Screening and evaluation of Lactobacillus spp. tightening the intestinal barrier

Abstract

The intestinal barrier plays an important role in the interactions between host and microbiome for nutrient absorption and immune regulation while also acting as the physical barrier preventing the transport of a variety of harmful substances from the gut to the bloodstream. Lactobacillus spp. has been researched a lot as probiotics for an alternative treatment in inflammatory gut diseases, while further sustainable evidence of their beneficial effects is required. The intestinal barrier consists of tight junction proteins such as ZO-1 and Occludin and acts as a shield against harmful bacteria while providing a suitable habitat for beneficial bacteria. Decreased barrier functionality has been shown to be a casual effect in inflammatory gut diseases. While there has been a multitude of research related to probiotics as an alternative treatment for these diseases, further sustainable evidence of their beneficial effects is required. This study aimed to 1) screen probiotic candidates of Lactobacillus spp. isolated from Korean feces using a transepithelial/transendothelial electrical resistance (TEER) assay, 2) evaluate their ability on tight junction proteins ZO-1 and Occludin, 3) find if supernatant or heat-killed bacteria sustained the beneficial effects as live probiotics. For their ability to induce enhancement in intestinal barrier functionality and to investigate the potential mechanisms underlying the beneficial effects of the probiotic candidates. A TEER assay was conducted with 21 strains of Lactobacillus spp. to screen probiotic candidates and species of L. rhamnosus were shown to increase relative TEER change significantly in comparison to the control. We selected 3 strains of Lactobacillus spp. (KBL363, KBL365 and KBL385) which demonstrated the most increasing effects in TEER change. Repeated TEER assay was conducted to investigate sustainable beneficial effects on the intestinal barrier and the assay results showed that probiotic candidates sustained beneficial effects for 24 h, with the most significant tightening effects seen in the first 12 h period. In accordance to TEER results, 3 selected probiotic strains significantly increased tight junction proteins ZO-1 and Occludin. We further investigated the effects of bacteria culture supernatant and heat-killed bacteria on intestinal barrier functionality using the 3 selected probiotic strains. However, unlike other studies describing beneficial effects of culture supernatant, there was no significant up regulation in TEER change observed in response to treatment with culture supernatant. In contrast to supernatant treatment, heat-killed probiotic candidates increased intestinal barrier functions in TEER assay and also enhanced tight junction proteins significantly, indicating that outer membrane vesicles may play a role in the tightening effects, although clear mechanisms are not yet understood. In conclusiongeneral, this study determined 3 strains of Lactobacillus spp. with probiotic potential to increase intestinal barrier functionality and also provided a mechanical suggestion underlying which bacterial components are responsible for tightening effects in intestinal barrier.