ISOLATION OF CASEIN PROTEIN FROM MILK

Project based lab report submitted for the partial fulfillment of requirement for
the award of B.Tech in Biotechnology

Project based lab work carried out by:

V.Sunandini(160010119)

B.Vaishnavi(160010166)

B.Manideepthi(160010181)

Guided by:

Dr. G.V.S.Ramakrishna

Associate Professor, Department of Biotechnology,

K L University, Vaddeswaram, Guntur District

Academic Year: 2018-19

 CERTIFICATE

This is to certify that V.Sunandini (160010119), B.Vaishnavi (160010166), B.Manideepthi

(160010181)of Biotechnology Department, KL University has done this mini project entitled
“ISOLATION OF CASEIN PROTEIN FROM MILK” during their B.Tech third year in
academic year 2018-2019.

Dr. K.Giridhar Dr. G.V.S.Rama krishna

Head of the Department Course Incharge

 ACKNOWLEDGEMENT

We are greatly indebted to Dr. K.Giridhar, Head - Department of

Biotechnology for giving us moral support and also permitting us to do this project
based lab experiments.

We would like to express our gratitude for Dr.G.V.S.Rama Krishna, our

guide, for his perpetual moral support. Without his guidance, it would have been
impossible for us to complete this mini project.

We would also like to convey our heartful thanks to SWATHI, Lab Technician
for their support in every step of the project.

We are also thankful to the management and our colleagues for their moral
support and encouragement.

V.Sunandini

B.Vaishnavi

B.Manideepthi
 DECLARATION

We, V.Sunandini(160010119),B.Vaishnavi(160010166),B.Manideepthi(160010181) of third

year Biotechnology Department, KL University hereby declare that the project based lab work
entitled “ISOLATION OF CASEIN PROTEIN FROM MILK” was carried out by us under the guidance of
Dr.G.V.S.Ramakrishna. This work has not been submitted in part or whole for award of any Degree or
Diploma from any other University / Institute.

Place: Vaddeswaram

Date:

V.Sunandini(160010119)

B.Vaishnavi(160010166)

B.Manideepthi(160010181)
INTRODUCTION:

Milk is the most nutritionally complete food found in nature it contains 3.3% of the total
protein .All kinds of milk, human or animal, contain vitamins (principally thiamine,
riboflavin, pantothenic acid, and vitamins A, B12, and D), minerals (calcium, potassium,
sodium, phosphorus, and trace metals), proteins (mostly casein), carbohydrates (principally
lactose), and lipids (fats). The amounts of these nutrients present in different types of milk
differ greatly, however. Cows' milk and goats' milk are almost identical in every respect.
Human milk contains less than half of the proteins and minerals of cows' or goats' milk, but
almost 1.5 times as much sugar. Horses' milk is quite low in proteins and fats compared with
the others, whereas reindeer milk is very high in proteins, fats, and minerals, but quite low in
carbohydrates. The average composition of whole cows' milk is 87.1% water, 3.4% protein,
3.9% fats, 4.9% carbohydrates, and 0.7% minerals. The only important nutrients lacking in
milk are iron and vitamin C.

Whole milk is an oil-in-water emulsion, containing its 3.9% fat dispersed as micron-sized
globules. The fat emulsion is stabilized by complex phospholipids and proteins that are
adsorbed on the surfaces of the globules. Because the fat in milk is so finely dispersed, it is
digested more easily than fat from any other source. The globules are lighter than water, and
thus coalesce on standing and eventually rise to the surface of the milk as cream. Vitamins A
and D are fat-soluble substances and are thus concentrated in the cream. The fats in milk are
primarily triglycerides, which are esters of saturated and unsaturated carboxylic acids with
glycerol, a tri-alcohol. About two thirds of the fatty acids in milk are saturated, and consist
primarily of C12, C14, and C16 acids. Milk is unusual in that about 12% of the fatty acids are
short-chain fatty acids (C2-C10) like butyric, caproic, and caprylic acids. Additional lipids
(fats and oils) in milk include small amounts of cholesterol, phospholipids, and lecithins. The
phospholipids help to stabilize the whole milk emulsion, as the phosphate groups help to
achieve partial water solubility for the fat globules. All the fat can be removed from milk by
extraction with petroleum ether or a similar organic solvent.

Milk proteins are synthesized in the mammary gland, but 60% of the amino acids used to
build the proteins are obtained from the cow's diet. Total milk protein content and amino acid
composition varies with cow breed and individual animal genetics.

There are three kinds of proteins in milk: caseins, lactalbumins, and lactoglobulins. All three

are globular proteins, which tend to fold back on themselves into compact, nearly spheroidal
units and are more easily solubilized in water as colloidal suspensions than fibrous proteins
are. They are "complete proteins", so-called because they contain all the amino acids essential
for building blood and tissue, and they can sustain life and provide normal growth even if
they are the only proteins in the diet. These proteins not only contain more amino acids than
plant proteins, but they contain greater amounts of amino acids than the proteins in eggs and
meats.
Casein is a naturally occurring macromolecule that accounts for approximately 80% of the
protein content of cow’s milk; Casein, the main protein in milk, is a phosphoprotein, meaning
that phosphate groups are attached to the hydroxyl groups of some of the amino acid side-
chains.that can be separated into various electrophoretic fractions, such as αs-casein, κ-
casein, β-casein, and γ-casein in which each constituent differs in primary, secondary, and
tertiary structure, amino acid composition, and molecular weight [5-7].

Casein exists in milk as the calcium salt, calcium caseinate. That can be separated into
various electrophoretic fractions, with a mixture of at least three similar proteins which differ
primarily in molecular weight and the amount of phosphorus groups they contain. Alpha- and
beta-casein have molecular weights in the 25,000 range and possess about 9 and 4-5
phosphate groups per molecule, respectively. They are both insoluble in water. Kappa-casein
has a molecular weight of about 8,000 and possesses 1-2 phosphate groups per molecule. It is
responsible for solubilizing the other two caseins in water by promoting the formation
of micelles.

Calcium caseinate has an isoelectric point of pH 4.6. Therefore, it is insoluble in solutions of

pH less than 4.6. The pH of milk is about 6.6; therefore, casein has a negative charge at this
pH and is solubilized as a salt. If acid is added to milk, the negative charges on the outer
surface of the casein micelles are neutralized (by protonation of the phosphate groups) and
the neutral protein precipitates, with the calcium ions remaining in solution: 

Ca-caseinate + 2H+ ---> casein + Ca2+

A natural example of this process occurs when milk sours. The souring of milk is an intricate
process started by the action of microorganisms on the principal carbohydrate in milk,
lactose. The microorganisms hydrolyse the lactose into glucose and galactose. Once galactose
has been formed, lactobacilli, a strain of bacteria present in milk, convert it to the sour-tasting
lactic acid. Since the production of the lactic acid also lowers the pH of the milk, the milk
clots when it sours due to the precipitation of casein.

When the fats and proteins have been removed from milk, the carbohydrates remain in the
whey, as they are soluble in aqueous solution. The main carbohydrate in milk is lactose.
Lactose (4-O-(-D-galactopyranosyl)-D-glucopyranose) is the only carbohydrate that
mammals synthesize. It is a dissacharide consisting of one molecule of D-glucose and one
molecule of D-galactose joined in 1,4'-fashion, and is synthesized in the mammary glands. In
this process, one molecule of glucose is converted to galactose and joined to another of
glucose. Galactose is thought to be needed by developing infants to build brain and nervous
tissue. It is more stable to metabolic oxidation than glucose and affords a better material for
forming structural units in cells. The digestion of lactose involves the enzyme lactase, which
hydrolyzes the disaccharide into its two component sugars.
In the first part of this experiment, we will isolate casein and lactose from cows' milk and
carry out a few chemical tests on the isolated casein and lactose. As implied above, these are
rather simple operations to carry out. Casein is precipitated by simply adjusting the pH of the
milk to be sufficiently acidic that the protein is insoluble, taking care not to acidify too much
so that the lactose does not hydrolyze. The other proteins remain water-soluble in acidic
solution, but they can also be precipitated and isolated by merely heating the acidic solution
and filtering. The isolated casein is insoluble in water, alcohol, and ether, but dissolves in
alkaline and some acidic solutions. Once the casein is removed, lactose can be isolated as the
alpha-anomer by addition of ethanol and crystallization from the resulting water-ethanol
mixture at room temperature.

Casein is isolated from milk commercially and is industrially important because after
dissolving in alkaline solutions and drying, it becomes a sticky substance that can be used in
glues, the coating of paper, and the binding of colours in paints and wallpaper. It is also used
as a coating for fine leather, and is cured with rennet to produce a plastic material used for
buttons. When isolated under sanitary conditions and dissolved in alkaline solutions, casein is
also employed in the manufacture of pharmaceutical and nutritional products.

Casein structure
Proteins are probably the most important class of biochemical molecules, along with lipids
and carbohydrates which are also essential for life. Casein is a protein that is found in milk
which is used independently in many foods as a binding agent. Its structure comprises of
presence of presence of amino acids. Amino acids have a variety of chemically reactive
groups like phenolic hydroxy groups, presence of peptide bonds. Casein also includes amino
groups, ketones and hydrazine groups. Its structure is shown as:
MATERIALS:

Milk

Beaker 

Test tube

Watch glass

Glacial Acetic acid

Electronic heater 

Funnels

Filter Paper

Aluminum foil

Methodology:

Isoelectric Precipitation

ISOELECTRICPRECIPITATION:
Protein itself can be either positively or negatively charged overall due to the terminal amine
-NH2 and carboxyl (-COOH) groups and the groups on the side chain. It is positively charged
at low pH and negatively charged at high pH. The intermediate pH at which a protein
molecule has a net charge of zero is called the isoelectric point of that protein. In general, the
net charge on the protein, either positive or negative, can interact with water molecules and
thus, more soluble. The protein is the least soluble when the pH of the solution is at its
isoelectric point.

The casein content of milk represents about 80% of milk proteins, has a molecular weight
of 25-35 kDa. The distinguishing property of casein is its low solubility at pH 4.6. The
common compositional factor is that, caseins are conjugated proteins mostly with
phosphate group(s) esterified to serine residues. These phosphate groups are important to
the structure of the casein micelle. Calcium binding by the individual caseins is
proportional to the phosphate content. Lowering the pH, leads to dissolution of calcium
phosphate until, at the isoelectric point (pH 4.6), all phosphate is dissolved and the casein
precipitates.
 pH<4.6 pH=4.6

SOLUBLE INSOLUBLE

pH=4.6 pH>4.6

SOLUBLE

As the pH falls the charge on the casein falls and it precipitates.Hence milk curdles as it
sours,or the casein precipitates completely at low pH.
CAESIN MICELLE:

Procedure:

Isolation of Casein

The older directions for the preparation of a pure casein simply stated that the casein was
precipitated from diluted fat-free milk by acidification. acetic acid was usually employed for
laboratory scale preparations and hydrochloric acid in large scale manufacture. measure 75ml
of fresh milk and pour into 3 different 100ml beakers containing 25ml each.bring the
temperature of 3 milk solutions to 25,60 and 100 respectively, at each temperature the milk
solution was again separated into 3 beakers to know the separation of milk protein at different
time intervals of 2,3 and 5 respectively and then add dropwise a solution of 10% acetic acid
while stirring with a stirring rod. do not add the acid too rapidly. continue the acid addition
(slightly less than 2 ml will be required), keeping the beaker on the hot plate, until the liquid
changes from milky to almost clear and the casein no longer separates. it is important not to
add too much acid, because it may hydrolyze some of the lactose in the milk and reduce your
yield stir the precipitated casein until it forms a large amophous mass; then remove it with a
stirring rod or tongs and place it in another beaker.

Collect the casein by suction filtration to remove as much water as possible. press the solid
with a spatula. place the casein between several layers of paper towels to help dry the
product, and let it stand in the air for 10-15 minutes. divide the wet product in half, and weigh
the two portions. place one portion in a 125 ml erlenmeyer flask with 35 ml of water and 0.5
ml of 1m naoh, stopper the mixture, shake it to ensure solution of as much of the casein as
possible, and save it for use in the chemical tests allow the second portion to dry completely
when dry, weigh this portion and calculate the total yield of casein from the powdered milk.
 TEMPERATURE TIME pI Weight of casein

60 2 4.05 1.79

3 3.56 0.95
5 3.22 0.62
Room temp(25) 2 4.12 1.42

3 4.04 1.12
5 3.57 0.95
100 2 4.47 1.39

3 3.29 0.81
5 3.22 0.63

Chemical Tests for Carbohydrates:

In this experiment, you will perform tests and reactions on the sample of lactose which you
isolated from milk and on samples of selected other mono- and disaccharides.

 Bradford's Test:

Bradford's test is similar to Benedict's test, but determines if a carbohydrate is a

monosaccharide or a disaccharide. Bradford's reagent reacts with monosaccharides to
produce cuprous oxide at a faster rate than disaccharides do:

RCHO  +  2Cu2+  +  2H2O ----->   RCOOH  +  Cu2O  +  4H+

Protein estimation by Bradford assay:

1. Take 0.1 ml of sample solution(protein solution) (tubes 1 to 9) and make

the volume to 1 ml with 0.1 M Phosphate buffer (pH 7.5).
2. Pipette appropriate aliquots of BSA solutions containing 0-100 µg
protein. Make the volume to 1 ml with 0.1M Phosphate buffer (pH 7.5)
in all the tubes.
3. Add 5 ml of Bradford reagent to all the tubes and mix thoroughly.
4. Record the absorbance at 595 nm against the reagent blank.
5.Plot a standard curve of A595 versus µg of proteins in standard.
5. Determine the protein content in the sample extract from the standard curve.
 6. Calculate the amount of protein per ml of sample.

OBSERVATION TABLE:

Volume of Volume of Absorbance

S.no sample phosphate buffer Bradford’s Reagent @595nm

B 0 1.0 5 0

1 0.1 0.9 5 0.054

2 0.2 0.8 5 0.112

3 0.3 0.7 5 0.142

4 0.4 0.6 5 0.182

5 0.5 0.5 5 0.205

6 0.6 0.4 5 0.165

7 0.7 0.3 5 0.188

8 0.8 0.2 5 0.225

9 0.9 0.1 5 0.250

10 1.0 0 5 0.234
 Bradford assay
0.3

0.25

0.2
OD at 595nm

0.15

0.1

0.05

0
b 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
Concentration of sample