The present invention used to detect the urea, adulteration in milk. More specifically this method describes the development of strip which is able to detect the presence of urea in milk sample. The is natural component of milk and it is found in the range of 0.1 to 0.2 mg based on the diet of the animal. Adulteration of urea in milk is done to increase the non-protein nitrogen content, fat value, and -■ whiteness and also increase the consistency and shelf-life of milk (Renny et al 2005; Lacks, 1979). Even after boiling the milk urea remain stable in milk and water. If the concentration of urea is greater than 0.2 mg it is considered as adulterated and the developed strip can be used for detection of urea greater than 0.2 mg/ml.
Background of the invention
Adulteration is common problem in developing countries due to lack of proper government policies. Generally-milk is adulterated by large number of adulterants due to increase in false properties such as increase in lactometer reading, increase in fat content, nitrogen content or as a preservative. One such commonly used adulterant is urea, which increase the nitrogen content, provide whiteness, increase the shelf-life of milk, and to standardize the solids not fat present in milk. Urea is a natural component of milk, it is reported by different researcher that the concentration of urea may varies from 0.1- 0.2 mg/L based on the proteinous nature of diet (Singh & Gandhi, 2015).The higher concentration of urea in milk is harmful for human being particularly pregnant women, children and sick individuals. Causes, indigestion, acidity, ulcer, cancer, malfunctioning of kidney, and osteoporosis (Trivedi et al, 2009; "Nikoleli et al, 2010). Various methods are available for detection of urea in milk or water one such method is p-dimethylaminobenzenaldehyde (PDMAB) test, which is based on the principal of formation of coloured complex by the urea with ^-dimethylaminobenzenaldehyde under acidic conditions. The presence of distinct yellow colour indicate urea presence in milk. (Food Safety and Standards Authority of India (FSSAI) approved recommended method). Another method which is approved by FSSAI is the

addition of soybean or arhar powder in to half teaspoon milk, and after 5 minute dip a red litmus paper for 30 second and change in colour from red to blue indicate the presence of urea in milk. Meisel (1995) develop a method in which 5 mL milk sample was mixed with 5 ml of 24% TCA to precipitate fat and proteins of milk. Their, 1 mL filtrate was mixed with 0.5 mL 2% sodium hypochlorite, 0.5 mL , 2% sodium hydroxide and add 0.5 mL 5% phenol solution'. A characteristic blue or bluish green colour develops in presence of added urea whereas pure milk remains colourless. Another method is based on enzyme reaction in 20 mg/ml urease enzyme added into sample mixture and then bromothymol blue added. Development of blue colour after 15-20 minutes indicate presence of urea in milk sample (http://face-cii.in/sites/default/files/dr_sitaram_dixit_-l.pdf). In another method discussed by S. Dixit. Mix the 5 ml milk with 5 ml paradimethyl amino benzaldehyde reagent and observe the colour change, in presence of urea colour of the above solution to yellow. And faint yellow colour indicate the presence of natural urea.
A strip based method for detection urea in milk sample was explained by Kumar H. et al (2000). In this method, they develop a strip which was light yellow incolour and upon-immersion in urea containing milk the colour will be change from light yellow to magenta. Developed strip can detect the urea concentration as low as 0.1 g/L.
Denis and Minot (1919) suggest the treatment of urea containing milk with urease extract and then using Van lyke and Cullen (1914) method for removal of ammonia formed. Then, cone. Of ammonia was can be determined by either nesslerization or titration; This method is performed using filtrate remaining after removal of casein protein.
Yang et al., in between 2011-13 used different variation of infra-red spectroscopy such as near infra¬red, Two-dimensional hetero-spectral mid-infrared and near-infrared correlation successful in determining the urea concentration of 0.01 g - 0.3 g/1 using two-dimensional infrared correlation spectra. Jha et al (2015) screen the different IR range for the detection of urea in milk and found 1670-1564 cm"1 comprising four smaller spectral regions (1670-1653, 1649-1621, 1615-1580, and 15?4-1564 cm"1) showed clear differences in absorption values for pure milk and urea containing milk with >900ppm urea.

Dinesh et al, 2013 developed a Resistance Temperature Detector (RTD) that is coated with Teflon. The Teflon have the property to attract ammonia. This increase the temperature, and the: increased temperature is into equivalent resistance change with the help of RTD that is used in a bridge circuit. The unbalance in the bridge circuit is proportional to the' level-of ammonia present in the milk. This method have the drawback of very low signal and need to be amplified every time. Another study by Valarrnathy et al, (2018) developed a sensor for the detection of mechanical pressure generated due to the production of gases such as ammonia and carbon dioxide. Production of gases exert certain pressure on the piezoelectric sensor which convert mechanical pressure into electric signal. The signal is further amplified and programmed to give quantity of ammonia present by using a calibration cure.
A potentiometric biosensor was developed by IndranilBasu et al, in 2004, Electrolyte-Insulator-Semiconductor capacitor (EISCAP) shows a shift in the measured CV with changes in the pH of the electrolyte; Ammonia and carbon dioxide liberated as a result of the en2ymatic reaction dissolve in water thereby producing ammonium hydroxide and carbonic acid respectively. Ammonium hydroxide is a strong base while carbonic acid is a weak acid. Hence pH of the electrolyte solution after the enzymatic reaction shifts towards the basic range. The change in pH is detected by CV measurements on the EISCAP and co-related to the concentration of urea.'This gives an estimation of urea content . present in milk.
Liquid Chromatography Isotope Dilution Mass Spectrometry (LC-1DMS) is used by Inaba et al, (2013) for determination of urea in milk. They first of all precipitate the milk protein with sequential addition of acetonitrile and chloroform and then separated with the help of liquid chromatography. To measure the urea concentration with accuracy, linearity, and very low uncertainty, exact matching of IDMS can be used.
Ramesh et al, (2015) proposed another method in which urea hydrolysis product can be measured with
the help of gas sensor. Valamurthy et al (2018) developed for the detection of ammonia gas based for
the detection of urea in milk. Their gas based sensor can detect a minimum of 2 mg/L of urea
adulteration in milk at 70°C. ,

Nie et al (2016) developed a method for detection of urea based on post postchemi luminescence; reaction was observed when urea injected into reaction mixture after chemi-luminance reaction of N-. bromo-succinimide and dichloro-fluorescein. The method was applied to determine the amount of urea in milk and provide satisfactory results.
A strip based test is developed by the Rajput et al (patent) for detection of urea in milk which have the
i
sensitivity upto 70mg/ml. this method involve the preparation of strip for detection of urea
contamination in milk.
Luther et al., (2017) developed a strip based test the detection multiple adulterants such as
starch, urea and sugar. Which is sucessfuUy able to detect the presence of these adulterants
with 90% sensitivity. But result can be obtained after 40 min of test and need cell-phone to
observe the results.
A method is discussed on the food lab site detect the urea in the milk on the basis of blue
colour development due.to reaction between ammonia and phenolic compound. The intensity
of colour is directly proportional to the concentration of urea present in the milk. The urea in
milk can be detected with this method in the range of 5 to 100 mg /dL.
Another method is discussed Sang Huachun and Han Dandanin (patent no. CN101900684A) in
2009, in this protected method, the para-dimethylaminobenzaldehyde reacts with urea and
converts into paradimethylaminobenzaldehyde urea which is lemon yellow colour.
In another patent (Patent No. CN1030544C), authors prepared the milk sample by mixing 50 g
of soya, hexamine (medicine) 2.5 g and 1000-1500 ml of water and soaked for 4-6 hours, then
ground soya and water together with large milk, muslin and filtered. The filtrate into vessel,
stand for 10 min to remove upper layer of foam ball, then used for detection urea in milk with
acid based indicator 1% bromothymol blue in ethanol solution turn the milk solution in blue or
green.
All the above discussed method have one or more limitation such as wastage of large amount
of chemicals, time consuming, usage of harmful chemical which increase the chance of human

contact and mostly methods are not sensitive enough and are laboratory based hence required ^ trained manpower. Major drawbacks of these techniques are the facts that these are valid for a limited range of concentrations and are not sufficiently precise.
Objectives of the Invention
• To develop a strip based test for detection of urea in milk.
• To develop a strip based test which can be done by the layman without proper training.
• To provide a strip which differentiates the pureimilk from urea adulterated milk by_visual distinction of colour changes.
• To provide a strip which can differentiate pure milk from urea containing milk without any specific laboratory instrument.
Description of Method
Method for preparation and use of strip for differentiation of normal milk from urea adulterated milk is described with the help of following example.
Example:
A). Materials required
i). Weighing balance, Make: Mettler Toledo, Modal no. ME204E/A04 ■ ■ ■
ii). Double distilled water
iii). Measuring cylinder, Make: Borosil, Code: 30260106
iv). Urea, Hi media, Product code: GRM3976-500g
v). Bromothymol blue, acid free, Make: CDH, Product code: 812170
vi). Soya flour.
vii). Sodium chloride, Hi media, Product code. GRM853-500g
viii). Centrifuge tube 50ml, make: Abdos, Product code. PI0432
ix). Filter paper, Make: Whatman™, Product code: 1001125
x). Funnel, Make Abdos, Product code: P70314
xi) Rotospin, Make: Tarson, Sr, No. 1501RR098

xii) Syringe filter 0.25m Make:- Axiva Sychem Biotech, Product code:-SFNY13RB
xiii). Disposable syringe 55x25 mm Make1. DISPO VAN
xiv). chromatography Sheet 46x 57 cm, Make:- Whatman, Product code: -1001917 .
xv). Petri plate 90 mm, Make:- Tarson, product code: 460090
Solution A: Sodium chloride solution was prepared by dissolving 0.9g Sodium chloride in 100 ml
water. Then, 3 g soya flour was added into 40 ml of above prepared normal saline solution and mixed
with the help of rotaspin for lh. After lh mixing at rotaspin, solution was filter with whatmann's filter
paper and then filter with 0.2um syringe filter. After filtration, filtrate is stored at 4°C until analysis.
Preparation of Indicator solution: The indicator solution was prepared by mixing solution A and
Dye solution in equal proportion (v/v).
B). Method
Stepwise preparation of Strip for urea detection is given in figure 1 and different steps involve in
preparation are given below.
1. Cut the chromatography sheet into 5* 6 cm pieces.
2. Take 10 ml indicator solution into 90 mm petri plate.
3. Take one piece of chromatographic sheet and dip into petri plate containing indicator solution.
4. After one minute, take out the sheet from indicator solution and dry at 35°C in BOD incubator for 10-15 minutes.
5. After completely drying, the sheet was further cut into pieces to make the strip of sized 0.5 x 6 cm.
6. Prepared strip is slightly yellowish in colour.
7. Now, strip is ready to use, and can be stored at room temperature upto 3 months.
Stepwise method for detection of urea in milk using strip is given in figure 2. Different steps involved
in urea detection using strip are also described below: .
i). Take 2 ml or more than 2 ml milk sample.
ii). Now dip 1/3 part of the above prepared strip into milk sample.

iii). Observe the colour change of strip for 5 minutes.
iv). Presence of light blue/sky blue colour on the strip within 5 minutes indicates the milk contains
urea equal to or greater than 0.5 mg/ml.
v). Absence of light blue/ sky blue colour within indicate urea less than 0.5 mg/ml.
References:
Denis, W., Minot, A.S., (1919) Methods For The Quantitative Determination Of The Non-Protein Nitrogenous Constituents Of Milk., J. Biol. Chem.3 37: 353..
Van Slyke, D. D., Cullen G.E., (1914) The Mode Of Action Of Urease And Of Enzymes In General; J. Biol.Chem., 19:211.
Laurinavicius, V., Razumiene, J., Gureviciene, V., (2013) Bio-electrochemical "conversion of urea on carbon black electrode and application. 13(6), 2208 -22.
Dinesh, V.P., Biji, P., Prasad, Aran K, Tyagi, A.K. (2013) Enhanced ammonia sensing properties using au decorated znonanorods. DOI:10.1109/icsens.2013.6688189 publication year: 2013, page(s): 1-4
Valarmathy R S, Haiitha J., Govrthaman S, Jawaharrajan B, (2018) Detection of urea adulteration in milk using Gas sensor, International Conference on New Horizons in Science Engineering Technology (NHSET-2018), International-Journal of Scientific Research in Computer Science, Engineering and Information Technology 4(5) ISSN : 2456-3307.
P. Singh and N. Gandhi, (2015) Milk preservative and adulterant: processing, regulatory and safety issues, Food reviews International, 31, (3) 236-261..
Meisel H. (1995) Application of fourth derivative spectroscopy to quantitation of whey protein and casein in total milk protein. Milchwissenschaft. 50:247-51. .
Sharma R., Rajput Y. S-, Barui A. K., & N., L. N. (2012) Detection of Adulterants in Milk, A Laboratory Manual. In N. D. R. Institute (Ed.)). Karnal-132001, Haryana, India. 2012 page 20.

Tanzina Azad and Shoeb Ahmed (2016), Common milk adulteration and their detection techniques, International Journal of Food Contamination, 3:22
Jamie L. Luther,3 Valentine Henry de Frahana and Marya Liebemian (2017) "Paper test card for detection of adulterated wXknAnaL Methods, 9,5674r5683.
Hemant Kumar, Ashok Kumar, Poonam Kumari, S. Jyotirmai N.B. Tulsani (2000)" A rapid estimation of ureain adultrated milk using dry reagent strip, Indian journal of Chemical Technology, 7; 146-147.
Shyam Narayan Jha, Pranita Jaiswal, Anjan Borah, Anuj Kumar Gautam, Neha Srivastava (2015) Detection and Quantification of Urea in Milk Using Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy, Food and Bioprocess Technology, 8(4), 926-933.
IndranilBasu, R. Venkata Subramanian, Arun Mathew, Anju Chadha and Enakshi Bhattacharya (2004) Department of Electrical Engineering , Indian Institute of Technology Madras,Chennai 600036, India "Potentiometric Biosensors Based on Silicon and Porous Silicon" , ISBN 0-9728422-7-6 , NSTI-Nanotech 2004V61. 1,2004.
Inaba, A., Yoo, G., Takei, Y., Matsumoto, K., Shimoyama, I., "A graphenefet gas sensor gated by ionic liquid" DOI: 10.1109/memsys.2013.6474408 publication year: 2013: 969 - 972.
Sang Huachun and Han Dandanin, (2009) Test paper for quickly detecting urea in liquid milk and preparation method thereof. Patent application No. CN101900684A.
Jayant Khandare, Debjani Paul, Shashwat Banerjee, Neetika Taneja, Mahesh Shidore (2012) "Rapid detection of urea in adulterated milk using thread based microfluidic system". US20140065712A1,
S. Dixit, http://face-cii.in/sites/default/files/dr_sitaram_dixit_-l.pdf

Claims:

A strip is developed for detection of urea in milk which differentiates pure milk from urea
adulterated milk when strip is immersed into urea adulterated milk sample. There is visible
difference in colour of strip when immersed into milk containing urea as compared to pure . milk within few minutes.
Strip as claimed in claim 1 is a paper strip prepared by immersion into indicator solution is
slightly yellowish in colour.
Indicator solution as claimed in claim 2 is a mixture of two component.
Two component as claimed in claim 3 are soya floor extract and dye
Dye as claimed in claim 4 is pH indicator dye. » , Time period as claimed in claim 1 is 0-6 minutes for milk sample. . Method as claimed in claim 1, can detect minimum 0. 4mg/ml of urea in milk. , The visible colour difference as claimed in claim 1 is slightly yellow in case ,of pure milk
and changes to sky blue in case of urea containing milk. . Colour difference as claimed in claim 1 can be observed with naked eyes. The application developed methods is not restricted to milk sample.