Tuesday, January 10, 2012

Isolation of Casein and Lactose from Milk- Proteins and Carbohydrates. Isolation of Casein and Lactose from Milk

Proteins and Carbohydrates. Isolation of Casein and Lactose from Milk


Milk is the most nutritionally complete food found in nature. All kinds of milk, human or animal, contain vitamins (principally thiamine, riboflavin, pantothenic acid, and vitamins A, B12, and D), minerals (calcium, potassium, sodium, phosphorus, and trace metals), proteins (mostly casein), carbohydrates (principally lactose), and lipids (fats). The amounts of these nutrients present in different types of milk differ greatly, however. Cows' milk and goats' milk are almost identical in every respect. Human milk contains less than half of the proteins and minerals of cows' or goats' milk, but almost 1.5 times as much sugar. Horses' milk is quite low in proteins and fats compared with the others, whereas reindeer milk is very high in proteins, fats, and minerals, but quite low in carbohydrates. The average composition of whole cows' milk is 87.1% water, 3.4% protein, 3.9% fats, 4.9% carbohydrates, and 0.7% minerals. The only important nutrients lacking in milk are iron and vitamin C.

Whole milk is an oil-in-water emulsion, containing its 3.9% fat dispersed as micron-sized globules. The fat emulsion is stabilized by complex phospholipids and proteins that are adsorbed on the surfaces of the globules. Because the fat in milk is so finely dispersed, it is digested more easily than fat from any other source. The globules are lighter than water, and thus coalesce on standing and eventually rise to the surface of the milk as cream. Vitamins A and D are fat-soluble substances and are thus concentrated in the cream. The fats in milk are primarily triglycerides, which are esters of saturated and unsaturated carboxylic acids with glycerol, a tri-alcohol. About two thirds of the fatty acids in milk are saturated, and consist primarily of C12, C14, and C16 acids. Milk is unusual in that about 12% of the fatty acids are short-chain fatty acids (C2-C10) like butyric, caproic, and caprylic acids. Additional lipids (fats and oils) in milk include small amounts of cholesterol, phospholipids, and lecithins. The phospholipids help to stabilize the whole milk emulsion, as the phosphate groups help to achieve partial water solubility for the fat globules. All the fat can be removed from milk by extraction with petroleum ether or a similar organic solvent.

There are three kinds of proteins in milk: caseins, lactalbumins, and lactoglobulins. All three are globular proteins, which tend to fold back on themselves into compact, nearly spheroidal units and are more easily solubilized in water as colloidal suspensions than fibrous proteins are. They are "complete proteins", so-called because they contain all the amino acids essential for building blood and tissue, and they can sustain life and provide normal growth even if they are the only proteins in the diet. These proteins not only contain more amino acids than plant proteins, but they contain greater amounts of amino acids than the proteins in eggs and meats.

Casein, the main protein in milk, is a phosphoprotein, meaning that phosphate groups are attached to the hydroxyl groups of some of the amino acid side-chains. Casein exists in milk as the calcium salt, calcium caseinate. It is actually a mixture of at least three similar proteins which differ primarily in molecular weight and the amount of phosphorus groups they contain. Alpha- and beta-casein have molecular weights in the 25,000 range and possess about 9 and 4-5 phosphate groups per molecule, respectively. They are both insoluble in water. Kappa-casein has a molecular weight of about 8,000 and possesses 1-2 phosphate groups per molecule. It is responsible for solubilizing the other two caseins in water by promoting the formation of micelles.

Calcium caseinate has an isoelectric point of pH 4.6. Therefore, it is insoluble in solutions of pH less than 4.6. The pH of milk is about 6.6; therefore, casein has a negative charge at this pH and is solubilized as a salt. If acid is added to milk, the negative charges on the outer surface of the casein micelles are neutralized (by protonation of the phosphate groups) and the neutral protein precipitates, with the calcium ions remaining in solution:

Ca-caseinate + 2H+ ---> casein + Ca2+

A natural example of this process occurs when milk sours. The souring of milk is an intricate process started by the action of microorganisms on the principal carbohydrate in milk, lactose. The microorganisms hydrolyse the lactose into glucose and galactose. Once galactose has been formed, lactobacilli, a strain of bacteria present in milk, convert it to the sour-tasting lactic acid. Since the production of the lactic acid also lowers the pH of the milk, the milk clots when it sours due to the precipitation of casein.

When the fats and proteins have been removed from milk, the carbohydrates remain in the whey, as they are soluble in aqueous solution. The main carbohydrate in milk is lactose. Lactose (4-O-( -D-galactopyranosyl)-D-glucopyranose) is the only carbohydrate that mammals synthesize. It is a dissacharide consisting of one molecule of D-glucose and one molecule of D-galactose joined in 1,4'-fashion, and is synthesized in the mammary glands. In this process, one molecule of glucose is converted to galactose and joined to another of glucose. Galactose is thought to be needed by developing infants to build brain and nervous tissue. It is more stable to metabolic oxidation than glucose and affords a better material for forming structural units in cells. The digestion of lactose involves the enzyme lactase, which hydrolyzes the disaccharide into its two component sugars.

In the first part of this experiment, we will isolate casein and lactose from cows' milk and carry out a few chemical tests on the isolated casein and lactose. As implied above, these are rather simple operations to carry out. Casein is precipitated by simply adjusting the pH of the milk to be sufficiently acidic that the protein is insoluble, taking care not to acidify too much so that the lactose does not hydrolyze. The other proteins remain water-soluble in acidic solution, but they can also be precipitated and isolated by merely heating the acidic solution and filtering. The isolated casein is insoluble in water, alcohol, and ether, but dissolves in alkaline and some acidic solutions. Once the casein is removed, lactose can be isolated as the alpha-anomer by addition of ethanol and crystallization from the resulting water-ethanol mixture at room temperature.

Casein is isolated from milk commercially and is industrially important because after dissolving in alkaline solutions and drying, it becomes a sticky substance that can be used in glues, the coating of paper, and the binding of colours in paints and wallpaper. It is also used as a coating for fine leather, and is cured with rennet to produce a plastic material used for buttons. When isolated under sanitary conditions and dissolved in alkaline solutions, casein is also employed in the manufacture of pharmaceutical and nutritional products.

In the test section of the experiment, we will carry out a few chemical tests on the isolated casein and lactose, as well as on test samples of other representative amino-acids and carbohydrates. Historically, these tests were designed for the purpose of structure elucidation. Since we already know the structures of these substances, we will use the chemical tests to demonstrate various aspects of the chemical reactivity of the protein casein. Of course, these tests depend on the specific structural features present in the molecules.


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