Nutrition Diet: How do they calculate protein in food?

October 20, 2025 •

3 min read

While proteins are essential macromolecules for human health, the nitrogen they contain is the key to measuring their quantity. This is the fundamental principle behind how they calculate protein in food in laboratories worldwide, though the process is more complex than it appears.

Quick Summary

Protein content in food is primarily calculated by measuring total nitrogen using laboratory methods like Kjeldahl or Dumas. The result is then converted to crude protein using a conversion factor, but this approach has accuracy limitations due to non-protein nitrogen.

Key Points

Total Nitrogen Measurement: Most methods rely on measuring total nitrogen, as proteins are the primary nitrogen source in many foods.
Kjeldahl vs. Dumas: Two main laboratory methods exist; Kjeldahl is a classical wet-chemistry technique, while Dumas is a faster, automated combustion method.
Conversion Factor: Total nitrogen is converted to "crude protein" using a conversion factor, like the historical 6.25, but specific factors (Jones factors) are more accurate for different food types.
Non-Protein Nitrogen: Both Kjeldahl and Dumas methods measure all nitrogen, including non-protein nitrogen (NPN), which can lead to an overestimation of true protein.
Amino Acid Analysis: The most accurate method for determining "true protein" is by breaking down the food into individual amino acids and summing them, though it is more complex and expensive.
Nutritional Labeling: Accurate protein calculation is crucial for nutritional labeling, product quality, and preventing food fraud.

The Foundational Principle: Nitrogen Analysis

Proteins are composed of amino acids, all of which contain nitrogen, setting them apart from fats and carbohydrates. This nitrogen content is the basis for most protein calculations in food science. By measuring the total nitrogen in a food sample, scientists can estimate its protein content. However, this method is indirect and can be affected by other nitrogen-containing compounds, such as free amino acids, nucleotides, or nitrates, collectively known as non-protein nitrogen (NPN).

The Kjeldahl Method: The Classical Approach

The Kjeldahl method, developed in 1883, is a traditional wet-chemistry technique for determining nitrogen and, subsequently, protein content. It is known for its precision but involves the use of hazardous reagents and is time-consuming. The method involves digesting the sample with sulfuric acid, distilling the released ammonia, and titrating it to quantify nitrogen. This total nitrogen is then converted to "crude protein" using a conversion factor. While reliable, a drawback is that it measures all nitrogen, including NPN.

The Dumas Method: A Modern, Automated Alternative

The Dumas method, an automated combustion technique, offers a faster, safer, and more environmentally friendly alternative to Kjeldahl. The process involves burning a sample at high temperatures in an oxygen-rich environment and measuring the released nitrogen gas. This nitrogen content is then converted to protein using a factor. Like Kjeldahl, Dumas measures total nitrogen, including NPN, which can lead to similar overestimations. However, its speed and ease of use are advantageous for routine testing.

The Nitrogen-to-Protein Conversion Factor (NPCF)

Converting measured nitrogen to protein requires a conversion factor. The historical universal factor was 6.25, assuming proteins are 16% nitrogen. However, the actual nitrogen percentage varies depending on the amino acid composition of different proteins.

Limitations of the 6.25 Factor

Variable Nitrogen Content: Different proteins have varying nitrogen percentages.
Non-Protein Nitrogen (NPN): The factor doesn't account for NPN, which can inflate protein values.

Using Specific "Jones Factors"

To improve accuracy, specific conversion factors, known as "Jones factors," are used for different food types, such as 6.38 for dairy and 5.71 for soy.

The Gold Standard: Amino Acid Analysis (AAA)

For the most precise measure of "true protein," amino acid analysis directly identifies and quantifies individual amino acids. This involves hydrolyzing the protein into amino acids, separating them using chromatography, and quantifying each one. The sum of the amino acids provides the true protein content. While highly accurate, AAA is more complex, time-consuming, and expensive than nitrogen-based methods.

Comparative Analysis of Protein Determination Methods


Feature	Kjeldahl Method	Dumas Method	Amino Acid Analysis (AAA)
Principle	Measures total nitrogen via wet chemistry (digestion, distillation).	Measures total nitrogen via combustion.	Identifies and sums individual amino acids.
Chemical Use	Uses hazardous concentrated sulfuric acid.	Automated, uses no hazardous reagents.	Uses strong acid for hydrolysis, requires specialized reagents.
Speed	Slow (30 min - 2 hours per sample).	Fast (less than 5 minutes per sample).	Time-consuming due to hydrolysis and separation.
Accuracy	Measures total nitrogen (protein + NPN), requiring conversion factors.	Measures total nitrogen (protein + NPN), requiring conversion factors.	Measures "true protein" directly, highest accuracy.
Cost	Lower initial cost, but higher ongoing cost for chemicals and waste disposal.	High initial cost for equipment, but lower operational cost.	High investment cost for equipment and expertise.
Primary Use	Traditional standard, still used for regulatory checks.	High-throughput, modern alternative for routine testing.	Specialized testing for novel foods, formulas, and precise quality control.

The Importance of Accurate Protein Measurement

The method used to calculate protein significantly impacts nutritional labeling, product claims, and food safety. Inaccurate measurements can mislead consumers and facilitate food fraud, such as adding non-protein nitrogen compounds to inflate apparent protein content. Accurate protein determination is essential for product quality, regulatory compliance, and maintaining consumer trust.

Conclusion

How they calculate protein in food involves various analytical approaches, from nitrogen measurement to direct amino acid analysis. While Kjeldahl and Dumas methods estimate "crude protein" based on total nitrogen, they can be affected by non-protein nitrogen. Using specific Jones factors can improve accuracy, but amino acid analysis offers the most precise determination of "true protein," albeit at a higher cost. The differences in these methods highlight the importance of transparency and selecting the appropriate technique to ensure nutritional integrity and food safety.

Food and Agriculture Organization (FAO): Methods of Food Analysis

Frequently Asked Questions

Crude protein is a measure of a food's total nitrogen content converted to a protein value. It includes non-protein nitrogen (NPN). True protein, in contrast, measures only the nitrogen bound within actual amino acids, excluding NPN, and provides a more accurate value.

The 6.25 factor is based on the assumption that all proteins contain an average of 16% nitrogen. This is often inaccurate because the nitrogen content varies between different types of proteins and the calculation includes non-protein nitrogen (NPN).

Non-protein nitrogen (NPN) refers to nitrogen from sources other than proteins. Examples include free amino acids, nucleotides, creatine, and urea. Measuring NPN can cause a protein value to be overestimated if not accounted for.

Jones factors are specific nitrogen-to-protein conversion factors developed for different food types to provide a more accurate protein calculation than the universal 6.25 factor. For example, soy has a specific factor of 5.71, while milk uses 6.38.

Amino acid analysis directly measures the quantity of individual amino acids, providing the most accurate 'true protein' value. Nitrogen methods, in contrast, measure total nitrogen and convert it, which is an indirect estimate that can include non-protein sources.

Yes, the Kjeldahl method is still widely used, especially for regulatory compliance and as a standard for comparison. However, its use of hazardous chemicals and longer analysis time has led to the adoption of faster alternatives like the Dumas method for routine testing.

The Dumas method is increasingly popular because it is faster, more automated, and does not require the hazardous chemicals used in the Kjeldahl method, making it more environmentally friendly.

A significant risk is food fraud, where non-protein nitrogenous compounds like melamine are added to artificially inflate the measured protein content, as detected by Kjeldahl or Dumas analysis. This poses a serious food safety concern.