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Proteins are highly essential compounds that are involved in many important biochemical process in our bodies and have important implications on our health. They help to build our bodies, act as antibodies to protect us from foreign antigens, involved in muscle movement, hormonal activities, provide mechanical support and provide coverings like hair, nails etc. They are made up of amino acids, which consist of nitrogen, carbon, hydrogen and oxygen as the main molecules.
Proteins are present in varying quantities in many different types of foods and it is important that we understand the nutritional content of the foods that we eat. Using techniques that help us determine the amount of protein in foods is a good way to know our protein intake. Apart from understanding the nutritional value, determination of protein content can help increase the market value of a particular food and give adequate knowledge of total protein content.
This review covers only a few, but important analytical techniques out of a plethora of many other protein analysis techniques.
The Kjeldahl Method:
The Kjeldahl method was first introduced by Johan Kjeldahl in 1883. and has been adopted by many national and international organisations. Although the Kjeldahl method has been modified a number of times, the principal procedure remains the same: digestion, distillation and titration.
Digestion: The nitrogen present in the food sample is decomposed by boiling with concentrated sulphuric acid (H2SO4) to obtain ammonium sulphate [(NH4)2SO4].
Distillation: The ammonium sulphate formed is converted to ammonia (NH3) by addition of sodium hydroxide (NaOH). The ammonia is then boiled and condensed in a receiving solution containing boric acid, which captures the ammonia gas resulting in the formation of ammonium borate complex. As the ammonia collects in the receiving solution, the colour of the solution changes.
Titration: The ammonium borate from the previous step is titrated with sulphuric acid (H2SO4) or hydrogen chloride (HCl). The strong acid neutralizes the ammonium borate and a reverse colour change indicates the end of the reaction. By knowing the amount of acid used in the titration, the amount of ammonia can be determined, which corresponds to the amount of nitrogen in the sample.
The traditional Kjeldahl method is cost effective.
Very low uncertainty. Anglov, Petersen and Kristiansen, 1999, showed that the relative standard uncertainty of Kjeldahl method was 0.19%.
This method has undergone modifications over the years resulting in high sensitivity, increased automation and time reduction.
Introduction of colorimetric method has led to simplification of Kjeldahl process and increased sample throughput.
The Kjeldahl method produces accurate results irrespective of the sampleâ€™s physical state.
This method determines the total nitrogen content, which means it does not differentiate between proteinacious and non-proteinacous nitrogen.
Use of hazardous chemicals that could have implications for the environment and pose a threat to the operator.
Toxic wastes are produced
Low precision. Anglov et al., (1999), proved that the relative intermediate precision of Kjeldahl method was 0.085%.
Errors could arise due to:
Precipitation of salt
Contamination in sample, reagents, standards used etc.
Foaming or bumping during digestion
Inadequate or excessive digestion or distillation
Inadequate addition of sodium hydroxide
(A Guide to Kjeldahl Nitrogen Determination Methods and Apparatus)
UV Spectrophotometric Method:
Spectrophotometer is an instrument that measures the ability of test samples to absorb or transmit light that passes through them. When a solution is turbid, that is, has high concentration of a particular substance or solute, it absorbs more light and transmits less. Consequently its absorbance reading will be high. As the turbidity decreases, absorbance too decreases and transmission of light through the solution is more. The absorbance readings are taken at specific wavelengths of light, usually measured in nanometres.
When the reading of the sample is taken at wavelengths between 10 nm and 400 nm, which is the wavelength of ultra-violet light, the method is known as Ultra-violet spectrophotometry or simply UV Spectrophotometry.
The protein is dissolved in a buffer or an alkali and absorbance of the protein solution read at a wavelength of 280 nm. From the absorbance readings, the concentration of protein can be calculated.
Tryptophan and tyrosine are two amino acids that show maximum absorbance of UV light at 280 nm. Given that many proteins are found to contain relatively constant levels of tryptophan and tyrosine, their absorbance readings can be used to determine the amount of protein present in food (Nielson, 2003).
Sensitive. Can detect proteins with concentrations as low as 100Âµg.
Presence of buffer salts do not cause interference with the results.
Does not entail the use of special dyes or reagents
The procedure does not destroy the sample.
Recovery of the sample is possible
(Nielson, 2003; Gomes, 2005).
Lower maintenance costs
Can be used for analysis of other food constituents too.
Nucleic acids too absorb light at 280 nm and cause interference. However this setback can be dealt with by taking absorbance readings at another wavelength in addition to that at 280 nm.
Presence of unwanted particles could increase turbidity, which would in turn give false readings (false positive).
Suitable for pure protein solutions. Or proteins should be extracted by using appropriate protein denaturating solutions or alkali.
Highly concentrated samples should be sufficiently diluted so as to obtain results that fall well within the detection levels of the instrument.
Noise can be generated from the light source or the electronic components of the instrument. Hence accuracy of results could be compromised.
Improper sample preparation
Contamination of samples could occur if not properly handled or stored.
The Bradford Method:
This method is based on the binding of Coomassie Brilliant Blue, a dye, with proteins. Here a low pH (acidic) of the protein solution is created which causes the negatively charged dye to bind to the protein. The amount of protein present in food can then be determined spectrophotometrically as the absorption maximum of the dye changes from 465nm to 595 nm when it binds to the protein molecules (Nielson, 2003).
Gives more rapid results than Kjeldahl (requires only two minutes).
Can be used to measure large molecular weight proteins
Shows greater sensitivity than Kjeldahl method. Sensitivity is greater at low pH (Chial and Splittgerber, 1993)
Low interferences from ATP, DNA, EDTA, potassium chloride, thymidine, tyrosine, valine etc.
Simple sugars, if present in sample, do not affect Bradford results.
Determines the proteinacious nitrogen only .
Kamizake et al, showed that the Bradford method can also be used to find out the total protein content in food samples without having the need to extract any lipids.
Protein-dye complex formed is stable (Seevaratnam et al., 2009).
Interference in results due to various detergents, acetone, ethanol, glycerol, magnesium chloride, phenol, potassium chloride, SDS (sodium dodecyl sulphate), sodium chloride (common salt), sucrose and so on. The errors arising due to interferences from these substances can be lowered by using suitable controls (Owusu-Apentin, 2002, Nielson, 2003).
This method involves usage of standard solutions. (Standard solutions are those solution that are highly comparable in character and composition to that of the sample). Proper selection of standard is required depending on the type of food to be analysed.
Using quartz cuvettes (a small rectangular container specifically crafted for spectrophotometric samples) as the dye has a tendency to bind to the walls of the quartz container. So, only glass or plastic cuvettes should be used. (Nielson, 2003)
Using impure dye. This could lead to low sensitivity, accuracy and preciseness (Owusu-Apentin, 2002).
Every method mentioned in this review has its own merits and demerits. The selection of the method to be used has been based on certain important criteria such as costs involved: that while high initial costs are acceptable, the running costs should be low. Other factors taken in to account are simplicity of technique, sensitivity and rapidness of the technique since we have to take into consideration the fact that several tonnes of powder is to be delivered on a daily basis. Keeping all these in mind, I recommend the UV Spectrophotometric Method as the best method for determination of protein content in food samples as it has low maintenance costs, it is sensitive, simple to perform and rapid.