Literature review: 'in vitro digestibility tests for allergenicity assessment'
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A systematic review of in silico and in vitro digestibility tests has been undertaken which identified a lack of harmonised test conditions that made comparison of test results difficult. Specific issues that should be considered further include a need for a clear description of the source and specific activity of the proteases used for the assays. Many reports only describe the pepsin:protein ratio on a (w:w) basis and do not take account differences in enzyme activity despite being a key descriptor for any digestion test. Given that that “batch” assays cannot replicate the dynamic digestive process it may be necessary to consider including several analyses undertaken using pH conditions and pepsin:protein ratios that represent physiological ranges. In this way the tests can be readily adapted to take into account individuals with impaired digestive capacity or the widespread use of ant-acids. In addition, the time course and sampling regime employed need to be harmonised and standardised methodology identified for monitoring protein digestion. Digestion needs to be followed using methods suitable for profiling of both large resistant fragments and lower molecular weight peptides. Such standardisation could build on the harmonised approaches developed by the proteomics community regarding, for example, optimal SDS-PAGE and trypsin digestion conditions and replication. This is a prerequisite before a consensus can be agreed as to what is meant by “resistance” to digestion. There was a lack of inclusion of comparator and standardised proteins to be included in tests. Based on published data a suggestion is made regarding a potential “reference” set of allergens, most of which can be purchased. However, whilst “non-allergenic” comparator proteins can be identified they are less readily sourced.