INTRODUCTION

Consumption of milk is important in the human diet. Milk from cows provides nutrients

such as fats, proteins, carbohydrates, and mineral which are essential for growth and

development. Proteins are classified into three types: fibrous, globular, and membrane proteins.

Globular proteins are one of the commonly known proteins. They are spherical when they are in

its tertiary structure and they are water-soluble forming a suspension when submerged (Berg, et

al., 2015, p. 44). Proteins have an average composition of 3.4% in milk. It contains all the

essential amino acids required by humans and there are three globular proteins found in milk,

namely caseins, lactalbumins, lactoglobulins (Fox, et al., 2015).

Commonly used as a food additive and for cheesemaking, casein is the major protein in

milk, constituting approximately 82% (Bylund, 2015). It exists as salt in the milk as calcium

caseinate and has an isoelectric point of 4.6. It is a family of protein divided into several types:

Alpha, beta, and kappa caseins, with each type of casein, which has different amino acid

composition and properties. Alpha and beta-casein are insoluble in water. Kappa-casein

however, is soluble in water by forming micelles, which responsible dissolving the other groups

of casein. It is because of its amount of phosphorus which makes them coagulate with water

(Fox, et al., 2015). This coagulation is the principle for cheese production through the use of

rennet enzymes as the coagulant (“Casein,” 2019).

Protein isolation is an essential step in studying a particular protein. It is a technique that

involves the separation of the protein of interest from all the non-protein material within the

same biological material (Franks, 1993). Casein can be isolated from milk through the method

known as isoelectric precipitation. It is a method of isolation where a protein is set to its

isoelectric point to make soluble and allowing a certain amino acid to separate to the solution

(Donald, 1990). An isoelectric point is a pH where an amino acid carries no electrical charge. By

setting the milk’s pH into 4.6, casein will precipitate. After isolating the protein, the next step in
analyzing the protein is to break down the isolate into amino acids through the use of a strong

acid or base. This step in separating peptide bonds in proteins is called hydrolysis. The

objectives of this experiment are to isolate casein from powdered non-fat milk by performing the

isoelectric precipitation, subject it to alkaline hydrolysis, and by neutralizing the hydrolysate

(Dave, et al., 1991).

METHODOLOGY

Casein Isolation

In a 100mL beaker, 5.0639 grams of non-fat powdered milk was dissolve in 20mL warm

distilled water, with the hot plate set at 55°C. After dissolving the milk, 2.8mL of 10% acetic acid

solution was added dropwise until the isoelectric point of casein was reached. The protein

coagulated in the milk and the precipitate was filtrated by gravity filtration. Filter paper were

used to dry the protein isolate. The were weighted and the percent yields was calculated. It was

divided into two portions with one used in the alkaline hydrolysis. The other portion was

wrapped in aluminum foil which will be used in the future experiment.

Alkaline hydrolysis and neutralization

The protein was cut into smaller pieces and was placed in a 50mL Erlenmeyer flask.

5mL of boiling water 2.0 grams of Ba(OH) 2 was added. The flask was covered with a cotton plug

and wrapped with aluminum foil before it was subjected in the autoclave for 5 hours at 15 psi.

After 5 hours in the autoclave, the hydrolysate was neutralized by adding drops of concentrated

16N sulfuric acid until pH reached 8.0. By the time the pH reached 8.0, 8N of sulfuric acid was

used instead until the pH reached 7.0. The hydrolyzed protein was filtered, discarding the

precipitate.
RESULTS AND DISCUSSION

Table 1: Gathered data of the protein isolate and hydrolysate

Data Measurements
Amount of non-fat powdered milk 5. 0639g at 52.6°C

dissolved
Initial pH of milk 6.40
Final pH of milk 4.65
Amount of acetic acid used 2.8mL
Amount of isolated casein 3.2510g
Final pH of hydrolysate 6.99

Isolated proteins should be pure in order to analyzes its structure and functionality. The

steps should be followed carefully to achieve a pure protein isolate. Non-fat milk was used

instead of regular milk because fats can hinder the isolation process. When fat is not separated

in milk, it will go with casein as it coagulates, producing a contaminated protein (Ezzat, n.d.). A

thermometer was used to measure the temperature when the milk was heated. It was removed

from the hot plate at 52.6°C because the temperature of the solution should not exceed more

than 55.0°C. More than the said temperature might denature casein and may alter its properties

(Campbell, et al., 2018).

Charged amino acid side chains in proteins provides polar interaction with water .

Setting the pH of the protein into its isoelectric point will cease these interactions, forming a

coagulate (Campbell, et al., 2018). A handheld pH meter was used to measure the pH. Its

isoelectric point is 4.6 and the final pH of the isolate in the experiment was 4.65. The amount of

acetic acid depends on the amount of the milk used and its pH value. Only 2.8mL of 10% acetic

was used to protonate the phosphate groups of casein, therefore neutralizing the negative

charge on the micelle of the protein. The isolate is white in color and has a clay-like texture

when formed. Moreover, the percent yield of the isolate was calculated using this equation:
 grams of protienisolate
%yeild = ×100 %
grams of non−fat powdered milk

By inputting the data, therefore:

3.2510 gramsisolated casein

%yeild = ×100 %=64.20 % of casein
5.0639 grams of non−fat powdered milk

The percent yield of the isolated casein is not accurate because of systematical errors

done in the experiment. The amorphous solid formed by casein was not completely dried when

it was weighted, thus a small fraction of water is still present. Drying the isolate and transferring

it from filter papers causes small protein parts to pulverized causing it to lose some weight. Also,

the isolate was not filtered properly and traces of it were discarded.

The moment that boiling water and Ba(OH)2 was added in the protein, alkaline

hydrolyzation occurred because of the presence of the bubbling reaction in the flask.

Autoclaving the isolate for a long time speeds up the reaction as it is evident in the appearance

of the hydrolysate after it was autoclaved. The transition of the color of the hydrolysate in a

yellowish-brown appearance indicates that alkaline hydrolysis is done. Amino acids are now

separated into the protein.

The next step after hydrolyzing casein using a strong base is to neutralize it using a

strong acid. Neutralizing casein is important in eliminating the base which hydrolyzed it because

it may hinder the results when the hydrolysate is analyzed using various chemical tests. A

strong acid such as sulfuric acid in barium hydroxide will form barium sulfate which does not

react with the hydrolysate and can be easily filtered, producing a pale-yellow pure casein

hydrolysate (Pavia, et al., 1990). The final pH of the hydrolysate is 6.99.

CONCLUSION

The experiment effectively isolated casein from non-fat powdered milk via isoelectric

precipitation. Reducing the milk’s pH into casein’s isoelectric point is the essential step in

protein analysis. It was hydrolyzed using barium hydroxide and was neutralize using sulfuric

acid. The alkaline hydrolysis and neutralization were successful enough to produce a pure

hydrolysate. The produced casein hydrolysate can now be subjected to various chemical tests

to identify its amino acid components, structure, and functionality. The purity of the isolate and

the hydrolysate can be further improved by minimizing the systematical errors. Sources of

systematical errors lie in having inaccurate measurements, a damp casein isolate, and

unfiltered residues.

REFERENCES

Books

Berg, J. M., Tymoczko, J. L., Gatto, G. J., & Stryer, L. (2015). Biochemistry (8th ed.). W. H.
Freeman.
Bylund G. (2015). Dairy processing handbook. Lund: Tetra Pak Processing Systems AB.
Campbell, M. K., Farrell, S. O., & McDougal, O. M. (2018). Biochemistry (8th ed.). Boston, MA:
Cengage Learning.
Fox, P. F., Uniacke-Lowe, T., McSweeney, P. L. H., & OMahony, J. A. (2015). Dairy chemistry
and biochemistry. Cham: Springer.
Franks, F. (Ed.). (1993). Protein biotechnology: isolation, characterization, and stabilization.
Springer Science & Business Media.
Pavia, D. L., Lampman, G. M., Kriz, G. S., & Engel, R. G. (1990). Introduction to organic
laboratory techniques: a microscale approach. Philadelphia: Saunders College Publishing.
Journal Articles
Dave, R.I., Joshi, N.S., Patel, J.R. and Thakar, P.N. (1991). Protein hydrolysates: A review.
Indian J. Dairy Sci., 44: 9.

Electronic References
Casein. (2019, October 31). Retrieved February 15, 2020, from
https://dairyprocessinghandbook.tetrapak.com/chapter/casein
Ezzat, M. (n.d.). Isolation of Casein, Lactose, and Albumin from Milk. Retrieved February 15,
2020, from
https://www.academia.edu/35060501/Isolation_of_Casein_Lactose_and_Albumin_from_M
ilk