Determination of protein content in food and organic materials

Accurate measurement of protein content is critical in food and organic material analysis, serving key roles in nutritional labeling, quality control, and regulatory compliance. Various industries, including food manufacturing, agriculture, and pharmaceuticals, rely on precise protein quantification to ensure product consistency and adherence to legal requirements.

Nitrogen-based protein quantification methods

Two of the most widely used techniques for determining protein content are the Kjeldahl Method and the Dumas Method, both of which measure nitrogen levels as a proxy for protein content. Each method has its advantages and limitations, making the choice dependent on factors such as sample type, required accuracy, throughput, and regulatory acceptance.

The Kjeldahl method, a classical approach, involves digestion of the sample in concentrated sulfuric acid, followed by neutralization, distillation, and titration to determine nitrogen content. It is considered highly reliable but can be labor-intensive and time-consuming.

The Dumas method, a combustion-based technique, offers faster results by oxidizing the sample at high temperatures, converting nitrogen into measurable gaseous compounds. While rapid and automation-friendly, it requires specialized instrumentation and may not always be suitable for all sample types.

Nitrogen-to-protein conversion and its limitations

The calculation of protein content from nitrogen levels is based on conversion factors, with the standard factor being 6.25 for most foods. This is derived from the assumption that protein contains approximately 16% nitrogen (100 ÷ 16 = 6.25). However, the nitrogen composition of proteins varies due to differences in amino acid profiles, leading to alternative conversion factors for specific food sources.

For example:

• Wheat and cereal proteins often use a conversion factor of 5.7.
• Dairy proteins (casein, whey) typically have a factor closer to 6.38.
• Soy protein may use 5.71 due to its unique nitrogen composition.

While nitrogen-based methods provide a standardized approach for protein estimation, they come with inherent assumptions and limitations:

• They assume all nitrogen originates from protein, which is not always the case. Some foods contain non-protein nitrogen sources (e.g., free amino acids, nucleic acids, ammonia), potentially leading to overestimation of protein content.
• The methods are susceptible to adulteration, as seen in food safety incidents like melamine contamination, where non-protein nitrogen was intentionally added to falsely elevate protein readings.

The Kjeldahl method

The Kjeldahl method is a classical technique for determining total nitrogen content in organic materials, which is subsequently converted into protein content using a specific conversion factor.

The sample undergoes digestion using concentrated sulfuric acid (H₂SO₄), potassium sulfate (K₂SO₄), and a catalyst (such as selenium or copper). The process involves:

• Sulfuric acid decomposing the organic material and converting nitrogen into ammonium sulfate.
• Potassium sulfate raising the boiling point of sulfuric acid, facilitating digestion.
• Catalysts accelerating the digestion reaction.

After digestion, the sample is neutralized with sodium hydroxide (NaOH), converting ammonium sulfate into ammonia gas (NH₃). The ammonia gas is distilled and captured in a receiving flask containing excess boric acid (H₃BO₃), forming ammonium borate.

The residual boric acid is titrated with a standard acid solution (e.g., hydrochloric acid or sulfuric acid) using an indicator such as methyl red or bromocresol green. The amount of acid consumed determines the nitrogen content, which is then converted to protein content using a conversion factor.

The standard nitrogen-to-protein conversion factor is 6.38 for dairy-based proteins, as dairy proteins contain approximately 15.67% nitrogen. Different food matrices require different conversion factors to account for variations in nitrogen content (amino acid sequences). The Kjeldahl method may slightly overestimate protein content due to the inclusion of non-protein nitrogen (NPN) in the total nitrogen measurement. True Protein Content is calculated by subtracting the non-protein nitrogen from the crude protein determined by the Kjeldahl method.

The Dumas Method

The Dumas method, also known as the combustion method, is a modern technique for nitrogen determination that operates on the principle of high-temperature combustion.

The sample is combusted in an oxygen-rich environment at high temperatures (typically 900–1000°C), converting all nitrogen-containing compounds into nitrogen oxides (NOx).

The combustion gases, including NOx, pass through a reduction column containing copper or similar reducing agents, converting nitrogen oxides into elemental nitrogen (N₂). Any excess oxygen is chemically bound to avoid interference in detection.

The gases then pass through a system containing magnesium perchlorate and sodium hydroxide to remove unwanted components such as water vapour and carbon dioxide.

The purified nitrogen gas is measured using a thermal conductivity detector (TCD), using helium as a carrier and reference gas. Quantitation is against multipoint external calibration. The measured nitrogen content is converted to protein content using an appropriate conversion factor.

At AsureQuality, protein content is determined using a Nitrogen/Protein Analyzer, manufactured by Leco. This software-controlled system ensures accurate nitrogen measurements across various organic matrices. The instrument operates under strict manufacturer guidelines to maintain precision and reliability.

Based on the Dumas combustion method, results are generated in approximately 4 minutes and minimizes chemical waste compared to the Kjeldahl method. The instrument automate the entire process for high-throughput sample processing.

The limit of reporting for protein by Kjeldahl or Dumas methods is 0.1% m/m (absolute value).

Comparison of the Kjeldahl and Dumas Methods

Conclusion

Both the Kjeldahl and Dumas methods are effective for protein quantification. The Kjeldahl method is widely used for regulatory and reference purposes, while the Dumas method provides a faster, more environmentally friendly alternative suitable for high-throughput laboratories. AsureQuality employs both Kjeldahl and Dumas methods for protein measurement to ensure accurate and efficient protein determinations for diverse food and organic samples. Both methods are accredited by IANZ and are applicable to a wide range of food and dairy products, including milk, milk powders, meat, pet foods, and similar matrices.

Analysis codes

The table below outlines the test codes associated with each sample type, along with the corresponding method references upon which they are based.