Suppressing isovaleric acid off-flavor in Bacillus subtilis fermentation and its consequential effects on secondary metabolism.

Bacillus subtilis-fermented soybean foods (BFSFs) are nutritious East Asian staples. However, off-flavors dominated by isovaleric acid (IVA), a branched-chain fatty acid, limit consumer acceptance and industrial adoption. Although leucine- and acetolactate-derived pathways have been proposed, key regulatory nodes for IVA in soymilk remain unclear. This study systematically evaluated the key gene nodes controlling IVA biosynthesis during soymilk fermentation by B. subtilis, including bcd (leucine dehydrogenase, Bcd), bkdAB (branched-chain α-keto acid dehydrogenase complex, Bkd), ptb (phosphotransbutyrylase, Ptb), and alsS (acetolactate synthase, ALS). This study utilized B. subtilis 168 (BS168) and a gene-editing approach to construct BS168 mutants lacking bkdAB and alsS, which were combined with previously generated BS168Δbcd and BS168Δptb. IVA was significantly reduced in all mutants, most markedly in BS168Δbcd (-81.85%) and BS168ΔbkdAB (-70.31%). The volatile profile shifted toward higher levels of fruity esters and alcohols and lower levels of acids and ketones, with improved sensory acceptability, particularly in BS168ΔbkdAB. Enzyme assays confirmed Bcd, Bkd, and Ptb as key pathway nodes, while ALS modulated upstream pyruvate flux toward branched-chain amino acid (BCAAs) biosynthesis. Collectively, this work genetically validates IVA pathway control in a real food matrix and shows that targeted disruption of bcd, bkdAB, and ptb is an effective strategy to suppress IVA off-flavor and improve sensory acceptability in B. subtilis-fermented soymilk, providing mechanistic targets for subsequent development and evaluation in food-grade or industrial strains.