Effects of in Vitro Culture with Chlorophyll a or b on the Fecal Flora Composition of High-Fat-Fed Mice and Metabolomic Analysis of Fermentation Products

The objective of this study was to investigate the effect of chlorophyll a versus b on the intestinal flora of mice induced by a high-fat diet (HFD) metabolomics combined with microbial diversity analysis. Results demonstrated that chlorophyll was transformed and degraded by the intestinal flora after 48 h culture in vitro. Its major derivatives included oxides (13²-hydroxy chlorophyll a, 15¹-hydroxy-lactone chlorophyll b, and 13²-hydroxy chlorophyll b) and magnesiumfree derivatives (pheophytin a and 13²-hydroxy pheophytin b). The reduced chlorophyll was degraded into non-fluorescent metabolites (m/z 666.2895), fluorescent metabolites (m/z 614.2735, 618.2684, and 792.3212) and pyrrole derivatives of propionate (m/z 223.0713). Microbial diversity analysis showed that the addition of chlorophyll down-regulated Escherichia-Shigella and up-regulated Acinetobacter, and chlorophyll a and chlorophyll b had different effects on the microbial abundance and species. Targeted metabolomics showed no significant difference in fatty acid contents among the control and chlorophyll addition groups. Untargeted metabolomics showed that chlorophyll addition resulted in differences in fecal metabolites (both chlorophyll a and b led to the synthesis of more antibiotics). These metabolites were mainly enriched in the caprolactam degradation pathway and the macrolide biosynthesis pathway 12, 14 and 16 for the chlorophyll a and b groups, respectively, with 15 shared pathways being found between the two groups. The results of this study will help to understand the effect of chlorophyll supplementation on the gut microbiota of HFD-induced mice, and provide useful information for understanding the interaction between chlorophyll and the intestinal flora.