Abstract
The susceptibility of newly expressed proteins to digestion by gastrointestinal proteases (e.g., pepsin) has long been regarded as one of the important endpoints in the weight-of-evidence (WOE) approach to assess the allergenic risk of genetically modified (GM) crops. The European Food Safety Authority (EFSA) has suggested that current digestion study protocols used for this assessment should be modified to more accurately reflect the diverse physiological conditions encountered in human populations and that the post-digestion analysis should include analytical methods to detect small peptide digestion products.The susceptibility of two allergens (beta-lactoglobin (β-Lg) and alpha-lactalbumin (α-La)) and two non-allergens (hemoglobin (Hb) and phosphofructokinase (PFK)) to proteolytic degradation was investigated under two pepsin digestion conditions (optimal pepsin digestion condition: pH 1.2, 10 U pepsin/μg test protein; sub-optimal pepsin digestion condition: pH 5.0, 1 U pepsin/10 mg test protein), followed by 34.5 U trypsin/mg test protein and 0.4 U chymotrypsin/mg test protein digestion in the absence or presence of bile salts. All samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in conjunction with Coomassie Blue staining and, in parallel, liquid chromatography tandem mass spectrometry (LC-MS) detection. The results provide following insights: 1) LC-MS methodology does provide the detection of small peptides; 2) Peptides are detected in both allergens and non-allergens from all digestion conditions; 3) No clear differences among the peptides detected from allergen and non-allergens; 4) The differences observed in SDS-PAGE between the optimal and sub-optimal pepsin digestion conditions are expected and align with kinetics and properties of the specific enzymes; 5) The new methodology with new digestion conditions and LC-MS detection does not provide any differentiating information for prediction whether a protein is an allergen. The classic pepsin resistance assay remains the most useful assessment of the potential exposure of an intact newly expressed protein as part of product safety assessment within a WOE approach.
Highlights
Assessment of the potential of a newly express protein (NEP) to be allergenic is one component of food and feed safety assessment for genetically modified (GM) foods
The results provide following insights: 1) liquid chromatograph-mass spectrometry (LC-MS) methodology does provide the detection of small peptides; 2) Peptides are detected in both allergens and non-allergens from all digestion conditions; 3) No clear differences among the peptides detected from allergen and non-allergens; 4) The differences observed in SDS-PAGE between the optimal and sub-optimal pepsin digestion conditions are expected and align with kinetics and properties of the specific enzymes; 5) The new methodology with new digestion conditions and LC-MS detection does not provide any differentiating information for prediction whether a protein is an allergen
SDS-PAGE followed by Coomassie blue staining analysis demonstrated that intact Hb, a nonallergen, was degraded under pepsin optimal conditions, pH 1.2 pepsin-to-protein ratios (PPR) 10 for 60 minutes, as evidenced by the complete disappearance of the protein band at ~14 kDa on the SDS-PAGE gel (Fig 2)
Summary
Assessment of the potential of a newly express protein (NEP) to be allergenic is one component of food and feed safety assessment for genetically modified (GM) foods. A weight-of-evidence (WOE) approach is utilized to assess the allergenic potential of a NEP [1,2,3]. This approach includes an evaluation of the history of safe use of the protein, or closely related homologs, comparisons of the NEP amino acid sequence to those of known allergens, an understanding of the mode-of-action of the NEP, and assessments of the NEP’s susceptibility to degradation by gastrointestinal proteases.
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