MILK AND MILK PRODUCTS 2016

S.NO. TITLE/METHOD PAGE NO.

BIS method

20 DETERMINATION OF MELAMINE AND CYANURIC ACID IN 191

MILK, MILK PRODUCT AND INFANT FORMULAE –
GUIDELINES

20.1 Method 1. Detection of Non-Protein Nitrogen (NPN) 192

Containing Substances in Milk

20.2 Method 2. ELISA based Methods 194

20.3 Method 3. ISO Method 194

Note: The test methods given in the manuals are validated/ standardized test methods. However,
it would be the responsibility of the respective testing laboratory to confirm that the above
methods are validated in its laboratory and gives proper result in their laboratory.

……….

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1. LIQUID MILK

Buffalo milk, cow milk, goat milk, sheep milk, mixed milk, standardised milk, full
cream milk, recombined milk, toned milk, double toned milk, and skimmed milk as laid
down under FSSAI Rules.

1.1. Preparation of Sample of Milk

Samples are received after few days of drawl and contain preservative (0.4%
formalin). Warm the sample to 37- 40°C by transferring it to the beaker and keeping it
in a water bath maintained at 40 - 45°C. Stir slowly for proper homogenisation. Mix
sample thoroughly by pouring back into the bottle, mixing to dislodge any residual fat
sticking to the sides and pour it back in the beaker. During mixing do not shake the
bottle vigorously. Allow the sample to come to room temperature (26- 28°C) and
withdraw immediately for analysis. If small clots or lumps are observed in the sample
which cannot be dispersed, a few drops of liquor ammonia may be used during
homogenisation. If even after homogenisation the sample shows lumps or clots or
droplets of oil are visible suggestive of curdling /splitting of milk, the sample should be
deemed unfit for analysis and rejected.

(Ref:- IS 1479 (Part II) – 1961 (Reaffirmed 1997) Methods of test for Dairy Industry -
Chemical Analysis of Milk. Bureau of Indian Standards, New Delhi).

1.2. Detection of Adulterants in Milk

1.2.1. Detection and Quantification of Cane Sugar in Milk

Sucrose is absent in milk and its presence in milk indicate adulteration. Presence
of sucrose in milk can be determined by the following method.

1.2.1.1. Qualitative Method: Modified Seliwanoff’s Method

Fructose in cane sugar (sucrose) reacts with resorcinol in HCl to give red colour.

1.2.1.1.1. Reagent

A. Resorcinol Solution (0.5%): Weigh 0.5 g of resorcinol in about 40 ml of distilled

water. Add 35 ml of concentrated HCl (12 N) to it and make up the volume to 100
ml using distilled water.

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Note: The resorcinol flakes should be white in colour.

1.2.1.1.2. Procedure

Take 1 ml of milk in a test tube. Add 1 ml of Resorcinol Solution and mix. Place
the tube in boiling water bath for 5 min. Withdraw the tube and observe the colour.
Appearance of deep red colour indicates presence of sucrose, or a ketose sugar. In pure
milk samples no such red color is developed and sample remains white in nature. The
limit of detection of method is 0.1%.

(Ref :- IS 1479 ( Part I) 1961 (Reaffirmed 2003) Methods of test for Dairy Industry – Rapid
Examination of Milk. Bureau of Indian Standards, New Delhi).

1.2.1.2. Quantitative Determination of Cane Sugar in Milk

If the test for Cane Sugar (Sucrose) is positive, the quantitative estimation of
sucrose is necessary for determination of SNF in milk sample.

The sucrose content in milk sample can be determined by volumetric method

(Lane-Eynon method). In this method, the milk sample is curdled with zinc acetate and
potassium ferrocyanide (Carrez Solution 1 and 2). Determine the sugar content before
and after inversion. The value before inversion indicates lactose and after inversion
total sugars.

1.2.1.2.1. Reagents

Same reagents those mentioned in Section 9.4. (Determination of sucrose

content in condensed/evaporated milk).

1.2.1.2.2. Procedure

Proceed as in described in Section 9.4 (Determination of sucrose content in

condensed/evaporated milk) and instead of condensed milk, take 40 g of milk sample.
Apply the factor 0.95 for calculating the sugar content.

(Ref:- IS 1166 – 1986 (Reaffirmed 1997) Specification for Condensed milk, Partly Skimmed
and Skimmed Condensed Milk(Appendix C: Determination of sucrose). Bureau of Indian
Standards, New Delhi; IS 4079 – 1967(Reaffirmed 1995) Specification for Canned
Rasogolla (Appendix C: Determination of sucrose). Bureau of Indian Standards, New Delhi
(Appendix C) Pearson’s Composition and Analysis of Foods, 9thedn, 1991 Modification of

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para 1 page 297).

1.2.2. Detection and Quantification of Starch in Milk

1.2.2.1. Qualitative method

1.2.2.1.1. Reagent

A. Iodine Solution: Dissolve 2.6 g of iodine and 3 g of potassium iodide in a sufficient

quantity of water and make up to 200 ml.

1.2.2.1.2. Procedure

Take about 5 ml of milk in a test tube. Bring to boiling condition and allow the
test tube to cool to room temperature. Add 1-2 drops of iodine solution to the test tube.
Development of blue colour indicates presence of starch which disappears when sample
is boiled and reappears on cooling. The limit of detection of method is 0.02%.

(Ref :- IS 1479 ( Part I) 1961 (Reaffirmed 2003) Methods of test for Dairy Industry – Rapid
Examination of Milk. Bureau of Indian Standards, New Delhi).

1.2.2.2. Quantitative Determination of Starch in Milk

If test for starch is positive, quantitative estimation of starch is to be carried out

for determination of SNF in milk sample. The sample of milk is curdled with alcohol, and
made free from lactose which is naturally present in milk. The precipitated starch is
washed with 50% alcohol to free it from lactose. The precipitated starch is hydrolysed
to convert it into reducing sugars. Reducing sugar is determined by Lane and Eynon
method (Section 9.4 (Determination of sucrose in condensed milk) and multiplied with
0.9 to get the starch content in milk.

1.2.2.2.1. Reagents

A. Ethanol (98%).

B. 10% sodium hydroxide.

C. Sodium carbonate.

1.2.2.2.2. Procedure

Weigh approximately 25 g sample in a 250 ml beaker. Add 20 ml of ethanol to

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curdle the milk. Filter the precipitate on a filter paper and wash the precipitate with
50% ethanol till the precipitate is free from lactose/sugar i.e. when the washings give a
negative test with resorcinol. Transfer the precipitate to a 500 ml flask with about 200
ml water and add 10 ml concentrate HCl to hydrolyse the starch by refluxing in a boiling
water bath for 2.5 hours. Cool and neutralise with 10% sodium hydroxide and sodium
carbonate towards the end using litmus paper. Make up to 500 ml with water. Shake
well and filter if necessary. Determine reducing sugar by Lane and Eynon method
(Section 9.4). Calculate starch as follows:

% starch = % reducing sugar x 0.9

(Ref:- Modified Method, A.O.A.C 17thedn, 2000 Official Method 925.50, Starch in
Confectionery, last para).

1.2.3. Detection of Cellulose in Milk

Cellulose in milk gives blue colour with Iodine – Zinc Chloride reagent.

1.2.3.1. Reagent

A. Iodine – Zinc Chloride reagent: Dissolve 20 g ZnCl2 in 8.5 ml water and when cool,

introduce the iodine solution (3 g potassium iodide and 1.5 g iodine in 60 ml water)
drop by drop until iodine begins to precipitate.

1.2.3.2. Procedure

Take about 10 g of milk in a 100 ml beaker. Add 50 ml of hot water and stir
thoroughly for about 2 min. Pour the mixture on a nylon cloth and wash the residue
with 50 ml of hot water twice. Scrape the residue with a spatula and place it in a
spotting plate. Stain a part of residue with Iodine-Zinc Chloride reagent and another
part with iodine solution (see Reagent 1.2.2.2.1.). Development of blue colour in Iodine-
Zinc Chloride reagent and absence of blue colour in Iodine Solution confirms presence
of cellulose. The method is also applicable to milk products like curd, rabri and
evaporated milk.

(Ref:- Manual Methods of Analysis for Adulterants & Contaminants in Foods. I.C.M.R 1990
page 27).

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1.2.4. Detection of Added 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 content in
natural milk varies from 20 mg/100 ml to 70 mg/100 ml. However, urea content above
70 mg/100 ml in milk indicates milk containing ‘added urea’. The addition of urea to
milk can be detected by using para-dimethylaminobenzaldehyde (DMAB). This method
is based on the principle that urea forms a yellow complex with DMAB in a low acidic
solution at room temperature.

1.2.4.1. Qualitative Method

This method is based on the principle that urea forms a yellow complex with
DMAB in a low acidic solution at room temperature.

1.2.4.1.1. Reagent

A. DMAB reagent (1.6%, w/v): Dissolve 1.6 g DMAB in 100 ml ethyl alcohol and add 10
ml concentrate HCl.

1.2.4.1.2. Procedure

Mix 1 ml of milk with 1 ml of 1.6% DMAB reagent. Distinct yellow colour is

observed in milk containing added urea. The control (normal milk) shows a slight
yellow colour due to presence of natural urea. The limit of detection of method is 0.2%.

1.2.4.2. Quantitative Estimation of Urea in Milk

Urea is a natural constituent of milk and is present to the extent of 70 mg per 100
ml (700 ppm). The test based on the use of para-dimethylamino benzaldehyde can be
used for the estimation of urea in milk after precipitation of milk proteins using
trichloroacetic acid.

1.2.4.2.1. Reagents/Apparatus

A. p-Dimethyl amino benzaldehyde (DMAB) solution: Dissolve 1.6 g DMAB in 100 ml

ethyl alcohol and add 10 ml concentrate HCl. The reagent is stable for 1 month.
Prepare new standard curve with each new batch of reagent.

B. Phosphate Buffer pH 7.0: Dissolve 3.403 g anhydrous potassium dihydrogen

orthophosphate (KH2PO4) and 4.355 g anhydrous dipotassium monohydrogen

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orthophosphate (K2HPO4) separately in 100 ml of distilled water. Combine solutions

and dilute to 1 litre with water.

C. Trichloroacetic acid (TCA) 24%, w/v: Freshly prepared. 24.0 g TCA is dissolved in
distilled water and volume made up to 100 ml.

D. Diluting Reagent: Equal volumes of 24% TCA and phosphate buffer (pH 7.0) are
mixed to make the diluting reagent.

E. Urea Standard Solution: (a) Stock solution: 5 mg / ml. Dissolve 5 ± 0.001 g reagent
grade urea in water and dilute to 1 litre with water. (b) Working solution:– Pipette 2,
4, 6, 8, 10, 12, 14, 16, 18 and 20 ml stock solution into 250 ml volumetric flask and
dilute to volume with phosphate buffer. (c) Reference solution -Use standard
solution containing 1.0 mg urea / 5 ml as reference standard. Store at less than 24°C.
The reagent is stable for 1 week.

1.2.4.2.2. Apparatus

A. Spectrophotometer – Instrument with maximum band width 2.4 nm at 420 nm, with
1 cm cells

B. Whatman filter paper: Grade 42.

C. Funnels.

D. Test tubes.

1.2.4.2.3. Procedure

1.2.4.2.3.1. Preparation of standard curve

Pipette 5 ml aliquots of working standard solutions into 20 x150 mm (25 ml) test tubes
and add 5 ml DMAB solution to each. Prepare reagent blank of 5 ml buffer and 5 ml
DMAB solution. Shake tubes thoroughly and let stand for 10 minutes. Read A in 1 cm cell
at 420 nm with reagent blank at zero A. Plot A against concentration urea Plot should be
straight line

1.2.4.2.3.2. Estimation

10 ml of milk sample is mixed with 10 ml of TCA to precipitate the proteins and filtered
using Whatman 42 filter paper. 5 ml of filtrate is then treated with 5 ml of DMAB

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reagent to develop the colour. Blank is prepared by taking 5 ml of diluting reagent and
treating with 5 ml of DMAB reagent. The optical density of the yellow colour is
measured at 420 nm. From standard curve the amount of urea in milk is calculated.

(Ref:- IS.1479 (Part I) – 1960 (Reaffirmed 2003) Methods of test for Dairy Industry, Part –
I Rapid examination of Milk. Bureau of Indian Standards, New Delhi; Bector, B.S., Ram, M.
and Singhal, O.P. (1998). Rapid platform test for the detection /determination of added
urea in milk. Indian Dairyman, 50(4): 59-62).

1.2.5. Detection of Ammonium Compounds in Milk

1.2.5.1. Method 1.

1.2.5.1.1. Reagents

A. 2% Sodium hydroxide.

B. 2% Sodium hypochlorite.

C. 5% Phenol solution.

1.2.5.1.2. Procedure

Take 1.0 ml of milk add 0.5 ml of 2% sodium hydroxide, 0.5 ml of 2% sodium

hypochlorite and 0.5 ml of 5% phenol solution. Heat for 20 seconds in boiling water
bath, bluish colour turns deep blue in presence of ammonium sulphate. The
development of pink colour shows that the sample is free from Ammonium sulphate.

(Ref:- Milk and Milk products Vol. 5 Published by N.C.E.R.T.).

1.2.5.2. Method 2.

1.2.5.2.1. Reagents

A. 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

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upper layer of the solution and store in a stoppered glass bottle.

1.2.5.2.2. Procedure

Take 5 ml of milk sample in a test tube. Add 1 ml of Nessler’s reagent. Mix the
contents of the tube thoroughly. Observe and note the color. The control milk sample
gives slight grayish colour. At low concentration of ammonium compounds, brownish
shade appears which is distinguishable at 0.15% followed by yellowish colour and then
orange colour development at higher concentration. The limit of detection of method is
0.15%.

(Ref:- Guleria, V. (1998). Detection of added ammonium salts in milk with and without the
addition of formalin. M. Sc. Thesis. NDRI, Karnal, India; Sharma, R.; Rajput, Y.S. and Naik,
N.L. (2012). Detection of adulterants in milk – a laboratory manual. NDRI Publication No.
88/2012, NDRI, Karnal, page 49-51).

1.2.6. Tests for Presence of Sulphates in Milk

Presence of sulfate salts, which may be added to milk to raise its SNF level in
milk, can be detected by using barium chloride.

1.2.6.1. Reagents

A. Barium chloride (BaCl2.2H2O) 5% (w/v) aqueous solution: Dissolve 5.0 g barium

chloride in distilled water and make the final volume to 100 ml.

B. Trichloroacetic acid (TCA), 24% (w/v, aq.): Dissolve the 24 g of TCA into distilled
water and make the final volume to 100 ml obtain 24% TCA.

1.2.6.2. Procedure

Take 10 ml of milk in a 50 ml stoppered test tube. Add 10 ml of TCA

solution. Filter the coagulated milk through Whatman filter paper Grade 42. Take 5 ml
of clear filtrate. Add few drops of barium chloride solution. Observe for any visible
precipitates in the tube. Formation of milky-white precipitates indicates the presence of
added sulfates like ammonium sulfate, sodium sulfate, zinc sulfate and magnesium
sulfate etc. to milk. The limit of detection of method is 0.05%.

(Ref:- Sharma, R.; Rajput, Y.S. and Naik, N.L. (2012). Detection of adulterants in milk – a
laboratory manual. NDRI Publication No. 88/2012, NDRI, Karnal, page 20-21).

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1.2.7. Detection and Estimation of Added Glucose in Milk

1.2.7.1. Qualitative Method

1.2.7.1.1. Reagents

A. Modified Barford’s reagent: Dissolve 24 g of Copper acetate in 450 ml of boiling

distilled water. Add 25 ml of 8.5% acetic acid, shake, cool to room temperature and
make up to 500 ml. After sedimentation filter the reagent and store in dark coloured
bottle.

B. Phosphomolybdic acid: 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. Check removal of
ammonia with the help of red litmus paper. Cool, dilute with water to about 350 ml.
Add 125 ml concentrated H3PO4 (85%) and dilute further to 500 ml.

1.2.7.1.2. Procedure

Take 1 ml of milk sample in a test tube. Add 1 ml of modified Barford’s reagent.

Heat the mixture for exact 3 min in a boiling water bath. Rapidly cool under tap water.
Add one ml of phosphomolybdic acid reagent to the turbid solution. Observe the colour.
Immediate formation of deep blue color after adding phosphomolybdic acid reagent
indicates the presence of added glucose in the milk sample. In case of pure milk, only
faint bluish color can be observed due to the dilution of Barford’s reagent. The limit of
detection of method is 0.1%.

1.2.7.2. Quantitative method

The qualitative method can be further extended for the estimation of glucose
content in milk. The natural level of glucose in milk is around 10 mg/100 ml.

1.2.7.2.1. Reagents

A. Modified Barford’s reagent: Dissolve 24 g of Copper acetate in 450 ml of boiling

distilled water. Add 25 ml of 8.5% acetic acid, shake, cool to room temperature and
make up to 500 ml. After sedimentation filter the reagent and store in dark coloured
bottle.

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B. Phosphomolybdic acid: 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. Check removal of
ammonia with the help of red litmus paper. Cool, dilute with water to about 350 ml.
Add 125 ml concentrated H3PO4 (85%) and dilute further to 500 ml.

C. Acetate buffer: 1 N Sodium acetate and 1N acetic acid in equal volume having 4.75
pH.

1.2.7.2.2. Procedure

To 1 ml of milk sample or 1 ml of reconstituted milk powder in a test tube add

equal volume of acetate buffer and filter. To 0.2 ml of filtrate add 2.8 ml water and 2 ml
of modified Barford’s reagent. Heat the tube in boiling water for 4 minutes. After cooling
for 2 minutes add 3 ml of phosphomolybdic acid and mix the contents. Development of
deep blue colour indicates the presence of glucose. Filter the contents of the tube
through Whatman No 42 filter paper. Collect the filtrate in a colorimetric tube, after
discarding first 1 ml. Measure the absorbance in a photoelectric colorimeter, using red
filter or determine absorption maxima in a spectrophotometer between 620- 780 um
against blank prepared identically from a pure milk sample. The concentration of
glucose in the sample can be determined with the help of a standard curve prepared
from milk samples containing known amounts of added glucose i.e., 0.5, 1.0, 2.0, 5.0
percent glucose in milk.

(Ref:- Manual Methods of Analysis for Adulterants and Contaminants in Foods, I.C.M.R
1990, page 28; Roy, N.K. and Sen, D.C. (1991) Rapid analysis of milk. In: Textbook of
Practical Dairy Chemistry. Vol. I. Chemical analysis of fluid milk. Kalyani Publishers, New
Delhi, India).

1.2.8. Detection of Sodium Chloride in milk

The presence of extraneously added sodium chloride in milk can be detected by

silver nitrate and potassium chromate reagent.

1.2.8.1. Reagents

A. Silver nitrate (AgNO3) solution: 0.1 N, aqueous.

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B. Potassium chromate (K2CrO4) solution: 10% (w/v) aqueous.

1.2.8.2. Procedure

Take 5.0 ml of milk sample and add 1.0 ml of 0.1 N silver nitrate solution (10%).
Mix the content thoroughly and add 0.5 ml of 10% potassium chromate solution and
observe the colour. Appearance of chocolate brown precipitate indicates the absence of
dissolved chloride in milk and appearance of yellow colour indicates presence of
dissolved chloride. The limit of detection of method is 0.02%.

(Ref :- Pearson’s Composition and Analysis of Foods, 9thedn,1991 – Modified Mohr method,
page 14).

1.2.9. Detection of Presence of Foreign Fat in Milk

In Indian context, among the various milk constituents, milk fat is the costliest.
Often, unscrupulous traders, remove the milk fat from milk and admix milk with
vegetable oil/refined oil with help of detergent or by adding other emulsifiers. In the
following test, milk fat is isolated from given milk sample and is subject to butyro-
refractometer reading (B.R.). Since, most of vegetable fat/oils have higher B.R.
compared to milk fat, any increase in B.R. reading above reference value of milk fat
indicate adulteration of milk with vegetable/refined oil. For isolation of milk fat from
milk, modified Gerber butyrometer can be used where both ends of the butyrometer are
open. Stem side opening of the butyrometer (which is generally closed) is closed with a
good quality removable silicon stopper. After the milk fat test, silicon stopper is
removed and milk fat is removed with the help of a syringe and same is subjected to B.R.
at 40°C. Since Gerber sulfuric acid causes some hydrolysis of fatty acids/triglycerides,
the B.R is multiplied by a factor to obtain corrected B.R. The fat in suspected milk
sample can also be isolated by solvent extraction method in which case correction of
B.R. is not required.

1.2.9.1. Method 1. Using modified Gerber Method

1.2.9.1.1. Reagents: Gerber sulphuric acid, Iso-amyl alcohol.

1.2.9.1.2. Procedure

Isolate the fat from milk by Gerber method using specially designed milk

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butyrometer, which is open at both ends. Close the stem side opening with a good
quality acid resistant silicon stopper. Add 10 ml of Gerber sulphuric acid, 10.75 ml milk
and 1 ml iso-amyl alcohol. Close the neck side with lock stopper; mix the content and
centrifuge at 1200 rpm, 5 min to get a clear fat column. Remove the silicon stopper from
the stem side and take out the fat from the stem of the butyrometer using a capillary or
a syringe.

For taking B.R. reading of the milk fat, 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.

1.2.9.1.3. Calculation

Calculate the Corrected B.R. reading of isolated fat as follows:

Corrected B.R. = Observed B.R. x 1.08

1.2.9.1.4. Interpretation

If the BR reading differs from the prescribed limit of variability (not more than
42 in case of non-cotton tract area and not more than 45 in case of cotton tract area),
presence of foreign fat in the milk may be suspected.

1.2.9.2. Method 2. Other Methods for Extraction of Milk fat

A. Solvent Extraction Method

Extract fat from the milk sample by Rose-Gottlieb method (See Section 1.3.4.2). Take the
B.R. reading at 40°C of the extracted fat and interpret the results as indicated in Section
1.2.11.1.4.

A. Cream Extraction Method

Separate the milk fat from the milk sample by centrifugation (5000 g, 10 min, 4°C).

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Carefully remove the cream plug and collect the cream in an aluminium dish. Heat the
dish over burner till ghee formation takes place. Filter the ghee residues and take the
B.R. reading of ghee and interpret the results as indicated in Section 1.2.11.1.4.

Note:

1. If fat is extracted using method listed in section 1.2.11.1, correction factor is

required. Result can be interpreted directly from the B.R. reading, if fat is extracted
using other method (Section 1.2.11.2).

2. Further check for presence of extraneous fat can be done by checking the fatty acid
profile of the extracted fat by GLC (See Manual on Oils and Fats for determination of
fatty acid composition of oils and fats).

(Ref:- IS.1479 (Part I) – 1960 (Reaffirmed 2003) Methods of test for Dairy Industry, Part –
I Rapid examination of Milk. Bureau of Indian Standards, New Delhi; Arora, K.L.; Lal, D.;
Seth, R. and Ram, J. (1996). Platform test for detection of refined mustard oil adulteration
in milk. Indian J. Dairy Sci., 49 (10): 721-723.; Lal, D.; Seth, R.; Arora, K.L. and Ram, J.
(1998). Detection of vegetable oils in milk. Indian Dairyman, 50 (7): 17-18).

1.2.10. Detection of Nitrates (Pond Water) in Milk

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.

1.2.10.1. Reagent

A. Diphenylamine (2%, w/v, in sulfuric acid): Weigh 2 g of diphenylamine and dissolve

it in sulfuric acid to obtain final volume of 100 ml.

1.2.10.2. 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.
Note the developed colour. Deep blue colour will be formed in presence of nitrate in the
milk sample. Pure milk sample will not develop any colour.

(Ref:- Sharma, R.; Rajput, Y.S. and Naik, N.L. (2012). Detection of adulterants in milk – a

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laboratory manual. NDRI Publication No. 88/2012, NDRI, Karnal, page 47-48).

1.2.11. Detection of Neutralizers in Milk

Neutralizers (NaOH, 0.1% for Na2CO3 and 0.2% for NaHCO3) are added to milk to
neutralize the developed acidity in milk. Rosalic acid method can be used for the
detection of presence of these neutralizers in milk. The other method available for
detection of neutralizers in milk is through determination of alkalinity of ash.

1.2.11.1. Method 1 (Rosalic acid Method)

There are two variants of this method. Both the variants are capable of detecting
neutralizers in milk.

Version 1.

1.2.11.1.1.Reagents

A. Rosalic acid solution (0.1%, w/v): Weigh 100 mg of rosalic acid powder and
dissolve it in the 30 ml ethyl alcohol and make up the volume with distilled water to
obtain final volume of 100 ml.

B. Ethyl alcohol (95%): Take 95 ml of ethyl alcohol in a 100 ml volumetric flask and
make the volume up to the mark with distilled water and mix well.

1.2.11.1.2.Procedure

To 10 ml of milk add equal volume of 95% alcohol in a test tube. Add a few drops
of 0.1% alcoholic solution (w/v) rosalic acid. If alkali is present a rose red colour
appears whereas pure milk shows only a brownish colour. The limit of detection of
method is 0.1% for NaOH, 0.1% for Na2CO3 and 0.2% for NaHCO3.

Version 2.

1.2.11.1.3.Reagent

Rosalic acid solution (0.05%, w/v): First prepare 60% (v/v) ethyl alcohol solution by
mixing 60 ml ethyl alcohol (95%) and 40 ml distilled water. Weigh 50 mg of rosalic acid
powder and dissolve it in small quantity of 60% ethyl alcohol and make up the volume
to 100 ml with 60% ethyl alcohol.

1.2.11.1.4.Procedure

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Take 2 ml milk sample in a test tube and add 2 ml rosalic acid solution. Mix the contents.
If alkali is present in milk, a rose red colour appears whereas pure milk shows only a
brownish colour.

1.2.11.2. Method 2. (Alkalinity of ash)

1.2.11.2.1.Reagent: 0.1N HCI.

1.2.11.2.2.Procedure

Neutralisation with lime water/sodium bicarbonate/ caustic soda increases ash

content and alkalinity of ash. Take 20 ml of milk in a silica dish, evaporate on a water
bath and keep in muffle furnace at 550ºC to get white ash. Dissolve the ash obtained in
10 ml of water and titrate with 0.1 N HCI. The titre of more than 1.2 ml indicates the
presence of neutralizers in milk.

(Ref:- IS 1479 ( Part II) 1961 (Reaffirmed 1997) Methods of test for Dairy Industry –
Chemical Analysis of Milk. Bureau of Indian Standards, New Delhi).

1.2.12. Detection of Hypochlorites and Chloramines in Milk

1.2.12.1. Method 1. Detection of Hypochlorite

In this test yellowish fluorescence is produced due to the presence of chlorate

(potassium or sodium chlorate) in the hypochlorite solution and is proportional to the
amount present. Stannous chloride in the reaction acts as reducing agent.

1.2.12.1.1.Reagent

A. Stannous chloride (SnCl2.2H2O) solution: 0.025%, (w/v) in 73.5% sulphuric acid

(prepared by mixing three volumes of concentrated sulphuric acid and one volume
of distilled water).

1.2.12.1.2.Apparatus

A. Centrifuge

B. Tubes for centrifuge: 12.5 ml capacity

C. Mercury vapour lamp fitted with a Wood’s filter.

1.2.12.1.3.Procedure

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 MILK AND MILK PRODUCTS 2016

Cool about 3 ml of milk sample in a test tube in a freezing mixture of ice and salt
to 2 to 5°C. In another test tube, take an equal volume of the stannous chloride solution
and similarly cool and add to milk. Shake the tube whilst in freezing mixture and hold
for 3 min. Place the mixture in a centrifuge tube and centrifuge for 3 min at 2500 rpm. A
yellow-green colour is produced in the presence of hypochlorite. Alternatively, after
centrifuging, examine the tube in ultraviolet light from a mercury vapour lamp fitted
with Wood’s filter for the presence of any yellow fluorescence.

1.2.12.2. Method 2. Detection of Hypochlorites and Chloramines

1.2.12.2.1.Reagents

A. Potassium Iodide (KI) solution: Prepare fresh by dissolving 7 g of KI in 100 ml

water.

B. Dilute HCl: To 200 ml of water, add 100 ml Concentrated Hydrochloric acid (sp. gr.
1.16).

C. Starch solution: - Boil 1 g starch in 100 ml water. Cool before using.

1.2.12.2.2.Procedure

A. To 5 ml of sample in a test tube add 1.5 ml of Potassium Iodide solution, mix

thoroughly and observe colour.

B. If unaltered, add 4 ml of dilute HCl, mix thoroughly with a glass rod flattened at one
end and note colour of curd.

C. Subsequently, place the tube in a water bath previously heated to 85°C and allow it
to remain for 10 minutes. The curd will rise to the surface. Cool the tube rapidly by
placing in cold water. Note the colour of the curd and the liquid.

D. Next add 0.5.to1.0 ml of starch solution to the liquid below curd and note the colour.

1.2.12.2.3.Interpretation

The proportion of available chlorine may be ascertained from Table 1.

Table.1. Reactions with various tests to detect residual chlorine in milk.

Test Concentration of available chlorine

No* 1:1000 1:2000 1:5000 1:10000 1:25000 1:50000
A Yellowish Deep Pale yellow - - -

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 MILK AND MILK PRODUCTS 2016

brown yellow
B Yellowish Deep Light - - -
brown yellow yellow
C Yellowish Deep Yellow Yellow Pale Yellowish
brown yellow yellow
D Blue purple Blue purple Blue, dark Dark Red Pale red-
purple purple purple purple
* Indicates the step number followed in the above procedure.

Note: The method is not reliable in the presence of more than 2.5 ppm of copper.

(Ref:- IS 1479 ( Part II) 1961 (Reaffirmed 1997) Methods of test for Dairy Industry –
th
Chemical Analysis of Milk. Bureau of Indian Standards, New Delhi; A.O.A.C 17 edn, 2000
Official Method 922.08 Hypochlorites and Chloramines in milk).

1.2.13. Test for Quaternary Ammonium Compounds in Milk

Quaternary ammonium compounds (QAC) may be present in milk due to some

residual detergent solution remaining after bottle washing. The following method
detects about 5 mg / Kg in milk and is included in B.S 1741: Part II.

1.2.13.1. Qualitative method

1.2.13.1.1.Reagents

A. Indicator solution: Prepare a stock solution by dissolving 0.05 g eosin in 100 ml

acetone. Shake 10 ml of stock solution with 90 ml of tetrachloroethane and 1 g citric
acid and filter before use.

B. Buffer: Dissolve 25 g citric acid in 100 ml water and adjust to pH 3.5 with 50%
Sodium Hydroxide solution (approximately 15 ml required).

1.2.13.1.2.Procedure

To a centrifuge tube add 1 ml milk, 5 ml water, 1 ml indicator solution and 0.2 ml

buffer and shake hard for 10 seconds. Centrifuge for 5 minutes at 3200 rpm. If QAC is
present the bottom layer assumes a red or pink colour. Samples containing about 1 mg /
kg of QAC show a faint pink colour. If the colour is deep pink or red, the amount of QAC
can be approximately determined by titration with a standard anionic detergent
solution.

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 MILK AND MILK PRODUCTS 2016

(Ref :- B.S. 1741: Part II, Pearson’s Composition and Analysis of Foods 9thedn 1991, page
548).

1.2.14. Test for Presence of Anionic Detergent in Milk

1.2.14.1. Qualitative method

Alkyl benzene sulphonic acid (ABS) or anionic detergent may be present in milk
due to intentional addition of detergent in milk or due to insufficient rinsing of dairy
equipments. The following method is based on the ionic interaction between the anionic
detergent and cationic dye. Anionic detergents have a property to form a complex with
cationic dyes. The solubility of dye and dye-detergent complex differs significantly
as dye-detergent complex is relatively less polar in comparison to dye alone. Formation
of dye-detergent complex between cationic dye and anionic detergents and
subsequently its extraction into the hydrophobic solvent layer (lower) is the principle
behind the method. The method is performed by addition of methylene blue dye
solution and chloroform to milk, mixing of the content followed by centrifugation. This
results in distribution of dye colour in upper layer and lower layers. Relative intensity of
the colour is noticed in these layers. Appearance of relatively more blue colour in lower
layer indicates the presence of detergent in milk. The developed test is sensitive to
detect anionic detergent up to 0.0125% (12.5 mg/100 ml).

1.2.14.1.1.Reagents

A. Methylene blue dye: 12.5 mg is dissolved in 100 ml of distilled water. Protect the
solution against direct sunlight.

B. Chloroform (Inflammable and toxic on inhalation. Mouth pipetting is not

recommended).

1.2.14.1.2.Procedure

Pipette 1 ml of suspected milk sample into a 15 ml test tube. Add 1 ml of

dye solution followed by addition of 2 ml chloroform. Vortex the contents for about 15
sec and centrifuge at about 1100 rpm for 3 min. Note the intensity of blue color in lower
and upper layer. Relatively, more intense blue color in lower layer indicates presence of
detergent in milk. Relatively more intense blue color in upper layer indicates absence of

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 MILK AND MILK PRODUCTS 2016

detergent in milk. The method can detect presence of 0.15% level of laboratory grade
detergent (e.g. labolene) in milk.

Ref:- Rajput, Y.S.; Sharma, R. and Kaur, S. (2006) A kit for detection of detergent in milk.
Indian Patent Office file no. 1970/Del/2006.

1.2.15. Test for Presence of Skimmed milk Powder in Natural milk (Cow, buffalo,
goat, sheep)

As per the law, use of skimmed milk powder (SMP) is not allowed for adjustment
of SNF in case of sale of cow/buffalo or mixed milk. A method has been developed for
the detection of presence of SMP in liquid milk. The method is based on the fact that the
coagulum obtained from reconstituted skim milk powder by addition of acetic acid,
gives intense blue colour on boiling with phosphomolybdic acid due to certain reducing
groups present in the proteins of milk powder which are able to cause reduction of
molybdenum blue resulting in formation of blue colour.

1.2.15.1. Reagents

A. Acetic acid:4%.

B. Phosphomolybdic acid: 1% solution in water.

1.2.15.2. Procedure

Take 50 ml of milk in a 60 ml centrifuge tube. Place the tube in the centrifuge and
balance it properly. Centrifuge at 5000 rpm for 15 minutes. Decant the supernatant
creamy layer carefully. Add 0.5 ml of 4% acetic acid to skim milk portion for coagulation
of protein. Centrifuge the tubes at 5000 rpm for 5 min. Decant the supernatant and
wash the precipitate with distilled water twice. Discard the washings. Then, add 2 ml of
1% phosphomolybdic acid to the washed precipitates. Mix the contents thoroughly and
heat in a water bath at boiling temperature for 15 minutes and then cool. The curd
obtained from pure milk shall be greenish in colour whereas the curd of sample
containing skimmed milk powder shall be bluish in colour. The intensity of bluish
colour depends on the amount of the skim milk powder present in the sample

(Ref :- Journal of Food Science and Technology, Vol 22 (1985) page 207-208).

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 MILK AND MILK PRODUCTS 2016

1.2.16. Test for Detection of Gelatine in Milk

1.2.16.1. Reagents: Mercury, Conc. HNO3, Saturated Picric Acid solution.

1.2.16.2. Procedure

Take 10 ml of sample, add 10 ml acid Hg(NO3)2 solution (Hg dissolved in twice its
weight of HNO3 and this solution diluted to 25 times its volume with water). Shake
mixture, add 20 ml water, shake again, let stand 5 minutes and filter. If much gelatine is
present, filtrate will be opalescent and cannot be obtained quite clear. To portion of
filtrate in test tube add equal volume of saturated aqueous picric acid solution. Yellow
precipitate is produced in the presence of considerable amount of gelatine, smaller
amounts are indicated by cloudiness.

Note: The test is applicable to milk products also. In applying this test to sour,
fermented, cultured, or very old samples of milk, cream or butter milk ; to sterilized
cream or evaporated milk or to cottage cheese, use care to recognize precipitate
produced by picric acid when added to the Hg(NO3)2filtrates from these materials in
absence of gelatine. Such samples with or without rennet and entirely free from
gelatine, give on standing distinct precipitate when treated as above. In every case,
however these precipitates differ in character than those produced by picric acid with
gelatine. Gelatine picric acid precipitate is finely divided, more apt to remain in
suspension, settles only slowly and adheres tenaciously to the bottom of the container,
from which it is rinsed with difficulty. Precipitates produced by picric acid in the
absence of gelatine are flocculent, separate readily (leaving serum practically clear) do
not adhere to walls of container and are easily removed by rinsing with water. When
gelatine is present in sample gelatine picric acid precipitate will remain in suspension
long after flocculent precipitate has settled, but on standing overnight the characteristic
sticky deposit will be found adhering tenaciously to bottom and sides of the test vessel.
If gelatine is present in relatively high concentration (1%) gelatine, picric acid
precipitate will be voluminous and will settle rather quickly.

(Ref :- A.O.A.C 17thedn ,2000 Official Method - 920.106. Gelatine in Milk and Milk products).

1.2.17. Test for Presence of Formalin in Milk

1.2.17.1. Method 1:Hehner’s Test

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 MILK AND MILK PRODUCTS 2016

1.2.17.1.1.Reagent: Concentrated sulphuric acid.

1.2.17.1.2.Procedure

Take milk sample (2 ml) in a test tube and add 2 ml of 90 percent

H2SO4containing traces of ferric chloride from the side of the test tube slowly.
Formation of purple ring at the junction indicates formaldehyde is present in milk. If
sucrose is present, distil the milk sample (25 ml) and then carry out the test on the
distillate by taking 2-3 ml of distillate and adding 2 ml of formaldehyde free milk. The
violet coloration does not appear usually when relatively large quantities of
formaldehyde are present.

Precaution: If H2SO4 is added from the top and not from the side of the test tube, it may
burn the milk solids and affect the end result.

(Ref:- Pearson’s Composition and Analysis of foods, 9th edition, 1991 page 90; IS 1479 (
Part II) 1961 (Reaffirmed 1997) Methods of test for Dairy Industry – Chemical Analysis of
Milk. Bureau of Indian Standards, New Delhi).

1.2.17.2. Method 2. Chromotropic Acid Test

1.2.17.2.1.Reagent

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

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

1.2.17.2.2.Procedure

Take one ml of milk sample in a test tube. Add 1 ml of the chromotropic acid reagent
and mix well. Appearance of yellow color confirms the presence of formalin in the
sample, whereas; control sample will remain white (translucent).

(Ref :- IS 1479 ( Part I) 1961 (Reaffirmed 2003) Methods of test for Dairy Industry – Rapid
Examination of Milk. Bureau of Indian Standards, New Delhi).

1.2.18. Test for Presence of Hydrogen Peroxide in Milk

1.2.18.1. Method 1.Vanadium pentoxide Test

1.2.18.1.1.Reagent

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 MILK AND MILK PRODUCTS 2016

A. Vanadium pentoxide solution: Dissolve 1 g of vanadium pentoxide (V2O5) in 100 ml

dilute sulphuric acid (6 ml concentrated sulphuric acid diluted to 100 ml).

1.2.18.1.2.Procedure

Add 10 to 20 drops of vanadium pentoxide reagent in 10 ml milk sample and mix. Note
the colour of the sample. Appearance of pink or red colour indicates the presence of
hydrogen peroxide in milk.

(Ref:- A.O.A.C 17thedn, 2000 Official Method 957.08 Hydrogen Peroxide in milk).

1.2.18.2. Method 2. Para-phenylenediamine Test

1.2.18.2.1.Reagent

A. Para-phenylenediamine solution: Weigh 2.0 g of para-phenylenediamine and

dissolve it in distilled water to obtain 100 ml solution i.e. 2% aqueous solution, w/v.
Dissolution of para-phenylenediamine in water is difficult and require thorough
mixing. The solution will appear pale yellow.

1.2.18.2.2.Procedure

Add about 2 ml of milk in a test tube. Add equal volume of raw milk. Add two
drops of 2 % of para-phenylenediamine reagent. Mix well. Observe the color of the
solution in the tube. Blue color will developed in the presence of H2O2, whereas pure
milk sample remain white in color.

(Ref:- IS 1479 ( Part I) 1961 (Reaffirmed 2003) Methods of test for Dairy Industry – Rapid
Examination of Milk. Bureau of Indian Standards, New Delhi).

1.2.18.3. Method 3. Using Potassium Iodide and Starch

1.2.18.3.1.Reagents

A. Potassium iodide solution: Weigh 20 g of potassium iodide and dissolve it in

distilled water to obtain a 100 ml solution.

B. Starch solution: Take 1 g starch powder and dissolve it in distilled water by heating
and make up the volume to 100 ml.

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 MILK AND MILK PRODUCTS 2016

C. Potassium iodide–starch reagent: Mix equal volumes of 20% potassium iodide

solution and 1% starch solution.

1.2.18.3.2.Procedure

Take 1 ml of milk sample in a test tube. Add 1 ml of the potassium iodide-starch

reagent and mix well. Observe the color of the solution in the tube. Blue color will
developed in the presence of H2O2, whereas pure milk sample remain white in color.

(Ref:- Sharma, R.; Rajput, Y.S. and Naik, N.L. (2012). Detection of adulterants in milk – a
laboratory manual. NDRI Publication No. 88/2012, NDRI, Karnal, page 10).

1.2.19. Test for Presence of Boric acid and Borates

1.2.19.1. Method 1.

1.2.19.1.1.Reagents

A. Turmeric Paper: Weigh 1.5 to 2.0 g of turmeric powder in 250 ml Erlenmeyer flask
and add 100 ml 80% (v/v) ethanol. Shake for 5 min and filter. Collect the filtrate in
a flat bottom dish. Dip Whatman filter paper Grade 2 in the clear filtrate. Remove
the paper and hang to dry in air. After 1 h, cut the paper into 6 X 1 cm strips and
store in tightly stoppered bottle protected from light.

B. Conc. Hydrochloric acid (sp. gr. 1.16).

C. Ammonium hydroxide (sp. gr. 0.88).

D. Lime water or caustic soda.

1.2.19.1.2. Procedure

Take 20 ml of milk in a porcelain dish and add 1.4 ml of conc. hydrochloric acid
and mix it thoroughly. Dip a strip of turmeric paper in the acidified milk. Appearance of
characteristic red colour on the turmeric paper indicates the presence of boric acid or
borax (Na2B4O7.10H2O). The red colour changes to dark blue green on adding
ammonium hydroxide, but reappears on re-acidification with hydrochloric acid.

1.2.19.2. Method 2.

1.2.19.2.1. Reagents: Concentrated hydrochloric acid.

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 MILK AND MILK PRODUCTS 2016

1.2.19.2.2. Procedure

Take 25 ml of milk sample in a porcelain dish. Make the sample alkaline with
lime water or caustic soda and evaporate it to dryness on water bath. Ignite the dry
residue in the dish by heating over low flame to complete charring of the organic
matter. Cool the charred residue, re-digest with 15 ml distilled water and add
concentrated hydrochloric acid, drop by drop until the ignited residue is dissolved. Then
add 1 ml of concentrated hydrochloric acid in excess. Immerse a strip of turmeric paper
in the solution and dry it in air. Observe the colour change. Appearance of characteristic
red colour indicates the presence of boric acid or borax. A further check is made by
adding a few drops of ammonium hydroxide on the red turmeric paper. It will turn into
deep blue-green colour but red colour reappears on re-acidification with hydrochloric
acid.

(Ref :- IS 1479 ( Part I) 1961 (Reaffirmed 2003) Methods of test for Dairy Industry – Rapid
Examination of Milk. Bureau of Indian Standards, New Delhi).

1.2.20. Test for Presence of Salicylic acid in Milk

1.2.20.1. Reagents: Dilute HCl, Ether, 0.5% (v/v) neutral Ferric Chloride solution.

1.2.20.2. Procedure

Place 50 ml of sample in a separating funnel. Add 5 ml of dilute HCl (1+ 3) and

extract with 50 ml ether, If mixture emulsifies add 10-15 ml petroleum ether and shake.
If this treatment fails to break emulsion centrifuge or let stand until considerable
portion of aqueous layer separates, drain latter, shake vigorously and again let separate.
Wash ether layer with two 5 ml portions of water, evaporate ether in a porcelain dish
and add 1 drop of 0.5 % (v/v) neutral Ferric Chloride solution. A violet colour indicates
presence of Salicylic acid.

(Ref :- A.O.A.C 17th, edn Official method 975. 30 Salicylic acid in Food and Beverages / IS
1479 ( Part II) 1961 (Reaffirmed 1997) Methods of test for Dairy Industry – Chemical
Analysis of Milk. Bureau of Indian Standards, New Delhi).

1.3. Other Tests for Chemical Analysis of milk

1.3.1. Alkaline Phosphatase Test for Checking Efficiency of Pasteurisation in

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 MILK AND MILK PRODUCTS 2016

Liquid Milk

Alkaline phosphatase is an indigenous milk enzyme. The enzyme activity is

destroyed at pasteurization temperature and has been adopted as an index of the
efficiency of pasteurization. Since milk is a proven vector for a number of pathogenic
bacteria, including Salmonella, Campylobactor and Listeria, the test is of very great
significance to the dairy industry as a means of policing the thoroughness of heat
treatments or the addition of raw milk to heated or unheated products. In the following
method, a solution of disodium p-nitrophenyl phosphate in a buffer of pH 10.2 is used as
substrate. This compound, colourless in solution, is hydrolyzed by alkaline phosphatase
of milk to liberate p-nitrophenol, which under alkaline condition gives an intense yellow
colouration to the solution. The liberated p-nitrophenol is measured by direct
comparison with standard colour discs in a Lovibond comparator. The test does not
apply to sour milk and milk preserved with chemical preservatives.

1.3.1.1. Reagents/Apparatus

All reagents should be of analytical grade.

A. Buffer solution: 1.5 g of sodium bicarbonate and 3.5 g of anhydrous sodium

carbonate dissolved in water and made up to one litre. Store in a refrigerator and
discard after 1 month.

B. Disodium p- nitrophenylphosphate. The solid substrate must be kept in the

refrigerator.

C. Buffer-substrate solution - Weigh accurately 0.15 g of substrate (disodium p--

nitrophenyl phosphate) into a 100 ml measuring cylinder and make up to 100 ml
with buffer solution. The solution should be stored in refrigerator and protected
from light. The solution should give a reading of less than the standard marked 10
on comparator disc APTW or APTW 7 when viewed through a 25 mm cell (distilled
water is used as a blank). The solution must be discarded after one week.

D. A Lovibond Comparator with stand for work in reflected light.

E. A lovibomd comparator disc APTW or APTW 7.

F. Two Fused glass cells of 25 mm depth.

33
 MILK AND MILK PRODUCTS 2016

G. A water bath or incubator capable of being maintained at 37.5±0.5°C.

H. 1 ml pipette and 5 ml pipette.

I. 1 litre graduated flask.

J. 100 ml measuring cylinder.

K. Test tubes, nominal size 150/16 mm with rubber stoppers.

1.3.1.2. Procedure

Into a test tube pipette 5 ml of buffer substrate solution, stopper and bring the
temperature to 37°C. Add 1 ml of test milk to it shake and replace stopper, incubate at
37°C for 2 hrs. Incubate one blank prepared from boiled milk of the same type as that
undergoing the test with each series of sample. Remove the tubes after 2 h and the
content should be well mixed. Place the boiled milk blank on left hand side of the
comparator stand and test sample on the right. Take reading in reflected light by
revolving the disc until the test sample is matched. Record readings falling between two
standards by affixing a plus or minus sign to the figure for the nearest standard.

Interpretation:- The test is considered satisfactory if it gives a reading of 10 µg or less of

p-nitrophenyl per ml of milk. Properly pasteurized milk gives no discernible colour.

Note: Precautions

1. All glassware must be cleaned before use. Cleaning should be done by soaking in
Chromic acid solution prepared by slowly adding 4 volumes of concentrated H 2SO4
to 5 volumes of 8% potassium dichromate. After cleaning in chromic acid glassware
must be rinsed in warm water and distilled water and finally dried. Glassware used
for the test must not be used for any other purpose and must be kept apart from
other apparatus in the laboratory.

2. A fresh pipette must be used for each sample of milk. Pipettes must not be
contaminated with saliva.

3. The sample of milk should be examined as soon as possible after arrival at the
laboratory. If not examined immediately it must be kept at a temperature between
3°C and 5°C until examined. The sample must be brought to room temperature
immediately before being tested.

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 MILK AND MILK PRODUCTS 2016

(Ref:- IS 1479 (Part II) 1961 (Reaffirmed 1997) Methods of test for Dairy Industry –
Chemical Analysis of Milk. Bureau of Indian Standards, New Delhi; F.A.O Manuals of Food
Quality control 14 /8 page 23; BIS (1977) IS: 8479 (Part II), Methods for determination of
phosphatase activity in milk and milk products. Part I. Routine method, Bureau of Indian
Standards, New Delhi).

1.3.2. Turbidity Test for Checking Efficiency of Sterilization in Liquid Milk

The turbidity test depends upon the denaturation of proteins of milk especially
albumin after sterilisation. When solutions of inorganic salts or acids are added albumin
separates with the casein. The sample after treatment with ammonium sulphate is
filtered and heating of the filtrate shows turbidity due to presence of albumin on
account of insufficient heat treatment. If milk has been sterilised properly all albumin
will have been precipitated and no turbidity will be produced. The test is not suitable
for UHT milk.

1.3.2.1. Reagents/Apparatus

A. Ammonium sulphate AR.

B. Conical flask, 50 ml.

C. Graduated cylinder, 25ml.

D. Test tubes 150 /16 mm.

E. Funnels, 6 cm dia.

F. Beaker, 400 ml.

G. Whatman No. 12 or equivalent, 12.5 cm folded filter paper.

H. Pipette, 20 ml.

1.3.2.2. Procedure

Pipette 20 ml of milk in a 50 ml conical flask, add 4.0±0.1 g of ammonium

sulphate. Shake the flask till the ammonium sulphate is completely dissolved. Allow the
mixture to settle for 5 min, filter through a folded filter paper in a test tube. Keep about
5 ml of the above filtrate in a boiling water bath for 5 min. Cool the tube in a beaker of
cold water and examine the contents for turbidity by moving the tube in front of an

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 MILK AND MILK PRODUCTS 2016

electric light shaded from the eyes of the observer.

Interpretation:- The milk is considered sterilized when the filtrate shows no turbidity.

(Ref:- IS 1479 ( Part II) 1961 (Reaffirmed 1997) Methods of test for Dairy Industry –
Chemical Analysis of Milk. Bureau of Indian Standards, New Delhi; F.A.O Manual of Food
Quality Control 14 / 8 page 26).

1.3.3. Determination of Total Solids (Gravimetric method)

In this procedure, a known quantity of milk is dried on a boiling water bath.

Subsequently sample is dried in hot air oven at 102 ±2°C and from the weight of the
residue, the total solids content in milk is determined.

1.3.3.1. Reagents/Apparatus

A. Analytical Balance having sensitivity of 0.1 mg.

B. Desiccator provided with an efficient desiccant (for example freshly dried silica gel
with a hydrometric indicator.

C. Boiling water bath provided with openings of adjustable size.

D. Drying oven, ventilated capable of being maintained thermostatically at 102 ± 2°C

throughout the total working space.

E. Flat bottomed dishes of height 20 – 25 mm, dia 50 - 75 mm and made of appropriate

material (stainless steel, nickel or aluminium) provided with well fitted readily
removable lids.

F. Water bath capable of being maintained at 35° - 40°C.

1.3.3.2. Procedure

Transfer sample to a beaker, warm slowly to 35° - 40°C on a water bath with
careful mixing to incorporate any cream adhering to the sample. Cool the sample
quickly to room temperature. Heat a dish with its lid alongside in the drying oven at
least 1 hour. Place the lid on the dish and immediately transfer to a desiccator. Allow to
cool to room temperature (at least 30 mins) and weigh to the nearest 0.1 mg. Add 5 ml
of prepared sample, place the lid on the dish and weigh again. Place the dish without
the lid on the vigorously boiling water bath in such a way that the bottom of the dish is

36
 MILK AND MILK PRODUCTS 2016

directly heated by the steam. Continue heating till most of the water is removed.
Remove the dish from the water bath, wipe the underside and place it in the oven
alongside the lid and dry in the oven for 2 hours. Place the lid and transfer to the
desiccator. Allow the dish to cool and weigh to the nearest 0.1 mg. Again heat the dish
with its lid alongside in the oven for 1 hour. Place the lid on the dish and immediately
transfer to the desiccator. Allow to cool and weigh again. Repeat the operation again
until the difference in the two consecutive weighing does not exceed 1 mg. Record the
lowest mass.

1.3.3.3. Calculation
M M0
Total Solid Content =M x 100
1 M0

Where

M0 = mass in g of dish + lid

M1 = mass in g of dish + lid and test portion

M2 = mass in g of dish + lid and dried test portion

Round the value obtained to nearest 0.01 % (m/m)

(Ref: - IS 12333 - 1997 / ISO 6731: 1989 Milk, Cream and Evaporated milk. –
Determination of total Solids Content -reference method. Bureau of Indian Standards, New
Delhi).

1.3.4. Determination of Fat in Milk

1.3.4.1. Method 1. Gerber Method

The milk is mixed with sulphuric acid and iso-amyl alcohol in a special Gerber
tube, permitting dissolution of the protein and release of fat. The tubes are centrifuged
and the fat rising into the calibrated part of the tube is measured as a percentage of the
fat content of the milk sample. The method is suitable as a routine or screening test. It is
an empirical method and reproducible results can be obtained if procedure is followed
correctly.

1.3.4.1.1. Reagents /Apparatus

A. Sulphuric acid with a density of 1.807 to 1.812 g/ml at 27°C corresponding to a

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 MILK AND MILK PRODUCTS 2016

concentration of sulphuric acid from 90-91 percent by weight.

B. Amyl alcohol for milk testing (furfural free). It should have density between
0.808 to 0.818 g/ml at 27°C.

C. Gerber sulphuric acid: Sulphuric acid shall have a density of 1.807 to 1.812 g/ml at
27°C corresponding to a concentration of sulphuric acid from 90 to 91% by mass.

Preparation of Gerber Sulfuric acid: Take required volume of water in a pyrex flask
(generally 100 ml of water is required for 900 ml of concentrated sulfuric acid) kept in
a basin of ice cold water. Carefully add the commercial sulfuric acid in small quantities
at a time keeping the container sufficiently cold and mix gently. Observe the following
precautions while performing the above experiment.

- Sulfuric acid is very corrosive. Handle it with care.

- Add acid to water. Add small quantities of acid to water at a time and cool the
mixture by stirring. Never add water to acid.

- Use heat resistant flask for dilutions.

After cooling the flaks, check the specific gravity of Gerber acid with hydrometer and if
necessary adjust the Gerber acid to the correct specific gravity with addition of water
or acid taking same precautions as before till specific gravity is in the range of 1.807 to
1.812 g/ml at 27°C (or 1.815 to 1.820 g/ml at 20°C). Store the prepared acid in a glass
stoppered bottle to avoid absorption of water.

D. Iso-amyl alcohol (C5H11OH): The iso-amyl alcohol shall have density of 0.803 to
0.805 g/ml at 27°C and should be furfural free.

E. Gerber butyrometer; 6, 8 and 10 percent (ISI marked).

F. Pipette: 10 ± 0.25 ml or automatic measure or tilt measure for sulphuric acid.

G. Pipette: 10.75 ± 0.03 ml for milk.

H. Pipette: 1 ± 0.05 ml or automatic measure or tilt measure for iso-amyl alcohol.

I. Lock stoppers for butyrometers.

J. Lock stopper key.

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