Isolation of Casein from Milk ( Dr. Huda HANIA )

Isolation of casein from milk

  • simple protein

  • hydrolyze to yield only amino acids e.g.: albumins , globulins.

  • conjugated protein

  • is a protein that functions in interaction with other chemical groups attached by covalent bonds or by weak interactions.

  • Some examples of conjugated proteins are lipoproteins, glycoproteins, phosphoproteins, hemoproteins

Phosphoproteins are proteins that are physically bonded to a substance containing phosphoric acid. Ionized phosphate groups by calcium ion increases hydrophobic interaction, which leads to precipitation.


Whole milk contains vitamins (vitamins A, D, and K), minerals (calcium, potassium, sodium, phosphorus,), proteins (which include all the essential amino acids), carbohydrates (chiefly lactose), and lipids.

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.

There are three kinds of proteins in milk:



3.lactoglobulins. All are globular

Structure  of casein

Is a phosphoprotein, which has phosphate groups attached to some of the amino acid side chains. Mostly these amino acid are serine and threonine.

 casein is a mixture of at least three similar proteins, which differ primarily in molecular weight and amount of phosphorus they contain (number of phosphate groups).

Casein is made up of the main 3 types of proteins are α-casein, β-casein, and κ-casein.

all casein proteins have different hydrophobic and hydrophilic regions along the protein chain.

  • α-Caseins are the major casein proteins. Its containing 8-10 phosphate groups,

  • β- casein contains about 5 phosphate residues,

  • β- casein it is more hydrophobic than α-caseins and κ-casein

  • Because α-caseins and β-caseins are highly phosphorylated, they are very sensitive to the concentration of calcium salts, that is, they will precipitate with excess Ca2+ ions

  • Unlike other caseins, κ-caseins are glycoproteins, and they have only 2 phosphate group.

  • Hence, they are stable in the presence of calcium ions, and they play an important role in protecting other caseins from precipitation and make casein more soluble forming casein micelles.

 Neither the α nor the β casein is soluble in milk, singly or in combination.

If κ casein is added to either one, or to a combination of the two, however, the result is a casein complex that is soluble owing to the formation of the micelle

Carbohydrate that found k casein :

The main carbohydrate present in milk is the sugar lactose.

Lactose is a disaccharide containing the monosaccharaides galactose and glucose.

when bacteria get into milk, they digest the lactose and form the acid lactic acid. This causes  lowering the pH        and cause a precipitation of the protein casein.

The word “micelle” is a chemical term. It is used to describe the structure that certain very large molecules

  • Very large molecules are considered to be too large to be truly soluble in water.

  • Instead, these large molecules will form structures that allow them to remain suspended in water

  • The dispersion of these large structures in water is known as a colloidal suspension.

  • The structures that allow large molecules to remain colloidally suspended in water are termed micelles.

  • In the case of casein, the parts of the casein molecules that have an affinity for water form the outside of the casein micelle.

  • Conversely, the parts of the casein molecule that are hate the water form the inner core of the micelle spheres

  • Casein micelles consist of water, protein, and salts.

  • Casein is present as a caseinate, which means that it binds primarily calcium and magnesium.

The pI of most proteins ranges between the                pH 4 to 6.

So pI for casein is 4.6 and the pH of milk is 6.6 at this pH the casein is have negative charge and soluble by bacteria action or by adding acid that lowering the PH cause ppt of casein

Casein can be precipitated by rennin

Renin enzyme :

Found in calves and gout

Is hydrolysis enzyme (peptidase) so cleave the peptide bond between amino acid


1.Enzyme attack the k- casein and breaking the peptide bond and release small part of peptide bond

2.This destroy cause left k-casein the  α and              B casein   which protect the casein from ppt  and ppt it as Para kappa casein

3.Unlike kappa the Para kappa casein cannot prevent the casein from  ppt in the present of calcium ion

If calcium removed from milk the renin cannot form  ppt to milk

nWhen the pH level decreases the milk forms Curds or whey.

nCurds are a dairy product obtained by ppt of  milk with renin or an acidic substance and then draining off the liquid portion.

Whey or milk serum is the liquid remaining after milk has been coagulation and remove curds


1.Place 20 ml (20 g) of milk into a 125 ml flask and heat at 40 oC in a water bath.

2.Add 5 drops of glacial acetic acid and stir for about                1 min.

3.Filter the resulting mixture through filter paper held in a funnel and gently squeeze out most of liquid.

4.Remove the solid (casein and fat) from the cheesecloth, place it into a 100 ml beaker and add 10 ml of 95% ethanol.

5.Stir well to break up the product. Pour off the liquid and add 10 ml of 1:1 ether-ethanol mixture to the solid.

6.Stir well and filter through filer paper

7.Let the solid drain well, then scrape it into a weighed filter paper and let it dry in the air.

8.Calculate the casein percentage in milk as follows:

    % Casein =  (grams of casein \ grams of milk) x 100

     Normal Range 3-5 %

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