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. 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.
That pH value is known as the isoelectric point IEP of the protein and is generally the pH at which the protein is least soluble. For casein, the IEP is approximately 4. 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. While the tests and the chemistry involved are described briefly below, you should read Ege, Chapters 25 and 26 for a more complete introduction and the necessary background.
Procedure: Weigh out 5 grams of powdered non-fat dry milk and dissolve it in 20 mL of warm water in a mL beaker. Do not add the acid too rapidly. Continue the acid addition slightly less than 2 mL will be required , keeping the beaker on the hot plate, until the liquid changes from milky to almost clear and the casein no longer separates. It is important not to add too much acid, because it may hydrolyze some of the lactose in the milk and reduce your yield in Experiment 11B.
Stir the precipitated casein until it forms a large amophous mass; then remove it with a stirring rod or tongs and place it in another beaker. Immediately add 0. The separation of lactose should be done as soon as possible during the same laboratory period. Collect the casein by suction filtration to remove as much water as possible.
Press the solid with a spatula. Stir the casein in the ether for a few minutes, decant the ether, and repeat the process with a second 5 mL portion of ether. After the second washing with ether, suction filter the product. The ether washings remove any small quantities of fat that may have precipitated with the casein. Place the casein between several layers of paper towels to help dry the product, and let it stand in the air for minutes.
Divide the wet product in half, and weigh the two portions. Place one portion in a mL Erlenmeyer flask with 35 mL of water and 0. You may carry out the chemical tests for the protein during this lab period if you have time, or in your next lab period. Allow the second portion to dry in your locker over the following two weeks.
When dry, weigh this portion and calculate the total yield of casein from the powdered milk. Show your calculations. Procedure: Gently boil the original liquid to which the calcium carbonate was added after isolation of casein. Bumping will not be a problem so long as you stir the solution constantly and vigorously with a glass rod.
The solution will foam somewhat as it refluxes. This procedure precipitates the remaining proteins lactalbumin and lactoglobulin. Suction filter the hot mixture to remove the proteins and calcium carbonate, and transfer the hot, slightly yellow filtrate to a mL Erlenmeyer flask. Concentrate the filtrate to a volume of about 5 mL by heating with constant swirling, again being careful to avoid bumping.
Foaming can be controlled by heating the liquid less vigorously and gently blowing onto it. Put this mixture aside and prepare a slurry of about 1 gram of Celite and 7.
Suction filter the slurry into a Hirsch funnel containing a correct sized filter paper to obtain a filter pad of Celite, and discard the alcohol in the filter flask. To the slightly cooled ethanol mixture containing the lactose, add 1 mL water. Suction filter the mixture through the Celite filter pad, making sure the filtrate is clear. If the filtrate is cloudy, heat it up and. Transfer the filtrate to a mL Erlenmeyer flask, heat it until it clears, then allow to cool slowly.
Stopper the flask and allow it to stand in your locker until your next lab period. Thoroughly dry the lactose and determine its weight and melting point. Determine the percentage yield of lactose from the powdered milk, and show your calculations. In this experiment, you will perform chemical tests on the sample of casein which you isolated from milk, in order to determine the presence of specific amino acids in this type of protein.
The tests will also be carried out on the amino acids, to help you identify a positive test with your sample, and on egg albumin, which is the main protein present in egg whites and is similar to the lactalbumin found in milk.
While there are literally dozens of tests that are characteristic for only certain amino acids, we will carry out only three. You will use the aqueous solution of casein which you prepared above suction filter it if it is cloudy , along with stock solutions of egg albumin, tyrosine, glycine, and cysteine which have been prepared for you.
Since we have more students than fume hoods, you will have to apportion your time carefully and stagger the amino acid tests with the carbohydrate tests, which you can carry out at the bench. Millon's test is given by any compound containing a phenolic hydroxy group. Consequently, any protein containing tyrosine will give a positive test of a pink to dark-red colour. The red colour is probably due to a mercury salt of nitrated tyrosine.
Add 3 drops of Millon's reagent and immerse the tubes in a boiling water bath for 5 minutes. Cool the tubes and record the colours formed. The ninhydrin reaction is used to detect the presence of -amino acids and proteins containing free amino groups.
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