Isolation and hydrolysis of casein

from milk magic (non-fat milk)

Academia, Mary Joyce M.

Biag, Irian Joseph
Cruz, Eleazar John
Dela cruz, Dave Ryan
Gaerlan, Gerard Matthew
3BIO2

INTRODUCTION

PROTEINS
-

Long chain of amino acids with a particular function

Consist of amide bonds (peptide bonds)that links the alpha

amino group of one AA and a carboxyl group of the other AA

Types of protein:
Globular= (somewhat) water- soluble; forms colloid in water
Fibrous = water- insoluble; form rod/wire like shapes

Foods/Beverages where proteins are abundant: Egg, Pork, Milk,

Beef, Fish

MILK
Nutrient-rich= serve as good sources ofcalciumandvitamin
Das well asproteinand other essential nutrients.
Isoelectric point: 6.6
Types of milk: (whole milk, skim milk , organic milk, soya milk)
* Some milks contain fats
SKIM MILK/NON-FAT MILK
= PREVENT fats from contaminating protein
Types of Protein in Milk
Casein =Main protein of milk (Most abundant protein)
Lactalbumins
Lactoglobulins
Fig.1 Non-Fat milk
* All are globular proteins
http://manila-na.all.biz/milk-magiclow-fat-250ml-g4766#.Vrnmilh9600

CASEIN

(Latin Caseus) =Cheese

A phospoprotein
Casein function as Storage protein
Commonly found on Mammalian milk; 80% protein in cow milk, 20% to 45% Human milk
Casein exists in milk as the calcium salt,calcium caseinate.
isoelectric point of pH 4.6
A mixture of ALPHA (
), BETA (
), KAPPA (
) CASEIN
* KAPPA casein =Hydrophilic
= Negatively charged
= solubilizes the other 2 casein to favour formation and stabilization of micelle

Casein micelle= colloidal suspension

= large molecules will form when dispersed in a solvent
= responsible for the opaque color of milk

Fig. 2 Casein micelle

Fig.3 Alpha, beta, kappa casein

make up casein micelle

ISOLATION TECHNIQUE

Isoelectric Precipitate
- Precipitation of protein at pI (0= net charge)
- Proteins become least soluble
- NO NET CHARGE = leads to reduced solubility
=protein is unable to interact w/ the medium

OBJECTIVES

OBJECTIVES
To isolate casein from powdered Milk Magic (non-fat milk) by
isoelectric precipitation
To subject isolated casein to acid and alkaline hydrolysis
To neutralize acid and base hydrolyzates

METHODOLOGY

ISOLATION OF CASEIN

~5g Milk Magic powder

Dissolve in 20mL distilled
HO
Heat to 40 C maximum
Milk Magic solution
Heat to 55 C maximum
Remove from hot plate
*Note initial pH
Add 10% CHCOOH while
stirring
Continue until pH reaches
4.6
*Note the volume of
CHCOOH used
Let stand for 3-4 minutes
Amorphous mass of casein

Amorphous mass of casein

Filter precipitate by filtration
Or decant if amorphous
mass is
separated from
solution

casein

filtrate

Dry between filter papers, and tissue

Weigh
Compute for the percent yield
casein

Isolated casein
Cut the isolated casein into
half.

Half of the isolated

casein

Half of the isolated

casein
Cover with Al foil
Keep for second
experiment.
Half of the isolated
casein

ACID HYDROLYSIS AND

NEUTRALIZATION OF ISOLATED
CASEIN

Half of the isolated

casein
Cut into small pieces.
Put into a 50 mL Erlenmeyer
flask.
Add 4 mL 8N HSO4
Casein solution
Put cotton plug to flask.
Cover with Al foil.
Label flask.
*Note appearance.
Autoclave at (121) 15 psi
for 5 hours.= Faster
hydrolysis
*Note appearance.
Acid Hydrolyzate

Acid Hydrolyzate
Add 1 spatula
Ba(OH)Dissolve
Check pH using pH meter
Filter

Precipitate

Neutralized hydrolyzate
(Filtrate)
If volume is less than 7 mL,
make up to 7 mL with
distilled water.
Hydrolyzate

ALKALINE HYDROLYSIS AND

NEUTRALIZATION OF ISOLATED
CASEIN

Half of the isolated

casein
Cut into small pieces.
Put into a 50 mL Erlenmeyer
flask.
Add 5 mL boiling water and
2.0 g Ba(OH)
Casein solution
Put cotton plug to flask.
Cover with Al foil.
Label flask.
Warm flask to dissolve the
base.
*Note appearance.
Autoclave at 15 psi for 5
hours.
*Note appearance.
Base hydrolyzate

Base hydrolyzate
Dilute with 15 mL distilled
water.
Transfer to 250 mL beaker.
Add 1.0 mL of 16N HSO4
dropwise
to neutralize.
Check pH using pH meter.
If not neutralized, add 8N
HSO4 dropwise until pH
7 is reached.
Neutralized hydrolyzate

Neutralized hydrolyzate
Filter

Precipitate

Hydrolyzate
(Filtrate)
If volume is less than 7 mL,
make up to 7 mL with
distilled water.
Hydrolyzate

DATA AND RESULTS

Group
1
2
3
4
5
6
7
8
9

Percent (%) Yield of

Casein
71.84%
88.88%
75.30%
77.75%
75.63%
91.80%
74.94%
66.47%
84.43%

ERRORS
Percentage yield
90% up = Did not dried; water was included in isolated casein
65 % below= some of proteins were left in the erlenmayer flask

Acid Hydrolysis
Group

Appearance before
Autoclaving

White clumps in
colorless solution

3
5

White solid with

colorless liquid
Clear liquid and intact
protein
Clear, colorless
solution with white,
lumpy solid
Suspension with
cloudy liquid and

Appearance after
Autoclaving
Brownish solution with
brown translucent
flakes on the surface
Brownish solution with
solid particles
Dark brown liquid
Disappearance of
white, lumpy solid.
Black liquid solution
observed with darkcolored oil-like
droplets
Black liquid

Base Hydrolysis
Group

Appearance before
Autoclaving

Appearance after
Autoclaving

Cloudy liquid with

yellow precipitate

Brownish liquid with

solid particles

Whitish turbid liquid

with yellowish solid

Brown liquid with

brown precipitate

Turbid caramel colored

Turbid yellow solution
solution with solid
particles at the bottom
Turbid solution and
yellowish in color

Turbid solution and

caramel in color

DISCUSSION

CONCEPTS

Isolation

Isoelectric Point (pI) and Protein Solubility

Isoelectric Precipitation

Hydrolysis

Neutralization

ISOLATION

Isolation is a term used in chemistry to separate

substances from each other

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 no net charge.

ISOELECTRIC PRECIPITATION
A precipitate is a deposit of solid particles settled outofasolution.
The milk containing the protein casein has an initial pH ranging from
6 to 7 (6.6).

Casein was heated to 55C (max) to avoid denaturing the protein or

lose quaternary, tertiary, secondary structure of protein; Or simply
unfolding the protein.

Acetic acid (C2H4O2) was added for it to reach the protein's isoelectric
point, which is at pH 4.6.

Calcium caseinate has an pI of pH 4.6.

This means it is insoluble in solutions with a pH less than 4.6.
The pH of milk is 6.6, therefore, casein has a negative charge at this pH
and is solubilized as a salt.
If an acid is added to milk, the negative charges on the outer surface of the
casein micelles are neutralized, by protonation of the phosphate groups.
The casein micelles are destabilized or aggregate because the electric
charge is decreased to that of the isoelectric point (0= net charge)
The casein micelles disintergrate and the casein (the neutral
protein)precipitates because it is no longer polar, with the calcium ions
remaining in solution

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.

DISCUSSION: ACID HYDROLYSIS

AND NEUTRALIZATION OF ISOLATED
CASEIN

ACID HYDROLYSIS

Utilizes sulfuric acid (H2SO4)

Less amino acids (S,T,R,C) are destroyed

Acid also serves as catalyst

IMPORTANT Amino acid DESTROYED IS

Tryptophan to HUMIN (Black pigment after

autoclave)

NEUTRALIZATION

Acid hydrolysis: Ba(OH)2

Sulfuric Acid + Barium Hydroxide = Barium Sulfate

(Salt) formed in the hydrolyzate for further
characterization of Amino acid in EXPT 2

Isolates other free amino acids

DISCUSSION: ALKALINE
HYDROLYSIS AND NEUTRALIZATION
OF ISOLATED CASEIN

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)

NEUTRALIZATION

Base hydrolysis: 8N H2SO4

Sulfuric Acid + Barium Hydroxide = Barium Sulfate (Salt)

formed in the hydrolyzate for further characterization of A.A. in
EXPT 2

CONCLUSION

CONCLUSION
Casein was isolated from non-fat milk by isoelectric
precipitation(pH=4.6) which was achieved by lowering the
pH of the milk solution by adding 10% acetic acid.
Hydrolysis breaks the protein into smaller peptides and free
amino acids.
The acid hydrolyzates generally produced a brown to black
liquids with particles in some flasks, while the base
hydrolyzates generally produced brown liquid with
precipitate after autoclaving.

REFERENCES

REFERENCES
Kavitha, S. (2015). Viva Voce in Biochemistry (1st ed.). U.S.A.: Jaypee
Brothers Medical Publishers, Ltd.
Nehete, J. Y., Bhambar, R. S., Narkhede, M. R., & Gawali, S. R. (2013).
Natural proteins: Sources, isolation, characterization and applications.
Pharmacognosy Reviews, 7(14), 107116. http://doi.org/10.4103/09737847.120508
McMaster University. (1997). Experiment 11: Proteins and carbohydrates,
isolation of casein and lactose from milk. In Chem2006 Lab Manual.
Pavia D., Lampman G., Kriz G., & Engel R. (2005). Introduction to Organic
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vlab.amrita.edu,. (2011). Isoelectric Precipitation of Proteins: Casein from
Milk. Retrieved 10 February 2016, from vlab.amrita.edu/?
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