CEBU DOCTORS’ UNIVERSITY COLLEGE OF ARTS AND SCIENCES Physical Sciences Department Laboratory Activity Sheet No. 4.1 Extraction and Characterization of Casein from Milk Group Members: fumayat, (gira Laboratory Schedule: TE 9 PM — 6° o9PUl 2 an a apt ul wo Program and Year: BSI |- 1 Score; I. Intended Learning Outcomes: '. Demonstrate and explain extraction of casein from milk using acid denaturation; 2. Characterize the presence of different components in casein using different color reactions, I Background Milk is the most nutitionally 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, carbohydrates (principally lactose), and lipids (fats). The amounts of these nutrients present in different types of milk differ greatly, however, Cows’ milk and goats’ mik are almost identical in every respect. Human milk contains less than haif ofthe proteins and minerals of cows or goats' milk, but almost 1.5 times as much sugar. The only important nutrients lacking in milk are iron and vitamin C. ‘There are three kinds of proteins in milk: casein, lactalbumin, and lactoglobulin. All three are lobular proteins, which tend to fold back on themselves into compact, nearly spheroidal units ‘and are mote 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 lfe 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 is the main protein in mik. tis a phosphoprotein, meaning that phosphate groups are attached to the hydroxyl groups of some of the amino acid side-chains. Its actualy a mixture of at least three similar proteins which differ primarily in molecular weight and the amount of phosphorus groups they contain, These are a-casein, B-casein, and x-casein. a-casein and B- ‘casein are both insoluble in water. x-casein is responsible for solubilizing the other two caseins in water by promoting the formation of micelles. Casein exists in milk as the calcium salt, calcium caseinate... Calcium caseinate has an Isoelectric point of pH 4.6. Therefore, itis insoluble in solutions of pH less than 4.8. The pH of rik 's 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 calclum 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 intvicate process started by the action of microorganisms on the principal carbohydrate in mik, lactose, ‘The microorganisms hydrolyse the lactose into glucose and galactose. Once galactose has been formed, lactobacill, a strain of bacteria present in milk, convert it to the sour-tasting lactic acid, 1|Page Scanned with CamScannerCEBU DOCTORS’ UNIVERSITY COLLEGE OF ARTS AND SCIENCES Physical Sciences Department ‘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 casei The second most abundant protein types in milk are the lactalbumin. Lactalbumin, like any albumins, is a globular protein that is soluble in water and in dilute salt solutions. ‘They are, however, denatured and coagulated by heat. Once the caseins have been removed, and the ‘solution has been made acidic, the lactalbumin can be isolated by heating the mixture to Precipitate them. AA third type of protein in milk is the lactoglobulins. They are present in smaller amounts than the ‘albumins and generally denature and precipitate under the same conditions as the albumins, The lactoglobulins carry the immunological properties of milk. They protect the young mammal until its own immune system has developed. For this activity, we will attempt to extract casein from nonfat milk, IIL. Matertals and Equipment: nonfat milk * glacial acetic acid + graduated cylinder 10% acetic acid (previously © beaker (CHsCOOH) exposed to + funnel Biuret reagent sunlight) ‘= stirring rod Ninhydrin solution + Molsch’s reagent © spatula concentrated nitric, = concentrated acid (HNOs) sulle ackd = concentrated (80) ammonia + filter paper Iv. Procedures: Extraction of Casein 1. Mix 50 mL of nonfat milk and 50 mL. of distilled water it in a 250-ml. beaker. 2. Add the 10% acetic acid dropwise to the mixture with vigorous stirring. Do not add the acid too rapidly. Continue the acid addition (slightly less than 3 mL will be required), until a flocculent precipitate forms. Avoid adding excess acid. ir grndunhy | 3. Allow the precipitated casein to settle down for a few minutes. Decant the supernatant liquid through a fiter paper in a funnel. ‘4. Then, pour all the precipitate into the funnel. Remove the excess moisture from the casein by pressing the precipitate between the fiter paper. Color Reactions Bluret test 4. Place a small portion (size of mongo bean) of casein in a test tube. Add 3 mL water. 2. Add 1 mL of Biuret reagent to the mixture in the test tube and wait for any color change. 3, Record your observation on the table provided. Ninhydrin test 1, Place a small portion (size of mongo bean) of casein in a test tube. Add 3 mL water. 2. Add 1 ml of Ninhydrin reagent to the mixture in the test tube and heat in boling water bath for 5 minutes. 3. Record your observation on the table provided. 2|Page Scanned with CamScannerCEBU DOCTORS’ UNIVERSITY COLLEGE OF ARTS AND SCIENCES Physical Sciences Department Xanthoprotelc Test 1. Place a small portion (size of mongo bean) of casein in a test tube. Add 3 mL. water, 2. Add 5 drops concentrated nitric acid (HNOs) to the mixture in the test tube and heat gently over a Bunsen flame until a yellow color produced. 3. Cool the solution. Add ammonia and observe color change. 4, Record your observation on the table provided. Adamkiewicz Test 1. Place a small portion (size of mongo bean) of casein in a test tube. Add 3 mL water, 2. Add 10 drops of glacial acetic acid (previously exposed to sunlight) to the mixture in the test tube. Mix well. 3. Tilt the test tube at 45° and superimpose 1 mL of concentrated sulfuric acid, Do not mix. ‘4. Note the color produced at the junction between 2 liquids. Record your observation on the table provided. Molisch’s Test 1. Place a small portion (size of mongo bean) of casein in a test tube. Add 3 mL water. 2. Add 1 mL of Molsich reagent into each test tube, and then run down concentrated H2SO, at the side of each test tube. 3. Wait for any color change and record your observation on the table provided. V. Results: Table 1. Result of the Color Reactions Name of Test Result Biuret test Eagle Ninhydrin test dete Nae, Xanthoproteic test chy ‘Adamkiewicz’s test aieg ‘Molisch’s test Pwpig ti Mi. Guide Question: 1. What process causes the precipitation of casein? Does this process affect its amino acid composition? I hudealyats in soluble in water produting att_calid odin AR Guo ar ning aud gerne, Thy ypu in gar cabin bs prepa fu mnik NU be ert so thal WS ddic RHeIF For Ae pokin ¥e bee Tyvehuue THE inglabllhy op Te Prvain Tea rea ok ys 2. In the precipitation of casein, why is it important to add the acid dropwise and not add casein excess acid? css thon oe avid is ortant. This is because exer Ge con hydrolyze and — reduce —Tavhe_"Tn milk, 3. Based from the result of the experiment, what component of milk is responsible for its white color? ed tra sult Of the experiment , the Castin gives _ the Scanned with CamScannerCEBU DOCTORS’ UNIVERSITY COLLEGE OF ARTS AND SCIENCES Physical Sciences Department 4. Based on the color reactions results, identify whether casein has the following listed Components. If the component is present, Just write present in blanks provided, And then, at the side write down the test that is sued to confirm this. a. peptide bonds? q b. free amino group? Presen’ inh ¢. phenyl-containing amino acids? —-Praent anthoprattic Tes d. tryptophan? > _Kownt 5. Is casein a glycoprotein? Explain why or why not based on the result of the color reaction. lr _r it in {ited postive in the Nalitch’ 6. Aside from bein, 1g a protein source, what other industrial application does casein have? Eatin ttn std in_couhugs tar pote aluts, pnicty plastics tnd _vunthel i Med jcorhen Lond cormelies all “an i wtot Prowssors orn Ut in F__agrove He an adaed nutri Ronal valve.” cheele Mabars use CATIA — Protein eT For and Water While Fauilrtahng matrix “poration. Scanned with CamScanner