Download Isolation of casein from milk

• simple protein
• hydrolyze to yield only amino acids e.g.: albumins , globulins.
• conjugated protein
• is a protein that functions in interaction with other chemical
groups attached by covalent bonds or by weak interactions.
• Some examples of conjugated proteins are lipoproteins,
glycoproteins, phosphoproteins, hemoproteins
 Phosphoproteins are proteins that are physically bonded
to a substance containing phosphoric acid. Ionized
phosphate groups by calcium ion increases hydrophobic
interaction, which leads to precipitation.
MILK COMPOSITION
 Whole milk contains vitamins (vitamins A, D, and K),
minerals (calcium, potassium, sodium, phosphorus,),
proteins (which include all the essential amino acids),
carbohydrates (chiefly lactose), and lipids.
 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.
 There are three kinds of proteins in milk:
1. caseins,
2. lactalbumins
3. lactoglobulins. All are globular
• α-Caseins are the major casein proteins. Its containing 8-10
phosphate groups,
• β- casein contains about 5 phosphate residues,
• β- casein it is more hydrophobic than α-caseins and κ-casein
• Because α-caseins and β-caseins are highly phosphorylated,
they are very sensitive to the concentration of calcium salts,
that is, they will precipitate with excess Ca2+ ions
• Unlike other caseins, κ-caseins are glycoproteins, and they
have only 2 phosphate group.
• Hence, they are stable in the presence of calcium ions, and
they play an important role in protecting other caseins from
precipitation and make casein more soluble forming casein
micelles.
 Neither the α nor the β casein is soluble in milk, singly or in
combination.
 If κ 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
 Carbohydrate that found k casein :
 The main carbohydrate present in milk is the sugar lactose.
 Lactose is a disaccharide containing the monosaccharaides
galactose and glucose.
 when bacteria get into milk, they digest the lactose and form
the acid lactic acid. This causes lowering the pH
a precipitation of the protein casein.
and cause
 The word “micelle” is a chemical term. It is used to describe
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the structure that certain very large molecules
Very large molecules are considered to be too large to be truly
soluble in water.
Instead, these large molecules will form structures that allow
them to remain suspended in water
The dispersion of these large structures in water is known as a
colloidal suspension.
The structures that allow large molecules to remain colloidally
suspended in water are termed micelles.
• In the case of casein, the parts of the casein molecules that
have an affinity for water form the outside of the casein
micelle.
• Conversely, the parts of the casein molecule that are hate the
water form the inner core of the micelle spheres
• Casein micelles consist of water, protein, and salts.
• Casein is present as a caseinate, which means that it binds
primarily calcium and magnesium.
 The pI of most proteins ranges between the
pH 4
to 6.
 So pI for casein is 4.6 and the pH of milk is 6.6 at this pH
the casein is have negative charge and soluble by bacteria
action or by adding acid that lowering the PH cause ppt of
casein
 Casein can be precipitated by:
 1. Calcium ions
 2. HCl
 3. Renin
 4. Bacteria
Casein can be precipitated by rennin
 Renin enzyme :
 Found in calves and gout
 Is hydrolysis enzyme (peptidase) so cleave the peptide bond
between amino acid
 Mechanism:
Enzyme attack the k- casein and breaking the peptide bond
and release small part of peptide bond
2. This destroy cause left k-casein the α and
B casein
which protect the casein from ppt and ppt it as Para kappa
casein
3. Unlike kappa the Para kappa casein cannot prevent the
casein from ppt in the present of calcium ion
1.
 If calcium removed from milk the renin cannot form ppt to
milk
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When the pH level decreases the milk forms Curds or
whey.
Curds are a dairy product obtained by ppt of milk with renin
or an acidic substance and then draining off the liquid
portion.
 Whey or milk serum is the liquid remaining after milk has
been coagulation and remove curds
 he curds are the solid white at the bottom, they whey is on
the top.
 Taking the curds out of the kettle to put them in
cheesecloth to drain
Procedure
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6.
Place 20 ml (20 g) of milk into a 125 ml flask and heat at
40 oC in a water bath.
Add 5 drops of glacial acetic acid and stir for about
1 min.
Filter the resulting mixture through filter paper held in a
funnel and gently squeeze out most of liquid.
Remove the solid (casein and fat) from the cheesecloth,
place it into a 100 ml beaker and add 10 ml of 95%
ethanol.
Stir well to break up the product. Pour off the liquid and
add 10 ml of 1:1 ether-ethanol mixture to the solid.
Stir well and filter through filer paper
7. Let the solid drain well, then scrape it into a
weighed filter paper and let it dry in the air.
8. Calculate the casein percentage in milk as follows:
% Casein = (grams of casein \ grams of milk) x
100
Normal Range 3-5 %