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Casein: The Isolation, Hydrolization, and

Neutralization of it from Non-fat Milk (Milk Magic)

*Buan, Daniel Carlos S.,* Carasig, Patricia., Conception, Klein., De Joya, Melissa
Group 2
College of Science, University of Santo Tomas
Espaa Boulevard, Manila

Casein is present in milk and cheese which is also used in processed foods and other industrial
products that has an isoelectric point of pH 4.6. Casein was isolated, hydrolyzed and neutralized
from non-fat powdered Milk Magic in this experiment. Adding drop-wise of 10% acetic acid first
isolated the casein until pH of solution reaches 4.6 then formed an amorphous mass which was
weighted and getting the percentage yield which was 70.6%. The isolated casein was then
hydrolyzed and then neutralized for further explanation.

The word protein specifies to a type of molecule in food that can be broken
down into amino acids. Proteins are big and convoluted molecules that play profuse
critical parts in the body. They are long chains of amino acids that control physiological
activities of cells and are necessary factors of cellular structure. (Molnar, 2003) Proteins
do most of the endeavor in cells and are needed for the function, structure and
regulation of the bodys tissues and organs. Antibodies, hormones, and globulins, they
regulate, catalyze, and shield the body chemistry in the form of enzymes. In the form of
tendons and ligaments, cartilage, callus, hair, muscles, and skin, proteins administer

structure to the body of a multi-celled organism; and then they involve the transport of
other substances and oxygen within an organism in the form of hemoglobin, myoglobin,
and diverse lipoproteins. (Moran, 1994)
The proteins of milk are classified into three essential groups of proteins on the
basis of their widely distinctive behaviours and system of existence. They are caseins,











mammalian milk, making up 20% and 45% of the proteins in human milk and 80% of the
proteins in cow milk (Ventimiglia, 2012). Casein exists in milk as calcium salt and
calcium caseinate. Casein is also the main protein in milk. Caseins have a suitable
amino acid composition that is essential for growth and progress of the nursing young.
This high characteristic protein in cow milk is one of the crucial reasons why milk is such
a necessary human food.
Caseins are made up of of several identical proteins which build a multimolecular, granular structure which is named casein micelle. An inclusion to casein
molecules, the casein micelle has water and salts. There are also enzymes that are
associated with casein micelles too. The micellar structure of casein in milk is crucial
part of the mode of digestion of milk in the stomach and intestine, the basis for many of
the milk products industries, and the basis for our ability to easily separate some
proteins and other components from cow milk (Murray, 2012)
Caseins are high feature origin of amino acids and highly digestible in the
intestine. Despite all of them are digested to any degree, most whey proteins are less
digestible in the intestine (Kasper, 2015). When vast whey protein is not digested

completely in the intestine, some of the intact protein may excite a localized intestinal or
a systemic immune feedback.
To isolate casein from non-fat milk by isoelectric precipitation and to hydrolyze
casein with a base (assigned to the group) were the objectives of the experiment.

In order to start the experiment, 5 grams of powdered non-fat milk (Milk Magic)
was dissolved in 20mL warm distilled water, for the isolation of casein. The solution was
heated on a hot plate in 55 degrees Celsius and then the beaker was removed. The
initial pH was noted. While stirring using a stirring rod, a solution of 10% acetic acid was
added. The 10% acetic acid was added dropwise until the pH of the solution reaches
4.6. The casein was stirred until it formed a large amorphous mass. The mass was then
filtered by suction to remove liquids that arent needed and then filtered by gravity. The
casein was then divided into two. The first half was for hydrolysis and the other half was
wrapped in a foil and then labelled.
Basic 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. 5 ml of distilled boiling water and 2.0 g of Ba(OH) 2 was placed
inside the flask. Plugged with cotton and then covered with aluminium foil, the flask was
then labelled. The flask was warmed in order to dissolve the base. The appearance was
noted before autoclaving.

On the next meeting, the appearance was then noted after its autoclaved form.
The hydrolysate was then neutralized by adding 1.0 ml of 16N H2SO4. In order to
neutralize it, its pH must be 7. When it was not yet neutralized, 8N H2SO4solution was
added dropwise. The pH meter was used in order to check the pH of the solution while it
was being neutralized. When it was already neutralized, the precipitate formed was
filtered off.

Results and Discussion

In the isolation of casein from non-fat Milk Magic, there are four principles which
are Isoelectric Precipitation, Acid Hydrolysis, Basic Hydrolysis, and Neutralization. The
first step in the experiment was the isolation of casein from non-fat milk (Milk Magic).
The casein was precipitated by warming the powdered milk with 20 ml distilled water in
55 degrees Celsius on a hot plate and adding 10% acetic acid. 10% acetic acid was
used in order to carefully reach the pH of casein which is 4.6 without overstepping. It is
crucial to be mindful regarding the temperature not to be too high or the acid to be too
strong, inasmuch these conditions also hydrolyze lactose into its element, galactose
and glucose. It is also crucial to avoid too much acid because galactose dissolves
some of the protein. Until the pH reaches 4.6, the 10% acetic acid was added dropwise.
Calcium Caseinate has its isoelectric point at pH 4.6. It is therefore insoluble in solutions
of pH less than 4.6. The milk (Milk Magic) has a pH of 6.6; for that reason casein is
solubilized as a salt and has a negative charge at this pH.

Weight of sample (milk powder)

5.00 g
Initial pH of milk solution
pH = 6.20
Volume of 10% acetic acid has
2.6 ml
Weight of empty watch glass
61.13 g
Weight of watch glass + dry casein
64.66 g
Weight of dried casein
3.53 g
Table 1. The results of the experiment
The weight of the powdered non-fat milk (Milk Magic) used in the experiment was
exactly 5.00 g.
The weight of the dried casein was 3.53 g which has a percentage yield of 70.60%.

3.53 g
x 100=70.60
5.00 g
Caseins calculation of its Percentage yield
The next step afterwards the isolation of casein, hydrolysis and neutralization
came in line. The isolated protein dealt with basic hydrolysis in the experiment. 5ml of
boiling distilled water and 2 grams of Ba(OH) 2 were added to the isolated casein and
then was autoclaved. The appearance of the casein was a cloudy white solution before
autoclaving. After autoclaving, it looked like a yellowish cloudy solution. Autoclaving is
used to speed up the hydrolysis reaction and to sterilize the casein. In order to
neutralize the hydrolyzate, 1.0 ml of 16N H 2SO4. The pH of the solution must be 7 in
order to neutralize it using a pH meter. Because it wasnt neutralized yet, 8N H 2SO4 was
added dropwise. The precipitate was then filtered off using a filter paper. H 2SO4


Ba(OH)2 were used instead of other strong bases and strong acids so that it could
prevent interference to the succeeding experiment.
Basic hydrolysis destroys more amino acids than acid hydrolysis which also
promotes racemization, which is process of changing from an optically active compound

into a racemic compound or mixture. In basic and acid hydrolysis, the acid and the base
acts as a catalyst, or a substance that hastens the rate of chemical reaction.

Isoelectric precipitation was used to isolate the casein with the use of 10% acetic
acid, which is a weak acid. The casein was isolated using a strong base that is Ba(OH)2
and a strong acid that is H2SO4. In base hydrolysis, Serine, Arginine, Threonine, and
Cysteine were destroyed while in acid hydrolysis, only the Tryptophan was destroyed.

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