DESC:FORM-2
THE PATENTS ACT, 1970
&
THE PATENTS RULES, 2003

COMPLETE SPECIFICATION

RAPID TESTS FOR QUALITATIVE &SEMI-QUANTITATIVE DETECTION OF ADULTERANTS IN MILK

NAGAPPA KARABASANAVAR,
PRASHANTH S. BAGALKOTE,
SANTOSH A. SAJJAN,
DHOOLAPPA MELINAMANI
and R.V. PRASAD

DEPARTMENT OF VETERINARY PUBLIC HEALTH & EPIDEMIOLOGY
VETERINARY COLLEGE, VINOBANAGAR, P.B. 53,
SHIVAMOGGA- 577 204, KARNATAKA, INDIA

The following specification particularly describes and ascertains the nature of this invention and the manner in which it is to be performed
FIELD OF INVENTION
The invention relates to therapid test kits and method for qualitative & semi-quantitative detection of adulterants in milk. It particularly relates to thedevelopment of paper based strips as test kits for qualitative & semi-quantitative detection of adulterants in milk. It more specifically relates tothe development of efficient dry paper based strips as test kits for qualitative & semi-quantitative detection of five major adulterants such as starch, soap/ detergent, neutralizers, hydrogen peroxide and urea in milk. It also relates to the development of a simple method for qualitative & semi-quantitative detection of five major adulterants such as starch, soap/ detergent, neutralizers, hydrogen peroxide and urea in milk by dry paper based strips as test kits.
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BACKGROUND OF THE INVENTION

Milk in its natural form is a unique food for the nourishment of human being from long back as it supplies nutrients like proteins, fat, carbohydrates, vitamins and minerals in moderate amounts in an easily digestible form than any other single food (Neumann et al., 2002, Nutr. Res., Vol. 22, pp. 193-220). It has all the substances required by the humans in its easiest assimilable form. Therefore, due to its nutritive value, milk is recommended to the young and old people alike (Kandapalet al., 2012, Indian Journal of Community Health, Vol. 24, No. 3, pp. 188-192). However, the quality of milk gets deteriorated due to its adulteration occurring at different marketing channels. Adulteration of milk is usually done by the addition of inferior and cheaper materials (elements) like the water, cane sugar, powdered milk, etc (Prasad et al., 1999,Principles and Practices of Animal Nutrition,1st Edition, Kalyani Publishers, New Delhi) and several of such reports of adulteration have been reported in dairy products in many parts of the India (Sharma, 1996, The Hindustan Times, Vol. 4, July 6, Newspaper from Delhi; and Choudhary, 1997,Navbharat Times, Vol. 3, September 28, News Paper from Delhi). Milk is adulterated by the addition of substances such as urea, fat, sugar, neutralizers, salt, hydrogen peroxide, etc. so as to maintain the desired viscosity and the specific gravity of the milk (Varley, 1969, Practical Clinical Biochemistry, 4th Edition, CBS Publishers, Delhi). Detergents in particular are added to emulsify and dissolve the oil in water so as to impart the frothy (foamy) appearance; likewise, for the characteristic white color of milk (Centre for Science and Environment, 2006, FSSAI Watch, Available from www.cseindia.org/category/thesarus/national-survey-milk-adulteration, 2011). Urea or sugar is added to mask the decreased SNF (solid-not-fat) content, to provide whiteness to the milk and to improve the milk taste. However, presence of the urea in milk overburdens kidneys as they have to filter out more urea content from the body. Caustic soda is added to the blended milk to neutralize the acidity, thereby preventing it from turning sour during the transportation (Hemanth et al., 2000, Indian Journal of Chemical Technology, Vol. 7, pp. 146-147 and Kandapal et al., 2012, Indian Journal of Community Health, Vol. 24, No. 3, pp. 188-192). Starch is also used as an adulterant into the milk; nevertheless, high amounts of starch could lead to diarrhea since it is indigestible in colon. Accumulation of starch in the body may also prove detrimental for diabetic patients. Hydrogen peroxide is added to milk to prolong its freshness, but peroxides damages the gastrointestinal cells which can lead to gastritis and inflammation of the intestine (Singuluri and Sukumaran, 2014, Journal of Chromatography Separation Technique, Vol. 5, No. 1, p. 212). Such adulterated milk is harmful for all, but is more dangerous for pregnant women, fetus and persons who are already having heart and kidney problems.
In recent years, with the advances in technology and the living standards of people, there has been a significant increase in the consumption of milk. However, it is most common phenomenon in the dairy industry to include adulterants either for cost-cutting purposes or for increasing the shelf-life of dairy products. Inclusion of such adulterants lowers the quality of milk, reduces its nutritional value, flavor, and may have several deleterious or harmful effects on human health. Previously, the Government of India promulgated the Prevention of Food Adulteration Act in 1954 to safeguard the public health by the adulteration of food. The Food Safety and Standards Act-2006 (FSSA-2006) has now taken the responsibility of food quality and safety since 2011. It prohibits the manufacture, sale and distribution of not only adulterated foods but also foods contaminated with toxicants. Despite food legislation, adulteration remains uncontrolled; furthermore, legal steps laid down in the Act are extremely difficult to maintain due to inadequate and untrained man power and laboratory facilities (Monika and Gupta, 2008, Recent Trends in Detection of Adulteration of Milk and Dairy Products, 8th Edition, pp. 43-52, Dairy Book, Varanasi).
In the prior art there are reports for the detection of adulterants in the milk, the interested ones include Indian patent application no. 744/MUM/2014 (WO2015132648 A2) which discloses a kit to detect at least one adulterant in milk selected from a group of cane sugar, hydrogen peroxide, malt dextrin, glucose, ammonia, urea, nitrates, sulfates, detergents, neutralizers, starch, and formalin. Also discloses a rapid detection method for identifying milk adulterant.
Mohit et al., 2014 (National Conference on Synergetic Trends in engineering and Technology – STET-2014, International Journal of Engineering and Technical Research ISSN: 2321-0869, Special Issue) disclose methods of detection and remedial measures for milk adulteration by various adulterants.
Hemanth et al., 2000 (Indian Journal of Chemical Technology, Vol. 7, pp. 146-147) reported rapid estimation of urea in adulterated milk using dry reagent strip.
CN 101398388 A discloses a preparation method of starch detection test paper in milk and milk solution and a detection method of starch; firstly, water solution containing alkali, iodide and oxidant is prepared, sufficiently dissolved and uniformly mixed, thus forming a stable test paper iodine original solution; the test paper iodine original solution is uniformly fixed on a carrier by the forms of coating, printing or soaking and dried so as to obtain the detection test paper. The liquid to be detected together with the developer is dripped on the test paper; if the liquid to be detected contains matters such as dextrin, starch and the like, the colour of the starch detection test paper changes to amaranth or navy blue, otherwise, the colour of the starch detection test paper changes to alabaster to colourless; the qualitative or semi-quantitative detection of matters such as starch and the like can be carried out by matching with a colorimetric card for use.
Indian patent application no. 1970/DEL/2006 discloses a kit for detection of detergent in milk mainly with synthetic milk.
Indian patent application no. 3677/DEL/2011 discloses a rapid and sensitive test kit and assay method for the qualitative and quantitative detection of an adulterant mainly anionic detergent in milk.
Indian patent application no. 3472/DEL/2013 discloses a strip for the detection of added urea in milk and method for its use in detecting the added urea in milk.
CN 101329280 A relates to a detection method, in particular to a quick detection method and a detection kit for residual hydrogen peroxide in milk, belonging to the field of chemical composition detection technique. The quick detection method for detecting the residual hydrogen peroxide in the milk comprises the steps as follows: quantitative potassium iodide and amylum are added into the milk sample; according to the colour change of the milk sample, whether residual hydrogen peroxide exists in the milk sample is judged and the content of the hydrogen peroxide is measured.
CN 2033904 U discloses an on-site quick test device for the adulterated milk, comprising of a box, a box cover, a fresh milk sampler, a combined test tube, a meter assembly, a combined reagent bottle, indicator paper, an alcohol lamp, a dropper bottle and tablets. The test device is applied for testing adulterated substance on site during the fresh milk purchasing.
Ramya et al., 2015 (Int. J. Agric.Sc& Vet.Med., Vol. 3, No. 1, February 2015) reported the detection of adulterants in retail milk samples procured in Proddatur town, YSR Kadapa (Dt), Andhra Pradesh.
Though there are prior arts in the literature that reported various kits and methods for the detection of a variety of adulterants in the milk, yet there exists drawbacks in the prior art for the rapid and efficient kit to detect the different adulterants present in milk. Moreover, none of the prior art discloses or teaches paper based rapid and efficient kit along with a method to qualitative & semi-quantitative detection of adulterants in milk. Therefore, there exists a need to develop a paper based rapid and efficient kit for the qualitative & semi-quantitative detection of adulterants present in the milk in an efficient manner. Thus, the present inventors aim is to overcome the drawbacks of the prior art by developing a simple, rapid and efficient paper based kit along with a method of qualitative & semi-quantitative detection of five major adulterants such as starch, soap/ detergent, neutralizers, hydrogen peroxide and urea in the milk (market milk).

OBJECTS OF THE INVENTION

The primary object of the present invention is the development of rapid test kits for qualitative & semi-quantitative detection of adulterants in milk.

The other object of the present invention is the development of paper based strips as test kits for qualitative & semi-quantitative detection of adulterants in milk.

The other object of the present invention is for the development of efficient paper based strips as test kits for qualitative & semi-quantitative detection of five major adulterants such as starch, soap/ detergent, neutralizers, hydrogen peroxide and urea in the milk.

Another object of the present invention is for a simple method for the qualitative & semi-quantitative detection of adulterants in milk by using paper based strips as test kits.

Another object of the present invention is for a simple method for the qualitative & semi-quantitative detection of five major adulterants such as starch, soap/ detergent, neutralizers, hydrogen peroxide and urea in milk in milk by using paper based strips as test kits.

Yet another object of the present invention is for the development of rapid paper based strips as test kits for qualitative & semi-quantitative detection of adulterants in milk which require little technical expertise and simple to interpret results.

Yet another object of the present invention is for the development of rapid paper based strips as test kits for qualitative & semi-quantitative detection of adulterants in milk which are eco-friendly and cost-effective.

Yet another object of the present invention is for the development of dry paper based strips as test kits for qualitative & semi-quantitative detection of adulterants in milk which replace the existing wet chemistries and have field applications to minimize the menace of milk adulteration which is illegal as per the law.

STATEMENT OF THE INVENTION
A rapid milk-adulteration detection test kit comprising one or more test paper strips is independently treated with reagents for detection of one or more milk adulterants selected from a group consisting of starch, soap/ detergent, neutralizers, hydrogen peroxide and urea. In one embodiment, the test paper strip is treated with iodine reagent for detection of starch.
In another embodiment, test paper strip is treated with phenolphthalein reagent for detection of soap/ detergent.
In another embodiment, the test strip is treated with rosalic acid reagent for detection of neutralizers.
In another embodiment, the test paper strip is treated with iodine-methylene blue reagent for detection of hydrogen peroxide.
In another embodiment, the test paper strip is treated with p-dimethyl amino benzaldehyde reagent for detection of urea.
A method for qualitative & semi-quantitative detection of adulterants in milk involves contact of milk sample with the detection test kit as claimed in any one of the preceding claims, drying the test paper strip and observing change in colour of the test strip and comparison of the developed colour with the colour comparator for the quantification of adulterant.
In one embodiment, the detection test kit is contacted with milk sample by comparing the developed colour with colour comparator as in figure 3 for detection and quantification of the starch.
In another embodiment, the detection test kit is contacted with milk sample by comparing the developed colour with colour comparator as in figure 4 for the detection and quantification of soap/ detergent.
In another embodiment, the detection test kit is contacted with milk sample by comparing the developed colour with colour comparator as in figure 5 for detection and quantification of neutralizers.
In another embodiment, the detection test kit is contacted with milk sample by comparing the developed colour with colour comparator as in figure 6 for detection and quantification of hydrogen peroxide.
In another embodiment, the detection test kit is contacted with milk sample by comparing the developed colour with colour comparator as in figure 7 for the detection and quantification of urea.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to the rapid test kits and method for qualitative & quantitative detection of adulterants in milk. It particularly relates to the development of paper based strips as test kits for qualitative & quantitative detection of adulterants in milk. It more specifically relates to the development of efficient dry paper based strips as test kits for qualitative & quantitative detection of five major adulterants such as starch, soap/ detergent, neutralizers, hydrogen peroxide and urea in milk. It also relates to the development of a simple method for qualitative & quantitative detection of five major adulterants such as starch, soap/ detergent, neutralizers, hydrogen peroxide and urea in milk by dry paper based strips as test kits.

The following examples are given by the way of illustration of the present invention and should not be construed to limit the scope of the present disclosure. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are intended to provide further explanation of the claimed subject matter.
The disclosure is further illustrated by the following examples which in no way should be construed as being further limiting. One skilled in the art will readily appreciate that the specific methods and results described are merely illustrative.

Preparation of reagents for detection of milk adulterants

1) Preparation of Phenolphthalein Reagent for detection of Soap/ Detergent in milk
• Ethanol 10 mL
• Phenolphthalein 0.1 g

Colour change
• Positive : Pink
• Negative : White

2) Preparation of Rosalic Acid Reagent for detection of Neutralizers in milk
• Ethanol 10 mL
• Rosalic acid 0.1 g

Colour change
• Positive : Pink
• Negative : White

3) Preparation of Iodine Reagent for detection of Starch in milk
• Potassium Iodide (KI) : 1.6 g dissolve in 10 mL of water
• Iodine : 1 g dissolve in above 10 mL KI solution
• Make volume to 100 mL by adding 90 mL of water

Colour change
• Positive : Blue
• Negative : Reddish brown
4) Preparation of Iodine-Methylene blue Reagent for detection of Hydrogen Peroxide in milk
• Starch : 1 g
• Iodine : 0.1 g
• Methylene blue : 0.1g
• Ethanol : 100 mL

Colour change
• Positive : Brown
• Negative : Blue

5) Preparation of Para Di-Methyl Amino Benzaldehyde (DMAB) Reagent for detection of Urea in milk
• DMAB : 1 g
• HCl : 2 N
• Ethanol : 10 mL

Colour change
• Positive : Yellow
• Negative : No colour change
Prepare reagents as described above, dry overnight in cool place in a Petri dish and pack into a labeled cover.

Paper strips were developed for the detection of adulterants in milk namely
1. Starch
2. Powdered soap/ Detergent
3. Neutralizers
4. Hydrogen peroxide
5. Urea
Developed paper strips were evaluated for performance under field conditions. Kits were validated at different laboratories for performance.
Paper strip kits can detect 5 types of milk adulterants
• Using this colour comparator (either one) anyone could say whether mentioned adulterants are present in milk or not.
• Qualitative detection i.e. ‘YES’ or ‘NO’

Example 1: Quantitative detection of STARCH adulteration in milk
• Take milk sample to be tested into a receptacle.
• Boil the milk for 10 minutes.
• Cool to room temperature.
• Takeout one starch strip and pour milk over the strip.
• Look for development of colour.
• By matching the developed colour with colour comparator (Figure 3) you will come to know approximate level of starch adulteration in milk.

Limit of detection (LOD) = 0.03%
• Blue Colour: + ve
• Iodine colour: -ve

Example 2: Quantitative detection of SOAP/ DETERGENT adulteration in milk
• Take milk sample to be tested into a receptacle.
• Takeout one paper strip and pour milk over the strip.
• Look for development of colour.
• By matching the developed colour with colour comparator (Figure 4) you will come to know approximate level of adulteration in milk.

Limit of detection (LOD) = 0.12%
• Pink Colour: + ve
• Colorless: -ve

Example 3: Quantitative detection of NEUTRALIZER(S) adulteration in milk
• Take milk sample to be tested into a receptacle.
• Takeout one paper strip and pour milk over the strip.
• Look for development of colour.
• By matching the developed colour with colour comparator (Figure 5) you will come to know approximate level of adulteration in milk i.e. KOH/ NaOH/ Sod. Carbonate/ Sod. Bicarbonate/ Detergent.

Limit of detection (LOD) = 0.06%
• Pink/ red Colour: + ve
• No Color change/ rose colour : -ve

Example 4: Quantitative detection of HYDROGEN PEROXIDE adulteration in milk
• Take milk sample to be tested into a receptacle.
• Takeout one paper strip and pour milk over the strip.
• Look for development of colour.
• By matching the developed colour with colour comparator (Figure 6) you will come to know approximate level of adulteration in milk.
Limit of detection (LOD) = 0.01%
• Brown Colour: + ve
• Blue Color : -ve

Example 5: Quantitative detection of UREA adulteration in milk
• Take milk sample to be tested into a receptacle.
• Takeout one paper strip and pour milk over the strip.
• Look for development of colour.
• By matching the developed colour with colour comparator (Figure 7) you will come to know approximate level of adulteration in milk.

Limit of detection (LOD) = 0.03%
• Deep Yellow Colour: + ve
• Faint yellow Color : -ve

Applications of rapid tests developed for the detection of adulterants in milk
1) Starch
2) Powdered soap / detergent
3) Neutralizers (Carbonates & Bicarbonates)
4) Hydrogen peroxide
5) Urea
Developed tests were validated for performance.

Novelty and applications/ advantages of the kits and method
• The test kits and methods can be used by anyone as even illiterate persons can be trained to use these test kits.
• The results after tests are easy and simple to interpret.
• The test kits and methods produce rapid/ instantaneous results.
• Dry paper based test kits and methods replace existing wet chemistries.
• The developed test kits and methods have field applications to minimize the menace of milk adulteration which is illegal as per the law.
BRIEF DESCRIPTION OF FIGURES

Figure 1: Paper strip kits which can detect 5 types of milk adulterants using this colour comparator (either one) anyone could say whether mentioned adulterants are present in milk or not.

Figure 2: Qualitative detection i.e. ‘YES’ or ‘NO’ of 5 types of milk adulterants.

Figure 3: Colour comparator for quantitative detection of STARCH adulteration in milk.

Figure 4: Colour comparator for quantitative detection of SOAP/ DETERGENT adulteration in milk.

Figure 5: Colour comparator for quantitative detection of NEUTRALIZERS adulteration in milk.

Figure 6: Colour comparator for quantitative detection of HYDROGEN PEROXIDE adulteration in milk.

Figure 7: Colour comparator for quantitative detection of UREA adulteration in milk.
Application no. 201641012071 Sheet 1 of 7
NAGAPPA KARABASANAVAR, PRASHANTH S. BAGALKOTE, SANTOSH A. SAJJAN, DHOOLAPPA MELINAMANI and R.V. PRASAD

Figure 1

Dated this 17th day of March 2017 SHARANABASAVA
PATENT AGENT
FOR APPLICANT(S)

Application no. 201641012071 Sheet 2 of 7
NAGAPPA KARABASANAVAR, PRASHANTH S. BAGALKOTE, SANTOSH A. SAJJAN, DHOOLAPPA MELINAMANI and R.V. PRASAD

Figure 2

Dated this 17th day of March 2017
SHARANABASAVA
PATENT AGENT
FOR APPLICANT(S)

Application no. 201641012071 Sheet 3 of 7
NAGAPPA KARABASANAVAR, PRASHANTH S. BAGALKOTE, SANTOSH A. SAJJAN, DHOOLAPPA MELINAMANI and R.V. PRASAD

Figure 3

Dated this 17th day of March 2017
SHARANABASAVA
PATENT AGENT
FOR APPLICANT(S)

Application no. 201641012071 Sheet 4 of 7
NAGAPPA KARABASANAVAR, PRASHANTH S. BAGALKOTE, SANTOSH A. SAJJAN, DHOOLAPPA MELINAMANI and R.V. PRASAD

Figure 4

Dated this 17th day of March 2017
SHARANABASAVA
PATENT AGENT
FOR APPLICANT(S)

Application no. 201641012071 Sheet 5 of 7
NAGAPPA KARABASANAVAR, PRASHANTH S. BAGALKOTE, SANTOSH A. SAJJAN, DHOOLAPPA MELINAMANI and R.V. PRASAD

Figure 5

Dated this 17th day of March 2017
SHARANABASAVA
PATENT AGENT
FOR APPLICANT(S)

Application no. 201641012071 Sheet 6 of 7
NAGAPPA KARABASANAVAR, PRASHANTH S. BAGALKOTE, SANTOSH A. SAJJAN, DHOOLAPPA MELINAMANI and R.V. PRASAD

Figure 6

Dated this 17th day of March 2017 SHARANABASAVA
PATENT AGENT
FOR APPLICANT(S)

Application no. 201641012071 Sheet 7 of 7
NAGAPPA KARABASANAVAR, PRASHANTH S. BAGALKOTE, SANTOSH A. SAJJAN, DHOOLAPPA MELINAMANI and R.V. PRASAD

Figure 7

Dated this 17th day of March 2017
SHARANABASAVA
PATENT AGENT
FOR APPLICANT(S)
,CLAIMS:We Claim,
1. A rapid milk-adulteration detection test kit comprising one or more test paper strips is independently treated with reagents for detection of one or more milk adulterants selected from a group consisting of starch, soap/ detergent, neutralizers, hydrogen peroxide and urea.
2. Milk-adulteration detection test kit as claimed in claim 1 wherein the test paper strip is treated with iodine reagent for detection of starch.
3. Milk-adulteration detection test kit as claimed in claim 1 wherein the test paper strip is treated with phenolphthalein reagent for detection of soap/ detergent.
4. Milk-adulteration detection test kit as claimed in claim 1 wherein the test strip is treated with rosalic acid reagent for detection of neutralizers.
5. Milk-adulteration detection test kit as claimed in claim 1 wherein the test paper strip is treated with iodine-methylene blue reagent for detection of hydrogen peroxide.
6. Milk-adulteration detection test kit as claimed in claim 1 wherein the test paper strip is treated with p-dimethyl amino benzaldehyde reagent for detection of urea.
7. A method for qualitative & semi-quantitative detection of adulterants in milk comprising contacting milk sample with a detection test kit as claimed in any one of the preceding claims, drying the test paper strip and observing change in colour of the test strip and compare the developed colour with colour comparator for quantification of adulterant.
8. Method as claimed in claim 7 wherein the detection test kit as claimed in claim 2 is contacted with milk sample by comparing the developed colour with colour comparator as in figure 3 for detection and quantification of starch.

9. Method as claimed in claim 7 wherein the detection test kit as claimed in claim 3 is contacted with milk sample by comparing the developed colour with colour comparator as in figure 4 for detection and quantification of soap/ detergent.
10. Method as claimed in claim 7 wherein the detection test kit as claimed in claim 4 is contacted with milk sample by comparing the developed colour with colour comparator as in figure 5 for detection and quantification of neutralizers.
11. Method as claimed in claim 7 wherein the detection test kit as claimed in claim 5 is contacted with milk sample by comparing the developed colour with colour comparator as in figure 6 for detection and quantification of hydrogen peroxide.
12. Method as claimed in claim 7 wherein the detection test kit as claimed in claim 5 is contacted with milk sample by comparing the developed colour with colour comparator as in figure 7 for detection and quantification of urea.

Dated this 17th day of March 2017 SHARANABASAVA
PATENT AGENT
FOR APPLICANT(S)