Natto, the richest known source of menaquinone 7 (MK7), is traditionally produced via Solid Substrate Fermentation (SSF) by Bacillus subtilis natto on cooked soy beans. In this work we report a threefold increase in MK7 concentration through the use of a mixture of soy protein granules and nixtamalized corn grits. The effects of fermentation processing factors were investigated and optimized in laboratory scale. These factors include initial moisture content, incubation temperature, incubation time, α-amylase pretreatment and solid substrate medium. Response surface methodology (RSM) was used to develop a mathematical model to identify the optimum values of key process variables to increase MK7 concentration; the model was also validated experimentally. The polynomial model fitted the experimental data well with R2 = 0.89 and R2 (adj) = 0.83. MK7 production was increased from 57.78 ± 1.57 mg/kg to 67.01 ± 0.18 mg/kg when the strain was cultivated at optimum conditions predicted by statistical approach (70% initial moisture, 35°C, 4 days, 10 µL/g amylase and equal substrate mix) as compared to basal conditions (60% initial moisture, 40°C, 6 days, 10 µL/g amylase and equal substrate mix).