In vitro digestion stability tests

Digestive stability testing was performed in a mock in vitro digestive environment simulating both a gastric and intestinal fluid. The goal of these tests is to estimate the rate of protein degradation or denaturation in the human digestive systems. Proteins that are rapidly denatured (by low pH) and enzymatically digested (by intestinal enzymes) have a lower probability of producing either toxicity since digestion products are common peptides or dietary amino acids or allergenicity because the protein is not present to elicit antibody production or allergic reactions. Cry1Ab, Cry1Ac, NPTII, and Bar are all degraded quickly in these in vitro mock digestive solutions, making them significantly less likely to exhibit local or systemic toxicity or allergenicity, as protein exposure is transient. The conclusions from the results of these tests, as concluded by EPA, for the Cry1Ab, Cry1Ac, NPTII, and Bar proteins, are provided below.

“The in vitro digestion assays confirm that the protein is being broken down in the presence of typical digestive fluids and is not unusually persistent in the digestive system. All were degraded in gastric fluid in 0–7 min” (Kough et al., 2010).

“The Cry1Ac protein was digested within 30 s in simulated gastric fluid containing pepsin. Small peptides remaining following gastric simulated digestion were completely degraded to amino acid residues in SIF (simulated intestinal fluid) upon contact” (Kough et al., 2010).

“NPTII degrades extremely readily in SGF (simulated gastric fluid). No NPTII protein was detected, by western blot analysis, at the first incubation time point of 10 s. In SIF (simulated intestinal fluid), NPTII also degrades readily with 50% degradation occurring after 2–5 min of incubation at 37°C.” (Fuchs et al., 1993)

“Bar proteins were degraded very rapidly and completely in the SGF (simulated gastric fluid) (pH 2) or SIF (simulated intestinal fluid) (pH 7.5), within few seconds of incubation, in the presence of pepsin or pancreatin, respectively. … In the SIF (simulated intestinal fluid) assay, the complete degradation of remaining 7-kDa fragments was achieved within 5 min rather than a few seconds.” (Hérouet et al., 2004).