Milk analysis

General introduction
If there is one substance which is used in cooking and dining all over the world, it
is probably milk. Milk is an everyday substance of great importance in our lives.
Therefore it becomes necessary for us to have as much information about it as
possible, regarding its composition, its uses and harmful effects (if any).
Role of milk in our lives
Value of milk in human nutrition
Milk, described as natures most perfect food is the sole source of nutrients in
most new born mammals. For the human infant, milk is the only source of
nutrients for the first two or three months of life and in many countries milk plays
a major role in the diet of growing child. Milk or milk substituents are important
during early growth of most domesticated mammals as well. In addition, milk can
be a valuable source of nutrients for the mature humans, especially the elderly.
Milk is important in the diet because of three of its ingredients, proteins,
calcium and riboflavin. Most important is protein, which provides many of the
essential amino acids that are often deficient in the cereal grains commonly used
for food.
Calcium is the nutrients most likely to be lacking in the dies of those who do not
consume milk or milk products. Adequate calcium is difficult to provide in the
human diet if milk is excluded. The daily calcium requirement of a nursing
mother is 1.3 g whereas that of a pregnant woman is 1.2 g. Calcium is especially
important in the diet of the elderly because calcium diffuses out of the bones,
porous bones develop, this condition is known osteoporosis, predispose elderly
people to their vertebrate and hips.
Riboflavin and Vitamin A are the vitamins most likely to be deficient in the
human diet. One quart of milk per day supplies all of the riboflavin requirements
of growing children and adults except pregnant and lactating females.
One quart of milk daily also supplies other nutrients like magnesium, fats,
calories, thiamine and Vitamin B.
Theory
Composition of milk
Although milk is generally believed to be a complete food, it lacks Iron and
Copper, which are essential nutrients. Milk contains Proteins, Fats,
Carbohydrates, Inorganic salts, Lactose, Fats and Minerals.
A. Milk protein
Milk contains three principle types of proteins: Casein, Lactalbumin,
Lactglobulins.

Milk
Cows
Goats
Buffalo
Human

%
2.8
2.7
3.8
0.5

B. Milk Carbohydrates
Lactose is the main carbohydrate present in the milk. Lactose is made up of two
simple sugars- Glucose and Galactose. The lactose content of milk is fairly stable.
Lactose is the major component responsible for osmotic pressure of milk.
C. Milk fats
Fats are present in the milk in the form of fine emulsion which is easily digestible
even without the presence of bile salts. #0% of total fatty acids are unsaturated
fatty acids. Almost all milk fats are made up of triglycerides. Triglyceride is
composed of the three fatty acids attached to a glycerol molecule.
Goats milk is more digestible than cows milk because of smaller fat droplets
and softer curds.
D. Calcium
Calcium in milk occurs in the form of free calcium ions. 75% of the calcium in
milk is in chemical or physical combination with casein, phosphate and citrate.
E. Phosphorus
More than one half of the phosphorus in the milk is combined with casein. It also
occurs in the form of inorganic phosphate.
F. Lactic Acid
It is produced by lactose fermentation in milk and helps in the absorption of
calcium ion.
G. Vitamins
Milk is rich in Riboflavin, Carotene and Vitamin A. It is poor in Vitamin C, Vitamin
D, Vitamin K. That is why cows milk is fortified with Vitamin D.
H. Casein
Casein is one of three major proteins in milk and is unique as there is no other
similar protein found naturally. It is one of the chief constituents of cheese.
Cheddar contains 33% of casein.
Experiment 1

Aim
The physical, microscopic and chemical analysis of milk.
I. Physical Examination
Colour
Odour

It is white in colour.
It has a characteristic odour. When it is
left out in a warm place it tends to turn
sour and smells due to production of
lactic acid.
It is colloidal. It is not transparent to
light.
It is denser than water.

State
Density
II. Microscopic analysis of milk
Apparatus
Procedure

Microscope, slide, milk drop.

A drop of milk taken on a clean slide is
observed under microscope.
Fat droplets and Brownian motion of
milk is observed.

Observation

III. Chemical analysis of milk

Chemicals required: 5% NaOH, 10% CuSO 4, 0.2% Ninhydrin, 1% acetic acid,
(NH4)2SO4 (Saturated), chlorophenol red, 2% Na2CO3, alcoholic alpha-napthol,
conc. H2SO4, Benedicts Reagent, phenylhydrazine, HCl, Glacial acetic acid, ethyl
alcohol, acetic anhydride, liquor NH3, Ca3(PO4)2, ammonium oxalate, ammonium
molybdate (solid), KCNS.
Test for protein
General Test
S.N
o.
1.

Experiment

Inference

Biuret Test
Take 6 mL of 5% NaOH in a test
tube. Add 2-3 drops of 10%
CuSO4 solution and divide into
two parts.
1st Part: Add 3mL of water
2

2.

Observation

nd

Part: Add 3mL of milk

Ninhydrin Test
To 1mL of milk add 2 drops of

No Change
Violet colour
appears
Bluish colour

Presence of protein
in given sample.

Protein present in

0.2% Ninhydrin. Mix well and

heat.

appears

the given sample.

Specific Test-Casein

S.N
o.
1.

2.

Experiment
Take 10 mL of milk and 10 mL
of water and a drop of 1%
acetic acid. Mix and filter.
Salting out of protein
To a small amount of above ppt.
add 5 mL of saturated
(NH4)2SO4. Mix well.

Observation

Inference

Precipitation
formed

Test is positive for

given sample.

Coagulum like
curd formed.

Presence of Casein
indicted

Test for Lactalbumin

S.N
o.
1.

2.

Experiment
To mL of filtrate obtained from
casein precipitation test add 1
drop of chlorophenol red.
Add 2% of Na2CO3 dropwise

Observation

Inference

Solution turn
yellow

Lactalbumin
present

Solution turn pink

Lactalbumin
present

Note: the term filtrate refers to the solution left after filtration done to obtain
casein. As mentioned later, the filtrate is used for all further tests.
Test for carbohydrate
S.N
o.
1.

2.

Experiment
Molisch Test
To 2 mL of filtrate add a drop of
alpha-naphthol + 2 mL of conc.
H2SO4
5 mL of filtrate is neutralised
with 2% Na2CO3 solution
dropwise. Divide the solution
into two parts:
1st Part: Benedicts
Qualitative Test
To 5 mL of qualitative reagent
add 8 drops of solution. Mix

Observation

Inference

Violet ring is seen

at the junction of
two layers

Carbohydrate
present

Green ppt. is
obtained

Reducing sugar in
the sample

well and heat in water bath.

2nd Part: Ozazone Test
Take phenylhydrazine hydrogen
chloride and add 5 mL of
solution in a test tube and add
one drop of glacial acetic acid.
Keep in water bath for 30
minutes.

Cotton ball
shaped crystals
are seen.

Lactose sugar
present

Test for fats

The ppt. obtained from casein filtration is used. Excess water is removed by
pressing the ppt. between dry filter paper.
S.N
o.
1.

Experiment

Observation

Inference

Take a dry test tube and put the

ppt. inti it. Add 5 mL of ethyl
alcohol. Extract liquid by
heating it in boiling water bath.
Filter and divide into two parts:
1st Part: Add 3 mL of distilled
water to one part.
2nd Part: Libberman Buchard
Reaction
To 2 mL of extract add 1 mL
acetic anhydride and 4-6 drops
of conc. H2SO4.

Fine emulsion
formed
Sample contains
cholesterol
Green colour
solution formed

Test for calcium

S.N
o.
1.
2.

Experiment

Observation

To 3 mL filtrate add 3-4 drops of

liquor NH3. Mix and boil.
In a separate test tube, take 4
mL of water and add 1 mL of
glacial acetic acid, mix and boil.
Now mix contents of both the
test tubes. Pour the hot solution
on filter paper. Collect filtrate in
a separate test tube. Add 5 mL
Ammonium Oxalate to the ppt.
left in the filter paper after

Gelatinous ppt. of
Ca3 (PO4)2 seen.
White colour
crystal of calcium
oxalate seen.

Inference
Ca2+ present.
Ca2+ present

separating the filter.

Test for phosphorus
S.N
o.
1.

Experiment
To 3 mL of filtrate add 2 mL of
conc. HNO3 and one spatula of
5% ammonium molybdate
(solid). Boil.

Observation
Yellow ppt.
formed

Inference
Inorganic
phosphate present

Test for Iron

S.N
o.
1.

Experiment
To 2 mL of filtrate add 1 mL of
conc. HNO3. Mix well. Add 2 mL
(NH4)CNS. Mix and boil.

Observation
Red ppt. formed.

Inference
Iron present

Inference
A. Milk was examined physically. It was found to be a white colloid (emulsion)
with characteristic odour.
B. Milk was examined microscopically. It was observed to contain fat droplets.
Brownian motion was also observed.
Experiment 2
Aim
Comparison of casein content in different milks.
Apparatus and reagents required
Dilute acetic acid, test tubes, beaker, filter paper and glass rod.
Source of milk
Mother dairy milk
Cows Milk
Soyabean Milk
Procedure
(1) 20 mL of the given milk sample is pipetted into a beaker.
(2) 10 mL of water is added to it

(3) Few drops of dilute acetic acid is added to the mixture so obtained and stirred
to precipitate casein.
(4) The obtained mixture is then filtered.
(5) The casein residue on filter paper is dried and filtered.
Observation table
Sample of Milk
Toned Milk (Mother Dairy)
Soyabean Milk (Sofit)
Cows Milk

Mass of Casein in 20 mL sample

445 mg
830 mg
915 mg

Inference
(1) (A) TONED MILK: Iron is present. Blood red ppt. with KCNS is seen. The
complex formed is [Fe(CNS)6)]3(B) (Mother Dairy) Very little cholesterol. Very little green coloured solution is
observed in Libberman Buchards test.
(2) COWS MILK: No iron present. All constituents otherwise present.
(3) SOYABEAN MILK: All constituents present.
Result
Casein Content

Cows Milk 915 mg

Toned Milk (Mother Dairy) 445 mg
Soyabean Milk (Sofit ) 830 mg