Study of survival and functionality of probiotic biofilms formulations using edible polysaccharide gel

L’objectif de cette thèse est d’encapsuler dans une matrice polyosidique des probiotiques sous forme de biofilm en utilisant comme modèle Lactobacillus paracasei ATCC334. La matrice d’encapsulation (billes) est obtenue par gélification ionique d’une pectine amidée et faiblement méthylée en présence d’ions calcium. Nous avons remarqué que le réseau de pectine dans ces billes d’un diamètre d’environ 500 µm permet la formation de microcolonies sphériques (biofilm-like) avec une taille de 25 µm de diamètre. Une augmentation de la concentration bactérienne de L. paracasei ATCC334 de 3 Log d’UFC après 24 h de croissance des bactéries immobilisées est observée pour atteindre au sein de ces billes une concentration supérieure à 10 Log d’UFC/g humide. Nous avons également noté une distribution homogène des microcolonies dans les billes et une organisation structurée des bactéries au sein des microcolonies. En effet, une adhésion des bactéries à la matrice pectine est observée ainsi que la présence de substances polymériques permettant de lier les bactéries les unes aux autres. Les résultats suggèrent donc des phénomènes d’interaction entre le réseau formé par la pectine et les bactéries dans les billes. Nous avons montré que L. paracasei ATCC334 formulée en biofilm dans ces billes de pectine présente une résistance accrue à un stress gastrique (pH 2) et au séchage par lyophilisation. La capacité d’adhésion des bactéries formulées à des cellules épithéliales est conservée et la pectine semble stimuler cette adhésion aux cellules de l’hôte. Des résultats in-vivo utilisant un modèle murin d’inflammation intestinale montrent que les biofilms de L. paracasei ATCC334 sont libérés au niveau intestinal où ils s’implantent notamment dans le côlon. Des microcolonies de tailles approchant les 20 µm sont retrouvées au niveau du côlon suggérant que les billes de pectine ont libéré les bactéries sous forme de biofilm. Par ailleurs, l’administration aux souris de la formulation à base de pectine avec des biofilms de probiotiques a entrainé chez les souris ayant reçu un traitement DSS (une molécule capable de déclencher une inflammation intestinale) : une perte de poids moindre, un état de santé général amélioré, une muqueuse colique moins altérée ainsi qu’une diminution de la réponse inflammatoire.The aim of this thesis is to encapsulate biofilm probiotics in a polysaccharide matrix using Lactobacillus paracasei ATCC334 as a model. The encapsulation matrix (beads) is obtained by the ionotropic gelation of amidated low-methoxylated pectin with calcium ions. We noticed that the pectin network in these beads with a diameter of approximately 500 microns, allow the formation of spherical microcolonies (biofilm-like) with a diameter of 25 microns. An increase of 3 Log CFU in the bacterial concentration of L. paracasei ATCC334 is observed after 24 hours of growth of the immobilized bacteria, while the observed concentration in these beads reaches more than 10 Log of CFU/g wet. We also noticed that the microcolonies within the beads are homogeneously distributed and the bacteria within the microcolonies are well structured. Moreover, a bacterial adhesion to the pectin matrix is observed as well as the presence of polymeric substances that bind the bacteria to each other. Our results suggest that interaction phenomenon may take place between pectin network and bacteria within beads. We also showed that biofilms of L. paracasei ATCC334 formulated in these pectin beads exhibit increased resistance to the gastric stress (pH 2) and to the freeze-drying process. In addition, the adhesion capacity of the formulated bacteria to epithelial cells is conserved and pectin seems to stimulate this adhesion to host cells. In-vivo results using a murine model presenting intestinal inflammation showed that L. paracasei ATCC334 biofilms are released in the intestinal level and are specifically implanted in the colon. Moreover, microcolonies of sizes approaching 20 μm are found in the colon suggesting that the bacteria are released in their biofilm form. In addition, the administration of pectinate beads containing probiotic biofilms to mice which have received a DSS treatment (inducing intestinal inflammation) resulted in: less weight loss of mice, improved their overall health status, less injured colonic mucosa and a decrease in the inflammatory response

Exopolysaccharide produced by Weissella confusa: Chemical characterisation, rheology and bioactivity

International audienceThe purpose of this study was to characterise the production of exopolysaccharide (EPS) by indigenous lactic acid bacteria (LAB) isolated from traditional Algerian dairy products and to evaluate their possible use in agri-foods. Among the collection of isolated strains, the strain Weissella confusa (W4) was selected for its ability to produce EPS once exposed to a sucrose culture medium. EPS produced were first isolated with a standardised method and further characterised in terms of molecular size, antioxidant activity, and rheological properties. Its direct implication in the texture and syneresis of acid milk gel was evaluated offering interesting industrial applications for its use during processes dealing with dairy products

Resveratrol Favors Adhesion and Biofilm Formation of Lacticaseibacillus paracasei subsp. paracasei Strain ATCC334

Bacterial strains of the Lactobacillaceae family are widely used as probiotics for their multifaceted potential beneficial properties. However, no official recommendations for their clinical use exist since, in many cases, oral administrations of these bacteria displayed limited beneficial effects in human. Additional research is thus needed to improve the efficiency of existing strains with strong potential. In this context, we assess in vitro the effects of nine polyphenols to stimulate biofilm formation by lactobacilli, a feature enhancing their functionalities. Among these polyphenols, we identify trans-Resveratrol (referred to hereafter as Resveratrol) as a potent inducer of biofilm formation by Lacticaseibacillus paracasei (formerly designated as Lactobacillus paracasei) ATCC334 strain. This effect is strain-dependent and relies on the enhancement of L. paracasei adhesion to abiotic and biotic surfaces, including intestinal epithelial cells. Mechanistically, Resveratrol modify physico-chemical properties of the bacterial surface and thereby enhances L. paracasei aggregation, subsequently facilitating adhesion and biofilm development. Together, our in vitro data demonstrate that Resveratrol might be used to modulate the behavior of Lactobacilli with probiotic properties. Combination of probiotics and polyphenols could be considered to enhance the probiotic functionalities in further in vivo studies

Intestinal release of biofilm-like microcolonies encased in calcium-pectinate beads increases probiotic properties of Lacticaseibacillus paracasei

International audienceIn this study, we show that calcium pectinate beads (CPB) allow the formation of 20 µm spherical microcolonies of the probiotic bacteria Lacticaseibacillus paracasei (formerly designated as Lactobacillus paracasei) ATCC334 with a high cell density, reaching more than 10 log (CFU/g). The bacteria within these microcolonies are well structured and adhere to a three-dimensional network made of calcium-pectinate through the synthesis of extracellular polymeric substances (EPS) and thus display a biofilm-like phenotype, an attractive property for their use as probiotics. During bacterial development in the CPB, a coalescence phenomenon arises between neighboring microcolonies accompanied by their peripheral spatialization within the bead. Moreover, the cells of L. paracasei ATCC334 encased in these pectinate beads exhibit increased resistance to acidic stress (pH 1.5), osmotic stress (4.5 M NaCl), the freeze-drying process and combined stresses, simulating the harsh conditions encountered in the gastrointestinal (GI) tract. In vivo, the oral administration of CPB-formulated L. paracasei ATCC334 in mice demonstrated that biofilm-like microcolonies are successfully released from the CPB matrix in the colonic environment. In addition, these CPB-formulated probiotic bacteria display the ability to reduce the severity of a DSS-induced colitis mouse model, with a decrease in colonic mucosal injuries, less inflammation, and reduced weight loss compared to DSS control mice. To conclude, this work paves the way for a new form of probiotic administration in the form of biofilm-like microcolonies with enhanced functionalities