67C-23 |
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M. VENKATACHALAM1, S. S. Teuber2, K. H. Roux3, and S. K. Sathe1. (1) Dept. of Nutrition, Food & Exercise Sciences, Florida State Univ., 402 Sandels Bldg., Mail Code 1493, Tallahassee, FL 32306-1493, (2) School of Medicine, Univ. of California, Davis, Div. of Rheumatology, Allergy & Clinical Immunology, 2221 Stockton Blvd., Sacramento, CA 95817, (3) Dept. of Biological Science, Florida State Univ., 335 Biology Unit 1, Mail Code 4370, Tallahassee, FL 32306-4370 Resistance to proteolysis has been suggested by some investigators as one of the tools for predicting protein allergenicity. The suggestion is based on the resistance of allergenic protein to proteolysis under in vitro simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) digestion conditions. Several contradictory results have been reported using such methods. However, in order to simulate human digestive system, variations in enzyme to test protein ratio, pH of the gastric fluid and incubation time need to be taken into account. We therefore determined effects of selected factors on antigenic stability of almond proteins. Proteins were extracted from defatted almond flour with deionized water (pH 8.5). Simulated gastric fluid (SGF) digestion was performed as per United States Pharmacopoeia (USP) protocol. Final digestion conditions were, digestion volume 220 ml, 0.25 mg of protein /ml of SGF, pepsin to protein ratio of 12.5:1, 1:1, 1:25, 1:100, and 1:500 w/w; pH 1.2, 2.0, 3.0, and 4.0; 37°C, and incubation times up to 4 h. Pepsin activity was terminated at the desired time interval by adding 10 ml 2 N NaOH. Substrate proteolysis was monitored by SDS-PAGE. Western blotting, using rabbit polyclonal antibodies raised against whole almond protein extract, was used to assess presence of detectable antigenic polypeptides. At a pepsin: protein ratio of 12.5:1 and a pH range of 1.2 - 4, no antigenic almond peptides could be detected > 5 min of digestion. However, western blotting of digested samples from pepsin:protein ratio (w/w) 1:25, 1:100, and 1:500 (pH range 1.2-4) indicated persistence of antigenic peptides. The data suggest incomplete proteolysis of antigenic proteins/polypeptides or the generation of antigenic proteins/polypeptides not present at the beginning of the substrate digestion. The simulated SGF digestion model as a tool to predict allergenicity of proteins is therefore unreliable and should be used with great caution.
Session 67C, Food Chemistry: Food analysis, irradiation and toxicology
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