Download Lab.3 Isolation of casein from milk

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
physically bonded to a substance
phosphoric acid. Ionized phosphate
calcium ion increases hydrophobic
which leads to precipitation.
that are
containing
groups by
interaction,
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
STRUCTURE OF CASEIN
Is a phosphoprotein, which has phosphate groups
attached to some of the amino acid side chains.
Mostly these amino acid are serine and threonine.
 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 is made up of the main 3 types of proteins
are α-casein, β-casein, and κ-casein.
all casein proteins have different hydrophobic and
hydrophilic regions along the protein chain.
• 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
and cause a precipitation of the protein casein.
The word “micelle” is a chemical term. It is used to
•
•
•
•
describe 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:
1. 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
If calcium removed from milk the renin cannot
form ppt to milk
 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
The 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
1.
Place 20 ml (20 g) of milk into a 125 ml flask and heat at 40 oC
in a water bath.
2.
Add 5 drops of glacial acetic acid and stir for about
min.
3.
Filter the resulting mixture through filter paper held in a funnel
and gently squeeze out most of liquid.
4.
Remove the solid (casein and fat) from the cheesecloth, place it
into a 100 ml beaker and add 10 ml of 95% ethanol.
5.
Stir well to break up the product. Pour off the liquid and add
10 ml of 1:1 ether-ethanol mixture to the solid.
6.
Stir well and filter through filer paper
1
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 %