Design of a new lyoprotectant increasing freeze-dried Lactobacillus strain survival to long-term storage.

BACKGROUND: Stabilization of freeze-dried lactic acid bacteria during long-term storage is challenging for the food industry. Water activity of the lyophilizates is clearly related to the water availability and maintaining a low aw during storage allows to increase bacteria viability. The aim of this study was to achieve a low water activity after freeze-drying and subsequently during long-term storage through the design of a lyoprotectant. Indeed, for the same water content as sucrose (commonly used lyoprotectant), water activity is lower for some components such as whey, micellar casein or inulin. We hypothesized that the addition of these components in a lyoprotectant, with a higher bound water content than sucrose would improve lactobacilli strains survival to long-term storage. Therefore, in this study, 5% whey (w/v), 5% micellar casein (w/v) or 5% inulin (w/v) were added to a 5% sucrose solution (w/v) and compared with a lyoprotectant only composed of 5% sucrose (w/v). Protective effect of the four lyoprotectants was assessed measuring Lactiplantibacillus plantarum CNCM I-4459 survival and water activity after freeze-drying and during 9 months storage at 25 °C. RESULTS: The addition whey and inulin were not effective in increasing Lactiplantibacillus plantarum CNCM I-4459 survival to long-term-storage (4 log reduction at 9 months storage). However, the addition of micellar casein to sucrose increased drastically the protective effect of the lyoprotectant (3.6 log i.e. 0.4 log reduction at 9 months storage). Comparing to a lyoprotectant containing whey or inulin, a lyoprotectant containing micellar casein resulted in a lower water activity after freeze-drying and its maintenance during storage (0.13 ± 0.05). CONCLUSIONS: The addition of micellar casein to a sucrose solution, contrary to the addition of whey and inulin, resulted in a higher bacterial viability to long-term storage. Indeed, for the same water content as the others lyoprotectants, a significant lower water activity was obtained with micellar casein during storage. Probably due to high bound water content of micellar casein, less water could be available for chemical degradation reactions, responsible for bacterial damages during long-term storage. Therefore, the addition of this component to a sucrose solution could be an effective strategy for dried bacteria stabilization during long-term storage.

Increased Survival of Lactococcus lactis Strains Subjected to Freeze-Drying after Cultivation in an Acid Medium: Involvement of Membrane Fluidity Cultivation in Acid Medium to Improve Bacterial Survival of Freeze-Drying.

RESEARCH BACKGROUND: Freeze-drying is the most widely used dehydration process in the food industry for the stabilization of bacteria. Studies have shown the effectiveness of an acid prestress in increasing the resistance of lactic acid bacteria to freeze-drying. Adaptation of bacteria to an acid stress is based on maintaining the properties of the plasma membrane. Indeed, the fatty acid composition of the membrane of lactic acid bacteria is often changed after an acid prestress. However, few studies have measured membrane fluidity after an acid stress during lactic acid bacterial strain cultivation. EXPERIMENTAL APPROACH: In order to use two pH profiles, the strains Lactococcus lactis NCDO 712 and NZ9000 were cultivated in two media, without any pH control. The two pH profiles obtained were representative of the initial medium composition, medium buffering properties and strain metabolism. Absorbance at 600 nm and pH were measured during bacterial cultivation. Then, the two strains were freeze-dried and their survival rates determined. Membrane fluidity was evaluated by fluorescence anisotropy measurements using a spectrofluorometer. RESULTS AND CONCLUSIONS: Cultivation under more acidic conditions significantly increased the survival during freeze-drying (p<0.05, ANOVA) of both strains. Moreover, in both strains of L. lactis, a more acidic condition during cultivation significantly increased membrane fluidity (p<0.05, ANOVA). Our results revealed that cultivation under such conditions, fluidifies the membrane and allows a better survival during freeze-drying of the two L. lactis strains. A more fluid membrane can facilitate membrane deformation and lateral reorganization of membrane components, critical for the maintenance of cellular integrity during dehydration and rehydration. NOVELTY AND SCIENTIFIC CONTRIBUTION: A better understanding of the involvement of membrane properties, especially of membrane fluidity, in bacterial resistance to dehydration is provided in this study.

Effect of ethanol perturbation on viscosity and permeability of an inner membrane in Bacillus subtilis spores.

In this work, we investigated how a combination of ethanol and high temperature (70°C), affect the properties of the inner membrane of Bacillus subtilis spores. We observed membrane permeabilization for ethanol concentrations ≥50%, as indicated by the staining of the spores' DNA by the cell impermeable dye Propidium Iodide. The loss of membrane integrity was also confirmed by a decrease in the peak corresponding to dipicolinic acid using infrared spectroscopy. Finally, the spore refractivity (as measured by phase contrast microscopy) was decreased after the ethanol-heat treatment, suggesting a partial rehydration of the protoplast. Previously we have used fluorescent lifetime imaging microscopy (FLIM) combined with the fluorescent molecular rotor Bodipy-C12 to study the microscopic viscosity in the inner membrane of B. subtilis spores, and showed that at normal conditions it is characterized by a very high viscosity. Here we demonstrate that the ethanol/high temperature treatment led to a decrease of the viscosity of the inner membrane, from 1000cP to 860cP for wild type spores at 50% of ethanol. Altogether, our present work confirms the deleterious effect of ethanol on the structure of B. subtilis spores, as well as demonstrates the ability of FLIM - Bodipy-C12 to measure changes in the microviscosity of the spores upon perturbation.

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

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.

Fisetin yeast-based bio-capsules via osmoporation: effects of process variables on the encapsulation efficiency and internalized fisetin content.

Osmoporation is an innovative method that can be used with food-grade yeast cells of Saccharomyces cerevisiae as natural encapsulating matrices. This technique overcomes barriers that difficult encapsulation and enables the internalization of fragile bioactive molecules such as fisetin into yeasts. In the present study, we assessed the effects of concentration, osmotic pressure, and temperature on the encapsulation efficiency (EE) and internalized fisetin content (IF). Two different quantification strategies were investigated: direct extraction (DE) without cell washing or freeze-drying steps and indirect extraction (IE) performed after washings with ethanol and freeze-drying. Our results showed that osmoporation improved EE (33 %) and IF (1.199 mg). The best experimental conditions were found by using DE. High-resolution images showed that the yeast cell envelope was preserved during osmoporation at 30 MPa and 84 % of yeast cells remained viable after treatment. Washing cells with organic solvent led to decreased EE (0.65 %) and IF (0.023 mg). This was probably due to either damages caused to yeast cell envelope or fisetin dragged out of cell. Overall, the results demonstrated the adequacy and relevant biotechnological potential of yeasts as encapsulating matrices for hydrophobic compounds. This fresh biotechnological approach has proven to be a promising tool for the production of bioactive-rich food products.

Conidiation of Penicillium camemberti in submerged liquid cultures is dependent on the nitrogen source.

OBJECTIVE: To study the ability of a commercial Penicillium camemberti strain, used for Camembert type cheese ripening, to produce conidia during growth in liquid culture (LC), in media containing different sources of nitrogen as, industrially, conidia are produced by growth at the surface of a solid state culture because conidiation in stirred submerged aerobic LC is not known. RESULTS: In complex media containing peptic digest of meat, hyphae ends did not differentiate into phialides and conidia. Contrarily, in a synthetic media containing KNO3 as sole nitrogen source, hyphae ends differentiated into phialides producing 0.5 × 10(7) conidia/ml. Conidia produced in LC were 25 % less hydrophobic than conidia produced in solid culture, and this correlates with a seven-times-lower expression of the gene rodA encoding hydrophobin RodA in the mycelium grown in LC. CONCLUSION: Conidiation of P. camembertii is stimulated in iquid medium containing KNO3 as sole source of nitrogen and therefore opens up opportunities for using liquid medium in commercial productions.

Innovative High Gas Pressure Microscopy Chamber Designed for Biological Cell Observation.

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 colonies as a function of time, and thus calculated colony expansion rates.

Surviving the heat: heterogeneity of response in Saccharomyces cerevisiae provides 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 recovery of entire populations once stresses are removed. The results show that plasma membrane permeability and potential are key factors involved in cell survival, but thermal resistance is not related to homeoviscous adaptation of the plasma membrane. These results have implications for growth and regrowth of populations experiencing environmental heat stress and our understanding of impacts at the level of the single cell. Given the important role of microbes in biofuel production and bioremediation, a thorough understanding of the impact of stress responses of populations and individuals is highly desirable.

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

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 4ns, upon viscosity increase from 1 to 1500cP 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 demonstrate that the bacterial spores possess an inner membrane that is characterized by a very high viscosity, exceeding 1000cP, where the lipid bilayer is likely in a gel state. We also show that this membrane evolves during germination to reach a viscosity value close to that of a vegetative cell membrane, ca. 600cP. The present study demonstrates quantitative imaging of the microscopic viscosity in hydrophobic layers of bacterial spores Bacillus subtilis and shows the potential for further investigation of spore membranes under environmental stress.

Absolute Humidity Influences the Seasonal Persistence and Infectivity of Human Norovirus.

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 binding patterns to A, B, and O group carbohydrates that might reflect capsid alterations. The responses of MNV and VLP to humidity were somewhat similar, with 10 and 100% RH exhibiting a strong conserving effect for both models, whereas 50% RH was detrimental for MNV infectivity and VLP binding capacity. The data analysis suggested that absolute humidity (AH) rather than RH is the critical factor for keeping NoV infectious, with an AH below 0.007 kg water/kg air being favorable to NoV survival. Retrospective surveys of the meteorological data in Paris for the last 14 years showed that AH average values have almost always been below 0.007 kg water/kg air during the winter (i.e., 0.0046 ± 0.0014 kg water/kg air), and this finding supports the fact that low AH provides an ideal condition for NoV persistence and transmission during cold months.

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

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 identify the nature of the resulting beneficial effects. Specific probiotic strains can act by modulating immune response, by producing particular molecules or releasing biopeptides, and by modulating nervous system activity. To date, the majority of studies have been conducted in animal models. New investigations on the related mechanisms in humans need to be carried out to better enable targeted and effective use of the broad variety of probiotic strains.

Survival kit of Saccharomyces cerevisiae for anhydrobiosis.

Yeast cells are well adapted to interfacial habitats, such as the surfaces of soil or plants, where they can resist frequent fluctuations between wet and dry conditions. Saccharomyces cerevisiae is recognized as an anhydrobiotic organism, and it has been the subject of numerous studies that aimed to elucidate this ability. Extensive data have been obtained from these studies based on a wide range of experimental approaches, which have added significantly to our understanding of the cellular bases and mechanisms of resistance to desiccation. The aim of this review is to provide an integrated view of these mechanisms in yeast and to describe the survival kit of S. cerevisiae for anhydrobiosis. This kit comprises constitutive and inducible mechanisms that prevent cell damage during dehydration and rehydration. This review also aims to characterize clearly the phenomenon of anhydrobiosis itself based on detailed descriptions of the causes and effects of the constraints imposed on cells by desiccation. These constraints mainly lead to mechanical, structural, and oxidative damage to cell components. Considerations of these constraints and the possible utilization of components of the survival kit could help to improve the survival of sensitive cells of interest during desiccation.

Osmoporation: a simple way to internalize hydrophilic molecules into yeast.

Internalization of hydrophilic molecules into yeast cytosol is required for different applications such as cell transformation or preservation of water soluble components by bioencapsulation. However, these molecules are not able to cross the plasma membrane and strategies have to be developed. Recent works revealed that osmotic perturbations could induce non-lethal transient permeabilization of the plasma membrane. In this work, we endeavored to clarify the phenomenon of permeabilization during rehydration after a mild hyperosmotic perturbation in order to evaluate the possibility of hydrophilic molecule internalization in yeast by this treatment. Rehydration step is particularly interesting because the large entry of water into the cells could help the internalization of molecules. The internalization of a fluorescent molecule [fluorescein isothiocyanate Dextran (FITC-Dextran), 20 kDa], added during the rehydration after a sublethal hyperosmotic treatment, was studied in Saccharomyces cerevisiae yeast cells. The internalization kinetic and the localization of the fluorescent molecules were studied by flow cytometry and fluorescence confocal microscopy. Our results show that the rehydration leads to the rapid internalization of FITC-Dextran due to a transient plasma membrane permeabilization. Thus, osmoporation, i.e. plasma membrane poration by modifications of osmotic pressure of the extracellular medium, could be a new and simple way to deliver molecules of particular interest into yeasts.

Production of conidia of Penicillium camemberti in liquid medium through microcycles of conidiation.

Microcycle conidiation is a survival mechanism of fungi encountering unfavorable conditions. In this phenomenon, asexual spores germinate secondary spores directly without formation of mycelium. As Penicillium camemberti conidia have the ability to produce conidiophores after germination in liquid culture induced by a thermal stress (18 and 30 °C), our work has aimed at producing conidia through this mean. Incubation at 18 and 30 °C increased the swelling of conidia and their proportion thereby producing conidiophores. Our results showed that the microcycle of conidiation can produce 5 × 10(8) conidia ml(-1) after 7 days at 18 °C of culture. The activity of these conidia was checked through culture on a solid medium. Conidia produced by microcycle conidiation formed a normal mycelium on the surface of solid media and 25 % could still germinate after 5 months of storage.

Sensitivity to vinyl phenol derivatives produced by phenolic acid decarboxylase activity in Escherichia coli and several food-borne Gram-negative species.

Ferulic, p-coumaric, and caffeic acids are phenolic acids present in soil, food, and gut, which have antimicrobial effects. Some Gram (+) bacteria metabolize these phenolic acids into vinyl derivatives due to phenolic acid decarboxylase activity (PAD) involved in the phenolic acid stress response (PASR). In this study, the antimicrobial activity of phenolic acids and their vinyl derivatives was tested on a panel of desirable and undesirable food-borne bacteria, especially Gram (-) species of Salmonella, Enterobacter, Klebsiella, and Pseudomonas, most of them without PAD activity. Native and engineered Escherichia coli strains either expressing or not PAD activity were included. Gram (-) bacteria of the panel were not significantly inhibited by phenolic acids at 3 mM, but were dramatically inhibited by the corresponding vinyl derivatives. On the contrary, Gram (+) bacteria displaying the PASR face the toxicity of phenolic acids by PAD activity and are not inhibited by vinyl phenols. In E. coli, the genes aaeB and marA, encoding efflux pumps for antimicrobial compounds, are upregulated by the addition of p-coumaric acid, but not by its derivative 4-vinyl phenol (p-hydroxystyrene). These results suggest that phenolic acids and their vinyl phenol derivatives produced by PAD (+) species could have a significant impact on undesirable or pathogenic food-borne Gram (-) bacteria in complex microbial ecosystems.

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

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 governs the mechanical behavior of the PM during hyperosmotic perturbation. The mutant strain, which accumulates ergosterol precursors, was more sensitive to osmotic fluctuations than the WT, which accumulates ergosterol. The hypersensitivity of erg6Δ was linked to modifications of the membrane properties, such as stretching resistance and deformation, which led to PM permeabilization during the volume variation during the dehydration-rehydration cycles. Anaerobic growth of erg6Δ strain with ergosterol supplementation restored resistance to osmotic treatment. These results suggest a relationship between hydric stress resistance and the nature of PM sterols. We discuss this relationship in the context of the evolution of the ergosterol biosynthetic pathway.

Air-drying kinetics affect yeast membrane organization and survival.

The plasma membrane (PM) is a key structure for the survival of cells during dehydration. In this study, we focused on the concomitant changes in survival and in the lateral organization of the PM in yeast strains during desiccation, a natural or technological environmental perturbation that involves transition from a liquid to a solid medium. To evaluate the role of the PM in survival during air-drying, a wild-type yeast strain and an osmotically fragile mutant (erg6Δ) were used. The lateral organization of the PM (microdomain distribution) was observed using a fluorescent marker related to a specific green fluorescent protein-labeled membrane protein (Sur7-GFP) after progressive or rapid desiccation. We also evaluated yeast behavior during a model dehydration experiment performed in liquid medium (osmotic stress). For both strains, we observed similar behavior after osmotic and desiccation stresses. In particular, the same lethal magnitude of dehydration and the same lethal kinetic effect were found for both dehydration methods. Thus, yeast survival after progressive air-drying was related to PM reorganization, suggesting the positive contribution of passive lateral rearrangements of the membrane components. This study also showed that the use of glycerol solutions is an efficient means to simulate air-drying desiccation.

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

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 occurred during the rehydration stage because of inadequacies in the membrane surface and led to cell death. Progressive dehydration conducted to the formation of some big PM pleats without membrane internalization. It also led to the modification of the distribution of the Sur7-GFP microdomains, suggesting that a lateral rearrangement of membrane components occurred. This event is a function of time and is involved in the particular deformations of the PM during a progressive perturbation. The maintenance of the repartition of the microdomains during rapid perturbations consolidates this assumption. These findings highlight that the perturbation kinetic influences the evolution of the PM organization and indicate the crucial role of PM lateral reorganization in cell survival to hydric perturbations.

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

We monitored the behavior of plasma membrane (PM) isolated from tobacco cells (BY-2) under hydrostatic pressures up to 3.5kbar at 30 degrees 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-beta-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 higher pressures (above 2 kbar) where both parameters reached a plateau value. The presence of a highly dehydrated gel state, insensitive to the sterol content, was thus proposed above 2.5 kbar. However, the F2N12S polarity parameter and the di-4-ANEPPDHQ intensity ratio showed strong effect on sterol depletion, even at very high pressures (2.5-3.5 kbar), and supported the ability of sterols to modify the electrostatic properties of membrane, notably its dipole potential, in a highly dehydrated gel phase. We thus suggested that BY-2 PM undergoes a complex phase behavior in response to the hydrostatic pressure and we also emphasized the role of phytosterols to regulate the effects of high hydrostatic pressure on plant PM.

Effect of sporulation conditions on the resistance of Bacillus subtilis spores to heat and high pressure.

Bacillus subtilis(B. subtilis) cells were placed in various environmental conditions to study the effects of aeration, water activity of the medium, temperature, pH, and calcium content on spore formation and the resulting properties. Modification of the sporulation conditions lengthened the growth period of B. subtilis and its sporulation. In some cases, it reduced the final spore concentration. The sporulation conditions significantly affected the spore properties, including germination capacity and resistance to heat treatment in water (30 min at 97°C) or to high pressure (60 min at 350 MPa and 40°C). The relationship between the modifications of these spore properties and the change in the spore structure induced by different sporulation conditions is also considered. According to this study, sporulation conditions must be carefully taken into account during settling sterilization processes applied in the food industry.