Isolation, Base Hydrolysis, and Neutralization of Casein from

Powdered Non-fat Milk

Kaitlyn Grace C. Vargas*, Mary Anne V. Villaflor, Dianne E.Yadao
College of Science, University of Santo Tomas, Espana Blvd., Manila

Abstract
The experiment involves isolation of casein through the use of isoelectric precipitation.
In this method, the milk solution was acidified to lower its pH in order to easily isolate
the casein from the milk. A percentage yield of 75.77% isolated casein was obtained.
Further analysis of the casein isolate was done through alkaline hydrolysis.

Introduction
Proteins are high molecular weight polymer mixtures of -amino acids that are
combined by peptide linkage ( -CO-N H-). They are the chief constituents of all living
matter. The sample used in this experiment, which is milk, contains high amount of
protein. It also contains fat, carbohydrate, mineral, and vitamins, which is why it can be
considered as a complete food. Milks pimary proteins are casein and lactalbumin (Deb,
2011).
Casein, or caseinogen, is classified under phosphoproteins, which contain phosphorus
as a prosthetic group or side chain. It contains about 85% of protein content of milk
(NIIR Project Consultancy Services [NPCS], 2012) and serves as a storage protein that
satisfies nutritional needs (Bettelheim and Landesberg, 2004). It is insoluble in water,
due to being highly hydrophobic, as well as in alcohol. In milk, it is dissolved in water in

the form of calcium caseinate, which is responsible for the white color of milk (NPCS,
2012).
According to NPCS (2012), casein has a relatively high number of noninteracting proline residues and also has no disulfide bridges, making it have a relatively
little tertiary structure, which is a three dimensional shape adopted by the entire peptide
chain (Smith, 2013). Having such characteristic makes casein unable to undergo
denaturation the disruption of secondary, tertiary, and quarternary structure of native
protein that results to physical, biological, and chemical modifications of the protein
(Deb, 2011). In milk, it is found as a suspension of particles, referred to as casein
micelles that is, similar to surfactant-type micelle, are spherical and have hydrophilic
parts at the surface. However, in contrast to surfactant micelles, its interior is particularly
hydrated. The pH of casein is 4.6, which will make it have a negative charge in milk,
which has a pH of 6.6. This pH value is also known as the isoelectric point of the protein
and is the pH at which the protein is least soluble. This guides the isoelectric
precipitation of casein involved in the isolation of casein from milk. (NPCS, 2012). In
this method, charge on the protein, either positive or negative, can interact with water
molecules, which makes a protein molecule more likely to dissociate itself from other
protein molecules, thus, more soluble. As a result, protein is the least soluble when the
pH of the solution is at its isoelectric point. To rapidly obtain the casein, a quantity of
acid is added to the milk solution (NPCS, 2012). The objectives of this experiment are to
isolate casein from non-fat milk by isoelectric precipitation and to subject the isolated
casein to alkaline hydrolysis. For further examination of the casein isolate, an additional

process called hydrolysis can be done. This is can be done with the aid of a strong acid
or base (Deb, 2011).

Methodology
For the isolation of casein, five grams of powdered non-fat dry milk was first
weighed and used as the sample in the experiment. It was then dissolved using 20 mL
of distilled water in a 100 mL beaker. The solution was heated to 55C using a hot plate.
The initial pH was noted after heating and 10% acetic acid was added in a dropwise
manner while stirring until pH reaches 4.6. The large amorphous mass, which is now
the casein that formed in the beaker, was filtered by gravity filtration or by a simple
decantation if the large amorphous mass separated from the solution. Then, the isolated
casein was dried between opposite layers of filter papers and paper towels. After drying,
it was placed on a watch glass for weighing and its percentage yield was calculated.
Lastly, the isolated casein was divided into two portions one for alkaline hydrolysis
and the other for another experiment. The latter half was wrapped in an aluminium foil
and stored in the refrigerator.
For the alkaline hydrolysis, the casein was cut into tiny pieces and placed it on a
50 mL Erlenmeyer flask. 5 mL of water and 2 g of powdered Ba(OH) 2 were added
before boiling the solution. After boiling, the flask was plugged with cotton and covered
with aluminium foil. The appearance before and after autoclaving were noted. After
autoclaving, a hydrolyzate was formed, and it was diluted with 15 mL of distilled water
and transferred to a 250 mL beaker. Then, it was neutralized by adding 1.0 mL of 16N

H2SO4 dropwise. Using a litmus paper, the pH was checked. 8N of H 2SO4 was added
dropwise if the solution was not yet neutralized after consuming 1.0 mL of 16N H 2SO4.
Then, the precipitate was filtered off. If the filtrate obtained was less than 7mL, distilled
water was added to make it reach such volume.
Results and Discussion
Table 1. Data gathered on casein isolation
Weight if powdered non-fat dry milk
Initial pH
Final pH
Volume of 10% acetic acid used
Weight of casein
Percent yield
Appearance of sample before autoclaving
Appearance of sample after autoclaving

4.9127 g
6.15
4.16
1.8 mL
3.7224 g
75.77%
presence of yellow globular precipitate in a
yellow liquid
orange liquid with a few white precipitate

In the earlier parts of the experiment, the milk solution was heated to 55 C to
avoid denaturation of the whey protein that is also present in milk, not of the casein that
is relatively heat-stable up to 71C. In this experiment, the isolated casein was obtained
by using isoelectric precipitation. Isoelectric precipitation is a method wherein a
precipitate is obtained with the aid of an acid. Acetic acid was used in the experiment to
charge the casein and become soluble. As mentioned earlier, a charge on the protein
leads to more solubility, which makes a pH of the solution at the isoelectric point of the
protein lead to less solubility, thus, formation of precipitate (NPCS, 2012). So, the
addition of acetic acid was intended to lower the pH of the milk solution and to
destabilize the casein micelles that led to the formation of casein isolate.

The second part of the experiment is the alkaline hydrolysis. Before autoclaving,
the casein has a yellow globular precipitate in a yellow liquid. However, after
autoclaving, the casein turned into an orange liquid with a few white precipitate.
Autoclaving speeds up the hydrolysis process of amino acids.
The isolated casein was cut into tiny pieces to aid in the rapid dissolving of the solution.
In the hydrolysis, Ba(OH)2 was used as the strong base to destroy the peptide bonds
between the amino acids. Ba(OH) 2 is known to being easily removed after hydrolysis
(Smith, 2013).

In the neutralization, After the hydrolysis, the hydrolyzate was

neutralized to prepare for a following experiment involving color reaction. H2SO4 was
used to react with Ba(OH)2 in order to form a solid, which is unnecessary in the next
experiment, that can be easily filtered off.

Conclusion
The casein precipitates when through isolectric precipitation, wherein an addition
of acetic acid lowers the pH of the milk solution down to the pH of the protein. An
alkaline hydrolysis helps in further analysis of the protein by breaking it down to amino
acids.

References
Bettelheim, F. A. & March, J. (2004). Introduction to organic & biochemistry (3rd ed.).
New York: Harcourt Brace College Publishers.
Deb, A. (2011). Fundamentals of biochemistry. London: New Central Book Agency.

Mundy, B. P., Armold, M. T., & Amend, J. R. (1993). Organic and biological chemistry.
Fort Worth: Saunders College Pub.

NIIR Project Consultancy Services Board. (2012). Detailed project profiles on dairy &
dairy products. New Delhi, India: Author.
Smith, J. (2013). Principles of general, organic, & biological chemistry. New York:
McGraw-Hill