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.

Egg-White Proteins Have a Minor Impact on the Bactericidal Action of Egg White Toward Salmonella Enteritidis at 45°C.

Salmonella enterica serovar Enteritidis is noted for its ability to survive the harsh antibacterial activity of egg white which is presumed to explain its occurrence as the major food-borne pathogen associated with the consumption of eggs and egg products. Liquid egg white is a major ingredient for the food industry but, because of its thermal fragility, pasteurization is performed at the modest temperature of 57°C (for 2-6 min). Unfortunately, such treatment does not lead to sufficient reduction in S. Enteritidis contamination, which is a clear health concern when the product is consumed without cooking. However, egg white is able to limit S. Enteritidis growth due to its alkaline pH, iron deficiency and multiple antimicrobial proteins. This anti-Salmonella activity of egg white is temperature dependent and becomes bactericidal once the incubation temperature exceeds 42°C. This property is exploited in the highly promising pasteurization treatment (42-45°C for 1-5 days) which achieves complete killing of S. Enteritidis. However, the precise mechanism and the role of the egg-white proteins are not fully understood. Here, the impact of exposure of S. Enteritidis to egg white-based media, with or without egg-white proteins (>10 kDa), under bactericidal conditions (45°C) was explored by measuring survival and global expression. Surprisingly, the bactericidal activity of egg white at 45°C was only slightly affected by egg-white proteins indicating that they play a minor role in the bactericidal activity observed. Moreover, egg-white proteins had minimal impact on the global-gene-expression response to egg white such that very similar, major regulatory responses (20% genes affected) were observed both with and without egg-white proteins following incubation for 45 min at 45°C. Egg-white proteins caused a significant change in expression for just 64 genes, including the psp and lysozyme-inhibitor responses genes which is suggestive of an early membrane perturbation effect. Such damage was supported by disruption of the proton motive force by egg-white proteins. In summary, the results suggest that low-mass components of egg white are largely responsible for the bactericidal activity of egg white at 45°C.

The Three Lipocalins of Egg-White: Only Ex-FABP Inhibits Siderophore-Dependent Iron Sequestration by Salmonella Enteritidis.

Salmonella Enteritidis is the most prevalent food-borne pathogen associated with egg-related outbreaks in the European Union. During egg colonization, S. Enteritidis must resist the powerful anti-bacterial activities of egg white (EW) and overcome ovotransferrin-imposed iron-restriction (the most important anti-bacterial mechanism of EW). Many pathogens respond to iron restriction by secreting iron-chelating chemicals called siderophores but EW contains a siderophore-sequestering "lipocalin" protein (Ex-FABP) that is predicted to limit the usefulness of siderophores in EW. S. Enteritidis produces two siderophores: enterobactin, which is strongly bound by Ex-FABP; and the di-glucosylated enterobactin-derivative, salmochelin (a so-called "stealth" siderophore), which is not recognized by Ex-FABP. Thus, production of salmochelin may allow S. Enteritidis to escape Ex-FABP-mediated growth inhibition under iron restriction although it is unclear whether its EW concentration is sufficient to inhibit pathogens. Further, two other lipocalins (Cal-γ and α-1-ovoglycoprotein) are found in EW but their siderophore sequestration potential remains unexplored. In addition, the effect of EW lipocalins on the major EW pathogen, S. Enteritidis, has yet to be reported. We overexpressed and purified the three lipocalins of EW and investigated their ability to interact with the siderophores of S. Enteritidis, as well as their EW concentrations. The results show that Ex-FABP is present in EW at concentrations (5.1 µM) sufficient to inhibit growth of a salmochelin-deficient S. Enteritidis mutant under iron restriction but has little impact on the salmochelin-producing wildtype. Neither Cal-γ nor α-1-ovoglycoprotein bind salmochelin or enterobactin, nor do they inhibit iron-restricted growth of S. Enteritidis. However, both are present in EW at significant concentrations (5.6 and 233 µM, respectively) indicating that α-1-ovoglycoprotein is the 4th most abundant protein in EW, with Cal-γ and Ex-FABP at 11th and 12th most abundant. Further, we confirm the preference (16-fold) of Ex-FABP for the ferrated form (K d of 5.3 nM) of enterobactin over the iron-free form (K d of 86.2 nM), and its lack of affinity for salmochelin. In conclusion, our findings show that salmochelin production by S. Enteritidis enables this key egg-associated pathogen to overcome the enterobactin-sequestration activity of Ex-FABP when this lipocalin is provided at levels found in EW.

Intracellular osmoprotectant concentrations determine Propionibacterium freudenreichii survival during drying.

Propionibacterium freudenreichii is a beneficial bacterium widely used in food as a probiotic and as a cheese-ripening starter. In these different applications, it is produced, dried, and stored before being used. Both freeze-drying and spray-drying were considered for this purpose. Freeze-drying is a discontinuous process that is energy-consuming but that allows high cell survival. Spray-drying is a continuous process that is more energy-efficient but that can lead to massive bacterial death related to heat, osmotic, and oxidative stresses. We have shown that P. freudenreichii cultivated in hyperconcentrated rich media can be spray-dried with limited bacterial death. However, the general stress tolerance conferred by this hyperosmotic constraint remained a black box. In this study, we modulated P. freudenreichii growth conditions and monitored both osmoprotectant accumulation and stress tolerance acquisition. Changing the ratio between the carbohydrates provided and non-protein nitrogen during growth under osmotic constraint modulated osmoprotectant accumulation. This, in turn, was correlated with P. freudenreichii tolerance towards different stresses, on the one hand, and towards freeze-drying and spray-drying, on the other. Surprisingly, trehalose accumulation correlated with spray-drying survival and glycine betaine accumulation with freeze-drying. This first report showing the ability to modulate the trehalose/GB ratio in osmoprotectants accumulated by a probiotic bacterium opens new perspectives for the optimization of probiotics production.

Data from a proteomic analysis highlight different osmoadaptations in two strain of Propionibacterium freudenreichii.

The article presents a proteomic data set generated by a comparative analysis of the proteomes of Propionibacterium freudenreichii, comparing the CIRM-BIA 129 and CIRM-BIA 1025 strains. The two strains were cultivated until the beginning of the stationary phase in a chemical defined medium (MMO), and in this medium in the presence of NaCl, with or without glycine betaine. Whole-cell proteins were extracted, trypsinolyzed and analyzed by nano LC-MS/MS, prior to identification and classification by function using the X!Tandem pipeline software and the proteomic data from NCBI.nlm.nigh.gov. Quantification of proteins was then carried out in order to detect change in their expression depending on the culture medium. This article is related to the research article entitled "Benefits and drawbacks of osmotic adjustment in Propionibacterium freudenreichii". The comparative proteomic analysis of the two strains reveal strain-dependent and medium-dependent stress proteomes in the probiotic P. freudenreichii.

Propionibacterium freudenreichii CIRM-BIA 129 Osmoadaptation Coupled to Acid-Adaptation Increases Its Viability During Freeze-Drying.

Propionibacterium freudenreichii is a beneficial bacterium with documented effects on the gut microbiota and on inflammation. Its presence within the animal and human intestinal microbiota was correlated with immunomodulatory effects, mediated by both propionibacterial surface components and by secreted metabolites. It is widely implemented, both in the manufacture of fermented dairy products such as Swiss-type cheeses, and in the production of probiotic food complements, under the form of freeze-dried powders. The bottleneck of this drying process consists in the limited survival of bacteria during drying and storage. Protective pre-treatments have been applied to other bacteria and may, in a strain-dependent manner, confer enhanced resistance. However, very little information was yet published on P. freudenreichii adaptation to freeze-drying. In this report, an immunomodulatory strain of this probiotic bacterium was cultured under hyperosmotic constraint in order to trigger osmoadaptation. This adaptation was then combined with acid or thermal pre-treatment. Such combination led to accumulation of key stress proteins, of intracellular compatible solute glycine betaine, to modulation of the propionibacterial membrane composition, and to enhanced survival upon freeze-drying. This work opens new perspectives for efficient production of live and active probiotic propionibacteria.

Taking Advantage of Bacterial Adaptation in Order to Optimize Industrial Production of Dry Propionibacterium freudenreichii.

Propionibacterium freudenreichii is a beneficial bacterium, used both as a probiotic and as a cheese starter. Large-scale production of P. freudenreichii is required to meet growing consumers' demand. Production, drying and storage must be optimized, in order to guarantee high P. freudenreichii viability within powders. Compared to freeze-drying, spray drying constitutes the most productive and efficient, yet the most stressful process, imposing severe oxidative and thermal constraints. The aim of our study was to provide the tools in order to optimize the industrial production of dry P. freudenreichii. Bacterial adaptation is a well-known protective mechanism and may be used to improve bacterial tolerance towards technological stresses. However, the choice of bacterial adaptation type must consider industrial constraints. In this study, we combined (i) modulation of the growth medium composition, (ii) heat-adaptation, and (iii) osmoadaptation, in order to increase P. freudenreichii tolerance towards technological stresses, including thermal and oxidative constraints, using an experimental design. We further investigated optimal growth and adaptation conditions, by monitoring intracellular compatible solutes accumulation. Glucose addition, coupled to heat-adaptation, triggered accumulation of trehalose and of glycine betaine, which further provided high tolerance towards spray drying and storage. This work opens new perspectives for high quality and fast production of live propionibacteria at the industrial scale.

Benefits and drawbacks of osmotic adjustment in Propionibacterium freudenreichii.

Propionibacterium freudenreichii is a beneficial bacterium used as a cheese starter and as a probiotic. Indeed, selected strains of P. freudenreichii combine both technological and health-promoting abilities. Moreover, during large-scale industrial production of dried bacteria and during consumption, P. freudenreichii may undergo different stressful processes. Osmotic adaptation was shown to enhance P. freudenreichii tolerance towards stresses, which are encountered during freeze-drying and during digestion. In this report, we compared the osmoadaptation molecular mechanisms of two P. freudenreichii strains. Both osmotolerance and osmoadaptation were strain-dependent and had different effects on multiple stress tolerance, depending on the presence of osmoprotectants. Availability of glycine betaine (GB) restored the growth of one of the two strains. In this strain, osmotic preadaptation enhanced heat, oxidative and acid stresses tolerance, as well as survival upon freeze-drying. However, addition of GB in the medium had deleterious effects on stress tolerance, while restoring optimal growth under hyperosmotic constraint. In the other strain, neither salt nor GB enhanced stress tolerance, which was constitutively low. Accordingly, whole cell proteomics revealed that mechanisms triggered by salt in the presence and in the absence of GB are different between strains. Osmotic adjustment may thus have deleterious effects on industrial abilities of P. freudenreichii. BIOLOGICAL SIGNIFICANCE: Propionibacteria are found in various niches including fodder, silage, rumen, milk and cheeses. This means adaptation towards different ecological environments with different physicochemical parameters. Propionibacterium freudenreichii, in particular, is furthermore used both as dairy starter and as probiotic and is thus submitted to high scale industrial production. Production and subsequent stabilization still need optimization. Drying processes like freeze-drying are stressful. Osmotic adjustments may modulated tolerance towards drying. However, they are strain-dependent, medium-dependent and may either reduce or increase stress tolerance. A case-by-case study, for each strain-medium thus seems necessary. In this work, we identify key proteins involved in osmoadaptation and give new insights into adaptation mechanisms in P. freudenreichii. This opens new perspectives for the selections of strains and for the choice of the growth medium composition.

Review: Adaptation of Beneficial Propionibacteria, Lactobacilli, and Bifidobacteria Improves Tolerance Toward Technological and Digestive Stresses.

This review deals with beneficial bacteria, with a focus on lactobacilli, propionibacteria, and bifidobacteria. As being recognized as beneficial bacteria, they are consumed as probiotics in various food products. Some may also be used as starters in food fermentation. In either case, these bacteria may be exposed to various environmental stresses during industrial production steps, including drying and storage, and during the digestion process. In accordance with their adaptation to harsh environmental conditions, they possess adaptation mechanisms, which can be induced by pretreatments. Adaptive mechanisms include accumulation of compatible solutes and of energy storage compounds, which can be largely modulated by the culture conditions. They also include the regulation of energy production pathways, as well as the modulation of the cell envelop, i.e., membrane, cell wall, surface layers, and exopolysaccharides. They finally lead to the overexpression of molecular chaperones and of stress-responsive proteases. Triggering these adaptive mechanisms can improve the resistance of beneficial bacteria toward technological and digestive stresses. This opens new perspectives for the improvement of industrial processes efficiency with regard to the survival of beneficial bacteria. However, this bibliographical survey evidenced that adaptive responses are strain-dependent, so that growth and adaptation should be optimized case-by-case.

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.

Global Gene-expression Analysis of the Response of Salmonella Enteritidis to Egg White Exposure Reveals Multiple Egg White-imposed Stress Responses.

Chicken egg white protects the embryo from bacterial invaders by presenting an assortment of antagonistic activities that combine together to both kill and inhibit growth. The key features of the egg white anti-bacterial system are iron restriction, high pH, antibacterial peptides and proteins, and viscosity. Salmonella enterica serovar Enteritidis is the major pathogen responsible for egg-borne infection in humans, which is partly explained by its exceptional capacity for survival under the harsh conditions encountered within egg white. However, at temperatures up to 42°C, egg white exerts a much stronger bactericidal effect on S. Enteritidis than at lower temperatures, although the mechanism of egg white-induced killing is only partly understood. Here, for the first time, the impact of exposure of S. Enteritidis to egg white under bactericidal conditions (45°C) is explored by global-expression analysis. A large-scale (18.7% of genome) shift in transcription is revealed suggesting major changes in specific aspects of S. Enteritidis physiology: induction of egg white related stress-responses (envelope damage, exposure to heat and alkalinity, and translation shutdown); shift in energy metabolism from respiration to fermentation; and enhanced micronutrient provision (due to iron and biotin restriction). Little evidence of DNA damage or redox stress was obtained. Instead, data are consistent with envelope damage resulting in cell death by lysis. A surprise was the high degree of induction of hexonate/hexuronate utilization genes, despite no evidence indicating the presence of these substrates in egg white.

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.

Osmoadaptative responses in the rhizobia nodulating Acacia isolated from south-eastern Moroccan Sahara.

Four strains of rhizobia nodulating Acacia were isolated from the Moroccan desert soil by trapping with seedlings of Acacia gummifera and Acacia raddiana, and were studied for their ability to tolerate high salinity and dryness conditions. The strains MDSMC 2, MDSMC 18 and MDSMC 50 were halotolerant (they tolerated up to 1 M NaCl) and they accumulated glutamate and mannosucrose. The synthesis of the latter solute, which is the major endogenous osmolyte, is partially repressed in the presence of glycine betaine. The strain MDSMC 34 was less halotolerant (growth inhibited by a concentration greater than 0.5 M NaCl), and accumulated trehalose (as the main endogenous osmolyte) and glutamate. Rhizobia from the Moroccan desert soil were highly resistant to desiccation and their tolerance to dryness was stimulated by osmotic pretreatment. Thus, the accumulation of mannosucrose or trehalose by desert rhizobia represents both an osmoadaptative response and a part of a desiccation tolerance mechanism.

Glutamine, glutamate, and alpha-glucosylglycerate are the major osmotic solutes accumulated by Erwinia chrysanthemi strain 3937.

Erwinia chrysanthemi is a phytopathogenic soil enterobacterium closely related to Escherichia coli. Both species respond to hyperosmotic pressure and to external added osmoprotectants in a similar way. Unexpectedly, the pools of endogenous osmolytes show different compositions. Instead of the commonly accumulated glutamate and trehalose, E. chrysanthemi strain 3937 promotes the accumulation of glutamine and alpha-glucosylglycerate, which is a new osmolyte for enterobacteria, together with glutamine. The amounts of the three osmolytes increased with medium osmolarity and were reduced when betaine was provided in the growth medium. Both glutamine and glutamate showed a high rate of turnover, whereas glucosylglycerate stayed stable. In addition, the balance between the osmolytes depended on the osmolality of the medium. Glucosylglycerate and glutamate were the major intracellular compounds in low salt concentrations, whereas glutamine predominated at higher concentrations. Interestingly, the ammonium content of the medium also influenced the pool of osmolytes. During bacterial growth with 1 mM ammonium in stressing conditions, more glucosylglycerate accumulated by far than the other organic solutes. Glucosylglycerate synthesis has been described in some halophilic archaea and bacteria but not as a dominant osmolyte, and its role as an osmolyte in Erwinia chrysanthemi 3937 shows that nonhalophilic bacteria can also use ionic osmolytes.

Erwinia chrysanthemi O antigen is required for betaine osmoprotection in high-salt media.

Cellular components necessary for osmoprotection are poorly known. In this study we show that O antigen is specifically required for the effectiveness of betaines as osmoprotectants for Erwinia chrysanthemi in saline media. The phenotype is correlated with the inability of rfb mutant strains to maintain a high accumulation level of betaines in hypersaline media.

Erwinia chrysanthemi at high osmolarity: influence of osmoprotectants on growth and pectate lyase production.

The mechanism of osmotic stress adaptation was investigated in the phytopathogen Erwinia chrysanthemi. Growth of the bacterium was inhibited by elevated medium osmolarity, and exogenous glycine betaine, proline, ectoine or pipecolate permitted recovery of growth at inhibitory osmolarity. Osmoprotectants were taken up by transporters induced by elevated osmolarity, and their level of accumulation within the cell was dependent on the osmolarity of the growth medium. The influence of osmolarity and osmoprotectants on the production of pectate lyases (PLs) was investigated. Increased medium osmolarity resulted first in an induction of PL activity, followed by a shift to the basal level at higher osmolyte concentrations. This induction was reversed by osmoprotectants in the medium. The increased PL activity was attributed in part to the induced transcription of the main PL gene, pelE, and all the osmoprotectants that were analysed were found to prevent pelE induction. PL activity was partially inhibited in vitro by high ionic strength but not by elevated concentrations of sugars, and the addition of osmoprotectants at 1 mM had no effect on PL activity in vitro.