Chemical Analysis of

UNIT 7 CHEMICAL ANALYSIS OF Milk and Milk Products

MILK AND MILK PRODUCTS

Structure

7.0 Objectives
7.1 Introduction
7.2 Test Methods
7.3 Let Us Sum Up
7.4 Key Words
7.5 Some Useful Books
7.6 Answers to Check Your Progress

7.0 OBJECTIVES
After reading this unit we should be able to:

l Perform different platform tests to accept or reject the milk

l Determine different milk constituents

l Analyze different milk products for their quality

l Analyze the water used in dairy plant for different attributes.

7.1 INTRODUCTION
Analysis is done to know the composition of a product. Analysis determines not
only the quality of the product but also the quantity of ingredients required in
manufacturing. Payment is based on certain important parameters of the product.
In case of milk it is Fat and SNF percent. In the industry Fat by Gerber Method
and SNF by using Lactometer are estimated. In some dairies Electronic Milk
Tester is used for determination of Fat in milk. Very few dairies use Milkoscans
for the determination Fat, Protein, Lactose and SNF. To determine the quality of
a product, tests for acidity, adulterants, preservatives etc have to be carried out.
Knowledge of methods, good laboratory practices and good testing skills are
required in the analysis of milk and milk products.

7.2 TEST METHODS

Title Sub-Titles
Testing of Milk Platform Tests:
Clot on Boiling (COB) Test
Alcohol Test (To determine heat stability of milk)
Determination of Titratable Acidity
Determination of Preservative and Adulterants 41
Chemical and Hydrogen Peroxide
Microbiological Analysis
of Milk and Milk Products Hypochlorites
Formaldehyde (Honnies Test)
Boric Acid and Borates
Maltodextrins
Urea
Neutralizers
Starch
Sugar
Salt
Mineral Oil
Other Tests:
Determination of milk fat (Gerber Method)
Testing Fat in Homogenized milk
Microscopic observation of fat globules size
Determination of SNF (Volumetric Method)
Determination of Total Solids
Phosphatase Test
Determination of Ash content in milk
Determination of Protein in milk
Testing of Milk Powder
Moisture Content by IMA
Moisture Content by drying method
Titrable Acidity
Rosalic Acid Test
Scorched Particles
Ash Content
Insolubility Index
Fat Percent (WMP)
Fat percent (SMP)
Bulk Density
Testing of Butter
Determination of Moisture
Determination of Curd
Determination of Fat
Titrable Acidity
Analysis of Salt in Table Butter
42
Testing of Ice Cream Chemical Analysis of
Milk and Milk Products
Determination of Fat
Determination of Protein
Titratable Acidity
Determination of Total Solids
Phosphtase Test
Titratable Acidity (Candy Mix)
Determination of Total Solids (Candy Mix)
Testing of Paneer
Determination of Moisture
Determination of Fat
Determination of Acidity
Testing of Ghee
Determination of Moisture
Free Fatty Acids Percent as Oleic Acid
Butyro Refractometer Reading
RM Test
Testing of Flavoured Milk (UHT Milk)
Determination of Fat
Determination of Total Solids
Determination of Acidity
Testing of Sterilized Cream
Determination of Fat
Determination of Acidity
Testing of Lassi Determination of Fat
Determination of Total Solids
Determination of Acidity
Testing of Curd Determination of Fat
Determination of Acidity
Testing of Water Hardness
PH
Sulphite Ions (For Boiler Water)
Phosphate Ions (For boiler water)
Residual Chlorine in Water by Tolidine Method
Residual Chlorine in Water by Chlorotex Method
Preparation Chemical Solutions/Reagents

43
Chemical and PLATFORM TESTS
Microbiological Analysis
of Milk and Milk Products
Clot on Boiling Test :

Apparatus:

Test Tubes, Spirit Lamp

Principle:

To detect in a rapid manner the presence of extent of developed acidity, which

might render the milk unsuitable for processing and distribution.

Procedure:

Conduct COB test by taking about 5 ml of milk in a test tube, boil on the flame
of a spirit lamp.

Observation:

Formation of clots in the test tube indicates COB positive milk and is unacceptable.

Alcohol Test (To determine heat stability of milk) :

Apparatus:

Test Tubes

Reagents:

80% ethyl alcohol; 60% ethyl alcohol

Principle:

This test is conducted to know the heat stability of milk. It is useful in the
manufacture of condensed milk, UHT, Dried and Pasteurized milk. 80% ethyl
alcohol is recommended for selection of milk to process under UHT system. For
selection of milk to pasteurize, 60% ethyl alcohol is recommended. One of the
factors causing heat instability of milk is disturbance in mineral balance.

Procedure:

Take 5 ml of milk in a test tube. Add the desired percent of ethyl alcohol in equal
quantity. Shake the contents. Observe for clots.

Observation:

Absence of clots indicate that the milk is suitable for respective heat treatment.

Determination of Titratable Acidity

Apparatus:

Burette, Conical flask (100 ml capacity), Stirring Rods, Pipette (10 ml) & Tilt
Measure (1 ml) for indicator.
44
Reagents: Chemical Analysis of
Milk and Milk Products
N/10 NaOH, Phenolphthalein indicator.

Principle:

When freshly drawn, milk contain very low acidity acid contributed by its
constituents, namely, carbondioxide, citric acid, albumin, casein and minerals.
Later during storage due to bacterial action on lactose acidity increases. Since
alkali neutralizes acid the acidity of milk is estimated through titration against
standard alkali using phenolphthalein as indicator. Normal acidity of milk range
from 0.14% to 0.16%. Lower range (less than 0.12%) indicate neutralization and
higher range (more than 0.16%) indicate development of acidity due to bacterial
action or mastitis milk.

Procedure:

Take 10 ml milk in 100 ml conical flask, add 10 ml distilled water. Add 1ml
phenolphthalein indicator and titrate against N/10 NaOH till a faint pink colour
appears to determine the percent lactic acid in milk.( The pink color has to match
with rosaniline acetate bench solution.)

Calculation:

Calculate the acidity % as volume of NaOH used X 0.09.

Check Your Progress - 1

1. Why COB test is conducted?

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2. What is the principle of Alcohol Test?

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3. Where this test is most useful?

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45
Chemical and 4. What substances are responsible for the Acidity in milk?
Microbiological Analysis
of Milk and Milk Products ……………………………..…………………………………………......
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5. What is the significance of developed acidity in milk?

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Detection of Preservatives and Adulterants

Note: For detection use Kits developed by NDDB. These Kits contain prepared
solutions and glassware that can be used at the site of testing. They are suitable
for detection of urea, ammonia fertilizers, nitrate pond water, starch and cereal
flours, sugar/sucrose, glucose, salt, neutralizers, hydrogen peroxide and formalin.
The Kits are easy to carry and cost effective. However for the benefit of students
some test methods are given below:

Hydrogen Peroxide

Apparatus:

Test Tubes

Reagents:

2% aqueous solution of paraphenyl diamine hydrochloride (fresh solution).

Principle:

It is a preservative. It is not permitted under the law to be added to milk or

milk products for consumption.

Procedure:

Method-1

Take 10 ml of milk in a test tube. Add 2 drops of PPDH. Mix thoroughly

and observe.

Observation:

Blue colour indicates the presence of hydrogen Peroxide.

46
Hypochlorites Chemical Analysis of
Milk and Milk Products
Apparatus:

Test tubes, water bath.

Reagents:

7% potassium iodide solution, 50% hydrochloric acid, 1% starch solution.

Principle:

Hypochlorites act as preservatives and sterilizing agent in milk. They are not
permitted under the law to be added to milk or milk products.

Procedure:

l Take 5 ml of milk and add 1.5 ml potassium iodide solution (7%) and mix. A
pale yellow or yellowish brown colour indicates the presence of hypochlorites.

l If no colour appears add 4 ml of hydrochloric acid to the above mix. Mix

thoroughly and keep in water bath at 85OC for 10 minutes. Cool by immersing
in cold water and add 1 ml of starch (1%) allowing it to flow down below the
curd.

Observation:

A blue to purplish red colour indicates the presence the presence of Hypochlorites.

Formaldehyde (Hehner Test)

Apparatus:

Test Tubes.

Reagents:

Ferric Chloride (1%), Conc. Sulphuric Acid.

Principle:

It acts as preservative. It is permitted only in samples to be used in chemical

analysis. Not permitted to be used in milk or milk products used for consumption.

Procedure:

Take 10 ml of milk in a test tube. Add 0.5 ml of Ferric Chloride (1%). Mix. Add
carefully 5 ml Conc. Sulphuric Acid down the side of the test tube in such a way
that it forms a separate layer at the bottom without mixing with the milk.

Observation:

Characteristic violet colour ring at the junction indicates presence of Formaldehyde.

47
Chemical and Boric Acid and Borates
Microbiological Analysis
of Milk and Milk Products Apparatus:

Test Tubes

Reagents:

Conc. HCI; Turmeric Paper; NH4OH (28%)

Principle:

It acts as preservative. Not permitted by the law to be added in milk and milk
products.

Procedure:

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

l Add about 1 ml of conc. HCI and mix well.

l Dip a strip of turmeric paper in the acidified milk.

l Dry the paper immediately and note the change in colour.

Observation:

Turmeric paper turning into red indicates the presence of Boric Acid and Borates.
Further confirmation is done by adding a drop of ammonium hydroxide. Colour
changes to dark green indicate the presence of Boric Acid.

Maltodextrins

Apparatus:

Test Tubes, beakers, pipettes, Whatman filter paper No.42, funnels

Reagents:

10% Citric acid solution

1% Iodine solution

Principle:

To increase the density of normal milk. It is an adulterant.

Procedure:

Take 20 ml of milk sample in a beaker. Boil & coagulate the milk using 10% citric
acid solution. Filter through Whatman filter paper No.42 & collect the filtrate.
Add 3 drops of iodine solution to 5 ml. filtrate & mix well.

Observations:

The appearance of chocolate brown colour indicates the presence of maltodextrins.

48
Urea Chemical Analysis of
Milk and Milk Products
Reagents:

P-Dimethylamino Benzaldehyde (DMAB) solution: Dissolve 16 gm in 1 litre of

alcohol and add 100 ml previously chilled Hcl. Stable for 1 month.

Principle:

It is added to increase the density of normal milk. It is an adulterant. Normal milk

in this test gives light yellow colour urea is a natural constituent of milk. However
more than 700 ppm is considered as added urea.

Procedure:

Take 2 ml milk in a test tube, add 2 ml DMAB solution and mix the contents.
Appearance of yellow colour indicates the presence of urea making the milk
unacceptable.

Rosalic Acid

Reagent:

Ethyl alcohol (95%), Rosalic Acid

Principle:

Rosalic develops a rose red colour with milk containing alklies, where as it gives
only a brownish colouration with pure milk.

Procedure:

(a) To 5 ml of milk in a test tube, add 5 ml of alcohol , Add few drops of one
percent (w/v) alcoholic solution of rosalic acid and mix. If neutralizer is present,
a rose red colour appears whereas pure milk shows only a brownish colouration.

(b) Take 2 ml rosalic acid solution (0.05% in 60:40 alcohol and distilled water) in a
test tube, add 2 ml of milk. Rose-Red colour development indicates neutralizer
presence in milk.

Note: Method (b) is commonly used in dairies to know heat stability and detection
of added neutralizers, in milk.

Starch

Reagent:

Iodine solution (1%)

Principle:

Iodine solution gives intense blue colour with starch due to formation of
unstable complex starch-iodo compound. Added to increase the density of
normal milk. (Adulterant).

49
Chemical and Procedure:
Microbiological Analysis
of Milk and Milk Products Take 3 ml. milk in a test tube, boil and cool under tap water. Add a drop of
1% Iodine solution.

Observation:

Presence of starch is indicated by the appearance of a blue colour which disappears

when the sample is boiled and re-appears on cooling.

Sugar

Reagent:

Conc. HCl, Resorcinol

Principle:

Resorcinol produces red colour with sucrose in acidic media. Sugar increases
specific gravity of milk. Unless permitted specifically under law it is considered as
an adulterant.

Procedure:

(a) To about 15 ml. of milk in test-tube, add one millilitre of concentrated

hydrochloric acid and 0.1gm of resorcinol and mix. Place the tube in boiling
water-bath for five minutes.

Observation:

In the presence of cane sugar, a red colour is produced.

(b) Take 3 ml of milk in a test tube and add 5 ml dilute HCl (1:2) containing
resorcinol (0.1 gm. resorcinol dissolved in 100 ml dilute HCl). Mix well and
keep the test tube in boiling water for 5 minutes.

Observation:

In the presence of cane sugar, a red colour is produced.

Salt

Reagent:

Silver nitrate solution (0.1341 %), Potassium chromate (10%)

Principle:

It is added to increase the density of normal milk (adulterant). Sometimes

brine solution can enter milk by accident during chilling.

Procedure:

Take 5 ml of silver nitrate (0.1341%) solution in a test tube and add two drops
of potassium chromate (10%) solution. It will give brick red colour. To this add
exactly 1 ml of milk and mix.
50
Observation: Chemical Analysis of
Milk and Milk Products
Appearance of yellow colour show the presence of salt.

Mineral Oil (Holde’s Test)

Reagent:

Alcoholic KOH.

(Dissolve 7 gms. KOH pallets in 10 ml distilled water and make the volume to
250 ml by adding rectified spirit).

Principle:

Mineral oil is an adulterant. It is added to give a false fat% during fat testing.

Procedure:

Take 1 gm of ghee extracted from milk in a 250 ml conical flask and to this add
22 ml alcoholic KOH and keep the flask on boiling water bath or hot plate and
saponify it till there is no oil droplets. To this add 25 ml of boiling distilled water
and swirl the flask. Development of turbidity indicates the presence of mineral
oil & such milk is unacceptable. No turbidity indicates absence of mineral oil.

Sample found unacceptable at any stage during platform tests renders the milk
unfit for acceptance. Confirm the results by drawing second sample. Reject such
milk

Check Your Progress - 2

1. Why preservatives and adulterants are added to milk?

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2. It maltrodexbin a preservative or adulterant?

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3. What is the objective in adding sugar and mineral oil to milk?

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51
Chemical and I. Testing of Milk
Microbiological Analysis
of Milk and Milk Products
i) Determination of Milk Fat :

Gerber Method:

Apparatus:

l Butyrometer 10% scale (0-10% scale with 0.1% mark)

l 10 ml automatic measure for sulphuric acid

l 10.75 ml pipette for milk

l 1 ml automatic measure for amyl alcohol

l Stoppers for butyrometer

l Gerber Centrifuge (1400 ± 70 RPM)

l Water bath (65 ± 2OC)

l Butyrometer stand

l Lock Stopper Key

Reagents:

Sulphuric acid (Specific Gravity 1.807 to 1.812 gm/ml at 27OC) corresponding

to a concentration of 90 to 91% by mass which is normally called as Gerber
Acid.

Amyl alcohol (Specific Gravity 0.810 to 0.812 gm/ml at 27OC) conforming to

grade 1 of IS:360:1964 of clear and colourless liquid shall distil between 130OC
to 132OC.

Principle:

Rapid Method of estimation of Fat in the fluid milk is known as “Gerber Method”.
It is based on the principle of measuring the volume of Fat released from a known
volume of the milk sample in a specially devised and accurately calibrated modified
form of glass cylinder called Butyrometer.

When a definite quantity of sulphuric acid and amyl alcohol are added to a definite
volume of milk, the proteins will be dissolved and fat globules will be set free with
the help of amyl alcohol which remains in liquid state due to heat produced by
the acid. During centrifugation fat being lighter (fat density 0.90 gm/ml at 40OC)
gets separated and comes to the top of the solution.

Procedure:

l Transfer 10 ml of sulphuric acid into the butyrometer by means of automatic

measure taking care not to wet the neck of the butyrometer with the sulphuric
acid.

52
l Warm the sample to approximately 27OC and mix thoroughly but do not shake Chemical Analysis of
Milk and Milk Products
it so vigorously as to cause churning of the fat. Allow the sample to stand for 3-
4 minutes after mixing to allow air bubbles to escape, invert the sample bottle 3-
4 times immediately prior to taking milk for test.

l Transfer 10.75 ml of sample into the butyrometer by using 10.75 ml milk pipette
by following the below mentioned procedure.

l Add 1 ml of amyl alcohol into the butyrometer by means of automatic measure

and close the neck of the butyrometer firmly with a stopper without disturbing
the contents. Shake the butyrometer carefully without inverting it until the contents
are thoroughly mixed, the curd is dissolved and no white particles are seen in the
liquid. Then invert the butyrometer few times to mix the contents thoroughly. (It
is always safe to use butyrometer stand while mixing/shaking the contents.)

l Transfer the butyrometer quickly in the waterbath at 65 ± 2OC and leave it there
for not less than 5 minutes.

l Take out the butyrometer out of the water bath and centrifuge at 1400 rpm for
4 minutes. Bring the centrifuge to stop gradually, transfer the butyrometers
(stoppers downwards) into the water bath at 65 ± 2OC and allow the butyrometer
to stand for not less than 3 minutes and not more than 10 minutes and take
down reading.

l Adjust the fat column within the scale on the butyrometer and take the reading.

l Use lock stopper key for fixing and removing the stopper into / from the
butyrometer and also for adjusting the fat reading.

Procedure for Testing Fat in Homogenized Milk

In case of homogenized milk, repeat the temperature adjustment and centrifuging

before taking the reading. If the second value does not exceed the first value by
more than half a smallest scale division of the butyrometer, the second value shall
be recorded as the fat content of the milk.

Microscopic Observation of Fat Globule size to assess Homogenization

Efficiency

Principle :

Homogenization of milk and cream is done to make fat globules into smaller and
equal size of about 2 microns. It helps in the distribution of fat uniformly in the
system and also delays/almost arrest the rise of fat to the top.

Apparatus:

l Microscope fitted with low, high and oil immersion objectives and 10 X eye
piece.

l Microscope Slides

l Cover glass.

l Graduated cylinder 100 ml. 53

Chemical and l Pipette 1 ml
Microbiological Analysis
of Milk and Milk Products l Test tubes 150 ml.

l Micrometer fitted in the eye-piece.

Procedure:

l Dilute the sample to be tested with distilled water so that the sample contain
from 0.1 to 0.2% Fat. The following table give the approximate dilution to use:

Milk -dilute 1 ml with 25 ml distilled water.

20% Cream -dilute 1 ml with 150 ml distilled water.

30% Cream- dilute 1 ml with 225 ml distilled water.

40% Cream -dilute 1 ml with 300 ml distilled water.

10% Ice Cream- dilute 1 ml with 75 ml distilled water.

20% Ice Cream -dilute 1 ml with 150 ml distilled water.

l Prepare a slide and observe under oil immersion. Note the size of the fat globules
through micrometer fitted in the eye-piece.

l Observations: In homogenized milk 90% of the fat globules should be within 2

micron size. No lumps should be present. No clumping formation should be
there.

ii) Determination of SNF

Volumetric Method

Apparatus:

Calibrated Lactometer at 15.5OC, Lactometer Jar (suitable to float the Lactometer),

Calibrated Thermometer and Enamel Tray.

Principle:

The constituents of milk are broadly divided into fat and solids-not-fat.The major
components of SNF are proteins and lactose. Fat is estimated more easily by
Gerber method. The specific gravity of milk is measured using a lactometer.
Corrections are made to the reading. Fat reading is taken using Gerber method.
A formula is used to workout SNF. Some constant factor derived from gravimetric
analysis is taken into calculation to get the results near to Gravimetric results.
Composition of milk (Cow/ Buffalo/ Breeds/ Season/ Feeding pattern) , the
constant factor, calibration of lactometer and Adulteration play a major role in
determing SNF using lactometer.With all these drawbacks it is still considered an
important test along with fat estimation in the Q.C. Laboratory.

Procedure:

l Warm the milk sample to 40OC to 45OC and maintain at this temperature for 5
54 minutes.
l Mix the contents by rotating and inverting the bottle, taking care to avoid the Chemical Analysis of
Milk and Milk Products
formation of air bubbles and froth.

l Cool the sample approx. near to the calibrated temperature of the Lactometer
(15.5OC).

l Invert the sample bottle two or three times, pour enough milk into the lactometer
jar taking care to avoid the formation of air bubbles, so that some milk overflows
when the lactometer is inserted.

l Insert the lactometer gently to wet the stem not more than a short length, about
3 mm beyond the position of equilibrium. The lactometer should float freely and
not touch the sides of the cylinder.

l Allow the lactometer to remain steady in the milk. Take the reading at 15.5OC
within 30 seconds. Note the reading of the lactometer corresponding to the top
of the meniscus on the stem without the error of parallax.

l Determine the fat percentage as per Gerber Test Method.

Calculation of Solids not Fat:

Formula :

CLR
SNF% = + 0.2 + 0.29
4

When CLR = Corrected lactometer reading

F = Fat Percentage

Note: The constant factor 0.29 is an example for lactometer at 15.5 degree C.
Lactometer at 840 F can also be used. By performing gravimetric analysis one has
to arrive at the constant factor specific to the area of operation.

iii) Determination of Total Solids

Gravimetric Method:

Apparatus:

Shallow flat bottomed dishes of aluminium alloy, nickel, stainless steel, porcelain
or silica, 7 to 8 cm diameter, about 1.5 cm in height and provided with easily
removable lid. Hot air oven maintained at 100 OC, ± 0C Hot water bath, Analytical
Balance.

Principle:

By evaporating water content in milk under controlled conditions the total solids
contents can be determined accurately.

Procedure:

l Weigh accurately the clean, dry empty dish with the lid. Pipette into the dish
about 5 ml of the well mixed sample of milk and weigh quickly with the lid on the 55
Chemical and dish. Place the dish uncovered on a boiling water-bath. Keep the base of the
Microbiological Analysis dish horizontal to promote uniform drying and protect it from direct contact with
of Milk and Milk Products
the metal of the water-bath. After at least 30 minutes, remove the dish, wipe the
bottom and transfer to a well ventilated oven at 98 to 100OC for about 3 hours,
placing the lid by side of the dish. The bulb of the thermometer shall be just
above the shelf carrying the dish. The dish shall not be placed near the walls of
the oven.

l After three hours, cover the dish and immediately transfer it to a desiccator.
Allow cooling for about 30 minutes and weigh. Return the dish uncovered, and
the lid to the oven and heat for one hour. Return to the desiccator, cool weigh as
before. Repeat if necessary until the loss of weight between successive weighing
does not exceed 0.5 mg. Note the lowest weight.

Calculation:
w
Total Solids, percent by weight = × 100
W

Where,

w = weight in gram of the residue after drying, and

W = weight in gram of the prepared sample taken for test.

iv) Phosphatase Test

Apparatus:

All purpose Lovibond comparator Pipettes: 1.0 ml /5ml.

Standard discs-APTW or APTW/7 Graduated flask: 1000ml.

Two 25 mm. fused glass cells. Measuring cylinder:100ml.

Water bath at 37 .5 +- 0.5degree centigrade Test tubes

Chemicals Required:

Phosphatase dye: Dissolve 0.15 gm. of 4-Nitrophenyl phosphate disodium salt in

100 ml. buffer solution.

Buffer solution: Dissolve 3.5 gm of Na2Co3 and 1.5 gm NaHCO3 to make 1 litre
of solution in distilled water. Keep the buffer solution and the dye in a cool place.

Principle:

Raw milk contains an enzyme called alkaline-phosphatase. It is destroyed at the

temperature necessary for efficient pasteurization. Pasteurisation indicate the
destruction of pathogens. But when milk containing phosphatase is incubated with
p-nitro-phenyl disodium orthophosphate, the liberated paranitro-phenol gives a
yellow colour under the alkaline conditions of the test. The colour is a measure
of the phosphatase content of the milk sample. Therefore, if phosphatase is
present it indicate that the milk is not properly- pasteurised or has been
contaminated after the heating process by raw milk.
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Procedure: Chemical Analysis of
Milk and Milk Products
Fill 5 ml solution of the phosphatase dye into test tubes marked at 10 ml . Add
1 ml of the milk to be tested, close the tubes with rubber stoppers and invert to
mix. Prepare in the same way a blank form a boiled milk of the same type of that
under test. Incubate all the tubes at 37 degree C. Read the yellow colour after
30 minutes, return to the bath, and take a second reading after incubation for a
further 90 minutes. The yellow colour is read in a Lovibond all-purpose comparator
on a resazurin stand, fitted with the disc calibrated in microgram p-nitrophenol.
The blank is placed on the left of the stand and the sample on the right. Readings
are taken by looking down on to the two apertures with the comparator facing
a good source of north daylight; the disc is revolved until the sample is matched;
readings falling between two standards are recorded to the nearest reading.

Interpretation of Results: Disc Reading after 30 minutes incubation

Interpretation

0 or trace Properly pasteurized

6 Doubtful

10 or ever Under pasteurized

Disc Reading after 2 Hours

Incubation Interpretation

0 to 10 Properly pasteurized

Over 10 Under pasteurized

The 30 minute test will reveal any serious fault in pasteurization, but to enable
minor errors to be detected, readings shall be taken after further incubation for
90 minutes.

Check Your Progess - 3

1. What are the time-temperature combination for destruction of phosphatase?

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2. Does the destruction of the enzyme phosphatase indicate the destruction of

pathogenic organisms and if so why?

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57
Chemical and v) Determination of Ash Content in Milk
Microbiological Analysis
of Milk and Milk Products
Apparatus:

Crucibles, Dessicator, Analytical Balance, Muffle Furnace, Electrical Heater

Principle:

Milk contains soluble substances containing salts like phosphates, citrates, sulphates,
chlorides and bicarbonates of calcium, magnesium, potassium, sodium etc. Heating
of milk at higher temperature decomposes organic matter and inorganic salts are
left behind in the form of ash. Addition of neutralizers increase the ash contents
in milk.

Procedure:

l Heat the crucible in order to remove any moisture from it.

l Cool the crucible in a dessicator to room temperature and weight it accurately.

l Weigh quickly and accurately about 10 g of milk in the weighed crucible.

l Evaporate the sample to dryness on a water bath, avoid spurting of the milk by
using a thin glass rod drawn to a point and remove any particle adhering to the
rod into the dish.

l Keep the evaporated milk sample in a muffle furnace at temperature not more
than 550OC until the ash is free from carbon. Normally it takes about 4 hours.
Switch off power supply, wait for an hour before the crucibles are shifted to
desiccator.

l Cool in a desiccator to room temperature and weigh quickly.

Observations:

Record the weights

l Weight of empty crucible = W g

l Weight of crucible with milk = W1 g

l Weight of crucible after ashing = W2 g

l Weight of milk taken = W1 – W g

l Weight of ash = W2 – W g

Calculation:

W2 − W
Percentage of ash by weight = × 100
W1 − W

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vi) Determination of Protein in Milk by Formal Titration (Pyne’s Chemical Analysis of
Milk and Milk Products
Method):

Apparatus:

Pipette – 10 ml, 2 ml and 1 ml graduated, Burette, flask 100 ml.

Reagents:

Neutral formalin, saturated potassium oxalate solution, N/10 sodium hydroxide,

phenolphthalein indicator

Principle:

When formaldehyde is added to milk which has previously titrated against standard
alkali to the end point of an indicator like phenolphthalein, it binds the amino
groups of the milk proteins and release an equivalent amount of proteins (H-ions),
which could be titrated against the alkali to the same end point. The amount of
alkali used in the second titration is a measure of the amino groups originally
present in the proteins.

Procedure:

l Pipette 10 ml of the well mixed sample of milk into a 100 ml of flask.

l Add 5 drops of phenolphthalein indicator

l Add 0.4 ml of saturated potassium oxalate and keep it aside for 2-4 minutes
without disturbing.

l Titrate the milk against the standard alkali to its end point.

l Add 2 ml of neutral formalin and mix well.

l Titrate against the standard alkali to the same end point as before.

l Record the volume of alkali used in the second titration (V).

Calculation :

The percentage of protein in the given sample of milk = V × 1.7

Note:

If neutral formalin is not available, determine the formaldehyde acidity correction

factor as given below and substract it from the second titration value and calculate
the percentage of protein.

Pipette 2 ml of formalin in a 100 ml flask, add 10 ml of distilled water and 5

drops of phenolphthalein. Neutralize with N/10 sodium hydroxide to the usual end
point. The volume of N/10 sodium hydroxide used gives the formaldehyde acidity
correction factor.

59
Chemical and II. Testing of Milk Powder
Microbiological Analysis
of Milk and Milk Products
Moisture Content

Routine Method

Use Infrared Moisture Analyser and note the Moisture percent reading.

Gravimetric Method

Apparatus :

Refer Gravimetric Method for Milk.

Procedure:

l Place the dish and its lid in the oven at 102±2OC for one hour. Transfer the dish
and its lid from the oven to the desiccator. Allow it to cool to room temperature
and weigh it.

l Mix thoroughly the milk powder sample and take approximately 1 gm. of the
powder in the dish, cover the dish and weigh the covered dish accurately and
quickly.

l Uncover the dish and put it with its lid in the oven at 102±2OC for two hours.

l Replace the lid, transfer the covered dish to the desiccator, allow it cool to room
temperature and weigh it accurately and quickly.

l Heat the uncovered dish and lid in the oven at 102±2OC for further 1 hour,
replace the lid, allow the covered dish to cool to room temperature in the
desiccator and weigh it.

l Repeat the process until successive weighings do not differ by more than 0.5
mg. It is usually found that drying is complete after the first two hours.

Calculation:

W −W
Moisture, percent by mass = W − W × 100
1 2

Where

M1 = initial mass in g of the dish and lid with the material taken for analysis.

M2 = the final mass in g of thed ish and lid with the material after drying;
and

M = Mass in g of the empty dish

TITRATABLE ACIDITY
Apparatus:

60
Refer Determination of Titratable Acidity for Milk.
Procedure: Chemical Analysis of
Milk and Milk Products
Weigh accurately 1 gm of the sample in a 100 ml beaker. Add 10 ml of warm
distilled water and make a solution using a glass rod. Cool to room temperature.
Add 1 ml phenolphthalein indicator and titrate against N/10 NaOH till a faint pink
colour persists. Calculate the acidity % as Volume of NaOH used X 0.9.

ROSALIC ACID TEST

Procedure:

To 5 ml of reconstituted powder in a test tube, add 5 ml of alcohol. Add few

drops of one percent (w/v) alcoholic solution of rosalic acid and mix. If neutralizer
is present, a rose red colour appears whereas negative test shows only a brownish
colouration.

Take 2 ml rosalic acid solution (0.05% in 60 : 40 alcohol and distilled water) in

a test tube, add 2 ml of reconstituted powder. Rose-Red colour development
indicates neutralizer presence in powder and formation of flakes indicates alcohol
test positive.

SCORCHED PARTICLES
Procedure:

Reconstitute 10 grams of SMP or 13 grams of WMP into 100 ml. of distilled

water in a flask. Observe the bottom of the flask in which powder is reconstituted
after keeping undisturbed for some time. Filter the reconstituted milk through
scorched particle tester and compare the filter pad with ADPI comparison card.
Powder with scorched particles more than Disk B reading is rejected.

ASH CONTENT
Procedure:

Weigh 3 gm. of powder in a well-dried silica crucible and heat on a heater till no
smoke comes out of the powder.

Keep it into a muffle furnace maintained at 550±20°C for three hours till grey ash
formation. Switch off the muffle furnace and let the temperature fall. Transfer the
crucible to a desiccator, cool completely and weigh. Heat the dish again at
550±20°C for 30 minutes. Cool the dish in a desiccator and weigh. Repeat this
process of heating for 30 minutes, cooling and weighing until the difference between
two successive weighings is less than one milligram. Record the lowest mass.

Calculate ash percentage as follows :

W2 − W
[A] Ash % by mass = × 100
W1 − W

M2 = Weight of crucible with ash.

M = Weight of empty crucible.

M1 = Weight of crucible with powder. 61

Chemical and
Ash% by mass
Microbiological Analysis [B] Ash % on dry matter basis = (100 − moisture%) × 100
of Milk and Milk Products

INSOLUBILITY INDEX
Apparatus:

Mixer, Centrifuge, 50 ml. graduated centrifuge tubes.

Procedure:

l The reconstituted temperature to be used in the insolubility index method will be

24OC for spray-dried products.

l Take 13 gm in case of WMP and 10 gm in SMP. Reconstitute the powder in

100 ml distilled water and 24±0.2OC. Add 3 drops of silicone anti-foaming
agent and mix it thoroughly in the blender for 90 seconds. Further add 3 drops
of silicone anti-foaming agent to the mixture and mix thoroughly with a spoon /
spatula for 10 seconds. Pour the mixture into a centrifuge tube up to the 50 ml
mark. Place the centrifuge tube in the centrifuge and rotate it for 5 minutes at 20
to 25OC. Hold the centrifuge tube in a vertical position and remove the
supernatant liquid. Add water upto 30ml mark in the centrifuge tube, completely
disperse the sediment with the stirring rod and make up the volume upto 50 ml
mark. Invert the centrifuge tube 5 times to mix its contents thoroughly. Place the
centrifuge tube in the centrifuge and rotate it for 5 minutes at 20 to 25OC.

Observation:

Remove the centrifuge tube from the centrifuge, hold the tube in a vertical position
and read the volume of the sediment to the nearest 0.05 ml

FAT PERCENT IN WMP

Procedure:

l Take 10 ml of Gerber Sulphuric acid in milk Butyrometer.

l Weigh 1.69 gm of WMP in a 50 ml beaker and dissolve it in approximate

10 ml water, transfer carefully into the Butyrometer, add 1 ml Amyl alcohol,
make the volume with water and centrifuge for 5 minutes.

l Record fat % by multiplying the Butyrometer reading with 20/3.

FAT PERCENT IN SMP

Procedure:

l Take 10 gm of SMP and reconstitute in 100 ml in distilled water.

l Take 10 ml of Gerber acid and 10.75 ml of reconstituted milk in the

butyrometer.

l Add 1 ml Amly alcohol, and centrifuge for 5 min after mixing.

62 l Record Fat% by multiplying the Butyrometer reading with 10.
 Chemical Analysis of
BULK DENSITY Milk and Milk Products

Principle:

It is important in selection of packaging material. Weight remaining same, volume

can affect the size of packing material, which in turn affect transportation. It can
also influence consumer’s perception about the product. Lost of packaging,
transportation and storage is influenced bulk density.

Procedure:

Weight 30 gm of powder in 100 ml cylinder. Fix the cylinder in the frame of bulk
density apparatus. Fix the apparatus for thirty strokes. Switch on the apparatus.
The apparatus will take 30 strokes. Note the volume of the powder in the
cylinder.

Calculate the density as follows:

Mass
Density =
Volume

Mass = Weight of the Powder

Volume = Volume of powder after 30 strokes.

III. Testing of Butter

Method for the determination of Fat in White Butter

The method involves determination of moisture, curd and fat.

Determination of Moisture

Apparatus:

l Drying Oven maintained at 100±1OC.

l Flat bottom moisture dish of Aluminium having 7 to 8 cm diameter and 2.5

cm depth.

l Glass Rods, Desiccator, Water Bath.

Procedure:

l Clean the dish and glass rod and dry in the oven maintained at 100±1OC for
at least one hour. Allow to cool to the room temperature in a desiccator
and weigh the dish.

l Accurately 3 to 4 gm of the prepared butter sample weigh into the dish.

Place the dish on a water bath for at least 20 minutes, stirring at frequent
intervals until no moisture can be seen at the bottom (inside) of the dish.
Wipe the bottom (outside) of the dish and transfer it to the oven maintained
at 100±1OC and keep it for 90 minutes. Allow the dish to cool in the
desiccator as before and weigh. Heat the dish again in the oven for 30
63
Chemical and minutes. Repeat the process of heating, cooling and weighing until the
Microbiological Analysis difference between two consecutive weights does not exceed 1 mg. Record
of Milk and Milk Products
the lowest weight.

l Preserve the residue for the determination of curd.

Calculation:
100( W1 − W2 )
Moisture, percent by weight = W1 − W
Where
W1 = weight in gm of the dish with the material before heating to constant
weight,
W2 = weight in gm of the dish with the material after heating to constant
weight, and
W = weight in gm of the empty dry dish.

Determination of Curd

Apparatus:

Glass Funnel - with folded 12.5 cm Whatman No.1 filter paper

Flat bottom flask - 250 ml capacity

Desiccator

b) Reagents:

Petroleum ether - Boiling range 400 - 60OC.

Procedure:

l Dry, cool and weigh glass funnel with folded 12.5 cm filter paper. Melt the
residue in the moisture dish from the moisture determination, add 25 to 50 ml of
petroleum ether and mix well. Place the funnel with the filter paper, wet the filter
paper with petroleum ether and decant the fatty solution from the dish into the
filter paper, leaving the sediment in the dish. Add 20 to 25 ml of petroleum ether
twice and decant the fatty solution into the filter paper, collect the filtrate in a
clean, dried, tared 250 ml flat bottom flask containing a glass bead. With the aid
of a wash bottle containing petroleum ether wash all the fat and sediment from
the dish into the filter paper.

l Finally wash the filter paper until free from fat, collecting all the filtrate in the
flask. Preserve the filtrate for the determination of fat.

l Dry the filter funnel in the oven maintained at 100±1OC for at least 30 minutes.
Cool in the desiccator and weigh. Repeat drying, cooling and weighing until the
loss of weight between the consecutive weighings does not exceed 1 mg.

Calculation:
100( W1 − W2 )
Curd, percent by weight =
W
Where
64 W1 = weight in gm of the filter funnel with residue
 W2 = weight in gm of the filter funnel alone, and Chemical Analysis of
Milk and Milk Products
W = weight in gm of the sample

Determination of Fat

Procedure:

Distill off the solution of fat in petroleum ether collected in a tared flask. After
removing all traces of solvent, dry the flask containing fat in an oven maintained
at 100±1OC for one hour, cool in a desiccator and weigh. Continue the drying,
cooling and weighing until the loss of weight between consecutive weighings does
not exceed 1 mg.

Calculation:
100( W1 − W2 )
Fat, percent by weight =
W
Where

W1 = weight in gm of the 250 ml flask with dried fat

W2 = weight in gm of the empty flask, and

W = weight in gm of the sample

Titratable Acidity :

Weigh accurately about 20 gm of the butter sample in a dry 250 ml conical flask.
Add 90 ml of hot, previously boiled water and shake the contents. While still hot
titrate with 0.02 N (N/50) sodium hydroxide, using 1 ml of phenolphthalein
indicator.

Calculate acidity percent (as lactic acid):

9XNXV
Percent by weight =
W
Where

N = normality of sodium hydroxide solution,

V = Volume of sodium hydroxide, and

W = Weight in gram of the sample.

Analysis of Salt in Table Butter

Principle:

The butter is melted in hot water and the chloride is titrated with a solution of
silver nitrate using potassium chromate as indicator.

Apparatus:

Conical flask- 250 ml capacity.

Burette – 50- ml graduated to 0.1 ml. 65

Chemical and Reagents:
Microbiological Analysis
of Milk and Milk Products Calcium carbonate (analytical grade, free from chloride)

Potassium chromate indicator (5 % (W/V) solution in water)

Standard silver nitrate solution – 0.1N

Procedure:

Weigh accurately about 5 gm of the sample into the 250 ml conical flask. Carefully
add 100 ml of boiling water. Allow to stand with occasional swirling for 5 to 10
minutes. After cooling to 50 to 55ºC (Titration temperature), add 2 ml of potassium
chromate solution. Mix by swirling. Add about 0.25 gm of calcium carbonate and
mix by swirling. Titrate at 50 to 55ºC with standard silver nitrate solution while
swirling continuously, until the brownish colour persists for half a minute. Carry
out a blank test with all the reagents in the same quantity except the sample
material. The maximum deviation between duplicate determination should not
exceed 0.02% of Na Cl.

Calculation:
5.85 N(V1 − V 2)
Sodium chloride, percent by weight =
W
Where
N = normality of silver nitrate solution
V1 = volume of silver nitrate in the sample titration
V2 = volume of silver nitrate in the blank titration, and
W = weight in gm of the sample.

IV. Testing of Ice cream

Method for Determination of Fat (Gerber Method):

Apparatus:

Analytical Balance, Hot Air Oven, Auto titrator, Gerber Centrifuge, Incubator
and Water Bath.

Reagents:

Sulphuric acid (Specific Gravity 1.807 to 1.812) corresponding to a concentration

of 90 to 91% by mass.

Amyl alcohol (Specific Gravity 0.810 to 0.812) conforming to grade 1of

IS:360:1964.

Procedure:

Take 10 ml Gerber Acid in a clean and dry ice-cream butyrometer (20% scale).

Add about 1-2 ml distilled water carefully from the walls of the butyrometer to
make a separate layer of water over the acid.
66
Take about 5gm of the sample (3gms. in case of chocolate mix/ chocolate ice Chemical Analysis of
Milk and Milk Products
cream) accurately weighed (it is convenient to weigh by difference) in the
butyrometer.

Add 2 ml Amyl alcohol.

Maintain the level in the butyrometer with warm distilled water and close the
butyrometer with a lock stopper.

Shake the butyrometer vigorously without inverting until the contents are thoroughly
mixed and no white particles are seen. Then invert the butyrometer few times.

Centrifuge the butyrometer at 1400 ± 70 RPM for 3 minutes.

Transfer the butyrometer keeping the stopper down-wards into a water bath at
a temperature 65± 2°C for 3 minutes, and then take out it and note the fat %
from the graduated scale Calculate fat % for 5 gm. of sample.

Method for Determination of Protein (Pyne’s Method)

Chemicals Required:

Phenolphthalein indicator solution.

Potassium oxalate solution: Saturated.

Formaldehyde: 40 % concentrated and neutral phenolphthalein.

Standard NaOH solution: N/10

Testing:

Take 10 gm. of the sample accurately weighted in a conical flask (it is convenient
to weigh by difference). Add 50 ml distilled water to it.

Add 1 ml. of phenolphthalein indicator solution to it followed by 0.4 ml. saturated

potassium oxalate solution mix the contents of the flask and set it aside for 2
minutes.

Neutralize the contents of the flask with standard N/10 NaOH solution to a pink
coloured end point.

Add 2 ml neutralized formaldehyde and again titrate with standard NaOH solution
to the same pink shade. Note the volume of NaOH solution used (V).

Calculation:

Protein %. = V X 1.7

Method for Determination of Titratable Acidity

Chemicals Required:

Phenolphthalein indicator solution.

Standard sodium hydroxide solution (N/10). 67

Chemical and Procedure:
Microbiological Analysis
of Milk and Milk Products l Weight accurately about 10gm (weight by difference) of the sample into a 100
ml flask.

l Add 50ml. of boiled and cooled distilled water to it. Mix properly.

l Add 1ml. Phenolphthalein indicator solution and titrate against standard sodium
hydroxide solution (N/10) to a light pink colour end point.

l Note the volume of NaOH solution used and calculate the titratable acidity as
follows.

9NV
l Titratable Acidity (as Lactic acid) % by weight =
W

Where,
N = Normality of the standard NaOH solution.
V = Volume of the standard NaOH solution.
W = Weight of the sample taken.

Method for Determination of Total Solids (Gravimetric method)

Apparatus: Shallow Flat bottom dishes of aluminum alloy having 7-8cm diameter
and about 105cm weight with a lid. Hot Air oven (1000 +10 C) water bath and
Analytical balance.

Procedure:

l Weigh accurately clean and dry metal dish (dried & cool in a desiccator).

l Take about 2 gm. (weight by difference) of sample in it and weigh it again.

l Add 2-3 ml. of distilled water to it, spread the sample properly in the dish and
dry it on a hot plate carefully.

l Transfer the dish in a well-ventilated hot air oven at 100±1°C for one and a half-
hour.

l Transfer the dish in a desiccator for cooling and then weigh again.

Calculation:

From the loss in weight calculate the total solids % by weight of the material
taken:
W3 − W1
Total solids % by weight = W − W ×100
1

Where,
W1 = Weight of empty dish.
W2 = Weight of dish with sample (before drying)
W3 = Weight of dish with sample (after drying)
68
Method for Phosphatase Test Chemical Analysis of
Milk and Milk Products
Chemicals Required:

Phosphatase dye: Dissolve 0.15 gm. of 4-Nitrophenyl phosphate disodium salt in

100ml. buffer solution.

Buffer solution: Dissolve 3.5gm. of Na2CO3 and 1.5gm. NaHCO3 to make 1 litre
of solution in distilled water. Keep the buffer solution and the dye in cool place.

Procedure:

l Take plain mix 1 ml each in two test tubes.

l Boil the contents of one of the test tubes to be used as control (reference).

l Add phosphatase dye 5 ml in each test tubes and mix the contents thoroughly.

l Plug the test tubes with cotton or rubber stoppers.

l Place the test tubes in an incubator/water bath maintained at a temperature

37(±1)°C and note for any change in colour. Development of yellow colour in
less than two and a half-hours time indicates a positive test & no colour change
indicates negative test.

Determination of Titratable Acidity (Candy Mix)

Chemicals Required:

Phenolphthalein indicator solution.

Standard sodium hydroxide solution (N/10).

Procedure:

Weight accurately about 10gm (weight by difference) of the sample into a 100ml.
flask.

Add 1ml. phenolphthalein indicator solution and titrate against standard

sodium hydroxide solution (N/10) to a light pink colour end point.

Note the volume of NaOH solution used and calculate the titrable acidity as
follows.
0.064 NV
Titrable Acidity (as citric acid) % by weight =
W
Where N = Normality of the standard NaOH solution.
V = Volume of the standard NaOH solution.
W = Weight of the sample taken.

Determination of Total Solids (Candy Mix)

Procedure:

l Take about 1 gm. Sea sand in an empty aluminium dish dried in a hot air oven
and place it again in the oven for 30 min. to eliminate moisture.
69
Chemical and l Take out the dish containing sand and cool it in a desiccator.
Microbiological Analysis
of Milk and Milk Products l Take about 2 gm. (weight by difference) of sample in it and weigh it again.

l Spread the sample properly in the dish and dry it on a hot plate carefully.

l Transfer the dish in a well-ventilated hot air oven at 102(±1)°C for one and a
half-hour.

l Transfer the dish in a desiccator for cooling and then weigh again.

Calculation:

From the loss in weight calculate the total solids % by weight of the material taken

W3 − W1
Total solids % by weight = ×100
W1 − W
Where
W1 = Weight of empty dish.
W2 = Weight of dish with sample (before drying)
W3 = Weight of dish with sample (after drying)

V. Testing of Paneer/Hard Cheese

Determination of Moisture

Procedure:

l Weigh accurately about 2 gm of shredded paneer into the previously dried and
weighed dish. Mix the material uniformly with 4 ml of hot distilled water with the
help of a small glass rod. Wash off the particles of material adhering to the glass
rod by pouring an additional 1 ml of hot distilled water. Heat the dish containing
the material after uncovering in the oven maintained at 102 ± 1OC for 4 hrs.

l Cool the dish in the desiccator and weigh with cover on.

l Replace the dish in the oven for 30 minutes until the difference between the two
consecutive weighings is less than one milligram.

l Record the lowest weight.

Calculation:
100( W3 − W1 )
Moisture percent by mass = × 100
W1 − W
Where
W = Mass in gm of empty dish
W1 = Mass in gm of dish with sample before drying
W2 = Mass in gm of dish with sample after drying.

70
Determination of Fat Chemical Analysis of
Milk and Milk Products
Reagents:

Gerber sulphuric acid

Amyl Alcohol

Procedure:

l Take 10 ml of gerber acid into butyrometer.

l Transfer accurately 1.69 gm of shredded / meshed paneer to the butyrometer.

l Add 1 ml of amyl alcohol with the help of tilt measure.

l Make up the volume in butyrometer by adding distilled water.

l Close the butyrometer with rubber - stopper and mix the contents by shaking
until the entire sample has been digested.

l Place the butyrometer in the centrifuge, balance the machine and centrifuge
for 5 minutes.

l Transfer the butyrometer in water bath at 65 ± 2OC for 5 minutes.

l Adjust the fat column within the scale and take reading.

Calculation:
20
Percent Fat =Butyrometer reading X
3
Pr esent Flat
Percent Fat by Mass (on dry basis) = 100 − Percent Moisture × 100

Determination of Titratable Acidity

Reagents:

Standard Sodium hydroxide solution (N/10).

Phenolphthalein indicator (0.5%)

Standard hydrochloric acid (N/10)

Procedure:

l Weigh 2 gms of the meshed paneer into the flask.

l Add 3 ml of boiling distilled water and mix well with a rod and further add 17 ml
of boiling water over the rod.

l Cool to room temperature and add 10 ml of N/10 NaoH and 1 ml

phenolphthalein.

l Titrate against N/10 HCl till disappearance of pink colour.

71
Chemical and Calculation:
Microbiological Analysis
10 − V
of Milk and Milk Products Acidity (Percent lactic acid) = × 0.9
W
Where,
V = Volume in ml of standard acid used in titration.
W = Weight in gm of sample.

VI. Testing Of Ghee

Determination of Moisture

Apparatus:

l Drying Oven maintained at 105±1OC.

l Flat bottom moisture dish of Aluminium having 7 to 8 cm diameter and 2.5 cm

depth.

l Glass Rods, Desiccator, Water Bath.

Procedure:

l Clean the dish and glass rod and dry in the oven maintained at 105±1OC for at
least one hour. Allow to cool to the room temperature in a desiccator and
weigh the dish.

l Accurately weigh into the dish 10 gm of the prepared ghee sample and transfer
it to the oven maintained at 105±1OC and keep it for 60 minutes. Allow the dish
to cool in the desiccator as before and weigh. Heat the dish again in the oven
for 30 minutes. Repeat the process of heating, cooling and weighing until the
difference between two consecutive weights does not exceed 1 mg.

l Preserve the residue for the determination of curd.

Calculation: 100 ( W 1 − W 2 )
Moisture, percent by weight = W1 − W
Where
W1 = weight in gm of the dish with the material before heating to constant
weight,
W2 = weight in gm of the dish with the material after heating to constant
weight, and
W = weight in gm of the empty dry dish.

Free Fatty Acids Percent as Oleic Acid

Reagents:

Standard Sodium Hydroxide (N/10)

Phenolphthalein Indicator (0.5%)

95 percent rectified spirit (Neutralised with NaOH using Phenolphthalein as

72
indicator).
Procedure: Chemical Analysis of
Milk and Milk Products
l Weigh exactly 10 gm of ghee in a dry 250 ml conical flask.

l To this add 50 ml of neutralised rectified spirit and heat to boiling on a hot plate.

l Titrate against N/10 NaOH solution using Phenolphthalein till pink colour persists.

Calculation:
V × 2.82
Percent FFA (as Oleic Acid) =
W

Where,
V= Volume of NaOH used in titration
W= Weight in gm of sample.

Butyro Refractometer Reading

Apparatus:

Refractometer (Digital)

Reagent:

Rectified Spirit, Glass distilled water

Procedure:

Clean the prism of digital refractometer gently. Check the value of glass distilled
water at 40°C. It should be zero. Dry the prism properly and put few drops of
melted ghee and take reading at 40°C. Instrument will show refractive index.
Convert R.I. reading into B.R. with the help of chart. After completion of work,
clean the prism with rectified spirit and glass distilled water by using soft tissue
paper. Switch off the instrument.

In case BR meter is used direct reading is taken.

Ghee
RI (1.4524 to 1.4545) B.R.(40 to 43)
1.4524 40.0
1.4525 40.1
1.4526 40.3
1.4527 40.4
1.4528 40.6
1.4529 40.7
1.4530 40.9
1.4531 41.0
1.4532 41.1
1.4533 41.3 73
Chemical and 1.4534 41.4
Microbiological Analysis
of Milk and Milk Products 1.4535 41.5
1.4536 41.7
1.4537 41.8
1.4538 42.0
1.4539 42.1
1.4540 42.3
1.4541 42.4
1.4542 42.5
1.4543 42.7

1.4544 42.8

1.4545 43.0

1.4548 43.5

1.4552 44.0

1.4555 44.5

1.4558 45.0

Determination of Reichert – Meissl Value (R.M.Value)

Reagents :

l Glycerine (analytical reagent grade )

l Concentrated sodium hydroxide solution 50 percent (weight/weight)

l Dilute sulphuric acid solution:- Approximately 1N (27.7 ml in one lit water)

l Sodium hydroxide solution :- N/10

l Phenolpthlein indicator :- Dissolve 0.1 gram of phenoppthalein in 100 ml of

ethyl alcohol.

l Ethylalcohol:- 90% by volume and neutral to phenolphthalein .

Procedure :

l Weigh accurately 5(+-0.01) gram filtered oil or fat sample in to clean, dry, 300ml
distilling flask.

l Add 20 gram of glycerine and 2 ml of concentrated sodium.hydroxide (50%

W/W) solution:

l Heat the flask with swirling over flame until completely saponified and mixture
becoming perfectly clear.

74 l Cool the content slightly and add 90 ml of boiling distilled water, which has been
 boiled for about 15 minutes. If the solution is not clear repeat the test with fresh Chemical Analysis of
Milk and Milk Products
sample.

l Add about 0.1 gram of pumice stone or 4-5 glass beeds, and 50 ml of dilute
sulphuric acid solution.

l Connect the flask to the distillation apparatus and heat very gently until the
liberated fatty acids melt and separate.

l Set the flame so that 110 ml of distillate shall be collected within 19 to 21 minutes.

l When the distillate exactly reaches the 110 ml mark on the flask, remove the
flame and quickly replace the flask by 25 ml measuring cylinder stopper the
graduated flask and without mixing placed it in a water bath maintained at 15
degree for 10 minutes so that the 110 ml graduated mark is 1 CM below the
water bath, dry the outside and mix the content gently by inverting the flask
4 or 5 times without shaking.

l Filter the liquid through a dry, 9cm whatman no.4 filter paper.

l Pipette 100 ml of the filtrate and add 5 drops of the phenolphtalein solution.

l Run a blank test without fat, but using the same quantities of the reagents.

l Calculation

l Reichert-meissl value = (A-B) X N X 11 or (A-B) x 1.1

A= Volume in ml of standard sodium hydroxide required for the test.

B= Volume in ml of the standard sodium hydroxide required for the blank.

N= Normality of standard sodium hydroxide solution

VII. Testing of FLAVOURED MILK (UHT Processed)

Determination of Fat

Follow same as in liquid milk

Determination of Total Solids

Follow same as in liquid milk

Determination of Acidity

Follow same as in liquid milk .Weigh exactly 10 gm of flavoured milk instead of

10 ml as mentioned in milk.

VIII. Testing of STERILIZED CREAM

Determination of Fat

Procedure:

l Take 10 ml of gerber sulphuric acid into cream butyrometer (0-40%) with the
help of tilt measure.
75
Chemical and l Weigh 5gm of cream into the butyrometer. To this add 1 ml amyl alcohol with
Microbiological Analysis the help of tilt measure.
of Milk and Milk Products
l Close the butyrometer with rubber stopper and mix the content by shaking until
all the curd has been digested.

l Place the butyrometer in the centrifuge, balance the machine and centrifuge for
5 minutes. Transfer the butyrometer in water-bath at 65 ± 2OC for 5 minutes.

l Adjust the fat column within the scale and take reading.

Determination of Acidity

Reagents:

Standard Sodium Hydroxide (N/10)

Phenolphthalein Indicator (0.5%)

Procedure:

l Weigh exactly 10 gm of cream in a dry 250 ml conical flask.

l To this add 20 ml of hot distilled water and titrate against N/10 NaOH solution
using Phenolphthalein till pink colour appears.

Calculation:
V × 0.9
Percent Acidity =
W
Where,
V = Volume of NaOH used in titration
W = Weight of sample in gm

IX. Testing of LASSI

Determination of Fat:

Follow same as per Dahi (As per11.1).

Determination of Total Solids

Follow same as in liquid milk (As per 4.3.3).

Determination of Acidity

Follow same as in liquid milk (As per 4.3.10).

Weigh exactly 10 gm lassi instead of 10 ml as mentioned in milk.

In acidometer, select curd.

X. Testing of for CURD (DAHI)

Determination of Fat
76
Procedure: Chemical Analysis of
Milk and Milk Products
l Weigh 100 gm of the curd in beaker

l Add 5 ml of strong ammonia to the weighed sample and shake well to make it
homogeneous.

l Pipette out 10.75 ml of well mixed sample of Dahi and transfer it to the
butyrometer.

l To this add 1 ml amyl alcohol with the help of tilt measure.

l Close the butyrometer with rubber stopper and mix the content by shaking
until all the curd has been digested.

l Place the butyrometer in the centrifuge, balance the machine and centrifuge
for 5 minutes.

l Transfer the butyrometer in water-bath at 65 ± 2OC for 5 minutes.

l Adjust the fat- column within the scale and take reading.

l Multiply the result obtained by the dilution factor (in this case 1/20) and add
the same to the obtained result to get the actual result.

Determination of Acidity

l Follow same procedure as in liquid milk

l Weigh exactly 10 gm dahi instead of 10 ml as mentioned in milk.

l In acidometer, select curd.

XI. Testing of WATER

Hardness:

Take 50 ml of water sample in a 250 ml conical flask, add one powdered total
hardness indicator tablet or 2 to 3 drops of 0.5% alcoholic Eriochrome black-
T solution (indicator) to it and shake well. Now add 2 ml of ammonia buffer
solution and shake well. Titrate this mixture with N/50 EDTA solution.

Note the volume, in ml, of EDTA solution used and multiply it by 20. The product
is the hardness, in ppm as calcium carbonate, of the water sample.

pH:

Use pH paper to check the pH

Sulphite Ions (For Boiler Water):

Take 50 ml of boiler water in a conical flask, neutralize it with about 2 ml of 50%

HCl solution and then add 1 ml of 1% starch solution and titrate it with iodate-
iodide solution taking blue color as the end point.

77
Chemical and Note the volume of iodate-iodide solution used and multiply with 16. The product
Microbiological Analysis is the ppm of Sulphite ions.
of Milk and Milk Products
Phosphate Ions (For Boiler Water):

To 1 ml of filtered boiler water add 1 ml of HNO3 solution and a pinch of

ammonium molybdate crystals and shake well.

Yellow colour development indicates presence of Phosphate ions.

Residual Chlorine in Water (For Processing):

It is tested by TOLIDINE method, generally, and also by CHLOROTEX method.

Acceptable limit is 0.1 to 0.2 ppm.

Tolidine Method:

To 50 ml water in a measuring cylinder add 1 ml of O-tolidine solution, observe

the colour and record in the register.

Chlorotex Method :

Take 5 ml chlorotex reagent in a cylinder and add 50 ml water. Compare the

colour with the standard colour chart given on the chlorotex reagent bottle.

Preparation of Chemical solutions / Reagents:

Alcoholic KOH: (Dissolve 7 gms. KOH pallets in 10 ml distilled water and make
the volume to 250 ml by adding rectified spirit).

DMAB (p-dimethylamine benzaldehyde): In a liter of Rectified spirit dissolve 16

gms p-dimethylamine benzaldehyde powder and add 100 ml of concentrated
HCl.

Ethylene Diamine Tetra Acetic Acid (N/50 E.D.T.A.): Dissolve 3.72 gms of
EDTA powder in distilled water and make volume to 1 liter.

Eryochrome Black t: Dissolve 0.5 gm eryochrome Black T in 100 ml rectified

spirit.

FERRIC chloride solution (1%): Dissolve 1 gm of Ferric chloride crystals in

distilled water and make volume to 100 ml.

Hydrochloric acid (50%) : Dilute 50 ml of concentrated hydrochloric acid with

50 ml of distilled water.

Hydrochloric Acid Solution (N/10) : Dilute 25 ml concentrated HCl to 2.5 liter

in distilled water (10 ml concentrated HCL in 1 litre distilled water).

Iodine Solution (1%): Dissolve 1 gm Iodine reagent and 1 gm KI in distilled water

and make volume to 100 ml.

Iodate Iodine solution: Dissolve 0.71 gm of potassium iodate (KIO3), 7.0 gm of

potassium iodate (KIO3), 7.0 gm potassium iodide (KI) and 0.5 gm Sodium
Bicarbonate (NaHCO3) in distilled water and make the volume to 1 liter.
78
Oxalic Acid 0.1N : Dissolve 6.30 gm in 1000 ml of distilled water. Chemical Analysis of
Milk and Milk Products
Phenolphthalein solution (0.5%) : Dissolve Phenolphthalein powder, 5 gms in
50% alcohol (500 ml) and then add 500 ml distilled water make up the volume
1 litre.

Potassium iodide solution (7%) : Dissolve 7 gms. of potassium iodide powder in

100 ml. distilled water.

Potassium chromate (10%) : Dissolve 10 gms. of potassium chromate powder in

100 ml of distilled water.

Phosphate Dye : Mix 3.5 gms Sodium Carbonate and 1.5 gms sodium Hydrogen
Carbonate in 1000 ml distilled water to prepare phosphate buffer solution. In 100
ml of this buffer mix 0.15 gm of 4-Nitrophenyl Disodium salt.

Potassium Hydroxide 0.1N : Dissolve 5.61 gm in 1000 ml of distilled water.

Potassium Chromate 0.1N : Dissolve 4.903 gm in 1000 ml of distilled water.

Paraphenylene diamine hydrochloride solution (2%) for H2O2 test : Dissolve 2 gm

of Paraphenylene diamine hydrochloride powder in 100 ml. distilled water.

Rosalic acid solution (0.05%) : Dissolve 2.5 gms of Rosalic acid powder in 5
litres of 60% alcohol (to 3.160 litres of rectified spirit and 1.840 litres of distilled
water). Density 0.91

Rosaniline Acetate Solution (Stock Solution): Dissolve 0.12 gm of rosaniline

acetate in approximately 50 ml of rectified spirit containing 0.5 ml of glacial acetic
acid. Make up to 100 ml with rectified spirit.

Rosaniline Acetate Solution (Bench Solution) : Dilute 1 ml of the stock solution

to 500 ml with a mixture of rectified spirit and distilled water in equal proportions
by volume.

Resorcinol Solution for sugar test : 0.1 gram Resorcinol dissolve in HCl (1:2) i.e.
35 ml concentrated HCl added to 70 ml distilled water.

Sodium Hydroxide solution (N/10 NaOH) : Dissolve 4 gms. of Sodium Hydroxide

pellets in distilled water and make up the volume to 1 liter. Standardized with N/
10 oxalic acid.

Sodium Hydroxide Solution (2%) : Dissolve 2 gms of NaOH pellets in 100 ml

of distilled water.

Starch Solution (1%) : Dissolve 1 gm of starch powder in 100 ml of warm

distilled water and cool.

Silver Nitrate solution (0.1341%) : Dissolve 0.1341 gm of AgNO3 powder in

distilled water and make volume to 100 ml. (For Salt Test)

Silver Nitrate N/10 (To check chloride in water) : Dissolve 1.698 gm silver nitrate
in distilled water and make up the volume upto 100 ml.

Sodium Carbonate 0.1N : Dissolve 5.3 gm. in 1000 ml. of distilled water.
79
Chemical and Alcoholic KOH Solution (For Mineral Oil Test) : Dissolve 28 gms of KOH in 1
Microbiological Analysis liter distilled alcohol.
of Milk and Milk Products
Check Your Progress – 4

1. Why ask content of milk is determined?

……………………………..…………………………………………......
……………………………..…………………………………………......
……………………………..…………………………………………......
……………………………..…………………………………………......

2. What is the Principle of determination of protein in milk by formal titration

method?

……………………………..…………………………………………......
……………………………..…………………………………………......
……………………………..…………………………………………......
……………………………..…………………………………………......

3. What is the importance of bulk density of dried?

……………………………..…………………………………………......
……………………………..…………………………………………......
……………………………..…………………………………………......
……………………………..…………………………………………......

4. What is the objective of determination of acid value and RM Value of ghee?

……………………………..…………………………………………......
……………………………..…………………………………………......
……………………………..…………………………………………......
……………………………..…………………………………………......

7.3 LET US SUM UP

Test methods most commonly used are included in this unit. The students are
advised to refer the BIS methods for more understanding. Since testing is very
critical for business utmost care is required in establishing a proper laboratory and
employing skilled personnel for running the same in the dairies.

7.4 KEY WORDS

Platform tests : when milk is received in the dairy, samples are taken
and tested in a laboratory, situated near the reception
area. The samples are kept on work bench or concrete
platform for testing. Hence the name.
80
B.R. Reading : a special value for Ghee. Legal requirement. Chemical Analysis of
Milk and Milk Products
R.M. Value : a special value to know the purity of Ghee.

Titratable Acidity : a value required in the keeping quality of milk and milk
product. Determine the shelf life of the product.

Under each method of testing apparatus, reagents and principle are explained
which include key words. Hence more words are not included here.

7.5 SOME USEFUL BOOKS

IS 1165:2002 Milk Powder (fourth revision)

IS 1223 2001 Apparatus for determination of fat by

gerber method (second revision)

IS 1224 (PT 1) 1977 Determination of milk fat by the gerber

method : Part 1 Milk (first revision)

IS 1224 (PT 2) 1977 Determination of milk fat by the gerber

method : Part 2 Milk Products

IS 1479 (PT 1): 1960 Methods of test for dairy industry : Part 1
Rapid examination of milk

IS 1479 (PT 1): 1960 Methods of test for dairy industry : Part 1
Rapid examination of milk

IS 1479 (PT 2): 1961 Methods of test for dairy industry : Part 2
Chemical Analysis milk

IS 1656 :1997 Milk cereal based weaning foods (third

revision)

IS 1806:1975 Malted milk foods (first revision)

IS 2311:1973 Fat extraction apparatus for milk and milk

products (first revision)

IS 2785:1979 Natural cheese (hard variety), processed

cheese, processed cheese spread and soft
cheese (first revision)

IS 2802 1964 Ice Cream

IS 4709:1968 Flavoured milk

IS 4883:1980 KHOA (first revision)

IS:4884:1968 Sterilized cream

IS 5162:1980 CHHANA (first revision)

IS 9070:1979 Method of determination of fat in cheese

81
by Van Gul method
Chemical and IS 9532:1980 Chakka and Shrikhand
Microbiological Analysis
of Milk and Milk Products IS 9585:1980 Lactometers

IS 9617:1980 DAHI

IS:10083:1982 Method of test for determination of SNF

(solids not fat) In milk by the use of
lactometers

IS 10484:1983 PANEER

IS 10501:1983 KULFI

IS 11202:1999/ Method for determination of lactic acid

and lactate content

ISO 3495:1997 in milk powder and similar products

IS 11622:1986 Method for determination of total solids

content in condensed milk

IS 11623:1986 Method for determination of moisture

content in milk Powder and similar
products

IS 11721:1986 Method for determination of fat content in

milk powder and ISO 1737:1985
similar products (reference method)

IS 11766:1986 Method for determination of titratable

acidity in milk

ISO 6092:1980 powder and similar (routine method)

IS:12299:1998 Dairy Whitener

IS:12333:1997 Method for determination of total solids

content in milk,

ISO 6731:1989 cream and evaporated milk (reference

method)

IS:12759:1989 Dried milk and dried milk products for

determination of

ISO 8156:1987 Insolubility index

IS:12760:1989 Dried milk - determination of sodium

and pottasium

ISO 8070:1987 contents by flame Emission Spectrometric

method

IS:12898:1989 Yoghurt
82
IS 13334(PT 1):1998 Skim Milk Powder – Standard Grade Chemical Analysis of
Milk and Milk Products
IS 13334(PT 2):1992 Skim Milk Powder – Extra Grade

IS 13500:1992 Spray dried milk powders – Scorched

particles –Determination

IS:13688:1999 Pasteurized milk

IS:13689:1992 Butter Oil (Butter fat)

IS:13690:1992 Pasteurized butter

IS:14433(PT 1):1997 Infant milk substitutes – Specification : Part

1 Milk protein based

IS:14542:1998 Partly skimmed milk powder

Manual in Dairy Chemistry published by NDRI, Bangalore

Testing manual by DGHS – Govt. of India.

7.6 ANSWERS TO CHECK YOUR PROGRESS

Your answer should include the following points.

Check Your Progress – 1

1. Quick determination of developed acidity in milk?

2. Alcohol test is done to determine the heat stability of milk. If milk is not sufficiently
stable it get coagulated when mixed in 1:1 ratio

3. In the manufacture of UHT milk, evaporated milk, sweetened condensed milk

and milk powder/

4. a) Natural acidity-proteins phosphate citrate.

b) Developed acidity - lactic acid.

5. Developed acidity make the milk unfit for human consumption and it get
coagulated.

Check Your Progress – 2

1. Preservative to prevent the growth of microorganisms and developed acidity

and to extend the shelf life.

Adulterants – For economical gains

2. Adulterant.

3. Sugar is added to increase the desity or specific gravity oil mineral is added to
indicate higher milk fat content.

83
Chemical and Check Your Progress – 3
Microbiological Analysis
of Milk and Milk Products 1. 62.8O C for 30 Min/71.7o for 15 seconds.

2. Yes, the temperature of inactivation of alkaline phosphatase enzyme in milk is

slightly high than the temperature at which most stable pathogen (microbacter
tuberclosis) is destroyed.

Check Your Progress – 4

1. Ash content is determined to ascertain wheather neutralizers are added to milk

or not or if the milk is of late lactation.

2. Formalin react with amino groups of proteins and release the equivalent No of
H+ ions which is proportional to the protein content in milk.

3. Effect the size of container transpiration cost and storage space which are
indirectly proportional to bulk density hence affect the cost of production.

4. Acid value of ghee indicate its quality higher the acid value poor is the quality of
ghee, R.M. value indicate the purity or adulteration of ghee with other fats.

84