The rapid expansion of commercially available fermented food products raises important safety issues particularly when infant food is concerned. In many cases, the activity of the microorganisms used for fermentation as well as what will be the immunological outcome of fermented food intake is not known. In this manuscript we used complex in vitro, ex-vivo and in vivo systems to study the immunomodulatory properties of probiotic-fermented products (culture supernatant and fermented milk without live bacteria to be used in infant formula). We found in vitro and ex-vivo that fermented products of Lactobacillus paracasei CBA L74 act via the inhibition of proinflammatory cytokine release leaving anti-inflammatory cytokines either unaffected or even increased in response to Salmonella typhimurium. These activities are not dependent on the inactivated bacteria but to metabolic products released during the fermentation process. We also show that our in vitro systems are predictive of an in vivo efficacy by the fermented products. Indeed CBA L74 fermented products (both culture medium and fermented milk) could protect against colitis and against an enteric pathogen infection (Salmonella typhimurium). Hence we found that fermented products can act via the inhibition of immune cell inflammation and can protect the host from pathobionts and enteric pathogens. These results open new perspectives in infant nutrition and suggest that L. paracasei CBA L74 fermented formula can provide immune benefits to formula-fed infants, without carrying live bacteria that may be potentially dangerous to an immature infant immune system.

Several recent reports describe a role of probiotics as a therapeutic approach for celiac disease (CD). Two undigested A-gliadin peptides, P31-43 and P57-68, are central to CD pathogenesis, inducing an innate and an adaptive immune response, respectively. They enter enterocytes and localize to vesicular compartment to induce their toxic/immunogenics effects. In this article, we tested the effect of probiotic Lactobacillus paracasei (LP) CBA L74 (International Depository Accession Number LMG P-24778), its supernatant and LP-fermented cereals on gliadin peptides, P31-43 and P57-68, entrance in Caco-2 cells. Both LP CBA L74 and its supernatant inhibit P31-43 (intensity of fluorescence; FI: 75%) and P57-68 (FI: 50%) entrance in Caco2 cells, indicating that this biological effect is due to some product included in LP CBA L74 supernatant. This effect was present also after fermentation of cereals. This study describes a novel effect of probiotics in the prevention of undigested gliadin peptides toxic effects.

Purpose Celiac disease (CD) is an autoimmune enteropathy, triggered by dietary gluten. The only treatment is a strict gluten-free diet. Oats are included in the list of gluten-free ingredients by European Regulation, but the safety of oats in CD is still a matter of debate. The present study examined the capability of different oat cultivars of activating the gliadin-induced transglutaminase-2 (TG2)-dependent events in some in vitro models of CD. In addition, we compared this capability with the electrophoresis pattern of peptic–tryptic digests of the proteins of the oat cultivars. Methods K562(S) cells agglutination, transepithelial electrical resistance of T84-cell monolayers, intracellular levels of TG2 and phosphorylated form of protein 42–44 in T84 cells were the early gliadin-dependent events studied. Results The results showed that the Nave oat cultivar elicited these events, whereas Irina and Potenza varieties did not. The ability of a cultivar to activate the above-described events was associated with the electrophoretic pattern of oat proteins and their reactivity to anti-gliadin antibodies. Conclusion We found significant differences among oat cultivars in eliciting the TG2-mediated events of CD inflammation. Therefore, the safety of an oat cultivar in CD might be screened in vitro by means of biochemical and biological assays, before starting a clinical trial to definitely assess its safety.

Celiac disease (CD) is an autoimmune disease characterized by an altered immune response stimulated by gliadin peptides that are not digested and cause damage to the intestinal mucosa. The aim of this study was to investigate whether the postbiotic Lactobacillus paracasei (LP) could prevent the action of gliadin peptides on mTOR, autophagy, and the inflammatory response. Most of the experiments performed were conducted on intestinal epithelial cells Caco-2 treated with a peptic-tryptic digest of gliadin (PTG) and P31-43. Furthermore, we pretreated the Caco-2 with the postbiotic LP before treatment with the previously described stimuli. In both cases, we evaluated the levels of pmTOR, p70S6k, and p4EBP-1 for the mTOR pathway, pNFkβ, and pERK for inflammation and LC 3 and p62 for autophagy. For autophagy, we also used immunofluorescence analysis. Using intestinal organoids derivate from celiac (CD) patients, we analyzed the effect of gliadin after postbiotic pretreatment with LP on inflammation marker NFkβ. Through these experiments, we showed that gliadin peptides are able to induce the increase of the inflammatory response in a more complex model of intestinal epithelial cells. LP postbiotic was able to induce autophagy in Caco-2 cells and prevent gliadin effects. In conclusion, postbiotic pretreatment with LP could be considered for in vivo clinical trials.

In vivo assays cannot always be conducted because of ethical reasons, technical constraints or costs, but a better understanding of the digestive process, especially in infants, could be of great help in preventing food-related pathologies and in developing new formulas with health benefits. In this context, in vitro dynamic systems to simulate human digestion and, in particular, infant digestion could become increasingly valuable. To simulate the digestive process through the use of a dynamic model of the infant gastroenteric apparatus to study the digestibility of starch-based infant foods. Using M.I.D.A (Model of an Infant Digestive Apparatus), the oral, gastric and intestinal digestibility of two starch-based products were measured: 1) rice starch mixed with distilled water and treated using two different sterilization methods (the classical method with a holding temperature of 121°C for 37 min and the HTST method with a holding temperature of 137°C for 70 sec) and 2) a rice cream with (premium product) or without (basic product) an aliquot of rice flour fermented by Lactobacillus paracasei CBA L74. After the digestion the foods were analyzed for the starch concentration, the amount of D-glucose released and the percentage of hydrolyzed starch. An in vitro dynamic system, which was referred to as M.I.D.A., was obtained. Using this system, the starch digestion occurred only during the oral and intestinal phase, as expected. The D-glucose released during the intestinal phase was different between the classical and HTST methods (0.795 grams for the HTST versus 0.512 for the classical product). The same analysis was performed for the basic and premium products. In this case, the premium product had a significant difference in terms of the starch hydrolysis percentage during the entire process. The M.I.D.A. system was able to digest simple starches and a more complex food in the correct compartments. In this study, better digestibility of the premium product was revealed.