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

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Trends in Food Science & Technology

journal homepage: www.elsevier.com/locate/tifs

Supercritical carbon dioxide technology: A promising technique for the non- T

thermal processing of freshly fruit and vegetable juices
Eric Keven Silvaa,b, M. Angela A. Meirelesb, Marleny D.A. Saldañaa,∗
a
Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB, T6G 2P5, Canada
b
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: Background: The new global trends for consuming natural products rich in bioactive compounds and health-
High-pressure carbon dioxide promoter phytochemicals have increased the modern consumer's interest in fruit and vegetable juices. But, the
Microbial inactivation current technologies based on thermal treatments reduce the nutritional value and degrade sensory attributes of
Enzymatic inactivation these products in relation to the fresh-like juices.
Sensory attributes
Scope and approach: Supercritical carbon dioxide (SC–CO2) technology has emerged as a potential non-thermal
Shelf-life
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 optimi-
zation, 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 phe-
nolics (flavonoids, tannins, quinones, among others), alkaloids (beta-
lain, theobromine, theophylline, among others), and terpenoids (car-
otenoids, 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 ster-
ilization 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 ve-
getable juices (Alongi, Verardo, Gorassini, & Anese, 2018; Cautela,
Castaldo, & Laratta, 2018; Su et al., 2019). However, thermal treat-
ments are associated with some undesirable effects on treated juices,
such as physicochemical, nutritional, rheological and sensorial dete-
rioration. 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: engerickeven@gmail.com (E.K. Silva), maameireles@lasefi.com (M.A.A. Meireles), marleny.saldana@ualberta.ca (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.
E.K. Silva, et al.
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-conven-
tional 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 com-
petitiveness of the food industry. Large industries represented by their
brands would only resist the changes in the food market through in-
novations in their production systems by providing safe and high-
quality products with preserved nutritional and sensory properties si-
milar 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 produc-
tion sector related to natural beverages like fruit and vegetable juices.
Therefore, the aim of this review was to discuss the potential of the SC-
CO2 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.
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 appli-
cations, such as extraction of phytochemical compounds (Moraes,
Zabot, & Meireles, 2015; Saldaña et al., 2002, 2015), particle en-
gineering loaded with biologically active substances (Rosa, Alvarez,
Albarelli, Santos, Meireles, & Saldaña, 2020; Saldaña, dos Reis
Coimbra, & Cardozo-Filho, 2015), impregnation of compounds of in-
terest 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
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Trends in Food Science & Technology 97 (2020) 381–390
addition, SC-CO2 technology is currently recognized as a fashion en-
vironmentally 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 for-
mulations, 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 character-
istics of the products. Therefore, non-thermal processes developed
using SC-CO2 technology can increase the overall quality of the pro-
cessed products and preserve their health-promoting properties.
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 non-
thermal 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 ve-
getable 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 em-
ployed in SC-CO2 treatments are usually below 50 °C. The HIUS pro-
cesses 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), per-
oxidase (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 Padilla-
Zakour (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.
3.2. CO2 solubility
A mechanism explaining how SC-CO2 acts on the microorganisms
and enzymes promoting their inactivation is not still fully understood.
E.K. Silva, et al.
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 acid-
pectin-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 so-
lubility 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 opera-
tional 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, García-
Alvarado, & 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) pro-
cessed freshly squeezed blood orange juice using a continuous high-
pressure 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 high-
pressure reactor. After the pumping step, the system reached the de-
sirable 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
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Trends in Food Science & Technology 97 (2020) 381–390
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 in-
activation, aiming to scaling up the process. Several patents have de-
scribed methods for the CO2 dispersion in liquid media based on mi-
crobubbles (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 im-
portant 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 high-
pressure reactors can work in parallel, simulating a continuous opera-
tional 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).
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 conven-
tional 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 litera-
ture. 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 micro-
organisms are increased due to exposure to CO2. The proposed me-
chanism corroborates with the results reported in the literature about
the strong influence that the processing time has on the microbial in-
activation. 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 Lacto-
bacillus casei cells inoculated in apple juice employing up to 55 °C in
their SC-CO2 treatments. Lactobacillus casei is known as a spoilage mi-
croorganism that promotes the physicochemical and sensory degrada-
tion of fruit juices. An expressive log reduction of 6.93 cycles was
E.K. Silva, et al. Trends in Food Science & Technology 97 (2020) 381–390

achieved at the maximum temperature level in the process conditions of

Spilimbergo, and Nicoli (2017)

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
Marszałek et al. (2015)

Porębska et al. (2017)

Torabian et al. (2018)

smallest thermal treatment known is the thermization, a sub-

Manzocco, Plazzotta,
Silva et al. (2018)
pasteurization 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
Reference

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

5.23 and 5.38-log reduction of mesophilic bacteria and

Up to 6.93 log reduction was observed at 10 MPa, 55 °C,
(50 °C) and up to 3.5 log reduction at thermal condition
Inactivation of 1.0 log cycle at non-thermal condition

lower microbial load. Fruits and vegetables naturally carry a microbial

yeasts, respectively, were achieved at 18 MPa, 45 °C

Up to 3.7 log reduction was observed in mesophilic

load after their harvest containing enteric pathogens such as Escherichia

Lactic acid bacteria and yeast were completely

coli, Campylobacter spp., Salmonella spp., and other pathogens besides
endogenous microorganisms responsible for the product degradation

inactivated at 12 MPa, 35 °C, and 10 min

(Esteban-Cuesta et al., 2018; Kechero, Baye, Tefera, & Tessema, 2019).
bacteria at 60 MPa, 45 °C, and 30 min

In this sense, food processing using any technology for the microbial
30 min and 70% CO2 volume ratio

inactivation assumes the use of high-quality raw materials.

Microbial inactivation results

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 in-
activation of their spores only applying SC-CO2 in a non-thermal pro-
and 90 min

cess are still scarce (Noman et al., 2018). Many studies have reported
(75 °C)

spores inactivation using SC-CO2 technology by associating this tech-

nology 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, Marín-
Pressure: 10–60 MPa Temperature: 35–65 °C

Pressure: 10–20 MPa Temperature: 35–55 °C

Process time: 15–150 min CO2 volume ratio:

Pressure: 8–18 MPa Temperature: 35–45 °C

Pressure: 6–18 MPa Temperature: 20–45 °C

Iniesta, and Calvo (2012) evaluated the effect of the high-pressure CO2
Temperature: 50 and 75 °C Process time:

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
CO2 volume ratio: 10–70%

cycles at 30 °C and 10 MPa. On the other hand, Porębska, Sokołowska,

Process time: 10–30 min

Process time: 10–30 min

Experimental conditions

CO2 volume ratio: 72%

CO2 volume ratio: 30%

Skąpska, and Rzoska (2017) also assessed the inactivation of Alicyclo-

* The percentage of CO2 volume was evaluated in relation to the total volume of the high-pressure reactor.
Process time: 30 min

bacillus acidoterrestris spores in apple juice using SC-CO2 technology. A

CO2 volume ratio:
Pressure: 60 MPa

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
20–40 min

way, an inactivation of 1.0 log cycle was reached while up to 3.5 log
95.83%

reduction was possible employing the higher temperature. However,

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

the authors used a CO2/juice ratio of 30% in relation to the total vo-
lume of the high-pressure reactor. The CO2/juice (v/v or w/w) ratio
Operational mode

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
Batch

Batch

Batch

Batch

Batch

process optimization with all variables (pressure, temperature, proces-

sing time/residence time, and CO2/juice ratio) are still needed to in-
crease the inactivation rate of spores in fruit and vegetable juices.

3.5. Effects of non-thermal SC-CO2 processing on endogenous enzymes

Vegetative cells

Vegetative cells

Vegetative cells

Vegetative cells
microorganism
Form of the

After postharvest, processing of fruits and vegetables, most en-

Spores

dogenous enzymes still remain in their active form. Therefore, the en-
zymatic inactivation in fruit and vegetable tissues using SC-CO2 tech-
nology is a critical step to prevent the degradation and changes in the
Mesophilic bacteria, yeasts and

Mesophilic bacteria, yeasts and

quality aspects of juices during storage, for instance, enzymatic

Alicyclobacillus acidoterrestris

bacteria, yeasts and moulds

browning and off-flavors formation. Thus, a high enzymatic inactiva-

Mesophilic and lactic acid

tion level is required aiming to guarantee the maintenance of the sen-

sory attributes and nutritional value similar to the fresh juices (Benito-
Lactobacillus casei

Román, Sanz, Illera, Melgosa, & Beltrán, 2019; Marszałek et al., 2016).
Microorganism

Enzymes are biocatalysts with their structures based on proteins

that present a high affinity to a specific substrate. The inactivation
moulds

moulds

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,
Strawberry

Elderberry

however, the mechanisms are substantially different. High-pressure

Table 1

Apple

Apple

Apple
Juice

treatments affect mainly the tertiary and quaternary structures of en-

zymes. The protein denaturation induced by high-pressure occurs due

384
 E.K. Silva, et al.

Table 2
Effect of SC-CO2 processing on endogenous enzyme inactivation in different fruit and vegetable juices.
Juice Enzyme Operational mode Experimental conditions Enzymatic inactivation results Reference

Apple PPO Batch Pressure: 10 and 25 MPa Temperature: 40 and 55 °C Process PPO was inactivated up to 80% at 25 MPa and 40 °C and 95% at 25 MPa and 55 °C Murtaza et al. (2019)
time: 20 min
CO2 volume ratio:

Quince PPO Batch Pressure: 20 MPa Temperature: 25–65 °C Process time: 20 min Total inactivation of PPO was reached at 65 °C and up to 35% residual activity was Iqbal et al. (2018)
CO2 volume ratio: observed at 55 °C

Apple PPO and PME Batch Pressure: 10–20 MPa Temperature: 35–45 °C Process time: PPO and PME inactivation rate increased with pressure and temperature. PME was Illera et al. (2018)
0–120 min CO2 volume ratio: 60% more resistant compared to PPO

Tomato PG, and PME Batch Pressure: 8.5–20 MPa Temperature: 35–55 °C Process time: PME was nearly complete inactivated at 55 °C and 20 MPa while PG was found to be Illera et al. (2018)
0–120 min CO2 volume ratio: 60% more resistant

385
Orange PME Batch Pressure: 10–30 MPa Temperature: 35–45 °C Process time: PME inactivation degree increased with pressure and temperature. After 60 min, PME Briongos et al. (2016)
3–60 min CO2 volume ratio: was effectively inactivated at 40 °C and 30 MPa

Beetroot PPO, POD, PG, and Batch Pressure: 10–60 MPa Temperature: 31–55 °C Process time: The lowest decimal reduction time was observed for PG and the highest for POD Marszałek et al. (2017)
PME 10–30 min CO2 volume ratio: 88%

Strawberry PPO and POD Batch Pressure: 10–60 MPa Temperature: 35–65 °C Process time: The process resulted in the total inactivation of PPO and on average 85% inactivation Marszałek et al. (2015)
10–30 min CO2 volume ratio: 72% of POD

Apple PPO and POD Batch Pressure: 10–60 MPa Temperature: 35–65 °C Process time: PPO and POD inactivation kinetics significantly depended on process parameters as Marszałek et al. (2017)
10–30 min CO2 volume ratio: 90% well as the origin of enzymes (commercial or endogenous)

Carrot and celery PPO, POD, PG, and Batch Pressure: 10–60 MPa Temperature: 31–55 °C Process time: The lowest and highest decimal reduction time was observed for PE and PG in celery Marszałek et al. (2016)
PME 10–30 min CO2 volume ratio: 88% juice were 200 and 1645 min, respectively

* 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.
Trends in Food Science & Technology 97 (2020) 381–390
E.K. Silva, et al.
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 sub-
mitted 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 demon-
strated 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 α-
helix 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 en-
zymatic inactivation of fruit and vegetable juices. Among the enzymes
inactivated are pectin methylesterase (PME), peroxidase (POD), poly-
galacturonase (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 re-
sponsible 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 en-
zymes. 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 im-
proved 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. 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 proper-
ties 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 re-
tention of ascorbic acid (vitamin C) in juices has been used as a reliable
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Trends in Food Science & Technology 97 (2020) 381–390
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 com-
parison 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 SC-
CO2 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) en-
riched apple juice with inulin, a prebiotic carbohydrate, and subjected
this functional beverage to the non-thermal SC-CO2 processing em-
ploying 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 com-
pared to the untreated beverage regarding phenolic compounds (pro-
tocatechuic acid, rutin, and chlorogenic acid), sugars (fructose, glucose,
sorbitol, and sucrose), and organic acids (citric and malic acid). The SC-
CO2-treated samples did not present differences in their nutritional
profile in relation to the unprocessed inulin-enriched apple juice. Be-
sides 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 cya-
nidin-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 an-
thocyanins, pelargonidin-3-rutonoside and cyanidin-3-glucoside , were
chemically stable to SC-CO2 treatment, however, pelargonidin-3-guco-
side was slightly degraded at the high-pressure level in comparison to
the unprocessed strawberry juice.
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 dis-
tribution 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 cal-
culated 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
E.K. Silva, et al.
width index was reduced from 26 ± 7 to 5.8 ± 0.1.
On the other hand, no significant changes were observed in physi-
cochemical and physical properties such as soluble solid content, pH, ξ-
potential, 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 prop-
erties 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 water-
melon 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 pre-
servation 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 en-
gineering aspects, aiming to improve the microbial and enzymatic in-
activation. 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 mi-
croorganisms, lactic acid bacteria, yeasts and moulds and a log reduc-
tion 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 com-
pared 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 sweet-
ness, 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.
3.6.4. Shelf-life
In addition to the previous quality parameters, shelf-life has an
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Trends in Food Science & Technology 97 (2020) 381–390
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, respec-
tively.
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 para-
meters of the juice such as freshness, flavor, acidity, bitterness, sweet-
ness, 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 nutri-
tional value, the ascorbic acid and total anthocyanin contents were kept
constant, similar to the fresh juice, in this cold storage time. The au-
thors 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 SC-
CO2-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 physicochem-
ical 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-CO2-
treated 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.
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 li-
quid media is required. Likewise, most studies performed to date have
still not evaluated the synergic effects among the main process vari-
ables. The CO2/juice ratio has been neglected as a process variable as
E.K. Silva, et al.
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 con-
cerning the effects of the continuous mode on the enzymatic inactiva-
tion are still needed.
Although technical feasibility of the SC-CO2 technology has been
demonstrated for the non-thermal production of fresh fruit and vege-
table 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.
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 stu-
dies are required to perform process optimization, exploring the sy-
nergism 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 conven-
tional thermal treatments and even other emerging technologies such as
high-pressure processing and high-intensity ultrasound had several
advantages, regarding nutritional, physicochemical, and sensory as-
pects. 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 non-
thermal 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 tech-
nology 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 post-
doctoral 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.
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