ISOLATION AND

HYDROLYSIS OF CASEIN
FROM NON FAT MILK
Acao, Praiseus
Awat, Ann Christine
Corpuz, Ma. Cristina
Escalona, Ropelio
Gianan, Amirah
3MB

Proteins
One

of the four biomolecules

Polymeric; the monomer is an amino acid
Amino acids are linked via Peptide bonds
Two types of proteins: Globular and Fibrous
Some functions of protein include:
Regulatory, Catalytic, Structural,
Defensive, Storage, Transport, Receptor

Milk

Common food which has high nutritional value

9 essential amino acids found in milk protein
Different kinds of milk: Non-fat, Skim Milk, Low
fat and pure fat
Three proteins found in milk: Casein,
lactalbumin and lactaglobulins. All are globular
proteins.
NON-FAT MILK was used to prevent FAT
from 'contaminating' CASEIN. Fats go
along with the protein isolation/hydrolysis

Casein

One of the major proteins in milk

Most abundant protein in milk (along with whey)
Phosphoprotein- contains one or more phosphate
groups attached in side chains (usu. serine or
threonine)
Slow-digesting protein (provides a sustained release of
amino acids)
A mixture of alpha, beta and kappa caseins form a
cluster called micelle which is responsible for the
white opaque appearance of milk.
Micelle - anywater-soluble aggregate, spontaneously
andreversibly,formedfromamphiphile
(possessesbothhydrophobicandhydrophilic
elements)molecules.
pI is at pH 4.6

OBJECTIVES

To isolate casein by Isoelectric

precipitation from non-fat milk
To subject the isolated casein to acid
and base hydrolysis
NEUTRALIZE!!

MATERIALS(NO NEED)

5g Milk Magic
10% Acetic acid
pH meter
8N H2SO4 (for acid hydrolysis)
Ba(OH)2 (for base hydrolysis)

METHODOLOGY
5g milk powder
Add 20mL distilled water
Stir warm to 55C
Remove from the hot plate

Warm milk solution

Note initial pH
Add dropwise of 10% Acetic acid(HAc) while
stirring until pH 4.6 (do not destroy the lumps
formed)
Note the volume of HAc used
Let it stand

Casein

Decant
Dry between filter and tissue papers
Compute for percent yield
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% Yield casein

Actual Yield (g Casein)

Theoretical Yield (g Milk)
100

Divide equally into two

Intact casein
Wrap with
Aluminum foil and
label
Refrigirate

Intact protein
for next
meeting

Casein

Cut in small pieces

Put in 50mL
Erlenmeyer flask

Acid
Base
Hydrolysis Hydrolysis

 Add 4ml of 8N H2SO4

Plug with cotton and
cover with aluminum
foil then label
Note appearance
Autoclave at 15psi for 5
hours

Acid
Hydrolyzate

Check pH using the pH

meter
If not yet neutralized, add
dropwise of saturated
Ba(OH)2
Check pH again using the
pH meter
Filter

Residue
(discard)

Dilute with 15ml distilled

H2O
Transfer into 250mL
beaker
Neutralize by adding 1
porcelain spatula of
Ba(OH)2 then dissolve all
of Ba(OH)2 by heating the
solution while stirring, do
not let it boil

Filtrat
e Transfer to

graduated
cylinder, make
to 7mL

7mL
Neutralized
Hydrolyzate
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Methodology

Non-fat milk was used so that it

would be easier to isolate casein as
whole milk contains more proteins.
The milk was only warmed so as not
to denature the milk.
Denature to cause the tertiary
structure of (aprotein) to unfold, as
with heat, alkali, or acid
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Methodology

Autoclave
This was done for the heat-catalysed
reaction.
This was to free the amino acids in
high pressure as the acid/base
catalyses the reaction.
HIGH PRESSURE = FASTER
HYDROLYSIS

Group
(Acid hydrolysis)

Before Autoclaving

After Autoclaving

Casein did not

dissolve; Casein
pieces with clear
liquid

Black Solid

Chunky, Does not

dissolve easily

Black solution with

thin residues on the
edges of the flask and
some are floating

Clear liquid with

white solids

Black Solid

Clear liquid with

white clumps of white
casein

Dark brown with

white paper-thin
brown casein

Clear solution with

white solids

Clear solution with

white solids

 Check pH using the pH

meter
If not yet neutralized, add
dropwise of 8N H2SO4 until
ph 7
Check pH again using the
pH meter
Filter

Base
Hydrolysis
Add 5mL of boiling
distilled H2O
Add 2.0g of Ba(OH)2
Plug with cotton and
cover with aluminum
foil then label
Warm gently to
dissolve all Ba(OH)2
Note appearance
Autoclave at 15psi for
5 hours

Residue
(discard)

Base
Hydrolyzate
Dilute with 15ml distilled H2O
Transfer into 250mL beaker
Neutralize by adding 1mL of
16N H2SO4 dropwise

Filtrat
e Transfer to

graduated
cylinder, make
to 7mL

7mL
Neutralized
Hydrolyzate
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Group
(Base hydrolysis)

Before Autoclaving

After Autoclaving

Clay creamy white

solid

Yellow orange with

white precipitate

Cloudy, light yellow

solution with lumps

Cloudy, light yellow

with small lumps

Darkish Yellow

Milk tea color brown

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Results
Percent Yield
Group 1

69%

Group 2

83.57%

Group 3

62.10%

Group 4

71%

Group 5

68.10%

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Results
Percent Yield
Group 6
Group 7

84.73%

Group 8

68.26%

Group 9

82.54

Group 10

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Discussion
Isoelectric Point (pI) and Protein Solubility
The pI of a protein is the pH at which protein is
least soluble and as a result it can be easily
'caught' in a mixture or solution.
The pH where a molecule has not net charge.

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Discussion
Isoelectric Precipitation
A

precipitate is a deposit of solid particles settled

outofasolution.
The milk containing the protein casein has an initial basic
pH ranging from 6 to 7 (6.6). Acetic acid was
added for it to reach the protein's isoelectric point, which
is at pH 4.6.
During the addition of the acid (donates H +), the negative
charges on the outer surface of the micelle are
neutralized (the phosphate groups are protonated), and
the neutral protein precipitates.
The casein peptides and micelle structures become
disturbed or denatured to form simpler structures which
become gelatinous.
Casein was obtained by reaching its pI.
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Discussion
Acid/Base Hydrolysis of Protein
(Casein)
Hydrolysis is the splitting or breaking
of a substance into smaller pieces with
the use of water.
Protein hydrolysis may involve
enzymes or chemical reagents and in
this case, we used either an acid H2SO4 or a base - Ba(OH)2 for splitting
proteins into smaller, amino acids.
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Acid Hydrolysis

Utilizes sulfuric acid (H2SO4)

Less amino acids are destroyed
Hydrolysis that is frequently used
over base hydrolysis
IMPORTANT A.A. DESTROYED IS W
Tryptophan to HUMIN (Black
pigment after autoclave)

Base Hydrolysis

Destroys more amino acids than acid

hydrolysis therefore it is less
frequently used. (Targets S, T, C and
R)
Utilizes Ba(OH)2 (barium hydroxide)
IMPT A.A. destroyed: R
Arginine to UREA and ORNITHINE
(No physical changes)
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Neutralization

Acid hydrolysis: Ba(OH)2

Base hydrolysis: 8N H2SO4
Sulfuric Acid + Barium Hydroxide
= Barium Sulfate (Salt) formed in
the hydrolyzate for further
characterization of A.A. in EXPT 2

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References

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1998, 2000, 2003).
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Clark, J. (2004). The hydrolysis of proteins. Retrieved from
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http://www.chemguide.co.uk/organicprops/aminoacids/proteinhydrolysis.html
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Jacobs, J. (2015, June 12). What is calcium caseinate? Retrieved from Livestrong.com:
http://www.livestrong.com/article/509796-what-is-calcium-caseinate/
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