Journal of Applied Microbiology ISSN 1364-5072
ORIGINAL ARTICLE
Antibacterial efficacy of 405, 460 and 520 nm light emitting
diodes on Lactobacillus plantarum, Staphylococcus aureus
and Vibrio parahaemolyticus
A. Kumar1, V. Ghate1, M.J. Kim1, W. Zhou1,2, G.H. Khoo3 and H.G. Yuk1,2
1 Food Science and Technology Programme, Department of Chemistry, National University of Singapore, Singapore, Singapore
2 National University of Singapore (Suzhou) Research Institute, Suzhou, Jiangsu, China
3 Post-Harvest Technology Department, Technology & Industry Development Group, Agri-Food & Veterinary Authority of Singapore, Singapore,
Singapore
Keywords
food preservation, foodborne bacteria,
Gompertz model, light emitting diode,
photodynamic inactivation.
Correspondence
Hyun-Gyun Yuk, Food Science and Technology Programme, Department of Chemistry,
National University of Singapore, 3 Science
Drive 3, 117543 Singapore, Singapore.
E-mail: [email protected]
2015/1625: received 17 May 2015, revised
24 September 2015 and accepted 12 October
2015
doi:10.1111/jam.12975
Abstract
Aims: Little information is available on a direct comparison of the
antibacterial efficacy of light emitting diode (LEDs) of different peak
wavelengths. Thus, the objective of this study was to evaluate the effect of
LEDs of three different wavelengths on bacterial inactivation.
Methods and Results: Lactobacillus plantarum, Staphylococcus aureus and
Vibrio parahaemolyticus were illuminated with 405, 460 and 520 nm LEDs at 4,
10 and 25°C respectively. Inactivation curves were plotted and fitted using
Gompertz Model. Illumination with 405 and 460 nm LED produced significant
inactivation (P < 005) in the population of V. parahaemolyticus (>4 log) while
Lact. plantarum and Staph. aureus showed relatively less susceptibility to the
LED illumination. The 520 nm LED produced negligible inactivation.
Conclusions: The 405 and 460 nm LEDs proved more effective in inactivating
the selected foodborne bacteria in this study compared to 520 nm LED. The
405 nm LED showed the greatest antibacterial effect at the same level of energy
dose.
Significance and Impact of the Study: The results in this study demonstrated
the antibacterial efficacy of 405 nm LED on Lact. plantarum and
V. parahaemolyticus, suggesting its potential for use in food industry for the
control of these micro-organisms.
Introduction
Light emitting diode (LED) is a semiconductor device
that is cost effective, and produces light within a narrow
bandwidth of wavelength through electroluminescence. In
recent times, LED technology has received attention in its
use in the area of food production and safety. In particular, the LED technology has been demonstrated to have
antibacterial effect on pathogenic bacteria. For example,
Maclean et al. (2009) showed that a 405 nm LED system
inactivated Methillin Resistant Staphylococcus aureus
(MRSA) in a clinical environment. About 56–90% reduction was observed in the population of bacteria.
Microbial inactivation by the LED technology is
generally referred to as photodynamic inactivation.
Photodynamic inactivation involves the interaction of
two nontoxic factors, photosensitizers and visible light, in
the presence of oxygen. Photosensitizers are photoactive
molecules that can produce reactive oxygen species
(ROS) upon absorption of light at a specific wavelength,
corresponding to the energy required to excite the photosensitizer. The excited photosensitizer molecule, on its
way back to the ground state, can produce ROS by either
of the two mechanisms known as Type I and Type II.
The type I mechanism produces hydroxyl radical and
superoxide anion by the direct transfer of an electron
from the excited photosensitizer to the molecular oxygen.
In the Type II mechanism, the excited photosensitizer
excites the triplet oxygen to the reactive singlet oxygen by
direct transfer of energy. These ROS generated by either
Journal of Applied Microbiology 120, 49--56 © 2015 The Society for Applied Microbiology
49
A. Kumar et al.
Photodynamic inactivation by LED
of these two mechanisms result in a number of cytotoxic
reactions and cause extensive damage to the cellular components. This eventually leads to the destruction of the
bacterial cells (Luksiene 2009; Luksiene and Brovko
2013).
In recent years, researchers have investigated the
antibacterial effect of LEDs of a visible range of wavelengths. Studies have found the LED of 405 nm to be
effective against the inactivation of bacteria and spores
(Maclean et al. 2009, 2013; Murdoch et al. 2012). Similarly, other studies have investigated the effect of LEDs
around the 460 nm range and concluded that narrow
wavelength visible light in this range is an effective
antibacterial tool. For example, the population of Escherichia coli O157:H7, suspended in TSB at pH 73, was
found to be reduced by about 51 log CFU ml1 after
illumination with an LED of wavelength 461 nm (Ghate
et al. 2013). The combined effect of more than one LED
of different wavelengths (405 and 880 nm) has also been
reported to produce significant antibacterial effect on
Staph. aureus and Pseudomonas aeruginosa. At a dose of
20 J cm2, Staph. aureus colonies reduced by 72% while
938% decrease was observed in the colonies of P. aeruginosa (Guffey and Wilborn 2006). Researchers have
claimed that individually, visible light of wavelength as
high as 630 nm produce an antibacterial effect against
selected micro-organisms (Nussbaum et al. 2002).
Staph. aureus is a common cause of food poisoning,
and some strains of Staph. aureus have acquired resistance against a range of antibiotics. Vibrio parahaemolyticus is known to cause gastrointestinal illnesses in humans
and it is one of the major bacteria that results in seafood-related foodborne disease. On the contrary, Lactobacillus plantarum is naturally found in raw fruits and
vegetables, and has been considered a spoilage bacterium
in canned and juice products since it can produce undesirable flavour and carbon dioxide (Tajchakavit et al.
1998).
To the best of our knowledge, the study on the direct
comparison of LEDs of varying wavelengths on the photodynamic inactivation of bacteria has been very limited.
Therefore, the objective of this study was to investigate
the antibacterial effect produced by LEDs of three different wavelengths on the selected foodborne bacteria. In
addition, the inactivation curves were modelled to compare the antibacterial effect produced by the three LEDs.
Materials and methods
Bacterial strains and culture conditions
Lact. plantarum ATCC 8014, Staph. aureus ATCC 6538
and V. parahaemolyticus ATCC 17802 were purchased
50
from the American Type Culture Collection (ATCC;
Manassas, VA). Frozen cultures were activated in culture
flasks containing 100 ml of sterile tryptic soy broth
(TSB; Oxoid, Basingstoke, UK) by incubating the cultures at 37°C for 24 h. The identities of the bacteria
were confirmed by growing the activated bacterial cells
onto MRS agar (Oxoid), Mannitol salt agar (Oxoid) and
Cholera medium TCBS (Oxoid) as selective media for
Lact. plantarum, Staph. aureus and V. parahaemolyticus,
respectively. To ensure strain purity a single bacterial
colony on the selective agar was cultured in TSB at
37°C for 24 h. The working cultures were prepared in
TSB with at least two consecutive transfers of 1% inoculum and incubation at 37°C for 24 h. The three bacterial strains received the same treatment unless otherwise
stated.
Light emitting diode illumination system
The LED illumination system was configured according
to a method described elsewhere with some modifications
(Ghate et al. 2013). Briefly, 405, 460 and 520 nm LEDs
were purchased from GETIAN (Shenzhen, China). Each
LED was installed in a circuit along with a resistor in series and a heat sink attached with a cooling fan in parallel
(Fig. 1a). In order to minimize the heating of bacterial
suspension underneath, the LED assembly was placed in
a housing that was open from top and allowed air circulation (Fig. 1b). The emission spectra of the LEDs were
measured using an Oriel spectrometer MS275 and an
Oriel enhanced UV photodiode (Oriel Instruments, Stratford, CT). The irradiance of each LED was measured
using a portable LED radiometer (UVATA, Shanghai,
China). The dose received by each bacterial sample was
calculated using the equation below. Equation 1 was
derived keeping in mind that the intensity of light and
other linear waves radiating from a point source follow
inverse-square law (Fig. 2).
0
1
1
B
C
P ¼ 2pIo h2 @1 qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiA
2
ð1 þ ðr=hÞ Þ
ð1Þ
where Io is the intensity measured at the centre of the
plate, r is the plate radius and h is the height of the LED
(point source) from the centre of the plate. P is the
amount of average energy falling per second on the plate
and the average dose D can be calculated by multiplying
P with the time of illumination and dividing by the total
plate area (A). In this study, the D10 value was defined as
the dose required for 90% inactivation while the 2D10
value was defined as the dose required for 99% inactivation in the population of bacteria.
Journal of Applied Microbiology 120, 49--56 © 2015 The Society for Applied Microbiology
A. Kumar et al.
Photodynamic inactivation by LED
(a)
Heat sink and cooling fan
LED
Resistor
Power supply
(b)
Cooling fan
LED
Bacterial
suspension
h
Heat sink
Io
r
Figure 1 Light emitting diode system (a) circuit diagram and (b) illumination setup.
Bacterial inactivation by LED illumination
The working cultures of bacteria were centrifuged
(Eppendorf, Hamburg, Germany) at 6000 g for 10 min.
The pellets were washed thrice with phosphate buffered
saline (PBS) and serially diluted in the same buffer to
achieve an initial population of approx. 106 CFU ml1.
Ten-millilitre of bacterial suspension (depth – 12 mm)
was placed in a sterile petri dish (30 mm radius) at a distance of 33 mm directly below the 460 and 520 nm LED
illumination systems and 12 mm below the 405 nm LED
illumination system to obtain comparable energy dose at
the same illumination time. Since at a concentration of
106 CFU ml1 the bacterial suspension was not turbid, it
was assumed that the effect of the depth (12 mm) on
the penetration of light was negligible. The suspension
was illuminated with the LEDs for 7 h at 25, 10 and 4°C.
Control experiments were performed under similar conditions but without LED illumination. Although, the temperature of the LED illuminated bacterial suspensions
increased slightly (≤2°C) due to heating effect of LED
radiation (Kumar et al. 2015), the increase in temperature was considered marginal and hence ignored. Bacterial inactivation by LED illumination was carried out
under aerobic condition for all the three species of bacteria. A 100 ll aliquot of LED illuminated or nonilluminated bacterial suspension was withdrawn at an interval
of 1 h and serially diluted with PBS. The diluents were
spiral plated (WASP 2; Don Whitley Scientific Ltd., West
Yorkshire, UK) onto tryptic soy agar (TSA; Oxoid) plates
and incubated at 37°C for 24–48 h, followed by counting
using an automatic colony counter (aCOLyte, Synbiosis,
Frederick, MD).
Modelling of inactivation kinetics
The inactivation kinetics was preliminarily modelled
using six different equations (Whiting–Buchanan,
Figure 2 Diagram representing the
illumination area of the light emitting diode
system. P, average dose; Io, intensity
measured at the centre of the plate.
Journal of Applied Microbiology 120, 49--56 © 2015 The Society for Applied Microbiology
51
A. Kumar et al.
Photodynamic inactivation by LED
NðtÞ
BM
BðtMÞ
Þ ¼ Cee Cee
log10 ð
N0
ð2Þ
with t as the exposure time to illumination from LED
(h), N as the microbial population after exposure to LED
(CFU ml1), N0 is initial inoculation level (CFU ml1),
and B, C and M are the parameters in the Gompertz
model.
Statistical analysis
Experiments were performed in independent triplicates
with duplicate sampling (n = 6). Significant differences in
the mean values were calculated at the 95% confidence
interval (P < 005) using one-way analysis of variance
(ANOVA) with the GRAPHPAD PRISM STATISTICAL software (ver.
6.00 for Windows; Graphpad Software, La Jolla, CA).
Results
The peak wavelength for the three LEDs was identified at
405, 460 and 520 nm respectively (Fig. 3). The 405 nm
LED had the narrowest spectrum profile with a full width
at half maximum (FWHM) value of 18 nm followed by
the 460 nm LED with an FWHM value of 21 nm. The
spectrum profile of the 520 nm LED was relatively
broader compared to the other two LEDs with an
FWHM value of 37 nm. The intensity recorded at the
centre of the plate Io was 120 mW cm2 for all three
LEDs. At the end of 7-h illumination by 405, 460 and
520 nm LEDs, the maximum dose received by the bacterial suspension underneath was calculated to be
60 9 102, 18 9 103, and 18 9 103 J cm2 respectively
(Table 1).
With the initial circuit and illumination setup design
(h = 32 mm), the irradiance at the centre of the plate
was 11 mW cm2 for the 405 nm LED system, while that
for the 460 and the 520 nm LED systems was approx.
120 and 108 mW cm2 respectively. This clearly reflected
that the intensity output of the 405 nm LED was much
lower compared to the other two LEDs. To offset for this
large variation in the output intensity, the 405 and the
520 nm LED systems were modified and the existing
resistors were replaced with relatively smaller resistors in
the circuit. The 405 nm LED system was further modified
and the distance between the bacterial suspension and the
52
LED bulb was reduced from 32 to 12 mm. These modifications compensated for the comparatively lower output
of the 405 and 520 nm LED systems and resulted in uniform output intensity (120 7 mW cm2) from the
three LED systems at the centre of the plate (Fig. 1).
However, besides Io and the radius of the plate (r), the
average dose is also a function of distance (h) between
the light source and the bacterial suspension (Eqn 1). As
the distance of 405 nm LED from the bacterial suspension was approximately one-third compared to the other
two LEDs, the average dose received by the bacterial suspension (across the plate) with the 405 nm LED system
was different from the dose with the 460 and the 520 nm
LED systems in spite of equal Io values (Table 1).
No considerable reduction in the population of any of
the three bacterial species took place for the nonilluminated control samples during the course of experiment
for 7 h (Fig. 4). In addition, illumination with the
520 nm LED system produced negligible inactivation in
the population of the three bacteria. Variation in the
temperature from 4 to 10 and 25°C did not result in any
noticeable effect either. On the contrary, the inactivation
Intensity (arbitrary units)
Weibull, Hom, Kamau, modified Gompertz, First-order
Kinetics) (Kumar et al. 2015). Based on Akaike’s information criterion (Yamaoka et al. 1978), the modified
Gompertz model proved most suitable to describe the
inactivation curves in this study. The modified Gompertz
equation is described as follows:
360
460
560
Wavelength (nm)
Figure 3 Emission spectra of the three light emitting diodes. (
405 nm, (
) 460 nm and (
) 520 nm.
)
Table 1 Characterization of light emitting diode systems
k
(nm)
r
(mm)
h
(mm)
Io
(mW cm2)
I
(mW cm2)
D
(J cm2)
405
460
520
30
30
30
12
32
32
120
120
120
24
74
74
60 9 102
18 9 103
18 9 103
k, peak wavelength; r, radius of the plate; h, distance between the
LED and bacterial suspension; Io, intensity measured at the centre of
the plate; I, average intensity across the plate; D, maximum dose at
the end of 7 h.
Journal of Applied Microbiology 120, 49--56 © 2015 The Society for Applied Microbiology
A. Kumar et al.
Photodynamic inactivation by LED
(b)
6
log (CFU ml–1)
log (CFU ml–1)
(a)
4
2
6
4
2
0
2
4
6
0
8
2
6
4
2
8
6
8
6
8
6
8
6
4
2
0
2
4
6
8
0
2
4
Time (h)
Time (h)
(e)
(f)
6
log (CFU ml–1)
log (CFU ml–1)
6
(d)
log (CFU ml–1)
log (CFU ml–1)
(c)
4
2
6
4
2
0
2
4
6
8
0
2
Time (h)
4
Time (h)
(h)
(g)
6
log (CFU ml–1)
log (CFU ml–1)
4
Time (h)
Time (h)
4
2
6
4
2
0
2
4
6
8
Time (h)
0
2
4
Time (h)
Figure 4 Inactivation curves for the
Staphylococcus aureus (a, b, c), Lactobacillus
plantarum (d, e, f), and Vibrio
parahaemolyticus (g, h, i) at 4, 10 and 25°C,
respectively. ( ) 405 nm, ( ) 405 nm, ( )
405 nm and (○) control.
log (CFU ml–1)
(i)
6
4
2
0
2
4
6
8
Time (h)
Journal of Applied Microbiology 120, 49--56 © 2015 The Society for Applied Microbiology
53
A. Kumar et al.
Photodynamic inactivation by LED
curves in Fig. 4 show that the 405 and the 460 nm LEDs
proved effective in producing bacterial inactivation.
The inactivation of Lact. plantarum with the 405 nm
LED system was significantly higher (P < 005) compared
to that of Staph. aureus at all three temperatures. The
D10 value for Lact. plantarum was 374, 394 and
353 J cm2 at 4, 10 and 25°C, respectively (Table 2). The
405 nm LED produced a maximum reduction of 36 log
in the population of Lact. plantarum, which was observed
at 25°C after 7 h. In contrast, the D10 value for
Staph. aureus exceeded the maximum dose of
60 9 102 J cm2 delivered by the system, at all three
temperatures. At the end of the experiment, the 405 nm
LED resulted in a 08, 05 and 06 log reduction in the
population of Staph. aureus at 4, 10 and 25°C, respectively. With the 460 nm LED system, a significant inactivation (P < 005) in the population of Lact. plantarum
was observed only at 25°C. In comparison, the 460 nm
LED system did not bring about any significant
(P > 005) inactivation in the population of Staph. aureus, at all three temperatures (Table 3).
V. parahaemolyticus exhibited faster rate of inactivation
compared to Staph. aureus and Lact. plantarum in the
presence of 405 as well as 460 nm LED illumination
(Tables 2 and 3). With the 405 nm LED, V. parahaemolyticus population reached below detection limit at
4 and 10°C, and recorded about 36 log reduction in its
population at 25°C. Similarly with the 460 nm LED, the
change in the population of V. parahaemolyticus at 25°C
was significantly lower (P < 005) compared to 4 and
10°C. The 2D10 values for V. parahaemolyticus were considerably smaller than the twice of the D10 values which
reflects the long lag phase observed (Fig. 4g–i) in the
photodynamic inactivation of the bacteria. Similarly, the
computed 2D10 values for Lact. plantarum were also considerably smaller than the twice of the D10 values.
Table 2 Parameter (‘C’, ‘B’ and ‘M’) values, D10 (J cm2) and 2D10 (J cm2) predicted by Gompertz model against 405 nm LED inactivation data
25°C
Lactobacillus plantarum
Staphylococcus aureus
Vibrio parahaemolyticus
10°C
Lactobacillus plantarum
Staphylococcus aureus
Vibrio parahaemolyticus
4°C
Lactobacillus plantarum
Staphylococcus aureus
Vibrio parahaemolyticus
C
B
M
D10
2D10
4980 0309
14E7 85E9
8190 1472
0504 0036
0010 0383
0218 0045
5260 0145
2793 13E4
3865 0402
374 3b
ND
170 5d
470 6b
ND
306 3c
2514 0284
1519 3936
5822 0369
0476 0076
0012 2985
0228 0056
4376 0292
4328 6122
2656 0114
394 15a
ND
147 6e
ND
ND
226 7e
4694 0872
3E10 6E10
6260 0540
0300 0051
0010 0001
0494 0613
5475 0632
3388 5123
2747 0156
353 20c
ND
142 3e
524 18a
ND
258 8d
ND, not determined.
Different letters (a–e) within a column indicate that the means are significantly (P < 005) different.
Table 3 Parameter (‘C’, ‘B’ and ‘M’) values, D10 (J cm2) and 2D10 (J cm2) predicted by Gompertz model against 460 nm inactivation data
C
25°C
Lactobacillus plantarum
Staphylococcus aureus
Vibrio parahaemolyticus
10°C
Lactobacillus. plantarum
Staphylococcus aureus
Vibrio parahaemolyticus
4°C
Lactobacillus plantarum
Staphylococcus aureus
Vibrio parahaemolyticus
B
M
D10
2D10
2561 0193
0591 2294
3854 0238
0492 0045
0141 005
0460 0049
5024 0184
6034 1904
3360 0144
1121 10a
ND
717 24d
ND
ND
1125 49b
0392 0283
0266 0342
5040 0334
0299 0220
0275 0416
0668 0081
4967 2374
4086 3428
4415 0135
ND
ND
958 12b
ND
ND
1175 19a
92E10 24
0526 2217
5287 0289
0009 0093
0233 0766
0613 0060
3639 4663
<00001
4458 0117
ND
ND
940 13c
ND
ND
1206 48a
ND, not determined.
Different letters (a–d) within a column indicate that the means are significantly (P < 005) different.
54
Journal of Applied Microbiology 120, 49--56 © 2015 The Society for Applied Microbiology
A. Kumar et al.
Discussion
As LED technology is being pronounced as a novel
method for the inactivation of bacteria, this study aimed
to compare the effect of different types of LED illumination on foodborne bacteria at different temperatures.
Two foodborne pathogens, Staph. aureus, and V. parahaemolyticus along with a spoilage bacterium Lact. plantarum were selected in this study as the model organisms
to test the effect of LEDs on different classes of bacteria
as well as different Gram-nature.
The results obtained in this study demonstrated that the
405 and the 460 nm LED systems produced considerable
inactivation in most cases, while the 520 nm LED did not
lead to any significant inactivation (P > 005) in the populations of the three bacteria. Photodynamic inactivation by
visible light depends on the absorption spectrum of a special class of compounds called photosensitizers (Nowis
et al. 2005). Endogenous photosensitizers such as porphyrins have a characteristic absorption band between 400
and 430 nm (Redi and Jori 1988) and photodynamic inactivation occurs when light of wavelength between 400 and
500 nm is absorbed (Maclean et al. 2009). This provides a
reason why negligible inactivation occurred for the three
bacterial strains in the 520 nm region. In addition, both
D10 and 2D10 values obtained with the 460 nm LED were
significantly higher (P < 005) compared to those obtained
with the 405 nm LED, regardless of bacterial species and
illumination temperature. This clearly demonstrated that
the 405 nm LED was much more effective in eliminating
bacteria compared to the 460 nm LED. The likely reason
for this observation is that a significant portion of the
405 nm LED spectrum fell in the UV range (Fig. 3). While
high-energy visible light may cause DNA and membrane
damage indirectly by the generation of ROS in the cells,
UV radiation may also produce direct damage to DNA (in
addition to ROS generation), enhancing the efficacy of the
process.
The variation in the D10 value across the temperature
was relatively small (approx. 10%) for Lact. plantarum
compared with other bacteria and did not show a consistent trend with the rise or fall in temperature. Nevertheless, the D10 value was highest for Lact. plantarum at
10°C and lowest at 4°C with the 405 nm illumination at
25°C occupying an intermediate value. However, a conclusion arising from this observation that the LED illumination at 4°C was more effective compared to that at
25°C for photodynamic inactivation of Lact. plantarum
would be misleading. This is reflected in the 2D10 values
of Lact. plantarum, which was significantly lower at 25°C
compared to 4°C. At 10°C, the 2D10 value was not computed due to relatively lesser inactivation and was presumably, higher than 60 9 102 J cm2. An unexpectedly
Photodynamic inactivation by LED
higher value of D10 in the population of Lact. plantarum
at 25°C compared to 4°C arose due to a longer lag phase
(the shoulder of inactivation curve) at 25°C (Fig. 4).
Other studies have shown that, in general, bacterial
decay in the presence of an antimicrobial increases with
increasing temperature (Liang et al. 2002). At higher temperature bacterial cells maintain higher degree of cellular
activity (Song et al. 2011) and thus have added metabolic
burden to account for, which may compromise the chances
for survival in the presence of an external antimicrobial
agent. Therefore, relatively higher level of inactivation in
the population of Lact. plantarum at 25°C may have arisen
due to the added metabolic burden and increased rate of
cytotoxic reactions at higher temperatures. Additionally,
further study is required to understand the reasons for a
longer lag phase in Lact. plantarum at 25°C (Fig. 4f).
Contrary to the case of Lact. plantarum, the D10 and
2D10 values for V. parahaemolyticus were significantly
higher at 25°C than those at 10 and 4°C, indicating more
effective inactivation at lower temperatures. While this
appears unexpected, it cannot be ruled out that some bacteria may be more susceptible to the LED illumination at
lower temperatures. This may be attributed to the
increased proportions of unsaturated fatty acids in bacterial membranes at lower temperatures (Ghate et al. 2013),
which the bacterial cells adjust in response to changes in
temperature so as to maintain proper membrane fluidity
(Beales 2004). Unsaturated fatty acids are more prone to
damage by ROS and this is the primary mechanism behind
the photodynamic inactivation of bacteria. However, it is
also possible that V. parahaemolyticus entered viable but
non culturable (VBNC) state at lower temperatures and
therefore, recorded higher levels of inactivation. Although,
the cells in the control sample as well as those illuminated
with 520 nm LED did not show any signs of entering
VBNC state (no significant reduction in cell number), a
combination of sub-optimal temperature and lethal LED
dose may have pushed the cells illuminated with 405 and
460 nm LED to enter VBNC state.
The results in this study indicate that the photodynamic
inactivation was more effective on Gram-negative V. parahaemolyticus compared to the two Gram-positive bacteria.
This is in contrary to few other studies, with Maclean et al.
(2009) reporting that Gram-positive bacteria required a
lesser dose of illumination for inactivation, while Ghate
et al. (2013) reported no significant difference between the
two. In particular, the experimental results for Staph. aureus seem to be in direct contrast to other studies on the
photodynamic inactivation of the pathogen. Studies by
Nitzan and Kauffman (1999) and Guffey and Wilborn
(2006) indicated that Staph. aureus responded well to photoinactivation. However, in this study both 405 and
460 nm LED produced negligible inactivation in the popu-
Journal of Applied Microbiology 120, 49--56 © 2015 The Society for Applied Microbiology
55
A. Kumar et al.
Photodynamic inactivation by LED
lation of Staph. aureus. One possible explanation is that
there could be variations in the amount and the type of
endogenous porphyrins present in the different strains of
Staph. aureus. This would have a significant effect on the
amount of photodynamic inactivation since the amount
and types of porphyrin compounds affect bacterial inactivation rates during LED illumination (Nitzan et al. 2004).
In conclusion, 405 nm LED illumination significantly
reduced the populations of all three bacteria, while
460 nm LED produced photodynamic inactivation only
in the populations of Lact. plantarum and V. parahaemolyticus. No significant inactivation was observed
with 520 nm LED illumination. The temperature did not
seem to produce a consistent effect on the photodynamic
inactivation of the three bacteria investigated. These
observations indicate that at higher temperature multiple
factors, such as metabolic burden and cytotoxic reactions
along with changes in the membrane fatty acid composition may aid or hinder bacterial inactivation. This study
demonstrated that at equal dose the photodynamic inactivation with the 405 nm LED, in general, was more
effective as compared to the 460 nm LED, indicating that
405 nm LED has the potential to be used as an antimicrobial control method for food preservation and safety.
Acknowledgements
This research was funded by A*STAR Nutrition and Food
Science grant (SERC 112 177 0035).
Conflict of Interest
No conflict of interest declared.
References
Beales, N. (2004) Adaptation of microorganisms to cold
temperatures, weak acid preservatives, low pH, and osmotic
stress: a review. Compr Rev Food Sci Food Saf 3, 1–20.
Ghate, V., Ng, K.S., Zhou, W., Yang, H., Khoo, G.H., Yoon,
W.B. and Yuk, H.G. (2013) Antibacterial effect of light
emitting diodes of visible wavelengths on selected
foodborne pathogens at different illumination
temperatures. Int J Food Microbiol 166, 399–406.
Guffey, J.S. and Wilborn, J. (2006) Effects of combined 405-nm
and 880-nm light on Staphylococcus aureus and Pseudomonas
aeruginosa in vitro. Photomed Laser Surg 24, 680–683.
Kumar, A., Ghate, V., Kim, M.J., Zhou, W., Khoo, G.H. and
Yuk, H.G. (2015) Kinetics of bacterial inactivation by
405 nm and 520 nm light emitting diodes and the role of
endogenous coproporphyrin on bacterial susceptibility.
J Photochem Photobiol B 149, 37–44.
56
Liang, Z., Mittal, G.S. and Griffiths, M.W. (2002) Inactivation
of Salmonella Typhimurium in orange juice containing
antimicrobial agents by pulsed electric field. J Food Prot
65, 1081–1087.
Luksiene, Z. (2009) Photosensitization for food safety.
Chemine Technologija 4, 62–65.
Luksiene, Z. and Brovko, L. (2013) Antibacterial
photosensitization-based treatment for food safety. Food
Eng Rev 5, 185–199.
Maclean, M., MacGregor, S.J., Anderson, J.G. and Woolsey,
G. (2009) Inactivation of bacterial pathogens
following exposure to light from a 405-nanometer
light-emitting diode array. Appl Environ Microbiol 75,
1932–1937.
Maclean, M., Murdoch, L.E., MacGregor, S.J. and Anderson,
J.G. (2013) Sporicidal effects of high-Intensity 405 nm
visible light on endospore-forming bacteria. Photochem
Photobiol 89, 120–126.
Murdoch, L.E., Maclean, M., Endarko, E., MacGregor, S.J. and
Anderson, J.G. (2012) Bactericidal effects of 405 nm light
exposure demonstrated by inactivation of Escherichia,
Salmonella, Shigella, Listeria, and Mycobacterium species in
liquid suspensions and on exposed surfaces.
ScientificWorldJournal 2012, 137805.
Nitzan, Y. and Kauffman, M. (1999) Endogenous porphyrin
production in bacteria by d-aminolaevulinic acid and
subsequent bacterial photoeradication. Lasers Med Sci 14,
269–277.
Nitzan, Y., Salmon-Divon, M., Shporen, E. and Malik, Z.
(2004) ALA induced photodynamic effects on Gram
positive and negative bacteria. Photochem Photobiol Sci 3,
430–435.
Nowis, D., Makowski, M., Stokłosa, T., Legat, M., Issat, T. and
Gołaz b, J. (2005) Direct tumor damage mechanisms of
photodynamic therapy. Acta Biochim Pol 52, 339–352.
Nussbaum, E.L., Lilge, L. and Mazzulli, T. (2002) Effects of 630-,
660-, 810-, and 905-nm laser irradiation delivering radiant
exposure of 1-50 J/cm2 on three species of bacteria in vitro.
J Clin Laser Med Sur 20, 325–333.
Redi, E. and Jori, G. (1988) Steady-state and time-resolved
spectroscopic studies of photodynamic sensitizers:
porphyrins and pthalocyanines. Res Chem Intermed 10,
241–268.
Song, L., Farrah, S.R. and Baney, R.H. (2011) Bacterial
inactivation kinetics of dialdehyde starch aqueous
suspension. Polymers 3, 1902–1910.
Tajchakavit, S., Ramaswamy, H.S. and Fustier, P. (1998)
Enhanced destruction of spoilage microorganisms in apple
juice during continuous flow microwave heating. Food Res
Int 31, 713–722.
Yamaoka, K., Nakagawa, T. and Uno, T. (1978) Application of
Akaike’s information criterion (AIC) in the evaluation of
linear pharmacokinetic equations. J Pharmacokinet
Biopharm 6, 165–175.
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