NAME : Dahwa Miracle-john Avinadab

REGISTRATION NUMBER : R1710069

PROGRAMME : MBChB (1.1)

DEPARTMENT : Biochemistry

PRACTICAL TITLE : Isolation Of Casein From Milk

PRACTICAL NUMBER : 08

DATE : 01 November 2017

GROUP MEMBERS: : Dhliwayo Tafadzwa R179945P

Diza Tafadzwa R1710002
Dhakwa Dan T R1710119
Dhokotera Paidamoyo R179926X
Dapira Chikomborero R179997Q
TITLE

Isolation Of Casein From Milk By Altering The pH Of The Milk.

INTRODUCTION

Milk is probably the most nutritionally-complete food that can be found in nature. This property
is important for milk, since it is the only food young mammals consume in the nutritionally
significant weeks following birth. Whole milk contains vitamins (principally thiamine,
riboflavin, pantothenic acid, and vitamins A, D, and K), minerals (calcium, potassium, sodium,
phosphorus, and trace metals), proteins (which include all the essential amino acids),
carbohydrates (chiefly lactose), and lipids (fats). The only important elements in which milk is
seriously deficient are iron and Vitamin C. Infants are usually born with a storage supply of
iron large enough to meet their needs for several weeks. Vitamin C is easily secured through
an orange juice supplement. The average composition of the milk of each of several mammals
is summarized in the accompanying table (Holme and Peck,1997).
Fig1: Average Percentage Composition of Milk from Various Mammals
Cow Human Goat Sheep Horse
Water 87.1 87.4 87.0 82.6 90.6
Protein 3.4 1.4 3.3 5.5 2.0
Fats 3.9 4.0 4.2 6.5 1.1
Carbohydrates 49 7.0 4.8 4.5 5.9
Minerals 0.7 0.2 0.7 0.9 0.4

Whole milk is an oil-water type of emulsion, containing about 4% fat dispersed as very small
(5-10 microns in diameter) globules. The globules are so small that a drop of milk contains
about a million of them. Because the fat in milk is so finely dispersed, it is digested more easily
than fat from any other source. The fat emulsion is stabilized to some extent by complex
phospholipids and proteins that are adsorbed on the surfaces of the globules (Keenan et al,
1980).

Mammalian milk is a complex fluid mixture of various proteins, minerals, and lipids, which
play an important role in providing nutrition and immunity to the new-born. Casein proteins,
which form about 80% of the bovine milk proteins, form large colloidal particles with calcium
phosphate to form casein micelles. Casein micelles are composed of four main types of
proteins: S1casein, S2casein, casein, and kcasein. These constituent casein proteins lack
welldefined secondary and tertiary structure due to large amount of propyl residues. These
micelles are being extensively studied because of their importance in functional behaviour of
milk and various milk products. These different casein proteins possess different functional
properties due to their primary amino acid sequence (Berg et al, 2005).

The term micelle has been applied to the dispersed phase of milk, that is the casein-protein
complex. Casein protein component of milk is made up of different proteins, which possess
different functions despite having no welldefined secondary and tertiary structure. These
proteins, which include S1, S2, , and kcasein, have a primary amino acid sequence
different from each other and occupy different positions in micelle and perform specific
functions (Vasudevan, 2011).

Milk contains three phases, namely liquid, colloid and emulsion. Proteins are present in every
phase. An emulsion is a suspension of droplets of one liquid into another liquid. Milk is an
emulsion of fat in water. Butter is an emulsion of water in fat. The solute is known as the
dispersed phase and the solvent is known as the continuous phase. Other examples of emulsions
include margarine, mayonnaise, cream, and salad dressing. A colloidal solution is when matter
exists in a state of division in between a true solution, which is sugar in water, and a suspension,
which is chalk in water. The characteristics of a colloid are small particle size, electrical charge,
and affinity of the particles for water molecules. In milk, the whey proteins are in colloidal
solution. The casein proteins are in colloidal suspension (Minard, 2000)

Milk fractions include the plasma, serum and solids-not-fat(SNF). The plasma has milk fat
usually for skimmed milk, the serum has casein micelles which is practically the whey and the
SNF contains the proteins, lactose, minerals, acids, enzymes and vitamins. It is the total solid
contents minus the fat content.

The main function of milk is to provide essential amino acids and minerals that are vital for the
development and therefore function of muscular and other tissues in new born mammals. It
also includes active proteins providing antibodies, metal and vitaminbinding proteins, and
several protein hormones. Milk proteins coagulate very rapidly in the stomach of newborn as
they are structurally built in a way that they form large complexes with calcium phosphate.
Normal bovine milk contains almost 3.23.7% protein which varies in composition and
concentration during different stages of lactation. Milk proteins are divided into two classes
and are no more thought to be a homogeneous protein. Caseins constitute about 7580% of
total protein and precipitate at pH 4.6 at 30C. The remaining fraction, serum or whey protein,
is soluble under similar conditions. The rest of proteins found in milk are trace fractions of
glycoprotein .
 Casein proteins and calcium phosphate form large colloidal particles called casein micelles.
The main function of the casein micelle is to provide fluidity to casein molecules and solubilize
phosphate and calcium. There is a very large flow of calcium through the mammary epithelial
tissue, and despite this, there is rarely any formation of calcium stones in the mammary gland.
It has been suggested that the calcification of the mammary gland is prevented by the formation
of caseinmicelle complex with calcium phosphate. The primary amino acid sequence of casein
proteins and their conformation in solution are therefore thought to prevent calcification of the
mammary gland in addition to providing nutrition. In addition to their biological role, which is
to provide nutrition, caseins are also studied for their role in human health and other
malfunctions such as stoneforming diseases in bovine animal . (Koolman and Roehm, 2005).

The main objective of this experiment was to isolate protein casein from milk by adding
hydrochloric acid.

MATERIALS AND METHOD

Skimmed milk amounting to 100ml was placed in a beaker and 10ml of 0.5M HCl were

added whilst stirring gently until the pH became 4.8. The contents of the beaker were left to

stand for about 7 minutes resulting in the formation of a flocculent precipitate upon standing.

This was followed by the addition of 100ml of distilled water, settling of the protein and

cautious decanting of the supernant fluid before the washing procedure was repeated for 3

more times. The precipitate was filtered off on a Buchner funnel using mutton cloth and

excess moisture was removed by covering precipitate with tissue and pressing gently. The

casein was suspended in 25ml water, stirred and the precipitate was collected in a Buchner

funnel whilst removing excess moisture. This was repeated 2 more times. Air was drawn

through the funnel for about 20 minutes and the precipitate was blotted with tissues. The

casein was transferred to a small beaker along with the addition of acetone just enough to

cover it well before stirring for several minutes. The product was filtered and the excess

acetone was removed with tissue. The acetone treatment procedure was repeated once more

to remove traces of water. The casein was then suspended in a small volume of diethyl ether

and the product, a white hygroscopic powder was covered with foil paper. A little of the
product was dissolved in water before the biuret test was performed on the solution. The

product was weighed afterwards and then wrapped in a paper.

RESULTS
A positive result for biuret test occurred since a purple solution emerged, thus, protein was
present.
The mass of product was 6.963g

DISCUSSION

Casein is a phosphoprotein which has phosphate groups attached to the hydroxyl groups of

some of the amino acid chains.It exists in milk as a calcium salt, calcium caseinate. It has an

isoelectric point of pH 4.6. Therefore it is insoluble in in solutions of withbn a pH less than

4.6. (Murray et al, 2003).

The Ph of milk is 6.6; henceforth casein has a negative charge at this Most proteins have

isoelectric pH (meaning the pH at which the net charge on protein is zero and it will have

positive and negative side chains; but the sum of those charges will be zero) close to 4.0-4.5

range. If the pH drops to these levels, most proteins will curdle and precipitate out of

solution. Acetic acid also does the same thing, lower the pH of the milk and cause the

proteins to precipitate out of milk (Vasudevan, 2011).

The casein is denatured by the acid and it comes out of solution. Acidification causes the

casein micelles to destabilize or aggregate by decreasing their electric charge to that of the

isoelectric point. At the same time, the acidity of the medium increases the solubility of

minerals so that organic calcium and phosphorus contained in the micelle gradually become

soluble in the aqueous phase. Casein micelles disintegrate and casein precipitates.
Aggregation occurs because of entropically driven hydrophobic interactions. Calcium

caseinate has its isoelectric (neutrality) point at pH 4.6. (Wilson and Walker, 1994).

When the pH was lowered to pH 4.8, a precipitate was formed. This was because the

isoelectric point of casein had been reached (about pH 4.6). Casein exists as a calcium salt,

calcium caseinate in milk and this soluble salt contributes to the milks white colour. Upon

addition of an acid like acetic acid, negative charges on the outside of the casein micelles are

neutralized by protonation of the phosphate groups. The casein (the neutral protein)

precipitates following the disintegration of casein micelles since it is no longer polar.

Calcium ions are present in solution. (Cowan et al, 1999).

The solution left behind is less white than before since there will be no longer any calcium

caseinate in solution. The solution gave a positive test result for the biuret test as evidence of

the presence of casein protein.

While the experiment was being conducted, some problems were met. Lowering the pH of

milk to 4.8 was prejudiced due to the absence of methyl red indicator; thus, the pH

was absolutely an approximation. Decanting was not fully accurate as it resulted in loss of

some precipitate due to the absence of suitable and appropriate apparatus. However, the

respective loss was significantly compensated for by decanting several times. A significant

amount of precipitate was lost in the mutton cloth used during filtration. Keeping the product

dry was difficult as it is hygroscopic.

CONCLUSION

Mass of isolated casein in typical bovine milk was 6.963g. Casein can be isolated from milk

by changing the pH so that it matches that of the isoelectric point which promotes

precipitation of the casein salt, henceforth easier isolation.

ANSWERS TO QUESTIONS

1) Most proteins have isoelectric pH (meaning the pH at which the net charge on protein
is zero, it will have positive and negative side chains, but the sum total of those
charges will be zero) close to 4.0-4.5 range. If the pH drops to these levels, most
proteins will curdle and precipitate out of solution. Acetic acid will also do the same
thing, lower the pH of the milk and cause the proteins to precipitate out of milk The
casein is denatured by the acid; it comes out of solution. Acidification causes the
casein micelles to destabilize or aggregate by decreasing their electric charge to that
of the isoelectric point. At the same time, the acidity of the medium increases the
solubility of minerals so that organic calcium and phosphorus contained in the micelle
gradually become soluble in the aqueous phase. Casein micelles disintegrate and
casein precipitates. Aggregation occurs because of entropically driven hydrophobic
interactions. Calcium caseinate has its isoelectric (neutrality) point at pH 4.6.
Therefore, it is insoluble in solutions of pH less than 4.0.

2) Calcium caseinate has its isoelectric (neutrality) point at pH 4.6. Therefore, it is

insoluble in solutions of pH less than 4.0.

REFERENCES

1) Stryer, L Berg, J and Tymoczko, J. (2002). Biochemistry. 5th edition. (W.H. Freeman

and Company: New York, USA). pgs 136-143

2) Keenan, C Kleinfelter, D and Wood, J. (1980). General College Chemistry. 6 th

edition. (Harper and Row Publishers, Inc: New York, USA) pgs 809-815

3) Ebbing, D. (1996). General Chemistry. 5th edition. (Houghton Mifflin Company:

Dallas, USA) pgs 1069-1070

4) Hill, J and Feigl, D. (1978). Chemistry and Life. (Burgess Publishing Company:

Florida, USA) pgs 506-508

5) Nelson, D and Cox, M. (2004). Lehninger Principles of Biochemistry. 4th edition. (W.

H. Freeman: New York, USA) pg 1036

6) D.M Vasudevan. (2011) Text Book Of Biochemistry For Medical Students. 6th edition
(Jaypee Brothers Medical Publishers, pvt ltd : New Delhi)