Food Analysis: Protein Determination and Lipid Analysis Report

This practical assignment details the determination of protein and lipid content in food samples using various analytical methods. The Kjeldahl method is used to determine protein content in flour by converting organic nitrogen to ammonium sulphate, which is then neutralized and distilled. The Biuret method measures albumin in egg white by complexing cupric ions with peptide bonds, producing a purple color that is quantified spectrophotometrically. The experiment involves preparing samples, conducting digestions, distillations, and spectrophotometric measurements. Results are analyzed to determine protein percentages and albumin content, comparing them with expected values and discussing potential sources of error and variations in food composition. Desklib provides a platform for students to access this assignment and other study resources.

INTRODUCTION:
PRAC 3:
Why use nitrogen for protein determination? – because the only element not present in
lipids/carbohydrates.
How can nitrogen be used to determine protein?
Determining protein methods: kjedahl & dumas. What’s the difference? How do they work/mechanism
of it/t heoretical aspect?
Expected protein content in flours. Why is there a range of protein content?
Why use biuret test with egg protein? What is the biuret test? How does biuret test work?
Expected protein content of eggs.
Protein is an essential part of life as it is found throughout the body – in the bone, skin,
muscles and every body part or tissue. It also makes up enzymes that are responsible for
chemical reactions and haemoglobin that carries oxygen in the blood. Generally, the body
makes amino acid (AA), which is the building blocks of protein (Wong, Aziz, and
Mohamed, 2008). However, a few AA needs to be obtained from food such as eggs, flour,
milk, yoghurt and fish. The protein content in flour is 10g/100g and in egg white is 11g/100g.
Nitrogen in protein is the most distinguishing element, where it typically ranges from 13.4%
to 19.1%, depending on the variation of specific AA composition in proteins. Proteins that
contain high amounts of basic AA have higher amount of nitrogen (Nielsen, 2010).
Numerous methods have been developed to measure protein content. Kjeldahl and Dumas are
two methods used to measure protein content in food. Kjeldahl method relies on the
conversion of protein nitrogen into ammonium ion by boiling sulphuric acid in the presence
of a catalyst whereas the Dumas method relies on the combustion of samples at a high
temperature and measurement of the resulting elemental nitrogen (Thompson et al., 2002).
Kjeldahl method is used in this experiment to determine the total organic nitrogen in the
sample. The nitrogen concentration of the food can determine the amount of protein present
in the food. In the Kjeldahl method, the total organic nitrogen is converted to ammonium
sulphate, which will then be neutralised with alkali and distilled with boric acid solution. The

borate anions formed are titrated with standardised acid that will be converted into nitrogen in
the sample. The result represents the crude protein content of the food.
The Kjeldahl method is carried out in three different but easy steps. Firstly, digestion takes
place where acid and catalyst is added to allow complete breakdown of all organic matters.
Non-volatile ammonium sulphate is formed from the reaction of nitrogen and sulphuric acid.
During digestion, protein nitrogen is liberated to form ammonium ions. Sulphuric acid then
oxidises organic matter and combine with the ammonium formed while carbon and hydrogen
elements are converted to carbon dioxide and water. Neutralisation and distillation will then
take place where the digest is diluted with water. Alkali containing sodium is added to the
sulphuric acid to allow neutralisation. The ammonia formed is distilled into boric acid
solution containing indicators methylene blue and methyl red. The advantage of using this
method is that its applicable to all types of food with high accuracy, thus being the official
method for crude protein analysis (Nielsen, 2010).
Biuret determination measures the albumin in the egg white, with the addition of copper salt
in an alkaline solution [i.e. sodium hydroxide (NaOH)]. A purple colour complex will be
produced when the cupric ion is complexed with peptide bonds. Steps taken in order to carry
out this procedure is by mixing protein solution with reagents that include copper sulphate,
NaOH and potassium sodium tartrate, which is used to stabilise the cupric ion in the alkaline
solution. The absorbance of the colour is read at 550nm against a reagent blank, where the
intensity is proportional to the protein content in the sample. A standard curve of
concentration versus absorbance is constructed using bovine serum albumin (BSA).
Comparing to Kjeldahl method, the biuret method takes a shorter time and is the simplest
form of protein analysis. Other than that, it doesn’t detect nitrogen from non-peptide and non-
protein sources (Nielsen, 2010).
MATERIALS AND METHODS:
Part A: Macro Kjeldahl – Determination of Protein in Flour
Different kinds of flour and its corresponding blanks were prepared and analysed.
Approximately 1g of flour was weighed out onto two joined Tallyho cigarette papers then
twisted into a ball and added to a digestion tube. Approximately 5g anhydrous sodium
sulphate and two catalyst tablets (each containing 1g sodium sulphate and 100mg copper
sulphate) were added to the same digestion tube. In a fume cupboard and using a face shield,

12mL concentrated sulphuric acid was added to the tube and it was gently shaken to wet the
samples. The digestion tubes were loaded onto a preheated digestion unit (420°C) with the
vacuum manifold attached to it. The samples were digested for approximately an hour until
they were a clear blue/green colour.
After an hour of digestion, the tubes were left to cool down before 50mL of distilled water
(diH2O) was added using a measuring cylinder. For each digestion tube, a 250mL conical
flask containing 30mL of 4% boric acid solution (containing bromocresol green and methyl
red indicators) was prepared. Both the digestion tube and conical flask were placed in the
distillation unit whereby the distillate tube was submerged into the flask. The distillation unit
automatically added 50mL of 40% sodium hydroxide (NaOH) to the distillation tube. It then
boiled the solution in the tube with steam. The distillate from the steam was collected in the
conical flask. The waste liquid in the digestion tube was automatically removed into a waste
bottle. The empty digestion tube was placed in the sink while the solution in the conical flask
was titrated against 0.1M hydrochloric acid (HCl) until the solution turned pink. The nitrogen
content and percentage protein were calculated.
Part B: Biuret Determination of Soluble Protein in Egg White
Approximately 20g of commercially pure liquid egg white was weighed into a beaker.
Approximately equal amount of 0.1M NaOH was added to the beaker to dissolve the egg
white using a magnetic stirrer. The dissolved egg white was washed into a 100mL volumetric
flask using diH2O. The beaker was rinsed several times to ensure all egg white had been
washed into the flask. The solution in the flask was diluted to volume with diH2O and the
flask was shaken to mix the solution. A standard bovine serum albumin (BSA) protein
solution with 10mg protein/mL was prepared. Twelve test tubes in duplicated pairs were set
up following Table 1 with the biuret reagent added last using a safety dispenser. The test
tubes were vortex and left to stand for 30 minutes. The absorbance of the solution in each test
tubes was read on the spectrophotometer at 550nm. A calibration curve was plotted using the
standards’ average absorbance value against its protein content (mg/mL).
Table 1: Reagents added to each pair of test tubes.
Reagents Blank Standard 1 Standard 2 Standard 3 Standard 4 Egg White
Sample
diH2O 2 mL 1.6 mL 1.2 mL 0.8 mL 0.4 mL 1.8 mL
Egg White
Solution - - - - - 0.2 mL

BSA
Solution 0 mL 0.4 mL 0.8 mL 1.2 mL 1.6 mL -
Biuret
Reagent 8 mL 8 mL 8 mL 8 mL 8 mL 8 mL
Protein
Content 0 mg/mL 0.4 mg/mL 0.8 mg/mL 1.2 mg/mL 1.6 mg/mL ??
RESULTS:
Part A: Macro Kjeldahl – Determination of Protein in Flour
The protein percentage of the flour sample was determined by calculating the nitrogen
percentage and multiplying it by the appropriate factor (5.7). The nitrogen percentage
calculation is shown and Table 2 shows the compiled result of different flour samples
including their nitrogen and protein percentage as well as their labelled values.
% Nitrogen (w /w)= ( mL HCl for sample−mL HCl for blank ) ×0.1 ×14
mg sample ×100
Table 2: Different flour samples with their analysis values and corresponding protein
percentage.
Sample Weight
(mg)
Sample
HCl (ml)
Blank
HCl (ml) % Nitrogen % Protein Label
(g/100g)
Bread & Pizza
Plain Flour 1000 15.1 0.3 2.072 11.810 12.5
Wholemeal Atta
Flour 1000 14.3 0.3 1.96 11.172 11.3
Coconut Flour 1000 16.5 0.3 2.268 12.928 18
Plain Flour 1007 13 0.5 1.738 9.906 10
Part B: Biuret Determination of Soluble Protein in Egg White
Using the average absorbance reading of the BSA protein standards, it was plotted against its
known protein content (mg/mL) and a linear trend line was fitted into it. The standards’
calibration curve is shown in Figure 1.

0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
f(x) = 0.263583333333333 x
R² = 0.999849741244629
BSA Standards Calibration Curve
Protein Content (mg/mL)
Absorbance @550nm
Figure 1: BSA Standards Calibration Curve. Average absorbance of the BSA protein standard was
plotted against known protein concentration (mg/mL). A linear trendline was determined with the
intercept set as zero along with its corresponding equation. The R2 value shows how close the data is
to the fitted line, with R2 = 1 being the ideal perfect straight line.
The protein concentration of the pure egg white was determined using the average
absorbance value of the pure egg white and the equation from the trend line. The albumin
percentage was then determined using the protein concentration. Table 3 shows the
absorbance values as well as the protein percentage expressed as g/100g of pure egg white.
The commercial pure egg white had 11.2g/100g of protein.
Table 3: (a) Absorbance values of standards and samples obtained from the spectrophotometer.
(b) Protein determination of pure egg white and its albumin percentage.
(a) (b)
Abs. 1 Abs. 2 Ave. Abs.
Blank 0 0 0 Protein content
(mg/mL) 0.522
Standard 1 0.103 0.112 0.108 Protein(mg) in 10mL
(0.2mL) 5.216
Standard 2 0.216 0.213 0.215 Protein(mg) in 100mL
(20.24g) 2608.118
Standard 3 0.321 0.318 0.320 Protein(mg) in 100g
pure EW 12885.960
Standard 4 0.415 0.419 0.417 Protein(g) in 100g
pure EW (% Protein) 12.886
Sample 0.137 0.138 0.138 % Albumin 8.634

DISCUSSION:
The protein content of the flour and egg are 10g/100g and 11g/100g respectively. The
nitrogen content in protein is the most differentiating element, which ranges from13.4% to
19.1%, reliant on the differences of particular amino acid composition in protein (Nielsen,
2010). It is worth noting that protein that contains high amounts of basic AA have a higher
amount of nitrogen. Kjeldahl and Dumas are two methods used to measure protein content in
food. The Kjeldahl method depends on the change of nitrogen into ammonium ion by
steaming sulphuric acid in the existence of a catalyst. The Dumas method relies on the
combustion of samples at a high temperature and then measurement of the resultant elemental
nitrogen (Thompson et al., 2002). In this experiment, the Kjeldahl method is utilised to
determine the total organic nitrogen in the sample. The nitrogen concentration can determine
the amount of protein present in the food. Biuret determinations measure the albumin in the
egg white, by adding copper salt into the sodium hydroxide solution. The purple colour
complex will appear when the cupric ion is complexed with the peptide bonds. The
procedures taken in this experiment is by mixing the protein solution with the reagent that
comprises the potassium sodium tartrate, copper sulphate and NaOH (Nielsen, 2010).
Bread & pizza plain flour shows low nitrogen and protein contents almost to the standard
level recommended (10g/100g). Additionally, the flour does not correspond to the label of
12.5g/100g and therefore, fails to meet the nitrogen standard of 13.4-19.1%. Wholemeal Atta
flour also show low nitrogen range. The protein content is 11.172% which is below the
amount indicated in the label (11.3g/100g). Coconut flour show low nitrogen range but it is
highest in four flour samples examined. The protein per cent is almost the recommended of
100g/100g. However, the content is contrary to the label of 18g/100g. Plain flour shows the
lowest nitrogen range of 1.738% and lower nitrogen content of 9.90g/100g from the
recommended standard of 10g/100g. On the biuret determination of the pure egg white
protein content, it shows lower protein per cent of 8.634 from the recommend 11g/100g. The
percentage is also below the commercial pure egg white content of 11.2g/100g. The results
above illustrate that coconut flour can be recommended for a person who needs high protein
content while the plain flour can be recommended for an individual with need of lower
protein content like high blood pressure patients.

REFERENCES:
NIELSEN, S. 2010. Food Analysis, Springer US.
SOURCE, T. N. Protein [Online]. Available:
https://www.hsph.harvard.edu/nutritionsource/what-should-you-eat/protein/
[Accessed].
THOMPSON, M., OWEN, L., WILKINSON, K., WOOD, R. & DAMANT, A. 2002. A
comparison of the Kjeldahl and Dumas methods for the determination of protein in
foods, using data from a proficiency testing scheme. Analyst, 127, 1666-8.
Wong, K.H., Aziz, S.A. and Mohamed, S. (2008).
Sensory aroma from Maillard reaction of individual
and combinations of amino acids with glucose in
acidic conditions. International Journal of Food Science and Technology,
43, 1512-1519.