FORM 2 THE PATENTS ACT 1970 (39 of 1970)
&
The Patents Rules, 2003
Provision/Complete Specification
(See section 10 and rule 13)
1, Title of Invention: Rapid Strip based test to detect adulteration in milk.
2. Applicant(s)
(a). NAME: College of Veterinary Science & Animal Husbandry.
(b).NATIONALITY: Indian
(c). ADDRESS: College of Veterinary Science & Animal Husbandry
Nanaji Deshmukh Veterinary Science University,
Jabalpur-482001(MP) India

Milk is one of the best food produced by nature as it contains proteins, carbohydrates, fats, minerals, vitamins etc. It is consumed by one and all including infants, adults and elderly people thereby making it versatile food for all. At present, India stands first with 18.5% (146.3 million tonnes) of milk production across globe and the per capita milk consumption is 322gms/day (GOI, 2016). However, due to poor hygienic conditions, lack of knowledge and lack of stringent food safety regulations, milk adulteration in India remains a common problem faced by people and is of great public health importance. As per Food Safety and Standard Authority of India (2012) investigation, 70% of milk sold in the country is adulterated by adding one or more chemical components and water. Still, there are no rapid tests available that can be used by general public to detect the common adulterants of milk. In the present study, different strips of have been developed for rapid and easy detection of various milk adulterants like starch, detergents, urea, preservatives and neutralizers.
These strips are chemically impregnated with various chemicals like iodine and potassium iodide (starch), bromothymol blue (urea), bromocresol purple (detergents), rosalic acid (neutralizers), turmeric powder and potassium iodide (preservatives). The detection is based on colour development has been described by FSSAI (2015) and Sherikar et. al. (2015) with certain modification. The color development is based on simple principle of diffusion of milk in the solid matrix made up of qualitative filter paper similar to Grade I (100 µm, filter speed medium, fast retention medium - crystalline precipitates smooth surface, normal hardness). The time required for each tests on these strips is very less and handy. These strip based tests don't require any specialised laboratory. The strip based tests can be performed in room temperature by adding as low as 10-20 µl of milk.

Procedure for development of Rapid strips for detection of adulteration in Milk
1. For development of Strip S for detection of starch, qualitative filter paper similar to Grade I was impregnated in solution of iodine - 1.5g & potassium iodide - 3g dissolved in 100ml of water and strip was dried in dark place in dessicator.
2.For development of Strip U for detection of Urea, qualitative filter paper similar to Grade I was impregnated in 0.5% bromothymol blue solution and strip was air dried. 3.For development of Strip D for detection of detergent, qualitative filter paper similar to Grade I was impregnated in 1% bromocresol purple solution and strip was air dried. 4.For development of Strip N for detection of detergent, qualitative filter paper similar to Grade I was impregnated in 1% rosalic acid dissolved in analytical grade ethanol and strip was air dried.
5. For development of Strip B for detection of detergent, qualitative filter paper similar to Grade I was impregnated in the filtrate of turmeric powder dissolved in 10 ml analytical grade ethanol and mixed with 0.7ml hydrochloric acid (35.4% with specific gravity 1.18) and strip was air dried.
6. For development of Strip H for detection of detergent, qualitative filter paper similar to Grade I was impregnated in 20g potassium iodide mixed with 1g of starch powder and strip was air dried.
7. For development of Strip SDS for detection of detergent, qualitative filter paper similar to Grade I was impregnated in 1% bromocresol purple solution and strip was air dried.

The procedure for detection of adulteration with different Strips are described as under-
1. For the detection of Starch with Strip S, the parboiled and precooled 20 µl of milk is poured on strip. The blue color of Strip S immediately and coffee color on drying is an indication of presence/adulteration of starch in milk. The detection limit of strip S is 0.3% of starch adulteration in milk.
2. For the detection of Urea in milk with Strip U, 200 mg of Arhar dal powder is mixed with 1 ml of milk followed by pouring of 20 µl mixed milk with Arhar on Strip U. The change in Strip U color to green in 30sec - 1min will confirm the presence of urea. The detection limit of Strip U is 0.004% of urea adulteration in milk.
3. For the detection of Detergents in milk with Strip D, pour 10- 20 µl of milk on strip D. The change in color of strip D to violet in 30 seconds will indicate presence of detergents. The detection limit of Strip D is 0.3% of detergent adulteration in milk.
4. For the detection of Sodium Bicarbonate in milk with Strip N, pour 10-20 µl of milk on strip N. Rose red color of strip in 30 seconds confirms the presence of sodium bicarbonate. The detection limit of Strip N is 0.15% of sodium bicarbonate adulteration in milk.
5. For the detection of Boric acid in milk with Strip B, pour 10-20 µl of milk on Strip B. Observance of orange color on strip in lmin - 2min indicates presence of boric acid. The detection limit of Strip B is 0.1% of boric acid adulteration in milk.
6. For the detection of Hydrogen peroxide in milk with Strip H, pour 10-20 µl of milk on Strip H. Brown color on strip will confirm the presence in milk. The detection limit of Strip B is 0.4% of hydrogen peroxide adulteration in milk.

7. For the detection of sodium dodecyl sulfate in milk with Strip SDS, pour 10-20 µl of milk on Strip SDS. Brown color on strip will confirm the presence in milk. The detection limit of Strip SDS is 0.75% of sodium dodecyl sulfate adulteration in milk.
Detection of different adulterants can be visualised by change in color of strips as described below-

S.No. Name of Colour of the In case of presence of In case of absence of
adulterants strip adulterants, colour changes to adulterants, colour changes to
1 Starch Brown Blue but changes to coffee colour on drying White (colourless)
2 Urea Golden
yellow Dark green Olive green
3 Detergents Yellow Violet Light blue
4 Sodium Bicarbonate Orange red Rose Red Discoloured
5 Boric acid Yellow Orange No colour change
6 Hydrogen peroxide White Brown coloured White (discoloured)
7 Sodium dodecyl sulfate Yellow Purple/violet Light blue

Claims
Procedure
1. For development of Strip S for detection of starch, qualitative filter paper similar to Grade I was impregnated in solution of iodine - 1.5g & potassium iodide - 3g dissolved in 100 ml of water and strip was dried in dark place in dessicator.
2. For development of Strip II for detection of Urea, qualitative filter paper similar to Grade I was impregnated in 0.5% bromothymol blue solution and strip was air dried.
3. For development of Strip D for detection of detergent, qualitative filter paper similar to Grade I was impregnated in 1% bromocresol purple solution and strip was air dried.
4. For development of Strip N for detection of detergent, qualitative filter paper similar to Grade I was impregnated in 1% rosalic acid dissolved in analytical grade ethanol and strip was air dried.
5. For development of Strip B for detection of detergent, qualitative filter paper similar to Grade I was impregnated in the filtrate of turmeric powder dissolved in 10 ml analytical grade ethanol and mixed with 0.7ml hydrochloric acid (35.4% with specific gravity 1.18) and strip was air dried.
6. For development of Strip H for detection of detergent, qualitative filter paper similar to Grade I was impregnated in 20g potassium iodide mixed with 1g of starch powder and strip was air dried.
7. For development of Strip SDS for detection of detergent, qualitative filter paper similar to Grade I was impregnated in 1% bromocresol purple solution and strip was air dried.
Detection Limit
1. The detection limit of Strip S is 0.3% of starch adulteration in milk.

2. The detection limit of Strip U is 0.004% of urea adulteration in milk.
3. The detection limit of Strip D is 0.3% of detergent adulteration in milk.
4. The detection limit of Strip N is 0.15% of Sodium bicarbonate adulteration in milk.
5. The detection limit of Strip B is 0.1% of Boric acid adulteration in milk.
6. The detection limit of Strip B is 0.4% of Hydrogen peroxide adulteration in milk.
7. The detection limit of Strip SDS is 0.75% of sodium dodecyl sulfate adulteration in milk.