Françoise Nau

High Intensity Pulsed Electric Fields Applied to Egg White: Effect on Salmonella Enteritidis Inactivation and Protein Denaturation

High-intensity electric fields have been successfully applied to the destruction of Salmonella Enteritidis in diaultrafiltered egg white. The effects of electric field strength (from 20 to 35 kV x cm(-1)), pulse frequency (from 100 to 900 Hz), pulse number (from 2 to 8), temperature (from 4 to 30 degrees C), pH (from 7 to 9), and inoculum size (from 10(3) to 10(7) CFU x ml(-1)) were tested through a multifactorial experimental design. Experimental results indicate that, for Salmonella inactivation, the electric field intensity is the dominant factor with a strongly positive effect, strengthened by its positive interaction with pulse number. Pulse number, temperature, and pH have also significant positive effects but to a lesser extent. In the most efficient conditions, the pulsed electric field (PEF) treatment is capable of 3.5 log10 reduction in viable salmonellae. Simultaneously, the measure of surface hydrophobicity does not indicate any increase after PEF treatment. These results suggest that no protein denaturation occurs, unlike what is observed after comparable heat treatment in terms of Salmonella inactivation (55 degrees C for 15 min).

Transport of particles in intestinal mucus under simulated infant and adult physiological conditions: impact of mucus structure and extracellular DNA.

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.

Charge and size drive spontaneous self-assembly of oppositely charged globular proteins into microspheres.

Controlled interactions and assembly of proteins with one another promise to be a powerful approach for generating novel supramolecular architectures. In this study, we report on the ability of oppositely charged globular proteins to self-assemble into well-defined micrometer-sized spherical particles under specific physicochemical conditions. We show that microspheres were spontaneously formed in all binary protein mixtures tested once the physicochemical conditions were optimized. The optimal pH value, initial protein concentrations needed to form microspheres, and protein stoichiometry in these microspheres varied and depended on the structural features of the mixed proteins. We show that charge compensation is required but not sufficient to guide optimal protein assembly and organization into microspheres. Size difference between protein couples (acidic and basic) is a key element that defines optimal pH value for microsphere formation and the protein molar ratio in the formed microspheres. Our findings give new elements that can help to predict the assembly behavior of various proteins in mixed systems.

The extent of ovalbumin in vitro digestion and the nature of generated peptides are modulated by the morphology of protein aggregates

The impact of heat-induced aggregation on the extent of ovalbumin digestion and the nature of peptides released was investigated using an in vitro digestion model. The extent of hydrolysis, estimated by the disappearance of intact ovalbumin and the appearance of soluble peptides, was greater for the linear aggregates as compared to the spherical aggregates. The latter result may be due to differences in the surface area to volume ratio of the aggregates, or the degree of unfolding of the proteins during aggregate preparation. Peptide identification using LC-MS/MS highlighted that ovalbumin aggregation rendered a number of peptide bonds accessible to digestive proteases which were not accessible in native ovalbumin. Moreover, the peptide bonds that were cleaved appeared to be specific depending on the morphology of the aggregates. This work illustrates the links existing between food structure and their breakdown during the digestive process. Such quantitative and qualitative differences may have important nutritional consequences.

Effect of dry heating on the microbiological quality, functional properties, and natural bacteriostatic ability of egg white after reconstitution.

Spray-dried egg white (powder) is widely used in the food industry because of its variety of functional properties and its practical advantages. Moreover, egg white powder is generally considered safe because it can withstand high temperatures that allow for the destruction of all pathogens, especially Salmonella. In France, two types of treatments are used to improve the functional properties (whipping and gelling) of dried egg white: standard storage at 67 degrees C for about 15 days and storage at 75 to 80 degrees C for 15 days. The objective of this study was to investigate the effects of two dry-heating treatments (storage at 67 and 75 degrees C for 15 days) on the subsequent ability of egg white to resist Salmonella growth after reconstitution. The impact on the endogenous microflora of the powder and on its functional properties was also considered. Both dry-heating treatments were efficient in destroying a large number of Salmonella. Dry heating at 75 degrees C affected the bacteriostatic ability of reconstituted egg white to a greater extent than did dry heating at 67 degrees C. This loss of bacteriostatic ability could be attributable to the thermal denaturation of ovotransferrin, resulting in a reduction in its activity as an iron chelator. However, dry heating at 75 degrees C resulted in improved functional properties. Ultimately, no complete compromise between better functional quality and the preservation of the bacteriostatic ability of egg white after reconstitution is possible. Our results underline the importance of the use of hygienic conditions with egg white powder, especially with powder subjected to high-temperature treatments.

Hen Egg White Lysozyme Permeabilizes Escherichia coli Outer and Inner Membranes

Natural preservatives answer the consumer demand for long shelf life foods, synthetic molecules being perceived as a health risk. Lysozyme is already used because of its muramidase activity against Gram-positive bacteria. It is also described as active against some Gram-negative bacteria; membrane disruption would be involved, but the mechanism remains unknown. In this study, a spectrophotometric method using the mutant Escherichia coli ML-35p has been adapted to investigate membrane disruption by lysozyme for long durations. Lysozyme rapidly increases the permeability of the outer membrane of E. coli due to large size pore formation. A direct delayed activity of lysozyme against the inner membrane is also demonstrated, but without evidence of perforations.

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.

Dietary Proteins Contribute Little to Glucose Production Even Under Optimal Gluconeogenic Conditions in Healthy Humans

Dietary proteins are believed to participate significantly in maintaining blood glucose levels, but their contribution to endogenous glucose production (EGP) remains unclear. We investigated this question using multiple stable isotopes. After overnight fasting, eight healthy volunteers received an intravenous infusion of [6,6-2H2]-glucose. Two hours later, they ingested four eggs containing 23 g of intrinsically, uniformly, and doubly [15N]-[13C]–labeled proteins. Gas exchanges, expired CO2, blood, and urine were collected over the 8 h following egg ingestion. The cumulative amount of dietary amino acids (AAs) deaminated over this 8-h period was 18.1 ± 3.5%, 17.5% of them being oxidized. The EGP remained stable for 6 h but fell thereafter, concomitantly with blood glucose levels. During the 8 h after egg ingestion, 50.4 ± 7.7 g of glucose was produced, but only 3.9 ± 0.7 g originated from dietary AA. Our results show that the total postprandial contribution of dietary AA to EGP was small in humans habituated to a diet medium-rich in proteins, even after an overnight fast and in the absence of carbohydrates from the meal. These findings question the respective roles of dietary proteins and endogenous sources in generating significant amounts of glucose in order to maintain blood glucose levels in healthy subjects.

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.

Dry-heating of lysozyme increases its activity against Escherichia coli membranes.

For food as well as for medical applications, there is a growing interest in novel and natural antimicrobial molecules. Lysozyme is a promising candidate for the development of such molecules. This protein is largely studied and known for its muramidase activity against Gram-positive bacteria, but it also shows antimicrobial activity against Gram-negative bacteria, especially when previously modified. In this study, the activity of dry-heated lysozyme (DH-L) against Escherichia coli has been investigated and compared to that of native lysozyme (N-L). Whereas N-L only delays bacterial growth, DH-L causes an early-stage population decrease. The accompanying membrane permeabilization suggests that DH-L induces either larger pores or more pores in the outer membrane as compared to N-L, as well as more ion channels in the inner membrane. The strong morphological modifications observed by optical microscopy and atomic force microscopy when E. coli cells are treated with DH-L are consistent with the suggested disturbances of membrane integrity. The higher hydrophobicity, surface activity, and positive charge induced by dry-heating could be responsible for the increased activity of DH-L on the E. coli membranes.