High pressure carbon dioxide process conditions: comparisons and some disparities in food processing

Due to the growing and intense demand of the consumer for high quality food, in the last years, an amount of research has been focused on minimizing nutritional losses and remaining the fresh-like feature, often affected by conventional thermal treatment. Among the emerging technologies, high-pressure carbon dioxide (HP-CO 2 ) has shown efficient results for food stabilization. Some disparities regarding the effect of process parameters in the treatment of foods with HP-CO 2 were observed in the literature. Thus, in this work, the data found in a bibliographic survey were organized into the five most discussed in topics the consulted works: the thermodynamic state, temperature, pressure, CO 2 ratio, and treatment time. In general, it was observed that enzymes and microorganisms have different resistance to process parameters, which can vary even longer with the change of environment components, the type of microorganism, or enzyme source. Therefore, results observed in a given food matrix may be different if the same treatment is applied to another different matrix.


Introduction
Heat treatment for food preservation represents a set of well-established techniques used by the food industry to avoid changes in food and ensure its quality at the time of consumption (Zhang et al., 2018). However, although conventional heat treatments are effective in their role of inhibiting enzymatic and microbiological activity, they are often associated with changes in the nutritional and organoleptic quality of food (Smigic et al., 2019). Then, due to the increasingly intense consumer demand for high-quality foods with a satisfactory shelf-life, in the last fifty years, research has been developed to minimize nutritional losses and maintain the fresh-like characteristic (Wang et al., 2020;Silva et al., 2020a;Yu et al., 2020). In this way, non-thermal processing technologies, such as High-Pressure Carbon Dioxide (from now on HP-CO2), High Hydrostatic Pressure (HHP), pulsed electric fields, UV-light, and ultrasound, have gained increasing credibility in the food sector (Zhang et al., 2018;Silva et al., 2020b;Roobab et al., 2021;Alvarenga et al., 2021).
In the 20's, the possible bactericidal effects of carbon dioxide (from now on CO2) were already being discussed (Valley & Rettger, 1927) and later, in the 50's, Fraser (1951) observed the inactivation effect of pressurized CO2 on Escherichia coli. Since then, the interest in these types of investigation as a possible alternative for microbiological inactivation to replace thermal pasteurization has grown considerably (Yu et al., 2020;Rao et al., 2015).
More recently, many authors have been devoted to understanding the mechanism of CO2 inactivation and the real influence of treatment conditions (Fleury et al., 2018;Martín-Muñoz et al., 2022;Marszałek et al., 2019). In general, the bactericidal effect of the CO2 can be attributed to some factors, such as damage to the phospholipid layer and, consequently, to the cell membrane; the fluidity and permeability of the membrane increased by hydrophobic interactions; the dissociation of carbonic acid in water and in the cytoplasm which reduces extracellular and intracellular pH (Yang et al., 2022), etc. In the case of enzymatic inactivation, it can also be attributed to a few reasons: in common with microorganisms, the acidification of the medium; the conformational changes of the enzyme caused by the process; and the inhibitory effect of molecular CO2 on its activity (Hu et al., 2013).
In non-thermal pasteurization technology with HP-CO2, the food is subjected to contact with pressurized CO2, either in the subcritical or supercritical phase, for a sufficient time for the necessary inactivation. This process can occur in batch mode, pseudo-continuous mode, or a continuous flow, depending on its specificities (Garcia-Gonzalez et al., 2007).
Often, beyond being pressurized, CO2 is elevated to conditions above its critical point and has characteristics typical of supercritical carbon dioxide (from now on SC-CO2). In addition to SC-CO2 having its conditions relatively accessible for the industrial context, with 7.38 MPa and 32.2 °C, it is inert, GRAS (generally recognized as safe), found naturally in the atmosphere, is not flammable, toxic, or corrosive and is easily removed by depressurization or degassing (Silva et al., 2020;Hu et al., 2013). Furthermore, when compared to other substances, it showed satisfactory performance as a pasteurizing agent (Enomoto et al., 1997;Gasperi et al., 2009). Due to the properties of CO2, the SC-CO2 treatment can be developed under mild conditions of temperature and pressure, so its effects tend to be less aggressive for substances of interest in the food (e.g. vitamins, antioxidants, flavonoids) (Silva et al., 2018). Due to these positive features, it has been investigated to produce higher sensory and nutritional quality foods (Porębska et al., 2017;Manzocco et al., 2017).
In the last years, the work by Garcia-Gonzalez et al. (2007) discussed some process conditions that still have no role and influence completely elucidated, such as: the influence of pressure and decompression ratio and the proportion of CO2 in the treatment. After a bibliographic survey of recent literature, it was observed that some process conditions with HP-CO2 in different studies exert different influences, sometimes not following a trend. Motivated by this scenario, this work aims to Research, Society andDevelopment, v. 11, n. 12, e23111234068, 2022 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v11i12.34068 3 discuss the current data made available regarding the most studied parameters with HP-CO2, they are temperature; pressure and depressurization ratio; CO2 ratio; exposure time. In this sense, the similarities and differences of the results obtained were shown in a discussion of potential interest to the industry and food researchers.

Methodology
This work was carried out on the systematic literature review context, as described by Donato & Donato (2019).
Complementarily, the publication by Smigic et al. (2019), a review in the specific area of this research, which also served as a model. After an adaptation of the methodology described by both references, the methodology of this work consists of the following six steps: 3. Bibliographic researchfind papers: Only research articles and literature reviews were included, preferably from the last five years (2017)(2018)(2019)(2020)(2021)(2022). For the search, the following terms were used: "supercritical carbon dioxide", "high-pressure carbon dioxide", combined with the term's "pasteurization", "food" and "inactivation". From the results found, papers were selected which discussed the treatment with HP-CO2 for food stabilization.
4. Inclusion and exclusion criteria: As exclusion criteria, works regarding the treatment of HP-CO2 for drying or compound extraction were not considered. 5. Assessment of the quality of the studies and data extraction: Based on the criteria of steps so far, the papers were read to select only the works that discussed the treatment with HP-CO2 for food stabilization. To discuss the results regarding trends and disparities, 22 recent articles dealing with process conditions were selected.
6. Data synthesis and quality of evidence assessment: The data found were grouped by similarities in relation to the four main parameters of the HP-CO2 process (temperature; pressure and depressurization ratio; CO2 ratio; treatment time) and gathered in two tables (Table 2 -enzymatic inactivation; Table 3 -microbiological inactivation) for discussion and more effective visualization of results.

Results and Discussion
After the initial search for the works, Table 1 presents the research corpus, which allows the visualization of the results found in total, by date, and after selection and exclusion. Research, Society andDevelopment, v. 11, n. 12, e23111234068, 2022 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v11i12.34068 4 This section will be discussed the disparities found in the five selected parameters. From the data collected in this bibliographic survey, it was observed that about a third of the studies considered that the temperature parameter influenced the results of stabilization by HP-CO2. This proportion was very similar for the pressure condition, followed by the treatment time, while among the four parameters discussed here, the CO2 ratio was the least discussed in the literature (around 10 % of papers). The discussion regarding the data from the 22 works used for this summary will be started by the section that discusses the CO2 thermodynamic state, which provides a brief discussion of the properties that can influence the effectiveness of the inactivation process. The following one will be discussed the temperature, pressure, CO2 ratio, and time influence.
In order to provide an overview of the reviewed works, two tables were also prepared, which are presented below. Table 2 shows the main parameters and results of enzyme inactivation in different matrices. Table 3 presents the parameters and results of the treatment with HP-CO2, in which SC-CO2 is included, for microbial inactivation in different matrices.
Research, Society and Development, v. 11, n. 12, e23111234068, 2022 (CC BY 4.0)    With the data presented in this way, it is possible to make comparisons and observe disparities. For example, while temperature possibly contributes to the inactivation of the researched enzymes and presented in Table 2, with the researched microorganisms (Table 3) the effects are more variable according to other process parameters, such as pressure, pH or treatment time.
It is important to note that the enzymes and investigated microorganism varied, despite the fact that polyphenol oxidase has been investigated in several works, just as Escherichia coli was a frequently researched microorganism. Although there are several works with the same objective, establishing comparisons is a challenge, because even in studies that used the same matrix, small variations were able to generate different results. In addition to the comparisons, the table systematizes the data to give an overview of the main results of works involving this topic.

CO2 thermodynamic state
Herein will be discussed results that show that the properties (e.g., diffusivity and solubility) linked to the CO2 thermodynamic state can influence the yields of the inactivation process. Although several results go towards the same trends, some disparities are found and will be pointed out below. Some authors realized that the properties of CO2 physical, physical-chemistry and thermophysical), in addition to other parameters, also influence the effectiveness of the treatment (Marszałek et al., 2019;Gasperi et al., 2009).
The SC-CO2, like other substances in the same state, behaves between liquid and gas. It is dense like a liquid and viscous like a gas, lacking surface tension and its diffusion coefficient is intermediate between the two phases (Amaral et al., 2017). In this sense, the combination of these properties has been widely explored for it provides good penetration into biological structures such as membranes and cell walls (Wang et al., 2020;Chen et al., 2022;Barbosa et al., 2020;Soares et al., 2020;Soares et al., 2019). On the other hand, Kincal et al. (2006) compared the inactivation of Escherichia coli, Salmonella typhimurium, and Listeria monocytogenes with subcritical and supercritical CO2 in orange juice varying the temperature from 25 (below the critical point) to 34.5 °C (above the critical point) and found that the addition of heat (consequently the change to supercritical phase) was not the determining factor for microbial reduction in this system being, therefore, a disparity in relation the recent results mentioned.

Temperature
Temperature is another important factor and one of the most cited in HP-CO2 and SC-CO2 pasteurization processes literature. It´s worth the comment that the temperature of the process is one of the more cited parameters by literature. Was already been reported that higher temperatures can stimulate inactivation due to increased solution vapor pressure, increased CO2 diffusivity that becomes easier the penetration and increased cell membrane fluidity (Buszewski et al., 2021;Fleury et al., 2018;Perrut, 2012;Spilimbergo & Bertucco, 2003). However, it is important to note that the  Tables 2 and 3, the temperature range generally used for the inactivation of enzymes and microorganisms is 25 to 55 °C, that in turn is below the temperature value that characterizes a heat treatment (<60 °C) (Illera et al., 2018) and it is lower than what often occurs in other treatments such as ultrasound or microwaves (Gallo et al., 2018;Kutlu et al., 2021).
For enzyme inactivation processes, an efficiency of more than 80% has often been achieved (Manzocco et al., 2017;Illera et al., 2018;Liao et al., 2019). One of the exceptions to this trend was the enzyme polygalacturonase, which presented resistance to treatment with HP-CO2 in tomato juice. The enzyme was treated at 55 °C, 20 MPa for 90 min and had only 59% of its activity reduced (Illera et al., 2018). Likewise, the peroxidase enzyme from apple juice showed more resistance to treatment temperature than polyphenol oxidase (Marszałek et al., 2017). Other studies conducted with microorganisms (see Table 3), such as Escherichia coli, Saccharomyces cerevisiae, and Lactobacillus casei, achieved considerable inactivation -up to 6.93 log by Silva et al. (2018) or 6.89 log by Feng et al. (2022) -that were attributed to the optimization of synergistic effects of temperature (Silva et al., 2018;Fleury et al., 2018, Bourdoux et al., 2022.
There is a disparity found in temperature parameters about the ones mentioned so far. In the treatment of

Pressure and depressurization ratio
The pressure parameter, as well as temperature, is one of the most cited parameters regarding the inactivation process as realized in Tables 2 and 3. High-pressure mechanisms, as in HHP processes, are able of inactivating vegetative cells by damaging the cell membrane or, from a molecular point of view can occur changes in the electrostatic, hydrophobic, and ionic interactions become the functioning of the structures infeasible (Sehrawat et al., 2020). However, the pressures adopted in the HP-CO2 treatment (generally <25 MPa) are considerably lower than those of the processes with HHP (around 300 -600 MPa) (Wang et al., 2020).
Due to the substance features the HP-CO2 technology becomes a low implementation cost alternative when compared to systems operating with other substances in the supercritical phase (Amaral et al., 2017). However, beyond financial concerns, due to the lower intensity of pressures used in HP-CO2 processes, these effects only will be achieved with the synergistic interaction between pressure and CO2 (Fleury et al., 2018), in this sense, only the low pressures cited are not enough to food stabilization.
Regarding the inactivation mechanism, it has already been proposed that cellular infeasibility could be caused by the blast from the decompression and expansion of CO2 in the intracellular environment. However, a higher depressurization rate did not always increase inactivation (Enomoto et al., 1997). Although HP-CO2 can promote cell wall disruption (Fraser, 1951;Nakamura et al., 1994), these effects have not been considered determinants of microbial reduction efficiency (Garcia-Gonzalez et al., 2007).
Analyzing the synergistic effect of process parameters, Illera et al. (2018) noticed an increase in the rate of pectinmethylesterase inactivation with increasing treatment pressure that varying from 8.5 to 20 MPa at 45 °C in tomato juice, while polygalacturonase was more resistant. One similar behavior was also observed in apple juice peroxidase and polyphenol oxidase enzymes that demonstrated sensitivity to pressure, however, peroxidase was more resistant and, therefore, exhibited different inactivation kinetics (Murtaza et al., 2020).
As well as the juices mentioned, mushroom polyphenol oxidase and horseradish peroxidase also had their residual activities reduced with the pressure increased from 10 to 65 MPa (Marszałek et al., 2019). The authors observed that even working at considerably lower pressures, CO2 treatment was much more effective than HHP in inactivating peroxidase (Marszałek et al., 2019). Moreover, pressure and depressurization ratio were the most relevant parameters in the inactivation of Staphylococcus aureus in salmon (Barbosa et al., 2020) and in the microbiological inactivation of Mongolian cheese, which went from 2.03 log at 10 MPa to 6.89 log at 20 MPa (Feng et al., 2022). However, a lower limit for its effect was observed, below 3.5 MPa, Escherichia coli, Saccharomyces cerevisiae, and Enterococcus faecalis appeared insensitive to the HP-CO2 process (Debs-Louka et al., 1999).
In this sense, the pressure proved to be a significant parameter and the processes commented on so far were sensible to its changes. On other hand, opposite trends were found that can be considered disparities. In the SC-CO2 study conducted by Silva et al. (2018), no influence of pressure (10 -20 MPa) was observed on hm the reduction of Lactobacillus casei in apple juice. It was argued that the effects of pressure may be more relevant in processes with HHP.
Similar results by Silva et al. (2018) were obtained by Chen et al. (2022) in their study with almonds. The authors found that increasing pressure from 15 to 20 MPa did not increase the Escherichia coli K12 inactivation. Despite the increase in pressure from 10 to 20 MPa causing an increase in the destruction of species of the genus Legionella in aqueous solution, this behavior was not observed with the variation from 20 to 30 MPa (Martín-Muñoz et al., 2022).
Although pressure influences the increase in solubility, its effect may be limited due to the saturation capacity of CO2 in the medium (Barbosa et al., 2020). It has been observed that the CO2 solubility increases appreciably with increasing pressure up to 10 MPa. Above that, there is a trend towards stabilization in which a slight increase in solubility can mean considerable additional costs with increasing pressure (Illera et al., 2019;Spilimbergo et al., 2005). In addition to the interference of the saturation of the medium, microorganisms may have different susceptibilities to pressure. Of those studied by Debs-Louka et al. (1999), Escherichia coli was the most susceptible, followed by Saccharomyces cerevisiae, while Enterococcus faecalis suffered only slightly from this effect.

CO2 ratio influence
The role of CO2 as a pasteurizing agent is related to its diffusivity through the food cellular material (Silva et al., 2020). Knowing its solubility and diffusivity in the matrices and working conditions provides an assertive choice of the necessary amount to be used to guarantee an efficient process. Matrices with high lipid content may induce a low inactivation via HP-CO2 due to their lipophilic behavior, which tends to reduce their action on the phospholipid bilayer in cells (Garcia-Research, Society andDevelopment, v. 11, n. 12, e23111234068, 2022 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v11i12.34068 In general, the CO2 solubility showed a strong dependence on the sugar content of the matrix (Illera et al., 2019;Ferrentino et al., 2010) while occurred a slight variation with citric acid content as noticed by Illera et al. (2019). Thus, even in treatments with the same conditions in a matrix (apple juice) with different soluble solids content (see Table 3), the inactivation efficiency can be greatly impacted due to the CO2 solubilization capacity (Porębska et al., 2017).
The majority of the works presented in Tables 2 and 3 do not include the CO2 ratio (mass or volume) in their discussions. However, Silva et al. (2018) observed that the CO2 volumetric ratio was the most significant parameter for the Lactobacillus casei inactivation in apple juice. The authors achieved inactivation of more than 6 log operating at 15 MPa, 55 °C at 70% CO2 volume for 30 min. Due to the synergistic effect of the SC-CO2 ratio, below 30% only a treatment above 55 °C would provide a 6 log reduction. Furthermore, in the enzymatic treatment of polyphenol oxidase conducted by Benito-Román et al. (2019), a significant effect attributed to the CO2: volume ratio was observed, and the authors achieved almost total inactivation by treating the polyphenol oxidase solution with CO2 ratios up to 3 g/mL.
For the treatment of Escherichia coli in pumpkin puree, Santos Júnior et al. (2021) observed that among the volumetric ratios of 1:0.5, 1:0.75, and 1:1 (puree: CO2), the one with the highest CO2 proportion favored the best Escherichia coli inactivation condition. In the study for inactivation of species of the genus Legionella in continuous flow water, total inactivation was achieved at a ratio of 1.0 CO2/suspension flow rate, which was not observed at 0.5 (Martín-Muñoz et al., 2022). Likewise, among the mass ratios 1:0.2; 1:0.6, and 1:1 (human milk: CO2) evaluated, the greatest results for reducing mesophilic aerobic and Escherichia coli amounts were achieved with the highest CO2 content (1:1) (Michelino et al., 2018). In the same trend, Casas et al. (2012) reduced 4 logs of Alicyclobacillus acidoterrestris in apple cream with CO2 at 30 °C and 10 MPa and it was noticed that an increase in inactivation was achieved by doubling the CO2 flow rate to 4g/min. However, the treatment efficiency was only increased when the increase in flow was associated with the increase in turbulence.
Although the commented data so far for this parameter shows it sensible to CO2 ratio, different trends were found in other works. For example, when the Salmon: CO2 mass ratio varied from 1:0.2 to 1:1.0, Barbosa et al. (2020) did not observe a significant increase in the inactivation of Staphylococcus aureus in raw salmon. Likewise, Kincal et al. (2006) did not observe the influence of the CO2 mass ratio: orange juice on the inactivation of Escherichia coli, Salmonella typhimurium, and Listeria monocytogenes. Ceni et al. (2016) observed only a slight difference, from 94.5 to 98.2%, in alkaline phosphatase inactivation with an increasing CO2:milk mass ratio from 0.05 to 0.45 and besides that, was noticed by Ceni et al. (2016) that this same higher ratio caused the milk coagulation due to the higher concentration of carbonic acid in the medium. In this sense, this issue must be taken into account in the treatment of raw materials sensitive to acidic media.

Time treatment influence
Likewise that in the conventional pasteurization treatments, the contact time between CO2 and the cellular material has shown a significant effect on the efficiency result achieved as well (Ceni et al., 2016;Barbosa et al., 2020). A longer process time provides a more efficient diffusion of CO2 in the aqueous medium and, consequently, greater contact with the matrix components and microbiological reduction (Silva et al., 2018). Despite this, in general, it is desired to reduce the process time.
As realized in Tables 2 and 3, approximately 26% of the works considered that the process time influenced the inactivation or directed the endeavor to achieve satisfactory inactivation in a possible shorter time. For this, some studies optimized other parameters so that their synergistic effect resulted in a decrease in the time required (Martín-Muñoz et al., 2022;Ceni et al., 2016;Silva, 2018;Paniagua-Martínez et al., 2018).
Was realized in the last paragraph that quite often it's expected that the achieved inactivation increases as much as the process time increases as well. On the other hand, in the results found in this review, process times for HP-CO2 treatments vary widely, from some minutes to a few hours (Benito-Román et al., 2019;Paniagua-Martinez et al., 2018;Castillo-Zamudio et al., 2021;Santos-Júnior et al., 2021;Barbosa et al., 2020). However, as seen in Tables 2 and 3, most of the works achieve their greatest results in around 20 and 30 min (Rao et al., 2016;Sikin et al., 2016;Silva et al., 2018;Marszałek et al., 2019;Murtaza et al., 2019;Bourdoux et al., 2022;Chen et al., 2022).
Even though the process larger time provides the greatest contact between the CO2 and the matrix compounds, ultrasound-assisted HP-CO2 studies achieved effective inactivation of Escherichia coli with treatment times of 3 and 5 minutes (Paniagua-Martinez et al., 2018;Castillo-Zamudio et al., 2021). Chen et al. (2022) conducted their study with SC-CO2 in almonds and noticed an increase in Escherichia coli inactivation with processing time as well. However, the authors observed a peak behavior in the results and after that, at 45 min, no increase in log reduction was noticed. It has been suggested that, in this case, the CO2 inactivation capacity was complete by that time (Chen et al., 2022). Similar behaviors were also noticed by Fleury et al. (2018) and Santos Júnior et al. (2021). Manzocco et al. (2017) achieved to reduce time for polyphenol oxidase inactivation in apple juice with increasing temperature and pressure up to 35 °C and 12 MPa, from which, the trend of time reduction did not remain. Although not a food study, in some of the trials by Hossain et al. (2015), the relevance of the synergistic effect of process time was suppressed by pressure and temperature parameters in the treatment of Enterococcus faecalis and Escherichia coli in clinical solid waste.
Therefore, some results showed that the action of time can be limited or even suppressed by the influence of other parameters.

Final Considerations
Based on this discussion, it is possible to understand that process parameters have different effects in different situations. The temperature, even if below the value that characterizes a heat treatment, in most of the studies presented in this research, this parameter played a meaningful role in food preservation. This is possibly due to the increase in fluid diffusivity and the stimulation of permeability of membranes and other structures, although there are a few cases where this parameter does not influence the process yields.
The pressure was also one of the parameters most cited as significant in this process and its effects were relevant in the stabilization of solid and non-solid foods. It induces the solubilization of CO2 until its saturation in the medium and was a meaningful parameter in some works, although in some others with HP-CO2, slight variations (up to 10 MPa) have not increased significantly in the treatment of microorganisms.
In most of the data discussed in this work, assays with a higher CO2 ratio were better in terms of efficiency, when compared to the same treatments with smaller amounts. Despite this, around only 10 % of the studies mentioned its effects. It is possible that the amount (or ratio) of CO2 used in the treatment is not directly related to its solubility in the matrix. In this case, to become the process more affordable, the choice of treatment ratio must also be evaluated in terms of its solubility.
Despite large variations, most of the studies reported the variation from 20 to 30 min as the needed time for their greatest inactivation. It is possible that this time can be reduced if HP-CO2 is combined with other technologies such as ultrasound.
In order to make clear comparisons and conclude HP-CO2 treatment is still a challenge, as different equipment and process parameters have been used in different studies. As in other conventional heat treatments, in general, it was observed that enzymes and microorganisms present different resistances to the HP-CO2 processes parameters which can vary even more with the change of the components of the matrix and the source of the microorganism or enzyme. Therefore, results observed in a given food matrix may be observed differently with the same treatment applied to another matrix. Furthermore, the HP-CO2 treatment has shown to be efficient for the inactivation of enzymes and microorganisms in a series of works, showing itself as a technology with potential applicability for the stabilization of foods without heat treatment.
In this sense, some disparities occurs in the main parameters of the inactivation process being more drastic or not that in turn must be assessed depending on the case. Therefore, further research is essential to demonstrate and explain the effects and the overall expected results for of each one of HP-CO2 process parameters and variables to food stabilization.