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 was 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.

Inactivation of Salmonella, Shiga Toxin-producing E. coli, and Listeria monocytogenes in Raw Diet Pet Foods Using High-Pressure Processing.

Pet food formulated with raw meat can pose health risks to pets and humans. High-pressure processing (HPP) was evaluated to achieve a 5-log reduction ofSalmonella,E. coliSTEC, andL. monocytogenesin commercial raw pet foods and maintain a 5-log reduction throughout post-HPP storage.Three formulation types that varied in the amounts of striated meat, organ meat, bone, seeds, and other ingredients (fruits, vegetables, and minor ingredients) designated as A-, S-, and R-formulations were used. Eight raw diet pet foods, consisting of three beef formulations (A-, S- and R-Beef), three chicken formulations (A-, S-, and R-Chicken), and two lamb formulations (A- and S-Lamb), were inoculated with 7 log CFU/g cocktails ofSalmonella,E. coliSTEC orL. monocytogenes, HPP at 586 MPa for 1-4 min, and stored refrigerated (4°C) or frozen (-10 to -18°C) for 21 days with microbiological analyses at various time intervals. A- formulations (20-46% meat, 42-68% organs, 0.9-1.3% seeds, and 10.7-11.1% fruits, vegetables, and minor ingredients) inoculated withSalmonellaand treated at 586 MPa for at least 2 min achieved a 5-log reduction 1 day post-HPP and maintained that inactivation level during frozen storage. A- and S-formulations inoculated withE. coliSTEC and treated at 586 MPa for at least 2 min achieved a 5-log reduction from day 6 of frozen storage. L. monocytogeneswas more HPP resistant thanSalmonellaandE. coliSTEC.S-formulations containing chicken or beef and stored frozen post-HPP had lower inactivation of L. monocytogenes compared to A-formulations containing chicken or beef. S-Lamb had higher frozen storage inactivation (5.95 ± 0.20 log CFU/g) compared to chicken (2.52 ± 0.38 log CFU/g) or beef (2.36 ± 0.48 log CFU/g). HPP coupled with frozen storage time was effective in achieving and maintaining a 5-log reduction ofSalmonellaandE. coliSTEC whileL. monocytogeneswas more resistant and requires further optimization to achieve a 5-log reduction.

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.

Evidence for Bacillus cereus Spores as the Target Pathogen in Thermally Processed Extended Shelf Life Refrigerated Foods.

The microbial safety concern associated with thermally processed extended shelf life (ESL) refrigerated foods is based on adequate elimination of spore-forming pathogens such as nonproteolytic Clostridium botulinum types B, E, and F. These pathogens are traditionally regarded as targets for validation of thermally processed ESL foods. However, their use for research is restricted due to their designation as select agents. In this study, the thermal resistances of spores of 10 nonproteolytic C. botulinum types B and F and seven psychrotrophic Bacillus cereus strains were evaluated in ACES (N-(2-acetamido)-2-aminoethanesulfonic acid) buffer (0.05 M, pH 7.00) and compared to determine whether any of the B. cereus strains could serve as a nonselect agent for establishing thermal processes for ESL refrigerated foods. Thermal decimal reduction times (DT-values) of both nonproteolytic C. botulinum types B and F and psychrotrophic B. cereus strains decreased as process temperature increased from 80 to 91°C, and the highest values were obtained at 80°C. All psychrotrophic B. cereus strains tested were more thermally resistant than nonproteolytic C. botulinum types B and F. DT-values of nonproteolytic C. botulinum types B and F decreased to <1.0 min at 87°C, whereas all psychrotrophic B. cereus strains had higher DT-values (i.e., 52.35 to 133.69 min) at the same temperature. Among all psychrotrophic B. cereus strains tested, BC-6A16 had the highest DT-values at any given temperature. The DT-values indicated that the psychrotrophic B. cereus strains were more thermally resistant than the nonproteolytic C. botulinum strains and therefore may be potential target pathogens for thermal process validation of ESL refrigerated foods. However, further comparative challenge studies are needed with a model food system or an ESL refrigerated food to confirm these results.

The use of High-Pressure Processing (HPP) to improve the safety and quality of raw coconut (Cocos nucifera L) water.

This research investigated the use of high-pressure processing (HPP) for inactivating vegetative pathogens and spoilage microbiota in fresh unfiltered coconut water (Cocos nucifera L) from nuts obtained from Florida and frozen CW from Brazil with pH >5.0 and storage at 4 °C. Additionally, CW was evaluated to determine if it supported the growth and toxin production of Clostridium botulinum with or without the use of HPP when stored at refrigeration temperatures. Samples of fresh unfiltered CW were inoculated to 5.5 to 6.5 logs/mL with multiple strain cocktails of E. coli O157:H7, Salmonella spp. and Listeria monocytogenes and HPP at 593 MPa for 3 min at 4 °C. HPP and inoculated non-HPP controls were stored at 4 °C for 54 and 75 days for Florida CW and Brazil CW, respectively. Results of analyses showed HPP samples with <1 CFU/mL and no detection (negative/25 mL) with enrichment procedures for the 3 inoculated pathogens for all analyses. The non-HPP control samples did not show growth of the pathogens but a gradual decrease in levels to ca. 3-Logs/mL by day 54 in the fresh Florida CW and similarly in frozen Brazil CW by Day 75. Microbial spoilage of uninoculated samples was evaluated for normal spoilage microbiota through 120 days storage at 4 °C. Microbial counts remained at ca. 2-logs with no detectable signs of spoilage for HPP samples through 120 d. The non-HPP control samples spoiled within 2 weeks of storage at 4 °C with gas production, cloudiness, and off-odors. To evaluate if CW supports the growth and toxin production of C. botulinum, samples of unfiltered and filtered (0.2 µm) CW were inoculated with either proteolytic or non-proteolytic C. botulinum spores at 2 log CFU/mL that were processed at 593 MPa for 3 min and stored at 4 °C and 10 °C for 45 days. Inoculated positive and non-inoculated negative controls were prepared and stored as the HPP treated and non-HPP samples. No growth of C. botulinum or toxin production was detected in either the unfiltered or filtered CW regardless if products were HPP treated or not. All inoculated samples with C. botulinum spores were enriched at Day-45 in PYGS media to determine the viability of the inoculated spores at the end of shelf-life and screened for C. botulinum toxins. In all samples, C. botulinum toxin Types A, B and E were detected indicating spores were viable throughout the storage. Type F toxin was not detected possibly due to inherent conditions in the samples that may affected toxin screening.