Nissin, natamycin, and epsilon-poly-l-lysine (epsilon-PL) are three safe, microbial-produced food preservatives used today in the food industry. However, current industrial production of epsilon-PL is only performed in several countries. In order to realize large-scale epsilon-PL production by fermentation, the effects of seed stage on cell growth and epsilon-PL production were investigated by monitoring of pH in situ in a 5-L laboratory-scale fermenter. A significant increase in epsilon-PL production in fed-batch fermentation by Streptomyces sp. M-Z18 was achieved, at 48.9 g/L, through the optimization of several factors associated with seed stage, including spore pretreatment, inoculum age, and inoculum level. Compared with conventional fermentation approaches using 24-h-old shake-flask seed broth as inoculum, the maximum epsilon-PL concentration and productivity were enhanced by 32.3 and 36.6 %, respectively. The effect of optimized inoculum conditions on epsilon-PL production on a large scale was evaluated using a 50-L pilot-scale fermenter, attaining a maximum epsilon-PL production of 36.22 g/L in fed-batch fermentation, constituting the first report of epsilon-PL production at pilot scale. These results will be helpful for efficient epsilon-PL production by Streptomyces at pilot and plant scales.