IntroductionTwo of the major unanswered questions in food allergy research are what makes one person, and not another, become allergic and what are the attributes of some foods and food proteins that make them more allergenic than others? Seeking to answer these questions is much more difficult than investigating the allergenic potency of inhalant or contact allergens since the proteins involved in sensitising or elicting allergic reactions may have undergone extensive modification during food processing and be presented within complex structures within food.These physicochemical changes will alter the way in which they are broken down during digestion and may modify the form in which they are taken up across the gut mucosal barrier and presented to the immune system. Certainly the structure of the food matrix can have a great impact on the elicitation of allergic reactions and fat-rich matrices may affect the kinetics of allergen release, potentiating the severity of allergic reactions (Grimshaw et al., 2003). However, because of its complex nature the impact of food processing and the food matrix on allergenicity of proteins has only recently become a subject of research. Such investigations are fraught with difficulties, not least the fact that food processing often renders food proteins insoluble in the simple salt solutions frequently employed in serological or clinical studies. As a consequence our understanding of the impact of food processing on allergenicity is limited to the more soluble and extractable residues in foods and the allergenic potential of insoluble protein complexes is virtually unstudied despite representing the vast bulk of food proteins consumed.
Proteins in fabricated food structuresMuch of our understanding of the effects of food processing on food protein structure and the fabrication of different types of food structure has been gained from studying model food,
Mucus forms a protective layer across a variety of epithelial surfaces, presenting a barrier to the uptake of particulates, including bacteria. In the gastrointestinal (GI) tract the barrier has to permit uptake of nutrients, whilst excluding potential hazards, such as bacteria. We have investigated the abilities of an exemplar food-derived particulate and a model bacterium to diffuse through porcine intestinal mucus as a model system. Transport was dramatically enhanced by adsorption of bile salts (BS), diffusion of 500-nm latex beads through mucus increased by three orders of magnitude over 2 min compared to the absence of BS. The diffusion coefficients, a probe of local mucus microrheology, showed a range of apparent viscosity from 1 mPas to at least 10 PaS. Similar effects of BS adsorption on diffusion were observed for model food emulsion droplets after simulated gastrointestinal digestion. In contrast, a non-motile bacterium, E. Coli, was found neither to diffuse through the mucus nor adhere to the mucus, regardless of the presence of BS. The negative charge imparted by BS adsorption significantly changed the electrostatic interactions with the mucus network, which is also primarily negatively charged. Thus, interfacial BS have a dominant role in determining transport of colloidal particles, including digested fat through the intestinal mucus but do not reduce the ability of the mucus to act as a barrier to bacteria. This information is important for the targeted delivery of bioactive molecules, including nutrients and pharmaceuticals and for understanding how GI health is maintained.
Pru p 1 (a Bet v 1 homologue) and Pru p 3 (a nonspecific lipid transfer protein; nsLTP) are major allergenic proteins in peach fruit, but differ in their abundance and stability. Pru p 1 has low abundance and is highly labile and was purified after expression as a recombinant protein in Escherichia coli. Pru p 3 is highly abundant in peach peel and was purified by conventional methods. The identities of the proteins were confirmed by sequence analysis and their masses determined by MS analysis. The purified proteins reacted with antisera against related allergens from other species: Pru p 1 with antiserum to Bet v 1 and Pru p 3 with antiserum to Mal d 3 (from apple). The presence of secondary and tertiary structure was demonstrated by circular dichroism (CD) and high field NMR spectroscopy. CD spectroscopy also showed that the two proteins differed in their stability at pH 3 and in their ability to refold after heating to 95 degrees C. Thus, Pru p 1 was unfolded at pH 3 even at 25 degrees C but was able to refold after heating to 95 degrees C at pH 7.5. In contrast, Pru p 3 was unable to refold after heating under neutral conditions but readily refolded after heating at pH 3.
The final boundary between digested food and the cells that take up nutrients in the small intestine is a protective layer of mucus. In this work, the microstructural organization and permeability of the intestinal mucus have been determined under conditions simulating those of infant and adult human small intestines. As a model, we used the mucus from the proximal (jejunal) small intestines of piglets and adult pigs. Confocal microscopy of both unfixed and fixed mucosal tissue showed mucus lining the entire jejunal epithelium. The mucus contained DNA from shed epithelial cells at different stages of degradation, with higher amounts of DNA found in the adult pig. The pig mucus comprised a coherent network of mucin and DNA with higher viscosity than the more heterogeneous piglet mucus, which resulted in increased permeability of the latter to 500-nm and 1-µm latex beads. Multiple-particle tracking experiments revealed that diffusion of the probe particles was considerably enhanced after treating mucus with DNase. The fraction of diffusive 500-nm probe particles increased in the pig mucus from 0.6% to 64% and in the piglet mucus from ca. 30% to 77% after the treatment. This suggests that extracellular DNA can significantly contribute to the microrheology and barrier properties of the intestinal mucus layer. To our knowledge, this is the first time that the structure and permeability of the small intestinal mucus have been compared between different age groups and the contribution of extracellular DNA highlighted. The results help to define rules governing colloidal transport in the developing small intestine. These are required for engineering orally administered pharmaceutical preparations with improved delivery, as well as for fabricating novel foods with enhanced nutritional quality or for controlled calorie uptake.
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