Pickles Juliette, Rafiq Samera, Cochrane Stella A, Lalljie Anja
Safety and Environmental Assurance Centre, Unilever, Colworth Science Park, Sharnbrook MK44 1LQ, Bedfordshire, UK.
Toxicol Rep. 2014 Oct 16;1:858-870. doi: 10.1016/j.toxrep.2014.10.008. eCollection 2014.
The introduction of novel proteins to food products carries with it the need to assess the potential allergenicity of such materials. Resistance to pepsin digestion is one parameter considered in such a risk assessment using a weight of evidence approach; however, the methodology used to investigate this has not been fully standardised. pepsin resistance assays typically involve SDS-PAGE performed under reducing conditions, with limited published data available on the assay using non-reducing conditions despite the need to consider non-reducing analysis techniques having been highlighted by regulatory bodies such as the European Food Safety Authority (EFSA). The purpose of the work reported here was to investigate the applicability of (and additional insight provided by) non-reducing analyses, by digesting a set of proteins using a ring-trial validated method, with analysis by SDS-PAGE under both reducing and non-reducing conditions. prediction of digest fragments was also investigated. Significant differences were observed between results under reduced and non-reduced conditions for proteins in which disulphide bonds have a major role in protein structure, such as ribulose 1,5-diphosphate carboxylase (RUBISCO) and bovine serum albumin. For proteins with no or few disulphide bonds, no significant differences were seen in the results. Structural information such as disulphide bond numbers and positions should be considered during experimental design, as a non-reduced approach may be appropriate for some proteins. The approach was a useful tool to suggest potential digest fragments, however the predictions were not always confirmed and should be considered a guide only.
将新型蛋白质引入食品需要评估此类物质的潜在致敏性。在使用证据权重法进行的此类风险评估中，对胃蛋白酶消化的抗性是一个考虑参数；然而，用于研究此参数的方法尚未完全标准化。胃蛋白酶抗性测定通常涉及在还原条件下进行的SDS-PAGE，尽管欧洲食品安全局（EFSA）等监管机构强调需要考虑非还原分析技术，但关于使用非还原条件进行测定的已发表数据有限。本文报道的工作目的是通过使用环试验证的方法消化一组蛋白质，并在还原和非还原条件下通过SDS-PAGE进行分析，研究非还原分析的适用性（以及提供的额外见解）。还研究了消化片段的预测。对于二硫键在蛋白质结构中起主要作用的蛋白质，如核酮糖1,5-二磷酸羧化酶（RUBISCO）和牛血清白蛋白，在还原和非还原条件下的结果之间观察到显著差异。对于没有或几乎没有二硫键的蛋白质，结果没有显著差异。在实验设计过程中应考虑二硫键数量和位置等结构信息，因为非还原方法可能适用于某些蛋白质。该方法是提示潜在消化片段的有用工具，然而预测结果并不总是得到证实，应仅将其视为一种指导。
