In Situ Determination of Clostridium Endospore Membrane Fluidity during Pressure-Assisted Thermal Processing in Combination with Nisin or Reutericyclin

Abstract

bThis study determined the membrane fluidity of clostridial endospores during treatment with heat and pressure with nisin or reutericyclin. Heating (90°C) reduced laurdan (6-dodecanoyl-2-dimethylaminonaphthalene) general polarization, corresponding to membrane fluidization. Pressure (200 MPa) stabilized membrane order. Reutericyclin and nisin exhibit divergent effects on heat-and pressure-induced spore inactivation and membrane fluidity. P ressure-assisted thermal sterilization (PATS) of food is an alternative to thermal processing (1). PATS releases dipicolinic acid (DPA) from endospores, rehydrates the core of endospores, and allows for spore inactivation (2,3,4). A combination of 200 to 800 MPa with 120°C eliminates resistant spores of Clostridium botulinum (2, 5). At or above 120°C, however, pressure may fail to accelerate thermal inactivation or even exerts protective effects (2, 5). Antimicrobials acting in concert with PATS may enhance the inactivation of endospores, thus ensuring product safety at reduced treatment intensity. Nisin and reutericyclin exhibit antimicrobial activity against endospores (6, 7). Nisin is a pore-forming lantibiotic (6), reutericyclin is a proton ionophore (8). Remarkably, nisin enhances pressure-induced spore inactivation (9, 10), whereas reutericyclin had no effect or even attenuated pressure-induced inactivation of bacterial endospores (11).Endospores have multiple, distinct layers that contribute to resistance and metabolic dormancy (2, 12, 13). Dehydration of the spore core contributes to endospore resistance (12). Endospores possess an outer membrane, a remnant of sporulation, and an inner membrane separating the dehydrated core from the hydrated exterior (14, 15). Lipids of the inner membrane are compressed, and the surface area expands during germination without lipid synthesis (16). Disruption of the inner membrane of endospores rehydrates the core and allows inactivation of endospores by antimicrobials (17, 18). An improved understanding of the effect of pressure on endospore membranes will improve the control of endospores by pressure and facilitate the selection of antimicrobials to support pressure-assisted sterilization; however, little is known about the Ϫ1 nisin (B), or in the presence of 6.4 mg liter Ϫ1 reutericyclin (C). Measurements were taken every 10 min as samples were heated to 90°C at a rate of 5°C/10 min. The graph highlights the area corresponding to the CH 2 asymmetrical stretching absorbance; i and ii denote the wavenumbers corresponding to gel state and liquid-crystalline membranes, respectively. Comparable results were obtained with C. beijerinckii (data not shown).

Involvement of Coat Proteins in Bacillus subtilis Spore Germination in High-Salinity Environments

“…Spores were also germinated by a higher pressure of 550 MPa. This treatment is thought to directly induce Ca 2ϩ -DPA release via Ca 2ϩ -DPA channels (16,18,19) and/or to induce a phase shift of the inner spore membrane (40)(41)(42). Again, it could be shown that germinations induced by 550 MPa were inhibited by NaCl: increasing NaCl concentrations clearly decreased the amount of DPA released, with 3.6 M NaCl no longer allowing any detectable DPA release (Fig.…”

“…Interestingly, the germination triggered by 550 MPa was also inhibited by NaCl. As treatments with a 550-MPa pressure directly induce Ca 2ϩ -DPA release, likely by activation of SpoVA channels and/or a phase shift of the inner spore membrane (16,18,19,(40)(41)(42), NaCl probably does not inhibit germination at the levels of signal integration and transduction from the germinosome.…”

The Copy Number of the spoVA 2mob Operon Determines Pressure Resistance of Bacillus Endospores

“…A pressure of 400 to 800 MPa at ambient temperature triggers release of Ca-DPA to induce germination independent of nutrient receptors (6,7). Treatment at 600 MPa and 80°C or higher inactivates spores without triggering physiological processes involved in spore germination (6,8,19,41). Previous studies hypothesized that the insertion of spoVA 2mob operon into B. subtilis 168 resulted in higher DPA concentrations in spores and consequently a high-level heat resistance (13,16).…”

“…The resistance of spores to pressure-assisted thermal sterilization may depend on the spores' ability to retain DPA and on the heat resistance of the DPA-free spores (6,11). The likely mechanisms for spore inactivation at more than 600 MPa and more than 80°C are a compromised inner spore membrane and the inactivation of integral membrane proteins (17)(18)(19). This therefore facilitates Ca-DPA release in the absence of physiological activity (6,20).…”

Microbiological efficacy of pressure assisted thermal processing and natural extracts against Bacillus amyloliquefaciens spores suspended in deionized water and beef broth

“…Although the mechanism of spore killing is unclear, disruption of inner membrane of endospores during PATP might rehydrate the core and allows inactivation of endospores by antimicrobials (Melly et al, 2002). A treatment of clostridial endospores at 600 MPa and 90 • C for 8 min reduced viable spore counts by more than 3 log cycle and resulted in the release of more than 95% DPA but did not affect the membrane fluidity (Hofstetter et al, 2013). Also, antimicrobial substances (nisin and reutericyclin) had a divergent effect on endospore membrane structure during thermal and pressure assisted thermal processing.…”