A novel experimental setup and pH methodology for studying the impact of O2 on Listeria monocytogenes growth under high CO2 MAP.

Listeria monocytogenes, the causative agent of listeriosis, is a pathogenic bacterium capable of growing both in cold and low-oxygen, conditions commonly found in Modified Atmosphere Packaging (MAP). Ensuring the efficacy of MAPs in inhibiting pathogen’s growth during cold storage is crucial for food safety. However, despite efforts to study L. monocytogenes growth under different gas compositions, research on the pathogen growth under constant atmosphere conditions is lacking. This study addresses this gap by using the Gas Washing Bottle Incubation System (GBIS), a novel experimental setup designed to minimize dynamic atmosphere changes usually occurring within MAP systems. In this study, L. monocytogenes was inoculated (3.0 Log CFU/mL) into BHI-filled bottles and stored for 13-days at 7°C under two high CO2 atmospheres (O2%/CO2%/N2%: 0/60/40, 20/60/20). The GBIS comprises three gas washing bottles connected to each other in series using plastic tubes. The setup was flushed daily with gases to maintain constant the target concentration. Samples for growth curve were collected daily before flushing and analysed by spread plate method, whereas pH samples were collected in days 0, 4, 8, and 12, and analysed by a novel non-destructive methodology. The growth curves were fitted by a linear growth model and the experiments were performed twice. Results showed a significant increase in bacterial count for both conditions, with a 4.4 Log increase under 0% O2 and a 3.8 Log increase under 20% O2. Statistical analysis revealed a significant difference in growth, indicating that anaerobic environments may enhance the growth of L. monocytogenes. Despite an initial drop, pH remained stable throughout the experiment, demonstrating the effectiveness of the GBIS setup in controlling and stabilizing pH over the duration of the experiments.