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Dense Phase Carbon Dioxide Research: Current Focus and Directions
Available online at www.sciencedirect.com
ScienceDirect Agriculture and Agricultural Science Procedia 2 (2014) 2 – 9
“ST26943”, 2nd International Conference on Agricultural and Food Engineering, CAFEi2014”
Dense Phase Carbon Dioxide Research: Current Focus and Directions Murat O. Balabana,*, Trang Duongb a
Chemical and Material Engineering Department, University of Auckland, 22 Symonds Street, Auckland, 1010, New Zealand b School of Chemical Sciences,University of Auckland, 23 Symonds Street,Auckland, 1010, New Zealand
Abstract The non-thermal processing method of dense phase carbon dioxide (DPCD) for heat sensitive foods preserves quality, nutrients while inactivating microorganisms and enzymes in liquid foods. There is increasing interest in solid foods. Challenges associated with processing solids with DPCD will be discussed. Use of gases other than CO2 is possible. Finally, the combination of other non-thermal technologies such as pulsed electric fields, high pressure processing, high power ultrasound, and irradiation with DPCD opens new possibilities of more reduction of microorganisms, better inactivation of enzymes at milder process conditions, while offering more effective preservation of quality and nutrients.
© by Elsevier Elsevier B.V. B.V. This is an open access article under the CC BY-NC-ND license © 2014 2014 The The Authors. Authors. Published Published by (http://creativecommons.org/licenses/by-nc-nd/3.0/). Peer-review under responsibility of the Scientific Committee of CAFEi2014. Peer-review under responsibility of the Scientific Committee of CAFEi2014 Keywords: Dense phase carbon dioxide; non-thermal food processing; combination of technologies; solid foods
1. Introduction The non-thermal food processing technology of Dense Phase Carbon Dioxide (DPCD) uses pressurized CO2 in the liquid, gaseous or supercritical fluid states. Fraser (1951) and Haas et al. (1989) reported the effects of DPCD on microorganisms. Since then the effects of DPCD on microorganisms, enzymes and quality attributes in liquid foods have been studied. More recently, interest and accumulated knowledge has been growing in the applications of DPCD to solid foods. Also, the synergistic effects of simultaneous application of different non-thermal technologies together with DPCD are becoming the centre of research focus in this area.
* Corresponding author. Tel.: +64-09-373-7599; fax: +64-09-373-7463. E-mail address: [email protected]
2210-7843 © 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/). Peer-review under responsibility of the Scientific Committee of CAFEi2014 doi:10.1016/j.aaspro.2014.11.002
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This document will briefly review the applications of DPCD to liquid foods, and then will discuss current knowledge in the area of solid foods. The possibilities of using gases other than CO 2, as well as combinations of gases will be mentioned. Finally, the combined effects of other non-thermal technologies together with DPCD will be discussed. An outlook towards potential industrial applications will be presented. 2. Brief overview of the application of DPCD to liquid foods The application of DPCD to liquid foods to cold-pasteurize and extend their shelf life without heating, and therefore preserving their nutritional and quality attributes has been reviewed (Damar and Balaban 2006; GarciaGonzalez et al. 2007). Many juices, beverages and other liquid foods have been studied. Microbial and enzymatic reduction, and on quality attributes including taste, nutrients, and other quality parameters have been researched. The advantages of using DPCD to cold-pasteurize liquid foods are: Mild temperature ranges (30-50 oC). This allows retention of quality attributes, nutrients and other beneficial components such as anthocyanins and polyphenols. The pressures used in DPCD are about 1/10 th of the required pressures for high hydrostatic pressure (HHP). The operation can be continuous. Carbon dioxide is an inexpensive gas in industrial quantities. Exclusion of oxygen is beneficial in reducing oxidation reactions. The reduction of pH by dissolved CO2 may be beneficial in reducing microorganisms, inactivating enzymes, and preserving nutrients such as vitamin C. Automatic carbonation of the product is possible if needed. The main disadvantages are: DPCD is not used commercially. There is no continuous commercial equipment with the throughput of the existing thermal treatment equipment. Compared to high-volume thermal pasteurization, DPCD is more expensive, and requires specialized high-pressure equipment and parts that result in higher initial capital expenditures. In view of the increasing concerns regarding global warming, CO2 needs to captured and recycled during the operation. 3. Emerging interest in the application to solid foods There are several difficulties in treating solids with DPCD compared with liquid foods: Solids currently cannot be processed on a continuous basis, the diffusion of CO2 into the bulk of the solids is slow, there is a concern of cellular damage and therefore texture and other quality changes at the surface of solids, much reduced levels of free water at the surface may limit the solubility of CO2 into the food with its implications regarding bacterial and enzymatic inactivation. The following sections will briefly discuss reported applications of DPCD to solid food classes. 3.1. Meat Wei et al. (1991) treated chicken strips inoculated with microorganisms at 14 MPa and 35 oC to obtain less than 2 log reductions after 2 hrs. Sirisee et al. (1998) observed that microorganisms in ground beef treated with DPCD (31 MPa, 42.5 oC) required more time to inactivate compared with the same organisms in liquid medium. Erkmen (2000) inoculated microorganisms to minced and skinned beef and observed that DPCD at 6 MPa, 45 oC for 150 min resulted in 5 and 1 log reduction in skinned and minced beef, respectively. Meurehg (2006) worked with Escherichia coli and Salmonella inoculated to beef trimmings before grinding. He defined the term “Controlled phase” to describe sub-critical, then supercritical pressures, and rapid decompression that results in “ice crystals” in microorganisms. About 10 MPa for 15 min caused about 1 log reduction of E.coli. He also studied the effect of treatment on colour, protein solubility and tenderness of the resulting product. Choi et al. (2008, 2009a, 2009b) treated porcine muscle to inactivate bacteria, and treated marinated pork and fresh pork for the inactivation of E.coli, Listeria, and Salmonella. For marinated pork treated at 14 MPa, 45 oC for 40 min, the reduction of Listeria was about 2.5 and 1.9 logs in soy sauce and hot pepper marinades, respectively. Ferrentino et al. (2013) treated dry cured ham pieces at 8 and 12 MPa, 35 to 50 oC for 5-60 min. Listeria was totally inactivated (initial level 107 CFU/g) at 50 oC, 12 MPa, 15 min. Lower initial loads required less severe conditions for total inactivation. The process slightly affected the colour and sensory attributes of the product.
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3.2. Seafood Wei et al. (1991) treated shrimp spiked with Listeria and reduced the surviving numbers by 2 logs when treating with DPCD at 6.18 MPa, 35 oC, 2 hrs. They reported that N2 under the same conditions had no effect on the microorganism. Meujo et al. (2010) studied the reduction of the trapped microorganisms in the digestive system of oysters by exposing them to two treatments: 1) 10 MPa, 37 oC for 30 min, 2) 17.2 MPa 60 oC for 60 min. Total aerobic plate counts showed that treatments 1 and 2 reduced the initial loads by 2 and 3 logs, respectively. A 6 logs reduction was reported for a strain of Vibrio. They also observed that treatment 2 resulted in the release of the adductor muscle, which may be useful in shucking the oyster. Sensory analysis revealed no significant difference regarding physical appearance, smell and texture of treated and untreated oysters. Ji et al. (2012) used a neural network of 3 x 5 x 2 layers to optimize the process parameters of pressure, temperature and treatment time of shrimp to reduce microbial loads. They found that 15 MPa, for 26 min at 55 oC reduced the microbial load by 3.5 logs. They also observed a cooked appearance of the final product. De Matos et al. (2013) treated oysters with DPCD to reduce Vibrio parahaemolyticus. The amount of CO2 used was 1:0.8 CO2:product by mass. Pressures from 8 to 20 MPa, were used at 34 oC. Using exposure times from 0.25 to 6 hr, they developed the inactivation kinetics of the organism. At 0.5 hr 75% of the initial load was inactivated. Total reduction of the initial load was observed around 3 hr (about 2 logs). They also used pressure cycles and different depressurization rates. 3.3. Plant matter Haas et al. (1989) treated herbs (chives, thyme, oregano, parsley, mint) at 5.4 MPa and 45 oC for 2 hrs. From a maximum initial load of 108 CFU/g, they reported total inactivation of aerobic counts. They noted some positive or negative consequences on aromas depending on the herb. The same authors also treated whole strawberries, melon, and cucumber to delay surface molding. Although “positive” results were observed for strawberries, “gross tissue destruction” occurred. Kuhne and Knorr (1990) treated celery sticks and leaves at 62.8 MPa and 40 oC for 30 min. They reported that the total plate count was reduced by about 4 logs from an initial load of about 107 CFU/g. Mazzoni et al. (2001) treated alfalfa seeds with DPCD at 27.6 MPa and 50 oC for 60 min. They reported a decrease of E.coli of about 1 log, and total aerobic bacterial count of less than 1 log. Calvo et al. (2007) treated cocoa powder with DPCD at 30 MPa and 65 oC for 40 min. The effect of treatment on aerobic thermophilic and mesophilic organisms was measured. With dry powder, no effects were observed. When 5 to 10% water was added then high temperatures (65 or 80 oC) resulted in total inactivation. In addition spiked fungi spores were inactivated at 30 MPa, 80 oC, for 30 min after addition of 5% water. Dehghani et al. (2008) tested different DPCD treatment conditions on heavily contaminated ginseng (about 107 CFU/g). The best results were obtained at 10 MPa, 60 oC for 15 hr, where less than 3 log reductions was observed, not enough to comply with minimum regulatory levels. When small amounts of water/ethanol/H 2O2 were added to CO2, complete inactivation of bacteria and fungi was achieved at 10 MPa, 60 oC and 6 hr. Zhong et al. (2008) treated fresh spinach leaves with DPCD at 7.5 to 10 MPa, 40 oC for 40 min to reduce E.coli. They reported 5 logs reduction to undetectable levels. Subcritical state was not nearly as effective. They also observed discoloration of the leaves and changes in texture (decreased firmness). Jung et al. (2009) treated alfalfa seeds with DPCD to reduce E.coli, Listeria and Salmonella. Pressures from 10 to 20 MPa, temperatures from 35 to 45 oC for 5 to 15 min were applied. Germination percentage was also measured. 20 MPa, 45 oC for 15 min reduced the levels of the pathogens by 7 logs. However, at the severe treatment conditions germination was impaired. If conditions were optimized for germination capability, then at 15 MPa, 35 oC for 10 min the reduction of E.coli was about 3.5 logs. Calvo and Torres (2010) treated paprika powder with DPCD. Pressures from 6-30 MPa, temperatures less than 95 o C, and exposure times from 10-150 min in a continuous CO2 flow system. Various initial moisture contents < 35% were tried. Initial water content and temperature were the influential parameters for microbial reduction. Around 35% water and 6 MPa, about 1.5 logs reduction was obtained. When pressure was increased to 30 MPa, 5 logs reductions were reached. They concluded that higher pressures should be avoided to minimize extraction of oleoresins. The final paprika retained its colour.
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Valverde et al. (2010) treated fresh cut conference pears with DPCD to reduce Saccharomyces cerevisiae. Continuous CO2 flow at pressures 6-30 MPa, 25-55 oC for 10-90 min was used. DPCD at 55 oC achieved 5 logs reduction, while it was necessary to go to 70 oC for the same reduction with heat. Relatively low (<6 MPa) pressures and exposure times in the order of minutes were required. They observed some darkening at the surface, and some texture loss. Niu et al. (2010) treated apple slices by DPCD and mild heat. Then they extracted apple juice from these slices. Temperatures from 25 to 65 oC were used at 20 MPa for 20 min in a batch system. They investigated the residual activities of pectin methylesterase (PME) and polyphenol oxidase (PPO) between DPCD and mild heat (65 oC). PPO was totally inactivated by DPCD, and its residual activity by mild heat was 36%. DPCD also resulted in a residual activity of PME of 18%. Ferrentino et al. (2012) treated fresh-cut coconut pieces with DPCD. They evaluated the microbial, textural and microstructure effects at 8-12 MPa, 24-45 oC, for 5 to 60 min. Treatment of 15 min at 45 oC and 12 MPa resulted in 4 logs CFU/g reduction of mesophilic microorganisms, lactic acid bacteria, total coliforms, and yeasts and molds. Although hardness was not affected, the microstructure exhibited irregularities. 4. Nitrous oxide (N2O) Nitrous oxide has critical properties close to that of CO2 (critical temperature: 36.4 °C and critical pressure: 7.25 MPa). It is a colourless, non-flammable gas at room temperature, with a slightly sweet odour and taste. It is used in dentistry for anaesthetic and analgesic effects. Liquid nitrous oxide is a good solvent for many organic compounds. Some properties of N2O cause concern: at higher temperatures it is an oxidizer similar to molecular oxygen. Its pressure curve is very sensitive to temperature, and it can reach decomposition temperatures due to adiabatic heating with pressure. N2O is a greenhouse gas 300 times more potent than CO2. It causes ozone depletion. It oxidizes the cobalamin form of vitamin B12. Animal studies show adverse reproductive effects on pregnant females. Qadir and Hashinaga (2001) used N2O mixed with oxygen to treat climacteric and non-climacteric fruits inoculated with various fungi. Ratios of 80:20 of N 2O:O2 were used with storage of the fruits at 20 °C. They found that that regardless of the physiological nature of the fruits or group of fungi, N 2O delayed the appearance of disease and reduced the lesion growth rate. This response to N2O was dose and time dependent. Spilimbergo et al. (2007) used pressurized N2O to inactivate yeasts in fresh apple juice. Pressures of 6-20 MPa, at 36 °C for 1–25 min were used in a multi-batch equipment. They reported that at 10 MPa they achieved complete inactivation of the yeasts (initial load 103 to 105 CFU/ml) in 5 min. Later, Gasperi et al. (2009) continued this work, and treated fresh apple juice with pressurized CO2 or N2O at 10 MPa, 36 °C for 10 min with stirring, and evaluated the quality of the resulting product by sensory tests. Although chemical parameters (total solids, organic acids, polyphenols, etc.) did not show any difference with untreated juice, analysis of headspace showed a loss of volatiles. Peach and kiwi juices were also treated with CO2 or N2O (Spilimbergo and Ciola, 2010). Treatment conditions of 35 oC, 10 MPa for 15 min resulted in total inactivation of natural flora, as well as Saccharomyces cerevisiae (105 CFU/ml) both with CO2 and N2O. They did not detect any significant differences between treated and untreated juices regarding physic-chemical attributes of sensory properties. When milk is treated with high pressure CO2, it may coagulate due to the lowering of pH by CO2 dissolving in water. Spilimbergo (2011) used pressurized N 2O at 12 MPa, with temperatures from 40-50 °C to process milk. She reported that at 50 °C, in slightly more than 10 min there were no viable organisms detected after the treatment. There was no significant difference between the pH and acidity values of the untreated samples and samples treated for 60 min. This is an advantage in cold-pasteurizing milk and other pH sensitive products. 5. Combinations of non-thermal processes The promise of the simultaneous use of non-thermal technologies is that their synergistic effect may lower the severity of the process and/or the treatment time compared with either technology applied alone. Of course if one technology were continuous, it would be beneficial if its combination technology also were continuous. In this section, examples of the combination of DPCD with other non-thermal technologies will be discussed.
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5.1 DPCD and ultrasound The use of high-power ultrasound (HPU) in combination with DPCD is a relatively new concept. The promise of ultrasound is very effective mixing, and therefore reducing the time needed to reach CO 2 saturation especially in batch systems. Although not well understood, there is also a possibility that cavitation may help in the saturation of CO2 in the liquid phase. Since ultrasound consists of high pressure and low pressure regions, during the compression cycle the solubility of CO2 in the liquid will increase by increasing the driving force for saturation. Then in the lowpressure cycle, the dissolved CO2 may supersaturate due to the decrease of pressure. Ortuño et al. (2012) combined high power ultrasound (HPU) with batch DPCD to inactivate E.coli in broth and in orange juice. They used pressures from 10 to 35 MPa, temperatures from 31 to 41 °C, and measured the process times necessary to inactivate E.coli using DPCD only, HPU only, and DPCD + HPU. At similar treatment conditions of pressure and temperature, DPCD by itself required 50 min to reduce E.coli by 8 logs. Using both DPCD + HPU reduced the time required by 95%. This time achieved in a batch system is shorter than that in a continuous system (between 5 to 10 min). The authors concluded that the combination of DPCD + HPU can achieve the same inactivation rate in a much shorter time, and using milder process parameters (pressure and temperature). Ortuño et al. (2013a) also used the combination of DPCD + HPU in broth, apple juice, and orange juice to inactivate Saccharomyces cerevisiae in a batch system. Pressures from 10 to 35 MPa, temperatures from 31 to 41 °C were used. With DPCD alone, 6.7 logs reduction was obtained at 35 MPa, 36 °C for 140 min. DPCD + HPU under the same pressure and temperature reached the same inactivation level in 2 min. They reported that the medium affected the rate of inactivation. The solids content could affect viscosity, which affects cavitation. Ortuño et al. (2014) also investigated the effect of using DPCD + HPU on enzyme inactivation in a batch system. The kinetics of inactivation of PME in orange juice, as well as those of E.coli and S. cerevisiae were determined. An increase in temperature and pressure increased the inactivation rate of E. coli, S. cerevisiae and PME. The medium influenced the effect of pressure and temperature increase on the inactivation rate. Total inactivation of PME was not achieved. The possible advantages of using the combination of continuous DPCD with HPU are exciting. If the same drastic time requirement for inactivation can be achieved as in the case of batch systems, this will potentially reduce the residence time of DPCD, and therefore reduce capital costs of building continuous DPCD systems. 5.2 DPCD and high hydrostatic pressure The advantage of high hydrostatic pressure (HHP) is to process foods that are already packaged, and therefore do not require concern about post-processing contamination. Although HHP effectively eliminates microorganisms and stabilizes liquid foods, it does not inactivate key enzymes that reduce the quality. In fact, HHP may increase the activities of enzymes such as e.g. PPO. On the other hand, DPCD as a continuous operation needs aseptic filling to containers, but can inactivate enzymes that remain after HHP processing. Therefore it is logical to combine these technologies to benefit from their individual advantages. Corwin and Shellhammer (2002) used fresh squeezed orange juice to measure the PME activity after carbonation of the juice at 1 atm added pressure, and treatment with HHP at 500 MPa (3 min) or 800 MPa (1 min) at either 25 °C or 50 °C. Also, tyrosinase, Lactobacillus plantarum and E. coli were prepared in buffer systems, and carbonated as described before. The average CO 2 concentration in the PME liquid was 2.1 volumes of CO 2 / volume of sample. They calculated this as a 0.2% molar basis. The effects of carbonation alone, HHP alone, and HHP + carbonation were measured. After 500 MPa for 3 min, HHP only resulted in % remaining activity of PME of 86%. With CO2, this was reduced to 56%. At 800 MPa, there was no significant difference between HHP alone and HHP +CO2 (7.8% and 6.8%). At 50 °C, there was no significant difference between HHP only and HHP + CO2 at both 500 and 800 MPa. The effect of added CO2 to PPO residual activity was significant: At 25 °C and 500 MPa, HHP only resulted in a residual activity of 98.5%, when CO 2 was added this was reduced to 59%. At 800 MPa, HHP alone resulted in 91% residual PPO activity, addition of CO 2 reduced this to 51%. At 50 °C, and 800 MPa, HHP alone resulted in 73% residual PPO activity, and addition of CO 2 reduced this to 21%. Addition of CO2 further reduced the Lactobacillus count by 2 logs at 25 °C compared to HHP alone, at 365 and 455 MPa. However, for E.coli both at 500 and 600 MPa, there was no additional reduction with the addition of CO 2. In a similar study, Boff et al. (2003) used CO2-assisted HHP to process orange juice, and evaluated quality attributes during 4 months of storage at 4 and 30 °C. HPP + CO2 produced a cloud-stable orange juice with more ascorbic acid and flavour volatiles than thermally processed juice (p < 0.05).
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Park et al. (2002) studied the effects of sequential application of DPCD and HHP on the safety and shelf life of carrot juice. A combined treatment of 4.9 MPa DPCD (5 oC, 5 min) and 300 MPa-HHP (25 oC, 5 min) completely inactivated the aerobic microorganisms. Also, a process of 4.9 MPa DPCD and 600 MPa-HHP effectively inactivated PPO, PME and lipoxygenase (LOX) enzymes, with residual enzyme activities of less than 11%, 35%, and 9%, respectively. These levels were 2-3 folds lower than that of DPCD or HHP treatments alone. At low HHP pressure of 200 MPa, addition of 4.9 MPa-DPCD (5 oC, 5 min) treatment prior to HHP (25 ºC, 5 min) improved the inactivation of the PPO, LOX and PME enzymes in carrot juice with a residual activity of 35%, 17% and 45%, respectively, compared with the residual activity of DPCD (40%, 20% and 50%, respectively) and HHP (83%, 78% and 80%, respectively) treatments. The enzyme activities had strong correlation with pH, which was dependent on the pressure of CO2. The colour and cloud of treated carrot juice were significantly affected by DPCD but not by HHP. However, sequential application is not desirable for the industry since it requires more processing time. Ortuño et al. (2013b) used simultaneous application of DPCD and HHP on feijoa puree. Samples were treated with HHP only (HHP), or HHP+carbonated (HHPcarb), or HHP+carbonated with added 8.5mL CO 2/g sample (HHPcarb+CO2) at 300, 450 and 600MPa, 25 oC, for 5 min. The results showed that HHP increased the residual enzyme activity (REA) of POD in HHP sample at 300 MPa to 140 ± 5%, but decreased to 60 ± 9% and 22 ± 13% at 450 MPa and 600 MPa, respectively. The addition of CO2 affected the REA of POD in samples at all tested pressures. The REA of PME significantly decreased in HHPcarb+CO 2 samples from 73 ± 14% at 300 MPa to 53 ± 3% at 600 MPa. The addition of CO2 enhances enzyme inactivation with HHP, which did not affect the colour of the puree, compared with puree treated with HHP only. It was recommended that more research needed to be conducted to understand the mode of enzyme inactivation by the simultaneous HHP and DPCD treatments. We recently investigated the effects of pressure (200-600 MPa), process time (1-13 min), pH (3.0-3.6), and CO2 levels (no CO2, CO2 saturation, and saturation with addition of 8.5 mL CO2/g sample) in a simultaneous HHP and DPCD process on POD, PPO and PME of feijoa puree, using response surface methodology (27 treatments). Increasing treatment time reduced all enzyme activities, while increasing CO 2 levels decreased PME activity, and pressure influenced PPO activity. The lower pH values tended to increase enzyme inactivation. The optimal process conditions of 13 min, 600 MPa, pH = 3, CO2 saturation, resulted in 74.3 ± 3.3%, 70.9 ± 2.6% and 53.9 ± 0.9% residual activity for POD, PPO and PME respectively. Combination of CO 2 with HHP induced synergistic enzyme inactivation, and reduction of pH did not prove to be the main factor. 5.3 DPCD and pulsed electric field The combination of pulsed electric field (PEF) and DPCD was studied with promising results. The hypothesis for the mechanism is that PEF aids the diffusion of CO 2 into the cell by causing electroporation, in which the lipid bilayer and proteins of cell membrane can temporarily destabilize due to the high voltage electric field. The cell plasma membrane becomes penetrable to small particles (i.e. CO2), resulting in swelling and then rupture of the cell membrane. Also, once the amount of CO2 builds up to a critical level within the cells, it influences the intracellular pH and extracts constituents that alter the structure of bio-membrane or disrupt the biological system. Spilimbergo et al. (2003) investigated the effects of combining pulsed electric field (PEF) and DPCD on inactivation of Escherichia coli, Staphylococcus aureus and Bacillus cereus. The microorganisms were first treated with PEF at up to 25 KV/cm (1-20 pulses, 5 second intervals) and then with DPCD at below 40 oC, and 20 MPa. The increasing electrical field strength and number of pulses decreased the microbial viability. A PEF treatment at 25 KV/cm, 10 pulses resulted in 2.9 and 3.3 log reductions of E. coli and S. aureus, respectively while B. cereus spores had a strong resistance to PEF (0.5 log reduction). Further treatment with DPCD (20 MPa, 34 oC, for 10 min) completely inactivated the bacterial species (7-8 logs reductions). A combined treatment of PEF at 25 KV/cm and 20 pulses and then DPCD at 20 MPa, 24 h at 40 oC partial inactivated B. cereus spores (3 logs reduction). The inactivation was increased with the increasing the DPCD treatment time (ie. contact time between CO 2 and the samples). PEF and DPCD had synergistic effect on all treated microorganisms. Pataro et al. (2010) applied the sequential treatment of PEF (6, 9, 12 KV/cm electric field strength and 10, 20 and 40 J/ml) and then DPCD (8, 11 and 14 MPa, for 3-30 min) treatment on Saccharomyces cerevisiae, and compared with samples treated with PEF or DPCD treatments alone. The maximum inactivation was achieved for PEF treatment at 12 kV/cm and 20 J/ml with 0.35 logs reduction, while for DPCD the maximum reduction was at 8 MPa, 35 oC, 3 min (3.13 logs reduction). The increasing pressure and time did not increase the yeast cell inactivation. Meanwhile, combined DPCD (8 MPa, 25 oC, 5 min) application after PEF resulted in total inactivation
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regardless the electric field level of PEF pre-treatment. Increasing the electric field strength and energy input significantly increased the inactivation effect of DPCD. The Peleg kinetics model was a good fit for the inactivation kinetics of this combination PEF and DPCD treatment, which takes into account the treatment time of DPCD. Other parameters were functions of the electrical field strength and the energy input. Further studies on real food materials were recommended to investigate the combined process on the physical and nutritional attributes of the products. 5.4 DPCD and irradiation Urbain et al. (1969) obtained a patent involving the use of irradiation of food containers with a pressurized headspace of CO2. Their claims stated pressure of 0.2 to 6 MPa, and gave examples of canned meat with CO2 pressures of 0.8 MPa. In an example given in the patent, defatted soybean meal (30% solids in water) was inoculated with Salmonella (104 CFU/ g), canned under a CO2 pressure of 0.68 MPa, and irradiated at 5 KGy. After incubation for 24 hr at 37 oC, the following results were obtained: control: 3.08 104 CFU/g, CO2 only 8.5 103 CFU/g, irradiation only 1.4 103 CFU/g, combined irradiation and CO2 <100 CFU/g. 6. Outlook The commercialization of the DPCD technology will depend on mathematically predicting microbial reductions and/or enzyme inactivation at process conditions (pressure, temperature, process time, CO 2 ratio, solubility of CO2, synergistic processes, etc.). In addition to the regulatory requirements, a demonstrated advantage over other processes in terms of quality, nutrients, and cost is needed for DPCD or its combined methods. Finally, the design and availability of continuous solids treatment will expand the application base of this technology. References Boff, J.M., Truong, T.T., Min, D.B., Shellhammer, T.H., 2003. Effect of Thermal Processing and Carbon-Dioxide Assisted High Pressure Processing on Pectinmethylesterase and Chemical Changes in Orange Juice. Journal of Food Science 68, 1179-1184. Calvo, L., Muguerza, B., Cienfuegos-Jovellanos, E., 2007. Microbial Inactivation and Butter Extraction in a Cocoa Derivative Using High Pressure CO2. Journal of Supercritical Fluids 42, 80-87. Calvo, L., Torres, E., 2010. Microbial Inactivation of Paprika Using High-Pressure CO2. Journal of Supercritical Fluids 52, 134-141. Choi, Y.M., Ryu, Y.C., Lee, S.H., Go, G.W., Shin, H.G., Kim, K.H., Rhee, M.S., Kim, B.C., 2008. Effects of Supercritical Carbon Dioxide Treatment for Sterilization Purpose on Meat Quality of Porcine Longissimus Dorsi Muscle. Lebensmittel-Wissenschaft und-Technologie 41, 317-322. Choi, Y.M., Bae, Y.Y., Kim, K.H., Kim, B.C., Rhee, M.S., 2009a. Effects of Supercritical Carbon Dioxide Treatment Against Generic Escherichia coli, Listeria monocytogenes, Salmonella typhimurium, and Escherichia coli O157:H7 in Marinades and Marinated pork. Meat Science 82, 419-424. Choi Y M, Kim O Y, Kim K H, Kim B C, Rhee M. S. 2009b. Combined Effect of Organic Acids and Supercritical Carbon Dioxide Treatments Against Nonpathogenic Escherichia coli, Listeria monocytogenes, Salmonella typhimurium and Escherichia coli O157:H7 in Fresh Pork. Letters in Applied Microbiology, 49, 510-515. Corwin, H., Shellhammer, T.H., 2002. Combined Carbon Dioxide and High Pressure Inactivation of Pectin Methyl Esterase, Polyphenol Oxidase, Lactobacillus plantarum and Escherichia coli. Journal of Food Science 67(2), 697-701. Damar, S., Balaban, M.O., 2006. Review of Dense Phase CO2 Technology: Microbial and Enzyme Inactivation, and Effects on Food Quality. Journal of Food Science 71(1), R1-11. De Matos, K.H.O., Lerin, L.A., Soares, D., Smania, A., de Oliveira, J.V., Monteiro, A.R., 2013. Inactivation Kinetics of Vibrio Parahaemolyticus Naturally Occurring in Oysters Using Supercritical CO2 Treatment. Ibero American Conference on Supercritical fluids Cartagena de Indias, Colombia. Dehghani, F., Annabi, N., Titus, M., Valtchev, P., Tumilar, A., 2008. Sterilization of Ginseng Using a High Pressure CO2 at Moderate Temperatures. Biotechnology and Bioengineering 102, 569-576. Erkmen, O., 2000. Antimicrobial Effects of Pressurised Carbon Dioxide on Brochothrix thermosphacta in Broth and Foods. Journal Science of Food and Agriculture 80, 1365-1370. Ferrentino, G., Balzan, S., Dorigato, A., Pegoretti, A., Spilimbergo, S., 2012. Effect of Supercritical CO2 Pasteurization on Natural Microbiota, Texture and Microstructure of Fresh-Cut Coconut. Journal of Food Science 77(5), E137-E143. Ferrentino, G., Balzan, S., Spilimbergo, S., 2013. Supercritical CO2 Processing of Dry Cured Ham Spiked with Listeria monocytogenes: Inactivation Kinetics, Color, and Sensory Evaluations. Food Bioprocess Technology 6, 1164-1174. Fraser, D., 1951. Bursting Bacteria By Release of Gas Pressure, Nature 167, 33-34.
Murat O. Balaban and Trang Duong / Agriculture and Agricultural Science Procedia 2 (2014) 2 – 9 Garcia-Gonzalez, L., Geeraerdm A.H., Spilimbergo, S., Elst, K., Van Ginneken, L., Debevere, J., Van Impe, J.F., Devlieghere, F., 2007. High Pressure CO2 Inactivation of Microorganisms in Foods: the Past, the Present and the Future. International Journal Food Microbiology 117, 128. Gasperi, F., Aprea, E., Biasioli, F., Carlin, S., Endrizzi, I., Pirretti, G., Spilimbero, S., 2009.Effect of Supercritical CO2 and N2O Pasteurization on the Quality of Fresh Apple Juice. Food Chemistry 115, 129-136. Haas, G.J., Prescott, H.E., Dudley, E., Dik, R., Hintlian, C., Keane, L., 1989. Inactivation of Microorganisms by CO2 Under Pressure. Journal of Food Safety 9, 253-265. Ji, H., Zhang, L., Liu, S., Qu, X., Zhang, C., Gao, J., 2012. Optimization of Microbial Inactivation of Shrimp by Dense Phase CO2. International Journal Food Microbiology 156, 44-49. Jung, W.Y., Choi, Y.M., Rhee, M.S., 2009. Potential Use of Supercritical Carbon Dioxide to Decontaminate Escherichia coli 0157:H7, Listeria monocytogenes and Salmonella typhimurium in Alfalfa Sprouted Seeds. International Journal Food Microbiology 136(1), 66-70. Kuhne, K., Knorr, D., 1990. Effects of High Pressure Carbon Dioxide on the Reduction of Microorganisms in Fresh Celery. Internationale Zeitschrift für Lebensmittel-Technik, Marketing, Verpackung und Analytik 41, 55-57. Mazzoni, A.M., Sharma, R.R., Demirci, A., Ziegler, G.R., 2001. Supercritical Carbon Dioxide Treatment to Inactivate Aerobic Microorganisms on Alfalfa Seeds. Journal Food Safety 21, 215-223. Meujo, D.A.F., Kevin, D.A., Peng, J., Bowling, J.J., Liu, J., Hamann, M.T., 2010. Reducing Oyster-Associated Bacteria Levels Using Supercritical Fluid CO2 As an Agent of Warm Pasteurization. International Journal of Food Microbiology 138, 63-70. Meurehg, C.A.T., 2006. Control of Escherichia coli O157:H7, Generic Escherichia coli, and Salmonella spp. on Beef Trimmings Prior to Grinding Using a Controlled Phase Carbon Dioxide (cpCO2) System. PhD Thesis, Manhattan, Kansas: Kansas State University. Niu, S., Xu, Z., Fang, Y., Zhang, L., Yang, Y., Liao, X., Hu, X., 2010. Comparative Study on Cloudy Apple Juice Qualities from Apple Slices Treated by High Pressure Carbon Dioxide and Mild Heat. Innovative Food Science and Emerging Technologies 11, 91-97. Ortuño, C., Martínez Pastor, M.T., Mulet, A.J., Benedito, A.J., 2012. An Ultrasound-Enhanced System for Microbial Inactivation Using Supercritical Carbon Dioxide, Innovative Food Science and Emerging Technologies 15, 31–37. Ortuño, C., Martínez Pastor, M.T., Mulet, A.J. Benedito, A.J., 2013a. Application of High Power Ultrasound in the Supercritical Carbon Dioxide Inactivation of Saccharomyces cerevisiae, Food Research International 51, 474–481. Ortuño, C., Duong, T., Balaban, M.O., Jose Benedito, J., 2013b. Combined High Hydrostatic Pressure and Carbon Dioxide Inactivation of Pectin Methylesterase, Polyphenol Oxidase and Peroxidase in Feijoa Puree. Journal Supercritical Fluids 82, 56-62. Ortuño, C., Balaban, M., Benedito, J., 2014. Modeling the Inactivation Kinetics of Escherichia coli, Saccharomyces cerevisiae and Pectin Methylesterase in Orange Juice Treated with Ultrasonic-Assisted Supercritical Carbon Dioxide. Journal Supercritical Fluids 90, 18-26 Park, S.J., Lee, J.I., Park, J., 2002. Effects of a Combined Process of High-Pressure Carbon Dioxide and High Hydrostatic Pressure on the Quality of Carrot Juice. Journal Food Science 67, 1827–1834. Pataro, G., Ferrentino, G., Ricciardi, C., Ferrari, G., 2010. Pulsed Electric Field Assisted Microbial Inactivation of Saccharomyces cerevisiae Cells by High Pressure Carbon Dioxide. Journal Supercritical Fluids 54,120-128. Qadir, A., Hashinaga, F., 2001. Inhibition of Postharvest Decay of Fruits by Nitrous Oxide, Postharvest Biology and Technology 22, 279-283. Sirisee, U., Hsieh, F., Huff, H.E., 1998. Microbial Safety of Supercritical Carbon Dioxide Processes. Journal of Food Processing and Preservation 22, 387-403. Spilimbergo, S., Dehghani, F., Bertucco, A., Foster, N.R., 2003. Inactivation of Bacteria and Spores by Pulsed Electric Field and High Pressure CO2 at Low Temperature. Biotechnology and Bioengineering 82(1), 118-125. Spilimbergo, S., Mantoan, D., Caazza, A., 2007. Yeast Inactivation in Fresh Apple Juice by High Pressure Nitrous Oxide. International Journal of Food Engineering 3(6), 1556. DOI: 10.2202/1556-3758.1134 Spilimbergo, S., Ciola, L., 2010. Supercritical CO2 and N2O Pasteurisation of Peach and Kiwi Juice. International Journal of Food Science and Technology 45, 1619-1625. Spilimbergo, S., 2011. Milk Pasteurization at Low Temperature Under N2O Pressure. Journal Food Engineering 105, 193-195. Urbain, W. M., Shank, J.L., Kauffman, F.L., 1969. Irradiation with CO2 Under Pressure. US Patent: 3,483,005. Valverde, M.T., Marin-Iniesta, F., Calvo, L., 2010. Inactivation of Saccharomyces cerevisiae in Conference Pear with High Pressure Carbon Dioxide and Effects on Pear Quality. Journal of Food Engineering 98, 421-428. Wei, C.I., Balaban, M.O., Fernando, S.Y., Peplow, A.J., 1991. Bacterial Effect of High Pressure CO2 Treatment on Foods Spiked with Listeria or Salmonella. Journal of Food Protection 54, 189-193. Zhong, Q., Black, D.G., Davidson, P.M., Golden, D.A., 2008. Non Thermal Inactivation of Escherichia coli K-12 on Spinach Leaves, Using Dense Phase Carbon Dioxide. Journal of Food Protection 71, 1015-1017.
Accepted for oral presentation in CAFEi2014 (December 1-3, 2014 – Kuala Lumpur, Malaysia) as paper 320.
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ScienceDirect Agriculture and Agricultural Science Procedia 2 (2014) 2 – 9
“ST26943”, 2nd International Conference on Agricultural and Food Engineering, CAFEi2014”
Dense Phase Carbon Dioxide Research: Current Focus and Directions Murat O. Balabana,*, Trang Duongb a
Chemical and Material Engineering Department, University of Auckland, 22 Symonds Street, Auckland, 1010, New Zealand b School of Chemical Sciences,University of Auckland, 23 Symonds Street,Auckland, 1010, New Zealand
Abstract The non-thermal processing method of dense phase carbon dioxide (DPCD) for heat sensitive foods preserves quality, nutrients while inactivating microorganisms and enzymes in liquid foods. There is increasing interest in solid foods. Challenges associated with processing solids with DPCD will be discussed. Use of gases other than CO2 is possible. Finally, the combination of other non-thermal technologies such as pulsed electric fields, high pressure processing, high power ultrasound, and irradiation with DPCD opens new possibilities of more reduction of microorganisms, better inactivation of enzymes at milder process conditions, while offering more effective preservation of quality and nutrients.
© by Elsevier Elsevier B.V. B.V. This is an open access article under the CC BY-NC-ND license © 2014 2014 The The Authors. Authors. Published Published by (http://creativecommons.org/licenses/by-nc-nd/3.0/). Peer-review under responsibility of the Scientific Committee of CAFEi2014. Peer-review under responsibility of the Scientific Committee of CAFEi2014 Keywords: Dense phase carbon dioxide; non-thermal food processing; combination of technologies; solid foods
1. Introduction The non-thermal food processing technology of Dense Phase Carbon Dioxide (DPCD) uses pressurized CO2 in the liquid, gaseous or supercritical fluid states. Fraser (1951) and Haas et al. (1989) reported the effects of DPCD on microorganisms. Since then the effects of DPCD on microorganisms, enzymes and quality attributes in liquid foods have been studied. More recently, interest and accumulated knowledge has been growing in the applications of DPCD to solid foods. Also, the synergistic effects of simultaneous application of different non-thermal technologies together with DPCD are becoming the centre of research focus in this area.
* Corresponding author. Tel.: +64-09-373-7599; fax: +64-09-373-7463. E-mail address: [email protected]
2210-7843 © 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/). Peer-review under responsibility of the Scientific Committee of CAFEi2014 doi:10.1016/j.aaspro.2014.11.002
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This document will briefly review the applications of DPCD to liquid foods, and then will discuss current knowledge in the area of solid foods. The possibilities of using gases other than CO 2, as well as combinations of gases will be mentioned. Finally, the combined effects of other non-thermal technologies together with DPCD will be discussed. An outlook towards potential industrial applications will be presented. 2. Brief overview of the application of DPCD to liquid foods The application of DPCD to liquid foods to cold-pasteurize and extend their shelf life without heating, and therefore preserving their nutritional and quality attributes has been reviewed (Damar and Balaban 2006; GarciaGonzalez et al. 2007). Many juices, beverages and other liquid foods have been studied. Microbial and enzymatic reduction, and on quality attributes including taste, nutrients, and other quality parameters have been researched. The advantages of using DPCD to cold-pasteurize liquid foods are: Mild temperature ranges (30-50 oC). This allows retention of quality attributes, nutrients and other beneficial components such as anthocyanins and polyphenols. The pressures used in DPCD are about 1/10 th of the required pressures for high hydrostatic pressure (HHP). The operation can be continuous. Carbon dioxide is an inexpensive gas in industrial quantities. Exclusion of oxygen is beneficial in reducing oxidation reactions. The reduction of pH by dissolved CO2 may be beneficial in reducing microorganisms, inactivating enzymes, and preserving nutrients such as vitamin C. Automatic carbonation of the product is possible if needed. The main disadvantages are: DPCD is not used commercially. There is no continuous commercial equipment with the throughput of the existing thermal treatment equipment. Compared to high-volume thermal pasteurization, DPCD is more expensive, and requires specialized high-pressure equipment and parts that result in higher initial capital expenditures. In view of the increasing concerns regarding global warming, CO2 needs to captured and recycled during the operation. 3. Emerging interest in the application to solid foods There are several difficulties in treating solids with DPCD compared with liquid foods: Solids currently cannot be processed on a continuous basis, the diffusion of CO2 into the bulk of the solids is slow, there is a concern of cellular damage and therefore texture and other quality changes at the surface of solids, much reduced levels of free water at the surface may limit the solubility of CO2 into the food with its implications regarding bacterial and enzymatic inactivation. The following sections will briefly discuss reported applications of DPCD to solid food classes. 3.1. Meat Wei et al. (1991) treated chicken strips inoculated with microorganisms at 14 MPa and 35 oC to obtain less than 2 log reductions after 2 hrs. Sirisee et al. (1998) observed that microorganisms in ground beef treated with DPCD (31 MPa, 42.5 oC) required more time to inactivate compared with the same organisms in liquid medium. Erkmen (2000) inoculated microorganisms to minced and skinned beef and observed that DPCD at 6 MPa, 45 oC for 150 min resulted in 5 and 1 log reduction in skinned and minced beef, respectively. Meurehg (2006) worked with Escherichia coli and Salmonella inoculated to beef trimmings before grinding. He defined the term “Controlled phase” to describe sub-critical, then supercritical pressures, and rapid decompression that results in “ice crystals” in microorganisms. About 10 MPa for 15 min caused about 1 log reduction of E.coli. He also studied the effect of treatment on colour, protein solubility and tenderness of the resulting product. Choi et al. (2008, 2009a, 2009b) treated porcine muscle to inactivate bacteria, and treated marinated pork and fresh pork for the inactivation of E.coli, Listeria, and Salmonella. For marinated pork treated at 14 MPa, 45 oC for 40 min, the reduction of Listeria was about 2.5 and 1.9 logs in soy sauce and hot pepper marinades, respectively. Ferrentino et al. (2013) treated dry cured ham pieces at 8 and 12 MPa, 35 to 50 oC for 5-60 min. Listeria was totally inactivated (initial level 107 CFU/g) at 50 oC, 12 MPa, 15 min. Lower initial loads required less severe conditions for total inactivation. The process slightly affected the colour and sensory attributes of the product.
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3.2. Seafood Wei et al. (1991) treated shrimp spiked with Listeria and reduced the surviving numbers by 2 logs when treating with DPCD at 6.18 MPa, 35 oC, 2 hrs. They reported that N2 under the same conditions had no effect on the microorganism. Meujo et al. (2010) studied the reduction of the trapped microorganisms in the digestive system of oysters by exposing them to two treatments: 1) 10 MPa, 37 oC for 30 min, 2) 17.2 MPa 60 oC for 60 min. Total aerobic plate counts showed that treatments 1 and 2 reduced the initial loads by 2 and 3 logs, respectively. A 6 logs reduction was reported for a strain of Vibrio. They also observed that treatment 2 resulted in the release of the adductor muscle, which may be useful in shucking the oyster. Sensory analysis revealed no significant difference regarding physical appearance, smell and texture of treated and untreated oysters. Ji et al. (2012) used a neural network of 3 x 5 x 2 layers to optimize the process parameters of pressure, temperature and treatment time of shrimp to reduce microbial loads. They found that 15 MPa, for 26 min at 55 oC reduced the microbial load by 3.5 logs. They also observed a cooked appearance of the final product. De Matos et al. (2013) treated oysters with DPCD to reduce Vibrio parahaemolyticus. The amount of CO2 used was 1:0.8 CO2:product by mass. Pressures from 8 to 20 MPa, were used at 34 oC. Using exposure times from 0.25 to 6 hr, they developed the inactivation kinetics of the organism. At 0.5 hr 75% of the initial load was inactivated. Total reduction of the initial load was observed around 3 hr (about 2 logs). They also used pressure cycles and different depressurization rates. 3.3. Plant matter Haas et al. (1989) treated herbs (chives, thyme, oregano, parsley, mint) at 5.4 MPa and 45 oC for 2 hrs. From a maximum initial load of 108 CFU/g, they reported total inactivation of aerobic counts. They noted some positive or negative consequences on aromas depending on the herb. The same authors also treated whole strawberries, melon, and cucumber to delay surface molding. Although “positive” results were observed for strawberries, “gross tissue destruction” occurred. Kuhne and Knorr (1990) treated celery sticks and leaves at 62.8 MPa and 40 oC for 30 min. They reported that the total plate count was reduced by about 4 logs from an initial load of about 107 CFU/g. Mazzoni et al. (2001) treated alfalfa seeds with DPCD at 27.6 MPa and 50 oC for 60 min. They reported a decrease of E.coli of about 1 log, and total aerobic bacterial count of less than 1 log. Calvo et al. (2007) treated cocoa powder with DPCD at 30 MPa and 65 oC for 40 min. The effect of treatment on aerobic thermophilic and mesophilic organisms was measured. With dry powder, no effects were observed. When 5 to 10% water was added then high temperatures (65 or 80 oC) resulted in total inactivation. In addition spiked fungi spores were inactivated at 30 MPa, 80 oC, for 30 min after addition of 5% water. Dehghani et al. (2008) tested different DPCD treatment conditions on heavily contaminated ginseng (about 107 CFU/g). The best results were obtained at 10 MPa, 60 oC for 15 hr, where less than 3 log reductions was observed, not enough to comply with minimum regulatory levels. When small amounts of water/ethanol/H 2O2 were added to CO2, complete inactivation of bacteria and fungi was achieved at 10 MPa, 60 oC and 6 hr. Zhong et al. (2008) treated fresh spinach leaves with DPCD at 7.5 to 10 MPa, 40 oC for 40 min to reduce E.coli. They reported 5 logs reduction to undetectable levels. Subcritical state was not nearly as effective. They also observed discoloration of the leaves and changes in texture (decreased firmness). Jung et al. (2009) treated alfalfa seeds with DPCD to reduce E.coli, Listeria and Salmonella. Pressures from 10 to 20 MPa, temperatures from 35 to 45 oC for 5 to 15 min were applied. Germination percentage was also measured. 20 MPa, 45 oC for 15 min reduced the levels of the pathogens by 7 logs. However, at the severe treatment conditions germination was impaired. If conditions were optimized for germination capability, then at 15 MPa, 35 oC for 10 min the reduction of E.coli was about 3.5 logs. Calvo and Torres (2010) treated paprika powder with DPCD. Pressures from 6-30 MPa, temperatures less than 95 o C, and exposure times from 10-150 min in a continuous CO2 flow system. Various initial moisture contents < 35% were tried. Initial water content and temperature were the influential parameters for microbial reduction. Around 35% water and 6 MPa, about 1.5 logs reduction was obtained. When pressure was increased to 30 MPa, 5 logs reductions were reached. They concluded that higher pressures should be avoided to minimize extraction of oleoresins. The final paprika retained its colour.
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Valverde et al. (2010) treated fresh cut conference pears with DPCD to reduce Saccharomyces cerevisiae. Continuous CO2 flow at pressures 6-30 MPa, 25-55 oC for 10-90 min was used. DPCD at 55 oC achieved 5 logs reduction, while it was necessary to go to 70 oC for the same reduction with heat. Relatively low (<6 MPa) pressures and exposure times in the order of minutes were required. They observed some darkening at the surface, and some texture loss. Niu et al. (2010) treated apple slices by DPCD and mild heat. Then they extracted apple juice from these slices. Temperatures from 25 to 65 oC were used at 20 MPa for 20 min in a batch system. They investigated the residual activities of pectin methylesterase (PME) and polyphenol oxidase (PPO) between DPCD and mild heat (65 oC). PPO was totally inactivated by DPCD, and its residual activity by mild heat was 36%. DPCD also resulted in a residual activity of PME of 18%. Ferrentino et al. (2012) treated fresh-cut coconut pieces with DPCD. They evaluated the microbial, textural and microstructure effects at 8-12 MPa, 24-45 oC, for 5 to 60 min. Treatment of 15 min at 45 oC and 12 MPa resulted in 4 logs CFU/g reduction of mesophilic microorganisms, lactic acid bacteria, total coliforms, and yeasts and molds. Although hardness was not affected, the microstructure exhibited irregularities. 4. Nitrous oxide (N2O) Nitrous oxide has critical properties close to that of CO2 (critical temperature: 36.4 °C and critical pressure: 7.25 MPa). It is a colourless, non-flammable gas at room temperature, with a slightly sweet odour and taste. It is used in dentistry for anaesthetic and analgesic effects. Liquid nitrous oxide is a good solvent for many organic compounds. Some properties of N2O cause concern: at higher temperatures it is an oxidizer similar to molecular oxygen. Its pressure curve is very sensitive to temperature, and it can reach decomposition temperatures due to adiabatic heating with pressure. N2O is a greenhouse gas 300 times more potent than CO2. It causes ozone depletion. It oxidizes the cobalamin form of vitamin B12. Animal studies show adverse reproductive effects on pregnant females. Qadir and Hashinaga (2001) used N2O mixed with oxygen to treat climacteric and non-climacteric fruits inoculated with various fungi. Ratios of 80:20 of N 2O:O2 were used with storage of the fruits at 20 °C. They found that that regardless of the physiological nature of the fruits or group of fungi, N 2O delayed the appearance of disease and reduced the lesion growth rate. This response to N2O was dose and time dependent. Spilimbergo et al. (2007) used pressurized N2O to inactivate yeasts in fresh apple juice. Pressures of 6-20 MPa, at 36 °C for 1–25 min were used in a multi-batch equipment. They reported that at 10 MPa they achieved complete inactivation of the yeasts (initial load 103 to 105 CFU/ml) in 5 min. Later, Gasperi et al. (2009) continued this work, and treated fresh apple juice with pressurized CO2 or N2O at 10 MPa, 36 °C for 10 min with stirring, and evaluated the quality of the resulting product by sensory tests. Although chemical parameters (total solids, organic acids, polyphenols, etc.) did not show any difference with untreated juice, analysis of headspace showed a loss of volatiles. Peach and kiwi juices were also treated with CO2 or N2O (Spilimbergo and Ciola, 2010). Treatment conditions of 35 oC, 10 MPa for 15 min resulted in total inactivation of natural flora, as well as Saccharomyces cerevisiae (105 CFU/ml) both with CO2 and N2O. They did not detect any significant differences between treated and untreated juices regarding physic-chemical attributes of sensory properties. When milk is treated with high pressure CO2, it may coagulate due to the lowering of pH by CO2 dissolving in water. Spilimbergo (2011) used pressurized N 2O at 12 MPa, with temperatures from 40-50 °C to process milk. She reported that at 50 °C, in slightly more than 10 min there were no viable organisms detected after the treatment. There was no significant difference between the pH and acidity values of the untreated samples and samples treated for 60 min. This is an advantage in cold-pasteurizing milk and other pH sensitive products. 5. Combinations of non-thermal processes The promise of the simultaneous use of non-thermal technologies is that their synergistic effect may lower the severity of the process and/or the treatment time compared with either technology applied alone. Of course if one technology were continuous, it would be beneficial if its combination technology also were continuous. In this section, examples of the combination of DPCD with other non-thermal technologies will be discussed.
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5.1 DPCD and ultrasound The use of high-power ultrasound (HPU) in combination with DPCD is a relatively new concept. The promise of ultrasound is very effective mixing, and therefore reducing the time needed to reach CO 2 saturation especially in batch systems. Although not well understood, there is also a possibility that cavitation may help in the saturation of CO2 in the liquid phase. Since ultrasound consists of high pressure and low pressure regions, during the compression cycle the solubility of CO2 in the liquid will increase by increasing the driving force for saturation. Then in the lowpressure cycle, the dissolved CO2 may supersaturate due to the decrease of pressure. Ortuño et al. (2012) combined high power ultrasound (HPU) with batch DPCD to inactivate E.coli in broth and in orange juice. They used pressures from 10 to 35 MPa, temperatures from 31 to 41 °C, and measured the process times necessary to inactivate E.coli using DPCD only, HPU only, and DPCD + HPU. At similar treatment conditions of pressure and temperature, DPCD by itself required 50 min to reduce E.coli by 8 logs. Using both DPCD + HPU reduced the time required by 95%. This time achieved in a batch system is shorter than that in a continuous system (between 5 to 10 min). The authors concluded that the combination of DPCD + HPU can achieve the same inactivation rate in a much shorter time, and using milder process parameters (pressure and temperature). Ortuño et al. (2013a) also used the combination of DPCD + HPU in broth, apple juice, and orange juice to inactivate Saccharomyces cerevisiae in a batch system. Pressures from 10 to 35 MPa, temperatures from 31 to 41 °C were used. With DPCD alone, 6.7 logs reduction was obtained at 35 MPa, 36 °C for 140 min. DPCD + HPU under the same pressure and temperature reached the same inactivation level in 2 min. They reported that the medium affected the rate of inactivation. The solids content could affect viscosity, which affects cavitation. Ortuño et al. (2014) also investigated the effect of using DPCD + HPU on enzyme inactivation in a batch system. The kinetics of inactivation of PME in orange juice, as well as those of E.coli and S. cerevisiae were determined. An increase in temperature and pressure increased the inactivation rate of E. coli, S. cerevisiae and PME. The medium influenced the effect of pressure and temperature increase on the inactivation rate. Total inactivation of PME was not achieved. The possible advantages of using the combination of continuous DPCD with HPU are exciting. If the same drastic time requirement for inactivation can be achieved as in the case of batch systems, this will potentially reduce the residence time of DPCD, and therefore reduce capital costs of building continuous DPCD systems. 5.2 DPCD and high hydrostatic pressure The advantage of high hydrostatic pressure (HHP) is to process foods that are already packaged, and therefore do not require concern about post-processing contamination. Although HHP effectively eliminates microorganisms and stabilizes liquid foods, it does not inactivate key enzymes that reduce the quality. In fact, HHP may increase the activities of enzymes such as e.g. PPO. On the other hand, DPCD as a continuous operation needs aseptic filling to containers, but can inactivate enzymes that remain after HHP processing. Therefore it is logical to combine these technologies to benefit from their individual advantages. Corwin and Shellhammer (2002) used fresh squeezed orange juice to measure the PME activity after carbonation of the juice at 1 atm added pressure, and treatment with HHP at 500 MPa (3 min) or 800 MPa (1 min) at either 25 °C or 50 °C. Also, tyrosinase, Lactobacillus plantarum and E. coli were prepared in buffer systems, and carbonated as described before. The average CO 2 concentration in the PME liquid was 2.1 volumes of CO 2 / volume of sample. They calculated this as a 0.2% molar basis. The effects of carbonation alone, HHP alone, and HHP + carbonation were measured. After 500 MPa for 3 min, HHP only resulted in % remaining activity of PME of 86%. With CO2, this was reduced to 56%. At 800 MPa, there was no significant difference between HHP alone and HHP +CO2 (7.8% and 6.8%). At 50 °C, there was no significant difference between HHP only and HHP + CO2 at both 500 and 800 MPa. The effect of added CO2 to PPO residual activity was significant: At 25 °C and 500 MPa, HHP only resulted in a residual activity of 98.5%, when CO 2 was added this was reduced to 59%. At 800 MPa, HHP alone resulted in 91% residual PPO activity, addition of CO 2 reduced this to 51%. At 50 °C, and 800 MPa, HHP alone resulted in 73% residual PPO activity, and addition of CO 2 reduced this to 21%. Addition of CO2 further reduced the Lactobacillus count by 2 logs at 25 °C compared to HHP alone, at 365 and 455 MPa. However, for E.coli both at 500 and 600 MPa, there was no additional reduction with the addition of CO 2. In a similar study, Boff et al. (2003) used CO2-assisted HHP to process orange juice, and evaluated quality attributes during 4 months of storage at 4 and 30 °C. HPP + CO2 produced a cloud-stable orange juice with more ascorbic acid and flavour volatiles than thermally processed juice (p < 0.05).
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Park et al. (2002) studied the effects of sequential application of DPCD and HHP on the safety and shelf life of carrot juice. A combined treatment of 4.9 MPa DPCD (5 oC, 5 min) and 300 MPa-HHP (25 oC, 5 min) completely inactivated the aerobic microorganisms. Also, a process of 4.9 MPa DPCD and 600 MPa-HHP effectively inactivated PPO, PME and lipoxygenase (LOX) enzymes, with residual enzyme activities of less than 11%, 35%, and 9%, respectively. These levels were 2-3 folds lower than that of DPCD or HHP treatments alone. At low HHP pressure of 200 MPa, addition of 4.9 MPa-DPCD (5 oC, 5 min) treatment prior to HHP (25 ºC, 5 min) improved the inactivation of the PPO, LOX and PME enzymes in carrot juice with a residual activity of 35%, 17% and 45%, respectively, compared with the residual activity of DPCD (40%, 20% and 50%, respectively) and HHP (83%, 78% and 80%, respectively) treatments. The enzyme activities had strong correlation with pH, which was dependent on the pressure of CO2. The colour and cloud of treated carrot juice were significantly affected by DPCD but not by HHP. However, sequential application is not desirable for the industry since it requires more processing time. Ortuño et al. (2013b) used simultaneous application of DPCD and HHP on feijoa puree. Samples were treated with HHP only (HHP), or HHP+carbonated (HHPcarb), or HHP+carbonated with added 8.5mL CO 2/g sample (HHPcarb+CO2) at 300, 450 and 600MPa, 25 oC, for 5 min. The results showed that HHP increased the residual enzyme activity (REA) of POD in HHP sample at 300 MPa to 140 ± 5%, but decreased to 60 ± 9% and 22 ± 13% at 450 MPa and 600 MPa, respectively. The addition of CO2 affected the REA of POD in samples at all tested pressures. The REA of PME significantly decreased in HHPcarb+CO 2 samples from 73 ± 14% at 300 MPa to 53 ± 3% at 600 MPa. The addition of CO2 enhances enzyme inactivation with HHP, which did not affect the colour of the puree, compared with puree treated with HHP only. It was recommended that more research needed to be conducted to understand the mode of enzyme inactivation by the simultaneous HHP and DPCD treatments. We recently investigated the effects of pressure (200-600 MPa), process time (1-13 min), pH (3.0-3.6), and CO2 levels (no CO2, CO2 saturation, and saturation with addition of 8.5 mL CO2/g sample) in a simultaneous HHP and DPCD process on POD, PPO and PME of feijoa puree, using response surface methodology (27 treatments). Increasing treatment time reduced all enzyme activities, while increasing CO 2 levels decreased PME activity, and pressure influenced PPO activity. The lower pH values tended to increase enzyme inactivation. The optimal process conditions of 13 min, 600 MPa, pH = 3, CO2 saturation, resulted in 74.3 ± 3.3%, 70.9 ± 2.6% and 53.9 ± 0.9% residual activity for POD, PPO and PME respectively. Combination of CO 2 with HHP induced synergistic enzyme inactivation, and reduction of pH did not prove to be the main factor. 5.3 DPCD and pulsed electric field The combination of pulsed electric field (PEF) and DPCD was studied with promising results. The hypothesis for the mechanism is that PEF aids the diffusion of CO 2 into the cell by causing electroporation, in which the lipid bilayer and proteins of cell membrane can temporarily destabilize due to the high voltage electric field. The cell plasma membrane becomes penetrable to small particles (i.e. CO2), resulting in swelling and then rupture of the cell membrane. Also, once the amount of CO2 builds up to a critical level within the cells, it influences the intracellular pH and extracts constituents that alter the structure of bio-membrane or disrupt the biological system. Spilimbergo et al. (2003) investigated the effects of combining pulsed electric field (PEF) and DPCD on inactivation of Escherichia coli, Staphylococcus aureus and Bacillus cereus. The microorganisms were first treated with PEF at up to 25 KV/cm (1-20 pulses, 5 second intervals) and then with DPCD at below 40 oC, and 20 MPa. The increasing electrical field strength and number of pulses decreased the microbial viability. A PEF treatment at 25 KV/cm, 10 pulses resulted in 2.9 and 3.3 log reductions of E. coli and S. aureus, respectively while B. cereus spores had a strong resistance to PEF (0.5 log reduction). Further treatment with DPCD (20 MPa, 34 oC, for 10 min) completely inactivated the bacterial species (7-8 logs reductions). A combined treatment of PEF at 25 KV/cm and 20 pulses and then DPCD at 20 MPa, 24 h at 40 oC partial inactivated B. cereus spores (3 logs reduction). The inactivation was increased with the increasing the DPCD treatment time (ie. contact time between CO 2 and the samples). PEF and DPCD had synergistic effect on all treated microorganisms. Pataro et al. (2010) applied the sequential treatment of PEF (6, 9, 12 KV/cm electric field strength and 10, 20 and 40 J/ml) and then DPCD (8, 11 and 14 MPa, for 3-30 min) treatment on Saccharomyces cerevisiae, and compared with samples treated with PEF or DPCD treatments alone. The maximum inactivation was achieved for PEF treatment at 12 kV/cm and 20 J/ml with 0.35 logs reduction, while for DPCD the maximum reduction was at 8 MPa, 35 oC, 3 min (3.13 logs reduction). The increasing pressure and time did not increase the yeast cell inactivation. Meanwhile, combined DPCD (8 MPa, 25 oC, 5 min) application after PEF resulted in total inactivation
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regardless the electric field level of PEF pre-treatment. Increasing the electric field strength and energy input significantly increased the inactivation effect of DPCD. The Peleg kinetics model was a good fit for the inactivation kinetics of this combination PEF and DPCD treatment, which takes into account the treatment time of DPCD. Other parameters were functions of the electrical field strength and the energy input. Further studies on real food materials were recommended to investigate the combined process on the physical and nutritional attributes of the products. 5.4 DPCD and irradiation Urbain et al. (1969) obtained a patent involving the use of irradiation of food containers with a pressurized headspace of CO2. Their claims stated pressure of 0.2 to 6 MPa, and gave examples of canned meat with CO2 pressures of 0.8 MPa. In an example given in the patent, defatted soybean meal (30% solids in water) was inoculated with Salmonella (104 CFU/ g), canned under a CO2 pressure of 0.68 MPa, and irradiated at 5 KGy. After incubation for 24 hr at 37 oC, the following results were obtained: control: 3.08 104 CFU/g, CO2 only 8.5 103 CFU/g, irradiation only 1.4 103 CFU/g, combined irradiation and CO2 <100 CFU/g. 6. Outlook The commercialization of the DPCD technology will depend on mathematically predicting microbial reductions and/or enzyme inactivation at process conditions (pressure, temperature, process time, CO 2 ratio, solubility of CO2, synergistic processes, etc.). In addition to the regulatory requirements, a demonstrated advantage over other processes in terms of quality, nutrients, and cost is needed for DPCD or its combined methods. Finally, the design and availability of continuous solids treatment will expand the application base of this technology. References Boff, J.M., Truong, T.T., Min, D.B., Shellhammer, T.H., 2003. Effect of Thermal Processing and Carbon-Dioxide Assisted High Pressure Processing on Pectinmethylesterase and Chemical Changes in Orange Juice. Journal of Food Science 68, 1179-1184. Calvo, L., Muguerza, B., Cienfuegos-Jovellanos, E., 2007. Microbial Inactivation and Butter Extraction in a Cocoa Derivative Using High Pressure CO2. Journal of Supercritical Fluids 42, 80-87. Calvo, L., Torres, E., 2010. Microbial Inactivation of Paprika Using High-Pressure CO2. Journal of Supercritical Fluids 52, 134-141. Choi, Y.M., Ryu, Y.C., Lee, S.H., Go, G.W., Shin, H.G., Kim, K.H., Rhee, M.S., Kim, B.C., 2008. Effects of Supercritical Carbon Dioxide Treatment for Sterilization Purpose on Meat Quality of Porcine Longissimus Dorsi Muscle. Lebensmittel-Wissenschaft und-Technologie 41, 317-322. Choi, Y.M., Bae, Y.Y., Kim, K.H., Kim, B.C., Rhee, M.S., 2009a. Effects of Supercritical Carbon Dioxide Treatment Against Generic Escherichia coli, Listeria monocytogenes, Salmonella typhimurium, and Escherichia coli O157:H7 in Marinades and Marinated pork. Meat Science 82, 419-424. Choi Y M, Kim O Y, Kim K H, Kim B C, Rhee M. S. 2009b. Combined Effect of Organic Acids and Supercritical Carbon Dioxide Treatments Against Nonpathogenic Escherichia coli, Listeria monocytogenes, Salmonella typhimurium and Escherichia coli O157:H7 in Fresh Pork. Letters in Applied Microbiology, 49, 510-515. Corwin, H., Shellhammer, T.H., 2002. Combined Carbon Dioxide and High Pressure Inactivation of Pectin Methyl Esterase, Polyphenol Oxidase, Lactobacillus plantarum and Escherichia coli. Journal of Food Science 67(2), 697-701. Damar, S., Balaban, M.O., 2006. Review of Dense Phase CO2 Technology: Microbial and Enzyme Inactivation, and Effects on Food Quality. Journal of Food Science 71(1), R1-11. De Matos, K.H.O., Lerin, L.A., Soares, D., Smania, A., de Oliveira, J.V., Monteiro, A.R., 2013. Inactivation Kinetics of Vibrio Parahaemolyticus Naturally Occurring in Oysters Using Supercritical CO2 Treatment. Ibero American Conference on Supercritical fluids Cartagena de Indias, Colombia. Dehghani, F., Annabi, N., Titus, M., Valtchev, P., Tumilar, A., 2008. Sterilization of Ginseng Using a High Pressure CO2 at Moderate Temperatures. Biotechnology and Bioengineering 102, 569-576. Erkmen, O., 2000. Antimicrobial Effects of Pressurised Carbon Dioxide on Brochothrix thermosphacta in Broth and Foods. Journal Science of Food and Agriculture 80, 1365-1370. Ferrentino, G., Balzan, S., Dorigato, A., Pegoretti, A., Spilimbergo, S., 2012. Effect of Supercritical CO2 Pasteurization on Natural Microbiota, Texture and Microstructure of Fresh-Cut Coconut. Journal of Food Science 77(5), E137-E143. Ferrentino, G., Balzan, S., Spilimbergo, S., 2013. Supercritical CO2 Processing of Dry Cured Ham Spiked with Listeria monocytogenes: Inactivation Kinetics, Color, and Sensory Evaluations. Food Bioprocess Technology 6, 1164-1174. Fraser, D., 1951. Bursting Bacteria By Release of Gas Pressure, Nature 167, 33-34.
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Accepted for oral presentation in CAFEi2014 (December 1-3, 2014 – Kuala Lumpur, Malaysia) as paper 320.
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