Isolation of Casein from Milk ■ Kinds of proteins in milk: Casein Lactalbumin Lactoglobulin They are complete proteins.

Molecular surface model of Casein The main protein in milk Present at a concentration of about 35g/l. It is a phosphoprotein. Not a single compound, it is a heterogeneous mixture of αS1 , αS2 , β, κ. Casein exists in milk as the calcium salt, calcium caseinate. Molecular surface model of K-Casein.

* Principle ■ The isoelectric point of a protein (I.E.P.) : is the PH at which the molecule is electrically neutral (net charge is zero). When a protein is charged e.g. with a –ve charge, the –ve molecules repel each other and thus remain in the solution and do not precipitate. When a protein is neutral at the I.E.P. they tend to precipitate due to the absence of the repelling forces and, the high molecular weight of protein. Most proteins show minimum solubility at their isoelectric point PH of milk is 4.5-4.8 (its isoelectric point). Casein is also insoluble in ethanol and this property is used to remove unwanted fat from the preparation. Milk is present at a high PH than the isoelectric point of casein

Measure the pH of the milk 60 30 15 45 5 10 20 25 35 40 50 55 60 30 15 45 5 10 20 25 35 40 50 55 Add 2 ml of CH3COONa Add 2 ml of CH3COOH 10% Measure the pH of the milk 40 ºC 25 ºC 30 ml Distilled Water 30 ml Milk pH=4.5

Shake well ph.ph. 10 ml 0.1N KOH 80 ml D.w. 10 ml H2O Precipitate 10 ml 95% alcohol 10 ml ether 10 ml ether-alcohol mix. 10 ml H2O

0.1N HCl VB VT Blank 10 ml 0.1N KOH 80 ml D.W. 3drops ph.ph.

* Calculations 1L (1 N) KOH = Mwt of casein 1 ml (0.1N) KOH = Mwt. Of Casein 10×1000 1ml (0.1N) KOH = 0.105 g casein = 1 ml HCl Volume of Casine = VB - VT (VB-VT) x 0.105 = ? g casein/30ml milk Y = ? g casine % Y = (VB-VT) x 0.105 x 100 30 Samar A. Damiati