4. Testing introduced proteins for potential denaturation by heat treatment that occurs during food processing
The potential for a protein to be denatured by heat treatment can be readily assessed in vitro where the protein is heated in aqueous solution at different temperatures ranging from 25 to 100 C for a duration of 15–30 min. A protocol for measuring the heat stability of proteins has been proposed by the Indian Department of Biotechnology (DBT, 2010). Once the protein solution has returned to room temperature, the functional activity of the protein can be assessed through measurement of catalytic activity (enzyme) or biological activity, such as an insect bioassay that measures insect survival and growth after exposure to an insect control protein. It is important to assess functional activity at room temperature to rule out the possibly that the protein might re-nature when cooled, which has been reported for certain proteins (Albillos et al., 2009). SDS–PAGE can also be used to assess degradation and/or post-heating solubility. Enzymologists traditionally use similar in vitro methods to determine the optimum temperature for enzyme function as well as the melting temperature when an enzyme denatures and loses functional activity (Kristjansson and Kinsella, 1991). For proteins where there is no bioassay to measure biological function, such as for structural proteins, it may not be possible to ascertain whether heat treatment causes a loss of function, although the biophysical properties may be indicative of denaturation (e.g., shift in protein solubility, migration on chromatography).