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[Display omitted] • Heat, acidity and long-term cultivation preadaptation promote VBNC formation by decreasing the intracellular ATP level. • Cross-stress protection exhibited organism-specific characteristics and could promote VBNC E. coli formation. • Food materials should avoid acidic environment, hot environment and long-term storage to reduce VBNC generation. As a novel non-thermal pasteurization technology, high pressure carbon dioxide (HPCD) has been used in food processing. However, it could induce microorganisms into a viable but nonculturable (VBNC) state, posing a potential risk to food safety and public health. In this study, we attempted to investigate the effect of various preliminary stresses including cold, heat, osmosis, acidity and oxidation on HPCD-induced VBNC formation. The results indicated that there was no effect of preliminary stresses on VBNC Staphylococcus aureus induction. However, heat, acidity and long-term (24 h) cultivation preadaptation could significantly increase the VBNC E. coli production induced by HPCD. Transcriptome analysis revealed that genes involved in ATP production were significantly decreased in these three stress-treated cells, and further ATP levels determination revealed that the ATP levels of the cell were significantly decreased after heat, acidity and long-term cultivation preadaptation, implying the decrease of ATP level caused by these stresses might be the reason for increasing VBNC production. To further study the relationship between ATP level and VBNC cell ratios after preadaptation. We artificially decreased the ATP levels, and detect their VBNC ratios after HPCD treatment. We found that with the ATP concentration decreasing after exposure to carbonyl cyanide m-chlorophenyl hydrazine (CCCP), the VBNC ratios were increased after HPCD treatment, indicating that the ATP contents were highly negatively correlated with VBNC ratios. This study proved that the preadaptation of heat, acidity and long-term cultivation could promote VBNC induction by decreasing the intracellular ATP level. In general, the obtained result gave the instruction about the storage environment for food materials, helped to further develop and optimize the HPCD processing to prevent VBNC formation and accelerate the development of HPCD technology in food industry. [ABSTRACT FROM AUTHOR]

[Display omitted] • pH ex3 is the main factor of HPCD for VBNC induction. • HP promotes HPCD-induced VBNC formation by increasing cell membrane permeability. • HP reduced the count of HPCD-induced VBNC cells by destroying the membrane. • Genes involved in transport and localization are crucial for VBNC induction. High pressure carbon dioxide (HPCD) has been used in food processing as a non-thermal pasteurization technology. However, the potential of HPCD to induce viable but non-culturable (VBNC) cells limits its application. The objective of this study was to explore the roles of extracellular pH of 3 (pH ex3) and high pressure (HP) of HPCD during VBNC induction and the underlying molecular mechanism. By using the model organism Escherichia coli O157:H7, we found that the combined effects of pH ex3 and HP could mimic the effect of HPCD for VBNC induction. Further investigation of the individual effect of pH ex3 and HP on VBNC induction revealed that pH ex3 could induce a higher proportion of VBNC cells with a slower induction rate compared with HPCD, whereas HP was unable to induce VBNC formation. Notably, the cells treated by pH ex3 and HPCD had similar morphological changes, and VBNC cells induced by pH ex3 and HPCD had similar stress resistance characteristics. These results strongly indicated that pH ex3 introduced by HPCD was a main factor for VBNC induction. Additionally, we found that HP played the role in accelerating VBNC formation in the process of HPCD treatment. Transcriptomic analysis revealed 85, 263 and 529 differentially expressed genes (DEGs) for HP-, pH ex3 - and HPCD-treated cells compared with untreated ones. 59 DEGs shared by pH ex3 and HPCD treatment might be responsible for VBNC induction, and they were mainly involved in cellular transport and localization. [ABSTRACT FROM AUTHOR]