| Summary: | <i>Bacillus cereus</i> is a food-borne pathogen that can produce cereulide in the growth period, which causes food poisoning symptoms. Due to its resistance to heat, extreme pH, and proteolytic enzymes, cereulide poses a serious threat to food safety. Temperature, pH, and a<sub>w</sub> can influence cereulide production, but there is still a lack of research with multi-environmental impacts. In this study, the effects of temperature (15~45 °C), pH (5~8), and a<sub>w</sub> (0.945~0.996) on the emetic reference strain <i>B. cereus</i> F4810/72 growth, cereulide production, relevant <i>ces</i> genes (<i>cesA</i>, <i>cesB</i>, <i>cesP</i>), and transcription regulators genes (<i>codY</i> and <i>abrB</i>) expression at transcription level were studied. <i>B. cereus</i> survived for 4~53 h or grew to 6.85~8.15 log<sub>10</sub> CFU/mL in environmental combinations. Cereulide accumulation was higher in mid-temperature, acidic, or high a<sub>w</sub> environments. Increased temperature resulted in a lower cereulide concentration at pH 8 or aw of 0.970. The lowest cereulide concentration was found at pH 6.5 with an increased a<sub>w</sub> from 0.970 to 0.996. Water activity had a strong effect on transcriptional regulator genes as well as the <i>cesB</i> gene, and temperature was the main effect factor of <i>cesP</i> gene expression. Moreover, environmental factors also impact cereulide synthesis at transcriptional levels thereby altering the cereulide concentrations. The interaction of environmental factors may result in the survival of <i>B. cereus</i> without growth for a period. Gene expression is affected by environmental factors, and temperature and pH may be the main factors influencing the correlation between <i>B. cereus</i> growth and cereulide formation. This study contributed to an initial understanding of the intrinsic link between the impact of environmental factors and cereulide formation and provided valuable information for clarifying the mechanism of cereulide synthesis in combined environmental conditions.
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