Colonic fermentation of prebiotics and dietary protein: pH effects, metabolite immunomodulation, and implications for endurance athletes

The human colon contains trillions of microbes, a community known as the gut microbiome (GM) which exhibits a wide range of physiological effects on the host. Many immune cells (both innate and adaptive) reside in the lamina propia under the epithelium in the large colon, interacting with dietary components, metabolites, and resident bacteria to shape the immune system. Diet is one of the most important factors interacting with GM and influencing metabolism and health conditions. Despite much-established evidence that prebiotics modulate GM, promote short-chain fatty acid (SCFA) production, and exhibit multidirectional influences, little is known about the environmental factor pH in shaping GM and metabolism and improving bone mineralization. Additionally, the knowledge of the immunomodulatory ability of protein fermentation metabolites is scarce when compared to the well-investigated butyrate.

This PhD thesis carried out two in vitro studies and explored the effect of increasing colonic pH gradients on prebiotic fermentation outcomes and the immunomodulation of proteolytic metabolites in bacteria-stimulated dendritic cells (DCs). As part of collaborations, this PhD thesis investigated the lowering pH effect from prebiotic fermentation and how this can be reflected in calcium absorption and bone mineralization in ovariectomized (OVX) rats. Based on a human diet intervention study, this PhD thesis analyzed the fecal pH, GM, and plasma cytokine in twenty endurance athletes following six-week interventions with either a reduced protein (RP) diet (~1 g/kg of body weight per day) or maintaining the habitual high protein diet (~2 g/kg of body weight per day).

The results showed that: 1) The influence of increasing colonic pH gradients on producing SCFAs is related to substrate and donor inoculum; the beneficial effects of prebiotics are not only attributed to the high SCFA production but also to the decreased pH which could again alter GM composition and activity. 2) Calcium and yogurt both showed positive effects on bone mineralization which is observed with decreasing gastrointestinal tract (GI) pH, high SCFA production, and GM changes. However, prebiotics (inulin and potential lactose) showed very limited effects on bone markers despite clear changes in the gut, suggesting the intricate mechanisms in the prebiotic-pH/SCFA-bone axis. 3) Protein fermentation metabolites showed different influences on the viability of DCs and bacteria-induced cytokine production; SCFAs exhibited the most prominent effects on influencing cytokine production, which was related to the type of bacteria stimulations; the unbranched fatty acid valerate stood out with a strong influence but was weaker relative to butyrate in short-term treatment, which can be explained by the different reactive oxygen species (ROS) production and gene expression between these two fatty acids; valerate and butyrate showed comparable influences in long-term treatment (2 days), suggesting the potential immunomodulating ability of other metabolites from protein fermentation. 4) Reducing dietary protein while increasing carbohydrates showed beneficial effects on the GM of endurance athletes with several species containing great metabolic capacities being promoted. Besides, the endurance-associated inflammation was alleviated in the athletic low-protein diet. Together, this PhD project contributes to a better understanding of microbial fermentation of prebiotics and dietary protein in the gut. The findings provide valuable information for assessing the health effects of prebiotics and protein sources and developing dietary guidelines for endurance athletes with special nutrition needs.