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Applied and Environmental Microbiology, March 1999, p. 1312-1315, Vol. 65, No. 3
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Pulsed-Light Inactivation of Food-Related
Microorganisms
N. J.
Rowan,1,*
S. J.
MacGregor,2
J. G.
Anderson,1
R. A.
Fouracre,2
L.
McIlvaney,2 and
O.
Farish2
Department of Bioscience & Biotechnology,1 and the Department of
Electronic & Electrical Engineering,2
University of Strathclyde, Glasgow, Scotland
Received 17 August 1998/Accepted 25 November 1998
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ABSTRACT |
The effects of high-intensity pulsed-light emissions of high or low
UV content on the survival of predetermined populations of
Listeria monocytogenes, Escherichia coli,
Salmonella enteritidis, Pseudomonas aeruginosa,
Bacillus cereus, and Staphylococcus aureus were
investigated. Bacterial cultures were seeded separately on the surface
of tryptone soya-yeast extract agar and were reduced by up to 2 or 6 log10 orders with 200 light pulses (pulse duration, ~100
ns) of low or high UV content, respectively (P < 0.001).
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TEXT |
Despite significant advances made
towards a better understanding of bacterial transmission and
pathogenicity in foods, the incidence of reported food-borne illnesses
associated with bacterial enteropathogens continues to be a major
problem in the United Kingdom (5) and North America
(7). It is generally accepted that contamination of both
unprocessed and uncooked foods with pathogenic bacteria is a major
source of concern, and any method of either reducing or eliminating
food contamination will have a significant effect on the incidence of
food-borne disease (5). A possible approach to reducing the
level of microbial contamination on food surfaces in slaughterhouses,
and in other food preparation environments, is UV irradiation
(1-3, 5, 8, 9). Stermer et al. (8) indicated
that the bacterial load on fresh meat can be effectively reduced by UV
irradiation, while Wallner-Pendleton and coworkers (9)
showed that this method of disinfection reduced Salmonella
surface contamination without adversely affecting poultry carcass color
or increasing meat rancidity. These studies indicate that if an
effective and economic method of UV generation can be developed, then
UV irradiation may have a practical application in the disinfection of
food and contact surfaces.
While conventional alternating-current systems produce light with a
power dissipation in the range of 100 to 1,000 W per device, a pulse
power energization technique (PPET) can dissipate many megawatts of
electrical power in the light source (4). PPET also tends to
produce a greater intensity of the shorter bactericidal wavelengths of
light, and, by using this approach, it is possible to design an
extremely short energization time of the light source (~100 ns). For
modest energy input (e.g., 3 J), this results in high peak power
dissipation (~35 MW). Here we present evidence that PPET may lend
itself to surface disinfection since it significantly reduces large
populations of various food-related microorganisms on laboratory-based media.
The effectiveness of PPET, with two different light sources, in
reducing predetermined microbial numbers on agar surfaces was
determined by using a variety of proven bacterial pathogens, namely,
Escherichia coli NCTC 12079 (serotype O157:H7),
Listeria monocytogenes NCTC 11994 (serotype 4b),
diarrheagenic Bacillus cereus NCTC 11145, Salmonella
enteritidis NCTC 4444, Staphylococcus aureus NCTC 4135, Pseudomonas aeruginosa NCTC 8203, and the yeast Saccharomyces cerevisiae NCTC 10716, obtained from the
National Collection of Type Cultures, Colindale, London, United
Kingdom. Bacterial cultures were grown at 30°C and maintained on
tryptone soya agar supplemented with 0.6% yeast extract (TSYEA). The
yeast culture was grown at 25°C and maintained on malt extract agar supplemented with 0.3% yeast extract, 1% glucose, and 0.3%
mycological peptone (MYGPA). Analysis of variance, balanced model
(Minitab software release 11; Minitab Inc., State College, Pa.), was
used to compare the effects of pulsed-light irradiation, number of pulses applied, and the type of microorganism treated. The studies were
performed in quadruplicate, and all significant differences are
reported at 95% (P < 0.05) and 99.9% (P < 0.001) confidence intervals.
Pulsed-light inactivation of food-related microorganisms.
The
bacterial test strains were inoculated into 100 ml of tryptone soya
broth supplemented with 0.6% yeast extract (TSYEB) and cultivated on a
shaker at 125 oscillations per minute to a population density of
~109 cells ml
1 (confirmed via plate
counts). A 0.1-ml aliquot of a 105 dilution of this
culture (the diluent used was 0.01 M sodium phosphate [pH 7.2]-0.15
M NaCl) was transferred to 100 ml of fresh TSYEB. The bacterial test
strains were again grown to a density of ~109 cells
ml1. S. cerevisiae was grown to a cell density
of ~109 cells ml1 (confirmed via plate
counts) in 100 ml of malt extract broth supplemented with 0.3% yeast
extract, 1% glucose, and 0.3% mycological peptone (MYGPB) at 25°C.
Ten samples, each containing 20 µl of a bacterial or yeast test
culture, were surface inoculated on separate TSYEA or MYGPA plates,
respectively, by using the Miles and Misra method (6).
The test assembly consisted of a rectangular polyvinyl chloride
housing, a pulse generator, and associated switching with controlling
circuitry as shown in Fig. 1. The light
source was mounted 4.5 cm above two sample holders that were set at a
position 45 degrees to the horizontal. This allowed two samples to be
irradiated simultaneously, with each sample receiving the same average
exposure. Two light sources were employed. The first of these was a
Heraeus Noblelight XAP series (type NL4006) constructed from a clear
fused-quartz tube (UV transparent) filled with xenon to a pressure of
59 kPa. The second light source was a Heraeus Noblelight XFP series
(type NL4320) with a cerium-doped quartz envelope, again filled with xenon to a pressure of 59 kPa. The envelope of this tube (NL4320) restricted the light output in the UV region. Both light sources produced a broad spectrum of white light with peak spectral emissions at wavelengths of ~550 nm, as shown in Fig.
2A for the low-UV-content tube and in
Fig. 2B for the high-UV-content tube. The major differences in emission
spectra occur between 200 and 450 nm.
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FIG. 1.
Schematic layout of experimental facility for microbial
inactivation with a pulsed-light source.
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FIG. 2.
Emission spectra (range, 200 to 900 nm) from two
different light sources; one shows a low UV content (A), and the other
shows a higher UV content (B).
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A single-stage, inverting, pulse-forming-network Marx generator was
used to create a high-peak-power discharge. The generator
was charged
to a DC voltage of 30 kV for all experiments and was
discharged
directly into the light source by using a plasma switch
triggered via a
high-voltage autotransformer. The generator source
capacitance was 6.4 nF, and the source impedance, when fired,
was 6.25
. A fiber-optic
link and timing control circuit were
used to activate the pulse
generator at a pulse repetition rate
of 1 pulse per s. The generator
was charged by using a Brandenburg
50-kV, 1-mA DC supply, and, at full
voltage, the pulse-forming-network
Marx generator contained a stored
energy of 3 J. The nominal duration
of the output pulse was 85 ns,
representing an average peak electrical
power, per pulse, of 35 MW.
This should be compared with the ~100-W
average power rating of the
light source when operated continuously.
At 1 pulse per s, the average
power consumption of the system
was 3 W, and consequently no
discernible increase in sample temperature
was observed during
treatment. The electrical diagnostics consisted
of a high-voltage DC
probe, used to measure the charging voltage,
and a high-speed transient
probe to monitor the pulsed voltage
applied to the light source. With
the line-source geometry employed,
the light intensity profile varied
by
30% from the center to
the edge of the sample. The light emission
was monitored with
a four-channel Ocean Optics SQ2000 fiber-optic
spectrometer. The
spectral resolution was 1.25 nm for each channel
(50-mm slit width),
and the detectors (Sony 1LX511) were enhanced to
allow UV detection.
Continuous monitoring of the optical emissions
verified that the
emission spectra were constant throughout the
experiment.
Surface-inoculated TSYEA and MYGPA plates containing approximately
10
9 cells ml of test culture
1 were positioned
in the PPET assembly (Fig.
1). Samples of each
test culture were
treated with either 100 or 200 pulses of high-intensity
light which
contained either a low or high level of UV. Following
treatment, the
plates were wrapped in aluminium foil to prevent
photoreactivation and
were incubated for 48 h at 30°C. The study
was carried out in
quadruplicate with duplicate plates for each
set of exposures. The
surviving populations were enumerated and
expressed as
log
10 CFU per
plate.
Initial experiments with
E. coli (Fig.
3) showed that the type of light source
used had a significant effect on the level
of inactivation
(
P < 0.001). A 5- and 6-log
10-order
inactivation
occurred after treatment with 100 and 200 pulses,
respectively,
with the higher UV light source, whereas 300 pulses of
low-UV
light gave a reduction of only 1 log
10 order.
Subsequently, 100
and 200 pulses were selected to test
Salmonella
enteritidis,
Staphylococcus aureus,
Escherichia
coli,
Pseudomonas aeruginosa,
Bacillus
cereus,
Listeria monocytogenes, and the yeast
Saccharomyces cerevisiae.
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FIG. 3.
Pulsed-light inactivation of surface-inoculated E. coli using two light sources which contained either a low- or
high-UV content.
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The results showed with each test culture that significantly greater
levels of microbial inactivation (
P < 0.001) occurred
with light pulses of high-UV content. With 200 pulses of high-UV
light,
all of the microbial populations treated were reduced by
5 to 6 log
10 orders, whereas with low-UV light, only a 1- to
2-log
10-order
reduction in cell numbers occurred. It was
found that there were
variations in the susceptibility of test cultures
(Table
1).
The levels of resistance of
the following bacteria differed (and
are listed in order of decreasing
resistance):
L. monocytogenes,
Staphylococcus
aureus,
Salmonella enteritidis,
E. coli,
B. cereus,
Saccharomyces cerevisiae, and
P. aeruginosa (the levels of resistance
between
S. aureus
and
S. enteritidis and those between
B. cereus,
and
S. cerevisiae did not significantly differ at the
P < 0.05
level). These findings are in agreement with
the work of Jay (
2),
in which gram-positive bacteria were
shown to be more resistant
to the effects of UV light than
gram-negative bacteria and pseudomonads
and flavobacteria were shown to
be the most sensitive.
By using this PPET approach for high-intensity light generation, it was
possible to produce significant levels of peak power
in the light
source which are not achievable under conventional
continuous
excitation (
4). This in turn results in a greater
relative
production of light in the shorter biocidal wavelengths.
It has been
well documented that UV is effective in killing microorganisms
contaminating the surfaces of a variety of materials, including
food
(
2,
3,
8,
9). The lethal action of high-intensity
broad-spectrum light is due predominantly to either photothermal
and/or
photochemical mechanisms (e.g., the formation of lethal
thymine dimers
on the microbial DNA) (
1,
2). Since only
a negligible rise
in temperature (i.e., less than 1°C) occurred
in the treated
agar, there were no appreciable photothermal effects.
Therefore, it is
likely that the lethality of this PPET approach
can be attributed to
the photochemical action of the shorter UV
wavelengths. This is
supported by the data in Fig.
2, which compares
the spectral emissions
produced by both PPET light sources and
shows that they differ in the
level of the shorter wavelengths
in the range of 200 to 450
nm.
Bank and coworkers (
1) have shown previously that a
6-log
10 decrease in viable bacterial numbers, which had
been surface
inoculated onto trypticase soy agar plates, could be
achieved
by using a computer-controlled modulated UV-C light source
(100
to 280 nm). Their light system used exposures of up to 60 s
at
40-W peak power compared to an 85-ns exposure at 35-MW peak power
in
the present study, demonstrating the antimicrobial effectiveness
of the
PPET. This approach may be further improved by optimizing
the emission
spectra to enhance the antimicrobial UV-C
content.
In conclusion, this study has shown that a 6-log
10
reduction in microbial populations was achieved after exposure to 200 pulses
of light containing high-intensity UV. The energy delivery
system
has modest energy requirements, ~3 J, and if this is delivered
in a short period of time, with a high repetition rate, then rapid
treatment can be
achieved.
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ACKNOWLEDGMENTS |
We thank S. M. Turnbull, Y. Koutsoubis, F. A. Tuema, and
D. A. Currie (University of Strathclyde) for their assistance in this study.
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Bioscience and Biotechnology, University of Strathclyde, Royal College, 204 George St., Glasgow G1 1XW, Scotland. Phone: 44 141 548 2531. Fax:
44 141 553 1161. E-mail: n.j.rowan{at}strath.ac.uk.
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Applied and Environmental Microbiology, March 1999, p. 1312-1315, Vol. 65, No. 3
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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