ESTIMATION OF CASEIN PROTEIN FROM SELECTED SKIMMED

MILK SAMPLES

Submitted to the

DEPARTMENT OF CHEMISTRY

MESIHA.A

GIRIJA.E

UMA MAHESWARI.V

SWETHA.C

III BSc Chemistry

Under the Guidance of

Dr.S.M.SAMEENA BANU M.Sc., M.Phil.,Ph.D

Assistant Professor
Department of Chemistry

J.B.A.S COLLEGE FOR WOMEN (AUTONOMOUS)

TEYNAMPET, CHENNAI - 600 018

APRIL – 2023

1
ESTIMATION OF CASEIN PROTEIN FROM SELECTED SKIMMED
MILK SAMPLES

Submitted to the

DEPARTMENT OF CHEMISTRY

MESIHA.A

GIRIJA.E

UMA MAHESWARI.V

SWETHA.C

III BSc Chemistry

Under the Guidance of

Dr.S.M.SAMEENA BANU M.Sc., M.Phil.,Ph.D

Assistant Professor
Department of Chemistry

J.B.A.S COLLEGE FOR WOMEN (AUTONOMOUS)

TEYNAMPET, CHENNAI - 600 018

APRIL – 2023

2
 DECLARATION

We Mesiha.A, Girija.E, Uma Maheswari.V, Swetha.C, bonafide students of

B.Sc. Chemistry in JUSTICE BASHEER AHMED SAYEED COLLEGE FOR WOMEN
(AUTONOMOUS) (Forenoon Session) would like to declare that this project entitled
“ESTIMATION OF CASEIN PROTEIN FROM SELECTED SKIMMED MILK
SAMPLES” is our original work, for the Partial fulfilment of the award of Bachelor in
Science in Chemistry.

Signature of the Candidates

Mesiha.A -

Girija.E -

Uma Maheswari.V -

Swetha.C -

Place: Chennai
Date: 06.04.2023

CERTIFICATE

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 This is to certify that Mesiha.A, Girija.E, Uma Maheswari.V, Swetha.C of III Year
B.Sc (Bachelor of Science) have done their group project in "ESTIMATION OF CASEIN
PROTEIN FROM SELECTED SKIMMED MILK SAMPLES” in partial fulfillment of
the requirements for the degree of bachelor of science, JUSTICE BASHEER AHMED
SAYEED COLLEGE FOR WOMEN (AUTONOMOUS), Forenoon Session.

SIGNATURE OF HEAD OF THE DEPARTMENT

GUIDE IN CHARGE

Date: 06-04-2023

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 CONTENTS

CHAPTERS TITLE PAGE.NO

I Introduction 6
II Scope of the Project 9
III Materials and Methods 11
IV Results and Discussion 13
V Conclusion 21
REFERENCES 23

LIST OF TABLES

Table No. Title of the Table Page No.

1. Yield of casein precipitated 15

2. Percentage of Lactic Acid 18
3. presence of protein [casein] by Xanthoproteic test 20

LIST OF FIGURES

Figure No. Title of the Figure Page No.

1. Structure of Casein 8
2. Structure of Lactic Acid 8
3 (a). (b). Yield of casein 15
3 (c). Yield of casein 16
4 (a). (b). (c). Lactic acid percentage 17
5 (a). (b). (c). Xanthoproteic test 19

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Chapter – I
GENERAL
INTRODUCTION

6
INTRODUCTION

Milk is a multi-nutrient fluid and it is the primary source of

nutrition for humans. It consists of 80% of proteins. The Protein in the milk is classified into
casein and whey protein. Milk protein consists of 80% of casein and 20% whey Protein. The
function of casein is to provide energy to the human body. The name casein is related to the
family of Phosphoproteins. These proteins are commonly found in mammalian milk. This
study deals with the precipitation of Casein estimation of lactic acid percentage from the
various skimmed milk samples. This technique of precipitation of casein and estimation of
lactic acid percentage is used to predict the Protein content and the lactic acid content in the
milk samples.

The composition of milk varies according to the animals from which it

comes, providing the correct growth rate and development for the young of that species [1].
Casein is a slow-digesting protein and it was suspended in the milk in a complex called a
micelle. The micelles are spherical and are 0.04 to 0.03 m in diameter. Milk composition
varies with the stage of location, age, and breed [2]. Milk is colloidal in nature due to the
presence of proteins. The proteins are heavy molecules; they form colloids when dispersed in
a water medium. The primary function of protein in living cells is to promote growth and
maintenance [3].

The nitrogen content of milk is distributed among casein at 76% while

protein and non-protein nitrogen is 6% [4]. The structure of protein consists of a polypeptide
chain of amino acids joined together by peptide linkages. Around the world, there are more
than six billion consumers of milk and milk products. Over 750 million people live in dairy
farming households [5]. It is used in paints for fast-drying water-soluble medium (Figure 1).
Casein-based glues are formulated from a mixture of casein, water, hydrated lime, and
sodium hydroxide [6].

STRUCTURE OF CASEIN

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 and is used independently in many foods as a binding agent. Its structure
comprises the presence of amino acids. Amino acids have a variety of chemically reactive

7
groups like phenolic hydroxy groups, and the presence of peptide bonds. Casein also includes
amino groups, ketones, and hydrazine groups. Its structure is shown as:

Figure 1

LACTIC ACID

Lactic acid is usually the principal acid produced when milk sours. For
this reason, in America, the titratable acidity of milk is often expressed as lactic acid. The
increase in acidity due to souring is called “developed” or “real” acidity, as contrasted to the
“original” acidity of the fresh milk. It has been stated many times in the literature that fresh
milk contains no lactic acid. Our results indicate that if fresh milk contains lactic acid, the
amount present is not over about 0.002 percent. A qualitative test showing the presence of
more than this amount would indicate souring.

A quantitative determination of lactic acid would serve as an indication

of the degree of souring. The titratable acidity of fresh milk usually falls between the limits of
0.12 and 0.20 percent, although extreme variations ranging from 0.05 to 0.50 have been
reported (2). The great variation in the original acidity shows the unreliability of the titratable
acidity as an indication of the presence of small amounts of developed acidity in unknown
samples

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 Figure -2

Chapter – II
SCOPE OF THE PROJECT

 The objective of this project is to analyse, estimate and detect the presence of casein,
lactic acid percentage and protein respectively in three different skimmed milk
samples.

 The main objective of this project is the usage of skimmed milk and milk products
with appropriate knowledge.

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10
 Chapter – III
MATERIALS AND
METHODS

PROTEINS

These natural products are especially rich in proteins, such as casein, β-

lactoglobulin, α-lactalbumin, lactoferrin, immunoglobulins, lactoperoxidase, lysozyme, and
growth factors, and possess various antibacterial, antifungal, antiviral, anticancer,
antioxidant, immunomodulatory properties, etc. In this study, the presence of proteins such as
casein in milk is detected by the xanthoproteic test.

TEST FOR ANALYSIS OF MILK

 QUANTITATIVE ANALYSIS OF CASEIN:

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 Quantitative analysis of casein precipitation from various skimmed milk samples.

 ESTIMATION OF LACTIC ACID PERCENTAGE:

To estimate the percentage of lactic acid present in various skimmed milk samples.

TEST FOR PROTEIN:

Presence of protein in the various skimmed milk samples is confirmed by

xanthoproteic test.

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 Chapter – IV
RESULTS AND DISCUSSION

EXPERIMENT 1

 QUANTITATIVE ANALYSIS OF CASEIN PRECIPITATION FROM

VARIOUS SKIMMED MILK SAMPLES.

AIM: To analyse the quantity of casein protein present in various skimmed milk samples.

CHEMICALS REQUIRED:

Skimmed milk samples [each 50ml]

 Amul
 Nandini [good life]
 Nestle
Acetic acid
Ethanol [25ml]
Di-ethyl ether [25ml]

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 Distilled water

APPARATUS REQUIRED

 Beaker
 Funnel
 Glass rod
 Watch glass
 Thermometer
 Conical flask
 Analytical balance
 Whatman filter paper

PROCEDURE:

The collected skimmed milk samples are filtered for the removal of
insoluble impurities like hair, dirt, etc. The filtered 50ml milk samples are taken in a beaker
and it was heated to 55C and treated with 10ml of acetic acid followed by continuous stirring.
The samples are kept for 10 minutes and undisturbed. The casein molecule in the skimmed
milk sample begins to precipitate at the negative charge in the milk samples permits the
dispersion of casein in it, when the positively charged, acid is added to the milk, it neutralizes
the negatively charged casein. The milk sample reached to the PH 4.7. The precipitate was
formed. The formed precipitates are filtered and washed with distilled water. Then it is
washed with diethyl ether and ethanol in 1:1 ratio [each 25ml] to remove the minute
quantities of fat presence in the milk sample. The collected casein is dried and weighed
accurately.

ADVANTAGES:

 This enables us to find the milk sample with high protein content.
 The quantitative analysis of casein precipitated from the various skimmed milk
samples provide the ample scope cottage cheese manufacture.

Sample Yield Of Casein (In Grams)

Amul 1.761

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 Nandini [good life] 1.698

Nestle 1.514

TABLE:1 Yield of casein precipitated

Figure 3 (a) Figure 3 (b)

Figure 3 (c)

EXPERIMENT 2

 ESTIMATION OF LACTIC ACID PERCENTAGE IN VARIOUS SKIMMED

MILK SAMPLES

AIM: To estimate the percentage of lactic acid present in various skimmed milk samples.

CHEMICALS REQUIRED:

 Skimmed milk samples [each 10ml]

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 Amul
Nandini [good life]
Nestle
 0.1 NaOH
 Phenolphthalein indicator

APPARATUS REQUIRED:

 Beaker
 Burette
 Burette stands
 Pipette [10ml]

PROCEDURE:

About 10ml of the collected skimmed milk sample is taken in 100ml

beaker. Few drops of phenolphthalein indicator is added to it. Then it is titrated against the
0.1N NaOH taken in the burette. The end point is detected by the appearance of pale pink
colour and the volume of NaOH consumed is noted down and lactic acid percentage is
calculated as follows.

Figure 4 (a) Figure 4 (b)

Figure 4 (c)

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CALCULATION:

 Sample A:
N1V1 (lactic acid) =N1V1 (NaOH)
N1 = N2V2 ÷ V1
N1 = 0.1 × 1.3 ÷ 10
N1 = 0.013N
% of lactic acid = Normality × gram equivalent weight × 100 ÷ 1000
= 0.013 × 90 × 100 ÷1000
= 0.117%

 Sample B:

N1V1 (lactic acid) =N1V1 (NaOH)

N1 = N2V2 ÷ V1
N1 = 0.1 × 1.2 ÷ 10
N1 = 0.012N
% of lactic acid = Normality × gram equivalent weight × 100 ÷ 1000
= 0.012 × 90 × 100 ÷1000
= 0.108%

 Sample C:

N1V1 (lactic acid) =N1V1 (NaOH)

N1 = N2V2 ÷ V1
N1 = 0.1 × 1.5 ÷ 10
N1 = 0.015N
% of lactic acid = Normality × gram equivalent weight × 100 ÷ 1000
= 0.015 × 90 × 100 ÷1000
= 0.135%

Sample Percentage Of Lactic Acid

Amul 0.117%

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 Nandini [good life] 0.108%

Nestle 0.135%

TABLE :2 Percentage of Lactic Acid

EXPERIMENT 3

TEST FOR PROTEIN IN MILK

AIM: To detect the presence of protein [casein] in the given milk samples.

CHEMICALS REQUIRED:

 Conc. Nitric acid

 Sodium hydroxide [NaOH]

TEST FOR PROTEIN: Presence of protein in the various skimmed milk samples is
confirmed by the xanthoproteic test.

PROCEDURE:

About 2g of the casein sample is taken in a test tube and 2ml of Conc. Nitric
acid is added to it and the solution is heated for about 2 minutes and cooled under tap water.
A yellow colour is obtained due to the nitration of aromatic ring. When few drops of NaOH
solution is added, the yellow colour obtained initially changes to orange indicating the
presence of protein
Figure 5 (a) Figure 5 (b)

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 Figure 5 (c)

Samples Xanthoproteic Test

Amul Positive

Nandini [good life] Positive

Nestle Positive

TABLE:3 presence of protein [casein] by Xanthoproteic test

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 Chapter – V
CONCLUSION

CONCLUSION:

Milk is an important diet in our daily life. It is an inevitable source

of proteins, carbohydrates, calcium, etc. This study deals with the presence and quantity of
casein protein and lactic acid percentage in various skimmed milk samples.
From this study, it may be suggested that the amounts of casein precipitated from sample A
(Amul) milk was higher than that of the other skimmed milk samples. Also from this study,
the lactic acid percentage of sample C (Nestle) is higher among all three samples.

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 If lactic acid levels get too high, it can lead to a life-threatening
condition known as lactic acidosis which may result in rapid breathing, excessive sweating,
cool and clammy skin, sweet-smelling breath, belly pain, nausea or vomiting, confusion, and
coma. Hence, we should be very careful about consuming milk which is inevitably part of our
diet that suits our body’s nutritional requirements and consume it according to it.

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 REFERENCES

REFERENCES

[1] Jung J and Lidfors L. 2001. Effects of amount of milk, milk flow And access to a rubber
teat on cross-sucking and nonnutritive Sucking in dairy calves. Applied Animal Behavior
Science 72:201–13.

[2] Aoki, T., H. Suzuki, and T. Imamura. 1974. Formation of soluble casein in whey protein-
free milk heated at temperature. Milchwissenscbaft 29:589

[3] Patricia Trueman, Nutritional Biochemistry, India, 2011.

[4] Farrell, H. M, and M P. Thompson. 1971. Biological significance of milk protein

polymorphism. I. Daily Sci. 54:1219

[5] T Hemma, J Otte. Status and prospects for small holder milk production. A global
perspective (PDF) Food and Agricultural Organization of the United Nations, 2010

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[7] G. Urbach, “Contribution of Lactic Acid Bacteria to Flavour Compound Formation in
Dairy Products,” International Dairy Journal, Vol. 5, No. 8, 1995, pp. 877-903.

[8] A. A. Ali, “Beneficial Role of Lactic Acid Bacteria in Food Preservation and Human
Health,” Research Journal of Microbiology, Vol. 5, No. 12, 2010, pp. 1213-1221.

[9] R. Sharma, B. S. Sanodiya, D. Bagrodia, M. Pandey, A. Sharma and P. S. Bisen,

“Efficacy and Potential of Lactic Acid Bacteria Modulating Human Health,” International
Journal of Pharma and Bio Sciences, Vol. 3, No. 4, 2012, pp. 935-948. [Citation Time(s):1]

[10]Shekarr D. 2009. ‘Performance of Vrindavani calves fed on liquid Whey and soy-
supplemented liquid whey’. M.V.Sc. Thesis Deemed University, Indian Veterinary Research
Institute,Izatnagar, India. 44 p.
[11] Aoki, T., Umeda, T. and Kako, Y. (1990) Cleavage of the linkage between colloidal
calcium phosphate and casein on heating milk at high temperature. J. Dairy Res., 57, 349–54.

[12] Aoki, T., Yamada, N., Tornita, I., et al.,(1987) Caseins are cross-linked through their
ester phosphate groups by colloidal calcium phosphate, Biochim. Biophys. Acta,911 238–43.

[13] M. Ouadghiri, M. Vancanneyt, P. Vandamme, S. Naser, D. Gevers, K. Lefebvre, J.

Swings and M. Amar, “Identification of Lactic Acid Bacteria in Moroccan Raw Milk and
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No. 2, 2008,

[14] Burks AW, Williams LW, Casteel HB, et al. Antibody Response to milk proteins in
patients with milk-protein Intolerance documented by challenge. J Allergy Clin Immunol
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[15] Madera C, Monjardin C, Suarez JE: Milk contamination and resistance to processing
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