Bacillus subtilis spores' inactivation kinetics by hyperbaric storage at room temperature and dependence of pH and nutrient availability

Raw and pasteurized food products may contain bacterial spores, which represent a food safety or spoilage threat. One of the most used strategies to temporarily inhibit spore germination and outgrowth at pH values above 4.6, in which generally bacterial spores can grow, is refrigeration (RF). Hyperbaric storage at room temperatures (HS/RT) is a new food preservation methodology that is being studied as an alternative to RF, especially for highly perishable food products. HS/RT (25-250 MPa) for up to 30 days was studied to evaluate the behaviour of Bacillus subtilis spores in McIlvaine buffer (nutrient-free matrix) and BHI-broth (nutrient-rich matrix) at three different pH levels (4.5, 6.0 and 7.5) to evaluate the effects of pH and nutrient availability on spores' behaviour under HS/RT compared with RF. The results showed that both pH and nutrient availability play a major role in the spores' fate under HS. At pH 4.5, in both nutrient conditions and up to 75 MPa, neither spore development nor inactivation occurred for at least 30 days (similarly to RF), but this behaviour was changed at pressures above 150 MPa, where at least 2.5 log units' inactivation was observed, regardless of the matrix. At pH 6.0 and 7.5, no spore development was found under HS (except at 25 MPa), and spore inactivation (particularly in BHI at pH 7.5) with at least 5 log units' reduction was observed. For RF at pH 6.0 and 7.5, the spores were able to develop after 5 and 15 days, respectively, in BHI-broth, with no changes being observed in McIlvaine buffer. The inactivation curves were mathematically described by nonlinear kinetic models (Biphasic, Weibull and Log-logistic), despite a few cases where the first-order inactivation kinetics described the experimental data more adequately. Phase-contrast microscopy revealed mostly bright-phase spores (ungerminated, viable spores) for all HS conditions at pH 4.5 (up to 150 MPa), indicating that a considerable fraction of spores remained ungerminated for 30 days, while at 4.5 (≥200 MPa), 6.0 and 7.5 dark-phase spores (germinating or death spores) were predominant, suggesting that the spores were unable to complete the germination process. These findings indicate that B. subtilis spores' inactivation during HS/RT is dependent on the presence of nutrients and on the pH of the matrix, being observed an inactivation of at least 5-log units, without applying any thermal treatment. Differently, RF allowed spore germination and outgrowth at pH 6.0 and 7.5 and so, HS/RT may be an interesting new methodology to destroy spores at RT, opening novel opportunities to preserve perishable foods.