Abstract The multi-component lactoperoxidase system (LPSys) is used for improving food safety and requires thiocyanate (SCN − ) as a substrate for the generation of antimicrobial hypothiocyanite (OSCN − ). Production of OSCN − is usually limited by intrinsic concentrations of SCN − or else SCN − must be supplied from a synthetic source. Our objective was to optimize the production of OSCN − in the LPSys using SCN − derived from glucosinolates present in yellow mustard ( Sinapis alba ) seed meal. Thiocyanate was produced from S. alba seed meal by shaking a 1:20 (w:w) meal:H 2 O mixture at 70 °C for 10 min. Increasing the exogenous H 2 O 2 to an equimolar concentration (500 μM) of SCN − further increased OSCN − production, whereas H 2 O 2 concentrations in excess of SCN − enhanced OSCN − decay. Glucose and glucose oxidase (GOx) were substituted for H 2 O 2 to sustain OSCN − production resulting in an optimized LPSys containing 4.92 U/mL lactoperoxidase, 64 mM glucose, 342 U/L GOx, and 500 μM SCN − . Maximum OSCN − in the optimized system was 40 μM, a concentration that is predicted to have antimicrobial effects, thus demonstrating the potential of the low value S. alba seed meal as source of SCN − for preparing a generally recognized as safe LPSys.