Protein measurements

Proteins are essential macronutrients that play a vital role in the growth, repair, and maintenance of the human body. Alongside carbohydrates and lipids, proteins can also serve as a source of energy in the diet when other energy sources are insufficient. Given their biological importance, evaluating the protein quality of food is a key aspect of nutritional science and food regulation. Protein quality can be assessed through various parameters, including the total protein content, the profile and balance of essential amino acids, the protein digestibility and bioavailability of amino acids. Accurate quantification of protein in food and food raw materials is crucial for ensuring both nutritional quality and regulatory compliance. Several analytical methods are currently used to measure protein content; however, these methods often yield varying results due to differences in their underlying principles. The most commonly applied techniques today, such as the Kjeldahl and Dumas methods, determine protein concentration indirectly by measuring the total nitrogen content of the sample. While useful, these methods lack analytical selectivity, as they cannot differentiate between nitrogen derived from protein and nitrogen originating from non-protein sources. This can lead to inaccuracies, especially in foods containing significant amounts of non-protein nitrogenous compounds. Understanding the limitations and applications of these methods is essential not only for accurate nutritional labeling but also for the broader evaluation of food quality. According to the Food and Agriculture Organization (FAO), the assessment of protein quality should go beyond total content and also consider the amino acid composition, the bioavailability of indispensable amino acids, and the protein’s ability to support optimal physiological function.

First developed in 1883 by Johan Kjeldahl, the Kjeldahl method remains one of the most widely used analytical techniques for determining total nitrogen content in organic substances. Although it has been significantly refined over the past century, its fundamental principles have stayed the same and it is still considered the international reference method for the determination of crude protein in food and industrial applications. The method is based on the assumption that most nitrogen in food is associated with amino acids and, by extension, with proteins. The procedure consists of three main steps. In the digestion phase, organic nitrogen is broken down by boiling the sample in concentrated sulfuric acid, forming ammonium sulfate. This is followed by distillation, where an excess base is added to convert ammonium ions (NH₄⁺) into ammonia gas (NH₃), which is then distilled and captured in a receiving solution. Finally, the amount of ammonia is quantified via titration, providing a measure of the total nitrogen present in the sample.

To convert the measured nitrogen content to an estimate of total protein, a conversion factor, typically 6.25, is applied. This value originates from the 19th century and is based on the assumption that proteins contain 16% nitrogen. However, this generalization overlooks the fact that the nitrogen content of proteins can vary significantly, depending on their amino acid composition. In reality, protein nitrogen content ranges from approximately 13% to 19%, and using a fixed factor of 6.25 can lead to systematic over- or underestimation of true protein content. Errors of 15–20% are not uncommon. Moreover, the Kjeldahl method lacks the ability to distinguish between protein nitrogen and non-protein nitrogen. Foods often contain various nitrogenous compounds such as nucleic acids, amines, nitrates, alkaloids, urea, and certain vitamins, all of which can inflate the apparent protein content. For instance, mushrooms have a high and variable content of non-protein nitrogen from compounds like chitin and chitosan, which can lead to overestimations of protein content by up to 23% when compared to amino acid-based methods. In one study, the protein content of certain foods was overestimated by 44–71% using the Kjeldahl method, highlighting its limitations when used without complementary techniques. Despite its shortcomings, the Kjeldahl method remains a standard in food industry and regulatory laboratories due to its robustness, reproducibility, and long-standing validation history. However, for applications requiring precise nutritional profiling, especially in protein quality assessment, more selective and modern analytical methods may be necessary.

Another widely used method for determining protein content is the Dumas method, which, like the Kjeldahl method, is based on the measurement of total nitrogen. First developed in the 19th century, the Dumas method has gained popularity as a modern alternative, primarily due to its faster analysis time, simplicity, and the fact that it avoids the use of hazardous chemicals like concentrated sulfuric acid. In the Dumas process, a food sample is combusted at high temperatures in the presence of oxygen, converting all nitrogen-containing compounds into nitrogen gas (N₂). The resulting gas is then measured, and the nitrogen content is converted into a crude protein estimate using a similar conversion factor as in the Kjeldahl method (most often 6.25). In contrast to Kjeldahl, the Dumas method does not involve digestion or titration, making it easier to automate and more environmentally friendly. However, while the Dumas method improves efficiency and safety, it does not enhance the analytical selectivity for true protein content. Just like Kjeldahl, it cannot distinguish between nitrogen from protein and nitrogen from non-protein sources such as nucleic acids, urea, or nitrates. As a result, the Dumas method is equally susceptible to overestimation and adulteration, particularly in complex or plant-based food matrices where non-protein nitrogen can be present in significant quantities (see Figure 1).