DETECTION OF NEUTRALIZERS,

PRESERVATIVES AND ADULTERANTS

IN MILK AND MILK PRODUCTS
 Introduction
 Now-a-days, milk producer or supplier or middle man do
adulteration in order to have high profit without caring about
the quality of milk.

 There are different kind of adulterants that are used for

adulteration depending upon situation.

 Adulteration of milk cheats the customer and may pose

serious risk to health in some cases .

 Adulteration is punishable offence under the provision of

Prevention of Food Adulterant act 1954 (PFA)[this act
was replaced by FSSAI, 2006]
 Adulteration

As per the definition of FSSAI, “Adulterant”

means any material, which is or could be
employed for the purposes of adulteration.

If any article contains any prohibited

preservatives or permitted preservatives in
excess of prescribed limits considered as
adulteration.
 Adulteration
• If quality of the article falls below the prescribed
standard or its constituents are present in
quantities not within the prescribed limits of
variability and also consider as adulteration.

• FSSAI states that no person shall either by

himself or by any servant or agent sell milk which
contains any added water and also milk
containing a substance not found in milk.
 Purposes of Adulteration
 Adulteration is Done for :-
 To increase the quantity of milk.

 Extending shelf life.

 Maintaining SNF.

 Avoiding the detection of sour milk

 To earn more profit

 Different kinds of adulterants like
sugar, starch, wheat flour, common salt,
baking soda, washing soda, urea,
hydrogen peroxide and formalin are
being used for the purpose of
adulteration depending on the situation.
 Synthetic Milk
• Synthetic milk is a milk prepared by combining liquid
soap, urea , vegetable oil , washing soda was a new
concept in dairy Industry.

• Time to time whole country is in panic due to news of

synthetic milk being mixed with natural milk.

• A lot of queries received from different dairy industries,

educationists, military dairy plants and consumers
regarding the detection of synthetic milk in natural
milk.
 Synthetic Milk
• The artificially prepared milk may cause a threat
to the life of infants. Such milk mixed to the
natural milk was difficult to detect .

• It is not checked immediately, this menace

would do incalculable harm not only to public but
to the dairy industry in a big way.
 Categories of Common
Adulterants
A. Carbohydrates: Sugar, glucose, starch, malto-dextrin,
etc.

B. Salts and fertilizers: Urea, ammonium sulphate,

potassium sulphate, sodium chloride, etc.

C. Neutralizers : Sodium carbonate, sodium bicarbonate,

sodium hydroxide, calcium hydroxide, etc.

D. Detergents : Easy and other liquid detergents,

washing powders, etc.
 Categories of Common Adulterants

E. Preservatives: Hydrogen peroxide,

formalin, etc.

F. Pond water: Heavier than tap water,

preferred for adulteration.

G. Oils and paints: Vegetable fats and oils,

mineral oil, white paint etc.
 Purpose of Adulteration
Adulterants Purpose

Starch, glucose, sugar, Increases the SNF of milk

maltodextrin
Sodium carbonate Decreases the developed acidity and
increases the shelf life
Caustic soda (NaOH) Decreases the acidity of milk
Urea Increases the nitrogen content and increases
the shelf life
Pond water Increases the specific gravity of milk
Detergents To dissolve the fats in water and component of
synthetic milk
Hydrogen peroxide, as a preservatives and increases the shelf life
Formalin
Vegetable oil Increases the fat
Methods for the detection of
adulterants:

The methods for the detection of

some of the common adulterants,
preservatives and neutralizers have been
discussed below:
 Neutralizers

• In milk NaOH, Na2CO3 and/or NaHCO3

are added as neutralizers, however, such
practice is not permissible.
• These are added by adulterators to
neutralize the developed acidity in milk.
• (a) Rosalic acid test: Take 2 ml milk in a test
tube. Add 2 ml Rosalic acid solution (0.05%; w/v
in ethanol). Mix the contents.
• If carbonate or bicarbonate is present a red
colour will appear. If NaOH is present a deep
rose red colour will appear.
• In the absence of all these or in case of pure
milk, only a brownish/orange colouration will be
present
• This test will only be applicable if the
neutralizers are present in the milk.
• If the added neutralizers were neutralized
by the developed acidity then this test will
be negative.
• Under these conditions, test the alkalinity
of ash content of the milk sample, as
follow:
(b) Alkalinity of ash test:
• Take 20 ml milk in a silica crucible.

• First evaporate the water to dryness, and then

burn the content to ash in a muffle furnace.

• Disperse the ash in 10 ml distilled water and

titrate the ash content against N/10 HCl using
phenolphthalein as indicator.

• If the volume of N/10 HCl exceeds 1.20 ml, then

the milk contains the added neutralizers.
 Carbohydrate based adulterants
Starch
• Starch is cheaply available in various forms such as wheat
flour, corn flour, and commercially manufactured starch.
• Being cheaper, it is sometimes added in the milk by
adulterators to raise the SNF.

Procedure:
• To 2 ml milk in a test tube, few drops of 1% iodine solution
(prepare by dissolving 2.5 g potassium iodide in 100 ml
water, add to it 1 g pure iodine crystal; shake well to give a
clear solution) are added.
• Blue colour developed indicates the presence of starch.
 Carbohydrate based adulterants
Sugar
• 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.
Procedure:
• Take 1 ml of milk sample and add 1 ml of 0.5%
resorcinol solution(0.5% resorcinol solution in
3N Hydrochloric acid).
• Mix the contents and heat in boiling water bath
for 5 min.
• If sugar is present in the sample, the red
colour will be produced. Blank shows pale
yellow or brownish colour.
 Carbohydrate based adulterants
Glucose
Glucose being a reducing sugar poses many problems
in its detection. Moreover, it is easily available in commercial
form as concentrated syrup.

Reagents:
• Modified Barford’s Reagent: Dissolve 24 gm of cupric
acetate in 450 ml boiling distilled water and immediately add
25 ml of 8.5% lactic acid to the hot solution. Cool and dilute
to 500 ml.

• Phosphomolybdic acid reagent: Take 35 gm of

Ammonium molybdate and 5 gm of sodium tungstate in a
one liter beaker. Add 200 ml of 10% (w/v) NaOH and 200 ml
of distilled water. Boil vigorously for Min. 20 min. Cool, dilute
to about 350 ml and add 125 ml of concentrated (85%)
phosphoric acid. Dilute to 500 ml.
Procedure for glucose detection
• Take one ml adulterated milk sample in a test
tube and add one ml of Barford’s reagent.

• Heat the mixture for 3 min. in a boiling water

bath and cool for 3 min. under tap water.

• Add one ml of phosphomolybdic acid

reagent and mix.

• Carry out a test with pure milk sample.

• Development of deep blue colour indicates

the presence of glucose while in case of
control, only faint bluish colour due to diluted
Barford’s reagent appears.
 Carbohydrate based adulterants

Maltodextrin
• To 2 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.
 Formalin
• Formalin is generally used by Public Health Departments to
preserve the milk samples for chemical analysis purpose.
• Formalin 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 35oC) for longer time, did not sour,
then that milk must be tested for formaldehyde by the following
simple method.
 FORMALIN
HEHNER TEST
Reagent:
Conc. sulphuric acid
Ferric chloride
Procedure:
• Take 5ml of suspected milk sample in a test tube.
• Add gently 2 ml of sulfuric acid containing trace of Ferric
chloride along the side of the tube (the added acid should
form a bottom layer of the mixture without mixing the
milk).
• Formation of a violet to purple coloured ring at the
junction of the two liquids indicates the presence of
formalin in the milk sample
 FORMALIN
LEECH TEST
Reagent:
HCl and 10% ferric chloride

Procedure:
• Take about 5.0 ml of milk in a test tube. Add to it equal volumes of
concentrated HCl containing 1.0 ml of 10% ferric chloride solution to
each 500 ml of the acid.
• Heat over a flame for about five minutes. Rotate the tube to breakup
the curd, and observe the colour. Development of violet colour
indicates the formalin added as a preservative.
 FORMALIN

CHROMOTROPIC ACID TEST

Reagent: It is prepared by dissolving 500 mg of chromotropic acid (1, 8-
dihydroxynapthalene-3, 6 disulphonic acid) in 72 per cent sulphuric acid.

Procedure:
• Take one ml of milk in a test tube and add one ml of
chromotropic acid reagent.
• Mix the contents well.
• Presence of formalin in milk is confirmed by the
appearance of bluish grey colour; whereas, control
sample remains colourless.
 Hydrogen peroxide
Hydrogen Peroxide is a preservative, but as per PFA rule it is
not permitted to be added to milk.

Procedure:
• Take 2 ml milk in a test tube and few drops of p-
phenylenediamine hydrochloride solution (2%, w/v, aq).
• The content will be blue in colour if hydrogen peroxide is
added or present in milk. In this case there will be no blue
colour.
 Hydrogen Peroxide
Reagent:
• Potassium iodide solution: Weigh 20 g of potassium iodide and
dissolve it in distilled water to obtain a 100 ml solution.
• Starch solution: Take 1 g starch powder and dissolve it in distilled
water by heating and make up the volume to 100 ml.
• Potassium iodide–starch reagent: Mix equal volumes of 20%
potassium iodide solution and 1% starch solution
Procedure :
• Take 1 ml of milk sample in a test tube.
• Add 1 ml of the potassium iodide-starch reagent and mix well.
• Blue colour will developed in the presence of H2O2, whereas pure milk
sample remain white in colour
 Salts

• Detection of common salt is based on the principle of argentometric

titrations.
• In this case excess of silver nitrate is made to react with potassium
chromate and resulted in the formation of reddish brown
precipitates of silver chromate.
Preparation of reagent
• Silver nitrate solution (0.1N): dissolve 1.6987g of silver nitrate in
100 ml of distilled water.
• 10% (w/v) potassium chromate: Dissolve 10 g of potassium
chromate in 100 ml distilled water.
.
 Salts

Procedure:
• Take 2 ml of milk and 1 ml of silver nitrate solution
(0.1N).
• Mix well and add two drops of a solution of 10%
potassium chromate.
• Yellow colour indicates the presence of added salt.
• Red colour will indicates the absence of sodium
chloride.
 Urea
• 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 content in natural milk varies from 20-70 mg/100ml.
• Urea content above 70 mg/100ml in milk indicates milk containing
‘added urea’.
• The addition of urea to milk can be detected by using para-
dimethyl aminobenzaldehyde (DMAB).
• This method is based on the principle that urea forms a yellow
complex with DMAB in a low acidic solution at room temperature.
 Urea
Preparation of DMAB (1.6% w/v) reagent
Dissolve 1.6 g DMAB in 100 ml ethyl alcohol and add 10
ml concentrated HCl.
Procedure
• Take 2 ml of milk in a test tube.
• Add 2 ml of 1.6 % DMAB reagent
• Appearance of distinct yellow color indicates the
presence of added urea.
• Slightly yellow color in unadulterated milk due to
natural urea in milk.
 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.

• Method I: Nessler’s reagent

• Method II:- Turmeric paper method
 Ammonium Salts
Preparation of Nessler’s reagent :
Dissolve the following chemicals separately.
 A- 8.0 g of mercuric chloride in 150ml distilled water.
 B- 60.0 g of sodium hydroxide in 150ml 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.
 Ammonium Salts
Procedure
• Pipette 2 ml of suspected milk sample into a test tube and add 0.5
ml of Nessler’s reagent.
• Mix the contents of the tube thoroughly.
• Appearance of brown colour confirms the presence of added
ammonium salts in milk.
 AMMONIUM SALTS

TURMERIC PAPER METHOD(METHOD II)

• 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.

PREPARATION OF REAGENT:
 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.
 Sodium hydroxide solution: 10%
 Ammonium Salt
Procedure
• Pipette 5ml 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.
 POND WATER
• Pond water is heavier than the tap water; some unscrupulous
persons for adulteration in milk usually prefer it.
• This method actually detects nitrates present in the pond water.
• In the pond water nitrates may come from fertilizers used in the
fields.

• PREPRATION OF REAGENT:-
• Diphenylamine (2% w/v, in sulfuric acid):- Weigh 2 g of
diphenylamine and dissolve it in conc. sulfuric acid to obtain final
volume of 100 ml.
 Pond Water
Procedure
• Take 2 ml of milk in a test tube.
• Rinse the test tube with the milk and drain the milk from the
test tube.
• Add 1-2 drops of 2% solution of diphenylamine.
• The sides of the test tube will turn blue if the milk sample
contains pond water.
 Sulfate salts
Presence of sulfates in milk can be detected by using barium chloride.

Reagents:
• Barium chloride (BaCl2.2H2O) solution: 5% (w/v, aq.)
• Trichloroacetic acid (TCA): 24% (w/v, aq.).
 Sulfate salts
Procedure:
• Take 10 ml of milk in a 50 ml stoppered test tube and add 10 ml of
TCA solution.

• Filter the coagulated milk through Whatman filter paper Grade 42.
Take 5 ml of clear filtrate and add few drops of barium chloride
solution.

• Formation of milky-white precipitates indicates the presence of

added sulfates like ammonium sulfate, sodium sulfate, zinc sulfate
and magnesium sulfate etc. to milk.
 Detergents

• Detergents are not present in natural milk.

• However, they gain entry into natural milk when synthetic milk is
added to it, as they are used in the preparation of synthetic milk.
• Detergents can be detected in milk by using indicators like
bromocresole purple or methylene blue.
• Detergents can be anionic, cationic and non-ionic, but in general,
anionic detergents are most commonly used commercially.
 Detergents
Principle:
• This method is based on the principle that methylene blue, which is
soluble in an aqueous phase, but form a complex with anionic
detergents, which is soluble in chloroform.
• Therefore, if methylene blue and chloroform are mixed with milk,
the concentration of methylene blue in the chloroform layer would
be directly proportional to the concentration of detergent in milk.
Reagents
• Ethanol (95% v/v)
• Methylene blue: Dissolve 25 mg of methylene blue in water and
make volume to 100 ml.
• Chloroform
 Detergents
Procedure
• Take 2.5 ml of suspected milk sample and 2.5 ml of pure milk in
separate test tubes.
• Add 7.5 ml of ethanol in each tube to precipitate the protein. Mix
well and filter the content through Whatman filter paper Grade 4.
• Take 2.0 ml of sample filtrate and add 2 ml of methylene blue
solution. Shake the contents of the tubes well and then add 4.0 ml
chloroform to each tube. Shake the tubes thoroughly again and
allow the chloroform layer (bottom layer) to separate from the
aqueous layer.
• Observe and compare the colour of chloroform layer in both the
tubes.
• If the intensity of blue colour in chloroform layer of suspected
milk sample is more than that of control (pure milk), the presence
of detergent is suspected.
 Detection of vegetable oil in milk using
butyro-refractometer (B.R.)

• This method is based on the principle that B.R. reading of

milk fat is comparatively lower than that of most of the
vegetable oils.

• Its adulteration with vegetable oils significantly increases

the B.R. reading.
Procedure:
• Clean the prism of the butyro-refractometer with diethyl ether.
• Allow the ether to evaporate to dryness. Maintain the temperature
of the prism at 40°C by circulating water using a thermostatically
controlled water-bath.
• Calibrate the butyro-refractometer by applying standard liquid
solution of known B.R reading.
• Again clean the prism with diethyl ether; apply 1-2 drops of clear,
extracted fat between the prism.
• Wait for 2 min before taking the reading so that sample should
attain temperature of 40°C.
• A correction of 0.55 is added to the observed B.R. reading for each
degree above 40°C or subtracted for each degree below 40°C to get
corrected B.R. reading of the sample.
• Interpretation: B.R reading of pure milk fat isolated by any of the
method falls in the range of 40-43. Any deviation from the
standard value indicates adulteration of milk with vegetable oil.

• Note: If the fat is isolated by Gerber method, B.R. reading is

decreased due to hydrolytic effect of sulphuric acid on fat. So, in
this case, observed B.R. reading is corrected as follows:
• Corrected B.R. reading = 1.08 X Observed B.R. reading.
 Detection of vanaspati in ghee
• The development of a permanent pink colour in a sample of ghee
with furfural solution in the presence of hydrochloric acid, indicates
the presence of sesame oil and the test is known as Baudouin test.

• According to the Govt. of India, Ministry of Agriculture Notification

(1962), the edible hardened oil (vanaspati) shall contain raw or
refined sesame (til) oil not less than 5% by weight i.e. addition of
sesame oil in other oils is a must for the manufacture of
hydrogenated edible fat.
 Detection of vanaspati in ghee

• This has been made compulsory because the specific chromogenic

constituents of sesame oil can be easily detected, and hence
adulteration of ghee with hydrogenated fats can be easily
established.

• The unsaponifiable matter of sesame oil contains two chromogenic

constituents namely sesamolin and sesamol which are not found in
other fats. The sesamol on condensation with furfural produces the
pink colour in the Baudouin test.
 Detection of vanaspati in ghee
Reagents:
– Hydrochloric acid (Sp. gr. 1.19)
– Furfural solution: 2 percent solution of furfural in ethanol, distilled
from rectified spirit not earlier than 24 h prior to the test.
Procedure:
• Take 5 ml of the melted ghee in 25 ml measuring cylinder provided
with a glass stopper, and add 5 ml of hydrochloric acid and 0.4 ml of
furfural solution.
• Insert the glass stopper and shake vigorously for 2 min. Allow the
mixture to separate.
• The development of a pink colour in the acid layer indicates presence
of sesame oil.
• Confirm by adding 5 ml of water and shaking again. If the colour in
acid layer persists, sesame oil is present. If the colour disappears, it is
absent.