Isolation and Characterization of Casein from Nonfat Powdered Milk

Jena Karynne Adelaide U. Go, Noelle Courtney T. Gutierrez,​ ​Maria Francesca Ysabel V.
Jahrling, and Gabriella E. Javier*
College of Science, University of Santo Tomas, Manila, Philippines

Abstract

The aim of the experiment was to isolate casein from nonfat powdered milk, and
characterize the isolated intact protein afterwards through the use of five different color reaction
tests. The tests served as indicators of the different protein present in the substances that were
given and prepared. The five used tests were namely: t​he Biuret test, Sakaguchi test, Ninhydrin
test, Xanthoproetic test, and the Hopskin-Cole test.

Introduction

Proteins are macromolecules consisting of one or more polypeptides (Walsh, 2002).

They are among the four major classifications of biomolecules that are important for the survival
of all living organisms. These proteins carry structures that are unique from other biomolecules
in terms of their difficulty to be separated. This is due to the similarities in structure and
properties that exist among their binding groups​—t​heir peptide bonds. These peptide bonds
establish the covalent linkages between the amino and carboxyl groups present in the amino
acid structure. Hence, the free functional​—or chemically reactive—nature of these polypeptides
to neighboring molecules during a reaction.

Given that separating one protein from another poses a difficulty due to their similar
properties, a common method used to separate them is through isoelectric precipitation.
According to ​Jaczyns​ki (2008), th​e isoelectric point (pI) of a protein is a pH level at which the
protein maintains a zero net electrostatic charge. In terms of separating biomolecules such as
protein, this method is used through adjusting the pH of a solution until it reaches a value where
it can be isolated from the rest of the solution. Isoelectric precipitation is then convenient when
fractioning a protein mixture (Harrison, et.al., 2015). In procedures where it will be used, the pH
is either adjusted above the highest pI, or below the lowest pI of the proteins present. Once the
pH level is adjusted, this allows precipitation to form which can now be isolated and removed
from the original mixture, then may be subjected to further experimentation and identification.

In studying separation and molecular structures of proteins, caseins found in milk are
usually the variable that is experimented on. Caseins are the major family of proteins in milk;
they are derived from three to five distantly related ​gene products (Thorn, Ecroyd, & Carver,
2014). It is the main protein present milk and it exists as calcium salt and calcium caseinate.
Casein, in the molecular level, contains a composition that would explain its hydrophobic and
soluble nature. High amounts of nonpolar amino acids such as valine, leucine, isoleucine,
phenylalanine, tyrosine, and proline were observed in casein in a study conducted by Otter in
2003. It was also mentioned that because of the high phosphate content present in casein, it
counteracts with the nonpolar amino acids, which would explain their reasonable solubility in
water. Furthermore, casein was observed to be rich in the amino acid, lysine; therefore it can be
used to supplement various lysine-deficient food sources such as cereal proteins.

Characterizing proteins and their respective amino acids are also essential in
discovering their functions and relative importance to organisms and the processes they
undergo. Proteins may be observed then eventually characterized as intact or solidified
products that must be mixed and turned into a suspension, or, as a hydrolyzate after undergoing
a hydrolysis procedure. One method to do so is through color reactions. Color reactions of
proteins are due to the properties of the side chains present in amino acids (Das, 2012).
Proteins produce specific colors when mixed and reacted with certain solutions. The change in
color then indicates the presence of a certain protein in the solution. Common color reactions
tests that are used are the Biuret test, Sakaguchi test,Ninhydrin test, Xanthoproetic test, and
the Hopskin-Cole test.

Discussion of Results

Isolation of Casein from Nonfat Milk

Pre-weighed 100mL beaker 62.5230g

Pre-weighed 100ml beaker w/ nonfat 67.0896g

powdered milk

Initial pH 6.8

pH (after adding 150 drops of acetic acid) 4.66

Volume of acetic acid used: 4.0mL

Weight of protein isolate (isolated Casein) 2.4195g

Table 1. Results of isolated casein from nonfat milk

Table 1 presents the values recorded from isolating casein from nonfat milk powder
solution. The recorded pH level of the original prepared powdered milk was equal to 6.8. After
adding 150 drops of acetic acid the pH then decreased to 4.66, a near level to the pI (isoelectric
point) of casein which was noted to be 4.6. According to the earlier mentioned process of
isoelectric precipitation, the pH of the given protein solution was indeed adjusted to the pI of the
isolate to be collected. Acetic acid, CH 3 COOH , was the solution added to decrease the pH as it
is an acid.
 Characterization of Protein Suspension (Isolated Intact Casein)

Color Reaction Visible Results

Biuret Test Purple clear solution, no particles visible

Sakaguchi Test Yellow clear solution, no particles visible

Ninhyrdin Test Transparent solution, particles left on the

bottom, suspended cloudiness

Xanthoproetic Test Yellow solution, with clear bottom

Hopkins-Cole Test Bluish cloudy solution

Table 2. Results of characterization of protein suspension

Table 2 presents the results from the color reactions the isolated intact casein
underwent. A total of 5 different color reaction tests were utilized to indicate the presence of
different proteins. The Biuret test results show that the protein suspension turned into a purple
solution. This would indicate the presence of peptide bonds, the chemical bond units that are
the bases of amino acids. It is similar to Eckersall (2008)’s study wherein it is stated that in t​he
Biuret reaction, a protein forms a complex with copper (Cu 2+ ) in alkaline solution, is dependent
on the presence of peptide bonds, and is blue-purple color.) The color change is then due to the
copper ions original charge of 2+ being reduced to a charge of 1+ in the presence of the peptide
bonds.

Sakaguchi test results reveal a yellow clear solution after the addition of 1 drop of 10%
N aOH and 0.02% naphthol solution, respectively. This would represent a positive Sakaguchi
test result, indicating the presence of arginine in the solution. Similar to ​Das (2012),‘s findings
that on boiling protein solution with Sakaguchi’s reagent containing sodium hypochlorite and
naphthol​, a red colored solution was produced. The data presented indicates a close
warm-enough color of yellow that is similar to the red solution produced in a similar study.

Ninhydrin test results present the solution to change into a clear transparent solution.
Having some particles left on the bottom of the test tube, while a suspended cloudiness was
also observed. Supposedly, proteins form blue or purple complexes on being heated with
ninhydrin (Das, 2012). Although this supposed reaction was not evident in the experiment, it
could have been manifested in the data through the suspended cloudiness that was recorded;
therefore, indicating still, the possible presence of amino acids in the solution.

For the Xanthoproetic test, the results reveal a change in the color of the solution to be
yellow and clear, with no observed particles floating about the substance. The test would be an
indicator of the aromatic amino acids such as tyrosine, tryptophan, and phenylalanine. These
when present in proteins, would give yellow precipitate when heated with concentrated nitric
acid ​(Joshi, 2006).

And finally, for the Hopkins-Cole test, results showed that a bluish cloudy solution
formed. Das (2012) stated that a protein solution mixed with Hopkins-Cole reagent, which
contains glyoxylic acid, when added with concentrated sodium hydroxide forms a separate
lower layer. This layer then forms a purple ring between the junction of the two layers formed;
indicating the presence of tryptophan in the protein.

Experimental

To isolate casein from the nonfat powdered milk, 5g of the nonfat powdered milk was
mixed with 20mL warm distilled water. The mixture was then heated to 55 degrees Celsius on a
hot plate. The original pH level of the powdered milk mixture was noted. Afterwards, drops of
acetic acid were added while stirring the mixture, until its pH reached 4.6. pH levels were taken
note using the pH meter. The mixture was then decanted and the formed precipitate, the
isolated intact casein, was weighed, and the percent yield was computed. The isolated product
was divided into the intact product and the protein to be hydrolyzed, respectively. Both protein
samples were then subjected to the five different color reaction tests, namely ​the Biuret test,
Sakaguchi test,Ninhydrin test, Xanthoproetic test, and the Hopskin-Cole test.
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