Food Bioprocess Technol (2009) 2:213–221
DOI 10.1007/s11947-008-0069-7
ORIGINAL PAPER
Effects of Water Activity in Model Systems on High-Pressure
Inactivation of Escherichia coli
Ilona Setikaite & Tatiana Koutchma & Eduardo Patazca &
Brian Parisi
Received: 7 September 2007 / Accepted: 7 February 2008 / Published online: 28 February 2008
# Springer Science + Business Media, LLC 2008
Abstract The objectives of the study were to measure the
effect of water activity (aw) and to quantitatively evaluate
the effect of the selected humectants under high-pressure
processing (HPP) in combination with processing parameters such as treatment time, temperature, and pressure on
the inactivation of Escherichia coli K12 in solid and liquid
model systems. Glycerol was used in liquid and solid
models to vary aw at 0.90, 0.95, and 0.99 levels. The model
systems samples and transmitting media were preconditioned to initial temperatures of 4 and 20 °C to compensate
for adiabatic heating upon compression to ensure that HPP
treatments at 400 and 600 MPa were performed at final
temperatures not higher than 40 °C. Decrease of aw from
0.99 to 0.90 in glycerol-based models caused considerably
less inactivation of E. coli K12 at tested pressures and
temperatures. Effect of different humectants at aw 0.95 and
0.99 on the inactivation of E. coli K12 was studied
comparing glycerol, fructose, sodium chloride, and sorbitol.
Among four types of solutes tested in the study, sodium
chloride appeared the least protective, with glycerol and
fructose being approximately equal, and sorbitol showed
the most protective effects on inactivation of E. coli K12.
The obtained data of E. coli K12 inactivation by HPP at
varied aw levels in different solutes demonstrated similar
effects of aw on microbial inactivation by thermal treatMention of trade names and commercial products in this article is
solely for the purpose of providing specific information and does not
imply recommendation or endorsement by the National Center for
Food Safety and Technology.
I. Setikaite : T. Koutchma (*) : E. Patazca : B. Parisi
National Center for Food Safety and Technology,
Illinois Institute of Technology,
6502 South Archer Rd,
Summit-Argo, IL 60501-1933, USA
e-mail: t_koutchma@hotmail.com
ments. The results must be taken into account when HP
preservation process and foods are developed.
Keywords Water activity . Model systems .
High-pressure processing . Microbial survival
Introduction
High-hydrostatic-pressure processing (HPP) has been demonstrated to be an effective processing technology for the
production of extended shelf-life (ESL) foods. Effects of
water activity (aw) have been found to be a determinant
factor of the microbial growth and inactivation in ESL
foods. The limiting aw for the growth of some pathogenic
microorganisms is approximately 0.9. There are numerous
reports showing the protective effect of low aw against heat
inactivation. Scott (1955) found that low aw protects
proteins and whole cells against heat. Microbial inactivation by HPP depends on a number of interacting factors
including processing conditions, composition of food, and
aw. The effects of processing conditions such as initial
sample temperature, pressurizing medium, and holding time
on the level of microbial inactivation as well as the overall
effect on the nutritional and sensory characteristics of food
were extensively reported (Cheftel 1995). However, limited
research is available on the effect of aw and humectants on
HPP inactivation (Hoover et al. 1989; Palou et al. 1997;
Knorr 1994).
A number of reported studies demonstrated similar
protective effects of low aw against HPP inactivation.
Hoover et al. (1989) stated that many food constituents
appear to protect microorganisms from the lethal effects
of HPP. Oxen and Knorr (1993) observed that a reduction
of aw from 0.98–1.0 to 0.94–0.96 resulted in a reduction of
214
inactivation rates under HPP treatment. They reported a 7log10 reduction in Rhodotorula rubra counts during
treatment at 25 °C and 400 MPa for 15 min when aw was
greater than 0.96, while no reduction was observed with aw
below 0.91.
Palou et al. (1997) studied the combined effect of HPP
and aw on the inactivation of Zygosaccharomyces bailii.
Complete inactivation of yeast at aw greater than 0.98 and
an increase in the surviving fraction with a decrease in aw
was observed. The addition of sucrose (to decrease aw) was
baroprotective and prevented inactivation of yeast even at
high pressures. Palou et al. (1997) also showed that the
required aw necessary to prevent growth depends on the
solutes present; e.g., glycerol lowers aw efficiently but still
may allow microbial growth. Baroprotective effects of
sodium chloride and sugars have been observed in studies
with Escherichia coli, Saccharomyces cerevisiae, and
Lactococcus lactis (Knorr 1994; Molina-Gutierrez et al.
2002).
Thus, reducing aw appears to protect microorganisms
against HPP inactivation. However, the parameters governing pressure tolerance are not the same for every bacterial
species. They vary from one bacterium to another and,
according to Hoover et al. (1989), may even be different for
a single species grown under different conditions or in
different growth media. However, limited information was
reported on the effects of aw on the inactivation of food
pathogens. Apart from general osmotic effect of water
activity, aw specific effects depend on the type of the solute
or humectants such as sodium chloride, sorbitol, sucrose, or
glycerol. Effect of challenge temperature and solute type
(glucose–fructose, sucrose, and sodium chloride) on heat
tolerance of Salmonella serovars at low aw was investigated
and reported by Mattick et al. (2001). However, the effects
of humectants at the same aw levels on HPP tolerance have
not been evaluated and reported except for protective effect
of sucrose and sodium chloride for L. lactis (MolinaHoppner et al. 2004).
The objectives of this study were: (1) to measure the
combined effect of water activity (aw) and HPP parameters
such as treatment time, temperature, and pressure on the
inactivation of E. coli K12 in solid and liquid models; and (2)
to quantitatively evaluate the effect of the selected humectants
on the inactivation of E. coli K12 at aw of 0.95 and 0.99.
Food Bioprocess Technol (2009) 2:213–221
Culture Preparation
Stock culture of E. coli K12 ATCC 35695 was maintained
at 4 °C on tryptic soy agar plates (TSA; Difco, Fisher
Scientific, Hanover Park, IL, USA). One colony was
transferred to tryptic soy broth (TSB; Becton Dickinson,
Sparks MD, USA), and culture was grown for 18 h in TSB
with 0.75% glucose in 250 ml Erlenmeyer flasks at 37 °C.
Stationary phase culture typically reached a final concentration of approximately 109 CFU/ml. Cells were harvested
by centrifuging once at 500 rpm (Eppendorf 5804R
Refrigerated Centrifuge, Hamburg, Germany), washed with
0.1% peptone water (Becton Dickinson) and suspended to
initial level of concentration about 106–107 CFU/ml. For
inoculation to a model system to a final concentration of
106–107 CFU/ml, culture was added directly without prior
harvesting or washing.
Preparation of Solid Model Gels
Glycerol was used for the preparation of solid model gels.
The composition of the model gels with varied aw levels
from 0.9 to 0.99 is shown in Table 1. Glycerol (enzyme
grade, Fisher Scientific) and kelcogel (industrial grade
Gellan Gum, CP Kelco US, Chicago, IL, USA) were
dissolved in distilled water at 75 °C. The solution was
autoclaved at 121 °C for 30 min. Solution of 1 M CaCl2
salt was prepared and autoclaved for 30 min. Bacterial
suspension was added directly to a final concentration of
106 CFU/ml. To obtain inoculated solid model gels,
600 μl of CaCl2 solution was added to the inoculated
mixture of glycerol, distilled water, and kelcogel to induce
gelatinization.
Preparation of Liquid Model Systems
Glycerol, fructose, sorbitol, and sodium chloride (NaCl; all
enzyme grade, Fisher Scientific) in different concentrations
were used to prepare the liquid model systems with varied
aw from 0.9 to 0.99 (Table 2). Liquid solutions were
prepared by mixing distilled water and the four different
solutes. After mixing, solutions were autoclaved for 30 min
at 121 °C.
Table 1 Composition of glycerol-based solid model gels
Materials and Methods
Composition
Water activity
0.99
0.95
0.90
96
4
1
600
81
19
1
600
72
28
1
600
Target Microorganism
E. coli K12 ATCC 35695 was used as test bacterium due to
its ability to survive at a low pH and aw and as a surrogate
for E. coli O157:H7.
Distilled water (ml)
Glycerol (ml)
Kelcogel (g)
CaCl2 (μl)
Food Bioprocess Technol (2009) 2:213–221
215
Table 2 Composition of liquid models
Composition (ml)
Glycerol (ml)
awa
Distilled water (ml)
Solute (ml)
0.90
72
28
a
0.95
81
19
Sorbitol (g)
0.99
96
4
0.95
65
35
Fructose (g)
0.99
72
28
0.95
81
19
Sodium chloride (g)
0.99
96
4
0.95
65
35
0.99
72
28
After autoclaving
Inoculation
For inoculation of solid model gels, overnight culture of E. coli
K12 was added into prepared liquid mixture of glycerol,
distilled water, kelcogel and CaCl2 at 35 °C. The amount of
alginate used was 1 g/100 ml of water. This temperature was
chosen to avoid gelatinization at lower temperatures. Ten
milliliter of inoculated alginate liquid was added to sterile
Petri plates and cooled to ambient temperatures to induce
gelatinization. Inoculated gel models were transferred to
individual 5×8 cm plastic bags (Ampac LLC, Cincinnati,
OH, USA). The plastic bags were vacuum sealed using a
Multivac seam sealer (Centurian Int’l, Lebanon, PA, USA).
For inoculation of the liquid models, 1 ml of the
overnight culture of E. coli K12 was added to 200 g of
test solutions resulting in a concentration of 106 to
107 CFU/ml in the sample. Ten milliliters of inoculated
liquid medium was transferred to individual sterile plastic
bags at ambient temperature and vacuum sealed. In order to
test the effect of the models on the bacterial growth at low
aw levels, inoculated gel and liquid models were held at 4
and 20 °C for 24 h. Samples were taken after 4, 14, and
24 h and surface plated on TSA.
Enumeration
For enumeration of E. coli K12 in the solid glycerol-based
model, 90 ml of buffered peptone water and 10 g of solid
model were homogenized at the highest speed for 60 s
using a Stomacher 400 (Seward Lab System, Nortfolk,
UK). For enumeration, appropriate dilutions were made
with 0.1% peptone water and surface plated in duplicate on
TSA. Plates were incubated for 24 h at 37 °C and counted.
The Autoplate 4000 Automated Spiral (Spiral Biotech,
Norwood, MA, USA) was used for enumeration. The unit was
calibrated before use and used according to manufacturer’s
instructions. Spiral plating using the Autoplate 4000 is
approved by the AOAC and is listed in the Bacteriological
Analytical Manual 2001 as a method for enumerating bacteria.
Water Activity and pH Measurements
The aw of the model systems was determined using a
calibrated water activity meter, Aqualab series 3 (Decagon
Devices, Pullman, WA, USA). Samples were analyzed in
triplicate with negligible variability (coefficient of variation
<0.1%) among measurements. aw was determined before
and after autoclaving in control non-inoculated samples.
The pH of all model solutions was measured with a
portable pH meter (model 230A, Orion Research, Beverly,
MA, USA).
High-Pressure Treatment
A hydrostatic food processor, QFP-6 (Quintus Food
Processing Cold Isostatic Press, ABB Autoclave Systems,
Columbus, OH, USA), was used for pressure treatments.
Glycol/distilled water 50% solution was used as a pressuretransmitting medium. The pressure come-up time depended
on the final pressure and varied from 95 to 130 s for
pressures from 400 to 600 MPa, respectively. Depressurization time was less than 6 s. The holding time for each
treatment was selected and programmed with increments of
0.01 min. The system was equipped with one type K
thermocouple attached to a data logger to monitor and
record temperature in the pressure-transmitting medium
throughout the process. In addition, the temperature of the
samples was measured in glycerol solid and liquid models.
The thermocouple was first attached to the plastic pouch,
then pouch was filled with the sample model and then
vacuum sealed. A data-acquisition set-up (Agilent 34970A,
Agilient, Palo Alto, CA, USA) was used to record pressure,
temperature, and time. LabVIEW software (LabVIEW
version 6, National Instruments, Austin, TX, USA) was
used to convert the signals to be read and recorded by a
computer.
The samples and transmitting media were preconditioned
to initial temperatures of 4 and 20 °C to compensate for
adiabatic heating upon compression to ensure that all HP
treatments were performed at final temperatures not higher
than 40 °C. Treatments were performed at 400 and
600 MPa. Treatment times varied from 1 to 10 min.
Pressure, temperature of pressure-transmitting medium, and
time were recorded at 1-s intervals during treatment. The
beginning of the holding time was taken at the time at
which the set pressure was reached. Depressurization
occurred at the end of the preprogrammed holding time.
After HPP, samples were quickly removed and held in the
216
Food Bioprocess Technol (2009) 2:213–221
a
ice-water until enumeration. The untreated inoculated
samples held in ice-water during experiments served for
enumeration of initial concentration of bacteria (N0).
a w=0.95 T=20C
8
7.8
Design of Experiments and Statistical Analysis
7.6
Log 10 N
7.4
The Microsoft Excel 7.0 and Minitab 14 statistical software
(Minitab, State College, PA, USA) were used for mathematical and statistical analysis. General full factorial design
was used for series of the experiments using solid and
liquid glycerol-based models at aw of 0.90, 0.95 and 0.99
and liquid model solutions at aw of 0.95 and 0.99. The
effect of pressure (400 and 600 MPa), initial temperature (4
and 20 °C), holding time (60 to 600 s), water activity, and
type of the solutes on survival fraction of E. coli K12 were
studied. Three replicate runs were performed for each
processing condition. Analysis of variances of effects of
pressure, temperature, time, water activity, and type of
solutes on survival of E. coli K12 was conducted for each
series of experiments in solid and liquid glycerol-based
models and liquid model solutions using Minitab 14.
7.2
7
6.8
6.6
6.4
6.2
6
0
5
10
15
20
25
Time, h
glycerol
sodium chloride
sorbitol
fructose
b
T=20C
8.5
8
Effect of Model Systems on Microbial Survival
The first step in the study was to develop and test model
systems for the range of aw from 0.9 to 0.99 that would not
inactivate the test bacterium. After E. coli K 12 was
introduced into model solutions, the solutions were held at
4 and 20 °C for 24 h. The data at 20 °C are shown in
Fig. 1a. Viable counts indicate that exposure to aw of 0.95
in itself was not toxic to E. coli. Glycerol was selected for
further studies of water activity in a gel-based solid system
since it was very effective in reducing aw. No inactivating
effect of glycerol was found at the lowest level of aw =0.9
at 20 °C (Fig. 1b). No difference of E. coli K12 growth was
detected in solid and liquid glycerol model systems.
The obtained results for E. coli K12 growth in glycerolcontaining solutions were in agreement with those reported
for Bacillus cereus by Jakobsen and Murell (1977) and for
Clostridium botulinum by Baird-Parker and Freame (1967),
who concluded that glycerol appeared less inhibitory than
other test solutes.
Effect of Water Activity on Inactivation of E. coli K12
Glycerol-Based Solid Model Systems
In order to minimize heating effects on HPP inactivation,
the effect of the initial temperature on the adiabatic
compression heating of the model systems was evaluated
7.5
Log 10 N
Results and Discussion
7
6.5
6
0
5
10
15
20
25
Time, h
Glycerol aw=1.0
Glycerol aw=0.90
Glycerol aw=0.95
Fig. 1 a Effect of solutes on growth of E. coli K12 in liquid models;
b growth curve of E. coli K12 in glycerol-based model system at 20 °C
at selected process pressures and aw levels. When model
samples were pre-cooled to 4 °C, the final process
temperature did not exceed 14 °C at 400 MPa and 20 °C
at 600 MPa in all tested models. When initial temperature
of the glycerol-based solid samples was pre-equilibrated to
20 °C, the final process temperatures rose to approximately
31±1 and 32±1 °C when pressure reached, respectively,
400 and 600 MPa. As it was reported by Patazca et al.
(2007), during the hold time, this final temperature can be
affected by external factors such as heat loss through the
wall of the vessel, which leads to a non-uniform temperature during HPP.
Table 3 presents pressure inactivation of E. coli K12 in
glycerol-based solid models at aw of 0.99, 0.95, and 0.90
under pressures of 400 and 600 MPa and initial temperatures of 4 and 20 °C. Analysis of the obtained inactivation
Food Bioprocess Technol (2009) 2:213–221
217
Table 3 Effect of aw, pressure, time of treatment, and initial temperature on the inactivationa of E. coli K12 in glycerol-based solid model systems
aw
0.90
Time, min
Initial temperature (°C)
400 MPa
1
3
5
8
600 MPa
1
3
5
8
0.95
0.99
4
20
4
20
4
0.03 (0.00)
0.06 (0.01)
0.15 (0.0)
0.38 (0.074)
0.11 (0.04)
0.17 (0.01)
0.07 (0.01)
0.17 (0.05)
0.041 (0.011)
0.51 (0.14)
0.12
0.47
0.63
0.98
–
0.08 (0.03)
0.15 (0.05)
0.48 (0.07)
–
0.80 (0.45)
1.20 (0.19)
1.47 (0.04)
–
0.30 (0.23)
0.88 (0.05)
1.42 (0.39)
–
1.39 (0.23)
2.53 (0.76)
3.69 (0.37)
(0.03)
(0.20)
(0.01)
(0.17)
20
0.31
1.40
2.55
4.08
(0.20)
(0.09)
(0.31)
(0.45)
2.21 (0.51)
3.70 (0.24)
6.8 (0.5)
–
2.30 (0.06)
6.17 (1.24)
–
–
6.8 (0.5)
–
–
–
Number in () is standard deviation.
(–) No inactivation data were generated at these processing conditions.
a
(log10 N/N0)
Glycerol-Based Liquid Model Systems
Time–temperature profiles in Fig. 2 show the behavior of
glycerol liquid models at aw of 0.99 under pressure of 400
and 600 MPa and initial temperatures T0 =4 and 20 °C. It
can be seen that the maximum process temperature of 40±
1 °C was achieved at 600 MPa and initial temperature of
T0 =20 °C compared to final temperature of 33±1 °C
achieved at 400 MPa.
Inactivation data of E. coli K 12 in glycerol liquid
models are summarized in Table 4. Analysis of variance for
glycerol-based liquid models of effect of pressure, temperature, time, and water activity on survival of E. coli K12
showed that each single factor was significant (P=0.0).
According to the F values, the aw of glycerol liquid models
had the largest effect on inactivation. A consistent increase
in the number of surviving of E. coli K12 was observed
with decrease of the aw at all processing conditions.
Specifically, at a HPP treatment of 400 MPa and initial
temperature 4 °C, after 3 min of holding a 2.24-log10
reduction was observed at a aw of 0.99. However, no
significant inactivation was observed at a aw of 0.95 and
0.90 at this processing condition. The results further
confirmed a baroprotective effect at a aw of 0.90 on E.
coli K12 compared to a aw of 0.95 and 0.99.
45
40
35
Temperature, oC
data led to the following general observations: decrease of
aw to 0.90 protected E. coli K12 from inactivation at both
pressures and temperatures; increase of pressure caused
more rapid inactivation at constant temperatures and aw;
higher inactivation was also achieved by increasing the
initial temperature of the test model systems at the constant
pressure. It can be seen that after 3 min holding time, at
initial temperature of 20 °C and 400 MPa pressure, the
extent of E. coli K12 inactivation increased by 2.3-log10 at
a aw of 0.99 and 0.3-log10 at a aw of 0.95 compared to
inactivation at the initial temperature of 5 °C. For a solid
glycerol model at 600 MPa at 20 °C (Table 3b), the increase
of holding time from 5 to 8 min in solid and liquid model
systems at aw of 0.90 resulted in less than 0.5-log
reduction, while in the first 3 min of pressurization,
0.8-log10 and 0.5-log10 reductions were achieved. Analysis
of variances for glycerol-based solid models of effects of
pressure, temperature, time, and aw on survival of E. coli
K12 showed that all single factors were significant in
inactivation except for time (P=0.069) at aw of 0.90 and
0.95 at 400 MPa. However, at a aw of 0.90, there was no
significant change in the inactivation with increased initial
temperature.
30
25
20
15
10
5
0
0
100
200
300
400
500
600
700
Time, s
600 MPa, 4°C
400 MPa, 4°C
600 MPa, 20°C
400 MPa, 20°C
Fig. 2 Time–temperature profiles within glycerol liquid models at aw
of 0.99 at 400 to 600 MPa
218
Food Bioprocess Technol (2009) 2:213–221
Table 4 Effect of aw, pressure, time of treatment, and initial temperature on the inactivationa of E. coli K12 in glycerol-based liquid models
aw
0.90
Time (min)
Initial temperature (°C)
0.95
4
400 MPa
1
3
5
8
10
600 MPa
1
3
5
8
10
20
4
0.99
20
4
20
–
0.03
0.08
0.15
0.27
(0.0)
(0.04)
(0.18)
(0.2)
–
0.02 (0.05)
0.11 (0.04)
0.19 (0.10)
0.43 (0.15)
–
0.04
0.16
0.21
0.30
(0.00)
(0.10)
(0.05)
(0.11)
–
0.09
0.34
0.78
2.63
(0.08)
(0.22)
(0.38)
(0.05)
0.31
2.24
4.10
7.09
–
–
0.08
0.15
0.48
0.80
(0.011)
(0.06)
(0.34)
(0.3)
–
0.52 (0.04)
0.67 (0.05)
0.79 (0.190
1.2 (0.35)
–
0.56 (0.07)
1.24 (0.13)
1.63 (0.63)
2.1 (0.45)
–
0.89
1.21
2.50
3.43
(0.41)
(0.02)
(0.26)
(0.1)
1.76 (0.68)
3.86 (0.54)
6.63 (0.35)
–
–
(0.04)
(0.22)
(0.80)
(0.55)
1.05
2.23
5.65
6.72
–
(0.03)
(0.23)
(0.11)
(0.77)
2.63 (0.26)
7.31 (1.08)
–
–
–
Number in () is standard deviation.
(–) No data was generated.
a
(log10 N/N0)
o
600 MPa 20 C
45
temperature, oC
40
35
30
25
20
0
100
200
300
400
500
tim e,s
600
0.99 liquid
700
0.95 liquid
400 MPa 20o C
45
Temperature, oC
It was also found that the change of aw of the glycerol
liquid model systems resulted in variations of final temperatures under identical pressures at aw of 0.95 and 0.99
(Fig. 3). For example, at 600 MPa in the glycerol liquid
model at a aw of 0.99, the temperatures reached approximately 40.7 °C while at a aw of 0.95, final measured
temperature was only 37.3 °C. At 400 MPa, the temperature reached 33.8 °C at a aw of 0.99, while for a aw of 0.95,
the temperature increased only to 32.0 °C (Fig. 3). The
observed temperature differences in adiabatic compression
heating can be best explained by the differences in
concentrations of glycerol that affected physical properties
of model solutions. The change in temperature as a result of
physical compression depends on the compressibility of the
substance, temperature, specific volume, and specific heat
capacity (Patazca et al. 2007).
The increase of initial temperature to 20 °C at 400 MPa
resulted in increase of inactivation at a aw of 0.99 and 0.95.
The maximum inactivation of 7.31-log10 in 3 min was
achieved for aw of 0.99 at 20 °C. On the contrary, at 4 °C
and 600 MPa, only a 3.86-log10 reduction was obtained. As
expected, increasing the pressure to 600 MPa resulted in an
increase in microbial inactivation at both temperatures.
Only slight increase of microbial inactivation (less than
1-log10 after 10 min) was observed at low aw of 0.90 with
increasing holding time at both pressures at initial temperature of 4 °C. The increase of initial temperature to 20 °C
did not result in significant increase of inactivation at this
aw level at 400 MPa. However, at 600 MPa and 20 °C, the
effect of holding time was more pronounced when 10 min
of holding resulted in 1.2-log 10 reduction. In the
40
35
30
25
20
0
100
200
300
Time,s
400
500
0.99 liquid
600
0.95 liquid
Fig. 3 Comparison of time–temperature profiles in the glycerol liquid
models at aw of 0.95 and 0.99 at 600 MPa and 400 MPa
Food Bioprocess Technol (2009) 2:213–221
219
application of HPP to foods, it has been reported that, while
higher pressure accelerates microbial inactivation, longer
exposure durations do not necessarily increase the lethal
effect of pressure (Cheftel 1995; Palou et al. 1997).
Intrinsic conditions of media, such as aw, and presence of
nutrients influence the duration of pressure treatments.
Data for models with aw levels of 0.95 and 0.90 also are
in agreement with the report of Doesburg et al. (1970), who
investigated a combined effect of temperature and aw on
survival of salmonellae in fish meal. For a aw of 0.71, the
increase in temperature from 15 to 30 °C reduced the
survival time by fivefold. The effects of temperature and aw
during the heat treatment reported are similar to the effects
of these factors found in our study for HPP. The results of
this study confirm the baroprotective effect of low aw
which are in agreement with those reported by Palou et al.
(1997) for Z. bailii.
coli K12 by pressure treatment depended upon the type of
solutes used to control aw. Among solutes tested to vary aw,
the addition of sorbitol at aw of 0.95 had pronounced
protective effect on E. coli K12 inactivation under pressure.
In contrast, sodium chloride at both aw levels promoted
higher inactivation. In the sodium chloride model at aw of
0.95, at 20 °C, 600 MPa, and 8 min holding time, a
reduction of a 6.03-log10 was observed. Whereas, a similar
treatment of inoculated sorbitol solution at a aw of 0.95
resulted in 0.38-log10 reduction. According to the obtained
data, the action of glycerol and fructose were similar in the
effect (Table 5).
The increase in pressure from 400 to 600 MPa resulted
in greater inactivation of E. coli K12 compare with increase
in initial temperature from 4 to 20 °C in all models.
However, at the pressure of 600 MPa, the increase in initial
temperature had more pronounced inactivation effect due to
adiabatic heating.
It was observed that higher inactivation of E. coli K12
was achieved by increasing time under pressure at aw of
0.99. However, the inactivation of E. coli K12 did not
increase significantly with increase of holding time for the
fructose and sorbitol models with the lower aw of 0.95.
With increasing exposure time up to 10 min of inoculated
sorbitol models to HPP, no statistically significant increase
in inactivation was found. Cheftel (1995) and Palou et al.
(1997) have reported that HPP accelerates microbial
inactivation in food applications; however, longer exposure
durations do not necessarily increase the lethal effect of
pressure.
In general, the obtained data of E. coli K12 inactivation
by HPP in different solutes are similar to the earlier
findings of researchers investigating the inactivation
Effect of Type of Solutes on Inactivation of E. coli K12
The effect of solute type on pressure inactivation of E. coli
K12 was studied by comparing the survival fraction at aw
of 0.95 and 0.99, at pressures of 400 and 600 MPa, and
initial temperatures of 4 and 20 °C and summarized in
Tables 5 and 6. Analysis of variance at aw of 0.99 and 0.95
(Table 7) of effect of four factors tested such as pressure,
temperature, time, and solutes on inactivation of E. coli
K12 showed that all single factors and their two-way and
three-way interactions were significant (P=0).
The differences in physical and chemical properties of
the solutes and their behavior under pressure had considerable influence on the ability of E. coli K12 to tolerate
suboptimal aw levels. It was found that inactivation of E.
Table 5 Effect of solute, initial temperature, pressure, and time on the inactivationa of E. coli K12 in liquid model systems at aw of 0.95
Type of solute
Fructose
Time of treatment (min)
Initial temperature (°C)
4
400 MPa
3
5
8
10
600 MPa
3
5
8
10
Sorbitol
20
4
Sodium chloride
20
4
20
0.09
0.18
0.31
0.75
(0.03)
(0.03)
(0.06)
(0.16)
0.16
0.55
0.78
1.48
(0.1)
(0.08)
(0.26)
(0.44)
0.05
0.13
0.17
0.17
(0.03)
(0.20)
(0.09)
(0.03)
0.04 (0.0)
0.15 (0.15)
0.017 (0.0)
0.24 (0.07)
0.48
0.84
1.07
2.22
(0.02)
(0.22)
(0.51)
(0.31)
1.02
1.45
2.30
2.80
0.72
0.91
1.32
2.60
(0.04)
(0.05)
(0.10)
(0.27)
1.42
1.97
2.92
3.11
(0.04)
(0.43)
(0.20)
(0.24)
0.16
0.34
0.40
0.40
(0.06)
(0.09)
(0.10)
(0.10)
0.27
0.26
0.38
0.55
1.65
2.08
2.34
3.82
(0.06)
(0.17)
(0.39)
(0.64)
2.44 (0.38)
3.49 (0.63)
6.03 (0.25)
–
Number in () is standard deviation.
(–) No inactivation data were generated at processing condition.
a
(log10 N/N0)
(0.20)
(0.14)
(0.12)
(0.18)
(0.22)
(0.01)
(0.22)
(0.6)
220
Food Bioprocess Technol (2009) 2:213–221
Table 6 Effect of solute, initial temperature, pressure and time on the inactivationa of E. coli K12 in liquid model systems at aw of 0.99
Type of solute
Fructose
Time of treatment, min
Initial temperature (°C)
4
400 MPa
3
5
8
10
600 MPa
3
5
8
10
Sorbitol
Sodium chloride
20
4
(0.17)
(0.02)
(0.06)
(0.16)
1.26 (0.25)
1.97 (0.0)
5.28 (0.0)
_
0.90
1.44
2.50
4.40
(0.02)
(0.05)
(0.02)
(0.18)
1.31
1.87
3.84
5.71
1.92 (0.01)
3.11 (0.06)
5.65 (0.4)
8.94 (0.06)
3.44 (0.32)
4.16 (0.12)
4.3 (0.23)
4.5 (0.25)
1.76
1.79
3.96
5.65
(0.05)
(0.070
(0.03)
(0.04)
2.63 (0.09)
4.09 (0.0)
6.45 (0.42)
_
1.20
1.77
3.74
7.09
20
4
(0)
(0.3)
(0.42)
(0.63)
1.42
1.95
3.44
7.17
20
(0.21)
(0.19)
(0.31)
(0.31)
3.08 (0.23)
7.32 (0.12)
_
_
1.73
2.36
5.58
7.18
(0.27)
(0.71)
(0.19)
(0.19)
4.17 (0.80)
7.25 (0.13)
_
_
Number in () is standard deviation.
(–) No inactivation data were generated.
a
log10 (N/N0)
kinetics by heat treatment. Heat resistance is generally
increased at reduced aw but strongly depended on the solute
used to reduce water activity. Many ionic solutes including
sodium chloride actually caused a decrease in heat
resistance when present at relatively low levels. At high
concentration, they may sometimes show considerable
protection. Glycerol frequently caused only slight increase
of heat resistance. Like in a case of heat treatments, sorbitol
and fructose appear to exhibit a similar protective effect on
E. coli K12 resistance in contrast to accelerating effects of
sodium chloride at the tested levels during HPP. The
observed data also agreed with Oxen and Knorr (1993)
who found a protective effect of high sucrose concentrations against pressure inactivation of R. rubra in
laboratory model systems. Hayert et al. (1996) also
observed baroprotective effects of glycerol and sorbitol on
S. cerevisiae at low aw values. For purposes of comparison
and quantification of the observed effects of water activity,
mathematical modeling of microbial inactivation kinetics
under HPP is required as a next research step.
Conclusions
The results suggest that high-pressure inactivation of
microorganisms depends not just on pressure level, treatment time, and process temperature but also on the aw
levels and type of solute used to adjust the aw in the model
system. Combination of these factors may be used to
accelerate the process, reduce the required pressure, and
Table 7 Analysis of variances of the single factors of pressure, temperature, time, solutes, and their two and three-way interactions at aw of 0.95
Source
DF
Seq SS
Adj SS
Adj MS
F
P
Time, C1
Temperature, C2
Solute, C3
Pressure, C4
C1 × C2
C1 × C3
C1 × C4
C2 × C3
C2 × C4
C3 × C4
C1 × C2 × C3
C1 × C2 × C4
C1 × C3 × C4
C2 × C3 × C4
C1 × C2 × C3 × C4
3
1
3
1
3
9
3
3
1
3
9
3
9
3
9
37.19
32.12
86.93
39.75
6.35
12.93
2.46
15.75
5.34
14.98
4.79
0.49
1.89
3.28
3.49
37.19
32.12
86.93
39.75
6.35
12.93
2.47
15.75
5.34
14.98
4.80
0.49
1.89
3.28
3.49
12.39
32.13
28.98
39.76
2.12
1.44
0.82
5.25
5.34
4.99
0.53
0.16
0.21
1.09
0.38
874.41
2265.7
2043.6
2803.6
149.40
101.32
58.00
370.33
376.91
352.25
37.59
11.61
14.79
77.04
27.39
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
Food Bioprocess Technol (2009) 2:213–221
reduce the cost of the process. The effect of come-up time
during HPP on inactivation of E. coli K12 and recovery of
injured cells during treatments should be evaluated in the
future.
It was observed that the inactivation of E. coli K12
bacterium under pressure decreased with reduction of aw
levels from 0.99 to 0.9 in the tested solid and liquid glycerol
model systems. The inactivation level was also affected by
the type of solute used to control that aw level. Among four
types of solutes tested in the study, sodium chloride appeared
the least protective, with glycerol and fructose being
approximately equal, and sorbitol showed the most protective effects on inactivation of E. coli K12. Baroprotective
effect of sorbitol was more pronounced as the aw decreased
from 0.99 to 0.95 than effects of glycerol, fructose, and salt.
The observed tendency of E. coli K12 inactivation by HPP at
varied aw levels in tested solutes were similar to those effects
of aw and tested humectants on microbial inactivation by
thermal treatments. The variations in measured time–
temperature profiles of the glycerol liquid models with aw
levels of 0.95 and 0.99 showed a difference in their adiabatic
heating behavior under pressure. The differences in physical
and chemical properties of the solutes and their concentrations under pressure and their behavior under compression
require further investigation. In addition, the phenomenon of
aw in foods under compression may play a critical role in
establishment of a high-pressure process. More research is
required to determine the response of other microorganisms
to various aw levels achieved by the addition of other solutes
to control aw.
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