Isolation and after autoclaving. The hydrolyzate was neutralized

Isolation and Hydrolysis of Casein from Milk
Angelica Carlos, Ymarie Catacutan, Arriah Celicious*, Russel Dauigoy
Department of Psychology, University of Santo Tomas, Manila, Philippines

Abstract: Casein, a protein, was isolated
from non-fat milk through isoelectric
precipitation. A percent yield of 69.89% was
obtained from this process. An acid
hydrolyzate of casein was acquired from the
method of acid hydrolysis. The isolated
casein was observed to have undergone a
qualitative change before and after
autoclaving. The hydrolyzate was
neutralized for the subsequent color

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During lactation, mammals
produce milk through their mammary
glands. Milk from cows consists of
87.1% water, 3.4% protein, 3.9% fats,
4.8% lactose, and 0.7% minerals.6 It is a
source of nutrients for many animals,
and it is used in the synthesis of many
dairy products in the market today.
Milk is a mixture of various types
of proteins—most of which are present
in small amounts. The basic constituents
of living organisms are proteins. This is
a macromolecule, having one or more
long chains of amino acids bonded
together by peptide bond. A linear chain
of bonded amino acids is called a
polypeptide.1 Proteins can further be
categorized into two types: globular and
fibrous proteins. Fibrous proteins are
elongated, filamentous proteins that
often provide strength and protection to
tissues and cells. Globular proteins, on
the other hand, are compact, spherical
proteins that are involved in metabolic
functions. While fibrous proteins are
water-insoluble, globular proteins are
soluble in water.
The proteins found in milk are
classified into three main groups, sorted
based on their behaviors and forms:
lactalbumins, lactogiobulins, and
caseins. These are all considered to be
globular proteins. Furthermore, they are
deemed to be complete proteins
because they contain the amino acids
imperative for building blood and tissue.
Casein is considered the main protein. It

is a heterogeneous mixture of proteins
in milk that contain phosphorous, and it
is exists in milk as calcium salt and
calcium caseinate. It forms micelle
complexes, and constitutes 80% of
mammalian milk.3 Alpha, beta, and
kappa-casein constitute the family of
proteins that casein has. Aside from
kappa-caseins, they are all naturally
hydrophobic. With high amounts of
phosphate, kappa-caseins can render
water-soluble casein.
Proteins are relatively easy to
isolate from other biomolecules—
carbohydrates, lipids, and nucleic acids.
Nevertheless, it is difficult to separate
them from each other due to their similar
structures and properties. It is useful,
therefore, to utilize their miniscule
differences to be able to isolate them.
One method of protein isolation is
isoelectric precipitation, wherein pH
amounts and molecular charges come
into play. At a certain pH value, also
known as the isoelectric point, an amino
acid does not migrate in an electric field
and has a neutral charge—a net charge
equal to zero.5 This is the pH where
amino acids in a mixture can be
separated. At 20?C, casein’s isoelectric
point is 4.6. A protein can be further
broken down to its individual amino
acids that make it up. The breaking
down of protein into its component
amino acids is a method called
This experiment aims to isolate
casein from non-fat milk through
isoelectric precipitation, and treat the
isolated casein with acid or alkaline

A. Isolation of Casein
5 grams of powdered non-fat dry milk
was dissolved in 20 milliliter of warm
distilled water inside a 100 milliliter
beaker. The solution was warmed to
55?C on a hot plate. A thermometer was
used to monitor the temperature of the
solution. The beaker was removed from
the hot plate. The initial pH of the
solution was taken note of. As the
solution was being stirred with a stirring
rod, 7 drops of 10% CH3COOH or acetic
acid were added. A large amorphous
mass was waited to form in the solution.
The mass was separated from the
solution via decantation. The crude
caffeine was dried with the use of filter

papers. The crude casein was weighed,
and the percent yield was calculated.
The crude casein was equally split into
two portions—one portion was for the
preparation of acid hydrolysis, and the
other portion was encased with
aluminum foil and stored for the
following experiment’s color reactions
B. Acid Hydrolysis and
The crude casein was cut into small
pieces. The casein fragments were
transferred into a 50 milliliter Erlenmeyer
flask. 4 moles 8 normal sulfuric acid was
added in the Erlenmeyer flask. The
Erlenmeyer flask was plugged with
cotton. The flask was covered with
aluminum foil at the opening, and
labeled. The appearance of the casein
in the solution was taken note of. The
solution was autoclaved at 15 psi for 5
hours. After autoclaving, the appearance
of the sample was taken note of. The
autoclaved sample was diluted with 15
milliliter of distilled water, and all the
contents were transferred to a 250
milliliter beaker. The sample was
neutralized by adding half a spatula of
solid barium hydroxide. Saturated
barium hydroxide was added until the
pH read was around 7.00. The
neutralized solution was filtered. 7
milliliter of the filtrate was collected.
Results and discussion:
Weight of Non-fat
5.0024 grams
Initial pH 4.62
Final pH 4.60
Drops of 10%
Acetic Acid Used
Weight of Crude
3.4960 grams
Percent Yield 69.89%
Appearance before
suspended in turbid
Appearance after
Black solution with
black precipitate

The data measured and observed
are shown on the table above. The
percent yield was computed as such:
crude casein g x 100 = percent yield of
non-fat milk g casein

3.4960 grams x 100 = 69.89% casein
5.0024 grams
Table 1. Results on Casein
Isolation and Hydrolysis

The isolation process of casein
was commenced by dissolving the non-
fat milk in 15 mL warm distilled water. A
non-fat milk was used to prevent casein
from binding with the fat molecules
when heated. Fat is present in whole
milk, and as the name of the non-fat milk
implies, it does not contain any fat
molecule. This absence of fat gives way
to an easier isolation of casein.
The non-fat milk solution was
heated to 55?C, but not higher, because
this prevented the exposure of proteins.
Beyond this temperature, the other
proteins present in milk—lactalbumins
and lactogiobulins—will become
denatured. In this instance, the folds of
globular proteins become disorganized
and untangled. These uncoiled and
disorganized proteins then coalesce and
generate a solid mass in the solution,
rendering an impure casein isolation.2
The isolation, as well as
coagulation, of casein from non-fat milk
utilized the alteration of pH. At
isoelectric pH, the protein is uncharged.
This means that the charges at the
amino side chains are equal to zero.
Normally, proteins have charged amino
side chains that have polar interactions
with water. Due to the neutral charge
brought about at isoelectric pH, the
intermolecular repulsions are minimized,
and the protein molecules coagulate,
forming a solid mass that displays
minimum water solubility. Since the pH
of milk is around 6.60, and the
isoelectric pH of casein is 4.60, a dilute
solution of acetic acid was used to lower
the pH to the isoelectric point of casein.
The isolated casein furthermore
underwent acid hydrolysis. The peptide
bonds present between amino acids are
broken down with the use of a strong
acid in the process of acid hydrolysis. In
contrast to alkaline hydrolysis, this
proceeds without racemization and with
less destruction of certain amino acids.
In this process, H2SO4 or sulfuric acid
was used over other strong acids due to
it being easily removed after hydrolysis.
Prior to hydrolysis, the isolated casein
was cream-colored, suspended in a
turbid solution. A black solution with
black precipitate was observed after
hydrolysis. This change is of great
importance because it signified the
destruction of the protein Tryptophan.
When tryptophan is destroyed, it is
converted to humin, a black pigment that
gives the black color to the casein

solution after hydrolysis.4 The proteins
threonine and serine were also
denatured in the process. Moreover,
acid hydrolysis was accompanied by
autoclaving during the experimentation.
The process of autoclaving served as a
catalyst during hydrolysis; the heat and
pressure from autoclaving accelerate
the hydrolysis of amino acids.

In order to isolate casein from
other proteins in powdered non-fat milk,
the solution was subjected to a method
called isoelectric precipitation. A percent
yield casein of 69.89% was obtained
from the experiment. Acid hydrolysis
broke down the crude casein into amino
acids. It converted and destroyed certain
amino acids such as tryptophan to
humin, and denatured threonine and

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