Combining heat treatment, control of water activity and pressure to preserve foods

Vesicle formation in the membrane of onion cells (Allium cepa) during rapid osmotic dehydration

BACKGROUND AND AIMS Optimization of osmotic dehydration in different plant cells has been investigated through the variation of parameters such as the nature of the sugar used, the concentration of osmotic solutions and the processing time. In micro-organisms such as the yeast, Saccharomyces cerevisiae, the exposure of a cell to a slow increase in osmotic pressure preserves cell viability after rehydration, while sudden dehydration involves a lower rate of cell viability, which could be due to membrane vesiculation. The aim of this work is to study cytoplasmic vesicle formation in onion epidermal cells (Allium cepa) as a function of the kinetics of osmotic pressure variation in the external…

Drying parameters greatly affect the destruction of Cronobacter sakazakii and Salmonella Typhimurium in standard buffer and milk

International audience; 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 re…

Cloning and expression of genes involved in conidiation and surface properties of Penicillium camemberti grown in liquid and solid cultures.

International audience; Based on bioinformatic data on model fungi, the rodA and wetA genes encoding, respectively, a RodA hydrophobin protein and the WetA protein involved in conidiation mechanisms, were PCR-cloned and characterized for the first time in Penicillium camemberti. These results, completed by a sequence of the brlA gene (available in GenBank), which encodes a major transcriptional regulator also involved in the conidiation mechanism, were used to compare, by qRT-PCR, the expression of the three genes in liquid and solid cultures in a synthetic medium. While expression of the brlA and wetA genes increased dramatically in both culture conditions after 4 days of growth, expressio…

Study and optimization of freeze-drying cycles of a model probiotic strain

This work is based on the experimental study of the freeze-drying process to understand the impact of numerous factors on the survival rates of a model probiotic strain of Lactobacillus casei type. With the aim to find out if cell density in the matrix and survival rates are linked, we have studied the location of the cells after freeze drying inside a porous  matrix composed of a lactose basis with a polymer, the polyvinylpyrrolidone (PVP) in various amounts. The best survival rate were obtained at slow freezing rate for a formulation containing 5% (m/V) of lactose and 5% (m/V) of PVP. Keywords: Freeze-Drying; Freezing; Probiotics; L. Casei ATCC 393 

Effect of Combined Physical Stresses on Cells: The Role of Water

International audience; The role of water in microorganism viability was assessed through the application of combined physical perturbations. The combination of different physical parameters could allow to balance the properties variations (especially water related) resulting from the increase of one parameter alone. Thus, it is possible to optimize the survival of cells in controlling these parameters. This was tested through two different examples. The first example shows that combination of osmotic level and temperature can allow optimizing yeast cell survival in following membrane fluidity variation. Moreover this analysis has allowed a better comprehension of cell inactivation during r…

Damage in Escherichia coli Cells Treated with a Combination of High Hydrostatic Pressure and Subzero Temperature

ABSTRACT The relationship between membrane permeability, changes in ultrastructure, and inactivation in Escherichia coli strain K-12TG1 cells subjected to high hydrostatic pressure treatment at room and subzero temperatures was studied. Propidium iodide staining performed before and after pressure treatment made it possible to distinguish between reversible and irreversible pressure-mediated cell membrane permeabilization. Changes in cell ultrastructure were studied using transmission electron microscopy (TEM), which showed noticeable condensation of nucleoids and aggregation of cytosolic proteins in cells fixed after decompression. A novel technique used to mix fixation reagents with the c…

Surviving the heat: heterogeneity of response inSaccharomyces cerevisiaeprovides insight into thermal damage to the membrane

Environmental heat stress impacts on the physiology and viability of microbial cells with concomitant implications for microbial activity and diversity. Previously, it has been demonstrated that gradual heating of Saccharomyces cerevisiae induces a degree of thermal resistance, whereas a heat shock results in a high level of cell death. Here, we show that the impact of exogenous nutrients on acquisition of thermal resistance differs between strains. Using single-cell methods, we demonstrate the extent of heterogeneity of the heat-stress response within populations of yeast cells and the presence of subpopulations that are reversibly damaged by heat stress. Such cells represent potential for…

Phenolic Acid-Mediated Regulation of the padC Gene, Encoding the Phenolic Acid Decarboxylase of Bacillus subtilis

ABSTRACT In Bacillus subtilis , several phenolic acids specifically induce expression of padC , encoding a phenolic acid decarboxylase that converts these antimicrobial compounds into vinyl derivatives. padC forms an operon with a putative coding sequence of unknown function, yveFG , and this coding sequence does not appear to be involved in the phenolic acid stress response (PASR). To identify putative regulators involved in the PASR, random transposon mutagenesis, combined with two different screens, was performed. PadR, a negative transcriptional regulator of padC expression, was identified. padR is not located in the vicinity of padC , and the expression of padR is low and appears const…

Screening of lactic acid bacteria for reducing power using a tetrazolium salt reduction method on milk agar.

WOS:000315703100020 ; www.elsevier.com/locate/jbiosc; International audience; Reducing activity is a physiological property of lactic acid bacteria (LAB) of technological importance. We developed a solid medium with tetrazolium dyes enabling weakly and strongly reducing LAB to be discriminated. It was used to quantify populations in a mixed culture (spreading method) and screen strains (spot method).

Inactivation of conidia of Penicillium chrysogenum, P. digitatum and P. italicum by ethanol solutions and vapours

A fractional factorial design, 2(5-1) experiments, was used for assessing the influence of 5 factors: water activity, aw [0.7, 0.9], temperature, T ( degrees C) [10, 30], mode of application, A [liquid, vapour], ethanol concentration, E (% w/w) [5, 10] and time, t (d) [1, 4] on the inactivation of spores of Penicillium chrysogenum, P. digitatum and P. italicum. Survival was determined by germination at optimal conditions within 3d. The experimental response was log (N 0/Nt), where N 0 and Nt (spore ml(-1)) the concentrations of viable spores at t=0 and t respectively. By a decreasing order of sensitivity to ethanol, moulds were ranked as followed: P. digitatum, P. italicum and P. chrysogenu…

Impact of oxidoreduction potential and of gas bubbling on rheological properties of non-fat yoghurt

International audience; The aim of this work was to study the effect of different gaseous conditions on the physico-chemical properties of yoghurt. Four conditions of oxidoreduction potential (Eh7), +433 mV (milk gassed with air), +405 mV (ungassed milk), +283 mV (milk gassed with N2) and −349 mV (milk gassed with N2–H2) were applied to milk. The rheological properties and microstructure of these yoghurts were determined by measuring apparent viscosity, whey separation (WS) and using confocal laser scanning microscopy. Exopolysaccharide (EPS) production by lactic acid bacteria (LAB) was also studied, and production was increased for extreme Eh7 values: the highest values were obtained for a…

Inactivation of dried spores of Bacillus subtilis 168 by a treatment combining high temperature and pressure

International audience; Specific treatments combining high temperatures of up to 150 °C and moderate pressure of up to 0.6 MPa have been applied to Bacillus subtilis 168 spores conditioned at different aw levels (between 0.10 and 0.70) corresponding to different residual water contents within the spore core. The spores were treated as a dry powder in a pressurized nitrogen environment or in water/glycerol solutions.These thermodynamic conditions were intended to prevent any water evaporation from the spore core during time/temperature treatments.Our results clearly show that retaining liquid water in the core by applying pressure during the treatment resulted in greater spore destruction (b…

Involvement of osmotic cell shrinkage on the proton extrusion rate in Saccharomyces cerevisiae

Saccharomyces cerevisiae has been subjected to hyperosmotic shocks by using permeating (sorbitol, xylitol, glycerol, NaCl) and nonpermeating (PEG 600) solutes. The proton extrusion rate decreased as the osmotic pressure increased, whichever solute was used. However, the total inhibition of the cellular H+ extrusion depended on the solute used. A total inhibition was observed at about 20 MPa with glycerol, xylitol and sorbitol. With PEG 600, a total inhibition of extracellular acidification was obtained at 8.5 MPa. NaCl, with an extracellular pressure of 37.8 MPa (near saturation), did not completely inhibit the extracellular acidification. These results showed that the total inhibition of p…

Study and optimization of freeze-drying cycles of model probiotic strain of lactobacillus casei type

International audience

Inactivation of PadR, the repressor of the phenolic acid stress response, by molecular interaction with Usp1, a universal stress protein from Lactobacillus plantarum, in Escherichia coli

ABSTRACT The phenolic acid decarboxylase gene padA is involved in the phenolic acid stress response (PASR) in gram-positive bacteria. In Lactobacillus plantarum , the padR gene encodes the negative transcriptional regulator of padA and is cotranscribed with a downstream gene, usp1 , which encodes a putative universal stress protein (USP), Usp1, of unknown function. The usp1 gene is overexpressed during the PASR. However, the role and the mechanism of action of the USPs are unknown in gram-positive bacteria. Therefore, to gain insights into the role of USPs in the PASR; (i) a usp1 deletion mutant was constructed; (ii) the two genes padR and usp1 were coexpressed with padA under its own promo…

Recovery Estimation of Dried Foodborne Pathogens Is Directly Related to Rehydration Kinetics.

International audience; Drying is a common process which is used to preserve food products and technological microorganisms, but which is deleterious for the cells. The aim of this study is to differentiate the effects of drying alone from the effects of the successive and necessary rehydration. Rehydration of dried bacteria is a critical step already studied in starter culture but not for different kinetics and not for pathogens. In the present study, the influence of rehydration kinetics was investigated for three foodborne pathogens involved in neonatal diseases caused by the consumption of rehydrated milk powder: Salmonella enterica subsp. enterica serovar Typhimurium, Salmonella enteri…

Interaction Between Osmotic and Hydrostatic Pressure in Yeast Inactivation

The protective effect of an osmotic perturbation on high-pressure inactivation has been investigated using a yeast strain:Saccharomyces cerevisiae. Osmotic shocks have been carried out with binary medium (glycerol/sorbitol and water) with or without glucose. Variations of osmotic level, osmotic shift kinetics and time between osmotic and hydrostatic treatment (350 MPa/10 min) have allowed the characterization of cell baroprotection. Two different cumulative baroprotection effects have been distinguished: one conferred by the solute on biological structures and the other related to the osmotic stress, cell volume variations and stress metabolism response (HSP, trehalose). These experiments h…

Nature of sterols affects plasma membrane behavior and yeast survival during dehydration.

International audience; The plasma membrane (PM) is a main site of injury during osmotic perturbation. Sterols, major lipids of the PM structure in eukaryotes, are thought to play a role in ensuring the stability of the lipid bilayer during physicochemical perturbations. Here, we investigated the relationship between the nature of PM sterols and resistance of the yeast Saccharomyces cerevisiae to hyperosmotic treatment. We compared the responses to osmotic dehydration (viability, sterol quantification, ultrastructure, cell volume, and membrane permeability) in the wild-type (WT) strain and the ergosterol mutant erg6Δ strain. Our main results suggest that the nature of membrane sterols gover…

Modeling the heat inactivation of foodborne pathogens in milk powder : High relevance of the substrate water activity

International audience; 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 afte…

A way to follow the viability of encapsulated Bifidobacterium bifidum subjected to a freeze-drying process in order to target the colon: Interest of flow cytometry

The aim of this work was to apply flow cytometry in order to assess and compare the viability of freeze-dried entrapped bacteria with an usual technique by quantification by plate count techniques. It also aimed at studying the effect of various cryoprotectants on the viability of an entrapped Bifidobacterium bifidum subjected to freeze-drying to check their ability to be delivered all along the gastro-intestinal tract. The alginate-pectinate beads were chosen as the encapsulation matrix added with different protectants. The beads were characterized by scanning electron microscopy and the viability was checked by both methods. The best combination to improve viability of entrapped bacteria …

Effect of oxidoreduction potential on aroma biosynthesis by lactic acid bacteria in nonfat yogurt

 ; The aim of this study was to investigate the effect of oxidoreduction potential (Eh) on the biosynthesis of aroma compounds by lactic acid bacteria in non-fat yogurt. The study was done with yogurts fermented by Lactobacillus bulgaricus and Streptococcus thermophilus. The Eh was modified by the application of different gaseous conditions (air, nitrogen, and nitrogen/hydrogen). Acetaldehyde, dimethyl sulfide, diacetyl, and pentane-2,3-dione, as the major endogenous odorant compounds of yogurt, were chosen as tracers for the biosynthesis of aroma compounds by lactic acid bacteria. Oxidative conditions favored the production of acetaldehyde, dimethyl sulfide, and diketones (diacetyl and pen…

L’imagerie microscopique et la spectroscopie de fluorescence : outils essentiels pour l’étude de modèles cellulaires sous haute pression

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Synergistic and Antagonistic Effects of Combined Subzero Temperature and High Pressure on Inactivation of Escherichia coli

ABSTRACT The combined effects of subzero temperature and high pressure on the inactivation of Escherichia coli K12TG1 were investigated. Cells of this bacterial strain were exposed to high pressure (50 to 450 MPa, 10-min holding time) at two temperatures (−20°C without freezing and 25°C) and three water activity levels (a w ) (0.850, 0.992, and ca. 1.000) achieved with the addition of glycerol. There was a synergistic interaction between subzero temperature and high pressure in their effects on microbial inactivation. Indeed, to achieve the same inactivation rate, the pressures required at −20°C (in the liquid state) were more than 100 MPa less than those required at 25°C, at pressures in t…

Caco-2 Invasion by Cronobacter sakazakii and Salmonella enterica Exposed to Drying and Heat Treatments in Dried State in Milk Powder

International audience; 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 hea…

Antioxidant Properties of Ergosterol and Its Role in Yeast Resistance to Oxidation

International audience; Although the functions and structural roles of sterols have been the subject of numerous studies, the reasons for the diversity of sterols in the different eukaryotic kingdoms remain unclear. It is thought that the specificity of sterols is linked to unidentified supplementary functions that could enable organisms to be better adapted to their environment. Ergosterol is accumulated by late branching fungi that encounter oxidative perturbations in their interfacial habitats. Here, we investigated the antioxidant properties of ergosterol using in vivo, in vitro, and in silico approaches. The results showed that ergosterol is involved in yeast resistance to tert-butyl h…

Conservation à long terme de systèmes biologiques viables et fonctionnels

National audience; La congélation et la déshydratation permettent la conservation de systèmes biologiques sur de longues périodes par le ralentissement des réactions de dégradation cellulaire. Ces opérations impliquent des transferts de chaleur et de masse pouvant conduire à l'altération des structures cellulaires. La maîtrise de la cinétique de ces transferts, associée à l'utilisation de différents protectants cellulaires, permet la mise en place de protocoles de congélation et de déshydratation spécifiques en fonction du système biologique à conserver.

Proton dynamics in bacterial spores, a neutron scattering investigation

International audience; Results from first neutron scattering experiments on bacterial spores are reported. The elastic intensities and mean square displacements have a non-linear behaviour as function of temperature, which is in agreement with a model presenting more pronounced variations at around 330 K (57 • C) and 400 K (127 • C). Based on the available literature on thermal properties of bacterial spores, mainly referring to differential scanning calorimetry, they are suggested to be associated to main endothermic transitions induced by coat and/or core bacterial response to heat treatment.

Contribution of exofacial thiol groups in the reducing activity of Lactococcus lactis

Lactococcus lactis can decrease the redox potential at pH 7 (E(h7)) from 200 to -200 mV in oxygen free Man-Rogosa-Sharpe media. Neither the consumption of oxidizing compounds or the release of reducing compounds during lactic acid fermentation were involved in the decrease in E(h7) by the bacteria. Thiol groups located on the bacterial cell surface appear to be the main components that are able to establish a greater exchange current between the Pt electrode and the bacteria. After the final E(h7) (-200 mV) was reached, only thiol-reactive reagents could restore the initial E(h7) value. Inhibition of the proton motive force showed no effect on maintaining the final E(h7) value. These result…

Effect of oxidoreduction potential and of gas bubbling on rheological properties and microstructure of acid skim milk gels acidified with glucono-delta-lactone

Milk oxidoreduction potential was modified using gases during the production of a model dairy product and its effect on gel setting was studied. Acidification by glucono-delta-lactone was used to examine the physicochemistry of gelation and to avoid variations due to microorganisms sensitive to oxidoreduction potential. Four conditions of oxidoreduction potential were applied to milk: milk was gassed with air, nongassed, gassed with N(2), or gassed with N(2)H(2). The rheological properties and microstructure of these gels were determined using viscoelasticimetry, measurement of whey separation, and confocal laser scanning microscopy. It appeared that a reducing environment led to less-aggre…

Recombinant hydroperoxide lyase for the production of aroma compounds: Effect of substrate on the yeast Yarrowia lipolytica

International audience; The aim of this study was to investigate the action mechanism of linoleic acid hydroperoxides (HPOD), which are the major substrates of hydroperoxide lyase for the production of flavour compounds, on the yeast Yarrowia lipolytica by evaluating their effect on the oxidative state of the cells. The total antioxidant capacity (TAC) and the activity of the main antioxidant enzymes, such as glutathione reductase, glutathione peroxidase and superoxide dismutase, of cells treated with HPOD were studied. The potential role of intracellular glutathione, including reduced glutathione (GSH) and oxidized glutathione (GSSG), in conferring HPOD resistance was also been examined. T…

Lateral reorganization of plasma membrane is involved in the yeast resistance to severe dehydration

International audience; In this study, we investigated the kinetic and the magnitude of dehydrations on yeast plasma membrane (PM) modifications because this parameter is crucial to cell survival. Functional (permeability) and structural (morphology, ultrastructure, and distribution of the protein Sur7-GFP contained in sterol-rich membrane microdomains) PM modifications were investigated by confocal and electron microscopy after progressive (non-lethal) and rapid (lethal) hyperosmotic perturbations. Rapid cell dehydration induced the formation of many PM invaginations followed by membrane internalization of low sterol content PM regions with time. Permeabilization of the plasma membrane occ…

High gas pressure: An innovative method for the inactivation of dried bacterial spores

In this article, an original non-thermal process to inactivate dehydrated bacterial spores is described. The use of gases such as nitrogen or argon as transmission media under high isostatic pressure led to an inactivation of over 2 logs CFU/g of Bacillus subtilis spores at 430 MPa, room temperature, for a 1 min treatment. A major requirement for the effectiveness of the process resided in the highly dehydrated state of the spores. Only a water activity below 0.3 led to substantial inactivation. The solubility of the gas in the lipid components of the spore and its diffusion properties was essential to inactivation. The main phenomenon involved seems to be the sorption of the gas under pres…

Ergosterol biosynthesis: a fungal pathway for life on land?

International audience; Sterols, essential lipids of most eukaryotic cells, ensure important structural and signaling functions. The selection pressure that has led to different dominant sterols in the three eukaryotic kingdoms remains unknown. Here, we investigated the influence of the progression in the different steps of the ergosterol biosynthetic pathway (EBP) on the yeast resistance to transitions from aqueous to aerial media, typical perturbations of the higher fungi habitats. Five mutants of the EBP (ergΔ), accumulating different sterol intermediates in the EBP, and the wild-type (WT) strain were exposed to drying under atmospheric air or nitrogen and wetting. Results show that the …

The role of water in bacteria spore resistance

Industrial production of dried yeast: plasma membrane as a survival indicator of air drying process

International audience; Preservation of microorganisms by desiccationis a major industrial interest.However, study of cell survival mechanismsthat occur during desiccation is complex. In this work, the impact of the magnitude and the kinetics of dehydration on yeastsurvival wereevaluatedin either hyperosmotic liquid medium or a gaseous environment. Asame lethal magnitude of dehydration and a same lethal kinetic effect were found. As previously shown for osmotic stress, this work demonstrate that yeast survival after drying was also related to plasma membrane disorganization, suggesting a similar passive rearrangement of the membrane components.Hyperosmotic treatment in liquid medium represe…

Direct investigation of viscosity of an atypical inner membrane of Bacillus spores: A molecular rotor/FLIM study

Abstract We utilize the fluorescent molecular rotor Bodipy-C12 to investigate the viscoelastic properties of hydrophobic layers of bacterial spores Bacillus subtilis. The molecular rotor shows a marked increase in fluorescence lifetime, from 0.3 to 4 ns, upon viscosity increase from 1 to 1500 cP and can be incorporated into the hydrophobic layers within the spores from dormant state through to germination. We use fluorescence lifetime imaging microscopy to visualize the viscosity inside different compartments of the bacterial spore in order to investigate the inner membrane and relate its compaction to the extreme resistance observed during exposure of spores to toxic chemicals. We demonstr…

Cellular Injuries in Cronobacter sakazakii CIP 103183T and Salmonella enterica Exposed to Drying and Subsequent Heat Treatment in Milk Powder

International audience; Because of the ability of foodborne pathogens to survive in low-moisture foods, their decontamination is an important issue in food protection. This study aimed to clarify some of the cellular mechanisms involved in inactivation of foodborne pathogens after drying and subsequent heating. Individual strains of Salmonella Typhimurium, Salmonella Senftenberg, and Cronobacter sakazakii were mixed into whole milk powder and dried to different water activity levels (0.25 and 0.58); the number of surviving cells was determined after drying and subsequent thermal treatments in closed vessels at 90 and 100 degrees C, for 30 and 120 s. For each condition, the percentage of unc…

Cryopreservation of Escherichia coli K12TG1: Protection from the damaging effects of supercooling by freezing

Injuries in living cells caused by water freezing during a freeze-thaw process have been extensively reported. In particular, intracellular water freezing has long been incriminated in cell death caused by a high cooling rate, but this supposition could not always be demonstrated. This work aims to discriminate the role of water freezing, dehydration and cold-induced injuries in cellular damage occuring during cryopreservation. For this purpose, Escherichia coli K12TG1 suspensions were maintained in a supercooled or frozen state at -20°C for times ranging from 10 min to 5 h. The supercooled state was maintained for a long period at -20°C by applying a non-injurious isostatic pressure (P<40 …

The Impact of Drying on Foodborne Pathogens Salmonella enterica and Cronobacter sakazakii

International audience; [Technical Session 1 – Intervention Strategies] Introduction: Salmonella enterica and Cronobacter sakazakii are foodborne pathogens responsible for severe infant illness. Their ability to survive in harsh environmental conditions make these species a matter of concern for the low moisture food industry. Purpose: This study aimed to evaluate and understand the impact of drying conditions on survival, physiology, and invasion capacity of Salmonella Typhimurium and C. sakazakii.Methods: Salmonella Typhimurium and C. sakazakii were mixed into whole milk powder and dried at different water activity (aw) levels (0.25, 0.58 and 0.80). For each strain, the impact of each dry…

Use of gases to improve survival of Bifidobacterium bifidum by modifying redox potential in fermented milk

The aim of this work was to study the effect of the oxidoreduction potential, modified using gas, on the growth and survival of a probiotic strain, Bifidobacterium bifidum, and 2 yogurt strains, Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus. Three fermented milks were manufactured with an initial oxidoreduction potential value adjusted to +440 mV (control milk), +350 mV (milk gassed with N(2)), and -300 mV [milk gassed with N(2) plus 4% (vol/vol) H(2) (N(2)-H(2))]. Acidification profiles, growth during milk fermentation and survival during storage at 4 °C for 28 d were determined. This study showed that fermented probiotic dairy products made from milk gassed with…

Fluorescent probes to evaluate the physiological state and activity of microbial biocatalysts: A guide for prokaryotic and eukaryotic investigation

International audience; Many fluorescent techniques are employed to evaluate the viability and activity of microbial cells used in biotechnology. These techniques are sometimes complex and the interpretation of results opened to misunderstanding. Moreover, new developments are constantly proposed especially concerning a more accurate evaluation of the state of the cells including eukaryotic microorganisms. This paper aims at presenting to biotechnologists unfamiliar with fluorescence the principles of these methods and the related possible pitfalls. It focuses on probes of the physical (integrity and fluidity) and energetical (intracellular pH and membrane potential) state of the cell membr…

Hydrodynamic characterization of a new small-scale reactor mixed by a magnetic bar

International audience; The purpose of this study was to evaluate the mixing characteristics of a new milliliter-scale (mL-scale) reactor developed for studying enzymatic activity or physiological cell response. The mL-scale reactor was designed to enable the integration of several sensors to carry out dynamic measurements in a controlled environment. Rapid homogeneity of the entire system is essential to ensure reproducible and reliable results, consequently the reactor was stirred to optimize both mass and heat transfers. A comparative study using three different techniques was undertaken to study mixing performances in the system. Firstly, mixing time (4,) was estimated in the reactor us…

Exploration of lipid metabolism in relation with plasma membrane properties of Duchenne muscular dystrophy cells: influence of L-carnitine.

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 functi…

A small HSP, Lo18, interacts with the cell membrane and modulates lipid physical state under heat shock conditions in a lactic acid bacterium

International audience; The small heat shock proteins (sHSP) are characterized by a chaperone activity to prevent irreversible protein denaturation. This study deals with the sHSP Lo18 induced by multiple stresses in Oenococcus oeni, a lactic acid bacterium. Using in situ immunocytochemistry and cellular fractionation experiments, we demonstrated the association of Lo18 with the membrane in O. oeni cells submitted to heat shock. The same result was obtained after exposure of cells to ethanol or benzyl alcohol, agents known to have an influence on membranes. For the different stresses, the protein was located on the periphery of the cell at membrane level and was also found within the cytopl…

High gas pressure survival/reactivation of soil microorganisms

International audience; Deep sea sediments constitute a considerable reserve of microorganisms belonging to different microbial communities. Our researches aimed to better understand cellular mechanisms related to cellular plasticity involved in resistance of such microbial communities to extreme conditions and more particularly to high level of pressure (> 50 MPa). Obviously, the first step is to isolate microorganisms present in deep sea sediments and then cultivate. The comparison of the cultivation of such microorganisms under atmospheric conditions and under pressure conditions will afford a possible reactivation of specific piezotolerants and/or piezophiles organisms from dormancy. Th…

Impact of probiotics on risk factors for cardiovascular diseases. A review.

International audience; Probiotic microorganisms have historically been used to rebalance disturbed intestinal microbiota and to diminish gastrointestinal disorders, such as diarrhea or inflammatory bowel diseases (e.g., Crohn's disease and ulcerative colitis). Recent studies explore the potential for expanded uses of probiotics on medical disorders that increase the risk of developing cardiovascular diseases and diabetes, such as obesity, hypercholesterolemia, arterial hypertension, and metabolic disturbances such as hyperhomocysteinemia and oxidative stress. This review aims at summarizing the proposed molecular and cellular mechanisms involved in probiotic-host interactions and to identi…

Thermodynamics of yeast cell osmoregulation: Passive mechanisms

International audience; The response of yeast cells to osmotic pressure variations of the medium were studied through the kinetics of cell-volume modifications corresponding to the mass transfer of water and solutes. Osmotic variations were made by modification of the concentration of an external binary solution (polyol/water) without nutritive components. Two phases were distinguished in the thermodynamic response. A transient phase following an osmotic shift, which is characterised by rapid water transfer across the cell membrane and whose kinetics determine cell viability; then, a steady-state phase is reached when the cell volume becomes quasi-constant. The response of the cell during t…

Optimized tableting for extremely oxygen-sensitive probiotics using direct compression

International audience; Faecalibacterium prausnitzii was previously recognized for its intestinal anti-inflammatory activities and it has been shown less abundant in patients with chronic intestinal diseases. However, the main problems encountered in the use of this interesting anaerobic microorganism are firstly its high sensitivity to the oxygen and secondly, its ability to reach the large intestine alive as targeted site. The aim of this study was to investigate the effect of direct compression on the viability of this probiotic strain after different compression pressure and storage using three different excipients (MCC, HPMC and HPMCP). The effect of compression process on cell viabili…

Drying process strongly affects probiotics viability and functionalities

International audience; Probiotic formulations are widely used and are proposed to have a variety of beneficial effects, depending on the probiotic strains present in the product. The impact of drying processes on the viability of probiotics is well documented. However, the impact of these processes on probiotics functionality remains unclear. In this work, we investigated variations in seven different bacterial markers after various desiccation processes. Markers were composed of four different viability evaluation (combining two growth abilities and two cytometric measurements) and in three in vitro functionalities: stimulation of IL-10 and IL-12 production by PBMCs (immunomodulation) and…

Behavior of plant plasma membranes under hydrostatic pressure as monitored by fluorescent environment-sensitive probes.

International audience; We monitored the behavior of plasma membrane (PM) isolated from tobacco cells (BY-2) under hydrostatic pressures up to 3.5 kbar at 30 °C, by steady-state fluorescence spectroscopy using the newly introduced environment-sensitive probe F2N12S and also Laurdan and di-4-ANEPPDHQ. The consequences of sterol depletion by methyl-β-cyclodextrin were also studied. We found that application of hydrostatic pressure led to a marked decrease of hydration as probed by F2N12S and to an increase of the generalized polarization excitation (GPex) of Laurdan. We observed that the hydration effect of sterol depletion was maximal between 1 and 1.5 kbar but was much less important at hig…

Etude, expérimentation et optimisation des cycles de lyophilisation d'une souche de probiotiques

International audience

Viability of lactic bacteria submitted to dehydration: Importance of the dehydration kinetics \textbar Servie des bactéries lactiques soumises à la déshydratation : Importance de la cinétique de déshydratation

International audience; no abstract

Applications des hautes pressions hydrostatiques en agroalimentaire

Anhydrobiosis: Inside yeast cells

International audience; Under natural conditions yeast cells as well as other microorganisms are regularly subjected to the influence of severe drought, which leads to their serious dehydration. The dry seasons are then changed by rains and there is a restoration of normal water potential inside the cells. To survive such seasonal changes a lot of vegetative microbial cells, which belong to various genera and species, may be able to enter into a state of anhydrobiosis, in which their metabolism is temporarily and reversibly suspended or delayed. This evolutionarily developed adaptation to extreme conditions of the environment is widely used for practical goals - for conservation of microorg…

Absolute Humidity Influences the Seasonal Persistence and Infectivity of Human Norovirus

ABSTRACT Norovirus (NoV) is one of the main causative agents of acute gastroenteritis worldwide. In temperate climates, outbreaks peak during the winter season. The mechanism by which climatic factors influence the occurrence of NoV outbreaks is unknown. We hypothesized that humidity is linked to NoV seasonality. Human NoV is not cultivatable, so we used cultivatable murine norovirus (MNV) as a surrogate to study its persistence when exposed to various levels of relative humidity (RH) from low (10% RH) to saturated (100% RH) conditions at 9 and 25°C. In addition, we conducted similar experiments with virus-like particles (VLPs) from the predominant GII-4 norovirus and studied changes in bin…

Modeling hexanal production in oxido-reducing conditions by the yeast Yarrowia lipolytica

International audience; Hexanal produced by cells of a recombinant Yarrowia lipolytica yeast expressing the hydroperoxide lyase (HPL) from green bell pepper fruit was studied under oxido-reducing conditions using the reducing dithiotreitol and oxidizing potassium ferricyanide compounds. The combined effect of pH, linoleic acid 13-hydroperoxides concentration, temperature and oxido-reducing molecules on the hexanal production was studied. Significant positive effects for the hexanal production were found using high concentrations of hydroperoxides (100 mM, 30 g/L). Adding reducing molecules enhanced significantly hexanal production while the oxidizing molecules had an inhibitory effect. Comb…

A new alternative in vitro method for quantification of Toxoplasma gondii infectivity.

An in vitro method to determine the infectious potency of an unknown suspension of the protozoan parasite Toxoplasma gondii based on kinetics of host cells lysis was developed. Mic1-3KO a mutant strain of T. gondii RH tachyzoites was inoculated in 25-cm² flasks containing a 90% confluent monolayer of human foreskin fibroblasts. Lysis kinetics was monitored for infection ratios ranging from 1∶10⁶ to 1∶10; we defined 10⁶ tachyzoites/ml⁻¹ as the threshold value for parasite egress. Results allowed us to build a calibration curve relating the initial infection ratios to the time needed to reach 10⁶ tachyzoites/ml⁻¹. Finally, we validated the method using a known mixture of dead and live parasit…

Cell inactivation and membrane damage after long-term treatments at sub-zero temperature in the supercooled and frozen states.

The survival of cells subjected to cooling at sub-zero temperature is of paramount concern in cryobiology. The susceptibility of cells to cryopreservation processes, especially freeze-thawing, stimulated considerable interest in better understanding the mechanisms leading to cell injury and inactivation. In this study, we assessed the viability of cells subjected to cold stress, through long-term supercooling experiments, versus freeze-thawing stress. The viability of Escherichia coli, Saccharomyces cerevisiae, and leukemia cells were assessed over time. Supercooled conditions were maintained for 71 days at -10 degrees C, and for 4 h at -15 degrees C, and -20 degrees C, without additives or…

Survie et/ou réactivation de microorganismes du sol sous hautes pressions gazeuses

National audience; Le sol constitue une réserve considérable de microorganismes représentatifs de nombreuses communautés cellulaires. Les recherches reposent sur l’hypothèse qu’il existe, dans les échantillons de sol, des communautés microbiennes adaptées à des conditions physiques (pression, température) très différentes de celles rencontrées dans nos laboratoires : soit parce que les conditions de milieu ont changé (sol), soit parce que les conditions de prélèvement sont très différentes des conditions de culture (grands fonds marins). Dans cette optique, l’utilisation des hautes pressions gazeuses (200-1000 bars dans un premier temps) est envisagée afin d’essayer de réactiver des microor…

Extremely rapid acclimation of Escherichia coli to high temperature over a few generations of a fed-batch culture during slow warming

This study aimed to demonstrate that adequate slow heating rate allows two strains of Escherichia coli rapid acclimation to higher temperature than upper growth and survival limits known to be strain-dependent. A laboratory (K12-TG1) and an environmental (DPD3084) strain of E. coli were subjected to rapid (few seconds) or slow warming (1 degrees C 12 h(-1)) in order to (re) evaluate upper survival and growth limits. The slow warming was applied from the ancestral temperature 37 degrees C to total cell death 46-54 degrees C: about 30 generations were propagated. Upper survival and growth limits for rapid warming (46 degrees C) were lower than for slow warming (46-54 degrees C). The thermal l…

Toxicity of fatty acid hydroperoxides towards Yarrowia lipolytica: Implication of their membrane fluidizing action

Abstract Linoleic acid hydroperoxide (HPOD), substrate of hydroperoxide lyase, an enzyme of the lipoxygenase pathway, can be transformed into many aromatic compounds, the so-called “green notes”. The presence of linoleic acid hydroperoxide in the culture medium of Yarrowia lipolytica, the yeast expressing the cloned hydroperoxide lyase of green bell pepper, undoubtedly exerted an inhibition on the growth and a toxic effect with 90% of yeast cells died after 120 min of exposition in 100 mM HPOD solution. The increase in cell membrane fluidity evaluated by measuring fluorescence generalized polarization with the increasing concentration of HPOD in the medium confirmed the fluidizing action of…

Can pressure-induced cell inactivation be related to cell volume compression? A case study for Saccharomyces cerevisiae

In this paper, emphasis has been put on the relationship between volume compression and cell inactivation for the Saccharomyces cerevisiae strain CBS 1171 submitted to high hydrostatic pressure treatments. The influence of cell dehydration on pressure inactivation was first investigated. Inactivation was found to be strongly limited, or even completely prevented for cells with a water content of 60% w/w or below. Moreover, the volume compression undergone by a single yeast cell was assessed as a function of pressure and hydration conditions using a high-pressure setup for pressure-volume-temperature measurements. Direct measurements of volume compression were performed on cell pellets after…

Data from: Ergosterol biosynthesis: a fungal pathway for life on land?

Sterols, essential lipids of most eukaryotic cells, ensure important structural and signaling functions. The selection pressure which has led to different dominant sterols in the three eukaryotic kingdoms remains unknown. Here we investigated the influence of the progression in the different steps of the ergosterol biosynthetic pathway (EBP) on the yeast resistance to transitions from aqueous to aerial media, typical perturbations of the higher fungi habitats. Five mutants of the EBP (ergΔ), accumulating different sterol intermediates in the EBP, and the WT strain were exposed to drying under atmospheric air or nitrogen and wetting. Results show that the progression in the EBP parallels an …