Stéphane Guyot - Academia.edu (original) (raw)

Papers by Stéphane Guyot

Microbial Biotechnology

SummaryAlthough mechanisms involved in response of Saccharomyces cerevisiae to osmotic challenge ... more SummaryAlthough mechanisms involved in response of Saccharomyces cerevisiae to osmotic challenge are well described for low and sudden stresses, little is known about how cells respond to a gradual increase of the osmotic pressure (reduced water activity; aw) over several generations as it could encounter during drying in nature or in food processes. Using glycerol as a stressor, we propagated S. cerevisiae through a ramp of the osmotic pressure (up to high molar concentrations to achieve testing‐to‐destruction) at the rate of 1.5 MPa day‐1 from 1.38 to 58.5 MPa (0.990–0.635 aw). Cultivability (measured at 1.38 MPa and at the harvest osmotic pressure) and glucose consumption compared with the corresponding sudden stress showed that yeasts were able to grow until about 10.5 MPa (0.926 aw) and to survive until about 58.5 MPa, whereas glucose consumption occurred until 13.5 MPa (about 0.915 aw). Nevertheless, the ramp conferred an advantage since yeasts harvested at 10.5 and 34.5 MPa (...

Microscopy and Microanalysis, 2019

In the context of microbiology, recent studies show the importance of ribonucleo-protein aggregat... more In the context of microbiology, recent studies show the importance of ribonucleo-protein aggregates (RNPs) for the understanding of mechanisms involved in cell responses to specific environmental conditions. The assembly and disassembly of aggregates is a dynamic process, the characterization of the stage of their evolution can be performed by the evaluation of their number. The aim of this study is to propose a method to automatically determine the count of RNPs. We show that the determination of a precise count is an issue by itself and hence, we propose three textural approaches: a classical point of view using Haralick features, a frequency point of view with generalized Fourier descriptors, and a structural point of view with Zernike moment descriptors (ZMD). These parameters are then used as inputs for a supervised classification in order to determine the most relevant. An experiment using a specific Saccharomyces cerevisiae strain presenting a fusion between a protein found i...

Food Research International, 2017

Due to the ability of foodborne pathogens to survive in low moisture foods, the decontamination o... more Due to the ability of foodborne pathogens to survive in low moisture foods, the decontamination of these products is an important issue in food hygiene. Up to now, such decontamination has mostly been achieved through empirical methods. The intention of this work is to establish a more rational use of heat treatment cycles. The effects of thermal treatment cycles on the inactivation of dried Salmonella Typhimurium, Salmonella Senftenberg, Cronobacter sakazakii and Escherichia coli were assessed. Bacteria were mixed with whole milk powder and dried down to different water activity levels (0.11, 0.25, 0.44 and 0.58). The rate of inactivated bacteria was determined after thermal treatment at 85°C, 90°C, 95°C and 100°C, from 0 s to 180 s in closed vessels, in order to maintain a w during treatment. In a first step, logarithmic bacterial inactivation was fitted by means of a classical loglinear model in which temperature and a w have a significant effect (p < 0.05). D T,aw values were estimated for each T, a w condition and the results clearly showed that a w is a major parameter in the thermal decontamination of dried foods, a lower a w involving greater thermal resistance. In a second step, Bigelow's law was used to determine z T , a classical parameter relative to temperature, and y aw values, a new parameter relative to a w resistance. The values obtained for z T and y aw showed that the bacterium most resistant to temperature variations is Salmonella Typhimurium, while the one most resistant to a w variations is Escherichia coli. These data will help design decontamination protocols or processes in closed batches for low moisture foods. Among low a w food products, whole milk powder (a w generally comprised between 0.20 and 0.45) production represents a very large volume (2.5 million tons per year in the European Union) which is directly consumed or included in the formulation of various food products such as dairy foods, cakes, cereal products, chocolate and infant formula (Forsythe, 2014). The milk used to make the powder was pasteurized but the further drying and conditioning processes could result in cross-contamination of the obtained powder (Carrasco,

Frontiers in Microbiology, 2017

Due to the ability of foodborne pathogens to survive in low moisture food, the decontamination of... more Due to the ability of foodborne pathogens to survive in low moisture food, the decontamination of milk powder is an important issue in food protection. The safety of food products is, however, not always insured and the different steps in the processing of food involve physiological and metabolic changes in bacteria. Among these changes, virulence properties may also be affected. In this study, the effect of drying and successive thermal treatments on the invasion capacity of Salmonella Typhimurium, Salmonella Senftenberg, and Cronobacter sakazakii was assessed. Bacteria were dried on milk powder at three different water activity levels (0.25, 0.58, and 0.80) and heated at two different temperatures (90 • C and 100 • C) for 30 and 120 s. After recovery, stressed bacterial populations were placed in contact with Caco-2 cells to estimate their invasion capacity. Our results show that drying increases the invasion capacity of foodborne pathogens, but that heat treatment in the dried state did not exert a selective pressure on bacterial cells depending on their invasion capacity after drying. Taken together, our findings add to the sum of knowledge on food safety in dried food products and provide insight into the effects of food processing.

Chemistry and physics of lipids, Jan 10, 2017

Increased levels of 7-ketocholesterol (7KC), which results mainly from cholesterol auto-oxidation... more Increased levels of 7-ketocholesterol (7KC), which results mainly from cholesterol auto-oxidation, are often found in the plasma and/or cerebrospinal fluid of patients with neurodegenerative diseases and might contribute to activation of microglial cells involved in neurodegeneration. As major cellular dysfunctions are induced by 7KC, it is important to identify molecules able to impair its side effects. Since consumption of olive and argan oils, and fish is important in the Mediterranean diet, the aim of the study was to determine the ability of oleic acid (OA), a major compound of olive and argan oil, and docosahexaenoic acid (DHA) present in fatty fishes, such as sardines, to attenuate 7KC-induced cytotoxic effects. Since elaidic acid (EA), the trans isomer of OA, can be found in hydrogenated cooking oils and fried foods, its effects on 7KC-induced cytotoxicity were also determined. In murine microglial BV-2 cells, 7KC induces cell growth inhibition, mitochondrial dysfunctions, r...

International Journal of Food Microbiology, 2017

Listeria monocytogenes, a bacterium that is responsible for listeriosis, is a very diverse specie... more Listeria monocytogenes, a bacterium that is responsible for listeriosis, is a very diverse species. Desiccation resistance has been rarely studied in L. monocytogenes, although it is a stress that is largely encountered by this microorganism in food-processing environments and that could be managed to prevent its presence. The objective of this study was to evaluate the resistance of 30 L. monocytogenes strains to moderate desiccation (75% relative humidity) and evaluate the correlation of such resistance with the strains' virulence, serotype and genotype. The results showed a great heterogeneity of strains regarding their ability to survive (loss of cultivability between 0.4 and 2.0 log). Strains were classified into three groups according to desiccation resistance (sensitive, intermediate, or resistant), and the strain repartition was analyzed relative to serotype, virulence level and environmental origin of the strains. No correlation was found between isolate origin and desiccation resistance. All serotype 1/2b strains were classified into the group of resistant strains. Virulent and hypovirulent strains were distributed among the three groups of desiccation resistance. Finally, a genomic comparison was performed based on 31 genes that were previously identified as being involved in desiccation resistance. The presence of those genes was localized among the genomes of some strains and compared regarding strainresistance levels. High nucleotide conservation was identified between resistant and desiccationsensitive strains. In conclusion, the findings regarding the strains of serotype 1/2b indicate potential serotype-specific resistance to desiccation, and thus, to relative humidity fluctuations potentially encountered in food-related environments. The genomic comparison of 31 genes associated to desiccation tolerance did not reveal differences among four strains which have different level of resistance to desiccation.

Food Microbiology, 2017

Salmonella Typhimurium and Cronobacter sakazakii are two foodborne pathogens involved in neonatal... more Salmonella Typhimurium and Cronobacter sakazakii are two foodborne pathogens involved in neonatal infections from milk powder and infant formula. Their ability to survive in low-moisture food and during processing from the decontamination to the dried state is a major issue in food protection. In this work, we studied the effects of the drying process on Salmonella Typhimurium and Cronobacter sakazakii, with the aim of identifying the drying parameters that could promote greater inactivation of these two foodborne pathogens. These two bacteria were dried under different atmospheric relative humidities in milk and phosphate-buffered saline, and the delays in growth recovery and cultivability were followed. We found that water activity was related to microorganism resistance. C. sakazakii was more resistant to drying than was S. Typhimurium, and milk increased the cultivability and recovery of these two species. High drying rates and low final water activity levels (0.11-0.58) had a strong negative effect on the growth recovery and cultivability of these species. In conclusion, we suggest that effective use of drying processes may provide a complementary tool for food decontamination and food safety during the production of low-moisture foods.

European Journal of Inorganic Chemistry, 2016

The new ligand L was prepared and features a 13‐membered tetraaza macrocyclic ring with a 1,8‐nap... more The new ligand L was prepared and features a 13‐membered tetraaza macrocyclic ring with a 1,8‐naphthalimide fluorophore appended to a C atom of its backbone. The protonation constants of L as well as its complexation constants with Zn2+ ions were determined in 1:1 water/methanol solutions by potentiometric titrations. Fluorimetric pH titrations were performed with L alone and L in the presence of Zn2+ ions (1:1), and the species distributions (%) versus pH were compared. A window‐shaped fluorescence trend was observed with pH for the L/Zn2+ system, which behaves as an off–on–off pH sensor. The on window is centred in the 6.5–7.5 pH range, in correspondence with the formation of [ZnL]2+. The ligand L also works as a sensor for Zn2+ ions in this pH window, which includes the physiological pH value (7.4). Accordingly, L was used as a probe for the visualization of the Zn2+ pools in Saccharomyces cerevisiae yeast cells at pH 7.4 and has the additional ability to distinguish between livi...

PLOS ONE, 2016

Relative air humidity fluctuations could potentially affect the development and persistence of pa... more Relative air humidity fluctuations could potentially affect the development and persistence of pathogenic microorganisms in their environments. This study aimed to characterize the impact of relative air humidity (RH) variations on the survival of Listeria monocytogenes, a bacterium persisting on food processing plant surfaces. To assess conditions leading to the lowest survival rate, four strains of L. monocytogenes (EGDe, CCL500, CCL128, and LO28) were exposed to different RH conditions (75%, 68%, 43% and 11%) with different drying kinetics and then rehydrated either progressively or instantaneously. The main factors that affected the survival of L. monocytogenes were RH level and rehydration kinetics. Lowest survival rates between 1% and 0.001% were obtained after 3 hours of treatment under optimal conditions (68% RH and instantaneous rehydration). The survival rate was decreased under 0.001% after prolonged exposure (16h) of cells under optimal conditions. Application of two successive dehydration and rehydration cycles led to an additional decrease in survival rate. This preliminary study, performed in model conditions with L. monocytogenes, showed that controlled ambient RH fluctuations could offer new possibilities to control foodborne pathogens in food processing environments and improve food safety.

Microscopy and Microanalysis, 2016

An original high-pressure microscopy chamber has been designed for real-time visualization of bio... more An original high-pressure microscopy chamber has been designed for real-time visualization of biological cell growth during high isostatic (gas or liquid) pressure treatments up to 200 MPa. This new system is highly flexible allowing cell visualization under a wide range of pressure levels as the thickness and the material of the observation window can be easily adapted. Moreover, the design of the observation area allows different microscope objectives to be used as close as possible to the observation window. This chamber can also be temperature controlled. In this study, the resistance and optical properties of this new high-pressure chamber have been tested and characterized. The use of this new chamber was illustrated by a real-time study of the growth of two different yeast strains – Saccharomyces cerevisiae and Candida viswanathii – under high isostatic gas pressure (30 or 20 MPa, respectively). Using image analysis software, we determined the evolution of the area of colonie...

Microscopy and Microanalysis, 2015

In biology, hemocytometers such as Malassez slides are widely used and are effective tools for co... more In biology, hemocytometers such as Malassez slides are widely used and are effective tools for counting cells manually. In a previous work, a robust algorithm was developed for grid extraction in Malassez slide images. This algorithm was evaluated on a set of 135 images and grids were accurately detected in most cases, but there remained failures for the most difficult images. In this work, we present an optimization of this algorithm that allows for 100% grid detection and a 25% improvement in grid positioning accuracy. These improvements make the algorithm fully reliable for grid detection. This optimization also allows complete erasing of the grid without altering the cells, which eases their segmentation.

PloS one, 2012

Duchenne muscular dystrophy (DMD) arises as a consequence of mutations in the dystrophin gene. Dy... more Duchenne muscular dystrophy (DMD) arises as a consequence of mutations in the dystrophin gene. Dystrophin is a membrane-spanning protein that connects the cytoskeleton and the basal lamina. The most distinctive features of DMD are a progressive muscular dystrophy, a myofiber degeneration with fibrosis and metabolic alterations such as fatty infiltration, however, little is known on lipid metabolism changes arising in Duchenne patient cells. Our goal was to identify metabolic changes occurring in Duchenne patient cells especially in terms of L-carnitine homeostasis, fatty acid metabolism both at the mitochondrial and peroxisomal level and the consequences on the membrane structure and function. In this paper, we compared the structural and functional characteristics of DMD patient and control cells. Using radiolabeled L-carnitine, we found, in patient muscle cells, a marked decrease in the uptake and the intracellular level of L-carnitine. Associated with this change, a decrease in t...

Microscopy and Microanalysis, 2014

In biology, cell counting is a primary measurement and it is usually performed manually using hem... more In biology, cell counting is a primary measurement and it is usually performed manually using hemocytometers such as Malassez blades. This work is tedious and can be automated using image processing. An algorithm based on Fourier transform filtering and the Hough transform was developed for Malassez blade grid extraction. This facilitates cell segmentation and counting within the grid. For the present work, a set of 137 images with high variability was processed. Grids were accurately detected in 98% of these images.

The Journal of Nutritional Biochemistry, 2009

Among the oxysterols accumulating in atherosclerotic plaque, 7-ketocholesterol (7KC) is a potent ... more Among the oxysterols accumulating in atherosclerotic plaque, 7-ketocholesterol (7KC) is a potent apoptotic inducer, which favours myelin figure formation and polar lipid accumulation. This investigation performed on U937 cells consisted in characterizing the myelin figure formation process; determining the effects of 7KC on the PI3-K/PDK-1/Akt signalling pathway; evaluating the activities of vitamin E (Vit-E) (alpha-tocopherol) on the formation of myelin figures and the PI3-K/PDK-1/Akt signalling pathway and assessing the effects of PI3-K inhibitors (LY-294002, 3-methyladenine) on the activity of Vit-E on cell death and polar lipid accumulation. The ultrastructural and biochemical characteristics of myelin figures (multilamellar cytoplasmic inclusions rich in phospholipids and 7KC present in acidic vesicles and the reversibility of these alterations) support the hypothesis that 7KC is an inducer of phospholipidosis. This oxysterol also induces important changes in lipid content and/or organization of the cytoplasmic membrane demonstrated with merocyanine 540 and fluorescence anisotropy, a loss of PI3-K activity and dephosphorylation of PDK-1 and Akt. It is noteworthy that Vit-E was able to counteract phospholipidosis and certain apoptotic associated events (caspase activation, lysosomal degradation) to restore PI3-K activity and to prevent PDK-1 and Akt dephosphorylation. When Vit-E was associated with LY-294002 or 3-methyladenine, impairment of 7KC-induced apoptosis was inhibited, and accumulation of polar lipids was less counteracted. Thus, 7KC-induced apoptosis is a PI3-K-dependent event, and Vit-E up- and down-regulates PI3-K activity and phospholipidosis, respectively.

International Journal of Food Microbiology, 2007

The effects of electric field (EF) treatments on Saccharomyces cerevisiae viability were investig... more The effects of electric field (EF) treatments on Saccharomyces cerevisiae viability were investigated using a PG200 electroporator (Hoefer Scientific Instrument, San Fransisco, CA, USA) with specific attention to induced thermal effects on cell death. Lethal electric fields (1.5 kV cm − 1 for 5 s) were shown to cause heat variations in the cell suspension medium (water + glycerol), while corresponding classical thermal treatments at equivalent temperatures had no effect on the cells viability. Variations of the electrical conductivity of the intra-and extracellular matrix caused by ions and solutes transfer across the membrane were shown to be involved in the observed heating. The results permitted to build a theoretical model for the temperature variations induced by electric fields. Using this model and the electrical conductivity of the different media, a plausible explanation of the cell death induced by low-intensity electric fields with long-duration pulses has been proposed. Indeed, cell mortality could in part be caused by direct and indirect effects of electric fields. Direct effects are related to well known electromechanical phenomena, whereas indirect effects are related to secondary thermal stress caused by plasma membrane thermoporation. This thermoporation was attributed to electrical conductivity variations and the corresponding intracellular heating.

Biotechnology and Bioengineering, 2005

We studied the mechanisms involved in heat gradient-induced thermotolerance of Saccharomyces cere... more We studied the mechanisms involved in heat gradient-induced thermotolerance of Saccharomyces cerevisiae. Yeasts were slowly heated in a nutrient medium from 25 to 508C at 0.58C/min or immediately heat shocked at 508C, and both sets of cultures were maintained at this temperature for 1 h. Cells that had been slowly heated showed a 50-fold higher survival rate than the rapidly heated cells. Such thermotolerance was found not to be related to protein synthesis. Indeed Hsp104 a known protein involved in yeast thermal resistance induced by a preconditioning mild heat treatment, was not synthesized and cycloheximide addition, a protein synthesis inhibitor, did not affect the thermoprotective effect. Moreover, a rapid cooling from 50 to 258C applied immediately after the heat slope treatment inhibited the mechanisms involved in thermotolerance. Such observations lead us to conclude that heat gradient-induced thermal resistance is not directly linked to mechanisms involving intracellular molecules synthesis or activity such as proteins (Hsps, enzymes) or osmolytes (trehalose). Other factors such as plasma membrane phospholipid denaturation could be involved in this phenomenon.

Journal of Agricultural and Food Chemistry, 2006