Strategies for effective high-pressure germination or inactivation of Bacillus spores involving nisin

The major challenge in employing high pressure (HP) at moderate temperature for sterilization is the remarkable resistance of bacterial spores. High isostatic pressure can initiate spore germination, enabling subsequent inactivation under mild conditions. However, not all spores could be triggered to germinate under pressure at temperatures ≤80°C so far. In this study, germination treatment combinations were evaluated for Bacillus spores involving moderate HP (150 MPa, 37°C, 5 min), very HP (vHP, 550 MPa, 60°C, 2.5 or 9 min), simple and complex nutrient germinants [L-valine, L-alanine, and tryptic soy broth (TSB)], nisin, and incubation at atmospheric pressure (37°C). The most effective combinations for Bacillus subtilis resulted in a reduction of culturable dormant spores by 8 log10 units. The combinations involved nisin, a nutrient germinant (L-valine or TSB), a first vHP treatment (550 MPa, 60°C, 2.5 min), incubation at atmospheric pressure (37°C, 6 h), and a second vHP treatment (550 MPa, 60°C, 2.5 min). Such treatment combination with L-valine reduced Bacillus amyloliquefaciens spores by only 2 log10 units. B. amyloliquefaciens, thus, proved to be substantially more HP-resistant compared to B. subtilis, validating previous studies. Despite combining different germination mechanisms, complete germination could not be achieved for either species. The natural bacteriocin nisin did seemingly not promote HP germination initiation under chosen HP conditions, contrary to previous literature. Nevertheless, nisin might be beneficial to inhibit the growth of HP-germinated or remaining ungerminated spores. Future germination experiments might consider that nisin could not be completely removed from spores by washing, thereby affecting plate count enumeration.