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    Isolation and Characterization of Casein From Milk

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    Valent Tambunan
    Casein is the main protein in milk and was isolated through acidification with acetic acid until the mixture reached a pH of 4.6, the isoelectric point of casein. This caused the negatively charged casein micelles to become neutralized and precipitate out of solution. A series of tests were performed on the isolated casein that characterized it through reactions with reagents like copper sulfate and ninhydrin. Casein forms colloidal complexes in milk due to its ionizable groups, giving milk its opaque white color. It can be denatured or have its shape altered by various substances like heat, acids, bases, and heavy metal salts.

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    Isolation and Characterization of Casein From Milk

    Uploaded by

    Valent Tambunan
    2K views4 pages
    Casein is the main protein in milk and was isolated through acidification with acetic acid until the mixture reached a pH of 4.6, the isoelectric point of casein. This caused the negatively charged casein micelles to become neutralized and precipitate out of solution. A series of tests were performed on the isolated casein that characterized it through reactions with reagents like copper sulfate and ninhydrin. Casein forms colloidal complexes in milk due to its ionizable groups, giving milk its opaque white color. It can be denatured or have its shape altered by various substances like heat, acids, bases, and heavy metal salts.

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    ISOLATION AND CHARACTERIZATION OF CASEIN FROM MILK

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    Casein is the main protein in milk and was isolated through acidification with acetic acid until the mixture reached a pH of 4.6, the isoelectric point of casein. This caused the negatively charged casein micelles to become neutralized and precipitate out of solution. A series of tests were performed on the isolated casein that characterized it through reactions with reagents like copper sulfate and ninhydrin. Casein forms colloidal complexes in milk due to its ionizable groups, giving milk its opaque white color. It can be denatured or have its shape altered by various substances like heat, acids, bases, and heavy metal salts.

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    2K views4 pages

    Isolation and Characterization of Casein From Milk

    Uploaded by

    Valent Tambunan
    Casein is the main protein in milk and was isolated through acidification with acetic acid until the mixture reached a pH of 4.6, the isoelectric point of casein. This caused the negatively charged casein micelles to become neutralized and precipitate out of solution. A series of tests were performed on the isolated casein that characterized it through reactions with reagents like copper sulfate and ninhydrin. Casein forms colloidal complexes in milk due to its ionizable groups, giving milk its opaque white color. It can be denatured or have its shape altered by various substances like heat, acids, bases, and heavy metal salts.

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    NUTRITIONAL BIOCHEMISTRY LABORATORY

    Experiment #4. ISOLATION AND CHARACTERIZATION OF


    CASEIN FROM MILK
    GROUP #2

    DATE : April 30,2016

    MEMBERS : - Libunao, Genesis


    -Tambunan, Valentina

    Introduction : When milk is acidified, it is transformed into a solid


    component, called curd, and a liquid component called whey. The curds contain the
    butterfat and a protein called casein. The carbohydrate, lactose, is present in the
    whey. In casein the main protein in milk is phosporotein, casein exist in milk as the
    calsium, salt calcium caseinate, it has an isoelectric point of pH 4.6 therefore
    casein has negative charge at this pH and issobilized as a salth.the way we can
    know or we can check the pH is first put 20.0g non-fat milk to Erlenmeyer flask
    and add 50.0ml of water, stir it well and heat the mixture to about 40c cant be
    exceed 40c in water bath, before heat the mixture, make sure to put the
    thermometer to Erlenmeyer flask so it will help to have the certain 40c and add
    few drops of acetic acid until it reach 4.6 pH. If acid is added to milk, the negative
    charges on the outer surface of the casein micelles are neutralized and the neutral
    protein precipitates with the calsium ions remaining the solation.
    Material & reagents :
    powdered non-fat milk/ skim milk
    thermometer
    pH meter and pH indicator
    funnel and filter paper
    hot plate and water bath
    150mL Erlenmeyer flask
    100 & 500 beaker
    Spatula and stiring rod
    Pipette and aspirators
    Test tube rack and test tubes

    -HCl
    -NaOH and 10 % NaOH
    - 70% C2H5OH
    -distilled water
    -5% CuSO4
    -2% glycine
    -2% gelatin
    -1% tyrosine
    -2% albumin
    - conc. HNO3

    10% acetic acid


    Ninhydrin reagen
    (NH4)2SO4

    - PB (NO3)2
    -Sn(IV) NO3

    Experimental :
    (A) isolation of protein : Place the 20.0g of non-fat milk and 50 mL of water in
    150mL Erlenmeyer flask and stir it well. And put the thermometer inside the
    Erlenmeyer and heat it in water bath up to 40C make sure it will not go more than
    40c. add 10% acetic acid for 5 drops and stir it well, continue it until it reach 4.6
    pH using the pH meter. Filter the mixture using filter paper, removing excess water
    from the mixture, discard the filter and transfer to watch glass. And weight the
    dried casein and calculate the percent by mass casein.
    (B) Identification test of casein:
    (B.1) Qualitative test :prepare 5 test tubes and in a clean and labelled test tube
    place 1.50mL of 2% glycine,2% gelatin ,1% tyrosine, 2% albumin, and casein
    prepared in part A ( spatula +distilled water) to each test tube. Add 0.5 mL
    10% NaOH solution and 0.20 mL 5% CuSO4 solution.
    (B.2) Ninhydrin test : prepare 5 test tubes and in a clean and labelled test tube
    place 1.50mL of 2% glycine,2% gelatin ,1% tyrosine, 2% albumin, and casein
    prepared in part A ( spatula +distilled water) to each test tube add 0.50mL of
    ninhydrin test and heated for 5 minutes.
    (B.3) prepare 5 test tubes and in a clean and labelled test tube place 1.50mL of
    2% glycine,2% gelatin ,1% tyrosine, 2% albumin, and casein prepared in part
    A ( spatula +distilled water) to each test tube add 1.0 mL conc.HNO3 while
    swirling. Heat the test tubes on the warm water bath. Observe a change in color
    (C) Denuration test :
    (C.1) heavy metal Ion test : place 2.00 mL skimmed milk in each of labelled test
    tube. Add a few drops of each of the ff heavy metal ions to corresponding labelled
    test tube as indicated :
    1. PB2+ as Pb( NO3)2
    2. Hg2+ as Hg( NO3)2
    3. Sn4+ as Sn(IV) NO3
    (C.2) Acid/base : prepare 5 test tubes and in a clean and labelled test tube place
    1.50mL of 2% glycine,2% gelatin ,1% tyrosine, and add 1.0mL HCL. And

    1.0NaoH to 2% albumin, and casein prepared in part A ( spatula +distilled


    water).
    (C.3) Heat : prepare 5 test tubes and in a clean and labelled test tube place 1.50mL
    of 2% glycine,2% gelatin ,1% tyrosine, 2% albumin, and casein prepared in
    part A ( spatula +distilled water) and heat it.
    Result and discussion: Casein is the protein that was isolated fromnon-fat milk
    through adding acetic acid. Whenacetic acid was added to the non-fat milkmixture at a
    controlled pH level, yellowish whiteprecipitate was produced. That process is
    calledisoelectric precipitation. Precipitation occurredbecause the protein has
    already reached its isoelectric point wherein its net charge equalledto zero. Intact
    proteins, acid hydrolysates and basichydrolysates were all tested to characterize anddetermine
    the functional groups that theycontain.. Each test that was performed has differentconcepts
    behind them. The reactions of theintact proteins and hydrolysates to the reagentsof each test,
    depends on their characteristics.They can be positive or negative in a particulartest. Biuret test is
    a general test for proteins and atest for detecting peptide linkage. Its principle iscomplexation
    reaction. The intact protein shouldbe positive in this test since its peptide linkage isnot broken
    unlike the two other samples thathad undergone hydrolysis. As seen in theresults, only the intact
    protein produced a violetsolution which is a positive indication of theBiuret test. Ninhydrin test
    is a test for detecting free alphaamino groups. Proline is the only amino acidthat is negative for
    the said test. Its principle isoxidative deamination and decarboxylation. Apositive indication of
    this test would be a blueviolet coloration in the solution. Intact proteincasein and the two other
    hydrolysates mustyield a positive result in this test. Xanthoproteic test is a test for presence of
    aromatic rings which includes tryptophan andtyrosine. Although phenylalanine is
    consideredone of them, it will not have a positive resultbecause it is inactive. Its principle is
    thenitration of the phenyl group. To know that thesubstance is positive for Xanthoproteic
    test,yellow to orange solution must be achieved.Intact proteins, acid hydrolysates and
    basichydrolysates are positive for this test. In theresult for this test, there had been a slight errorin
    the color of the result of acid hydrolysate.
    Conclusion : Casein is a phosphoprotein, which has phosphate groups are attached to some of
    the amino acid sidechains. These are attached mainly to the hydroxyl groups of the serine and
    threonine moieties. Calcium caseinate has its isoelectric (neutrality) point at 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 micelle are neutralized (the phosphate groups are protonated)
    and the neutral protein precipitates.

    Milk is white because of its reflectance properties. The opacity of milk is due to its
    content of suspended particles of fat, proteins (mainly Casein which is a group name for the
    dominant class of proteins in milk. The caseins easily form polymers containing several
    identical or different types of molecules. Due to the abundance of ionisable groups and
    hydrophobic and hydrophilic sites in the casein molecule, the molecular polymers formed by
    the caseins are very special. The polymers are built up of hundreds and thousands of individual
    molecules and form a colloidal solution) and certain minerals. The colour of milk varies from
    white to cream (depend on natural coloring pigment). Skimmilk is more transparent, (due to
    absent of fat portion) and the colloidal solution, which gives skimmilk its whitish-blue tinge.
    Casein is a milk protein and has a isoelectric point( the pH where net charge on the
    molecule is 0) of 4.6 I.e. it is insoluble in any a solvent whose pH is less than 4.6 .pH of milk is
    around 6.6 so it is present as calcium caseinate. On the addition of acid , anionic form of casein
    gets neutralized and due to decrease in the pH precipitates out
    Since Proteins can be denatured (distorted in shape) by heat, alcohol, acids, bases, or the
    salts of heavy metals. Milk used an antidote in cases of heavy metal poisoning. poisons are salts
    of heavy metals such as mercury and silver; these denature the protein strands wherever they
    touch them.

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