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