The Nile tilapia fishes (weight ranging from 100 to 150 g each) were purchased alive from the local market. All fishes were slaughtered, gutted, headed, and cleaned with tap water. These cleaned fishes were cut into two pieces, inserted into a polyethylene plastic pouch (20 × 15 cm), and subsequently placed into the pressure vessel. The plastic pouch tips were folded and unsealed to allow CO₂ exchange as well as prevented any external solid or liquid entering the pouch container.

The pressure vessel was developed in Gadjah Mada University with gross volume of 500 mL. Its lid was equipped with 180 bar analog pressure gauge with five bar resolution. A calibrated digital thermostat (Elitech STC-3000) with a 10 KΩ negative-temperature-coefficient (NTC) thermistor was used to control barrel temperature of the pressure vessel. Food grade CO₂ was supplied into the pressure chamber by inverting the 25 kg-type CO₂ cylinder. This equipment could provide a maximum pressure of 90 bar at 40 °C (CO₂ density = 0.486 g/mL at 40 °C), which was the upper limit of this experiment. The schematic installation of the pressure vessel is shown in Fig. 1.

Figure 1.  

Schematic installation of pressure vessel.

The CO₂ pressure and density affected the reduction of protease activity and microbial loads significantly as shown in Fig. 3 and Fig. 4, respectively. All significant reductions of protease activity and microbial counts were achieved under supercritical phase of CO₂. Subcritical pressure was considered inadequate to reduce both muscle protease activity and raw fish microbial counts significantly. At 70 bar, residual activity of protease was remaining high at 85.47% and total microbial counts reduction was still less than 1-log. Two logs reductions of microbial counts was achieved under 80 bar or more. Therefore, 80 bar was considered as minimum pressure to achieve significant reduction for both protease inactivation and microbial loads of raw fillets.

Figure 3.  

Protease residual activity as a function of CO₂ pressure at 40 °C.

Figure 4.  

Protease residual activity as a function of CO₂ density at 40 °C.

Since the relationship between pressure and density of CO₂ at 40 °C is not linear as shown in Fig. 5⁠ (Lemmon et al. 2020), the density differences among treatments were increased along with pressure increase (Fig. 4). The relationship between CO₂ pressure and protease residual activity was found to follow zero order reaction (Fig. 3) and the relationship between CO₂ density and protease residual activity was found to follow first order reaction (Fig. 4).

Figure 5.  

The relationship between CO₂ pressure and CO₂ density at 40 °C.

As shown in Fig. 6, 70 and 75 bar were found inadequate to make significant effect on reduction of microbial counts. After 90 bar exposure, the reduction of microbial loads was still less than 4-logs. The relationships between CO₂ pressure and CO₂ density with residual microbial counts were found to follow first order reaction, as shown in Fig. 6 and Fig. 7, respectively.

Figure 6.  

Residual microbial counts as a function of CO₂ pressure at 40 °C.

Figure 7.  

Residual microbial counts as a function of CO₂ density at 40 °C.

The relationship between pressure and treated fillet texture was not linear, as shown in Fig. 8. In the subcritical stage of HPCD, the texture of treated samples was softened significantly compared to untreated samples. The texture was improved in the supercritical phase (75-85 bar). However, it was softened again at 90 bar. Ninety bar CO₂ pressure resulted fully cooked appearance of fillet and destroyed texture. Though the texture and visual appearance of 90 bar treated fillet was similar to that of fully cooked fillet, the odor of raw fish was still left.

Figure 8.  

Effect of HPCD (40 °C and 15 min) of hardness of fillet. Bars with the same letter were not significantly different. Vertical lines indicate standard deviation.

In the range from 70 to 90 bar, only 80 and 85 bar treatments could retain texture that were not significantly different than that of untreated samples. All other treatments showed significant softening compared to untreated samples. Hence, 85 bar and 15 min HPCD treatment was considered as maximum level of CO₂ pressure that tilapia fillet could withstand without suffering severe textural degradation.

The HPCD pressure was found to have very significant effect on Lightness values (L*), as shown in Table 1. The values of a* (redness/greenness) and b* (yellowness/blueness) did not show significant differences than that of untreated samples. Higher pressure of applied CO₂ was found to increase the lightness (L*) of fillet while both redness/greenness(a*) and yellowness/blueness (b*) remain unaltered.

For consumers’ perception, color is one of the most important sensory characteristics of fish muscles in determining their purchasing decision (Liu et al. 2013). In HPP processing, possibly one of the most detrimental sensory attributes of HPP fish muscles is its cooked appearance due to the increase in the whiteness or the loss of translucency, of pressurized fish flesh (Matser et al. 2000). Similar to HPP treated fish, the whiteness increase also appeared on HPCD pasteurized Nile tilapia. Discoloration remains a drawback for food processing that using high pressure.

As shown in Table 2, all treated samples suffered weight loss compared to untreated samples. The increase of pressure itself did not result in significant differences among treated samples. The moisture contents of all samples were not significantly difference compared to that of untreated samples. For all three lowest residual protease activity (80, 85, and 90 bar), 80 bar sample resulted the lowest protein and fat contents, and experienced the lowest post-pressurization pH.

Compared to the untreated samples, 80 and 85 bar treatments did not experience hardness change significantly (Fig. 8). The significant differences between 85 bar samples and untreated samples were only color change and fat contents. For 85 and 90 bar treatments, the weight loss was mostly contributed by moisture loss. Meanwhile, weight loss in 80 bar treatment was mostly contributed by solid loss, indicated by higher moisture, lower fat and lower protein contents.

As shown in Fig. 8, there were only texture of 80 and 85 bar treated samples, which were not significantly different than that of untreated samples. Untreated, 80, and 85 bar treated samples were stored at 4 °C for 14 days and recorded their softening progress. The softening development of untreated fillets, 80 bar, and 85 bar of the treated fillets is displayed in Fig. 9. All the untreated samples showed that softening was increased with time, with the exception from 6 to 8 days of storage. Meanwhile, for both 80 and 85 bar, there were some observations, which had the same level of its initial hardness and the rest were softer than their initial hardness. The softening development of pasteurized samples were not as fast as untreated samples. Since each observed sample was from individual fish, it developed independent behavior to each other. For an example, observation on 10^th day of 80 bar treated samples was higher than observation on 8^th day. Both observations were from different fish; therefore, the onset of softening performed by individual sample might be different to each other.

Figure 9.  

The fillet softening development in 14 days storage at 4 °C.

On the final day of observation, untreated samples lost 52.9% of its initial hardness. This finding was in agreement with the softening of climbing perch (Anabas testudineus), which lost 50.1% of its initial hardness after 15 days stored in ice (Varghese and Mathew 2017⁠). The hardness of 80 bar treated samples was at the same level to its initial hardness, but still lost 18.8% hardness of fresh untreated samples. Eighty-five bar treated samples lost 33.1% hardness of fresh samples, which was better than untreated samples on 14^th day of chilled storage.

In this work, the CO₂ pressure significantly affected the reduction of protease activity and microbial counts. The mechanisms of protease inactivation by HPCD were proposed by several researchers. The pressure itself is not adequate to reduce protease activity as reported by HPP application. Bilbao-Sáinz et al. (2009) reported 70% residual activity of milk protease after subjected to HPP application at 600 MPa, 15 min, and 60 °C. Leite Júnior et al. (2015) reported no change of protease activity from Rhizomucor miehei after subjected to 190 MPa and 25 °C. Permanent inactivation of an enzyme is due to the changes of secondary structure of the enzyme and only achievable by pressure not less than 700 MPa (Tauscher 1995), Therefore, beside pressure, there must be one or more factors of HPCD that affect protease inactivation.

Ishikawa et al. (2014)*1 reported inactivation of alkaline and acid protease with supercritical CO₂ micro-bubbling method from 40 to 250 bar. In order to produce micro-bubble scCO₂, this report injected scCO₂ through 10 μm filter into the enzyme solution. Micro-bubbling application was reported to inactivate glucoamylase three folds than without micro-bubbling methods. Carbon dioxide is known to dissolve in water and lowering its pH. The correlation between CO₂ pressure increase and pH decrease was reported by Meyssami et al. (1992). However, effect of pH was reported insufficiently to reduce protease activity (Damar and Balaban 2006). Chen et al. (1993) reported that the combination of low pH solution of polyphenol oxidase (PPO) and N₂ pressure resulted much higher residual activity than that of scCO₂ application on buffered PPO solution. The significance of CO₂ presence in the mechanism of enzyme inactivation during gas pressure pasteurization was reported by Ishikawa et al. (1996). It was reported that scCO₂ decomposed α-helix structures and left 31.3% of residual α-helix structures after exposed to micro-bubble supercritical CO₂ at 250 bar for 30 min. This decomposition of α-helix would result irreversible inactivation of the enzyme.

The solubility of CO₂ and its dissolution rate into the cell cytoplasm and muscle tissue liquid when HPCD applied on muscle tissue is still unknown and need further investigation. The cell size of eukaryotes is within 10-100 μm (Campbell and Reece 2005)⁠ and the cell membrane pores are much smaller, 110 ± 40 nm (Zhou et al. 2009)⁠. Therefore, it was possible that muscle tissue acted as the micro-filter and cell membranes play its role as the nano-filter, similar to the micro-filter used in Ishikawa et al. (2014). Smaller bubble size of scCO₂ increase its solubility and dissolution rate in pure water was reported by Suzuki et al. (2013).⁠ Higher dissolved CO₂ is expected to yield higher inactivation of enzymes, since it increase the availability of CO₂ for altering secondary structure of the enzyme.

It could be expected that if subjected to HPCD pressure higher than 90 bar (at 40 °C), the fish texture would be deteriorated severely. Since this work was to search the balance between low residual protease activity and acceptable textural properties, complete inactivation of protease should be avoided. Elimination of muscle protease activity would result in severe degradation of fillet textural properties. In this regard, 80 and 85 bar of HPCD pressure at 40 °C can be considered as optimum pressure to reduce activity of muscle protease, as well as maintaining good textural properties of tilapia fillet.

Beside enzymatic activity, microbial activity also plays significant role on food deterioration. The bactericidal effect of scCO₂ could be contributed by one or more mechanisms. It could be cell disruption (Fraser 1951 and Lin et al. 1992⁠), extraction of cellular components (Kamihira et al. 2014 and Lin et al. 1991), and cell membrane modifications that could cause cytoplasm leakage (Hong and Pyun 2001). Cellular disruption usually requires short compression and decompression stage, which cause cell rupture. For the solid foodstuffs, which requires texture stability, this approach should be avoided. Bactericidal effect due to cellular components extraction and membrane modification could be exploited for the effort of microorganism reduction on solid foodstuff while maintaining acceptable texture. This work only applied single cycle of compression and decompression, therefore, the pasteurization effect on the reduction of microbial loads might be the accumulated effect of cell disruption by compression and decompression stage, extraction of cellular components, and cytoplasm leakage due to cell membrane modification.

Acceptable residual microbial counts in this work was achieved by 90 bar application. Unfortunately, 90 bar application was failed to maintain acceptable texture (Fig. 8). Both 80 and 85 bar were able to maintain most of their original texture, but their microbial counts exceeded maximum limit of 5·10⁵ CFU/g. Since 80 bar application could reduce microbial counts down to 2-logs, it could be expected that the raw fish with 10⁵ CFU/g could be reduced to 10³ CFU/g with 80 bar or more of scCO₂ pressure. Therefore, any additional treatment for reducing microbial loads prior to HPCD treatment is necessary in the future.

Maximum aerobic microbial counts of frozen raw fish was recommended at 5·10⁵ CFU/g (International Commission on Microbiological Specifications for Foods. 1986⁠ and Badan Standarisasi Nasional 2009). Normally, fish skin contains 10²–10⁷ CFU/cm², and between 10³–10⁹ CFU/g in intestine and gills (Huss 1993). The fresh fishes used in this study were fallen under 10⁹ CFU/g, which was normal but below standard required by safety regulation. Cleaning the fish with flowing tap water before HPCD treatment was found insufficient to reduce initial microbial counts.

The effect of HPCD pressure on texture was found significant but did not produce stable trend. Some researchers reported the effect of pressure of scCO₂ on texture of some seafoods qualitatively (de Matos et al. 2018, Ji et al. 2012, Meujo et al. 2010, Wei et al. 1991), but those works did not measure the effect of CO₂ pressure on seafood texture. However, the reports of high-pressure effect on seafood texture are available from HPP pasteurization and in agreement with the result in this work. Ashie and Simpson (1996) reported that at 1,000 atm, the strength and elasticity of bluefish muscle was better than original across all holding time. At 2,000 and 3,000 atm, until 10 min holding time; 2,000 atm treatment was better than original texture and 3,000 atm pressure. However, after 10 min holding time; 3,000 atm was better than 2,000 atm results. Kaur et al. (2016) reported the increase of hardness, springiness, chewiness, gumminess, and cohesiveness of Penaeus monodon along with the HPP pressure increase. Therefore, the increase of pressure did not increase or decrease textural quality of fish muscle consistently.

At 90 bar application, scCO₂ pressure resulted severe adverse effect on textural property and muscle integrity. Maximum pressure for the fish fillet could withstand CO₂ pressure was considered at 85 bar. Ninety bar CO₂ pressure or more might be justified for cooking application to eliminate completely both enzymatic and microbiological activities. HPCD might be a prospective application for the meat better than for the fish. Consumers preference for raw meat is freshness and tenderness (Ma and Ledward 2013), ⁠ while for fresh fish they prefer freshness and firmness (Sriket 2014).

Compared to HPP, color change induced by HPCD was initiated at much lower pressure. Color change of HPP treatment on carp fillet was reported to be initiated at 1,000 bar (Meuhreg 2004,Sequeira-Munoz et al. 2006, and Yoshioka et al. 1996). In this study, color change began from the lowest pressure of the experiment (70 bar). Wei et al. (1991)⁠ reported that shrimp and chicken color was changed after treated with 137 bar supercritical CO₂ for two hours. In this study, color changes occurred in lower pressures (90 bar or less) and shorter holding time. It should be noted that for economic consideration, commercial processing time is typically limited to 20 min (Truong et al. 2014⁠), therefore 15 min exposure time was acceptable but preheating and decompression time needs further improvement.

The color changes affected by high pressure is likely due to degradation of muscle myoglobin. The required temperature to denature myoglobin of tilapia significantly was reported at 60 °C (Chen et al. 2004). Myoglobin degradation is started at neutral and mildly acidic pH due to oxidation. The denaturation of myoglobin is increased along with the decrease of pH and completely denaturated at pH 2.0 (Pandiscia 2013). Therefore, the decrease of pH due to dissolved CO₂ might cause most of the myoglobin degradation.

As it was explained previously, scCO₂ extracted cellular components and in this work, also squeezed out these components out of the cells and muscle tissues. This extraction and squeezing effect reduced fillet mass, especially during decompression stage. Lipids also well known dissolved in scCO₂. Gupta and Shim (2007)⁠ reported the solubility of fish oil in scCO₂ from 90 to 200 bar at 313.2 K to 353.2 K, respectively. Other investigator, Hammam (1992)⁠ reported the solubility of pure lipids in 150-350 bar scCO_2. In this study, which used lower pressure (90 bar or lower), there was no correlation between fat solubility and pressure increase. Possibly, required density of CO₂ at 40 °C for dissolving fat should be equal to or more than the density of CO₂ at 90 bar. On the one hand, fat loss was not caused by its solubility in supercritical CO₂, but mostly by compression and decompression effects of pressure that squeezed out fat and other solids from its biological matrices. On the other hand, from 80 to 90 bar, protein was more concentrated along with pressure surge. This result indicated that protein was embedded firmly in its biological matrices.

In the non-thermal food processing technology, HPP and HPCD are two novel technologies that use high pressure. HPP employs ultrahigh hydrostatic pressure, up to 10,000 bar (Heinz and Buckow 2009), while HPCD uses significantly lower CO₂ pressure. The pressure of HPCD is the function of interaction between CO₂ density and CO₂ temperature (Span and Wagner 1996), while HPP pressure is the product of hydraulic pressure. Since the inactivation of the enzyme and microbial loads in HPCD is mostly contributed by the penetration and concentration of CO₂ into the cell and tissue, the pressure produced by CO₂ density is much lower than that of HPP. However, the disadvantage of HPCD over HPP is material losses as shown in Table 2. Beside product weight loss, due to diffusivity properties of CO₂, HPCD is prone to gas leakage during pasteurization. To achieve stable pressure during long HPCD application, gas leakage is compensated by CO₂ reinjection. Meanwhile, HPP process is applied on top of final vacuum packed product (Hogan et al. 2005) , which prevent product leakage. Since hydraulic liquid could not penetrate both the product packaging and pressure vessel seal, HPP is relatively capable to maintain product quantity throughout compression, holding, and decompression time. In HPCD pasteurization, CO₂ must be diffused into treated products, which require open or perforated container of the product. This open or slightly open package is the source of material losses during HPCD application.

The operational pressure of HPCD, which is significantly lower than that of HPP, requires less pressure resistant assembly than that of HPP equipments. HPP pasteurization requires 7,000 bar or more to achieve permanent inactivation of enzymes and need ultra-high pressure capable installation. Though HPCD requires lesser pressure resistant installation than that of HPP installation, it requires seal that impenetrable from scCO₂ diffusion. Soft metal seal such as aluminum might be impenetrable from scCO₂ but requires much more tightening force to be well sealed; while polymer seals, e.g. rubber and silicone, could not withstand scCO₂ penetration during long or repetitive HPCD application. HPP requires much more rugged installation than that of HPCD, including its seals, while HPCD requires lesser pressure resistant installation but need impenetrable seal from high diffusivity CO₂. If the same product is pasteurized by both HPP and HPCD, HPP requires more pumping power to deliver hydraulic pressure but need lower material input than that of HPCD, while the HPCD requires less power input to pump CO₂ but need more material input than that of HPP. Furthermore, products subjected to HPP do not need post treatment after pressurization, since it is already packed in final packaging; while HPCD products need immediate post pressurization handling since its packaging is remaining open after pasteurization.