Search

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.

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.

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.

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.

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.

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.

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.

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: 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.

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 effect of basic peptides on the gelation of a pectin from the cell wall of tomato was examined through the determination of gel stiffness, and swelling behaviour of the gel in water. Poly-L-lysine, poly-L-arginine, and a synthetic peptide, designed to mimic a sequence of basic amino acids found in a plant cell wall extensin, act as crosslinking agents. Circular dichroism studies on the interaction of synthetic extensin peptides with sodium polygalacturonate demonstrated that a conformational change was induced as a result of their complexation. In addition to their effect as crosslinking agents, the polycationic peptides reduced the swelling of the pectin network in water.

A specific, chemical degradation of the methyl esterified galacturonic acid residues of pectins is described. These residues are converted, with hydroxylamine, to hydroxamic acids, and then, with a carbodiimide, to isoureas; the latter undergo a Lossen rearrangement on alkaline hydrolysis. The isocyanates formed are hydrolysed to 5-aminoarabinopyranose derivatives, which spontaneously ring open to give 1,5-dialdehydes. The latter are reduced, in situ, to avoid peeling reactions, with sodium borohydride to give substituted arabitol residues. Thus, overall, partially esterified pectins are specifically cleaved to generate a series of oligogalacturonic acids bearing an arabitol residue as aglycone. Analysis of oligomers so generated discloses the pattern of contiguous nonesterification in a variety of pectins of differing degrees of esterification. Other potential applications are described.

The swelling of tomato pectin and isolated tomato pericarp cell wall material was investigated in aqueous media under different ionic conditions, pH, and external osmotic stress. Conditions were chosen to include those that would be encountered in vivo. Swelling in these systems was strongly influenced by the polyelectrolyte nature of the polymer and the extent of cross-linking with divalent counterions.

Oligogalacturonates were produced by the limited enzymic hydrolysis of polygalacturonic acid and purified by ion-exchange chromatography. The fractions obtained were of limited polydispersity, determined by analytical ion-exchange chromatography. Oligomers with an average degree of polymerization of 10–15 were readily crystallized from aqueous salt solutions at neutral pH as single crystals. Crystal morphology of the salts examined, Na + , K + and Ca 2+ were characteristic of the salt. The wide-angle X-ray diffraction patterns obtained for the sodium salt were consistent with published fibre diffraction data of this salt form.

Selected fractions of cauliflower stem, obtained after ethanol graded precipitation of 0.5 M KOH cell wall extractions, were further purified by anion-exchange and gel-permeation chromatographic methods. Pure xyloglucans and various xylan-rich polysaccharide complexes were obtained, particularly from the most mature section of the stem. A pectic-xylan–xyloglucan complex, tentatively characterised by methylation analysis, was digested separately with purified and highly specific endo -xylanase and also endo -polygalacturonase. Digestions degraded the xylan and pectic polysaccharides moieties, and caused a decrease in the molecular weight of the xyloglucan moieties, thus demonstrating the strong, probably covalent interactions between the different polymer species. The formation of polysaccharide complexes during secondary thickening and the structure of the pectic-xylan–xyloglucan complexes in cauliflower stem tissues are discussed.

A series of singly branched α -dextrins with dp ≥ 5 was prepared by treating amylopectin with an α -amylase (Termamyl) and then with β -amylase. The dextrins were separated by gel filtration chromatography and the composition of the fractions was checked by HPAEC. Each fraction gave essentially a single peak by HPAEC but NMR spectroscopy indicated that only the fraction corresponding to dp5 consisted of one compound. The fractions corresponding to dp6, 7 and 8 were found to be mixtures of isomers. Three isomers of dp6 were separated by reversed phase HPLC and were characterised by NMR spectroscopy. The isomers were identified as 6 2 - α -maltotriosyl-maltotriose, 6 3 - α -maltosyl-maltotetraose and 6 2 - α -maltosyl-maltotetraose. 1 H and 13 C NMR parameters of these compounds and of dp5, 6 2 - α -maltosyl-maltotriose, are discussed in relation to the linkage pattern of the different glucose units and compared with similar parameters in the linear maltodextrins.

An alcohol-insoluble residue from carrot root, analysed by an established method, appeared to contain partially non-methyl esterified pectic polysaccharides. Digestion with Driselase, to which such esters are thought to be resistant, gave small amounts of acidic oligosaccharides. Treatment with alkali at room temperature hydrolysed some of these components and gave more acidic 1,4-linked oligogalacturonic acids as products. Since Driselase is known to cleave pectic methyl esters, these components appeared to contain non-methyl galacturonoyl esters. However, the predominant component of this type was shown to be a monomethyl esterified, singly-acetylated tetragalacturonic acid. It was converted to galacturonic acid when re-treated with Driselase and, thus, was a product of incomplete digestion. Other, more minor components with similar properties were either other methyl esterified oligomers or were present in such small amounts that their characterization was not possible.

We hypothesize that the rate of release of lipids from salmon muscle during in vitro digestion is altered by additional meal components. In vitro digestion of salmon was performed using a mixture of porcine gastrointestinal enzymes and bile salts. Broccoli and barley were also added to the digestion simulating a meal. The extent of lipolysis was determined by measuring the release of fatty acids (FAs) during sampling at the simulated gastric phase endpoint (60 minutes) and 20, 40, 60, 80, 110 and 140 minutes simulated small intestinal phase, using solid phase extraction and GC-FID. Adding barley resulted in a lower overall release of FA from salmon, whereas broccoli caused an initial delay followed by increased release from 80–140 min when lipid digestion of salmon alone plateaued. The impact of broccoli and barley on the release of peptides and digesta viscosity were also measured. The effect of different components in the meal shown by this in vitro study suggests that it would be possible to make dietary changes affecting the lipolysis, further triggering specific responses in the gastrointestinal tract. However, these observations need to be validated in vivo, and the mechanisms need to be further examined.

Cell-wall material was prepared from unripe tomato fruit, and a pectic polysaccharide extracted with cyclohexanediaminetetraacetic acid. The structure of the purified pectic polysaccharide was examined by sugar and methylation analysis, and was typical of a rhamnogalacturonan from the primary cell wall. The physicochemical properties of the isolated polysaccharide were characterised by viscometry and size-exclusion chromatography. The polysaccharide was polydisperse, but of large molecular size as indicated by an intrinsic viscosity of 810 ml. g −1 . At concentrations above ∼ 0.2–0.6% w/w, coil entanglement was observed as an increase in the dependency of viscosity on concentration. For these concentrated solutions, clear elastic gels were formed on addition of calcium ions. At concentrations in the range 0.6–2.8% w/w the shear modulus of the gel showed a c 1.9 dependence on concentration. The modulus of the gel increased linearly with absolute temperature in a rubberlike way, enabling an estimate of cross-link density to be made.

In the present paper we have investigated the impact of microbial transglutaminase on thermally treated ovalbumin. In fact, ovalbumin was modified by microbial transglutaminase following heat treatment for 1 h at 80 °C. The protein was both intra- and inter-molecularly crosslinked as shown by the formation of high molecular weight polymers and a monomer having similar molecular weight but higher electrophoretic mobility when compared to unmodified ovalbumin. Analysis of simulated digestion under physiological conditions has demonstrated that the biopolymers obtained after transglutaminase treatment were more resistant to both gastric and duodenal digestion. Whilst they started gradually being digested after 5 min of incubation with pepsin, some of them were still present even after 60 min incubation with the duodenal enzymes, trypsin and chymotrypsin. Furthermore, incubation of 1.5% (w/v) ovalbumin gel in the presence of transglutaminase led to the formation of a well-developed viscoelastic gel network with higher modulus and lower phase angle values. These results suggest a possible use of transglutaminase-modified ovalbumin in the food industry as a potential ingredient to enhance both the functional properties of many food-based products, such as digestibility, and mechanical properties, such as viscoelasticity and gel strength.

Recombinant human gastric lipase (hGL) was transiently expressed in Nicotiana benthamiana leaves using the CPMV-HT expression system. Expression levels of up to 0.5 mg recombinant hGL per gram of infiltrated leaf tissue were obtained. Proteins expressed from two hGL constructs, wild type (wt-hGL) and with a Histidine tag at its C terminal (hGL-His), were purified from leaf tissue using Immobilized Lectin Affinity chromatography and Immobilized Metal Affinity chromatography. Both variants were glycosylated, enzymatically active, and had an apparent molecular weight similar to native hGL (approx. 50 kDa). The recombinant hGLs were stable under acidic conditions and in the presence of gastric pepsin. Moreover, as found with the naturally occurring enzyme, the activity of recombinant hGL on the short chain lipid, tributyrin, was higher than on long chain Intralipid 30% emulsion. The maximum specific activity measured on tributyrin was 310 U/mg of protein and the maximum yield was 193 U/g of infiltrated leaf tissue. These results show that transient expression in plants can be used to produce active hGL that could be efficiently purified using established techniques. The approach provides a means of generating large quantities of hGL that could be of use for a number of applications both in vitro and in vivo.

International audience; Background: Peanut allergy is one of the most common and severe food allergies, and processing is known to influence the allergenicity of peanut proteins. We aimed to establish the effect of heating and glycation on the IgE-binding properties and biological activity of 2S albumins (Ara h 2/6) from peanut. Methodology/Principal Findings: Native Ara h 2/6 was purified from raw peanuts and heated in solution (15 min, 110 degrees C) in the presence or absence of glucose. Ara h 2 and 6 were also purified from roasted peanut. Using PBMC and sera from peanut-allergic patients, the cellular proliferative potency and IgE reactivity (reverse EAST inhibition) and functionality (basophil degranulation capacity) of allergens were assessed. Heating Ara h 2/6 at 110 degrees C resulted in extensive denaturation, hydrolysis and aggregation of the protein, whilst Ara h 2 and 6 isolated from roasted peanut retained its native conformation. Allergen stimulation of PBMC induced proliferation and Th2 cytokine secretion which was unaffected by thermal processing. Conversely, IgE reactivity and functionality of Ara h 2/6 was decreased by heating. Whilst heating-glycation further reduced the IgE binding capacity of the proteins, it moderated their loss of histamine releasing capacity. Ara h 2 and 6 purified from roasted peanut demonstrated the same IgE reactivity as unheated, native Ara h 2/6. Conclusions/Significance: Although no effect of processing on T-cell reactivity was observed, heat induced denaturation reduced the IgE reactivity and subsequent functionality of Ara h 2/6. Conversely, Ara h 2 and 6 purified from roasted peanut retained the structure and IgE reactivity/functionality of the native protein which may explain the allergenic potency of this protein. Through detailed molecular study and allergenicity assessment approaches, this work then gives new insights into the effect of thermal processing on structure/allergenicity of peanut proteins.

Initially the resistance to digestion of two cow's milk allergens, beta-casein, and beta-lactoglobulin (beta-Lg), was compared using a "high-protease assay" and a "low-protease assay" in a single laboratory. The low-protease assay represents an alternative standardised protocol mimicking conditions found in the gastrointestinal tract. For the high-protease assay, both proteins were incubated with either pepsin or pancreatin and digestion monitored by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and reverse phase-high performance liquid chromatography. The low-protease assay involved gastroduodenal digestion in the presence or absence of phosphatidylcholine (PC). Both beta-casein and beta-Lg were susceptible to hydrolysis by pepsin and pancreatin in the high-protease assay. In contrast, the kinetics of beta-casein digestion in the low-protease assay were slower, beta-Lg being pepsin resistant. During duodenal digestion, beta-Lg was gradually degraded and addition of PC slowed digestion. Subsequently, the reproducibility of the low-protease assay was assessed in 12 independent laboratories by visual assessment of the gels and densitometric analysis: the inter- and intra-laboratory variability was affected by sampling and electrophoresis method employed. The low-protease assay was shown to be reproducible. Future studies will extend these findings using a broader panel of proteins.

Peanut is a major cause of type 1 hypersensitive reactions including anaphylaxis. This results from the presence of a number of protein allergens, six of which are being studied as part of the EU FP6 EuroPrevall programme. These are Ara h 1 (7S globulin), Ara h 2, Ara h 6 (2S albumins), Ara h 3/4 (11S globulins) and Ara h 8 (Bet v 1 homologue). Methods for the purification of Ara h 1, Ara h 3/4, Ara h 2 and Ara h 6 from peanut seeds and for the production of recombinant Ara h 8 in Escherichia coli are described with spectroscopic analyses being used to confirm that they are authentically folded. N-terminal sequencing of the proteins purified from peanut seeds also revealed details of the differences between isoforms and their generation by proteolytic processing within the seed. Preliminary IgE binding studies of the purified allergens confirmed that they retained their immunological properties indicating their suitability for use in allergy diagnosis.

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.

Proteomic approaches have been used to characterise the main 2S albumin isoforms from Brazil nuts (Bertholletia excelsa). Whilst most isoforms (∼10 discrete protein species) exhibited molecular masses of around 12 kDa with a high amino acid sequence homology, important charge heterogeneity was found, with pIs varying between 4.6 and 6.6, with one ≥7.0. Proteomic analysis showed that these corresponded to a total of six National Center for Biotechnology Information (NCBI) accessions and that three isoforms had been purified to homogeneity corresponding to gi/384327, 112754 and 99609. The latter sequence corresponds to an isoform, previously only identified at the nucleotide sequence level, had a slightly higher molecular weight (13.4 kDa), and with noticeable differences in the primary structure. Proteins corresponding to six different NCBI accessions were identified, the heterogeneity of which had been increased by posttranslational processing. Evidence was found of cyclization of the N-terminal glutamine residue in two isoforms, together with ragged C-termini, indicative of carboxypeptidase activity within the vacuole following posttranslational processing. No evidence of glycosylation was found. Circular dichroism (CD) and Fourier transform-infrared (FT-IR) spectroscopy indicated all the studied isoforms were predominantly α-helical in nature, but that the Mr 13 400 species was structurally distinct, with a higher proportion of α-helical structure., This research has been supported by a Marie Curie Fellowship of the European Community programme “Quality of Life and Management of Living Resources” under contract number QLK1-CT-2001-51997 and the BBSRC competitive strategic grant for IFR.

BackgroundIgE-binding of process-modified foods or proteins is the most common method for examination of how food processing affects allergenicity of food allergens. How processing affects sensitization capacity is generally studied by administration of purified food proteins or food extracts and not allergens present in their natural food matrix.ObjectivesThe aim was to investigate if thermal processing increases sensitization potential of whole peanuts via the oral route. In parallel, the effect of heating on sensitization potential of the major peanut allergen Ara h 1 was assessed via the intraperitoneal route.MethodsSensitization potential of processed peanut products and Ara h 1 was examined in Brown Norway (BN) rats by oral administration of blanched or oil-roasted peanuts or peanut butter or by intraperitoneal immunization of purified native (N-), heated (H-) or heat glycated (G-)Ara h 1. Levels of specific IgG and IgE were determined by ELISA and IgE functionality was examined by rat basophilic leukemia (RBL) cell assay.ResultsIn rats dosed orally, roasted peanuts induced significant higher levels of specific IgE to NAra h 1 and 2 than blanched peanuts or peanut butter but with the lowest level of RBL degranulation. However, extract from roasted peanuts was found to be a superior elicitor of RBL degranulation. Process-modified Ara h 1 had similar sensitizing capacity as NAra h 1 but specific IgE reacted more readily with process-modified Ara h 1 than with native.ConclusionsPeanut products induce functional specific IgE when dosed orally to BN rats. Roasted peanuts do not have a higher sensitizing capacity than blanched peanuts. In spite of this, extract from roasted peanuts is a superior elicitor of RBL cell degranulation irrespectively of the peanut product used for sensitization. The results also suggest that new epitopes are formed or disclosed by heating Ara h 1 without glucose.

Atomic force microscopy (AFM) has been used to investigate the nature of the long branches attached to pectin which were described in a previous report [Round, A. N.; MacDougall, A. J.; Ring, S. G.; Morris, V. J. Carbohydr. Res. 1997, 303, 251-253]. Analysis of the AFM images and comparison with neutral sugar and linkage analyses of the two pectin fractions suggest that the distribution and total amount of branches observed do not correspond with the pattern of neutral sugar distribution. It is thus postulated that the long chains consist of polygalacturonic acid, attached via an as yet undetermined linkage to the pectin backbone, with the neutral sugars present as short, undetected branches. This explanation would have important implications for the nature of 'in situ' pectin networks within plant cell walls and models of gelation in commercial extracted pectin. and the existence of significant branching will markedly influence the viscosity of extracted pectins. (C) 2001 Elsevier Science Ltd. All rights reserved.

We have studied the movement and metabolism of oligogalacturonides through shoots of tomato (Lycopersicon esculentum L. cv Rutgers). Oligomers of polygalacturonic acid were prepared by enzyme digestion and gel filtration. These were end-reduced with [(3)H]NaBH4, using an improved reaction method, to yield oligoalditols. The radiolabelled oligomer of degree of polymerisation 6 was supplied to tomato shoots through their transpiration stream. Analysis of the distribution of radiolabel in the plant, and TLC of radiolabelled material recovered from the plant revealed the following: a) material recovered from the plant could be identified as an oligogalacturonide from its behaviour on TLC and susceptibility to digestion with polygalacturonase; b) end-reduced oligogalacturonides moved freely through the plant and were not complexed to high-molecularweight compounds and immobilised; c) during passage through the plant, modifications to the oligogalacturonide occurred, presumably as a consequence of metabolism in the apoplastic space. We found evidence of i) esterification of the molecule, and ii) shortening of the oligogalacturonide chain. The results show that in the assay for protease-inhibitor-inducing factor using cut shoots, oligogalacturonide elicitors can move into the leaves and act directly on the cells producing protease inhibitor.

Apple (Malus domestica) is the most widely cultivated fruit crop in Europe and frequently causes allergic reactions with a variable degree of severity. So far, four apple allergens Mal d 1, Mal d 2, Mal d 3 and Mal d 4 have been identified. Mal d 1, a Bet v 1 related allergen, and Mal d 4, apple profilin, are sensitive to proteolytic degradation, whereas Mal d 2, a thaumatin-like protein and Mal d 3, a nonspecific lipid transfer protein, are rather stable to proteolytic processes. Mal d 1 and Mal d 4 were purified after expression in Escherichia coli expression system, while Mal d 2 and Mal d 3 were purified from apple fruit tissue. All purified proteins were subjected to detailed physicochemical characterisation to confirm their structural integrity and maintained IgE binding capacity. Detailed investigations of carbohydrate moieties of Mal d 2 demonstrated their involvement in the overall IgE binding capacity of this allergen. It was concluded that the folded structure and IgE binding capacity of all four allergens were preserved during purification.