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

Optimized tableting for extremely oxygen-sensitive probiotics using direct compression

International audienceFaecalibacterium 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 viability was studied and a strategy was proposed to improve probiotic viability. Results showed that cell viability decreased almost linearly with compression pressure. MCC and HPMC seemed the most favorable carriers and after storage, each tablet exhibited a survival above108 CFU. Storage stability was obtained with a pressure of 201 MPa after 28 days at 25 °C, in anaerobic condition and with 11% relative humidity. Compression after a pre-consolidated stage improved clearly the survival rate due to lower temperature increase and lower shearing force. Thus, direct compression seems to be suitable in producing probiotics tablets with extremely oxygen-sensitive strains, and could provide sufficient protection during storage to expect therapeutic efficiency

Nouvelles stratégies de stabilisation des bactéries extrêmement sensibles à l’oxygène : cas du probiotique Faecalibacterium prausnitzii.

International audienceIntestinal bacteria are of growing interest in the areas of health and wellness [1, 3]. The intestinal ecosystem is characterized by its complex composi-tion and its very low oxygen content. Therefore, nu-merous bacterial species isolated from this environ-ment are extremely sensitive to oxygen. This feature is responsible for the difficulty of implementation of these microorganisms for probiotic large-scale appli-cations, particularly during processing and storage. In this presentation, we present the main steps that have been overcome to conserve and design a stabilization strategy for the bacteria Faecalibacterium prausnitzii, recognized for their anti-inflammatory activities intestinal and their beneficial effects on Crohn’s disease and ulcerative colitis [3]. The first objective was to look for a medium and conditions optimized to stabilize the bacteria to the dry state in powder form, with a high survival rate (FUI FPARIS). Indeed, stabiliza-tion processes by dehydration lead to mechanical and oxidative perturbations [4] affecting survival and functionality of cells [5]. The second objective was to develop a specific protection protocol for the conservation of the probiotic in a dry solid form (capsules or microgranules) and to maintain vi-ability during passage through the gastrointestinal tract. Encapsulation has been considered since this technique protects molecules or cells against various environmental perturbations [6-8]...Les bactéries intestinales suscitent un intérêt croissant dans les domaines de la santé et du bien-être [1, 2]. L’écosystème intestinal est caractérisé par sa composition complexe et sa très faible teneur en oxygène. De ce fait, de nombreuses espèces bacté-riennes isolées de cet environnement présentent une sensibilité extrême à l’oxygène. Cette particularité est à l’origine de la difficulté de mise en œuvre de ces micro-organismes dans le cadre d’applications probiotiques à grande échelle, notamment au cours du procédé de fabrication et de leur conservation. Dans cette communication, nous présentons les principales étapes qui ont été franchies en vue de conserver et de concevoir une stratégie de stabili-sation/protection de la bactérie Faecalibacterium prausnitzii, reconnue pour ses activités anti-in-flammatoires intestinales et ses effets bénéfiques sur la maladie de Crohn et la rectocolite hémorra-gique [3]. Le premier objectif a été de concevoir un milieu et des conditions optimisées pour stabiliser la bactérie à l’état sec, sous forme de poudre, avec un taux de survie élevé (FUI FPARIS). En effet, les procédés de stabilisation par déshydratation sont à l’origine de contraintes mécaniques et oxydatives [4] pouvant altérer la viabilité et la fonctionnalité les cellules [5]. Le deuxième objectif est de développer un protocole de protection spécifique permettant la conservation du probiotique sous une forme sèche (microgranules ou capsules) et de maintenir la viabilité au cours du passage dans le tractus gastro-intestinal. L’encapsulation a été envisagée puisque cette technique permet la protection de molécules ou de cellules contre des perturbations environne-mentales diverses [6-8]..

Nouvelles stratégies de stabilisation des bactéries extrêmement sensibles à l’oxygène : cas du probiotique Faecalibacterium prausnitzii.

International audienceIntestinal bacteria are of growing interest in the areas of health and wellness [1, 3]. The intestinal ecosystem is characterized by its complex composi-tion and its very low oxygen content. Therefore, nu-merous bacterial species isolated from this environ-ment are extremely sensitive to oxygen. This feature is responsible for the difficulty of implementation of these microorganisms for probiotic large-scale appli-cations, particularly during processing and storage. In this presentation, we present the main steps that have been overcome to conserve and design a stabilization strategy for the bacteria Faecalibacterium prausnitzii, recognized for their anti-inflammatory activities intestinal and their beneficial effects on Crohn’s disease and ulcerative colitis [3]. The first objective was to look for a medium and conditions optimized to stabilize the bacteria to the dry state in powder form, with a high survival rate (FUI FPARIS). Indeed, stabiliza-tion processes by dehydration lead to mechanical and oxidative perturbations [4] affecting survival and functionality of cells [5]. The second objective was to develop a specific protection protocol for the conservation of the probiotic in a dry solid form (capsules or microgranules) and to maintain vi-ability during passage through the gastrointestinal tract. Encapsulation has been considered since this technique protects molecules or cells against various environmental perturbations [6-8]...Les bactéries intestinales suscitent un intérêt croissant dans les domaines de la santé et du bien-être [1, 2]. L’écosystème intestinal est caractérisé par sa composition complexe et sa très faible teneur en oxygène. De ce fait, de nombreuses espèces bacté-riennes isolées de cet environnement présentent une sensibilité extrême à l’oxygène. Cette particularité est à l’origine de la difficulté de mise en œuvre de ces micro-organismes dans le cadre d’applications probiotiques à grande échelle, notamment au cours du procédé de fabrication et de leur conservation. Dans cette communication, nous présentons les principales étapes qui ont été franchies en vue de conserver et de concevoir une stratégie de stabili-sation/protection de la bactérie Faecalibacterium prausnitzii, reconnue pour ses activités anti-in-flammatoires intestinales et ses effets bénéfiques sur la maladie de Crohn et la rectocolite hémorra-gique [3]. Le premier objectif a été de concevoir un milieu et des conditions optimisées pour stabiliser la bactérie à l’état sec, sous forme de poudre, avec un taux de survie élevé (FUI FPARIS). En effet, les procédés de stabilisation par déshydratation sont à l’origine de contraintes mécaniques et oxydatives [4] pouvant altérer la viabilité et la fonctionnalité les cellules [5]. Le deuxième objectif est de développer un protocole de protection spécifique permettant la conservation du probiotique sous une forme sèche (microgranules ou capsules) et de maintenir la viabilité au cours du passage dans le tractus gastro-intestinal. L’encapsulation a été envisagée puisque cette technique permet la protection de molécules ou de cellules contre des perturbations environne-mentales diverses [6-8]..

Comparison of two encapsulation processes to protect the commensal gut probiotic bacterium Faecalibacterium prausnitzii from the digestive tract

International audienceAnaerobic commensal gut bacteria are among the most promising future treatments for many diseases. Because of their sensitivity to oxygen and to the harsh conditions of the gut, few studies on formulations that deliver bacteria in a viable and functional form to the intestine have been done. The aim of this study was to develop a reliable and easy-to-scale-up process meeting these requirements. The designed process consisted of mixing a freeze-dried bacterium, Faecalibacterium prausnitzii, with a melted hydrophobic matrix (Gelucire® 43/01) to encapsulate them into a solid lipid base after cooling. These formulations were compared with freeze-dried beads containing bacteria encapsulated by ionotropic gelation. This work demonstrates that both formulations improve the survival of bacteria in the stomach and distal jejunum buffers during a digestive exposure test. However, only lipid inclusion provided sufficient protection suitable for therapeutic application. Nevertheless, Gelucire® poorly stabilizes bacteria during storage, unlike the freeze-dried beads