Establishing objective detection limits for the pepsin digestion assay used in the assessment of genetically modified foods.
BACKGROUND
Guidelines for assessing the potential allergenicity of genetically modified (GM) organisms recommend testing the digestibility of the introduced protein by pepsin. Previous studies detailed the digestion procedure but have not described a simple objective measurement of the extent of digestion nor evaluated the impact of variation in pepsin activity.


METHODS
Samples of eight proteins were digested by pepsin at pH 1.2 and 2.0 using standard conditions (10,000 U of pepsin activity per mg test protein) as well as 5000 and 20,000 units per mg of test protein. An independent digestion assay of hemoglobin was used to verify pepsin activity for each assay. Digestion was stopped in timed samples between 0.5 and 60 min. Digestion samples and undigested protein (10% and 100%) were separated by SDS-PAGE. Residual stained protein bands were measured by image analysis.


RESULTS
The differences in pH and pepsin concentration only had minor effects on digestion of intermediately stable proteins: concanavalin A, ovalbumin, and lysozyme, but not on rapidly digested or stable proteins.


CONCLUSIONS
Verification of pepsin activity and measurement of an objective endpoint of digestion (e.g. (90%) should provide more comparable results for the safety assessment of novel food proteins.
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Food Allergy Research and Resource Program (FARRP), Department of Food Science & Technology, University of Nebraska-Lincoln, 143 Food Industry Complex, Lincoln, NE 68583-0955, USA.
Ariyarathna
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Chen
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Lee
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HL
Pramod
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SN
Goodman
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D000485:Allergens / Q000276:immunology / Q000633:toxicity; D000818:Animals; D004063:Digestion; D004591:Electrophoresis, Polyacrylamide Gel; D005512:Food Hypersensitivity / Q000175:diagnosis* / Q000209:etiology; D030841:Food, Genetically Modified / Q000633:toxicity*; D017408:Guidelines as Topic; D006454:Hemoglobins / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007091:Image Processing, Computer-Assisted; D010434:Pepsin A / Q000378:metabolism*; D013552:Swine; D013997:Time Factors; D018675:Toxicity Tests / Q000379:methods
D000485:Allergens; D006454:Hemoglobins; D010434:Pepsin A