Proteins, from the Greek proteios, meaning first, are a class of organic compounds which are present in and vital to every living cell. In the form of skin, hair, callus, cartilage, muscles, tendons and ligaments, proteins hold together, protect, and provide structure to the body of a multicelled organism. In the form of enzymes, hormones, antibodies, and globulins, they catalyze, regulate, and protect the body chemistry. In the form of hemoglobin, myoglobin and various lipoproteins, they affect the transport of oxygen and other substances within an organism.
The total protein component of milk is composed of numerous specific proteins. The primary group of milk proteins are the caseins. There are 3 or 4 caseins in the milk of most species; the different caseins are distinct molecules but are similar in structure. All other proteins found in milk are grouped together under the name of whey proteins. The major whey proteins in cow milk are beta-lactoglobulin and alpha-lactalbumin. The major milk proteins, including the caseins, ? -lactoglobulin and a-lactalbumin, are synthesized in the mammary epithelial cells and are only produced by the mammary gland.
The immunoglobulin and serum albumin in milk are not synthesized by the epithelial cells. Instead, they are absorbed from the blood (both serum albumin and the immunoglobulins). An exception to this is that a limited amount of immunoglobulin is synthesized by lymphocytes which reside in the mammary tissue (called plasma cells). These latter cells provide the mammary gland with local immunity. Caseins have an appropriate amino acid composition that is important for growth and development of the nursing young. This high quality protein in cow milk is one of the key reasons why milk is such an important human food.
Caseins are highly digestible in the intestine and are a high quality source of amino acids. Most whey proteins are relatively less digestible in the intestine, although all of them are digested to some degree. When substantial whey protein is not digested fully in the intestine, some of the intact protein may stimulate a localized intestinal or a systemic immune response. This is sometimes referred to as milk protein allergy and is most often thought to be caused by ? -lactoglobulin. Milk protein allergy is only one type of food protein allergy.
Caseins are composed of several similar proteins which form a multi-molecular, granular structure called a casein micelle. In addition to casein molecules, the casein micelle contains water and salts (mainly calcium and phosphorous). Some enzymes are associated with casein micelles, too. The micellar structure of casein in milk is an important part of the mode of digestion of milk in the stomach and intestine, the basis for many of the milk products industries (such as the cheese industry), and the basis for our ability to easily separate some proteins and other components from cow milk.
Casein is one of the most abundant organic components of milk, in addition to the lactose and milk fat. Individual molecules of casein alone are not very soluble in the aqueous environment of milk. However, the casein micelle granules are maintained as a colloidal suspension in milk. If the micellar structure is disturbed, the micelles may come apart and the casein may come out of solution, forming the gelatinous material of the curd. This is part of the basis for formation of all non-fluid milk products like cheese.
Casein protein, like other protein sources, provides a rich amino acid supply to the body. Current data suggest that exercise can increase protein needs and that increased protein intakes can improve the response to exercise training. Casein protein is slowly digested and this property of casein makes it optimal for consuming during the day as snacks in the form of dairy products or as a protein shake. Since casein slowly enters the blood stream, it has a negligible impact on protein synthesis. However, casein does have a powerful effect in suppressing protein breakdown.
This may promote a better protein status over time. Casein protein makes up approximately 80% of the protein in milk. The beneficial properties of casein are partly a result of the amino acid composition and partly a result of the active peptides (the unique amino acid chain configurations that make up casein). In order to prevent the denaturing (or destruction) of the interesting peptides, appropriate processing techniques are required. Since milk protein isolates contain 80% casein, often people will use the terms milk protein isolate as casein interchangeably.
The objectives of the experiment were to isolate casein from milk by isoelectric precipitation and to obtain information about the composition of the proteins using hydrolysis and neutralization. Hydrolysis is a chemical process in which a certain molecule is split into two parts by the addition of a molecule of water. One fragment of the parent molecule gains a hydrogen ion (H+) from the additional water molecule. The other group collects the remaining hydroxyl group (OH? ). The most common hydrolysis occurs when a salt of a weak acid or weak base (or both) is dissolved in water.
Water autoionizes into negative hydroxyl ions and positive hydrogen ions. The salt breaks down into positive and negative ions. However, under normal conditions, only a few reactions between water and organic compounds occur. In general, strong acids or bases must be added in order to achieve hydrolysis where water has no effect. The acid or base is considered a catalyst. They are meant to speed up the reaction, but are recovered at the end of it. •Methodology Procedure for Acid Hydrolysis and Neutralization In a 50-mL Erlenmeyer flask, 5mL of 8N of H2SO4 was added to ? f the protein isolate. The flask was labelled and plugged with a piece of cotton. The flask was covered with aluminium foil. Appearance of the sample was noted before autoclaving. The flask was autoclaved at 15 psi for 5 hours. The appearance of the sample was noted after autoclaving. Hydrolyzate was diluted with 15mL of distilled water and then contents were transferred to a 250mL beaker. Hydrolyzate was neutralized by adding one spatulaful of Ba(OH)2. It was made sure that all the Ba(OH)2 has dissolved and then pH was checked using litmus paper (redapurple).
If the hydrolyzate is not yet neutral, saturated was added with Ba(OH)2 solution and the pH is again checked pH using litmus paper. pH 7 was confirmed using pH of water. Color Reaction Tests For Biuret test, a drop of 2. 5M NaOH was added to 3 drops of the hydrolyzate. It was mixed well. A drop or more of . 01M CuSO4 solution was added. It was mixed well and the color was noted. For Sakaguchi test, a drop of 10% NaOH and a drop of . 02% napthol solution to 5 drop of the protein suspension or hydrolyzate and was mixed well. After 3 minutes, a drop of freshly-prepared 2% NaOBr. The color produced was noted.
For Ninhydrin test,. 5mL of . 1% ninhydrin solution. It was mixed well. It was heated in a boiling water bath for 2-3 minutes. Color produced was noted. For Xanthoproteic test, 1mL was added without the dilution of water. 3 drops was added slowly of conc. HNO3. it was mixed well. Color produced was noted. It was heated in a boiling water bath for a minute. The solution was cooled For Hopkins-cole test, 2mL of Hopkins-Cole reagent was added to 2 drops of hydrolyzate. It was mixed well. The tube was inclined and 2mL of conc. H2SO4 was added slowly down the side of the tube until two layers are formed. Color produced was noted.