Effects of potato fiber and soluble corn fiber on the fecal microbiome of dogs with implications to inflammation

Dietary fiber provides numerous benefits to the health of both companion animals and humans including improved digestive health (increases stool weight and promotes normal laxation) and systemic health (promotes satiety and decreases incidences of obesity and type II diabetes). Novel dietary fibers often are evaluated for their ability to induce prebiotic effects. Prebiotics are defined as non-digestible food ingredients that, when consumed in sufficient amounts, are fermented and selectively stimulate the growth, activity, or both, of one or a limited number of microbial genera or species in the gut microbiota that ultimately benefits health of the host. Not all dietary fibers are the same in terms of their physico-chemical properties, which ultimately affects their fermentability. The overarching objectives of this thesis were to assess two novel dietary fiber sources, potato fiber (PF) and soluble corn fiber (SCF), for their inclusion in commercial dog diets, and ultimately, their prebiotic potential and implications for host health. Specifically, PF and SCF were evaluated for their chemical composition, in vitro fermentability, in vivo characteristics (nutrient digestibility and fecal fermentation characteristics), and ability to change fecal microbiota concentrations. Previous research in our laboratory found that feeding graded concentrations of dietary PF to dogs elicited linear increases (P < 0.05) in fecal acetate, propionate, butyrate, and total short-chain fatty acids (SCFA) without causing detriments to nutrient digestibility. We investigated this further by studying the prebiotic potential of PF by analyzing the fecal microbiome. We predicted that our previous results on fecal fermentation characteristics would be associated with shifts in the fecal microbiome. Based on previous studies involving dietary fiber in pets and humans, we expected that at the phylum level, an increase in fecal Firmicutes and a decrease in fecal Fusobacteria would occur. More specifically, we also predicted increases in bifidobacteria and other SCFA-producing genera (e.g., Blautia, Lachnospira, and Faecalibacterium spp.) with increasing dietary PF concentrations. As anticipated, with increasing concentrations of dietary PF, there were significant (P < 0.05) changes in fecal concentrations of key SCFA-producing bacteria. Butyrate-producing genera, such as Blautia and Faecalibacterium spp. increased (P < 0.05) with increasing dietary PF concentrations, which was confirmed by qPCR. Our sequencing data also confirmed an increase (P < 0.05) in Firmicutes and a decrease in Fusobacteria (P < 0.05) with increased dietary PF consumption. Prebiotics often are evaluated by their ability to produce butyrate (a butyrogenic effect) or to stimulate the growth of bifidobacteria (a bifidogenic effect); however, the definition of a prebiotic is not limited to this genus. We found that with ingestion of PF, Bifidobacterium spp. were positively correlated with butyrate (R = 0.49; P < 0.05) and lactobacilli (R = 0.82; P < 0.05) concentrations. Bifidobacteria’s main carbohydrate fermentation products are acetate and lactate when substrate is available, while lactobacilli produce lactic acid. This has previously been explained by bacterial cross-feeding where primary fermenters of carbohydrate will produce secondary metabolites (acetate and lactate) or hydrolyzed substrate that can be utilized by other bacteria. Overall, this research suggests to us that PF is beneficial to gut health and should be investigated as a novel functional ingredient in dog diets. Soluble corn fiber (NUTRIOSE®) also was evaluated for its efficacy as a fiber source in dog foods; however, the objective was to determine a minimum dose to elicit a prebiotic effect. We found that SCF was highly fermentable in vitro showing increases (P < 0.05) in acetate, propionate, and butyrate concentrations over 12 h. When tested in vivo from 0-1.25% of the diet, we found no detrimental effects on nutrient digestibility or fecal consistency; however, no changes were shown in fecal fermentation characteristics. Furthermore, this translated into no appreciable differences in the fecal microbiome. Overall, SCF was fermentable in vitro; however, more research is needed to evaluate a more effective dose to elicit in vivo effects. The changes we observed with increasing dietary PF in the dog presented the most intriguing results. Faecalibacterium, and more specifically, Faecalibacterium prausnitzii, a well-characterized butyrate-producing species, was increased by feeding graded dietary levels of PF to dogs. This bacteria is often found in low concentrations in humans and dogs with inflammatory bowel diseases (IBD). These previous results led us to investigate whether PF, with its in vivo fermentation characteristics and ability to modulate the microbiome, would be efficacious in attenuating the inflammatory response in the dextran-sodium sulfate (DSS)-induced colitis mouse model. We hypothesized that a moderately fermentable PF would be more effective at attenuating the symptoms and inflammatory response in DSS colitis than non-fermentable cellulose (Cell). Mice provided the PF/DSS treatment exhibited decreased (P < 0.05) symptoms of DSS colitis by showing a delayed loss in body weight compared with mice provided the Cell/DSS treatment. Furthermore, fermentation of PF during DSS colitis was found to be anti-inflammatory by showing suppression (P < 0.05) of distal colon gene expression of inflammatory cytokines (TNF-α, IL-1β, IL-6, and IL-17A). What was particularly interesting were the changes in distal colon gene expression of CXCL1, which is a neutrophil chemoattractant. Mice provided non-fermentable Cell had a 7-fold increase (P < 0.05) in CXCL1 expression compared with all other treatment groups. Pathology scores from an independent observer also showed decreases (P < 0.05) in leukocyte infiltration scores in distal colon. In this acute-colitis model, neutrophils would be the predominant innate immune cell type at the site of inflammation. Therefore, this suggests that fermentation of PF to produce SCFA may be a driving force in controlling leukocyte recruitment to the site of inflammation. As a potential mechanism linking SCFA concentrations and leukocyte recruitment, we analyzed free fatty acid receptor 2 (FFAR2) expression in distal colon. Leukocytes, and in particular neutrophils, have FFAR2 on their cell surface. Short-chain fatty acids can bind to this receptor, causing changes in chemotaxis and reactive oxygen species concentrations. We found that DSS caused an increase (P < 0.05) in FFAR2 expression in distal colon; however, there was no main effect of diet or an interactive effect. One alternative mechanism proposed was the role of SCFA suppressing innate T-helper 17 (Th17) cells, which recruit neutrophils and monocytes through the concentrations of IL-17A and chemokines. Short-chain fatty acids have been found to induce both effector and regulatory T cells by suppression of histone deacetylases that is independent of FFAR2 or 3. Inhibition of histone deacetylases by SCFA and, in particular, acetate, has been shown to regulate the mTOR pathway required for the generation of Th17 cells. Collectively, these data show that moderately fermentable fiber intake during DSS colitis is anti-inflammatory, which could potentially be due to SCFA decreasing leukocyte recruitment. Overall, the ability of a dietary fiber to elicit positive effects on fermentation and health benefits is due to the physico-chemical properties of the fiber. Here, we found that ingestion of a moderately fermentable fiber elicited increases in fecal SCFA concentrations, and modulated the microbiome. This dietary fiber elicited anti-inflammatory effects in the DSS-model, which may have been a result of SCFA affecting leukocyte recruitment to the site of inflammation. These data show that ingestion of a moderately fermentable prebiotic fiber could affect the acute inflammatory response through controlling leukocyte recruitment. More studies are needed to assess the importance of moderately fermentable fibers in the diet during in controlling inflammatory bouts noted in IBD