Characterization of Bacillus amyloliquefaciens spores after thermal and pressure-assisted thermal processing by infrared microspectroscopy and multivariate analysis

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Pressure-assisted thermal processing (PATP) utilizes combination of pressure (500- 800MPa) and heat (90-120°C) to inactivate bacterial spores but limited studies have been documented regarding its mechanism of inactivation. Fourier transform infrared (FT-IR) microspectroscopy was used to compare and contrast biochemical changes occurring in Bacillus amyloliquefaciens spores during thermal processing (TP: 105°C-0.1MPa) and PATP (105°C-600MPa). Spore crops were prepared using two different media (TSAYE and NAYE). Surviving spores were enumerated after incubation at 32°C up to 72 hours. Spectra were collected by attenuated total reflectance (ATR) in mid-infrared region (4000-700 cm-1). A SIMCA model (900-1800 cm-1) could differentiate pressure resistance/sensitivity of different spore crops before they were further treated. D105°C-0.1MPa of spores grown on TSAYE and NAYE were 24.21±0.25 and 36.79±1.34 min, whereas D105°C-600MPa were 1.32±0.38 and 1.65±0.21 min, respectively. Surviving spores after treatments could be precisely predicted by PLSR models with r-value >0.98. Changes in acidic proteins and dipicolinic acid (DPA) structure modification were primarily detected during TP and PATP. Bands associated to spore inactivation were identified as 1381, 1415, and 1442 cm-1, indicating the contribution of carboxylate (COO-) vibration of calcium dipicolinate (DPA-Ca2+), the interaction of Ca2+ with COO-, and pyridine ring vibration of DPA/acid peptides, respectively. PATP showed additional effects on amide bands (1570-1650 cm-1) of protein. ATR-IR microspectroscopy is a powerful tool to discriminate the combined pressure-heat spore resistances and obtain further information related to the contribution of DPA during spore inactivation by TP and PATP.



pressure-assisted thermal processing, bacterial spores, FT-IR, infrared microspectroscopy