本研究探讨了马铃薯胰蛋白酶抑制剂（PTI）抑菌活性及其抑菌机理。采用滤纸片扩散法测定抑菌圈直径，微量二倍稀释法测定最低抑菌浓度（MIC）、最低杀菌浓度（MBC），以此评价PTI的抑菌性。通过扫描电镜观察并测定胞膜通透性、胞膜完整性、胞内活性氧含量及对细菌蛋白酶的抑制率等实验，探讨了PTI的抑菌机理。结果表明：PTI对枯草芽孢杆菌、蜡样芽孢杆菌和铜绿假单胞菌有较明显的抑制作用，其抑菌圈直径分别为12.56 mm、12.35 mm和11.76 mm，MIC为1.88~3.75 mg/mL，MBC均为3.75 mg/mL。PTI破坏了菌体细胞的形态，导致菌悬液中相对电导率升高和核酸的泄漏，影响了细菌的膜通透性；通过荧光显微镜观察，1 MIC的PTI使菌体胞膜的完整性遭到破坏并促进胞内ROS的产生，造成了菌体氧化损伤；PTI对菌体蛋白酶的活性表现出一定的抑制作用。通过实验得出PTI可能通过这种多靶点协同增效作用达到其抑菌效果。

In this study, the antibacterial activity and underlying mechanism of the potato trypsin inhibitor (PTI) was investigated. To evaluate the antibacterial activity of PTI, the inhibition zone diameter (DIZ) was determined through the filter paper diffusion method, and the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined by the double microdilution method. The antibacterial mechanism of PTI was explored through observations by scanning electron microscopy, and measurements of cell membrane’s permeability and integrity, intracellular reactive oxygen content as well as inhibition rate of bacterial protease. The results showed that PTI exhibited significant inhibitory effects against Bacillus subtilis, Bacillus cereus and Pseudomonas aerimonas with the corresponding diameters of bacteriostatic zone as 12.56 mm, 12.35 mm and 11.76 mm, respectively, MIC ranging from 1.88 to 3.75 mg/mL, and MBC being the same for the three bacteria (3.75 mg/mL). PTI destroyed the morphology of bacterial cells and led to the increases of relative conductivity and leakage of nucleic acid in the bacterial suspension, which affected the membrane permeability of bacteria. Fluorescence microscopic images showed that PTI at the concentration of MIC destroyed the integrity of the bacterial cell membrane, promoted the production of intracellular ROS, causing oxidative damage to the bacterial cells. PTI showed a certain inhibitory effect on the activity of bacterial protease. Based on the experimental results, it is concluded that PTI probably exerts its antibacterial activity through such a multi-target synergistic effect.

广东省国际合作项目（2019A050510005；2018A050506016）；广州市经济技术开发区国际合作项目（2018GH21）；广州市科学研究项目（201904010240）；中央高校基本科研业务费项目（2018KZ08；2019MS099）