Food Bioprocess Technol
DOI 10.1007/s11947-013-1197-2
ORIGINAL PAPER
Effect of Hyperbaric Pressure Treatment on the Growth
and Physiology of Bacteria that Cause Decay
in Fruit and Vegetables
Pansa Liplap & Vicky Toussaint & Peter Toivonen &
Clément Vigneault & Jérôme Boutin & G. S. Vijaya Raghavan
Received: 11 July 2013 / Accepted: 22 September 2013
# Springer Science+Business Media New York 2013
Abstract The response of bacteria to hyperbaric pressure
treatment was investigated. Three selected bacteria which
cause fruit and vegetable decay (i.e., Pseudomonas cichorii,
Pectobacterium carotovorum, and Pseudomonas marginalis)
were inoculated onto BIOLOG microplates and subjected to
different pressure and temperature conditions including 100,
200, 400, 625, and 850 kPa at 20 °C and 100 kPa at 4 °C.
Changes in microplate color, which corresponds to carbon
source utilization of bacteria or their growth, were monitored
every 24 h for 7 days. Results showed that the bacterial growth
was affected by both hyperbaric pressure and temperature. As
hyperbaric pressure increased, the bacterial growth signifi-
cantly decreased and the extent was dependent on bacterial
species. The 850-kPa pressure treatment reduced maximum
growth by 71, 56, and 43 % for Pseudomonas cichorii,
Pectobacterium carotovorum, and Pseudomonas marginalis,
respectively. Among these bacteria, Pseudomonas cichorii
was the most pressure-sensitive, while the most temperature-
sensitive was Pectobacterium carotovorum . In general, an
increase in hyperbaric pressure caused bacteria to utilize car-
bon sources similar to those when they were exposed to low
temperature. Overall, hyperbaric treatment has the potential to
P. Liplap (*) : C. Vigneault : G. S. V. Raghavan
Department of Bioresource Engineering, Macdonald Campus,
McGill University, Sainte-Anne-de-Bellevue, Montreal, QC,
Canada H9X 3V9
e-mail: pansa.liplap@mail.mcgill.ca
P. Liplap : V. Toussaint : C. Vigneault : J. Boutin
Horticulture Research and Development Centre, Agriculture and
Agri-Food Canada, Saint-Jean-sur-Richelieu, QC, Canada
P. Toivonen
Pacific Agri-Food Research Centre, Agriculture and Agri-Food
Canada, Summerland, BC, Canada
directly reduce bacterial growth in fruit and vegetables after
harvest.
Keywords Low temperature . BIOLOG . Carbon source
utilization . Bacterial growth
Introduction
Several studies have shown the close relationship between a
high consumption of fruit and vegetables and lower incidence
of some chronic diseases (Lir et al. 2000; Van't Veer et al.
2000). Thus, demand for fresh fruit and vegetables has in-
creased over recent decades. In contrast, their availability in
the form of processed fruit and vegetable products is falling
behind the gross consumption (Barth et al. 2009). However,
increased fruit and vegetable production is not the solution to
this problem of availability because approximately 15–40 %
of the production volume is lost between harvest and con-
sumption (James and James 2010) with spoilage being one of
the major causes for these losses (Kantor et al. 1997). Since
fruit and vegetable tissues provide environments conductive
for survival and growth of microorganisms, they will inevita-
bly succumb to microbiological attack and spoilage. Although
decay-causing microorganisms may be present on fruit and
vegetables at harvest, rots generally develop during transport
and storage (Barth et al. 2009). Loss due to decay by rot-
causing pathogens during postharvest handling of fruit and
vegetables is estimated to 20–25 % in developed countries,
and the situation is much worse for developing countries
(EI-Ghaouth et al. 2004; Korsten 2006; Sharma et al. 2009).
Given this situation, improving management of freshly
harvested fruit and vegetables to control rots is crucial to
provide commodities with high quality standards.

Several different strategies have been used to control decay
pathogens in fresh fruit and vegetables. Among them, biolog-
ical control of postharvest disease using microbial antagonists
like yeasts and bacteria has been reported to inhibit decay of
fruit effectively (Sharma et al. 2009; Korsten 2006; Droby
et al. 2003). However, the application of antagonistic micro-
organisms is limited due to difficulty in management of the
storage environment and other postharvest treatments required
to achieve sufficient protection from the biocontrol agent
(Janisiewicz and Korsten 2002). Synthetic chemicals, includ-
ing pesticides and preservatives, have been widely applied to
fresh produce. Nevertheless, use of chemicals is not the most
desirable means of disease control because they are generally
expensive, can cause environmental pollution, and may in-
duce pathogen resistance. But most importantly, the presence
of chemical residues leads to harmful effects on the human
health by interfering with the reproductive systems and fetal
development as well as in their capacity to cause cancer and
asthma (Gilden et al. 2010). Therefore, use of chemical treat-
ments as postharvest treatments has been so far restricted.
Recently, physical treatments, either alone or in combination,
such as heat (such as water and vapor), irradiation, UV-B/C,
modified/controlled atmosphere, pressure, etc., have drawn
great attention in the postharvest field because they not only
control insect and storage diseases but can also maintain or
even improve eating qualities of stored fruit and vegetables
(Charles and Arul 2007; Vigneault et al. 2012)
Of the physical means, hyperbaric treatment has shown to
improve quality preservation on some horticultural produce,
i.e., tomato (Goyette et al. 2012; Liplap et al. 2013b, c), lettuce
(Liplap et al. 2013a), mume fruit (Baba et al. 1999), cherry, and
grape (Romanazzi et al. 2008). The basic operation consists of
subjecting produce to a pressurized air environment ranging
from 0.1 to 1.0 MPa (1–10 atm), in which the proportion of air
composition is maintained (Goyette et al. 2007). It is different
from conventional high pressure treatment, where the pressure
is set between 400 and 1,200 MPa (Ahmed and Ramaswamy
2006). An application of such high pressures is generally not
suitable for fruit and vegetables because they can cause irre-
versible damage to cell structure (Ahmed and Ramaswamy
2006; Baba and Ikeda 2003). The main advantage of hyperbaric
pressure treatment over the other physical treatments is the
homogeneity of response, as it acts instantaneously and uni-
formly throughout a mass of product independently of the size
and the shape (Goyette et al. 2007).
The inhibitory effects of high pressure on the growth of
several microorganisms have been widely reported (San
Martín et al. 2002). In general, very high pressure is required
to kill or inactivate the growth of microorganisms. Nevertheless,
recent studies have shown that microorganisms were affected by
hyperbaric treatment as well. Romanazzi et al. (2008) showed
that hyperbaric treatment (150 kPa) applied for 4 h significantly
reduced the incidence of brown rot, gray and blue molds, and
Food Bioprocess Technol
total rots (including Alternaria sp., Rhizopus sp., and
Cladosporium sp. rots) of sweet cherries. They also found that
wounded table grapes, subjected to 150 kPa pressure for 24 h
and then inoculated with Botrytis cinerea, had significantly
fewer berries infected and lesions were less severe. Similar
results were observed from our previous study (Liplap et al.
2013a), where the lettuce was treated with varying pressures
ranging from 100 to 850 kPa at 20 °C for 5 days. The devel-
opment of decay was dramatically delayed when lettuce was
stored under hyperbaric pressure, especially at 850 kPa, in
comparison with ambient pressure. Nevertheless, the mode of
action of hyperbaric treatment on the growth of microorgan-
isms has not been clearly elucidated. It is probably due to the
direct impact of elevated pressure itself on the microorganisms
(Segovia-Bravo et al. 2012), or the elevated O2, causing O2
toxicity to microorganisms, bacteria, yeasts, and molds (Kader
and Ben-Yehoshua 2000; Bean 1945), or the enhancement of
the host pathogen defense compound synthesis, induced by
mild stress as demonstrated for UV-C and heat treatments
(Charles and Arul 2007; Lu et al. 2010). To date, very limited
information is available concerning hyperbaric pressure ef-
fects on decay-causing microorganisms of fruit and vegeta-
bles. Studies are required to understand the mechanisms of
hyperbaric treatment on microorganism inactivation.
In this study, the physiology of three selected bacteria
causing fruit and vegetable decay (i.e., Pseudomonas cichorii,
Pectobacterium carotovorum, and Pseudomonas marginalis)
under different hyperbaric pressures (from 100 to 850 kPa)
was investigated. BIOLOG microplates (GEN III) containing
71 carbon sources were used to follow bacteria development
under the different hyperbaric pressure treatments, thus elim-
inating the potential interference of a plant self-defense mech-
anism. The hyperbaric treatments were applied at 20 °C and
their carbon source utilization behaviors were compared to
that of storage at 4 °C at ambient atmospheric pressure.
Materials and Methods
Species, Bacterial Inoculum, and Microplates
Pseudomonas marginalis , Pseudomonas cichorii , and
Pectobacterium carotovorum used in this study were isolated
from rotting lettuce tissues (Liplap et al. 2013a). For isolation
of bacteria, pieces of lettuce from different samples with
rotting symptoms were sequentially washed under running
distilled water, cut into small pieces, and macerated in
10 mM of phosphate buffer (pH 7.0) for 30 min. Macerate
was then diluted 1:10 and streaked onto King's B agar medium
(KB) (King et al. 1954) for Pseudomonas spp. recovery and
Nutrient Agar (NA) (Becton Dickinson and Company,
Sparks, MD, USA) for other species, both media were sup-
plemented with 50 mg L−1 of cycloheximide (Sigma Aldrich
Food Bioprocess Technol
Co., St. Louis, MO, USA) to avoid fungal growth. Bacteria
were grown on media at 28 °C for 48 h. After the incubation
period, predominant colonies on plates were purified by streak-
ing on KB (for fluorescent colonies) or NA. Fluorescent
Pseudomonas isolates were identified based on LOPAT profile
and identification confirmed using the GEN III Microbial
Identification System (BIOLOG, Hayward, CA, USA).
Pectobacterium was identified using GEN III and the pectolytic
activity was verified using surface sterilized potato slices (De
Boer and Kelman 2001).
The bacterial inocula were prepared for each species. A
bacterial suspension was adjusted to A 600 =0.09 in 10 mM
phosphate buffer pH 7.0 which corresponds to about 108 CFU
mL-1. The final concentrations of these inocula were verified
by dilution plating onto KB medium. These bacterial suspen-
sions were used immediately to inoculate BIOLOG GEN III
microplates.
For microplate preparation, the inoculating protocol of the
manufacturer for the GEN III was followed as described in the
user guide, with the exception that the inoculation fluid was
inoculated using 10 μL of the bacterial suspension prepared as
described above. The inoculated microplates were then transferred
into each hyperbaric vessel with three plates per species and three
species per treatment. The experiment was conducted twice.
Hyperbaric Treatment System
Hyperbaric pressure system used during the tests is illustrated
in Fig. 1. The system is similar to the one previously used for
horticultural produce storage, where pressure, air flow rate,
and relative humidity are controlled (Liplap et al. 2013a, b).
Briefly, it consists of a compressed air tank, three low pressure
vessels, three high pressure vessels, and an infrared gas ana-
lyzer. The low pressure vessels are made from painted steel
apparatus (PRO-TEK, Mirabel, Quebec, Canada), 220 mm in
height and 265 mm inside diameter, whereas the high pressure
vessels are made from stainless steel (GracoTM, Minneapolis,
MN, USA), 225 mm high and 235 mm inside diameter. The
net volume of each vessel is 10.75 L. A 12.7-mm flat rubber
ring is used to ensure air tightness between the cover and the
chamber. Each vessel is equipped with a pressure regulator
and a flow control needle valve to individually regulate the
pressure and air flow rate, respectively. A safety relief valve is
used to prevent pressure overload. Two compression fittings
are fastened to the vessel to quick connect the airflow inlet and
outlet using plastic tubes of 3.2 mm inner diameter. The air
inlet of the vessel is connected to a compressed air tank
equipped with a regulating manometer. A channel selector
(a manifold equipped with controlled valves) is installed to
connect the air outlet of the selected vessel to a CO2 infrared gas
analyzer (Guardian® Plus, Kirkton Capus, Livingston, UK) and
the electronic air flow meter (Bronkhorst TM , Ruurlo,
Netherlands). The manifold equipped with controlled valves,
air flow meter, and CO2 gas analyzer are all connected to a data
acquisition and control card (Personnel DAQ 3000, Cleveland,
Ohio, USA) and a personal computer.
Experimental Procedure
Prior to the experiment, the hyperbaric system was sanitized to
avoid contamination of other microorganisms from the air. The
pressurized air was filtered through a series of filters: a 20-μm, a
0.03-μm, and a 0.01-μm filter. The air supply line was
disinfected with a 10 % v/v sodium hypochlorite solution.
The storage vessels were cleaned with 70 % v/v ethanol solu-
tion prior to use. Each of the inoculated BIOLOG GEN III
microplates were submitted to one of the following pressure
and temperature conditions: 100, 200, 400, 625, or 850 kPa at
20 °C or 100 kPa at 4 °C. The 100 kPa at 20 °C treatment was
the control and 100 kPa at 4 °C was the cold treatment. The
vessels were then sealed and pressurized for 7 days. The
microplates were taken out and read every 24 h up to 168 h
(day 7) using a microplate reader (Model ELx808BLG, BioTek
instruments, Winooski, VT). Absorbance was read at 590 nm
since respiration of the carbon source in each well induces the
tetrazolium dye color development (purple) with a peak of
absorbance at this wavelength. After the 7th day, the plates
were kept at 100 kPa, 20 °C (ambient conditions) and the
changes in color were monitored until 240 h (day 10).
BIOLOG Data Treatment
BIOLOG is a 96-well microplate technique originally devel-
oped for a rapid identification of bacteria (Garland and Mills
1991). In addition to that role, it is found useful in investigat-
ing the response of bacteria to different nutrients and environ-
mental conditions. The BIOLOG microplate GEN III model
analyzes microorganisms in 94 phenotypic tests: 71 carbon
source utilization assays and 23 chemical sensitivity assays,
with one negative control (no carbon source) and one positive
control used as a reference for chemical sensitivity assays. The
microbial activity in the cells is determined based on the
exchange of electrons generated during respiration, leading
subsequently to tetrazolium-based color changes. Therefore,
an increase in color development indicates the nutrient utili-
zation, which corresponds to bacterial growth.
Each microplate provides a set of 95 data per reading; con-
sequently, to allow easier interpretation, the analysis was carried
out through groups of substrates: (1) all wells (n =95), all carbon
sources (n =71), sugars (n =26), sugar alcohols (n =5), amino
acids (n =11), hexose acids (n =9), and the others (carboxylic
acids, esters, and fatty acids; n =18). Overall changes in color
development were expressed as average well color development
(AWCD) based on the classified substrate groups. AWCD at a
given time was derived from the mean difference among absor-
bance values of the selected carbon sources (C ) and the

Fig. 1 Hyperbaric system for evaluation of the effect of different pressure levels on bacterial growth
absorbance value of the control well (R: no carbon source) as
shown in Eq. 1. The negative absorbance values obtained from
C-R term were set to zero prior to calculating a mean.
X bial safety or product shelf life (Zwietering et al. 1990; Cayre
ðC−RÞ
AWCDsub:group ¼
n
where:
AWCDsub.group average well color development (AWCD)
of each substrate group
C absorbance value of each well
R absorbance value of the control well
N number of wells in the substrate group
Mean AWCD values were plotted over time for each sub-
strate group. As the area under the curve (AUC) corresponds to
the growth of bacteria (asymptote, growth rate, lag time, etc.)
and explains combined effect of color development (Preston-
Mafham et al. 2002), it was used to quantify the effect of
different treatment conditions. In this study, AUC was estimated
using trapezoid approach (Eq. 2). The AUC method is particu-
larly useful when the data do not fit the logarithmic form.
Xn
Trapezoidal area ¼ ½ðV i þ V i−1 Þ  ðt i −t i−1 Þ
i¼1
where:
v absorbance value (absorbance unit)
t incubation time (hour)
n number of reading
Food Bioprocess Technol
The predictive modeling was performed to describe the
behavior of bacterial growth under different hyperbaric treat-
ment conditions. Also, the model allows prediction of micro-
et al. 2005). In general, bacterial growth shows a phase in
ð1Þ which the specific growth rate (μ M) starts a value of zero and
then accelerates to a maximum value (A) in a certain period of
time, resulting in a lag time (λ) (Fig. 2). Due to the sigmoidal
shape of the color development, logistic curve is used as the
basis for curve fitting models. A number of microbial growth
models are found in literature, such as Richards, Stannard,
Schute, logistic model and others (Zwietering et al. 1990). In
this study, the AWCD of all carbon sources was fitted to the
modified Gompertz equation (Eq. 3), a widely used asymmet-
ric logistic model in which the exponential increase is
corrected by an exponential term (Zwietering et al. 1990;
Vandepitte et al. 1995). The obtained kinetic model parame-
ters (A , μ M, and λ) are useful in investigating the behavior of
bacteria.
n hμ e io
yðt Þ ¼ Aexp −exp M ðλ−t Þ þ 1 ð3Þ
A
where:
ð2Þ
y (t) average well color development of all carbon sources
(absorbance value)
A maximum absorbance value (absorbance unit)
μM specific growth rate (absorbance unit per hour)
λ lag time (hour)
e = exp (1)
Food Bioprocess Technol

wells) utilization is shown in Fig. 3. It is clearly shown that

Fig. 2 An example of bacterial growth curve expressed by average well
color development (AWCD). The fitting Gompertz equation parameters
A, μ M, and λ indicate maximum rate color development, specific growth
rate, and lag time, respectively
temperature was the most significant factor affecting the growth
of bacteria. The bacteria treated at 4 °C grew slowly throughout
the 7 days of treatment and did not fully develop to the sigmoi-
dal curve. This is not surprising because of reduced microbial
activity at low temperature. Interestingly, an increase in sur-
rounding pressure at 20 °C clearly showed a positive impact on
the growth behavior of the bacteria. The higher the pressure
applied, the lower the microbial growth. In general, at 20 °C,
the curves followed a sigmoidal curve with different extents of
maximum growth and their corresponding times. The exponen-
tial growth phase at 20 °C ranged from 24 to 72 h and was also
dependent on the types of bacteria.

Principal component analysis (PCA) was also performed 1.0

Pseudomonas cichorii
on AUC data of all substrates to distinguish growth pattern of
0.8
treatment conditions. PCA reduces a multivariate data set and
projects original data onto a small number of principal com- 0.6

AWCD
ponents (PCs). Each PC extracts a portion of the variance from
the original data, with the greatest amount of variance 0.4
extracted by the first axis. Relationships among treatment
0.2
conditions were obtained by plotting scores of the first two
PCs in two dimensions, allowing comparison on the basis of 0.0
different patterns of carbon source utilization. 0 24 48 72 96 120 144 168
1.0
Pectobacterium carotovorum
Statistical Analysis 0.8

The experiments were conducted in a two-way factorial design 0.6

AWCD

(bacterium and treatment) with three replications and conducted

0.4
twice. An analysis of variance was performed on the results (i.e.,
AUC and fitting Gompertz equation parameters: A, μ M, λ) 0.2
using the General Linear Model with SAS software (SAS
Institute, Cary, NC, USA), and significant differences between 0.0
0 24 48 72 96 120 144 168
treatment means were determined using Duncan's multiple 1.0
range test with a 95 % confidence interval (P <0.05). Pseudomonas marginalis
PCA was conducted on the AUC using XLSTAT software 0.8
(Version 2011.2.4, Addinsoft, France). PCA was then used to
0.6
AWCD

evaluate separation of treatment conditions based on the 95-

substrate utilization. Scores for variable loading were evaluated 0.4
to determine which substrate contributed the largest discrimi-
nation power. 0.2

0.0
0 24 48 72 96 120 144 168
Results Time (h)
Fig. 3 A typical of average well color development (AWCD) of all
Pattern of Carbon Source Utilization chemical and carbon sources (95 wells) for Pseudomonas cichorii,
Pectobacterium carotovorum, and Pseudomonas marginalis under dif-
The response of bacteria was found to vary differently depend- ferent hyperbaric pressure and temperature conditions: gray square =
100 kPa, 20 °C, gray circle = 200 kPa, 20 °C, gray triangle = 400 kPa,
ing on nutrient group, but the same trend was observed under 20 °C, white square = 625 kPa, 20 °C, white circle = 850 kPa, 20 °C, and
the treatment conditions. A typical bacterial growth curve of white triangle = 100 kPa, 4 °C. Values represent an average and standard
each bacterium based on all chemical and carbon source (95 deviation (S.D.) of six replicates
 Food Bioprocess Technol

Quantification of Bacterial Growth Using Area
Under the Curve
The results on bacterial growth are quantified using AUC of the
classified groups and presented in Table 1. This interpretation is
useful because it explains the combined effect of color devel-
opment throughout the entire period of experiment (Preston-
Mafham et al. 2002). In general, the bacterial growth was found
to be strongly affected by bacterial type (P< 0.0001), treatment
condition (P< 0.0001), and their interaction (P< 0.0001), ex-
cept for sugar alcohol and hexose acid interactions (P< 0.05).
To compare the effect of nutrient groups on microbial
growth, the ambient condition at 100 kPa, 20 °C was taken into
account. There were differences in consumption of sugar and
sugar alcohol groups among bacteria. Pseudomonas cichorii
and Pseudomonas marginalis utilized very little amount of
sugar with a maximum AUC of ~18 and ~48, respectively.
Although they utilized only little sugars, they were instead found
to consume a lot of sugar alcohols, as indicated by a high AUC
(maximum AUC ~128 and 158 for Pseudomonas cichorii and
Pseudomonas marginalis, respectively). For Pectobacterium
carotovorum, it utilized both sugars and sugar alcohols well
with a maximum AUC of 97 and 127, respectively.
Pseudomonas cichorii and Pectobacterium carotovorum were
found to respond differently to amino acids and hexose acids
(Table 1). Pseudomonas cichorii preferentially used amino
acids but not hexose acids. In contrast, Pectobacterium
carotovorum preferred hexose acids rather than amino acids.
However, Pseudomonas marginalis consumed both amino and
hexose acids equally well. Finally, the remaining group contain-
ing carboxylic acids, esters, and fatty acids was consumed well
by Pseudomonas marginalis and less so by Pseudomonas
cichorii and Pectobacterium carotovorum.
Considering the effect of treatment conditions, in all cases
(bacterial species and nutrient group), the control (100 kPa,
20 °C) treatment showed the highest AUC, indicating the
highest growth compared with all the other treatment condi-
tions. The 100-kPa, 4 °C treatment resulted in the lowest AUC,
representing the most effective response to inhibiting microbial
growth. When hyperbaric pressures were applied at 20 °C, the

Table 1 Effect of hyperbaric pressure and temperature conditions on the growth of bacteria (represent by area under the curve: AUC) under different
substrate groups. Values represent an average and standard deviation (S.D.) of six replicates

Bacteria Treatment AUC

Sugars Sugar alcohols Amino acids Hexose acids Carboxylic acid,

esters, fatty acids

Pseudomonas cichorii 100 kPa, 4 °C 5.3±3.5 c 16.3±16.2 d 12.7±10.5 f 15.6±11.4 e 9.1±6.5 f

100 kPa, 20 °C 18.2±6.2 a 127.5±14.6 a 84.5±9.6 a 62.0±3.6 a 59.0±5.1 a
200 kPa, 20 °C 15.8±7.3 a 99.7±26.1 b 68.0±3.7 b 47.4±4.1 b 49.8±1.0 b
400 kPa, 20 °C 12.7±5.0 ab 81.8±13.2 b 55.8±2.3 c 39.5±1.8 c 38.0±1.7 c
625 kPa, 20 °C 8.9±3.3 bc 54.8±25.9 c 38.0±1.9 d 34.8±3.5 c 28.9±1.5 d
850 kPa, 20 °C 3.1±2.5 c 23.5±19.8 d 23.6±1.7 e 27.9±3.0 d 20.5±3.3 e
Pectobacterium carotovorum 100 kPa, 4 °C 4.8±4.3 e 5.8±8.5 d 8.0±8.4 d 3.5±4.9 f 1.4±1.7 f
100 kPa, 20 °C 96.8±11.7 a 126.5±24.0 a 42.6±4.2 a 86.2±6.6 a 26.3±2.5 a
200 kPa, 20 °C 88.2±10.4 b 115.3±17.1 a 40.5±2.2 a 75.6±6.3 b 21.9±1.8 b
400 kPa, 20 °C 70.3±3.4 c 87.8±6.8 b 26.4±5.7 b 59.4±5.7 c 16.6±1.5 c
625 kPa, 20 °C 62.7±3.8 c 70.7±4.4 c 14.6±1.6 c 46.2±4.0 d 10.1±1.6 d
850 kPa, 20 °C 50.4±4.7 d 60.6±6.9 c 12.0±1.6 cd 30.6±11.2 e 7.3±1.4 e
Pseudomonas marginalis 100 kPa, 4 °C 12.7±4.6 c 36.2±13.7 d 29.7±10.0 d 27.5±8.5 d 14.8±2.2 d
100 kPa, 20 °C 47.9±3.0 a 158.2±25.4 a 128.7±12.4 a 114.1±7.8 a 76.2±6.9 a
200 kPa, 20 °C 46.7±4.7 a 131.6±20.7 b 112.0±10.1 b 98.8±22.6 ab 57.6±8.7 b
400 kPa, 20 °C 44.3±4.8 a 131.0±29.8 b 101.0±13.7 b 86.9±23.4 b 46.5±4.2 c
625 kPa, 20 °C 38.8±2.2 b 111.5±17.2 bc 81.5±9.0 c 78.3±17.9 bc 36.7±5.1 d
850 kPa, 20 °C 35.5±4.0 b 99.5±17.7 c 71.3±1.0 c 62.8±17.0 c 30.7±2.4 d
Treatment (T) ** ** ** ** **
Bacteria (B) ** ** ** ** **
T ×B ** * ** * **

Data with the same letter for each bacterium within a column are not significantly different at P <0.05 (Duncan's multiple range test)
*P< 0.001; **P< 0.0001 (significant difference)
Food Bioprocess Technol

AUC was found to significantly decrease (P< 0.05) for all
bacteria. This indicates that an increase in pressure effectively
reduced the ability of bacteria to metabolize nutrients (sugars,
sugar alcohols, amino acids, hexose acids, and the other
groups). There were significant differences between the control
(100 kPa, 20 ° C) and at pressure treatments ≥625 kPa.
Interestingly, there were no significant differences in AUC
between the 100-kPa, 4 °C treatment and 850-kPa, 20 °C
treatment for sugars and sugar alcohols for Pseudomonas
cichorii, amino acids for Pectobacterium carotovorum, and
the others group (carboxylic acids, esters, and fatty acids) for
Pseudomonas marginalis. These indicate that hyperbaric treat-
ment has the potential of being used to reduce bacterial growth.
However, the combined effect of hyperbaric treatment and
lowered temperature would be of interest for further study.
Quantification of Bacterial Growth Using Kinetic Model
Even though the AUC gives a complete picture of differences
in carbon source utilization, the use of kinetic model can
provide additional information since it describes three different
aspects of the color response (A, μ M, and λ) and allows finer
separation of bacterial community (Preston-Mafham et al.
2002). In this study, all carbon sources are presumed to be
nutrients contained in a food product. The kinetics of microbial
growth is studied using the modified Gompertz equation and
the fitting results are presented in Table 2. It was found that the
growth behavior of the three selected bacteria fitted well with
Gompertz equation with high coefficient of determination in all
cases (R 2 =0.930–0.999). In general, the growth behavior of
bacteria (A , μ M, and λ ) is affected by bacterial species
(P <0.0001), pressure (P <0.0001), and their interaction
(P <0.0001), except for the interaction of the A parameter
(P> 0.05). For the different bacteria, on average, Pseudomonas
marginalis had the highest A values and λ , followed by
Pectobacterium carotovorum and Pseudomonas cichorii, while
Pectobacterium carotovorum grew faster than Pseudomonas
marginalis and Pseudomonas cichorii with the highest μ M. In
all cases of bacteria, the maximum growth was observed for the
control (100 kPa) and decreased significantly when increasing
the hyperbaric pressure. The 850-kPa pressure treatment reduced
the maximum growth by 71, 56, and 43 % for Pseudomonas
cichorii, Pectobacterium carotovorum , and Pseudomonas
marginalis , respectively, indicating that the physiology of

Table 2 Comparison of the estimated Gompertz fitting parameters from development, μ M = the specific color development rate, and λ = the lag
average well color development (AWCD) of carbon sources for different time. Values represent an average and standard deviation (S.D.) of six
hyperbaric pressure and temperature conditions: A = the maximum color replicates

Bacteria Treatment Gompertz fitting parameter

A μM λ R2
(Absorbance) (Absorbance h−1) (h)

Pseudomonas cichorii 100 kPa, 20 °C 0.389±0.040 a 0.008±0.001 a 18.4±1.6 bc 0.994-0.999

200 kPa, 20 °C 0.315±0.052 b 0.007±0.001 b 15.0±4.3 c 0.992-0.998
400 kPa, 20 °C 0.244±0.026 c 0.006±0.001 b 18.1±1.2 bc 0.994-0.998
625 kPa, 20 °C 0.176±0.025 d 0.006±0.001 b 21.3±1.0 b 0.945-0.998
850 kPa, 20 °C 0.112±0.025 e 0.003±0.001 c 25.7±5.4 b 0.988-0.997
Pectobacterium carotovorum 100 kPa, 20 °C 0.564±0.043 a 0.024±0.003 a 23.1±0.9 a 0.990-0.997
200 kPa, 20 °C 0.489±0.041 b 0.026±0.004 a 22.6±0.2 a 0.988-0.997
400 kPa, 20 °C 0.381±0.011 c 0.027±0.005 a 22.5±0.5 a 0.978-0.994
625 kPa, 20 °C 0.323±0.019 d 0.019±0.003 b 22.7±0.3 a 0.953-0.999
850 kPa, 20 °C 0.250±0.011 e 0.015±0.002 b 22.9±0.2 a 0.930-0.999
Pseudomonas marginalis 100 kPa, 20 °C 0.688±0.010 a 0.015±0.001 ab 30.8±1.6 a 0.998-0.999
200 kPa, 20 °C 0.648±0.009 b 0.016±0.002 a 29.2±1.8 a 0.996-0.999
400 kPa, 20 °C 0.561±0.005 c 0.014±0.001 b 24.0±0.9 b 0.991-0.998
625 kPa, 20 °C 0.461±0.015 d 0.014±0.001 b 24.3±0.4 b 0.993-0.999
850 kPa, 20 °C 0.391±0.009 e 0.014±0.001 b 25.6±1.6 b 0.971-0.993
Treatment (T) ** ** **
Bacteria (B) ** ** **
T ×B NS ** **

Data with the same letter for each bacterium within a column are not significantly different at P <0.05 (Duncan's multiple range test)
*P< 0.001; **P< 0.0001 (significant difference)
 Food Bioprocess Technol

Table 3 Effect of hyperbaric pressure and temperature conditions on the growth of bacteria (represented by area under the curve: AUC) under different
chemical sensitivity assays. Values represent an average and standard deviation (S.D.) of six replicates

Bacteria Chemical sensitivity assays 4 °C 20 °C

100 kPa 100 kPa 200 kPa 400 kPa 625 kPa 850 kPa

Pseudomenas pH 6 25.3±23.8 d 275.8±12.0 a 265.1±29.0 a 267.3±16.8 a 243.0±6.5 b 213.9±9.4 c

cichorii pH 5 3.2±3.5 c 248.6±34.9 a 264.8±18.3 a 257.7±29.5 a 257.8±10.3 a 201.4±31.9 b
1 % NaCl 20.5±18.0 e 242.9±17.3 a 248.8±13.2 a 212.7±6.4 b 184.9±8.0 c 148.3±5.3 d
4 % NaCl 4.7±5.6 d 131.2±30.4 a 84.2±28.6 b 81.0±24.0 b 33.3±33.6 c 0.3±0.3 d
a a a a a a
8 % NaCl
1 % sodium lactate 32.5±34.5 b 229.8±39.0 a 241.5±40.3 a 223.6±67.5 a 171.2±137.5 a 141.1±154.8 ab
a a a a a a
Fusidic acid
a a a a a a
D -Serine
a a a a a a
Troleandomycin
Rifamycin SV 37.5±34.7 c 244.9±38.6 a 265.1±21.6 a 245.8±10.9 a 209.2±4.2 b 198.8±7.3 b
a a a a a a
Minocycline
Lincomycin 39.9±29.9 d 214.8±19.1 bc 250.5±8.7 a 223.5±6.9 b 203.4±7.1 c 199.3±4.7 c
Guanidine HCl 6.4±5.7 c 107.6±29.6 a 84.9±19.6 a 98.7±11.3 a 99.0±16.1 a 40.8±41.8 b
Niaproof 4 6.8±4.6 c 211.2±34.8 a 207.1±27.3 a 218.1±22.9 a 204.8±18.7 a 169.4±27.1 b
Vancomycin 41.8±33.4 d 232.7±25.9 b 265.8±9.3 a 235.2±4.7 b 205.8±5.5 c 199.2±6.0 c
Tetrazolium violet 66.0±43.0 b 337.5±103.5 a 340.8±91.7 a 278.8±54.4 a 285.0±58.6 a 295.0±76.7 a
Tetrazolium blue 39.5±24.9 c 391.2±61.9 a 391.6±64.0 a 337.6±65.5 a 270.5±46.8 b 238.5±27.2 b
a a a a a a
Nalidixic acid
a a a a a a
Lithium chloride
Potassium tellurite 3.3±3.7 c 223.4±50.3 a 248.5±29.2 a 243.5±18.9 a 182.6±15.8 b 150.5±30.7 b
a a a a a a
Aztreonam
a a a a a a
Sodium butyrate
Sodium bromate 2.1±1.7 d 162.9±6.8 a 138.5±7.8 b 126.4±11.0 c 9.4±13.2 d 0.1±0.1 d
Pectobacterium pH 6 4.0±2.5 c 212.8±30.9 a 206.0±16.7 a 164.7±22.9 b 154.7±8.8 b 155.3±6.1 b
carotovorum pH 5 0.3±0.4 c 159.4±14.0 a 168.6±15.7 a 155.7±18.0 a 131.0±4.3 b 134.2±13.9 b
1 % NaCl 5.7±6.6 c 216.6±12.8 a 220.6±15.3 a 180.7±25.1 b 178.6±12.1 b 190.2±15.4 b
4 % NaCl 2.7±3.7 c 199.2±9.6 a 198.3±8.3 a 182.0±12.4 a 176.1±2.3 a 79.5±60.3 b
8 % NaCl 0.3±0.2 c 27.7±7.8 a 24.0±4.9 a 17.7±7.4 b 2.5±1.4 c 0.0±0.0 c
1 % Sodium lactate 12.4±9.2 c 234.8±21.3 a 255.8±9.7 a 239.2±8.3 a 217.4±6.2 a 161.4±93.4 b
Fusidic acid 4.0±3.6 d 106.0±14.2 a 110.4±5.7 a 90.1±7.6 b 84.0±5.4 b 55.8±9.4 c
a a a a a a
D -Serine
Troleandomycin 8.5±12.5 c 159.3±17.1 b 180.9±3.1 a 178.7±13.1 a 159.2±19.1 b 150.7±18.3 b
Rifamycin SV 5.2±5.9 d 197.3±14.3 b 221.7±11.2 a 195.8±7.7 b 177.3±12.5 c 175.9±15.4 c
a a a a a a
Minocycline
Lincomycin 8.5±7.6 d 193.9±16.7 b 220.8±11.9 a 202.0±5.0 b 188.5±5.6 bc 174.5±18.9 c
Guanidine HCl 5.6±5.1 c 152.1±21.0 a 141.6±4.3 ab 149.7±4.3 a 151.2±5.0 a 136.2±10.9 b
Niaproof 4 5.5±2.3 b 162.8±25.9 a 157.0±14.6 a 157.1±10.9 a 173.3±8.1 a 165.1±20.0 a
Vancomycin 10.2±11.5 e 190.9±24.8 bc 220.8±15.6 a 196.3±19.3 b 172.8±9.3 cd 168.4±17.6 d
Tetrazolium violet 28.6±12.4 e 447.7±15.3 a 402.9±58.3 b 307.0±43.6 c 248.4±28.3 d 211.3±25.8 d
Tetrazolium blue 23.7±7.8 e 344.2±28.5 a 280.3±29.5 b 223.4±22.0 c 184.1±12.4 d 181.2±19.6 d
a a a a a a
Nalidixic acid
a a a a a a
Lithium chloride
a a a a a a
Potassium tellurite
a a a a a a
Aztreonam
Sodium butyrate 2.4±2.5 e 183.5±9.9 a 177.5±8.5 a 137.9±6.0 b 91.0±15.2 c 18.8±3.2 d
a a a a a a
Sodium bromate
Food Bioprocess Technol

Table 3 (continued)

Bacteria Chemical sensitivity assays 4 °C 20 °C

100 kPa 100 kPa 200 kPa 400 kPa 625 kPa 850 kPa

Pseudomonas pH 6 115.7±13.2 c 218.8±19.7 b 240.2±37.0 b 268.4±28.5 a 288.7±19.0 a 288.1±14.1 a

marginalis pH 5 64.1±11.9 d 209.8±44.3 c 248.6±22.2 b 279.5±15.7 a 297.3±16.5 a 297.6±16.8 a
1 % NaCl 98.0±19.0 d 264.8±20.3 c 292.3±13.2 ab 306.8±15.0 a 275.8±21.7 bc 275.6±12.1 bc
4 % NaCl 5.7±3.0 d 188.5±24.9 a 178.0±5.9 ab 161.5±49.0 abc 108.5±76.5 c 126.1±63.4 bc
8 % NaCl 0.1±0.1 b 7.7±5.9 ab 17.4±14.4 a 13.1±15.1 a 0.0±0.0 b 0.0±0.0 b
1 % Sodium lactate 131.9±15.8 d 242.2±29.8 c 276.4±13.1 b 289.8±5.1 b 323.6±17.3 a 333.2±13.6 a
Fusidic acid 18.3±10.0 c 219.3±27.6 b 269.0±37.1 a 288.1±23.4 a 287.3±18.2 a 257.9±21.8 a
D -Serine 0.1±0.1 b 0.8±2.0 b 6.2±9.7 b 31.1±18.9 a 6.9±9.6 b 0.4±0.9 b
Troleandomycin 86.4±15.9 b 218.3±38.2 a 242.2±51.9 a 235.6±23.7 a 241.0±13.0 a 219.4±10.8 a
Rifamycin SV 111.6±8.7 d 234.0±26.8 c 262.3±54.6 bc 278.0±25.3 ab 305.5±22.1 a 281.3±22.6 ab
Minocycline 0.0±0.0 c 173.1±20.8 a 165.4±37.7 a 168.5±44.3 a 153.6±39.5 a 93.6±45.6 b
Lincomycin 128.7±10.3 c 228.5±27.4 b 254.0±56.0 ab 266.3±33.3 ab 272.3±15.1 a 270.0±26.1 a
Guanidine HCl 11.9±3.3 c 225.1±6.8 a 230.8±11.7 a 228.2±9.0 a 224.9±7.0 a 212.1±5.8 b
Niaproof 4 32.4±6.0 c 206.8±11.6 b 208.9±14.7 b 212.2±10.0 ab 223.0±4.3 a 202.0±11.6 b
Vancomycin 120.5±10.2 b 250.0±31.1 a 250.0±37.0 a 271.7±28.4 a 283.3±27.8 a 268.0±17.2 a
Tetrazolium violet 196.4±21.7 b 450.8±2.5 a 451.6±2.5 a 455.1±2.1 a 453.2±2.5 a 450.9±4.9 a
Tetrazolium blue 143.7±39.7 b 432.9±13.1 a 429.1±29.7 a 445.9±2.2 a 443.5±4.8 a 439.5±8.5 a
a a a a a a
Nalidixic acid
Lithium chloride 0.0±0.0 c 2.2±5.3 c 0.0±0.0 c 30.2±73.9 c 105.6±16.1 b 150.1±41.7 a
Potassium tellurite 0.0±0.0 c 214.8±43.9 b 252.1±10.7 a 251.6±24.1 a 263.4±14.1 a 262.8±10.9 a
Aztreonam 17.5±3.6 e 185.3±12.6 d 225.3±7.6 ab 239.5±9.0 a 235.7±12.1 ab 211.6±17.1 c
Sodium butyrate 0.1±0.1 b 19.5±5.7 a 2.4±1.4 b 0.4±0.7 b 0.0±0.0 b 0.1±0.1 b
Sodium bromate 0.1±0.1 b 4.8±4.0 ab 45.7±58.2 a 34.4±57.1 ab 2.9±3.2 b 0.0±0.0 b

Data with the same letter within each row are not significantly different at P <0.05 (Duncan's multiple range test)
a
No color development

Pseudomonas cichorii is highly influenced by hyperbaric pres-
sure, followed by Pectobacterium carotovorum and
Pseudomonas marginalis . For Pseudomonas cichorii and
Pectobacterium carotovorum, hyperbaric pressure, especially
850 kPa, caused a significant reduction in specific growth rate
compared with the control (100 kPa) but it was not the case for
Pseudomonas marginalis. For λ, it depended on the species
of bacteria, which could be either increase or decrease or
be stable over the applied pressure levels. The hyperbaric treat-
ment tended to increase λ for Pseudomonas cichorii, decrease
for Pseudomonas marginalis , and remain constant for
Pectobacterium carotovorum.
nine (i.e., 8 % NaCl, fusidic acid, D -serine, troleandomycin,
minocycline, nalidixic acid, lithium chloride, aztreonam, and
sodium butyrate) and seven (i.e., D -serine, minocycline,
nalidixic acid, lithium chloride, potassium tellurite, aztreonam,
and sodium bromate) chemical sensitivity assays, respectively.
Pseudomonas marginalis did not grow on nalidixic acid.
Generally, hyperbaric pressure and lowered temperature treat-
ments caused a reduction in bacterial growth on the various
chemicals, except for some on which Pseudomonas marginalis
seemed to have enhanced growth with increasing pressure.
Multivariate Analysis

Effect of Hyperbaric Pressure and Temperature Condition
on the Response of Bacteria to Chemical Sensitivity Assays
The response of bacteria to chemical sensitivity was found to
vary greatly for both bacterial species and treatment condi-
tions (Table 3). The letter ( a ) in the table indicates that bacteria
did not grow in the presence of those chemicals. Pseudomonas
cichorii and Pectobacterium carotovorum did not respond to
The PCA on carbon source utilization profile allowed assessing
differences in the patterns of bacterial behavior among treat-
ment conditions (Fig. 4). It is found that the treatments were
clearly divided into distinct groups with first (PC1) and second
(PC2) principal components, accounting for 60.6, 74.3, and
59.9 % of the total variance in the data set for Pseudomonas
cichorii, Pectobacterium carotovorum , and Pseudomonas
marginalis , respectively. The separation of the treatment
 Food Bioprocess Technol

4 Pseudomonas gradually turned to negative values with increasing pressure,

3 cichorii
and approaching values similar to 100 kPa, 4 °C treatment,
2 especially in Pseudomonas cichorii. In other words, the PC1
PC2 (21.02 %)

1 explained the variation caused by the hyperbaric pressure treat-

0 ment. In general, the response of bacteria at 100 kPa, 4 °C
-1 (cold) linked to both PC1 and PC2. These results confirm that
-2 treatment conditions greatly influence the carbon source utili-
-3 zation, and increasing the hyperbaric pressure causes bacteria to
-4
alter their metabolism in a manner similar to that found when
-3 -2 -1 0 1 2 3 placed into cold temperatures.
PC1 (39.56 %)
5 Pectobacterium
4 carotovorum Discussion
3
PC2 (23.22 %)

2 Little information of the effect of hyperbaric pressure on

1
microbial growth on produce is available. According previous
0
-1
studies, hyperbaric treatment has been shown to reduce mi-
-2 crobial growth on some horticultural produce, such as sweet
-3 cherries and table grapes (Romanazzi et al. 2008) and Boston
-4 lettuce (Liplap et al. 2013a). There are three hypothesized
-5 modes of action involved in the reduction of bacterial growth,
-3 -2 -1 0 1 2 3
i.e., direct impact of pressure (Segovia-Bravo et al. 2012), a
PC1 (51.06 %)
toxicity caused by high O2 level (Kader and Ben-Yehoshua
3
Pseudomonas 2000; Bean 1945), or mild stress-induced self-defense mech-
marginalis
2 anism within the plant tissue (Romanazzi et al. 2008). The use
PC2 (27.96 %)

1
0 ment, eliminating the possibility of a plant self-defense mech-
-1
-2
-3
-3 -2 -1 0 1 2 3
PC1 (31.93 %)
Fig. 4 Multivariate classification of different pressure and temperature
conditions on Pseudomonas cichorii, Pectobacterium carotovorum, and
Pseudomonas marginalis bacteria using area under the curve of both all
carbon sources and chemical sensitivity: gray square = 100 kPa, 20 °C,
gray circle = 200 kPa, 20 °C, gray triangle = 400 kPa, 20 °C, white
square = 625 kPa, 20 °C, white circle = 850 kPa, 20 °C, and white
triangle = 100 kPa, 4 °C
conditions in PC space can be related to differences in carbon
utilization by examining the factor loading of the original
variables to the PCs. The most important carbon sources in
treatment differentiation were defined as those which had at
least half of their variance explained by PC1 and PC2 (Table 4).
Separation in treatment conditions based carbon source utiliza-
tion was found to be dependent on the species of bacteria
(indicated by different types of carbon source utilization). In
all cases, the separation among hyperbaric pressure treatments
was separated by PC1. The bacterial growth at ambient condi-
tions (100 kPa, 20 °C) positively correlated with PC1 but
of BIOLOG plate as nutrient simulation of a commodity can
reveal the growth behavior of bacteria under hyperbaric treat-
anism. Results (growth profile, AUC, kinetic parameters)
indicate that hyperbaric treatment itself significantly affected
the growth behavior of decay-causing bacteria. However, as
the hyperbaric treatment involves two phenomena taking
place simultaneously (increasing pressure and increasing O2
level), the understanding of which factor is most critical for
bacterial growth still remains unresolved. Arroyo et al. (1997)
found that to reduce the growth of Gram-negative bacteria by
1 log at 20 °C requires a pressure as high as 300 MPa for
10 min. This implies that the reduction of bacterial growth
found in this study is due unlikely to pressure effects and
hence the increase of O2 level must be the critical factor for
response. Bert (1878) found that many microorganisms such
as bacteria, yeasts, and molds were affected by high O2, but
their sensitivity depends greatly on species of microorganism,
which is in line with our study. The hypothesis is supported by
Harrison (1976), who found that the growth behavior (growth
rate, growth efficiency, and respiration rates) of living organ-
isms is dependent on O2 tension. Nevertheless, contradictory
results have been reported, showing that O2 alone did not
prevent microbial growth and may enhance decay in fruits
(Kader and Ben-Yehoshua 2000). A combination of high O2
(50 kPa O2) and high CO2 (30 kPa CO2) was required to
inhibit growth of microorganisms (Amanatidou et al. 1999).
Perhaps, the reduction of bacterial growth found in this study
Food Bioprocess Technol

Table 4 Substrate loading from PCA using area under the curve of all BIOLOG data. “Loading” is the correlation between a new PC variable and the
original variable. Higher correlation indicates that substrate was important in distinguishing between treatment conditions

Chemical group Substrate Pseudomonas Pectobacterium Pseudomonas

cichorii carotovorum marginalis

PC1 PC2 PC1 PC2 PC1 PC2

Sugars Dextrin – 0.68 – – – –

D -Maltose – 0.73 – – – –
D -Trehalose – 0.60 0.94 – 0.74 –
D -Cellobiose 0.52 – 0.95 – – –
Gentiobiose – – 0.95 – – –
Sucrose – – 0.92 – – 0.76
D -Turanose – – – – –
Stachyose – – 0.98 – – 0.62
D -Raffinose – – 0.97 – – 0.71
a-D -Lactose – 0.62 0.97 – – –
D -Melibiose – 0.68 0.96 – – –
β-Methyl-D -glucoside – 0.61 0.97 – – –
D -Salicin – 0.55 0.96 – – –
N-Acetyl-D -glucosamine – 0.78 0.76 – – 0.93
N-Acetyl-b-D -mannosamine – 0.50 – 0.80 – –
N-Acetyl-D -galactosamine – 0.83 – 0.75 – –
N-Acetyl neuraminic acid – 0.84 – 0.93 – –
α-D -Glucose 0.93 – 0.94 – 0.85 –
D -Mannose 0.93 – 0.94 – 0.87 –
D -Fructose 0.80 – 0.97 – 0.74 –
D -Galactose 0.84 – 0.95 – 0.84 –
3-Methyl glucose – 0.72 – 0.93 – –
D -Fucose – 0.72 – 0.93 – –
L -Fucose – 0.87 – 0.86 – –
L -Rhamnose – 0.84 0.83 – 0.82 –
Inosine 0.69 – 0.73 – – 0.94
Sugar alcohols D -Sorbitol – – – – 0.66 –
D -Mannitol 0.86 – 0.97 – 0.77 –
D -Arabitol 0.91 – – 0.79 0.66 –
myo-Inositol 0.93 – 0.94 – 0.76 –
Glycerol 0.95 – 0.97 – 0.86 –
Hexose-PO4's D -Glucose-6-PO4 – 0.75 0.94 – – –
D -Fructose-6-PO4 – 0.81 0.95 – – –
Amino acids Gelatin – – – – 0.50 –
Glycyl-L -Proline – 0.75 – 0.98 0.51 –
L -Alanine 0.75 – – 0.95 – −0.72
L -Arginine 0.91 – – 0.90 – –
L -Aspartic acid 0.93 – 0.94 – 0.95 –
L -Glutamic acid 0.91 – 0.83 – 0.95 –
L -Histidine 0.77 – – 0.75 0.91 –
L -Pyroglutamic acid 0.85 – – 0.80 – 0.76
L -Serine 0.83 – 0.84 – 0.84 –
D -Aspartic acid 0.93 – 0.92 – – –
D -Serine – 0.76 – 0.83 – –
Hexose acids Pectin – – 0.90 – – 0.94
 Food Bioprocess Technol

Table 4 (continued)

Chemical group Substrate Pseudomonas Pectobacterium Pseudomonas

cichorii carotovorum marginalis

PC1 PC2 PC1 PC2 PC1 PC2

D -Galacturonic acid 0.68 – 0.96 – 0.68 –

L -Galactonicacid lactone – 0.74 0.82 – – –
D -Gluconic acid 0.91 – 0.83 – 0.92 –
D -Glucuronic acid – 0.61 – 0.78 0.69 –
Glucuronamide 0.71 – – 0.86 0.76 –
Mucic acid 0.76 – 0.92 – 0.95 –
Quinic acid 0.89 – – 0.82 0.89 –
D -Saccharic acid 0.82 – 0.96 – 0.92 –
Carboxylic acids p-Hydroxy-phenylacetic acid – – – – 0.84 –
esters, and Methyl pyruvate 0.61 – 0.75 – 0.47 –
fatty acids D -Lactic acid methyl ester – 0.81 – 0.92 – –
L -Lactic acid 0.85 – – 0.82 0.92 –
Citric acid 0.61 – 0.94 – 0.94 –
α-Keto-glutaric acid 0.74 – – 0.88 0.78 –
D -Malic acid 0.90 – – 0.80 – –
L -Malic acid 0.91 – 0.90 – 0.90 –
Bromo-Succinic acid 0.88 – 0.92 – 0.54 –
Tween 40 0.71 – 0.78 – 0.50 –
g-Amino-butryric acid 0.80 – – – – 0.89
α-Hydroxy-butyric acid – – – – 0.87 –
β-Hydroxy-D,Lbutyric acid 0.89 – – 0.60 0.79 –
α-Keto-butyric acid – – – – 0.95 –
Acetoacetic acid 0.55 – 0.60 – – –
Propionic acid 0.86 – – 0.73 0.90 –
Acetic acid 0.81 – 0.85 – 0.76 –
Formic acid 0.76 – 0.79 – – –
Chemical sensitivity pH 6 0.71 – 0.92 – – 0.95
pH 5 – – 0.86 – – 0.95
1 % NaCl 0.82 – 0.85 – – 0.90
4 % NaCl 0.92 – 0.87 – – –
8 % NaCl 0.54 – 0.84 – – –
1 % Sodium lactate 0.73 – 0.82 – – 0.95
Fusidic acid – 0.81 0.93 – – 0.95
D -Serine – 0.64 −0.74 – – 0.28
Troleandomycin – – 0.81 – – 0.88
Rifamycin SV 0.76 – 0.84 – – 0.94
Minocycline – 0.53 0.27 – 0.69 –
Lincomycin 0.68 – 0.83 – – 0.91
Guanidine HCl 0.80 – 0.79 – – 0.88
Niaproof 4 – −0.66 0.69 – – 0.85
Vancomycin 0.73 – 0.85 – – 0.92
Tetrazolium violet 0.75 – 0.98 – – 0.90
Tetrazolium blue 0.87 – 0.97 – – 0.90
Nalidixic acid – 0.45 – 0.90 – –
Lithium chloride – 0.79 – 0.87 – –
Potassium tellurite 0.67 −0.61 −0.55 – – 0.92
Food Bioprocess Technol
Table 4 (continued)
Chemical group Substrate Pseudomonas
cichorii
PC1
Aztreonam – –
Sodium butyrate −0.56
Sodium bromate 0.89
was a result of synergistic effects between elevated pressure
and O2 level as well. Bert (1878) found that an increase in air
pressure at 1.5–4.4 MPa (15–44 atm) preserved meat and raw
eggs for several days at room temperature. In our study, a
smaller increase in total air pressure (200–850 kPa) causes
significant impact on growth behavior of bacteria compared
with atmospheric pressure (100 kPa), especially the A and μ M
features of the growth curve. This is in accordance with our
previous study on hyperbaric treatment of lettuce, in which the
incidence of rots reduced with an increase in hyperbaric
pressure (200–850 kPa).
The response of bacterial growth is dependent on nutrients
available. Pseudomonas cichorii and Pseudomonas marginalis
grew greatly in an environment rich with sugar alcohols, follow-
ed by amino acids and hexoses acids but not in sugars group,
while the growth of Pectobacterium carotovorum grew well in
sugars, sugar alcohols, hexose acids but not in amino acids.
Thus, the severity of rot incidence taking place in a commodity
may be predictable from nutrient availability and species of
bacteria. Also, the resistance of bacterial growth to pressure
and temperature treatments varies among bacteria. The pressure
treatment seems to be the most effective method in inhibiting the
growth of Pseudomonas cichorii, followed by Pectobacterium
carotovorum as observed from a small discrepancy of the
growth curve compared to the treatment at 4 °C (Fig. 3).
Pseudomonas marginalis showed a greater resistance to hyper-
baric treatment than the other two species. This may be due
partly to their relation to O2 tension (Krejzar et al. 2008).
Although the 100-kPa, 4 °C treatment is very effective in
delaying the λ and slowing down the μ M during the 7 days of
experiment, Pseudomonas cichorii and Pseudomonas
marginalis had a tendency to grow as much under this treatment
as in the 850-kPa hyperbaric treatment at 20 °C after 7 days. This
implies that although low temperature delays the growth of some
bacteria, their population will eventually increase to those held at
20 °C in long-term storage. Pectobacterium carotovorum seems
to be the most temperature-sensitive bacteria among the three,
since its growth was very low over the entire 7 days of treatment.
After the 7-day treatment, the growth of bacteria was
monitored at 100 kPa, 20 °C for another 3 days (data not
shown). It was found that Pseudomonas cichorii treated at
elevated pressure and lowered temperature generally restored
Pectobacterium Pseudomonas
carotovorum marginalis
PC2 PC1 PC2 PC1 PC2
– 0.74 – 0.93
– 0.93 – 0.71 –
– – – – –
their growth similar to those kept at ambient conditions. The
growth resumption of some bacterial was also observed after
1.0-MPa O2 treatments for 8 h (Caldwell 1965). However, in
our study, neither Pectobacterium carotovorum nor
Pseudomonas marginalis resumed their growth after hyper-
baric treatment. This implies that the hyperbaric treatment not
only provides direct inhibition bacterial growth but the treat-
ment may also have residual effects on bacterial growth and
the magnitude of this residual effect is dependent on the
species of bacteria. The residual effect may be attributed to
the damage of vital cellular macromolecules of bacteria
caused by surplus active oxygen radical species at high O2,
thus permanently inhibiting bacterial growth (Bean 1945;
Zobell and Hittle 1967).
Conclusions
Three bacteria causing fruit and vegetables decay (i.e.,
Pseudomonas cichorii , Pectobacterium carotovorum , and
Pseudomonas marginalis ) were tested with different hyper-
baric pressure and temperature conditions using BIOLOG
microplates. Hyperbaric pressure treatment at 20 °C was
found to significantly inhibit the growth of all selected
bacteria as indicated by AUC and growth kinetic model
parameters (A , μ M , and λ ). The response of bacteria to
hyperbaric pressure was dependent on bacterial species,
among which the Pseudomonas cichorii was the most
pressure-sensitive bacterium, followed by Pectobacterium
carotovorum and Pseudomonas marginalis . Multivariate
analysis showed that an increase in hyperbaric pressure caused
bacteria to utilize carbon sources similar to those subjected to
low temperature (4 °C). Overall, hyperbaric pressure having
characteristics of high pressure and O2 has the potential of
being used to reduce bacterial growth in fruit and vegetables
after harvest. Further research on enhancement of fruit and
vegetable self-defense compound synthesis by hyperbaric
pressure is recommended.
Acknowledgments The authors are grateful to Agriculture and Agri-
Food Canada for the financial support. The Royal Thai Government is
gratefully acknowledged for Mr. Pansa Liplap's PhD Scholarship.

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