DESC:Priority Claim:
[0001] The application claims priority from the provisional specification numbered 202211015324 filed with the Indian Patent Office, Delhi on 21st March 2022 entitled “A paper-based rapid test card for the detection of adulterants in milk”, the entirety of which is expressly incorporated herein by reference.
Preamble to the description:
[0002] The following specification describes the invention and the manner in which it is to be performed:
DESCRIPTION OF THE INVENTION
Technical field of the invention
[0003] The present invention relates to a paper-based rapid test card for the detection of adulterants in milk. More particularly, the paper-based rapid test card is capable of simultaneously detecting and multiple adulterants along with the concentration.
Background of the invention
[0004] Milk is a wholesome food with good source of proteins, fats, carbohydrates, vitamins, and minerals. Milk is easily digested and quickly absorbed. Milk being a good source of protein contains many essential amino acids which is required for appropriate growth of individuals. Milk is consumed by people of all age groups.
[0005] The adulteration of milk is due to the increasing demand worldwide, supply gap, perishable nature of milk, lack of adequate testing and to increase profit. Milk is frequently adulterated with water, melamine, sugar, urea, starch etc. The reason behind adulteration of milk is to mimic the colour, consistency, and nitrogen content. Adulterated milk is considered as a food risk.
[0006] The addition of adulterants such as urea, formalin, detergents, ammonium sulphate, boric acid, caustic soda, salicylic acid, benzoic acid, hydrogen peroxide, melamine has a catastrophic effect on health.
[0007] Milk is adulterated in order to enhance the parameters such as fat percentage, SNF (Solid-not-Fat), protein content, shelf life etc. The addition of cane sugar, sulphate salts, urea is to increase the SNF content, Urea is added to increase the non-protein nitrogen content, ammonium sulphate is added to increase the lactometer reading and formalin, salicylic acid, benzoic acid and hydrogen peroxide are added as preservatives. The milk fat has high commercial value and hence is removed from the milk and replaced by adding non-milk fat like vegetable oil. The detergents are added as emulsifiers to dissolve the oil and give a frothy appearance.
[0008] The consumption of adulterated milk in long run impacts health severely. The ingestion of formalin and melamine above safety limit causes renal failure. Gastro-intestinal problems such as gastritis, inflammation is caused by consumption of peroxides and detergents. The carbonates and bicarbonates disrupt the hormonal signalling. The excessive starch in the adulterated milk causes diarrhoea and accumulation of starch can be fatal for diabetic patients.
[0009] The Patent Application No. IN201641012071 entitled “Rapid tests for qualitative & semi-quantitative detection of adulterants in milk” discloses paper-based test strips as rapid test kits and method for qualitative and quantitative detection of adulterants in milk. The rapid test kits detect starch with iodine as reagent, soap, and detergents with phenolphthalein as reagent, neutralizers with rosalic acid as detergent, hydrogen peroxide with iodine-methylene blue as reagent and urea with p-dimethyl amino benzaldehyde as reagent. The method includes 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 for all the adulterants.
[0010] The Patent Application No. WO2015IB00276 entitled “Kit to detect milk adulteration” discloses a kit and a rapid method to detect milk adulteration. The adulterants comprising cane sugar, hydrogen peroxide, malt dextrin, glucose, ammonia, urea, nitrates, sulphates, detergents, neutralizers, starch, and formalin. The kit comprises test strips which are independently with reagents for the detection of adulterants. The test strip can be coated with reagents such as a mixture of reagents citric acid, citric hydroxide, glucose oxidase, haemoglobin and o-toridine where off-white to blue colour change indicates presence of glucose; a mixture of haemoglobin and o-toridine dissolved in phosphate buffer where off-white to navy blue colour change indicates the presence of hydrogen peroxide; a mixture of reagents citric acid, citric hydroxide, glucose oxidase, haemoglobin, o-toridine and glucoamylase where off-white to navy blue color change indicates presence of cane sugar; a mixture of reagents citric acid, citric hydroxide, glucose oxidase, haemoglobin, o-toridine and alpha glucosidase where off-white to navy blue color change indicates presence of malt dextrin, a mixture of urease and bromothymol blue dissolved in buffer where orange to navy blue color change indicates presence of urea; dimethylsulfide dissolved in water where dark blue to pink color change indicates the presence of sulphate, rosalic acid dissolved in water where orange to pink color change indicates the presence of neutralizer; acidified Schiff s reagent where white to pink color change indicates the presence of formalin; crystal violet reagent dissolved in water, the crystal violet reagent solution is optionally acidified where white to purple color change indicates the presence of detergents; a mixture of iodine and potassium iodide dissolved in water where white to blue color change indicates the presence of starch; a three layered sandwiched test strip, having first layer coated with alkaline solution where navy blue color indicates the presence of ammonia; third layer coated with bromothymol solution and placing intermediate layer a blank LH-004 gas filter paper; and a zinc powder test strip, GR reagent and 1M-HCl where white to red purple color change indicates the presence of nitrates.
[0011] The Patent Application No. IN201821008795 entitled “Rapid strip based test to detect adulteration in milk” discloses a rapid method to detect common adulterants in milk. For the detection of iodine, solution of iodine 1.5g & potassium iodide 3g dissolved in 100 ml of water and strip was dried in a dark place in the desiccator. For the detection of urea, 0.5% bromothymol blue solution and strip was air dried. For the detection of detergents, 1% bromocresol purple solution and strip was air dried. Starch has a detection limit of 0.3%, urea-0.004%, Detergent-0.3% and sodium bicarbonate 0.15% as an adulterant in milk.
[0012] The Patent Application No. CN2011190532 entitled “Test paper slip for testing adulterated milk rapidly” discloses a test paper slip for rapid detection of four different adulterants simultaneously. The test paper slip detects starch using 10 ml of an iodine-potassium iodide solution with an I2 concentration of 0.032 mol/mL and a KI concentration of 0.126 mol/mL, diluting it with equal amount of a solvent, immersing completely quantitative test paper into the diluted iodine-potassium iodide solution for 5 minutes, airing the test paper at a temperature of 5 to 60 C in darkness, clipping, and preserving it in a dark place; carbamide by taking 6 ml of a sodium nitrite hydrochloric acid solution with a sodium nitrite content of 0.04%, adding 0.4 g of a Griess reagent into the sodium nitrite hydrochloric acid solution, mixing well, immersing completely quantitative test paper into the mixture for 10 minutes, taking out the immersed test paper, airing it in the shade, clipping, and preserving it in a dark place; an edible alkali by taking 10 ml of a prepared Bromothymol Blue ethanol indicator with a Bromothymol Blue content of 0.04%, placing the indicator on a disk, immersing completely quantitative test paper into the indicator for 10 to 15 minutes, taking out it, airing it, clipping, and preserving it in a dry place; and a salt test by mixing 15 ml of a silver nitrate solution with a silver nitrate concentration of 0.01 mol/mL and six drops of a potassium chromate solution with a potassium chromate content of 10% quickly, immersing completely quantitative test paper into the mixture for about 20 minutes, taking out it, airing the immersed test paper, clipping, and preserving it in a dry place. The test paper slip has high sensitivity and accuracy.
[0013] Although, various rapid tests for the detection of adulterants in milk exist, the concentration of adulterant is not determined. Milk is consumed by millions of people every day. The rate of adulteration of milk is increasing and the testing facilities are limited. The detection of various adulterants along with concentration rapidly is crucial.
Summary of the invention
[0014] The present invention discloses a paper-based rapid test card which overcomes the drawbacks of existing rapid test kits. The paper-based rapid test card detects several adulterants in the milk sample simultaneously. The paper-based rapid test card is accurate, cost effective and user friendly.
[0015] The paper-based rapid test card comprises four layers including a base structure, a holding structure, a chemical layer comprising paper or blotting paper or absorbent paper and a coating layer comprising whitening agent and sodium alginate. The chemical layer comprises reagents to detect the presence of adulterants in milk.
[0016] The paper-based rapid test card detects adulterants including detergent, urea, starch, neutralizer, preservatives, and salt. The present invention discloses paper-based rapid test card to detect single adulterant, 3 adulterant and 6 adulterants simultaneously.
[0017] The adulterant in milk is detected upon the change in colour of the paper-based rapid test card by the addition of adulterated milk. The degree of change in colour corresponds to the concentration of adulterant and can be determined by the scale provided on the paper-based rapid test card.
[0018] The present invention discloses a paper-based rapid test card for the detection of adulterants. The paper-based rapid test card detects single adulterant as well as multiple adulterants simultaneously. The paper-based rapid test card is accurate, economical, user-friendly and biodegradable in nature.
Brief description of the drawings
[0019] The foregoing and other features of embodiments will become more apparent from the following detailed description of embodiments when read in conjunction with the accompanying drawings.
[0020] FIG 1 illustrates the paper-based rapid test card for the detection of detergent as an adulterant in milk
[0021] FIG 2 illustrates the paper-based rapid test card for the detection of urea as an adulterant in milk
[0022] FIG 3 illustrates the paper-based rapid test card for the detection of starch as an adulterant in milk.
[0023] FIG 4 illustrates the paper-based rapid test card for the detection of soda as an adulterant in milk.
[0024] FIG 5 illustrates the paper-based rapid test card for the detection of preservative as an adulterant in milk.
[0025] FIG 6 illustrates the paper-based rapid test card for the detection of common salt as an adulerant in milk.
[0026] FIG 7 illustrates the paper-based rapid test card for the detection of preservative, detergent and common salt as adulterants in milk.
[0027] FIG 8 illustrates the paper-based rapid test card for the detection of starch, soda and urea as adulterants in milk.
[0028] FIG 9 illustrates the paper-based rapid test card for the detection of preservative, soda, common salt, starch, detergent and urea as adulterants in milk.
[0029] FIG 10 tabulates the percentage limit of detection of adulterants in milk by the paper-based rapid test card.
Detailed description of the invention
[001] In order to make the matter of the invention clear and concise, the following definitions are provided for specific terms used in the following description.
[002] The term “Adulterant” refers to substances added to food items which alters the original form for technical and economic advantages.
[0030] The invention discloses a paper-based rapid test card for the detection of adulterants in milk. The paper-based rapid test card detects various adulterants in one go. The paper-based rapid test card detects adulterants including detergent, urea, starch, neutralizer, preservatives, and salt.
[0031] The presence of adulterant is determined by the reaction of adulterated milk with the reagent present in the card and respective colour change. The concentration of adulterant is determined by comparing with the colour scale present on the card. The degree of colour change is directly proportional to the concentration of adulterant.
[0032] The paper-based rapid test card comprises four layers including a base structure comprising laminated or coated plastic layered card, a holding structure comprising non-toxic plastic, chemical layer comprising paper or blotting paper or absorbent paper and a coating layer comprising whitening agent and sodium alginate. The chemical layer comprises crushed enzymes and reagents.
[0033] The adulterated milk is added in a dropwise manner preferably 1-2 drops to the card, the milk enters the micro-capillaries and surface pipes of the chemical layer and reacts with the crushed enzymes and reagents. The change in color indicates the presence of the adulterant. The concentration of the adulterant is determined by comparing it with the color scale present on the card.
[0034] The present invention discloses a paper-based rapid test card for the detection of detergent, urea, starch, neutralizer, preservatives, and salt as adulterants. FIG 9 illustrates the paper-based rapid test card for the detection of preservative, soda, common salt, starch, detergent and urea as adulterants in milk.
[0035] The present invention discloses single adulterant testing paper-based rapid test card for the detection of detergent, urea, starch, neutralizer, preservatives, and salt as adulterants.
[0036] The present invention also discloses paper-based rapid test card for the simultaneous detection of three adulterants where preservative, detergent and common salt are detected as adulterants. FIG 7 illustrates the rapid test paper card for the detection of preservative, detergent and common salt as adulterants in milk.
[0037] The present invention discloses yet another paper-based rapid test card for the detection of three adulterants where starch, soda and urea as adulterants are detected as adulterants. FIG 8 illustrates the rapid test paper card for the detection of starch, soda and urea as adulterants in milk.
[0038] The following examples are offered to illustrate various aspects of the invention. However, the examples are not intended to limit or define the scope of the invention in any manner.

Example 1: Detection of detergent as an adulterant in milk
[0039] The detergents are chemical substances with cleansing properties used to remove grease and grime. The detergents are added to milk as an adulerant to increase the shelf life of milk and prevent the formation of curd. Typically, the detergents are added in the form of caustic soda, sodium carbonate and sodium bicarbonate to impart odd color and taste to the milk.
[0040] FIG 1 illustrates the paper-based rapid test card for the detection of detergent as an adulterant in milk.
[0041] In order to detect the presence of detergents as adulterant in milk, phenolpthalein is used as an indicator. The adulterated milk adulterated with detergent comprises sodium carbonate, which reacts with water to form hydroxide ions. The adulterated milk is added dropwise to the paper-absed rapid test card for the detection of detergent as an adulterant. Phenolpthalein is an organic acid-base indicator, which reacts with the hydroxide ions of the adulterated milk and the color changes to pink. The change of color from white to pink on the paper-based rapid test card indicates the presence of detergent as an adulterant in milk. The concentration of detergent as an adulterant is determined by the degree of color change from white to pink.
Example 2: Detection of urea as an adulterant in milk
[0042] Urea is an organic, metabolic compound. Urea is used as a raw material in the manufacture of chemicals. Urea is added to milk as an adulterant to increase the solids-not-fat (SNF) value, non-protein nitrogen content, inhibit microbial growth and adds shelf life.
[0043] FIG 2 illustrates the paper-based rapid test card for the detection of urea as an adulterant in milk.
[0044] In order to detect the presence of urea as an adulterant, the reagent is prepared by dissolving p–dimethylaminobenzaldehyde (DMAB) at a concentration range of 0.5g –2.0 g in 100 mL of 95% ethanol and 12.5 mL of concentrated hydrochloric acid. The adulterated milk is added dropwise to the paper-based rapid test card for the detection of urea as an adulterant. The adulterated milk comprising urea as an adulterant reacts with the reagent DMAB and a yellow complex is formed. The change of color from white to yellow on the paper-based rapid test card indicates the presence of urea as an adulterant in milk. The concentration of urea as an adulterant is determined by the degree of color change from white to yellow.
Example 3: Detection of starch as an adulterant in milk
[0045] Starch is a glucose polysaccharide used for the storage of energy in plants. The polymeric units of starch are amylose and amylopectin. Starch is a white, tasteless powder which is insoluble in cold water. Starch is added as an adulterant to increase the carbohydrate content and density of milk. Starch stabilizes the milk emulsion.
[0046] FIG 3 illustrates the paper-based rapid test card for the detection of starch as an adulterant in milk.
[0047] In order to detect the presence of starch as an adulterant, iodine at a concentration of 1% is used as an indicator. The adulterated milk is added dropwise to the paper-based rapid test card for the detection of starch as an adulterant. The adulterated milk comprising starch as an adulterant reacts with the indicator iodine of the paper-based rapid test card and forms starch-iodo compound. The starch-iodo complex is deep blue-black in color. The change of color from white to deep blue-black on the paper-based rapid test card indicates the presence of starch as an adulterant in milk. The concentration of starch as an adulterant is determined by the degree of color change from white to deep blue-black.
Example 4: Detection of neutralizer as an adulterant in milk
[0048] Neutralizers are substances used to neutralize acidic materials. Neutralizers such as sodium hydroxide, sodium carbonate and sodium bicarbonate are added to regulate the acidity of the milk. The presence of neutralizers indicate unhygenic storage of milk. The addition of neutralizers to the milk are not permitted.
[0049] FIG 4 illustrates the paper-based rapid test card for the detection of neutralizer as an adulterant in milk.
[0050] In order to detect the presence of neutralizer as an adulterant, aurin is used as an indicator. The indicator is prepared by mixing aurin (para-rosalic acid) at a concentration of 0.02g with ethanol at a concentration of 95%. The adulterated milk is added dropwise to the paper-based rapid test card for the detection of neutralizer as an adulterant. The adulterated milk adulterated with neutralizer comprising sodium hydroxide reacts with aurin to obtain a pinkish-red color. The change of color from white to pinkish-red on the paper-based rapid test card indicates the presence of neutralizer as an adulterant in milk. The concentration of neutralizer as an adulterant is determined by the degree of color change from white to pinkish red.
Example 5: Detection of preservative as an adulterant in milk
[0051] Hydrogen peroxide is a chemical substance used as a disinfectant, bleaching and cleaning agent. Hydrogen peroxide is an antimicrobial agent. The addition of hydrogen peroxide to milk increases the shelf life of milk by reducing the rate of microbial contamination.
[0052] FIG 5 illustrates the paper-based rapid test card for the detection of preservative as an adulterant in milk.
[0053] In order to detect the presence of hydrogen peroxide as an adulterant, potassium iodide-starch is used as an indicator. The indicator is prepared by mixing potassium iodide at a concentration of 3g with starch solution at a concentration of 2%. Hydrogen peroxide is an oxidizing agent which oxidizes iodide present in the reagent potassium iodide to iodine. The adulterated milk is added dropwise to the paper-based rapid test card for the detection of hydrogen peroxide as an adulterant. The adulterated milk comprising hydrogen peroxide as an adulterant reacts with oxidized iodine to obtain blue color. The change of color from white to blue on the paper-based rapid test card indicates the presence of hydrogen peroxide as an adulterant in milk. The concentration of preservative as an adulterant is determined by the degree of color change from white to blue.
Example 6: Detection of common salt as an adulterant in milk
[0054] The milk which is sold is often diluted. Adulterant such as common salt salt are added to increase the thickness and viscosity of the milk.
[0055] FIG 6 illustrates the paper-based rapid test card for the detection of common salt as an adulterant in milk.
[0056] In order to detect the presence of common salt as an adulterant, potassium chromate and silver nitrate are used as reagents. The reagent is prepared by mixing silver nitrate at a concentration of 1.68g dissolved in 100ml of distilled water with potassium dichromate at a concentration of 1g dissolved in 10ml of distilled water thoroughly. The adulterated milk is added dropwise to the paper-based rapid test card for the detection of common salt as an adulterant. Silver nitrate reacts with potassium chromate to form silver dichromate, a red solid. The silver dichromate reacts with sodium chloride present in the adulterated milk to form yellow colored sodium chromate. The change of color from white to yellow on the paper-based rapid test card indicates the presence of common salt as an adulterant in milk. The concentration of common salt as an adulterant is determined by the degree of color change from white to yellow.
Example 7: Analysis of accuracy of paper-based rapid test card in the detection of adulterants
[0057] The paper-based rapid test card is evaluated for the accuracy at which the adulterants are detected. The minimum concentration of adulterants present in the milk to be detected by the paper-based rapid test card is analyzed. The analysis of accuracy of paper-based rapid test card aids in determinng percentage of limit of detection of adulterants.
[0058] The accuracy of the paper-based rapid test card is evaluated by indivdually adding the adulterants including urea, starch, detergent, neutralizer, preservative and salt to the milk sample. To determine the minimum percentage limit of detection, the adulterants urea, detergent, neutralizer, preservative and salt are individually added to the milk sample at the least concentration and increased gradually. To determine the minimum percentage limit of detection of the adulterant starch, starch is boiled with the milk sample and tested by gradual increase in concentration. The limit of detection is determined for the least concentration at which the adulterant is detected by the paper-based rapid test card.
[0059] FIG 10 tabulates the percentage limit of detection of adulterants in milk by the paper-based rapid test card. The adulterant urea is detected at a percentage limit of detection of 0.02% at a concentration of 15mg/100ml. The adulterant starch is detected at a percentage limit of detection of 0.02% at a concentration of 20mg/100ml. The adulterant detergent is detected at a percentage limit of detection of 0.1% at a concentration of 100mg/100ml. The adulterant neutralizer is detected at a percentage limit of detection of 0.01% at a concentration of 5mg/100ml. The adulterant preservative is detected at a percentage limit of detection of 0.05% at a concentration of 50mg/100ml. The adulterant common salt is detected at a percentage limit of detection of 0.02% at a concentration of 20mg/100ml.
[0060] The paper-based rapid test card detects the presence of adulterants including urea, starch, detergent, neutralizer, preservative and salt at a percentage accuracy of 99.7%.
[0061] The present invention discloses a paper-based rapid test card, which is simple, accurate and useful for the detection of common adulterants in milk. The rapid test paper-based card aids in simultaneous detection of adulterants such as preservative, soda, common salt, starch, detergent and urea. The concentration of adulterants are determined by the degree of color change on the rapid test paper-card. The paper-based rapid test card of the present invention allows the detection of multiple adulterants using a single drop of milk. The present invention is economical, user-friendly and gives an instant result. The paper-based rapid test card is biodegradable in nature thus avoiding any disposal problems into the environment.

,CLAIMS:We Claim:

1. A paper-based rapid test card for the detection of one or more adulterants in milk, the test card comprises a base structure, a holding structure, a chemical layer and a coating layer.

2. The paper-based rapid test card as claimed in claim 1, wherein the chemical layer is composed of an adsorbent paper or a blotting paper.

3. The paper-based rapid test card as claimed in claim 1, wherein the coating layer comprises a whitening agent and sodium alginate.

4. The paper-based rapid test card as claimed in claim 1, wherein the adulterants are selected from a group comprising soda, common salt, starch, detergent, urea and preservative in milk.

5. The paper-based rapid test card as claimed in claim 1, wherein the adsorbent paper or blotting paper of the chemical layer comprises one or more reagents including phenolphthalein to detect detergent as an adulterant, p–dimethylaminobenzaldehyde (DMAB) in ethanol and concentrated hydrochloric acid to detect urea as an adulterant, iodine to detect starch as an adulterant, aurin to detect neutralizer as an adulterant, potassium iodide-starch to detect preservative as an adulterant and potassium chromate and silver nitrate to detect common salt as an adulterant.

6. The paper-based rapid test card as claimed in claim 1, wherein the presence of the adulterants is detected by change of color after the addition of 1-2 drops of adulterated milk on the paper-based rapid test card.

7. The paper-based rapid test card as claimed in claim 1, wherein the concentration of adulterant is determined by comparison of the depth of color change with the paper-based rapid test card.

8. The paper-based rapid test card as claimed in claim 1, wherein the paper-based rapid test card detects 1 adulterant, 3 adulterants and 6 adulterants simultaneously.