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Introduction
Protein content is a critical parameter in human food, pet food, and feed analysis, influencing nutritional labelling, ingredient selection, and processing decisions.
One of the tests is the determination of protein content of food. The exact determination of the amount of protein, through the determination of the nitrogen content, is fundamental to define the nutritional features of animal feed and for the safety of final food products intended for human and animal consumption.
Methods available for nitrogen/protein analysis
There are some analytical techniques to determine protein/nitrogen content:
❖ NIR (near-infrared reflectance)
❖ wet-chemical Kjeldahl
❖ combustion-based Dumas
While spectroscopic techniques like near-infrared reflectance (NIR) can estimate protein and other constituents, they are secondary methods requiring extensive calibration and are highly sensitive to matrix variations.
Primary quantitative protein determination traditionally relies on measuring nitrogen using either the wet-chemical Kjeldahl method or the combustion-based Dumas method, with protein values calculated through nitrogen-to-protein conversion factors that vary by material type.
Kjeldahl method
For over a century, the Kjeldahl method was the most frequently applied method and the industry standard for the determination of the total protein content of food products by measuring the total organic Nitrogen content.
The Kjeldahl method was originally designed for the brewing industry, for monitoring protein changes in grain during germination and fermentation (Kjeldahl, 1883; Bradstreet, 1954). First published in 1883, the method has been accepted with modifications as the standard for the determination of nitrogen content.
The Kjeldahl method and devices have been significantly modified over the past 100 years, but the basic principles are still valid and include three main steps:
- Digestion – the decomposition of nitrogen from organic samples by boiling in concentrated sulfuric acid resulting in an ammonium sulphate solution
- Distillation – adding excess base to the acid digestion mixture, which converts methane (NH4+) to ammonia (NH3), followed by boiling and condensation of the NH3 gas in a receiving solution
- Titration – the amount of ammonia in the receiving solution is quantified
Although semi-automatic or automatic Kjeldahl systems are available today, the various processing steps still involve cumbersome procedures that may not fully comply with the most recent safety regulations, due to the use of potentially hazardous substances, like Sulfuric acid and Sodium hydroxide.
As an analysis involving wet chemicals, it requires excessive time and labour costs.
Nowadays, this is undesirable in relation to safety-based reasons and from an economic standpoint.
Dumas method
The Dumas method is considered a direct method for determination of total nitrogen content. It was developed in 1831 by Dumas, from the observation that nitrogenous compounds heated with alkali give ammonia, which can be determined volumetrically.
Currently, the method consists of combusting a sample of known mass in a high-temperature (range, 800–900°C) chamber in the presence of oxygen, producing carbon dioxide (CO2), water and nitrogen. The gases are then passed over special columns that absorb CO2 and water.
A column containing a thermal conductivity detector at the end is then used to separate the nitrogen from residual CO2 and water, and the remaining nitrogen content is measured. The instrument must initially be calibrated by analysing a pure material containing a known nitrogen concentration.
The measured signal from the thermal conductivity detector for the unknown sample is converted into a nitrogen content.
Dumas systems are therefore easy to use, fast, and do not require the use of potentially hazardous chemical reagents.
Kjeldahl and Dumas comparison
As the Dumas method determines total Nitrogen including inorganic fractions like nitrite and nitrate and the Kjeldahl method only organic nitrogen and ammonia, differences in results occurred in the comparative studies.
The Kjeldahl method does not recover all organic nitrogen and has problems especially with the recovery of heterocyclic N-compounds like e.g. nicotinic acid. This has not been seen as a problem, as in crude protein determinations the main issue was the conversion of alpha-amino nitrogen from amino acids into ammonia.
Historically, the nitrogen to protein conversion factors for the traditional Kjeldahl method, have been established based on the amino acid pattern of the sample. For feed and food samples with varying composition, a general factor of 6,25 has been agreed on. When using the same conversion factors for techniques with different nitrogen recoveries, differences in results may occur.
According to ISO/TS 16634-2:2009 the generally agreed conversion factor for the product analysed, is equal to 5.7 for wheat, rye and their milled products and 6.25 for all other products falling within the scope of this ISO standard.
Dumas and Kjeldahl methods will lead to different results, depending on the non-protein-nitrogen content of the analysed sample and to what degree they are recovered by respective methods.
The AAFCO (American Association of Feed Control Officials) PTS is one of the most comprehensive proficiency testing schemes with some 300 participating labs and more than 100 reported methods.
For Kjeldahl the values for the reference method AOAC 2001.11 have been selected. As can be seen, the standard deviations of reported results (in parenthesis) are comparable, but the Dumas method results in higher values.
During five harvest years (2000 – 2004) the Max Rubner Institute in Detmold, Germany, performed a comprehensive study with more than 800 wheat samples comparing the crude protein results between Kjeldahl and Dumas methods.
They found that some 2% of “Dumas protein” was not determined by the Kjeldahl method and presented the following relationship between Dumas and Kjeldahl protein values:
Kjeldahl = 0.959*Dumas + 0.258
As the difference between the methods not only depends on growing year and cultivar but also on growing conditions (i.e. rain, fertilisation) this formula could not generally be used for converting results.
Numerous other comparative studies have been reported. Some studies come to the conclusion that the Dumas method may replace the Kjeldahl method for the determination of Crude Protein in selected food groups when appropriate coefficients are used.
They suggested correction factors of 1.01 for dairy, 1.00 for oilseeds, 0.99 for feed, 0.98 for infant formulas, 0.95 for cereals, 0.94 for meats, 0.89 for vegetables, 0.80 for fish and 0.73 for fruits when calculating Crude Protein using Dumas method results.
After several comparative studies, there are now various international standards that provide for the use of the Dumas method as an alternative to the Kjeldahl method.
Dumas advantages
- Sample throughput
The analysis time for the Kjeldahl method is a minimum of 100 minutes. Using the batch method, a maximum of 100 samples can be analyzed in a day, but a single sample takes the same time as analyzing the entire batch. The method is time-consuming and labour-intensive due to the many manual steps involved in sample preparation and processing
With the Dumas method of nitrogen determination, the analysis time per sample is four to five minutes, i.e. results (even for individual samples) are available more quickly than with the Kjeldahl method.
- Cost per analysis
The cost per analysis for the Kjeldahl method is approximately € 6*. The main cost factors are the cost of chemicals, their proper disposal and personnel costs. In addition, it requires fume cupboard space, which is usually limited and expensive. In comparison, the cost per analysis with the Dumas method is around € 0.49*, depending on the analyzer and the carrier gas used (*costs may vary by country).
- Eco friendliness
Nitrogen determination according to Kjeldahl uses concentrated sulfuric acid and catalysts that are hazardous to the user and the environment. In addition, the liquid waste from the analysis process is harmful to the environment and requires costly disposal.
The Dumas method, on the other hand, requires no harmful or toxic chemicals.
