Food matrix structure (from Biscuit to Custard) has an impact on folate bioavailability in healthy volunteers.

PURPOSE: We examined the impact of matrix food structure on post-prandial folate bioavailability (and other macronutrients) in human volunteers using a randomized, controlled, crossover experimental design. METHODS: Twelve healthy male volunteers (22.6 ± 0.4 years old) were offered four food models (differing in matrix structure: Custard, Pudding, Sponge cake and Biscuit) to which 1 mg of folic acid was added, according to a randomized, controlled, crossover experimental design. Plasma folates, glucose, insulin, alpha amino nitrogen and triglycerides were measured over the post-prandial period (from T0 to T480 min). RESULTS: Food matrix structure was capable of altering folate plasma availability. The highest folate availability was observed for pudding and to a lesser extent Sponge cake whereas the lowest was for the two matrices presenting extreme rheological properties: Custard (liquid) (P < 0.05 total AUC) and to a lesser extent Biscuit (hard solid) (P < 0.05, AUC 180 min). The analysis of plasma kinetics of appearance of other nutrients/metabolites helps to understand/explain the lower bioavailability of folates in Custard and Biscuit. CONCLUSION: A least overall efficient bio-accessibility of all macronutrients and folic acid is observed in the gut lumen for Biscuit (delayed/incomplete destructuration of biscuit along the digestive tract). On the contrary, the lower folic acid absorption observed with custard does not fit with the rapid plasma appearance of other nutrients and should require further investigation.

Food material properties as determining factors in nutrient release during human gastric digestion: a review.

The fundamental mechanisms of nutrient release from solid foods during gastric digestion consists of multiple elementary processes. These include the diffusion of gastric juice into the food matrix and its simultaneous enzymatic degradation and mechanical breakdown by the peristaltic activity of the stomach. Understanding the relative role of these key processes, in association with the composition and structure of foods, is of paramount importance for the design and manufacture of novel foods possessing specific target behavior within the body. This review covers the past and current literature with respect to the in-stomach processes leading to physical and biochemical disintegration of solid foods and release of nutrients. The review outlines recent progress in experimental and modeling methods used for studying food disintegration mechanisms and concludes with a discussion on potential future research directions in this field. Information from pharmaceutical science-based modeling approaches describing nutrient release kinetics as a result of food disintegration in the gastric environment is also reviewed. Future research aimed at understanding gastric digestion is important not only for setting design principles for novel food design but also for understanding mechanisms underpinning dietary guidelines to consume wholesome foods.

Relative reactivity to egg white and yolk or change upon heating as markers for baked egg tolerance.

BACKGROUND: Hen's egg food allergy is frequent in childhood and phenotypically heterogeneous. Some children can tolerate extensively heated egg. We investigated whether individual relative responses could differentiate children who tolerate baked egg. METHODS: Reactivities to raw, pasteurized or hard-boiled egg (E), egg white (EW), and egg yolk (EY) fractions were tested by skin prick test (SPT) in 54 egg-allergic children. IgE-sensitization to EW and EY was determined by ImmunoCAP and IgE-binding to EW and 8 EW proteins and to EY and 4 EY sub-fractions by ELISA. Population heterogeneity was assessed by hierarchical ascending classification upon individual variations of reactivity and links between classifications and clinical features by analyzing the contingency tables. RESULTS: All children had positive SPT to raw E and raw EW and 72% to raw EY. Heating decreased SPT-reactivity for some children, pasteurization being less effective than hard-boiling. Children were classed into three classes from relative SPT-reactivity to raw fractions, two from variations of SPT-reactivity with each thermal processing or EW/EY ratio of sensitization, and four from their sensitization pattern. Classifications according to heating were found independent of each other. SPT variations with hard-boiling, IgE-sensitization (ratio or pattern) were linked to allowance by the physicians of egg in baked products. CONCLUSIONS: Egg-allergic children were often both sensitized to EY and EW, and heterogeneous patterns of relative responses were evidenced. Irrespective of age and level of sensitization, a low EW/EY ratio or SPT getting null with hard-boiling was found in children allowed to eat baked egg.

The anti-bacterial iron-restriction defence mechanisms of egg white; the potential role of three lipocalin-like proteins in resistance against Salmonella.

Salmonella enterica serovar Enteritidis (SE) is the most frequently-detected Salmonella in foodborne outbreaks in the European Union. Among such outbreaks, egg and egg products were identified as the most common vehicles of infection. Possibly, the major antibacterial property of egg white is iron restriction, which results from the presence of the iron-binding protein, ovotransferrin. To circumvent iron restriction, SE synthesise catecholate siderophores (i.e. enterobactin and salmochelin) that can chelate iron from host iron-binding proteins. Here, we highlight the role of lipocalin-like proteins found in egg white that could enhance egg-white iron restriction through sequestration of certain siderophores, including enterobactin. Indeed, it is now apparent that the egg-white lipocalin, Ex-FABP, can inhibit bacterial growth via its siderophore-binding capacity in vitro. However, it remains unclear whether Ex-FABP performs such a function in egg white or during bird infection. Regarding the two other lipocalins of egg white (Cal-γ and α-1-glycoprotein), there is currently no evidence to indicate that they sequester siderophores.

Antimicrobial activity of lysozyme isoforms: Key molecular features.

Increasing bacterial resistance towards antibiotics has stimulated research for novel antimicrobials. Proteins acting on bacterial membranes could be a solution. Lysozyme has been proven active against E. coli by disruption of both outer and cytoplasmic membranes, with dry-heating increasing lysozyme activity. Dry-heated lysozyme (DH-L) is a mixture of isoforms (isoaspartyl, native-like and succinimide lysozymes), giving rise to two questions: what effects does each form have, and which physicochemical properties are critical as regards the antibacterial activity? These issues were investigated by fractionating DH-L, analyzing structural properties of each fraction, and testing each fraction in vivo on bacteria and in vitro on membrane models. Positive net charge, hydrophobicity and molecular flexibility of the isoforms seem key parameters for their interaction with E. coli membranes. The succinimide lysozyme fraction, the most positive, flexible and hydrophobic, shows the highest antimicrobial activity, induces the strongest bacterial membrane disruption and is the most surface active on model lipid monolayers. Moreover, each fraction appears less efficient than DH-L against E. coli, indicating a synergetic cooperation between lysozyme isoforms. The bacterial membrane modifications induced by one isoform could facilitate the subsequent action of the other isoforms.

Native lysozyme and dry-heated lysozyme interactions with membrane lipid monolayers: lateral reorganization of LPS monolayer, model of the Escherichia coli outer membrane.

Lysozyme is mainly described active against Gram-positive bacteria, but is also efficient against some Gram-negative species. Especially, it was recently demonstrated that lysozyme disrupts Escherichia coli membranes. Moreover, dry-heating changes the physicochemical properties of the protein and increases the membrane activity of lysozyme. In order to elucidate the mode of insertion of lysozyme into the bacterial membrane, the interaction between lysozyme and a LPS monolayer mimicking the E. coli outer membrane has been investigated by tensiometry, ellipsometry, Brewster angle microscopy and atomic force microscopy. It was thus established that lysozyme has a high affinity for the LPS monolayer, and is able to insert into the latter as long as polysaccharide moieties are present, causing reorganization of the LPS monolayer. Dry-heating increases the lysozyme affinity for the LPS monolayer and its insertion capacity; the resulting reorganization of the LPS monolayer is different and more drastic than with the native protein.

Native and dry-heated lysozyme interactions with membrane lipid monolayers: Lipid packing modifications of a phospholipid mixture, model of the Escherichia coli cytoplasmic membrane.

Antimicrobial resistance is currently an important public health issue. The need for innovative antimicrobials is therefore growing. The ideal antimicrobial compound should limit antimicrobial resistance. Antimicrobial peptides or proteins such as hen egg white lysozyme are promising molecules that act on bacterial membranes. Hen egg white lysozyme has recently been identified as active on Gram-negative bacteria due to disruption of the outer and cytoplasmic membrane integrity. Furthermore, dry-heating (7 days and 80 °C) improves the membrane activity of lysozyme, resulting in higher antimicrobial activity. These in vivo findings suggest interactions between lysozyme and membrane lipids. This is consistent with the findings of several other authors who have shown lysozyme interaction with bacterial phospholipids such as phosphatidylglycerol and cardiolipin. However, until now, the interaction between lysozyme and bacterial cytoplasmic phospholipids has been in need of clarification. This study proposes the use of monolayer models with a realistic bacterial phospholipid composition in physiological conditions. The lysozyme/phospholipid interactions have been studied by surface pressure measurements, ellipsometry and atomic force microscopy. Native lysozyme has proved able to absorb and insert into a bacterial phospholipid monolayer, resulting in lipid packing reorganization, which in turn has lead to lateral cohesion modifications between phospholipids. Dry-heating of lysozyme has increased insertion capacity and ability to induce lipid packing modifications. These in vitro findings are then consistent with the increased membrane disruption potential of dry heated lysozyme in vivo compared to native lysozyme. Moreover, an eggPC monolayer study suggested that lysozyme/phospholipid interactions are specific to bacterial cytoplasmic membranes.

Egg white versus Salmonella Enteritidis! A harsh medium meets a resilient pathogen.

Salmonella enterica serovar Enteritidis is the prevalent egg-product-related food-borne pathogen. The egg-contamination capacity of S. Enteritidis includes its exceptional survival capability within the harsh conditions provided by egg white. Egg white proteins, such as lysozyme and ovotransferrin, are well known to play important roles in defence against bacterial invaders. Indeed, several additional minor proteins and peptides have recently been found to play known or potential roles in protection against bacterial contamination. However, although such antibacterial proteins are well studied, little is known about their efficacy under the environmental conditions prevalent in egg white. Thus, the influence of factors such as temperature, alkalinity, nutrient restriction, viscosity and cooperative interactions on the activities of antibacterial proteins in egg white remains unclear. This review critically assesses the available evidence on the antimicrobial components of egg white. In addition, mechanisms employed by S. Enteritidis to resist egg white exposure are also considered along with various genetic studies that have shed light upon egg white resistance systems. We also consider how multiple, antibacterial proteins operate in association with specific environmental factors within egg white to generate a lethal protective cocktail that preserves sterility.

The satiating effects of eggs or cottage cheese are similar in healthy subjects despite differences in postprandial kinetics.

Studies have reported a better satiating effect of eggs when compared with common cereal-based breakfasts, an effect that can be attributed to their macronutrient composition. Our aim was to compare the satiating power of an omelette and cottage cheese, both being common food snacks with similar nutrient compositions (containing proteins and lipids) but in different food forms. Thirty healthy volunteers participated in a randomized crossover trial. On each test day, the subjects consumed one of the two snacks, both providing 1346 kJ, 26 g protein, 21 g lipids, and 8 g lactose. The elapsed time between the snack and lunch request, their food intake at lunch, and their satiety scores were recorded. In a subgroup of 10 volunteers, blood was sampled to measure plasma metabolites and hormones. The two preloads were similar in terms of the time between the snack and a request for the buffet (167 ± 8 min), energy intake at the buffet (3988 ± 180 kJ) and appetite ratings. Plasma amino acid and urea concentrations indicated a marked delay in kinetic delivery after the eggs compared with the cottage cheese. In contrast, glucose, triglycerides and cholesterol displayed similar profiles after the snack. GIP and insulin secretions increased significantly after the cottage cheese, while glucagon and GLP-1 secretions were delayed with the omelette. We conclude that despite important differences in protein kinetics and their subsequent effects on hormone secretion, eggs and cottage cheese had a similar satiating power. This strongly suggests that with dose of proteins that is compatible to supplement strategies, i.e. 20-30 g, a modulation of protein kinetics is ineffective in increasing satiety.

Peptide bonds cleaved by pepsin are affected by the morphology of heat-induced ovalbumin aggregates.

The study aimed to assess the extent to which protein aggregation, and even the modality of aggregation, can affect gastric digestion, down to the nature of the hydrolyzed peptide bonds. By controlling pH and ionic strength during heating, linear or spherical ovalbumin (OVA) aggregates were prepared, then digested with pepsin. Statistical analysis characterized the peptide bonds specifically hydrolyzed versus those not hydrolyzed for a given condition, based on a detailed description of all these bonds. Aggregation limits pepsin access to buried regions of native OVA, but some cleavage sites specific to aggregates reflect specific hydrolysis pathways due to the denaturation-aggregation process. Cleavage sites specific to linear aggregates indicate greater denaturation compared to spherical aggregates, consistent with theoretical models of heat-induced aggregation of OVA. Thus, the peptides released during the gastric phase may vary depending on the aggregation modality. Precisely tuned aggregation may therefore allow subtle control of the digestion process.

Quantitative magnetic resonance imaging of in vitro gastrointestinal digestion of a bread and cheese meal.

The monitoring of food degradation during gastrointestinal digestion is essential in understanding food structure impacts on the bioaccessibility and bioavailability of nutrients. Magnetic Resonance Imaging (MRI) has the unique ability to access information on changes in multi-scale structural features of foods in a spatially resolved and non-destructive way. Our objective was to exploit various opportunities offered by MRI for monitoring starch, lipid and protein hydrolysis, as well as food particle breakdown during the semi-dynamic in vitro gastrointestinal digestion of complex foods combined in a meal. The meal consisted of French bread, hard cheese and water (drink), with a realistic distribution of bolus particle sizes. The MRI approach was reinforced by parallel chemical analysis of all macronutrients in the supernatant. By combining different imaging protocols, quantitative MRI provided insights into a number of phenomena at the level of the cheese and bread particles and within the liquid phase that are hard to access through conventional approaches. MRI thus revealed the progressive ingress of fluids into the bread crust and the release of the gas trapped in the crumb, the erosion of cheese particles, the creaming of fat, the disappearance of small food particles and changes in liquid phase composition. Excellent agreement was obtained between the quantitative parameters extracted from the MRI images and the results of the chemical analysis, demonstrating the strong potential of MRI for the monitoring of in vitro gastrointestinal digestion. The present study proposes further improvements to fully exploit the capabilities of MRI and constitutes an important step towards the extension of quantitative MRI to in vivo studies.

A pilot study for the intrinsic labeling of egg proteins with 15N and 13C.

The aim of this study was to produce intrinsically and uniformly doubly (15)N-(13)C-labeled proteins. These proteins can be used as intrinsic tracers of dietary amino acids, both α-amino groups and carbon skeletons, during postprandial metabolic utilization. Two (Rhodes) laying hens were fed for 16 days with a standard poultry diet supplemented with 0, 0.2% or 0.4% of a mixture of 20 doubly (15)N-(13)C-labeled AAs. A third hen was given a non-enriched diet, as the control. The eggs laid were collected over 24 days, from 3 days before to 4 days after supplementation. The (15)N and (13)C enrichments in proteins from white and yolk were measured by EA-IRMS and GC-C-IRMS for enrichment in individual amino acids. After 10 days of supplementation, the (15)N enrichment reached an isotopic plateau at 1500 to 3000 ‰, depending on the supplementation level, in both white and yolk while the (13)C enrichment was 220 to 650 ‰ in white and was 100 to 250 ‰ in yolk. The (15)N enrichment was similar among the amino acids, except for the aromatic ones in which the enrichment was lower. The δ(13)C values were variable among amino acids in both white and yolk, ranging from 77 ‰ for tyrosine to 555 ‰ for proline with the 0.2 % supplementation level. In conclusion, the incorporation of 0.2 % labeled amino acids in the hen diet allowed us to achieve sufficient enrichment for metabolic studies. However, due to the non-homogeneity of the (13)C labeling, adequate (13)C enrichment of individual amino acids must be considered depending on the investigated metabolic pathway.

Spatial-temporal mapping of the intra-gastric pepsin concentration and proteolysis in pigs fed egg white gels.

While there is a consensus that food structure affects food digestion, the underlying mechanisms remain poorly understood. A previous experiment in pigs fed egg white gels of same composition but different structures evidenced such effect on food gastric disintegration. In this study, we detailed the consequences on intra-gastric pH, pepsin concentration and proteolysis by sampling throughout the stomach over 6 h digestion. Subsequent amino acid absorption was investigated as well by blood sampling. While acidification was almost homogeneous after 6 h digestion regardless of the gel, pepsin distribution never became uniform. Pepsin started to accumulate in the pylorus/antrum region before concentrating in the body stomach beyond 4 h, time from which proteolysis really started. Interestingly, the more acidic and soft gel resulted in a soon (60 min) increase in proteolysis, an earlier and more intense peak of plasmatic amino acids, and a final pepsin concentration three times higher than with the other gels.

Strong improvement of interfacial properties can result from slight structural modifications of proteins: the case of native and dry-heated lysozyme.

Identification of the key physicochemical parameters of proteins that determine their interfacial properties is still incomplete and represents a real stake challenge, especially for food proteins. Many studies have thus consisted in comparing the interfacial behavior of different proteins, but it is difficult to draw clear conclusions when the molecules are completely different on several levels. Here the adsorption process of a model protein, the hen egg-white lysozyme, and the same protein that underwent a thermal treatment in the dry state, was characterized. The consequences of this treatment have been previously studied: net charge and hydrophobicity increase and lesser protein stability, but no secondary and tertiary structure modification (Desfougères, Y.; Jardin, J.; Lechevalier, V.; Pezennec, S.; Nau, F. Biomacromolecules 2011, 12, 156-166). The present study shows that these slight modifications dramatically increase the interfacial properties of the protein, since the adsorption to the air-water interface is much faster and more efficient (higher surface pressure). Moreover, a thick and strongly viscoelastic multilayer film is created, while native lysozyme adsorbs in a fragile monolayer film. Another striking result is that completely different behaviors were observed between two molecular species, i.e., native and native-like lysozyme, even though these species could not be distinguished by usual spectroscopic methods. This suggests that the air-water interface could be considered as a useful tool to reveal very subtle differences between protein molecules.

Succinimidyl residue formation in hen egg-white lysozyme favors the formation of intermolecular covalent bonds without affecting its tertiary structure.

Protein chemical degradations occur naturally into living cells as soon as proteins have been synthesized. Among these modifications, deamidation of asparagine or glutamine residues has been extensively studied, whereas the intermediate state, a succinimide derivative, was poorly investigated because of the difficulty of isolating those transient species. We used an indirect method, a limited thermal treatment in the dry state at acidic pH, to produce stable cyclic imide residues in hen lysozyme molecules, enabling us to examine the structural and functional properties of so modified proteins. Five cyclic imide rings have been located at sites directly accessible to solvent and did not lead to any changes in secondary or tertiary structures. However, they altered the catalytic properties of lysozyme and significantly decreased the intrinsic stability of the molecules. Moreover, dimerization occurred during the treatment, and this phenomenon was proportional to the extent of chemical degradation. We propose that succinimide formation could be responsible for covalent bond formation under specific physicochemical conditions that could be found in vivo.

Statistical modeling of in vitro pepsin specificity.

The specificity of pepsin, the major protease of gastric digestion, has been previously investigated, but only regarding the primary sequence of the protein substrates. The present study aimed to consider in addition physicochemical and structural characteristics, at the molecular and sub-molecular scales. For six different proteins submitted to in vitro gastric digestion, the peptide bonds cleaved were determined from the peptides released and identified by LC-MS/MS. An original statistical approach, based on propensity scores calculated for each amino acid residue on both sides of the peptide bonds, concluded that preferential cleavage occurred after Leu and Phe, and before Ile. Moreover, reliable statistical models developed for predicting peptide bond cleavage, highlighted the predominant role of the amino acid residues at the N-terminal side of the peptide bonds, up to the seventh position (P7 and P7'). The significant influence of hydrophobicity, charge and structural constraints around the peptide bonds was also evidenced.

The Role of Ovotransferrin in Egg-White Antimicrobial Activity: A Review.

Eggs are a whole food which affordably support human nutritional requirements worldwide. Eggs strongly resist bacterial infection due to an arsenal of defensive systems, many of which reside in the egg white. However, despite improved control of egg production and distribution, eggs remain a vehicle for foodborne transmission of Salmonella enterica serovar Enteritidis, which continues to represent a major public health challenge. It is generally accepted that iron deficiency, mediated by the iron-chelating properties of the egg-white protein ovotransferrin, has a key role in inhibiting infection of eggs by Salmonella. Ovotransferrin has an additional antibacterial activity beyond iron-chelation, which appears to depend on direct interaction with the bacterial cell surface, resulting in membrane perturbation. Current understanding of the antibacterial role of ovotransferrin is limited by a failure to fully consider its activity within the natural context of the egg white, where a series relevant environmental factors (such as alkalinity, high viscosity, ionic composition, and egg white protein interactions) may exert significant influence on ovotransferrin activity. This review provides an overview of what is known and what remains to be determined regarding the antimicrobial activity of ovotransferrin in egg white, and thus enhances understanding of egg safety through improved insight of this key antimicrobial component of eggs.

Identification of New Antimicrobial Peptides that Contribute to the Bactericidal Activity of Egg White against Salmonella enterica Serovar Enteritidis at 45 °C.

A recent work revealed that egg white (EW) at 45 °C exhibits powerful bactericidal activity against S. enterica serovar Enteritidis, which is surprisingly little affected by removal of the >10 kDa EW proteins. Here, we sought to identify the major EW factors responsible for this bactericidal activity by fractionating EW using ultrafiltration and nanofiltration and by characterizing the physicochemical and antimicrobial properties of the resulting fractions. In particular, 22 peptides were identified by nano-LC/MS-MS and the bactericidal activities of representative peptides (with predicted antimicrobial activity) were further assessed. Two peptides (FVPPVQR and GDPSAWSWGAEAHS) were found to be bactericidal against S. enterica serovar Enteritidis at 45 °C when provided in an EW environment. Nevertheless, these peptides contribute only part of this bactericidal activity, suggesting other, yet to be determined, antimicrobial factors.

In vitro static digestion reveals how plant proteins modulate model infant formula digestibility.

Infant formulas (IFs) are the key nutritional source for infants who cannot be breastfed. There is currently a growing interest in these sensitive products in order to control their quality and to design their composition with regard to nutritional balance. In a context of sustainable development and increasing growth of the world population, it seems essential to search for alternative to animal protein in food today. Plant proteins offer interesting nutritional and functional benefits thanks to the latest improvement through research and development. In this context, five model IFs were developed with identical composition, except that 50% of the proteins were either whey proteins in the "milk-reference IF", pea, faba bean, rice or potato proteins in the four "plant IFs" tested. The IFs were evaluated using an in vitro static gastro-intestinal model simulating infant conditions. The protein hydrolysis degree (DH) and the amino acid bioaccessibility (AAB) were used as indicators of protein digestibility. Results showed that both DH and AAB were very similar between the milk-reference IF, pea and faba bean IFs, but significantly lower for the rice and potato IFs. This study provides new insights into the impact of protein sources on IF digestibility.

Role of biochemical and mechanical disintegration on ß-carotene release from steamed and fried sweet potatoes during in vitro gastric digestion.

The role of biochemical and mechanical disintegration on ß-carotene release from steamed sweet potatoes (SSP) and fried sweet potatoes (FSP) during in vitro gastric digestion was investigated. Results revealed that, in the absence of mechanical forces generated by the stomach, biochemical digestion did not have a great effect on the breakdown of cell walls within the sweet potato food matrix and the release of ß-carotene was similar in both SSP and FSP. Cell wall in the plant-food may act as a physical 'barrier' towards the action of gastric juice and to the release of nutrients into the gastric digesta. However, FSP underwent quicker softening and collapse during in vitro gastric digestion compared to the compact and denser structure of SSP. This may explain the faster cell wall breakdown and subsequent ß-carotene release from FSP cellular matrix than SSP when mechanical forces are applied as in the human gastric simulator (HGS).