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Nonthermal Preservation of Foods Using Combined

Processing Techniques
a b
Javier Raso & Gustavo V. Barbosa-Cánovas
a
Tecnología de los Alimentos, Facultad de Veterinaria, Universidad de Zaragoza, C/ Miguel
Servet, 177, 50013 Zaragoza. SPAIN
b
Department of Biological Systems Engineering, Washington State University, Pullman, WA
99164-6376
Published online: 03 Jun 2010.

To cite this article: Javier Raso & Gustavo V. Barbosa-Cánovas (2003) Nonthermal Preservation of Foods Using Combined
Processing Techniques, Critical Reviews in Food Science and Nutrition, 43:3, 265-285, DOI: 10.1080/10408690390826527

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 Critical Reviews in Food Science and Nutrition, 43(3):265–285 (2003)

Nonthermal Preservation of Foods Using

Combined Processing Techniques
Javier Rasoa and Gustavo V. Barbosa-Cánovas*b
a Tecnología de los Alimentos, Facultad de Veterinaria, Universidad de Zaragoza, C/ Miguel Servet, 177,

50013 Zaragoza. SPAIN; bDepartment of Biological Systems Engineering, Washington State University,
Downloaded by [UZH Hauptbibliothek / Zentralbibliothek Zürich] at 22:21 13 September 2013

Pullman, WA 99164-6376

Referee: Dr. Jorge Welti-Chanes, Vicerrectoria Academica, Universidad de las Americas Pubela, Santa Catarine Martir, 72820
Cholula, Pue., Mexico

* To whom correspondence should be sent: Gustavo V. Barbosa-Cánovas, Phone: 509 3356188. Fax: 509 3352722, e-mail:
barbosa@mail.wsu.edu

ABSTRACT: In the last 2 decades, consumer demand for fresher, higher quality, and safer food has promoted
research on nonthermal methods of food preservation for the inactivation of microorganisms and enzymes as an
alternative to thermal processes. However, the high resistance of certain enzymes and microorganisms to nonthermal
processes, especially bacterial spores, limit their application. To expand the use of nonthermal processes in the
food industry, combinations of these technologies with traditional or emerging food preservation techniques are
being studied. The use of nonthermal processes in combination with other preservation technologies presents a
number of potential benefits to food preservation. The purpose of this article is to review some successful
combinations of different nonthermal technologies, such as high hydrostatic pressure, ultrasound, pulsed electric
fields, and irradiation, with traditional or emerging food preservation technologies.

I. INTRODUCTION Most food preservation procedures inhibit

Foods begin to lose their quality the moment
they are harvested, through changes resulting from
physical, chemical, enzymatic, or microbiological
reactions. Food preservation prevents these deterio-
rative reactions, extending a food’s shelf life and
assuring its safety. Microorganisms and enzymes
are the main agents responsible for food spoilage
and therefore the targets of preservation techniques.
An “ideal” method of food preservation has
the following characteristics:
• It improves shelf life and safety by inactivating
spoilage and pathogenic microorganisms.
• It does not change organoleptic and nutritional
attributes.
• It does not leave residues.
• It is cheap and convenient to apply.
• It encounters no objections from consumers
and legislators.
deteriorative agents. Some of these technologies
(e.g., chilling or freezing) maintain a food’s fresh
character. However, applications of others, such
as lower aw or pH, also affect freshness. Overall,
these technologies are incapable of assuring food
safety, since modifications of preservation condi-
tions, such as breakdown in the chill chain or food
rehydration, could lead to the growth of patho-
genic and spoilage microorganisms.
Currently, heat is the only food preservation
technique to act via inactivation, which is used
substantially in the food industry. Inactivation of
microorganisms and enzymes results in a shelf-
stable, safe product. Thermal processing has most
of the characteristics of an “ideal” food preserva-
tion method. However, in some foods the high
thermotolerance of certain enzymes and microor-
ganisms, mainly bacterial spores, makes neces-
sary the application of extreme heat treatments,
which affects the nutritional and organoleptic food

1040-8398/03/$.50
© 2003 by CRC Press LLC
265
 properties. Therefore, alternatives to thermal pro-
cessing as the main means of inactivating patho-
genic and spoilage microorganisms are being
sought by the food industry.
In the last 20 years, consumer demand for
high-quality foods that are microbiologically safe
and stable has awakened a growing interest in
nonthermal preservation techniques capable of
inactivating microorganisms and enzymes
(Mertens and Knorr 1992; Barbosa-Cánovas et al.
1998). During nonthermal processing, the tem-
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perature of foods is held below the temperature
normally used in thermal processing; therefore, a
minimal degradation of food quality is expected.
However, nonthermal technologies must not only
improve food quality, but also promote an equiva-
lent or, preferably, an enhancement of safety lev-
els, when compared with other procedures they
replace.
Overall, most nonthermal preservation tech-
niques are highly effective in inactivating vegeta-
tive cells of bacteria, yeast, and molds (Cheftel
1995; Wouters and Smelt 1997; Smelt 1998).
However, bacterial spores and most enzymes re-
main difficult to inactivate with these procedures,
thus their use is limited to foods where enzymatic
reactions do not affect food quality or where spore
germination is inhibited by other prevailing con-
ditions, such as low pH.
To extend the use of nonthermal processing
in the food industry, combinations of these tech-
nologies with traditional or emerging food preser-
vation techniques are being studied. This approach,
known as “hurdle technology”, has already been
applied successfully using traditional techniques
of food preservation (Leistner and Gorris 1995).
Food preservation using combined methods in-
volves successive or simultaneous applications of
various individual treatments. Combined treat-
ments are advantageous, principally because many
individual treatments alone are not adequate to
ensure food safety or stability. In some circum-
stances, combined treatments allow a milder use
of single treatments, with consequent improve-
ment in food quality. The total preservation effect
of combining several preservation techniques can
be merely additive, but in terms of food quality
and safety a synergistic effect is preferable. How-
ever, combining technologies is not always ad-
266
vantageous, because in some circumstances an
antagonistic effect may result (Figure 1).
Combining nonthermal methods with other
food preservation techniques can (1) enhance the
lethal effects of nonthermal processing, (2) re-
duce the severity of nonthermal treatment needed
to obtain a given level of microbial inactivation,
and/or (3) prevent the proliferation of survivors
following treatment (Table 1). The purpose of
this article is to review some successful combina-
tions of given nonthermal technologies with other
preservation techniques.
II. COMBINATIONS WITH HIGH
HYDROSTATIC PRESSURE
During high hydrostatic pressure (HHP) pro-
cessing, foods are subjected to pressures in the
range 100 to 1000 MPa. These treatments are
capable of inactivating microorganisms and en-
dogenous enzymes, while retaining nutrients and
flavors (Tauscher 1995). The first studies on HHP
preservation of foods were conducted at the end
of the 19th century (Hite 1899). However, this
technology has not had commercial application
until recent years. The renewed interest in HHP
processing in the last decade is not only due to its
preservation effects but also to its potential to
modify the functional properties of foods (Tewari
et al. 1999).
Overall, HHP is very effective in inactivating
vegetative cells of microorganisms, but pressure
treatment alone does not achieve a substantial
inactivation of spores and reduction in activity of
certain enzymes (Smelt 1998; Hendrickx et al.
1998). The majority of HHP-processed products
available in the market are high acid products like
fruit juices or sauces (Tewari et al. 1999). These
products are good candidates for HHP preserva-
tion, because due to low pH, they are mainly
spoiled by microorganisms relatively sensitive to
HHP (yeast, molds, and lactic bacteria) and do
not support the germination of pressure-resistant
bacterial spores.
Interest in using high-pressure technology to
extend the shelf-life of low-acid foods (e.g., poul-
try and red meat, foie-gras, goat milk cheese,
liquid whole egg, or cooked ham) is increasing
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FIGURE 1. Possible effects obtained by combining different preservation technologies.
(Carlez et al. 1993; Crawford et al. 1996; El
Moueffak et al. 1995; Obrien and Marshall 1996;
Pushpam et al. 1997; Ponce et al. 1997; López-
Caballero et al. 1999). Preservation of such foods
with HHP requires choosing the appropriate com-
bined treatment that can achieve enough levels of
microbial inactivation and shelf-life extension.
A. Combining HHP with Heat
The combined effect of HHP and heat on
microbial inactivation has been widely investi-
gated among various microorganisms suspended
in different media, and in solids and liquids foods
(Tables 2 and 3).
The resistance of vegetative cells of bacteria
to HHP appears to be greatest at temperatures
approximately 20 to 30˚C. At higher and lower
temperatures, microorganisms are more sensi-
tive to HHP. The resistance to HHP by vegeta-
tive cells of bacteria decreases even when pres-
sure is combined with heat at nonlethal tempera-
tures. This combination allows substantial
inactivation (>6 log10 cycles) of spoilage and
pathogenic microorganisms at lower pressures
and/or shorter times than that required when
pressurization is carried out at room temperature
(Table 2). For example, the inactivation of List-
eria monocytogenes in UHT milk did not occur
at 200 MPa and temperatures up to 45˚C. In
contrast, at 200 MPa and 55˚C, a near 6-log10-
cycle reduction in cell count was obtained after
15 min of treatment (Simpson and Gilmour 1997).
Another benefit of this combination is that varia-
tion in pressure resistance among strains is much
lower when HHP is combined with moderate
temperature. Resistance by different strains of
L. monocytogenes, Staphylococcus aureus, Es-
cherichia coli O157:H7, and Salmonella species
267
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TABLE 1

268
Main Combinations That Include Nonthermal Methods of Food Preservation Investigated and Objectives Reached with These
Combinations
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TABLE 2
Inactivation of Bacteria Vegetative Cells with Combination of HHP and Heat

269
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270
TABLE 3
Inactivation of Bacterial Spores with Combination of HHP and Heat
 to 345 MPa varied widely at 25°C, but these
differences were greatly reduced at 50°C (Alpas
et al. 1999).
In general, the kinetics of inactivation of most
vegetative cells by HHP at low temperatures shows
an initial exponential rate, followed by pronounced
tailing (Smelt 1998). This tail tends to disappear
when HHP is combined with heat (aKalchayanand
et al. 1998). Patterson and Kilpatrick (1998) fitted
inactivation curves of combined pressure and heat
treatments on E. coli O157:H7 and S. aureus in
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milk and poultry using the Gompertz equation.
They devised a simple model that predicted the
inactivation of these microorganisms at various
pressure-temperature combinations. This model
can be very useful in commercial applications in
establishing processing conditions that achieve
sufficient inactivation levels and assure food safety
and stability.
Inactivation of vegetative forms of yeast and
molds in combination with HHP and heat has
been scarcely investigated, probably because mi-
croorganisms involved are very sensitive to mild
HHP treatments at room temperature (Ogawa et
al. 1990; Pandya et al. 1995; Palou et al. 1997;
Parish 1998; aRaso et al. 1998). However, inacti-
vation of ascospores in molds requires combining
HHP and moderate temperatures (60 to 70°C)
(Butz et al. 1995).
Inactivation of bacterial spores with HHP,
unlike inactivation of vegetative bacteria, occurs
in two steps. Pressure initially causes the germi-
nation of spores and then inactivates the germi-
nated forms (Clouston and Wills 1969; Gould and
Sale 1970). In general, bacterial spores appear to
be resistant to HHP treatments at room tempera-
ture; it has been reported they can resist pressures
as high as 800 MPa for many hours. However,
pressures as low as 10 MPa can initiate germina-
tion of bacterial spores, thus sensitizing them to
heat, radiation, chemical agents, and even HHP
treatments (Gould 1973; Wuytack et al. 1998).
Initiation of germination and inactivation of
bacterial spores by HHP are highly temperature
dependent, thus increase at higher temperatures
(Sale et al. 1970; bRaso et al. 1998). Table 3
shows the combinations of pressure and tempera-
ture capable of inactivating bacterial spores. At
low temperatures, the treatments cause spore ger-
mination, but in general the pressure is insuffi-
cient to cause appreciable inactivation of germi-
nated forms. Combined HHP and heat is espe-
cially effective at temperatures allowing
inactivation of germinated spores (>60˚C), sug-
gesting that spores germinated by HHP are di-
rectly inactivated by heat. However, recently it
has been observed that inactivation of B. subtilis
and Bacillus stearothermophilus spores by HHP
at 70 and 90°C, respectively, did not involve spore
germination (Mönch et al. 1999).
Hayakawa et al. (1994) found that inactiva-
tion of B. stearothermophilus by combining HHP
and heat was more effective when an oscillatory
treatment (6 cycles for 5 min) was applied. At
70˚C and 600 MPa, 4 log cycles of inactivation
were obtained with continuous treatment and more
than 6 log cycles of inactivation were achieved
with oscillatory treatment.
The effect of simultaneous applications of
moderate pressure (1 to 30 MPa) and heat (115 to
125˚C) on the inactivation of B. stearothermophilus
has also been investigated (Mallidis and Drizou
1991), but the effect of pressure on the reduction of
heat resistance was insignificant; Dt values only
decreased by a factor of 2.
The effect of HHP on enzymes varies widely
for different enzymes, probably due to the struc-
tural differences of individual enzymes. In gen-
eral, combinations of pressure with moderate tem-
peratures increase the level of enzyme inactivation,
but in some cases an increase in enzyme activity
has been reported (Hendrickx et al. 1998).
Temperatures between 45 and 55˚C and pres-
sures between 600 and 900 MPa can inactivate
pectinesterase, lipase, polyphenoloxidase (PPO),
lipoxygenase, peroxidase (POD), lactoperoxidase,
phosphatase, and catalase in different extensions
(Seyderhelm et al. 1996).
Cano et al. (1997) studied the inactivation of
POD, PPO, and pectinmethylesterase (PME) in
strawberry puree and orange juice treated with
combined HHP and heat at pressures lower than
400 MPa. POD activity in strawberry puree only
decreased 25% when using the best combination
(230 MPa at 43˚C), and PPO showed the same
activity before and after treatment at 400 MPa
and 60˚C. In orange juice, 400 MPa at 32˚C pro-
duced the greatest inactivation (50%) of POD,
271
 and at higher temperatures this enzyme was acti-
vated. PME activity in orange juice also increased
with increasing temperature in the range of pres-
sures investigated. Similar results on inactivation
of PME and PPO in tomato puree were obtained
(Hernandez and Cano 1998).
The high resistance of endogenous enzymes
to HHP or to combined HHP heat treatments
emphasizes the necessity of combining pressur-
ization with other techniques, such as storage at
low temperature, chemical modification of en-
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zymes, and use of enzymes (“killer enzymes”) or
naturally occurring protein inhibitors (Ashie et al.
1996) to maintain food quality and extend shelf-
life.
Combining HHP and heat could be of great
practical interest, especially in preserving low-
acid foods. Combining mild temperatures and pres-
sure allows the pasteurization/sterilization of foods
at lower temperatures. For example, microbial
inactivation in duck foie-gras after a combined
high-pressure heat treatment (400 MPa, 50˚C)
was similar to that following traditional thermal
pasteurization (85˚C at the coldest point)
(Elmoueffak et al. 1995). Another practical ben-
efit of this combination would be the inactivation
of microorganisms at moderate pressures, which
is economically feasible and does not induce qual-
ity loss in the treated product.
B. Combining HHP with low pH
In general, microbial inactivation of both
vegetative cells and bacterial spores with HHP is
scarcely affected by the pH of the treatment me-
dium, and results reported by different authors are
contradictory. Maggi et al. (1993) studied the
HHP inactivation of four strains of Salmonella
and seven other strains of Enterobacteriaceae,
and found that Salmonella and four of the Entero-
bacteriaceae strains were less pressure sensitive
at acid pH, while the other three Enterobacteri-
aceae were more sensitive at neutral pH.
Several authors (Sale et al. 1970; Fornari et
al. 1995; cRaso et al. 1998) have reported that
inactivation of bacterial spores by combining
pressure and temperature was greatest when the
pH was near neutrality. However, a higher in-
272
activation of spores in B. coagulans and
Clostridium sporogenes PA 3679 was obtained
when the pH of the treatment medium was re-
duced from 7 to 4. (Roberts and Hoover 1996;
Stewart et al. 2000).
Different yeasts and molds showed the same
pressure resistance in sutsuma mandarin juice
acidified between 2.5 and 4.5 with citric, tartaric,
lactic, or acidic acid (Ogawa et al. 1990). How-
ever, lowering the pH of citrate buffer from 5 to 3
enhanced the lethality in Saccharomyces cerevisiae
(225 MPa, 25˚C, 30 min) and Zygosaccharomyces
bailii (200 MPa, 25˚C, 30 min), 2 and 1.5 log10
cycles, respectively (Pandya et al. 1995).
Although microbial sensitivity to HHP is not
increased by lowering pH, the key factor in this
combination is the prevention of microbial growth
and the germination of spores that can survive
HHP treatment at acidic pH. García-Graells et al.
(1998) observed that high-pressure-resistant mu-
tants of E. coli surviving a pressure treatment
resulted in accelerated inactivation during subse-
quent storage at low pH.
C. Combining HHP with Antimicrobials
Interest in natural antimicrobials has expanded
in recent years in response to consumer demand
for less “artificial” additives. The potential of
these natural antimicrobials is substantial, espe-
cially in combination with other food preserva-
tion techniques (Gould 1996).
Recent studies have indicated that HHP inflicts
sublethal injury on microorganisms, even at lower
pressures required for their death (Patterson et al.
1995). Sublethally injured cells are more susceptible
to antimicrobial components (Kalchayanand et al.
1994). This fact is especially of value in Gram-
negative bacteria because their outer membrane acts
as an efficient barrier against hydrophobic solutes
and macromolecules, such as bacteriocins (Halander
et al. 1997).
Hauben et al. (1996) observed that during
HHP exposure (>180 MPa) E. coli cells became
sensitized to lysozyme and nisin, two antimicro-
bial proteins excluded by the outer membrane at
ambient pressure. HHP treatment (320 MPa,
15 min, 23˚C) reduced viable counts of E. coli to
 4.06 log10, inactivating 5.7, 5.4, and 6.9 log10 in
the population, respectively, when nisin (100 IU/ml),
lysozyme (10 µg/ml), or both antimicrobials were
present during pressurization. However, adding
these antimicrobials immediately after pressure
treatment did not cause any viable count reduc-
tion. E. coli pressure-resistant mutants were also
sensitized to nisin and lysozyme by HHP at the
same pressure levels required for nonpressure-
resistant strains. An oscillatory treatment (3 cycles
for 10 min) at 400 MPa in the presence of both
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antimicrobials allows a reduction of at least
6 log10 cycles of E. coli MG 1615, which is one of
the most pressure-resistant vegetative cells de-
scribed to date (Masschalck et al. 2000).
The application of a cyclic pressure treatment
(3 cycles for 10 min at 550 Mpa, 20°C) in combi-
nation with nisin (100 IU/ml) and lysozyme (100
µg/ml) strongly enhances the inactivation of four
E. coli strains (MG1655 and three pressure-resis-
tant mutants obtained from this strain) suspended
in skim milk. During the cyclic treatment, inactiva-
tion obtained in the four strains studied was at least
3 log10 cycles higher than that obtained with con-
tinuous treatment for 30 min under the same con-
ditions (García-Graells et al. 1999).
The presence of pediocin AcH and nisin
during pressurization also increased the lethal
effect of HHP in both Gram-positive and Gram-
negative pathogens. When S. aureus,
L. monocytogenes, S. typhimurium, or E. coli
were pressurized at 345 MPa at 25˚C for 10 min
in the presence of a mixture of pediocin AcH
(3000 IU/ml) and nisin (3000 IU/ml), additional
inactivation ranging between 1.3 and 5.1 log10
cycles resulted (bKalchayanand et al. 1998).
The combination of antimicrobials with pres-
surization at moderate temperatures has also been
studied in pathogenic and spoilage microorgan-
isms. The presence of pediocin AcH (3000 AU/ml)
during pressurization at 450 MPa and 45 to 50˚C
for 5 min produced an additional reduction of 3 to
4 log10 cycles in the viability of S. aureus, L.
monocytogenes, S. typhimurium, E. coli, Lacto-
bacillus sake, Leuconostoc mesenteroides, Serra-
tia liquefaciens, and Pseudomonas fluorescens.
These combinations inactivated more than 8 log10
cycles in all species investigated (bKalchayanand
et al. 1998).
A strong synergistic effect against Lactoba-
cillus plantarum and E. coli has also been ob-
served when combining nisin with moderate HHP
at reduced temperatures (<15°C). When nisin was
present during treatment and in the recovery me-
dium at a concentration of 0.1 µg/ml, more than
6 log10 reductions in the population of L. plantarum
was achieved with HHP treatment at 200 MPa
and 10°C for 10 min. This treatment caused the
same inactivation for E. coli when nisin was
present at 2 µg/ml (Ter Steeg et al. 1999).
Combining antimicrobials with HHP can en-
hance the effectiveness of pressurization with
advantages in product quality and safety. Results
obtained by different authors indicate that this
combination at reduced or moderate temperatures
can enhance the effectiveness of HHP treatments.
The inhibition of microbial growth following treat-
ment is an additional advantage of combining
HHP and antimicrobials and should be investi-
gated more deeply.
D. Combining HHP with Modified
Atmospheres
HHP at low temperatures combined with
modified atmosphere packaging (MAP) has been
used to extend the shelf-life of salmon and prawns.
Amanatidou et al. (2000) compared the shelf-life
of vacuum or MAP (50% CO2+ 50% O2) packed
salmon, with the shelf life of HHP-processed
salmon under vacuum, HHP-processed salmon
and subsequently packed under MAP (50% CO2+
50% O2), and HHP-processed salmon with com-
pressed gases (50% CO2+ 50% O2). This last
treatment was more effective in delaying micro-
bial growth during storage at 5°C. However, the
best treatment for salmon in terms of shelf-life
extension and quality retention was a pressuriza-
tion treatment of 150 MPa for 10 min followed by
MAP. This treatment extended the shelf-life at
least 5 days, compared with the vacuum-packed
and chill-stored salmon usually sold. A combina-
tion of vacuum packaging and HHP also pro-
longed the shelf life of prawns (López-Caballero
et al. 2000). Pressurization at 200 MPa or 400
MPa for 10 min at 7°C under vacuum extended
the shelf-life of vacuum-packaged prawns 7 and
273
 14 days, respectively, in terms of microbiological
indices. However, pressurization treatments in-
duced the appearance of some dark spots during
storage of prawns.
Results from these studies indicate that com-
bining HHP and MAP packaging could be useful
in extending the shelf-life of fresh products dur-
ing refrigerated storage. Specific studies should
be conducted to discover the best combination for
any given food.
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E. Combining HHP with Carbon Dioxide
The antimicrobial effect of carbon dioxide
(CO2) is well known (Dixon and Kell 1989), and
it has been observed that this effect increases
when CO2 is applied under pressure. In general,
pressures used in this combination are below 50
MPa. Microbial inactivation with this combina-
tion has been observed in natural flora and with
specific spoilage and pathogenic microorganisms
in model systems and foods (Kamihira et al. 1987;
Haas et al. 1989; Wei et al. 1991; Arreola et al.
1991; Lin et al. 1994; Hong et al. 1997). How-
ever, pressurized CO2 does not appear to have a
significant effect on bacterial spores and fungal
ascospores at temperatures below 80˚C (Ballestra
and Cuq 1998).
Time, pressure, temperature, water activity, and
pH are the critical parameters in this combination.
An increase in temperature and/or pressure and a
decrease in pH enhances the antimicrobial effects of
CO2 under pressure (Haas et al. 1989; Ballestra and
Cuq 1998). On the other hand, inactivation decreases
at low water activity (Haas et al. 1989).
The exact mechanism behind microbial inac-
tivation using CO2 under pressure is still unclear.
Some authors suggest that the quick release of
carbon dioxide following compression plays a
significant role in microbial reduction (Nakura et
al. 1994). However, others suggest that the inac-
tivation of microorganisms occurs during the pres-
surization step, not during decompression. It is
thought that, under pressure, more CO2 molecules
can pass through the membrane and lower the
internal pH, thereby affecting the key enzymes in
cell metabolism (Haas et al. 1989). The inactiva-
tion of pressurized CO2 has also been related to
274
the extraction of cell wall constituents, such as
phospholipides and hydrophobic compounds (Lin
et al. 1992).
Pressurized CO2 is a promising alternative to
improving food quality by the reduction of micro-
bial loads especially present on food surfaces.
However, a long treatment period is generally
needed to achieve substantial microbial inactiva-
tion.
III. COMBINATIONS WITH ULTRASOUND
Ultrasound is defined as sound waves with
frequencies above that of human hearing (typi-
cally higher than 16 kHz). These waves can be
propagated in a liquid media as alternating com-
pression. If ultrasound has sufficient energy, a
phenomenon known as cavitation occurs. Cavita-
tion involves the formation, growth, and rapid
collapse of microscopic bubbles. Based on theo-
retical considerations, extremely high tempera-
tures and pressures are momentarily delivered to
the liquid media during the collapse of bubbles
(Suslick 1988). Electrical discharge and produc-
tion of free radicals have also been associated
with extreme conditions occurring inside the col-
lapsing bubble (Leighton 1998). However, the
ultimate reason for microbial inactivation via ul-
trasound is believed to be the mechanical damage
caused by cavitation (dRaso et al. 1998).
Ever since the lethal effects of ultrasound on
microorganisms were first observed (Harvey and
Loomis 1929), its use has been continually sug-
gested for disinfection and food preservation (Paci
1953; Jacobs and Thornley 1954; Boucher 1980;
Gaboriaud 1984). However, this technology has
not been adopted, probably due to the long treat-
ment needed for substantial microbial inactiva-
tion.
Recently, it was demonstrated that microbial
inactivation with ultrasound increases when treat-
ment is applied under pressure (Manosonication,
MS) (dRaso et al. 1998). For example, an increment
of hydrostatic pressure from 0 to 600 kPa at constant
amplitude (150 µm) decreased the decimal reduc-
tion time (D value) of Yersinia enterocolitica eight
times. The increased lethality of ultrasound under
higher static pressure was more remarkable within a
 given pressure range (0 to 300 kPa). At constant
hydrostatic pressure, microbial inactivation depended
on the amplitude of the ultrasonic waves. At 200
kPa, the D value of Y. enterocolitica decreased 11
times when the amplitude increased from 21 to 150 µm.
An exponential relationship was observed between
the lethality of ultrasound and the amplitude. The
influence of hydrostatic pressure and the amplitude
on the lethality of MS treatments in different Gram-
negative and Gram-positive bacteria was the same
and has been described by two mathematical equa-
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tions (aPagán et al. 1999). Combinations of MS with
other food preservation technologies have been in-
vestigated.
A. Combining MS with Low pH
The influence of treatment medium pH on
MS (117 mm, 200 kPa, 40°C) resistance to
L. monocytogenes was studied by Pagán et al.
(1999). The DMS value of this microorganism did
not change when pH decreased from 7 to 4. Simi-
lar results on the influence of pH on MS resis-
tance were obtained for Aeromonas hydrophila
and Yersinia enterocolitica (unpublished data).
B. Combining MS with Low aw
In general, the presence of solutes in the treat-
ment medium prevents microbial inactivation by
different lethal agents. However, this influence
was greater for heat than for MS treatment. Add-
ing 57% sucrose to the decreased aw in the treat-
ment medium, until 0.94, increased the heat resis-
tance at 62°C of L. monocytogenes 25 times, while
its MS (117 mm, 200 kPa, 40°C) resistance in-
creased only two times (bPagán et al. 1999). There-
fore, MS could be a very useful alternative to heat
treatment for the inactivation of microorganisms
in foods with low aw.
C. Combining MS with Heat
(Manothermosonication)
Different authors have investigated combina-
tions of heat and ultrasound to decrease the intensity
of heat treatments. The heat resistance of B. cereus,
Bacillus licheniformis, B. stearothermophilus, and
thermoduric streptococci decreased following
ultrasonication treatment at 20 kHz (Burgos et al.
1972; Sanz et al. 1985; Ordoñez et al. 1984). This
effect was even bigger when heat and ultrasound
were applied simultaneously (Ordoñez et al. 1987;
García et al. 1989).
The application of MS treatment simultaneously
with heat treatment (Manothermosonication) also
led to higher microbial inactivation. While in most
vegetative cells the lethal effect of MTS was addi-
tive, on Enterococcus faecium and B. subtilis spores,
a synergistic effect was observed (Figure 2) (eRaso
et al. 1998; aPagán et al. 1999).
MTS treatments have also been very effec-
tive in the inactivation of different enzymes re-
lated to food quality, such as POD, lipoxigenase,
PPO, lipase, protease, or PME (López et al. 1994;
Vercet et al. 1995; Vercet et al. 1999). For ex-
ample, simultaneous applications of heat (72°C)
and ultrasound (20 kHz, 117 µm) under moderate
pressure (200 kPa) increased the inactivation rate
of orange juice PME 25 times in buffer, and more
than 400 times in orange juice.
In conclusion, the application of ultrasound
under pressure simultaneously with heat treat-
ment results in higher microbial and enzyme in-
activation. Therefore, the same inactivation level
is achieved over a shorter treatment period or at
lower temperature. Consequently, this combina-
tion could be advantageous, due to the minimiza-
tion of heat-induced damage in product quality.
IV. Combinations with Pulsed Electric
Fields
Microbial inactivation by pulsed electric
fields (PEF) was first observed in the early
1960s (Doevenspeck 1961). This technology
involves applications of short duration (micro-
seconds), high-intensity electric field pulses.
PEF has received increased attention in the last
decade as a food preservation technique be-
cause of its potential to inactivate microorgan-
isms at temperatures below those adversely
affecting food quality (Castro et al. 1993; Qin
et al. 1995).
275
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FIGURE 2. Microbial inactivation by heat treatment (HT), Manosonication (MS) and Manothermosonication (MTS).
Experimental conditions:
Bacillus subtilis spores: MS (20 kHz, 117 µm, 200 kPa, 6 min), HT (90°C, 6 min), MTS (20 kHz, 117 µm, 200 kPa,
90°C, 6 min) Enterococcus faecium: MS (20 kHz, 117 µm, 200 kPa, 3 min), HT (62°C, 3 min), MTS (20 kHz, 117
µm, 200 kPa, 62°C, 3 min)Yersinia enterocolitica: MS (20 kHz, 117 µm, 200 kPa, 0.5 min), HT (54°C, 0,5 min), MTS
(20 kHz, 117 µm, 200 kPa, 54°C, 0.5 min).

PEF is highly effective in killing vegetative
cells of bacteria, yeast, and molds (Wouters and
Smelt 1997). However, PEF inactivation of bac-
terial spores and enzymes as related to food quality
is unclear. While some authors have reported the
inactivation of bacterial spores (Marquez et al.
1997) and enzymes by PEF (Ho et al. 1997; Giner
et al. 2000), others have observed that treatments
were unsuccessful (Pagán et al. 1997; Grahl and
Markl 1996).
PEF technology is not being used to preserve
foods commercially at present. However, the fea-
sibility of PEF technology extending the shelf-
life of different foods without apparent change in
product properties, as in apple juice, skim milk,
beaten eggs, green pea soup, or orange juice, has
been demonstrated on a pilot plant scale (Qin et
al. 1995; Qiu et al. 1998). Following PEF treat-
ment, these products must be stored under refrig-
eration to prevent enzymatic reactions and spore
germination.
The combinations of PEF with other preser-
vation technologies are being investigated to in-
crease the lethal effect of this nonthermal process
and to extend its application to different liquid
foods.
A. PEF and Heat
In general, the heat generated during PEF
treatment resulting from the sample’s resistance
to current flow is removed. However, it has been
observed that lethality of PEF treatments increases
with an increase in processing temperature
(Hülsheger et al. 1981; Wouters et al. 1999). Sev-
eral authors have demonstrated that PEF treat-
ments applied at higher start temperatures increase
the inactivation effect in a batch treatment system
(Jayaram et al. 1992; Pothakamury et al. 1996;
Hülsheger et al. 1981), or in a continuous-treat-
ment system (Wouters et al. 1999). This incre-

276
 ment in the lethality effect of combined treat-
ments has been observed at both lethal and non-
lethal temperatures for microorganisms investi-
gated. For example, inactivation of E. coli by
PEF, with a field strength of 36 kV/cm and pulse
duration of 2 µs, increased from 2 to 3 log cycles
when temperature was increased from 7 to 40˚C
(Pothakamury et al. 1996). The higher suscepti-
bility of microorganisms to PEF has been related
to the temperature effect on membrane fluidity
properties. While phospholipids are closely packed
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in a rigid gel structure at low temperatures, at
high temperatures they are less ordered, the mem-
brane has a liquid-crystalline structure, and its
thickness is reduced (Jayaram et al. 1992).
Of interest to the orange juice industry, the
inactivation of different microorganisms with PEF
treatment at 50 and 55°C in a continuous system
has been investigated. At 55°C and 30 kV/cm,
E. coli, L. mesenteroides, and Listeria innocua
were inactivated by as much as 5 log10 units after
6.2 pulses. However, only a 3 log10 unit reduction
was achieved for S. cerevisiae ascospores after
7 pulses of 50 kV/cm at the same temperature
(McDonald et al. 2000).
Further studies are necessary to determine an
optimal heat-PEF combination that can inactivate
the maximum level of PEF-resistant microbial
species possible with a minimal effect on food
quality; studies on the effect of this combination
on bacterial spores and enzymes are needed as
well.
B. PEF and Low pH
The influence of pH on bacterial inactivation
by PEF is unclear. Sale and Hamilton (1967) and
later Hülsheger et al. (1981) reported no influence
of pH on the inactivation of E. coli. However,
Vega-Mercado et al. (1996) found that inactiva-
tion of this microorganism was more significant
at pH 5.69 than at 6.82. Wouters et al. (1999)
studied the influence of treatment medium pH on
inactivation of L. innocua, S. cerevisiae, and
L. plantarum with PEF continuous treatment, ob-
serving that lower pH values resulted in increased
inactivation. For example, a PEF treatment that
inactivated 0.6 Log10 cycles of L. innocua at pH 6
was found to inactivate more than 6 log cycles at
pH 4. Surprisingly, Alvarez et al. (2000) found
that Salmonella Senftenberg was more PEF resis-
tant at acidic pH than at neutral pH when studying
the inactivation of this microorganism in different
buffers with the same conductivity (2 mS/cm).
Independent of this pH influence on micro-
bial inactivation by PEF, acid foods such as fruit
juices are good candidates for processing with
this technology because bacterial spores cannot
germinate at low pH.
C. PEF and Antimicrobials
It is well known that PEF induces reversible
or irreversible structural changes in cell mem-
branes, resulting in pore formation and loss of
selective permeability properties (Wouters et al.
1997). On the other hand, some antimicrobials
such as lysozyme or nisin act on the cell mem-
branes, and others such as organic acids cross the
microbial membranes and access the cytoplasm
where they act. According to the mechanisms of
action, for both preservation techniques, a power-
ful synergistic effect should be expected when
they are combined: antimicrobials could increase
the susceptibility of membranes to dielectric break-
down and/or PEF could facilitate the access of
antimicrobials to the membranes or cytoplasm.
Nisin is the antimicrobial most studied in
combination with PEF. It has been demonstrated
that presence of nisin in the treatment medium
can increase the inactivation effect of PEF for
both Gram+ and Gram– bacteria. A synergistic
effect has been observed when bacteria are incu-
bated with nisin after PEF treatment, when nisin
is present during PEF treatment, or when nisin is
added to the bacterial suspension after PEF treat-
ment. The lethality of PEF treatment (1 single
pulse at 12.5 kV/cm) in the presence of 50 IU/ml
of nisin on three pathogenic microorganisms,
L. monocytogens Scott A, E. coli O157:H7, and
S. typhimurium M1, increased 3.2, 0.7, and 0.7
log10 units, respectively (Kalchayanand et al.
1994). When PEF treatment (100 µs, 16.7 kV/cm)
was spread over a 10-min time interval in the
presence of nisin (0.06 µg/ml), the total inactiva-
tion (3.8 log units) was 1.8 log10 units larger than
277
 the sum of reductions with single treatments (Pol
et al. 2000). Dutreux et al. (2000) studied the
inactivation of Micrococcus luteus by combining
PEF treatment (100 µs, 33 kV/cm) with nisin
treatment (100 IU/ml, 2 h). The inactivation ef-
fect of individual treatments was 2.4 and 1.4 log10
units, respectively. The total inactivation effect of
combined treatment was slightly higher when PEF
treatment was applied prior to the incubation pe-
riod with nisin (5.2 log10), when compared with
incubation with nisin prior to PEF treatment
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(4.9 log10).
Combining PEF with nisin also affects mi-
croorganisms suspended in different foods. The
combination effect of PEF treatment on L. innocua
suspended in liquid whole eggs or skim milk,
followed by exposure to nisin, was additive or
synergistic, depending on the intensity of PEF
treatment. The synergistic effect was observed at
the highest electric field intensity studied (50 kV/cm)
(Figure 3) (Calderón-Miranda et al 1999ª;
Calderón-Miranda et al. 1999b).
Combining organic acids with PEF resulted
in higher inactivation of E. coli O157:H7. How-
ever, a synergistic effect was observed at pH 3.4,
but not at pH 6.4. At the lower pH, the presence
of benzoic or sorbic acid (1000 ppm) during a
single high-voltage electric pulse (12.5 kV/cm)
resulted in an inactivation of around 2 log10 units
greater than that with individual treatments. As
the undissociated fraction of acids in solution is
much higher at pH 3.4 than pH 6.4, the synergis-
tic effect at low pH may indicate that PEF en-
hanced the entry of the undissociated fraction but
not the dissociated one (Liu et al. 1997).
Results indicated that PEF in combination
with antimicrobials increases bacterial inactiva-
tion and extends the spectrum of action of some
bacteriocins. However, more investigation is
needed to understand the mechanisms of action
for treatment optimization to enhance the safety
and shelf-life of foods processed by PEF.
D. Combining PEF with HHP
Heinz and Knorr (2000) designed laboratory-
size equipment to study the inactivation effects of
simultaneous applications of HHP and PEF. The
278
lethality of PEF treatment applied simultaneously
with sublethal HHP treatment (200 MPa for less
than 1 min) on B. subtilis vegetative cells was
lower than the lethality of PEF treatment applied
under atmospheric pressure. Thus, the sublethal
pressure treatment produced a stabilizing effect
on cells of B. subtilis against the PEF treatment.
However, a synergistic effect was observed when
the cells were exposed to a treatment of 200 MPa
for 10 min, followed by PEF treatment (24.7 kV/cm,
300 µs) immediately before the pressure
release. The additional inactivation effect was
2 log10 units.
V. COMBINATIONS WITH IRRADIATION
Irradiation is a nonthermal food preservation
process that has been reexamined in recent years.
The process involves applications of electrons or
electromagnetic waves to foods. Sources of ioniz-
ing radiation used to irradiate food include gamma
rays, electron beams, and X-rays. Although the
mechanisms of bacterial inactivation by irradia-
tion are not completely understood, it is thought
that irradiation inactivates microorganisms mainly
by causing lesions in DNA. Irradiation is consid-
ered the only known technique capable of ensur-
ing the hygienic quality of raw food in a fresh or
frozen state (Loaharanu 1995).
From the beginning, safety and wholesome-
ness of irradiated foods has been an issue of
concern. In 1999, WHO stated that according
to established good manufacturing practices,
irradiated food products can be considered safe
and nutritionally adequate. Currently, many
countries have approved irradiation of a variety
of foods, such as fruits, vegetables, spice, pork,
beef, poultry, fish, and seafood (Olson 1998).
Irradiation enhances the shelf-life of foods and
ensures their innocuousness because most mi-
croorganisms in vegetative form are extremely
sensitive to irradiation at low doses (Radomyski
et al. 1994). However, in some circumstances
combined treatments are more satisfactory since
the dose required for complete sterilization can
induce undesirable changes in food flavor or is
at higher than permitted levels (Thakur and
Singh 1995).
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FIGURE 3. Sensitization of Listeria innocua to nisin with PEF treatment in liquid whole egg and skim milk.
Experimental conditions
Nisin: 10 IU/ml
PEF: 50 kV/cm, 32 pulses of 2 µs.
A. Combining Irradiation with Low-
Temperature and Modified Atmospheres
Irradiation of various fresh foods at doses
significantly affecting organoleptic properties, in
combination with storage at chill temperatures,
improves food safety and extends shelf-life
(Radomyski et al. 1998; Thayer and Rajkowski
1999). To avoid the growth of surviving microor-
ganisms during storage at chill temperatures, the
combination of irradiation with modified atmo-
sphere packaging has been investigated in differ-
ent foods, such as pork, beef, poultry, turkey,
carrots, and lettuce (Lambert et al. 1992; Lee et
al. 1996; Thayer and Boyd 1999; Murano et al.
1998; Hagenmaier and Baker 1998; Prakash et al.
2000). In general, combining low-dose irradia-
tion (≤ 3 kGy) with modified atmosphere packag-
ing (vacuum or gas packaging) widely extended
the shelf-life of fresh foods in refrigeration. For
example, irradiation (1.75 kGy) of pork chops
packaged in a modified atmosphere (75% N2,
25% CO2) extended the shelf-life at 4°C from 8 to
12 days, and significantly improved microbio-
logical safety (Grant and Patterson 1991).
As the effects of combining irradiation with
MPA on pathogenic and spoilage microorgan-
isms, and sensory quality, depend on food and
atmosphere composition, specific studies should
be conducted to find an optimal gas composition
and irradiation level.
B. Combining Irradiation with Heat
A combination of irradiation and heat has
been proposed for required lethality while pre-
serving food quality, thereby reducing the detri-
mental effects of heat or irradiation alone on food.
While the inactivation effect of heat treatment
followed by irradiation is additive (Kim and
Thayer 1996), a synergistic effect has been ob-
279
 served when heat treatment is applied after irra-
diation or when both treatments are simultaneously
applied (thermoradiation). Several studies have
established that irradiation sensitizes vegetative
cells and bacterial spores to a subsequent heat
treatment. This heat-sensitizing effect has been
observed in both vegetative cells and bacterial
spores suspended in aqueous or lipid systems or
foods (Kempe 1955; Gombas and Gomez 1978;
Thayer et al. 1991; Shamsuzzaman and Lucht
1993; Grant and Patterson 1994; Kim and Thayer
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1996). For example, in minced roast beef, the D70
value of a strain of L. monocytogenes decreased
from 22.4 s to 5.5 s after a preirradiation treat-
ment (0.8 kGy) (Grant and Patterson 1994).
A synergistic effect on the inactivation of veg-
etative bacteria and bacterial spores can be achieved
with thermoradiation (Pallas and Hamdy 1976;
Fisher and Pflug 1977). For example, Pallas and
Hamdy (1976) observed that the D value (dose
inactivating 90% of population) of S. aureus de-
creased from 0.098 to 0.053 kGy, while the irradia-
tion temperature increased from 35 to 45°C. This
synergistic effect of thermoradiation has also been
observed in the inactivation of microorganisms in
several foods. Schaffner et al (1989) reported that
thermoradiation caused greater inactivation of Sal-
monella enteritidis in liquid whole egg than either
heat or radiation alone. The D of this microorgan-
ism was 0.26, 0.237, and 0.078 kGy at 19, 50, and
60°C, respectively. A synergistic effect was also
observed during thermoraditation of Vibrio
vulnificus in buffer, fresh oyster, and fresh fish at
40˚C (Ama et al. 1994).
C. Combining Irradiation with HHP
HHP in combination with irradiation has also
been studied. Clouston and Wills (1969) observed
that hydrostatic pressure greater than 500 atm
decreased the resistance of Bacillus pumilus spores
to gamma-irradiation. This decrease in resistance
was associated with the initial germination of
bacterial spores by HHP. Also, an irradiation treat-
ment sufficient to inactivate a proportion of
B. coagulans spores increased the pressure sensi-
tivity of the survivors (Sale et al. 1970). When
pressure and radiation were applied simulta-
280
neously, the effect obtained in B. pumillus was
additive, rather than synergistic (Wills 1974).
The combined effect of low-dose irradiation
and HHP on the microbiological quality of differ-
ent species of meat has also been investigated. A
value of 2.0 kGy for D was calculated for
Clostridium sporogenes inoculated into samples
of chicken breast meat exposed to irradiation,
followed by pressurization at 680 MPa at 80˚C
for 20 min. This was one-half the D value of
samples that were only irradiated (4.1 kGy)
(Crawford et al. 1996).
Staphylococcal counts in lamb meat were
reduced by 1 log cycle in samples subjected to
irradiation (1 kGy) or HHP (200 MPa, 30 min).
However, when both treatments combined were
applied, an inactivation of more than 4 log cycles
was achieved (Pushpa et al. 1997). Therefore,
combinations of HHP and irradiation can lower
the intensity of treatment required for any process
when used alone, and as a consequence can im-
prove the sensorial quality and microbial safety
of meat.
CONCLUSION
During the last 2 decades, different nonthermal
technologies have been explored extensively.
These technologies have been shown to inactivate
microorganisms and enzymes without the adverse
effects on organoleptic and nutritional properties
caused by heat. However, the high resistance to
nonthermal processes of some enzymes and mi-
croorganisms, especially bacterial spores, has lim-
ited their application as an alternative to heat
treatments. The use of nonthermal processes in
combination with other preservation technologies
presents a number of potential benefits to food
preservation. Consumer demand for fresh, natu-
ral, and minimally processed products has re-
sulted in an increasing number of refrigerated
foods in the market. Shelf-life and product safety
is limited due to the growth of psycropilic micro-
organisms (some of them pathogens) in refriger-
ated foods. Nonthermal processing is an efficient
means to achieve significant reductions in psy-
chrophilic pathogens and spoilage microorgan-
isms, with a minimum of detrimental effects on
 food product quality. Irradiation and HHP have
already been used commercially with this pur-
pose in mind. For example, irradiated strawber-
ries and packaged poultry have become commer-
cially available in the United States (Marcotte
1992; Pszczola 1993), and HHP-processed or-
ange juice and sliced cooked ham have been
marketed in France and Spain, respectively
(Tewari et al. 1999).
Furthermore, in addition to improving the
shelf-life and safety of traditional foods,
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nonthermal preservation methods or combinations
of such processes with traditional or emerging
technologies is very attractive to the food indus-
try, because it provides the opportunity to intro-
duce new products in the food market. HHP-
processed guacamole, a dip of Mexican origin
sold in United States supermarkets, is an example.
While use of irradiation or HHP has already
been established in the industry, others such as
ultrasound or PEF must continue to be developed
and evaluated for commercial application. Al-
though many studies have been conducted in the
area of nonthermal processing, more research is
needed to understand the mechanisms of action
behind different technologies, in order to find the
most convenient nonthermal technology or com-
binations applicable to specific foods.
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