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High-pressure CO2(HPCD) is a nonthermal technology that can effectively inactivate the vegetative forms of pathogenic and spoilage bacteria, yeasts, and molds at pressures less than 30 MPa and temperatures in the range of 20°C to 40°C. However, HPCD alone at moderate temperatures (20–40°C) is often insufficient to obtain a substantial reduction in bacterial spore counts because their structures are more complex than those of vegetative cells. In this review, we first thoroughly summarized and discussed the inactivation effect of HPCD treatment on bacterial spores. We then presented and discussed the kinetics by which bacterial spores are inactivated by HPCD treatment. We also summarized hypotheses drawn by different researchers to explain the mechanisms of spore inactivation by HPCD treatment. We then summarized the current research status and future challenges of spore inactivation by HPCD treatment. [ABSTRACT FROM PUBLISHER]

The objective of this study was to investigate the inactivation of the Bacillus subtilis spores by high pressure CO 2 combined with high temperature (HPCD + HT) and to analyze the clumping effect of the spores on their HPCD + HT resistance. The spores of B . subtilis were subjected to heat at 0.1 MPa and HPCD at 6.5–25 MPa, and 82 °C, 86 °C, and 91 °C for 0–120 min. The spores were effectively inactivated by HPCD + HT, but a protective effect on the spores was also found, which was closely correlated to the pressure, temperature and time. The spores treated by HPCD + HT at 6.5 and 10 MPa exhibited a two-stage inactivation curve of shoulder and log-linear regions whereas the spores at 15–25 MPa exhibited a three-stage inactivation curve of shoulder, log-linear and tailing regions, and these curves were well fitted to the Geeraerd model. Approximately 90% of pyridine-2,6-dicarboxylic acid (DPA) was released after HPCD + HT and the 90% DPA release time depend on the pressure and temperature. Moreover, the spore clumping in suspensions was examined by dynamic light scattering. The particle size of the spore suspensions increased with the increase of pressure, temperature and time, indicating the spore clumping. 0.1% Tween 80 as a surfactant inhibited the spore clumping and increased the inactivation ratio of the spores by HPCD + HT. These results indicated that the spore clumping enhanced the spores' resistance to HPCD + HT and induced a protective effect. [ABSTRACT FROM AUTHOR]

[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] • Pressure resistant, sublethal injury and viable but non-culturable cells existed during the HPP treatment. • The pressure resistant property of different species was positively correlated with the sublethal injury cell count. • The pressure resistant property of different species was negatively correlated with the VBNC cell count. • The combination of HPP with CO 2 or Nisin could reduce the formation of pressure resistant, sublethal injury and VBNC cells. This study aimed to analyze and reduce the pressure resistance (PR), sublethal injury (SLI), and viable but non-culturable (VBNC) populations during HPP. Escherichia coli , Staphylococcus aureus , Bacillus amyloliquefaciens and Lactiplantibacillus plantarum were selected for evaluation of PR, SLI and VBNC cell counts and proportions during HPP. The results revealed that the bactericidal efficiency against these strains gradually improved as the processing pressure increased. However, viable bacteria could still be detected, suggesting that there may involve the presence of resistant population that difficult to be killed or revived from SLI. Further detecting the quantity and proportion of PR, SLI and VBNC bacteria found that these state of cells were present during whole HPP treatment. Additionally, the more resistant a bacterial species was to high pressure, the fewer SLI and more resuscitable VBNC (RVBNC) populations it generated, and vice versa. Therefore, correlation analysis was also employed to make the relationship between log reduction, SLI and RVBNC population ratios clearer. The results demonstrated that the log reduction was highly positive correlation with SLI population ratios, and negative correlation with RVBNC population within our detected species at 500 MPa. Furthermore, CO 2 and Nisin were employed to combined with HPP to reduce these survivors. Comparing with 233, 218, 241 and 259 MPa for HPP treatment, it took 37, 89, 135 and 229 MPa for HPP + CO 2 , and 189, 161, 199 and 292 MPa for HPP + Nisin to the first decimal reduction for E. coli , S.aureus , B. amyloliquefaciens and L. plantarum , respectively. The results indicated that HPP combined with CO 2 or Nisin could significantly reduce the quantity of PR, SLI, and RVBNC cells during HPP, and provide better bactericidal effects. In conclusion, we quantified the presence of PR, SLI, and VBNC bacteria after high pressure treatment and investigate the effectiveness of HPP combined with CO 2 or Nisin to enhance the inactivation of bacteria and reduce the occurrence of PR, SLI, and RVBNC bacteria. [ABSTRACT FROM AUTHOR]

During the pandemic of coronavirus disease 2019, the fact that frozen foods can carry the relevant virus raises concerns about the microbial safety of cold-chain foods. As a non-thermal processing technology, high pressure carbon dioxide (HPCD) is a potential method to reduce microbial load on cold-chain foods. In this study, we explored the microbial inactivation of low temperature (5-10 °C) HPCD (LT-HPCD) and evaluated its effect on the quality of prawn during freeze-chilled and frozen storage. LT-HPCD treatment at 6.5 MPa and 10 °C for 15 min could effectively inactivate E. coli (99.45%) and S. aureus (94.6%) suspended in 0.85% NaCl, SARS-CoV-2 Spike pseudovirus (>99%) and human coronavirus 229E (hCoV-229E) (>1-log virus tilter reduction) suspended in DMEM medium. The inactivation effect of LT-HPCD was weakened but still significant when the microorganisms were inoculated on the surface of food or package. LT-HPCD treatment at 6.5 MPa and 10 °C for 15 min achieved about 60% inactivation of total aerobic count while could maintain frozen state and quality of prawn. Moreover, LT-HPCD treated prawn exhibited significant slower microbial proliferation and no occurrence of melanosis compared with the untreated samples during chilled storage. A comprehensive quality investigation indicated that LT-HPCD treatment could maintain the color, texture and sensory of prawn during chilled or frozen storage. Consequently, LT-HPCD could improve the microbial safety of frozen prawn while maintaining its original quality, and could be a potential method for food industry to improve the microbial safety of cold-chain foods. • Low temperature HPCD (LT-HPCD) can achieve certain microbial inactivation. • LT-HPCD can maintain the frozen state and the quality of frozen prawn. • LT-HPCD inhibits microbial proliferation and melanosis of prawn during chill storage. [ABSTRACT FROM AUTHOR]

Under stressful conditions, bacteria can enter viable but non-culturable (VBNC) state to survive. VBNC cells lost ability to grow on routine culture medium but are still alive and may revive in suitable conditions. The revived cells can consume nutrients or produce toxins, leading to food spoilage or human illness, posing great risk to food safety and public health. Previously, we have reported that high pressure carbon dioxide (HPCD), an environment-friendly sterilization technology, can induce VBNC formation. However, the underlying mechanism is unclear. By performing a comprehensive transcriptomic analysis, we revealed that HPCD initiated high expression of asr , encoding an acid shock protein, could promote VBNC formation of E. coli O157:H7. Quantitative reverse transcription PCR analysis suggested that high expression of asr (i) inhibited acid resistance (AR) systems, resulting in endogenous proton accumulation; (ii) inhibited hchA expression, a protein stabilizing factor. The two effects resulted in endogenous protein aggregation, which was highly correlated to VBNC formation. Accordingly, HPCD-stressed cells exhibited decreased efficiency of electron transfer chain and ATP production, which was also contributory to cytoplasmic protein aggregation. Taken together, HPCD-initiated high expression of Asr coupled with decreased ATP production led to protein aggregation, finally promoted the cells to enter VBNC state. • Molecular responses of E. coli O157:H7 to HPCD were investigated to explore crucial factors regulating VBNC formation. • Acid shock protein Asr was found to be involved in regulating HPCD induced VBNC formation. • Asr inhibited AR systems / protein stabilizer HchA to facilitate endogenous protein aggregation to promote VBNC formation. [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]