Synbiotic efficacy of probiotic and prebiotic food ingredients for gut health

The main objective of this study was to test the efficacy of synbiotic food combinations carrying probiotic and prebiotic dietary fibres (DF) components for mitigating colonic inflammation that is associated with gut health issues such as inflammatory bowel disease (IBD). Although the exact aetiology of IBD is yet to be elucidated, emerging evidence supports the involvement of a recurrent tripartite pathophysiological circuit encompassing dysregulated immune responses, altered epithelial integrity and microbial dysbiosis. Therefore, the potential of dietary interventions incorporating food combination synergism to mitigate the inflammatory circuit, and thereby resolve or prevent the severity of colonic inflammation, was investigated. Whole plant prebiotic sugar cane fibre (PSCF) and green banana resistant starch (GBRS) flour prebiotics were evaluated for their individual as well as synbiotic efficacy in combination with probiotic Bacillus coagulans MTCC 5856 spores (B. coagulans) for ameliorating chemically-induced acute colitis and spontaneous chronic colitis in mice models of IBD. The research initially determined the stability and the bioefficacy of B. coagulans spores in-vitro by evaluating their ability to survive simulated digestion, adhesion to human colonic epithelial cells and immunomodulatory capacity. The tolerance of the probiotic B. coagulans spores to simulated digestion was tested by exposure to simulated saliva, gastric and intestinal juices. There was a high survival rate of 92% to the simulated digestion process. There was also substantial adherence to human colonic cells HT-29 (86%) and LS174T (81%). Furthermore, the spores exerted marked immunomodulatory effects in HT-29 cells by suppressing IL-8 and increasing IL-10 secretion. The B. coagulans spores also induced a pronounced differential immunomodulatory efficacy in response to lipopolysaccharideinduced inflammation under co-treatment (increased IL-10 and reduced IL-8) relative to posttreatment (suppressed IL-8 with no IL-10 detection) in HT-29 cells. These observations support the application of B. coagulans spores prior to or during the onset of inflammation to maximise the probiotic benefits in treating inflammatory bowel conditions. The prophylactic efficacy of dietary supplementation with B. coagulans spores and PSCF alone or as synbiotic combination was then evaluated for their ability to attenuate dextran-sulfate sodium (DSS)-induced acute colitis in C57BL/6J mice. The study also aimed to analyse the beneficial effects of pre-conditioning the gut with supplemented diets prior to induction of chemical colitis in imparting protection and amelioration against DSS-induced acute colitis. The mice were fed a normal chow diet supplemented with either whole plant PSCF alone, B. coagulans or its synbiotic combination (PSCF-synbiotic). The mice in control group (DSS-control) received normal chow. After the first seven days of supplementation, acute colitis was induced with 2% DSS administered in drinking water for seven days with the continuation of the supplementations. The disease activity indices (DAI), macroscopic markers, histological colonic damage, expressions of tight junction (TJ) proteins, mucus staining were analysed. The profile of colonic and serum cytokines and other inflammatory mediators (colonic iNOS activity and serum C-reactive protein level) were determined. Additionally, the faecal metabolomic and short-chain fatty acids (SCFA) (caecal, mucosalassociated and faecal samples) profiles were also measured. Synbiotic supplementation ameliorated DAI and histological score (72% reduction, 7.38, respectively), more effectively than either B. coagulans (47% reduction, 10.1) and PSCF (53% reduction, 13.0) alone relative to the DSS-control. PSCF-synbiotic supplementation also significantly (P < 0.0001) preserved the expression of TJ proteins and modulated the altered serum IL-1˜í‚⧠(‚Äöv†v¿40%), IL-10 (+26%), and C-Reactive protein (CRP) (‚Äöv†v¿39%) levels compared to the unsupplemented DSS-control. DSS insult in control mice resulted in decreased expression of TJ proteins and altered immune responses. Moreover, B. coagulans spores alone induced extra butyrate production in the caecum (+81%), but only +17% in mucosal-associated samples and +44% in faeces relative to DSS-control group. In contrast, the synbiotic combination resulted in substantial increase in butyrate levels across the whole length of colon with +80% in caecum, +57% in mucosalassociated sample and +54% in faeces. The ability of B. coagulans spores and GBRS alone and as synbiotic combination (GBRS-synbiotic) was then examined for prophylactic efficacy in influencing the onset and disease outcomes of DSS-induced acute colitis in C57BL/6J mice. This study employed the same design where, after the first seven days of supplementation, acute colitis was induced with 2% DSS administered in drinking water for seven days with the continuation of the supplementations. The GBRS-synbiotic supplementation alleviated the DAI and histological damage score (67% reduction, 8.8 respectively) more than B. coagulans (52% reduction, 10.8 respectively) or GBRS (57% reduction, 13.6 respectively) alone. Compared to the DSScontrol, synbiotic supplementation significantly (P < 0.0001) maintained the expression of TJ proteins. Moreover, synbiotic effects accounted for approximately 40% suppression of IL-1˜í‚⧠and 29% of the increase in serum IL-10 while also reducing CRP (37%) to that of the DSScontrol. Additionally, relative to that of DSS-control, GBRS-synbiotic supplementation also significantly raised the SCFA profile especially faecal butyrate level (+66%) more extensively compared to B. coagulans supplementation alone (+46%). Both the studies demonstrated marked prophylactic efficacy of the synbiotic supplementations (PSCF-synbiotic and GBRSsynbiotic) in ameliorating the acute colitis in mice. A further study employed a spontaneous colitic Winnie (Muc2 mutant) mice model of IBD to determine if the previous results were specific to the DSS model or more generally applicable. In the Winnie model the chronic colonic inflammation results from a primary intestinal epithelial defect conferred by a missense mutation in Muc2 mucin gene. Winnie mice were fed normal chow diet supplemented with either B. coagulans, PSCF or its synbiotic combination for 21 days. Prominent features of the spontaneous colitis in Winnie, such as severe clinical manifestations, colonic histological alterations, dysregulated immune responses, altered SCFA levels and microbial dysbiosis, were examined to determine the therapeutic effect of the supplementations in mitigating chronic inflammation. All three supplementations reduced diarrheic stools as well as prevented body weight loss. PSCFsynbiotic supplementation significantly ameliorated histological score in both proximal (P = 0.0443) and distal (P < 0.0001) colon sections more effectively than B. coagulans and PSCF alone. Moreover, PSCF-synbiotic supplementation substantially modulated the altered colonic and serum cytokine levels as well as lowered serum CRP level by 29% compared to the unsupplemented Winnie-control. While, PSCF favoured the abundance of the bacterial genus Akkermansia, PSCF-synbiotic was effective in restabilising the depleted levels of Prevotella in Winnie colitic mice. PSCF-synbiotic was also markedly effective in elevating and normalising the levels of short-chain fatty acids along the length of the colon compared to that in unsupplemented Winnie-control mice. The potentiated health outcome effects of the synbiotic combinations observed in these studies may be associated with a synergistic direct immune-regulating efficacy of the probiotic and prebiotic components. The synbiotic combinations may also exert their effects by improved ability to protect epithelial integrity, stimulation of probiotic spores by the respective prebiotic fibre and/or with stimulation of higher levels of fermentation of fibres releasing SCFAs that mediate the reduction in colonic inflammation. Thus, the synergism between the probiotic and prebiotic components used in these studies could be attributed to the observed augmented beneficial effects. The knowledge obtained from thesis not only warrants investigation of synbiotic supplementations as an adjuvant therapy in human IBD, but the results could also be applied to design novel functional food products targeted at improving gut health and enhanced eating practice.