DETECTION OF ADULTERATNS IN MILK

Dairy Chemistry Division

National Dairy Research Institute, Karnal

Introduction
Addition of neutralizers and adulterants in milk has become a common feature for fulfilling the milk
demands of over populated country. Now for dairy industry it seems to be difficult to run the plant
without neutralization of milk. For milk vendors and shop-keepers, adulteration of milk with water to
increase the quantity in order to supply milk in large number of house-holds also has become a common
practice. The lack of timely action against the adulterators by the Public Health Departments and lack of
easier and rapid methods for detection of adulteration further encouraged this menace. Common man i.e.
consumers are not aware of the methods and chemicals used in the methods. Now in NDRI Karnal, the
procedures for the detection of various adulterants and neutralizers have been simplified to be easily
adopted by the dairy industry. The prepared reagents as well as a KIT for the detection of adulterants and
neutralizers are available in the Dairy Chemistry Division of NDRI, Karnal.

Preservatives

A. Test for formaldehyde

Formalin (40% water solution of formaldehyde) is generally used by Public Health Departments to
preserve the milk samples for chemical analysis purpose. Formaldehyde is very poisonous chemical.
Though, it can preserve the milk for very long time, it should never be added to milk meant for
processing due to its poisonous property. Moreover, it affects the quality of the milk products. If milk
kept at room temperature (25 to 35 oC) for longer time, did not sour, then that milk must be tested for
formaldehyde by the following simple method:

Method 1: Leach test

1. Take about 5 ml of milk in a test tube.

2. Add to it equal volume of Conc. HCl containing 1 ml of 10% ferric chloride solution to each 500 ml
of the acid.
3. Keep the tube in boiling water bath for about 3-4 min.
4. Observe the colour of the solution in the tube. The tube containing pure sample will turns yellowish.
The positive sample (i.e. containing HCHO) will turn violet to brown black.

Method II: Chromotropic acid test

Reagent: Saturated solution of 1,8-dihydroxynaphthalene-3,6-disulphonic acid in about 72% sulphuric

acid (about 500 mg/100 ml). Light straw-coloured solution should result.

1. Take one ml of milk sample in a test tube. Add 1 ml of the Chromotropic acid reagent and mix well.
2. Appearance of yellow colour confirms the presence of formalin in the sample, whereas; control
sample will remain colourless.

B. Test for hydrogen peroxide

Hydrogen Peroxide is a preservative, but as per PFA rule it is not permitted to be added in milk. Hence if
it is found, then milk is said to be adulterated.

Method I

Reagent: Para-phenylenediamine hydrochloride solution (2%, aq, w/v).

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Procedure:

1. Add to about 5 ml of milk in a test tube, an equal volume of raw milk, followed by five drops of a 2 %
of para-phenylenediamine.
2. A blue colour is developed in the presence of hydrogen peroxide.

Note: It is unlikely that the addition of less than 0.1% of H2O2 to milk could be detected after 24 h, owing
to the action of peroxidase and catalase which stimulate its conversion into water. If  0.2% H2O2 is
added, some will persist for considerable long time. Owing to the fact that larger amount of H 2O2 are
known to destroy peroxidase, it is always advisable to add to the sample an equal volume of raw
unpreserved milk and to follow with a few drops of a 0.2% solution of para-phenylenediamine. Under
these circumstances a blue colour will develop immediately if H2O2 is added.

Method II

A method using potassium iodide and starch was standardized for the detection of hydrogen
peroxide in milk.

Procedure: Take one ml milk sample in a test tube. Add one ml of potassium iodide-starch reagent (mix
equal volumes of 20% potassium iodide solution and 1% starch solution) to the test tube. Appearance of
blue colour indicates the presence of hydrogen peroxide in the milk sample whereas control samples
remain colourless.

C. Detection of Neutralizers

Alkali in various forms like sodium carbonate, sodium bicarbonate, sodium hydroxide and lime are used
to neutralize developed acidity in milk. Detection of such neutralizers can be made by the following two
tests.

Method I. Rosalic Acid Test:

Reagents: Ethanol (95%), Rosalic acid solution (0.1% in alcohol).

Procedure:

1. Take in test tube about 5 ml milk and mix with 5-ml ethanol followed by 2-3 drops of rosalic acid
solution.
2. Formation of rose red colouration indicates the presence of alkali as neutralizer. Pure milk produces
brownish or brownish yellow colour only.

Rosalic acid is an organic dye, which is used as an indicator-changing colour at pH 7.0 to 8.0. Hence,
milk made even faintly alkaline by addition of neutralizers can be detected due to formation of rose red
colour with rosalic acid solution.

Method II. Ash alkalinity test

Neutralization of milk whether with lime, soda, or caustic soda, invariably increases the ash content and
the total alkalinity of the ash from a fixed quantity.

Reagent: HCl (standard, 0.1 N), Phenolphthalein indicator.

Procedure:

1. Pipette 20 ml of milk into a porcelain basin and evaporate to dryness on boiling water bath.

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2. Remove the basin, cool to room temperature and ignite the residue by heating over Bunsen flame
until gray-white ash is obtained.
3. Cool the basin to room temperature. Add to the residue 10-ml of water and disperse the ash in water
by stirring with a glass rod.
4. Titrate the ash dispersate by standard HCl using phenolphthalein indicator. If the volume of 0.1 N
HCl required to neutralize the ash dispersate exceeds 1.20 ml; the milk is suspected to contain
neutralizers.

D. Detection of starch or cereal flours

Reagent: Iodine solution (1%), Dissolve 2.5 g potassium iodide in 100 ml water, add to it 1 g pure iodine
crystal, shake well to give a clear solution.

Procedure:

1. Take about 3 ml of well-mixed milk sample in a test tube.

2. Heat the milk to just boiling by holding the tube over flame, and thereafter cool to room time.
3. Add 1-2 drops of 1% iodine solution.
4. Observe the development of colour. Formation of blue-violet colour indicates presence of starch
cereal flours.

E. Detection of cane sugar

Sugar or cane-sugar, is generally added to milk in order to raise the lactometer reading of the milk
which was diluted with water, so that by lactometer reading, the detection of added water is prevented. In
suspected samples, sugar can be easily detected by following method:

Reagent: Resorcinol, conc. HCl. (or prepare sucrose detecting reagent by dissolving 0.5 g of resorcinol in
100 ml of 35%. HCl prepared in distilled water.)

Procedure:

1. To about 5 ml of milk in a test tube, add 1 ml of conc. HCl and 0.1 g of resorcinol and mix.
2. Place the tube in boiling water bath for 5 min.
3. In the presence of cane sugar, red colour is produced.

Note: The test can be simplified by taking 1 ml of suspected sample of milk is a test tube followed by the
addition 1 ml of sucrose detecting reagent. In the presence of cane sugar, red colour is produced.

F. Detection of glucose

Glucose being a reducing sugar poses many problems in its detection. Moreover, it is easily available
in commercial form as concentrated syrup. These days adulteration of milk with glucose is increasing.
Now it has become possible to detect Glucose in milk by the following method:

Reagents:

1. Barfoed's reagent: Dissolve 24 g cupric acetate in 450 ml boiling water and immediately add 25 ml of
8.5% lactic acid to the hot solution. Shake to dissolve almost all precipitate, cool and dilute with
water to 500 ml. If necessary decant of filter to get a clear solution.
2. Phosphomolybdic acid reagent: Take 35 g ammonium molybdate and 5 g sodium tungstate in a large
beaker; add 200 ml of 10% NaOH solution and 200 ml water. Boil vigorously (20-60 min) so as to
remove nearly whole of ammonia. Cool, dilute with water to about 350 ml. Add 125 ml conc. H 3PO4
(85%) and dilute further to 500 ml.

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Procedure:

1. Take 1 ml of milk sample in a test tube. Add 1 ml of modified Barefoed's reagent.

2. Heat the mixture for exact 3 min in a boiling water bath and then rapidly cool under tap water.
3. Add one ml of phosphomolybdic acid reagent to the turbid solution and observe the colour.
4. Immediate formation of deep blue colour indicates the presence of added glucose. In case of pure milk
only faint bluish colour is formed due to the dilution of Barefoed's reagent.

G. Detection of nitrates (pond water)

Pond water is heavier than the tap water; some unscrupulous persons for adulteration in milk usually
prefer it. However, it can be easily detected by the following method. This method actually detects
nitrates present in the pond water. In the pond water nitrates may come from fertilizers used in the fields.

Reagent: Diphenylamine: Prepare 2% solution of diphenylamine in conc. sulfuric acid.

Procedure:

Take 2 ml of milk in a test tube. Rinse the tube with the milk and drain the milk from the test tube.Add
two-three drops of the reagent along the side of the test tube. Deep blue colour will be formed in presence
of nitrate.

H. Detection of Urea in milk

Urea is a natural constituent of milk and it forms a major part of the non-protein nitrogen of milk.
Urea concentration in milk is variable within herd. Urea is one of the ingredients of synthetic milk along
with caustic soda, detergent, sugar and foreign fats. Adulteration of natural milk with synthetic milk
increases the level of urea to such an extent that on consumption of this adulterated milk causes
toxicological hazards. Estimation of urea concentration in milk may serve as a tool for checking the
menace of adulteration of natural milk with synthetic milk. The average urea content in milk of Karan
Swiss, Karan Fries and Sahiwal cows was reported to be 28.57, 28.79 and 25.39 mg/100 ml (range 20 to
35 mg/100 ml). In buffalo milk, the average urea content was found to be 35.10 mg (range 25 to 40
mg/100 ml). The addition of urea to milk can be detected by using DMAB method. This method is based
on the principle that urea forms a yellow complex with p-dimethyl aminobenzaldehyde (DMAB) in a low
acidic solution at room temperature. The intensity of yellow colour is measured at 425 nm. Here only
qualitative method is described

Alcohol
Urea + DMAB  Yellow complex
HCl
Reagent:

1.6% DMAB reagent: Dissolve 1.6 g DMAB in 100-ml ethyl alcohol and add 10-ml conc. HCl.

Procedure:

1. Take equal quantity of milk and equal quantity of 24% TCA in a glass stoppered test tube. Mix and
filter it.
2. Take 3 ml of filtrate in a test tube and add 3 ml of 1.6% DMAB reagent in ethyl alcohol and HCl.
Note the colour obtained.
3. The occurrence of distinct yellow colour indicates the presence of added urea in milk.

Note: The control (milk sample containing no added urea) showed a slight yellow colour due to the
presence of natural urea in milk.

I. Sodium chloride

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 Take 5 ml of milk and 1 ml of silver nitrate solution (0.1 N). Mix well and add two drops of a
solution of 10% potassium chromate. Yellow colour indicates the presence of added salt. Otherwise, red
colour will appear.

J. Ammonium salts

The added ammonium salts e.g ammonium chloride, ammonium sulfate, ammonium nitrate and
ammonium dihydrogen orthophosphate can be detected in milk by two methods i.e Nessler’s reagent
method and turmeric paper method.

Method I: Nessler’s reagent method

Reagent : Nessler’s reagent: Dissolve the following chemicals separately.

a. 8.0 g of mercuric chloride in 150 ml distilled water.
b. 60.0 g of sodium hydroxide in 150 ml distilled water.
c. 16.0 g of potassium iodide in 150 ml distilled water.

Add reagent a to reagent b and mix well. To this mixture, add reagent c, mix and dilute the
contents to 500 ml. Leave this solution undisturbed and decant the clear upper layer of the solution and
store in a stoppered glass bottle.

Procedure: Pipette 5 ml of suspected milk sample into a test tube and add 1 ml of Nessler’s reagent. Mix
the contents of the tube thoroughly. Appearance of yellowish or grey colour confirms the presence of
added ammonium salts in milk

Method II. Turmeric paper method

This method is based on the principle that ammonium salts on addition of strong alkali liberate
ammonia and the liberated ammonia turns turmeric paper to pinkish red.

Reagents:

a. Turmeric paper: Dissolve 10 g of pure turmeric powder in 100 ml distilled water and dip Whatman
filter paper Grade 1 strips into it for 2 min. Dry the paper at room temperature. The dried filter paper
is wetted with distilled water before use.
b. Sodium hydroxide solution: 10% (aq.)

Procedure: Pipette 5 ml of suspected milk sample in a test tube and add 1 ml of 10% sodium hydroxide
solution in such a manner that should not touch the rim of the test tube while adding. Mix the contents of
the tube. Place a piece of wet turmeric paper on the rim of the test tube and keep the test tube
undisturbed. Observe the change in the colour of the turmeric paper. Appearance of pinkish red colour
confirms the presence of ammonium salt in milk.

K. Detection of Vanaspati n ghee

Procedure: Take 5 ml of melted ghee in a test tube. Add 5 ml co concentrated HCl. Mix well and add 5
drops of colorless furfural solution. Mix and allow to stand for 5 min. Appearnce of pink colour in the
lower layer shows the presence of vanaspati addition in ghee.

L. Maltodextrin

To 5 ml milk sample in a test tube, 2 ml of dilute iodine solution (0.05 N) is added. Appearance of
chocolate red brown colour developed indicates the presence of maltodextrin.

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Quantitative method for the detection of added urea in milk by a colorimetric method

Reagents:

a. Trichloroacetic acid (TCA) 24% w/v: Twenty-four grams of TCA is dissolved in distilled water and
the total volume is made to 100 ml.
b. Phosphate buffer (pH 7.0): Anhydrous potassium dihydrogen orthophosphate (3.403 g) and
anhydrous di-potassium monohydrogen orthophosphate (4.355 g) were dissolved in distilled water
and the volume was made to one litre.
c. Diluting reagent: Equal volumes of 24% (w/v) TCA and phosphate buffer, pH 7.0 are mixed to make
the diluting reagent.
d. p-dimethyl amino-benzaldehyde (DMAB) reagent (1.6% w/v): p-dimethyl amino benzaldehyde (1.6%
w/v) in ethyl alcohol containing 10% (v/v) concentrated hydrochloride acid.
e. Standard urea solution (1 mg/ml): Weigh 100 mg of urea (AR grade) and dissolve in phosphate
buffer, pH 7.0 and make up the volume to 100 ml.

Procedure: Ten ml of well mixed sample of milk is mixed with 10 ml of TCA (24% w/v) to
precipitate the protein and filtered through Whatman No.42 filter paper. Five ml of filtrate was treated
with 5 ml of 1.6% DMAB reagent to develop the yellow colour in a test tube. Reagent blank is prepared
by taking 5 ml of diluting reagent and treating with 5 ml of DMAB reagent. The optical density of
yellow colour of the sample is measured at 425 nm in a spectrophotometer against reagent blank. From
standard curve, as described below, the amount of urea in milk is calculated.
Preparation of standard curve: Standard urea solution (1 mg/ml) was prepared in phosphate buffer pH
7.0. Different concentrations of urea solution ranging from 0.1 mg to 2.0 mg were separately taken in
different test tubes and the total volume was made to 5 ml of each case with diluting reagent solution.
Then 5 ml of 1.6% DMAB reagent was added to each test tube to develop the colour. The optical density
of the yellow colour thus obtained was measured at 425 nm and is plotted against concentration.