Selected approaches for enhancing pressure-assisted thermal processing (PATP) lethality on bacterial spore inactivation were investigated using high pressure microbial kinetic testing equipment. Bacillus amyloliquefaciens TMW 2.479 spores were used as the test organisms. Efficacy of pressurization rate and double-pulse treatment in enhancing PATP lethality was examined. Pressurization rate influenced the PATP lethality as a function of pressure-holding time. During short pressure holding times (≤2 min), PATP treatment with the slow pressurization rate (3.75 MPA/s) provided enhanced spore reduction over that of fast pressurization rate (18.06 MPa/s). Regardless of the pressurization rate, after 5-min treatment at 105°C-600 MPa, 6 log reduction of B. amyloliquefaciens spores were observed. Double-pulse treatment enhanced PATP lethality by approximately 2.4 to 4 log CFU/ml, in comparison to single pulse for a given pressure holding time.
The efficacy of combining organic acids (acetic, citric, and lactic; 100 mM, pH 5.0) with PATP treatment in enhancing B. amyloliquefaciens inactivation was studied. In combination with 2-min PATP treatment at 700 MPa-105°C, acetic and citric acids were found to provide synergistic effect on inactivating B. amyloliquefaciens spores than the spores suspended in lactic acid or deionized water. Organic acids in combination with PATP treatment also induced spore germination (33% to 80%) as a function of pressure holding time and type of organic acid used. Combining organic acids with PATP also inhibited the growth and recovery of the remaining survivor population during 28-day storage at 32°C in a low-acid food model system (carrot puree, pH 5.0).
Biochemical changes in B. amyloliquefaciens spores grown in two different sporulation media (TSAYE and NAYE) as influenced by PATP, high pressure processing (HPP), and thermal processing (TP) were investigated using Fourier-transform infrared spectroscopy (FT-IR). FT-IR spectra of the bacterial spores were not only influenced by the processing conditions, but also by the sporulation media. PATP and TP treatments acted upon specific targets of calcium dipicolinate (CaDPA), which were identified by bands at 1381, 1415, and 1442 cm-1. Changes in secondary protein bands were also observed during PATP and TP. Surviving spore populations of B. amyloliquefaciens after PATP and TP treatments could be estimated by cross-validated PLSR models using spectra region (900-1800 cm-1). However, such a developed model based on single-pulse data could not be used for predicting double-pulse lethality especially during 2nd pulse pressure holding time, possibly due to differences in respective spore inactivation mechanisms.
In conclusion, various physical- and chemical-based approaches can be used for enhancing PATP lethality of highly pressure-thermal resistant B. amyloliquefaciens spores. Different processing conditions and sporulation media caused changes in FT-IR spectra especially in CaDPA and secondary protein bands. Organic acids, citric and lactic acids in particular, in combination with PATP treatment enhanced PATP lethality and further inhibited spore recovery in the treated carrot puree during extended storage.