2S albumin storage proteins are becoming of increasing interest in nutritional and clinical studies as they have been reported as major food allergens in seeds of many mono- and di-cotyledonous plants. This review describes the main biochemical, structural and functional properties of these proteins thought to play a role in determining their potential allergenicity. 2S albumins are considered to sensitize directly via the gastrointestinal tract (GIT). The high stability of their intrinsic protein structure, dominated by a well-conserved skeleton of cysteine residues, to the harsh conditions present in the GIT suggests that these proteins are able to cross the gut mucosal barrier to sensitize the mucosal immune system and/or elicit an allergic response. The flexible and solvent-exposed hypervariable region of these proteins is immunodominant and has the ability to bind IgE from allergic patients´ sera. Several linear IgE-binding epitopes of 2S albumins spanning this region have been described to play a major role in allergenicity; the role of conformational epitopes of these proteins in food allergy is far from being understood and need to be investigated. Finally, the interaction of these proteins with other components of the food matrix might influence the absorption rates of immunologically reactive 2S albumins but also in their immune response.

This document provides supplementary guidance on specific topics for the allergenicity risk assessment of genetically modified plants. In particular, it supplements general recommendations outlined in previous EFSA GMO Panel guidelines and Implementing Regulation (EU) No 503/2013. The topics addressed are non-IgE-mediated adverse immune reactions to foods, in vitro protein digestibility tests and endogenous allergenicity. New scientific and regulatory developments regarding these three topics are described in this document. Considerations on the practical implementation of those developments in the risk assessment of genetically modified plants are discussed and recommended, where appropriate. (C) 2017 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority

2S storage albumins occur in a diverse range of plant seeds, are members of the prolamin superfamily [1] and constitute one of the most important major plant food allergens that sensitize via the gastrointestinal (GI) tract [2]. Among the tree nuts, Brazil nut is frequently associated with immunoglobulin E (IgE)-mediated food allergy [3], the 2S albumin, known as Ber e 1, being the major allergen [4]. 2S albumins are considered to be structurally homologous, typically heterodimeric (small and large subunits of 4000 and 9000 M r , respectively) globular proteins. They have a conserved skeleton of cysteine residues (typical of members of the prolamin superfamily), which form four intermolecular disulphide bonds that hold the two subunits together and contribute to their stability and compactness [5]. This rigid The major 2S albumin allergen from Brazil nuts, Ber e 1, was subjected to gastrointestinal digestion using a physiologically relevant in vitro model system either before or after heating (100°C for 20 min). Whilst the albumin was cleaved into peptides, these were held together in a much larger structure even when digested by using a simulated phase 1 (gastric) followed by a phase 2 (duodenal) digestion system. Neither prior heating of Ber e 1 nor the presence of the physiological surfactant phosphatidylcholine affected the pattern of proteolysis. After 2 h of gastric digestion, 25% of the allergen remained intact, 50% corresponded to a large fragment of M r 6400, and the remainder comprised smaller peptides. During duodenal digestion, residual intact 2S albumin disappeared quickly, but a modified form of the 'large fragment' remained, even after 2 h of digestion, with a mass of 5000 Da. The 'large fragment' comprised several smaller peptides that were identified, by using different MS techniques, as deriving from the large subunit. In particular, sequences corresponding to the hypervariable region (Q37-M47) and to another peptide (P42-P69), spanning the main immunoglobulin E epitope region of 2S albumin allergens, were found to be largely intact following phase 1 (gastric) digestion. They also contained previously identified putative T-cell epitopes. These findings indicate that the characteristic conserved skeleton of cysteine residues of 2S albumin family and, particularly, the intrachain disulphide bond pattern of the large subunit, play a critical role in holding the core protein structure together even after extensive proteolysis, and the resulting structures still contain potentially active B-and T-cell epitopes.Abbreviations GI, gastrointestinal; IgE, immunoglobulin E; SGF, simulated gastric fluid; PtdCho, egg l-phosphatidylcholine.

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Interactions with food components may alter the resistance of food proteins to digestion, a property thought to play an important role in determining allergenic properties. The kinetics of breakdown of the bovine milk allergen alpha-lactalbumin during in vitro gastrointestinal digestion was found to be altered by interactions with physiologically relevant levels of phosphatidylcholine (PC), a surfactant that is abundant both in milk and is actively secreted by the stomach. Breakdown during gastric digestion was slowed in the presence of PC and accompanied by small alterations in the profile of resulting peptides, with little effect being observed during subsequent duodenal digestion. alpha-Lactalbumin was found to unfold at gastric (acid) pH, giving a CD spectrum similar to that obtained for the partially folded state it is known to adopt at pH values below its isoelectric point. Fluorescence polarization studies performed at low pH indicated that this partially unfolded form of the protein was able to penetrate into the PC vesicles. These interactions are probably responsible for the slowing of gastric digestion by reducing the accessibility of the protein to pepsin. These findings show that interactions with other food components, such as lipids, may alter the rate of breakdown of food proteins in the gastrointestinal tract. It underlines the importance of the food matrix in affecting patterns of food allergen digestion and hence presentation to the immune system and that in vitro digestion systems used for assessing digestibility of allergens must take account of surfactants.

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24Resistance to gastrointestinal conditions is a requirement for bacteria to be 25 considered probiotics. In this work, we tested the resistance of six different Lactobacillus 26 strains and the effect of carbon source to four different gastrointestinal conditions: presence 27 of α-amylase, pancreatin, bile extract and low pH. Novel galactooligosaccharides 28 synthesized from lactulose (GOS-Lu) as well as commercial galactooligosaccharides 29 synthesized from lactose (GOS-La) and lactulose were used as carbon sources and 30 compared with glucose. In general, all strains grew in all carbon sources, although after 24 31 h of fermentation the population of all Lactobacillus strains was higher for both types of 32 GOS than for glucose and lactulose. No differences were found among GOS-Lu and GOS-33La. α-amylase and pancreatin resistance was retained at all times for all strains. However, a 34 dependence on carbon source and Lactobacillus strain was observed for bile extract and 35 low pH resistance. High hydrophobicity was found for all strains with GOS-Lu when 36 compared with other carbon sources. However, concentrations of lactic and acetic acid 37 were higher in glucose and lactulose than GOS-Lu and GOS-La. These results show that 38 the resistance to gastrointestinal conditions and hydrophobicity is directly related with the 39 carbon source and Lactobacillus strains. In this sense, the use of prebiotics as GOS and 40 lactulose could be an excellent alternative to monosaccharides to support growth of 41 probiotic Lactobacillus strains and improve their survival through the gastrointestinal tract. 42 43

Bowman-Birk inhibitors (BBI) from soybean and related proteins are naturally occurring protease inhibitors with potential health-promoting properties within the gastrointestinal tract. In this work, we have investigated the effects of soybean BBI proteins on HT29 colon adenocarcinoma cells, compared with non-malignant colonic fibroblast CCD-18Co cells. Two major soybean isoinhibitors, IBB1 and IBBD2, showing considerable amino acid sequence divergence within their inhibitory domains, were purified in order to examine their functional properties, including their individual effects on the proliferation of HT29 colon cancer cells. IBB1 inhibited both trypsin and chymotrypsin whereas IBBD2 inhibited trypsin only. Despite showing significant differences in their enzyme inhibitory properties, the median inhibitory concentration values determined for IBB1 and IBBD2 on HT29 cell growth were not significantly different (39.9+/-2.3 and 48.3+/-3.5 microM, respectively). The cell cycle distribution pattern of HT29 colon cancer cells was affected by BBI treatment in a dose-dependent manner, with cells becoming blocked in the G0-G1 phase. Chemically inactive soybean BBI had a weak but non-significant effect on the proliferation of HT29 cells. The anti-proliferative properties of BBI isoinhibitors from soybean reveal that both trypsin- and chymotrypsin-like proteases involved in carcinogenesis should be considered as potential targets of BBI-like proteins.