Lesson 3

CHEMISTRY OF MILK PROTEINS

3.1 Introduction
About two-thirds of all cells consist of proteins and they have functional and
structural roles in human body such as catalyst (enzyme), regulator (hormone), protection
(immunoglobulin), carrier proteins (lactoferrin, haemoglobulin) and as structural proteins
(collagens). Proteins are body building constituent, essential for growth and repair of tissue.
Proteins are the complex organic compounds containing Carbon, Hydrogen, Oxygen and
Nitrogen and sometimes Sulphur, Phosphorous or other elements.
Proteins are defined as high molecular weight polymers of α-amino acids that are
formed by living organisms. On hydrolysis proteins yield a mixture of amino acids.1 g
protein gives 4.1 food calories of energy.

3.2 Structure of Proteins

Proteins are large molecules made up of smaller units called amino acids. The
primary structure of proteins consists of a polypeptide chain of amino acids residues joined
through peptide bonds. A protein molecule contains around 100-200 linked amino acids. The
type and order of amino acids in the protein molecule determines the nature of protein. Any
change of amino acids regarding type or position in the molecular chain results in a protein
with different properties.

3.2.1 Amino Acids

Amino acids are the building blocks of proteins. They are characterised by the
presence of one amino group (NH2) and one carboxyl group (COOH) attached to the α-
Carbon atom. Hence they are also known as α-amino acids. Amino acids are produced by
substitution of an amino group (NH2) for a hydrogen atom in a molecule of fatty acid.

CH2HCOOH → CH2NH2COOH

The amino acids can emit hydronium (H+) ions in alkaline solutions and absorbs
hydronium (H+) ions in acid solutions. Such compounds are known as amphoteric substances
or ampholytes.An amino group and a carboxyl group can react with each other to yield a
peptide linkage by releasing a water molecule. Therefore, a linear peptide chain can be
formed from a number of amino acids. If the chain length is short it is known as peptides and
if it is long it is known as polypeptides or proteins.

NH2 R1

R – CH – COOH +H HN – CH -- COOH

NH2 R1

R – CH – CO – NH –CH – COOH + H2O

Peptide linkage

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 Proteins are made up of 22 amino acids and 18 of these amino acids are present in
milk proteins. 8 of the amino acids (9 for infants) cannot synthesize by human beings and
these amino acids are known as essential amino acids and all of them are present in milk.

3.2.2 Classification of Proteins

1. Simple proteins: The proteins which yields only a mixture of amino acids or their
derivatives on hydrolysis. Eg: Whey proteins

2. Complex/ Conjugated proteins: These are simple proteins containing a non-protein radical.
Eg: Casein (phosphoric acid as a non-protein radical)

3. Derived proteins: These are the break down products of simple or conjugated proteins. Eg:
Proteoses, peptones.

3.3 Milk Proteins

Milk proteins are among the most valuable proteins to man. They contain all the
essential amino acids. Milk contains about 3.5% protein. The principal proteins found in milk
are Casein and Whey Proteins.

3.4 Classification of Milk Proteins

Milk proteins can be broadly divided into two major categories- casein and whey
proteins. Casein is dispersed in milk in the colloidal state while whey proteins are present in
solution phase. The nitrogen content of milk is average 0.5%. the average percentage of
protein nitrogen in milk is 90%, whereas non protein nitrogen is 10%. Of the 90%of protein
nitrogen, casein contributes 73.5% and the rest 16.5% is due to albumin, globulin and
proteose-peptone.
Casein family contains phosphorus and it coagulates or precipitates at pH 4.6. Whey
proteins donot contain phosphorus and they remain in solution at pH 4.6. In cow milk
approximately 82% of the milk protein is casein and 18% is whey protein.

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3.5 Casein
Casein is the principal milk proteins. Casein in milk form complexes known as
micelles, which are dispersed in the water phase of milk. Casein micelle consists of subunits
of different caseins (αS1, αS2, β and γ) held together by calcium-phosphate bridges on the
inside surrounded by a layer of κ casein, which helps to stabilize the micelle in solution.
Casein micelles are spherical in shape and they are 0.04- 0.3µ in diameter. They are
porous structures that allow the water phase to move freely in and out of the micelle. β-casein
along with some calcium phosphate will migrate in and out of the micelle with changes in
temperature. The casein family consists of several types of casein such as αS1, αS2, β, γ and
κ. Each type of casein has its own amino acid composition, genetic variation and functional
properties. Casein molecules are suspended in milk in a complex known as micelles. The
high phosphate content in the casein family allows it to associate with calcium and form
calcium-phosphate salts.

3.5.1Structure of Casein

Core coat model of Casein (sub micellarmodel)

Casein is dispersed in milk in the colloidal state, as tertiary calcium phosphate
caseinate complex or micelle. Micelle is made of sub micelles of casein molecules (spherical
aggregates of several casein molecules). These micelles contain about 10-100 casein
molecules. The colloidal casein is present in milk as tertiary calcium phosphate caseinate
complex or micelle. Each submicelle of calcium phosphate caseinate complex is bridged
together by tertiary calcium phosphate by an ester linkage with the serine hydroxyl groups.
On removal of colloidal calcium phosphates the micelles will disintegrate.k-casein
has hydrophilic has a C- terminal and a hydrophobic N-terminal. Sub micelles located
towards the interior of the structure are deficient in k-casein. k-casein rich sub micelles are
located towards the exterior of the micelle. Hydrophilic C-terminal ofk-casein protrudes from
surface giving a hairy appearance to the structure. This hair gives stability to the
micelle.Almost all regions of casein molecules are severely restricted in mobility.Unlike any
other proteins, milk proteins are very stable.

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3.5.2 Preparation of Casein
The pH of diluted skim milk is brought down to 4.6, the isoelectric pH of casein with
the addition of 10% HCl or 1N acetic acid or acetate buffer pH 4.6. The precipitated casein is
removed by centrifugation or filtration. It is followed by washing with water, acetone and
ultimately dried with solvent ether. Casein may also be prepared by replacement of 10% HCl
with 10% acetic acid.
Another method to obtain casein is by means of rennet coagulation. Skim milk is first
warmed to 350C and rennet is added at the rate of approximately 115g per 460 litres of milk
to coagulate casein in 15-20 minutes. The curd is cut, heated to 650C and the whey drained.
Further washing of curd takes place at 300C and the curd is dried at 40-500C. Rennet casein is
also known para casein.
Rennin changes casein into para casein which has same composition as that of casein
but differs in physical properties. Para casein is easily precipitated by calcium ions. The
concentration of calcium in normal milk is sufficient to para-casein. Calcium para caseinate
(curd) is used for the manufacture of many varieties of cheeses.

3.6 Whey Proteins

Whey protein is the name commonly applied to milk serum proteins. Casein is
removed from skim milk by precipitation method, the remaining solution contains milk
serum proteins.
Lactalbumin and Lactoglobulin are the principal whey proteins. They constitute about
80% of the total whey proteins. In the rest 20% are proteoses, peptones, blood serum
albumins, immunoglobulins, lactoferrin, etc. Lactalbumin is the albumin present in milk and
Lactoglobulins are the globulin present in milk.
Whey proteins do not contain phosphorus but contain a large amount of sulphur
containing amino acids. These amino acids form disulphide bonds within the protein and give
a compact spherical shape. These disulphide bonds can be broken leading to loss of compact
structure by a process known as denaturation. Higher heat treatment causes denaturation of
whey proteins. Denaturation causes change in the physical structure of proteins, but generally
does not affect the amino acid composition and nutritional properties.

3.6.1 β-Lactoglobulin
This is the major whey protein present in milk. When milk is heated to above 60oC
denaturation is initiated. Sulphur containing amino acid of β-Lactoglobulin play a major role
in the denaturation. Sulphur bridges form between β-Lactoglobulin molecules and κ-Casein
molecules and also between β-Lactoglobulin and α-Lactalbumin molecules.
At higher temperature, sulphurous compounds such as hydrogen sulphide are
gradually released. These sulphurous compounds are responsible for the cooked flavour of
heat treated milk.

3.6.2 α-Lactalbumin
It accounts for 3.7% of total protein. α-Lactalbumin plays a significant role in the
synthesis of lactose in udder. It contains no phosphorus but rich in sulphur.

3.6.3 Immunoglobulins
These are antibodies synthesized in response to stimulate by specific antigens. There
are five major classes of immunoglobulins namely IgG, IgA, IgM, IgE and IgD. Of these,
IgG is the predominant fraction in cow and buffalo milk. It accounts for 90% of the total
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 immunoglobulins.Immunoglobulins (I.G) are the proteins which provide immunity. Milk
contains around 1g I.G/litr. Colostrum contains 100 g I.G/ litre.

3.6.4 Lactoferrin
This glycoprotein displays a strong inhibitory effect towards Gram negative
enteropathogenic bacteria. ie., pathogens that effect gastroenteritis. Because of its ability to
bind free iron which is required for the growth of enteropathogenic bacteria. The average
lactoferrin content in cow milk is 0.15 to 0.32 mg/ml and that of buffalo milk is 0.32 to 0.50
mg/ml.

3.6.5 Proteose-peptone
Proteose and peptones are the polymers of amino acids which are of lower molecular
weight than proteins and are formed by the partial hydrolytic degradation of proteins. They
are usually not heat denaturable. The proteins of milk which remain soluble in acid after
heating are proteoses and peptones. The average level of proteose-peptone in cow milk is
240mg/100ml and buffalo milk is 330mg/100ml.

3.7 Physico-Chemical Properties of Milk Proteins

3.7.1 Physical Properties
1. Colour: Industrial casein appears to be yellowish white, but in pure state casein is snow-
white.
2. Smell & Taste: In pure state casein is smell-less and tasteless.
3. Texture: Industrial casein has granular texture. Casein in pure state is a non-crystalline
powder.
4. SpecificGravity: S G of casein varies from 1.26 – 1.31.
5. Solubility: Casein is only 0.1 % soluble in water and insoluble in common organic solvents.
6. Opticalactivity: Except glycine, all the amino acids are optically active. Alkaline solution
casein is laevo rotatory (rotates the plane of plane polarised light to left).

3.7.2 Chemical properties

1. Amphoteric property: Casein can behave both as an acid and a base.
Casein + HCl → Casein hydrochloride (at pH < 4.6)
Casein + NaOH → Sodium caseinate(at pH > 4.6)
2. Hydrolysis: On hydrolysis with acids, bases and enzymes, casein yields a mixture of α-amino
acid and phosphoric acid.
3. Coagulation: Casein is coagulated by acid, rennet, alcohol, heavy salts (such as mercuric
chloride) and heat (131-138 0C). Casein is coagulated at higher temperature than whey
proteins in fresh milk having low acidity. In case of high acidity of milk, casein can be
coagulated at lower temperature.
4. Oxidation: Amino acids up on oxidation yields imino acids and on hydrolysis formketo
acids.
5. Reduction: When disulphide group of casein is reduced by agents like H2S, Na2S, sulphidal
groups are formed.
S-S → S- H

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3.8 Non- Protein Nitrogenous Constituents
A large number of nitrogen containing compounds of low molecular weight are not
precipitated with proteins by 12% trichloroacetic acid. Some small peptides are included in
this group. These non protein nitrogen (NPN) constituents aggregate about 1g/litre and
account for about 6% of the total N (250-350mg of Nitrogen/litre).
Casein, α-Lactalbumin, β-Lactoglobulin and proteose-peptone precipoitate when 12%
trichloroacetic acid is added to milk and NPN is present in the filtrate. The compouns in the
filtrate are creatin, creatinine, urea, ammonia, uric acid, α-amino nitrogen (amino acids),
hippuric acid, orolic acid, etc.The compounds present in the urine of dairy animals have a
remarkable similarity between those present in non protein nitrogenfractions of milk. As a
matter of fact the compounds or substances present in the urine of dairy animals are the
resultant waste metabolites of dairy animal’s body. It is apparent that the bulk of these waste
metabolites in the urine of dairy animals originate from the blood and hence this entry or
appearance and levels in milk or urine are due to the protein metabolism of the animals.
Intake of feed by the animals is directly proportional to the presence of these compounds or
substances in milk.
NPN in milk varies from season to season and has no biological value as protein. It
cannot be utilized by the body as a substitute of protein nor can it increase the cheese yield.
Pasteurization by itself has no effect. However pasteurization with homogenization causes an
increase in the non protein and amino nitrogen content. Increase in NPN was observed when
concentrated milk is stored.

3.9 Effect of Heat on Milk Proteins

Heat stability of casein
 The stability of casein is influenced by the time and temperature combination of heating.
 Heat treatment causes a release in colloidal calcium to the milk serum.
 In heat treated milk, when stored, changes occur which are not due to bacteriological or
enzymatic activities but due to the behavior of casein micelles.
 Aggregation of heat treated milk occurs, when stored.
 Casein micelles are remarkably stable at a temperature up to 140oC. Heating at 140oC for
more than 20 minutes leads to destabilization of casein micelles and gel formation.
 Drastic heat treatment is necessary to coagulate casein.
 Heat treatment causes destabilization of casein particles that aggregate to form a three
dimensional structure entrapping some of the milk serum.
 Together with heat, the stability of the casein break down of casein micelles with
concomitant release of esterified phosphate and hydrolysis of peptide linkages.

Heat stability of whey proteins

 Heat treatment causes denaturation of whey proteins. Such coagulation is more sensitive
to electrolytes with decrease in solubility.
 Whey proteins are heat labile and heat denaturation occurs at 80oC. Denaturation is
accompanied by extensive breaking and randomization of the stabilizing disulphide
bonds. ß-Lactoglobulin is more heat labile than α-Lactalbumin.
 Heat treatment inactivates the enzymes.
 Heat denaturation of whey proteins results in the evolution of H2S, formation of cooked
flavor.

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  Normal pasteurization does not cause denaturation of whey proteins. It needs more
drastic heat treatment. When milk is heated to 118oC, heat denaturation of whey proteins
results in production of amino sugar condensation with formation of a brown pigment
called melanoidin (maillard reaction).

3.10 Enzymes
Enzymes are biological secretion of a living cell, they are protein in nature and serve
as a biological catalyst. Some enzymes like pepsin, trypsin and ribonuclease are simple
proteins since they consist entirely of amino acid units. Others, however, contain non-protein
portions and are, therefore, conjugated proteins. Apoenzyme is the polypeptide segment of
an enzyme. The non-protein organ moiety which can easily be dissociated from the
proteinaceous part of the enzyme is known as the co-enzyme. When the organic moiety
cannot easily be removed from the apoenzyme, it is termed as the prosthetic group. The
substances on which the enzymes act are known as substrates.

Enzymes are a group of protein that have the ability to catalyse chemical reaction and
the speed of such reactions. Enzymes are the complex organic compounds secreted by the
living cells which stimulate chemical reactions without getting themselves used up in the
reaction.

3.10.1 Properties of Enzymes

1. Enzymes are protein in nature

2. They are specific in their action. Eg: proteinases acts only upon proteins.
3. They are relatively unstable-inactivated by high temperature, pH and chemicals
4. They require very restricted range of pH for their activity
5. Enzyme reactions are reversible
6. They stimulate chemical reactions without getting themselves used up in the reaction
3.10.2 Classification of Enzymes
1. Hydrolytic group of enzymes: Under this group, lipase and esterases are involved in fat
hydrolysis, thereby causing rancidity in milk and milk products. Proteases proteolyze
protein thereby causing gelation in milk and milk products particularly UHT milk.
2. The second group consists of catalase and lysozyme. Catalase is synonymous with the
udder infection and the lysozyme is related to the immune properties of milk.
3. Xanthine oxidase, cytochrome C reductase and alkaline phosphatase are associated with
the microsomal particles of milk. Destruction of pathogenic bacteria in ilk is indicated
by the inactivation of alkaline phosphatse.
4. The role of ribonuclease, salonase, rhodanase and carbonic anhydrase is not yet known.

3.10.3 Enzymes in Milk

1. Proteinase: It is a casein hydrolysing enzyme. It is of great importance in cheese

manufacturing. Eg: Rennet.

2. Lipase: It is a fat splitting enzyme. It brings about hydrolysis of fat liberating fatty
acids, including the volatile one (Butyric acid) which gives rancid flavour and glycerol. The
cause of hydrolytic rancidity in butter fat, ghee and milk products containing fat is lipase. The

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systematic name of lipase is glycerol esterhydrolase. The lipase in milk can be divided into
two main categories viz. plasma lipase and membrane lipase.

3. Phosphatase: Raw milk contains phosphatase, but it is inactivated by time-

temperature combination necessary for efficient pasteurization. Its absence in milk indicates
effective pasteurization. There is acid as well as alkaline phosphatase. Inactivation of alkaline
phosphatase by pasteurization is an index of destruction of Coxiellaburnetti.The systematic
name of this enzyme is orthophosphoric monoesterphosphohydrolase.

4. Catalase: It is an oxidizing enzyme. It splits H2O2 into H2O and O2. Milk contains
small quantity of catalase. But high ell content of milk due to mastitis or udder infection
increase catalase value. Its systematic name is hydrogen peroxide oxidoreductase. An index
of udder disease is the increase in activity of catalase.

5. Amylase: It is starch splitting enzyme. It occurs in two forms. Α-amylase splits

starch into dextrin whereas β amylase into maltose. Amylase content of infected udder is
more than that of normal milk.

6. Reductase: It is the general term used for enzymes which have the power of
reducing or decolourising methylene blue and other oxidizing-reducing dyes. The amount of
this enzyme present in milk determines the quality of milk expressed in terms of Methylene
Blue Reduction Time (MBRT). When milk contains more microbial load, time for reducing
methylene blue to colourless will be lower. Thus good quality milk will have higher MBRT.

7. Peroxidase: It is an oxidizing enzyme. It acts on peroxides and liberates active

oxygen. It occurs in milk and especially in abnormal milk. It is a heat resistant enzyme and
does not destroy by the pasteurization temperature.

8. Lactase: It hydrolyses lactose to glucose and galactose. Glucose and galactose are
later converted into lactic acid by lactic bacteria.

9. Lysozyme: immunity of young and old is related to this enzyme. Also the keeping
quality of milk products may be related to it. The systematic name is mucopeptide-n-acetyl
neutrminyl hydrolase.