Biochemistry Laboratory Formal Report

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Alexandra Veronica T. Rebosa*, Carlos Diego A. Rozul, Jose Ricardo
P. Samson, Murielle F. Santiago
Department of Psychology, College of Science

* e-mail: lexierebosa@yahoo.com
Abstract
The protein casein was isolated from nonfat milk by means of isoelectric
precipitation. gradually adding acetic acid to the warmed milk in order to
reach the isoelectric pH of milk, which is 4.6, and by filtering the resulting
amorphous mass (casein). The obtained casein weighed 2.3382g and its
percent yield was 46%.
Keywords: milk, protein, casein, isoelectric pH, isoelectric precipitation
Introduction
Milk is a very vital substance necessary for the nourishment of animals. It is also
a rich source of nutrients such as B vitamins, calcium, fat, and protein. It is especially
rich in protein, providing ten essential amino acids necessary for growth and
development.
A protein is a biomolecule made up of smaller monomers known as amino acids,
which are joined together by peptide bonds (McGuire & Beerman, 2012). A peptide
bond is a type of covalent bond which is formed when there is a loss of a water
molecule between an amino group and a carboxylic acid (Petsko & Ringe, 2004). Amino
Isolation of Casein from Milk
CHEMISTRY
600L
EXPT 01
PAGE 01-02
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acids, also known as the building block of proteins, have three very important parts: (1)
a central carbon (C) atom connected to a hydrogen (H) atom, (2) an amino group (----
NH
2
), and (3) a carboxylic group (---COOH or ---COO
-
). Amino acids also contain a side
chain connected to the amino group known as the R group (McGuire & Beerman, 2012).
There are three different kinds of proteins found in milk. These proteins are
known as caseins, lactalbumins, and lactoglobulins, all of which are globular (Menguito,
et. al, 2014). Casein was the only milk protein isolated in the experiment via isoelectric
precipitation.
Casein is a type of protein found in milk when its pH reaches its isoelectric point
at 4.6. It is obtained via the process of isoelectric precipitation, which involves the
gradual acidification of the milk using acids such as acetic acid (HAc), in order for the
milk to reach its isoelectric pH, the point in which the compound has no net charge (Fox
& McSweeney, 2003). The basis for this is because at the milks isoelectric point, the
protein loses its solubility to interact with its solvent.
5g of powdered nonfat milk and 10% acetic acid were used in isolating the casein
in this experiment. This experiment aims to isolate casein from nonfat milk by isoelectric
precipitation.
Results and discussion
For this experiment, the weight of the nonfat milk powder and casein, the initial
pH of the milk, the volume of the acetic acid used, and the percent yield of the extracted
casein were recorded. The obtained measurements can be found in Table 1 (see
below):
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Mass of nonfat milk powder 5.0410g
Initial pH of milk 6.55
Volume of acetic acid used 0.18 mL
Weight of casein 2.3382g
% yield of extracted casein 46.38%

Table 1. Obtained data from the isolation of casein
Based on the data above, the nonfat milk used in the experiment is slightly
acidic, though almost neutral given its pH. As the milk was heated and maintained to
about 55C, acetic acid was gradually added in order for the milk to reach its isoelectric
pH, which is 4.6. Once the milk reached its isoelectric point, the milk started to
coagulate into chunks of an amorphous solid due to its diminished solubility.
The obtained percent yield of the extracted casein in the experiment was
46.38%, which is to say that the nonfat milk used in the experiment consisted 46%
protein. Cows milk is known to have around 80% casein protein while human milk is
known to have about 40% casein (Breedon, 2012). Although human milk is known to
have more or less the same amount of casein as the milk used in the experiment, milk
can also have a lower casein content when its fat content is lower.
Experimental methodology
For the experiment, the protein casein was isolated from nonfat milk. 5g of
powdered non-fat milk was dissolved in 20mL of warm distilled water in a 100mL
beaker. Afterwards, the solution was heated to 55C on a hot plate, after which the
initial pH of the milk was recorded. 10% acetic acid solution was added to the dissolved
mix in drops gradually while being stirred with a stirring rod. The acid solution was
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continuously dropped into the nonfat milk mixture until the pH of the nonfat milk reached
4.6. The volume of the acid used was recorded. The mixture was left to stand until an
amorphous mass was formed. The amorphous mass, casein, was decanted, pressed,
and dried between two pieces of filter paper. Afterwards, the casein was weighed, and
its percent yield was computed. Lastly, the casein was split into two portions; the first
portion was saved for hydrolysis (the next experiment), and the second portion was
stored in the refrigerator.
References
Fox, P.F. & McSweeney, P.L.H. Advanced Dairy Chemistry: Volume 1: Proteins, Parts
A&B; Springer Science & Business Media: New York, USA, 2003; pp 2-6
McGuire, M. & Beerman, K. Nutritional Sciences: From Fundamentals to Food;
Cengage Learning: USA, 2012; p 163
Menguito, C.A, Albano, P.B.S., Macabeo, A.P.G., Manansala, T.L., & Santiago, K.S.
Basic Laboratory Experiments in Biochemistry; University of Santo Tomas College of
Science: Manila, 2014; p 1
Petsko, G.A. & Ringe, D. Protein Structure and Function; New Science Press: London,
UK, 2004; p 8
http://www.rpi.edu/dept/chem-eng/Biotech-Environ/PRECIP/precpph.html (retreived on
August 6, 2014)
http://ons.wvdhhr.org/Portals/20/PDFs/Aunt%20C%20Choosing%20Infant%20Milks%20
andFormulas%20Part2%20commercial%20formulas%204-12%20THIS.pdf` (retrieved
on August 6, 2014)