Influence of immune activity of Cor a 9 from raw and roasted hazelnuts after gastric digestion

Cor a 9 is one of the most common hazelnut allergen, a non-glycosylated protein, consisting of two subunits, an acidic (ranging between 35-40 kDa) and a basic subunit (ranging between 20-25 kDa). Very important fact is that the acid chain carries the immunoreactivity, according to literature. The survival of large fragments of Cor a 9 is necessary for its ability to sensitize individual. The aim of this study was to investigate Cor a 9, and to compare the digestive stability and allergenicity of large and small peptides released after pepsin digestion of whole raw and roasted hazelnut grains in standardized and physiologically relevant in vitro conditions, after heat treatment (roasting as the most abundant type of heat treatment). In vitro simulated phases of oral and gastric digestion were performed with ground raw and roasted hazelnut kernels according to the 1.0 INFOGEST protocol. After digestion proteins were extracted from the digestion mixture and analysed by 1D and 2D SDS-PAGE, while their IgE test was examined in the sera of allergic patients using ELISA and 2D immunoblot. The focus of the research was on the analysis of the 2DE map by Image 2D Master Platinum 7.0 software, comparing region of acid and basic Cor a 9 from raw and roasted hazelnut. Cor a 9 peptides are resistant to gastric digestion, and are able to bind IgE patients. Roasted hazelnuts are more prone to digestion in the stomach than the raw sample and cause a milder IgE response in patients. The gastric digestion phase of raw and roasted hazelnut grains resulted in partial extraction and digestion of Cor a 9 into digestion-resistant peptides with preserved IgE-binding epitopes. These results show significant resistance of Cor a 9 raw and roasted hazelnuts to digestion in the stomach, as they remained mostly intact after 2 hours of gastric (pepsin) phase and retained their allergenicity

Influence of Raw and Roasted Hazelnut Food Matrix on Ige Binding Activity After Application of the Harmonized Static Digestion Protocol

Most of the hazelnut proteins are resistant to proteolysis in the gastrointestinal tract, and the survival of their large fragments are essential for their sensitizing capacity. Usually, studies were carried out on purified proteins, paying no attention to the potential impact of the food matrix and thermal treatment on allergenicity. Obtained hazelnut peptides after gastric digestion, especially those with potential IgE binding epitopes, highlight the need for further studies on their IgE reactivity. The aim of this study was to investigate and compare digestion stability and allergenicity of large and small peptides released after pepsin digestion of whole raw and roasted hazelnut kernels under standardized and physiologically relevant in vitro conditions, after thermal treatment (roasting as most abundant type of thermal treatment). In vitro simulated oral and gastric phase digestion was carried out with ground raw and roasted hazelnut kernels according to INFOGEST protocol. Digested proteins were extracted from the digestion mixture and analysed by 1D and 2D SDS-PAGE, while their IgE biding was probed with allergic patients’ sera via ELISA and 2D immunoblot. The most abundant hazelnut allergens within 2DE map were acidic and basic chains of Cor a 9 and Cor a 11. Digestion-resistant peptides of Cor a 11 and Cor a 9 were able to bind patients’ IgE. Roasted hazelnut is more prone to gastric digestion than the raw sample, and cause milder IgE response in patients. Gastric phase digestion of raw and roasted hazelnut kernels resulted in partial extraction and digestion of Cor a 11 and Cor a 9 into digestion-resistant peptides with preserved IgE-binding epitopes. These results demonstrate substantial resistance of raw and roasted hazelnut allergens to gastric digestion since they remained mostly intact after 2 h of gastric (pepsin) phase and retained their allergenicity

Allergome of oral-gastric in vitro digest of roasted hazelnut shows stronger IgE binding compared to the raw counterpart

Background: In vitro pepsin digestion is important factor when assessing protein food allergenicity. Roasted hazelnut is more common in human nutrition than a raw hazelnut; however, all studies were focused on Cor a 9 allergen obtained from a raw hazelnut. There are only two studies employing in vitro INFOGEST digestion harmonized protocol on hazelnut with its full matrix. The aim of this study was to assess immunoreactivity of raw and roasted hazelnut gastric digests and to compare secondary/tertiary structure of Cor a 9 allergen purified from these two sources. Methods: Digestion resistant protein fragments were analysed by 1D/2D electrophoresis. Following digestion, IgE binding from patients’ pooled sera and by specific antibodies, were assessed in ELISA and immunoblot. CD spectroscopy was applied for Cor a 9 structural analyses. Results: Cor a 11 and acidic forms of Cor a 9 were more prone to pepsin proteolysis, yet their large fragments survived partially. Cor a 8 was protected by lipids, retaining capability to bind its specific antibody. Roasting did not significantly affect secondary structure of the most abundant hazelnut allergen, Cor a 9. Conclusion: Roasting of hazelnut seems to boost IgE binding derived from pooled sera of hazelnut allergic patients with oral-gastric allergen digests.Related to Book of Abstracts: [https://www.cost-infogest.eu/content/download/4051/35805/file/V-ICFD%20Book%20of%20Abstracts.pdf]Related to record of lecture: [https://www.youtube.com/watch?v=Dj0_1fyY724

INFOGEST Digestion Assay of Raw and Roasted Hazelnuts and Its Impact on Allergens and Their IgE Binding Activity

Most of the food allergens sensitized via the gastrointestinal tract resist thermal treatments and digestion, particularly digestion by pepsin. Roasted hazelnuts are more commonly consumed than raw ones. Since no studies have characterized gastric digestion protein fragments of raw and roasted hazelnuts nor their IgE binding properties, we compared these aspects of raw and roasted hazelnuts’ gastric digesta obtained by INFOGEST protocol. Their electrophoretically resolved profiles were probed with hazelnut allergic patients’ sera in 1D and 2D immunoblots. Electrophoretic profiles demonstrated pepsin digestion of all hazelnut allergens to varying extents. While 2D immunoblots indicated that roasting slightly reduced allergenicity, IgE ELISA with the pool of sera showed a slight significant (10%) increase in IgE binding in both gastric digesta. Cor a 9 isolated from the raw and roasted hazelnuts, characterized by far and near CD, remained stable after roasting, with preserved IgE reactivity. Its immunoreactivity contribution by inhibitory ELISA was noticeable in raw and roasted hazelnut digesta; its activity was slightly stronger in the roasted preparations. Roasting has a visible impact on proteins; however, it did not affect overall IgE reactivity. Gastric digestion slightly increases the overall IgE reactivity in raw and roasted hazelnuts, and may therefore impact the profiles of allergens and their fragments available to interact with the immune system in the small intestine

Expression, Purification and Crystallization of Wheat Profilin (Tri a 12)

Profilin from wheat (Triticum aestivum) has been identified as an allergen (Tri a 12). The recombinant 14 kDa protein was produced in Escherichia coli, purified and crystallized using the hangingdrop vapour-diffusion method. A diffraction-data set was collected in-house from a single crystal to a resolution of 3.3 Å. The crystals belonged to space group P3221, with unit-cell parameters a = b = 58.9 Å, c = 82.5 Å, α = β = 90° and γ = 120°. (doi: 10.5562/cca1790

Digestomics of raw and roasted hazelnut according to harmonized static digestion method suitable for solid food and characterization of gastric-phase products

Introduction Stability to gastric digestion represents a very important parameter of food protein allergenicity. Usu- ally digestion experiments are carried out on purified proteins or protein extracts; however, use of solid food is far closer to the in vivo situation, taking into account food protein interactions with other food components, such as polyphenols and lipids. Objective The aim of this study was to investigate and compare digestion stability and allergenicity of large and small peptides released after pepsin digestion of whole raw and roasted hazelnut kernels under standardized and physiologically relevant in vitro conditions. Methodology In vitro simulated oral and gastric phase digestion was carried out with ground raw and roasted hazelnut kernels. Digested proteins were extracted from the mixture and analyzed by SDS-PAGE, 2D-PAGE, and compared with Image Master 2D Platinum 7.0. Western blot probed with allergic patients’ sera and specific antibodies for Cor a 8. Main findings Several important hazelnut seed storage digestion resistant proteins and peptides have been identi- fied and characterized. Most abundant hazelnut allergens were resolved on a 2DE map, for instance, acidic and basic chains of Cor a 9, and Cor a 11. Digestion-resistant peptides of Cor a 11 and Cor a 9 were able to bind IgE. Lipid transfer protein (Cor a 8) was highly resistant to gastric proteolysis. Conclusion To conclude, roasted hazelnut is more prone to gastric digestion than raw, and cause milder IgE response in patients. Gastric phase digestion of raw and roasted hazelnut kernels results in partial extraction and digestion of Cor a 11 and Cor a 9 into digestion-resistant peptides with preserved IgE-binding epitopes. These results demonstrate substantial resistance of raw and roasted hazelnut allergens to gastric digestion since they remained mostly intact after 2 h of gastric (pepsin) digestion and retained their allergenicity

Digestomics of raw and roasted hazelnut according to Infogest protocol and characterization of gastric-phase products

Brief introduction: Stability to gastric digestion represents a very important parameter of food protein allergenicity. Usually digestion experiments are carried out on purified proteins or protein extracts; however, use of solid food is far closer to the in vivo situation, taking into account food protein interactions with other food components, such as polyphenols and lipids. Objective: The aim of this study was to investigate and compare digestion stability and allergenicity of large and small peptides released after pepsin digestion of whole raw and roasted hazelnut kernels under standardized and physiologically relevant in vitro conditions. Methodology: In vitro simulated oral and gastric phase digestion was carried out with ground raw and roasted hazelnut kernels. Digested proteins were extracted from the mixture and analyzed by SDS-PAGE, 2D-PAGE, and compared with Image Master 2D Platinum 7.0. Western blot probed with allergic patients’ sera and specific antibodies for Cor a 8. Main findings: Several important hazelnut seed storage digestion resistant proteins and peptides have been identified and characterized. Most abundant hazelnut allergens were resolved on a 2DE map, for instance acidic and basic chains of Cor a 9, and Cor a 11. Digestion-resistant peptides of Cor a 11 and Cor a 9 were able to bind IgE. Lipid transfer protein (Cor a 8) was highly resistant to gastric proteolysis. Conclusion: To conclude, roasted hazelnut is more prone to gastric digestion than raw, and cause milder IgE response in patients. Gastric phase digestion of raw and roasted hazelnut kernels results in partial extraction and digestion of Cor a 11 and Cor a 9 into digestion- resistant peptides with preserved IgE-binding epitopes. These results demonstrate substantial resistance of raw and roasted hazelnut allergens to gastric digestion since they remained mostly intact after 2 h of gastric (pepsin) digestion and retained their allergenicity

Non-specific lipid-transfer proteins: Allergen structure and function, cross-reactivity, sensitization, and epidemiology

Al·lèrgia; Epidemiologia; Proteïna de transferència de lípidsAlergia; Epidemiología; Proteína de transferencia de lípidosAllergy; Epidemiology; Lipid transfer proteinBackground Discovered and described 40 years ago, non-specific lipid transfer proteins (nsLTP) are present in many plant species and play an important role protecting plants from stressors such as heat or drought. In the last 20 years, sensitization to nsLTP and consequent reactions to plant foods has become an increasing concern. Aim The aim of this paper is to review the evidence for the structure and function of nsLTP allergens, and cross-reactivity, sensitization, and epidemiology of nsLTP allergy. Materials and Methods A Task Force, supported by the European Academy of Allergy & Clinical Immunology (EAACI), reviewed current evidence and provide a signpost for future research. The search terms for this paper were “Non-specific Lipid Transfer Proteins”, “LTP syndrome”, “Pru p 3”, “plant food allergy”, “pollen-food syndrome”. Results Most nsLTP allergens have a highly conserved structure stabilised by 4-disulphide bridges. Studies on the peach nsLTP, Pru p 3, demonstrate that nsLTPs are very cross-reactive, with the four major IgE epitopes of Pru p 3 being shared by nsLTP from other botanically related fruits. These nsLTP allergens are to varying degrees resistant to heat and digestion, and sensitization may occur through the oral, inhaled or cutaneous routes. In some populations, Pru p 3 is the primary and sole sensitizing allergen, but many are poly-sensitised both to botanically un-related nsLTP in foods, and non-food sources of nsLTP such as Cannabis sativa, Platanus acerifolia, (plane tree), Ambrosia artemisiifolia (ragweed) and Artemisia vulgaris (mugwort). Initially, nsLTP sensitization appeared to be limited to Mediterranean countries, however more recent studies suggest clinically relevant sensitization occurs in North Atlantic regions and also countries in Northern Europe, with nsLTP sensitisation profiles being broadly similar. Discussion These robust allergens have the potential to sensitize and provoke symptoms to a large number of plant foods, including those which are raw, cooked or processed. It is unknown why some sensitized individuals develop clinical symptoms to foods whereas others do not, or indeed what other allergens besides Pru p 3 may be primary sensitising allergens. It is clear that these allergens are also relevant in non-Mediterranean populations and there needs to be more recognition of this. Conclusion Non-specific LTP allergens, present in a wide variety of plant foods and pollens, are structurally robust and so may be present in both raw and cooked foods. More studies are needed to understand routes of sensitization and the world-wide prevalence of clinical symptoms associated with sensitization to these complex allergens