Isolation and Identification of Casein From Milk Course Notes

Milk Milk is the probably the most nutritionally complete food found in nature. Whole milk contains vitamins (principally thiamine, riboflavin, panthothenic acid and vitamins A, B12 and D), minerals (calcium, sodium, phosphorus, potassium, and trace minerals), proteins (which include all the essential amino acids), carbohydrates (mostly lactose), and lipids (fats). Whole milk is an oil in water emulsion, containing approximately 4% fat dispersed as very small (micron sized) globules. The fat emulsion is stabilized by complex phospholipids and proteins that are absorbed on the surface of the emulsion. Since the fat in milk is so finely dispersed it is more easily digested than fats from any other source.

Proteins and Casein A protein is a naturally occurring, unbranched polymer in which the monomer units are amino acids. More specifically a protein is a peptide in which at least 50 amino acids residues are present. Proteins can be classified into two types: fibrous and globular. Fibrous proteins are proteins in which peptide chains are arranged in long strands or sheets. Globular proteins are proteins that tend to fold back on themselves into compact spheroidal shaped units. Globular proteins do not form intermolecular interactions between protein units and are more easily solubilized in water as colloidal suspensions than fibrous proteins are. They are "complete proteins" or "storage proteins" because they contain all the amino acids essential for building blood and tissue, and can sustain life and provide normal growth even if they are the only proteins in the diet. Milk contains three kinds of proteins: caseins, lactalbumins, and lactoglobulins, all of which are globular proteins. The main protein in milk is casein. Casein is a phosphoprotein which has phosphate groups attached to the hydroxyl groups of some of the amino acids side-chains. Casein exists in milk as a calcium salt, calcium caseinate. It is actually a mixture of three similar proteins, alpha, beta and kappa caseins which form a micelle. Alpha- and beta-casein are both insoluble in water and

Calcium caseinate has an isoelectric point of pH 4. The casein micelles are destabilized or aggregate because the electric charge is decreased to that of the isoelectric point (pH at which there is no net charge because there are equal number of positive and negative charges present).6. This means it is insoluble in solutions with a pH less than 4. differing only in the structure of the R-group or the side chain. The casein micelles disintergrate and the casein (the neutral protein) precipitates because it is no longer polar. Ca+2Caseinate + 2HCl -> Casein + CaCl2 Amino Acids All amino acids found in proteins have the basic structure. Amino Acid Structure and pH . with the calcium ions remaining in solution. If an acid is added to milk. therefore. The kappa-casein which has a hydrophilic portion is responsible for solubilizing the other two caseins by promoting the formation of and stabilizing the micelles.are solubilized by the micelle surrounding them. shown below.6. by protonation of the phosphate groups. the negative charges on the outer surface of the casein micelles are neutralized. The pH of milk is 6. casein has a negative charge at this pH and is solubilized as a salt.6.

this structure may change so that a proton from the COOH. resulting in a net neutral charge because the number of protonated ammonium groups with a positive charge and deprotonated carboxylate groups with a negative charge are equal. the carboyxlic acid (-COOH) is deprotonated by the removal of a proton. This ion is called a zwitterion (German meaning mongrel ion or hybrid ion). Zwitterions In an aqueous solution at a certain compound-specific pH. The pH where the zwitterion is formed is known as the isoelectric point of the amino acid.An amino acid can have several forms depending on the pH of the system. transfers to the NH2.The zwitterion form of amino acids is the most stable form in the human body. At low ph or acid conditions. amino group. leaving an ion with both a negative charge and a positive charge. The isoelectric point is the point where the net overall charge is zero. the amino group (-NH2) is protonated by the addition of a proton (H+) from the acid. carboxylic acid group. At high pH or basic conditions. Isolation of Casein .

The violet color is a positive reaction in a Biuret test. Casein is insoluble in ethanol so this property is used to remove the unwanted fat from the preparation. Heating the milk causes the micelles to dissociate more readily when the pH is lowered. taking approximately 7 hours for complete digestion. drop by drop. If too much acetic acid is added the protein precipitate will dissolve. gelatin and glutamic acid. It is important to not heat the milk too hot because over the optimal temperature the curds dissociate quickly into fine particles and are no longer curds. The casein will then precipitate out of the milk making the protein available for digestion. in approximately forty minutes to an hour. The liquid will change from milky to almost clear when no more casein separates. The casein is then dried by using vacuum filtration. then acetic acid is added. The casein and butterfat are separated from the whey by straining the precipitate through cheesecloth. The whey protein is readily digested allowing for a speedy increase of amino acids level and protein synthesis. to determine the presence of specific amino acids. providing a slow steady release of amino acids to your muscles. The digestion of casein in the stomach is a very slow process. The curds will need to be broken up by mashing to remove as much liquid as possible.The milk is heated to 40 oC. When you drink milk. to adjust the pH to the isoelectric point of casein. your stomach acid will drop the pH of the milk to the isoelectric point of casein. These copper ions will form a complex with the nitrogens and carbons of the peptide bonds in an alkaline solution causing the pale blue color of Cu +2 to change to violet (the complex color). Biurets Reagent contains copper ions. Chemical Analysis of Casein and Other Proteins and Amino Acids You will perform chemical tests on your isolated casein as well as on glycine. freeing up the amino acids. . the optimal temperature to denature the milk into curds. This will cause the casein to "clot" and preciptate out along with butterfat leaving a liquid component called whey. Proteins give a strong biuret reaction because they contain a large number of peptide bonds. Biuret test You will perform the general test for the presence of protein by using the Biuret Test.

Biuret Test for Proteins POSITIVE: violet NEGATIVE indicator: color of copper ion Ninhydrin Test The ninhydrin test is used to detect the presence of alpha amino acids and proteins that contain free amino groups. these molecules give characteristic deep purple-blue color. These amino acid secretions that make up latent fingerprints are stable compounds that do not migrate through dry paper with time. latent fingerprints on paper that has been protected from the elements have been developed after 30 years. When using ninhydrin to detect latent fingerprints the amines left over from peptides and proteins sloughed off in fingerprints will react with ninhydrin. As a result. Ninhydrin spray is most commonly used at crime scenes to detect latent fingerprints on porous surfaces such as paper. . When heated with ninhydrin. The deep purpleblue color is a positive indicator of a free amino acid group when using the ninhydrin test. (-NH2).

blue NEGATIVE indicator Xanthoprotein Test Proteins and amino acids that contain phenyl rings will form a yellow colored compound when concentrated nitric acid is used. The yellow stains on skin caused by nitric acid are the result of the xanthoprotein reaction. The yellow colored product upon the addition of nitric acid is the test for the presence of tyrosine and tryptophan (phenyl rings) in a protein.POSITIVE: purple . POSITI VE indicator of xanthroprotein test Heavy Metal Ions .

however once the heavy metal ions leave the stomach and are absorbed through the intestines they will cause havoc to the body's chemistry. The cyanide anion. Cu2+ and Ag+ are all poisonous enzyme inhibitors because they irreversibly bind to the -SH groups of the cysteine amino acid residues of enzymes. their protein content readily combines with the heavy metal ions to form an insoluble solid. heart.Heavy metal ions such as Hg2+. If it is reproduced. Pb2+. These enzymes may be inhibited but they will quickly be expelled from the body and replaced. written permission must be granted by the author: D. which can lead to cancer. as is . In the brain they can cause alzheimers. and the brain. Egg Whites and Milk Antidotes Egg whites and milk are used as antidotes for heavy metal poisoning because if taken immediately after the ingestion of the metal poison.also belongs to this class of poisons because it binds to iron atoms that are cofactors of many enzymes. The resulting insoluble matter is removed from the stomach by the use of an emetic . In the kidneys thay can cause renal failure. loss of vision and hearing. liver. This results in a significant alteration of the tertiary structure of the enzyme and diminishes its catalytic activity. and coma. Spurlock 1.Mercury can even alter the chemical structure of proteins. The heavy metal binds tightly to proteins and enzymes thus interfering with their functions and diminishing their catalytic activity. there is no real hazard as far as the digestive enzymes are concerned. In the gastrointestinal tract they disrupt carbohydrate metabolism. Heavy metals act as catalysts which increases the production of free radicals. tremors. Once the heavy metals leave the stomach they congregate in the liver. CN. When a heavy metal ion is ingested. kidneys. and kidney diseases. cause ulcers and necrosis (cell death). blocking the bodies ability to absorb nutrients. The literature on the soluble protein fraction of milk has been reviewed to show the difficulties that are encountered in attempting to decide whether the fraction consists. thus preventing the digestive enzymes from destroying the denatured protein and once again liberating This material may be copied for personal use only. gastrointestinal tract.

usually supposed. It has been shown that on boiling the casein-free filtrate of milk. and the amount of albumin and globulin N coagulated represents about 70 per cent of the total soluble protein N of milk. and 24 per cent proteosepeptone substances. 4–7. 2.centrations of trichloracetic acid. The maximum amount of albumin and globulin rendered coagulable by heating milk itself has been determined by precipitating denatured albumin and globulin along with casein at pH. . prolonged boiling does not cause hydrolysis. maximum coagulation of albumin and globulin occurs at a pH. 3. 5.By precipitation of the casein-free nitrate of milk with various con. The conclusion has been reached that the soluble protein fraction of normal fresh milk is composed of approximately 76 per cent albumin and globulin. of lactalbumin and lactoglobulin only. This maximum represented an average of 76 per cent of the total soluble protein N in a series of normal milk samples. of 4–75–4–80. or whether it consists partly of secondary proteins of a proteose-peptone nature. a partition of the soluble proteins has been effected which strongly suggests the presence of proteose-peptone substances to an extent in agreement with the results of heat coagulation. At this pS. 4. and reasons have been given for considering this maximum as the total of true albumin and globulin present.

Sign up to vote on this title
UsefulNot useful

Master Your Semester with Scribd & The New York Times

Special offer for students: Only $4.99/month.

Master Your Semester with a Special Offer from Scribd & The New York Times

Cancel anytime.