Isolation, Alkaline Hydrolysis and Neutralization of Casein from Nonfat Dry Milk

Jacqueline C. To*, Mark Aljon G. Valdez, Julia Mae P. Viduya, Angelica Isabel R.
Zaragoza
College of Science, University of Santo Tomas Espaa Blvd. Manila
Abstract
Casein is a very important component of milk. It is the main protein that milk has. In this experiment we
isolated this protein that is casein from non-fat milk where the protein separates into a solid form that can
be easily separated from the liquid solution. Taking half of the yield and subjecting it through alkaline or
base hydrolysis, noting the appearance before and after autoclaving.

Introduction
Milk is good for your bones, this statement is always what we associate milk
with and it is since it is always known for being rich in calcium that our bones so need to
stay strong and healthy, but milk is not fully made up of just calcium as an element. Milk
is composed of multiple compositions mainly water, protein, fats, carbohydrates and
other minerals. So where does milk come from, it is the product of the mammary glands
of mammals mainly produced to give nutrition to the said mammals young before they
are able to properly digest food and it also helps build up the youngs immune system.
Milk is made up of approximately 3.4% protein, 82% of its total protein is made up of
casein. Proteins are naturally occurring chains of amino acids connected by peptide
bonds. There are two types of proteins fibrous and globular, casein is a type of globular
protein. Globular proteins fold back on themselves making a sort of spheroidal shape
and they do not form intermolecular interactions between protein units.

Isolation is a term used in chemistry to separate

substances from each other, in this experiment we
isolated casein from non-fat milk with 10% Acetic Acid
(C2H4O2).

Figure 1: Acetic
Acid

Casein is the main phosphoprotein found in milk, it is a protein that has

phosphate groups attached to hydroxyl groups. It is the major component in cheese,
which we all know is made from milk. Casein has multiple uses apart from making
cheese, it is also known to have been used in the production of plastic, paint, glue, and
medical uses such as protein supplements and mineralization of teeth. In milk it also
exists as calcium caseinate and calcium salt.
Hydrolysis is the term used when water is added to a compound in which a
reaction will occur to produce other compounds. For this experiment we used Alkaline or
base hydrolysis. For the base hydrolysis Barium hydroxide (Ba(OH)2) was used.
An autoclave is a pressure chamber usually
used for sterilization of medical equipment and
medical waste before disposal using high-pressure,
high-temperature steam.

After autoclaving the

hydrolyzate is neutralized to pH 7 by adding sulfuric

acid (H2SO4).

Figure 2: Sulfuric
acid

Methodology
Approximately 5g of non-fat powdered milk was dissolved in 20mL distilled water
in a beaker and was heated to 55C on a hot plate. The initial pH was measured using
the pH meter. Once the initial pH was recorded 10% acetic acid was added to the milk
in a drop wise manner until the pH drops to 4.6, the volume of acetic acid used was
noted. The amorphous mass was decanted and the remaining liquid was disposed of.
The casein was dried by being sandwiched between two filter papers and having the
remaining liquid pressed out. The researchers now had the isolated casein. The casein
was weighed and the percent yield was solved. The isolated casein was cut in half and
one portion was wrapped in aluminum foil and stored, the other half was used in base
hydrolysis.
Half of the casein was cut into very small pieces and was added to a solution of
2g of Ba(OH)2 that was completely dissolved in 5mL of boiling distilled water in a 50mL
Erlenmeyer flask. The Erlenmeyer flask was plugged with a piece of cotton then
covered with aluminum foil and was secured with masking tape then labeled. The flask
was warmed gently and the appearance of the casein before autoclaving was noted.
The flask was then autoclaved at 15psi for 5 hours; the appearance of the casein after
autoclaving was noted. The sample after autoclaving was known as the hydrolyzate.
The sample was diluted with15mL distilled water and transferred into a 250mL beaker.
1mL of 16N H2SO4 was added drop wise to neutralize the sample. The pH meter was
used to check if the sample was neutralized, if the sample was not yet neutral with a pH
of 7 8N H2SO4 was added drop wise until the hydrolyzate reached pH7. The precipitate
that had formed was filtered off and disposed of. The volume of the filtrate was

measured using a graduated cylinder and was made sure to have reached at least 7mL
by adding distilled water. The samples were then put aside to be used for the color
reactions.
Results and Discussion
Table 1: results for Isolation of Casein from Milk

Weight of non-fat milk

5.0581g

Volume of 10% Acetic Acid

2.82mL

Initial pH

6.12

Final pH

4.42

Weight of dried casein

3.9402g

Percent yield

77.8988%

Appearance of casein

White cheese like solid

Table 1 shows the results for the first part of the experiment, which is the isolation
of the protein casein from non-fat milk. Non-fat milk was used in this experiment instead
of regular milk because the fat in milk does not contain any casein, meaning if regular
milk with fat was used the casein yield would be noticeably lesser than that of the nonfat milk. The milk dissolved in water should not be heated to more than 55C because a
higher temperature can affect the structure of the protein we are aiming to isolate. The
caseins pH is brought down to 4.6 because pH4.6 is the isoelectric point of casein,
meaning that the molecule has no electrical charge. Unfortunately in our experiment the
pH of our casein reached below 4.6 and it could have been one of the sources of errors
through out the rest of our experiment. The casein was dried and all liquid in the

amorphous mass was pressed out to prevent having a bigger yield than we should have
had since the liquid is not part of casein.

Table 2: results for Alkaline/Base Hydrolysis of Casein

Appearance of Sample Before Autoclaving

Yellowish clump of solid in a murky white

liquid

Appearance of Sample After Autoclaving

Golden-orange/caramel color liquid with

golden-orange/Caramel precipitate at the
bottom.

The protein was cut into very small pieces to make it easier to react with the
Ba(OH)2 in the boiling water. Ba(OH)2 was used instead of NaOH because though both
of them are strong bases NaOH is not as soluble as Ba(OH) 2 in water. Autoclaving
completely destroys the Serine, Threonine, Cystein and Arginine. Adding 16N H 2SO4
neutralizes the hydrolyzate near to pH 7 and slowly adding 8N H 2SO4 drop wise helps to
reach pH 7. The precipitate is filtered out because what is needed is the neutral
solution. The groups solution reached 7mL so distilled water was not added anymore.

Conclusion
Casein was isolated by the addition of 10% acetic acid, a weak acid and then
having the casein reach its isoelectric pH of 4.6. Casein was then hydrolysized using

alkaline or base hydrolysis with a strong base Ba(OH)2 . Serine, Threonine, Cystein and
Arginine were all destroyed in the process of base hydrolysis.
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