Structural analysis of high pressure treated Bacillus subtilis spores

The aim of this study was to investigate the internal structural changes in high pressure treated Bacillus subtilis spores and to correlate these changes with results from plate counts, flow cytometry and analysis of dipicolinic acid (DPA) release. For the scanning electron microscopic (EM) evaluation, a focused ion beam sectioning method was developed and compared with transmission EMs. Based on this set of methods, a mechanism was proposed in which 150 MPa at 37 °C induces a physiological-like DPA release, followed by degradation of the cortex and the DNA-binding small acid-soluble proteins (SASPs). Sectioned spores looked like hollow spheres and were inactivated prior to outgrowth. A pressure increase to 550 MPa retarded completion of germination and a porous internal network structure was visible by scanning EM, possibly indicating the lack of SASPs degradation. Higher pressures (600–900 MPa) and temperatures (60–80 °C) may inhibit the degradation of the spore cortex.Industrial relevanceThe high pressure thermal sterilization (HPTS) process is an emerging technology to produce high quality low acid food products, which are shelf-stable at ambient temperature. However, an industrial scale process has not yet been implemented. The mechanisms suggested in this paper may help to understand the mechanisms of inactivation of bacterial spores treated with various pressure and temperature combinations. In addition, a focused ion beam milling procedure was developed that enables the analysis of internal structure changes in bacterial endospores. These three-dimensional scanning electron micrographs (EMs), which have a reasonable resolution, could allow individual spore tomography, and not only for pressure treated spores.

► A fast focused ion beam sectioning method for SEM imaging of spores was developed.
► Pressure and temperature dependent spore inactivation mechanisms were proposed.
► 150 MPa and 37 °C induce a physiological-like germination including SASP degradation.
► 550 MPa and 37 °C disable the completion of stage II of germination.
► Above 600 MPa and 60 °C, structural changes in the inner spore membrane may occur.