[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: As a 1st step, this study aimed at investigating the microbial quality of liquid egg white in a French egg
processing company. Thirty raw and 33 pasteurized liquid egg white samples were analyzed. Pasteurization was globally found efficient on mesophilic contaminants (1.7 ± 1.6 and 0.8 ± 0.9 log CFU/mL in raw and pasteurized samples, respectively), including for the control of Salmonella. However,Gram-positive enterococci were still detected in the pasteurized samples. As a 2nd step, a representative bacterial collection was built for exploring the spoilage issue in egg-based chilled desserts. Custard cream was chosen as growth medium since this food is widely used for the production of French chilled desserts. All of the 166 isolates of the bacterial collection were shown to be able to grow and to induce spoilage of the custard cream at refrigeration temperature (10 °C). Several spoilage types were highlighted in the custard cream, on the basis of changes regarding pH, consistency, production of holes or gas. As a 3rd step, bacterial enzymatic activities were explored on custard cream-based agar media. The bacterial collection was reduced to 43 isolates, based on further selection regarding the genera and the spoilage types previously highlighted. Albeit to different degrees, all these isolates were able to produce proteases. A large part of these isolates also expressed lipolytic and amylolytic activities. This study
emphasizes the need to control egg white contamination and especially with Gram-positive heat-resistant Enterococi, in order to guarantee the shelf life of egg-based chilled desserts.
[Show abstract][Hide abstract] ABSTRACT: The reduction of wastes due to food spoilage may help save food resources among the current worldwide food production. Industrial egg products, obtained after egg breaking, recovery and pasteurisation of the internal egg components, are used in a wide range of food, including chilled egg- and milk-based food particularly susceptible to microbial spoilage due to their low cooking temperatures. One of the main bacteria involved in the spoilage of pasteurized egg and dairy products belongs to the spore-former B. cereus group. Several members of this group may lead to food spoilage due to their enzymatic activities, even at low temperatures. However, the risk of spoilage has mainly been assessed by targeting enzymatic activities in dairy products or in vegetables foods that do not require chilled storage.In order to consider more accurately the food spoilage issue at both optimal and low temperatures, this work proposes to characterize the enzymatic potential of a collection of 68 strains of the B. cereus group, coming from pasteurized liquid egg products collected in the French egg breaking industry at two consecutive seasons. An egg-based medium was designed, allowing the kinetic characterisation of bacterial lipases and proteases at optimal and refrigeration temperatures. The results contribute to further investigate a comprehensive bacterial collection that was already characterised for its cold adaptation, its ability to grow in whole egg at higher levels than in optimum laboratory medium, to colonize the industrial surfaces, and to induce phase, texture and colour shifts of whole egg.This study contributes to the search for early markers that could help the producers of pasteurized liquid egg and dairy products to better assess the microbial quality of their products for their orientation towards specific applications.
[Show abstract][Hide abstract] ABSTRACT: Although egg white is known as expressing efficient antimicrobial properties under physiological conditions, little is known on the microbial quality of industrial egg white. This issue is of great importance when egg white enters into the composition of highly perishable products, such as chilled egg-based desserts. Due to the high thermo-sensitivity of its constitutive proteins, liquid egg white is stabilized at low heat-treating times and temperatures (2 to 6 min at 55°C to 57°C in France). The remaining of heat-resisting bacteria may shorten the shelf-life of the desserts, with damaging health and economical consequences. The objective of this study was to investigate the microbial quality of raw and pasteurized liquid egg white products collected in a French company at two consecutive warm and cold seasons. A total of 63 samples were analyzed, including 30 raw and 33 pasteurized (at 57°C for 6 min) liquid egg white products. The level and type of bacterial contamination was evaluated by pour-plating in PCA (incubation for 24h at 30°C under aerobic conditions) and 16SrDNA sequencing of the colonies. The average counts were fairly low and a broad range of population was highlighted from one sample to another, namely 1.7 ± 1.6 log CFU/mL
[Show abstract][Hide abstract] ABSTRACT: Antibiotic resistance causes public health problems and stimulates research for novelantimicrobials. Particular attention is given to molecules that limit drug resistancedevelopment.1Hen egg white lysozyme acting on the bacterial cell envelope through itsphysico-chemical properties is thus a good candidate.2,3However, its antimicrobial effectcaused by membrane permeabilization on Gram-negative bacteria remains limited. But somephysico-chemical modifications of the lysozyme can modify its membrane activity, increasinglysozyme antimicrobial properties against E. coli ; dry-heating is able to induce suchmodifications. 4 Especially, we previously highlighted that native (N-L) and dry-heatedlysozyme (DH-L) disrupt the outer membrane of E. coli , but in different ways.3,4 The mode of insertion into the bacterial outer membrane and molecular interactions remains unknown.This was thus investigated using an E. coli lipopolysaccharide monolayer (LPSM) membranemodel, mimicking the outer leaflet of the bacterial outer membrane. The interactions betweenlysozyme and LPSM were studied by tensiometry, ellipsometry, atomic force microscopy(AFM) and Brewster angle microscopy (BAM). Both N-L and DH-L are able to insert into aLPSM. As expected, electrostatic interactions between the negatively charged LPSM and bothpositively charged forms of lysozyme were observed. Furthermore, we could establish that N-L and DH-L insertion into the LPSM depends on the presence of the polysaccharide moieties.These polysaccharide chains might increase the space between the lipid headgroups, enablinglysozyme insertion. Moreover, dry-heating increases the lysozyme affinity for the LPSM.Microscopic observations (BAM and AFM) show that the LPSM reorganizes and reorients inthe presence of DH-L, in contrast to N-L. Dry-heating thus improves the lysozyme insertion,which might explain the increased activity on the outer membrane of E. coli , resulting in ahigher antimicrobial effec
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Quatrième Edition du Congrès Maghrébin sur les Toxi-infections alimentaires "Sécurité sanitaire des aliments face aux mutations sociétales et à la mondialisation"
Fès (Maroc) - 16-18 Avril 2014
Le Maroc accueille du 16 au 18 Avril 2014 à la ville de Fès, la quatrième édition du Congrès Maghrébin sur les Toxi-infections alimentaires sous le thème : «Sécurité sanitaire des aliments face aux mutations sociétales et à la mondialisation».
Deux ateliers en marge du congrès sont organisés les 15 et 16 Avril 2014 sous les thèmes :
1) Analyse critique des principes, des étapes de mise en place et de l’audit du système HACCP dans les établissements de produits alimentaires et
2) Microbiologie Prévisionnelle.
[Show abstract][Hide abstract] ABSTRACT: Bovine abortion of unknown infectious etiology still remains a major economic problem. Thus, we investigated whether Brucella spp., Listeria monocytogenes, Salmonella spp., Campylobacter spp. and Coxiella burnetii are associated with abortion and/or stillbirth in Tunisian dairy cattle. Using a pan-Chlamydiales PCR, we also investigated the role of Chlamydiaceae, Waddlia chondrophila, Parachlamydia acanthamoebae and other members of the Chlamydiales order in this setting. Veterinary samples taken from mid to late-term abortions from twenty dairy herds were tested. From a total of 150 abortion cases collected, infectious agents were detected by PCR in 73 (48.66%) cases, 13 (8.66%) of which represented co-infections with two infectious agents. Detected pathogens include Brucella spp (31.3%), Chlamydiaceae (4.66%), Waddlia chondrophila (8%), Parachlamydia acanthamoebae (5.33%), Listeria monocytogenes (4.66%) and Salmonella spp. (3.33%). In contrast,
Campylobacter spp. and Coxiella burnetii DNA were not detected among the investigated veterinary samples. This
demonstrates that different bacterial agents may cause bovine abortion in Tunisia. This is the first report suggesting the role of Parachlamydia acanthamoebae in bovine abortion in Africa. Further studies with a larger number of samples are necessary to confirm whether this emerging pathogen is directly linked to abortion in cattle.
PLoS ONE 03/2014; 9(3):e91549. DOI:10.1371/journal.pone.0091549 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To evaluate the food safety and spoilage risks associated with psychrotrophic Bacillus cereus group bacteria for the egg product industry, and to search for relevant risk markers.
A collection of 68 psychrotrophic B. cereus group isolates, coming from pasteurized liquid whole egg products, was analyzed through a principal component analysis (PCA) regarding their spoilage and food safety risk potentials. The principal component analysis showed a clear differentiation between two groups within the collection, one half of the isolates representing a safety risk and the other half a spoilage risk.
Relevant risk markers were highlighted by PCA, i.e. (i) for the food safety risk, the presence of the specific 16S rDNA-1m genetic signature and the ability to grow at 43°C on solid medium; and (ii) for the spoilage risk, the presence of the cspA genetic signature.
This work represents a first step in the development of new diagnostic technologies for the assessment of the microbiological quality of foods likely to be contaminated with psychrotrophic B. cereus group bacteria. This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: For food as well as for medical applications, there is a growing interest for 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 E. 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 AFM 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 would be responsible of the increased activity of DH-L on the E. coli membranes.
Journal of Agricultural and Food Chemistry 01/2014; 62(7). DOI:10.1021/jf405155p · 2.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: All over the world, the incidence of Salmonella spp contamination on different food sources like broilers, clams and cow milk has increased rapidly in recent years. The multifaceted properties of Salomnella serovars allow the microorganism to grow and multiply in various food matrices, even under adverse conditions. Therefore, methods are needed to detect and trace this pathogen along the entire food supply network. In the present work, PFGE and ERIC-PCR were used to subtype 45 Salmonella isolates belonging to different serovars and derived from different food origins. Among these isolates, S. Enteritidis and S. Kentucky were found to be the most predominant serovars. The Discrimination Index obtained by ERIC-PCR (0.85) was slightly below the acceptable confidence value. The best discriminatory ability was observed when PFGE typing method was used alone (DI = 0.94) or combined with ERIC-PCR (DI = 0.93). A wide variety of profiles was observed between the different serovars using PFGE or/and ERIC-PCR. This diversity is particularly important when the sample origins are varied and even within the same sampling origin.
PLoS ONE 12/2013; 8(12):e81315. DOI:10.1371/journal.pone.0081315 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Abstract This article reports the prevalence and antibiotic resistance of the Bacillus cereus group isolated from different foods (milk and dairy products, spices, and rice salad) in Morocco. In total, 402 different food samples collected from 2008 to 2010 were analyzed by microbiological methods to isolate B. cereus. The strains were subjected to a polymerase chain reaction test in order to verify whether they belonged to the B. cereus group. Sixty-four of all isolates (15.9%) were found to be positive. Among the sources, B. cereus strains from milk and dairy products constituted the largest proportion of isolates (33/64; 51.6%) followed by spices (22/64; 34.4%) and salad with rice (9/64; 14.1%). The genetic diversity of the strains of B. cereus group was examined by pulsed-field gel electrophoresis (PFGE) of chromosomal DNA digested with SmaI. The enzyme restriction profiles showed a high degree of polymorphism among the strains. The results showed that PFGE analysis could reveal the genetic differences among B. cereus strains. Investigation of antibiotic-resistance profiles showed that isolates were resistant to ampicillin (98.4%), tetracycline (90.6%), oxacillin (100%), cefepime (100%), and penicillin (100%), and were susceptible to chloramphenicol (67.2%), erythromycin (84.4%), and gentamicin (100%). The results of this study indicated that B. cereus could be a significant etiological agent of food poisoning in Morocco because of its high prevalence. Also, we demonstrated that the majority of strains came from milk and dairy products. However, additional research involving cytotoxicity tests is needed to more evaluate this sanitary risk.
[Show abstract][Hide abstract] ABSTRACT: Natural preservatives answer the consumer demand for long shelf-life foods, considering synthetic molecules are perceived as a health risk. Lysozyme is already used because of its muramidase activity against Gram-positive bacteria. It is also described active against some Gram-negative bacteria; membrane disruption would be involved, but the mechanism remains unknown. In the presently reported 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.
Journal of Agricultural and Food Chemistry 09/2013; 61(41). DOI:10.1021/jf4029199 · 2.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Bacterial membranes are often thought to be the main targets of the antimicrobial activity of egg white. In order to test this hypothesis, the state of the membranes of Escherichia coli K-12 cells during either bactericidal (45°C) or bacteriostatic (30°C) incubation in
egg white at natural alkaline pH was studied by biochemical methods. Namely, the permeability of the outer membrane was evaluated through its ability to incorporate a hydrophobic fluorescent probe (1-N-phenylnaphthylamine), and the permeability of the cytoplasmic membrane was evaluated
through the release of a specific intracellular enzyme (β-galactosidase). The bacteria were observed by atomic force microscopy in order to support the biochemical results. At 45°C, the outer membrane of E. coli K-12 incorporated the hydrophobic probe, suggesting that it was
disrupted. In addition, the cytoplasmic β-galactosidase was released at this temperature. The atomic force microscopy analysis revealed the formation of spheroplasts, which provided further evidence of the cell wall disruption and a progressive release of cellular contents. At 30°C,
biochemical and micrographic experiments confirmed that membrane integrity was preserved. These techniques provide a useful approach for studying the mechanisms of bacterial cell death in egg white.
[Show abstract][Hide abstract] ABSTRACT: Rennes – France) Quelques mois après l’autorisation de BACTOCELL® DRINK en eau de boisson, Lallemand Animal Nutrition a organisé sa deuxième journée technique BACTOCELL® pondeuses. Cet évènement a eu lieu le 26 Juin à Rennes où plus d’une trentaine de professionnels du monde avicole étaient présents.
Le temps d’une demi-journée, différents sujets autour de la microflore des pondeuses ont été abordés. Plusieurs intervenants se sont succédés, apportant des présentations variées et complémentaires : Sophie Jan ( INRA-Agrocampus) Rennes, Hervé Morvan (LDA 22), Adeline Huneau (ANSES).