Protein Determination: Why Use Nitrogen And Different Methods

Methods: Macro Kjeldahl – Determination of Protein in Flour

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).

Biuret Determination of Soluble Protein in Egg White

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.

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	??

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.

Results

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

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.

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

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.