Other methods

While the Kjeldahl and Dumas method determines total Nitrogen, there are also methods available which determine protein directly. Since these methods are almost never used to determine values for FCDB, these methods are not covered in depth.

UV-(N)IR-Spectroscopy

A number of spectroscopy methods can be used to measure protein content directly. The basic principles behind these methods are similar: They use the ability of protein to absorb or scatter light in the ultraviolet (205 - 280 nm) and/or infrared (0.75-16 µm) or near infrared region. First a calibration curve of the absorbance of known protein concentrations is prepared. Then the absorbance of the solution being analysed is measured and its protein concentration is determined from the calibration curve. These approaches are in general rapid and non destructive. The calibration curves to be used depend on the type of protein that has to be determined.

Colorimetric methods

Colorimetric assays are based on the interaction between characteristic groups in amino acids or peptide bonds with a reagent.

As a result, a color is formed; the intensity of the color is proportionally to the protein contents. Common used colorimetric methods are:

• The Biuret Method: Biuret reagent (contain, among others, cupric (Cu2+) ions. These ions interact with peptide bonds and produce a violet-purplish color (more information).

• The Lowry method: This method uses Biuret reagent + another reagent (Folin-Ciocalteu) that interacts with tyrosine and tryptophan. This gives a bluish color. This method is more sensitive to low protein concentrations than the Biuret method.

• Dye binding methods: A known amount of a negatively charged dye is added to a protein solution. The proteins form an insoluble complex with the dye. The unbound dye remains soluble. The amount of unbound dye is determined by measuring its absorbance (after removal of the insoluble protein-dye complex), and is a measure of the amount of protein.

• Turbimetric methods: Proteins that are normally soluble in solution can be made to precipitate by addition of certain chemicals. Protein precipitation causes the solution to become turbid (). The degree of turbidity is a measure of protein concentration.

General remarks

UV-(N)IR-spectroscopy and colorimetric techniques are rapid and can be used routinely. These methods are sensitive to low concentrations of protein. However there are no ISO standards available for these methods. A disadvantage of these methods is the condition of transparent solutions which for some foods mean an unavoidable significant amount of sample preparation before analysis. Furthermore, interfering compounds in protein determination are carbohydrates, salts, fats and detergents. Also the amino acids composition of a particular protein can influence the response of protein, e.g. on colour development in a dye- binding assay. Therefore the calibration of the assay using an appropriate protein mixture is recommended in order to obtain accurate measurements. The Kjeldahl method is applied as a reference method to calibrate direct methods of protein determination. Therefore, in fact these methods can be seen as Kjeldahl methods.

These cupper ions in the Biuret reagent interact with peptide bonds and produce a violet-purplish color