Trends in Food Science & Technology 22 (2011) 427e441
Review
High pressure carbon
dioxide
pasteurization of
solid foods: Current
knowledge and
future outlooks
Giovanna Ferrentino* and
Sara Spilimbergo
Department of Materials Engineering and Industrial
Technologies, University of Trento, via Mesiano 77,
Trento 38050, Italy (Tel.: D39 0461 282 485;
e-mail: giovanna.ferrentino@ing.unitn.it)
High pressure carbon dioxide (HPCD) technology applied to
foods has gained a particular scientific interest considering
the number of publications and patents published in the last
decades. Although the antimicrobial effect of HPCD has
been demonstrated mainly for liquid foodstuffs, very few
research papers investigated the possibility to exploit the treat-
ment on solid foods. In this concern, the main objective of the
present review is to give a general survey of the published
knowledge concerning the HPCD applied to solid foods.
Remarks and future outlooks will be highlighted with the
aim to suggest a research line to follow for future studies.
Introduction
Manufacturers of food products are currently under in-
creasingly stringent demands concerning the production
processes. This is essentially caused by the growing interest
of consumers for high-quality and “minimal processing”
products, as well as for energy saving and safer production
processes. Minimally processed foods are obtained with
methods of food safety and preservation that are designed
* Corresponding author.
0924-2244/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tifs.2011.04.009
to retain the natural and fresh properties of foods
(Manvell, 1997).
Most of the food products contain high levels of nutri-
ents or a high water activity; therefore they are particularly
susceptible of microbial spoilage which results in a deterio-
ration of their organoleptic characteristics, and may even
risk the health of immune-compromised individuals
(Tournas, Heeres, & Burgess, 2006).
Preservation of food products reduces the number of un-
desirable microorganisms below a specific critical value
during the shelf storage period of the product. It is usually
performed through different methods as thermal pasteuriza-
tion up to 80
C and sterilization up to 120
C (e. g. fruits,
food powders and meat), drying (e. g. vegetables, and
herbs), freezing (e. g. meats, fishes, and vegetables), addi-
tion of preservatives (e. g. meats, and vegetables), autoclav-
ing (e. g. herbs), gamma irradiation (e. g. vegetables), or
ethylene oxide and methyl bromide exposure (e. g. vegeta-
bles; spices). These treatments are successful to eliminate
the degenerative effects of enzymes and microorganisms,
but on the other hand they may also reduce the food quality
by causing alterations in the taste and in the organoleptic
features of food products. Several authors observed a reduc-
tion in vitamin C retention, color degradation, and changes
in other quality indicators due to the thermal treatments
applied to fruits (Awuah, Ramaswamy, & Economides,
2007; Vikram, Ramesh, & Paprulla, 2005). Others reported
that the application of both methyl bromide and ethylene
oxide is extremely toxic. Methyl bromide is potentially
capable of depleting the atmospheric ozone layer. Ethylene
oxide has been banned in Europe because of safety and en-
vironmental concerns, and its use for the treatment of
ground spices has been revoked in the United States
(Thayer, Josephson, Brynjolfsson, & Giddings, 1996).
Autoclaving, freezing, drying or exposure to steam have
been described as methods which cause degradation of bio-
active compounds and the destruction of the delicate tissue
of the foods (Howard, 2008).
Alternatives to traditional treatments as the use of
ozone, radical species, and high hydrostatic pressure
have been proposed in these years (San Martin,
BarbosaeCanovas, & Swanson, 2004). The latter has
been already used for few years at commercial scale for
the decontamination of solid foods such as meat products
(Shigehisa, Ohmori, Saito, Taji, & Hayashi, 1991) or
ready to eat meals (Cheftel, 1995). Pressures ranging
428 G. Ferrentino, S. Spilimbergo / Trends in Food Science & Technology 22 (2011) 427e441
from 300 to 700 MPa are usually applied. The process re-
sulted effective on different microbial species but the high
equipment cost, the difficulty of controlling and managing
an operating pressure in such extreme range of values, the
safety concerns represent the potential drawbacks to be
overcome.
Among the other innovative preservation methods, high
pressure carbon dioxide process (HPCD) has gained great
interest in the scientific field. Since the 1980s it has been
increasingly investigated as a promising technique to in-
duce a pasteurizing/sterilizing effect when applied both to
solid and liquid matrixes. The HPCD preservation method
provides several advantages. Carbon dioxide (CO2) used
in this process is not only a powerful solvent for a wide
range of compounds of interest in food processing, but is
relatively inert, inexpensive, nontoxic, nonflammable, recy-
clable and readily available in high purity leaving no resi-
due when removed after the process (Clifford &
Williams, 2000). Furthermore, it is considered to be
a GRAS (Generally Recognized as Safe) solvent, which
means it can be used in food products.
So far most of the research has been focused on suspen-
sions of pure cultures of different microorganisms inocu-
lated or naturally occurring in liquid food products (e.g.
fruit juices, beer, wine and milk) which affect the food
spoilage and give main concern to health (Arreola,
Balaban, Wei, Peplow, & Marshall, 1991; Del Pozo-
Insfran, Balaban, & Talcott, 2006). The method seems to
be very promising because HPCD (or dense phase CO2)
is able of killing bacteria, yeast and fungi at moderate pres-
sure and low temperature preserving more quality attributes
of the products than traditional treatments (Ferrentino,
Plaza, Ramirez-Rodrigues, Ferrari, & Balaban, 2009;
Spilimbergo & Ciola, 2010).
Three recent reviews compile the relevant current
knowledge about the potential of HPCD treatment and
summarize the most significant state of the art, including
the most important applications and data for the treatment
applied to a wide range of microorganisms mainly in liquid
substrates (Damar & Balaban, 2006; Garcia-Gonzalez
et al., 2007; Spilimbergo & Bertucco, 2003).
Compared to liquids, the HPCD process applied to solid
foods is less studied due to the complexity of the matrix,
which can make the CO2 bactericidal action more difficult,
and to the lack of information about the inactivation mech-
anism which is almost obscure and scarcely studied.
The aim of the present review is to provide the status of
the art of HPCD process applied to solid foodstuffs with the
examination of almost all the literature published on this
subject including the most relevant patents.
The review has been constructed as follows: a description
of the application of the process for each category of solid
foods tested so far (meats, fruits and vegetables, food pow-
ders, sprouts seeds, spices and herbs, fishes) has been
addressed. For each category of solid foods a discussion
of the main effects of the HPCD treatment on microbial
inactivation and quality attributes of the foods has been re-
ported considering all significant articles published in the
field.
A brief section of the review is dedicated to the descrip-
tion of the main results obtained by different authors when
the HPCD process is carried out in combination with some
pretreatments or additives.
Finally a reflection on the important aspects of the pro-
cess, its limits and potentials, the lack of scientific informa-
tion to fill up, and a direction to orientate the research will
be evidenced.
Application of HPCD treatment to solid foods
The effects of the HPCD treatment on the microbial
inactivation and quality attributes of food matrixes are
reported in the following sections. The process has been ap-
plied to meats (chicken, pork, and beef), vegetables (celery,
and spinach), seeds and food powders (alfalfa seeds, cocoa
powder, and ginseng), fruits (cut pieces of pears, straw-
berries, honeydew melon, and cucumber), spices and herbs
(chives, thyme, oregano, parsley, and mint), and fish
(shrimp, and oyster). Considering the different characteris-
tics and behaviors of each substrate to the treatment, the lit-
erature review has been reported separately for each
category of solid matrix.
Table 1 is a compilation of the experimental results that
can be found in the literature with indication of the type of
food, microorganism, the present industry conventional
treatment, and the conditions of HPCD treatment with the
corresponding achieved microbial inactivation. Table 2 re-
ports the desired level of microbial inactivation required
by law (Regulation (EC) No. 2073/2005) for each type of
microorganism and foodstuffs mentioned. Table 3 summa-
rizes the description and the main observations of the ef-
fects of the treatment on the appearance, and some
quality attributes of the foods subjected to the process.
Meats
Microbial inactivation
The HPCD technology in particular in supercritical state
(31
C and 7.4 MPa) has been widely investigated and ap-
plied to meat considering the ability of CO2 to extract and
fractionate fats from ground beef into lower melting tem-
perature components, as well as for the removal of choles-
terol (Chao, Mulvaney, Bailey, & Fernando, 1991). Apart
from this application, the treatment has been also applied
to test the efficacy on the inactivation of bacteria strains
to induce pasteurization. Sirisee, Hsieh, and Huff (1998)
tested the tolerance of Escherichia coli and Staphylococcus
aureus in ground beef to HPCD treatment carried out at
42.5
C and 31.03 MPa. Ground beef samples were mixed
with the suspended microbial cultures and then placed in
the reactor to be processed. The experimental results high-
lighted the longer treatment time needed to inactivate both
target microbes compared to the same treatment carried out
on the same microorganisms in a liquid phosphate buffer
Table 1. Application of HPCD treatment on microbial forms detected on solid substrates. Type of food, microorganism, conventional treatment, and HPCD process conditions with the
corresponding microbial inactivation and references are reported.

Food Target microorganism Process conditions Microbial inactivation Reference Conventional treatment


Flour Mold 6.2 MPa, 23 C, 2 h 99.8% Steam, microwaves, Joule effect, thermal treatments
Bacteria 99.6%
Strawberries 6.2 MPa, 22
C, 2 h 99% Chemical preservation (acidulants, antioxidants, chlorine or
antimicrobials); gas and controlled modified atmosphere;
refrigeration; moisture reduction


Mozzarella cheese Bacteria 6.2 MPa, 23 C, 16 h 87% (Haas et al., 1989) Antimicrobial packaging, gas and controlled modified

G. Ferrentino, S. Spilimbergo / Trends in Food Science & Technology 22 (2011) 427e441

Parmesan cheese 1.4 MPa, 23 C, 168 h 50% atmosphere, refrigeration


Romano cheese 1.4 MPa, 23 C, 168 h 99%


Onions 5.5 MPa, 23 C, 2h 90% Chemical preservation (acidulants, antioxidants, chlorine or
antimicrobials); gas and controlled modified atmosphere;
refrigeration; moisture reduction
Dry peppers 5.5 MPa, 23
C, 2 h 90% Sterilization with ethylene oxide; ionizing radiation; steam
(30% moisture added) treatment
Chives 5.5 MPa, 45
C, 2 h Total Chemical preservation (acidulants, antioxidants, chlorine or
Thyme inactivation antimicrobials); gas and controlled modified atmosphere;
Oregano refrigeration; moisture reduction
Parsley
Mint
Fresh celery leaves Natural microorganisms 6.9, 31.4 and 4 Log (cfu/g) (Kuhne & Knorr, Chemical preservation (acidulants, antioxidants, chlorine or
and leafstalks 62.8 MPa, 1990) antimicrobials); gas and controlled modified atmosphere;
40 or 60
C, 30 or refrigeration; moisture reduction
60 min
Chicken meat Salmonella typhimurium 13.7 MPa, 35
C, 2 h 94e98% (Wei et al., 1991) Addition of organic acids (acetic and lactic acids); ionizing
Listeria monocytogenes 79e84% radiation; steam pasteurization
ATCC15313
Shrimp L. monocytogenes 99% Quick freezing, heat e cool pasteurization
ATCC15313
Ground beef Escherichia coli 31.03 MPa, 42.5
C, 1 Log (cfu/g) (Sirisee et al., Addition of organic acids (acetic and lactic acids); ionizing
systems 180 min 1998) radiation; steam pasteurization
Staphylococcus aureus 31.03 MPa, 42.5
C, 3 Log (cfu/g)
120 min
Kimchi vegetables Lactic acid bacteria 6.9 MPa, 10
C, 4 Log (cfu/ml) (Hong & Park, Chemical preservation (acidulants, antioxidants, chlorine or
24 h 1999) antimicrobials); gas and controlled modified atmosphere;
refrigeration; moisture reduction
Skinned beef meat Brochothrix thermosphacta 6.1 MPa, 45
C, 5 Log (Erkmen, 2000) Addition of organic acids (acetic and lactic acids); ionizing
Minced beef meat 150 min 1 Log radiation; steam pasteurization
(continued on next page)

429
Table 1 (continued )

430
Food Target microorganism Process conditions Microbial inactivation Reference Conventional treatment


Alfaalfa seeds Escherichia coli K12 27.6 MPa, 50 C, 92.8% (Mazzoni et al., Chemical preservation (chlorine compounds; acidified sodium
Total aerobic bacteria 60 min 85.6% 2001) chlorite, hydrogen peroxide, trisodium phosphate, peracetic
acid, ethanol and commercial cleaning solutions)
Beef trimmings Total plate count 10.3 MPa, 36
C, 0.83 Log (Meurehg, 2006) Addition of organic acids (acetic and lactic acids); ionizing
E. coli O157:H7 15 min 0.93 Log radiation; steam pasteurization
E. coli 1.00 Log
Salmonella spp. 1.06 Log
Ground beef Total plate count 10.3 MPa, 36
C, 0.78 Log
E. coli O157:H7 15 min 0.94 Log

G. Ferrentino, S. Spilimbergo / Trends in Food Science & Technology 22 (2011) 427e441

E. coli 0.94 Log
Salmonella spp. 1.23 Log
Cocoa powder Aerobic mesophilic 30.0 MPa, 65
C, Total (Calvo et al., 2007) Steam, microwaves, Joule effect, thermal treatments
spores 40 min inactivation
Aerobic thermophilic
spores
Mesophilic thermo
resistant spores
Thermophilic
thermo resistant spores
Total plate count
Fresh Spinach E. coli K12 10 MPa, 40
C, 5 Log (cfu (Zhong et al., 2008) Chemical preservation (acidulants, antioxidants, chlorine
Leaves 10 min per leaf) or antimicrobials); gas and controlled modified atmosphere;
refrigeration; moisture reduction
Ginseng Powder Total aerobic 10 MPa, 60
C, 2.67 Log (cfu/g) (Dehghani et al., 2008) Sterilization with ethylene oxide; ionizing radiation; steam
microbial count 15 h treatment
Alfaalfa seeds E. coli O157:H7 15 MPa, 35
C, 3.51 Log (cfu/g) (Jung et al., 2009) Chemical preservation (chlorine compounds; acidified sodium
10 min chlorite, hydrogen peroxide, trisodium phosphate, peracetic
Listeria Monocytogenes 10 MPa, 45
C, 2.65 Log (cfu/g) acid, ethanol and commercial cleaning solutions)
S. typhimurium 5 min 2.48 Log (cfu/g)
Boneless pork loins Escherichia coli 12 MPa, 35
C, 1.5 Log (cfu/cm2) (Choi et al., 2009b) Addition of organic acids (acetic and lactic acids); ionizing
L. monocytogenes 30 min 1.4 Log (cfu/cm2) radiation; steam pasteurization
S. typhimurium 1.56 Log (cfu/cm2)
E. coli O157:H7 1.0 Log (cfu/cm2)
Soy sauce paste Escherichia coli 14 MPa, 45
C, 33.81% (Choi et al., 2009a)
marinated pork L. monocytogenes 40 min 37.96%
loins S. typhimurium 34.48%
E. coli O157:H7 36.84%
Hot e pepper Escherichia coli 26.42%
paste marinated L. monocytogenes 27.59%
pork loins S. typhimurium 32.74%
E. coli O157:H7 28.28%
Pears S. cerevisiae 10 MPa, 50
C, 4 Log (cfu/g) (Valverde et al., 2010) Chemical preservation (acidulants, antioxidants, chlorine
10 min or antimicrobials); gas and controlled modified atmosphere;
refrigeration; moisture reduction
Oyster Aerobic Plate Count 17.2 MPa, 60
C, 3 Log (cfu/g) (Meujo et al., 2010) Quick freezing, heat e cool pasteurization
60 min
Paprika powder Mesophilic aerobic 30.0 MPa, 90
C, 5.5 Log (cfu/g) (Calvo & Torres, 2010) Sterilization with ethylene oxide; ionizing radiation;
microorganisms 45 min steam treatment
 G. Ferrentino, S. Spilimbergo / Trends in Food Science & Technology 22 (2011) 427e441 431

Table 2. Desired level of microbial inactivation based on the type of food and microorganism.

Inactivation target degree (cfu/g)

Food Total plate Aerobic thermo-resistant Yeasts/Moulds Enterobacteriacee E. coli Salmonella Listeria
count spores
Cocoa product <103 <10 <10 <10 Total absence Total absence Total absence
Ginseng powder <104 ND <102 ND Total absence Total absence Total absence
Fruits and vegetables <104 ND ND <102 <20 Total absence Total absence
Meat products <105 ND ND <102 <50 Total absence Total absence
Dairy products <105 ND ND <102 102 < 103 Total absence Total absence
Fishery products <106 ND ND <102 <20 Total absence Total absence
Sprouted seeds <103 ND ND <102 <20 Total absence Total absence
Spices and herbs <103 ND ND <102 <20 Total absence Total absence
ND ¼ not defined.

solution (Sirisee et al., 1998). One log cycle reduction of E.
coli in ground beef took 178 min, but only 1.7 min was
needed to achieve the same inactivation level in liquid
phosphate buffer solutions. Same experimental evidences
were observed for S. aureus inoculated in ground beef
system and in liquid phosphate buffer solutions. The expla-
nation was related to the presence of fats and proteins in
ground beef which could play an important role in protect-
ing microorganisms from high pressure CO2 bactericidal
action and to the lower moisture content (72%) which
reduced the amount of CO2 able to dissolve in the food
matrix.
The protective effect of carbohydrate and other or-
ganic compounds in foods was also reported by
Erkmen (2000) who inoculated Brocothrix thermos-
phacta microbial cells on minced and skinned beef
meat and carried out the HPCD treatment (6.1 MPa,
45
C for 150 min) in a batch device. Also in this study,
the results demonstrated that the treatment was not as ef-
fective as for a liquid substrate. B thermosphacta sus-
pended in brain heart infusion broth was completely
inactivated under 6.1 MPa after 80, 50 and 30 min at
25, 35 and 45
C respectively. The sterilization effect
at 6.1 MPa and 45
C on B thermosphacta was observed
after 150 min in skinned meat. The increase of microbial
resistance to the treatment was attributed to the nature of
the matrix and compounds (carbohydrates and fats) as
previously observed by Sirisee et al. (1998). However
a reduction equal to 5 and 1 log cycles was observed
for B. thermosphacta inoculated on skinned and minced
meat, respectively.
In a further study, the treatment was applied to chicken
meat strips (breast meat with no skin) to inactivate Salmo-
nella and Listeria cultures in which the chicken samples
were dipped (Wei, Balaban, Fernando, & Peplow, 1991).
The samples treated at 13.7 MPa and 35
C for 2 h and
spiked with Salmonella were shown to reduce the bacterial
numbers by 94e98% while those spiked with Listeria were
only reduced by 79e84%.
Choi, Bae, Kim, Kim, and Rhee (2009a) conducted
a study to evaluate the effects of supercritical carbon
dioxide treatment in marinated pork products (boneless
pork loins) in soy sauce and hot e pepper paste for the in-
hibition of generic E. coli, Listeria monocytogenes, Salmo-
nella typhimurium and E. coli O157:H7. Published
inactivation kinetics showed that two stages were observed
in the microbial survival curves. The early stage was char-
acterized by a slow rate of microbial reduction which then
sharply decreased during the later stage. The results dem-
onstrated that the inactivation rate increased with increas-
ing pressure, temperature, and exposure time but that it
was also depended on the initial number of cells and the
suspending medium. Carrying out a treatment at 14 MPa,
45
C for 40 min, the observed reduction levels were equal
to 37.96% for L. monocytogenes, 34.48% for S. typhimu-
rium, 33.81% for generic E. coli, and 36.84% for E. coli
O157:H7 in soy sauce marinated pork. Conversely, when
the hot e pepper paste marinated pork was subjected to
the HPCD treatment, the reduction level was 26.42% for
generic E. coli; 27.59% for L. monocytogenes; 32.74% for
S. typhimurium and 28.28% for E. coli O157:H7. However
the inactivation results were too low if compared with stan-
dard pasteurization methods. As shown in Table 2 total ab-
sence of Listeria was needed to consider the food safe for
human consumption.
Effect on quality attributes
Only visual observations of the ground beef sample
treated by HPCD were reported by Sirisee et al. (1998). Ac-
cording to these authors, the color of ground beef changed
after the treatment and looked like cooked ground beef.
This observation was also supported by the experimental
results reported by Brown and Mebine (1969) who stated
that high concentrations of CO2 could cause darkening in
tissues by combining with myoglobin to form metmyoglo-
bin. This effect could be limited overcome if this technique
has to be applied to beef meats which contains high levels
of myoglobin (myoglobin content: 0.05e0.20% for white
chicken meat, 0.1e0.3% for pork and veal, and
0.4e2.0% for beef).
Qualitative analyses were performed on the samples of
chicken treated by HPCD (Wei et al., 1991). Also in this
432 G. Ferrentino, S. Spilimbergo / Trends in Food Science & Technology 22 (2011) 427e441

Table 3. Description of the effect of HPCD treatment on some quality attributes of treated foods.

Food Observations Reference

Strawberries, honeydew melon, cucumber Tissue destruction (Haas et al., 1989)

Chives, oregano Enhanced aroma

Parsley Similar taste of the untreated sample
Slight off aroma
Thyme Worst taste compared to the untreated sample
Mint Better taste compared to the untreated sample
Enhanced aroma
Chicken meat and shrimp Color change to whitish (Wei et al., 1991)
Cooked appearance
Loss of liquid

Ground beef Color change to dark (Sirisee et al., 1998)

Cooked appearance

Kimchi Higher pH (Hong & Park, 1999)

Lower titratable
Better sensory properties
No significant color, flavor and texture changes

Alfaalfa seeds No detrimental effect on viability of the seeds (Mazzoni et al., 2001)
No detrimental effect on germination rate of the seeds

Ground beef Higher tenderness (Meurehg, 2006)

No significant changes in juiciness, flavor intensity
and off flavor intensity

Cocoa powder Decreased water content (Calvo et al., 2007)

No effect on physical aspect

Meat of porcine longissimus dorsi muscle No effect on muscle pH (Choi et al., 2008)
No effect on tenderness
No effect on water e holding capacity
Increased lightness
Sarcoplasmic protein denaturation

Spinach leaves Discoloration (Zhong et al., 2008)

Decreased leaf firmness

Cabbage, lettuce, mizuna Soften structure (Matsufuji et al., 2009)

Soy sauce and hot pepper marinated No differences in the surface color intensity (Choi et al., 2009a)
paste marinated pork Overall acceptability

Alfalfa seeds No effect on germination rate (Jung et al., 2009)

Paprika powder Slight reduction of color intensity (Calvo & Torres, 2010)
Decrease of water content

Pears Consistency loss (Valverde et al., 2010)

Softer aspect
Loss of liquid
Transition to brown coloring

Oyster Retention of the overall acceptability (Meujo et al., 2010)

case the authors reported a color change of the samples which A more complete analysis was recently performed by
turned whitish and seemed to be cooked or soaked in acid. In Choi et al. (2008) who investigated the effect of supercrit-
addition, the treated samples showed a liquid loss from the ical carbon dioxide treatment for sterilization purpose on
tissue which was completely absent in the untreated sample. meat quality and protein denaturation of the porcine
 G. Ferrentino, S. Spilimbergo / Trends in Food Science & Technology 22 (2011) 427e441
longissimus dorsi muscle. During the treatment, CO2 was
easily adsorbed into the meat lowering the meat pH with
the formation of carbonic acid and its subsequent dissocia-
tion in bicarbonate and hydrogen ions (Jacobsen &
Bertelsen, 2002). It was observed that the pressure and tem-
perature of the HPCD treatment could affect molecular in-
teractions and proteins conformation, leading to protein
denaturation and aggregation in the meat (Messens, Van
Camp, & Huyghebaert, 1997). In the same study, the
HPCD treatment (7.4 MPa at 31.1
C for 10 min) was re-
ported not to affect the muscle pH, total weight, tenderness
and water holding capacity of the treated meat. However
the treated samples had a higher lightness and a lower red-
ness value. The change in color was correlated to a denatur-
ation process of proteins occurred during the treatment. The
authors also verified that, while the HPCD treatment had no
effect on myofibrillar protein solubility including myosin
protein, the SDS e PAGE analysis revealed a denaturation
of sarcoplasmic protein (phosphorylase b, creatine kinase,
triosephophate isomerase and one unknown protein) which
masked the red color of the sarcoplasm, causing the muscle
to become pale.
Despite these findings, sensory evaluations of soy sauce
and hot-pepper paste marinated pork samples revealed no
differences in the surface color intensity and an overall ac-
ceptability of controls and samples treated with supercriti-
cal CO2 at 15.2 MPa and 31.1
C for 10 min (Choi et al.,
2009a).
Several studies demonstrated the efficiency of the su-
percritical carbon dioxide process which increased the
quality of meats inducing the extraction of cholesterol
and other lipids. This potential is becoming more and
more attractive due to consumer concern over dietary in-
take of fat and cholesterol. It has been reported that CO2
under supercritical conditions solubilizes a portion of the
lipid components and removes them from the food ma-
trix. Chao et al. (1991) found that up to 36.9% of the
cholesterol and 71.2% of total lipids in the meat could
be removed during the process. However, such process
had limited effects on fat extraction of meat products
containing high levels of fats (Clarke, 1991). A negative
effect of the extraction with CO2 was associated to the
dehydratation of the muscle tissue which resulted in a de-
creased solubility of some muscle proteins and other
changes in functional properties (Hardardottir &
Kinsella, 1988).
Fruits and vegetables
Microbial inactivation
Very few researches have been addressed applying the
HPCD treatment to fruits. The first study was carried out
in 1989 by Haas, Prescott, Dudley, Dik, Hintlian, & Keane
on fresh strawberries, honeydew melon and cucumber in
order to delay surface molding. The treatment had a positive
effect on molding especially for strawberries showing a pre-
servative action.
433
Apart from this study, only recently the HPCD technology
has been applied and tested more in deep on fresh e cut con-
ference pears to test the inactivation of Saccharomyces cere-
visiae isolated from the natural flora of the pear puree
(Valverde, Mar
ın-Iniesta, & Calvo, 2010). The results dem-
onstrated the key role of the temperature in the inactivation
rate and how the nature of the substrate influenced the choice
of the process conditions. The yeast survival ratio did not lin-
early depend on temperature. The survival curves presented
two stages: a first stage up to 35
C, where inactivation was
minimal, and a second one from this temperature to 55
C,
where the inactivation rate rapidly increased. The reason
for a two stage dependence on temperature could be related
to the growth mechanism of mesophile microorganisms,
since their optimal temperature for multiplication occurs
between 25 and 40
C. Above that temperature, proteins
denature, cytoplasmic membranes collapse, and cells lyse
are deactivated. In addition the results demonstrated that an
increase in pressure from 6 to 30 MPa did not significantly
increase the inactivation rate and the same happened for
the treatment time. Complete microbial inactivation occurred
at 10 MPa, 55
C for 10 min. The lesser water activity of the
solid pear could justify the lower inactivation rate of S. cere-
visiae in comparison to liquid media, aside from their differ-
ent compositions and physical states (Lin, Yang, & Chen,
1992).
The HPCD treatment has been also applied to some veg-
etables. Kuhne and Knorr (1990) carried out experiments
on fresh celery and leafstalks showing a substantial inacti-
vation effect on total count. Their data indicated that a treat-
ment carried out for 30 min at 40
C and 62.8 MPa induced
a reduction in the total plate count of approximately
104 cfu g1 (from 1.7$107 cfu g1 in the untreated sample
to 9.8 103 cfu g1 after the treatment). They also reported
the inefficiency to inactivate spores on the same substrates
and under the same experimental conditions.
The HPCD treatment (7.0 MPa for 24 h) performed on
the fermented Baechu kimchi, a Chinese cabbage from
a group of traditional vegetable foods in Korea at about
pH 4.45, brought insufficient reduction of lactic acid bacte-
ria (Hong & Park, 1999).The results were significantly
different from previous studies for pure cultures of lactic
acid bacteria. As a matter of fact, the same research group
isolated Lactobacillus sp. from the late stages of kimchi fer-
mentation and showed that the HPCD treatment induced
inactivation suspending the microbial cells in the culture
broth (Hong & Park, 1997). The different result was attrib-
uted to the lowered diffusivity and the limited mass transfer
of CO2 due to the compact structure of the salted Chinese
cabbage and to the lack of kimchi juice in the solid sample
which could act as an easy penetration medium.
The last application of the HPCD treatment to vegetables
was reported by Zhong, Black, Davidson, and Golden (2008)
who evaluated the potential of the treatment on the inactiva-
tion of inoculated E. coli Ke12 and background microflora
on fresh spinach leaves. The study demonstrated that the
434 G. Ferrentino, S. Spilimbergo / Trends in Food Science & Technology 22 (2011) 427e441
microbial reduction obtained in CO2 supercritical conditions
(7.5 and 10 MPa at 40
C and 40 min) was significantly higher
than that in subcritical state (5 MPa at 40
C and 40 min). At
5 MPa and 10 min, the microbial reduction of E. coli and
background microflora was about 2 and 1 log cycles respec-
tively while in supercritical conditions a reduction to unde-
tectable level (w 5 Log cycles) was obtained. However no
inactivation evidences were reported to highlight the
efficacy of the treatment against pathogens such as E. coli
O157:H7 and Salmonella, very common microorganisms
on this substrate.
Effect on quality attributes
Results on the evaluation of the physical attributes of
fruits treated by HPCD showed negative effects of the treat-
ment on the products. Although just a visual observation
was addressed, the treatment resulted in gross tissue de-
struction of strawberries, honeydew melon and cucumber
(Haas et al., 1989).
Quantitative analyses were performed on the HPCD
pears treated by Valverde et al. (2010) in terms of pH,
sugar content and color. The results showed that the pro-
cess did not reduce the pH and did not alter the sugar con-
tent of the pears although a loss of consistency was
observed and manifested as a softer aspect and a loss of
liquid. A change in the color parameters (decrease of the
luminosity L, increase of the a value to the red and reduc-
tion of the b value to the blue) of the fruits was also de-
tected mainly due to the reduced vitamin C
concentrations and to the partial and ineffective inactiva-
tion of the peroxidase enzyme activity.
From these early findings, it is evident that the primary
problem of applying this technology to the preservation of
fruits is the adverse effect this process produces on the re-
duction of fruit firmness and texture due to the use of the
high pressure. It is possible to conclude that the HPCD pro-
cess is more adequate for products where a firm texture is
not essential, such as fruit cocktails, creams, juices, or other
types of fruit preserves.
Few analyses have been also performed to evaluate the
impact of the process on the quality attributes of vegetables.
Results of sensory analyses performed by Hong and Park
(1999) on kimchi samples during storage at 10
C showed
that the treated vegetables had a higher pH, lower titratable
acidity and better sensory properties. No differences were
detected for color, off e flavor and hardness comparing
the samples with the untreated.
The study carried out by Zhong et al. (2008) showed the
importance of the choice of the process conditions. Digital
photographs of HPCD spinach leaves demonstrated that the
treatment performed at longer processing time (40 min) and
higher pressure (10 MPa) resulted in a greater leaf discolor-
ation and decrease of leaf firmness. Discoloration was
likely caused by the CO2 dissolution into leaf tissues, which
acidified the leaves and degraded the chlorophylls
(Mingotaud, Chauvet, & Patterson, 1996).
Food powders
Microbial inactivation
In the field of food powders, the HPCD treatment has been
only applied for the inactivation of natural microflora of a non
fermented high polyphenol cocoa powder with a butter con-
tent of 10.5% obtained from the grinding of cocoa seeds
(Calvo, Muguerza, & Cienfuegos-Jovellanos, 2007). The ef-
ficacy of the HPCD treatment was tested for the inactivation
of mesophilic and thermophilic aerobic microorganisms. In
addition considering that the natural contamination of the ca-
cao was due to bacteria spores, the efficiency of the treatment
was also tested on Aspergillus niger (CECT 2574, ATCC
16404) and Aspergillus ochraceus spores spiked on cocoa
samples. Results demonstrated that an increase in pressure
from 13 to 30 MPa at 65
C for 40 min did not show any effect
on the microbial inactivation of mesophilic and thermophilic
aerobic microorganisms. No microbial inactivation was also
observed operating the treatment at 30 MPa although the
temperature was increased to 40, 65 and 80
C. Same results
were also obtained applying twelve cycles of compression/
decompression up to 30 MPa to the cocoa sample heated at
80
C. The inactivation target degree was only achieved
with the combination of higher temperatures (80 or 65
C)
and some extra humidity (5 or 10%) at a pressure of
30 MPa concluding that the initial water content was one of
the key factors affecting the inactivation mechanism. Results
on cocoa samples contaminated with spores demonstrated
that the inactivation of both types of fungi spores was fully
achieved at 30 MPa, 80
C for 30 min after the addition of
5% water.
Effect on quality attributes
Analysis on the quality attributes of the cocoa powder
were performed in particular to detect the effect of the
HPCD treatment on polyphenolic compounds as (þ) - cat-
echin, () - epicatechin, proanthocyanidins and anthocya-
nins. These compounds could be extracted if wet CO2 is
used as solvent. On the other hand, the temperatures ap-
plied to achieve sterilization could provoke the flavanols
polymerization. Contrary to what it could be expected,
the polyphenolic compounds of the samples treated at
30 MPa, 80
C with 10% water addition were not extracted.
The chromatographic profiles of the extracts of the treated
samples did not show the presence of phenolic acids or fla-
vonoids. Moreover, the water content of the sample after
the treatment decreased to about the initial level. This as-
pect is of great importance for the storage, shelf life, and
physical aspect of the cocoa powder, as well for its further
applications, since the humidity content of the final product
should not exceed 9%, as specified by law for this type of
compounds (Calvo et al., 2007).
Sprouts seeds
Microbial inactivation
Few papers have been published reporting the effect of
HPCD on microorganisms occurred in sprouts seeds, in
 G. Ferrentino, S. Spilimbergo / Trends in Food Science & Technology 22 (2011) 427e441
particular alfalfa seeds considered a valuable dietary food
and commonly consumed raw or slightly cooked in salads
or sandwiches (Jung, Choi, & Rhee, 2009; Mazzoni,
Sharma, Demerci, & Ziegler, 2001). Mazzoni et al.
(2001) demonstrated the efficacy of the treatment on the
natural indigenous aerobic microorganisms and on
a non e pathogenic strain of E. coli K12 inoculated on
the seeds. For the aerobic plate count, at a fixed temperature
of 50
C, the treatment carried out at 13.8 MPa for 15 min
resulted in less than 10% inactivation, while 85.6% inacti-
vation was achieved increasing the treatment time to
60 min and the pressure to 27.6 MPa. Results demonstrated
that the treatment was also effective on E. coli K12 with re-
ductions equal to 26.6, 68.1, and 81.3% at 13.8, 20.7 and
27.6 MPa for 15 min and 50
C, respectively.
The high level of microbial inactivation obtained in the
study of Mazzoni et al. (2001) showed that the supercritical
CO2 was not serving as only bactericidal agent but it might
be reaching microorganisms in hidden in crevices or be-
tween the cotyledon and the head of the sprouts due to
the high pressure applied.
An extensive inactivation study was also carried out by
Jung et al. (2009) who contaminated alfalfa sprouts with
E. coli O157:H7, L. monocytogenes and S. typhimurium mi-
crobial strains. The results demonstrated the efficiency of
the treatment, although a limited step of the process was
the low CO2 solubility due to the solid nature of the matrix
(Choi et al., 2009a; Wei et al., 1991). An HPCD treatment
performed at 20 MPa, 45
C for 15 min determined more
than 7 log reductions for the three tested pathogens.
Effect on quality attributes
The rate of germination is the quality attribute moni-
tored during the treatment of alfalfa seeds indicating the
nutritional value of seeds: fats and carbohydrates are bro-
ken down and the starch digestibility is increased during
the germination process (Vidal-Valverde et al., 2002).
The study carried out by Mazzoni et al. (2001) showed
that the HPCD treatment did not have any detrimental
effect on the viability of alfalfa seeds. The percent of
germination for all treatment conditions was over 90%
and no significant differences in the germination rate
were detected between the treated and untreated seeds,
indicating that the HPCD process could be effective for
the treatment of alfalfa seeds at commercial level without
compromising the germination quality.
Jung et al. (2009) demonstrated that treating seeds with
HPCD at high treatment conditions (20 MPa at 40 and
45
C) impaired the germination capability of alfalfa seeds.
To not influence the germination capability of alfalfa seeds
after three days of germination it was necessary to apply
process conditions which did not guarantee the microbial
safety (>5 log reductions of foodborne pathogens). The
contrasting conclusions obtained by the only two papers
published on this matter, suggest that further studies are
435
needed to gain more insights on the effects of the treatment
both on germination rate and sprouting conditions.
Spices and herbs
Microbial inactivation
One of the first applications of the HPCD treatment to
spices and herbs has been published by Dehghani,
Annabi, Titus, Valtchev, and Tumilar (2008) for the sterili-
zation of ginseng powder. Considering that the cultivation
of ginseng usually takes up to 6 years and the species are
submerged in wild soil for the entire period, over than
78% of the extracted ginseng products are highly contami-
nated with fungi and bacteria. The common sterilization
methods used for this product include autoclaving, ethylene
oxide exposure, and g-irradiation. These methods have
been proven to achieve complete removal of many microor-
ganisms including several bacteria classes such as E. coli,
S. aureus, and Bacillus cereus (Byun et al., 1998). The gin-
seng powder used in the study of Dehghani et al. (2008)
could not be released into the market due to the high level
of total bacteria (5$107 cfu g1) and also to the presence of
fungi. The acceptable levels for oral use by the Therapeutic
Goods Administration in Australia (TGA) are contamina-
tion less than 104 cfu g1 and 102 cfu g1 for total aerobic
microbial count and fungi, respectively. HPCD treatment
was applied to evaluate the possibility to achieve complete
inactivation of fungi and bacteria. However the results of
the study confirmed that the only bactericidal action of
CO2 was not efficient to completely inactivate microorgan-
isms in ginseng samples.
There was not significant effect of changing the process-
ing time from 15 min to 2 h at 15.0 MPa and 60
C on total
aerobic microbial count reduction. At 15.0 MPa and pro-
cessing time of 2 h, increasing the temperature from
25 to 60
C slightly decreased the CO2 efficiency for total
aerobic count reduction. Increasing the processing time to
15 h at 60
C only decreased the total aerobic count to
2.67 log cycle that is still beyond the limit for oral admin-
istration by TGA (e.g., total aerobic microbial count
decreased from 5$107 cfu g1 to 1.10$105 cfu g1). In
addition such long processing times are not desirable and
practical for commercial purposes.
More recently Calvo and Torres (2010) applied the HPCD
treatment for the inactivation of natural microflora of paprika
powder, the dried and milled product of selected varieties of
red pepper from the genus Capsicum. The effects of the treat-
ment were tested on the initial microbial load (aerobic mes-
ophilic count, Enterobacteriaceae, E. coli, S. aureus, moulds
and yeasts) and on spores isolated from paprika powder by
cultivation. Results demonstrated that the microbial inactiva-
tion was a strong function of the water activity of paprika
samples reaching a value of 1.5 log cycle at 6.0 MPa and
80
C for 30 min with a water percentage of 35%.
Although the beneficial effects of the initial water mois-
ture were evident, higher amounts of water activity were
not investigated because the powder became a paste and
436 G. Ferrentino, S. Spilimbergo / Trends in Food Science & Technology 22 (2011) 427e441
was difficult to handle also with the addition of anti e cak-
ing agents in the vessel. Information about the behavior of
the inactivation rate with the change of the process param-
eters were also reported in the study. Slight variations of the
inactivation rate occurred increasing the pressure from 6.0
to 30.0 MPa obtaining less than a 1 log cycle reduction
difference.
The higher resistance of the natural microflora of pa-
prika powder to the HPCD treatment could be linked to
the protective effect due to the interaction with the food in-
gredients or to the insufficient contact between the CO2 and
the microbial cells. To investigate the possible protective
effect of the matrix, the authors inoculated the natural mi-
croflora isolated from paprika powder on different sterilized
materials (ground coffee, cocoa powder and flour) whose
appearance and texture was similar to that of paprika.
The results indicated that no important differences were
obtained independently of the chosen inoculated product.
Furthermore to improve the contact between the CO2 and
the microorganisms and get better results in the microbial
inactivation some options were taken into account. The first
attempt was the use of an anti e caking agent (CaCO3) widely
applied in the food industry in proportions less than 2% to
avoid the formation of lumps. The intercalation of inert but
highly porous particles such as perforated beads, bentonite
and activated carbon was also taken into account to increase
the porosity of the bed in order to prevent the possible forma-
tion of preferential channels that could lead to “dry or dead”
zones. The last attempt was the increase of the flow rate by
tripling the total mass of CO2 in contact with the paprika.
The experimental results demonstrated no remarkable
improvements for any of these attempts.
The authors concluded that the only factor influencing the
sterilization process was the resistance of the natural micro-
flora which was higher in a low water activity environment.
Optimizing the initial moisture between 25 and 30%, temper-
ature between 85 and 90
C, pressure between 6.0 and
10.0 MPa and treatment times between 30 and 45 min, it
was possible to achieve the disinfection and the total count
reduction required by law (as shown in Table 2).
The moisture content was found to be an important pa-
rameter to be controlled also for the application of HPCD
treatment to wheat flour. Haas et al. (1989) reported that
at higher moisture level equal to 28%, the inactivation of
bacterial and molds populations in wheat flour was en-
hanced operating in a temperature range of 25 and 50
C
at 6.2 MPa for two hours. Good microbial reduction was
also observed when flour with the addition of 25% of glyc-
erin (to increase the water activity to 0.91) was inoculated
with S. senftenberg, S. aureus and E. coli. A further in-
crease of glycerin to 75% corresponding to water activity
equal to 0.61 greatly reduced the killing power. In the
same study experiments carried out on dry peppers and
remoistened pepper (30%) showed similar results. Testing
dry materials with high microbial counts such as rosemary,
onion powder and bell pepper demonstrated that without
rehydration no killing effect due to the CO2 treatment
was evident while there was complete kill in fresh spices
of thyme, mint, chives, and oregano.
Effect on quality attributes
Few studies have been performed to characterize the
quality of food powders after the HPCD treatment. The
only results were the one published on paprika powders
and expressed in ASTA units which take into account the
color intensity of the powder (Calvo & Torres, 2010).
The authors reported that only a slight color reduction, an
average of 6 ASTA units, was detected in the treated sam-
ples independently from the temperature and the final water
content of the powder. However, some browning was ob-
served when treatment times were prolonged over 60 min
at temperatures over 90
C. Considerable color loss oc-
curred at the highest pressures over 25 MPa due to the ex-
traction of oleoresin and pigments. In conclusion the study
reported that factors influencing paprika quality were: the
water content and the process temperature. In the study
of Haas et al. (1989) quality analyses were performed on
fresh herbs exposed to 5.5 MPa, 45
C for 2 h. The results
demonstrated that thyme, mint, chives, and oregano had en-
hanced aromas after the treatment. However some herbs
showed a different taste after the treatment. Parsley tasted
similar to the untreated but developed a slight off aroma af-
ter treatment while untreated thyme and mint tasted better
than the treated sample. The results seemed to be dependent
on the variety, and maturity of the herbs which on the other
hand affected the taste, aroma and the palatability
evaluations.
Fishes
Microbial inactivation
The first attempt to apply the HPCD treatment for the
preservation of fish was carried out by Wei et al. in 1991.
In this study, shrimp samples spiked with Listeria were
treated by HPCD at 5.85 MPa, 35
C for 2 h. The results
demonstrated that the treatment reduced the bacterial
counts by only 35e45% but increasing the pressure to
13.7 MPa a microbial reduction equal to 99% was obtained.
More recently an innovative approach to post harvest
processing of oyster has been introduced focusing on the
effects of supercritical carbon dioxide on bacterial contam-
inants trapped in the digestive system of oysters (Meujo
et al., 2010). To observe the effects of CO2 as warm pas-
teurization technique, experiments were performed
in vitro on bacterial culture of a non e pathogenic strain
of Vibrio used as a model for Vibrio spp. and several bac-
terial isolates from an oyster homogenate. The authors re-
ported that the level of total bacterial inactivation
achieved with the HPCD treatment (100 bar for 30 min at
37
C or 172 bar for 60 min at 60
C), was comparable
to that achieved with several FDA approved post harvest
processing for oysters, namely, high hydrostatic pressure
and quick frozen (Prapaiwong, Wallace, & Arias, 2009).
Table 4. Application of HPCD treatment coupled with additives or pretreatments on microbial forms in food system. Type of food, microorganism, process conditions, and additives or
pretreatments employed are reported with the corresponding microbial inactivation and references.

Additives/Pretreatments Target microorganism Food system Process conditions Microbial inactivation Reference
100% CO2 at atmospheric pressure Anaerobe spores Fresh celery HPCD (30 min at 40
C and 62.8 MPa) 50% (Kuhne & Knorr, 1990)
leaves and CO2 flushed on the sample for 60 min

G. Ferrentino, S. Spilimbergo / Trends in Food Science & Technology 22 (2011) 427e441

leafstalks at 27
C, 0.1 MPa

H2O2 Thermo e resistant spores Cocoa HPCD (2 h at 40
C and 30.0 MPa) No inactivation (Calvo et al., 2007)
powder (CO2 saturated in H2O2 as solvent)

Ethanol HPCD (2 h at 40
C and 30.0 MPa) CO2

saturated in ethanol
Acetic acid Escherichia coli Boneless HPCD (30 min at 35
C and 12 MPa) 3% 2.58 Log (cfu/cm2) (Choi et al., 2009b)
Lactic acid Listeria monocytogenes pork loins solution of acetic acid (samples immersed 2.60 Log (cfu/cm2)
Salmonella typhimurium for 1 min at 4
C) 3% solution of lactic 2.33 Log (cfu/cm2)
E. coli O157:H7 acid (samples immersed for 1 min at 4
C) 2.10 Log cfu/cm2)

NaOCl Natural microflora Red paprika HPCD (10 min at 35
C and 6.0 MPa) 4 Log (cfu/g) (Matsufuji et al., 2009)
Green pepper 100 ppm NaOCl solution (samples 3.8 Log (cfu/g)
Cucumber immersed for 10 min at room temperature) 4.2 Log (cfu/g)
Cabbage 3 Log (cfu/g)
Lettuce 3 Log (cfu/g)
Mizuna (Brassica) 4 Log (cfu/g)
Celery 3.5 Log (cfu/g)
Onion 2.5 Log (cfu/g)
Carrot 2 Log (cfu/g)
Bean sprout 7 Log (cfu/g)

Water Total aerobic Ginseng powder HPCD (1 h at 60
C and 10.0 MPa) 1 mL 0.1 Log (cfu/g) (Dehghani et al., 2008)
microbial count of water in 70 mL of CO2

Ethanol HPCD (1 h at 60
C and 10.0 MPa) 1 mL 0.2 Log (cfu/g)

of ethanol in 70 mL of CO2

H2O2 HPCD (1 h at 60
C and 10.0 MPa) 1 mL 0.4 Log (cfu/g)

of H2O2 in 70 mL of CO2

Water þ ethanol þ H2O2 HPCD (1 h at 60
C and 10.0 MPa) 1 mL 1.7 Log (cfu/g)
of water þ1 mL of ethanol þ 1 mL of
H2O2 in 70 mL of CO2

437
438 G. Ferrentino, S. Spilimbergo / Trends in Food Science & Technology 22 (2011) 427e441

Effect on quality attributes pathogenic bacteria in fresh pork. Experimental results

Quality observations of shrimp samples treated by Wei
et al. (1991) showed some color change after the HPCD
treatment carried out at 13.7 MPa, 35
C for 2 h. As ob-
served for the chicken samples, the authors reported that
the outer layer of the shrimps turned whitish and gave the
appearance of being cooked rapidly at low temperature or
soaked in acid. Also loss of small amount of liquid from
the tissue was observed giving the idea of some tissue dam-
aging caused by the treatment.
The study on oysters treated by supercritical CO2 re-
ported a sensory analysis in terms of physical appearance,
smell and texture assessed by a panel of 13 people
(Meujo et al., 2010). The treated oysters were compared
with two standards: a freshly shucked, untreated oyster
and an oyster left at room temperature for about two days
to develop a mild odor associated with spoiled oysters.
The sensory analysis revealed that oysters remained accept-
able considering their physical appearance, texture and
smell after the exposure to CO2 process (10 and 20 MPa
for 20 and 50 min at 37
C).
Additives and treatments combined with HPCD
treatment
Few research papers have been published demonstrating
an increase of the microbial inactivation rate by adding
some co e solvents or coupling the effects of HPCD treat-
ment with a pretreatment. Table 4 is a compilation of the ex-
perimental results found in literature concerning the effect of
coupling additives and pretreatments to the HPCD treatment.
Indications of the types of food, microorganism, process con-
ditions, and employed additives or pretreatments are reported
with the corresponding microbial inactivation.
The addition of co e solvents or additives to pressurized
CO2 has been proposed in a consistent number of publica-
tions mainly applied to liquid substrates. The synergistic or
antagonist effect of the process has been explained based
on the nature of the co e solvent able to increase the
amount of CO2 in contact with the substrate and in turn
available to act as antimicrobial agent (Ferrentino,
Balaban, Ferrari, & Poletto, 2010; Has & Herman, 1977;
Haas et al., 1989).
Choi, Kim, Kim, Kim, and Rhee (2009b) reported the
antimicrobial effects of coupling organic acids (acetic or
lactic acid solutions for 1 min) and HPCD treatments
(12 MPa and 35
C for 30 min) under various conditions
on the reduction of a nonpathogenic E. coli and three
Table 5. Relevant patents accomplished for the application of HPCD treatment to solid foods.
showed that the combined treatments had no synergistic ef-
fect and no significant differences were observed between
the samples treated coupling acetic acid and HPCD or lac-
tic acid and HPCD.
Combination treatments applied to solid foods is a com-
pletely undiscovered field. Only two papers investigated the
efficacy of the HPCD treatment combined with pretreat-
ments (Calvo et al., 2007; Kuhne & Knorr, 1990) for the
inactivation of spores as shown in Table 4.
Since spores could not be inactivated exposing fresh cel-
ery leaves and leafstalks to only HPCD treatment at
62.8 MPa for 30 min at 40
C, Kuhne and Knorr (1990)
proposed a pretreatment with the exposure of the samples
to 100% CO2 at atmospheric pressure and 27
C for
60 min. At the end of the combined treatments, 50% reduc-
tion of anaerobe spores was observed.
The use of combined treatments was also suggested by
Calvo et al. (2007) to inactivate thermo e resistant spores
in cocoa powder coupling the effect of ethanol and hydro-
gen peroxide (H2O2) with HPCD (30.0 MPa and 40
C for
2 h). The results showed that the application of a combined
treatment was useless with the presence of both ethanol and
H2O2. Hemmer, Drews, LaBerge and Matthews (2006) pub-
lished experimental data demonstrating that adding H2O2
(<100 ppm), 6 log reductions on populations of Geobacil-
lus stearothermophilus and Bacillus atrophaeus spores
strips (impregnated on cellulose supports) were completely
deactivated using supercritical CO2 in 1 h at 40
C and
20.0 MPa. In the same year, Zhang et al. (2006) showed
with transmission electron microscopy images (TEM) that
B. atrophaeus spore envelope (growth on strips and glass
vials) was damaged by supercritical CO2 allowing the pen-
etration of H2O2 and the oxidation of some vital internal
structures causing spore death.
Patents
Patents related to the use of HPCD pasteurization/steril-
ization of solid foods are scarce. Two are the most relevant
shown in Table 5 where is reported the number of the pat-
ent, the assignee and the description of the method. The
first patent described the coupled effect of pressurized
CO2 with another non e thermal technique (Urbain,
Shank, & Kauffman, 1969). The food, mainly meat, was
exposed to an atmosphere of pressurized carbon dioxide
and subsequently to relatively low energy ionizing radia-
tion. The patent reported that the combined process resulted

Assignee Number Observations Reference

Swift & Company, Chicago, Ill.
Air Liquid, Houston, TX
US 3,483,005 Method of sterilizing food products which are (Urbain et al., 1969)
susceptible to the development of flavor degradation
US 2008/01711 A1 Method to pasteurize pre-packaged food at, (Rasanayagam & Yuan, 2008)
or near room temperature, using supercritical
carbon dioxide
 G. Ferrentino, S. Spilimbergo / Trends in Food Science & Technology 22 (2011) 427e441
in a truly sterile food product free from spoilage, with an
extended storage life, without discoloration and with un-
changed flavor.
Rasanayagam and Yuan (2008) granted a patent to pas-
teurize pre e packed foods at, or near room temperature,
using supercritical CO2. The invention described a method
to use supercritical CO2 to inactivate pathogenic microor-
ganisms in foods, and at the same time, to carbonate the
food in a pre e packed pouch or container with a pre de-
fined polymer structure that allowed CO2 to permeate while
discouraging the permeation of other substances. The
method was able to induce a substantial pasteurization (3
or 4 log cycles), and to inhibit enzymes in the foodstuff.
Additionally the polymer pouch was able to retain CO2 to
a predetermined level (between 0 and 5 volumes of CO2)
in a way to establish a modified atmosphere suitable for
the storage of the pasteurized food.
Potentials of HPCD technology and future outlooks
The review clearly shows that the treatment has poten-
tials in reducing microbial loads improving the safety of
the food. However a great challenge is the control of pro-
cess parameters (pressure, temperature and treatment
time) considering that they can affect molecular interac-
tions and protein conformation causing color and structural
changes in food products. This aspect seems to be a serious
concern to extensively apply the technology. It is evident
that the treatment seems suitable not for all solid foodstuffs
due to the physical consistency of the products and their
ability to positively react to the process in terms of micro-
bial inactivation and retention of quality attributes. The ef-
fects of the treatment on the quality attributes reported so
far clearly evidence that the process cannot be applied to
soft foods or leafy vegetables. It seems suitable for foods
which retaine their structure or maintaine an appeal for
the consumers although in soft form (i.e. fruits in syrup).
A further fundamental aspect to take into account is the
weakest knowledge of mechanistic studies and mathemati-
cal modeling of the inactivation kinetics which are essential
to understand the fundamentals of the HPCD treatment, to
optimize the process parameters and propose a safe, cheap
and time consuming technique. In addition it is evident that
a great challenge for the application of the technology is the
need to investigate and understand the mechanisms by
which HPCD inactivates pathogens and microbes and
how the food matrix interferes with the inactivation.
The overview of the scientific works published so far re-
veals that most of the results on the effects of the HPCD
treatment on solid products are just qualitative. Very few
systematic or quantitative analyses have been performed
to investigate the chemical and physical effects of the treat-
ment. To confirm the potentials of the treatment from
a quantitative point of view more scientific evidences are
needed.
In this concern, the definition of the foods suitable for
the treatment is necessary carrying out screening tests
439
with a wide range of product types to enable the selection
of suitable candidate products. The achievement of kinetics
of microbial and enzymatic inactivation at different process
conditions are necessary at lab scale testing both typical
pathogens but also natural microbes occurring in foods.
In addition, extensive studies need to be performed to
evaluate the shelf life and long term safety of the best can-
didate products to the treatment. Once all these missing in-
formation will be filled, the subsequent step will be the
scaling of such novel technology into production processes,
including design of appropriate equipment and screening of
processing parameters after scale e up suitable for food
matrixes.
Only with scientific results, convincing data, and provi-
sion of clear, objective and unbiased information, including
the potentially negative aspects of the technology and their
limitations, the HPCD treatment could become one of the
most available emergent technologies applied to solid
foods.
References
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Awuah, G. B., Ramaswamy, H. S., & Economides, A. (2007). Thermal
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Brown, W. D., & Mebine, L. B. (1969). Autoxidation of
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