High-Pressure-Mediated Survival of Clostridium botulinum and Bacillus amyloliquefaciens Endospores at High Temperature

Abstract

Endospores of proteolytic type B Clostridium botulinum TMW 2.357 and Bacillus amyloliquefaciens TMW 2.479 are currently described as the most high-pressure-resistant bacterial spores relevant to food intoxication and spoilage in combined pressure-temperature applications. The effects of combined pressure (0.1 to 1,400 MPa) and temperature (70 to 120°C) treatments were determined for these spores. A process employing isothermal holding times was established to distinguish pressure from temperature effects. An increase in pressure (600 to 1,400 MPa) and an increase in temperature (90 to 110°C) accelerated the inactivation of C. botulinum spores. However, incubation at 100°C, 110°C, or 120°C with ambient pressure resulted in faster spore reduction than treatment with 600 or 800 MPa at the same temperature. This pressure-mediated spore protection was also observed at 120°C and 800, 1,000, or 1,200 MPa with the more heat-tolerant B. amyloliquefaciens TMW 2.479 spores. Inactivation curves for both strains showed a pronounced pressure-dependent tailing, which indicates that a small fraction of the spore populations survives conditions of up to 120°C and 1.4 GPa in isothermal treatments. Because of this tailing and the fact that pressure-temperature combinations stabilizing bacterial endospores vary from strain to strain, food safety must be ensured in case-by-case studies demonstrating inactivation or nongrowth of C. botulinum with realistic contamination rates in the respective pressurized food and equipment.High hydrostatic pressure (HHP) is a powerful tool for inactivating food pathogens and spoilage organisms and thus achieving food safety, with flavors, colors, and nutrients being affected to a lesser extent than in conventional heat treatments (6, 28). It is generally assumed that the introduction of HHP to a heat-mediated inactivation process enhances the inactivation of bacterial endospores compared to heat treatment alone and that the rise of pressure or temperature will result in accelerated and steady inactivation. This is of particular interest because bacterial endospores are not inactivated by pressure treatment at ambient temperature, and endospores of the genera Bacillus and Clostridium tolerate pressures over 1,000 MPa at 25°C (28). For inactivation of these organisms, combinations of high pressure and heat are required (27).Changes in pressure are thermodynamically coupled to changes in temperature, and the adiabatic heating of samples during compression makes it difficult to distinguish pressure effects on bacterial spores from temperature effects. Spores in a pressure-treated sample inevitably experience a dynamic temperature curve, which depends on the pressure difference, the compression rate, and the heat transfer rate from the sample to the pressure vessel. A nearly constant temperature during pressure holding times can be achieved by minimizing or eliminating heat transfer throughout the process (adiabatic treatments). With the aim of a fast, energy-saving process for the inactivation of bacter...

Construction of Nontoxigenic Mutants of Nonproteolytic Clostridium botulinum NCTC 11219 by Insertional Mutagenesis and Gene Replacement

“…However, the increasing consumer demand for fresh-tasting, healthy ready-to-eat foods that have been minimally processed and contain less salt and no artificial preservatives yet have a long shelf-life represents a challenge for the food industry in view of these botulinum safety guidelines (10,11,12). Furthermore, novel food processing and preservation technologies (e.g., high-pressure or pulsed electric field treatment and natural preservatives) have found their way to commercial food production, but data regarding their efficiency in controlling gIICb is scarce or lacking (13,14,15,16,17). This is in sharp contrast to the attention given in this context to other pathogens, like Listeria monocytogenes, Salmonella, and enterohemorrhagic Escherichia coli.…”

Strong and Consistently Synergistic Inactivation of Spores of Spoilage-AssociatedBacillusandGeobacillusspp. by High Pressure and Heat Compared with Inactivation by Heat Alone

“…Contrary to the case for proteolytic C. botulinum, there are a number of published studies suggesting that the most heat-resistant spoilage bacterium of concern for LASSF, Geobacillus stearothermophilus, is not nearly as resistant to thermal processing under high pressure as its heat resistance would predict (2,14,17,21,24). In comparison, strains of the aerobic mesophilic species Bacillus amyloliquefaciens, which produces spores with intermediate heat resistance, have been shown to produce highly HPTresistant spores (1,17,18,23) that under some conditions appear to be stabilized by high pressure against thermal inactivation (16). B. amyloliquefaciens is closely related to Bacillus subtilis and Bacillus licheniformis, but to date, the spores of these related bacteria have been found to be relatively sensitive to HPT processing (17,18,26).…”

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

“…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.…”

“…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).…”