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ChemistryProjectAnalysisofthe
differentsamplesofmilkfor
presenceofCasein
Friday,6December2013

BlogArchive
2013(2)

ABSTRACTOF
REPORT

December(2)
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AboutMe
PatitPawanBarik

Milk is the best and cheapest source of nutrition and an


article of daily diet, easily accepted and used by all the age
groups in rural as well as in urban areas. It provide appreciable
amount of fats and protein and also provides body building
vitamins along with furnishing energy giving lactose and many
other nutrients, therefore an ideal food for pregnant female
and infants. Milk can provide a wide range of readily available
nutrients to maintain health and normal growth of body. Milk
has no pronounced taste what is slightly sweet to most
persons. Milk is the largest and the single most important
commodity within the livestock sector. Although slightly varies
in composition and properties, the milk of different species
contain the same constituents in general. On average, milk is
made up of 87.4% water and 12.6% milk solids (3.7% fat, 8.9%
milk solids-not-fat). The milk solids-not-fat contain protein
(3.4%), lactose (4.8%), and minerals (0.7%).Milk fat often
called butter fat is commercially, the most valuable
constituent of milk. It is also of great importance from the
standpoint of the food value of the milk. The agreeable
flavour of rich milk and to a large extent of other dairy
products is largely due to the milk fat. Proteins are among the
most complex of organic substances. They contain carbon,
hydrogen, oxygen, nitrogen, sulphur and sometimes
phosphorus. The protein of milk is not a single compound but
includes two major proteins and small quantities of others.
Between them casein constitute about 80 % of the total and
lacto albumin 18%. A third protein recognized as present in

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milk is lacto globulin. It is present in very small amounts,


probably about 0.05 to 0.07 %. Ash constituents of milk are
extremely important in their relation to the heat stability of
the milk.

ANALYSISOF
DIFFERENTSAMPLES
OFMILKFOR
PRESENCEOFCASEIN
A File Report submitted to :

______________
______________
Internal Examiner

______________
_______________
Head Of Chemistry Department Principal

CERTIFICATE
This is to certify that this dissertation titled
Analysis of different samples of milk for presence
of Casein was submitted by Patit Pawan
Barikto

Head of Chemistry Department of


Kendriya Vidyalaya, Chittaranjan, W.B.
carried under guidance and supervision during
the
Academic year 2013-2014.

ACKNOWLEDGE
MENT
I wish to express my deep gratitude and
sincere thanks to thePrincipal,Kendriya
Vidyalaya, Chittaranjan, W.B. for his
encouragement and for all the facilities that
he provided for this project work. I sincerely
appreciate this magnanimity by taking me into
his fold for which I shall remain indebted to
him. I extend my hearty thanks toMr.
K.Batabyal (PGT Chemistry), who guided me to
the successful completion of this project. I
take this opportunity to express my deep
sense of gratitude for his invaluable guidance,
constant encouragement, constructive
comments, sympathetic attitude and immense
motivation, which has sustained my efforts at
all stages of this project work.

DECLARATION
I do hereby declare that this project work has
been
originally carried under the guidance and
supervision
of
Mr. K. Batabyal (PGT Chemistry),
Head of Chemistry Department,
Kendriya Vidyalaya, Chittaranjan, W.B.

Patit Pawan Barik


XII A
Roll no.- 32

TABLEOF
CONTENTS
Introduction

Aim

Requirement

Theory

Procedure

Observations

Conclusion

Reference

INTRODUCTION
Milk is a food of exceptional interest. Not only is milk an
excellent food for the very young, but humans have also
adapted milk, specifically cows milk, as a food substance for

adapted milk, specifically cows milk, as a food substance for


persons of all ages. Many specialised milk products like
cheese, curd, butter and ice cream are staples of our diet.
Milk is probably the most nutritionally-complete food that
can be found in nature. This property is important for milk,
since it is the only food young mammals consume in the
nutritionally significant weeks following birth.
Whole milk contains Vitamins (principally Thiamine,
Riboflavin, Pantothenic acid, and Vitamins A, D, and K),
Minerals (Calcium, Potassium, Sodium, Phosphorus, and trace
metals), proteins (which include all the essential amino
acids), carbohydrates (chiefly Lactose), and Lipids (fats).
The only important elements in which milk is seriously
deficient are iron and Vitamin C. Infants are usually born
with a storage supply of iron large enough to meet their
needs for several weeks. Vitamin-C is easily secured through
an orange juice supplement. Milk is the largest and the single
most important commodity within the livestock sector.
Although slightly varies in composition and properties, the
milk of different species contain the same constituents in
general. On average, milk is made up of 87.4% water and
12.6% milk solids (3.7% fat, 8.9% milk solids-not-fat). The
milk solids-not-fat contain protein (3.4%), lactose (4.8%),
and minerals (0.7%).Milk fat often called butter fat is
commercially, the most valuable constituent of milk. It is
also of great importance from the standpoint of the food
value of the milk. The agreeable flavour of rich milk and to a
large extent of other dairy products is largely due to the
milk fat. Proteins are among the most complex of organic
substances. They contain carbon, hydrogen, oxygen,
nitrogen, sulphur and sometimes phosphorus. The protein of
milk is not a single compound but includes two major proteins
and small quantities of others. Between them casein
constitute about 80 % of the total and lacto albumin 18%. A
third protein recognized as present in milk is lacto globulin.
It is present in very small amounts, probably about 0.05 to
0.07 %. Ash constituents of milk are extremely important in
their relation to the heat stability of the milk.

Previous
Experimental
Observations&

Results
OBSERVATIONS
Average Percentage Composition of Milk from Various
Mammals:

Source of milk

Water(%)

Minerals(%)

Proteins(%)

Fats(%)

Carbohydrates
(%)

Cow

87.1

0.7

3.4

3.9

4.9

Human

87.4

0.2

1.4

4.0

4.9

Goat

87.0

0.7

3.3

4.2

4.8

Sheep

82.6

0.9

5.5

6.5

4.5

RESULTS
The Buffalos milk has highest casein presence in all
the samples of milk. Therefore, buffalos milk is most
suitable for nutrition.

CASEIN
Casein is a phosphoprotein, which has phosphate groups are
attached to some of the amino acid side chains. Casein
exists in milk as the calcium salt, calcium caseinate. This salt
has a complex structure. It is composed of , , and
caseins which form a micelle, or a solubilized unit. Neither
the nor the casein is soluble in milk, singly or in
combination. If casein is added to either one, or to a
combination of the two, however, the result is a casein
complex that is soluble owing to the formation of the
micelle. The casein is thought to stabilize the micelle.
Since both and casein are phosphoproteins, they are
precipitated by calcium ions.
The casein protein, however, has fewer phosphate groups
and a high content of carbohydrate bound to it. It is also
thought to have all its serine and threonine residues (which
have hydroxyl groups), as well as its bound carbohydrates,

on only one side of its outer surfaces. This portion of its


outer surface is easily solubilized in water since these polar
groups are present. The other portion of its surface binds
well to the water-insoluble and caseins and solubilizes
them by forming a protective colloid or micelle around them.
Since the entire outer surface of the micelle can be
solubilized in water, the unit is solubilized as a whole, thus
bringing the and caseins, as well as casein, into
solution. Calcium caseinate has its isoelectric (neutrality)
point at pH 4.6. Therefore, it is insoluble in solutions of pH
less than 4.6. The pH of milk is about 6.6 therefore casein
has a negative charge at this pH and is solubilized as a salt.
If acid is added to milk, the negative charges on the outer
surface of the micelle are neutralized (the phosphate
groups are protonated) and the neutral protein precipitates:
Ca2+Caseinate + 2 HCl Casein + CaCl2
The calcium ions remain in solution. When milk sours, lactic
acid is produced by bacterial action and the consequent
lowering of the pH causes the same clotting reaction. The
isolation of casein from milk will be carried out in this
experiment.
The casein in milk can also be clotted by the action of an
enzyme called rennin. Rennin is found in the fourth stomach
of young calves. However, both the nature of the clot and
the mechanism of clotting differ when rennin is used. The
clot formed using rennin, calcium paracaseinate, contains
calcium.
Ca2+Caseinate + Rennin Ca2+Paracaseinate + A Small
Peptide
Rennin is a hydrolytic enzyme (peptidase) and acts
specifically to cleave peptide bonds between phenylalanine
and methionine residues. It attacks the casein, breaking
the peptide chain so as to release a small segment of it. This
destroys the water-solubilizing surface of the casein,
which protects the inner and caseins and causes the
entire micelle to precipitate as calcium paracaseinate. Milk
can be decalcified by treatment with oxalate ion, which
forms an insoluble calcium salt. If the calcium ions are
removed from milk, a clot will not be formed when the milk
is treated with rennin.

The clot, or curd, formed by the action of rennin is sold


commercially as cottage cheese. The liquid remaining is
called the whey. The curd can also be used in producing
various types of cheese. It is washed, pressed to remove any
excess whey, and chopped. After this treatment, it is
melted, hardened and ground. The ground curd is then
salted, pressed into molds, and set aside to age .It is a
major protein constituent in milk & is a mixed phosphorprotein. Casein has isoelectric pH of about 4.7 and can be
easily separated around this isoelectric pH. It readily
dissolves in dilute acids and alkalies. Casein is present in milk
as Calcium Caseinate in the form of micelles. These
micelles have negative charge and on adding acid to milk the
negative charges are neutralized.
Natural milk is an opaque white fluidsecreted by the
mammary glands of female mammal. The main constituents
of natural milk are Protein, Carbohydrate, Minerals,
Vitamins, Fats and Water and are a complete balanced diet.
Fresh milk is sweetish in taste. However, when it is kept for
long time at a temperature of 5 it become sour because of
bacteria present in air. These bacteria convert lactose of
milk into lactic acid which is sour in taste. In acidiccondition
casein of milk starts separating out as a precipitate. When
the acidity in milk is sufficient and temperature is around
36, it forms semi-solid mass, called curd.

AIM
To study the quantity of Casein in different samples
of milk.

REQUIREMENTS
Beakers (250 ml)
Filter-paper
Glass rod
Weight box
Filtration flask

Filtration flask
Buchner funnel
Test tubes
Porcelain dish
Different samples of milk
1 % Acetic acid solution

Ammonium sulphate solution

CHEMICAL
REACTION
Ca2+Caseinate2- + 2 CH3COOH(aq) Casein +
(CH3COO)2Ca

THEORY
Casein is a phosphoprotein, which has phosphate groups are
attached to some of the amino acid side chains.
These are attached mainly to the hydroxyl groups of the
serine and threonine moieties. Actually, casein is a mixture
of at least three similar proteins, which differ primarily in
molecular weight and amount of phosphorus they contain
(number of phosphate groups). Casein exists in milk as the
calcium salt, calcium caseinate. This salt has a complex
structure. It is composed of , , and caseins which form a
micelle, or a solubilized unit. Neither the nor the casein
is soluble in milk, singly or in combination. If casein is
added to either one, or to a combination of the two,
however, the result is a casein complex that is soluble owing
to the formation of the micelle. The casein is thought to
stabilize the micelle. Since both and casein are
phosphoproteins, they are precipitated by Calcium ions.
The casein protein, however, has fewer phosphate groups
and a high content of carbohydrate bound to it. It is also
thought to have all its serine and threonine residues (which
have hydroxyl groups), as well as its bound carbohydrates,

on only one side of its outer surfaces. This portion of its


outer surface is easily solubilized in water since these polar
groups are present. The other portion of its surface binds
well to the water-insoluble and caseins and solubilizes
them by forming a protective colloid or micelle around them.
Since the entire outer surface of the micelle can be
solubilized in water, the unit is solubilized as a whole, thus
bringing the and caseins, as well as casein, into
solution. Calcium caseinate has its isoelectric (neutrality)
point at pH 4.6. Therefore, it is insoluble in solutions of pH
less than 4.6. The pH of milk is about 6.6 therefore casein
has a negative charge at this pH and is solubilized as a salt.
If acid is added to milk, the negative charges on the outer
surface of the micelle are neutralized (the phosphate
groups are protonated) and the neutral protein precipitates:
Ca2+Caseinate + 2 HCl Casein + CaCl2
The calcium ions remain in solution. When milk sours, lactic
acid is produced by bacterial action and the consequent
lowering of the pH causes the same clotting reaction. The
isolation of casein from milk will be carried out in this
experiment.
The casein in milk can also be clotted by the action of an
enzyme called rennin. Rennin is found in the fourth stomach
of young calves. However, both the nature of the clot and
the mechanism of clotting differ when rennin is used. The
clot formed using rennin, calcium paracaseinate, contains
calcium.
Ca2+Caseinate + Rennin Ca2+Paracaseinate + A Small
Peptide
Rennin is a hydrolytic enzyme (peptidase) and acts
specifically to cleave peptide bonds between phenylalanine
and methionine residues. It attacks the casein, breaking
the peptide chain so as to release a small segment of it. This
destroys the water-solubilizing surface of the casein,
which protects the inner and caseins and causes the
entire micelle to precipitate as calcium paracaseinate. Milk
can be decalcified by treatment with oxalate ion, which
forms an insoluble calcium salt. If the calcium ions are
removed from milk, a clot will not be formed when the milk
is treated with rennin.

The clot, or curd, formed by the action of rennin is sold


commercially as cottage cheese. The liquid remaining is
called the whey. The curd can also be used in producing
various types of cheese. It is washed, pressed to remove any
excess whey, and chopped. After this treatment, it is
melted, hardened and ground. The ground curd is then
salted, pressed into molds, and set aside to age .It is a
major protein constituent in milk & is a mixed phosphorprotein. Casein has isoelectric pH of about 4.7 and can be
easily separated around this isoelectric pH. It readily
dissolves in dilute acids and alkalies. Casein is present in milk
as Calcium Caseinate in the form of micelles. These
micelles have negative charge and on adding acid to milk the
negative charges are neutralized.

PROCEDURE
1. A clean dry beaker has been taken, followed by
putting 20 ml of cows milk into it and adding 20 ml of
saturated ammonium sulphate solution slowly and with
stirring. Fat along with Casein was precipitate out.
2. The solution was filtered and transferred the
precipitates in another beaker. Added about 30 ml of
water to the precipitate. Only Casein dissolves in water
forming milky solution leaving fat undissolved.
3. The milky solution was heated to about 40oC and add
1% acetic acid solution drop-wise, when casein got
precipitated.
4. Filtered the precipitate, washed with water and the
precipitate was allowed to dry.
5. Weighed the dry solid mass in a previously weighed
watch glass.
6. The experiment was repeated with other samples of
milk.

OBSERVATIONS
According to above experiment :
Sl. No.

Source of Milk

Weight of Dried Solid Mass

CONCLUSION
The __________ milk has highest value of the dried solid
mass i.e. the composition of Casein is highest in the sample
of ________ milk.

SOLUTIONTOTHE
ANALYSIS
The ________ milk has the highest presence of casein in
milk. Therefore, ________ milk is the best for nutrition.

REFERENCES
Wikipedia
Encyclopedia Encarta
Britannica
icbse.nic.in
Practical Chemistry books

This is the project created by me in Class 12th for


the external practical exam.
Readmore
PostedbyPatitPawanBarikat18:46

Nocomments:

Recommend this on Google

ABSTRACTOF
REPORT

Milk is the best and cheapest source of nutrition and an


article of daily diet, easily accepted and used by all the age
groups in rural as well as in urban areas. It provide appreciable
amount of fats and protein and also provides body building
vitamins along with furnishing energy giving lactose and many
other nutrients, therefore an ideal food for pregnant female
and infants. Milk can provide a wide range of readily available
nutrients to maintain health and normal growth of body. Milk
has no pronounced taste what is slightly sweet to most
persons. Milk is the largest and the single most important
commodity within the livestock sector. Although slightly varies
in composition and properties, the milk of different species
contain the same constituents in general. On average, milk is
made up of 87.4% water and 12.6% milk solids (3.7% fat, 8.9%
milk solids-not-fat). The milk solids-not-fat contain protein
(3.4%), lactose (4.8%), and minerals (0.7%).Milk fat often
called butter fat is commercially, the most valuable
constituent of milk. It is also of great importance from the
standpoint of the food value of the milk. The agreeable
flavour of rich milk and to a large extent of other dairy
products is largely due to the milk fat. Proteins are among the
most complex of organic substances. They contain carbon,
hydrogen, oxygen, nitrogen, sulphur and sometimes
phosphorus. The protein of milk is not a single compound but

includes two major proteins and small quantities of others.


Between them casein constitute about 80 % of the total and
lacto albumin 18%. A third protein recognized as present in
milk is lacto globulin. It is present in very small amounts,
probably about 0.05 to 0.07 %. Ash constituents of milk are
extremely important in their relation to the heat stability of
the milk.

ANALYSISOF
DIFFERENTSAMPLES
OFMILKFOR
PRESENCEOFCASEIN
A File Report submitted to :

______________
______________
Internal Examiner

______________
_______________
Head Of Chemistry Department Principal

CERTIFICATE
This is to certify that this dissertation titled
Analysis of different samples of milk for presence
of Casein was submitted by Patit Pawan

Barikto
Head of Chemistry Department of
Kendriya Vidyalaya, Chittaranjan, W.B.
carried under guidance and supervision during
the
Academic year 2013-2014.

ACKNOWLEDGE
MENT
I wish to express my deep gratitude and
sincere thanks to thePrincipal,Kendriya
Vidyalaya, Chittaranjan, W.B. for his
encouragement and for all the facilities that
he provided for this project work. I sincerely
appreciate this magnanimity by taking me into
his fold for which I shall remain indebted to
him. I extend my hearty thanks toMr.
K.Batabyal (PGT Chemistry), who guided me to
the successful completion of this project. I
take this opportunity to express my deep
sense of gratitude for his invaluable guidance,
constant encouragement, constructive
comments, sympathetic attitude and immense
motivation, which has sustained my efforts at
all stages of this project work.

DECLARATION
I do hereby declare that this project work has
been
originally carried under the guidance and
supervision
of
Mr. K. Batabyal (PGT Chemistry),
Head of Chemistry Department,
Kendriya Vidyalaya, Chittaranjan, W.B.

Patit Pawan Barik


XII A
Roll no.- 32

TABLEOF
CONTENTS
Introduction

Aim

Requirement

Theory

Procedure

Observations

Conclusion

Reference

INTRODUCTION
Milk is a food of exceptional interest. Not only is milk an
excellent food for the very young, but humans have also
adapted milk, specifically cows milk, as a food substance for

adapted milk, specifically cows milk, as a food substance for


persons of all ages. Many specialised milk products like
cheese, curd, butter and ice cream are staples of our diet.
Milk is probably the most nutritionally-complete food that
can be found in nature. This property is important for milk,
since it is the only food young mammals consume in the
nutritionally significant weeks following birth.
Whole milk contains Vitamins (principally Thiamine,
Riboflavin, Pantothenic acid, and Vitamins A, D, and K),
Minerals (Calcium, Potassium, Sodium, Phosphorus, and trace
metals), proteins (which include all the essential amino
acids), carbohydrates (chiefly Lactose), and Lipids (fats).
The only important elements in which milk is seriously
deficient are iron and Vitamin C. Infants are usually born
with a storage supply of iron large enough to meet their
needs for several weeks. Vitamin-C is easily secured through
an orange juice supplement. Milk is the largest and the single
most important commodity within the livestock sector.
Although slightly varies in composition and properties, the
milk of different species contain the same constituents in
general. On average, milk is made up of 87.4% water and
12.6% milk solids (3.7% fat, 8.9% milk solids-not-fat). The
milk solids-not-fat contain protein (3.4%), lactose (4.8%),
and minerals (0.7%).Milk fat often called butter fat is
commercially, the most valuable constituent of milk. It is
also of great importance from the standpoint of the food
value of the milk. The agreeable flavour of rich milk and to a
large extent of other dairy products is largely due to the
milk fat. Proteins are among the most complex of organic
substances. They contain carbon, hydrogen, oxygen,
nitrogen, sulphur and sometimes phosphorus. The protein of
milk is not a single compound but includes two major proteins
and small quantities of others. Between them casein
constitute about 80 % of the total and lacto albumin 18%. A
third protein recognized as present in milk is lacto globulin.
It is present in very small amounts, probably about 0.05 to
0.07 %. Ash constituents of milk are extremely important in
their relation to the heat stability of the milk.

Previous
Experimental
Observations&

Results
OBSERVATIONS
Average Percentage Composition of Milk from Various
Mammals:

Source of milk

Water(%)

Minerals(%)

Proteins(%)

Fats(%)

Carbohydrates
(%)

Cow

87.1

0.7

3.4

3.9

4.9

Human

87.4

0.2

1.4

4.0

4.9

Goat

87.0

0.7

3.3

4.2

4.8

Sheep

82.6

0.9

5.5

6.5

4.5

RESULTS
The Buffalos milk has highest casein presence in all
the samples of milk. Therefore, buffalos milk is most
suitable for nutrition.

CASEIN
Casein is a phosphoprotein, which has phosphate groups are
attached to some of the amino acid side chains. Casein
exists in milk as the calcium salt, calcium caseinate. This salt
has a complex structure. It is composed of , , and
caseins which form a micelle, or a solubilized unit. Neither
the nor the casein is soluble in milk, singly or in
combination. If casein is added to either one, or to a
combination of the two, however, the result is a casein
complex that is soluble owing to the formation of the
micelle. The casein is thought to stabilize the micelle.
Since both and casein are phosphoproteins, they are
precipitated by calcium ions.
The casein protein, however, has fewer phosphate groups
and a high content of carbohydrate bound to it. It is also
thought to have all its serine and threonine residues (which
have hydroxyl groups), as well as its bound carbohydrates,

on only one side of its outer surfaces. This portion of its


outer surface is easily solubilized in water since these polar
groups are present. The other portion of its surface binds
well to the water-insoluble and caseins and solubilizes
them by forming a protective colloid or micelle around them.
Since the entire outer surface of the micelle can be
solubilized in water, the unit is solubilized as a whole, thus
bringing the and caseins, as well as casein, into
solution. Calcium caseinate has its isoelectric (neutrality)
point at pH 4.6. Therefore, it is insoluble in solutions of pH
less than 4.6. The pH of milk is about 6.6 therefore casein
has a negative charge at this pH and is solubilized as a salt.
If acid is added to milk, the negative charges on the outer
surface of the micelle are neutralized (the phosphate
groups are protonated) and the neutral protein precipitates:
Ca2+Caseinate + 2 HCl Casein + CaCl2
The calcium ions remain in solution. When milk sours, lactic
acid is produced by bacterial action and the consequent
lowering of the pH causes the same clotting reaction. The
isolation of casein from milk will be carried out in this
experiment.
The casein in milk can also be clotted by the action of an
enzyme called rennin. Rennin is found in the fourth stomach
of young calves. However, both the nature of the clot and
the mechanism of clotting differ when rennin is used. The
clot formed using rennin, calcium paracaseinate, contains
calcium.
Ca2+Caseinate + Rennin Ca2+Paracaseinate + A Small
Peptide
Rennin is a hydrolytic enzyme (peptidase) and acts
specifically to cleave peptide bonds between phenylalanine
and methionine residues. It attacks the casein, breaking
the peptide chain so as to release a small segment of it. This
destroys the water-solubilizing surface of the casein,
which protects the inner and caseins and causes the
entire micelle to precipitate as calcium paracaseinate. Milk
can be decalcified by treatment with oxalate ion, which
forms an insoluble calcium salt. If the calcium ions are
removed from milk, a clot will not be formed when the milk
is treated with rennin.

The clot, or curd, formed by the action of rennin is sold


commercially as cottage cheese. The liquid remaining is
called the whey. The curd can also be used in producing
various types of cheese. It is washed, pressed to remove any
excess whey, and chopped. After this treatment, it is
melted, hardened and ground. The ground curd is then
salted, pressed into molds, and set aside to age .It is a
major protein constituent in milk & is a mixed phosphorprotein. Casein has isoelectric pH of about 4.7 and can be
easily separated around this isoelectric pH. It readily
dissolves in dilute acids and alkalies. Casein is present in milk
as Calcium Caseinate in the form of micelles. These
micelles have negative charge and on adding acid to milk the
negative charges are neutralized.
Natural milk is an opaque white fluidsecreted by the
mammary glands of female mammal. The main constituents
of natural milk are Protein, Carbohydrate, Minerals,
Vitamins, Fats and Water and are a complete balanced diet.
Fresh milk is sweetish in taste. However, when it is kept for
long time at a temperature of 5 it become sour because of
bacteria present in air. These bacteria convert lactose of
milk into lactic acid which is sour in taste. In acidiccondition
casein of milk starts separating out as a precipitate. When
the acidity in milk is sufficient and temperature is around
36, it forms semi-solid mass, called curd.

AIM
To study the quantity of Casein in different samples
of milk.

REQUIREMENTS
Beakers (250 ml)
Filter-paper
Glass rod
Weight box
Filtration flask

Filtration flask
Buchner funnel
Test tubes
Porcelain dish
Different samples of milk
1 % Acetic acid solution

Ammonium sulphate solution

CHEMICAL
REACTION
Ca2+Caseinate2- + 2 CH3COOH(aq) Casein +
(CH3COO)2Ca

THEORY
Casein is a phosphoprotein, which has phosphate groups are
attached to some of the amino acid side chains.
These are attached mainly to the hydroxyl groups of the
serine and threonine moieties. Actually, casein is a mixture
of at least three similar proteins, which differ primarily in
molecular weight and amount of phosphorus they contain
(number of phosphate groups). Casein exists in milk as the
calcium salt, calcium caseinate. This salt has a complex
structure. It is composed of , , and caseins which form a
micelle, or a solubilized unit. Neither the nor the casein
is soluble in milk, singly or in combination. If casein is
added to either one, or to a combination of the two,
however, the result is a casein complex that is soluble owing
to the formation of the micelle. The casein is thought to
stabilize the micelle. Since both and casein are
phosphoproteins, they are precipitated by Calcium ions.
The casein protein, however, has fewer phosphate groups
and a high content of carbohydrate bound to it. It is also
thought to have all its serine and threonine residues (which
have hydroxyl groups), as well as its bound carbohydrates,

on only one side of its outer surfaces. This portion of its


outer surface is easily solubilized in water since these polar
groups are present. The other portion of its surface binds
well to the water-insoluble and caseins and solubilizes
them by forming a protective colloid or micelle around them.
Since the entire outer surface of the micelle can be
solubilized in water, the unit is solubilized as a whole, thus
bringing the and caseins, as well as casein, into
solution. Calcium caseinate has its isoelectric (neutrality)
point at pH 4.6. Therefore, it is insoluble in solutions of pH
less than 4.6. The pH of milk is about 6.6 therefore casein
has a negative charge at this pH and is solubilized as a salt.
If acid is added to milk, the negative charges on the outer
surface of the micelle are neutralized (the phosphate
groups are protonated) and the neutral protein precipitates:
Ca2+Caseinate + 2 HCl Casein + CaCl2
The calcium ions remain in solution. When milk sours, lactic
acid is produced by bacterial action and the consequent
lowering of the pH causes the same clotting reaction. The
isolation of casein from milk will be carried out in this
experiment.
The casein in milk can also be clotted by the action of an
enzyme called rennin. Rennin is found in the fourth stomach
of young calves. However, both the nature of the clot and
the mechanism of clotting differ when rennin is used. The
clot formed using rennin, calcium paracaseinate, contains
calcium.
Ca2+Caseinate + Rennin Ca2+Paracaseinate + A Small
Peptide
Rennin is a hydrolytic enzyme (peptidase) and acts
specifically to cleave peptide bonds between phenylalanine
and methionine residues. It attacks the casein, breaking
the peptide chain so as to release a small segment of it. This
destroys the water-solubilizing surface of the casein,
which protects the inner and caseins and causes the
entire micelle to precipitate as calcium paracaseinate. Milk
can be decalcified by treatment with oxalate ion, which
forms an insoluble calcium salt. If the calcium ions are
removed from milk, a clot will not be formed when the milk
is treated with rennin.

The clot, or curd, formed by the action of rennin is sold


commercially as cottage cheese. The liquid remaining is
called the whey. The curd can also be used in producing
various types of cheese. It is washed, pressed to remove any
excess whey, and chopped. After this treatment, it is
melted, hardened and ground. The ground curd is then
salted, pressed into molds, and set aside to age .It is a
major protein constituent in milk & is a mixed phosphorprotein. Casein has isoelectric pH of about 4.7 and can be
easily separated around this isoelectric pH. It readily
dissolves in dilute acids and alkalies. Casein is present in milk
as Calcium Caseinate in the form of micelles. These
micelles have negative charge and on adding acid to milk the
negative charges are neutralized.

PROCEDURE
1. A clean dry beaker has been taken, followed by
putting 20 ml of cows milk into it and adding 20 ml of
saturated ammonium sulphate solution slowly and with
stirring. Fat along with Casein was precipitate out.
2. The solution was filtered and transferred the
precipitates in another beaker. Added about 30 ml of
water to the precipitate. Only Casein dissolves in water
forming milky solution leaving fat undissolved.
3. The milky solution was heated to about 40oC and add
1% acetic acid solution drop-wise, when casein got
precipitated.
4. Filtered the precipitate, washed with water and the
precipitate was allowed to dry.
5. Weighed the dry solid mass in a previously weighed
watch glass.
6. The experiment was repeated with other samples of
milk.

OBSERVATIONS
According to above experiment :
Sl. No.

Source of Milk

Weight of Dried Solid Mass

CONCLUSION
The __________ milk has highest value of the dried solid
mass i.e. the composition of Casein is highest in the sample
of ________ milk.

SOLUTIONTOTHE
ANALYSIS
The ________ milk has the highest presence of casein in
milk. Therefore, ________ milk is the best for nutrition.

REFERENCES
Wikipedia
Encyclopedia Encarta
Britannica
icbse.nic.in
Practical Chemistry books

This is the project created by me in Class 12th for


the external practical exam.
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