Role of dipicolinic acid in heat resistance of spores of Clostridium botulinum and Clostridium sporogenes PA3679 by thermal and pressure-assisted thermal processing.

Dipicolinic acid (DPA) is a major constituent of spores and reportedly provides protection against inactivation by various thermal processes, however the relationship between DPA and resistance towards pressure-assisted thermal processing is not well understood. Thermal and pressure-assisted thermal inactivation studies of Clostridium botulinum nonproteolytic strains QC-B and 610-F, proteolytic strain Giorgio-A, and thermal surrogate Clostridium sporogenes PA3679 spores suspended in ACES buffer (0.05 M, pH 7.0) were performed to determine if a relationship exists between DPA release and log reduction of spores. Thermal inactivation at 80, 83, and 87°C for nonproteolytic strains and 101, 105, and 108°C for the proteolytic strain and thermal surrogate were conducted. Pressure-assisted thermal inactivation for nonproteolytic strains at 83°C/600 MPa and for the proteolytic strain and thermal surrogate at 105°C/600 MPa were performed. Surviving spores were enumerated by 5-tube MPN method for log reductions and analyzed for released DPA by liquid chromatography-tandem mass spectrometry. The correlation between MPN log reductions, released DPA, and D-values were calculated. A positive correlation between released DPA and log reduction of spores was observed for QC-B and 610-F at 80 and 83°C (r = 0.6073 - 0.7755; P <0.01). At 87°C, a positive correlation was detected for 610-F (r = 0.4242, P < 0.05) and no correlation observed for QC-B (r = 0.1641; P > 0.05). A strong, positive correlation (r = 0.8359 - 0.9284; P < 0.05) between released DPA and log reduction of spores was observed for Giorgio-A at 101, 105, and 108°C and a strong, positive correlation (r = 0.8402; P < 0.05) was observed for PA3679 at 101°C. A positive correlation (r = 0.5646 - 0.6724; P <0.01) was observed for QC-B, 610-F, and Giorgio-A after pressure-assisted thermal treatment. No correlation (r = 02494; P > 0.05) was found for PA3679 after pressure-assisted thermal treatment. These results suggest a correlation exists between DPA release and heat resistance, however, the level of correlation varied between strains and temperatures. The findings from this research may aid in the development of spore inactivation strategies targeting the thermal resistance profiles of various strains of C. botulinum spores.

Barotolerance of Acid-adapted and Cold-adapted Bacterial Isolates of E. coli O157:H7, Salmonella spp., and L. monocytogenes in an Acidic Buffer Model.

The fruit and vegetable juice industry has shown a growing trend in minimally processed juices. A frequent technology used in the production of functional juices is cold pressure, which refers to the application of high pressure processing (HPP) at low temperatures to inactivate foodborne pathogens. HPP juice manufacturers are required to demonstrate a 5-log reduction of the pertinent microorganism to comply with FDA Juice HACCP. However, there is no consensus on validation study approaches for bacterial strain selection or their preparation. Individual bacterial strains were grown using three different growth conditions: neutral, cold-adapted, and acid-adapted. Approximately 6.0-7.0 log CFU/mL of the matrix-adapted bacterial strains were inoculated individually into buffered peptone water (BPW) at pH 3.50 ± 0.10 (HCl adjusted) and treated at sublethal pressures of 500 MPa for Escherichia coli O157:H7 and 200 MPa for Salmonella spp. and Listeria monocytogenes (180 s, 4°C). Analyses were conducted at 0, 24, and 48 h (4°C storage) post-HPP on nonselective media. E. coli O157:H7 exhibited greater barotolerance than Salmonella spp. and L. monocytogenes. In neutral growth conditions, E. coli O157:H7 strain TW14359 demonstrated the greatest resistance (2.94 ± 0.64 log reduction), and E. coli O157:H7 strain SEA13B88 was significantly more sensitive (P < 0.05). Salmonella isolates, neutral and acid-adapted, expressed similar barotolerance to one another. Cold-adapted S. Cubana and S. Montevideo showed greater resistance compared to other cold-adapted strains. Acid-adapted L. monocytogenes strain MAD328 had <1.00 ± 0.23 log reduction while acid-adapted L. monocytogenes strains CDC and Scott A were significantly more sensitive (P < 0.05) with reductions of 2.13 ± 0.48 and 3.43 ± 0.50 log CFU/mL, respectively. These results suggested, under the conditions tested, bacterial strain and preparation methods influence HPP efficacy and should be considered when conducting validation studies.

Rapid detection and quantitation of dipicolinic acid from Clostridium botulinum spores using mixed-mode liquid chromatography-tandem mass spectrometry.

Analysis of the dipicolinic acid (DPA) released from Clostridium botulinum spores during thermal processing is crucial to obtaining a mechanistic understanding of the factors involved in spore heat resistance and related food safety applications. Here, we developed a novel mixed-mode liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for detection of the DPA released from C. botulinum type A, nonproteolytic types B and F strains, and nonpathogenic surrogate Clostridium sporogenes PA3679 spores. DPA was retained on a mixed-mode C18/anion exchange column and was detected using electrospray ionization (ESI) positive mode within a 4-min analysis time. The intraday and interday precision (%CV) was 1.94-3.46% and 4.04-8.28%, respectively. Matrix effects were minimal across proteolytic type A Giorgio-A, nonproteolytic types QC-B and 202-F, and C. sporogenes PA3679 spore suspensions (90.1-114% of spiked DPA concentrations). DPA recovery in carrot juice and beef broth ranged from 105 to 118%, indicating limited matrix effects of these food products. Experiments that assessed the DPA released from Giorgio-A spores over the course of a 5-min thermal treatment at 108 °C found a significant correlation (R = 0.907; P < 0.05) between the log reduction of spores and amount of DPA released. This mixed-mode LC-MS/MS method provides a means for rapid detection of DPA released from C. botulinum spores during thermal processing and has the potential to be used for experiments in the field of food safety that assess the thermal resistance characteristics of various C. botulinum spore types.