Author/Authors :
Thomas، نويسنده , , K. and Aalbers، نويسنده , , M. and Bannon، نويسنده , , G.A. and Bartels، نويسنده , , M. and Dearman، نويسنده , , R.J. and Esdaile، نويسنده , , D.J. and Fu، نويسنده , , T.J. and Glatt، نويسنده , , C.M. and Hadfield، نويسنده , , N. and Hatzos، نويسنده , , C. and Hefle، نويسنده , , S.L. and Heylings، نويسنده , , J.R. and Goodman، نويسنده , , R.E. and Henry، نويسنده , , B. and Herouet، نويسنده , , C. W. Holsapple، نويسنده , , M. and Ladics، نويسنده , , G.S. and Landry، نويسنده , , T.D. and MacIntosh، نويسنده , , S.C. and Rice، نويسنده , , E.A. and Privalle، نويسنده , , L.S. and Steiner، نويسنده , , H.Y. and Teshima، نويسنده , , R. and van Ree، نويسنده , , R. and Woolhiser، نويسنده , , M. and Zawodny، نويسنده , , J.، نويسنده ,
Abstract :
Rationale. Evaluation of the potential allergenicity of proteins derived from genetically modified foods has involved a weight of evidence approach that incorporates an evaluation of protein digestibility in pepsin. Currently, there is no standardized protocol to assess the digestibility of proteins using simulated gastric fluid. Potential variations in assay parameters include: pH, pepsin purity, pepsin to target protein ratio, target protein purity, and method of detection. The objective was to assess the digestibility of a common set of proteins in nine independent laboratories to determine the reproducibility of the assay when performed using a common protocol.
s. A single lot of each test protein and pepsin was obtained and distributed to each laboratory. The test proteins consisted of Ara h 2 (a peanut conglutin-like protein), β-lactoglobulin, bovine serum albumin, concanavalin A, horseradish peroxidase, ovalbumin, ovomucoid, phosphinothricin acetyltransferase, ribulose diphosphate carboxylase, and soybean trypsin inhibitor. A ratio of 10 U of pepsin activity/μg test protein was selected for all tests (3:1 pepsin to protein, w:w). Digestions were performed at pH 1.2 and 2.0, with sampling at 0.5, 2, 5, 10, 20, 30, and 60 min. Protein digestibility was assessed from stained gels following SDS–PAGE of digestion samples and controls.
s. Results were relatively consistent across laboratories for the full-length proteins. The identification of proteolytic fragments was less consistent, being affected by different fixation and staining methods. Overall, assay pH did not influence the time to disappearance of the full-length protein or protein fragments, however, results across laboratories were more consistent at pH 1.2 (91% agreement) than pH 2.0 (77%).
sions. These data demonstrate that this 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.
