CHEMISTRY INVESTIGATORY

PROJECT

CHEMISTRY INVESTIGATORY
PROJECT

CASEIN PRESENT IN DIFFERENT SAMPLES

OF MILK

NAME : KISHAN KUMAR SINGH

CLASS : XII-A
ROLL NO. : 4606574
BATCH : 2018-2019
GUIDE : DR.S.VASUDHEVAN, PGT CHEMISTRY
SCHOOL : KENDRIYA VIDYALAYA D.G.Q.A
 Casein in Milk

CERTIFICATE

This is to certify that KISHAN KUMAR SINGH of Class XII-A and Roll no:
4606574 has satisfactorily completed his chemistry project prescribed by
the Central Board of Secondary Education (CBSE) for AISSCE course during
the academic year 2016-2017.

Teacher in charge

External examiner internal examiner

 Casein in Milk

ACKNOWLEDGEMENT

I would like to sincerely and profusely thank our chemistry teacher

Dr.S.VASUDHEVAN PGT (Chemistry) and our lab attendant for their able
guidance and for their support for completing our project.
We would also like to extend our gratitude to our Principal
Mrs.R.Vijayalakshmi for providing us with the facilities that were required.

Signature of the candidate

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 Casein in Milk

Topic Page No.

Introduction

Aim

Required Materials

Theory

Procedure

Conclusion

Bibliography

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 Casein in Milk
INTRODUCTION
Casein is the name for a family of related phosphoproteins (αS1, αS2, β, κ). These
proteins are commonly found in mammalian milk, making up 80% of the proteins
in cow milk and between 20% and 45% of the proteins in human milk. Casein has a
wide variety of uses, from being a major component of cheese, to use as a food
additive, to a binder for safety matches. As a food source, casein supplies amino
acids, carbohydrates, and the two inorganic elements calcium and phosphorus.

Composition
Casein contains a fairly high number of proline residues, which do not interact. There
are also no disulfide bridges. As a result, it has relatively little tertiary structure. It is
relatively hydrophobic, making it poorly soluble in water. It is found in milk as
a suspension of particles called "casein micelles" which show only limited resemblance
with surfactant-typemicellae in a sense that the hydrophilic parts reside at the surface
and they are spherical. However, in sharp contrast to surfactant micelles, the interior
of a casein micelle is highly hydrated. The caseins in the micelles are held together
by calcium ions and hydrophobic interactions. Any of several molecular models could
account for the special conformation of casein in the micelles. One of them proposes
the micellar nucleus is formed by several sub micelles, the periphery consisting of
microvellosities of κ-casein. Another model suggests the nucleus is formed by casein-
interlinked fibrils. Finally, the most recent model proposes a double link among the
caseins for gelling to take place. All three models consider micelles
as colloidal particles formed by casein aggregates wrapped up in soluble κ-casein
molecules. The isoelectric point of casein is 4.6. Since milk's pH is 6.6, casein has a
negative charge in milk. The purified protein is water-insoluble. While it is also
insoluble in neutral salt solutions, it is readily dispersible in dilute alkalis and
in salt solutions such as sodium oxalate and sodium acetate. The enzyme trypsin can
hydrolyze off a phosphate-containing peptone. It is used to form a type of
organic adhesive.

Uses of Casein
Paint
Casein paint is a fast-drying, water-soluble medium used by artists. Casein paint has
been used since ancient Egyptian times as a form of tempera paint, and was widely
used by commercial illustrators as the material of choice until the late 1960s when,
with the advent of acrylic paint, casein became less popular. It is still widely used by
scene painters, although acrylic has made inroads in that field as well.

Glue
Casein-based glues, formulated from casein, water, hydrated lime and sodium
hydroxide were popular for woodworking, including for aircraft, as late as the de
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 Casein in Milk
Havilland Albatross airliner. Casein glue is also used in transformer manufacturing
(specifically transformer board) due to its oil permeability. While largely replaced with
synthetic resins, casein-based glues still have a use in certain niche applications, such
as laminating fireproof doors and the labeling of bottles.
Cheese making
Cheese consists of proteins and fat from milk, usually the milk of cows, buffalo, goats,
or sheep. It is produced by coagulation of casein. Typically, the milk is acidified and
then coagulated by the addition of rennet, containing a proteolytic enzyme, typically
obtained from the stomachs of calves. The solids are separated and pressed into final
form. Unlike many proteins, casein is not coagulated by heat. During the process of
clotting, milk-clotting proteases act on the soluble portion of the caseins, κ-casein, thus
originating an unstable micellar state that results in clot formation. When coagulated
with chymosin, casein is sometimes called paracasein. Chymosin (EC 3.4.23.4) is an
aspartic protease that specifically hydrolyzes the peptide bond in Phe105-Met106 of κ-
casein, and is considered to be the most efficient protease for the cheese-making
industry (Rao et al., 1998). British terminology, on the other hand, uses the term
caseinogen for the uncoagulated protein and casein for the coagulated protein. As it
exists in milk, it is a salt of calcium.
Plastics and fibre
Some of the earliest plastics were based on casein. In particular, galalith was well
known for use in buttons. Fiber can be made from extruded casein. Lanital, a fabric
made from casein fiber (known as Aralac in the United States), was particularly
popular in Italy during the 1930s. Recent innovations such as QMilch are offering a
more refined use of the fiber for modern fabrics.
Protein supplements
An attractive property of the casein molecule is its ability to form a gel or clot in the
stomach, which makes it very efficient in nutrient supply. The clot is able to provide a
sustained slow release of amino acids into the blood stream, sometimes lasting for
several hours. Often casein is available as hydrolyzed casein, whereby it
is hydrolysed by a protease such as trypsin. Hydrolysed forms are noted to taste bitter
and such supplements are often refused by infants and lab animals in favor of intact
casein.
Medical and dental uses
Casein-derived compounds are used in tooth remineralization products to
stabilize amorphous calcium phosphate (ACP) and release the ACP onto tooth surfaces,
where it can facilitate remineralization.

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Manufacturing Paint with the help of Casein

Cheese Making
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Plastics and Fibres

Protein Supplements
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Medical and dental uses

MILK
Milk is a complete diet as it contains in its Minerals, Vitamins Proteins, Carbohydrates,
Fats and Water. Average composition of milk from different sources is given below:

SOURCE WATER MINERALS PROTEINS FATS CARBOHYDRATES

OF MILK (%) (%) (%) (%) (%)
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
Milk is a complex biological mixture of chemicals that serves as the primary food
source for infant mammals. Milk contains most of the biological molecules necessary to
sustain life including water, a variety of vitamins, minerals, proteins, sugars, and lipids
(DNA, although necessary for life, is made by your body and therefore is not needed on
a dietary level). Although most mammals stop drinking milk upon maturity, many (but
not all) human cultures continue drinking milk and consuming milk products (for
example, cheese, butter, and cream) throughout their entire life. It would be quite
challenging to isolate every component of milk because some of the chemicals are
present in very small quantities. You can very easily, however, isolate the most
abundant components from each other. A common example of this is removing the fat
from cow’s milk to make skim milk or removing the sugar from milk to make lactose-
free milk. The Table given below shows the average composition of milk from several
mammals consumed by humans.
 Casein in Milk
 Milk components (% composition)
Cow Human Goat
Water 87.8 87.4 87.0
Protein 3.0 1.4 3.3
Lipids 3.9 4.0 4.2
Sugars 4.6 7.0 4.8
Minerals 0.7 0.2 0.7

Information about the Milk Components

Lipids:
If you were asked if fat mixes with water, chances are you would say “no”. However,
milk contains around 4% fat in water. This means that in 100 mL of water, about 4 mL
of fats would be present. This is possible because milk contains, in addition to fats, a
number of lipids called phospholipids. These molecules are similar to the fatty acids
you studied in class, but have a negatively charged phosphate group attached to one
end. The negatively charged phosphate group, like other charged, polar groups, is quite
soluble in water. The non-polar, uncharged hydrocarbon area is completely insoluble
in water. The dual hydrophobic (water hating) area hydrophilic (water loving) area
personality of the phospholipids (chemists would call it amphipathic) lets the milk fat
be soluble in the milk water by aligning a number of its hydrophobic areas with the
other fats, with the phosphates facing out toward the water. This makes a mixture
called an emulsion, meaning a fat dissolved in water by means of an amphipathic
molecule, called an emulsifying agent (soaps are emulsifying agents that work to
dissolve grease). In this lab, you will be using powdered milk that has already had the
fat removed.
Proteins:
There are three main proteins in milk; casein (case-in), lactalbumin (lact-al-bumin),
andlactoglobulin (lact-o-glob-ulin). In this lab you will be isolating the casein, and a
mixture of thelactalbumin and lactogobulin proteins (they isolate under the same
conditions and are difficult to separate from each other).
Chances are you have consumed casein without knowing it. Casein can precipitate
from water when its normal structure is destroyed. We call this process protein
denaturation. The curds in cottage cheese are precipitated casein protein (the enzyme
rennin is used in this case to precipitate the protein) and the Indian cheese paneer is
made by adding an acid to milk to precipitate the curds. Many cheeses
Are made by precipitating casein and removing it from the left over liquid, the whey.
The lactalbuminproteins are easily precipitated by heating. After casein proteins are
removed, heating the whey solution can provide the solid protein. Although a minor
component, the lactoglobulin proteins are the immune proteins present in milk that
protect a baby from illness until it can develop its own immune system.

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Sugars:
The main carbohydrate present in milk is the sugar lactose. Lactose is a disaccharide
containing the monosaccharide’s galactose and glucose. Mammals produce an enzyme
called lactase that breaks the disaccharide into its monomers during digestion. Many
mammals stop producing lactase after maturity leading them to be lactose intolerant—
they lack the ability to digest the sugar in milk. Although most people can digest
lactose upon maturity, there are many who can’t. These people can buy lactose-free
products, or buy the enzyme supplement lactase to aid in digestion of the sugar. On a
practical note, when bacteria get into milk, they digest the lactose and form the acid
lactic acid. This causes a precipitation of the protein casein. This is what happens when
old milk “sours”.
CASEIN
Casein is a major protein constituent in milk & is a mixed phosphor-protein. 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.

Casein is a protein that is found in milk and used independently in many foods as a
binding agent. Technically, it is part of a group called phosphoproteins, collections of
proteins bound to something containing phosphoric acid. It may also be called
caseinogens, particularly in European foods.
A salt, meaning it has no net ionic charge, of the element calcium; casein has a number
of interesting properties that make it useful in foods and cooking. Many people believe
proteins are healthier if consumed when not denatured one of the major lines of
reasoning used in supporting a raw food diet. Denaturing occurs when a protein loses
its inherent structure, due to high heat or acid for example, at which point it no longer
acts in the ordinary manner. Casein, because of its structure, is not susceptible to
denaturing.
Casein can be found in two main types: edible and technical. Edible casein is widely
used in both medicine and food, both for nutritional value and as a binder. The
technical type is used in an enormous range of products, including paints, cosmetics,
and many types of adhesives. A significant number of people are allergic to this protein
and may find themselves experiencing reactions both to food products and to products
such as nail polish that contain it.
People with allergies or who are vegan, and therefore avoid animal products
altogether, are not always aware of the prevalence of casein in foods. For these people,
it is important to note that, although a product may be labeled lactose free, it may still
contain casein for other reasons. Soy cheeses, for example, often contain protein
derived from milk, which may stimulate allergic reactions in people who assume that
they are dairy free.

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 Casein in Milk
Casein has also been linked to negative effects in people with autism. While
in most people, this protein is easily broken down by the digestive system
into peptides known as casomorphins, and then further processed into
basic amino acids, some evidence suggests that in autistics, this process
does not occur fully. The resulting casomorphins, which fail to break down
completely, may have an effect on the body similar to that of morphine or
other opiates. For this reason, some experts on autism recommend that
people suffering from autism avoid products containing this protein.
Controversies of Casein
 Autism
Although research has shown high rates of use of complementary and
alternative therapies for children with autism, including gluten and/or
casein exclusion diets, as of 2008 there is a lack of evidence for the efficacy
of these diets. A 2006 review of seven studies indicated that, although all
reported benefits of exclusive diets in reducing autism symptoms, all
suffered design flaws, and there was not enough evidence overall to justify
recommending exclusion diets to patients.
 A1/A2 beta caseins in milk-
According to Food Standards Australia New Zealand (FSANZ), “Milk
contains many types of proteins can be quite different in the milk from
different breeds of cows and in the milk from other animals. Of the six
major protein types in cow’s milk, four are casein proteins and the other
two are whey proteins. The caseins usually make up about 80% of the
protein in cow’s milk. One of the major caseins is beta casein. There are
different beta casein types, but the most common are beta casein 17 A1
(milk high in this type is known as A2 milk). Certain breeds of cows, such as
Friesians, produce mostly A1 milk, whereas other breeds, such as
Guernsey’s, as well as sheep and goat, produce mostly A2 milk. Milk
produced in Australia and New Zealand is normally a mix of A1 and A2
milks. The European Food Safety Authority carried out a literature review
in 2009 concluding “a cause and effect relationship is not established
between the dietary intake BCM7, related peptides or their possible protein
18 precursors and non-communicable diseases”. Studies supporting these
claims have had significant flaws, and the data are inadequate to guide
autism treatment recommendations.

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Cancer-
T.Colin Campbell’s The China Study (2005), a book, describes a direct
correlation between casein administered torats and the promotion of
cancer cell growth when exposed to carcinogens. Aflatoxin (a potent
carcinogen) was adminis19tered to these rats over a 2 week dosing period.
The rats were given a 1 week post dosing period before beginning the test
(promotion period). During the promotion period, one group of rats was
put on a 5%casein protein diet and another group on a20% casein protein
diet. None of the rats on 5% casein protein developed foci, precursors to
cancerous cell growth, and every raton 20% casein protein developed the
precancerfoci. It should be noted that all test groups were fed a 20% casein
diet for a total of 5 weeks(2-wk acclimation, 2-wk dos20ing, 1-wk post-
dosing)prior to the 12 week promotion period in order to survive the initial
aflatoxin B1(AFB1) dosing, regardless of whether they were in the 5% or
20%test groups. Campbell has performed additional studies using a range
of different carcinogens and other experimental animals, and claims to have
found a consistent correlation between cancer growth and the amount of
casein protein in diet. A2001 study suggests another milk protein, when
protein, may play a protective role against colon tumors in rats. According
to21a study from the Australian Dairy Council, casein has antimutagenic
effects.
Casein isolation
1. Place about 4 g of powdered nonfat milk and 42 mL of water into a
beaker and record the mass.
Heat the milk to 40oC.
2. When the milk has reached 40oC, start adding the acetic acid solution 5
drops at a time. After every 5 drops, stir the solution with a spatula. You will
see a white solid forming. This is the casein. Push the casein to the side of
the beaker above the solution and push the liquid out and to compress the
protein. Transfer to a watch glass as you go. Stop after 20 drops of the acetic
acid (1 mL). If you see any liquid on the watch glass, pipette it back into the
beaker.
3. Add 0.2 g of calcium carbonate to the milk solution in the beaker to
neutralize the pH.
4. Transfer the casein to a section of nylon hose. Wrap the protein and
gently squeeze over the top of the beaker to remove as much of the milk
solution as possible.
5. Weigh a plastic boat and record. Add the casein and record the mass on
the data sheet.
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STRUCTURE OF CASEIN
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STRUCTURE OF MILK
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PROTEIN-
Proteins are essential nutrients for the human body. They are one of the building
blocks of body tissue, and can also serve as a fuel source. As a fuel, proteins contain 4
kcal per gram, just like carbohydrates and unlike lipids, which contain 9 kcal per gram.
The most important aspect and defining characteristic of protein from a nutritional
standpoint is its amino acid composition.

Proteins are polymer chains made of amino acids linked together by peptide bonds.
During human digestion, proteins are broken down in the stomach to smaller
polypeptide chains via hydrochloric acid and protease actions. This is crucial for the
synthesis of the essential amino acids that cannot be biosynthesized by the body.

There are nine essential amino acids which humans must obtain from their diet in
order to prevent protein-energy malnutrition. They are phenylalanine, valine,
threonine, tryptophan, methionine, leucine, isoleucine, lysine, and histidine. There are
five dispensable amino acids which humans are able to synthesize in the body. These
five are alanine, aspartic acid, asparagine, glutamic acid and serine. There are six
conditionally essential amino acids whose synthesis can be limited under special
pathophysiological conditions, such as prematurity in the infant or individuals in
severe catabolic distress. These six are arginine, cysteine, glycine, glutamine, proline
and tyrosine.

Humans need the essential amino acids in certain ratios. Some protein sources contain
amino acids in a more or less 'complete' sense. This has given rise to various ranking
systems for protein sources.

Sources of protein include grains, legumes and nuts, as well as animal sources such as
meats, dairy products, fish and eggs. Vegetarians and vegans can get enough essential
amino acids by eating a variety of plant proteins. It is commonly believed that athletes
should consume a higher-than-normal protein intake to maintain optimal physical
performance.
 Casein in Milk

Protein can be found in a wide range of food. The best combination of protein sources
depends on the region of the world, access, cost, amino acid types and nutrition
balance, as well as acquired tastes. Some foods are high in certain amino acids, but
their digestibility and the anti-nutritional factors present in these foods make them of
limited value in human nutrition. Therefore, one must consider digestibility and
secondary nutrition profile such as calories, cholesterol, vitamins and essential
mineral density of the protein source. On a worldwide basis, plant protein foods
contribute over 60 percent of the per capita supply of protein, on average. In North
America, animal-derived foods contribute about 70 percent of protein sources. Meat,
products from milk, eggs, soy, and fish are sources of complete protein.

Whole grains and cereals are another source of proteins. However, these tend to be
limiting in the amino acid lysine or threonine, which are available in other vegetarian
sources and meats. Examples of food staples and cereal sources of protein, each with a
concentration greater than 7 percent, are (in no particular order) buckwheat, oats, rye,
millet, maize (corn), rice, wheat, sorghum, amaranth, and quinoa.

Vegetarian sources of proteins include legumes, nuts, seeds and fruits. Legumes, some
of which are called pulses in certain parts of the world, have higher concentrations of
amino acids and are more complete sources of protein than whole grains and cereals.
Examples of vegetarian foods with protein concentrations greater than 7 percent
include soybeans, lentils, kidney beans, white beans, mung beans, chickpeas, cowpeas,
lima beans, pigeon peas, lupines, wing beans, almonds, Brazil nuts, cashews, pecans,
walnuts, cotton seeds, pumpkin seeds, sesame seeds, and sunflower seeds.

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Plant sources of protein-

Food staples that are poor sources of protein include roots and tubers such as yams,
cassava and sweet potato. Plantains, another major staple, are also a poor source of
essential amino acids. Fruits, while rich in other essential nutrients, are another poor
source of amino acids. The protein content in roots, tubers and fruits is between 0 and
2 percent. Food staples with low protein content must be complemented with foods
with complete, quality protein content for a healthy life, particularly in children for
proper development.

A good source of protein is often a combination of various foods, because different

foods are rich in different amino acids. A good source of dietary protein meets two
requirements:

The requirement for the nutritionally indispensable amino acids (histidine,

isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and
valine) under all conditions and for conditionally indispensable amino acids (cystine,
tyrosine, taurine, glycine, arginine, glutamine, proline) under specific physiological and
pathological conditions. The requirement for nonspecific nitrogen for the synthesis
of the nutritionally dispensable amino acids (aspartic acid, asparagine, glutamic acid,
alanine, serine) and other physiologically important nitrogen-containing compounds
such as nucleic acids, creatine, and porphyrins.

Healthy people eating a balanced diet rarely need protein supplements. Except for a
few amino acids, most are readily available in human diet. The limiting amino acids are
lysine, threonine, tryptophan and sulfur-containing amino acids.

The table below presents the most important food groups as protein sources, from a
worldwide perspective. It also lists their respective performance as source of the
commonly limiting amino acids, in milligrams of limiting amino acid per gram of total
protein in the food source. The table reiterates the need for a balanced mix of foods to
ensure adequate amino acid source.

Food source lysine Threonine Tryptophan Sulfur

amino acids containing
Legumes 64 38 12 25
Cereals and 31 32 12 37
whole grains
Nuts and 45 36 17 46
seeds
Fruits 45 25 11 27
Animal 85 44 12 38
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 Casein in Milk

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

REQUIREMENTS
 Beakers (250 ml)
 Filter-paper
 Glass rod
 Weight box
 Filtration flask
 Buchner funnel
 Test tubes
 Porcelain dish
 Different samples of milk
 1 % acetic acid solution
 Ammonium sulphate solution

THEORY
Natural milk is an opaque white fluid Secreted by the mammary glands of Female
mammal. The main constituents of natural milk are Protein, Carbohydrate, Mineral
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 degree it become
sour because of bacteria present in air. These bacteria convert lactose of milk into
lactic acid which is sour in taste. In acidic condition casein of milk starts
separating out as a precipitate. When the acidity in milk is sufficient and temperature
is around 36 degree, it forms semi-solid mass, called curd.

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PROCEDURE –

1. A clean dry beaker has been taken, followed by putting 20 ml of cow’s 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

Sample No. Source Content of % of casein

protein
1. Cow milk 0.60 3.00
2. Goat milk 0.65 3.25
3. Buffalo milk 0.85 4.20
4. Amul milk 0.75 3.88

Conclusion/discussion
Cow's milk contains about 87% of water and 13% of solids. The observation that in
some mixtures which promote better growth, whole milk and casein protein which are
not statistically different.

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BIBLIOGRAPHY
1. ICBSE.COM
2. www.nutritionexpress.com>category>protein
3. https://en.wikipedia.org/wiki/Casein
4. https://www.google.co.in/?gws_rd=ssl#q=difference+between+protein+and+case
in+in+wikipedia
5. https://www.google.co.in/search?q=structure+of+casein&biw=1467&bih=716&tb
m=isch&tbo=u&source=univ&sa=X&ved=0ahUKEwjyi5iD4sjKAhVOC44KHUMmD
DUQsAQIHw#imgrc=kgFmUv

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