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.