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MEASUREMENT OF EGG WHITE PROTEIN

CONCENTRATION USING BIURET ASSAY


1.0

2.0

AIM
To determine protein levels in stored egg white and in fresh egg white by
spectrophotometry.
To compare the differences in protein levels of stored egg white and fresh egg
white.

INTRODUCTION

This study is an analysis of Egg white and its varying protein content based on age.
It is a common generalisation that the freshness of a food product directly affects its
nutritional properties and hence its value. Worldwide, there are 1.8 trillion chicken eggs
laid each year (McGuiness, 2014) and the average Briton eats about 2 to 3 eggs a week
(Taylor, 2011).
This experiment was designed to evaluate the average protein concentration of stored
egg white and fresh egg white and compare their differences (IF any). Prior to this
experiment I presumed that the concentration of protein in fresh egg white was much
higher than stored egg-white protein.
Albumen is an aqueous solution made of 90% water and 10% proteins. The pH of
albumen of freshly laid eggs is 7.6-7.9 and rises to 9.7 during storage due to diffusion of
solubilized CO2 through the shell (Belitz et al., 2009). Also the thinning of egg albumen
with age has been difficult to interpret in chemical terms and remains one of the main
problems in egg chemistry (Burley & Vadehra, 1989).
Using beer-lamberts law (which explains that the intensity of the colour of a food
material and hence the absorption at 540nm, is directly proportional to the protein
concentration), protein quantity of the stored and fresh egg white can be deduced from
a standard curve of a standard protein obtained via spectrophotometry.

3.0

MATERIALS AND METHODS

3.1
MATERIALS
This experiment was carried out using Bovine serum albumen as the standard protein,
biuret reagent as the indicator and Sainsburys free range grade A eggs were used for
our Stored and Fresh egg sample, all water used for preparing solutions was distilled.
Gilson Pippetes were used, spectrophotometer used was an Ultrospec 2100 pro, while
the vortex was an 80 watts Stuart scientific vortex.
3.2
PREPARATION OF THE STANDARD CURVE
A 10mg/ml stock solution of BSA was prepared by weighing 100mg of BSA and
dissolving it in 10ml of water. This was used to prepare the 9 samples of varying BSA
concentration ranging from 0-4.0mg/ml at 0.5mg/ml intervals, producing 8 sample tubes
and 1 reference tube.
Predetermined final volume for all tubes was 5ml i.e.
( ?BSA + ?H2O+ 3ml biuret reagent)=5ml solution.
Hence the final protein concentration was a factor of the ratio between BSA stock
solution and distilled water. BSA and water volumes were determined for all samples
using the formula.

CinitialVinitial= CfinalVfinal

Where Vfinal was the volume of BSA for its respective sample,
After appropriate volumes had been determined via calculation, the tubes were
numbered from T1-T9 based on their predicted final concentration. Appropriate amounts
of BSA and water were transferred into the numbered tubes and 3ml of biuret assay was
added to all the tubes, the sample was then mixed on the vortex mixer for 1 minute and
allowed to stand for 10 minutes. 1ml of samples from the 9 tubes was transferred into
1ml cuvettes and appropriately labelled.
The spectrophotometer was set to a wavelength of 550nm and tube 1 was set as
reference and tube 2 was read at the same wavelength (three readings were obtained
per tube). The same procedure was carried out for tubes 3-9. The absorbance was
plotted against final protein concentration for tubes 1-9(Table 1).
Table 1.Content of each tube for the determination of standard curve

3.3
PREPARATION OF EGG SAMPLES BY SERIAL DILUTION.
Tubes were labelled as S1-S5 and F1-F5, Stored eggs were cracked and their albumen
separated. 2ml of stored egg albumen was carefully transferred to a tube S1 and 2ml of
water was added to S1and thoroughly mixed using the vortex, this formed the first 1:2
dilution. After this a fivefold serial dilution was carried out by transferring 2ml of S1 to
tube S2 and 2ml of water was pipetted into S2 and thoroughly mixed using the vortex,
then 2ml of S2 was transferred to tube S3 and 2ml of water transferred to S3, this was
carried out until S5. The same procedure was used for fresh eggs for tubes F1-F5. At the
end of the serial dilution all tubes were made to be 2ml in volume.
3ml of biuret reagent was added in all tubes using a pippete. Before spectrophotometry
T1 from the standard curve was set as the reference at 550nm wavelength. 1ml from
tubes F1-F5 and S1-S5 was transferred into a cuvette and labelled appropriately. The
readings of the labelled cuvettes was then carried out at 550nm and three readings per
tube were obtained, the results and mean absorbance were then determined (table 3).
All readings were taken within 5 minutes.
Mean absorbance of dilutions which fell within the range of the standard curve was used
to calculate the protein concentration in mg/ml using the equation=ax+b (table 4). Mean
protein concentration of within calibration curve range dilutions were obtained from
two replicated tests on stored and fresh eggs from the same batch (means 2 & 3 in table
4). These values in addition to the computed protein concentration (means 1, 2&3) were
then used to compute the original protein concentration and% protein concentration in
the egg albumen (table 5). These three means of protein concentration values were used
in the statistical analysis.

3.4
STATISTICAL ANALYSIS
Using the Mean protein concentration of within calibration curve range dilutions.
Statistical tests were carried out to ascertain if there was a difference in the protein
concentration of Fresh and Stored eggs in either their means or variances. This was done
using F-test and a students t-test on Fresh and Stored eggs.

4.0

RESULTS AND DISCUSSIONS

4.1

STANDARD CURVE

Table 2.Absorbance values and protein concentration of standard protein.

A straight line graph was obtained from the standard curve data (fig 1).
1
0.9
0.8
0.7
0.6
Absorbance (550nm)

f(x) = 0.19x + 0.01


R = 1

0.5
0.4

Linear ()

0.3

Stored egg

0.2

Fresh eggs

0.1
0
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5
Protein concentration (mg/ml)

4.2

SERIAL DILUTION OF EGG SAMPLES

TABLE 3. ABSORBANCE READING FOR PROTEIN DETERMINATION IN STORED (S1-S5)


AND FRESH (F1-F5) EGGS (550NM)

Absorbance values for S5 and F5 fell within the range of the standard curve and were
chosen to represent the data.
Protein concentration (mg/ml) = (absorbance-0.0051)/0.1947
Protein concentration in S5 and F5 was calculated to be 2.35mg/ml and 1.98 mg/ml
respectively (Table 4).
TABLE 4.PROTEIN CONCENTRATIONS

Protein concentration in stored and fresh eggs was calculated to be 1.93mg/ml and 1.99
mg/ml respectively. Statistical analysis using F-test showed that the variances were

equal and the T- test proved that the means of Fresh and Stored eggs were not
statistically different.
4.3
CALCULATION OF %PROTEIN IN EGG-WHITE
Protein concentration in stored egg and fresh egg were converted to their total protein in
the egg-white by multiplying them by dilution factor (32). % protein in stored and fresh
egg was calculated by using this formula:

(% protein concentration in egg / egg density) x 100.

Where egg density =1.031 g/cm3 = 1.031g/ml= 1031mg/ml


Table 5. Undiluted protein concentrations1

eans in the same column followed by the same letter show no significant difference at p>0.05

5.0

DISCUSSION

The F-test showed that the variances were equal (P> 0.05) and the T- test proved that
the means of Fresh and Stored eggs were not statistically different (P> 0.05). This may
be due to the fact that during storage, n-ovalbumin which is the most abundant protein
in egg albumin is observed to change to s-ovalbumin which is a more stable form (Ana,
2006) and can withstand storage conditions leading to insignificant difference in protein
level of egg albumin during storage. The same author also reported significant increase
in S- ovalbumin levels from 5% in fresh egg to 81% in egg stored by refrigeration for six
months.
The protein content was measured at 5.99% and 6.17% for stored and fresh egg
respectively. Burley & Vadehra reported that the percentage protein in egg white is to be
10.5% which is approximately 5% higher than the computed result from this study for
both stored and fresh eggs, but this could be accounted for by the storage method used.
Storage environment also influences the internal quality of egg (Dudusola, 2009; Farhad
and Fariba, 2011).
PROBLEMS AND ASSUMPTIONS OF THE EXPERIMENT
Errors that occurred during this experiment could be as a result of one or all these
factors.
The incorporation of air bubbles while drawing egg albumen into the Gilson
pippete during the experiment.
The time lag between taking the readings from the egg samples.

6.0

CONCLUSION

From the results obtained during this experiment

The concentration of protein in the original undiluted sample of fresh and stored
eggs was calculated.
The total egg white protein of fresh and stored eggs was measured.
The % by weight protein of fresh and stored eggs was assessed and was found to
be slightly consistent with previous literature.

However no significant difference was found between the protein concentration of fresh
and stored eggs at 0.05 level of significance.

REFERENCES
Ana C.C.A.(2006). Albumen protein and functional properties of gelation and foaming:
Review. Sc.Agric.,63(3):291-298.
Belitz, H.D., Grosch, W., Schieberle, P. (2009). Food Chemistry.4th ed. Berlin: Springer
Verlag Berlin Heidelberg. 548-549.
Burley, R.W. and Vadehra, D.V., (1989). The Avian Egg: chemistry and biology. United
States of America: John Wiley & sons, Inc..71-72.
Dudusola, I.O. (2009). Effects of storage methods and length of storage on some quality
parameters of Japanese quail eggs.Tropicultura, 27(1):45-48.
Farhad, A.And Fariba, R. (2011). Factors affecting quality and quantity of egg production
in laying hens: A review. World Applied Sciences Journal, 12(3):372-384.
McGuiness, K. (2014) Business web summit, Irish Independent.
Taylor, V. (2011). British heart foundation.