Enhancement of thermal processing with food-grade antimicrobial compounds in low-moisture food matrix to improve food safety

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Heat resistant foodborne pathogens have been a concern in low-moisture foods and ingredients (LMFs). Increased thermal resistance of pathogens such as Escherichia coli O157:H7 and Salmonella Typhimurium at low water activity (aw) reduces the efficiency of thermal treatment in LMFs. Alternative methods are therefore needed to augment thermal processing and reduce food safety risk. This study investigated the enhancement of thermal treatment efficiency against pathogenic bacteria in LMF matrices at different aw by inclusion of food-grade antimicrobial compounds. Based on their similar target sites in cells, it is hypothesized that antimicrobial compounds may work synergistically with heat treatment in LMF matrices. The treatment effect may be dependent on both aw and matrix compositions. Physiological and transcriptional changes may take place within cells adapted to different environments and contribute to the varied bacterial resistance. A combination of Butylparaben (BP), a known antimicrobial and thermal treatment was tested to enhance inactivation rates of S. Typhimurium and E. coli O157:H7 in meat and bone meal (MBM) equilibrated to water activity (aw) of 0.4 and 0.7. Presence of BP significantly accelerated thermal inactivation of E. coli O157:H7 in MBM at both 0.4 and 0.7 aw. However, inactivation rate of S. Typhimurium was not affected by the combined treatment at either 0.4 or 0.7 aw. BP accelerated the inactivation of the thermal-resistant subpopulation in E. coli O157:H7 but not S. Typhimurium. Therefore, further studies were conducted to identify alternative antimicrobial compounds to enhance thermal treatment against S. Typhimurium in LMF matrices and investigate the resistance mechanisms. Trans-cinnamaldehyde (CA) and eugenol (EG) were selected from a screening study to assist thermal treatment against S. Typhimurium adapted to different aw in whey protein (WP), corn starch (CS) or peanut oil (PO). Although addition of CA or EG significantly accelerated thermal inactivation of S. Typhimurium in water and LMF components at 0.9 aw, similar effect was not observed in bacteria adapted to lower (0.4) aw in any of those matrices. The matrix effect on bacterial thermal resistance was observed at 0.9 aw and were ranked as WP > PO > CS. The combined heat treatments reduced bacterial metabolic activity, which was partially dependent on the food components as well. S. Typhimurium had lower membrane fluidity because of membrane fatty acid composition change when adapted to a lower aw, which contributed to resistance against the combined heat treatments. Based on the aw- and matrix- dependent bacterial resistance and physiological changes observed in the two previous studies, transcriptional analysis was conducted to further understand the molecular mechanism behind the bacterial resistance. Expression of nine stress-related genes in S. Typhimurium adapted to different aw in LMF components were analyzed with or without the CA-assisted heat treatment. The upregulation of rpoH and dnaK in S. Typhimurium was induced by the stress during bacterial adaptation to the low aw environments as well as the combined heat treatment, which contributed to the bacterial resistance to both desiccation and the combined treatment. Although its link to the desiccation response in bacteria is not fully understood, the downregulation of ompC during the combined treatment also partially contributed to the treatment resistance. The upregulation of rpoE, otsB, proV and fadA in S. Typhimurium was induced by the desiccation stress during incubation at the low aw environment but was not a major contributor to the resistance during the combined treatment. The observed upregulation of fabA and downregulation of ibpA could not be directly linked to either bacterial resistance to desiccation or the combined heat treatment. Differential expressions were also observed among different aw levels or in different matrices at the same aw. The inconsistencies between the expression profiles of dnaK and ompC and bacterial resistance during the combined treatment suggested the presence of additional stress-response pathways.In conclusion, the results have demonstrated the potential of using food-grade antimicrobial compounds to complement thermal treatment in LMF during processes that start with a relatively high aw (such as dehydration). However, their effectiveness may subside at low aw environments. Future works should focus on optimizing the parameters of the CA/ EG-assisted heat treatment for higher efficiency, studying the molecular mechanism behind the bacterial resistance at global scale, and exploring alternative non-thermal processing technologies to enhance thermal treatment efficiency in LMFs without raising thermal treatment intensity.