**4.2.1 Production of salt-tolerant acid protease by** *B. subtilis* **JM-3**

For the isolation of protiolytic bacteria, anchovy sauce fermented at 15 ± 3 °C for 3 years was inoculated on a brain–heart infusion (BHI) agar and incubated at 27.5 °C. The highest transparent colony was isolated and identified by the genetic mapping method to be *B. subtilis*. Five hundred milliliters of the medium in a 1-L wide-mouth culture flask was inoculated with 10 mL of *B. subtilis* JM-3 suspension prepared from BHI broth media cultivated at 37 °C for 3 days. The culture was incubated at 37 °C for 8 days in a shaking incubator at 150 rpm. The optimal protease production reached 1500 U/L (W.J. Kim and S.M. Kim, 2005).
