Isolation, Alkaline Hydrolysis, Neutralization of Casein from Non-Fat Milk

*Beatrice Ong, Jose Pontejos, Andrea Beatrice Ramos, Gendelline Rigor

College of Science, University of Santo Tomas, España Blvd., Manila

Abstract

Casein, a protein found in non-fat milk, was isolated by a method called isoelectric precipitation. A yield of
66.44% was obtained. Hydrolyzate from casein was produced by alkaline hydrolysis while. The appearance of the
hydrolyzate before and after autoclaving underwent a qualitative change. Neutralization of the hydrolyzate followed
in preparation for color reactions.

Introduction

Milk is a substance produced by the mammary glands of mammals during lactation.

(Damodaran, 2011, pg. 73) Not only is it a good source of nutrition but also a source of the

synthesis of other products such as cheese, cream, and other dairy products.

Milk contains a variety of important components such as fat, electrolytes, vitamins,

sugars, and proteins. Proteins are combinations of different amino acids which are joined

together by peptide bonds. They have a wide array of biologically significant functions. Proteins

can be classified into two types: fibrous proteins and globular proteins. Fibrous proteins are

composed of long rod-shaped or string-like molecules that intertwine to form strong fibers.

Fibrous proteins are water-insoluble and are found in connective tissue, elastic tissue, skin, and

hair. Globular proteins on the other hand are spherical and form suspension or are dissolved
when immersed in water. Examples of globular proteins are hemoglobin and transferrin. (Seager

& Slabaugh, 2010)

The three kinds of proteins that are found in milk are casein, lactalbumins, and

lactoglobulins. The major protein being casein. Casein is a globular phosphoprotein, which are

able to form micelle complexes, constitutes 80% of the protein content in bovine milk. (Treweek,

2012) Casein is a family of similar proteins. These proteins are called alpha, beta and kappa-

caseins. These proteins of the casein family are naturally hydrophobic except for kappa-caseins.

Kappa-caseins, along with the high amounts of phosphate, renders casein soluble in water.

Isolation of a protein is a starting approach in studying a certain protein. One method of

protein isolation is called isoelectric precipitation in which there is an interplay between the

concepts of pH and charge of a molecule. At a certain pH, also known as the isoelectric point, a

certain amino acid will have a neutral charge. (Amend, Armond, and Mundy, 1993) This concept

allows the separation of amino acids from a mixture. The isoelectric point of casein at 20˚C is

4.6. (Bonczar, Misztal, Molik, Zebrowska, & Zieba, 2012, pg 327) Further analysis on a certain

protein can also be done by breaking down the protein to the individual amino acids that make

up that said protein. The process of breaking down the protein in the presence of a strong base or

acid is hydrolysis. (Seager & Slaughbaugh, 2010)

The objectives of the experiment are: to isolate casein from non-fat milk via isoelectric

precipitation (1), to perform alkaline hydrolysis on the isolated casein (2), and to neutralize the

resulting hydrolyzate (3).

Methodology

A. Isolation of Casein

In a 100-mL beaker, 5 grams of powdered non-fat dry milk was mixed with 20-mL of

warm water. This solution was heated to 55˚C on a hot plate. As the temperature reached 55˚C, it

was removed from the hot plate. The initial pH of the solution was measured using a pH meter.

10% acetic acid was added dropwise to the solution while it was stirred using a stirring rod. As

soon as the pH of the solution is 4.6, the amount of 10% acetic acid used was taken note of. The

solution eventually formed a large amorphous mass. The amorphous mass was separated with the

solution via decantation. The newly separated amorphous mass or the isolated casein was dried

between filter papers. After drying, the mass of the isolated casein was measured using an

analytical balance. The mass of the isolated casein was utilized in determining the percent yield

of casein from the 5 grams of powdered milk. Isolated casein was divided into two portions: one

in preparation for the succeeding experiment’s color reaction tests and the other portion in

preparation for alkaline hydrolysis.

B. Alkaline Hydrolysis and Neutralization

The isolated casein was cut into very small pieces and placed in a 50-mL Erlenmeyer

flask. 5 mL of boiling water and 2.0 grams of Ba(OH)2 was also added to the 50-mL Erlenmeyer

flask. The flask was plugged with cotton and covered with aluminum foil. The appearance of the

isolated casein immersed in the solution was taken note of before autoclaving at 15 psi for 5
hours. After the process of autoclaving, the appearance of the sample was noted again. The

resulting hydrolyzate was neutralized by H2SO4. 16 N of H2SO4 was added until the pH read

was around 8. As the pH of 8 was reached, 8 N of H2SO4 was added until the hydrolyzate

reached pH 7. The neutralized solution was filtered off by gravity filtration. 7 mL of filtrate was

obtained from the solution. The filtrate was used as a sample for the color reactions test in the

succeeding experiment.

Results and Discussion

Table I. Data Table on Casein Isolation

Weight of Non-Fat Milk 5.0009 g
Initial pH of Solution 6.11
Final pH of Solution 4.61
Initial Volume of Acetic Acid Used 5.0 mL
Final Volume of Acetic Acid Used 3.0 mL
Volume of Acetic Acid Used 2.0 mL
Weight of isolated casein 3.3228 g
% Yield 66.44%

The data table above shows the results obtained during the experimentation. The volume

of 10% acetic acid was measured using a graduated cylinder by subtracting the initial volume of

acetic acid to the final volume after adding it to the sample drop wise. The % yield was

computed by this equation:

Data collection of other data such as the weight of non-fat milk, weight of isolated casein,

initial pH of the solution, and final pH of the solution was described in the methodology.
 Isolation of casein begins with the dissolving the powdered non-fat milk in 20-mL of

warm distilled water. Powdered non-fat milk was used rather than whole milk due to the

presence of fat in whole milk. The absence of fat in the powdered non-fat milk will allow an

easier isolation of casein rather than a fat-filled milk that would be a hindrance in isolation

casein. Warm distilled water will allow the non-fat milk to dissolve faster however it must be

noted not to exceed a temperature of 55˚C. Beyond this temperature, the other proteins such as

lactalbumins in the milk will become denatured. Denatured globular proteins have their tertiary

folds untangled and disorganized. These untangled proteins will eventually coagulate to form a

solid mass. (Caret, Denniston, Topping, 2004) Coagulated lactalbumins in the solution will

render an impure isolation of casein.

Rather than the use of heat, altering the pH allows for the isolation and coagulation of

casein. “Proteins normally have charged amino side chains on their surfaces that undergo

favorable polar interactions with the surrounding water.” (Parson, Vance, Zubay, 1995, pg 119)

At a certain pH called the isoelectric pH, the charges at the amino side chains of a protein are

neutral. Due to the neutral charge, each individual protein molecule cease to repel each other

thus they aggregate to form a coagulation which is insoluble in the solution. Since the milk

solution has a pH of 6.11, acetic acid was used to lower the pH to the isoelectric point of casein.

The isoelectric point of casein is 4.6. A dilute solution of acetic acid was added dropwise to

reach the isoelectric point of casein. 10% Acetic acid must be added drop wise to be able to

carefully monitor the pH. Also a sudden addition of acetic acid might make the solution have a
pH lower than 4.6. This will influence the casein to dissolve back into the solution since it will

gain a charge and hinder its coagulation.

After the isoelectric precipitation of casein, the casein was dried thoroughly to remove

any impurities. The weight of the isolated casein was measured and the % yield was computed

for:

Hydrolysis of the isolated casein was done via alkaline hydrolysis. Alkaline hydrolysis is

the use of a strong base as a means to destroy the peptide bonds between amino acids in the

casein. 2.0 grams of Ba(OH)2 was utilized as the strong base in the hydrolysis of casein.

Ba(OH)2, compared to other strong bases such as NaOH, is preferred due to to it being easily

removed after hydrolysis.

I II
I
Fig. I & II. Appearance of protein isolate before (I) and after (II) autoclaving
 Before autoclaving, the appearance of the casein isolate was a whitish yellow precipitate

immersed in a turbid solution while after autoclaving an orange solution with a only a few traces

of white precipitate were evident. This signifies that hydrolysis occurred since the casein isolate

was broken down into free amino acids (which are not visible in the solution). Alkaline

hydrolysis has its own disadvantages. Partial or complete destruction of amino acids such as

arginine, cystine, serine and threonine. Alkaline hydrolysis also causes racemization of the

remaining amino acids. During the experimentation, alkaline hydrolysis was also accompanied

by autoclaving. Pressure and heat from autoclaving speed up the process of hydrolysis of amino

acids.

Neutralization, following hydrolysis, was done to be able to use the hydrolyzate for the

color reactions test. In order for color tests such as ninhydrin test to give the expected results,

these tests require the casein hydrolyzate to be of neutral pH. In the process of neutralization,

H2SO4 when reacted with Ba(OH)2 will form a precipitate (barium sulfate) which is insoluble to

the solution and can be filtered off easily. Thus other strong acids or bases such as HCl and

NaOH are not utilized in neutralizing the hydrolyzate.

The percent yield of casein from the non-fat milk could be further maximized if the

following errors were not committed: Error due to exceeding the isoelectric pH, error due to

heating the non-fat milk solution over 55˚C, and error due to contamination and spillages.
Conclusion

Casein coagulates or precipitates when the pH of the solution is equal to the isoelectric

pH of casein. A percent yield casein of 66.44% was obtained. Alkaline hydrolysis, accompanied

with autoclaving, breaks down the protein isolate into amino acids. However alkaline hydrolysis

has its disadvantages in which it destroys some amino acids and triggers racemization. H 2SO4

and Ba(OH)2 are suitable reagents in hydrolysis and neutralization of casein hydrozylate since it

can be removed easily during final filtration.

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