Isolation of Casein Protein from Milk | Casein Protein Isolation| How to Isolate Casein Protein

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Milk is a mixture of many types of proteins, most of them present in very small amounts. Milk proteins are classified into three main groups of proteins on the basis of their widely different behaviors and forms of existence. They are caseins (80%), whey proteins and minor proteins. Casein is a heterogeneous mixture of phosphorous containing proteins in milk. Casein is present in milk as calcium salt and calcium caseinate. It is a mixture of alpha, beta and kappa caseins to form a cluster called micelle. These micelles are responsible for the white opaque appearance of milk.
Casein, like proteins, are made up of many hundreds of individual amino acids. Each may have a positive or a negative charge, depending on the pH of the [milk] system. At some pH value, all the positive charges and all the negative charges on the [casein] protein will be in balance, so that the net charge on the protein will be zero.
Principle:
Most proteins show a minimum solubility at their isoelectric pH and this principle is used to isolate casein by adjusting the pH of the milk to 4.6, its isoelectric point. The main bulk of the precipitate is the casein. The entrapped residual fat can be removed by repeated washing with the solvents such as ethanol and ether. Casein is insoluble in these solvents and this property can be advantageously used to remove the unwanted fat from the preparation.
Procedure:
1. Place 100 ml milk in 500 ml beaker and warm to 40⁰C
2. Warm 100 ml acetate to 40⁰C
3. Add acetate slowly to the milk. Stir the contents during addition.
4. Check the pH with pH meter. Adjust the amount of acetate, if needed, to reach a final pH of 4.6
5. Cool the contents to room temperature and leave the whole for a further 5 min.
6. Filter the precipitate carefully through a piece of clean muslin cloth.
7. Wash the precipitate several times with the water.
8. Suspend the precipitate in 30 ml ethanol. Stir the contents.
9. Filter through filter paper.
10. Wash the precipitate with a 50 ml mixture of ethanol and ether (1:1).
11. Wash with 50 ml ether.
12. Collect the precipitate in the dry watch glass and allow ether to evaporate. (leave overnight for drying or apply mild heat for an hour )
13. Cool, weigh the casein by difference, and express the result as percentage casein (w/v)
दूध कई प्रकार के प्रोटीन का मिश्रण होता है, उनमें से अधिकतर बहुत कम मात्रा में मौजूद होते हैं। दूध प्रोटीन को उनके व्यापक रूप से अलग व्यवहार और अस्तित्व के रूपों के आधार पर प्रोटीन के तीन मुख्य समूहों में वर्गीकृत किया जाता है। वे केसिन (80%), मट्ठा प्रोटीन और मामूली प्रोटीन हैं। कैसिन दूध में फॉस्फोरस युक्त प्रोटीन का एक विषम मिश्रण है। कैसिन कैल्शियम नमक और कैल्शियम केसिनेट के रूप में दूध में मौजूद है। यह अल्फा नामक क्लस्टर बनाने के लिए अल्फा, बीटा और कप्पा केसिन का मिश्रण है। ये माइकल दूध की सफेद अपारदर्शी उपस्थिति के लिए ज़िम्मेदार हैं।
प्रोटीन की तरह केसिन, कई सैकड़ों व्यक्तिगत एमिनो एसिड से बने होते हैं। [दूध] प्रणाली के पीएच के आधार पर प्रत्येक के पास सकारात्मक या नकारात्मक चार्ज हो सकता है। कुछ पीएच मान पर, सभी सकारात्मक शुल्क और [केसिन] प्रोटीन पर सभी नकारात्मक शुल्क संतुलन में होंगे, ताकि प्रोटीन पर शुद्ध चार्ज शून्य हो।
Source- vlab.amrita.edu...
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