After isolation and identification, the initial selection of microorganisms to be used as probiotics is based on in vitro and in vivo tests to prove some beneficial effects when administered. Subsequently, the biotechnological stage involves the development of a form of administration of the potential probiotic that allows its viability at high population levels during storage and also maintains its beneficial properties. However, the production process and delivery vehicle can change the efficiency of a probiotic (22,23). In this sense, the present study aimed to confirm whether the potential probiotic activity of S. cerevisiae UFMG A-905, demonstrated in previous studies (10–19), was maintained when administered as viable cells in a craft wheat beer brewed with this same yeast. S. Typhimurium is ideally suited for studying the interplay between the pathogen, the host, and a probiotic due to the availability of excellent animal models (24). Experimental S. Typhimurium infection in a murine model results in a disease that resembles human typhoid fever. Few hours after oral infection, the pathogenic bacterium invades enterocytes, M and dendritic cells in the distal ileum using the type III secretion system (T3SS) encoded by genes belonging to the pathogenicity island I (25). This secretion system consists of two components that allow the pathogen to invade the epithelial lining (T3SS-1) and survive in the host tissue (T3SS-2). Each T3SS injects several dozen proteins, called effectors, into the cytosol of epithelial cells (for T3SS-1) or macrophages (for T3SS-2) to induce bacterial entry or ensure the spread of bacteria in tissue, respectively. After translocation, invasion is characterized by a rapid bacterial multiplication in the liver and spleen, which results in hepato and splenomegaly, respectively. After a week, a plateau phase is established, characterized by the recognition of bacteria by the innate immune system through phagocytic cells. This leads to the production of several pro-inflammatory cytokines (TNF, IL-1, IL-6, IL-12, IFN-γ), as well as to a massive infiltration of monocytes and neutrophils into the sites of inflammation. Bacterial growth in these locations leads to the formation of abscesses containing predominantly polymorphonuclear leukocytes. In these lesions, S. Typhimurium resides intracellularly in macrophages. Later, effector mechanisms of adaptive immunity are induced, and anti-Salmonella antibody titers increase, in addition to the production of pro-inflammatory cytokines, especially IFN-γ (25). The death of mice is essentially a result of liver damage triggered by the production of pro-inflammatory cytokines and the induction of nitric oxide synthesis elicited by lipid A. The absence of S. Typhimurium translocation observed in mice of the 905/ST group was also described in our previous study (11) and could explain the lower number of liver inflammatory lesions in these animals seven days after challenge with the enteropathogenic bacteria. In the ST group of the present study, the results obtained in the histopathological analysis were consistent with the known kinetics of Salmonella invasion. The mild lesions observed in the ileum can be explained by the rapid passage of the pathogenic bacteria through the intestinal epithelium that occurred at the beginning of the invasion, while the more severe damage observed in the liver results from the tropism of the Salmonella for this organ after a week of infection, when the first deaths were noticed.
Many mechanisms of action have been proposed to explain how bacterial and yeast probiotics act, such as the production of antagonistic compounds (organic acids, bacteriocins or H2S), competition for nutrients, inhibition of pathogen adhesion to epithelium (by receptor competition and spatial exclusion, or by adhesion entrapment on the probiotic surface), inhibition of toxin action (by trapping on the probiotic surface, proteolytic degradation of the toxin molecule or its receptor) and modulation of the immune system (stimulation of IgA production or blood clearance, anti-inflammatory capacity during infective or inflammatory pathologies), interference on bacterial-induced signaling pathways, and actions on bacterial virulence factors (26). The production of antagonistic substances to pathogens or anti-inflammatory molecules requires that the probiotic is alive. On the other hand, co-aggregative or immunomodulation actions due to components of the probiotic structure do not depend on the viability. The International Scientific Association for Probiotics and Prebiotics (ISAPP) defined a postbiotic as a “preparation of inanimate microorganisms and/or their components that confers a health benefit on the host” (27). To meet this definition, postbiotics must contain inactivated microbial cells, cell components, or cell metabolites that induce health benefits. In the present work, the increase in survival to Salmonella infection in animals treated with unfiltered beer (from 33–75% in the ST and 905UFL/ST groups, respectively) was similar to that observed in the initial selection of S. cerevisiae UFMG A-905 as a potential probiotic (from 15–55% in the ST and 905/ST groups, respectively) (10). These results may be due to an action of probiotic yeast cells and/or their postbiotic extracellular metabolites. As this protective effect was not observed in filtered beer (905FL/ST), this seems to eliminate the second hypothesis. However, the in situ production of active metabolite in the intestine after ingestion cannot be ruled out. Indeed, the culture supernatant of Saccharomyces boulardii exhibits an anti-inflammatory effect by interfering with Salmonella-stimulated cell signaling pathways, suggesting that a soluble factor produced by yeast is implicated. This supernatant contains a small (1 kDa) heat-stable, water-soluble anti-inflammatory molecule that inhibited the activation of NF-κB by LPS, IL-1β and TNF (28).
sIgA, the dominant antibody isotype in the intestinal mucosa, is produced locally by activated B lymphocytes in the lamina propria. Activation of B cells for the production of IgA can occur either dependently or independently of T lymphocytes. In mice, most of the IgA is produced by T cell-independent mechanisms, contrary to what happens in humans. sIgA prevents bacterial access to the apical surface of epithelial cells, trapping bacteria in the mucus layer, which plays a crucial role in preventing the invasion of pathogenic microorganisms (29). In the present study, the reduction of sIgA levels in the intestinal fluid of mice treated with unfiltered beer when compared to the ST group may be due to the lower intestinal population of Salmonella through greater elimination of pathogenic bacteria by entrapment on the yeast surface as demonstrated previously (14).
Rodents lack a direct homologue of IL-8, but the chemokine CXCL-1/KC is regarded as functional homologue of IL-8. Chemokine ligand 1 (CXCL-1/KC) is a small peptide belonging to the CXC chemokine family that acts as a chemoattractant for various immune cells, mainly neutrophils, to the site of injury or infection and plays an important role in the inflammatory response. The simultaneous increases in CXCL-1/KC levels and MPO activity in the ST group are therefore coherent. As an anti-inflammatory cytokine, IL-10 serves to antagonize the pro-inflammatory effects of other cytokines and can thus keep inflammation in check. In the present study, the increase in its production, accompanying that of pro-inflammatory cytokines, was therefore expected.
As described in the study of Tiago et al. (14), electronic microscopy showed that S. Typhimurium cells bound preferentially to S. cerevisiae UFMG A-905 than to intestinal epithelial cells when the yeast was present. This may be a mechanism by which this probiotic yeast prevents the adhesion of pathogens to specific receptors in the intestinal epithelium and subsequent host invasion. The resulting lower load of adherent bacteria could explain the lower translocation to the spleen and liver and, consequently, the reduction of pro-inflammatory cytokines and chemokines and the lower recruitment of neutrophils and eosinophils in mice treated with unfiltered beer when compared with the group ST.
Before ending this discussion, it is important to remember that excessive beer consumption has deleterious effects on the human body, with an increased risk of diseases and important social problems such as addiction, accidents and violence. However, some data show that a moderate consumption of beer does not produce the main known chronic damage, and that even some benefits against cardiovascular diseases are observed (30). Recently, the cardiovascular health effects of alcohol have been classified as having a J-shaped curve, in which consumers with low to moderate intake are less at risk than non-drinkers throughout life, whereas those who drink exceedingly have the highest risk (31). It should also be remembered that beer contains, in addition to ethanol, vitamins, phenolic compounds, bitter components of hops, essential oils and biogenic amines (32). In addition, its moderate consumption appears to have positive effects for the body, as it increases the cholesterol associated with high density lipoproteins (HDL), reducing the risks of diseases and cardiovascular accidents (33–35). Finally, recent studies showed that changes observed in a few microbial taxa, and the higher butyric acid concentration in intestinal contents of consumers versus non-consumers of beer, suggest a potentially beneficial effect of moderate beer consumption on gut microbiota and intestinal health (36,37). Despite these properties, we must remember that beer like any alcoholic beverage should be consumed in moderation.