Isolation and Hydrolysis of Casein from a Non-fat Milk

Carl Antoni Mallillin*, Victor Michael Angelo Manimtim

Jasmin Marcos, Andrea Czarina Mariano
Department of Biological Sciences, College of Science,
University of Santo Tomas
Manila, Philippines
Abstract:
Isoelectric precipitation was used to isolate the protein, Casein, from a non-fat milk. Acetic
acid was added dropwise to the solution until it formed an amorphous mass. The amorphous mass
was then dried and weighed to calculate the percent yield which was 75.12%. The dried isolated
casein was then subjected to acid hydrolysis and neutralization.
Introduction:
Milk is a white liquid produced by the mammary glands. This biologically complex liquid
is a good source of calcium, phosphorous and magnesium (Tull, 1996). Though this liquid is rich
in various proteins, lipids and minerals, they are a poor source of iron and ascorbic acid. Milk is
used in various forms of consumption. For example, converting milk to curd, cheese, yoghurt.
Casein is found as a suspension of particles known as casein micelles.

Casein is the major protein found in milk and it compromises 80% of the protein found in
cow milk (Kunz, 1990). Generally, casein is a globular protein in which the proline residues do
not interact with each other (Figure 1). It contains all special amino acids including: Serine,
Proline, Cysteine, Lysine, Arginine, Histidine, Tryptophan, Threonine, Valine, etc (DRINC, n.d.).
The phosphate group of casein is attached to the hydroxyl groups of some of the amino acid side
chains. Thus, making casein related to phosphoproteins.

Figure 1. Structure of Casein Micelle

Retrieved from:
https://www.intechopen.com/books/
milk-proteins-from-structure-to-
biological-properties-and-health-
 In this experiment, isoelectric precipitation will be used to isolate Casein from a non-fat
milk. The isolated protein will then be subjected to acid hydrolysis and neutralization.

Results and Discussion:

In the experiment, whole milk was not used to isolate
Casein, because fat is difficult to separate from protein. Thus,
non-fat milk was used to avoid inaccurate results and
inconvenience. The milk solution was heated to 55°C in order to
facilitate precipitation. Temperature higher than this will
Figure 2. - Calcium Caseinate
denature casein (Denniston, 2004). The pH of the heated milk
Retrieved from:
solution was lowered with the use of Acetic Acid or CH 3COOH https://www.slideshare.net/shazi5
and formed an amorphous, white mass. This precipitate was due 250/isolation-of-casein-from-milk

to the insolubility of casein at pH 4.6 which also known as its isoelectric point. Generally, the pH
of milk is about 6.6 and the protein, casein, has a negative charge and is soluble as a salt. Casein
exist in milk as a calcium caseinate as seen in Figure 2.

The precipitating agent used to isolate casein is Acetic acid or CH 3COOH. A weak acid is
used to precipitate casein, as opposed to using a strong acid, because weak acids generally do not
dissociate completely. As a result, the acid protonates the carboxylate group and the negative
charge of the outer surface of the casein micelle will be neutralized and therefore, decreasing its
solubility (Pavia, 1995) (see figure 3).

Figure 3. – Addition of a weak acid to calcium caseinate.

Retrieved from: https://www.slideshare.net/shazi5250/isolation-of-casein-from-milk

In addition, lowering of the pH of drives hydrophobic interactions which causes casein

micelles to aggregate or destabilize (Pavia, 1995). It also increases the solubility of the of the
calcium and phosphorous in the micelle. Therefore, spoiled milk is an example of the coagulation
of casein. The sour taste of spoiled milk is the result of acidification and the shapeless mass
produced is casein.

The following table summarizes the results of the isolation of casein:

Table 1. - Results
Weight of powdered non-fat milk 5.0011 g
Weight of Casein 3.7572 g
% Yield 75.12%
Initial pH 6.4
Final pH 4.6
Drops or Volume of 10% HAC 125 drops or 6.25 ml
Appearance of the hydrolysate before autoclaving Colorless liquid with white solid lumps
Appearance of the hydrolysate after autoclaving Black liquid with paper-like suspension

About 6.25 ml of Acetic acid was used to bring casein to its isoelectric point. The results
indicate that casein is about 75.12% of the weight of the non-fat milk. In addition, this shows that
majority of protein contents of milk is casein. The appearance of the hydrolysate before
autoclaving is a colorless liquid with white solid lumps suspended (figure 4). The percent yield of
casein was computed by using the following formula:
𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝐶𝑎𝑠𝑒𝑖𝑛
% 𝑌𝑖𝑒𝑙𝑑 =
𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑃𝑜𝑤𝑑𝑒𝑟𝑒𝑑 𝑀𝑖𝑙𝑘

Acid hydrolysis is the process in which the peptide bonds of a protein are broken into its
amino acid components (Kurbanova, 2014). In the experiment, acid hydrolysis is performed by
adding a strong acid, in this case 4 mL of 8N H2SO4, to the isolated protein. The designated
concentration of the strong acid is vital as 8 % by volume is not suitable for completing and
uncomplicated hydrolysis of casein (Borchers and Berg, 1942).
On the other hand, high concentration would yield irreversible
denaturation of the protein isolate. The parameters of
autoclaving are as important as the concentration of the acid
added. For example, long durations of autoclaving could induce
Figure 4. – Hydrolysate
before autoclaving
racemization and destruction of the protein (Borchers and Berg, 1941). Moreover, higher
temperature and higher pressure could also denature the protein.

The figure shows that after autoclaving, the appearance of the

hydrolysate is now a black solution with paper-like solids
suspended. The black precipitate formed after autoclaving
indicates that the amino acid, Tryptophan, has been destroyed by
Figure 5. - Hydrolysate the acid hydrolysis. The precipitate that has formed is known as
after autoclaving Humin. By hydrolyzing casein, the tryptophan present condenses
with an aldehyde in the presence of strong acid while being heated (Burr and Gortner, 1924).
Proteins, apparently, have a small amount of unknown aldehyde which reacts with the indole side
chain of the Tryptophan which produces this black precipitate (Burr and Gortner, 1924). The acid
hydrolysate is then neutralized with Ba(OH)2(s) to be used for color reactions.

Methodology:
Isolation of Casein:
5 g of powdered non-fat dry milk was dissolved in 20 ml distilled water in a 100-ml beaker.
The solution was heated to 55°C on a hot plate and then removed. The initial pH of the milk
solution was noted and then a dropwise of 10% acetic acid was added while being stirred with a
stirring rod. 10% acetic acid was added until the pH reached 4.6 and the volume of the acid used
was noted. The amorphous mass formed was decanted from the solution. The isolated casein was
dried between two filter papers and then weighed. The percent yield was determined. The isolated
casein was divided into two portions. One portion was used for acid/base hydrolysis. The other
portion was wrapped in aluminum foil and stored.

Acid Hydrolysis and Neutralization:

The protein isolate was cut into very small pieces and was placed into a 50-ml Erlenmeyer
flask and then 4 ml 8N H2SO4 was added. The flask was plugged with a piece of cotton and covered
with aluminum foil. The flask was labeled and the appearance of the sample was noted before
autoclaving. The flask was autoclaved at 15 psi for 5 hrs. The appearance of the sample was noted
after autoclaving. The hydrolysate was diluted in 15 ml of distilled water and then transferred the
contents to a 250-ml beaker. The hydrolysate was neutralized by adding a spatulaful of Ba(OH)2(s).
The pH was checked by using a litmus paper. Saturated Ba(OH)2 (l) was added dropwise if the
hydrolysate is not yet neutral. The pH was confirmed using a litmus paper. The precipitate formed
was filtered off. Distilled water was added to the distillate if the volume is less than 7 ml.

Conclusion:

Isoelectric precipitation is utilized to coagulate and precipitate the protein, Casein. The use
of a weak acid is essential to lower the pH and bring the milk solution to its isoelectric pH 4.6.
Once the casein is insoluble enough to be isolated, it is subjected to acid hydrolysis. The addition
of a strong acid and autoclaving the hydrolysate cleaved the peptide bonds of the casein and
produced a black precipitate indicating the loss Tryptophan and formation of Humin.

References:
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