1. No category

LWT - Food Science and Technology xxx (2013) 1e5
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LWT - Food Science and Technology
journal homepage: www.elsevier.com/locate/lwt
Inactivation kinetics of Bacillus coagulans spores under ohmic and conventional
heating
Romel Somavat a, Hussein M.H. Mohamed b, Sudhir K. Sastry c, *
a
Abbott Nutrition, 3300 Stelzer Road, Columbus, OH 43219, USA
Department of Food Hygiene and Control, Faculty of Veterinary Medicine, Cairo University, Egypt
c
Department of Food, Agricultural and Biological Engineering, The Ohio State University, 206 Agricultural Engineering Building, 590 Woody Hayes Drive, Columbus, OH 43210, USA
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 2 May 2012
Received in revised form
21 February 2013
Accepted 5 April 2013
Bacillus coagulans spores are commonly involved in the spoilage of food of pH between 4 and 4.5. Recent
studies on ohmic heating have indicated the presence of an additional nonthermal effect of electricity on
the bacterial spores of Geobacillus stearothermophilus and Bacillus subtilis. We investigated the kinetics of
inactivation of B. coagulans spores (ATCC 8038) in fresh tomato juice under ohmic heating at frequencies
of 10 kHz and 60 Hz, and compared it with conventional heating using a specially designed experimental
setup that assured identical temperature histories for all treatments. Ohmic heating at 60 Hz showed
significantly lower D-values at 95, 100 and 105 C compared to conventional heating. While 10 kHz also
showed a similar trend of higher inactivation compared to conventional heating, the difference was
significant only at 105 C. Both ohmic treatments also showed higher inactivation than conventional
heating during the come-up time. In conclusion, ohmic heating resulted in accelerated inactivation of
B. coagulans spores compared to conventional treatment.
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Ohmic heating
Bacillus coagulans
Inactivation kinetics
1. Introduction
Bacillus coagulans is a spoilage microorganism commonly
associated with food acidified to between pH 4.0 and 4.5 (Palop,
Raso, Pagan, Condon, & Sala, 1999). This microorganism is specifically responsible for flat sour spoilage outbreaks in tomato
based products (Sandoval, Barreiro, & Mendoza, 1992; York et al.,
1975). B. coagulans is a non-pathogenic organism, but it can pose
a safety hazard because of its ability to increase the pH of a high
acid food, processed with a reduced treatment, to a level where
surviving Clostridium botulinum spores can germinate (Anderson,
1984; Fields, Zamora, & Bradsher, 1977). Hence, it is not only
relevant to the tomato industry because of its higher thermal
resistance than the other sporeformers involved with tomato
products (Mallidis, Frantzeskakis, Balatsouras, & Katsaboxakis,
1990), but also because in the past it has been linked to a few
cases of botulism through tomato juices (Fields et al., 1977). An
outbreak of C. botulinum in inherently high acid food can only
occur with an associated rise of the pH, which, in the case of a
* Corresponding author. Tel.: þ1 614 292 3508; fax: þ1 614 292 9448.
E-mail address: [email protected] (S.K. Sastry).
tomato based product, can be linked to the growth of
B. coagulans.
Ohmic heating is an alternate processing method shown to yield
higher quality foods than conventional heating (Kim et al., 1995). It
is classified as a purely thermal process, mainly because of an
inadequate understanding of the nonthermal effect of electricity on
microorganisms. Several past studies have shown an additional
effect of electricity during the ohmic heating of plant tissues (Jemai
& Vorobiev, 2002; Kulshrestha & Sastry, 2003), vegetative microorganisms (Guillou, Besnard, El Murr, & Federighi, 2003; Guillou &
El Murr, 2002; Loghavi, Sastry, & Yousef, 2007, 2009; Palaniappan,
Sastry, & Richter, 1992) and bacterial spores (Cho, Yousef, &
Sastry, 1999). Although studies indicating the additional effects of
alternating current on microorganisms date back to the time of
Tracy (1932), they failed to adequately control sources of errors.
Somavat, Mohamed, Chung, Yousef, and Sastry (2012) have presented an assessment of error in ohmic heating devices used for
microbial studies, and concluded that the relatively large size of
ohmic treatment devices and non-identical thermal histories between conventional and ohmic treatments might have resulted in
experimental errors. The basic design of ohmic devices used for
microbial study has remained almost the same since the time of
Tracy (1932), until as recently as Cho et al. (1999) and Guillou et al.
(2003). These devices were considerably larger and more complex
0023-6438/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.lwt.2013.04.004
Please cite this article in press as: Somavat, R., et al., Inactivation kinetics of Bacillus coagulans spores under ohmic and conventional heating,
LWT - Food Science and Technology (2013), http://dx.doi.org/10.1016/j.lwt.2013.04.004
R. Somavat et al. / LWT - Food Science and Technology xxx (2013) 1e5
Ohmic Treatment Chamber
Capillary cells
Electrode
than the simple capillary sized sample holders used by microbiologists for conventional heating studies.
Somavat et al. (2012) described a capillary sized treatment cell
which was able to deliver identical temperature histories for ohmic
and conventional treatments while using similar sized sample
holders. Using these cells they determined the inactivation kinetics
of Geobacillus stearothermophilus spores and found additional
nonthermal effects of electricity at the frequencies of 10 kHz and
60 Hz. The frequency of 10 kHz in pulsed mode with a square
waveform is known to reduce electrochemical reactions at the
foodeelectrode interface (Samaranayake, Sastry, & Zhang, 2005;
Jun, Sastry, & Samaranayake, 2007), whereas the frequency of 60 Hz
is associated with increased inactivation of vegetative microorganisms (Cho et al., 1999; Guillou & El Murr, 2002) and enhanced
rupture of plant tissues (Kulshrestha & Sastry, 2003; Schreier, Reid,
& Fryer, 1993). A study on inactivation kinetics of B. coagulans
spores is required to further our understanding of the ohmic
heating process, as well as to evaluate its potential for use by the
related industry. Hence, the main aim of this study was to investigate the effect of ohmic heating at the frequencies of 10 kHz and
60 Hz on the inactivation kinetics of B. coagulans spores (ATCC
8038) in comparison to conventional treatment.
Electrode
2
Threads
Isoconductive
solution
Cooling
Section
2. Materials and methods
Tomato juice samples inoculated with B. coagulans spores were
heated in ohmic and conventional capillary cells in a controlled
setting which ensured identical temperature histories regardless of
method of heating (Somavat et al., 2012). The samples were treated
ohmically at frequencies of 10 kHz and 60 Hz in pulse mode, or
conventionally to the temperatures of 95, 100, 105 and 110 C. The
samples were held at four different holding times at each temperature e 0, 10, 20 and 30 min for 95 C; 0, 2, 4 and 6 min for
100 C; 0, 1, 2 and 3 min for 105 C; and 0, 10, 20 and 30 s for 110 C.
Separate runs were conducted for ohmic and conventional treatments. Two sample-containing capillary cells mounted on each
capillary tube holder were used for each holding time. Three replicates were done at each condition. Data were analyzed by
regression and Analysis of Variance (ANOVA) to determine statistical significance. Details of experimental protocol are in the
following sections.
2.1. System design
2.1.1. Ohmic and conventional capillary cells
The ohmic and conventional capillary cells and the supporting
system described by Somavat et al. (2012) were used. Capillary tubes
holding 37 ml of tomato juice inoculated with B. coagulans spores
were plugged at both ends with tomato alginate (conductive) for
ohmic heating, or with nonconductive capillary tube sealant for
conventional heating. The capillary tubes were aligned parallel to the
electric field inside an external ohmic heating chamber containing
an iso-conductive salt solution and designed to provide rapid cooling
and pressurized conditions. To hold capillary cells in place, they were
mounted on cell holders (two cells per holder) snapped on the top
part of the treatment chamber (simplified schematic in Fig. 1). The
system accommodated 5 such cell holders, thus containing a total of
8 sample containing cells in addition to 2 thermocouple cells.
Thermocouple capillary cells were prepared by inserting a T-type
thermocouple inside a basic cell, which was then prepared similarly
to either ohmic or conventional cells. The achievement of a pure
ohmic heating effect inside the ohmic capillary cells was confirmed
through a temperature distribution study which showed that the
ohmic cells always remained at a slightly higher temperature than
the surroundings, thereby confirming the internal ohmic generation
Fig. 1. Typical arrangement of the capillary cells inside the treatment chamber.
of heat inside the cells. Because of this temperature gradient, separate runs of ohmic and conventional treatments were conducted to
ensure equal temperature histories in both cases. The system also
facilitated rapid post-treatment cooling through pulling of the
treated samples in the cooling section at w20 C with the help of an
attached thread. A more detailed description of the setup and procedures is provided by Somavat et al. (2012).
2.1.2. Power supply and control
A square pulsed waveform with a duty ratio of 0.5 was generated using a circuit containing an integrated gate bipolar transistor
(IGBT) serving as a high frequency switching device. A function
generator (Instek, Chino, CA) was used to deliver 10 kHz or 60 Hz
frequency input through the IGBT circuit. The waveform and duty
ratio had been previously shown effective in reducing electrochemical reactions at the electrodes (Samaranayake et al., 2005). A
data logger (Agilent Technologies, Inc., Santa Clara, CA) was used to
record the time, temperature, frequency, voltage and current data.
A waveform plot of the square pulsed waveform at 10 kHz frequency and 0.5 duty ratio is shown in Fig. 2.
Fig. 2. A representation of the square waveform; 10 kHz frequency and 0.5 duty ratio.
Please cite this article in press as: Somavat, R., et al., Inactivation kinetics of Bacillus coagulans spores under ohmic and conventional heating,
LWT - Food Science and Technology (2013), http://dx.doi.org/10.1016/j.lwt.2013.04.004
R. Somavat et al. / LWT - Food Science and Technology xxx (2013) 1e5
3
Fig. 4. Inactivation of B. coagulans spores under ohmic 10 kHz, ohmic 60 Hz and
conventional treatments at 95 C with holding times of 0, 10, 20 and 30 min. Key:
conventional
10 kHz;
60 Hz.
Fig. 3. Typical voltage and matching temperature histories of the ohmic and conventional treatments during the come-up and holding times for the 110 C treatment.
Key:
voltage;
ohmic - - - conventional.
An input voltage of 65 V was used across the electrodes of the
external ohmic chamber (13 V/cm across the capillary tubes) during
the come-up time. Voltage was subsequently reduced and manually adjusted during the holding period to maintain the specified
temperature. A graph for typical voltage and temperature histories
during the come up and holding time for the 110 C process is
presented in Fig. 3. On average, come-up times of around 158, 170,
180 and 192 s were obtained during heating from room temperature (21 C) to 95, 100, 105 and 110 C, respectively.
2.2. Microbiological experiments
2.2.1. Preparation of the B. coagulans spore suspension
The strain of B. coagulans, ATCC 8038, was obtained from the
American Type Culture Collection (Manassas, VA, USA). The strain
was grown in tryptic soy broth (TSB; Difco; Becton, Dickinson and
Co., Sparks, MD, USA) at 37 C for 48 h under aerobic conditions and
transferred at least three times before spore preparation. Spores of
the microorganism were obtained by plating 500 ml of actively
growing culture (24 h at 37 C) into nutrient agar (NA; Difco;
Becton, Dickinson and Co., Sparks, MD, USA) supplemented with
500 mg/L dextrose (BD, Difco) and 3 mg/L manganese sulfate
(Fisher Scientific, Pittsburgh, PA, USA) (Palop et al., 1999). The
inoculated plates were incubated at 50 C for 7 days, where more
than 90% of sporulation was obtained as verified by observing the
refractile spores under phase-contrast microscopy. Spores were
harvested by flooding plates with 5 ml of cold sterile deionized
water, and releasing the colony containing spores from the surface
of the agar with the use of a sterile disposable inoculation loop.
Collected spores were washed six times by centrifugation (8000 g
for 20 min) at 4 C. After the last centrifugation, the spore pellets
were resuspended in sterile deionized water. The spore suspensions were heated at 80 C for 15 min and checked microscopically
to ensure the absence of vegetative cells. The spore suspension was
stored at 4 C until used. A volume of 37 ml of tomato juice inoculated with 107 cfu/ml spores was filled in each capillary cell for
treatment.
2.2.2. Enumeration procedure
Treated capillary cells were washed with cold 1400 ppm hypochlorite solution and rinsed with cold sterile water. The capillary
washing protocol developed by Somavat et al. (2012) was followed
to eliminate any residual hypochlorite from affecting the final plate
count. The clean capillary cells were then crushed inside sterile
polypropylene tubes containing 0.1% peptone water using sterile
glass rods. A heat shock at 80 C for 15 min was given to inactivate
all vegetative cells. Further dilutions in peptone water were prepared and plated on TSA agar plates. Inoculated plates were incubated for 48 h at 37 C and colonies enumerated.
2.3. Tomato juice preparation
Fresh Roma tomatoes of bright red color (an ‘a’ value of 20)
bought from a local grocery store (Kroger Inc.) were used. Tomatoes
were cut in four quarters and then were blended to prepare the
tomato juice media. pH of the juice, inherently ranging from 4.1 to
4.3, was adjusted to a standard value of 4.4 using sodium citrate to
eliminate the varying acidity from affecting the thermal resistance
of the organism.
3. Results and discussion
Survivor plots at 95 C, 100 C, 105 C and 110 C are shown in
Figs. 4e7, respectively. D-values of 7.96, 1.63 and 0.91 min at the
temperatures 95, 100 and 105 C for ohmic heating at 60 Hz were
significantly lower than D-values of 10.1, 2.52 and 1.32 min for
conventional treatment (Table 1). Ohmic heating at 10 kHz frequency with a square pulsed waveform resulted in significantly
lower D-value than conventional heating only at 105 C with a Dvalue of 1.03 min; although it showed a general trend of extra
inactivation at other temperatures also. At 100 C, ohmic heating at
60 Hz showed significantly greater inactivation than the ohmic
sample at 10 kHz (D100 of 2.27 min). No significant difference between ohmic and conventional treatments was observed at the
highest temperature of 110 C. Z-values for 10 kHz, 60 Hz and
conventional treatments were 9.89, 11.18 and 8.68 C, respectively.
Fig. 5. Inactivation of B. coagulans spores under ohmic 10 kHz, ohmic 60 Hz and
conventional treatments at 100 C with holding times of 0, 2, 4 and 6 min. Key:
conventional
10 kHz;
60 Hz.
Please cite this article in press as: Somavat, R., et al., Inactivation kinetics of Bacillus coagulans spores under ohmic and conventional heating,
LWT - Food Science and Technology (2013), http://dx.doi.org/10.1016/j.lwt.2013.04.004
4
R. Somavat et al. / LWT - Food Science and Technology xxx (2013) 1e5
Table 1
D- and Z-values at 95, 100, 105 and 110 C for 10 kHz, 60 Hz and conventional
inactivation of B. coagulans spores.
Treatment
10 kHz
60 Hz
Conventional
Z-value, C
D-values, min
95 C
100 C
105 C
110 C
8.81a,b
7.96b
10.1a
2.27a
1.63b
2.52a
1.03b
0.91b
1.32a
0.13a
0.15a
0.16a
9.89a
11.2a
8.68a
Note: Different superscripts within the same column are significantly different
(p < 0.05).
Fig. 6. Inactivation of B. coagulans spores under ohmic 10 kHz, ohmic 60 Hz and
conventional treatments at 105 C with holding times of 0, 1, 2 and 3 min. Key:
conventional
10 kHz;
60 Hz.
Ohmic treatment at 60 Hz showed an additional killing effect,
significantly over a greater range of temperature than conventional
heating. While 10 kHz also showed a general trend of higher
inactivation than conventional heating, its effect was only significant at 105 C. The absence of any significant difference between
both ohmic treatments and conventional samples at 110 C indicates that at higher temperatures, pronounced thermal effects
overshadow the nonthermal changes of electricity. These results
clearly demonstrate accelerated inactivation of B. coagulans spores
under ohmic treatments as compared to conventional heating.
The overall trend of higher inactivation of bacterial spores
under the effect of ohmic heating is in agreement with the results
observed by Somavat et al. (2012) on G. stearothermophilus spores.
Thus, our study further supports the hypothesis presented by
them to explain the non-thermal effects of electricity on bacterial
spores. They hypothesized that during heat activation of dormant
spores, a synergistic effect of electric current and temperature
cause an increase in the release of ionic compounds like calcium
dipicolinic acid (DPA) molecules from the core, and fragments of
denatured spore protein enzymes from the coat. Interaction of
these released ionic molecules with the electric field would
further increase the rate of activation through a simultaneous
increase in the electrical conductivity of the spore, making it
more prone to additional nonthermal effects of electricity. We
note that electroporation, a mechanism often used to explain
inactivation of vegetative bacterial cells is likely not relevant here,
since the spore structure differs so greatly from that of vegetative
cells.
Another interesting point of note is the consistently (with only
the 110 C exception) greater efficiency of 60 Hz treatments over
the 10 kHz treatments. This is opposite to the findings of Somavat
et al. (2012) who observed that 10 kHz resulted in comparatively
Fig. 8. Spore reductions during come up times of 158, 170, 180 and 192 s to the
temperatures of 95, 100, 105 and 110 C, respectively, for conventional, 60 Hz and
10 kHz treatments. Key:
conventional
10 kHz;
60 Hz.
more inactivation than 60 Hz at lower temperatures for the thermophilic spores of G. stearothermophilus. However, this ambiguous
result is consistent with the hypothesis that different oscillating
electric fields cause different parts of a bacterial spore to react,
thereby resulting in effects which are not yet understood. In general, the response of spores to electric fields is poorly understood,
and it may be speculated that the polar components of the spore,
which form a significant proportion of the spore mass (DPA, small
acid-soluble proteins) may react to an applied electric field and
oscillate, creating a disruption of the spore structure. Differences
between spore structure may explain differences in observations;
but we must reemphasize that this is purely speculative and not
supported by experimental evidence at this time.
Both ohmic heating treatments also showed additional inactivation during the come-up time (CUT) from the room conditions to
the target temperature (Fig. 8). This is significant because the
electric field strength during the CUT (when the sample undergoes
heating) is higher during the holding periods, when the field is
periodically reduced to maintain a constant temperature (Fig. 3).
Thus, CUT inactivation would reflect the effect of a full-strength
electric field.
4. Conclusion
Fig. 7. Inactivation of B. coagulans spores under ohmic 10 kHz, ohmic 60 Hz and
conventional treatments at 110 C with holding times of 0, 10, 20 and 30 s. Key:
conventional
10 kHz;
60 Hz.
Ohmic heating at 60 Hz and 10 kHz result in accelerated inactivation of B. coagulans spores compared to conventional heating.
These results further confirm the presence of the additional
nonthermal effect of ohmic heating on bacterial spores as observed
by Cho et al. (1999) and Somavat et al. (2012). Ohmic heating also
resulted in considerably increased inactivation during the comeup-time than conventional heating, showing that full-strength
fields that occur during these periods have significant effects.
Please cite this article in press as: Somavat, R., et al., Inactivation kinetics of Bacillus coagulans spores under ohmic and conventional heating,
LWT - Food Science and Technology (2013), http://dx.doi.org/10.1016/j.lwt.2013.04.004
R. Somavat et al. / LWT - Food Science and Technology xxx (2013) 1e5
Acknowledgments
The authors gratefully acknowledge support from USDA-CSREES
Project No: 2009-55503-05198 titled Quality of Foods Processed
Using Selected Alternative Processing Technologies. Salaries and
research support also provided by the Ohio Agricultural Research
and Development Center (OARDC), The Ohio State University. References to commercial products and trade names are made with
the understanding that no endorsement or discrimination from the
Ohio State University is implied.
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LWT - Food Science and Technology (2013), http://dx.doi.org/10.1016/j.lwt.2013.04.004