度（通常為50 mg/L）對(duì)于脯氨酸含量的水果來(lái)說(shuō)，結(jié)果必須要乘上稀釋倍數(shù)（稀釋后體積/原體 積）對(duì)于濃縮果汁和蜂蜜來(lái)說(shuō)，結(jié)果必

7、須乘以稀釋倍數(shù)（稀釋后體積ml/預(yù) 案重量g）結(jié)果用mg/kg表示&參考資料A0AC官方分析方法出版時(shí)間 15th Ed. （1990）方法編號(hào)工作表脯氨酸樣品編號(hào)壯 Date.樣品類型 Analyst樣品標(biāo)準(zhǔn)稀釋度(w/v)稀釋度(v/v) 離心管編號(hào)#空白管號(hào)A520吸光度A520空白吸光度AA520如果50 mg/L標(biāo)準(zhǔn)溶液的口兒值 ,結(jié)果需經(jīng)實(shí)驗(yàn)室主管的核準(zhǔn),樣品 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；每天補(bǔ) 充新的) 2-methoxy ethanol2-甲氧基乙醇formic acid (96%)蟻

10、酸(96%)(甲酸)aqueous isopropanol(2- Propanol ) - diluted 1+1水：異丙醇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的帶螺帽的玻璃離心管(確定管子的邊沒(méi)有破損；除掉有破損的瓶子) spectrophotometer set to measure absorbance at 520 nm520 nm測(cè)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.蘋果,梨和 多數(shù)漿果類的汁液是直接做。Citrus juices are diluted 1+20 with water. 柑橘類的汁液要用水稀釋 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 應(yīng)該適當(dāng)?shù)南♂屖垢彼岬臐舛仍谙♂尯筮_(dá)到小于50 mg/L o Fruit juice concentrates are diluted to single strength with water, and then further diluted appropriately.濃縮果汁用水稀釋到原來(lái)的濃度，然后再適當(dāng)?shù)南♂?。Honey is diluted g to 50 mL with water.蜂蜜取 g 用 50 mL 的水稀釋。Centifuge turbid samples for approximately 5

13、 minutes prior to analysis to remove suspended material; use clear supernatant for analysis.渾濁的樣品，在分析前5分鐘離心，移去懸浮物，用清光的浮 在表面的去測(cè)。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.準(zhǔn)備脯氨酸標(biāo)準(zhǔn)儲(chǔ)備溶液(500 mg/L)：用水溶解g肺氨酸至100 mL,溶液保存 到冰箱，每月配制新的標(biāo)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.每天準(zhǔn)備新的脯氨酸標(biāo)準(zhǔn)溶液(50 mg/L),將5 mL脯氨酸標(biāo)準(zhǔn)儲(chǔ)備溶液(500 mg/L)用水稀釋到50 mLo用這個(gè)濃度做分析。DeterminationNumber a series of screw cap centrifuge tubes, and prepare a table correlating sample numbers to tube numbers. Pipet the following into tubes

17、:給一系列的帶有螺帽的離心管編號(hào)，準(zhǔn)備相關(guān)于管子編號(hào)的表，將下面的物質(zhì) 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顏色深的果汁如果不稀釋可能含有色素會(huì)干擾檢測(cè)。校正使用顏色空白，用于 蘋果、梨等漿果類和蜂蜜。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. 水空白為50 mg/L的標(biāo)準(zhǔn)溶液用于校正柑橘類等高稀釋度的果汁。Cap tubes tightly and shake well (wear latex gloves while handling ninhydrin solutions).蓋緊蓋子充分振搖(當(dāng)操作荀三酮溶液時(shí)戴上乳膠手 套)。Simultaneously place all tubes in a boiling water bath for exactly 15 minutes.同時(shí)放置所有的管子到沸水浴中15分鐘。Cool tubes for 5 minutes in a room temperature wat

20、er bath. 在室溫的水浴中冷卻管:5 分鐘。 Add 5 mL aqueous isopropanol (1+1) into each tube.力口 5 mL 水：異丙醇(1： 1)溶液到每個(gè)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處測(cè)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).(有絮狀物的樣品，例如芒 果，離心此管子，小心的用移液管將清液移到比色皿中，傾倒管子可能重新懸 浮絮狀物) 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測(cè)量前丟 棄這些漂洗液。Measure A52O of all

22、of the solutions within 35 minutesof cooling.在35分鐘之內(nèi)將所有溶液測(cè)量520 nm處吸光度。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.戴上乳膠手套避免給皮膚上濺上的三酮溶液，如果給皮膚 上濺上司三酮溶液，皮膚上會(huì)有深紫色的污點(diǎn)。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的標(biāo)準(zhǔn)溶液)< ,在分發(fā)前應(yīng)該經(jīng)過(guò)主管審核，樣品可能需 要復(fù)檢。Proline (mg/L) = (DAsample /DAstandard) x CWhere C is the concentration of the standard (typically 50 mg/