In Vitro Digestibility Assays

One biophysical aspect shared by many, but not all, food allergens is resistance to pepsin digestion in a low-pH environment. The premise on which this assay is based is that the allergen or fragments of the allergen that contain IgE-binding epitopes must be resistant to digestion in the human gastrointestinal (GI) tract and, thus, be available to interact with immune system cells. Standard laboratory conditions have been described whereby proteins are evaluated for their resistance to pH 1.2-2 in the presence of pepsin. Pepsin-digested proteins are loaded onto SDS-PAGE gels and stained with Coomassie blue protein dye to observe peptide fragments that may remain after exposure to acidic conditions. This standard method is performed as part of a multistep assessment of allergens and is referred to as an in vitro simulated gastric fluid (SGF) test.32,33 The purpose of the SGF test is to provide some physical correlation to the probability that a food protein could function as an allergen even after partial destruction during in vivo digestion/proteolysis.34 Food proteins that show complete breakdown would have little or no capacity to present intact IgE-binding epitopes or structures large enough to cause sensitization to the host immune system.

The evaluation of food protein allergens in the SGF test is considered an important aspect of determining protein stability and ability to retain allergenic structure during gut passage.17 As proteins have been introduced into GM crops, there has been interest in describing the stability of the proteins when processed as a food. Attempts to correlate stability of peptide fragments from food allergens with their allergenic potency became prominent as the first GM crop foods came to market in the mid-1990s.32 However, there can be variations in the measured stability of proteins observed in SGF test results due to different techniques, changes in pH, enzyme concentration, protein purity, and matrix.28,35 Although they are generally understood to be standard tests, digestion assays show only a limited feature of the biophysical properties important for a food protein to act as an allergen.

Conclusions as to the presence of stable fragments after in vitro digestion remain a function of the techniques used and the protein in question. To this end, a multisite study was performed by Thomas et al.35 that attempted to standardize the SGF method and evaluate consistency of performance. Results of the study indicated that exact methodology was critical; there was better agreement, 91% versus 77% for digestibility of full-length proteins, using pH 1.2 instead of pH 2.0, respectively. Conclusions from the Thomas et al.27 study indicated that a reproducible, standard method for SGF was possible. Correlating results of enzymatic digestion studies with allergenicity is inherently difficult and it remains prudent to not use these assays in isolation for attempting assessments of food protein allergenicity.36

It should be noted that SGF assays do not mimic the inherently complex digestive process found in the human GI tract. SGF assays are in vitro tests and address only one aspect of the digestion process, pepsin digestion in the stomach. If protein fragments are observed in SGF testing, then it may be appropriate to proceed with additional enzymatic testing such as the simulated intestinal fluid (SIF) assay. The SIF assay addresses another aspect of the digestion process, pancreatin digestion in the small intestine. Sequential enzymatic degradations (SGF followed by SIF digestion) can be utilized to determine whether a protein is likely to survive in the human GI tract long enough to interact with the immune system. Digestibility assays inherently test protein degradation out of context from the intact gut and under-represent the complete digestive process which would affect a protein in vivo. To date there is no validated human digestive model for safety assessments, although these test systems are being developed.

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