Stage-dependent inhibitory mechanism of ε-polylysine against Clostridium perfringens spores: inner membrane damage-mediated metabolic imbalance.

Clostridium perfringens (C. perfringens) spores pose a persistent food safety risk owing to their high resistance, yet the mechanisms governing their germination and outgrowth remain poorly understood. This study utilized ε-polylysine (ε-PL) as a model antimicrobial to systematically investigate its inhibitory effects and underlying mechanisms during the spore-to-vegetative cell transition. The results indicate that ε-PL had the least effect on early germination events; over 60 min, no significant differences were observed between the treatment and control groups in terms of OD₆₀₀ reduction or DPA release. In contrast, ε-PL significantly prolonged the lag phase and inhibited growth rates during the growth phase, suggesting a phase-dependent inhibitory effect. Mechanistic analyses revealed that the spore coat and cortex act as critical physical barriers during early germination, limiting antimicrobial access. As these outer layers progressively degraded, the inner membrane (IM) became exposed as the primary target. ε-PL disrupted IM lipid organization, significantly decreasing membrane order (GP value declined from 0.56 to 0.42), inducing membrane depolarization, and increasing permeability. These impairments led to intracellular protein leakage, ATP depletion, inhibition of Na+/K+-ATPase activity, and abnormal accumulation of pyruvate (0.3 g/L). Furthermore, excessive generation of reactive oxygen species (ROS) and DNA interference collectively signaled a collapse of energy metabolism and cellular integrity. The efficacy of ε-PL was validated in a ham sausage model, where microbial proliferation was significantly delayed. This study provides critical mechanistic insights into spore control and supports the application of ε-PL in food preservation.