Screening of probiotic strains to improve visceral hypersensitivity in irritable bowel syndrome by using in vitro and in vivo approaches.

Oral administration of probiotics has been proposed as a promising biotherapy to prevent and treat different diseases related to gastrointestinal disorders, such as irritable bowel syndrome (IBS). Due to the increasing research area on the characterisation of new probiotic bacterial strains, it is necessary to perform suitable in vitro experiments, using pertinent cellular models, in order to establish appropriate readout profiles based on IBS symptoms and subtypes. In this work, a collection of 30 candidate strains, belonging mainly to the Lactobacillus and Bifidobacterium genera, were screened using three different sets of in vitro experiments with different readouts to identify promising probiotic strains with: (1) the ability to inhibit the synthesis of IL-8 production by TNF-α stimulated HT-29 cells, (2) immunomodulatory properties quantified as increased IL-10 levels in peripheral blood mononuclear cell (PBMCs), and (3) the ability to maintain epithelial barrier integrity by increasing the trans-epithelial/endothelial electrical resistance (TEER) values in Caco-2 cells. Based on these criteria, three strains were selected: Lactobacillus gasseri PI41, Lacticaseibacillus rhamnosus PI48 and Bifidobacterium animalis subsp. lactis PI50, and tested in a murine model of low-grade inflammation induced by dinitrobenzene sulfonic acid (DNBS), which mimics some of the symptoms of IBS. Among the three strains, L. gasseri PI41 improved overall host well-being by preventing body weight loss in DNBS-treated mice and restored gut homeostasis by normalising the intestinal permeability and reducing pro-inflammatory markers. Therefore, the potential of this strain was confirmed in a second murine model known to reproduce IBS symptoms: the neonatal maternal separation (NMS) model. The PI41 strain was effective in preventing intestinal permeability and reducing colonic hypersensitivity. In conclusion, the set of in vitro experiments combined with in vivo assessments allowed us to identify a promising probiotic candidate strain, L. gasseri PI41, in the context of IBS.

Assessment of the safety of Levilactobacillus brevis CNCM I-5321, a probiotic candidate strain isolated from pulque with anti-proliferative activities.

Gut dysbiosis has been strongly correlated with colorectal cancer (CRC) development and the use of probiotics to modulate this imbalance represents a potential and promising therapy to prevent and treat CRC. For this reason, the identification of novel probiotic strains from diverse origins has widely increased in recent years, including traditional fermented foods. In this work we describe a new strain previously isolated from pulque (a traditional Mexican beverage), Levilactobacillus brevis CNCM I-5321, which may represent an interesting probiotic candidate to prevent and treat cancer. Indeed, our results show that CNCM I-5321 displays significant and specific antiproliferative capacities in human intestinal cancer cell lines (HT-29, HTC-116 and Caco-2 cells), but not in normal cells (FH cells). In addition, CNCM I-5321 is able to induce: (1) a pro-inflammatory immune response through stimulation of interleukin (IL)-2, IL-6, IL-12 and IL-17 cytokines and (2) apoptosis via activation of caspase 8. On the other hand, a minimum inhibitory concentration (MIC) assay revealed phenotypic resistance of this strain to ampicillin and chloramphenicol. However, no known transferable determinants were found in the genome of CNCM I-5321, thus this probiotic candidate presents no risk of horizontal transfer to the intestinal bacterial population. Finally, the safety status of CNCM I-5321 was evaluated using an innovative model of chicken embryo chorioallantoic membrane (CAM) to assess undesirable and/or toxic effects. Overall, our results support that CNCM I-5321 strain is non-pathogenic and safe for potential use as an anti-cancer candidate in human and animal medicine.

Improvement of bovine ß-lactoglobulin production and secretion by Lactococcus lactis

The stabilizing effects of staphylococcal nuclease (Nuc) and of a synthetic propeptide (LEISSTCDA, hereafter called LEISS) on the production of a model food allergen, bovine ß-lactoglobulin (BLG), in Lactococcus lactis were investigated. The fusion of Nuc to BLG (Nuc-BLG) results in higher production and secretion of the hybrid protein. When LEISS was fused to BLG, the production of the resulting protein LEISS-BLG was only slightly improved compared to the one obtained with Nuc-BLG. However, the secretion of LEISS-BLG was dramatically enhanced (~10- and 4-fold higher than BLG and Nuc-BLG, respectively). Finally, the fusion of LEISS to Nuc-BLG resulting in the protein LEISS-Nuc-BLG led to the highest production of the hybrid protein, estimated at ~8 æg/ml (~2-fold higher than Nuc-BLG). In conclusion, the fusions described here led to the improvement of the production and secretion of BLG. These tools will be used to modulate the immune response against BLG via delivery of recombinant lactococci at the mucosal level, in a mouse model of cow's milk allergy.