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Casein Extraction from various milk samples and its role as a viable substitute
for conventional plastics

Article  in  International Journal of Applied Engineering Research · August 2015

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 International Journal of Applied Engineering Research (IJAER)
Print ISSN 0973-4562

Casein Extraction from various milk samples and its role as a viable substitute for

conventional plastics

Neha Patni1*, Neha Tripathi2, Sweta Bosmia3

Chemical Engineering Department, Institute of Technology,
Nirma University, Ahmedabad
Email Id*: neha.patni@nirmauni.ac.in

Abstract – Plastic waste disposal is nowadays an inevitable
issue of concern because of defying biodegradation attributes
of conventional petroleum based plastics. These conventional
plastics are produced from fossil fuel reserves which are in
limited stocks again captures attention because of depleting
reserves. The scarcity of space for disposal of plastic waste
and growing environmental concerns for non-biodegradable
synthetic plastics have fuelled research towards development
of eco-friendly biopolymer materials. It is very difficult to
reduce the consumption of plastic products due to their
versatile properties, but it is possible to replace petroleum-
based plastics with alternative materials that have polymer-
like properties and that degrade after being discarded. In this
paper we present a concept of producing a biodegradable
polymer from casein which is protein extracted from milk.
Casein extracted from various milk samples is compared for
its yield and the sample with maximum yield is then blended
with the plasticizers to form a biodegradable polymer. The
formed polymer involves minimal use of carbon so is
biodegradable and can be used for various low density
polymer applications.
Keywords – Conventional petroleum based plastics, non-
biodegradable, Casein.
1. Introduction
Historically, petroleum-based plastics have been formulated to
increase the durability and reduce photo, chemical, and biological
degradation [1]. Popular chemical formulations include
polyethylene, polystyrene, and polyurethane. However, recent
concern about the persistence of plastic materials in the
environment, either as litter or through landfill disposal of
municipal solid wastes, has prompted a rethinking of the use of
plastic-based products [2]. In several states and communities,
legislation has been enacted requiring that biodegradable plastics
be used in common short-term applications such as packaging
materials [3]. The use of petroleum-based synthetic plastics have
led to solid waste management problems and secondary problems
such as global warming, which is caused by increased carbon
dioxide levels in the atmosphere during the manufacturing and
disposal processes. Conventional plastics are resistant to
biodegradation, as the surfaces in contact with the soil in which
they are disposed are characteristically smooth [4].
Microorganisms within the soil are unable to consume a portion
of the plastic, which would, in turn, cause a more rapid
breakdown of the supporting matrix. This group of materials
usually has an impenetrable petroleum based matrix, which is
reinforced with carbon or glass fibres. Biopolymers are
renewable, largely biodegradable and can have very similar
properties to conventional polyolefin polymers. These are all
desirable properties given that the use of conventional plastics is
wide spread. Polyolefins are produced from fossil fuels and
resource depletion may become a determining factor in future
production. Understanding their environmental impact is
important when comparing them to biopolymers.
2. Biodegradable plastic from milk protein:
Casein
Casein is a naturally occurring macromolecule that accounts for
approximately 80% of the protein content of cow’s milk; it is a
phosphoprotein that can be separated into various electrophoretic
fractions, such as αs-casein, κ-casein, β-casein, and γ-casein in
which each constituent differs in primary, secondary, and tertiary
structure, amino acid composition, and molecular weight [5-7].
The casein family of protein consists of several types of caseins
(α-s1, α-s2, ß-6) and each has its own amino acid composition,
genetic variations, and functional properties. The caseins are
suspended in milk in a complex called a micelle. The caseins have
a relatively random, open structure due to the amino acid
composition (high protein content). The high phosphate content
of the casein family allows it to associate with calcium and form
calcium phosphate salts. But casein is not very strong, and water
can wash it away. To beef up casein, and boost its resistance to
water, the scientists blended in a small amount of clay and
reactive molecule formaldehyde, which links casein's protein
molecules together.
2.1 Presence of casein in milk
Milk contains 3.3% total protein. Milk proteins contain all 9
essential amino acids required by humans. Milk proteins are
synthesized in the mammary gland, but 60% of the amino acids
used to build the proteins are obtained from the cow's diet. Total
milk protein content and amino acid composition varies with cow
breed and individual animal genetics. There are 2 major
categories of milk protein that are broadly defined by their
chemical composition and physical properties. The casein family
contains phosphorus and will coagulate or precipitate at pH 4.6.
The serum (whey) proteins do not contain phosphorus, and these
proteins remain in solution in milk at pH 4.6. The principle of
coagulation, or curd formation, at reduced pH is the basis for
cheese curd formation. In cow's milk, approximately 82% of milk
protein is casein and the remaining 18% is serum, or whey
protein.
2.2 Casein structure
Proteins are probably the most important class of biochemical
molecules, along with lipids and carbohydrates which are also
essential for life. Casein is a protein that is found in milk which is
used independently in many foods as a binding agent. Its structure
comprises of presence of presence of amino acids. Amino acids
have a variety of chemically reactive groups like phenolic
hydroxy groups, presence of peptide bonds. Casein also includes
amino groups, ketones and hydrazine groups. Its structure is
shown as:
Figure 1: Structure of casein [8]
3. Extraction of casein from milk
When a sample of non-fat milk is precipitated using acetic acid
after heating milk, the casein separates from the sample leaving a
clear solution. By stirring the solution while adding acid, the
casein separates from the milky solution as an amorphous mass
which could be removed using suction filtration. When acetic acid
is added into the milk sample it forms precipitates. The
precipitates are then washed 2 or 3 times using water after
decanting off the filtrate which is then rubbed with sufficient
amount of 0.1% sodium hydroxide solution and then the resulting
solution is filtered using a cloth. Precipitates termed as casein, are
then treated with absolute alcohol in a beaker. The separated
casein is then mixed with the ether solution for a few minutes, the
ether is allowed to settle, and the process is repeated with a
second portion of ether. After the second washing with ether,
again filter the product. The ether washings remove any small
quantities of fat that may have precipitated with the casein [9].
3.1 Confirmation of presence of casein
Since casein structure is composed of variety of functional
groups like carbonyl groups, phenolic hydroxyl group,
amino acids, peptide bonds it can be confirmed using
confirmatory tests for various functional groups present in
it.
4. Experimental work
The experiment for extraction of casein was carried out for three
milk samples with varying fat content. The results were compared
based upon the yield obtained after the extraction in which
precipitation was caused using glacial acetic acid manufactured
by High Purity Lab Chemicals Pvt. Ltd. Mumbai-02; and was
used without any further modification. Casein was extracted from
SAGAR Skimmed Milk Powder Spray Dried of non-fat protein
rich milk supplied by Gujarat Co-operative Milk Marketing
Federation Ltd. and AMUL milk samples of different fat content.
4.1 Nutritional information of milk samples
The milk samples which were used had difference in the amount
of fat content and protein content are: Sample A: non-fat milk
powder, Sample B: Amul Shakti millk, Sample C: Amul Cow
milk The nutritional difference in the taken samples are compared
below in Table 1.
Table 1: Nutritional information
Type of milk Fat Protein
sample
A 1g per 100g of milk 35g per
powder 100g of milk
powder
B 4.5 g per 2.89 g per
100ml 100ml
C 3.5 g per 3.2 g per
100ml 100ml
4.2 Casein Preparation
The milk solution is prepared by adding 80ml of distilled water
and 20g of non-fat milk powder by proper stirring. The formed
solution of non-fat milk is precipitated using 1.2 millilitres of
glacial acetic acid after heating it till a temperature of 55° C
(temperature should not be more than 55° C). The casein
separates as precipitates from the sample leaving a clear solution.
By stirring the solution while adding acid, the casein separates
from the milky solution as an amorphous mass which could be
removed using suction filtration. The precipitates are then washed
2 or 3 times using water after decanting off the filtrate which is
then rubbed with sufficient amount of 0.1% sodium hydroxide
solution and then the resulting solution is filtered using a cloth.
Precipitates termed as casein, are then treated with absolute
alcohol in a beaker. The separated casein is then mixed with the
ether solution for a few minutes, the ether is allowed to settle, and
the process is repeated with a second portion of ether. After the
second washing with ether, again filter the product. The ether
washings remove any small quantities of fat that may have
precipitated with casein.
 As the maximum yield was obtained for non fat milk powder
sample because of minimal fat proportion it was further checked
with confirmatory tests of the functional group and was used for
further polymer aerogel formation.

5. Tests for presence of casein

Confirmatory Test for presence of casein protein: Xanthoproteic
test: Dark Yellow colour confirming the positive test results.

Figure 2.a)
.

Figure 3: Confirmation of protein using Xanthoproteic test

Biuret Test: Confirmation of peptides bond in protein by purple

colour

Figure 2.b)
Figure 2: a.) Heating of milk sample b.) Separation of
Figure 4: Confirmation of peptide bond using Biuret test
precipitated casein

4.3 Process yield 6. Aerogel Formation using extracted casein

Weight of milk powder used initially = 20 gram Casein was solubilised and emulsified in an aqueous solution of
Weight of glacial acetic acid = 1.015 gram 0.05 N sodium hydroxide by heating the mixture at 100 °C for 2
Weight of product obtained = 16.74 gram hours. Once the casein formed a thick emulsion with aqueous
𝑃𝑟𝑜𝑐𝑒𝑠𝑠 𝑌𝑖𝑒𝑙𝑑 NaOH, it was blended with formaldehyde and then again heated
𝑑𝑟𝑦 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑝𝑟𝑜𝑑𝑢𝑐𝑡 𝑜𝑏𝑡𝑎𝑖𝑛𝑒𝑑 for 15 min at 100°C. The mixture is then air dried in a flat tray at
= ∗ 100 ambient temperature conditions (around 25 °C). A thin film
𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 (𝑚𝑖𝑙𝑘 𝑝𝑜𝑤𝑑𝑒𝑟 + 𝑔𝑙𝑎𝑐𝑖𝑎𝑙 𝑎𝑐𝑒𝑡𝑖𝑐 𝑎𝑐𝑖𝑑)
16.74 around 2-3mm thickness is then formed. The formed aerogel film
=
(20+1.015) is then kept for heat treatment for 24 hours at a high temperature
= 79.657 % around 130°C. The heat-treated films were then exposed to
Thus a process yield of 79.657 % is obtained for the production of relative humidity conditions of room air at room temperature for
casein. examination of the effect of moisture on mechanical properties of
On a similar basis the yield calculated for other samples is listed these films. The formed film is shown in figure 5.
below in Table 2.

Table 2: Calculated Process Yield for various milk samples

Milk Sample Calculated Yield ( in
percentage)

A 79.657

B 27.683

C 37.452
 4. Aminabhavi, T.M., Balundgi, R.H., Cassidy, P.E; Review on
biodegradable plastics, Polymer Plastics Technology and
Engineering, 29(3), 235-262, (1999).
5. Ghosh, A.; Ali, M. A.; Dias, G.; J. Biomacromolecules, 10,
1681-1688, (2009).
6. Audic, J. L.; Chaufer, B.; Daufin, G. Lait; 83, 417–438,
(2003).
7. Barreto, P. L. M.; Pires, A. T. N.; Soldi, V.; Polym. Degrad.
Stab.79, 147–152, (2003).
8. J.C. Garbosa, A.T. Garcia, R.S. Garcia, R.C. Gomez, D.A.
Gonong Group; Color Reactions of Casein Protein and
Basic Hydrolysate, # 4, 2CMT, Faculty of Pharmacy, UST.
9. Agarwal O.P., Advanced Practical Organic Chemistry, 371-
372.
Figure 5: Casein Aerogel polymer film
10. Pojanavaraphan T., Magaraphan R., Chiou B. S., Schiraldi
The casein protein is blended with formaldehyde as a cross D. A.; Development of Biodegradable Foamlike Materials
Based on Casein and Sodium Montmorillonite Clay,
linking agent, which combines with the lysine residue for the
Biomacromolecules, volume 11, No. 10, 2640–2646,
aerogel formation as per the Maillard reaction. The reaction in (2010).
figure 6 shows a reaction for glyceraledehyde molecule which has
a similar chemistry of bonding as that of formaldehyde and
casein. Lysine is the basic amino group which is present in
proteins.

Figure 6: Cross linking mechanism by Maillard reaction [10]

7. Conclusion
The casein extraction was carried out for milk samples with
different fat content. The conversion of casein from milk powder
of non-fat protein rich milk sample gave the highest yield out of
the three samples. The casein is a protein molecule with elastic
properties, high mechanical strength and also insoluble in water.
Due to such properties its suitability to be used as a polymer
increases. To improve upon the properties of casein it is blended
with formaldehyde which serves as a plasticizer and the aerogel is
formed. The formed aerogel can serve as a viable substitute for
conventional non-biodegradable polymers used for low density,
high temperature applications. These materials may have several
potential applications including the fabrication of bioscaffolds,
foams, contact lenses, drug delivery capsules, and numerous other
technological applications.

8. References
1. Rivard C. J.; Biodegradable plastics: further research needed
to meet environmental mandate, J. Environ. Health, 53, 24-
26, (1991).
2. Thayer A. M.; Solid waste concerns spur plastic recycling
efforts, Chem. Eng. News, 67, 7-15, (1989).
3. Smock D.; Are biodegradable plastics the answer to litter?,
Plastics World, 45, 28-31, (1987).

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