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Biochem writeup 8 - Copy

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TAVONGA C. P. S. TSVAKWI
R109091R
MBChB 1
BC201
EXPERIMENT 8
Theory
Milk is a food of exceptional interest. Not only is milk an excellent food for the very young, but
humans have also adapted milk, specifically cow’s milk, as a food substance for persons of all
ages. Many specialized milk products like cheese, yogurt, butter, and ice cream are staples of
our diet. Milk is probably the most nutritionally-complete food that can be found in nature. This
property is important for milk, since it is the only food young mammals consume in the
nutritionally significant weeks following birth. The average composition of the milk of each of
several mammals is summarized in the accompanying table. (Minard, 2000)
Table 1: Average Percentage Composition of Milk from Various Mammals
Cow
Human
Goat
Sheep
Water
87.1
87.4
87.0
82.6
Protein
3.4
1.4
3.3
5.5
Fats
3.9
4.0
4.2
6.5
Carbohydrates 4.9
7.0
4.8
4.5
Minerals
0.7
0.2
0.7
0.9
Horse
90.6
2.0
1.1
5.9
0.4
It is interesting to note that milk from different animals has the same constituents although
they are present in different amounts as shown in the table above. Milk is composed, in part, of
a variety of proteins. The protein components of milk are revealed by the technique of MALDITOF mass spectrometry, which separates molecules on the basis of their mass to charge ratio.
(Berg etal, 2005)
Figure 1: Mass Spectrometer of the Composition of Milk
There are three kinds of proteins in milk: caseins, lactalbumins, and lactoglobulins and all are
globular. Casein exists in milk as the calcium salt, calcium caseinate. This salt has a complex
structure. It is composed of α, β, and κ caseins which form a micelle, or a solubilized unit.
Neither the α nor the β casein is soluble in milk, singly or in combination. (Minard, 2000) Casein
has many phosphoserine groups that bind Ca2+. Calcium, phosphate, and amino acids are all
valuable to suckling young, so casein efficiently provides three essential nutrients. (Lehninger,
2005) Calcium salts of casein are primarily responsible for the white appearance of milk as milk
is particularly rich in calcium. (Koolman, Roehm, 2005) Calcium caseinate has its isoelectric
(neutrality) point at pH 4.6. Therefore, it is insoluble 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.
(Minard, 2000)
In this experimental procedure, casein is to be precipitated with HCL. Precipitation is widely
used in downstream process of biological products such as proteins. This unit operation serves
to concentrate and fractionate the target product from various contaminants. Protein
precipitate formation occurs in a stepwise process. The addition of a precipitating agent, in this
case HCL, and steady mixing destabilizes the protein solution. Mixing causes the precipitant and
the target product to collide. The mechanism of precipitation is to alter the salvation potential
of the solvent and thus lower the solubility of the solute by addition of a reagent.
(www.scribd.com/Isolation-of-Casein-From-Milk-Aim-To-Isolate-Casein) After decanting and
rinsing off the acid several times with distilled water, acetone is added to dry the precipitate.
Finally, diethyl ether is introduced to the precipitate. The ether removes any small quantities of
fat that may have precipitated with the casein and evaporates, leaving the casein extract in the
form of a white powder. (www.basicchemistrylab.blogspot.com/isolation-of-casein-and-lactosefrom-milk)
Aims and Objectives
a) To isolate casein from milk
b) To test for the presence of peptide bonds in casein
Method
Refer to Biochemistry Practical Schedule, 2010, pages 8.1 – 8.2
Alterations
a) No water was added to the 100ml of milk
b) No methyl red indicator paper was used; 20ml of HCL was added
c) The precipitate was centrifuged instead being filtered with a Buchner funnel
Results
Biuret Test – Color change of solution changed from colorless to purple
Mass of casein – 0.021g
Discussion
When the Biuret Test was performed on part of the product, the end color was purple. This is a
positive test for protein and it was expected because casein is a protein. The amount of casein
extracted, 0.021g, is small. This is probably because there were some difficulties encountered in
this experiment. The major difficulty was obtaining the precipitate on standing and decanting
the supernatant fluid. Minute quantities of precipitate were harvested on addition of acid and
this amount was further reduced by the washing procedure and subsequent decanting. Another
difficulty was encountered during the final drying procedure as the casein was more of a
compact substance than a powder. This can be due to the fact that the procedure for drawing
air through the funnel in order to remove the moisture was not performed. The Buchner funnel
was not used; therefore the moisture was not completely removed to leave the precipitate in
the form of a powder.
Conclusion
The casein was successfully extracted from milk and it was proven from the Biuret Test that
casein comprises protein.
Answers to Questions
1. If acid is added to milk, the negative charges on the outer surface of the micelle
are neutralized. In other words the phosphate groups are protonated and the neutral
protein precipitates:
Ca2+ Caseinate + 2HCl B Casein + CaCl2
The calcium ions remain in solution. When milk sours, lactic acid is produced by bacterial
action and the consequent lowering of the pH causes the same clotting reaction. (Minard,
2000)
2. Calcium caseinate has its isoelectric (neutrality) point at pH 4.6. Therefore, it is insoluble in
solutions of pH less than 4.0.
References
1. Berg J.M, Tymoczko J.L, Stryer L, Biochemistry, 2005, 5th Edition, W.H. Freeman And
Company, page 137
2. Koolman J, Roehm K.H, Color Atlas of Biochemistry, 2005, 2nd Edition, Thieme, Germany ,
page 342
3. Lehninger A, Principles of Biochemistry, 2005, 4th Edition, page 1063
4. http://www.scribd.com/doc/21788568/1-Isolation-of-Casein-From-Milk-Aim-To-IsolateCasein, last updated January 2010
5. Minard R, Introduction to Organic Laboratory Techniques: A Microscale Approach, 2000,
Pennsylvania State University, Experiment 112
6. http://basicchemistrylab.blogspot.com/2010/02/isolation-of-casein-and-lactosefrom_02.html, last updated February 2010
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