Experiment 1A: Isolation of Casein from

Milk + Color Reactions of Intact Casein

BUAN, Patnubay F.
DE MESA, Neil Louie
JOCSON, Luigi Miguel
REYES, Corwin Guile
TAN, Abraham
3B9
ABSTRACT
Casein is the principal protein found in milk. Casein is one of the proteins in milk which is
used as a binding agent. The experiment aims to isolate Casein from 5 grams of non-fat
milk. Through the Isoelectric precipitation method, casein may be obtained. The average
percent yield of casein is 78.00%. The isolated casein was then tested with various color
reaction tests, namely Biuret test, Ninhydrin test, Xanthoproteic test, and Hopkins-Cole
test. The colors of each reaction were noted and the structure of Casein was obtained.
INTRODUCTION
MILK
- Complex biological substance with high amount of proteins,
lipids, and minerals.

- Milk proteins contain all 9 essential amino acids needed by

humans.(Histidine, isoleucine, leucine, lycine,
methionine,phenylalanine, threonine, tryptophan and valine.)

- Other proteins in milk: caseins, lactalbumins, lactoglobulins

(casein being the most dominant)
PROTEINS
- Naturally occurring, unbranched polymer made of
monomer units called amino acids.
- Chains of amino acids linked by covalent peptide
bonds.
CASEIN
- Main protein in cows milk
- Classified as a Phosphoprotein
- has phosphate groups attached to the hydroxyl groups of some of
the amino acid side-chains.

- Exists in milk as a calcium salt (calcium caseinate)

- Has an isoelectric point of 4.6
- If pH = 4.6, casein has zero net charge, and its least soluble
form as a salt due to isoelectric precipitation, and can be
isolated.
COLOR REACTIONS
- Biuret Test :Test for presence of peptide bonds

- Ninhydrin Test Test for compounds with free alpha amino

group.

- Xanthoproteic Test: test for aromatic amino acid

- Hopkins-Cole Test: Test for the presence of indole group in

Trp
OBJECTIVES
OBJECTIVES
1) To isolate and characterize intact casein using various chemical
tests.
2) To analyze the functional groups from each casein from low-fat
milk used in color reactions.

3) To explain the principles behind each test.

EXPERIMENTATION
Isolation of Casein from Milk

MATERIALS
-Powdered non-fat milk (5g)
-10% Acetic acid

EQUIPMENT
-pH meter
-stirring rod
Isolation of Casein from Milk
20 mL Distilled water Non-fat powdered milk

-Measure 5g of non-fat milk

-Mix with distilled water in a 100 mL beaker
10% HAc Milk solution
-Measure 1mL HAc -Heat the solution to 55c hot plate

-Pour slowly the 1mL acetic acid

-Stir gently with stirring rod
-Measure the pH (initial pH)
-Rinse the pH meter with distilled water then dry with tissue paper
Solution
1 mL acetic acid Solution

-Add dropwise with gentle stirring

-Add untill white curds are formed
-Decant to a filter paper
-Press with tissue to dry

Casein
-Weigh the casein
-Compute the %yield
-Divide in to two

in mL EF wrap in foil (intact

casein)
ISOLATED CASEIN
Color Reactions of Intact Casein

Intact Casein
-Grind with mortar and pestle
-Add 10 ml distilled HO

Ninhydrin Test Xanthoproteic Test

Biuret Test Hopkins-Cole Test
-add 10 drops of -add 10 drops of
-add 3 drops of - add 2 drops of
casein casein suspension to
casein suspension casein suspension
suspension to a a test tube
to a test tube to a test tube
test tube -add 1 mL distilled
-add 2 mL
-add 1 drop of 2.5 HO
-add 1 mL Hopkins-Cole
NaOH distilled HO -add 3 drops of conc.
reagent
-add 1 drop of 0.01 HNO
-add 0.5 mL 1% -incline the test
CuSO -note color
ninhydrin solution tube while slowly
-mix well -heat in boiling water
-mix well adding 2 mL of
-note color bath for 1 min. conc. HSO down
-heat in boiling -cool the solution
produced the side of test
water bath for 2-3 -add NaOH dropwise
tube until two
minutes until solution is
layers form
-note color alkaline
-note color formed
produced -test with litmus paper
at interphase
 Ninhydrin

MIXTURE of Casein + H2O Glyoxylic acid (Hopkins-Cole

reagent)
RESULTS AND
DISCUSSIONS
EXPERIMENTAL
Why was non-fat milk chosen?
Non-fat milk was chosen due to the fact that the
purpose of this experiment is to isolate protein.
Choosing milk that contains lipids/fats would
further complicate the process of isolating protein.
Since it would not be removed in the process of
isoelectric precipitation method.
If however, milk with fat was chosen,
centrifugation is a necessary step to separate the
lipids prior to isolating protein.
Discussion
Isoelectric precipitation method
The precipitation of a protein at its isoelectric point, at which
proteins are generally least soluble
pI of casein = pH 4.6, pI of milk = approx. pH 6.5
10% HAc is added gradually to lower the milk pH to 4.6 (pI of
casein)
HAc (pKa 4.76) was chosen because of its common availability
and its weakly acidic properties, so as to not drastically lower
the pH if stronger acids were chosen.
Why does protein precipitate at its isoelectric point?

At other pH besides its pI, the net charge of the protein (+ or -) can interact with
nearby water molecules, therefore it is more likely to dissociate itself with other
protein molecules. Thus, more soluble.
The net negative charge of the micelles that surround the protein, helps in
solubilizing it, in pH outside pI.
at the pI of the protein, wherein its net charge is 0, the micelles lose their net
negative charge since their phosphate groups are protonated.
Once the micelles are neutralized, the protein is neutralized as well and breaks free
from the soluble solution, it escapes as precipitate.
Discussion
The average volume used in this experiment was 1.85 mL of HAc
At its pI, casein starts to solidify, forming a large amorphous mass
The mass is separated from the remaining residues (lactose, salts,
minerals, and water), which is discarded, by either filtration or
decantation
The separated mass is then weighed (average yield of 78%) and
subjected to the color reactions
Discussion of principles behind each test:
Biuret test
Used to detect presence of peptide bonds (minimum of 2),
positive results yield a violet color. Negative result yields the initial
color. Principle behind is the reaction of Cupric ions (Biuret
reagent) with the amino groups the amino acid.
Ninhydrin test
Used to detect presence of primary amines (all amino acids except
proline)or secondary amines (proline), positive results yield a violet
color.
Discussion of principles behind each test:
Xanthoproteic test
Used to detect presence of amino acids with aromatic groups
(Phe, Tyr, and Trp), positive results yield a yellow color. Negative
results will yield the initial color, (usually colorless). Principle
behind is the nitration of the aromatic rings.
Hopkins-Cole test
Used to detect the presence of tryptophan in proteins, positive
results yield a violet interphase (upon addition of conc. sulphuric
acid to form layers) .
Biuret test.
- Purple solution.

- Semi-transparent
Biuret test.
- The Biuret test is based on the
ability of Cu (II) ions to form a
violet-coloured chelate complex
with peptide bonds (-CONH-
groups) in alkaline conditions.

- More peptide bonds = more

concentrated the color
Ninhydrin
Test
Before heating (Left)

- Opaque, white cloudy

solution.

After heating (Right)

- Solution turnes into

purple.

- Transluscent.
Ninhydrin Test
- The ninhydrin test is positive for amino acid.
- Free amino acids can react with ninhydrin reagent yielding a
deep purple solution.
- Deep purple solution: RUHEMANNS PURPLE
- Ruhemanns purple: A blue-violet dye formed in the reaction
of ninhydrin with amino acids.

- Ninhydrin: oxidising agent.

- causes oxidative decarboxylation of amino acids
Xanthoproteic Test

Before heating
- White, cloudy solution.
- Yellow coloration was
seen on the bottom.

After Heating
- Bright Yellow clumps
was formed on top of the
mixture.
- Translucent fluid is seen
on the middle.
- Pale yellow clumps on the
bottom.
Xanthoproteic Test
- Yellow color: due to xanthoproteic acid
- Xanthoproteic acid is formed through
the nitration of amino acids such as
tryptophan.
- Nitric acid is used to be able to nitrate
the amino acids.
Hopkins-Cole Test
- Transparent solution.

- Cloudy upper layer.

- Purple interphase
Hopkins-Cole Test
- Protein solution + Hopkins Cole reagent,
- Hopkins Cole reagent: consists of oxalic acid.
- Concentrated sulfuric acid: slowly added to form two
layers.
- A purple ring appears between the two layers if the test is
positive for tryptophan
CONCLUSION
Conclusion
The isolation of casein from milk can be brought by using the isoelectric
precipitation method or the precipitation of proteins on its isoelectric point. By
doing this, the protein, casein, solidified and was isolated.
The tests that were done for the color reactions of casein detected its
functional groups. Ninhydrin test is done to check for the presence primary or
secondary amines in which the test yielded positive results. Xanthoproetic test
checks for the presence of amino acids with phenyl rings. Since intact casein has
tryptophan and tyrosine, it resulted to a positive result in the xanthoproetic test.
Hopkins-Cole test determines the presence of tryptophan in proteins and casein
yielded a positive result as shown in its violet interphase. Biuret test checks for
the presence of peptide bonds which are evident in intact casein for it gives the
casein its solid form.
References
Color Reactions of Amino Acids. (2016, May 05). Retrieved September
26, 2017, from http://www.biochemden.com/color-reactions-of-
amino-acids/
Isoelectric Precipitation. (2017, June 16). Retrieved September 25,
2017, from
http://www.oxfordreference.com/view/10.1093/oi/authori ty.20
110803100012784
Isoelectric Precipitation of Proteins: Casein from Milk. (n.d.).
Retrieved September 25, 2017, from
http://vlab.amrita.edu/?sub=3&brch=63&sim=158&cnt=1
Qualitative Analysis of Amino Acid. (n.d.). Retrieved September 26,
2017, from
http://vlab.amrita.edu/?sub=3&brch=63&sim=1094&cnt=1
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https://mbbsstudystuff.com/protein-biuret-test/

http://brilliantbiologystudent.weebly.com/biuret-test-for-protein.html