Supercritical carbon dioxide technology: A promising technique for the non-thermal processing of freshly fruit and vegetable juices

Trends in Food Science & Technology 97 (2020) 381–390

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Supercritical carbon dioxide technology: A promising technique for the nonthermal processing of freshly fruit and vegetable juices

T

Eric Keven Silvaa,b, M. Angela A. Meirelesb, Marleny D.A. Saldañaa,∗ a b

Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB, T6G 2P5, Canada LASEFI/DEA/FEA (School of Food Engineering)/UNICAMP (University of Campinas), Rua Monteiro Lobato, 80, Campinas, SP, CEP 13083-862, Brazil

A R T I C LE I N FO

A B S T R A C T

Keywords: High-pressure carbon dioxide Microbial inactivation Enzymatic inactivation Sensory attributes Shelf-life

Background: The new global trends for consuming natural products rich in bioactive compounds and healthpromoter phytochemicals have increased the modern consumer's interest in fruit and vegetable juices. But, the current technologies based on thermal treatments reduce the nutritional value and degrade sensory attributes of these products in relation to the fresh-like juices. Scope and approach: Supercritical carbon dioxide (SC–CO2) technology has emerged as a potential non-thermal technology for the inactivation of spoilage and pathogenic microorganisms and endogenous enzymes responsible for the deterioration of fruit and vegetable juices. Likewise, non-thermal SC-CO2 processing can preserve the compounds associated with beneficial health effects besides maintaining sensory attributes. Thus, the effects of the SC-CO2 technology on the microbial and enzymatic inactivation, nutritional compounds, physicochemical properties, sensory attributes and shelf-life of the fruit and vegetable juices are discussed. Key findings and conclusions: SC-CO2 technology is a promising technique for the processing of fresh fruit and vegetable juices in a non-thermal way. SC-CO2 processing is able to inactivate microbial and enzymatic load of plant-based juices in the temperature range of 35–55 °C and pressure range of 10–60 MPa. SC-CO2 treated juices are sensorially similar to the fresh-like products with their nutritional value and physicochemical characteristics very close to the unprocessed juices. Under cold storage conditions, the juices stabilized by SC-CO2 treatment achieved a microbial shelf-life of at least 20 days with quality attributes of freshly juice, depending on their processing parameters and type of juice. However, additional studies are required to perform process optimization, exploring the synergism among its main variables in the same way that economic viability studies are needed.

1. Introduction The global market for fruit and vegetable juices has increased in the last years due to new trends to consume natural beverages and food products. Modern consumers are more conscious of the relationship between the intake of bioactive compounds obtained from vegetable matrices with the maintenance of health and well-being. In this sense, fruit and vegetable juices meet the new worldwide demand. These natural beverages are rich in phytochemical compounds such as phenolics (flavonoids, tannins, quinones, among others), alkaloids (betalain, theobromine, theophylline, among others), and terpenoids (carotenoids, monoterpenoids, diterpenoids, triterpenes, sesquiterpenoids, among others). The health-promoting compounds obtained from plant matrices are associated with the prevention of hypertension, type 2 diabetes, depression, cancer, cardiovascular and several chronic

∗

diseases, including mental illnesses (Aune et al., 2017; X.; Liu, Yan, Li, & Zhang, 2016; Mamluk et al., 2017; Wang et al., 2017; Zhan et al., 2017). Conventional thermal technologies, such as pasteurization or sterilization treatments known as high-temperature and short-time (HTST), low-temperature long-time (LTLT), and ultra high temperature (UHT) processing, have been used to inactivate microorganisms and enzymes and thus extend the shelf-life of food products, including fruit and vegetable juices (Alongi, Verardo, Gorassini, & Anese, 2018; Cautela, Castaldo, & Laratta, 2018; Su et al., 2019). However, thermal treatments are associated with some undesirable effects on treated juices, such as physicochemical, nutritional, rheological and sensorial deterioration. Thermal processing results in off-flavors compounds besides promoting the degradation of nutritional compounds in several natural beverages from fruits and vegetables (Iqbal et al., 2019; Kaushik,

Corresponding author. 3-18A Agriculture/Forestry Ctr, 115 St - 90 Ave, Edmonton, AB, T6G 2P5, Canada. E-mail addresses: [email protected] (E.K. Silva), maameireles@lasefi.com (M.A.A. Meireles), [email protected] (M.D.A. Saldaña).

https://doi.org/10.1016/j.tifs.2020.01.025 Received 3 October 2019; Received in revised form 9 January 2020; Accepted 16 January 2020 Available online 23 January 2020 0924-2244/ © 2020 Elsevier Ltd. All rights reserved.

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E.K. Silva, et al.

addition, SC-CO2 technology is currently recognized as a fashion environmentally friendly technology (Sanli, Bozbag, & Erkey, 2012). However, the main advantage that makes SC-CO2 the most applied supercritical fluid in several research and production sectors is its low critical temperature, which allows the development of non-thermal processes. Pharmaceutical, cosmetic and food industries have a great interest in the development of non-thermal processes to increase the value or add value to their products. Many drugs used in pharmaceutical formulations, natural colorants-based cosmetics, and bioactive compounds responsible for the nutritional value of the food products are thermally labile (Chen et al., 2020; Purohit & Gogate, 2015). Besides, thermal treatments change the rheological properties and sensory characteristics of the products. Therefore, non-thermal processes developed using SC-CO2 technology can increase the overall quality of the processed products and preserve their health-promoting properties.

Gondi, Rana, & Srinivasa Rao, 2015; Putnik et al., 2019). The new challenge for the food industry worldwide in the next years is the investment, development, and implementation of non-conventional processing based on non-thermal and emerging technologies, such as supercritical carbon dioxide (SC–CO2) (Silva, Arruda, Eberlin, Pastore, & Meireles, 2019), high-pressure processing (Wibowo et al., 2019), high-intensity ultrasound (Dolas, Saravanan, & Kaur, 2019), cold plasma (Šimončicová, Kryštofová, Medvecká; Ďurišová, & Kaliňáková, 2019), pulsed electric field (Evrendilek, 2017), and others. Food product innovation has an important role to improve the competitiveness of the food industry. Large industries represented by their brands would only resist the changes in the food market through innovations in their production systems by providing safe and highquality products with preserved nutritional and sensory properties similar to non-processed foods (Martins, Oliveira, Rosenthal, Ares, & Deliza, 2019). Among many other technological applications, SC-CO2 technology has emerged as a potential technology for non-thermal processing of fruit and vegetable juices (Illera, Sanz, Beltrán, et al., 2018; Marszałek, Skąpska, Woźniak, & Sokołowska, 2015). Several food products and beverages were stabilized applying high-pressure CO2 treatment (Amaral et al., 2017; Guimarães, Silva, Freitas, Meireles, & Cruz, 2018). SC-CO2 processing was able to inactivate microorganisms and enzymes in liquid food products non-thermally, even using mild temperature conditions (Smigic, Djekic, Tomic, Udovicki, & Rajkovic, 2019). Thus, the development and improvement of this new promising emerging technology can bring opportunities to innovation in the food production sector related to natural beverages like fruit and vegetable juices. Therefore, the aim of this review was to discuss the potential of the SCCO2 technology as a novel non-thermal treatment for stabilization of fruit and vegetable juices, presenting their fundamentals, advantages, and challenges. In addition, the effects of the non-thermal SC-CO2 processing on the nutritional compounds, physicochemical properties, sensory attributes, and shelf-life of the plant-based juices as well as its microbial and enzymatic inactivation mechanisms were discussed.

3. Non-thermal SC-CO2 treatment of fruit and vegetable juices 3.1. General The consumer market demand for fresh-like natural beverages such as fruit and vegetable juices has driven food industries to invest in nonthermal technologies. In addition, emerging technologies have gained interest and acceptance as new food processing methods by modern consumers. Among these, SC-CO2 technology has been proposed as a promising non-thermal treatment for the production of fruit and vegetable juices with high-value-added compounds (Benito-Román et al., 2019; Murtaza et al., 2019). Various natural beverages were stabilized using SC-CO2 technology, such as strawberry juice (Marszałek et al., 2015), apple juice (Illera, Sanz, Beltrán, et al., 2018), orange juice (Briongos et al., 2016), beetroot juice (Marszałek, Krzyżanowska, et al., 2017), and carrot and celery juices (Marszałek, Krzyżanowska, Woźniak, & Skąpska, 2016). In relation to other non-thermal emerging technologies that have gained highlight in recent years such as high-pressure processing (HPP) and high-intensity ultrasound (HIUS), SC-CO2 technology has its main advantage to allow processing at lower temperatures than the HIUS and much lower pressures than the HPP. The process temperatures employed in SC-CO2 treatments are usually below 50 °C. The HIUS processes are classified according to the temperature range employed into sub-lethal (< 45 °C) and lethal (> 45 °C) (Anaya-Esparza et al., 2017). Aadil et al. (2015) processed grapefruit juice using HIUS technology for the inactivation of enzymes, such as pectinmethylesterase (PME), peroxidase (POD) and polyphenoloxidase (PPO) and microorganisms (total plate count, yeast and mould). The optimal HIUS process condition were obtained at 60 °C and 60 min applying 420 W. Saeeduddin et al. (2015) evaluated the HIUS processing of pear juice aiming to inactivate PME, POD and PPO and microorganisms. The HIUS treatment at 65 °C and 525 W for 10 min showed the best results in retention of ascorbic acid and reduction in enzyme activities and complete microorganism inactivation. While SC-CO2 technology works with pressures between 8 and 60 MPa, HPP requires pressure ranges from 100 to 800 MPa for juice processing (Barba et al., 2017). Buerman, Worobo, and PadillaZakour (2020) inactivated spoilage fungi employing 600 MPa for 3 min in apple juice. Stinco et al. (2019) treated carrot juice with HPP and observed that the highest inactivation of POD (31%) and PPO (57%) was achieved with 600 MPa and 300 MPa, applied in three cycles. Lower process temperatures and pressures reduce the operational cost and initial investment. Moreover, handling the pressurized CO2 system is easier and safer than the HPP system.

2. SC-CO2 technology fundamentals and applications Supercritical fluids are pure substances that are above their critical temperature and pressure. In the supercritical phase, pure substances exhibit advantages in relation to other thermodynamic phases (solid, liquid, and gas) because their physical and chemical properties are between the liquid and gas phase. Besides that, their properties are easily adjustable by small changes of temperature and pressure. Supercritical fluids have densities similar to the liquids and viscosities nearby to the gases. Their diffusion coefficients are greater than those presented by liquids. These physical properties combined with their absence of surface tension allow their quick penetration into the cells, particles, and polymeric structures (Brunner, 2005). The transport properties of supercritical fluids make them suitable for various applications, such as extraction of phytochemical compounds (Moraes, Zabot, & Meireles, 2015; Saldaña et al., 2002, 2015), particle engineering loaded with biologically active substances (Rosa, Alvarez, Albarelli, Santos, Meireles, & Saldaña, 2020; Saldaña, dos Reis Coimbra, & Cardozo-Filho, 2015), impregnation of compounds of interest into polymeric matrices (Buratto, Hoyos, Cocero, & Martín, 2019), reaction medium (Ciftci & Saldaña, 2012; dos Santos, Meireles, & Martínez, 2017), and microbial and enzymatic inactivation (Marszałek, Krzyżanowska, Woźniak, & Skąpska, 2017). Among the supercritical fluids, SC-CO2 is the most commonly used for different applications in several fields due to its critical properties, which are easily achieved. The critical pressure and temperature of the CO2 are 7.38 MPa and 31.2 °C, respectively. Carbon dioxide presents advantages such as being inert to oxidation reactions, non-flammable, non-corrosive, non-toxic, GRAS (generally recognized as safe) solvent, and low-cost because it is naturally available in the atmosphere. In

3.2. CO2 solubility A mechanism explaining how SC-CO2 acts on the microorganisms and enzymes promoting their inactivation is not still fully understood. 382

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the high pressure pump. The residence time was adjusted by setting the flow rate of the system (coconut water + CO2) flowing through the holding coil. Thus, technical solutions to enhance the CO2 dispersion in liquid media are needed to improve the enzymatic and microbial inactivation, aiming to scaling up the process. Several patents have described methods for the CO2 dispersion in liquid media based on microbubbles (Osajima, Shimoda, Kawano, & Okubo, 1998) and membrane contactor (Sims, 2001). On the other hand, the development of SC-CO2 processing in a pseudo-continuous operational mode could be a promising alternative to the non-thermal treatment of natural beverages obtained from fruit and vegetable juices since the operating time is one of the most important variables for the microbial and enzymatic inactivation. Working in a pseudo-continuous operational mode allows longer processing time resulting in a better CO2 diffusion into plant material cells than in a continuous mode. In a pseudo-continuous process, two or more highpressure reactors can work in parallel, simulating a continuous operational mode behavior. This operational mode enables the simulation of a continuous operational mode by intercalating the charge/processing/ discharge steps of each reactor. Thus, the fruit or vegetable juice is simultaneously charged and discharged from the process (Moraes et al., 2015).

However, knowledge of CO2 solubility in natural beverages like fruit and vegetable juices is of great interest for a better understanding of the CO2 action as a pasteurization agent (Illera, Sanz, Beltrán, & Melgosa, 2019). To date, few studies evaluated CO2 solubility in liquid foods or model systems similar to the fruit and vegetable juices. Ferrentino, Barletta, Donsì, Ferrari, and Poletto (2010) studied the CO2 solubility in a complex solution of water-malic acid-ascorbic acidpectin-glucose-sucrose, simulating the composition of apple juice, and in a commercial apple juice sample at the pressure and temperature range of 7.5-15.0 MPa and 35–60 °C, respectively. The authors observed that the CO2 solubility was inversely proportional to the glucose and sucrose concentration in both samples. Likewise, organic acids slightly affected CO2 solubility in the model system and real apple juice. Illera et al. (2019) evaluated the CO2 solubility in apple and carrot juice in the pressure range from 8 to 20 MPa and in the temperature range from 35 to 45 °C. They verified that the presence of sugars led to lower solubility values of CO2 in the juices. 3.3. Operational modes The equipment used for SC-CO2 processing of liquid foods is specific to each application and the process may be operated using any operational mode, such as batch, semicontinuous or pseudo-continuous and continuous. For a comprehensive review regarding the operational modes of the SC-CO2 processing applied to the pasteurization of liquid products refer to Perrut (2012). Aiming to process large volumes of natural beverages, the development of a continuous process is highly desirable, however, the continuous systems require long treatment times besides high pressures (Paniagua-Martínez, Mulet, GarcíaAlvarado, & Benedito, 2018). A continuous high-pressure CO2 system was used to treat orange juice in a non-thermal process at ambient conditions with pressures from 14 to 107 MPa, residence times from 3 to 10 min, and CO2/juice ratios from 0.1 to 1.0. A detailed study was carried out to evaluate the effects of process conditions on microbial inactivation and quality properties of the orange juice. Residence time followed by pressure had significant effects on microbial inactivation, whereas CO2/juice ratio did not exhibit influence on microbial load. The treatment resulted in a 5-log reduction of the natural flora of spoiled juice, and decreased up to 5 cycles of pathogenic Escherichia coli O157:H7, Salmonella typhimurium, and Listeria monocytogenes. Regarding orange juice's quality properties, the results for the sensory analysis demonstrated that no difference between fresh and treated juice after two weeks of refrigerated storage at 1.7 °C was observed. On the other hand, pectin methylesterase (PME) was not completely inactivated (Kincal et al., 2006; Kincal et al., 2005). Likewise, Fabroni, Amenta, Timpanaro, and Rapisarda (2010) processed freshly squeezed blood orange juice using a continuous highpressure CO2 pilot system at 23 MPa, 36 ± 1 °C, 5.08 L/h juice flow rate, and 3.91 L/h CO2 flow rate, which corresponded to a CO2 mass (g) per juice mass (g) ratio of 0.770. The residence time was 15 min. The total microbial inactivation was achieved at these conditions; however, the samples showed a lower inactivation of PME, with an average percentage remaining activity of 66.81%. The authors concluded that the continuous SC-CO2 treatment was not a promising alternative to conventional thermal processing but a new mild technology for the stabilization of blood orange juice. A process alternative to maximize the enzymatic inactivation during SC-CO2 treatment would be to increase the CO2 diffusion through the plant material cells by promoting good mixing and dispersion of CO2 during its contact with the juice. In the continuous system, CO2 and the juice were homogenized and pumped before flowing through the highpressure reactor. After the pumping step, the system reached the desirable pressure condition inside the reactor (Del Pozo-Insfran, Balaban, & Talcott, 2006). Damar, Balaban, and Sims (2009) processed coconut water using a continuous high-pressure CO2 system. The product and liquid CO2 were pumped separately and homogenized before reaching

3.4. Effects of non-thermal SC-CO2 processing on spoilage and pathogenic microorganisms SC-CO2 processing has been proposed as a novel non-thermal treatment technique for food products aiming to replace the conventional technique known as thermal pasteurization. However, many conflicts among the hypotheses regarding the microbial inactivation mechanisms used by this emerging technology are found in the literature. Spilimbergo, Elvassore, and Bertucco (2002) stated that the modification of the extracellular and intracellular pH is the most probable effect that causes the microbial inactivation using the SC-CO2 technology. Garcia-Gonzalez et al. (2007) proposed a detailed microbial inactivation mechanism for the process based on SC-CO2 technology. The start of the inactivation process depends on the CO2 diffusion time into the microbial cells. The lag phase and generation time of microorganisms are increased due to exposure to CO2. The proposed mechanism corroborates with the results reported in the literature about the strong influence that the processing time has on the microbial inactivation. The interaction time between CO2 and the product during the SC-CO2 treatment directly influences the microbial inactivation rates (Ceni et al., 2016). Where high temperatures cannot be employed aiming to perform non-thermal processing, the diffusion coefficients should be increased, promoting turbulence inside the high-pressure reactor during the inactivation process. The efficiency of the SC-CO2 technology to inactivate several types of microorganisms in different food products has been evaluated over the last decades (Amaral et al., 2017; Fleury, Savoire, Harscoat-Schiavo, Hadj-Sassi, & Subra-Paternault, 2018; Omar et al., 2018; Perrut, 2012). This review presents relevant information focusing on recent studies that used SC-CO2 technology for inactivation of microorganisms in fruit and vegetable juices in the last years (2015–2020). Table 1 presents the last studies regarding SC-CO2 technology as a microbial inactivation technique of natural beverages. To the best of our knowledge, few studies focusing on microbial inactivation applied to juice processing were made in this period, evidencing how this emerging technology still needs more investments in research and development. All studies have demonstrated the non-thermal effects of the SC-CO2 treatment on microbial inactivation. Silva, Alvarenga, Bargas, Sant'Ana, and Meireles (2018) evaluated the non-thermal inactivation of Lactobacillus casei cells inoculated in apple juice employing up to 55 °C in their SC-CO2 treatments. Lactobacillus casei is known as a spoilage microorganism that promotes the physicochemical and sensory degradation of fruit juices. An expressive log reduction of 6.93 cycles was 383

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Manzocco, Plazzotta, Spilimbergo, and Nicoli (2017)

achieved at the maximum temperature level in the process conditions of 10 MPa, 30 min, and 70% CO2/juice (v/v) ratio in relation to the total volume of the high-pressure reactor. The authors explained that the smallest thermal treatment known is the thermization, a subpasteurization treatment of the raw milk, in which the milk is heated between 57 and 68 °C for 10–20 s. Therefore, 55 °C could still be considered for the development of non-thermal processing. Likewise, a moderate log reduction of 3.52 cycles was obtained at 35 °C, 15 MPa, 10 min, and CO2/juice (v/v) ratio of 40%, demonstrating that lower temperatures and faster process may be used for the inactivation of a lower microbial load. Fruits and vegetables naturally carry a microbial load after their harvest containing enteric pathogens such as Escherichia coli, Campylobacter spp., Salmonella spp., and other pathogens besides endogenous microorganisms responsible for the product degradation (Esteban-Cuesta et al., 2018; Kechero, Baye, Tefera, & Tessema, 2019). In this sense, food processing using any technology for the microbial inactivation assumes the use of high-quality raw materials. Several studies have reported the SC-CO2 treatment efficiency on the inactivation of microorganisms in their form of vegetative cells (Li, Wang, Zhu, Xu, & Hu, 2013; Neagu, Borda, & Erkmen, 2014; Sara Spilimbergo & Ciola, 2010). However, studies where an expressive inactivation of their spores only applying SC-CO2 in a non-thermal process are still scarce (Noman et al., 2018). Many studies have reported spores inactivation using SC-CO2 technology by associating this technology with ethanol as a cosolvent (Park, Choi, Kim, & Kim, 2013), peracetic acid (Sikin, Walkling-Ribeiro, & Rizvi, 2016), and nisin (Silva, Araujo, Souza Ferreira, & Kieckbusch, 2016). Casas, Valverde, MarínIniesta, and Calvo (2012) evaluated the effect of the high-pressure CO2 processing on the inactivation of Alicyclobacillus acidoterrestris spores in apple cream at pressure levels of 10, 15, and 35 MPa and a temperature range from 30 to 80 °C. The authors observed a log reduction up to 4 cycles at 30 °C and 10 MPa. On the other hand, Porębska, Sokołowska, Skąpska, and Rzoska (2017) also assessed the inactivation of Alicyclobacillus acidoterrestris spores in apple juice using SC-CO2 technology. A non-thermal and thermal treatment was applied to the natural beverage at 50 °C and 75 °C, respectively. They observed that in a non-thermal way, an inactivation of 1.0 log cycle was reached while up to 3.5 log reduction was possible employing the higher temperature. However, the authors used a CO2/juice ratio of 30% in relation to the total volume of the high-pressure reactor. The CO2/juice (v/v or w/w) ratio process variable has been neglected in SC-CO2 batch and continuous processes. Many studies fixed the volume relationship between the CO2 and high-pressure reactor. Therefore, studies focusing on the SC-CO2 process optimization with all variables (pressure, temperature, processing time/residence time, and CO2/juice ratio) are still needed to increase the inactivation rate of spores in fruit and vegetable juices.

* The percentage of CO2 volume was evaluated in relation to the total volume of the high-pressure reactor.

Lactic acid bacteria and yeast were completely inactivated at 12 MPa, 35 °C, and 10 min Pressure: 6–18 MPa Temperature: 20–45 °C Process time: 30 min CO2 volume ratio: Mesophilic and lactic acid bacteria, yeasts and moulds Apple

Vegetative cells

Batch

Torabian et al. (2018) 5.23 and 5.38-log reduction of mesophilic bacteria and yeasts, respectively, were achieved at 18 MPa, 45 °C and 90 min Pressure: 8–18 MPa Temperature: 35–45 °C Process time: 15–150 min CO2 volume ratio: 95.83% Mesophilic bacteria, yeasts and moulds Elderberry

Vegetative cells

Batch

Silva et al. (2018) Pressure: 10–20 MPa Temperature: 35–55 °C Process time: 10–30 min CO2 volume ratio: 10–70% Lactobacillus casei Apple

Vegetative cells

Batch

Up to 6.93 log reduction was observed at 10 MPa, 55 °C, 30 min and 70% CO2 volume ratio

Porębska et al. (2017) Inactivation of 1.0 log cycle at non-thermal condition (50 °C) and up to 3.5 log reduction at thermal condition (75 °C) Pressure: 60 MPa Temperature: 50 and 75 °C Process time: 20–40 min CO2 volume ratio: 30% Alicyclobacillus acidoterrestris Apple

Spores

Batch

Marszałek et al. (2015) Up to 3.7 log reduction was observed in mesophilic bacteria at 60 MPa, 45 °C, and 30 min Pressure: 10–60 MPa Temperature: 35–65 °C Process time: 10–30 min CO2 volume ratio: 72% Mesophilic bacteria, yeasts and moulds Strawberry

Vegetative cells

Batch

Reference Microbial inactivation results Experimental conditions Operational mode Form of the microorganism Microorganism Juice

Table 1 Effect of SC-CO2 processing on microbial inactivation in different fruit and vegetable juices.

E.K. Silva, et al.

3.5. Effects of non-thermal SC-CO2 processing on endogenous enzymes After postharvest, processing of fruits and vegetables, most endogenous enzymes still remain in their active form. Therefore, the enzymatic inactivation in fruit and vegetable tissues using SC-CO2 technology is a critical step to prevent the degradation and changes in the quality aspects of juices during storage, for instance, enzymatic browning and off-flavors formation. Thus, a high enzymatic inactivation level is required aiming to guarantee the maintenance of the sensory attributes and nutritional value similar to the fresh juices (BenitoRomán, Sanz, Illera, Melgosa, & Beltrán, 2019; Marszałek et al., 2016). Enzymes are biocatalysts with their structures based on proteins that present a high affinity to a specific substrate. The inactivation mechanism of enzymes is associated with the disruption of interactions responsible for their secondary and tertiary structures. Heat, chemicals and high-pressure are the main inductors of the protein denaturation, however, the mechanisms are substantially different. High-pressure treatments affect mainly the tertiary and quaternary structures of enzymes. The protein denaturation induced by high-pressure occurs due 384

385

PPO and POD

PPO, POD, PG, and PME

Apple

Carrot and celery

Batch

Batch

Batch

Batch

Batch

Batch

Batch

Batch

Batch

Operational mode

Pressure: 10–60 MPa Temperature: 31–55 °C Process time: 10–30 min CO2 volume ratio: 88%

Pressure: 10–60 MPa Temperature: 35–65 °C Process time: 10–30 min CO2 volume ratio: 90%

Pressure: 10–60 MPa Temperature: 35–65 °C Process time: 10–30 min CO2 volume ratio: 72%

Pressure: 10–60 MPa Temperature: 31–55 °C Process time: 10–30 min CO2 volume ratio: 88%

Pressure: 10–30 MPa Temperature: 35–45 °C Process time: 3–60 min CO2 volume ratio:

Pressure: 8.5–20 MPa Temperature: 35–55 °C Process time: 0–120 min CO2 volume ratio: 60%

Pressure: 10–20 MPa Temperature: 35–45 °C Process time: 0–120 min CO2 volume ratio: 60%

Pressure: 20 MPa Temperature: 25–65 °C Process time: 20 min CO2 volume ratio:

Pressure: 10 and 25 MPa Temperature: 40 and 55 °C Process time: 20 min CO2 volume ratio:

Experimental conditions

* PME: pectin methylesterase; POD: peroxidase; PPO: polyphenol oxidase; PG: polygalacturonase. **The percentage of CO2 volume was evaluated in relation to the total volume of the high-pressure reactor.

PPO and POD

Strawberry

PG, and PME

Tomato

PPO, POD, PG, and PME

PPO and PME

Apple

Beetroot

PPO

Quince

PME

PPO

Apple

Orange

Enzyme

Juice

Table 2 Effect of SC-CO2 processing on endogenous enzyme inactivation in different fruit and vegetable juices.

Marszałek et al. (2017)

Marszałek et al. (2016)

PPO and POD inactivation kinetics significantly depended on process parameters as well as the origin of enzymes (commercial or endogenous) The lowest and highest decimal reduction time was observed for PE and PG in celery juice were 200 and 1645 min, respectively

Marszałek et al. (2015)

Marszałek et al. (2017)

The lowest decimal reduction time was observed for PG and the highest for POD

The process resulted in the total inactivation of PPO and on average 85% inactivation of POD

Briongos et al. (2016)

Illera et al. (2018)

PME was nearly complete inactivated at 55 °C and 20 MPa while PG was found to be more resistant PME inactivation degree increased with pressure and temperature. After 60 min, PME was effectively inactivated at 40 °C and 30 MPa

Illera et al. (2018)

PPO and PME inactivation rate increased with pressure and temperature. PME was more resistant compared to PPO

Iqbal et al. (2018)

Murtaza et al. (2019)

PPO was inactivated up to 80% at 25 MPa and 40 °C and 95% at 25 MPa and 55 °C

Total inactivation of PPO was reached at 65 °C and up to 35% residual activity was observed at 55 °C

Reference

Enzymatic inactivation results

E.K. Silva, et al.

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indicator of the processing severity and maintenance of the product quality (Aguilar, Garvín, Ibarz, & Augusto, 2017). The main process condition responsible for nutritional degradation is associated with high processing temperatures due to the thermal sensitivity of several phytochemical compounds, however, other process conditions such as high-pressures and high-shear stress may also result in degradation of bioactive compounds in fruit and vegetable juices (Dolas et al., 2019). Non-thermal SC-CO2 processing allows the preservation of nutritional compounds mainly due to the use of lower temperature even in comparison to the other non-thermal emerging technologies such as HPP and HIUS (Bhattacharjee, Saxena, & Dutta, 2019). Oulé, Dickman, and Arul (2013) evaluated the influence of the SCCO2 treatment at 25 MPa and 40 °C on the ascorbic acid retention in freshly squeezed orange juice in comparison to conventional thermal processing carried out at 90 °C for 60 s. The orange juice treated with SC-CO2 retained 88% of its ascorbic acid content in relation to the untreated sample, while the thermally processed juice preserved only 57% of its ascorbic acid content. Silva, Arruda, et al. (2019) and Silva, Guimarães, et al. (2019) enriched apple juice with inulin, a prebiotic carbohydrate, and subjected this functional beverage to the non-thermal SC-CO2 processing employing a constant temperature of 35 °C at pressure levels of 10, 15, and 20 MPa for 10 min. The effects of the SC-CO2 treatments were compared to the untreated beverage regarding phenolic compounds (protocatechuic acid, rutin, and chlorogenic acid), sugars (fructose, glucose, sorbitol, and sucrose), and organic acids (citric and malic acid). The SCCO2-treated samples did not present differences in their nutritional profile in relation to the unprocessed inulin-enriched apple juice. Besides that, the inulin molecular chain was not broken into short chain fructooligosaccharides units, evidencing the capacity of the SC-CO2 technology to preserve the chemical structure of prebiotic compounds. The influence of the SC-CO2 processing on the anthocyanin stability, such as pelargonidin-3-glucoside, pelargonidin-3-rutonoside, and cyanidin-3-glucoside, in strawberry juice was studied by Marszałek et al. (2015). They compared the untreated juice with the samples processed at 45 °C for 30 min for the pressure levels of 30 and 60 MPa. The anthocyanins, pelargonidin-3-rutonoside and cyanidin-3-glucoside , were chemically stable to SC-CO2 treatment, however, pelargonidin-3-gucoside was slightly degraded at the high-pressure level in comparison to the unprocessed strawberry juice.

to water penetration into the protein structure. Water penetration into the protein matrix leads to conformational transitions resulting in its unfolding (Fernández-Lucas, Castañeda, & Hormigo, 2017). Marszałek et al. (2019) compared the effects of SC-CO2 and HPP on structural changes and activity loss of oxidoreductive enzymes such as POD and PPO. The authors observed differences in the inactivation behavior between both enzymes, but a lesser degree for the same enzyme submitted to the different treatments. They explained that the differences are related to their properties such as chemical structure and molecular weights and highlighted the influence of the disruptive mechanism associated with the two techniques applied to the enzyme structures. Murtaza et al. (2019) evaluated the PPO inactivation in fresh apple Malus domestica juice using SC-CO2 technology. The authors demonstrated the effects of the SC-CO2 on the conformational changes in PPO. From the conformational maps of protein structures, they showed that the increase of the pressure and temperature results in the loss of αhelix conformation and concluded that SC-CO2 caused dissociation, conformation and aggregation in PPO. Table 2 presents the recent findings reported in the literature in the last years (2015–2020) for the effects of SC-CO2 processing on the enzymatic inactivation of fruit and vegetable juices. Among the enzymes inactivated are pectin methylesterase (PME), peroxidase (POD), polygalacturonase (PG), and polyphenol oxidase (PPO). These endogenous deteriorative enzymes are associated with changes in color, flavor, consistency, juice cloud, and nutritional losses in plant-based products. On the other hand, the inactivation of enzymes such as lipoxygenase (LOX) and hydroperoxide lyase (HPL) was not evaluated. LOX has an important role in the degradation of carotenoids by a co-oxidation mechanism in the presence of free fatty acids. Likewise, HPL is responsible for the flavor stability of fruit and vegetable juices (Rodrigo, Jolie, Loey, & Hendrickx, 2007). From the results presented in Table 2 for the enzymatic inactivation of fruit and vegetable juices using SC-CO2 technology, some remarks are highlighted. All studies have demonstrated the importance of the mild temperature conditions on the inactivation of endogenous enzymes. The effectiveness of enzymatic inactivation seems to be achieved only under temperature conditions very close to the limit of what is meant as a non-thermal process, i.e. 55 °C. CO2 solubility into fruit and vegetable juices depends on mass transfer coefficients. Besides the temperature increase, the CO2 diffusion coefficients can be improved by promoting high turbulence levels inside the reactor during the SC-CO2 treatment. Moreover, the variety of fruit and vegetable has an important role in enzymatic inactivation. Different varieties for the same fruit or vegetable can present enzymes with different resistance to high-pressure carbon dioxide treatment. Illera, Sanz, Trigueros, Beltrán, and Melgosa (2018) studied the PME and PG inactivation in tomato juice using SC-CO2 technology. They evaluated the effects of pressure (8.5–20 MPa), and temperature (35–55 °C) on the inactivation kinetics from 0 to 120 min, using a CO2/ juice ratio of 60% in relation of the total volume of the high-pressure reactor. Nearly complete inactivation of the PME (1.5 ± 0.5%) was obtained at 55 °C and 20 MPa after 90 min of treatment, however, in this same process condition, PG was more resistant, presenting residual activities of around 60 ± 2%.

3.6.2. Physicochemical and physical properties SC-CO2 processing such as other high-pressure treatments promotes the homogenization of the fruit and vegetable juices, resulting in modifications in their rheological behavior as well as particle size distribution and microstructure (Amaral et al., 2018; Silva, Guimarães, Costa, Cruz, & Meireles, 2019). The beverage during the pressurization and depressurization step under CO2 high-pressure conditions flows quickly inside the reactor and through valves and tubing, increasing significantly its velocity. Thus, the beverage is submitted to the high shear stress besides the cavitation phenomenon, which promotes high turbulence in the system, improving the homogenization and particle size reduction. The high mechanical stress deforms the cell fragments from fruit and vegetable, resulting in changes in their shape and surface area (Augusto, Ibarz, & Cristianini, 2012). Illera, Sanz, Beltrán, et al. (2018) evaluated the effects of SC-CO2 batch treatment on the particle size distribution of cloudy juice from Golden delicious apples. They compared the non-thermal SC-CO2 processing at 45 °C and 20 MPa for 60 min with the untreated fresh juice using the average diameter calculated based on the average diameter of a sphere of similar area, known as the Sauter mean diameter (D32), and average diameter of a sphere of the same volume, known as the Brouckere diameter (D43). The unprocessed apple juice had D32 = 1.7 ± 0.1 μm and D43 = 109 ± 3 μm and their values were 0.21 ± 0.01 μm and 2.4 ± 0.2 μm, respectively, after the SC-CO2 treatment. Moreover, the volume-based particle size distribution was narrowed. The distribution

3.6. Effects of non-thermal SC-CO2 processing on quality parameters 3.6.1. Nutritional compounds In addition to microbial and enzymatic inactivation, a novel natural beverage stabilization treatment must preserve the nutritional properties of the fruit and vegetable juices. The ascorbic acid, carotene, lutein, lycopene and phenolic compound contents are widely used by the food industry to define the nutritional value of the natural beverages (Fernandez, Bengardino, Jagus, & Agüero, 2020; Gao et al., 2019; González, Vegara, Martí, Valero, & Saura, 2015). In particular, the retention of ascorbic acid (vitamin C) in juices has been used as a reliable 386

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important role in the marketing potential of the fruit and vegetable juices. Few studies evaluated the shelf-life of natural juices processed by SC-CO2 technology, regarding microbial quality and other parameters (Fabroni et al., 2010; Ferrentino, Plaza, Ramirez-Rodrigues, Ferrari, & Balaban, 2009; Torabian et al., 2018; Zou et al., 2016). Zou et al. (2016) compared the shelf-life of the mulberry Morus alba L. juice processed by SC-CO2 technology (15 MPa/55 °C/10 min), HPP (500 MPa/5 min), and the conventional thermal treatment (110 °C/ 8.6 s) during 28 days of storage at 4 °C and 25 °C. The SC-CO2 treated juice at the evaluated process conditions resulted in a shorter shelf-life in comparison to the other treatments, considering the microbial quality, however, it retained more phenols and anthocyanins due to its non-thermal effects. Under refrigerated conditions, SC-CO2 treated mulberry juice resulted in acceptable microbial levels even after 21 days of storage, presenting a count of 2.80 ± 0.02 and 2.03 ± 0.1 log10 CFU/mL of mesophilic bacteria and yeasts and moulds, respectively. Fabroni et al. (2010) evaluated the shelf-life of freshly squeezed blood orange juice processed at 13 MPa and 36 ± 1 °C using 0.385 g CO2/g juice. The microbial quality was analyzed performing the count of mesophilic bacteria and spoilage microorganisms that typically contaminate orange juice. The profile method based sensory analyses using a trained panel were carried out to determine the quality parameters of the juice such as freshness, flavor, acidity, bitterness, sweetness, off-flavor, color, intensity of taste, and intensity of the scent during cold storage. Both analyses, microbial and sensorial quality, were performed right after processing (time zero) and every 5 days under cold storage at 4 ± 1 °C for 30 days. The samples achieved a shelf-life of at least 20 days of refrigerated storage. Regarding nutritional value, the ascorbic acid and total anthocyanin contents were kept constant, similar to the fresh juice, in this cold storage time. The authors suggested that freshly squeezed blood orange juice treated with SC-CO2 technology was a novel product for a new market segment within a retail framework for freshly squeezed juices with a 20-day shelf-life. Marszałek et al. (2018) assessed the influence of pressure levels of 10, 30, and 60 MPa (45 °C/30 min) on the long-term cold storage of SCCO2-treated cloudy apple juice. The authors demonstrated that the phenolic compounds chemical stability during the cold storage was influenced by the pressure applied in the juice treatment since higher polyphenol retention was achieved when a higher pressure was used. Also, SC-CO2 treatment inhibited the browning reactions in stored apple juice. The total color differences of the samples after 10 weeks did not exceed 20 units, whereas untreated juice after 15 h had 40 units. They concluded that SC-CO2 treatment may be a promising innovative process to increase the phytochemical, nutritional, and physicochemical quality of cloudy apple juices. Iftikhar, Wagner, and Rizvi (2014) processed orange juice with addition of ethanol to SC-CO2 at 2% (v/v). They verified a maximum PME inactivation of 97% at 30 MPa and 40 °C for 60 min. The SC-CO2treated orange juice after 14 days of cold storage at 4 °C maintained PME activity at the same value right after its processing. Thus, SC-CO2 treatment resulted in a stabilized juice with similar qualities to fresh juice.

width index was reduced from 26 ± 7 to 5.8 ± 0.1. On the other hand, no significant changes were observed in physicochemical and physical properties such as soluble solid content, pH, ξpotential, titratable acidity, and color parameters in fruit and vegetable juices processed using SC-CO2 technology (Briongos et al., 2016; Ferrentino & Spilimbergo, 2017; Gasperi et al., 2009; Illera, Sanz, Trigueros, Beltrán, & Melgosa, 2018; Murtaza et al., 2019; Silva et al., 2019). The maintenance of these physicochemical and physical properties corroborates with obtaining processed juices similar to fresh-like products after the SC-CO2 processing. Illera, Sanz, Beltrán, et al. (2018) also demonstrated that the pH values, color parameters and ξ-potential of the cloudy juice from Golden delicious apples did not modify after the SC-CO2 treatment. Liu, Hu, Zhao, and Song (2012) processed watermelon juice using SC-CO2 technology at 50 °C and evaluated the effects of pressure (10, 20, and 30 MPa) and processing time (15, 30, 45, and 60 min) on the physicochemical properties of the beverage. The SC-CO2 treatment at all process conditions evaluated did not change the pH values and soluble solids content of the watermelon juice. 3.6.3. Sensory attributes The consumer demand for high organoleptic quality food products such as fresh-like products are increasing worldwide every year. One of the main advantages associated to the SC-CO2 processing is the preservation of sensory attributes of the processed product due mainly to non-thermal treatment. Overall, the literature regarding the effects of SC-CO2 technology on the sensory properties as well as the acceptance of the non-thermally processed juices by the consumers is still scarce. Most studies published in recent years have focused on process engineering aspects, aiming to improve the microbial and enzymatic inactivation. Few studies have demonstrated the effects of the SC-CO2 technology on the sensory quality of fruit and vegetable juices (Amaral et al., 2018; Cappelletti et al., 2015; Damar et al., 2009; Fabroni et al., 2010; Gasperi et al., 2009; Gunes, Blum, & Hotchkiss, 2005; Oulé et al., 2013). Cappelletti et al. (2015) compared the SC-CO2 treatment and thermal processing to stabilize the fresh coconut water and their effects on the sensory quality. The optimal SC-CO2 process conditions of pressure, temperature, and processing time were 12 MPa, 40 °C, and 30 min, respectively, for a log reduction of 5 cycles in mesophilic microorganisms, lactic acid bacteria, yeasts and moulds and a log reduction of 7 cycles of total coliforms. Thermal processing was performed at 90 °C for 1 min. At this process condition, the coconut water natural microbial flora was completely inactivated. The sensory analysis compared the coconut water processed by SC-CO2 technology versus the conventional thermal treatment with the untreated fresh coconut water. Sensory analysis was carried out using a comparative descriptive flash profiling by a panel of 16 trained judges to assess how the non-thermal process based on SC-CO2 technology and thermal processing affected sensory properties. According to the spider plot of the average profile of the coconut water samples for each sensory attribute, such as sweetness, acidity, butter, cardboard, cooked, floral, hazelnut, toasted bread, and others, the sample treated with SC-CO2 technology presented a profile similar to the untreated fresh coconut water while the thermally processed sample had formation of off-flavor compounds. Amaral et al. (2018) studied the effects of the SC-CO2 processing on the sensory attributes of whey-grape juice drink processed at pressure levels of 14, 16, and 18 MPa in a non-thermal way (35 °C) for 10 min in comparison to HTST-treated sample (72 °C for 15 s). All samples were evaluated according to a 9-point hedonic scale (9 = extremely liked; 5 = neither liked nor disliked; and 1 = extremely disgusted). The fifty consumers aged 16–38 years were asked to score the attributes such as aroma, texture, appearance, and overall impression. The beverages thermally treated exhibited the lowest scores in almost all attributes.

4. Critical observations The main process variables impacting the SC-CO2 performance on the microbial and enzymatic inactivation of the fruit and vegetable juices are pressure, temperature, processing time/residence time and the CO2/juice ratio. CO2 solubility into juices is a key property to the success of the SC-CO2 technology as a non-thermal process. Thus, the application of technical solutions to enhance CO2 dispersion in the liquid media is required. Likewise, most studies performed to date have still not evaluated the synergic effects among the main process variables. The CO2/juice ratio has been neglected as a process variable as

3.6.4. Shelf-life In addition to the previous quality parameters, shelf-life has an 387

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well as its synergism with the other variables to enhance the SC-CO2 performance as a new reliable process to stabilize fresh juices in all operational modes. In the recent findings reported in the literature (2015–2020), only batch operational mode has been evaluated mainly due to long time required for CO2 to diffuse throughout the suspended fragments of fruit and vegetable tissues in the juices, promoting the desired inactivation results. The main studies demonstrating the promising results of the continuous high-pressure CO2 system for the fruit and vegetable juices processing were published until 2010. However, more studies concerning the effects of the continuous mode on the enzymatic inactivation are still needed. Although technical feasibility of the SC-CO2 technology has been demonstrated for the non-thermal production of fresh fruit and vegetable juices, there are no studies in which the economic viability of the process has been evaluated. Studies regarding the economic viability of a pseudo-continuous or continuous process line are required to attract investments from the industrial sector.

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Fruit and vegetable intake and the risk of cardiovascular disease, total cancer and allcause mortality—a systematic review and dose-response meta-analysis of prospective studies. International Journal of Epidemiology, 46, 1029–1056. Barba, F. J., Mariutti, L. R. B., Bragagnolo, N., Mercadante, A. Z., Barbosa-Cánovas, G. V., & Orlien, V. (2017). Bioaccessibility of bioactive compounds from fruits and vegetables after thermal and nonthermal processing. Trends in Food Science & Technology, 67, 195–206. Benito-Román, Ó., Sanz, M. T., Illera, A. E., Melgosa, R., & Beltrán, S. (2019). Polyphenol oxidase (PPO) and pectin methylesterase (PME) inactivation by high pressure carbon dioxide (HPCD) and its applicability to liquid and solid natural products. Catalysis Today In press. Benito-Román, Ó., Sanz, M. T., Illera, A. E., Melgosa, R., Benito, J. M., & Beltrán, S. (2019b). Pectin methylesterase inactivation by high pressure carbon dioxide (HPCD). 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5. Conclusions and perspectives This review has confirmed the potential of the SC-CO2 technology for the processing of fresh fruit and vegetable juices in a non-thermal approach. Overall, SC-CO2 processing is able to inactivate microbial and enzymatic load of plant-based juices in the temperature range of 35–55 °C and pressure range of 10–60 MPa. However, additional studies are required to perform process optimization, exploring the synergism among the main process variables (pressure, temperature, processing time/residence time, and CO2/juice ratio) in the same way that economic viability studies are needed. The juices processed by this emerging technology when compared to those obtained using conventional thermal treatments and even other emerging technologies such as high-pressure processing and high-intensity ultrasound had several advantages, regarding nutritional, physicochemical, and sensory aspects. SC-CO2 treated juices are similar to the fresh-like products with their nutritional value and physicochemical characteristics very close to the unprocessed juices. Under cold storage conditions (4 ± 1 °C), the juices stabilized by SC-CO2 treatment achieved a microbial shelf-life of at least 20 days with quality attributes of freshly juice depending on their processing parameters and type of juice. Therefore, the nonthermal SC-CO2 processing is a promising technology since it meets all trends of the global market demand for natural functional beverages, promoting good health and well-being. Also, currently SC-CO2 technology is recognized as a fashion environmentally friendly technology and the development of new processes has been encouraged in many industrial sectors and research fields. Acknowledgements Eric Keven Silva thanks FAPESP (2018/14550–6) for his postdoctoral assistantship at University of Alberta. M. Angela A. Meireles thanks CNPq (302423/2015–0) for her productivity grant. Marleny D.A. Saldaña thanks the 2017–2018 McCalla Professorship award and the Natural Sciences and Engineering Research Council of Canada (NSERC, #04371–2019) for funding her research program on emerging processing technologies. References Aadil, R. M., Zeng, X.-A., Zhang, Z.-H., Wang, M.-S., Han, Z., Jing, H., et al. (2015). Thermosonication: A potential technique that influences the quality of grapefruit juice. International Journal of Food Science and Technology, 50, 1275–1282. Aguilar, K., Garvín, A., Ibarz, A., & Augusto, P. E. D. (2017). Ascorbic acid stability in fruit juices during thermosonication. Ultrasonics Sonochemistry, 37, 375–381. Alongi, M., Verardo, G., Gorassini, A., & Anese, M. (2018). Effect of pasteurization on in vitro α-glucosidase inhibitory activity of apple juice. LWT, 98, 366–371. Amaral, G. V., Silva, E. K., Cavalcanti, R. N., Cappato, L. P., Guimaraes, J. T., Alvarenga,

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