Biofilm-associated heat resistance of Bacillus cereus spores in vitro and in a food model, Cheonggukjang jjigae

Bacillus cereus, a foodborne pathogen, is capable of forming spores and biofilms as methods to withstand environmental stresses. These bacterial structures are an issue for food safety as they aid the bacteria survive heat sterilisation processes of foods and food contact surfaces. This study was conducted to investigate the role of the biofilm structure in providing an extra layer of protection to spores against heat treatments. For this, heat resistance of B. cereus spores in intact biofilms was compared to that of planktonic spores in vitro and in a Cheonggukjang jjigae food model. Using methods developed in this study to measure the wet and dry heat resistance of spores in intact biofilms, it was found that B. cereus spores have significantly higher heat resistances when present in biofilms rather than as planktonic spores, and that dry heat is less effective than wet heat at killing spores in biofilms. In further detail, for wet heat treatments, spores in biofilms of the strain isolated from Cheonggukjang (Korean fermented whole soybean), B. cereus CH3, had generally higher wet heat resistances than the reference strain, B. cereus ATCC 10987, both in vitro and in the Cheonggukjang jjigae food model. However, the spores in biofilms of the two strains showed similar heat resistance to dry heat, with some exceptions, when biofilms were formed in vitro or in Cheonggukjang jjigae broth. Meanwhile, B. cereus ATCC 10987 spores in biofilms had higher or similar wet heat resistances in vitro compared to in Cheonggukjang jjigae broth. Wet heat resistances of B. cereus CH3 spores in biofilms were all statistically similar regardless of biofilm formation media (brain heart infusion and Cheonggukjang jjigae broths). For dry heat, spores in biofilms of both B. cereus strains were more heat resistant when biofilms were formed in the Cheonggukjang jjigae food model rather than in vitro. Altogether, heat resistances of spores in biofilms formed in vitro and in the food environment were found to be different depending on the tested B. cereus strain, but higher than planktonic spores in any case. This is the first study examining the heat resistance of B. cereus spores in intact biofilms matrices attached to the surface, both in vitro and in a food model. Therefore, this research is valuable to understand the protective effects of biofilms formed in food environments and to reduce the food safety risks associated with B. cereus.