Catherine Guérin-Dubiard

Egg yolk phosvitin: preparation of metal-free purified protein by fast protein liquid chromatography using aqueous solvents

Two chromatographic methods for hen egg yolk phosvitin purification avoiding organic solvents were evaluated. Hydrophobic interaction and ion-exchange chromatographies were applied to isolated phosvitin. Hydrophobic interaction chromatography has better capacity than ion-exchange chromatography to fractionate phosvitin in their different polypeptides, but its protein yield was lower (0.7 vs. 1.7% of egg yolk dry matter). Finally, ion-exchange chromatography was selected and allowed to fractionate phosvitin polypeptides, including the recovering of phosphoproteins with high electrophoretic mobility: phosvettes. Highly purified (>98%) and free metal protein was obtained in reduced time. Phosvitin polypeptide heterogeneity was evidenced.

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.

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.

Ovotransferrin Plays a Major Role in the Strong Bactericidal Effect of Egg White against the Bacillus cereus Group.

Bacillus cereus group bacteria are opportunistically pathogenic spore-forming microorganisms well known in the sector of pasteurized food products because of their involvement in spoilage events. In the sector of egg product processing, these bacteria may lead to important economic losses. It seemed then relevant to study their behavior in egg white, a widely used egg product usually recognized as developing different levels of antimicrobial activities depending on the environmental conditions. A strong bactericidal effect (decrease in the bacterial population of 6.1 ± 0.2 log CFU/ml) was observed for 68 B. cereus group isolates, independently incubated at 30°C in egg white at pH 9.3 (natural egg white pH). To determine which components could explain such a strong bactericidal effect, an experimental strategy was carried out, based on egg white fractionation by ultrafiltration and by anion-exchange liquid chromatography. The role of the protein fraction was thus demonstrated, and subsequent nano-liquid chromatography-tandem mass spectrometry analyses allowed identification of ovotransferrin as a major protein involved. The strong bactericidal effect was confirmed in the presence of commercial ovotransferrin. Such a bactericidal effect (i.e., a decrease in the bacterial population through cell death) had never been described because ovotransferrin is known for its bacteriostatic effect (i.e., inhibition of growth) due to its ability to chelate iron. Surprisingly, the addition of iron did not reverse the bactericidal effect of ovotransferrin under alkaline conditions (pH 9.3), whereas it completely reversed this effect at pH 7.3. Ovotransferrin was shown to provoke a perturbation of the electrochemical potential of the cytoplasmic membrane. A membrane disturbance mechanism could, hence, be involved, leading to the lysis of B. cereus group bacteria incubated in egg white.

The nutritional quality of eggs

Scientific evidence and consumer awareness of the importance of diet in combination with a healthy lifestyle has led to an increased focus on the nutritional evaluation of certain foods such as eggs. Nutritional evaluation includes the nutrient quantification (per 100 g or per portion) of the food, which conveys the contribution of the nutrient content to an individuals daily supply and the role played by the food as part of a balanced and healthy diet. With this in mind, this chapter describes the function of certain nutrients, taking into account beneficial or adverse effects of an average intake level. Based on this evaluation, the appropriate use of enrichment to improve the nutritional quality of eggs is discussed.

Egg Compounds with Antioxidant and Mineral Binding Properties

Ovotransferrin is a monomeric egg white glycoprotein, belonging to the transferrin family of proteins widely distributed in various biological fluids. It has the same structural characteristics as hen serum transferrin, because these proteins derive from the same gene and differ only by their attached carbohydrate (Mizutani et al. 2001 ). This protein has a molecular weight of 77 kDa and is made up of two domains, an N-domain and a C-domain, with a short linking region (Williams 1982). Each domain has a binding site for metal ion, tyrosyl, histidyl, arginyl residues being implicated as effective in this action; the site requires synergistic anion binding (Oe et al. 1989). Ovotransferrin binds iron very strongly, in particular Fe3+, since its dissociation constant (KDiss) is 10−24 M (Stevens 1991); it can bind two iron atoms per molecule. The order of iron binding is pH dependent; at pH 6.0 it binds first to the C-domain, but at pH 8.5 it first binds to the N-domain. The function of ovotransferrin is generally accepted as iron transport. It can bind other metal ions, including toxic ones. Mizutani et al. (2005) report that the aluminum-bound form is almost

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