Specific Gaseous Conditions Significantly Improve Lactobacillus casei and Escherichia coli Survival to Freeze Drying and Rehydration

 Background and objective: Presence of oxygen during production and rehydration of freeze-dried starters and probiotics can decrease viability of the bacteria. Indeed, removal of water from cells during freeze-drying can promote dysfunction in anti-oxidative mechanisms, resulting in oxidative stress by accumulation of reactive oxygen species. The aim of this study was to show how atmospheric or less oxidative gaseous conditions affect bacterial survival to freeze-drying and rehydration of two strains, including Lactobacillus casei, a widely used bacteria in biotechnology, and Escherichia coli, a laboratory model bacteria.Material and methods: Lactobacillus casei ATCC 334 and Escherichia coli K12 were freeze dried for 24h in 5% sucrose (m v-1). Two gaseous conditions (an oxygen-free gas and atmospheric air) were used during various steps of the process, including bacterial cultivation, mixing of the bacteria with the protectant and rehydration. Oxygen-free gas condition was obtained with an oxygen-free gas, composed of nitrogen, hydrogen and carbon dioxide (N2H2CO2)and an anaerobic chamber.Results and conclusion: Gaseous conditions included significant effects on bacterial survival rates (P<0.001 for Lactobacillus casei and Escherichia coli). Interestingly, for both bacteria, the optimal combination was atmospheric air during mixing of the bacteria with the lyoprotectant (P<0.001 for Lactobacillus casei and Escherichia coli) and N2H2CO2 during rehydration (P<0.001 for Lactobacillus casei and P<0.05 for Escherichia coli). Management of gaseous conditions during a freeze-drying process and rehydration (atmospheric air during mixing of the bacteria with lyoprotectant and oxygen-free gas during rehydration) enhances survival of the bacteria by preserving them from oxidative stress.Conflict of interest: The authors declare no conflict of interest

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

[EN] 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.Verlhac, P.; Vessot-Crastes, S.; Degobert, G.; Cogné, C.; Andrieu, J.; Beney, L.; Gervais, P. (2018). Study and optimization of freeze-drying cycles of a model probiotic strain. En IDS 2018. 21st International Drying Symposium Proceedings. Editorial Universitat Politècnica de València. 635-642. https://doi.org/10.4995/IDS2018.2018.7400OCS63564

The Yin-Yang of the Green Fluorescent Protein:Impact on Saccharomyces cerevisiae stress resistance

International audienceAlthough fluorescent proteins are widely used as biomarkers (Yin), no study focuses on their influence on the microbial stress response. Here, the Green Fluorescent Protein (GFP) was fused to two proteins of interest in Saccharomyces cerevisiae. Pab1p and Sur7p, respectively involved in stress granules structure and in Can1 membrane domains. These were chosen since questions remain regarding the understanding of the behavior of S. cerevisiae facing different heat kinetics or oxidative stresses. The main results showed that Pab1p-GFP fluorescent mutant displayed a higher resistance than that of the wild type under a heat shock. Moreover, fluorescent mutants exposed to oxidative stresses displayed changes in the cultivability compared to the wild type strain. In silico approaches showed that the presence of the GFP did not influence the structure and so the functionality of the tagged proteins meaning that changes in yeast resistance were certainly related to GFP ROS-scavenging ability (Yang)

Rôle de la membrane plasmique dans la survie des microorganismes à la déshydratation : Contribution à l’optimisation de procédés de conservation des microorganismes

Cette synthèse est consacrée au rôle de la membrane plasmique des microorganismes, à travers ses évolutions structurales et fonctionnelles, dans la survie à la déshydratation. Elle comprend une première partie dans laquelle sont regroupés les principaux résultats portant sur l’influence de la déshydratation sur la survie microbienne, sur le comportement membranaire in situ et sur les mécanismes physiques de la déstabilisation membranaire. La deuxième partie est consacrée à la transposition de cette compréhension aux applications de déshydratation industrielle des microorganismes. Elle porte sur le développement d’un procédé de déshydratation des microorganismes et sur la mise au point d’outils prédictifs pour le séchage des microorganismes

Use of Gases in Microorganism Preservation Processes

International audienc

Influence of the fluidity of the membrane on the response of microorganisms to environmental stresses

International audienceThe aim of this mini-review is to relate membrane physical properties to the adaptation and resistance of microorganisms to environmental stresses. In the first part, the effects of various stresses on the structure and dynamic properties of phospholipid and biological membranes are presented. The compensation of these effects, i.e., change in membrane fluidity, phase transitions, by the active cellular control of the membrane chemical composition, is then described. In this natural process, the change in membrane fluidity is viewed as the detecting "input" signal that initiates the regulation, activating proteic effectors that in turn may influence the chemical composition of the membrane (feedback). This adaptation system allows the maintenance of the physical characteristics of membranes and, thereby, of their functionality. When environmental stresses are extreme and occur abruptly, the regulation process may not compensate for the changes in the membrane physical characteristics. In such cases, important variations in the membrane fluidity and structure may induce cellular damages and cell death. However, the lethal consequences are not systematically observed because protective effects of changes in the membrane physical state on the resistance to stresses are also reported

Sélection et intégration d'une souche probiotique fonctionnelle dans une matrice sèche

Les probiotiques sont des micro-organismes vivants, capables d interagir avec le microbiote et les cellules de l organisme hôte. Par leurs présences ou leurs métabolismes, ils contribuent notamment à la régulation du système immunitaire. D un point de vue technologique, le développement d un complément alimentaire contenant des probiotiques nécessite la maitrise des étapes de sélection et de production d une bactérie fonctionnelle sous forme sèche et revivifiable. Au cours de ce travail, nous avons développé des tests de criblage en cytométrie en flux, permettant de déterminer l aptitude d une bactérie à être séchée et l origine de sa sensibilité. Pour mettre en place ces tests, nous nous sommes intéressés aux mécanismes mis en jeu lors du procédé de séchage. Ainsi, lors de la déshydratation, les micro-organismes vont subir simultanément un stress osmotique et un stress oxydant. Les résultats montrent un fort impact du séchage sur la viabilité et le potentiel immuno-modulateur avec une diminution de la composante pro-inflammatoire (IL-12). Pour protéger les bactéries, trois stratégies d optimisation ont été développées : le séchage par formation de mousse, l encapsulation dans une matrice alginate-protéines de pois, et le co-séchage d une bactérie fragile avec une bactérie résistante. Les connaissances acquises au cours de cette étude ont permis d optimiser la production industrielle de la souche finale du projet. Afin de répondre au nouveau cadre réglementaire européen, une étude clinique en double aveugle contre placébo est actuellement en cours (2011-2013)Probiotics are live microorganisms that can act on the immune system at specific sites in the gastro-intestinal tract. From a technological point of view, the development of a dietary supplement containing viable and functional probiotics requires the control of the selection and production steps. In a first time, we have developed specific and sensitive screening tests to determine the ability of bacteria to be dried and the origin of its sensitivity. To develop these tests, we investigated the mechanisms involved during drying process. Thus, microorganisms simultaneously undergo osmotic stress and oxidative stress. Results show a strong impact of drying on the survival rate and immunomodulatory potential with a decrease of proinflammatory cytokines (IL-12). To protect probiotic bacteria, three optimization strategies have been developed: drying by foam formation, encapsulation of probiotic bacteria using a pea-protein alginate matrix and drying of a sensitive probiotic strain with a resistant bacterium. Knowledge obtained during this study was used to optimize the industrial process of the final strain of the project. A randomized double blinded (RDB) trial is in progress (2011-2013) to test the bacteria in humansDIJON-BU Doc.électronique (212319901) / SudocSudocFranceF

Conservation à long terme de systèmes biologiques viables et fonctionnels

National audienceLa 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

Implication de la membrane plasmique dans la survie de Saccharomyces cerevisiae lors de perturbations hydriques (rôle clé de l'ergostérol)

La conservation de microorganismes d intérêt (ferments, probiotiques) sous forme sèche et revivifiable est très répandue dans l industrie. Cependant, les procédés de déshydratation conduisent à des taux de survie variables en fonction du groupe, de l espèce et de la souche de microorganismes considérée ainsi que du type de procédé utilisé (séchage, lyophilisation, congélation). La membrane plasmique (MP), de par sa position entre l environnement intra et extracellulaire, est une cible privilégiée des perturbations hydriques. Les modifications de cette structure lors de stress hydriques sont décrites pour être directement impliquées dans la mort des microorganismes. La compréhension des réponses membranaires se produisant pendant un cycle de déshydratation/réhydratation est essentielle afin d optimiser la survie des microorganismes lors des procédés de déshydratation. Les manipulations réalisées lors de cette étude ont visé à caractériser les modifications fonctionnelles (intégrité) et structurales (déformations, répartition latérale de microdomaines riches en stérols) de la MP de Saccharomyces cerevisiae lors de différents types de perturbations hydriques (déshydratations osmotiques de différentes amplitudes et cinétiques, séchage dans différentes ambiances gazeuses). L impact de la composition de la MP sur la survie des levures a également été étudié par l utilisation de mutants accumulant différents types de stérols au niveau membranaire. Ce travail a confirmé la forte implication de la MP dans la mort des cellules lors de perturbations hydriques. L étude des modifications membranaires a permis d élucider le lien entre la cinétique de déshydratation et la survie des levures. Il a également été montré que l ergostérol est une molécule clé dans la survie des levures aux perturbations hydriques.Preservation of microorganisms of interest (ferments, probiotics) in dry form is widespread in the industry. However, the dehydration processes lead to variable survival rates according to the group, species and strain of microorganism considered, but also according to the type of process used (drying, freeze drying, freezing). The plasma membrane (PM), by its position between the intra-and extracellular environment, is a target of hydric perturbations. Changes in this structure during hydric stresses are described to be directly involved in microorganism death. Understanding of membrane responses occurring during a dehydration/rehydration cycle is essential to maximize the survival of microorganisms in the process of dehydration.Manipulations performed in this study aimed to characterize functional (integrity) and structural (deformations, lateral distribution of sterol-rich microdomains) changes of the PM of Saccharomyces cerevisiae during different types of hydric perturbations (osmotic dehydration of different magnitudes and kinetics, drying in different gas atmospheres). The impact of the composition of PM on yeast survival was also studied by using mutants accumulating different types of sterols in membranes. This work confirmed the strong involvement of the PM in cell death during hydric perturbations. The study of membrane changes helped to elucidate the relationship between the kinetics of dehydration and survival of yeasts. It has also been shown that ergosterol is a key molecule for the survival of yeasts during hydric perturbations.DIJON-BU Doc.électronique (212319901) / SudocSudocFranceF

Cell Death Induced by Mild Physical Perturbations Could Be Related to Transient Plasma Membrane Modifications

International audienceAn understanding of membrane destabilization induced by osmotic treatments is important to better control cell survival during biotechnological processes. The effects on the membranes of the yeast Saccharomyces cerevisiae of perturbations similar in intensity (same amount of energy) but differing in the source type (heat, compression and osmotic gradient) were investigated. The anisotropy of the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene was measured before and after each treatment to assess the reversibility of the membrane changes related to each treatment. Except for heat shock at 75°C, changes in membrane fluidity were reversible after the return to initial conditions, showing that two kinds of physical stress can be distinguished regarding the reversibility of membrane changes: high and mild energy stresses. With the application of osmotic gradients, anisotropy was assessed during treatment with five osmotic pressure levels from 30.7 to 95.4 MPa with two different yeast strains and related to the rate of cell death caused by each stress. The exposure of cells to increasing osmotic pressures involved a progressive lowering of membrane anisotropy during lethal perturbations. Osmotic stresses associated with reversible fluidity changes of increasing intensity in the membrane led to proportional death rates and time-dependant cell death of increasing rapidity during the application of the stress. Finally, a hypothesis relating the extent of membrane structural changes to the kinetic of cell death is proposed