Isolation of casein from milk

proteins Simple protein : Hydrolyze to yield only amino acids e.g.: albumins and 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 and 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.

MILK COMPOSITION Milk composition Lipids Proteins Vitamins Minerals Carbohydrates Lipids Proteins Vitamins Minerals Water

MILK COMPOSITION Whole milk contains vitamins (vitamins A, D, and K), minerals (calcium, potassium, sodium and phosphorus), proteins (which include all the essential amino acids), carbohydrates (lactose: which is a disaccharide containing the monosaccharaides galactose and glucose) 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: Caseins. Lactoalbumins. 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: α-casein, β-casein, and κ-casein. All casein proteins have different hydrophobic and hydrophilic regions along the protein chain.

α-casein: are the major casein proteins, it’s contain 8-10 phosphate groups. β-casein: it’s contain about 5 phosphate residues. β-casein 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-casein are glycoproteins, and they have only 2 phosphate groups. 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, the result is a casein complex that is soluble owing to the formation of the micelle.

The word “micelle” is a chemical term 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 is present as a caseinate, which means that it binds primarily calcium and magnesium.

casein micelle under electron microscope

The pI of most proteins ranges between the pH 4 to 6. Why casein is soluble in milk? Because the pI for casein is 4.7 and the pH of milk is 6.6 (pH of milk > pI of casein) at this pH the casein is have negative charge and this mean it will be soluble in milk. Bacteria action or adding acid that lower the PH and cause ppt. of casein.

Casein can be precipitated by: Excess calcium ions: 2. Acid as: (HCl, acetic acid): 3. Renin enzyme. 4. Bacterial action.

:Casein can be precipitated by rennin Rennin enzyme : Found in calves and goat. Is hydrolysis enzyme (peptidase) so cleave the peptide bond between amino acid.

Note: If calcium removed from milk the renin can’t form ppt. to milk. Mechanism of renin: Enzyme attack the k- casein, breaking the peptide bond and release small part of peptide bond. This destroy k-casein which protect the casein from ppt and ppt. it as Para kappa casein. Unlike k-casein the Para kappa casein can’t prevent the casein from ppt. in the present of calcium ions. Note: If calcium removed from milk the renin can’t form ppt. to milk.

Bacterial action When bacteria get into milk, they digest the lactose and form lactic acid. This causes lowering the pH of milk from 6.6 to 4.7 (pI for casein) and cause a precipitation of the casein.

When the pH level decreases the milk forms curds and whey. Curds: 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 coagulated and removal of curds.

Procedure: Place 20 ml (20 g) of milk into a flask and heat at 40oC in a water bath. Add 5 drops of glacial acetic acid and stir for about 1 min. Filter the resulting mixture through filter paper held in a funnel and gently squeeze out most of liquid. Remove the solid (casein and fat) from the filter paper, place it into a beaker and add 10 ml of 95% ethanol. Stir well to break up the product. Pour off the liquid and add 10 ml of 1:1 ether-ethanol mixture to the solid. Stir well and filter through filer paper. Let the solid drain well, then scrape it into a weighed filter paper and let it dry in the air.

Calculate the casein percentage in milk as follows: Normal Range 3-5 %

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