Isolation and Hydrolyzation of Casein from Non-fat Milk

Vida Sison, JinetteSurot*, Coleen Sy, Denise Tan

College of Science, University of Santo Tomas

Abstract
Casein is a protein commonly found in milk. In this experiment, it was isolated and hydrolyzed. It

was isolated by adding, drop wise, 10% acetic acid until its pH reached 4.6, which is its

isoelectric point. Casein appeared as a white amorphous mass. It was decanted, dried and

weighed. A percent yield of 52.44% was computed. The isolated casein was divided into two. The

other half was kept as intact protein and the other half was hydrolyzed and neutralized. Both

intact protein and hydrolyzate were subjected to various color reactions in the later experiment.

Introduction
Milk is an essential food source. It is perhaps the most nutritionally-complete food that is

found in nature. It is a complex biological fluid that contains water, carbohydrates, vitamins,

minerals, proteins and lipids which are crucial for growth and development, especially for the

young mammals. It is a good source of calcium and phosphorus but it is very deficient in vitamin

c and iron. Milk proteins contain all 9 necessary amino acids that is required for the growth if the

newborn. These amino acids are connected by peptide bonds and make up the protein. There are

three main groups of these protein found in milk, namely caseins, lactalbumins and

lactoglobulins. These are all globular which means they form themselves into compact units and

appear as nearly spheroidal shapes.

Casein is the main protein found in milk. It is hydrophobic which means it is poorly

soluble in water. It is responsible for the white opaque appearance of milk because it combines
with calcium and phosphorus in order to form clusters of casein molecules, also known as

micelles. It has an isoelectric pH of 4.6 which means, at 4.6 pH the positive and negative charges

of amino acids are equal and the overall charge is zero. Casein proteins can also be broken down

into its component amino acid. This process of breaking down proteins into amino acids is called

hydrolysis.

Hydrolysis is a reaction that involves the breakdown of a bond and is achieved by

extreme heat in the presence of water. This reaction often changes the pH of a solution. There are

three types of hydrolysis but only two are used in this experiment namely, acid hydrolysis and

basic hydrolysis. Both types of hydrolysis are complete hydrolysis of peptide bonds. However,

both have disadvantages. In acid hydrolysis, the destruction of the amino acid tryptophan will

result in the formation of humin which is a black precipitate, while in basic hydrolysis, amino

acid arginine would result in the formation of ornithine and urea. Specific acid and base were for

each type of hydrolysis in order to minimize the loss of amino acids. In acid hydrolysis, sulfuric

acid was used while in basic hydrolysis, barium hydroxide was used.

 Objectives:

1. To isolate casein from non-fat milk by isoelectric precipitation

2. To subject the isolated casein to acid or alkaline hydrolysis

Results and Discussion

There are three principles behind this experiment. The first principle is the isolation via

isoelectric precipitation. The pI of casein is at 4.6 which mean at 4.6 it displays minimum water
solubility that is why it is at this pH value where the casein was precipitated. Prior to the addition

of 10% acetic acid, it was heated to 55° C because other proteins might also hydrolyze.

Figure 1. Structure of Amino Acid

Milk has a pH value of 6.6, as 10% acetic acid was added, it caused the phosphate groups

of casein was protonized and the neutral protein precipitated. As the drop of pH occurred, it also

caused the casein micelles to aggregate. Thus, increasing the solubility of phosphorous and

calcium in the micelle. Non-fat milk was used for the experiment and not whole milk because fat

can remain with the casein during precipitation. (Bollag, Rozycki & Edelstein, 1996)

Figure 2. Casein Micelle

The second principle is the hydrolysis. Acid hydrolysis since it was assigned to our

group. Racemization does not happen with this type of hydrolysis, unlike alkaline hydrolysis

wherein amino acid structures are altered. There is also lesser destruction of amino acids. The

only amino acid that is totally lost in the process of acid hydrolysis is the amino acid tryptophan.

Amino acids serine, cysteine and threonine are also partially broken down and glutamine and

aspargine are converted to their acidic forms, glutamic acid and aspartic acid, respectively. On
the other hand, four amino acids are totally lost in this type of hydrolysis. These are Cysteine,

Threonine, Serine, and Arginine. Acid hydrolysis is commonly used because it destroys less

amino acid compared to the alkaline hydrolysis. In addition, in acid hydrolysis yields a solution

with back precipitate called humin while in alkaline hydrolysis, arginine is converted into orthine

and urea giving it its brown color. (Copeland, 2004) Also, sulfuric acid was used in acid

hydrolysis in order to minimize the loss of certain amino acids (Kubanova, 2014) and barium

hydroxide was used in basic hydrolysis because the rate of hydrolysis increases by the presence

of barium. (Helleiner, 1955) To speed up hydrolysis and to break the peptide bonds, it was

autoclaved for 5 hours as 15 psi.

Weight of Milk 5.1300 g

Weight of Casein 2.6900 g
% Yield 52.44 %
Final pH 4.60
Drops % HAc 200 drops
Sample before acid hydrolysis White solid mass
Sample after acid hydrolysis Solution with black precipitate

Table 1. Result of the experiment

The third and last principle of this experiment is neutralization. Neutralization was done

by adding a strong base or strong acid to neutralize the hydrolyzate for the next experiment. In

our case, we added a strong base to neutralize the acidic hydrolyzate. Ba(OH)2 was used to

completely remove sulfate ions because the BaSO4 formed is poorly soluble in water and

precipitates out. Aqueous Ba(OH)2 was added until its pH reached 7.

Figure 3. Reaction during neutralization of acidic hydrolyzate

Experimental
5 grams of powdered non-fat milk was dissolved in 20mL warm distilled water. The
solution was heated until it reached exactly55°C. The initial pH was noted. 10% acetic acid was
added drop wise, papers to dry. It was then weighed. The isolated casein was then divided into
two. The first half was hydrolyzed and the other half was wrapped in a foil and then labeled and
will be used for the next experiment.
Acid hydrolysis was assigned to the group for the hydrolysis of protein. The casein that
will be used for hydrolysis was cut into small pieces and then placed inside a 50 ml Erlenmeyer
flask. 4ml of 8N H2SO4 was added into the erlenmeyer flask. It was plugged with cotton and then
covered with aluminium foil, the flask was then labeled. It was autoclaved at 15 psi for 5 hours.
The appearance of the hydrolyzate was noted. The hydrolyzate was then neutralized by adding a
spoonful of solid Ba(OH)2. The pH was checked after the Ba(OH)2 was dissolved. When the
hydrolyzate was not yet neutralized, aqueous Ba(OH)2 was added until its pH reached 7. It was
then filtered and the filtrate was used for the color reactions in the next experiment.

Conclusion
Isolation of casein from non-fat milk was done by adding 10% acetic acid which

decreased the pH of the casein to 4.6. It is a 4.6 where it displays minimum water solubility and

is also the explanation why casein was able to precipitate. The isolated casein was hydrolyzed

with the use a strong acid, H2SO4. Since racemization does not happen with this type of

hydrolysis, there was only one amino acid that was completely lost and that is tryptophan.

References:

Bollag, D. M., Rozycki, M. D., & Edelstein, S. J. (1996). Protein methods. New York et al.:

Wiley-Liss.

Copeland, R. A. (2004). Enzymes: a Practical introduction to structure, mechanism, and data

analysis. New Delhi, India: Wiley - VCH (Wiley - India).

Helleiner, C. W., & Butler, G. C. (1955). The Hydrolysis Of Phosphate Diesters With Barium

Hydroxide. Canadian Journal of Chemistry,33(4), 705-710. doi:10.1139/v55-085

Kurbanova, Marina & Maslennikova, Svetlana. (2014). Acid Hydrolysis of Casein. Foods and

Raw Materials. 2. 27-30. 10.12737/4124.