Experiment 2- Analysis of Milk and Dairy Product

Aim - To analyze milk by performing sampling techniques, organoleptic test,

COB test, Alcohol test and Lactometer test.

1. Sampling Techniques
Introduction
Sampling of milk and milk products is done for chemical or bacteriological
analysis or redressal of consumer complaints. The sampling procedures are laid
down to avoid or prevent the difficulties that are faced to meet the regulatory,
administrative, or technical standards involved in the sampling and during the
interpretation of the results. Proper collection of milk samples is of paramount
importance for identification of mastitis pathogens. For chemical analysis,
sample should be drawn with clean and dry sampling equipment and for
microbiological analysis, all the equipment like plunger, sampling bottles, corks
or rubber stoppers should be sterile and handled under aseptic conditions. The
drawn sample should be a true representative of the bulk to prevent false-
positive results.

Collection of Samples

The samples to be drawn for chemical analysis should be done carefully; like
the person involved in sampling should use gloves, clean sample bottles or bags.
The samples should be submitted to the laboratory in the original unopened
form. Containers that are leak-proof, dry, wide mouthed, and sufficient in size
to contain the required quantity of the sample should be used. The containers
should be airtight and should not contaminate the sample. The storage
conditions of the samples should be maintained as underlined for a product and
an additional sample should be drawn for liquid samples to check the
temperature of actual product. The record containing all the details of the
sample along with the results of tests performed should be thoroughly
maintained.

Sampling for Raw Milk

1. Sampling from an Individual Container - The milk to be sampled is
poured into another container, three to four times or mixed using a plunger.
During mixing the milk, the plunger should be inserted up to the bottom of
 the container followed by pulling it upwards as quickly as possible, follow
the same procedure of plunging for about 10 times. The plunger should be
held in a slanting position and its position in the container should be changed
to ensure that the milk in the vessel is properly mixed and agitated. The
agitation/mixing of milk should not be done at 26.5-29.5°C as the milk gets
churned at this temperature. Once thorough mixing is done, draw the sample,
and store it at refrigeration before analysis. The drawn sample should be
properly labelled and must contain information like the type of milk, society
code, and route number.
2. Sampling from Several Containers: The sample from different containers
is taken by pouring milk into a vat and mixing it. If vat is not available then a
composite sample is taken from the individual containers by thoroughly
mixing their contents. Proper labelling of the drawn sample should be done
and it should contain information like type of milk, society code, and route
number.
3. Sampling from Storage Tanks and Rail and Road Milk Tankers: Milk
that is meant to be sampled from tank or tanker should be mixed with a large
plunger. Plunger should be inserted into the manhole of tank. Once the
plunging is completed, the sample taken from the top of the tank or from the
stopcock in the tank door or by opening valve on the discharge line
connected to the tank. The drawn sample should be properly labelled and
should contain information like storage tank number/road or rail tanker
number, and temperature of milk recorded.

Sampling for Processed Milk

1. Sampling from Storage tank - The sampling of processed milk from a
storage tank or silo is done by starting the agitator at least 15 min before the
sampling. The person responsible for drawing a sample from the tank should
carry all the sampling equipment like bottle, thermometer, and marker with
him. After 15 min of starting the agitator, the stopcock from the storage tank
is opened and about 2-3 L of milk is allowed to flow. Then in a sampling
container let 1 L of milk be collected in it and close the stopcock. Record the
temperature of milk; it should be below 4°C. Then fill four sampling bottles
with at least 300 ml of milk and mark the storage tank from which the milk
sample is taken.
2. Sampling of the Packed Milk: The sampling can be done by taking four
consecutive packets of milk from each head of the packaging machine
(generally a prepack machine has two heads, i.e., two filling points). The
temperature of the milk at the time of filling should be recorded (filling
temperature should be 3 °C) along with its batch number, use by date, weight
of the milk contained in the packet, etc.
2. Organoleptic Test-
It is preliminary quality test conducted at the farm level, at the processing
plant's re platform or at the collection centre. It is basic sensory and visual test
where grader m good sense of sight, smell and touch.

The milk received at the dairy plant is assessed for any objectionable flavour or
colour or for the presence of any objectionable material in the milk. The milk
failing to pass such tests is rejected for further processing.

Procedure:

I. Remove the lid of the can or tanker and observe for the presence of any
extraneous matter.

II. Sniff the milk for any objectionable flavour that is unnatural to milk.

III. Put 20-30 ml of milk in the mouth and roll into the mouth cavity and the
palate for assessing taste and flavour of milk.

IV. If the taste, flavour, or colour of milk is found to be unnatural or

objectionable, it should be rejected.

3. COB (Clot-on-Boiling Test) Test:

This is a cheap, quick and easy to perform quality test that gives information on
the acidity of the milk. It is done to check the suitability of milk for
pasteurization or other heat treatment. This test however not quantitative but
does not detect any acidity level below 0.2% of lactic acid concentration.

Procedure:

1. Take 5 ml milk sample is taken in a test tube and placed in a boiling water
bath for 5min (or hold it the over flame and boil).

II. Observe the formation of clots or flakes on the wall of the test tube.

Development of clots or flakes on boiling indicates milk with a higher

developed acidity, thus not suitable for heat treatment like pasteurization. Milk
showing a positive COB test if pasteurized will lead to choking of the
pasteurizer.
4. Alcohol Test-

The alcohol test is used on fresh milk to indicate whether it will coagulate on
thermal processing. This test is especially important for the manufacture of
UHT milk, evaporated milk and milk powders. This test is more sensitive than
Clot-on Boiling (COB) test. It is based on tendency of milk protein to get
unstable as a result of disturbance in the mineral balance of milk. Milk with
high developed acidity, or having calcium and magnesium compounds in
greater than normal amounts, will coagulate when alcohol is added. Increased
levels of albumen (colostrum milk) and salt concentrates (mastitis) may also
results in a positive test.

Procedure: 

1. For routine testing take 5 ml milk and mix it with 5 ml of ethanol

solution.
2. Observe the formation of clots or flakes on the wall of the test tube.
3. If the tested milk is of good quality, there will be no coagulation, clotting
or precipitation. Presence of flakes or clots indicates poor quality milk.

In dairy industries, normally three different concentrations of ethanol solution

are used for the test, depending upon the further use of milk. These are 68% v/v,
65% v/v and 60% v/v. Milk which passes 68% ethanol test is considered as that
of superior quality. For manufacturing UHT milk and milk powders, the raw
milk should pass 68% ethanol test. Dairies generally consider 60% ethanol test
negative raw milk for manufacturing pasteurized polypack milk as it does not
have to undergo rigorous heat treatment as in case of UHT milk. The milk
which do not even pass 60% ethanol test is rejected at the processing units.

5. Lactometer Test:

Lactometer is a small glass instrument that is used to test the purity of the milk.
It works on the principle of specific gravity of milk (Archimedes' Principle). It
measures the relative density of milk with respect to water. This law states that
whenever a solid is immersed partially or fully inside a liquid it is acted upon by
an upward thrust, which is equal to the weight of the liquid displaced by it.
Lactometers are a type of hydrometers and are calibrated beforehand with a
liquid of known specific gravity.
The lactometer consists of a long-graduated test tube with a cylindrical bulb
with the graduation ranging from 15 at the top to 45 at the bottom. The test tube
is filled with air. This air chamber causes the instrument to float. The spherical
bulb is filled with mercury to cause the lactometer to sink up to the proper level
and to float in an upright position in the milk. Inside the lactometer there may
be a thermometer extending from the bulb up into the upper part of the test tube
where the scale is located. The correct lactometer reading is obtained only at the
temperature of 60°C. It measures cream content i.e., solids-not-fat (SNF)
content of milk.
Procedure:

I. Adjust the temperature of milk sample at the temperature prescribed for a

lactometer

II. Mix the sample well. Avoid incorporation of air or foam formation.

III. Pour sufficient milk into the lactometer jar.

IV. Dip the lactometer into the lactometer jar containing milk carefully so that
the lactometer does not touch the walls of the jar.

V. Wait until the lactometer attains a stable state and assumes a constant level.

VI. Read the lactometer reading and check the temperature of milk. If the
temperature of milk is higher or lower than that prescribed for the lactometer,
use the correction from the standardized table for the corresponding
temperature. This is called as corrected lactometer reading (CLR).

VII. Calculate the SNF using the formula mentioned.

Formula:

Specific gravity-1+ (CLR/1000)

Percent SNF- (CLR/4) (02x fat (1-01 (0.1 is the factor

The factor to be added in the calculation of SNF varies according to the type of
lactometer used (due to different temperature for different lactometer).
 Experiment 2- Detection of adulteration in milk

Aim - To analyze milk by perform contamination tests for water, urea, starch,
and detergent.

1. Water Test

Water is the most common adulterants used for increasing the milk volume by
dilution, resulting in the decrease of nutrition substances, such as protein and
solid content. A further concern about the water used to dilute milk is that it is
usually obtained from an unsafe and inexpensive source and may be
contaminated with heavy metals, agricultural chemicals, or microorganisms. If
milk of such poor standard is consumed it poses a serious threat to the health of
its consumers even if it doesn’t cause serious health effects on human body.
Further contamination is done to mask this change in water.

Procedure-

1. The presence of water can be detected by putting a drop of milk on a

polished slanting surface. The drop of pure milk flows slowly leaving a
white trail behind it, whereas milk adulterated with water will flow
immediately without leaving a mark.
2. Freezing point of normal milk is around -0.550 C, addition of any other
adulterants also decreases the freezing point indicating possible adulteration.

2. Starch Test

Starch can be added to milk as a thickener to give the appearance that the milk
is high in cream. Starch can also be added to adulterated milk to increase its
density; sugars are part of the total solids of milk and part of what makes milk
heavier than water. In doing so it can mask the effects of diluting milk with
water by giving the appearance that the adulterated milk has the same density as
unadulterated milk. Starch is a complex carbohydrate (polysaccharide) that
reacts with iodine, unlike the primary milk sugars lactose and galactose, which
are single and disaccharides: Thus, the presence of starch can easily be detected
by adding a small amount of iodine

The iodine test is used to test for the presence of Starch. Iodine solution-iodine
dissolved in an aqueous solution of potassium iodide-reacts with starch
producing a blue black colour. This reaction is the result of the formation of
polyiodide chains from the reaction of starch and iodine.

Apparatus
Milk sample. Test tubes Water bath (or other device that can hold boiling
water). Iodine solution (1%)

Procedure-

1. Add 5mL milk sample to a test tube that can be boiled.

2. Boil milk samples for 5 minutes.

3. Allow samples to cool to room temperature.

4. Add 2 to 3 drops of iodine solution to each sample. Observe colour changes.

3. Urea Test-

Urea, being a natural constituent of milk, constitutes the major portion of non-
protein nitrogen in milk. According to FSSAI act 2006 and PFA rules 1955,
maximum allowable limit for urea in milk is 70 mg/100 ml .Milk can be
adulterated with urea in two ways – by Intentional addition of urea and by
addition of unspecified synthetic milk to natural milk.

The urea is converted into ammonia by urease enzyme. The ammonium ions
react with a phenolic derivative and form a blue-green coloured complex whose
intensity, measured at 700 nm, is directly proportional to the concentration of
urea in the sample.

Procedure-
1. To 5ml milk add equal volume of 24 % trichloroacetic acid (TCA) to
precipitate fat and proteins of milk then filter and collect filtrate after that
take 1 ml.
 2. Filtrate and add 0.5 ml. sodium hypochlorite (2%), 0.5ml. sodium
hydroxide (2%) and 0.5 ml phenol solution (5%) and mix.
3. A characteristic blue or bluish green colour develops in presence of
added urea whereas pure milk remains colourless.

4. Detergent Test -

The detergent in milk can cause food poisoning and other gastrointestinal
complications. Its high alkaline level can also damage body tissue and destroy
proteins. Other synthetic components can cause impairments, heart problems,
cancer or even death. While the immediate effect of drinking milk adulterated
with urea, caustic soda and formalin is gastroenteritis, the long-term effects are
far more serious.

Procedure-

1. To find if your milk is laced with detergent, take some milk in a bottle and
add an equal amount of water.
2. Shake well. Adulterated milk will have a dense lather whereas pure milk will
have a thin layer of foam due to the shaking.

Take 2.0 ml of milk in a test tube and add 1.0 ml of 0.025% methylene blue
solution in water. Mix the contents and add 5.0 ml of chloroform and shake the
contents vigorous undisturbed for 15 min or centrifuge the contents for 10 min.
and observe the colour in lower layer. Intense blue colour in lower layer
indicates the presence of detergent. Keep the tubes undisturbed for 15 min or
centrifuge the contents for 10 min. And observe the colour in lower layer.
Intense blue colour in lower layer indicates the presence of detergent.