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 betta-casein are both insoluble in water and are solubilized by the micelle surrounding them. 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.

Calcium caseinate has an isoelectric point of pH 4.6. This means it is insoluble in solutions with a pH less than 4.6. The pH of milk is 6.6, therefore, casein has a negative charge at this pH and is solubilized as a salt. If an acid is added to milk, the negative charges on the outer surface of the casein micelles are neutralized, by protonation of the phosphate groups. 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 becasue there are equal number of positive and negative charges present). The casein micelles disintergrate and the casein (the neutral protein) precipitates because it is no longer polar, with the calcium ions remaining in solution.

Ca+2Caseinate + 2HCl -> Casein + CaCl2

Amino Acids

All amino acids found in proteins have the basic structure, shown below, differing only in the structure of the R-group or the side chain.

Amino Acid Structure and pH

An amino acid can have several forms depending on the pH of the system. At low ph or acid conditions, the amino group (-NH2) is protonated by the addition of a proton (H+) from the acid.

At high pH or basic conditions, the carboyxlic acid (-COOH) is deprotonated by the removal of a proton.

Zwitterions

In an aqueous solution at a certain compound-specific pH, this structure may change so that a proton from the COOH, carboxylic acid group, transfers to the NH2, amino group, leaving an ion with both a negative charge and a positive charge, 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. This ion is called a zwitterion (German meaning mongrel ion or hybrid ion). The pH where the zwitterion is formed is known as the isoelectric point of the amino acid. The isoelectric point is the point where the net overall charge is zero.The zwitterion form of amino acids is the most stable form in the human body.

Isolation of Casein

The milk is heated to 40 oC, the optimal temperature to denature the milk into curds, then acetic acid is added, drop by drop, to adjust the pH to the isoelectric point of casein. This will cause the casein to "clot" and preciptate out along with butterfat leaving a liquid component called whey. The liquid will change from milky to almost clear when no more casein separates. Heating the milk causes the micelles to dissociate more readily when the pH is lowered, freeing up the amino acids. It is important to not heat the milk to hot because over the optimal temperature the curds dissociate quickly into fine particles and are no longer curds. If too much acetic acid is added the protein precipitate will dissolve. The casein and butterfat are separated from the whey by straining the precipitate through cheesecloth. Casein is insoluble in ethanol so this property is used to remove the unwanted fat from the preparation.

The casein is then dried by using vacuum filtration. The curds will need to be broken up by mashing to remove as much liquid as possible.

When you drink milk, your stomach acid will drop the pH of the milk to the isoelectric point of casein. The casein will then precipitate out of the milk making the protein available for digestion. The whey protein is readily digested allowing for a speedy increase of amino acids level and protein synthesis, in approximately forty minutes to an hour. The digestion of casein in the stomach is a very slow process, taking approximately 7 hours for complete digestion, providing a slow steady release of amino acids to your muscles.

Chemical Analysis of Casein and Other Proteins and Amino Acids

You will perform chemical tests on your isolated casein as well as on glycine, gelatin and glutamic acid, to determine the presence of specific amino acids.

Biuret test

You will perform the general test for the presence of protein by using the Biuret Test. Biurets Reagent contains copper ions. 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). The violet color is a positive reaction in a Biuret test. Proteins give a strong biuret reaction because they contain a large number of peptide bonds.

Ninhydrin Test

The ninhydrin test is used to detect the presence of alpha amino acids and proteins that contain free amino groups, (-NH2). When heated with ninhydrin, these molecules give characteristic deep purple-blue color. The deep purple-blue color is a positive indicator of a free amino acid group when using the ninhydrin test. Ninhydrin

spray is most commonly used at crime scenes to detect latent fingerprints on porous surfaces such as paper. When using ninhydrin to detect latent fingerprints the amines left over from peptides and proteins sloughed off in fingerprints will react with ninhydrin. These amino acid secretions that make up latent fingerprints are stable compounds that do not migrate through dry paper with time. As a result, latent fingerprints on paper that has been protected from the elements have been developed after 30 years.

Xanthoprotein Test

Proteins and amino acids that contain phenyl rings will form a yellow colored compound when concentrated nitric acid is used. 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. The yellow stains on skin caused by nitric acid are the result of the xanthoprotein reaction.

Heavy Metal Ions

Heavy metal ions such as Hg2+, Pb2+, Cu2+ and Ag+ are all poisonous enzyme inhibitors because they irreversibly bind to the -SH groups of the cysteine amino acid residues of enzymes. This results in a significant alteration of the tertiary structure of the enzyme and diminishes its catalytic activity. The cyanide anion, CN- also belongs to this class of poisons because it binds to iron atoms that are cofactors of many enzymes.

When a heavy metal ion is ingested, there is no real hazard as far as the digestive enzymes are concerned. These enzymes may be inhibited but they will quickly be expelled from the body and replaced, 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 heavy metal binds tightly to proteins and enzymes thus interfering with their functions and diminishing their catalytic activity. Mercury can even alter the chemical structure of proteins. Once the heavy metals leave the stomach they congregate in the liver, kidneys, gastrointestinal tract, and the brain. In the kidneys thay can cause renal failure. In the gastrointestinal tract they disrupt carbohydrate metabolism, blocking the bodies ability to absorb nutrients, cause ulcers and necrosis (cell death). In the brain they can cause alzheimers, tremors, loss of vision and hearing, and coma. Heavy metals act as catalysts which increases the production of free radicals, which can lead to cancer, heart, liver, and kidney diseases.

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, their protein content readily combines with the heavy metal ions to form an insoluble solid. The resulting insoluble matter is removed from the stomach by the use of an emetic , thus preventing the digestive enzymes from destroying the denatured protein and once again liberating