| Summary: | <i>Bacillus amyloliquefaciens-9</i> (GBacillus-9), which is isolated from the intestinal tract of the white-spotted bamboo shark (<i>Chiloscyllium plagiosum</i>), can secrete potential antibacterial materials, such as β-1,3-1,4-glucanase and some antimicrobial peptides. However, the low fermentation production has hindered the development of GBacillus-9 as biological additives. In this study, the Plackett−Burman design and response surface methodology were used to optimize the fermentation conditions in a shake flask to obtain a higher yield and antibacterial activity of GBacillus-9. On the basis of the data from medium screening, M9 medium was selected as the basic medium for fermentation. The data from the single-factor experiment showed that sucrose had the highest antibacterial activity among the 10 carbon sources. The Plackett−Burman design identified sucrose, NH<sub>4</sub>Cl, and MgSO<sub>4</sub> as the major variables altering antibacterial activity. The optimal concentrations of these compounds to enhance antibacterial activity were assessed using the central composite design. Data showed that sucrose, NH<sub>4</sub>Cl, and MgSO<sub>4</sub> had the highest antibacterial activities at concentrations of 64.8, 1.84, and 0.08 g L<sup>−1</sup>, respectively. The data also showed that the optimal fermentation conditions for the antibacterial material production of GBacillus-9 were as follows: Inoculum volume of 5%, initial pH of 7.0, temperature of 36 °C, rotating speed of 180 rpm, and fermentation time of 10 h. The optimal fermentation medium and conditions achieved to improve the yield of antibacterial materials for GBacillus-9 can enhance the process of developing biological additives derived from GBacillus-9.
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