Isolation of
Casein
CHARISSE ANNE P. IGARAN, RMT
MILK COMPOSITION
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Procedure
1. To a 250-mL Erlenmeyer flask, add approximately 50 g of 2% milk.
2. Record the mass to the nearest 1/100th of a gram.
3. Heat the flask in a water bath while stirring the milk.
4. When the milk temperature has reached 40⁰C, remove the flask from the
water bath, and add 10 drops of glacial acetic acid while stirring.
5. Filter mixture by pouring it through 4 layers of cheesecloth held in a 100-
mL beaker.
6. Rinse the Erlenmeyer flask with 0.1 M acetic acid if necessary.
7. Remove most of the liquid from the solid (casein and fat) by squeezing
the cloth gently.
8. Discard the filtrate.
Procedure
• Place the solid into a 100-mL beaker and add 40-mL of 95% ethanol.
9.
10. After stirring the mixture for 5 min., allow the solid to settle.
11. Carefully decant the liquid containing the fat into a beaker.
12. Discard the liquid.
13. To the residue, add 15-mL of 95% ethanol and10-mL diethylether.
14. After stirring the mixture for 5 min., collect the solid by vacuum filtration
using pre-weighed filter paper.
15. Allow the casein to dry, weigh it, and calculate the percentage of casein
in the milk.
Casein has 4.6 and the pH of milk is
6.6 at this pH
Adding acid lowers the PH and
affects its solubility, causeing
precipitation of casein
The food industry takes advantage of this effect in
many of the processes used in the manufacture of
dairy products, due to the realization of desirable
properties of the finished products.
For instance, during the production of yoghurt,
casein coagulates and forms solid clumps in the
course of the gel formation due to the activity of
fermentative microorganisms.
Principle
• Most proteins show a minimum solubility at their isoelectric pH and
this principle is used to isolate casein by adjusting the pH of the
milk to 4.6, its isoelectric point.
• The main bulk of the precipitate is the casein.
• The entrapped residual fat can be removed by repeated washing
with the solvents such as ethanol and ether.
• Casein is insoluble in these solvents and this property can be
advantageously used to remove the unwanted fat from the
preparation.
Casein precipitates only at pH = 4.6. Why?
Casein precipitates at pH 4.6 because it is its
isoelectric point. It means that when the solutions'
pH drops below 4.6, the casein will eventually
precipitate forming insoluble salt. The negative
charge of casein in milk will be protonated,
aggregating the casein and precipitates.
Chemical Analysis
of Proteins
CHARISSE ANNE P. IGARAN, RMT
A. THE BIURET TEST PRINCIPLE:
• A Biuret test is a chemical test used to determine the
presence of a peptide bond in a substance. It is based
on the biuret reaction in which a peptide structure
containing at least two peptide links produces a violet
color when treated with alkaline copper sulfate. The
colored coordination complex is formed between Cu2+
ion and carbonyl oxygen (>C=O) and amide nitrogen
(=NH) of the peptide bond. Once this complex has been
formed, the solution turns from blue to purple.
B. NINHYDRIN TEST PRINCIPLE:
The test is performed as a result of the reaction between the amino
group of free amino acid and ninhydrin. The presence of ninhydrin,
a strong oxidizing agent, causes the amino acid to go through
oxidative deamination liberating ammonia and reduces the
formation of ninhydrin (hydrindantin). The NH3 formed from an
amino group reacts with another molecule of ninhydrin and is
reduced product (hydrindatin) to give a blue substance diketohydrin
(Ruhemanns complex). However, in case of amino acid like proline
and hydroxyproline, a different product having a bright yellow color
is formed. Asparagine, which has a free amide group, reacts to give
a brown colored product.
C. THE MILLON'S TEST PRINCIPLE:
In Millon’s test, when subjected to heat, the phenol
group of tyrosine is first nitrated by nitric acid
present in the Millon's reagent. The nitrated tyrosine
complexes the mercury (I) and mercury (II) ions in
the solution resulting to either a pink to brick red
precipitate or solution. The proteins, on the addition
of Millon’s reagent, form a white precipitate first due
to denaturation of proteins by mercury salts
D. THE XANTHOPROTEIC TEST
PRINCIPLE:
Uses a nitration reaction to determine presence of proteins
in a solution. Treating aromatic amino acids with
concentrated nitric acid leads to nitration of aromatic ring
and formation of yellow nitro products. It is used to detect
the amino acids containing aromatic nucleus (tyrosine,
tryptophan, and phenylalanine) in a protein solution which
gives yellow color nitro derivatives on heating with
concentration HNO3. When strong basic solution is added
the color of obtained products turns darker (yellow to
orange).
E. HOPKIN'S COLE TEST
PRINCIPLE:
The indole group of tryptophan reacts with
glyoxylic acid in the presence of conc. H2SO4 to
give a purple colored complex. Glyoxylic acid is
prepared by reducing Oxalic acid with
magnesium powder or sodium amalgam.
Glacial acetic acid which has been exposed to
the sunlight also contains glyoxylic acid and
can thus be used for this test.
F. LEAD ACETATE TEST
PRINCIPLE:
This test is specific for Sulphur containing amino
acids (Cysteine, Cystine and Methionine). The
purpose of this test is to detect the sulphide
group in cysteine and cystine. On boiling the
sulphur containing amino acids, the Sulphur is
split off from these amino acids as metallic
sulphides (K2S or Na2S). When a sulphide group
is detected, a brown-black color is formed.
TEST DETECTS POSTIVIE
BIURET PEPTIDE BOND VIOLET/PURPLE
NINHYDRIN AMMONIA (AMINO GROUP, BLUE-PURPLE
PRIMARY, SECONDARY
AMINE)
MILLON TYROSINE PINKISH RED/ BRICK
RED
XANTHOPROTEIC AROMATIC AMINO ACID YELLOW-ORANGE
(TYROSINE, TRYPTOPHAN,
PHENYLALANINE)
HOPKIN’S COLE INDOLE RING (TRYPTOPHAN) BLUE-VIOLET RING
LEAD ACETATE CYSTEINE, METHIONINE BROWN-BLACK
PRECIPITATE
METHODS PRINCIPLE
SERUM PROTEIN It uses an electrical field to separate the proteins in the blood serum
into groups of similar size, shape, and charge.
ELECTROPHORESIS
ISOELECTRIC FOCUSING Determines the isoelectric point of proteins. Protein migrate into the
point where its net charge is zero-isoelectric pH.
ION EXCHANGE In this type of chromatography is exchange of ions.The cationic and
anionic are exchangers. Positively charged molecules are attracted to a
CHROMATOGRAOHY negatively charged solid support in cation exchange while negatively
charged molecules are attracted to a positively charged solid support in
anion exchange.
TURBIDIMETRY Measures turbidity of solution by measuring the amount of light passing
through the solution.
SOLVENT A process of separating metabolites based on their different solubilities,
thus resulting to precipitation.
FRACTIONATION
• Examination of the proteins particularly in human plasma can provide
information reflecting disease states in many different organ systems.
• The most frequently performed measurement—that for total protein—is
usually performed on serum, which has no fibrinogen and no anticoagulant
that may slightly dilute proteins in plasma.
• Although total protein determination gives the physician some information
as to a patient’s general status regarding nutrition or severe organ disease
(as in protein-losing states), further fractionations yield far more clinically
useful information
