Villers-Bocage, France
Villers-Bocage, France
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Chatelard-Chauvin C.,POle fromager AOP Massif central | Pelissier F.,ACTALIA | Hulin S.,POle fromager AOP Massif central | Montel M.C.,French National Institute for Agricultural Research
Food Control | Year: 2015

The aim of this study was to run challenge tests to investigate the behaviour of Listeria monocytogenes in raw milk Cantal type uncooked pressed cheeses throughout the cheese process and during storage in different packagings (stretch film, vacuum packs and kraft paper) at 6°C during two-thirds of the storage period, then at 10°C; for kraft paper the last five days were at 15°C. Whatever the weight of the cheese (10 or 40kg), L.monocytogenes only increased during the first 24h of cheese making and decreased significantly in both cores and rinds during ripening at 9°C from day 45 to day 360. In cores, decay rate can depend on milk properties and in our experiment were associated with high levels of lactate and acetate during ripening. The decrease continued significantly in the cores during storage in all three packagings, more rapidly than during ripening. The populations of the pathogen were usually similar whatever the packaging. In rind, L monocytogenes decreased most significantly during storage in kraft paper; after 45 days of storage the counts were significantly lower in kraft paper than under vacuum whereas Gram positive non lactic acid bacteria and yeasts were at higher counts. The low aw (<0.90) in the rinds of kraft wrapped portions may be responsible for this decrease. High pH (6

PubMed | CNRS Laboratory of Physical Chemistry and Microbiology for the Environment, ACTIA, French National Institute for Agricultural Research and ACTALIA
Type: Journal Article | Journal: FEMS microbiology ecology | Year: 2016

Q phages infect Escherichia coli in the human gut by recognizing F-pili as receptors. Infection therefore occurs under reducing conditions induced by physiological agents (e.g. glutathione) or the intestinal bacterial flora. After excretion in the environment, phage particles are exposed to oxidizing conditions and sometimes disinfection. If inactivation does not occur, the phage may infect new hosts in the human gut through the oral route. During such a life cycle, we demonstrated that, outside the human gut, cysteines of the major protein capsid of Q phage form disulfide bonds. Disinfection with NaClO does not allow overoxidation to occur. Such oxidation induces inactivation rather by irreversible damage to the minor proteins. In the presence of glutathione, most disulfide bonds are reduced, which slightly increases the capacity of the phage to infect E. coli in vitro Such reduction is reversible and barely alters infectivity of the phage. Reduction of all disulfide bonds by dithiothreitol leads to complete capsid destabilization. These data provide new insights into how the phages are impacted by oxidizing-reducing conditions outside their host cell and raises the possibility of the intervention of the redox during life cycle of the phage.

PubMed | Plate Forme dInnovations Nouvelles Vagues, Aerial, ADIV, ACTALIA and 4 more.
Type: Journal Article | Journal: The ISME journal | Year: 2015

The microbial spoilage of meat and seafood products with short shelf lives is responsible for a significant amount of food waste. Food spoilage is a very heterogeneous process, involving the growth of various, poorly characterized bacterial communities. In this study, we conducted 16S ribosomal RNA gene pyrosequencing on 160 samples of fresh and spoiled foods to comparatively explore the bacterial communities associated with four meat products and four seafood products that are among the most consumed food items in Europe. We show that fresh products are contaminated in part by a microbiota similar to that found on the skin and in the gut of animals. However, this animal-derived microbiota was less prevalent and less abundant than a core microbiota, psychrotrophic in nature, mainly originated from the environment (water reservoirs). We clearly show that this core community found on meat and seafood products is the main reservoir of spoilage bacteria. We also show that storage conditions exert strong selective pressure on the initial microbiota: alpha diversity in fresh samples was 18958 operational taxonomic units (OTUs) but dropped to 2712 OTUs in spoiled samples. The OTU assemblage associated with spoilage was shaped by low storage temperatures, packaging and the nutritional value of the food matrix itself. These factors presumably act in tandem without any hierarchical pattern. Most notably, we were also able to identify putative new clades of dominant, previously undescribed bacteria occurring on spoiled seafood, a finding that emphasizes the importance of using culture-independent methods when studying food microbiota.

PubMed | French National Institute for Agricultural Research and ACTALIA
Type: Evaluation Studies | Journal: Journal of agricultural and food chemistry | Year: 2015

Chymosin is the major enzyme of natural rennet, traditionally used in cheese making for its high milk-clotting activity. For technical reasons, an accurate characterization of rennet should include its total clotting activity and also its enzymatic composition. Monoclonal antibodies specific to chymosin were obtained from mice immunized with purified bovine chymosin, and an inhibition enzyme-linked immunosorbent assay (ELISA) was developed for the quantification of chymosin in solution. No cross-reactivity was observed with other milk-clotting enzymes commonly used in cheese making. The limit of detection and limit of quantification were 125 and 400 ng/mL, respectively. The values of precision within and among runs were 7.23 and 7.39%, respectively, and satisfying recovery, from 92 to 119%, was found for spiked samples. The inhibition ELISA was successfully applied to commercial rennets, and the results were consistent with those obtained using the standard chromatographic method (IDF 110: A, 1987).

PubMed | Aerial, Montpellier University, ACTALIA, LNE and 2 more.
Type: | Journal: Food microbiology | Year: 2016

Predicting microbial safety of fresh products in modified atmosphere packaging implies to take into account the dynamic of O2, CO2 and N2 exchanges in the system and its effect on microbial growth. In this paper a mechanistic model coupling gas transfer and predictive microbiology was validated using dedicated challenge-tests performed on poultry meat, fresh salmon and processed cheese, inoculated with either Listeria monocytogenes or Pseudomonas fluorescens and packed in commercially used packaging materials (tray+lid films). The model succeeded in predicting the relative variation of O2, CO2 and N2 partial pressure in headspace and the growth of the studied microorganisms without any parameter identification. This work highlighted that the respiration of the targeted microorganism itself and/or that of the naturally present microflora could not be neglected in most of the cases, and could, in the particular case of aerobic microbes contribute to limit the growth by removing all residual O2 in the package. This work also confirmed the low sensitivity of L. monocytogenes toward CO2 while that of P. fluorescens permitted to efficiently prevent its growth by choosing the right combination of packaging gas permeability value and initial % of CO2 initially flushed in the pack.

PubMed | LABEO Manche, CNRS Laboratory of Physical Chemistry and Microbiology for the Environment, University of Caen Lower Normandy and Actalia
Type: Journal Article | Journal: Applied and environmental microbiology | Year: 2016

Human noroviruses (HuNoVs) are the main cause of shellfish-borne gastroenteritis outbreaks. In the absence of routine technical approaches allowing infectious particles to be detected, this viral pathogen is currently targeted by genome research, leading to difficult interpretations. In this study, we investigated the potential of F-specific RNA bacteriophages (FRNAPH) as fecal and viral contamination indicators in shellfish and water from a local harvesting area. FRNAPH were also used as microbial source tracking tools. Constraints imposed by detection limits are illustrated here by the detection of infectious FRNAPH in several samples in the absence of FRNAPH genomes. The opposite situation was also observed, likely explained by the persistence of the genomes being greater than infectivity. Similar considerations may be applied to HuNoVs, suggesting that HuNoV genome targeting is of limited relevance in assessing infectious risks. While FRNAPH did not provide any benefits compared to Escherichia coli as fecal pollution indicators in water, novel observations were made in shellfish: contrary to E. coli, a seasonal trend of infectious FRNAPH concentrations was observed. These concentrations were higher than those found in water, confirming bioaccumulation in shellfish. This study also underlines a relationship between the presence of HuNoV genomes and those of human-specific FRNAPH subgroup II (FRNAPH-II) in shellfish collected throughout Europe. Further research should be undertaken to evaluate FRNAPH potential as an indicator of the presence of infectious HuNoVs. To this end, shellfish involved in HuNoV-caused gastroenteritis outbreaks should be analyzed for the presence of infectious FRNAPH-II.This work provides new data about the use of F-specific RNA phages (FRNAPH) as a tool for evaluating fecal or viral contamination, especially in shellfish. In our case study, FRNAPH did not provide any benefits compared to E. coli as fecal pollution indicators in water but were found to be very useful in shellfish. Their concentrations in shellfish were higher than those found in the surrounding water, confirming bioaccumulation. This study also underlines a relationship between the presence of human norovirus genomes (HuNoVs) and those of FRNAPH subgroup II (FRNAPH-II). Considering that the two virus types have similar behaviors and since FRNAPH infectivity can be investigated, the specific detection of infectious FRNAPH-II could be regarded as an indication of the presence of infectious HuNoVs. The contribution of infectious human FRNAPH targeting for assessing the viral risk associated with HuNoVs in shellfish should thus be investigated.

PubMed | CNRS Laboratory of Physical Chemistry and Microbiology for the Environment and ACTALIA
Type: | Journal: Food and environmental virology | Year: 2016

Heat and free chlorine are among the most efficient and commonly used treatments to inactivate enteric viruses, but their global inactivation mechanisms have not been elucidated yet. These treatments have been shown to affect at least the capsid proteins of viruses and thus may affect the surface properties (i.e. electrostatic charge and hydrophobicity) of such particles. Our aim was to study the effects of heat and free chlorine on surface properties for a murine norovirus chosen as surrogate for human norovirus. No changes in the surface properties were observed with our methods for murine norovirus exposed to free chlorine. Only the heat treatment led to major changes in the surface properties of the virus with the expression of hydrophobic domains at the surface of the particles after exposure to a temperature of 55C. No modification of the expression of hydrophobic domains occurred after exposure to 60C, and the low hydrophobic state exhibited by infectious and inactivated particles after exposure to 60C appeared to be irreversible for inactivated particles only, which may provide a means to discriminate infectious from inactivated murine noroviruses. When exposed to a temperature of 72C or to free chlorine at a concentration of 50mg/L, the genome became available for RNases.

PubMed | CNRS Laboratory of Physical Chemistry and Microbiology for the Environment and ACTALIA
Type: Journal Article | Journal: Food and environmental virology | Year: 2016

The differences in physicochemical characteristics between infectious and non-infectious viral particles are poorly known. Even for heat, which is known as one of the most efficient treatments to inactivate enteric viruses, the global inactivation mechanisms have not been described yet. Such knowledge would help distinguish between both types of particles and therefore clarify the interpretation of the presence of viral genomes in food after heat treatment. In this study, we examined in particular the differences in electrostatic charge and hydrophobicity between the two particle types. MS2 phage, a common surrogate for enteric viruses, was used as a model virus. The heat-induced inactivation process of the infectious phages caused hydrophobic domains to be transiently exposed and their charge to become less negative. The particles also became progressively permeable to small molecules such as SYPRO Orange dye. The presence of non-infectious phage particles in which the genome was not accessible to RNases has been clearly demonstrated. These observations were done for MS2 phages exposed to a temperature of 60C. When exposed to a temperature higher than their critical temperature (72C), the particles were disrupted and the genome became available for RNases. At lower temperatures, 60C in this study, the transient expression of hydrophobic domains of remaining infectious phages appeared as an interesting parameter for improving their specific detection.

PubMed | CNRS Laboratory of Physical Chemistry and Microbiology for the Environment and ACTALIA
Type: | Journal: Journal of virological methods | Year: 2015

In recent years, foodborne viruses, especially human noroviruses (NoV) and hepatitis A virus (HAV), have been increasingly reported as the causes of foodborne disease outbreaks. Soft red fruits, especially raspberries, have a high incidence among the types of food concerned. Due to low infectious doses and low concentrations of enteric viruses in food samples, it is necessary to have an efficient and rapid detection method to implement prevention measures. A standard method for virus detection and quantification in food, including raspberries (XP CEN ISO/TS 15216-1 and -2, 2013) is currently available. This method proposes a consensus detection approach by RT-real time PCR (RT-qPCR) but also a virus extraction procedure based on the elution-concentration principle. In this study, an alternative method of extraction in which RNAs are directly extracted from food matrices (based on direct RNA extraction) has been optimized. First, each step was improved to make it a highly rapid, specific and simple method. Second, the standard virus concentration method was compared with the optimized direct RNA extraction one. Human enteric viral surrogates, Murine Norovirus (MNV) and F-specific RNA bacteriophage GA, were selected according to their adhesion properties and resistance to pH close to our main targets (NoV and HAV). Raspberries were artificially contaminated using two different techniques (immersion and spotting) in order to define a recovery rate and the amounts of virus recovered. Results showed that the direct RNA extraction method revealed significantly higher viral extraction efficiency (46.2%) than the elution-concentration method (20.3%), with similar proportions of inhibitors for both. In the same way with inoculation by spotting, the best recovery rate of GA phage (39.7% against 0.7%) and MNV (42.8% against 0.5%) was observed by direct RNA extraction. For the lowest concentrations of phage and virus in the immersion bath, only the direct RNA extraction method allowed their recovery. Direct RNA extraction proved to be more effective (best recovery rate), faster (<8h) and simpler (fewer steps) than the one proposed in the CEN ISO standard method when it came to detecting enteric viruses on raspberries.

PubMed | CNRS Laboratory of Physical Chemistry and Microbiology for the Environment and Actalia
Type: | Journal: Food microbiology | Year: 2017

F-specific RNA bacteriophages (FRNAPH) have been used as indicators of environmental fecal pollution for many years. While FRNAPH subgroup I (FRNAPH-I) are not host specific, some FRNAPH-II and -III strains appear specific to human pollution. Because a close relationship has been observed between FRNAPH-II genome and human norovirus (NoV) in shellfish, and because FRNAPH infectivity can easily be investigated unlike that of NoV, the detection of human infectious FRNAPH could therefore provide a valuable tool for assessing viral risk. In this study, an integrated cell culture real-time RT-PCR method has been developed to investigate infectious FRNAPH subgroup prevalence in oysters. This rapid screening method appears more sensitive than E.coli or NoV genome detection, and allows an FRNAPH subgroup present in low concentrations (0.05PFU/g of oyster) to be detected in the presence of another 1000 times more concentrated, without any dissection step. Its application to marketed oysters (n=135) over a 1-year period has allowed to identify the winter peak classically described for NoV or FRNAPH accumulation. Infectious FRNAPH were detected in 34% of batches, and 7% were suspected of having a human origin. This approach may be helpful to evaluate oysters depuration processes, based on an infectious viral parameter.

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