Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to strips for the detection of added urea in milk urea.
2. Description of Related Art
Milk is a white liquid produced by the mammary glands of mammals. It provides the primary source of nutrition for young mammals before they are able to digest other types of food. The early lactation milk is known as colostrum, and carries the mother's antibodies to the baby. Milk can reduce the risk of many diseases in the baby. The exact components of raw milk varies by species, but it contains significant amounts of saturated fat, protein and calcium as well as vitamin C. Cow's milk has a pH ranging from 6.4 to 6.8, making it slightly acidic. The constituents of milk that is most important for detection of adulteration of milk.Milk naturally contains enzymes, vitamins, pigments, salts, sugar, fat, and proteins.

Enzymes. The enzymes of cow's milk are reported as follows by Rogers; proteinases, lactase, diastase, lipase, salolase, catalase, peroxidase, and aldehydrase. Rogers states that the proteolytic enzyme, galactase, brings about slow decomposition of milk proteins into peptones, amino acids, and ammonia.
Vitamins:- All the vitamins recognized at the present time are contained in milk. Some are present in comparatively large and others in smaller amounts.
Pigments:- The appearance of milk is white. This is due to light rays reflected by the colloidally dispersed constituents of the milk, the calcium caseinate, and calcium phosphate.
Milk contains two classes of yellow or orange pigments. The water-soluble pigment, which imparts a yellow color with a green fluorescence to the whey of milk, was formerly called lactochrome. A name recently suggested for this pigment is lactoflavin. It is regarded as one flavin of a specific group, collectively to be called lyochromes. It is possible that lacto-flavin is composed of more than one pigment. Rogers says "lactoflavin forms compounds with saccharides, proteins, and purines (uric acid). These compounds possibly either occur naturally in milk or readily form during isolations, thus accounting for the several lactoflavins isolated from milk." It is probable that the pigment lactoflavin is one of the five fractions of vitamin G (B2). Milk is relatively rich in this vitamin.
A fat-soluble pigment, carotene, found in the fat gives the milk a more or less yellow tinge, which is more pronounced as the fat particles become more concentrated and form cream. The group of pigments called caroti-noids, which include carotene, xanthophyll, and related pigments, has been described in the chapter on fruits and vegetables. The chief pigment of butter fat is the carotene, but little xanthophyll being found. The depth of color depends upon the amount of pigment present. The color of carotene in solution varies from yellow to orange and to a deep red-orange as the concentration increases. The amount of carotene found in the butter fat depends upon the extent of carotene in the food of the cow. Green grasses, hay cured to retain its green color, green corn, and carrots are rich in carotene. The carotene content of milk fat is less rich during the winter months, if

the food of the cow is poor in carotene during this period. Only in cow's milk is the fat extensively pigmented. With the exception of the fat of human milk, which is often pigmented, the fat of the milk of other animals is either devoid of or contains little pigment.
Salts:- Milk contains salts of potassium, sodium, magnesium, calcium, phosphates, chlorides, and citrates. Traces of sulfates and carbonates are found. Iron is present in small amount. Iodides are also found in small amounts, the amount being greater in some localities than in others. Iodides may be easily transmitted from the feed to the milk. Supplee and Bellis have found copper to average about 0.52 part per million in freshly drawn milk. Brickner has reported that milk contains 3.6 to 5.6 parts of zinc per million parts of milk. Manganese in normal milk averages 0.02 to 0.06 parts per million. The greater part of the sulfur is found in the milk proteins. Barger and Coyne state that part of the sulfur in milk is found in the amino acids methionine and cystine of the proteins, but that all of the sulfur is not accounted for.
The salts of milk are found in milk in solution, in the colloidal state, and in combination with the proteins. The exact chemical combinations of the different salts in the milk are not fully determined. For this reason different authorities report different salt combinations. Thus formulas imply definite combination, whereas there is a complex salt equilibrium in milk, which has not been satisfactorily worked out. The citrates, the combinations of which may be trisodium and tripotassium citrate, tricalcium and trimagnesium citrate, are probably entirely in solution. The possible chlorides of potassium, sodium, and calcium are also in solution. Some authorities believe that the phosphates are present chiefly as dicalcium phosphate, CaHP04 others believe that tricalcium phosphate, Ca3(P04)2, is the principal phosphate combination.
The phosphates are partly in solution, with the greater portion in colloidal dispersion. When the particles of dicalcium phosphate are heated they become aggregated and partially precipitated.

Calcium and magnesium are in combination with the casein to form calcium and magnesium caseinates. Zoller states that there may be traces of sodium and potassium caseinates.
Lactose:- The solubility of lactose and its properties may be found in the chapter on sugar. It is caramelized by heat at rather low temperatures.
Fat:- Butter fat is composed of glycerol and fatty acids. Fatty acids of both the saturated and unsaturated series are present. The relative percentage of the unsaturated fatty acids varies with the feed, averaging higher in summer than in winter. Dean and Hilditch state that the oleic and linoleic acids increase by 4 per cent (mols), with a parallel diminution in butyric and stearic acids when cows return to pasture. They also report that with increased age of the cow the unsaturated acids increase at the expense of palmitic acid, which was lowered from 29 to 22-23 per cent by weight of the total fat in the four years of observations made on milk from the same group of cows. The saturated fatty acids are butyric, caproic, caprylic, capric, lauric, myristic, plamitic, and stearic. Arachadonic acid has been reported absent by some investigators. Butter fat contains a higher proportion of the first-named saturated acids than other food fats. The first ones in the series are quite volatile with steam, the volatility decreasing with increase in molecular weight of the acid. Hence, when butter is heated for several minutes, the percentage of the lower saturated fatty acids may be decreased. The unsaturated fatty acids include oleic, linoleic, and arachidic.
While reviewing the each component of milk we come to the conclusion that urea is most widely used as adulterant in milk and have harmful effect on humans. Due to the increasing demand, adulteration of milk is very common. Synthetic milk is prepared by emulsifying vegetable oils with appropriate amount of detergent and urea. Urea has been very most common and notorious adulterant in India. Urea is generally added in the preparation of synthetic milk to raise the SNF value of milk.

Continuous monitoring of urea has been accomplished by a number of electrochemical methods (Goldfinch, M. J.; Lowe, C. R. Anal. Biochem. 138: 430-436 (1983); Luo, S.; Walt, D. R. Anal. Chem. 61: 1069-1072 (1989)). Sensors utilizing such methods are created by immobilizing the enzyme urease onto the surface of an electrode. The enzymatic hydrolysis of urea produces ammonia and carbon dioxide, which are protonated at physiologic pH to form ammonium and carbonate ions, which increase the electrical conductivity of the solution proximal to the electrode.
Urea can also be monitored using an optical sensor. The detection of analytes by optical sensors usually requires the development of fluorescent transducers which are specific for different analytes. Optical transducers have also been coupled to the detection of urea via the urease driven hydrolysis of urea, with the optical transducer modulated by ammonium or ammonia.
There is also a drawback to designing a sensor based on detection of ammonia: namely the rapid protonation of ammonia at physiologic pH. The pKa of ammonium is 9.3, which is not a pH that supports maximum enzyme activity.
Various indicators for urea are known, many urea sensors exhibit problems with interferences from pH and CO2 effects, low sensitivity, slow response times and reversibility. From a manufacturing standpoint, it would therefore be desirable to develop an inexpensive test strips capable of detecting urea that has a high sensitivity, fast response time, and is reversible. It would also be advantageous for the sensor to be able to function in conjunction with sensors detecting other analytes.
Prior Art: - The need for specific and reliable systems for the control of milk
production opens the search for new analytical approaches with biological markers and electronic analyzers that are ever easier and faster to use.

Sadat et. al. 2006 determine the adulteration of natural milk with synthetic milk using ac conductance measurement. Samples of the natural milk with synthetic milk were analyzed by the method of electrical admittance spectroscopy. Measurements at 100 kHz and 8 °C show statisticallysignificant difference between the conductance (G).
Present and available method for the detection of adulterants in milk is composed of an analyzer that utilizes a sophisticated spectrophotometric technology and a series of specific reagents.
Milk Urea Nitrogen (MUN) content is related to the protein content of animal nourishment and is used to define an adequate diet. The chemical test of urea concentration in raw milk may help to detect fraudulent additions done to increase the protein content of milk.
No prior art discloses the detection of urea in the milk by colure test method .the present invention describes the protocol for the detection of urea in the milk in few seconds by the lay men at home .The present invention provides the test strips coated with chemical which is sensitive to the urea at ppm level.
SUMMARY OF THE INVENTION: - The present invention relates to development of simple test strip for the detection of urea in the adulterated milk. The present invention provides a urea sensing strip which forms ammonium ion, carbonic ion and hydroxyl ion in the presence of urea
For detection of urea, we have taken, cellulose paper and coated the paper with silver nitrate, after drying of paper the second layer was coated with potassium chomate . Urea present in the milk reacts with chemical on the on the strip to form ammonium ion, carbonic ion and hydroxyl ion on the strip, which is identified by a colour change on the strip from brown through light yellow with increasing concn. of urea in the milk. Min. detectable levels are 0.1% urea. These tests are recommended as standard platform tests for detection of added urea.

Synthetic milk is not milk but it is entirely a different component with a high degree of adulteration to increase the volume of milk and thereby the profit. Generally it is a mixture of water, pulverized detergent or soap, sodium hydroxide, vegetable oil, salt and urea. The simplicity and rapidity with which milk can be adulterated always tempted the unscrupulous milk vendors to indulge in fraudulent practices and adulterate the milk. The ever-rising greed has given way to the development of a new type of adulterated milk known as synthetic milk, solution of any one of the substances above mentioned acts as a source of solids not fat (SNF). The ingredients that go in to the making of synthetic milk are calculated in such a way that the fat and SNF percentage is similar to mixed milk. Hence it easily passes the platform tests carried out at the village level dairy cooperative society (fat and lactometer reading etc.) but from the health point of view of the consumers, it is highly dangerous. The taste is highly objectionable.
Similar to genuine milk production, the practice of preparing the synthetic milk too starts at the village level. The places notorious for the production of synthetic milk include parts of Rajasthan, Haryana, and Uttar Pradesh in India. Slowly but steadily the practice is spreading to other parts of India.
Process of synthetic milk production
Vegetable refined oil (any brand) whose butyrorefractometer reading is less than 42 is taken in a wide mouthed container along with a suitable emulsifier and thoroughly mixed so that the entire content is made in to a thick white paste. After this is achieved, water is slowly added to the paste until the density of the liquid is similar to that of milk. Then it is added with urea or sodium sulphate or glucose or maltose or sometimes any one of the commonly available fertilizers is added. These substances are usually dissolved in hot water and then added to the seemingly milk like solution. The refined oil in synthetic milk acts as a source of fat where as the hot

Common comparison between genuine and synthetic milk
(Table Removed)
EXAMPLE 1
A piece of cellulose paper was dipped in 0.1M solution of silver nitrate .Silver nitrate
is an inorganic compound with chemical formula AgNO3. This compound is a versatile precursor to many other silver compounds, such as those used in photography. It is far less sensitive to light than the halides. It was once called lunar caustic because silver was called luna by the ancient alchemists. After drying of strips we coated potassium chromate 5% w/w solution on the strips. The strips become colored due to the complex formation. Different concentration of silver nitrate and potassium chromate solution was used on the strips. The procedure for strip development is as follows.
(Equation Removed)
EXAMPLE 2
Urea or carbamide is an organic compound with the chemical formula (NH2)CO. The molecule has two amine (-NH2) groups joined by a carbonyl (C=O) functional group.
A strip was prepared as in Example 1, when one drop of urea solution dip onto the strip the color of the strip become colorless hence the strip showed response to urea.
(NH2)CO + strip → white precipetate

(Figure Removed)
FIG. 1 shows the color change in various concentrations of urea .fig.l shows that drop of various concentration of urea solution change the color from the strips.

Claims
What is claimed is:
1. A urea test strips material comprising:
a first layer comprising a sensitive chemical silver nitrate wherein it can react quantitatively with urea
a second layer comprising potassium carbonate ,a first layer was coated with second layer contacting potassium carbonate.
2. The sensor material of claim 1 wherein the added urea present in the milk reacts with chemical on the on the strip to form ammonium ion, carbonic ion and hydroxyl ion on the strip, which is identified by a color change on the strip from brown through light yellow with increasing concn. of urea in the milk.
3. The strips material of claim 2 wherein the Min. detectable levels are 0.1% urea. These tests are recommended as standard platform tests for detection of added urea.
4. A process for preparing a urea sensing strips material comprising:
forming a first layer of the first mixture on a substrate;
combining potassium carbonate and a second polymer to form a second mixture;
forming a second layer of the second mixture on the first layer; and
forming a third layer of a third polymer on the second layer.