High Pressure Carbon Dioxide Treatment Research Articles

Widely targeted metabolomics analysis reveals the potential metabolic network of high-pressure carbon dioxide regulating fresh-cut Chinese water chestnut yellowing

In this study, we applied widely targeted metabolomics and physiological analysis to investigate the non-volatile metabolites changes and regulatory mechanisms of high-pressure carbon dioxide (HPCD) treatment on the yellowing of fresh-cut Chinese water chestnut (CWC). A total of 349 non-volatile metabolites were identified and subjected to principal component analysis (PCA), OPLS-DA and KEGG enrichment analysis. The results showed that the yellowing of fresh-cut CWC was related to membrane lipid metabolism and flavonoid biosynthesis. Damage stress induced membrane lipid metabolism and flavonoid biosynthesis in surface tissues, and the increase of phenylalanine ammonia-lyase (PAL) activity in flavonoid biosynthesis might be triggered by signaling molecules from membrane lipid metabolism. On the one hand, HPCD treatment could directly inhibit the activity of PAL, and reduce the biosynthesis of flavonoids in the damaged tissues. On the other hand, HPCD could indirectly inhibit the generation and accumulation of potential signaling molecules by regulating membrane lipid metabolism, thus preventing signaling molecules from triggering flavonoid biosynthesis. This study proposed a potential network of HPCD regulating the yellowing of fresh-cut CWC and established the foundation for further control of yellowing in fresh-cut CWC.

Molecular dynamics simulation of interaction between high pressure carbon dioxide (HPCD) and phenylalanine ammonia-lyase (PAL)

High-pressure carbon dioxide (HPCD) could effectively suppress the yellowing phenomenon in Chinese water chestnut (CWC) by significantly inhibiting the activity of the key enzyme—phenylalanine ammonia-lyase (PAL). However, the molecular mechanism underlying this inhibition remains unclear. In this study, we investigated the structural changes and inhibition mechanisms of PAL from CWC subjected to HPCD treatment. Homology modeling was used to construct the 3D structure of CWC PAL. The results showed that CWC PAL was highly conserved and had higher α-helix content. Molecular dynamics simulations and molecular docking were utilized to investigate the non-covalent interaction between CO2 and PAL. Our findings revealed that CO2 induced conformational changes in PAL via hydrogen bonds, polar interactions, and hydrophobic interactions, thereby increasing PAL's α-helix structure and preventing the substrate L-Phe from entering its active site. Additionally, HPCD treatment caused the movement of the Phe-111 residue in the inner lid-loop, resulting in steric hindrance and destruction of the active center of PAL. These results provide a theoretical basis for expanding the application of HPCD in the food processing industry.

Effect of preliminary stresses on the induction of viable but non-culturable Escherichia coli O157:H7 NCTC 12900 and Staphylococcus aureus ATCC 6538

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.

Transcriptome Analysis of the Influence of High-Pressure Carbon Dioxide on Saccharomyces cerevisiae under Sub-Lethal Condition.

High-pressure carbon dioxide (HPCD), a novel non-thermal pasteurization technology, has attracted the attention of scientists due to its high pasteurization efficiency at a lower temperature and pressure. However, the inactivation mechanism has not been well researched, and this has hindered its commercial application. In this work, we used a sub-lethal HPCD condition (4.0 MPa, 30 °C) and a recovery condition (30 °C) to repair the damaged cells. Transcriptome analysis was performed by using RNA sequencing and gene ontology analysis to investigate the detailed lethal mechanism caused by HPCD treatment. RT-qPCR analysis was conducted for certain upregulated genes, and the influence of HPCD on protoplasts and single-gene deletion strains was investigated. Six major categories of upregulated genes were identified, including genes associated with the pentose phosphate pathway (oxidative phase), cell wall organization or biogenesis, glutathione metabolism, protein refolding, phosphatidylcholine biosynthesis, and AdoMet synthesis, which are all considered to be associated with cell death induced by HPCD. The inactivation or structure alteration of YNL194Cp in the organelle membrane is considered the critical reason for cell death. We believe this work contributes to elucidating the cell-death mechanism and providing a direction for further research on non-thermal HPCD sterilization technology.

High-pressure carbon dioxide treatment and vacuum packaging alleviate the yellowing of peeled Chinese water chestnut (Eleocharis tuberosa)

This work evaluated the effects of high-pressure carbon dioxide (HPCD) treatment and vacuum packaging on the yellowing of peeled Chinese water chestnut (CWC) during storage. The changes in color, enzymatic activities, total phenolic content and malondialdehyde contents were assessed in peeled CWC. The results showed that vacuum packaging alone, atmospheric packaging after HPCD treatment, and vacuum packaging after HPCD treatment (HPCD+VP) could significantly delay the yellowing of fresh-cut CWC to varying degrees. The total phenolic content was lower (68%) in HPCD+VP samples than that of control. Compared to control, the enzymatic activities such as phenylalanine ammonia-lyase, polyphenol oxidase and lipoxygenase decreased by 76% (12th day), 24% (6th day) and 19% (6th day) respectively in HPCD+VP samples. HPCD and vacuum packaging could efficiently inhibit several enzymes activities. Our study exhibited that HPCD+VP treatment delayed the yellowing of fresh-cut CWC by reducing the phenylpropanoid metabolism pathway activity and cell membrane peroxidation.

High-pressure carbon dioxide treatment alleviates browning development by regulating membrane lipid metabolism in fresh-cut lettuce

The influence of high-pressure carbon dioxide (HPCD) treatment on the browning of fresh-cut lettuce was investigated during storage at 4 °C. The browning degree, color, weight loss, browning-related enzymes, and components related to membrane lipid metabolism of fresh-cut lettuce were assessed during storage. The results showed that HPCD treatment slowed down the browning of fresh-cut lettuce. Polyphenol oxidase activity and total phenolic content of HPCD-treated group were reduced by 53.50% and 74.63%, respectively. In addition, HPCD treatment inhibited lipoxygenase activity and delayed membrane lipid peroxidation, thus decreasing PAL's stimulation by membrane lipid peroxidation products and further reducing the synthesis of phenolic. On the other hand, HPCD treatment maintained the structural integrity of the cell membrane and prevented the cell membrane compartmentalization from being broken due to cell membrane breakage by inhibiting phospholipase D activity, which avoided the contact between enzyme and phenol and delayed the oxidative browning.

The effect of high pressure carbon dioxide on the inactivation kinetics and structural alteration of phenylalanine ammonia-lyase from Chinese water chestnut: An investigation using multi-spectroscopy and molecular docking methods

The effect of high pressure carbon dioxide (HPCD) on the inactivation kinetic and structure of phenylalanine ammonia-lyase (PAL) from Chinese water chestnut (CWC) was studied in this paper. The inactivation kinetic of PAL treated by HPCD (1–4 MPa, 35–55 °C, and 5–60 min) were determined and fitted to the first order kinetics model with calculating kinetic parameters. As revealed by the circular dichroism spectral, the α-helix and β-turn content in secondary structure increased and the β-sheet content decreased. And the intensity of the fluorescence spectra reflecting tertiary structure decreased, together with the λmax blue-shifted with the increasing pressure. Fluorescence and circular dichroism spectral results indicated that the conformation of PAL was altered by HPCD. The findings of particle size distribution and ζ potential showed that HPCD could cause the aggregates of PAL particles. Moreover, molecular docking indicated the interactions between small molecules (CO2, H2CO3, HCO3−, and CO32−) and PAL might result in a decrease in PAL activity by forming steric hindrance, preventing substrate from binding. Finally, this paper proposed a potential mechanism for inactivation of PAL by HPCD treatment, where the loss in PAL activity was correlated to changes in secondary and tertiary structure of PAL, which was induced by aggregation effect of HPCD.

Extracellular pH decline introduced by high pressure carbon dioxide is a main factor inducing bacteria to enter viable but non-culturable state

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 (pHex3) 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 pHex3 and HP could mimic the effect of HPCD for VBNC induction. Further investigation of the individual effect of pHex3 and HP on VBNC induction revealed that pHex3 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 pHex3 and HPCD had similar morphological changes, and VBNC cells induced by pHex3 and HPCD had similar stress resistance characteristics. These results strongly indicated that pHex3 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-, pHex3- and HPCD-treated cells compared with untreated ones. 59 DEGs shared by pHex3 and HPCD treatment might be responsible for VBNC induction, and they were mainly involved in cellular transport and localization.

Effect of high-pressure carbon dioxide treatment on browning inhibition of fresh-cut Chinese water chestnut (Eleocharis tuberosa): Based on the comparison of damaged tissue and non-damaged tissue

In this paper, the effect of high-pressure carbon dioxide (HPCD) inhibiting the browning of fresh-cut Chinese water chestnut (CWC) was explored by comparing the differences between damaged and non-damaged tissues. The results showed that the browning of fresh-cut CWC could be effectively inhibited by 2 and 4 MPa treatment, and with these conditions, the microbial load was kept at a low and stable level during the whole storage period. In damaged tissues, the phenylalanine ammonia-lyase (PAL) activity was decreased, thus cutting off the accumulation of naringenin and eriodictyol (only slightly detected at 1 MPa). In non-damaged tissues, the activities of polyphenol oxidase (PPO), peroxidase (POD), and PAL were significantly increased to overcome the treatment stress. The structural change of PAL proved its activity reduction. The mechanism of browning inhibition could be explained by direct inactivation of PAL in damaged tissue and indirect regulation of stress resistance response in non-damaged tissue.

High-pressure supercritical carbon dioxide uses to inactivate Escherichia coli in pumpkin puree

Coli ATCC 25922 inactivation was studied to determine the effect of high-pressure carbon dioxide (HPCD) process on pumpkin puree. Experiments were performed using a batch HPCD system at three conditions of pressure (7.5 MPa, 17.5 MPa and 27.5 MPa) at 32 °C. Afterwards, at the best experimental condition (27.5 MPa – 275 bar), a kinetic was performed to assess inactivation of microorganisms over time (from 1 to 8 h). The physicochemical characteristics (pH, total soluble solids – TSS, titratable acidity – TA, total carotenoids, total reducing sugars – TRS, moisture and optical microscopy) of the pumpkin puree were also evaluated. HPCD with acidification increases bacterial efficacy of treatments, as well as significant changes in physicochemical parameters. HPCD treatment reduced the microbial load moderately in all experiments, by a maximum of approximately 3.17 log cycles in 8 h of process at 27.5 MPa (275 bar). Optical microscopy showed no difference in cell wall, just in starch which was expected by cooking.

Study of high pressure carbon dioxide on the physicochemical, interfacial and rheological properties of liquid whole egg

In this research, the impact of High Pressure Carbon Dioxide (HPCD) on the physicochemical, interfacial and rheological properties of liquid whole egg (LWE) was investigated. HPCD treatment increased the solubility, free sulfhydryl groups and surface hydrophobicity of LWE. The highest foaming ability (186%) and foaming stability (72%) were obtained at 60 min of HPCD treatment, which were 1.3-fold and 1.4-fold those of the control group (147% and 51%, respectively). Compared with the control group, the LWE emulsion had a minimum particle size (16.3 μm) when the treatment time was 75 min, and the highest absolute value of zeta potential reached 7.66 mV when the processing time was 60 min. HPCD treatment decreased the apparent viscosity of LWE at a low shear rate. Dynamic viscoelastic characteristics indicated that both the G’ and G’’ moduli of LWE under the HPCD process were lower than those of the control group at any frequency.

Critical damage to the cellular organelles of Saccharomyces cerevisiae under sublethal conditions upon high pressure carbon dioxide treatment

ABSTRACTHigh pressure carbon dioxide treatment is a non-thermal pasteurization technique. However, critical damage, resulting from the treatment, to microbial cells has not been observed directly, and the detailed mechanism of the microbicidal activity is not understood. In this study, we analyzed the damage to Saccharomyces cerevisiae organelles, which were visualized using organelle-specific GFP fusion proteins. Yeast strains were subjected to high pressure carbon dioxide treatments at 30°C and 4.0 MPa for 2–8 h. Reduction in yeast cell viability was accompanied by the disruption of endoplasmic reticulum, nuclear membrane, Golgi body, and nucleolus. However, visible damage to the cell membrane was not observed. Fluorescence microscopy was utilized to confirm that high pressure carbon dioxide treatment damaged membranes of major organelles, but not the cell membrane.

Effect of high pressure carbon dioxide on alkaline phosphatase activity and quality characteristics of raw bovine milk

Abstract Novel food processing techniques would always be pursuits of researchers and food industry to avoid unfavorable thermal effects, especially in dairy and milk processing. In this study, effects of high pressure carbon dioxide (HPCD) on the activity of alkaline phosphatase (ALP) and main quality indices of raw bovine milk at 20 MPa using a batch system were investigated. A complete inactivation of ALP activity as exposure to HPCD treatment at 50 °C and 20 MPa for 50 min was observed. The protein and lactose content of HPCD-treated bovine milk hold steady, while pH value and total solids content decreased, turbidity and average particle size increased significantly (p

Polyphenol oxidase (PPO) and pectin methylesterase (PME) inactivation by high pressure carbon dioxide (HPCD) and its applicability to liquid and solid natural products

In the present work, High Pressure Carbon Dioxide (HPCD) has been used to inactivate the enzymes Polyphenol Oxidase (PPO) and Pectin Methylesterase (PME). The work has been done in two stages: in the first one, fundamental knowledge about the inactivation mechanism induced by HPCD was obtained using pure commercial enzymes. The influence of the main process parameters was studied: pressure, temperature, time and ratio CO2/volume of enzymatic solution. The changes in the tertiary structure of the enzymes after the HPCD treatment were analyzed by fluorescence spectroscopy running two different tests: intrinsic fluorescence measurement and ANS binding. In the second stage, the acquired knowledge was used to study the inactivation pattern of these two enzymes in liquid and solid natural products, including fruit and vegetable juices or shrimps. The results revealed differences in the inactivation pattern of the enzymes in these substrates compared to the pure commercial enzymes, but the applicability of HPCD technology for the inactivation of enzymes in real substrates at industrial scale was also revealed.

Pectin methylesterase inactivation by High Pressure Carbon Dioxide (HPCD)

In the present work the effect of High Pressure Carbon Dioxide (HPCD) on the activity and structure of a commercial pectin methylesterase (PME) was studied and the results were compared to the conventional thermal inactivation technology. The use of supercritical CO2 (pressure 6–18 MPa, temperature 40–55 °C and time up to 75 min) increased dramatically the PME inactivation rate. The experimental data fitted the first order model and the inactivation kinetic study of PME was completed with the calculations of the activation energy and volume of activation.HPCD treatment induced important changes in the tertiary structure of the enzyme, as revealed the intrinsic fluorescence, KI quenching and ANS binding analyses. They showed that HPCD treatment rearranged the structure of the enzyme. These changes induced by HPCD were significantly different from those induced by mild thermal treatment. Finally, the structural changes detected correlated with the enzyme activity losses observed.

Inactivation, Aggregation and Conformational Changes of Polyphenol Oxidase from Quince (Cydonia oblonga Miller) Juice Subjected to Thermal and High-Pressure Carbon Dioxide Treatment.

Polyphenol oxidase (PPO) causes the browning reaction in fruits and vegetables and deteriorates the quality. Thermal treatment for enzyme inactivation may result in defects as opposed to high pressure CO2 (HPCD) processing. In this study, the changes in activity, dissociation, aggregation and conformation of purified PPO from thermal and HPCD treated juice were investigated. HPCD exhibited inactivation of PPO at 55–65 °C whereas thermal processing alone at the same temperature resulted in PPO still showing activity. Under thermal treatment at 25 and 65 °C, the browning degree was higher (0.39 and 0.24) than for HPCD-treated juice (0.23 and 0.12). Fluorescence and circular dichroism spectral results indicated that HPCD induced large decreases in intensities, revealing a rearrangement of the secondary structure and destruction of the native configuration of the PPO molecule. The particle size distribution (PSD) pattern revealed structural modification leading to initial dissociation and subsequent aggregation of PPO after HPCD treatment. Polyacrylamide gel electrophoresis (PAGE) analysis exhibited that molecular size of protein was 40 kDa. In conclusion, the HPCD method was found to be more effective than thermal treatment to inactivate PPO. Structural modifications provided better insights into the phenomena of activation and inactivation of PPO.

Effect of high pressure carbon dioxide on tomato juice: Inactivation kinetics of pectin methylesterase and polygalacturonase and determination of other quality parameters

Tomato juice, Lycopersicon esculentum cv. Canario, has been treated by high pressure carbon dioxide (HPCD) as non-thermal preservation treatment. The inactivation kinetics for pectinmethylesterase (PME) and polygalacturonase (PG) were determined at different pressures (8.5–20 MPa) and temperatures (35–55 °C). At the highest operating pressure and temperature essayed in this work, it was found that PME could be almost completely inactivated, whereas PG resulted to be more HPCD resistant at the working conditions. PME enzyme inactivation curves were properly described by a Weibull type model, while the fractional conversion model was the most appropriate for the PG with a sharp initial decrease in activity. On the contrary, high pressure processing (HPP) led to a nearly complete inactivation of PG while PME was very resistant at 600 MPa. It was also found that HPCD treatment led to a smaller particle size distribution of tomato juice.

Inactivation model and risk-analysis design for apple juice processing by high-pressure CO2.

Sigmoidal microbial survival curves are observed in high-pressure carbon dioxide (HPCD) pasteurization treatments. The objectives of this study were to use the Gompertz primary model to describe the inactivation in apple juice of the pathogen Escherichia coli CGMCC1.90 and to apply probabilistic engineering to select HPCD treatments meeting at least 5 log10 reductions (SV≥5) at 95% confidence. This required secondary models for the temperature (T, °C) and pressure (P, MPa) dependence of the Gompertz model parameters. The expressions [Formula: see text] and [Formula: see text] selected using goodness-of-fit measures and assessments based on Akaike and Bayesian information criteria were consistent with proposed mechanistic models for HPCD bactericidal effects. Monte Carlo simulations accounting for the variability and uncertainty of the parameter b and c estimates were used to predict SV values for a given time, temperature and CO2 pressure combination and desired confidence boundary. A similar approach used to estimate process times meeting SV≥5 at 95% confidence for a given temperature and CO2 pressure combination, showed that HPCD processes met this requirement only for relatively long processing times, i.e., 35-124min in the experimental range of 32-42°C and 10-30MPa. Therefore, further HPCD research is required to reduce processing time.

Evaluation of HPCD batch treatments on enzyme inactivation kinetics and selected quality characteristics of cloudy juice from Golden delicious apples

Cloudy apple juice has been treated by high pressure carbon dioxide (HPCD) as non-thermal technology to inactive polyphenoloxidase and pectinmethylesterase in batch mode. Stirring speed (from 200 to 600 rpm) induced an increase in the enzyme inactivation rate while a triple cycle of pressurization/depressurization led to the same enzyme inactivation efficiency. Enzyme inactivation kinetics were determined at different temperatures (from 35 to 45 °C) and pressures (from 10 to 20 MPa). Data were described by the first order kinetic model and the Weibull model. For the first order kinetic model, decimal reduction time for HPCD treatment was found to be smaller than for mild heating, in the same temperature range. The same tendency was observed for the first decimal reduction time in the Weibull model. HPCD treatment resulted in a homogenization effect reflected in the shifting of the particle size distribution towards smaller diameters after treatment. HPCD treatment did not result in a change of water and oxalate soluble pectin content, total phenolic compounds and hidroxymethylfurfural content.

Impact of high-pressure carbon dioxide on polyphenoloxidase activity and stability of fresh apple juice

Freshly-extracted apple juice was exposed to high pressure carbon dioxide (HP-CO2) treatment at 20, 35 and 45 °C at different pressure conditions (6.0, 12.0 and 18.0 MPa) for up to 30 min. Samples were analysed for residual enzymatic activity. The time needed for 90% enzyme inactivation (Dp) decreased when CO2 pressure increased, while the CO2 pressure sensitivity of the enzyme (zp) showed no variation with temperature. The HP-CO2 treatment at 12 MPa and 35 °C allowed the minimum residual enzyme activity (20%) to be reached in 10 min. Samples treated under these conditions showed lower polyphenoloxidase activity and higher microbial stability than untreated apple juice while presenting a sensory fresh-likelihood higher than thermally pasteurized apple juice.