Identification, source tracking, and targeted control of key spoilage bacteria in sub-high-temperature sterilized ham sausage

To improve sensory quality while maintaining microbial safety, the meat industry increasingly adopts mild thermal processing (105-115 °C) for ham sausage production. However, this reduced intensity allows the survival of heat-resistant bacterial endospores, increasing spoilage risks. This study combined high-throughput sequencing and culture-dependent methods to characterize the microbiota in spoiled mildly-heated ham sausages and trace contamination sources. Initially, Clostridium spp. dominated spoiled products (91.58%), and C. botulinum M1 was isolated. After intensified sterilization (escalated from 115 °C for 30 min to 118 °C for 28 min), Clostridium was eliminated, whereas Bacillus spp. (e.g., B. subtilis B5) became dominant (85.65%). Source-tracking identified spices as the primary Bacillus contamination vector. To control these spore-formers, a targeted hurdle strategy using ε-polylysine hydrochloride (ε-PLH) and potassium sorbate (Ksorb) was evaluated. This combination exhibited strong synergistic antibacterial activity (FICI = 0.16). Mechanistic analyses demonstrated substantial bacterial membrane disruption, intracellular leakage, and enhanced oxidative stress driven by reactive oxygen species accumulation ( P < 0.05). In a simulated spoilage model, the co-treatment effectively suppressed bacterial growth, stabilized pH, delayed total volatile basic nitrogen (TVB-N) accumulation, and extended shelf-life. These results provide a practical strategy to enhance the safety of mildly heat-processed meat products.