Act.

7
Quantitative Analysis of Protein
Estimation of Amount of Casein in Milk
Proteins are chains of amino acid
molecules connected by peptide bonds
 Amino acid chains are 20 different amino acids exist
called polypeptides

The amino acids within protein chains can bond

across the chain and fold to form 3-dimensional
structures.
This Photo by Unknown Author is licensed under CC BY-SA
Proteins can be relatively straight or form tightly
compacted globules or be somewhere in between.

 The term “denatured” is used when proteins unfold from

their native chain or globular shape.

 Denaturing proteins is beneficial in some instances.

 To allow easy access to the protein chain by enzymes
for digestion,
 To increase the ability of the whey proteins to bind water and
provide a desirable texture in yogurt production.
Milk Protein Chemistry
2 major categories of milk protein:
Casein family contains phosphorus and will
coagulate or precipitate at pH 4.6.
Serum (Whey) proteins do not contain
phosphorus, and these proteins remain in
solution in milk at pH 4.6.
Casein family of protein in milk
 consists of several types of
caseins (α-s1, α-s2 , ß, and 6)
and each has its own amino
acid composition, genetic
variations, and functional
properties. 
  The caseins are suspended in
milk in a complex called a
micelle.
The high phosphate
content of the casein
family allows it to
associate with calcium
and form calcium
phosphate salts.

This Photo by Unknown Author is licensed under CC BY-SA

Serum (Whey) Protein family
 consists of approximately 50% ß-lactoglobulin, 20% α-lactalbumin,
blood serum albumin, immunoglobulins, lactoferrin, transferrin,
and many minor proteins and enzymes.

 Like the other major milk components, each whey protein has its
own characteristic composition and variations.

 The functions of many whey proteins are not clearly defined, and
they may not have a specific function in milk but may be an
artifact of milk synthesis. 
 ß-lactoglobulin is thought to be a carrier of vitamin A.
Amount of Casein in milk

 Casein is a complete protein source. That means it

provides all the essential amino acids your body needs for
growth and repair.

Casein is a slow digesting protein

constitutes approximately 80% (29.5 g/L) of the total
protein in milk
Learning Objectives:

 Toprecipitate and isolate the casein curd from the

milk samples by acidification to reach casein’s
isoelectric point.
 Tocompare the mass of casein curd extracted from
the different samples.
 To find the milk with the highest casein content
Materials/Chemicals and Reagents

 20 mL milk sample (cow, carabao, goat) Water bath

 Acetic Acid Stirring rod
 Ethanol Graduated cylinder (100
mL)
 Watch Glass Filter Paper (Whatman
No.1)
 100 mL Beaker Buchner funnel with
suction (vacuum)
http://www.biosciencenotes.com/wp-content/uploads/2018/07/casein5-300x199.jpg
Procedure

Preparation of Milk Samples

1. Using the graduated cylinder, a 20 ml sample of
fresh milk was poured in a dry clean beaker.
2. Each sample was labeled properly.
Isolation of Casein
1.the beaker with milk sample was placed into the water
bath at 40⁰C for 5 minutes.
2.After 5 minutes, 10 drops of 5% acetic acid was slowly
added to the beaker with constant stirring.
3.The samples were kept undisturbed for 10 minutes.
4.Casein was seen precipitating and forming curd along with
fat.
5.The solution was filtered, and the precipitate was
transferred into another beaker.
PRINCIPLES:

Milk samples heated at 40 - 60 deg C.

The casein component of milk is relatively
heat-stable, capable of surviving pasteurization
at 62-71°C.
Conversely the whey protein component is
denatured at these temperatures.
Adding acetic acid dropwise to the milk
sample.
 Acidifyingthe milk – allows the casein to
precipitate.
 Toprecipitate the casein, acetic acid is added drop
by drop until the isoelectric point is reached.
 Theisoelectric point of casein is at pH 4.6 where
the protein shows the least solubility and gets
precipitated.
The isoelectric point of a protein (I.E.P.) is the pH at which
the molecule is electrically neutral (net charge is zero).

 When a protein is charged e.g. with a negative

charge, the negative molecules repel each other
and thus remain in the solution and do not
precipitate.
 But when these molecules are neutral, they tend
to precipitate due to the absence of the repelling
forces and, the high molecular weight of protein.
Also, at a low pH, casein protein denatures
(unfold).
The unfolded proteins are then free to
interact with each other and clump
together in a way they could not do when
they were properly folded.
The milk takes on a curdled appearance
from the lumps of proteins that are
binding one another.
Purification of Casein

1. To separate casein from fat in the curd, 10 ml of ethanol

was added with stirring.
2. The liquid of dissolved fat was poured out and the solid
casein was set aside.
3. The solid casein was washed with cold water and filtered
using Whatman No.1.
4. The excess water gently removed from the filter paper.
5. The precipitate was dried for five minutes.
Addition of ethanol to the precipitates

 The precipitate doesn’t dissolve in H2O, alcohol

or ether but dissolves in alkaline solutions (e.g.
KOH forming potassium caseinate).

 Caseinis insoluble in ethanol and this property

is used to remove fat from the preparation.
Weighing of Casein

1. The solid casein was transferred from filter paper to

the watch glass.
2. The weight of casein was measured using a top
loading balance.
RESULTS

Table 1. Casein in milk samples

Yield of Casein
Milk Sample
(Grams)/20ml
Cow 5.01

Carabao 3.14

Human 0.8
Kulkarni CP. 2017. Analysis of casein precipitation from the various milk
samples available in market. International Journal of Food Science and
Nutrition. 2(6): 215-216
The casein precipitated from the cow milk
contains more amount of casein protein than
the goat and buffalo milk samples.
Incow's milk, approximately 82% of milk protein is
casein and the remaining 18% is serum, or whey
protein (milkfacts.info).
The lower amount of casein in the buffalo milk could
be due to the more fat content in it (Kulkarni 2017).
Human milk contains approximately 40% casein,
and 60% whey.
Applications:
 For infant nutrition
 Knowing the protein ratio of human milk is considered
a guideline when manufacturing artificial milk, also
called formula
 Ifinfant formulas have greater percentage of casein, it
will be more difficult for the baby to digest.
 Approximately 60-80% of all protein in human milk is
whey protein.  These proteins have great infection-
protection properties.
Applications:

  Forfood technology applications and dairy food

production.
 The quantitative analysis of casein precipitated from the
various milk samples provide the ample scope to the
cottage cheese manufacture.
 Commercially available milk may have variations in casein
content which may indicate adulteration of milk by adding
water or other substances.
Applications:

• Non-food
applications of
casein
REFERENCES
 Milk Facts. http://milkfacts.info/Milk%20Composition/Protein.htm
 General Properties of Casein.
https://www.sigmaaldrich.com/life-science/metabolomics/enzyme-explorer/enzyme-r
eagents/casein.html
 Applied Biochemistry Lab.
https://walghazzawi.kau.edu.sa/Files/0007119/Files/119837_applied_biochemistry.pdf
 What’s in Breast Milk? American Pregnancy Association.
https://americanpregnancy.org/first-year-of-life/whats-in-breastmilk/
 Kulkarni CP. 2017. Analysis of casein precipitation from the various milk samples
available in market. International Journal of Food Science and Nutrition. 2(6): 215-216
 Lonnerdai B, Forsum E. 1985. Casein content of human milk. Am.J. Clin Nutr. 41(1):113-
120
 Jean-Luc Audic, Bernard Chaufer, Georges Daufin. Non-food applications of milk
components and dairy co-products: A review. Le Lait, INRA Editions, 2003, 83 (6),
pp.417-438. 10.1051/lait:2003027