Introduction
Products containing protein are required to list the protein content on their nutritional labels. The determination and calculation of this protein content are commonly performed using the Kjeldahl method. This method measures the amount of nitrogen in the product to estimate its protein content. This blog will explain the Kjeldahl method and assess its accuracy.
Kjeldahl method
Kjeldahl Method
The Kjeldahl method, used since 1883, determines the protein content in food products based on nitrogen levels. The Kjeldahl method is often used as a standard to calculate the protein content. By measuring nitrogen levels, carbohydrates and fats, which do not contain nitrogen, are excluded from the measurement. Nearly all nitrogen in our diets comes from amino acids in proteins.
The method involves breaking down the sample with concentrated sulfuric acid, which decomposes the organic material and converts nitrogen into ammonium sulfate. A catalyst, typically a mixture of potassium sulfate with copper or selenium, is added to enhance the reaction and raise the boiling point of the acid. The resulting digest is neutralized with a base, releasing ammonia gas, which is then distilled and captured in a trapping solution. Finally, the amount of ammonia is quantified by titration with a standard acid to determine the nitrogen content. The amount of acid used in the titration correlates directly with the amount of ammonia, and thus the original nitrogen content of the sample.
Nitrogen to protein
From nitrogen content to protein content
Once the Kjeldahl method is complete, protein content can be calculated from the nitrogen content. To determine protein content, the nitrogen amount is multiplied by a factor (N) that represents the average nitrogen content in proteins. With proteins containing approximately 16% nitrogen, the factor used is 6.25.
It is important to note that not all nitrogen in foods comes from proteins. Nitrogen can also be present in components such as free amino acids, nucleotides, creatine, and choline. Nitrogen from these non-protein sources is referred to as non-protein nitrogen. Furthermore, the number of nitrogen atoms varies among different amino acids, causing the protein nitrogen content to range from 13% to 19%, which relates to conversion factors between 5.26 and 7.69.
Additionally, when dealing with relatively large amino acid molecules, such as glutamine, discrepancies can occur between the measured protein content and the actual amino acid content. This can potentially lead to an overestimation of protein content when using the Kjeldahl method.
Jones factor
The Jones factors
To enhance accuracy in protein content determinations, Jones formulated specific N factors for different foods, known as ‘’Jones factors‘’. These factors have been widely employed. For animal proteins such as meat, milk, and eggs, the factors range from 6.25 to 6.38. In contrast, the factors for vegetable proteins, which are significant in cereal- and legume-based diets, generally fall between 5.7 and 6.25.
True protein
True Protein
For a more precise determination of protein content, methods such as ion-exchange, gas-liquid, or high-performance liquid chromatography can be used to measure total amino acids. This approach gives the “true protein” content by weight. The advantage of these methods is that no assumptions about the non-protein nitrogen content or the specific amino acid proportions are required. However, the disadvantage of these methods is that it requires more advanced equipment than the Kjeldahl method, which may be inaccessible to some laboratories, particularly those with limited analysis capabilities. Additionally, certain amino acids, like sulfur-containing amino acids and tryptophan, can be more challenging to measure and may require more expertise.
Protein content
Protein content
Determining protein content is crucial for accurate nutritional labeling. The Kjeldahl method, which measures nitrogen content, is commonly used but can be affected by non-protein nitrogen and varying nitrogen levels in proteins. Jones factors improve accuracy by providing specific conversion factors for different protein sources. Advanced methods like ion-exchange, gas-liquid, and high-performance liquid chromatography offer even more precise “true protein” measurements but require sophisticated equipment and expertise, making them less accessible than the Kjeldahl method.
We Advise on Protein content determination
When producing protein-rich sports nutrition, it’s essential to carefully consider the method used to determine protein content. Checking the protein source and calculating the total protein content using a Jones factor in case of the Kjeldahl method can be particularly useful, especially for vegetable proteins. Additionally, it is advisable to review the protein content determination for products high in specific amino acids. When the amino acid molecule is relatively large, discrepancies can occur between the measured protein content and the actual amino acid content, potentially leading to an overestimation of protein content when using the Kjeldahl method.
References
References
CHAPTER 2: METHODS OF FOOD ANALYSIS. (z.d.). https://www.fao.org/4/y5022e/y5022e03.htm
ISO 8968-1:2014. (z.d.). ISO. https://www.iso.org/standard/61020.html
Protein measurement standard expanded for greater consumer protection and harmonized trade. (2014, 6 februari). ISO. https://www.iso.org/news/2014/02/Ref1815.html
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