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ACKNOWLEDGEMENTS

I would like to express my sincere gratitude to my class teacher Ms.Kavitha


C.M as well as our principal Ms.Anila M. Jayachandran who gave me the
golden opportunity to do an investigatory project in chemistry. It has helped
me in doing a lot of research and know about so many new things. I would
also like to thank my parents and friends who helped me a lot in finishing this
project within the limited time. I also take this opportunity to express my
profound gratitude to all the lab technicians of the Amala Cancer Research
Centre for allowing us to use some of their equipments and machines for this
project. Last, but not the least, I would like to thank God, who made all the things
possible.

COMPARATIVE STUDY OF WHEY PROTEINS IN


DOMESTIC ANIMALS
Introduction
Milk is a highly nutritious fluid formed in the udders of domestic animals
like cow and goat, designed to sustain the newborn during its first months of life.
The nutritional composition of milk is highly complex, and it contains almost
every single nutrient that the human body needs. Milk is a rich source of proteins.
Proteins in milk can be divided into two groups based on their solubility in water.
Insoluble milk protein is called casein, whereas soluble proteins are known as
whey proteins. Both of these groups of milk proteins are considered to be of
excellent quality, with a high proportion of essential amino acids and good
digestibility. Casein forms the majority (80%) of proteins in milk. One important
property of casein is its ability to increase the absorption of minerals, such as
calcium and phosphorus. Casein may also promote lower blood pressure levels.
Whey is the liquid part of milk that separates during its coagulation. Whey
protein is a mixture of proteins isolated from whey, accounting for 20% of the
protein content in milk. When cheese is produced, the fatty parts of the milk
coagulate and the whey is separated from it as a by-product. Consumption of whey
protein is excellent for the growth and maintenance of muscles. It is particularly
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notable that human breast milk is actually 60% whey, compared to 20% in cow or
goat milk. As a result, it is a popular supplement among athletes and bodybuilders.
Whey protein is found in a lot of dairy products as well as energy bars and
powdered protein drinks, since it helps increase the rate at which your muscles
grow and aid in weight management. Packaged foods in international markets with
whey protein have whey protein isolate, whey protein concentrate or
hydrolyzed whey protein in the ingredients list. Whey protein powder can also
be added as a supplement to several common foods like oatmeal and soup. Whey
protein contains the complete group of essential amino acids your body cannot
make. Studies show that Leucine present in whey is the most anabolic (growth
promoting) amino acid and it may also improve glucose tolerance, prevent fatty
liver and reduce tissue inflammation due to obesity. Another essential amino acid,
Cysteine can help boost levels of the cellular antioxidant Glutathione. Whey
proteins in general are believed to enhance the bodys antioxidant defense, reduce
inflammation and lower blood pressure.
At the end of the day, whey protein is an exceptionally healthy way to add
more protein to your diet. It is a quality protein source that is absorbed and utilized
efficiently by the human body. This is particularly important for athletes, body
builders or people who need to gain muscle mass and strength, while losing fat.

When it comes to muscle gain and fat loss, protein is the king of nutrients and
whey protein seems to be even better than other forms of quality protein.
Cows milk and goats milk protein are both comprised of around 80%
casein milk protein and 20% whey protein. Because goats eat the same thing year
round, the fatty acid content remains consistent. On the other hand, cows often
feed on different grains depending on the season creating variations in this area.
Although the nutritional value of cow's milk and goat's milk are similar, the
supplementary quantity of whey proteins could have a greater health benefit.
Cholesterol level is on the higher side in cows milk, and goats milk has more of
vitamins A, B6 and vitamin D and also more potassium, manganese, chloride, and
copper. The calcium contents are almost the same. Levels of vitamin B12 and zinc
are generally a bit lower in goats milk. The higher potassium content contributes
to maintaining low blood pressure, essential in avoiding strokes and heart attacks.

Composition of milk of domestic animals


Constituents
Water
Protein
Fat
-Saturated fatty acids
-Monounsaturated
fatty
acids
-Polyunsaturated

Cow
87.8 g
3.2 g
3.9 g
2.4 g

Goat
88.9 g
3.1 g
3.5 g
2.3 g

1.1 g

0.8 g

0.1 g

0.1 g

4.8 g
14 mg
120 mg
66 kcal

4.4 g
10 mg
100 mg
60 kcal

fatty

acids
Carbohydrate
Cholesterol
Calcium
Energy

Objectives of the study


To quantitatively compare the non coaguable proteins from whey of different
domestic animal.
Materials required
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Milk from different sources, test tubes, rennin tablet, biuret reagent, standard
protein solution, beakers, spectrophotometer, centrifuge machine, micropipettes
Spectrophotometre
Spectrophotometer is an instrument commonly used in chemistry and
biology laboratories. It works on the principle of spectrophotometry, and is used
for the measurement of intensity of coloured and colourless solutions. Basically it
measures the absorbance or optical density of solutions used in them and this
information is used for the mathematical calculation of the concentration of that
substance in the given solution. Spectrophotometer thus can be used for the
quantitative analysis of dissolved substances in solutions.

Figure 1. Mechanism of quantitative measurement in spectrophotometer


The diagram illustrates the theory behind measurement of unknown
quantities of substances in a given solution. Monochromatic light is the light of a
single wavelength. Monochromatic light is used for the measurement in

spectrophotometer. Monochromatic light is produced with the help of a prism


which splits light coming from a light source. The light source commonly used in
spectrophotometer is a tungsten lamp. The monochromatic light is now passed
through the solution. This is called the incident light. The light passes through
solution and the light coming out is called emergent light. The emergent light will
have lesser intensity than incident light, since a part of the photon of incident light
will have absorbed by the substances present in the solution. Here the absorption of
light is determined by the Beers and Lamberts law, which explains that higher the
concentration of substances in the solution, greater the amount of light absorbed
and greater the absorbance recorded by the machine.
The reduced intensity of emergent light will be sensed by photodetectors and
this intensity will be inversely proportional to the amount of substances present in
the solution used. The machine is designed to measure the intensity of emergent
light and the value will be used to calculate absorbance. The concentration of the
substance present in the solution is mathematically found out from this absorbance
using a standard solution of known concentration. Standard solutions are very
important in finding out concentration of unknown concentration solutions.
The measured absorbance of a known concentration standard solution can be
related to the absorbance of an unknown solution using the formula

Concentration of protein = Absorbance of test concentration of standard


Absorbance of standard
Experimental procedures
Experiment involves coagulation of milk samples using rennin tablets and
separation of whey from coagulate by filtration. Rennin is an enzyme which
coagulates milk protein casein without affecting other protein. The whey is then
used for the quantitative estimation of noncoaguable milk proteins by biuret
method
A. Coagulation of milk samples
i.
Take 20 ml of milk in a beaker. Grind one rennin tablet and add it to
ii.
iii.
iv.
v.
vi.

milk
Warm the milk. The milk will coagulate
Filter the coagulated (solidified) milk
The liquid filterate obtained is whey
The procedure is repeated for all experimental samples of milk
Quantitative estimation of whey protein is done using biuret method

B. Biuret method of estimation of protein


PRINCIPLE: Proteins are formed of subunits namely aminoacids with the
formation of peptide linkage between them. The peptide bonds present in protein
react with copper ions (Cu+) in the biuret reagent to give a purple colour that can
be visually detected and quantitatively measured using appropriate instruments. So
the formation of final colour depends on the number of peptide bonds in the given
sample which directly relates to the concentration of proteins in the given sample.
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Biuret reagent: Biuret reagent consists of copper sulphate, sodium potassium


tartarate and potassium iodide made in sodium hydroxide solution. Commercially
purchased biuret reagent kit for protein quantitative determination was used for the
analysis.
Procedure
Reagents required:
a. Commercially purchased Biuret reagent kit.
b. Standard protein solution (500 mg/100ml) and distilled water: Prepared by
dissolving accurately weighed 500 milligrams of protein (Bovine serum albumin)
in exactly 100 ml of distilled water.
Analysis procedure: Three test tubes were marked as blank (B), standard (S) and
test (T). Added 1 ml of distilled water to B, I ml of standard to S and 1 ml of whey
sample to T. Added 3 ml of biuret reagent to each tube and mixed the contents.
Tubes are kept for incubation at 37C for 15 minutes. The concentration of purple
colour developed is read using spectrophotometre.
Calculation:
Concentration of protein = Absorbance of test concentration of standard
Absorbance of standard

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RESULTS
Labeled tubes
Standard S1
Standard S2

Absorbance
0.874
0.885

Average absorbance
0.879

Cow 1
Cow 2

C1
C2

0.114
0.110
0.112

Goat 1
Goat 2

G1
G2

0.136
0.135
0.135

Concentration of protein = Absorbance of test concentration of standard


Absorbance of standard

Concentration of whey protein in Cow milk - 0.637 g/dl


Concentration of whey protein in Goat milk - 0.768 g/dl
Whey proteins of milk samples from domestic animals cow and goat are as follows
ANIMAL

CONCENTRATION OF WHEY

Cow

PROTEIN
0.637 g/dl

Goat

0.768 g/dl

DISCUSSION
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The concentration of non coaguable whey proteins from different animal


sources was determined. Goats milk was found to have higher concentration of
noncoaguable proteins in their milk compared to cows milk which was found to
have a lower concentration of noncoaguable proteins in it. Several types of proteins
found in cow's and goat's milk act as enzymes, transport nutrients, increase growth
and help your body resist diseases. Whey proteins produced by the mammary
gland of cows and goats consist mainly of beta-lactoglobulin and alphalactalbumin, although other whey proteins also are present.
The presence of whey proteins in milk varies with the type of animal, the
lactation period and other factors. In a 2004 article in "The EFSA Journal," the
European Food Safety Authority evaluated goat's milk as a source of protein for
infant formulas. The group noted several differences specifically between the whey
proteins in goat's milk versus cow's milk and concluded that three of the main
whey proteins, alpha-lactalbumin, beta-lactoglobulin and serum albumin, were
more highly concentrated in goat's milk. The alpha-lactalbumin in cow's milk was
measured at 1.2 grams per liter, while in goat milk it was almost double at 2.2
grams per liter. Beta-lactoglobulin in goat's milk was just as significant at 4.9
grams per liter versus cow's milk at 3.0. The largest difference was in the serum
albumin, which was significantly higher in goat's milk, approximately three times
as high as in cow's milk at 1.2 versus 0.4 grams per liter.
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The higher concentration of whey proteins in goat's milk would seem to


indicate that it can provide more nutrition than cow's milk. However, if you have a
milk protein allergy, this may not be a good choice, as your intestines will have
more leftover proteins to deal with. In some cases, those who have been unable to
ingest cow's milk in the past have safely been able to drink goat's milk, although
scientists are still not sure about the reason behind.
It's considered a good choice for health-conscious adults too. A University
of Granada study showed drinking goat's milk regularly may help to reduce
cholesterol levels, and support those with iron deficiency anaemia, since it helps to
regenerate haemoglobin.

BIBLIOGRAPHY
1. Comprehensive practical chemistry for class XII, CBSE.

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2. Pehrsson PR, Haytowitz DB, Holden JM, Perry CR and Beckler DG


(2000). "USDA's National Food and Nutrient Analysis Program: Food
Sampling" . Journal of Food Composition and Analysis 13 (4): 379389.
3. Hemme T and Otte J (2010). Status and Prospects for Small holder Milk
Production: A Global Perspective . Food and Agriculture Organization of the
United Nations.
4. Studdert VP, Gay CC (2007). Saunders Comprehensive Veterinary Dictionary.
St. Louis, Missouri, USA: Saunders Elsevier. ISBN 0-7020-2789-8.
5. Basnet S, Schneider M, Gazit A, Mander G, Doctor A (2010). "Fresh Goat's
Milk for Infants: Myths and RealitiesA Review". Pediatrics 125 (4): 20091906.
6. Goff, Douglas (2014). "Introduction to Dairy Science and Technology: Milk
History, Consumption, Production, and Composition: World-wide Milk
Consumption and Production". Dairy Science and Technology 12(3): 12-16

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