22nd International Symposium on Plasma Chemistry
July 5-10, 2015; Antwerp, Belgium
Plasma inactivation of microorganisms on granular food products in a Dielectric
Barrier Discharge
D. Butscher1, D. Zimmermann 1,2, M. Schuppler2 and Ph. Rudolf von Rohr1
1
2
ETH Zurich, Institute of Process Engineering, Sonneggstr. 3, 8092 Zurich, Switzerland
ETH Zurich, Institute of Food Science and Nutrition, Schmelzbergstr. 7, 8092 Zurich, Switzerland
Abstract: Motivated by the frequent microbial contamination of granular food products
like sprouts and cereal grains, we constructed an atmospheric pressure dielectric barrier
discharge, which we successfully applied for the plasma inactivation of microorganisms on
polymeric model substrates, wheat grains and various plant seeds. Inactivation was shown
to be plasma based and not a result from mechanical, electrical or thermal stress.
Keywords:
seeds
plasma, decontamination, dielectric barrier discharge, wheat grains, plant
1. Introduction
Food safety and decontamination are crucial issues in
food industry. The EHEC outbreak in central Europe
2011, caused by contaminated sprouts, is only one of
many tragic examples [1]. Also, cereal grains are naturally
contaminated by a variety of bacteria and molds, which
can negatively influence product properties or even form
toxins [2]. Furthermore, contamination with pathogenic
microorganisms such as enteropathogenic E. coli or
Salmonella might occur from animal feces or during
processing [3]. For comestibles however, the application of
conventional thermal or chemical sterilization methods is
limited since many products are sensitive to heat,
moisture and a variety of chemicals. A promising
alternative to these methods is plasma sterilization, where
the synergetic combination of charged particles, reactive
neutrals and UV photons can effectively inactivate
microorganisms [4]. In a previous study we investigated
the reduction of bacterial endospores on wheat grains in a
low pressure plasma circulating fluidized bed reactor.
Here, we report the plasma inactivation of Geobacillus
stearothermophilus endospores on wheat grains as well as
the reduction of E. coli on various plant seeds (onion,
radish and alfalfa) in an atmospheric pressure dielectric
barrier discharge (DBD).
2. Experimental setup
For the plasma inactivation of microorganisms on
granular materials an atmospheric pressure DBD was
constructed. It consists of two planar aluminum plates
with dimensions of 100 x 200 mm, both embedded in a
polyoxymethylene housing and covered by dielectric
barriers made of polymethylmethacrylat with a thickness
of 2 mm. Polymethylmethacrylat spacers form a 5 mm
gas gap. Argon was used as a working gas, which
streamed the discharge in longitudinal direction. A
polymeric foam (PUR-Ester 20 PPI open-pored,
Vibroplast AG, Switzerland) was inserted at the gas in-
P-III-9-4
and outlet to ensure homogeneous gas distribution and to
keep particles inside the discharge (Fig. 1).
The entire discharge was installed on a vibrating table
to continuously rotate particles and guarantee
homogeneous treatment. To power the discharge, fast
high-voltage unipolar nanosecond square pulses were
generated by means of a transistor switch described in
detail elsewhere [5].
Fig. 1. Schematic of experimental setup.
3. Microbiological procedure
Wheat grain samples of 10 g were inoculated with
Geobacillus stearothermophilus endospores (ATCC 7953,
Merck, Germany) to a level of 10E7 colony forming units
(CFU) per gram of wheat grains. Therefore, wheat grains
were placed in a centrifugal tube, drizzled with 1 ml of
spore solution (10E8 CFU/ml) and homogenously mixed.
After a storage time of 60 to 90 h in closed Falcon tubes,
wheat grains were plasma treated in the DBD.
For the determination of the remaining spore
concentration after experiments wheat grain samples were
immersed into 90 ml of buffered NaCl-Peptone (BPW)
solution and allowed to soak for 20 min at room
temperature. The mixture was then homogenized in a
stomacher twice for 20 s and a heat treatment (85 °C, 15
1
4. Plasma decontamination of model substrates
To demonstrate the general feasibility of our discharge
to inactivate bacterial spores, we investigated the
reduction of Geobacillus stearothermophilus endospores
on flat and spherical PP substrates. The logarithmic spore
reduction over treatment time is shown in Fig. 2. All
experiments were conducted with 8 kV, 10 kHz, 500 ns
pulses and 2.8 norm liter per minute (nlm) of argon.
2
Experiments with PP substrates evidently demonstrate
that bacterial spores can be efficiently inactivated in our
discharge, and it can be assumed that vegetative bacteria
are even more susceptible to plasma treatment than
bacterial spores [6].
wheat grains
PP plates
PP spheres
Logarithmic spore reduction [log(CFU)]
0
-1
-2
-3
-4
-5
01
5
10
15
60
Treatment time [min]
Fig. 2. Logarithmic spore reduction as a function of
treatment time for various substrates.
Geobacillus stearothermophilus endospores on PP
spheres could be reduced by over 2.7 logarithmic units
(99.8%) within 1 minute and up to 4.8 log (99.998%)
after 10 minutes of plasma treatment. While reduction
was fast in the beginning the process slows down after the
fast initial phase. This might me the result of stacking,
where inactivated spores shield viable organisms
underneath.
The treatment efficiency on PP plates is slightly lower
than on PP spheres. This difference is supposed to result
from an enhanced electric field strength around spherical
particles as compared to plates in the discharge and, as a
consequence, a higher plasma intensity and elevated
concentration of reactive species.
To be able to clearly attribute the inactivation of spores
to the effect of plasma, we also investigated the influence
of the mechanical stress from the vibrating table,
electrical stress from the electric field and thermal stress
from plasma gas temperature separately (Fig. 3).
10
Spore concentration [CFU/g]
min) was applied for spore isolation and activation.
Finally, a decimal dilution series was conducted in
duplicate, plated on brain heart infusion agar and
incubated for 48 h at 55 °C.
To demonstrate the general feasibility of our discharge
to inactivate bacterial spores and to investigate the
influence of the substrate shape, we also inoculated flat
and spherical polypropylene (PP) substrates as an
alternative to wheat grains in a first step. PP plates with
dimensions of 76 x 26 x 1 mm were pretreated in a mild
plasma discharge (8 kV, 500 ns, 5 kHz pulses, 2.8 L/min
argon, 5 min) to increase their wettability, and 100 μL of
spore solution was distributed over their surface with an
inoculation loop. After a storage time of 60 h and
subsequent plasma treatment, the PP plates were
immersed into 30 mL BPW solution in Falcon tubes,
vortexed and agitated on a vertically rotating disk for 20
min. The subsequent procedure for CFU quantification
was as described before. In addition, we applied PP
granulate (PP Regranulat natur, Minger AG, Switzerland)
as an intermediate step between flat PP plates and wheat
grains. They have a smooth surface like the one of PP
plates but are of spherical-like shape similar to wheat
grains. After mild plasma treatment for wettability
increase (as described above) 10 g of PP granulate were
inoculated with 100 μL of spore suspension, plasma
treated and evaluated following the procedure of PP plates.
In a further study, the inactivation efficiency of our
discharge was also tested for various plant seeds, where
onion, radish and alfalfa sprout seeds were artificially
inoculated. Therefore, E. coli (1576, DSM, Netherlands)
was enriched in an overnight culture and diluted with
lysogeny broth (LB) by a factor of ten to reach a
concentration of 10E7 CFU/ml. Plant seeds were then
drizzled with 1 ml of this bacterial solution,
homogenously mixed and stored in open petri dishes for
approximately three hours. After plasma treatment,
samples were immersed into 90 ml of phosphate-buffered
saline (PBS) and homogenized in the stomacher for 3
minutes. A decimal dilution series was conducted, plated
on LB agar and incubated for 24 h at 37 °C.
Each inactivation experiment was conducted in
triplicate. For statistical analysis, mean as well as standard
error of the mean was calculated from the three
experimental results. Finally, the reduction of CFU is
expressed by the decimal logarithm of the ratio of final
CFU concentration and initial value from inoculated but
untreated transport controls.
10
10
10
6
4
2
0
Control
Vib + E-field
80°C
100°C
Plasma
Fig. 3. Influence of mechanical, electrical and thermal
stress on spore viability on PP spheres.
P-III-9-4
5. Plasma decontamination of wheat grains
Focusing on the spore inactivation on wheat grains,
experiments were conducted with 8 kV, 10 kHz, 500 ns
pulses and 2.8 nlm of argon. Approximately 85% (0.8 log)
reduction was reached after 5 minutes and more than 3
log after 60 minutes of plasma treatment (Fig. 2). This
demonstrates that spore reduction on wheat grains is
considerably more difficult than on flat and spherical PP
substrates. Presumably, this is a consequence of spores
being sheltered by the uneven surface, loose pieces of
bran or hidden deep inside the wheat grain crease.
Even though the plasma inactivation of spores on wheat
grains has shown to be challenging, there is still room for
improvement. The inactivation efficiency can be
improved by increasing the power density in the discharge,
which is mainly governed by pulse frequency and voltage.
While the pulse frequency acts in analogy to treatment
time and increases the total number of discharge pulses,
elevating the pulse voltage mainly augments the number
density of micro-discharges per pulse [7]. These beneficial
influences could also be proven experimentally. In
contrast, the influence of mechanical, electrical and
thermal stress on the spore reduction could also be
excluded for wheat grains, so that the inactivation can be
clearly attributed to the action of plasma species.
6. Plasma decontamination of sprout seeds
The plasma inactivation efficiency of our DBD was
also tested for other granular food products (Fig. 4).
Logarithmic bacteria reduction [log(CFU)]
In order to impose an electric field of the same strength
as during plasma treatment to contaminated PP spheres,
we streamed air through the discharge gap to avoid the
ignition of plasma due to its higher breakdown voltage as
compared to argon. No spore reduction could be
identified in an experiment with 10 kHz and 8 kV for 10
min. Furthermore, we used a fiber optic temperature
sensor to approximate the gas temperature in the
discharge and measured a maximum temperature of 78 °C
for the standard condition in our study (10 kHz, 8 kV, 10
min). Applying a temperature of 80 °C to contaminated
PP spheres in an oven for 10 min also did not result in any
spore inactivation, neither did a temperature of 100 °C.
Thus, we can clearly attribute the spore reduction in our
experiments to the action of plasma species.
onion
radish
alfalfa
0
-1
-2
-3
0
5
Treatment time [min]
10
Fig. 4. Logarithmic bacteria reduction as a function of
treatment time for various plant seeds.
Various plant seeds (onion, radish and alfalfa) were
artificially inoculated with E. coli and treated in the
discharge with 8 kV, 10 kHz 500 ns pulses and 5.6 nlm of
argon for 5 and 10 minutes, respectively. Results, given in
Fig. 4, show that it is possible to reduce E. coli by up to 3
log units (99.9%), but it also becomes evident that
inactivation efficiency strongly depend on the substrate.
Supposedly, surface properties (geometry, roughness,
porosity) but probably also the water content of the seed
affect the treatment efficiency.
7. Conclusion
Flat and spherical polypropylene substrates were
artificially
contaminated
with
Geobacillus
stearothermophilus endospores and treated in a pulsed
DBD operated with argon. These experiments
demonstrate the general feasibility of our discharge to
inactivate bacterial spores, and they show that spherical
substrates enhance the electric filed strength in the
discharge leading to a better inactivation. Furthermore,
the spore reduction could be clearly attributed to the
effect of plasma species while mechanical, electrical and
thermal effects could be excluded.
Wheat grains were also contaminated with Geobacillus
stearothermophilus endospores and treated in the DBD. A
comparison with flat substrates revealed the challenge to
sanitize the complex shape of wheat grains with its
uneven surface and its deep crease. The elevation of pulse
voltage and frequency could however improve the
inactivation efficiency.
Besides wheat grains, other plant seeds (onion, radish
and alfalfa) were inoculated with E. coli and plasma
treated in the DBD. All samples allowed to reduce the
bacterial load by orders of magnitude , but differences in
inactivation efficiency occured, which might result from
differences in surface properties or water content.
8. Acknowledgement
Financial support from CTI is gratefully acknowledged.
P-III-9-4
3
9. References
[1] H. Karch, E. Denamur, U. Dobrindt, B.B. Finlay, R.
Hengge, L. Johannes, E.Z. Ron, T. Tønjum, P.J.
Sansonetti, M. Vicente, EMBOmolecularMedicine
2012, 4, 841.
[2] A. Laca, Z. Mousia, M. Díaz, C. Webb, S.S.
Pandiella, Journal Food Engineering 2006, 72, 332.
[3] W. Sperber, TechnicalBulletin IAOM 2003, 3, 7929.
[4] A. Fridman, in (Ed: A. Fridman), Cambridge
University Press2008, 848.
[5] P. Peschke, S. Goekce, C. Hollenstein, P. Leyland, P.
Ott, AIAAPaper 2011, 3734, 27.
[6] K. Kelly-Wintenberg, A. Hodge, T.C. Montie, L.
Deleanu, D. Sherman, J. Reece Roth, P. Tsai, L.
Wadsworth, Journal Vacuum Science & Technology
A 1999, 17, 1539.
[7] U. Kogelschatz, PlasmaScience,IEEETransactions
2002, 30, 1400.
4
P-III-9-4