For the past several decades, protein analysis from food products has been performed by determining the nitrogen content after complete acid hydrolysis and digestion by the Kjeldahl method followed by an analytical step in which the resulting ammonium ion is quantified by titrimetry, colorimetry, or by the use of an ion-specific electrode.
The result is then multiplied by a pre-established protein conversion factor that determines the final protein content of the sample (Chang 1998, Dierckx and Huyghebaert 2000). While this “wet” analytical technique is still the gold standard in protein analysis, it is time-consuming and involves the use of many dangerous chemicals both to the analyst and to the environment. Its main advantage is that the food sample used in this analytical procedure is considered large enough to be a genuine representative of the entire product.
On the other hand, Dumas combustion is a more recent and faster “dry” analytical instrumental method of determining the protein content in foods and is based on the combustion of a very small sample at 900◦C in the presence of oxygen. The resulting liberated nitrogen gas is analyzed in three minutes by the equipment through built-in programmed processes with the resultant value also multiplied by pre-determined conversion factors, requiring no further analysis or the use of dangerous chemicals. Both of these methods assume that all the nitrogenous compounds in the sample are proteins, but other organic molecules such as nucleotides, nucleic acids, some vitamins, and pigments (e.g., chlorophyll) also contains nitrogen, overestimating the actual protein content of the sample. Both techniques measure crude protein content, not actual protein content.
While the Kjeldahl method is the internationally accepted method of protein determination for legal purposes, Dumas combustion is slowly becoming more acceptable as its accuracy and repeatability will soon be superior to that of the Kjeldahl method (Schmitter and Rihs 1989, Simonne et al. 1997). As far back as the turn of the century, colorimetric methods for protein determination became available with procedures such as the Biuret, Lowry (original and modified), bicinchoninic acid (BCA), Bradford, and ultraviolet (UV) absorption at 280 nm (Bradford 1976). These colorimetric methods exploit the properties of specific proteins, the presence of specific amino acid functional groups, or the presence of peptide bonds.
All require the extraction, isolation, and sometimes purification of the protein molecule of interest to attain an accurate absorbance reading. Considering that the nutrients in foods exist in complex matrices, these colorimetric methods are not practical for food analysis. Additionally, only a few dye-binding methods (official methods 967–12 and 975–17) have been approved for the direct determination of protein in milk (AOAC 1995).