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

conventional plastics

A project report

Submitted in partial fulfilment of the requirements

For the Degree of

Master of Science

In

Chemistry

Submitted by

ANKITA KUMARI

BU2019PGMCH04

Under the guidance of

Dr. Pooja puri

To the

DEPARTMENT OF CHEMISTRY

SCHOOL OF BASIC SCIENCES

BAHRA UNIVERSITY

JULY 2021

1
 STUDENT DECLERATION
I hereby, certify that the work, which is being presented in project, entitled
“Casein Extraction from milk samples and its role as a viable substitute for
conventional plastics” in fulfilment of requirement for the award of Master
of Science in Chemistry, submitted in Department of CHEMISTRY of
BAHRA UNIVERSITY, India, is an authentic record of my own carried out
during period from July 2021.

Signature of Student’s
Ankita Kumari

This is certify that above statement made by the student’s is correct to the best
of my knowledge.

Signature of Supervisor
Dr. Pooja Puri
Date : 10July 2021

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 CERTIFICATE

This is certify that these entitled “Casein Extraction from various milk sample and its role
as a viable substitute for conventional plastics” submitted by Ankita kumari having Roll no.
BU2019PGMCH04 in partial fulfilment of the requirements for the award of Degree of Master
of Science in Chemistry to BAHRA UNIVERSITY, Shimla Hills, Solan is a record of

student’s own work carried out under supervision and guidance of Dr. Pooja puri
To the best of my knowledge, this thesis has not been submitted in part or full elsewhere in any
other university or institution for the award of degree. It is further understood that by this
certificate the undersigned do not endorse or approve any statement made, opinion expression
or conclusion drawn here in but approve the thesis only for the purpose for which it is
submitted.

Dr. Sumita Sood

Associte Professor&H.O.D
Dept. Of Chemistry

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 ACKNOWLEDGEMENT
This dissertation was not done by me alone. There are lots of good spirits and angles who guided and motivated
me along the way. I would like to take this opportunity to express my heartiest gratitude to all of them.
First of all, I would like to thank H.O.D. Department of Chemistry, Dr. Sumita Sood for his encouragement for
completion of this project.
I am grateful and I would like to express my sincere gratitude to my supervisor Dr. Pooja Puri for his
germinal ideas, invaluable guidance, continuous encouragement and constant support in making this thesis possible.
They have trained me in their professional framework and they have never failed to do that. I am being honored by
studying under their guidance and being challenged by their professional standards not only in academia but also in
common life. I won’t forget any moments of my life with them.
I hope this thesis will be helpful for those who need reference in the field of production forecasting. Finally, I
would like to express my gratefulness to all of them who involved directly or indirectly in the completion of my
thesis.
Thank you.

ANKITA KUMARI

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 Index

1. Introduction 6
2. Biodegradable plastic from milk protein:casein 7-8
3. Extraction of casein from milk 9
4. Experimental work 10-12
5. Tests for presence of casein 13
6. Aregogel formation using extraction f casein 14
7. Conclusion 15
8. References 16

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 Introduction
Historically, petroleum- based plastics have been formulated to
increase the durability and reduce photo, chemical ,and biological
degradation (1). Popular chemical formulation include
polyethylene, polystyrene, and polyurethane. However, recent
concern about the persistence of plastic material in the
environment, either as litter or through landfill disposal of
municipal solid wastes, has promoted a rethinking of the use of
plastic-based products(2).In several states and communities,
legislation has been anacted requiring that biodegradable plastics
be used in common short-term applications such as packaging
material(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 with in the soil are
unable to consume a portion of plastic, which would, in turn, cause
a more rapid breakdown of the supporting matrix. This group of
materials usally 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.

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2. Biodegradable plastic from milk protein:Casein

Casein is 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, β-casein, and ϒ-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.

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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:

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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.

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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 sample Fat Protein

A 1g per 100g of milk powder 35g per 100g of milk powder

B 4.5 g per 100ml 2.89 g per 100ml

C 3.5 g per 100ml 3.2 g per 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

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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.

Figure 2.b)
Figure 2: a.) Heating of milk sample b.) Separation of precipitated casein

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4.3 Process yield
Weight of milk powder used initially = 20 gram
Weight of glacial acetic acid = 1.015 gram
Weight of product obtained = 16.74 gram =
ℎ ℎ (
+ )∗ 100
= 16.74(20+1.015)
= 79.657 %
Thus a process yield of 79.657 % is obtained for the production of
casein.
On a similar basis the yield calculated for other samples is listed
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

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.

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5. Tests for presence of casein

Confirmatory Test for presence of casein protein: Xanthoproteic

test: Dark Yellow colour confirming the positive test results.

Figure 3: Confirmation of protein using Xanthoproteic test

Biuret Test: Confirmation of peptides bond in protein by purple
colour

Figure 4: Confirmation of peptide bond using Biuret test

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Result

6. Aerogel Formation using extracted casein

Casein was solubilised and emulsified in an aqueous solution of
0.05 N sodium hydroxide by heating the mixture at 100 °C for 2
hours. Once the casein formed a thick emulsion with aqueous
NaOH, it was blended with formaldehyde and then again heated
for 15 min at 100°C. The mixture is then air dried in a flat tray at
ambient temperature conditions (around 25 °C). A thin film around
2-3mm thickness is then formed. The formed aerogel film is then
kept for heat treatment for 24 hours at a high temperature around
130°C. The heat-treated films were then exposed to relative
humidity conditions of room air at room temperature for
examination of the effect of moisture on mechanical properties of
these films. The formed film is shown in figure 5.

Figure 5: Casein Aerogel polymer film

The casein protein is blended with formaldehyde as a cross linking

agent, which combines with the lysine residue for the aerogel
formation as per the Maillard reaction. The reaction in 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]

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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.

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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).

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.
10. Pojanavaraphan T., Magaraphan R., Chiou B. S., Schiraldi D.
A.; Development of Biodegradable Foamlike Materials Based on
Casein and Sodium Montmorillonite Clay, Biomacromolecules,
volume 11, No. 10, 2640–2646, (2010).

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