Shrimp head by-product (SHP) has been increasingly studied for applications in bacterial fermentation and the production of secondary metabolites. In this work study, Bacillus velezensis EB.KN15 was grown via fermentation using SHP as a novel substrate on a large scale and its potential fungicidal effect against durian pathogen fungi was evaluated. SHP was found rich in protein content (29.18%) and several mineral elements suitable for EB.KN15 fermentation. The appropriate cultural medium for B. velezensis EB.KN15 growth in the small-scale fermentation using 250 mL-Erlenmeyer flask included 2% SHP/LB (7/3), 0.05% MnSO4, and 0.1% KH2PO4 at 34 °C, pH 7 for 36 h. When scaling up fermentation in a 14 L-bioreactor system, this strain exhibited better productivity (1.2 × 1012 CFU/mL) in a shorter fermentation time (only 10 h). Anti-fungal activity tests revealed for the first time, that EB.KN15 has potential fungicidal effect against Fusarium incarnatum, Phytophthora palmivora, and Fusarium solani with high inhibition values of 70%, 78.95%, and 80%, respectively. Gas chromatography mass spectrometry could identify 11 major volatiles produced by B. velezensis EB.KN15 in the supernatant. The molecular docking indicated effective interaction of these major volatiles with the target protein responsible for F. solani inhibition with good binding energy (DS ≤ 7.1 kcal/mol) and an acceptable root-mean-square deviation value (1.05–1.96 Å). This study suggests that SHP is a new substrate for the fermentation of B. velezensis EB.KN15 and recorded this strain as a novel potential candidate to effectively manage durian pathogen fungi.
