Novel probiotic evidence of lactobacilli on immunomodulation and regulation of satiety hormones release in intestinal cells
Introduction
Gut microbiota is a major constituent of the morpho-functional system including intestinal epithelium and mucosal immune system (Scarpellini et al., 2010). This complex system is continually on a dynamic balance and is responsible of the host local intestinal integrity (Scarpellini et al., 2010). Probiotics are live microorganisms that when consumed in adequate amounts, produce beneficial effects on both digestive and systemic levels. Probiotics are overall safe, acid and bile tolerant, able to adhere and colonize the intestinal tract (Sanders et al., 2010). However, the term “probiotic” should be dedicated to live microbes for which a health benefit has been shown in controlled studies (Gaurner et al., 2008). The most commonly used microorganisms as probiotics are lactic acid bacteria (LAB), including Lactobacillus, Enterococcus, Streptococcus and Bifidobacterium species. Nevertheless, further microbial species including Escherichia coli Nissle, Bacillus cereus and Saccharomyces cerevisiae are also recognized as probiotics (Fijan, 2014). Indeed, lactobacilli colonize the gut and vagina of mammalians. They are widely described as beneficial microorganisms because of their aptitudes to relieve irritable bowel syndrome, reduce diarrhoea, promote immunity and restore the balance of gut microflora (Akoglu et al, 2015, Aragon et al, 2010, Galdeano, Perdigón, 2006, Mitsuoka, 1996, Orel, Kamhi-Trop, 2014, Van Niel et al, 2002). Moreover, lactobacilli play an important role in the protection of the host against harmful microorganisms and also strengthen the immune system (Soccol et al., 2010). Some species were described to improve food digestibility and reduce metabolic disorders (Kumar et al., 2012). LAB and particularly lactobacilli were used, since centuries, for food preservation and food fermentation as beneficial agents. LAB are able to modify physicochemical properties of food by fermentation, enhancing their taste and their digestibility and to prevent contamination of potential pathogens by lowering the pH (Gaurner et al., 2008). The genus Lactobacillus is commonly found in the upper gastrointestinal tract of mammals. To express their beneficial effects in the host, probiotic strains must be able to survive the passage through the digestive tract. Tolerance to high concentration of bile salts is an important parameter to microbial survival in the intestinal tract. So far, research has mainly focused on strains sensitivity toward low pH, proteolytic enzymes and bile salts (Sanders et al., 2010). In several reports, human enterocyte-like Caco-2 cells have been used for in vitro studies to elucidate the mechanisms of cellular adhesion of nonpathogenic lactobacilli (Chauvière et al, 1992, Ferreira et al, 2011). The ability of lactobacilli to adhere to mucosal surfaces of the intestine, and the subsequent long or short-term colonization have been the most commonly encountered criteria for the selection of probiotic strains.
Another recent probiotic lactobacilli interest is their action on gastrointestinal hormones involved in food intake regulation. Among these hormones, two are the focus of different investigations: the glucagon like peptide-1 (GLP-1) and the cholecystokinin (CCK) (de Graaf, Blom, Smeets, Stafleu, & Hendriks, 2004). These hormones were described to be involved in the control of appetite as satiation signals secreted by the intestine in response to meal stimulation (Mars et al, 2012, Moran, Dailey, 2011, Walsh, 1994). GLP-1 is produced by enteroendocrine L cells, stimulates glucose-induced insulin secretion and inhibits glucagon secretion. This hormone has two active forms, GLP-1 (7–36) amide and GLP-1 (7–37), product of a posttranslational processing of a proglucagon precursor in the mammalian intestine (Oh, Lee, Ko, Choi, & Kim, 2003). CCK is produced in the gut by enteroendocrine I cells and is a member of a peptide hormone family, characterized by the same carboxyl-terminal pentapeptide sequence (-Gly-Trp-Met-Asp-Phe-NH2). This hormone is widely distributed throughout the central nervous system and the digestive tract (Oikonomou, Buchfelder, & Adams, 2008). The STC-1 plurihormonal cell line is derived from an endocrine tumour developed in the small intestine of a double transgenic mouse (Rindi et al., 1990). The STC-1, murine endocrine cells, are considered a suitable model for the in vitro study of gastrointestinal hormones release (Cordier-Bussat et al, 1997, Cordier-Bussat et al, 1998, Sufian et al, 2007). These endocrine cells are able to secrete both GLP-1 and CCK hormones (Cordier-Bussat et al, 1997, Cordier-Bussat et al, 1998, Cudennec et al, 2012, Geraedts et al, 2011).
This study provides further probiotics traits to recently proven beneficial lactobacilli isolated from vaginal origin of Lebanese women (Al Kassaa, Hamze, Hober, Chihib, & Drider, 2014). The resistance of these strains to the harsh conditions of the digestion process was evaluated throughout the buccal, gastric and duodenal phases simulated in vitro. Then the adhesion capacities of these lactobacilli strains were studied and their cytotoxicity toward Caco-2 cells was determined. Immunomodulatory effect of these strains, particularly on IL-6, IL-8 and IL-10 secretion, was also considered. Finally we look for the impact of these lactobacilli strains on the secretion and expression of CCK and GLP-1.
Section snippets
Bacterial strains
Lactobacillus strains used in this work were recently isolated by Al Kassaa et al. (2014). These strains were Lactobacillus gasseri CMUL34, L. fermentum CMUL54, L. gasseri CMUL57, L. acidophilus CMUL67, L. gasseri CMUL80, L. gasseri CMUL99 and L. plantarum CMUL140. Before each experiment, the strains were grown for 18–24 h at 37 °C in De Man–Rogosa–Sharpe (MRS) medium (De Man, Rogosa, & Sharpe, 1960).
Lactobacilli tolerance to the conditions of the modelled digestion process
To assess the survivability of the Lactobacillus strains, a static in vitro digestion model,
Lactobacilli strains are resistant to enzymes and pH of digestive environment
All lactobacilli strains used in this study showed a good resistance to the conditions occurring during the simulated digestion process. The initial number of living bacterial cells, of about 108 CFU mL−1 in the beginning of the process, decreased more or less, depending of the strain, during the passage through the stomach and duodenal compartments. The strains L. gasseri CMUL34, L. fermentum CMUL54 and L. gasseri CMUL57 showed a decrease of CFU number of less than one logarithmic unit. Other
Discussion
Lactobacilli used in this work were isolated from vaginal samples and were characterized for their probiotic traits (Al Kassaa et al., 2014). Besides their inhibitory activity against pathogens as Gardnerella vaginalis CIP7074T, Candida albicans ATCC10231, E. coli CIP103982 and Streptococcus aureus ATCC33862 (Al Kassaa et al., 2014), they were recently proven to be active against HSV-2 (Al Kassaa et al., 2015).
The present study provides insightful information regarding the metabolic activities
Conclusion
Seven strains of Lactobacillus, including L. gasseri (4 strains CMUL34, 57, 80 and 99), L. fermentum CMUL54, L. acidophilus CMUL67 and L. plantarum CMUL140 were selected for their probiotic potential and inhibitory action against various pathogens (Al Kassaa et al., 2014). These antagonistic properties of the lactobacilli strains used in the present study could be useful in probiotic products. These strains also show good ability to survive gastric conditions and colonize the intestine, at
Conflicts of interests
The authors disclose no conflicts of interest.
Acknowledgements
YB was a recipient of a postdoctoral fellowship obtained during the “emerging project” call funded by the Conseil Régional du Nord Pas de Calais. The authors are indebted to this local authority. The experiments were performed at Charles Viollette Institute, IUT “A” and Lille 1 University, which are gratefully acknowledged.
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