Insights into specific and nonspecific butyrate-producing pathways during the in vitro fecal fermentation of butyrylated starch

The esterase-mediated esterolysis is the key for specific butyrate production. • Carboxylesterase assay verified the involvement of esterases during the fermentation. • The classical metabolic pathway for nonspecific butyrate production is altered. Butyrylated starch is a special type-4 resistant starch with butyrate-carrying attribute. In this study, the unique butyrate-producing capability of butyrylated starch was deeply investigated by focusing on its specific and nonspecific butyrate-producing pathways, respectively, using specially designed substrates as controls. In vitro fermentation studies revealed that butyrylated and isobutyrylated starches generated high levels of butyrate and isobutyrate, respectively, highlighting the role of butyryl group metabolism in the specificity of butyrate production. Carboxylesterase assays have demonstrated that butyryl group metabolism is primarily facilitated by carbohydrate esterases expressed in the gut microbiota. Combined with 16S rRNA sequencing and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, it was found that butyrylated starch fermentation did not significantly enhance traditional butyrate synthesis pathways but modified the balance between the butyryl-CoA:acetyl-CoA transferase and butyrate kinase pathways by altering the gut microbiota composition, specifically by upregulating the relative abundance of indicator species such as Bacteroides , the Lachnospiraceae_NK4A136_group , and Parabacteroides . These insights offer theoretical guidance for designing butyrylated starch structures and regulating intestinal health. Download: Download high-res image (169KB) Download: Download full-size image