Protein Determination in Cereals and Seeds

Cereals and seeds are significant components of the human diet and the principal part of feeding stock for domestic animals. One of the most important nutrients present in these is protein. Functioning as enzymes, hormones, and antibodies, as well as transport and structural components, proteins are required for the body’s structure and proper function.1 In addition to its dietary importance, protein content has become a guideline for some cereal trade transactions as well as a means to assess quality. The monitoring of protein content through the measurement of nitrogen release must be accurate in order to determine the nutritional quality of produce.

The two most prevalent protein determination methods rely on the release of nitrogen from the amine groups found in the peptide bonds of protein’s polypeptide chains. Regulations for appropriate monitoring methods for grain and seed analysis vary throughout the world. In the United States, the Association of Official Analytical Chemists (AOAC) International Method 992.23 and the American Association of Cereal Chemists (AACC) Method 46-30 (1999) indicate that the suitable fineness of grind must be determined for each different material analyzed to achieve precision that gives a residual standard deviation (RSD) of �2% for 10 successive determinations of nitrogen.

The traditional Kjeldahl method relies on oxidation to release nitrogen, while the Dumas combustion method, as its name implies, breaks down the bonds in the peptide chains, permitting the release of nitrogen through complete combustion of the sample.

Food analysis is a growing and important market that requires superior accuracy in order to ensure optimum productivity. The dominance of the traditional Kjeldahl method for nitrogen measurement has recently come under threat by the challenge of safer, cleaner instruments employing the Dumas combustion principle.2 The combustion method offers a faster, safer, and more reliable method for the analysis of foodstuffs.

The Kjeldahl Method

The Kjeldahl method originates from the determination of the protein content of grains used in the brewing industry and relies on quantitative determination of nitrogen. This method can be broken down into three basic steps.

• Digestion of the sample: The most time-consuming step in the analysis, this is designed to break down the bonds that hold the polypeptides together and convert them to simpler chemicals such as water, carbon dioxide, and ammonia. Adding strong sulfuric acid and heating the mixture to about 370ºC to 400ºC for 60 to 90 minutes oxidizes the organic material and releases ammonium ions.
• Distillation: This separates the ammonia from the digestion mixture by raising the pH with sodium hydroxide, which changes the ammonium ions into ammonia gas. The ammonia is collected through boiling and distillation of the gas into a trapping solution of hydrochloric acid.
• Titration: As the ammonia dissolves into the trapping solution, it is back-titrated so that the quantity of distilled-off ammonia can be calculated and the amount of nitrogen in the protein determined.

Because the initial digestion of the sample is so slow, scientists have tried to speed up the process using many different catalysts, including mercury and selenium. The addition of a neutral substance, such as potassium sulfate, raises the boiling point of the digesting acid and the temperature of the reaction, promoting acceleration. Despite these measures, the reaction remains time-consuming, inefficient, and costly on a large scale.

The Combustion Method

The combustion method involves burning a sample in an oxygen-rich atmosphere at high temperatures and analyzing the resulting gases. This process has three stages.

• Combustion: Once the sample is weighed and purged of any atmospheric gases, it is heated in a high-temperature furnace and rapidly combusted in the presence of pure oxygen at about 1,000ºC. Cupric oxide may be used to complete the oxidation.
• Reduction and adsorption: The combustion products—mainly carbon dioxide, water, nitrogen dioxide, and nitrogen gas—are collected and allowed to equilibrate. An aliquot of the gas mixture is passed over hot copper to remove any oxygen and catalytically convert nitrogen dioxide to nitrogen. The sample is then passed through a trap that removes carbon dioxide and water.
• Quantitation: The total nitrogen is measured by thermal conductivity.

Historically, the original Dumas combustion method has proven time-consuming and laborious, with incomplete combustion giving nitrogen oxides and air contamination.3

A Comparison

The two methods achieve similar repeatability in a variety of feed samples, with the standard deviation for nitrogen content staying within AOAC and AACC guidelines.4 The Dumas combustion method was developed several years before the Kjeldahl technique but suffered from imprecise and inaccurate results. However, new higher quality instruments have dramatically improved the accuracy and reliability of this test.5 Analyzers based on the dynamic flash combustion of the sample, including the Thermo Scientific Flash 4000 N/Protein Analyzer, are now able to handle a wide array of laboratory requirements, such as accuracy, day-to-day reproducibility, and high sample throughput.