UNIVERSITY OF DAR ES SALAAM

COLLEGE OF NATURAL AND APPLIED SCIENCE

DEPARTMENT OF MOLECULAR BIOLOGY AND BIOTECHNOLOGY

NAME: SIMLEMBE AMAN G.

REGISTRATION NUMBER: 2019-04-12080

DEGREE: BSC. IN FOOD SCIENCE AND TECHNOLOGY

COURSE NAME: PRACTICALS IN BIOCHEMISTRY (BN 240)

COURSE INSTRUCTOR: 1. Dr.MOSHA

2.Mr. KISOKA

COURSE COORDINATOR; Dr, ROSE MASALU

TITLE: ISOLATION AND CHARACTERIZATION OF MILK CASEIN.

DATE OF EXPERIMENT; 12ST, MAY, 2021.

DATE OF SUBMITION; 18TH, MAY, 2021

ABSTRACT

The experiment was conducted so as to solate casein fraction from raw cow milk. Acid selective
precipitation method was used in which Glacial acetic acid was added so as to curdle the casein
then filtered to remove whey followed by addition of ethanol and diethyl ether into the curds to
remove the fat precipitated along with the casein. After isolating casein , the Biuret test and
Xanthoproteic acid test was performed as a confirmatory for the presence of casein. The results
obtained where the casein percentage yield was 18.8 percentage and recovery was 15.3
percentage. On Biuret test casein gave positive test of purple colour while glycine gave
puplishblue colour and on xanthoproteic acid test casein gave Yellowish-orange while glycine
retain its original colour. Therefore, casein from milk was effectively extracted as the results
shows postive tests.
INTRODUCTION

Casein is isolated from milk commercially and is industrially important because after dissolving
in alkaline solutions and drying ,it becomes a sticky substance that can be used inglues ,the
coating of paper,and the binding of colours in paints and wallpaper .it is also usedas a coating
for fine leather,and is cured with rennet to produce a plastic material used for buttons .when
isolated under sanitary conditions and dissolved in alkaline solutions,casein isalso employed n
the manufacture of pharmaceutical and nutritional products.

Casein has awide variety of uses, from being a major component of cheese, to use as a food
additive, to a binder for safety matches. As afood source,casein supplies amino
acids;carbohydrates;and two inorganic elements,calciumandphosphorusWhole milk is very
nutritious as it contains all the necessary nutrients one is required to have in what we call a
balanced meal. It contains vitamins such as thiamine, riboflavin, panthothenicacid and vitamins
A, B 12 and D, minerals like; calcium, sodium, phosphorus, potassium, andtrace minerals,
proteins which include all the essential amino acids, carbohydrates (mostlylactose), and lipids
(fats).

The protein present in milk is of three kinds namely: caseins, lactalbumins, and
lactoglobulins,all of which are globular proteins. Despite the existence of all this proteins, the
main protein inmilk is casein.Casein is a phosphoprotein which has phosphate groups attached
to the hydroxyl groups of someof the amino acids side-chains. Casein exists in milk as a calcium
salt, calcium caseinate. It isactually a mixture of three similar proteins, alpha, beta and kappa
caseins which form a micelle.Alpha- and beta-casein are both insoluble in water and are
solubilized by the micelle surroundingthem. The kappa-casein which has a hydrophilic portion is
responsible for solubilizing the other two caseins by promoting the formation of and stabilizing
the micelles because the kappa-caseinmolecules all have a negativeelectrical chargethus they
repel each other, keeping the micellesseparated under normal conditions and in a
stablecolloidalsuspensionin the water-basedsurrounding fluid.

Collectively the casein micelles form a large fluid portion of the ,milk asthey make up around
76– 86%of the protein in milk, by weight.Calcium caseinate has an isoelectric point of pH 4.6.
This means it is insoluble in solutions witha pH less than 4.6. The pH of milk is 6.6, therefore,
casein has a negative charge at this pH and issolubilized as a salt. If an acid is added to milk, the
negative charges on the outer surface of thecasein micelles are neutralized, by protonation of
the phosphate groups. The casein micelles aredestabilized or aggregate because the electric
charge is decreased to that of the isoelectric point(pH at which there is no net charge because
there are equal number of positive and negativecharges present).

The casein micelles disintergrate and the casein (the neutral protein) precipitates because it is
no longer polar, with the calcium ions remaining in solution.

Ca+2Caseinate + 2HCl -> Casein + CaCl2

On drinking milk, the stomach acid will drop the pH of the milk to the isoelectric point of
casein.The casein will then precipitate out of the milk making the protein available for
digestion. Thewhey protein is readily digested allowing for a speedy increase of amino acids
level and proteinsynthesis, in approximately forty minutes to an hour. The digestion of casein in
the stomach is a very slow process, taking approximately 7 hours for complete digestion,
providing a slow steadyrelease of amino acids to your muscles.The Biuret Test is a general test
for protein. When the pale blue Cu2+ion forms acomplex with adjacent amide nitrogens of the
peptide backbone, a very deep violet bluecolor forms.The Xantoperoteic Acid Test on the other
is a general test for the presence of thearomatic amino acids, tryptophan, phenylalanine and
tyrosine, in proteins. Aromaticgroups that have an amino group (tryptophan) or a hydroxyl
group (tyrosine) are easilynitrate by concentrated nitric acid to form yellow (xantho, Greek for
yellow) .

METHODOLOGY

Materials and equipment

Beaker

Conical flask
Measuring cylinder

Analytical balance

Water bath

Chemical reagents used

Ethanol

Diethyl ether

Acetate buffer

Hydrochloric acid

sodium hydroxide

Glycine

Milk

Distilled water

copper sulfate

Procedure of isolation of casein.

Empty flask was weighed and weght recorded, followed by addition of 150ml of milk into the
empty flask where the whole content in the flask were weighed and the weight of milk was
recorded.

About 100 ml of acetic acid was added into the milk in flask so as to lower the pH for curd
formation then left to stand for ten minutes at room temperature where filtration and
squeezing was performed to separate the whey and curds.

About 25ml of 95 percent ethanol was added into the curds obtained and the mixture was
left to stand for five minutes to remove the fat from the curd by decantation. Then after five
minutes, 25ml of 1:1 (v/v) mixture of diethyl ether and ethanol was added to dissolve additional
fat followed by vacuum filter to remove water and other volatile components.

The solids were measured and percent yield was calculated and compared with the theoretical
yield of 3.5%.
Procedure s of chemical analysis of proteins
Biuret Test:
About 4 mL of distilled water was added into a pea-sized amount of casein in a small test
tubeso as to dissolve the mixture.
Thoroughly mixed 4mL of the protein solution was mixed with 4mL of 3M sodium hydroxide
solution.
To the mixture 1 drop of 0.1% copper sulfate solution was added while noting and recording
the colour change with continuous, addition of the copper sulfate solution one drop at time,
noting and recording observations and stoped after adding 10 drops of the copper sulfate
solution and the procedure was repeated with 2% glycine.

Xanthoproteic acid test

A peas sized amount of prepare casein was added to test tube and dissolved in 4ml of i) distilled
water and ii)0.01M HCl then 1ml of concentrated nitric acid was added carefully and mixed
then, the appearance of heavy white precipitate was noted.

The mixture was warmed inhot water bath and the changes to yellow coloured solution was
noted

The mixture was cooled in stream of cold tap water bath and few drops of 3M of sodium
hydroxide was carefully.

The procedure was repeated by using 2% and the observation was recorded.
RESULTS

The protein suspension gave positive results for biuret test as purple-blue colour observed in both
hydrolysis and acid condition but for glycine sample light blue colour was observed as analysed
in the table bellow.

Table 1; RESULTS OF QUALITATIVE TEST OF PROTEIN (BIURET’S TEST)

Protein Reagents used Colour on biuret test inference

 Casein Water + Sodium Hydroxide Purple colour Casein present

Casein HCL + Sodium Hydroxide Light purple colour Casein present

Glycine Water + Sodium Hydroxide Puprish-blue colour Glycine present

Glycine HCL + Sodium Hydroxide Puprish-blue colour Glycine present

Figure 01: showing the positive results for caseij and glycine Buiret test.

XANTHOPROTEIC ACID TEST RESULTS

Casein sample gave positive results were a yellowish-orange colour was observed as displayed in
the result table below.

Protein Reagents used Colour on biuret test

Casein Water + Sodium Hydroxide + HNO Yellowish-orange colour

Casein HCL + Sodium Hydroxide + HNO Yellowish-orange colour

Glycine Water + Sodium Hydroxide + HNO Retain its original colour

 Glycine HCL + Sodium Hydroxide + HNO Retain its original colour

Figure 02; showing positive results for xanthoproteic test for casein and glycine

CALCULATION INVOLVED IN THE EXPERIMENT

Calculation of Casein yield

Mass of Milk = 153.7g

Mass of casein = 28.9g

Yield percentage (%)

% Yield =

% Yield = 18.8%

Therefore the casein yield was 18.8%

Percentage recovery =?

Percentage recovery = theoretical yield – actual yield

%Recovery = 3.5% -18.8% = -15.3%

DISCUSSION
In the results obtained milk gave a casein yield of 18.8% and recovery of 15.3% while the
qualitative test of casein by Biuret test gave positive results of purple colour and glycine gave
puprish-blue and on xanthoproteic acid test , milk casein gave yellowish-orange colour and
glycine retained its orignal colour which means the yield of casein 18.8% was greater compared
to the xpected value of 3.5% that may be due to fats and water in casein curds were not
completely removed by decantation and vacuum filter and pressing that have led to increased
weight of casein hence high yield.

Futhermore, he colour on Biuret test and xanthoproteic acid test shows the presence of casein
and glycine The inconsistency of results from this experiment is due to short drying time and
method used might have inaccurately dried the sample and fat incomplete separation from the
dissolved curds (Post, 2011). Principally, most proteins show a minimum solubility at their
isoelectric pH and this principle is used to isolate casein by adjusting the pH of the milk to 4.6,
its isoelectric point. Casein is insoluble in these solvents and this property can be
advantageously used to remove the unwanted fat from the preparation.

Answers to the given questions.

Casein has an isoelectric point of 4.6, what is the charge of casein in it is native environment
milk?

In native environment milk , casein has negative charge.

How to separate fat from the protein in the casein precipitate?

Fat is separated from the casein precipitate by addition of 90% of ethanol into casein
precipitate where by additional fat can also be removed by addition of diethyl ether into the
ethanol residue.

To make the casein precipitate in solution part B, between distilled water and 0.01M HCl which
one give the best result? The one that give the best result is 0.01M HCl because at normal pH
of milk, the net negative charge of the micelle (casein) will help in solubilizing it. At isoelectric
point, the net charge is zero, so at pH of 4.6 by adding acid will bring it closer to the isoelectric
point, thereby precipitating the protein.
CONCLUSION

Casein has an appropriate amino acid composition that is important for growth and
development of thenursing young. This high quality protein in cow milk is one of the key
reasons why milk is such animportant human food .Many people with an allergy to milk are
actually allergic to one of twocomponents of the milk: the casein or the lactose. Removal of
these products particularly thecasein allows individuals with allergies to tolerate milk. The
knowledge gained from this practical is absolutely helpful people who can`t eat immoderately
milk to be able to consume itafter the isolation of casein. Casein-free dairy products are also
used for children with autism.caseins are highly digestible in the intestine and are high quality
amino acids .Most whey proteinsare relatively less digestible in the intestine, although all of
them are digested to some degree. When substantialwhey protein is not digested fully in the
intestine, some of the intact protein may stimulate alocalized intestinal or a systemic immune
response. This is sometimes referred to as milk protein allergy andis most often thought to be
caused by ß-lactoglobulin. Milk protein allergy is only one type of food proteinallergy.

RECOMMENDATION

Casein protein is a milk protein extract recognized for its excellent amino acid
profile,slowdigestion, and interesting peptides (casomorphins, casokinins, casoxins, etc), to
improve isolation method in small scale practices, it’s better to use skim milk instead of whole
milk in which fat would not interfere with casein purity and drying time should be enough to
obtain pure casein.
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