Protein Determination in Cereals and Seeds

Alternatively, the Kjeldahl method’s universality, precision, and reproducibility had made it the internationally recognized method for estimating the protein content in foods and the standard method against which all others were judged. Unfortunately, for this method, different correction factors are needed for individual proteins to account for each protein’s unique amino acid sequence. Additional disadvantages, such as the need to use concentrated sulfuric acid at high temperature and the relatively long testing time, compare unfavorably with the combustion method for measuring crude protein content.6 With relatively recent technological advances, the combustion method now offers significant advantages over the Kjeldahl method, including a shorter analysis time—minutes as opposed to hours—along with lower costs and environmental suitability.7

Advances in Combustion Technology

New generation combustion methods are capable of taking a “sample-in, results-out” approach, operating in real time and requiring minimum sample preparation. The large sample size capacity of these instruments ensures optimum homogeneous analysis and results precision, reducing sample handling time and minimizing matrix effects. Thermal conductivity detectors feature an extensive working range to cover nitrogen/protein applications from low parts per million to high percentage concentrations. Contemporary software such as Thermo Scientific’s Eager Xperience allows 24/7 unattended operation, ensuring maximum output. And automatic carbon dioxide adsorber regenerating technology ensures that the self-cleaning filters never need to be changed, saving time and money.

A relative standard deviation of less than or equal to two for 10 successive determinations of nitrogen—as per AOAC and AACC regulations—must be achieved in order to substantiate the new combustion method tools offered.

For our experiment, we chose a variety of cereals and seeds to validate the accuracy and reproducibility of Flash 4000 N/Protein Analyzer according to AOAC and AACC guidelines with the pre-treatment of the sample. The instrument conditions were as follows:

• Temperature left reactor 950ºC
• Temperature right reactor 840ºC
• Temperature oven 50ºC
• Carrier flow (Helium) 300 ml/min
• Reference flow (Helium) 300 ml/min
• Standard 500 mg
• Ethylenediaminetetraacetic acid (9.59% Nitrogen)
• Sample weight 600 mg to 1.6 g

The Eager Xperience software automatically calculates the protein content using the default protein factor of 6.25 (5.70 for rice). The protein factor can be changed in accordance with the food type.

We determined nitrogen and protein levels in cereals samples first (see Table 1, p. 38). We homogenized barley and rice to particle sizes of 1 mm and 2 mm. The data obtained from 10 consecutive determinations showed excellent reproducibility. In all cases, the relative standard deviation was less than 2% according to official methods. We observed no memory effect when changing sample type, indicating complete detection of the nitrogen present in the sample. We observed no significant differences in the results when changing the sample particle size from 2 to 1 mm.

We then determined nitrogen and protein levels for sunflower seeds (see Table 2, below). Oil seeds, due to the sample nature of high fat content, require a proper optimization of the oxygen amount needed for combustion to obtain accurate data (see Figure 1, p. 38). We homogenized the sample at a particle size of 2 mm. The data were found to be reproducible, with an RSD percentage as per official method requirements. The left side of the table shows the reproducibility of 10 determinations using a weight of around 500 mg, while the right side of the table indicates the reproducibility of 10 determinations in a range from 700 to 1000 mg of the same sunflower sample. We observed no memory effect when changing sample weight.

The new combustion methods, combined with newer technologies like the Flash 4000, eliminate the need for sample preparation and are capable of protein determination that falls within the required accuracy determined by the AOAC and AACC guidelines. In addition, consumable costs associated with sample preparation are minimized and safety in the laboratory is increased. Improved accuracy, day-to-day reproducibility, and high sample throughput make the combustion method faster, safer, and more reliable than the Kjeldahl method.