Detection of added urea in milk

Detection of added urea in milk

• 1.
  Detection of AddedUrea in Milk F a r h a n g H a m i d I r a q - K u r d i s t a n - s u l a y m a n i y a h + 9 6 4 7 7 0 1 4 2 0 9 6 2 F a r h a n g p d k . h a m i d @ g m a i l . c o m 5 / 2 0 / 2 0 1 6 Farhang Hamid Milkurea isan approximate indicatorof the amountof crude proteinina cow's diet.Itisformedfromthe metabolism of absorbedaminoacidsand bodyprotein.The digestionof proteininthe rumenreleasesammonia.If there isexcessammoniaitisabsorbedthroughthe rumenwall intothe blo streamand convertedtoureain the liver.Mostof the ureais excretedint cow’surine althoughsome passesintothe milk.If the dietisdeficientin nitrogen,thenthe ureaisnot excreted,butrecycledbackintothe rumena convertedbackto ammonia.
• 2.
  Detection of AddedUrea in Milk Urea is a natural constituent of milk and it forms a major part of the non-protein nitrogen Urea concentration in milk is variable within herd. Urea content in natural milk varies from 20 mg/100 ml to 70 mg/100 ml. However, urea content above 70 mg/100 ml in milk indicates milk containing ‘added urea’. The addition of urea to milk can be detected by using para-dimethylaminobenzaldehyde (DMAB). This method is based on the principle that urea forms a yellow complex with DMAB in a low acidic solution at room temperature Qualitative Method:- This method is based on the principle that urea forms a yellow complex with DMAB in a low acidic solution at room temperature. Reagent:- DMAB reagent (1.6%, w/v): Dissolve 1.6 g DMAB in 100 ml ethyl alcohol and add 10 ml concentrate HCl. Procedure:- Mix 1 ml of milk with 1 ml of 1.6% DMAB reagent. Distinct yellow colour is observed in milk containing added urea. The control (normal milk) shows a slight yellow colour due to presence of natural urea. The limit of detection of method is 0.2%. Quantitative Estimation of Urea in Milk:- Urea is a natural constituent of milk and is present to the extent of 70 mg per 100 ml (700 ppm). The test based on the use of para-dimethylamino benzaldehyde can be used for the estimation of urea in milk after precipitation of milk proteins using trichloroacetic acid. Reagents/Apparatus:- A. p-Dimethyl amino benzaldehyde (DMAB) solution: Dissolve 1.6 g DMAB in 100 ml ethyl alcohol and add 10 ml concentrate HCl. The reagent is stable for 1 month. Prepare new standard curve with each new batch of reagent. B. Phosphate Buffer pH 7.0: Dissolve 3.403 g anhydrous potassium dihydrogen orthophosphate (KH2PO4) and 4.355 g anhydrous dipotassium monohydrogen orthophosphate (K2HPO4) separately in 100 ml of distilled water. Combine solutions and dilute to 1 litre with water.
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  C. Trichloroacetic acid(TCA) 24%, w/v: Freshly prepared. 24.0 g TCA is dissolved in distilled water and volume made up to 100 ml. D. Diluting Reagent: Equal volumes of 24% TCA and phosphate buffer (pH 7.0) are mixed to make the diluting reagent. E. Urea Standard Solution: (a) Stocksolution: 5 mg / ml. Dissolve 5 ± 0.001 g reagent grade urea in water and dilute to 1 litre with water. (b) Working solution:– Pipette 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 ml stock solution into 250 ml volumetric flask and dilute to volume with phosphate buffer. (c) Reference solution -Use standard solution containing 1.0 mg urea / 5 ml as reference standard. Store at less than 24°C. The reagent is stable for 1 week. Apparatus:- A. Spectrophotometer – Instrument with maximum band width 2.4 nm at 420 nm, with 1 cm cells B. Whatman filter paper: Grade 42. C. Funnels. D. Test tubes. Procedure:- Preparationof standard curve Pipette 5 ml aliquots of working standard solutions into 20 x150 mm (25 ml) test tubes and add 5 ml DMAB solution to each. Prepare reagent blank of 5 ml buffer and 5 ml DMAB solution. Shake tubes thoroughly and let stand for 10 minutes. Read A in 1 cm cell at 420 nm with reagent blank at zero A. Plot A against concentration urea Plot should be straight line. Estimation:- 10 ml of milk sample is mixed with 10 ml of TCA to precipitate the proteins and filtered using Whatman 42 filter paper. 5 ml of filtrate is then treated with 5 ml of DMAB reagent to develop the colour. Blank is prepared by taking 5 ml of diluting reagent and treating with 5 ml of DMAB reagent. The optical density of the yellow colour is measured at 420 nm. From standard curve the amount of urea in milk is calculated. (Ref:- IS.1479 (Part I) – 1960(Reaffirmed2003)Methods of test forDairyIndustry,Part – I Rapid examination of Milk. Bureau of Indian Standards, New Delhi; Bector, B.S., Ram, M. and Singhal, O.P. (1998). Rapid platform test for the detection /determination of added urea in milk. Indian Dairyman, 50(4): 59 -62).