Valérie Lechevalier

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.

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.

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.

Role of Incubation Conditions and Protein Fraction on the Antimicrobial Activity of Egg White against Salmonella Enteritidis and Escherichia coli

The mechanism of egg white antimicrobial activity involves specific molecules and environmental factors. However, it is difficult to compare the data from the literature because of the use of various bacterial strains and incubation conditions. The aim of our study was to determine the effect of temperature, pH, inoculum size, and egg white protein concentration on egg white antimicrobial activity and to investigate the putative interactions among these factors by conducting a complete factorial design analysis. The behavior of Salmonella Enteritidis and Escherichia coli was studied after precultivation in tryptic soy broth and Luria-Bertani broth, respectively, using three different egg white protein concentrations (0, 10, and 100%), five temperatures (37, 40, 42, 45, and 48°C), two pHs (7.8 and 9.3), and six inoculum levels (3 to 8 log CFU/ml). The essential role of temperature was identified. An inverse relationship was observed between bacterial growth and an increase in temperature. The role of egg white proteins was clearly demonstrated. In the absence of egg white proteins, bacterial growth occurred under most incubation conditions, whereas the presence of 10 and 100% protein produced bacteriostatic or bactericidal effects. The interaction between temperature and protein concentration was significant. At the highest tested temperatures, proteins were less involved in the bactericidal effect. Bacterial destruction was higher at pH 9.3 than at pH 7.8. Under our experimental conditions, Salmonella Enteritidis was more resistant to inactivation by egg white than was E. coli.

The food matrix affects the anthocyanin profile of fortified egg and dairy matrices during processing and in vitro digestion

The aim of the present study was to understand to what extent the inclusion of anthocyanins into dairy and egg matrices could affect their stability after processing and their release and solubility during digestion. For this purpose, individual and total anthocyanin content of four different enriched matrices, namely custard dessert, milkshake, pancake and omelettete, was determined after their manufacturing and during in vitro digestion. Results showed that anthocyanin recovery after processing largely varied among matrices, mainly due to the treatments applied and the interactions developed with other food components. In terms of digestion, the present study showed that the inclusion of anthocyanins into food matrices could be an effective way to protect them against intestinal degradation, and also the incorporation of anthocyanins into matrices with different compositions and structures could represent an interesting and effective method to control the delivery of anthocyanins within the different compartments of the digestive tract.

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.