Establishing objective detection limits for the pepsin digestion assay used in the assessment of genetically modified foods

Outcome of the public consultation on the draft guidance on allergenicity assessment of genetically modified plants

“…And the highly varied solute and enzyme forms do not provide a useful tool. The current model (Thomas et al, 2004;Ofori-Anti et al, 2008) use commonly available pepsin of specific activity and purity and use a standard set of conditions. If one wants to consider modifying conditions, it can be done, but to what end?…”

“…(Not higher). In addition, the protein should be tested at the recommended concentration (1 mg protein in 10 kUnits pepsin) and with activity measurement of the pepsin (Ofori-Anti et al, 2008). The protein quantity should be gauged to ensure high level digestion and survival of digestion resistant fragments should be noted.…”

Safety assessment of the calcium-binding protein, apoaequorin, expressed by Escherichia coli

“…The assay described by Astwood et al (1996) was refined further; adding an objective measurement of the extent of digestion as described by Ofori-Anti et al (2008). The assay was performed under standard conditions of 10 units of pepsin activity per microgram of test protein using a high purity form of pepsin from Worthington Biochemical, Corp., The mass ratio was approximately 3.6 mg pepsin per mg of test protein and other quality control measures as recommended by Ofori-Anti et al (2008) were followed.…”

“…Thomas et al (2004) published a method standardizing the pH, purity of the protease, ratio of pepsin to test article, and purity of the test protein. We (Ofori-Anti et al, 2008) added an objective measure of the degree of digestion to improve the evaluation for assessing digestibility. However, although a correlation exists between allergenicity of dietary proteins and pepsin resistance (Astwood et al, 1996) some unstable allergens may be protected in the food matrix of the gut in vivo (Polovic et al, 2007).…”

Enzymatic and Chemical Modifications of Food Allergens

“…Thomas et al (Thomas et al, 2008) demonstrated that common protocol for evaluating the in vitro digestibility of proteins is reproducible and yields consistent results when performed using the same proteins at different laboratories. However, digestibility assays of pure proteins may not be always relevant in assessing allergenicity potential as they do not account for the effects of food matrices (Ofori-Anti et al, 2008). Therefore, in vitro gastrointestinal digestion protocols should be preferably combined with immunological assays in order to elucidate the role of large digestion-resistant fragments and the influence of the food matrix (Moreno, 2007).…”