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Horse milk is a valuable raw material and a very attractive alternative for scientific research to address the issue of cow milk (CM) allergy due to its protein profile. A decrease in immunoreactive properties can be achieved by thermal, enzymatic, and hydrolytic processing. Therefore, the aim of this study was to explore the possibility of reducing the immunoreactivity of horse milk proteins by microbial transglutaminase (TG) polymerization. To determine how TG linking alters immunoreactivity under simulated digestion of the examined milk, analyses were performed before, during, and after digestion. The dose-dependent (1, 10, and 100 U) effects of microbial TG on horse and cow milk were analyzed. A consecutive 3-stage digestion was simulated with salivary, gastric, and intestinal fluids. The effects of digestion were analyzed by SDS-PAGE, particle size analysis, and size-exclusion chromatography. Immunoreactivity was assessed using competitive ELISA (β-lactoglobulin and α-casein) and immunodot (sera from 7 patients aged 3 to 13 years who are allergic to CM proteins). Horse milk contained almost half of the amount of total proteins in CM. The dose 1 U/g of total milk protein changed the immunoreactivity of both cow and horse milk. With increasing TG doses, α-casein immunoreactivity increased, and β-lactoglobulin decreased. After total digestion, horse milk was characterized by 2.4-fold lower average IgE and 4.8-fold lower IgG reactivity than CM. We found that TG alters the IgE and IgG reactivity of CM after in vitro digestion. Horse milk was less reactive to IgE and IgG than was CM, with animal and patient sera. The effect of TG on immunoreactivity depends on enzyme quantity and milk protein type. The diet based on modified horse milk proteins could be an alternative for some patients with CM protein allergy; however, confirmation through clinical trials is needed.

Determination of the cause of a biliary obstruction is often inconclusive from serum analysis alone without further clinical tests. To this end, serum markers as well as the composition of bile of 74 patients with biliary obstructions were determined to improve the diagnoses. The samples were collected from the patients during an endoscopic retrograde cholangiopancreatography (ERCP). The concentration of eight bile salts, specifically sodium cholate, sodium glycocholate, sodium taurocholate, sodium glycodeoxycholate, sodium chenodeoxycholate, sodium glycochenodeoxycholate, sodium taurodeoxycholate, and sodium taurochenodeoxycholate as well as bile cholesterol were determined by HPLC-MS. Serum alanine aminotransferase (ALT), aspartate transaminase (AST), and bilirubin were measured before the ERCP. The aim was to determine a diagnostic factor and gain insights into the influence of serum bilirubin as well as bile salts on diseases. Ratios of conjugated/unconjugated, primary/secondary, and taurine/glycine conjugated bile salts were determined to facilitate the comparison to literature data. Receiver operating characteristic (ROC) curves were determined, and the cut-off values were calculated by determining the point closest to (0,1). It was found that serum bilirubin was a good indicator of the type of biliary obstruction; it was able to differentiate between benign obstructions such as choledocholithiasis (at the concentration of >11 µmol/L) and malignant changes such as pancreatic neoplasms or cholangiocarcinoma (at the concentration of >59 µmol/L). In addition, it was shown that conjugated/unconjugated bile salts confirm the presence of an obstruction. With lower levels of conjugated/unconjugated bile salts the possibility for inflammation and, thus, neoplasms increase.

Numerous studies support the protective role of bioactive peptides against cardiovascular diseases. Cereals represent the primary source of carbohydrates, but they also contain substantial amounts of proteins, therefore representing a potential dietary source of bioactive peptides with nutraceutical activities. The analysis of wheat extracts purified by chromatographic techniques by means of HPLC‐UV/nanoLC‐nanoESI‐QTOF allowed the identification of a signal of about 7 kDa which, following data base searches, was ascribed to a nonspecific lipid‐transfer protein (nsLTP) type 2 from Triticum aestivum(sequence coverage of 92%). For the first time nsLTP2 biological activities have been investigated. In particular, in experiments with human umbilical vein endothelial cells (HUVEC), nsLTP2 displayed antioxidant and cytoprotective activities, being able to significantly decrease reactive oxygen species (ROS) levels and to reduce lactate dehydrogenase (LDH) release, generated following oxidative (hydrogen peroxide) and inflammatory (tumor necrosis factor α, interleukin‐1β, and lipopolysaccharide) stimulation. The obtained promising results suggest potential protective role of nsLTP2 in vascular diseases prevention. nsLTP 2 peptide is resistant to proteases throughout the gastrointestinal tract and exerts antioxidant and cytoprotective activities. These characteristics could be exploited in vascular diseases prevention.

The gastrointestinal hydrolysis of food proteins has been portrayed in scientific literature to predominantly depend on the activity and specificity of proteolytic enzymes. Human bile has not been considered to facilitate proteolysis in the small intestine, but rather to assist in intestinal lipolysis. However, human bile can potentially influence proteins that are largely resistant to gastric digestion, and which are mainly hydrolysed after they have been transferred to the small intestine. We used purified and food-grade bovine milk β-lactoglobulin (βLg) to assess the impact of bile salts (BS) on the in vitro gastrointestinal digestion of this protein. Quantitative analysis showed that the proteolysis rate increased significantly with increasing BS concentration. The effect was consistent regardless of whether individual BS or real human bile samples, varying in BS concentrations, were used. The total BS content of bile was more important than its BS composition in facilitating the proteolysis of βlg. We also show that the impact of human bile observed during the digestion of purified βLg and βLg-rich whey protein isolate can be closely replicated by the use of individual BS mixed with phosphatidylcholine. This could validate simple BS/phosphatidylcholine mixtures as human-relevant substitutes of difficult-to-obtain human bile for in vitro proteolysis studies.

The behaviour of oligosaccharides from lactulose (OsLu) included with milk was examined during in vitro gastrointestinal digestion using the Infogest protocol as well as some small intestine rat extract. The digestion was compared with commercial prebiotics GOS and Duphalac®. Electrophoretic analysis demonstrated that the prebiotic carbohydrates did not modify the gastric digestion of dairy proteins. Similarly, no significant effect of gastrointestinal digestion was shown on the prebiotic studied. In contrast, under the intestinal conditions using a rat extract, the oligosaccharides present in OsLu samples were less digested (, This work has been funded by MINECO of Spain Project AGL2014-53445-R, ALIBIRD-CM S-2013/ABI-2728 and Instituto Danone.

peer-reviewed The purpose of this study was to investigate in vitro the extent to which specific food structures alter gastric behaviour and could therefore impact on nutrient delivery and digestion in the small intestine. Results obtained from a specifically developed gastric digestion model, were compared to results from a previous human study on the same foods. The semi-dynamic model could simulate the main gastric dynamics including gradual acidification, lipolysis, proteolysis and emptying. Two dairy-based foods with the same caloric content but different structure were studied. The semi-solid meal comprised a mixture of cheese and yogurt and the liquid meal was an oil in water emulsion stabilised by milk proteins. Our findings showed similar gastric behaviour to that seen previously in vivo. Gastric behaviour was affected by the initial structure with creaming and sedimentation observed in the case of liquid and semi-solid samples, respectively. Lipid and protein digestion profiles showed clear differences in the amount of nutrients reaching the simulated small intestine and, consequently, the likely bioaccessibility after digestion. The semi-solid sample generated higher nutrient released into the small intestine at an early stage of digestion whereas nutrient accessibility from liquid sample was delayed due to the formation of a cream layer in the gastric phase. This shows the strong effect of the matrix on gastric behaviour, proteolysis and lipolysis, which explains the differences in physiological responses seen previously with these systems in terms of fullness and satiety. This work has funded by the Irish Dairy Levy Research Trust (project number MDDT6261). Ana-Isabel Mulet-Cabero was funded under Teagasc Walsh Fellowship scheme and BBSRC in the UK (grant BB/J004545/1).

Scope Allergies to lipid transfer proteins involve severe adverse reactions; thus, effective and sustainable therapies are desired. Previous attempts disrupting disulfide bonds failed to maintain immunogenicity; thus, the aim is to design novel hypoallergenic Pru p 3 variants and evaluate the applicability for treatment of peach allergy. Methods and results Pru p 3 proline variant (PV) designed using in silico mutagenesis, cysteine variant (CV), and wild‐type Pru p 3 (WT) are purified from Escherichia coli. Variants display homogenous and stable protein conformations with an altered secondary structure in circular dichroism. PV shows enhanced long‐term storage capacities compared to CV similar to the highly stable WT. Using sera of 33 peach allergic patients, IgE‐binding activity is reduced by 97% (PV) and 71% (CV) compared to WT. Both molecules show strong hypoallergenicity in Pru p 3 ImmunoCAP cross‐inhibition and histamine release assays. Immunogenicity of PV is demonstrated with a phosphate‐based adjuvant formulation in a mouse model. Conclusions An in silico approach is used to generate a PV without targeting disulfide bonds, T cell epitopes, or previously reported IgE epitopes of Pru p 3. PV is strongly hypoallergenic while structurally stable and immunogenic, thus representing a promising candidate for peach allergen immunotherapy., Engineering of the lipid transfer protein Pru p 3 from peach results in the generation of stable protein fold variants. These molecules demonstrate very low allergenic potency opposed to the wild‐type allergen. Proline variant presents enhanced integrity and elicits an immune response in a mouse model, thus representing a candidate molecule for allergen immunotherapy of peach allergic patients.

International audience; It is well recognised that the average UK diet does not contain sufficient fibre. However, the introduction of fibre is often at the detriment of the organoleptic properties of a food. In this study on the gastrointestinal fate of nanoparticles, we have used cellulose nano-crystals (CNCs) as Pickering stabilising agents in oil in water emulsions. These emulsions were found to be highly stable against coalescence. The CNC and control emulsions were then exposed to simulated upper gastrointestinal tract digestion and the results compared to those obtained from a conventional protein stabilised emulsion. Finally the digested emulsions were exposed to murine intestinal mucosa and lipid and bile absorption was monitored. Importantly, the results show that the CNCs were entrapped in the intestinal mucus layer and failed to reach the underlying epithelium. This entrapment may also have led to the reduced absorption of saturated lipids from the CNC stabilised emulsion versus the control emulsion. The results show the potential of CNCs as a safe and effective emulsifier.

4th International Conference on Food Digestion -- MAR, 2015 -- Naples, ITALY, WOS: 000384789000006, Within the active field of in vitro digestion in food research, the COST Action INFOGEST aimed to harmonize in vitro protocols simulating human digestion on the basis of physiologically inferred conditions. A harmonized static in vitro digestion (IVD) method was recently published as a primary output from this network. To validate this protocol, inter-laboratory trials were conducted within the INFOGEST network. A first study was performed using skim milk powder (SMP) as a model food and served to compare the different in-house digestion protocols used among the INFOGEST members. In a second inter-laboratory study applying the harmonized protocol, the degree of consistency in protein hydrolysis was investigated. Analysis of the hydrolyzed proteins, after the gastric and intestinal phases, showed that caseins were mainly hydrolyzed during the gastric phase, whereas beta-lactoglobulin was, as previously shown, resistant to pepsin. Moreover, generation of free amino acids occurred mainly during the intestinal phase. The study also showed that a few critical steps were responsible for the remaining inter-laboratory variability. The largest deviations arose from the determination of pepsin activity. Therefore, this step was further clarified, harmonized, and implemented in a third inter-laboratory study. The present work gives an overview of all three inter-laboratory studies, showing that the IVD INFOGEST method has led to an increased consistency that enables a better comparability of in vitro digestion studies in the future. (C) 2015 The Authors. Published by Elsevier Ltd.

Dietary fibre has long been recognised as healthy because of its prebiotic quality and a number of dietary fibres, especially beta glucan have been shown to lower levels of circulating LDL cholesterol. However, although EFSA allow health claims to be made for this, there is no fundamental understanding of the detailed mechanism involved. More recently dietary fibre has been shown to have a range of functionality in the upper GI tract. The presence of fibre can alter gastric emptying thus affecting fullness and satiety. These alterations are a result of differences in viscosity, nutrient release and nutrient sensing in the duodenum. The current proposed mechanisms for the cholesterol lowering effects involve disruption of the normal recycling of bile possibly by sequestering bile salts and fatty acids or by significantly decreasing the rate of absorption as a result of entanglement with intestinal mucus. The use of quantitative confocal microscopy methods such as fluorescence recovery after photobleaching (FRAP) and multiple particle tracking has provided evidence that dietary fibre can combine with intestinal mucus and produce a layer that significantly delays the transport of lipid digestion products. We have also used similar methods in conjunction with more conventional rheology to show that DNA from the gut epithelium can contribute significantly to the barrier properties of the intestinal mucus layer. The delay in the transport of nutrients to the gut epithelium has implications for the control of gastric emptying and through secretion of GI hormones such as CCK and thus for the satiating ability of foods. It may also have implications for the reabsorption of bile.

Highlights • Comparison was made between an enhanced β-glucan diet versus a control diet in pigs. • In vitro digestion showed that 90% of the β-glucan was solubilised in the proximal small intestine. • The permeability of the intestinal mucus of the pigs on the enhanced diet was decreased. • The results have implications for type two diabetes risk factors in humans., This study investigates the influence of the dietary fibre β-glucan on nutrient composition and mucus permeability. Pigs were fed a standard diet or a diet containing twice the β-glucan content for 3 days (n = 5 per group), followed by the collection of small intestinal mucus and tissue samples. Samples of the consumed diets were subjected to in vitro digestion to determine β-glucan release, nutrient profile and assessment of mucus permeability. In vitro digestion of the diets indicated that 90% of the β-glucan was released in the proximal small intestine. Measurements of intestinal mucus showed a reduction in permeability to 100 nm latex beads and also lipid from the digested enhanced β-glucan diet. The data from this study show for the first time that reducing mass transfer of bile and lipid through the intestinal mucus layer may be one way in which this decrease in bile reabsorption by soluble fibre is enabled.

Interactions between the surface of the intestinal mucus and the molecular components of a food, toxin or drug control the latter's bioabsorption. Understanding the colloidal basis of such interactions requires a thorough thermodynamic characterization of the mucus layer. Towards this aim, surface thermodynamics of porcine intestinal mucin are studied in this work by the aid of inverse gas chromatography (IGC), at infinite dilution and a temperature range (33–60 °C) well below the glass transition. The affinity of several molecular probes, both apolar and polar of different functionalities, onto the mucin surface is evaluated in terms of dispersive and specific interactions calculated by processing the chromatographic retention profiles. Well defined Gaussian peaks, typical of Henry's adsorption, have been obtained for apolar probes. The absence of any thermal transitions is confirmed by the linear drop of the surface free energy with temperature rise, within a typical energy range for carbohydrate polymers (γds = 33–40 mN m−1). For specific polar probes, however, non-Gaussian tailing peaks have been recorded, indicative of desorption retardation phenomena. The potential of polar probes to deploy specific interactions with mucin surface is interpreted on the basis of the homomorph concept. It is inferred that a kind of molecular sieving mechanism assisted by the high polarity of probes seems to favor retention. Evaluation of the surface acid–base interaction potential demonstrates that the mucosal barrier is Lewis amphoteric with predominant basic character. Complementary data obtained via TMDSC, TGA, XRD, and FTIR are discussed in conjunction.

Mucus provides a barrier to bacteria and toxins while allowing nutrient absorption and waste transport. Unlike colonic mucus, small intestinal mucus structure is poorly understood. This study aimed to provide evidence for a continuous, structured mucus layer and assess the diffusion of different sized particles through it. Mucus structure was assessed by histology and immunohistochemistry. Ultra-structure was assessed by scanning electron microscopy. Tracking of 100 nm and 500 nm latex beads was conducted using ex vivo porcine mucus. The porcine jejunum and ileum were filled with mucus. Layered MUC2 staining was visible throughout the small intestine, covering villus tips. Scanning electron microscopy showed net-like mucin sheets covering villi (211 ± 7 nm pore diameter). Particle tracking of 100 nm latex beads, showed no inhibition of diffusion through mucus while 500 nm beads displayed limited diffusion. These results suggest a continuous mucus layer exists throughout the small intestine, which is highly stratified adjacent to the epithelium. The network observed is consistent with previous observations and correlates with stratified MUC2 staining. Mucin pore size is consistent with free diffusion of 100 nm and limited diffusion of 500 nm particles. Small Intestinal mucus structure has important implications for drug delivery systems and prevention and treatment of conditions like mucositis and inflammatory bowel disease.

The analysis of the bioaccessibility and bioavailability of polyphenols from cauliflower waste was achieved using targeted metabolomics and chemometric approaches. Changes in phenolic profile throughout the in vitro digestion and Caco-2 transport were investigated using LC-MS combined with principal components analysis and orthogonal partial least squares–discriminant analysis, and diffusion of polyphenols through intestinal mucus was monitored using fluorescence recovery after photobleaching (FRAPb). Results showed that recovery of polyphenols in the gastric phase was approximately 76–106% whilst losses of up to 70% were observed after the intestinal phase. Kaempferol-3- O -diglucoside and kaempferol-3- O -diglucoside-7- O -glucoside were found to permeate intact through the Caco-2 cells in very small amounts (0.3% recovery). Polyphenols also diffused rapidly through intestinal mucus without altering their biophysical properties. We conclude that targeted metabolomics and FRAPb are rapid and convenient tools to study the recovery of polyphenols during in vitro digestion, mucosal diffusion and Caco-2 transport.

The effect of mucin hydrogen bonding on the structure of intestinal mucus has been studied with micro-differential scanning mirocalorimetry (μ-DSC), supported by spectroscopy. The experiments were performed in water–dimethyl sulfoxide (DMSO) solutions, using either water–DMSO mixtures of an appropriate DMSO content or water as blanks, as to isolate the effects of the solvent to hydrogen bonding. When using matched water–DMSO blanks, thermal events at low temperatures are linked to the negation of mucin–DMSO interactions, while events at higher temperatures are linked to the break-up of hydrogen bonds connecting the sugars of the individual macromolecules. When using a matched solvent as blank, alterations in Cp, such as increases at 10% and 15% DMSO, have been linked to the break-up and creation of quaternary structures. In the case of water as blank, a monotonic but not linear decrease in enthalpy, hence extent of hydrogen bonding, is observed. The above are complemented by UV spectroscopy: A blue shift of the conjugated aminoacids in the presence of DMSO suggests that the inherent stability of mucin is not only due to steric volume exclusions, but also due to extensive hydrogen bonding on behalf of the sugar moieties.

The aim of this study was to determine the extent to which oat particle size in a porridge could alter glucose absorption, gastric emptying, gastrointestinal hormone response, and subjective feelings of appetite and satiety. Porridge was prepared from either oat flakes or oat flour with the same protein, fat, carbohydrate, and mass. These were fed to eight volunteers on separate days in a crossover study, and subjective appetite ratings, gastric contents, and plasma glucose, insulin, and gastrointestinal hormones were determined over a period of 3 h. The flake porridge gave a lower glucose response than the flour porridge, and there were apparent differences in gastric emptying in both the early and late postprandial phases. The appetite ratings showed similar differences between early- and late-phase behavior. The structure of the oat flakes remained sufficiently intact to delay their gastric emptying, leading to a lower glycemic response, even though initial gastric emptying rates were similar for the flake and flour porridge. This highlights the need to take food structure into account when considering relatively simple physiological measures and offering nutritional guidance.NEW & NOTEWORTHY The impact of food structure on glycemic response even in simple foods such as porridge is dependent on both timing of gastric emptying and the composition of what is emptied as well as duodenal starch digestion. Thus structure should be accounted for when considering relatively simple physiological measures and offering nutritional guidance.

The aims of the present work were to assess digestibility of almond protein in the upper gastrointestinal tract, evaluate the effects of food matrix on protein release and assess the persistence of immunoreactive polypeptides generated during simulated digestion. Prunin, the most abundant protein in almond flour, was sensitive to pepsin, with complete digestion after 20 min in the gastric phase. Addition of the surfactant phosphatidylcholine did not affect the rate and kinetic of digestion, as observed by SDS-PAGE analysis and HPLC, in the stomach and the small intestine of either natural or blanched almond flour. However, incorporation of almond flour into a food matrix, such as chocolate mousse and Victorian sponge cake, decreased the rate of almond protein degradation by pepsin and immunoreactivity of almond polypeptides detected by dot blots and sandwich ELISA retained better. Most of the almond protein identified by in-gel tryptic digestion and MALDI-TOF analysis corresponded to prunin, with pI values of 5–7. Further human sera studies are warranted to investigate the relationship between food matrix and almond allergy.

a b s t r a c t Background: Development and maintenance of tolerance to food allergens appears to be associated with alterations in antigen specific IgE and IgG4 responses. Previous studies have focused only on comparing IgE and IgG4 linear epitope recognition patterns but take no account of conformational epitopes. Objective: The aim of this study was to compare Ara h 1-specific IgE and IgG4 epitope recognition patterns in patients with severe peanut allergy, applying a method allowing for identification of both linear and conformational epitopes. Methods: Polyclonal sera from three individual patients, suffering from severe allergic reaction to peanuts, including anaphylaxis, were used to analyse the IgE and IgG4 epitope recognition patterns of the major peanut allergen Ara h 1. Epitope identification was conducted by competitive immuno-screening of a phage-displayed random heptamer peptide library. Resulting epitope-mimicking sequences were aligned for identification of consensus sequences and localised on the surface of the Ara h 1 molecule by a computer-based algorithm. Results: All epitope-mimicking sequences identified were found to correspond to conformational epi- topes. Each individual patient had his/her own distinct IgE as well as IgG4 epitope recognition profile, though some important IgE epitopes were common to all patients. In general the IgG4 epitope pattern was more heterogeneous than the IgE pattern, did not coincide with IgE epitopes and had a lower affinity than IgE. Conclusions: This study demonstrated the usefulness of the phage-display technology in distinguishing between the epitope pattern of IgE and IgG4, giving detailed information on fine specificity and affinity. Competitive immuno-screening of phage-display random peptide libraries could be a future valuable tool to study the balance and dynamics of the IgE and IgG4 epitope recognition repertoire and provide a diagnostic tool giving information on the associated allergic phenotype.

We describe a simple protocol for identifying and quantifying the two components in binary mixtures of species possessing one or more similar proteins. Central to the method is the identification of 'corresponding proteins' in the species of interest, in other words proteins that are nominally the same but possess species-specific sequence differences. When subject to proteolysis, corresponding proteins will give rise to some peptides which are likewise similar but with species-specific variants. These are 'corresponding peptides'. Species-specific peptides can be used as markers for species determination, while pairs of corresponding peptides permit relative quantitation of two species in a mixture. The peptides are detected using multiple reaction monitoring (MRM) mass spectrometry, a highly specific technique that enables peptide-based species determination even in complex systems. In addition, the ratio of MRM peak areas deriving from corresponding peptides supports relative quantitation. Since corresponding proteins and peptides will, in the main, behave similarly in both processing and in experimental extraction and sample preparation, the relative quantitation should remain comparatively robust. In addition, this approach does not need the standards and calibrations required by absolute quantitation methods. The protocol is described in the context of red meats, which have convenient corresponding proteins in the form of their respective myoglobins. This application is relevant to food fraud detection: the method can detect 1% weight for weight of horse meat in beef. The corresponding protein, corresponding peptide (CPCP) relative quantitation using MRM peak area ratios gives good estimates of the weight for weight composition of a horse plus beef mixture.

Thermal and chemical modification of protein structures is known to affect their interfacial activity. We have looked in detail at the effect of heating on the structure and subsequently the surface properties of LTP1, a nonspecific lipid transfer protein from barley, both in the presence and in the absence of its lipid adduct. CD and NMR spectroscopy showed that some of the protein molecules refold back to the native state structure after being heated to 100 degrees C for 2 h. However, for a proportion of the molecules, the structure of the protein was irreversibly unfolded which resulted in an increase in surface activity irrespective of the presence of the lipid adduct. These molecules show an increase in surface activity, which is normally associated with an increase in molecular flexibility and surface hydrophobicity and is a property that has been shown to be highly sensitive to structural changes. This explains why thermal and chemical modification of LTP1 is important in optimizing the surface properties of the protein that are essential in diverse applications from biosensors to beer foam.

Our objective was to investigate the impact of EPA versus DHA on arterial stiffness and reactivity and underlying mechanisms (with a focus on plasma oxylipins) in the postprandial state. In a three-arm crossover acute test meal trial, men (n = 26, 35–55 years) at increased CVD risk received a high-fat (42.4 g) test meal providing 4.16 g of EPA or DHA or control oil in random order. At 0 h and 4 h, blood samples were collected to quantify plasma fatty acids, long chain n-3 PUFA-derived oxylipins, nitrite and hydrogen sulfide, and serum lipids and glucose. Vascular function was assessed using blood pressure, reactive hyperemia index, pulse wave velocity, and augmentation index (AIx). The DHA-rich oil significantly reduced AIx by 13% (P = 0.047) with the decrease following EPA-rich oil intervention not reaching statistical significance. Both interventions increased EPA- and DHA-derived oxylipins in the acute postprandial state, with an (1.3-fold) increase in 19,20-dihydroxydocosapentaenoic acid evident after DHA intervention (P < 0.001). In conclusion, a single dose of DHA significantly improved postprandial arterial stiffness as assessed by AIx, which if sustained would be associated with a significant decrease in CVD risk. The observed increases in oxylipins provide a mechanistic insight into the AIx effect.

Non-specific lipid transfer proteins (LTPs) are a family of lipid-binding molecules that are widely distributed across flowering plant species, many of which have been identified as allergens. They are highly resistant to simulated gastroduodenal proteolysis, a property that may play a role in determining their allergenicity and it has been suggested that lipid binding may further increase stability to proteolysis. It is demonstrated that LTPs from wheat and peach bind a range of lipids in a variety of conditions, including those found in the gastroduodenal tract. Both LTPs are initially cleaved during gastroduodenal proteolysis at three major sites between residues 39–40, 56–57 and 79–80, with wheat LTP being more resistant to cleavage than its peach ortholog. The susceptibility of wheat LTP to proteolyic cleavage increases significantly upon lipid binding. This enhanced digestibility is likely to be due to the displacement of Tyr79 and surrounding residues from the internal hydrophobic cavity upon ligand binding to the solvent exposed exterior of the LTP, facilitating proteolysis. Such knowledge contributes to our understanding as to how resistance to digestion can be used in allergenicity risk assessment of novel food proteins, including GMOs.

Background: Fish currently supplies only 40% of the eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) required to allow all individuals globally to meet the minimum intake recommendation of 500 mg/d. Therefore, alternative sustainable sources are needed. Objective: The main objective was to investigate the ability of genetically engineered Camelina sativa (20% EPA) oil (CO) to enrich tissue EPA and DHA relative to an EPA-rich fish oil (FO) in mammals. Methods: Six-week-old male C57BL/6J mice were fed for 10 wk either a palm oil–containing control (C) diet or diets supplemented with EPA-CO or FO, with the C, low-EPA CO (COL), high-EPA CO (COH), low-EPA FO (FOL), and high-EPA FO (FOH) diets providing 0, 0.4, 3.4, 0.3, and 2.9 g EPA/kg diet, respectively. Liver, muscle, and brain were collected for fatty acid analysis, and blood glucose and serum lipids were quantified. The expression of selected hepatic genes involved in EPA and DHA biosynthesis and in modulating their cellular impact was determined. Results: The oils were well tolerated, with significantly greater weight gain in the COH and FOH groups relative to the C group (P < 0.001). Significantly lower (36–38%) blood glucose concentrations were evident in the FOH and COH mice relative to C mice (P < 0.01). Hepatic EPA concentrations were higher in all EPA groups relative to the C group (P < 0.001), with concentrations of 0.0, 0.4, 2.9, 0.2, and 3.6 g/100 g liver total lipids in the C, COL, COH, FOL, and FOH groups, respectively. Comparable dose-independent enrichments of liver DHA were observed in mice fed CO and FO diets (P < 0.001). Relative to the C group, lower fatty acid desaturase 1 (Fads1) expression (P < 0.005) was observed in the COH and FOH groups. Higher fatty acid desaturase 2 (Fads2), peroxisome proliferator–activated receptor α (Ppara), and peroxisome proliferator–activated receptor γ (Pparg) (P < 0.005) expressions were induced by CO. No impact of treatment on liver X receptor α (Lxra) or sterol regulatory element-binding protein 1c (Srebp1c) was evident. Conclusions: Oil from transgenic Camelina is a bioavailable source of EPA in mice. These data provide support for the future assessment of this oil in a human feeding trial.

In the small intestine the nature of the environment leads to a highly heterogeneous mucus layer primarily composed of the MUC2 mucin. We set out to investigate whether the soluble dietary fibre sodium alginate could alter the permeability of the mucus layer. The alginate was shown to freely diffuse into the mucus and to have minimal effect on the bulk rheology when added at concentrations below 0.1%. Despite this lack of interaction between the mucin and alginate, the addition of alginate had a marked effect on the diffusion of 500 nm probe particles, which decreased as a function of increasing alginate concentration. Finally, we passed a protein stabilised emulsion through a simulation of oral, gastric and small intestinal digestion. We subsequently showed that the addition of 0.1% alginate to porcine intestinal mucus decreased the diffusion of fluorescently labelled lipid present in the emulsion digesta. This reduction may be sufficient to reduce problems associated with high rates of lipid absorption such as hyperlipidaemia., Graphical abstract, Highlights • There are no specific interactions between alginate and intestinal mucus. • Mucus rheology was hardly altered by small additions of alginate. • Alginate freely diffused into mucus and reduced diffusion of 500 nm beads. • Intestinal mucus permeability was decreased by a factor of two.

Dietary fibre has been shown to have a range of functionality in the upper GI tract. The presence of fibre can alter gastric emptying thus affecting fullness and satiety. These alterations are a result of differences in nutrient release and nutrient sensing in the duodenum. The current proposed mechanisms for the cholesterol lowering effects involve disruption of the normal recycling of bile possibly by sequestering bile salts and fatty acids or by significantly decreasing the rate of absorption as a result of entanglement with intestinal mucus. In experiments simulating the digestion of bread we studied the effect of the addition of beta glucan. Clearly in the early stage of digestion the starch provides most of the rheological properties of the system. The results show that although dietary fibre may significantly increase viscosity of digesta from the later stages of the simulation, it has minimal impact of the rates of hydrolysis of both protein and starch. However, using quantitative confocal microscopy methods such as FRAP and multiple particle tracking has provided evidence that dietary fibre can combine with intestinal mucus and produce a layer that significantly delays the transport of lipid digestion products. These results highlight the complexity of possible roles for dietary fibre in the upper GI tract.

The research was aimed at defining the impact of non-enzymatic glycosylation of pea globulin 7S on its biological properties. Glycation of pea globulin 7S was carried out at the temperature of 37 °C for 7 days and at 60 °C for 3 days. No glycation of the of the pea globulin 7S was observed when using high pressure of 500 MPa. Polyclonic rabbit antibodies were produced for immunochemical studies. The immunologic activity of non- and glycated pea globulin 7S was examined with the ELISA and Western blot. Also, excretion of IL-5, -10 and 13 as well as INF-γ by human peripheral blood lymphocytes along with symptoms of allergy to soy proteins under the influence of non- and glycated pea globulin 7S was assessed, as well their apoptosis and necrosis. Moreover, transportation of those proteins by the Caco-2 monolayer and their cytotoxicity were also studied. The results have proved that pea globulin 7S glycated in various physiochemical conditions is characterized by different properties to those of non-glycated ones. Lysine glycation entails a decrease in its nutritional value. Glycated pea globulin 7S manifests lowered affinity to antibodies, which suggests a limitation of its immunoallergic properties. On the other hand, the fact that glycated pea globulin 7S stimulates maturing of Th0 cells to Th2 ones, confirms its role in the process of food allergy induction. Decreased cytotoxicity of glycated pea globulin 7S and its transportation through the Caco-2 monolayer may stand for its allergenicity caused by its limited transportation to the cells of the immune system.

Mucus forms a protective layer across a variety of epithelial surfaces. In the gastrointestinal (GI) tract, the barrier has to permit the uptake of nutrients, while excluding potential hazards, such as pathogenic bacteria. In this short review article, we look at recent literature on the structure, location, and properties of the mammalian intestinal secreted mucins and the mucus layer they form over a wide range of length scales. In particular, we look at the structure of the gel-forming glycoprotein MUC2, the primary intestinal secreted mucin, and the influence this has on the properties of the mucus layer. We show that, even at the level of the protein backbone, MUC2 is highly heterogeneous and that this is reflected in the networks it forms. It is evident that a combination of charge and pore size determines what can diffuse through the layer to the underlying gut epithelium. This information is important for the targeted delivery of bioactive molecules, including nutrients and pharmaceuticals, and for understanding how GI health is maintained.

BACKGROUND: Allergen epitope characterization provides valuable information useful for the understanding of proteins as food allergens. It is believed that IgE epitopes in general are conformational, nevertheless, for food allergens known to sensitize through the gastrointestinal tract linear epitopes have been suggested to be of great importance. OBJECTIVE: The aim of this study was to identify IgE specific epitopes of intact and digested Ara h 1, and to compare epitope patterns between humans and rats. METHODS: Sera from five peanut allergic patients and five Brown Norway rats were used to identify intact and digested Ara h 1-specific IgE epitopes by competitive immunoscreening of a phage-displayed random hepta-mer peptide library using polyclonal IgE from the individual sera. The resulting peptide sequences were mapped on the surface of a three-dimensional structure of the Ara h 1 molecule to mimic epitopes using a computer-based algorithm. RESULTS: Patients as well as rats were shown to have individual IgE epitope patterns. All epitope mimics were conformational and found to cluster into three different areas of the Ara h 1 molecule. Five epitope motifs were identified by patient IgE, which by far accounted for most of the eluted peptide sequences. Epitope patterns were rather similar for both intact and digested Ara h 1 as well as for humans and rats. CONCLUSIONS: Individual patient specific epitope patterns have been identified for the major allergen Ara h 1. IgE binding epitopes have been suggested as biomarkers for persistency and severity of food allergy, wherefore recognition of particular epitope patterns or motifs could be a valuable tool for prevention, diagnosis, and treatment of food allergy.

Fundamental knowledge of physicochemical interactions in the gastrointestinal environment is required in order to support rational designing of protein-stabilized colloidal food and pharmaceutical delivery systems with controlled behavior. In this paper, we report on the colloidal behavior of emulsions stabilized with the milk protein sodium caseinate (Na-Cas), and exposed to conditions simulating the human upper gastrointestinal tract. In particular, we looked at how the kinetics of proteolysis was affected by adsorption to an oil–water interface in emulsion and whether the proteolysis and the emulsion stability could be manipulated by enzymatic structuring of the interface. After cross-linking with the enzyme transglutaminase, the protein was digested with use of an in vitro model of gastro-duodenal proteolysis in the presence or absence of physiologically relevant surfactants (phosphatidylcholine, PC; bile salts, BS). Significant differences were found between the rates of digestion of Na-Cas cross-linked in emulsion (adsorbed protein) and in solution. In emulsion, the digestion of a population of polypeptides of Mr ca. 50–100 kDa was significantly retarded through the gastric digestion. The persistent interfacial polypeptides maintained the original emulsion droplet size and prevented the system from phase separating. Rapid pepsinolysis of adsorbed, non-cross-linked Na-Cas and its displacement by PC led to emulsion destabilization. These results suggest that structuring of emulsions by enzymatic cross-linking of the interfacial protein may affect the phase behavior of emulsion in the stomach and the gastric digestion rate in vivo. Measurements of ζ-potential revealed that BS displaced the remaining protein from the oil droplets during the simulated duodenal phase of digestion. Diffusion of the postdigestion emulsion droplets through ex vivo porcine intestinal mucus was only significant in the presence of BS due to the high negative charge these biosurfactants imparted to the droplets. This implies that the electrostatic repulsion produced can prevent the droplets from being trapped by the mucus matrix and facilitate their transport across the small intestine mucosal barrier.

Background - Peanuts are often consumed after roasting, a process that alters the three-dimensional structure of allergens and leads to Maillard modification. Such changes are likely to affect their allergenicity. Objective - We aimed to establish the effect of thermal treatment mimicking the roasting process on the allergenicity of Ara h 1 and a mix of 2S albumins from peanut (Ara h 2/6). Methods - Ara h 1 and Ara h 2/6 were purified from raw peanuts and heated in a dry form for 20 min at 145 °C in the presence (R+g) or absence (R-g) of glucose, and soluble proteins were then extracted. Sera obtained from 12 well-characterized peanut-allergic patients were used to assess the IgE binding and degranulation capacities of the allergens. Results - Extensive heating at low moisture resulted in the hydrolysis of both Ara h 1 and Ara h 2/6. However, in contrast to Ara h 2/6, soluble R+g Ara h 1 formed large aggregates. Although the IgE-binding capacity of R+g and R-g Ara h 1 was decreased 9000- and 3.6-fold, respectively, compared with native Ara h 1, their capacity to elicit mediator release was increased. Conversely, both the IgE-binding capacity and the degranulation capacity of R-g Ara h 2/6 were 600–700-fold lower compared with the native form, although the presence of glucose during heating significantly moderated these losses. Conclusions - and Clinical Relevance Extensive heating reduced the degranulation capacity of Ara h 2/6 but significantly increased the degranulation capacity of Ara h 1. This observation can have important ramifications for component-resolved approaches for diagnosis and demonstrates the importance of investigating the degranulation capacity in addition to IgE reactivity when assessing the effects of food processing on the allergenicity of proteins.

We have studied the effect of the adsorption of milk proteins at the oil-water interface on their digestibility in simulated gastrointestinal environment. The investigations aimed to characterize how both the breakdown of the adsorbed proteins and the interactions with physiological surfactants, phosphatidylcholine (PC) and bile salts (BS), influence structural transformations of model, protein-stabilized food emulsions in the gastrointestinal track. Proteolysis of two contrasting proteins, β-casein (β-Cas) and β-lactoglobulin (β-Lg), was compared between the protein presented in solution or in emulsion, after adsorption at the oil-water interface. Digestion of β-Cas was faster when presented as an emulsion and led to the persistence of a 6 kD peptide not seen when the protein was presented in solution. Adsorption gave rise to a pepsin-susceptible form of β-Lg. Complex interactions were observed with PC introduced to the system in the vesicular form. Measurements of interfacial tension revealed that PC displaced the proteins from the oil droplets after only 30 s for β-Lg and 12 min for β-Cas, so that the gastric digestion largely took place in solution. Pepsinolysis of adsorbed β-Cas played a dominant role in emulsion destabilization. In contrast, collapse of β-Lg-stabilized emulsion under gastric conditions was mainly dependent on protein-PC interactions. β-Lg was significantly protected through simulated duodenal digestion as a result of a complex formed with the PC. In the absence of PC, the proteins were completely broken down after duodenal digestion, during which the duodenal surfactants, BS, displaced any remaining protein from the interface and governed the final structure of emulsion.

Water-soluble extracts of Teleme cheeses prepared from sheep, goat or cow milk and matured 120 days have been analysed for constituent peptides and proteins using proteomics. Techniques used include: (a) two-dimensional gel electrophoresis, with matrix-assisted laser desorption ionisation/mass spectrometry, (b) high-performance liquid chromatography in conjunction with mass spectrometry and Edman degradation and (c) tandem mass spectrometry. Gel electrophoresis showed species-specific differences in whey proteins and peptides from caseins but differences could not be resolved unambiguously using available databases. From high-performance liquid chromatography individual peaks were shown to be composed of a spectrum of peptides, with α-lactalbumin, β-lactoglobulin and a spectrum of casein-derived peptides most abundant. Tandem mass spectrometry detected casein-derived peptides with mass range 3000–1500 Da and identified species-specific differences. Overall, the peptide profile for Teleme cheese is typical of other cheeses and the species milk source can be resolved through proteomics.

A lipid transfer protein (LTP, Cor a 8) together with the 11S (Cor a 9) and 7S seed storage globulins (Cor a 11) are major food allergens present in hazelnut. Methods are described for their purification and characterisation using in-gel tryptic digestion mass spectrometry to confirm their identities and circular dichroism and Fourier-transform infrared spectroscopies to demonstrate that they are authentically folded. Preliminary immunochemical studies have also confirmed that the purified preparations retain their immunological properties in terms of immunoglobulin E binding, determined by immunoblotting using serum from hazelnut allergic patients. These preparations form a basis for development of improved methods of diagnosis of food allergy based on the concept of component-resolved diagnosis.

Standardized allergen extracts are needed for diagnosis and therapy purposes. For grapes, standardization is hampered by low protein and high tannin and pectin concentrations. The aim of the current study was to develop an optimized method for the extraction of grape proteins and possibly extend this to other fruits. Several existing or modified extraction methods were compared by means of protein concentration determination, SDS-PAGE, immunoblotting and radioallergosorbent test (RAST). An optimized extraction protocol was obtained in which we combined a high concentration of plant tissue, a concentrated, enriched and neutral buffer able to remove sugars and keep proteins soluble and a bivalent buffer for pectin removal. Both the quantitative (protein concentration) and qualitative parameters (SDS-PAGE protein patterns and IgE reactivity) were compared to standard protocols and commercial extracts used as diagnostic tools in the clinical practice. This method proved to be the most efficient mainly compared to the standard Björksten protocol in extracting the low molecular weight proteins, including the major grape allergen (lipid transfer protein, Vit v 1). It proved to be an easy, low cost and reproducible method proposed to prepare grape extracts that could replace the commercially available ones, used for diagnosis and possibly extend the method to other fruits especially in extracting LTPs., Vassilopoulou was supported through the IFR Marie Curie Training Site Fellowship (QLK1-CT-2000-60030) and an Iraklitos fellowship for PhD students (Ministry of Greece for Education and Religions E.P.E.A.E.K II KA 70/3/7148). C. Mills and N. Rigby were funded by the BBSRC competitive strategic grant to IFR. L. Zuidmeer and R. van Ree were (partly) funded by a grant from the EC: SAFE QLK1-CT-2000-01394.

A rapid multiple reaction monitoring (MRM) mass spectrometric method for the detection and relative quantitation of the adulteration of meat with that of an undeclared species is presented. Our approach uses corresponding proteins from the different species under investigation and corresponding peptides from those proteins, or CPCP. Selected peptide markers can be used for species detection. The use of ratios of MRM transition peak areas for corresponding peptides is proposed for relative quantitation. The approach is introduced by use of myoglobin from four meats: beef, pork, horse and lamb. Focusing in the present work on species identification, by use of predictive tools, we determine peptide markers that allow the identification of all four meats and detection of one meat added to another at levels of 1% (w/w). Candidate corresponding peptide pairs to be used for the relative quantification of one meat added to another have been observed. Preliminary quantitation data presented here are encouraging.

Nanoparticles (NPs) in biological fluids immediately interact with proteins forming a biomolecular corona (PC) that imparts their biological identity. While several studies on the formation of the PC in human plasma have been reported, the PC of orally administrated NPs has been less investigated, mostly in the presence of a food matrix. In fact, food matrixes when digested are subject of several dynamic changes that will certainly affect the PC formed on the NPs. The lack of studies on this topic is clearly related to the difficulty in isolating representative PC NPs from such a complex environment. In this work magnetite NPs were added to in vitro simulated digestion simultaneously with bread and PC NPs were isolated after gastric and duodenal phases by sucrose gradient ultracentrifugation (UC). The PC NPs were characterized in terms of size and protein composition. Translocation studies were then performed on Caco-2 monolayers in a serum free environment and cell morphology was characterized by confocal microscopy. PC NPs isolated from gastric and duodenal phases were different in size, surface charge and protein corona composition. NP cellular uptake was enhanced by the digestive PC inducing morphology changes in the cell monolayer. Overall, in this work we were able to isolate PC NPs from digested fluids in the presence of a food matrix and study their biological response on Caco-2 cells.

Nanoparticles (NPs) in contact with biological fluids are generally coated with environmental proteins, forming a stronger layer of proteins around the NP surface called the hard corona. Protein corona complexes provide the biological identity of the NPs and their isolation and characterization are essential to understand their in vitro and in vivo behaviour. Here we present a one-step methodology to recover NPs from complex biological media in a stable non-aggregated form without affecting the structure or composition of the corona. This method allows NPs to be separated from complex fluids containing biological particulates and in a form suitable for use in further experiments. The study has been performed systematically comparing the new proposed methodology to standard approaches for a wide panel of NPs. NPs were first incubated in the biological fluid and successively recovered by sucrose gradient ultracentrifugation in order to separate the NPs and their protein corona from the loosely bound proteins. The isolated NP–protein complexes were characterized by size and protein composition through Dynamic Light Scattering, Nanoparticle Tracking Analysis, SDS-PAGE and LC-MS. The protocol described is versatile and can be applied to diverse nanomaterials and complex fluids. It is shown to have higher resolution in separating the multiple protein corona complexes from a biological environment with a much lower impact on their in situ structure compared to conventional centrifugal approaches.

It is not possible to look in detail at the wide range of static digestion methods that have been used to date. However, this section looks at some of the general approaches that have been used to look at the digestion of various nutrients and bioactives. I have focussed on the two main nutrients that undergo digestion in the upper GI tract, namely protein and lipid. In the case of protein, the research has largely been driven by the need to assess allergenic potential and the parameters used in such an assessment are given along with the justification provided by the authors for their choice. For the lipid digestion, we have drawn heavily upon the work of Julian McClemments and colleagues who have been prolific in generating data in this area. The information provided highlights the fact that a wide range of methods are in use leading to a need for a single method, a role that can be filled by the Infogest method.

Background Sensitization to hazelnut allergens vary depending on the geographic origin and age of the patients. The objective of this study was to further investigate the allergenic activity of hazelnut allergens using sera from patients recruited in various European regions and presenting different sensitization patterns to hazelnut proteins. Methods Natural Cor a 11 and Cor a 9 were purified from hazelnut whereas Cor a 1 and Cor a 8 were produced as recombinant proteins (rCor a 1.04 and rCor a 8). Sera from hazelnut allergic patients were collected in France (n = 5), Switzerland (n = 2), Greece (n = 11) and Spain (n = 3), within the Europrevall project. Total and allergen-specific IgE were quantified by enzyme allergosorbent test and IgE immunoblot were performed using pooled sera from birch-pollen endemic region or from Greece. Histamine Release (HR) assays were performed with stripped basophils passively sensitized with individual sera and challenged by a hazelnut extract or the different hazelnut allergens. Results As previously described, hazelnut allergic patients from Mediterranean countries are mainly sensitized to the nsLTP Cor a 8 whereas patients from France and Switzerland are sensitized to pollen-related allergens. Interestingly, an intermediate profile was evidenced in patients from Madrid. Hazelnut 7S globulin (Cor a 11) and 11S globulin (Cor a 9) were found to be minor allergens, recognized only by patients from Mediterranean countries. The biologic activity of the 4 tested allergens, analysed by HR assay, further confirmed the sensitization patterns, but also demonstrated the very high elicitation potency of Cor a 8. Conclusions This work, extending previously published researches, represents a step towards the better understanding of the complexity of hazelnut allergy and provides new data on the biological activity of hazelnut allergens and extracts. Electronic supplementary material The online version of this article (doi:10.1186/s13601-015-0066-7) contains supplementary material, which is available to authorized users.

This section describes the consensus static digestion method developed within the COST Action InfoGest. Simulated gastro-intestinal digestion is widely employed in many fields of food and nutritional research. Various different digestion models have been proposed, which often impedes the possibility of comparing results across research teams. For example, a large variety of enzymes from different sources such as porcine, rabbit or human have been used and these differ in their activity and characterization. Differences in pH, mineral composition and digestion time that alter enzyme activity and other phenomena may also significantly alter results. Other parameters such as the presence of phospholipids, specific enzymes such as gastric lipase and digestive emulsifiers, etc. have also been discussed at length. In this section, a general standardised and practical static digestion method is given, based on physiologically relevant conditions that can be applied for various endpoints. A framework of parameters for the oral, gastric and small intestinal digestion is outlined and their relevance discussed in relation to available in vivo data and enzymes. Detailed, line-by-line guidance recommendations and justifications are given but also limitations of the proposed model. This harmonised static, in vitro digestion method for food should aid the production of more comparable data in the future.

Background: The FAST (food allergy-specific immunotherapy) project aims at developing safe and effective subcutaneous immunotherapy for fish allergy, using recombinant hypoallergenic carp parvalbumin, Cyp c 1. Objectives: Preclinical characterization and good manufacturing practice (GMP) production of mutant Cyp (mCyp) c 1. Methods:Escherichia coli-produced mCyp c 1 was purified using standard chromatographic techniques. Physicochemical properties were investigated by gel electrophoresis, size exclusion chromatography, circular dichroism spectroscopy, reverse-phase high-performance liquid chromatography and mass spectrometry. Allergenicity was assessed by ImmunoCAP inhibition and basophil histamine release assay, immunogenicity by immunization of laboratory animals and stimulation of patients' peripheral blood mononuclear cells (PBMCs). Reference molecules were purified wild-type Cyp c 1 (natural and/or recombinant). GMP-compliant alum-adsorbed mCyp c 1 was tested for acute toxicity in mice and rabbits and for repeated-dose toxicity in mice. Accelerated and real-time protocols were used to evaluate stability of mCyp c 1 as drug substance and drug product. Results: Purified mCyp c 1 behaves as a folded and stable molecule. Using sera of 26 double-blind placebo-controlled food-challenge-proven fish-allergic patients, reduction in allergenic activity ranged from 10- to 5,000-fold (1,000-fold on average), but with retained immunogenicity (immunization in mice/rabbits) and potency to stimulate human PBMCs. Toxicity studies revealed no toxic effects and real-time stability studies on the Al(OH)3-adsorbed drug product demonstrated at least 20 months of stability. Conclusion: The GMP drug product developed for treatment of fish allergy has the characteristics targeted for in FAST: i.e. hypoallergenicity with retained immunogenicity. These results have warranted first-in-man immunotherapy studies to evaluate the safety of this innovative vaccine.

Summary Background The process of gastro-duodenal digestion may play a role in determining the allergenic properties of food proteins. The sensitizing and allergenic potential of digestion products of highly degraded allergens, such as the major peanut allergen Ara h 1, is currently under debate. We evaluated the effect of in vitro gastro-duodenal digestion of Ara h 1 on T cell reactivity and basophil histamine release. Methods An in vitro model of gastro-duodenal digestion was used to investigate changes in the allergenic properties of Ara h 1 using in vitro assays monitoring T cell reactivity (proliferation, cytokine production) and histamine release of basophils from peanut allergic individuals. The digestion process was monitored using an SDS-PAGE gel. Results In vitro gastric digestion led to rapid degradation of Ara h 1 into small fragments Mr L5600. Gastric digestion did not affect the ability of Ara h 1 to stimulate cellular proliferation. Gastro-duodenal digestion significantly reduced its ability to stimulate clonal expansion (P

BACKGROUND: Severe grape allergy has been linked to lipid transfer protein (LTP) sensitization. LTPs are known to be resistant to pepsin digestion, although the effect of gastroduodenal digestion on its allergenicity has not been reported. OBJECTIVE: We sought to investigate the effect of gastric and gastroduodenal digestion on the allergenic activity of grape LTP. METHODS: The proteolytic stability of grape LTP was investigated by using an in vitro model of gastrointestinal digestion. The allergenicity of LTP and its digesta was assessed in vitro by means of IgE immunoblotting, RASTs, and in vivo skin prick tests in the same patients with grape allergy. RESULTS: Grape LTP was resistant to gastric digestion, and yielded a 6000-d relative molecular mass C-terminally trimmed fragment after duodenal digestion. This fragment retained the in vitro IgE reactivity of the intact protein. Inclusion of phosphatidylcholine during gastric digestion protected the LTP to a limited extent against digestion. Digestion did not affect the in vivo (skin prick test) biologic activity of LTP. CONCLUSION: The allergenic activity of grape LTP was highly resistant to in vitro digestion. This property might facilitate sensitization through the gastrointestinal tract and might also potentiate the ability of LTPs to elicit severe allergic reactions in sensitized individuals. CLINICAL IMPLICATIONS: Purified natural allergens will facilitate the development of component-resolved diagnostic approaches, including allergen chips. This study contributes to our understanding of the role digestion plays in symptom elicitation in true food allergy

Background Allergy to a plant food can either result from direct sensitization to that food or from primary sensitization to pollen, latex, or another food. Objective We sought to investigate the primary sensitizers in apple allergy across Europe, the individual allergens involved, and whether these differences determine the clinical presentation. Methods Patients (n = 389) with positive case histories and skin prick test responses to fresh apple were selected in the Netherlands, Austria, Italy, and Spain. Skin prick tests and RASTs to a panel of pollens and plant foods were performed, as well as RASTs to Bet v 1 and the apple allergens Mal d 1, 2, 3, and 4. Results In the Netherlands, Austria, and Italy apple allergy is mild (>90% isolated oral symptoms) and related to birch pollinosis and sensitization to Bet v 1 and its apple homologue, Mal d 1, which has an odds ratio of local reactions of 2.85 (95% CI, 1.47-5.55). In Spain apple allergy is severe (>35% systemic reactions) and related to peach allergy and sensitization to Mal d 3 (nonspecific lipid transfer protein), which has an odds ratio of systemic reactions of 7.76 (95% CI, 3.87-15.56). Conclusion The analysis of individual apple allergens in a clinical context has provided insight into the sensitization pathway and into the intrinsic risk an allergen bears to induce mild or severe food allergy. Clinical implications Information on the sensitization pathway is essential to develop preventive strategies in food allergy. The application of individual food allergens with a known intrinsic risk will improve the prognostic value of diagnostic tests.

Background: Non-specific lipid transfer proteins (LTPs) are involved in allergy to fresh and processed fruits. We have investigated the effect of thermal treatment and glycation on the physico-chemical and IgE-binding properties of the LTP from apple (Mal d 3). Methods: Mal d 3 was purified from apple peel and the effect of heating in the absence and presence of glucose investigated by CD spectroscopy, electrospray and MALDI-TOF mass spectrometry. IgE reactivity was determined by RAST and immunoblot inhibition, SPT and basophil histamine release test. Results: The identity and IgE reactivity of purified Mal d 3 was confirmed. Mild heat treatment (90°C, 20 min) in the absence or presence of glucose did not alter its IgE reactivity. More severe heat treatment (100°C, 2 h) induced minor changes in protein structure, but a significant decrease in IgE-binding (30-fold) and biological activity (100- to 1000-fold). Addition of glucose resulted in up to four glucose residues attached to Mal d 3 and only a 2- and 10-fold decrease of IgE-binding and biological activity, respectively. Conclusions: Only severe heat treatment caused a significant decrease in the allergenicity of Mal d 3 but glycation had a protective effect. The presence of sugars in fruits may contribute to the thermostability of the allergenic activity of LTP in heat-processed foods.

The miscibility, occurrence of glass formation, and microstructure of maltotriose mixtures with water, methanol, ethanol, propan-1-ol and butan-1-ol at 20°C were studied. Maltotriose is fully miscible with water, but in binary mixtures, it is only partially miscible with short-chain alcohols. In methanol mixtures, the maltotriose-rich phase is a highly viscous liquid, but it is glassy in ethanol, propan-1-ol and butan-1-ol mixtures. In ternary alcohol-water-maltotriose mixtures, increasing water content increased miscibility, resulting in the progressive transformation of two phase liquid-glass mixtures into low viscosity single phase solutions. At water contents below about 4% w/w, the state diagram of ethanol-water-maltotriose mixtures includes a region of liquid-glass coexistence. Quenching single phase solutions into this region of the state diagram yielded aggregated dispersions of glassy particles, a process we term “glassy precipitation.”

Although English walnut is a commonly allergenic tree nut, walnut allergens have been poorly characterized to date. The objective of this work was to characterize the natural, low molecular weight (LMW) allergens from walnut. A protocol was developed to purify LMW allergens (specifically 2S albumins) from English walnuts. In addition to 2S albumins, a series of peptides from the N-terminal region of the 7S seed storage globulin proprotein were also identified and characterized. These peptides comprised a four-cysteine motif (C-X-X-X-C-X10-12-C-X-X-X-C) repeated throughout the 7S N-terminal region. Upon IgE immunoblotting, 3/11 and 5/11 sera from walnut-allergic subjects showed IgE reactivity to the 7S N-terminal fragments and 2S albumin, respectively. The mature 7S protein and the newly described 7S N-terminal peptides represent two distinct types of allergens. Because the proteolytic processing of 7S globulins has not been elucidated in many edible plant species, similar protein fragments may be present in other nuts and seeds.

The digestion of dietary components in the human gastrointestinal (GI) tract is a complex, dynamic, inherently heterogeneous process. A key aspect of the digestion of lipid in the GI tract is the combined action of bile salts, lipase and colipase in hydrolysing and solubilising dispersed lipid. The bile salts are a mixture of steroid acid conjugates with surfactant properties. In order to examine whether the different bile salts have different interfacial properties their dynamic interfacial behaviour was characterised. Differences in the adsorption behaviour to solid hydrophobic surfaces of bile salt species were studied using dual polarisation interferometry and atomic force microscopy (AFM) under physiological conditions. Specifically, the cholates adsorbed more slowly and a significant proportion were irreversibly adsorbed following buffer rinsing; whereas the deoxycholates and chenodeoxycholates adsorbed more rapidly and desorbed to a greater extent following buffer rinsing. The conjugating groups (taurine, glycine) did not influence the behaviour. AFM showed that the interfacial structures that remained following buffer rinsing were also different between these two groups. In addition, the adsorption–desorption behaviour affected the adsorption of colipase to a solid surface. This supports the idea that cooperative adsorption occurs between certain bile salts and colipase to facilitate the adsorption and activity of pancreatic lipase in order to restore lipolytic activity in the presence of bile salts. This study provides insights into how differences in bile salt structure could affect lipase activity and solubilisation of lipolysis products and other lipid-soluble bioactive molecules.

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.

A dessert matrix previously used for diagnosis of food allergies was incurred with pasteurised egg white or skimmed milk powder at 3, 6, 15 and 30 mg allergen protein per kg of dessert matrix and evaluated as a quality control material for allergen analysis in a multi-laboratory trial. Analysis was performed by immunoassay using five kits each for egg and milk (based on casein) and six 'other' milk kits (five based on β-lactoglobulin and one total milk). All kits detected allergen protein at the 3 mg kg(-1) level. Based on ISO criteria only one egg kit accurately determined egg protein at 3 mg kg(-1) (p=0.62) and one milk (casein) kit accurately determined milk at 6 (p=0.54) and 15 mg kg(-1) (p=0.83), against the target value. The milk "other" kits performed least well of all the kits assessed, giving the least precise analyses. The incurred dessert material had the characteristics required for a quality control material for allergen analysis.