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INTRODUCTION
Proteins are large biomolecules, or macromolecules, consisting of one or
more long chains of amino acidresidues. Proteins perform a vast array of functions
within
living organisms,
including catalyzing
metabolic
reactions, DNA
replication,responding to stimuli, and transporting molecules from one location to
another. Proteins differ from one another primarily in their sequence of amino acids,
which is dictated by the nucleotide sequence of their genes, and which usually
results in protein folding into a specific three-dimensional structure that determines
its activity.
Milk is the most nutritionally complete food found in nature.Cow's milk and
goat's milk are almost identical in every respect.The only important nutrients
lacking in milk are iron and vitamin C. There are 3 main protein in milk:
casein,lactalbumin and lactoglobulin.
Eggs are one of the few foods that are used throughout the world regardless of
religion and ethnic group (Stadelman and Cotterill, 2001). The chicken egg is one of
the perfectly preserved biological items found in nature and is also considered as
the best source of protein, lipids, vitamins, and minerals.
Eggs consist of 3 main components: eggshell (912%), egg white (60%), and
yolk (3033%). Whole egg is composed of water (75%), proteins (12%), lipids (12%),
and carbohydrates and minerals (1%; Kovacs-Nolan et al., 2005). Proteins present in
egg are distributed among the egg white and yolk, whereas lipids are mainly
concentrated in the yolk. Yolk is covered with the vitelline membrane and mainly
consists of water (50%), protein (1517%), lipids (3135%), and carbohydrates (1%).
Protein present in egg yolk consists of lipovitellins (36%), livetins (38%), phosvitin
(8%), and low-density lipoproteins (17%). Also, yolk contains 1% carotinoides, which
makes it yellow in color (Stadelman and Cotterill, 2001). In this experiment we will
be isolating the casein from milk and egg albumin.
charges on the [casein] protein will be in balance, so that the net charge on the
protein
will
be
zero.
That pH value is known as the isoelectric point (IEP) of the protein and is
generally the pH at which the protein is least soluble. For casein, the IEP is
approximately 4.6 and it is the pH value at which acid casein is precipitated. In milk,
which has a pH of about 6.6, the casein micelles have a net negative charge and are
quite stable. During the addition of acid to milk, the negative charges on the outer
surface of the micelle are neutralized (the phosphate groups are protonated), and
the neutral protein precipitates.
The same principle applies when milk is fermented to curd. The lactic acid
bacillus produces lactic acid as the major metabolic end-product of carbohydrate
[lactose in milk] fermentation. The lactic acid production lowers the the pH of milk
to the IEP of casein. At this pH, casein precipitates. (McMaster University Chem Lab
Manual)
1.2 Objectives
The objective of this experiment is to isolate casein from milk and to isolate egg
albumin
2. METHODOLOGY
The elderly known technique for casein separation is the physicochernical one.
It even supports the casein definition (Gordon and Kaplan, 1972). Lowering milk pH
to 4.6 leads to casein precipitation. The precipitate is washed several times in order
to reach a satisfactory degree of purification. AlI types of acid can be employed as
precipitants but the most used ar hydrochloric and sulphuric acids. Because of po or
valorization of acid casein wheys, recent techniques of so called ionie acidification
(Triballat, 1979; Rialland and Barbier, 1980, were recently developed. They are
found on exchange of milk cations (Na t , K+, Ca++) with protons (H+) brought by
ion exchange resins. The resulting wheys have a lower mineraI content, especially
the ones coming from the Bridel process (Rialland et Barbier, 1980) which do not
contain any acid anions. Another advantage of this last process is an increase of
casein yield due to the retention in the curd of the main proteose-peptone
consequently of an hysteresis effect of solubility of this component (Pierre et Douin,
1984).
products
department.
2.1 Materials
and
services,
please
contact
our
Technical
Services
The materials used in this experiment were the following : test tube, beaker,
graduated cylinder, fitration paper, thermometer and stirring rod. While the
chemicals used were : 1% AgNO 3, dilute HNO3, 95% Alcohol, HCL, (NH)SO,
acetone, 1 N CH3COOH.
2.2 Procedure
Prepare a beaker to evaporize milk then dilute to water and heat for 40C, after
diluted, add 10% of hydrochloride solution dropwise and stir vigorously and
thoroughly. Wait for another 10 minutes for precipitation. Stir for 5 minutes and
pipette. A watch glass was then prepared for washing the curd in 5 mL of distilled
water, stir vigorously and decanting to remove the residue. Wash until the chloride
that was filtrate was free, if it was turn to yellow, the chloride is still present. Add 12 drops of HNO3 in the water to avoid losing of casein due to peptization. And let
the curd dry in filter paper and wash using 95% alcohol, stir vigorously for 3
minutes. Then filter and remove the alcohol left by pressing the precipitate between
filter paper extract and use acetone. Then weight the watch glass and calculate the
casein yield in g/ml.
The egg white was separated fom the yolk from one medium sized chicken egg. The
yolk was discarded and the volume of the egg white was measured using a
pre-weighed beaker and The weight was determined. 0.1 mL of 1N acetic acid was
added .It was stirred gently until no ovoglobulin precipitation was observed. A
cheese cloth was used to filter in 250ml beaker and buffered saturated ammonium
sulfate was added. After 30 minutes the mixture was transferred to the test tube
and it was centrifuge. The clear yellow centrifuge was discard and was transferred
to a beaker then buffered ammonium sulfate was added. It was stirred gently until
precipitation observed. The mixture then was refrigerated.
experiment
is
on
Casein
isolation
from
non-fat
milk,
hydrolysis
andneutralization. The first step in the experiment was the isolation of casein from
milk (highland pasteurized milk). The casein was precipitated by warming the milk
and adding10% acetic acid. It is important that the heating not be excessive or the
acid
too
strong,because
these
conditions
also
hydrolyze
lactose
into
its
the latter dissolves some of the protein. The 10% acetic acid was added
continuously until the pH reaches 4.6.Calcium caseinate has its isoelectric
(neutrality) point at pH 4.6. Therefore, it isinsoluble in solutions of pH less than 4.6.
The pH of milk is about 6.6; therefore casein has a negative charge at this pH and is
solubilized as a salt. If acid is added to milk, thenegative charges on the outer
surface of the micelle are neutralized (the phosphategroups are protonated) and the
neutral protein precipitates:
Ca-caseinate + 2H + ---> casein + Ca2+
Figure 1. Precipitation of Casein
As the pH falls the charge on casein falls and it precipitates. Hence milk curdles as
it sours, or the casein precipitates more completely at low pH.
Table 1. Results from the isolation of casein from milk
Mass of Residue +
Whey
paper (g)
highland
pasteurize
d milk
28.5 g
29.5 g
10.5 mL
Table 1 show the results from the experiment. However, casein % was not
calculated due to the reason that this experiment was not finished.
chicken egg
( mL )
( mL )
30 mL
19 mL
4. CONCLUSION
In this experiment, the isolation of casein from milk and the isolation of
egg albumin were unsuccessful. Due to the shortage of time, the study
resulted to a failure those prevent us to discuss the results.
5. REFERENCES
University,
Chem2o6
Lab
Manual,
1997,
http://www.chemistry.mcmaster.ca/chem2o6/labmanual/
Minard, B., Chem35 Synthetic Experiments, PennState University,
http://courses.chem.psu.edu/chem35/HTML/Experiments/Exp112.pdf
2002