Standardization of the C:N ratio in ileal digesta changes relationships among fermentation end-products during in vitro hindgut fermentation in pigs

Undigested proteins that become available for the microbiota in the hindgut can be used as building blocks for bacterial cells, or can enter various catabolic pathways. Degradation via protein fermentation pathways is least preferred, as several fermentation end-products released can be toxic for the host. Directing microbial protein metabolism towards protein synthesis or degradative pathways that result in less toxic end-products, for example through nutritional interventions, is an interesting strategy for improving health. We studied variation in protein fermentation patterns, resulting from variation in substrate composition. Ileal digesta, obtained from cannulated pigs fed different protein sources, were subjected to fermentation in vitro under different conditions; 1) ileal digesta were fermented as is, 2) ileal digesta were fermented after standardization to a constant high C:N ratio, by addition of high fermentable carbohydrates and 3) ileal digesta samples were incubated under limiting N concentrations. Gas production was monitored as an indirect measure of microbial activity, and fermentation end-products at different points in time were analyzed by gas chromatography and high resolution mass spectrometry. Using principal component (PC) analysis, we identified patterns in protein fermentation end-products and related them to the composition of ileal digesta. Protein-associated fermentation end-product concentrations of e.g. isovaleric-, isobutyric-, phenylacetic-acid and p-cresol were negatively affected by the available amount of high fermentable carbohydrates combined with a high C:N ratio. The aforementioned fermentation end-products positively correlated with NH3 concentrations and negatively with short chain fatty acids (SCFA) concentrations. Standardization to a constant high C:N ratio changed their relationship; isovaleric-, isobutyric-, phenylacetic-acid and p-cresol lost their correlation with NH3 concentrations, became positively correlated with SCFA concentrations, and now showed a positive correlation with available amounts of high fermentable carbohydrates. Our observations demonstrate an important role of the C:N ratio in the relationship between fermentation end-products. At constant C:N, protein fermentation end-products correlate with end-products of carbohydrate fermentation and NH3, often considered as a proxy for protein fermentation, loses its predictive power.