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

A.

Group 2

College of Science, University of Santo Tomas

España Boulevard, Manila

Abstract

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.

Introduction

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 body’s 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,

whey proteins and minor proteins. These proteins are regularly found in

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 multi-

molecular, 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.

Methodology

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 aren’t 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

Casein’s 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 H2SO4. The pH of the solution must be 7 in

order to neutralize it using a pH meter. Because it wasn’t neutralized yet, 8N H 2SO4 was

added dropwise. The precipitate was then filtered off using a filter paper. H2SO4 and

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.

Conclusion

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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