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A THESIS FOR THE DEGREE OF MASTER OF SCIENCE

Application of Innovative Processing of deep UV-LED

to Control Food-borne Pathogens

병원 미생물 저감화를 한 DUV-LED 적 최적화 연

February, 2015

Joo Yeon Shin

Department of Agricultural Biotechnology

Seoul National University

Joo Yeon Shin

사학 문

Application of Innovative Processing of deep UV-LED

to Control Food-borne Pathogens

병원 미생물 저감화를 한 DUV-LED 적 최적화 연

지도 수 강동현

논문 사학 논문 로 제출함

2015 년 2 월

울대학 대학원 농생명공학

신 주 연

신주연 사 학 논문 함

2015 년 2 월

원 문 태 화 ( )

원 강 동 현 ( )

원 진 호 ( )

III

ABSTRACT

The UV irradiation for treating food has been approved by the U.S. Food

and Drug Administration. So far, most UV treatment is performed by low-

pressure (LP) UV lamp of 253.7 nm. But these lamps have some potential

risks such as possibility of mercury leakage, short life time and requirement

of significant amount of energy. As an alternative to UV mercury lamps,

deep UV-LEDs (DUV-LEDs) are of great interest for disinfection. The

objectives of this study were to examine the basic spectral properties of

DUV-LEDs and the effects of UV-C irradiation for inactivating food-borne

pathogens on solid medium as well as in water. A cocktail of Escherichia

coli O157:H7, Salmonella Typhimurium and Listeria monocytogenes strains

was spread-plated onto each selective medium or inoculated to the sample of

water. As temperature increased, intensity of LED was slightly decreased

while LP lamp showed intensity increasing until it reaches to the peak

temperature around 30 °C. As the dose of UV radiation increased from 0 to

1.67 mJ/cm2, all kinds of food-borne pathogens showed great reduction by

IV

>5 log. When treatment temperature increases, reduction level of E. coli

O157:H7 and S. Typhimurium were gradually increased, but L.

monocytogenes showed no significant different (P > 0.05). Only for E. coli

O157:H7, the significant reduction (P < 0.05) was observed at 90% relative

humidity condition compared with other conditions such as 30 and 60 %. In

case of 10 ml of water treatment, levels of surviving cells of all pathogens

were reduced to below the detection limit (1.0 log CFU/g). At the scaled-up

test, surviving populations of general bacteria were reduced by 5.38 and 4.37

log CFU/ml for 2 L and 3 L of water, respectively, and lowered to below

detection limit for 1 L of water at the dose of 3 mJ/cm2. Linear correlation

between inactivation and dosage of UV irradiation was indicated at

continuous water decontamination system. The result of this study suggests

that a novel type of DUV-LED’s applicability was confirmed as an

alternative to the lamp type in inactivating food-borne pathogens.

Keywords: DUV-LED, ultraviolet irradiation, food-borne pathogens,

surface and water decontamination, environmental device

Student Number: 2013-21176

V

CONTENTS

ABSTRACT.................................................................................................. III

CONTENTS....................................................................................................V

LIST OF TABLES......................................................................................VIII

LIST OF FIGURES........................................................................................X

I. INTRODUCTION........................................................................................1

II. MATERIALS AND METHODS................................................................4

2.1. Spectral characteristics of DUV-LED

2.1.1. Collimated UV radiation design.................................................4

2.1.2. Experimental setup......................................................................6

2.1.3. Irradiance measurements.............................................................8

2.2. Effect of various conditions on inactivation of food-borne pathogens

by using DUV-LED

2.2.1. Bacterial strains and inoculum conditions..................................8

2.2.2. Culture preparation...................................................................9

2.2.3. Culture inoculation....................................................................9

2.2.4. UV treatment.............................................................................10

2.2.5. Bacterial enumeration...............................................................11

2.2.6. Statistical analysis.....................................................................11

VI

2.3. UV treatment for water decontamination

2.3.1. Bacterial strains and inoculum conditions................................12

2.3.2. Culture preparation...................................................................13

2.3.3. Culture inoculation....................................................................13

2.3.4. UV treatment.............................................................................14

2.3.5. Bacterial enumeration...............................................................16

2.3.6. Statistical analysis.....................................................................16

III. RESULTS................................................................................................17


3.1.1. Emission spectrum of DUV-LED.............................................17

3.1.2. Comparison of properties between DUV-LEDs and LP lamps

.............................................................................................................19

3.1.3. Assessment of the effective area by LED arrangements...........21


by using DUV-LED

3.2.1. Bactericidal effect by UV treatment on media..........................25

3.2.2. Effect of UV irradiation temperature........................................27

3.2.3. Effect of UV irradiation humidity.............................................29


3.3.1. Bactericidal effect by UV treatment at batch water system......31

3.3.2. Bactericidal effect by UV treatment at scale-up system...........33

VII

3.3.3. Bactericidal effect by UV treatment at continuous water system

.............................................................................................................37

IV. DISCUSSIONS.......................................................................................45

V. REFERENCES….....................................................................................51

VI. 문초록................................................................................................58

VIII

LIST OF TABLES

Table 1. Surviving populations of E. coli O157:H7, S. Typhimurium, and L.

monocytogenes on media following UV-C irradiation, 4cm distance between

petri dish and LED, for intensity of 5.57 μW/cm2….………........................26


monocytogenes on media following UV-C irradiation for 1 min at 0°C, 4 °C,

15 °C, 25 °C, or 37°C. 4 cm distance between petri dish and LED..............28


monocytogenes on media following UV-C irradiation for 1 min at the

humidity of 30%, 60% and 90%, 25℃, 12.5 cm distance between petri dish

and LED.........................................................................................................30


monocytogenes in batch water system following UV-C irradiation. Treated at

room temp, 4.5 cm distance between sample and LED.................................32

IX

Table 5. Reduction levels of Escherichia coli O157:H7 in continuous water

system following UV-C irradiation at various flow rates and intensities......39

Table 6. Reduction levels of Salmonella Typhimurium in continuous water


Table 7. Reduction levels of Listeria monocytogenes in continuous water


X

LIST OF FIGURES

Fig. 1. Spatial arrangements for DUV-LEDs (a set of four LEDs)................5

Fig. 2. Schematic diagram of DUV-LED irradiation system at Seoul National

University (Seoul, Korea)................................................................................7

Fig. 3. Continuous water decontamination system at Seoul National

University (Seoul, Korea)..............................................................................15

Fig. 4. External appearance (a) and emission spectrum (b) of the DUV-LED

module...........................................................................................................18

Fig.5. Comparison between DUV-LED and LP lamp for (a) warm-up time

and (b) variation of intensity in accordance with temperature. �, DUV-LED;

s, LP mercury lamp.....................................................................20

Fig. 6. Petri factor (a) and irradiance (b) for LEDs accordance with spatial

arrangements as a function of distance from the LED...................................22

XI

Fig. 7. Irradiance of four corner arranged LEDs as a function of distance

from

LED................................................................................................................24

Fig. 8. Survival curves for Escherichia coli O157:H7 (A), Staphylococcus

aureus (B) and General bacteria (C) by UV-C irradiation from DUV-LED

with water volume of �, 1L; ¡, 2L; q,

3L....................................................34

Fig. 9. Survival curves for Escherichia coli O157:H7 (A), Salmonella

Typhimurium (B) Listeria monocytogenes (C) by UV-C irradiation from

DUV-LED with power of �, 100mW; ¡, 150mW; q,

200mW....................42

1

I. INTRODUCTION

Food safety has definite correlation to the public health. Each year,

about more than 16% of people get an illness and 3,000 are killed by

consuming contaminated food in the United States (CDC, 2011). Also,

higher than 50 billion dollars of economic cost related with foodborne

illnesses is burdened in the United States per year (Scharff, 2012). For these

major causes of food-borne outbreaks are generally recognized as E. coli

O157:H7, Salmonella spp. and L. monocytogenes. E. coli O157:H7 has

become notifiable foodborne pathogen which causing severe hemorrhagic

colitis and hemolytic-uremic syndrome (Doyle, 1991; Sodha et al., 2014).

The symptoms of salmonella spp. infection are diarrhea, abdominal pain,

mild fever, blood-tinged stools and vomiting (Baird-Parker, 1990; Zansky et

al., 2002). L. monocytogenes can survive and grow at refrigeration

temperatures, which adversely affects pregnant woman and elderly people

like immunocompromised persons (Datta, 2003; Farber and Peterkin, 1991).

Ultraviolet light (UV) which covers the region of the electromagnetic

spectrum from 100 to 400 nm is classified as UV-A (320-400 nm), UV-B

(280-320 nm) and UV-C (200-280 nm) (Guerrero–Beltrán and Barbosa–

Cánovas, 2004). The UV-C light is considered as the most germicidal effect

2

region of the UV spectrum for inactivating microorganisms such as bacteria,

viruses, protozoa, fungi, yeasts and algae by formation of photoproducts in

the DNA (Bintsis et al., 2000; Yaun et al., 2004). The pyrimidine dimer is

the most major product, which formed between adjacent pyrimidine

molecules in the same DNA strand. These dimers can interrupt both proper

transcription and replication of DNA, eventually leading to cell death

(Guerrero–Beltrán and Barbosa–Cánovas, 2004; Franz et al., 2009; Lopez-

Malo et al., 2005)

The use of UV irradiation as a disinfectant to treat food has been

approved by the U.S. Food and Drug Administration (US FDA, 2000). So far,

the majority of UV treatment is performed by low-pressure mercury UV

lamp of 253.7 nm in academic as well as in the industry field. But these

lamps have some potential risks such as possibility of mercury leakage, short

life time and requirement of significant amount of energy (Bowker et al.,

2011). As an alternative to UV mercury lamps, the application form of UV-C

light emitting diodes (DUV-LEDs) have been developing. DUV-LED has

numerous advantages over than conventional type of mercury lamp. While

emitting wavelength of low-pressure mercury lamps is fixed at 253.7 nm, the

emission wavelength of DUV-LED can be tuned at various individual

wavelengths in all UV spectrum. Adjustment to match the most effective

wavelength for disinfection in wide range of environmental conditions can

3

enhance efficiency of inactivation rates (Bettles, 2007). Also DUV-LED has

fracture-resistance to external shock, flexible spatial application by compact

size and reduction of heat generation.

Several studies have been performed for resulting the efficacy of

DUV-LEDs in water (Hamamoto et al., 2007, Chatterley et al., 2011).

Wurtele et al. (2011) and Oguma et al. (2013) developed both static test and

flow-through test system for different figures to examine the inactivation

efficiency. Although they had developed each unique type of water

decontamination system, both established continuous system still has some

limits for applying to practical use. To be a meaningful level of treating

amounts of water, certain level of flow rate is required over than a unit of

liter per minute. Also, to see the effect at high flow rate system, high power

of DUV-LED module should be guaranteed at short UV exposure time. For

these reason new type of flow-through water disinfection system was created

in order to prevent the loss of treating UV radiation.

The objectives of this study were to examine the basic properties of

DUV-LEDs such as a spectrum and intensities in accordance with several

distances and arrangements of LEDs. Also, the effects of UV-C irradiation

for inactivating E. coli O157:H7, Salmonella Typhimurium and L.

monocytogenes on solid medium at various temperatures and humidities as

4

well as at the each batch and continuous water treatment system were

investigated.

5

II. MATERIALS AND METHODS


2.1.1. Collimated UV radiation design

Four DUV-LED modules (LG Innotek Co., Korea) were connected to the

electronic printed circuit board (PCB) to get a constant electric current of

20mA from the DC power supply (TPM series, Toyotech). All of these LEDs

emitted with a single wavelength, 275 ± 3 nm. Several spatial arrangements

of four DUV-LEDs were set and analyzed to clearly fix the optimal LED

configuration which leads collimated radiation. Fig. 2 shows the five kinds

of disposition of LEDs which are tested in this study. (Colleen Bowker et al.,

2011)

6

Fig. 1. Spatial arrangements for DUV-LEDs (a set of four LEDs).

Evenly spaced Original Straight line

4 Corners Staggered line

7

2.1.2. Experimental setup

Four LED modules were arranged at each corner, 6 cm distance from

each other with 4cm distance between LEDs and petri dish were set because

this method showed generally equal intensity on the whole petri dish (90mm

diameter) above 4 cm of the sample. More concisely, the petri factor was

higher than 0.9 that means an ideal configuration for UV irradiance (Bolton

and Linden, 2003). The PCB with LED and inoculated media were placed in

the constant temperature chamber (IL-11, Lab Companion, Daejeon, Korea)

to optimize treatment conditions. The petri dish was located at the right

down side of the LEDs to get the maximum UV exposure (Fig. 1).

8

Fig. 2. Schematic diagram of DUV-LED irradiation system at Seoul National

University (Seoul, Korea).

9

2.1.3. Irradiance measurements

Irradiance of DUV-LED was measured by spectrometer (AvaSpec-

ULS2048-USB2-UA-50, Avantes, Netherlands) calibrated for 200 to 400nm

range of all UV spectrum. Optical probe was placed 4 cm above the LEDs

and peak irradiance value of spectrum was read. In order to calculate petri

factor, the optical probe scanned every 5mm over the petri dish (Bolton and

Linden, 2003). The maximum intensity value was multiplied by obtained

petri factor to get corrected Irradiance which means an average fluence rate.


by using DUV-LED

2.2.1. Bacterial strains and inoculum conditions

Three bacterial strains of E. coli O157:H7 (ATCC 35150, ATCC 43889,

ATCC 43890), S. Typhimurium (ATCC 19585, ATCC 43971, DT 104), and

L. monocytogenes (ATCC 7644, ATCC 19114, ATCC 19115) were obtained

from the Bacterial Culture Collection at Seoul National University (Seoul,

Korea). Stock cultures were prepared at – 80 °C in 0.7ml of Tryptic Soy

Broth (TSB; Difco, Becton Dickinson, Sparks, MD, USA) and 0.3ml of 50%

10

glycerol solution. In this study bacteria were streaked onto Tryptic Soy Agar

(TSA; Difco), incubated at 37 °C for 24 h, and stored at 4°C before used.

2.2.2. Culture preparation

Each strain of E. coli O157:H7, S. Typhimurium, and L. monocytogenes

was cultured in 5 ml TSB at 37 °C for 24 h and harvested by centrifugation

at 4000 ×g for 20 min at 4 °C. Cell pellets were obtained by washing with

sterile 0.2% peptone water (Bacto, Sparks, MD) in three times and the final

pellets were resuspended in 9ml PW, corresponding to approximately 108 to

109 CFU/g. Resuspended pellets of each strain of all pathogen species were

combined to constitute a 3-pathogen mixed culture cocktail. By three times

of 10-fold serial dilution with 0.2% sterile peptone water, initial

concentration of prepared culture was approximately 5-6 log CFU/g.

2.2.3. Culture inoculation

Culture suspension was serially diluted with 0.2% sterile peptone water

and 0.1ml diluent was spread-plated onto selective media. Sorbitol

MacConkey Agar (SMAC; Difco), Xylose Lysine Desoxycholate Agar

(XLD; Difco), and Oxford Agar Base with antimicrobial supplement MB

11

Cell (MOX; MB Cell) were used as selective media to enumerate E. coli

O157:H7, S. Typhimurium and L. monocytogenes respectively. To get a

numerable number of colonies on the tested media, two kinds of sequential

diluents were spread-plated. After the inoculation, the media was dried for

30 min approximately.

2.2.4. UV treatment

In order to minimize photoreactivation, all of the UV-treated petri dishes

were covered with an aluminum foil. Inoculated samples were treated with

275nm LEDs, 5.57 μW/cm2 intensity for 0, 0.5, 1, 3, 5 min at adjusted

temperature. Doses of UV were calculated by multiplying UV intensities by

the UV irradiation times. For testing at different temperature, UV irradiation

was applied to samples for 1 min at 4, 15, 25, 37 °C. In all temperature tests,

samples were kept at the controlled temperature of chamber for 5 min to

accommodate for the environmental change. For examining of humidity

impact to the UV irradiation, temperature and humidity chamber (TH-TG-

300, JEIO TECH, Korea) was used to adjust humidity of 30, 60, 90% with

maintained temperature at 25°C.

12

2.2.5. Bacterial enumeration

For enumeration of E. coli O157:H7 and S. Typhimurium, and L.

monocytogenes, Sorbitol MacConkey Agar (SMAC; Difco) and Xylose

Lysine Desoxycholate Agar (XLD; Difco) and Oxford Agar Base with

antimicrobial supplement MB Cell (MOX; MB Cell) were used respectively.

All plates were incubated at 37 °C for 24 h and enumerated.

2.2.6. Statistical analysis

All experiments were duplicate-plated and replicated three times. All

data were analyzed with ANOVA using Statistical Analysis System (SAS

Institute, Cary, NC, USA) and Duncan`s multiple range test to determine if

there were significant differences (P < 0.05) in mean values of

microorganism populations.

13


2.3.1. Bacterial strains and inoculum conditions

Bacterial strains, E. coli O157:H7 (ATCC 35150, ATCC 43889, ATCC

43890), S. Typhimurium (ATCC 19585, ATCC 43971, DT 104), L.

monocytogenes (ATCC 7644, ATCC 19114, ATCC 19115, and

Staphylococcus aureus (ATCC 13565, ATCC 25923, ATCC 29213) were

obtained from the Bacterial Culture Collection at Seoul National University

(Seoul, Korea). Stock cultures were prepared at - 80℃ in 0.7ml of Tryptic

Soy Broth (TSB; Difco, Becton Dickinson, Sparks, MD, USA) and 0.3ml of

50% glycerol solution. In this study bacteria were streaked onto Tryptic Soy

Agar (TSA; Difco), incubated at 37°C for 24 h, and stored at 4°C before

used. For the scale-up water experiment, general bacteria were obtained from

a creek (Seoul, Korea).

14

2.3.2. Culture preparation

Each strain of E. coli O157:H7, S. Typhimurium, L. monocytogenes, and

Staphylococcus aureus was cultured in 5 ml TSB at 37 °C for 24 h and in

case of general bacteria, 1 ml of creek water was mixed with 20 ml TSB and

cultured at 37 °C for 24 h. Then harvested by centrifugation at 4000 ×g for

20 min at 4 °C. Cell pellets were obtained by washing with sterile 0.2%

peptone water (Bacto, Sparks, MD) in three times. The final pellets were

resuspended in 9 ml PW, corresponding to approximately 108 to 109 CFU/ml.

2.3.3. Culture inoculation

Sterile distilled water (DW) was used in this experiment. In case of small

batch system for water decontamination, mixed culture cocktail (0.1 ml) was

inoculated into 25 ml of DW at room temperature. For scale-up system, each

4 ml, 8 ml, 12 ml aliquot of mixed culture cocktail of E. coli O157:H7 and S.

aureus was inoculated into 1 L, 2 L and 3 L of DW respectively. Equal

amount of cultured general bacteria inoculum was separately inoculated into

the DW suspension to investigate the single decontamination effect of

15

general bacteria. 8 ml of culture cocktail was inoculated into 2 L of DW to

be used in the continuous type of water decontamination system.

2.3.4. UV treatment

For small scale of decontamination system, inoculated samples were

treated with a 278 nm LED at 4.5 cm distance between sample and the UV

source. 10 ml of inoculated water sample was contained in the petri dish (50

mm x 15 mm; internal dimension) and the sample was serially mixed by

stirring bar to irradiate evenly. Dosage of UV were calculated by multiplying

UV intensities by the UV irradiation times.

For the scale-up system, treatment method was roughly similar to the

previous small scale system, but in this case 4 LEDs were used in order to

irradiate uniformly on the large surface of water.

Continuous system (Fig. 3) consisted of a power supply (TPM series,

Toyotech, Japan), a peristaltic pump (JWS600, JenieWell, Korea), and a

manufactured quartz pipe (Kum-kang quartz, Korea) which is attached with

LED modules. Inoculated water sample was moved by the pump, along the

silicon tube and treated on the way of flowing. 2 to 4 LED modules which

have the intensity of 50 mW were attached to the quartz pipe and flow rates

were adjusted to the 0.5, 1, 1.5 and 2 liter per minute (LPM).

16

Fig. 3. Continuous water decontamination system at Seoul National University (Seoul, Korea).

Quartz line with

LED modules

Peristaltic Pump Before After

Power supply

supply

17

2.3.5. Bacterial enumeration

1-ml aliquots of sample were 10-fold serially diluted in 9-ml blanks of 0.2%

PW, and 0.1 ml of sample or diluent was spread-plated onto each selective

medium. Sorbitol MacConkey Agar (SMAC; Difco), Xylose Lysine

Desoxycholate Agar (XLD; Difco), Oxford Agar Base with antimicrobial

supplement MB Cell (MOX; MB Cell), Baird-Parker Agar (BP; Oxoid) with

egg-yolk tellurite enrichment, and Tryptic Soy Agar (TSA; Difco) were used

as selective media to enumerate E. coli O157:H7, S. Typhimurium, L.

monocytogenes, S. aureus and general bacteria respectively. All agar media

were incubated at 37°C for 24-48 h and typical colonies were counted.

2.3.6. Statistical analysis

All experiments were duplicate-plated and replicated three times. All

data were analyzed with ANOVA using Statistical Analysis System (SAS

Institute, Cary, NC, USA) and Duncan`s multiple range test to determine if

there were significant differences (P < 0.05) in mean values of

microorganism populations.

18

III. RESULTS


3.1.1. Emission spectrum of DUV-LED

Typical spectral irradiance of 275 nm UV-LED was measured by

spectrometer (AvaSpec-ULS2048-USB2-UA-50) as shown in fig.3. The full

width at half maximum (FWHM) which means the wavelength gap between

the half output of the peak intensity was 11.3 nm for the 275nm LED.

19

(A)

(B)

Wavelength (nm)

240 260 280 300 320 340 360

Norm

aliz

ed inte

nsi

ty

0.0

0.2

0.4

0.6

0.8

1.0

Fig. 4. External appearance (A) and emission spectrum (B) of the DUV-LED

module.

20

3.1.2. Comparison of properties between DUV-LEDs and LP lamps

Warm-up time of both DUV-LEDs and low pressure (LP) lamps was

determined by measuring the intensity over time (0 to 10 min). After 5 min,

intensity of DUV-LEDs was decreased about 5.45%, on the other hand

intensity of LP lamps was increased about 90.43%. Intensity change

accordance to temperature presented different patterns between DUV-LED

and LP lamp. As temperature increases, intensity of LED was slightly

decreased while LP lamp showed intensity increasing until it reaches to the

peak temperature around 30 °C and then decreased.

21

(A)

(B)

Fig. 5. Comparison between DUV-LED and LP lamp for (A) warm-up time

and (B) variation of intensity in accordance with temperature

time (min)

0 2 4 6 8 10 12

Irra

dia

nce

rate

0.0

0.2

0.4

0.6

0.8

1.0

1.2

DUV-LED

LP lamp

Temperature ( )℃

0 10 20 30 40 50

UV

outp

ut (%

)

0

20

40

60

80

100

DUV-LED

LP lamp

22

3.1.3. Assessment of the effective area by LED arrangements

Intensity at a specified distance from DUV-LED is measured and petri

factor of each point was calculated. Fig. 4 shows each spatial disposition of

four LEDs. In order to get collimated beam of UV irradiation, the petri factor

should satisfy the value of over than 0.9. For arrangement of 4 corners, the

petri factor was calculated as 0.48, 0.90, 0.82 and 0.80 at the distance of 2, 4,

6, 8 cm respectively. In other configuration, the petri factor was steadily

increased from around 0.5 to 0.9. All configuration showed decreasing

intensity with increasing distance between LED and probe. The 4 corner

configuration at 4cm distance had an intensity of 4.41 μW/cm2 while other

configuration at 8cm distance which have petri factor of 0.9, that means even

distribution measured as lower than 3.2 μW/cm2. Also, intensity at a

specified distance from DUV-LED is measured. Intensity varied from 7.1 to

0.7 μW/cm2 as distance from lighting sources increases from 2 to 20cm.

23

(A)

Distance from LEDs (cm)

1 2 3 4 5 6 7 8 9

Pe

tri f

act

or

0.0

0.2

0.4

0.6

0.8

1.0

Original Evenly spacedStraight line4 CornersStaggered line

24

(B)


1 2 3 4 5 6 7 8 9

Inte

nsi

ty (

μW

/cm

2)

0

2

4

6

8

10

12

14

OriginalEvenly spacedStraight line4 CornersStaggered line

Fig. 6. Petri factor (A) and irradiance (B) for LEDs accordance with spatial

arrangements as a function of distance from the LED.

25


0 5 10 15 20

Inte

nsi

ty (

μW

/cm

2)

0

1

2

3

4

5

6

7

8

y = 0.0255x2 - 0.9080x + 8.7243

Fig. 7. Irradiance of four corner arranged LEDs as a function of distance

from LED

26


by using DUV-LED

3.2.1. Bactericidal effect by UV treatment on media

The reduction in viability of food-borne pathogen by UV radiation is

presented in table 1. As the dose of UV radiation increased from 0 to 1.67

mJ/cm2, all kinds of food-borne pathogens such as E. coli O157:H7, S.

Typhimurium and L. monocytogenes showed great reduction by >5 log.

Initial populations of E. coli O157:H7, S. Typhimurium and L.

monocytogenes were 6.21, 6.11, and 5.75 log CFU/ml respectively. The

population (log CFU/ml) of surviving E. coli O157:H7 was decreased by

4.58 and 2.70 after UV irradiation of 0.17 and 0.34 mJ/cm2, >5 log reduction

was observed at the dose of 1 mJ/cm2. For S. Typhimurium, the overall

reduction pattern is similar to that of E. coli O157:H7. The number of S.

Typhimurium treated by UV-C irradiation was 4.45 and 3.46 log CFU/ml

after irradiation of 0.17 and 0.34 mJ/cm2 respectively. After 1 mJ/cm2 of UV

treatment, S. Typhimurium were also reduced below the detection limit. In

the response of L. monocytogenes to different UV doses, the level of

surviving population was 5.36, 4.82, 2.16 log CFU/ml after UV dose of 0.17,

27

0.34, 1.00 mJ/cm2 respectively. UV dose of 1.67 mJ/cm2 was required to

inactivate below the detection limit.

28

Table 1. Surviving populations a of E. coli O157:H7, S. Typhimurium, and L. monocytogenes on media following UV-

C irradiation, 4cm distance between petri dish and LED, for intensity of 5.57 μW/cm2

a Data represent means ± standard deviations from three replications.

b Values followed by the same letters within the column per parameter are not significantly different (P > 0.05).

Treatment

time (min)

Dose

(mJ/cm2)

Population (log10 CFU/g) by organism b

E. coli O157:H7 S. Typhimurium L. monocytogenes

0 0 6.21±0.15 A 6.11±0.10 A 5.75±0.34 A

0.5 0.17 4.58±0.10 B 4.45±0.33 B 5.36±0.43 AB

1 0.34 2.70±0.25 C 3.46±0.40 C 4.82±0.38 B

3 1.00 0.00±0.00 D 0.00±0.00 D 2.16±0.59 C

5 1.67 0.00±0.00 D 0.00±0.00 D 0.00±0.00 D

29

3.2.2. Effect of UV irradiation temperature

Table 2 shows the bactericidal effect of 1 min treatment of UV irradiation

against food-borne bacteria such as E. coli O157:H7, S. Typhimurium and L.

monocytogenes on different temperatures at 0, 4, 15, 25 or 37 °C. Survival

population of all three food-borne pathogens was decreased with increasing

temperatures. For E. coli O157:H7, the significant reduction (P < 0.05) of

4.22 log was observed at 0°C. As treatment temperature increases from 0 to

37 °C, reduction level of E. coli O157:H7 was gradually increased, in

particular at more than 25 °C, it reduced by >5 log. Populations of 3.61, 3.39,

3.20, 2.94, 2.22 log CFU/ml were observed in S. Typhimurium after 1 min of

UV treatment at 0, 4, 15, 25 or 37 °C, respectively. Also, treatment at 37 °C

significantly reduced (P < 0.05) levels of S. Typhimurium by > 4 log,

compared from the control. The surviving population of UV treated L.

monocytogenes was significantly (P < 0.05) different from the control level,

but among temperature conditions, irradiated samples were not significantly

different (P > 0.05).

30


C irradiation for 1 min at 0°C, 4 °C, 15 °C, 25 °C, or 37°C. 4 cm distance between petri dish and LED


b Values followed by the same letters within the column per parameter are not significantly different (P > 0.05).

Treatment

temperature (℃)



Control 6.42±0.17 A 6.26±0.02 A 5.35±0.13 A

0 2.20±0.64 B 3.61±0.16 B 4.33±0.22 B

4 1.98±0.19 BC 3.39±0.31 B 4.35±0.09 B

15 1.47±0.25 CD 3.20±0.10 BC 4.20±0.14 B

25 1.16±0.28 DE 2.94±0.43 C 4.35±0.09 B

37 0.53±0.40 E 2.22±0.17 D 4.31±0.09 B

31

3.2.3. Effect of UV irradiation humidity

Surviving populations of E. coli O157:H7, S. Typhimurium and L.

monocytogenes on media following UV treatment at the different humidity

of 30, 60, and 90 % is presented in Table 3. Only for E. coli O157:H7, the

significant reduction (P < 0.05) was observed at 90% relative humidity

condition compared with other conditions such as 30 and 60 %, about 0.5 log

of more reduction at 90% humidity. In case of S. Typhimurium and L.

monocytogenes, there were no significant (P > 0.05) differences in various

humidity conditions.

32


C irradiation for 1 min at the humidity of 30%, 60% and 90%, 25℃, 12.5 cm distance between petri dish and LED


b Values followed by the same letters within the column are not significantly different (P > 0.05).

Treatment

humidity (%)



Control 5.58±0.03 A 5.00±0.08 A 4.41±0.08 A

30 1.55±0.20 B 2.82±0.35 B 2.81±0.04 B

60 1.61±0.16 B 2.72±0.09 B 2.83±0.05 B

90 1.11±0.09 C 2.76±0.06 B 2.82±0.10 B

33


3.3.1. Bactericidal effect by UV treatment at batch water system

Populations (log CFU/g) of E. coli O157:H7, S. Typhimurium and L.

monocytogenes in 10 ml of water during UV treatment are depicted in Table

4. The levels of surviving cells of all pathogens were reduced to below the

detection limit (1.0 log CFU/g) after UV treatment at the dose of 3 mJ/cm2.

The initial population of E. coli O157:H7 was 6.72 log CFU/g and the

number of surviving cells was 5.18, 2.87, 1.69 and 1.07 log CFU/g after

irradiation of 0.2 to 2 mJ/cm2. For S. Typhimurium the overall inactivation

pattern is similar to that of E. coli O157:H7. Populations of 5.09. 3.75, 2.14

and 1.30 log CFU/g were observed in S. Typhimurium after UV dose of 0.2,

0.5, 1 and 2 mJ/cm2 respectively. In case of L. monocytogenes, the level of

surviving population was decreased as 4.84, 3.76 and 1.97 log CFU/g after

irradiation of 0.2 to 1 mJ/cm2. At the dosage of 2 mJ/cm2, treatment

significantly reduced (P < 0.05) below the detection limit

34

Table 4. Surviving populations a of E. coli O157:H7, S. Typhimurium, and L. monocytogenes in batch water system

following UV-C irradiation. Treated at room temp, 4.5 cm distance between sample and LED


b Values followed by the same letters within the column per parameter are not significantly different (P < 0.05).

Dose (mJ/cm2) Population (log10 CFU/g) by organism b


0 6.72±0.28 A 6.71±0.17 A 5.29±0.11 A

0.2 5.18±0.42 B 5.09±0.15 B 4.84±0.22 B

0.5 2.87±0.23 C 3.75±0.32 C 3.76±0.28 C

1 1.69±0.27 D 2.14±0.60 D 1.97±0.20 D

2 1.07±0.12 E 1.30±0.30 E <1.00 E

3 <1.00 E <1.00 E <1.00 E

35

3.3.2. Bactericidal effect by UV treatment at scale-up system

The survival of E. coli O157:H7, S. aureus and general bacteria in scale-

up water decontamination system following UV treatment is shown in Fig. 8.

In general, the reduction of E. coli O157:H7, S. aureus and general bacteria

decreased with increasing the amount of treating water 1 L to 3 L. Fig 8 (A)

shows the inactivation effect of UV irradiation against E. coli O157:H7 in

scaled-up water. At the UV dosage of 1.5 mJ/cm2, populations were reduced

below the detection limit (1.0 log CFU/ml) for the scale at 1 L and 2 L of

water. For the scale of 3 L, population was reduced below the detection limit

after treatment of 2.0 mJ/cm2. The inactivation of S. aureus in scale-up

system is shown in Fig. 8 (B), and the trend of reduction was similar to that

of E. coli O157:H7. At the UV dosage of 2.0 mJ/cm2, populations were

reduced below the detection limit (1.0 log CFU/ml) for the scale at 1 L and

reduced by 6.09 and 4.87 log CFU/ml at 2 L and 3 L of water. For the scale

of 3 L, population was reduced below the detection limit after treatment of

3.0 mJ/cm2. Fig. 8 (C) shows the bactericidal effect of UV treatment against

general bacteria. At the UV dosage of 3.0 mJ/cm2, surviving populations

were reduced by 5.38 and 4.37 log CFU/ml for 2 L and 3 L of water,

respectively, and was lowered to below detection limit for 1 L of water.

36

Dose (mJ/cm2)

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5

Lo

g C

FU

/ml

0

1

2

3

4

5

6

7

Dose (mJ/cm2)

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5

Log C

FU

/ml

0

1

2

3

4

5

6

7

(A)

(B)

37

Dose (mJ/cm2)

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5

Lo

g C

FU

/ml

0

1

2

3

4

5

6

7

(C)

Fig. 8. Survival curves for Escherichia coli O157:H7 (A), Staphylococcus

aureus (B) and General bacteria (C) by UV-C irradiation from DUV-LED

with water volume of �, 1L; ¡, 2L; q, 3L.

38

3.3.3. Bactericidal effect by UV treatment at continuous water system

The reduction of E. coli O157:H7, S. Typhimurium and L. monocytogenes

at continuous water system during UV irradiation is presented in Tables 5, 6

and 7, respectively. In general, the reduction level of E. coli O157:H7, S.

Typhimurium and L. monocytogenes increased with decreasing flow rate and

increasing intensity. Table 5 shows the inactivation effect of UV irradiation

against E. coli O157:H7 in continuous system. UV treatment for 100 mW

with lower than at the flow rate of 1.0 LPM, 150 mW with lower than at the

flow rate of 1.5 LPM and 200mW with lower than at the condition of 2.0

LPM accomplished more than 99.9% reduction. Table 6 shows the

inactivation effect of UV irradiation against S. Typhimurium in continuous

water system. UV treatment for 100 mW showed 1.20, 1.40, 1.87 and 2.87

log reductions at 2.0, 1.5, 1.0 and 0.5 LPM, respectively. For intensity of 150

mW at the flow rate of 0.5 LPM and 200mW with lower than at the

condition of 1.0 LPM accomplished more than 99.9% reduction. The

inactivation of L. monocytogenes in continuous water system is shown in

Table 7, and the trend of reduction was similar to those of E. coli O157:H7

and S. Typhimurium. But just at the condition as intensity of 200 mW with

the flow rate of 0.5 LPM showed 99.9% reduction level of L. monocytogenes.

39

Fig. 9 shows the reduction curves for E. coli O157:H7, S. Typhimurium

and L. monocytogenes in continuous water decontamination system

according to the dose of UV irradiation. As the dose increases, the amount of

reduction was increased for all three pathogens, and the graph presented the

greater resistance in order by L. monocytogenes, S. Typhimurium and E. coli

O157:H7.

40

Table 5. Reduction levels a of Escherichia coli O157:H7 in continuous water system following UV-C irradiation at

various flow rates and intensities

Flow rate (LPM) Log reduction [log10 (N0/N)] by treatment type b

100mW 150mW 200mW

2.0 1.97±0.32 Aa

(98.93%)

2.82±0.28 Ab

(99.85%)

3.36±0.30 Ab

(99.97%)

1.5 2.36±0.12 Aa

(99.56%)

3.81±0.31 Bb

(99.98%)

4.06±0.19 Bb

(>99.99%)

1.0 3.24±0.32 Ba

(99.94%)

4.20±0.11 Bb

(>99.99%)

4.90±0.10 Cc

(>99.99%)

0.5 4.69±0.40 Ca

(>99.99%)

5.02±0.57 Ca

(>99.99%)

6.38±0.06 Db

(>99.99%)


b Means with the same uppercase letter in the same column are not significantly different (P > 0.05). Means with the

same lowercase letter in the same row are not significantly different (P > 0.05).

41

Table 6. Reduction levels a of Salmonella Typhimurium in continuous water system following UV-C irradiation at



100mW 150mW 200mW

2.0 1.20±0.17 Aa

(93.74%)

1.38±0.39 Aa

(95.83%)

1.85±0.39 Aa

(98.59%)

1.5 1.40±0.19 Aa

(96.05%)

1.81±0.37 ABab

(98.46%)

2.27±0.38 ABb

(99.46%)

1.0 1.87±0.29 Ba

(98.66%)

2.11±0.35 Ba

(99.22%)

3.06±0.53 Bb

(99.91%)

0.5 2.87±0.25 Ca

(99.86%)

3.67±0.26 Cb

(99.98%)

5.81±0.43 Cc

(>99.99%) a Data represent means ± standard deviations from three replications.



42

Table 7. Reduction levels a of Listeria monocytogenes in continuous water system following UV-C irradiation at






100mW 150mW 200mW

2.0 0.42±0.14 Aa

(62.27%)

0.44±0.28 Aa

(63.97%)

0.93±0.27 Ab

(88.16%)

1.5 0.58±0.23 Aa

(73.56%)

0.60±0.27 Aa

(75.07%)

1.19±0.38 Aa

(93.49%)

1.0 0.63±0.15 Aa

(76.56%)

0.89±0.09 Aa

(87.02%)

1.49±0.41 Ab

(96.79%)

0.5 1.48±0.17 Ba

(96.69%)

2.31±0.42 Bb

(99.51%)

3.47±0.41 Bc

(99.97%)

43

Dose (mJ/cm2)

0 1 2 3 4 5 6 7 8

log

red

uction

(C

FU

/ml)

0

1

2

3

4

5

6

7

Dose (mJ/cm2)

0 1 2 3 4 5 6 7 8

log

re

du

ctio

n (

CF

U/m

l)

0

1

2

3

4

5

6

7

(A)

(B)

44

Dose (mJ/cm2)

0 1 2 3 4 5 6 7 8

log r

educt

ion (

CF

U/m

l)

0

1

2

3

4

5

6

7

(C)

Fig. 9. Survival curves for Escherichia coli O157:H7 (A), Salmonella

Typhimurium (B) Listeria monocytogenes (C) by UV-C irradiation from

DUV-LED with power of �, 100mW; ¡, 150mW; q, 200mW.

45

IV. DISCUSSION

Traditionally, UV irradiation has been used for disinfecting surface, air

and water for a long time. Also the decontamination effect of UV irradiation

has been widely verified by many studies until now. As one of the non-

thermal methods for reducing a broad range of microorganisms, including

some pathogens, it has been considered to be a safety assurable treatment. LP

lamp which had been commonly used has some side effects such as low

efficiency and potential of mercury leakage so it is really necessary to

replace this technology. Therefore in this study, I confirmed that the basic

spectral characteristics which associated with bactericidal power compared

to LP lamp. Also the real pasteurization effect of UV irradiation by DUV-

LED technology was investigated.

For this research the DUV-LED which has the wavelength of 275 ± 3 nm

was selected because of its cost and the germicidal range. Globally, the

technology of DUV-LED still in development stage and a few companies

such as Sensor Electronic Technology (SET) and LG innotek is now

producing the prototype of LED for lower UV-C range lower than 280nm.

That is the reason of high cost is set in production. 200 to 280 nm range of

UV-C radiation has germicidal effect which depends on the wavelength. The

46

maximum DNA absorption of UV-C peaks at the wavelength of 260-265 nm

(Kowalski, 2009; US EPA, 2006). By considering both cost and germicidal

effectiveness, the LED for the wavelength of 275nm was chosen for this

study.

It is important to minimize the amount of light loss by taking optimal

LED alignment and distance adjustment while lowering overall cost. Colleen

Bowker et al. (2011) used Comsol Multiphysics method to investigate an

optimal collimated irradiation design. They reported that the simulated four

corners configuration showed highest petri factor over other type of arrays,

but it had limitation for prediction result. Therefore actual evaluation of petri

factor and intensity for DUV-LED was required. By comparing each

arrangement, more than 0.9 of petri factor is measured at 4 cm distance in

four corner array and 8 cm distance in others. Taken together with irradiance

factor, as four corners arrangement at 4 cm distance showed higher intensity

than 8 cm distance by other arrangements, so it was selected for the most

proper irradiation design.

As shown in Fig. 7 (A), the intensity of UV lamp reached to maximum

range after 5 min or more so that there should be a possibility of delaying

disinfecting effect. But in case of DUV-LED, high intensity was measured

from beginning and it consistently maintained. For these reason, LP lamp’s

warm up time could be removed by alternating to DUV-LED technology. Fig.

47

7 (B) indicates another benefit of DUV-LED for wide range of working

temperatures, especially at cold temperature around 0 to 4 °C. By comparing

each intensity at 4 °C and room temperature, 4.5% higher irradiance power

was measured at cold condition. Peak intensity of LP lamp was showed at

the room temperature and decreased about 62.5% at 4 °C. Similar result

already had been reported by Crawford et al. (2005). There are plenty of

microorganisms which can grow at refrigeration temperatures, particularly

Listeria monocytogenes is one of the major food-borne pathogens (Donnelly

and Briggs, 1986; Rosenow and Marth, 1987). For inactivating these bacteria,

DUV-LED is more meaningful method than using a LP lamp.

The populations of surviving all three pathogens showed decreasing

tendency when treated UV energy increased. L. monocytogenes of gram-

positive bacterium had more resistance to UV radiation than gram-negative

bacteria such as E. coli O157:H7 and S. Typhimurium. Because a thick

peptidoglycan wall surrounds the cytoplasmic membrane of gram-positive

bacteria, while gram-negative bacteria possess an external membrane (Virto

et al., 2005). Many research for disinfecting food-borne pathogens at various

food samples reported that L. monocytogenes is considered as one of the

most UV-resistant bacteria (Lu et al., 2011; Guerrero-Beltran and Barbosa-

Canovas, 2006; Gabriel and Nakano, 2009).

48

Most of the UV-related studies have focused on the storage temperature

which following UV treatment (Lemoine et al., 2007; Gonzalez-Aguilar et

al., 2004). Along with storage temperature, treatment temperature condition

is one of the key factors. In present research, treatment temperature had a

profound effect on inactivating E. coli O157:H7 and S. Typhimurium. When

temperature increases from 0 to 37 °C the population of surviving both

pathogens were decreased. Although DUV-LED emits higher intensity of

radiation at lower temperature, inactivation effect showed opposite results.

The enhanced inactivation at higher temperatures might be explained by

phase transition of the phospholipid molecules which is in the cell membrane

(Jayaram et al, 1992). Thayer and Boyd (1995) stated that inactivation level

of bacteria to gamma radiation was directly related to chemical reactions at

treatment temperatures. Rather than interaction of irradiation, cellular

inactivation is due to interactions with radiolytic products of water. It can be

applied to temperature-dependent UV radiation, because of photochemical

reactions which can occur as a direct result of UV radiation energy (Gayan et

al., 2013). And also, the fluidity of the cell membrane was increased by

heating, making the affected cells more sensitive to UV exposure (Gayan et

al., 2014). On the other hand, UV treatment temperature dose not effect to

the reduction rate of L. monocytogenes because of its bacterial characteristics.

49

To adopt LED technology commercially, scale-up system of UV treatment

is necessary for evaluating at the actual effectiveness. For that reason, 10 ml

capacity of initial treatment was scaled–up by 1 L to 3L. As shown in Fig. 8,

overall inactivation tendency was similar to each amount of treated water but

when the volume of water is increased, slight more resistance to the same

dosage of UV radiation was investigated. The sensitivity to ultraviolet

irradiation was different between kinds of bacteria, it was hard to inactivate

in the order of E. coli O157:H7, S. aureus and general bacteria. The

preference of batch or continuous type of water treatment system would be

determined by the purpose and process of use. But in consideration of all

cases, continuous type of decontamination system deserves careful

examination. In Tables 5, 6 and 7, reduction population were determined by

the condition of each level of flow rate and intensity. As the flow rate

increased, reduction level of each pathogen was decreased because of the

shorter UV treatment time. By combination of both factors, flow rate and

intensity, the inactivation effect should be explained at continuous system as

a consequential in the sole of UV dosage factor. As a result, continuous

system conditions reduced the disinfection ability but the inactivation rate

showed dependently on the calculated dosage of UV irradiation. Therefore,

UV treatment system in order to inactivate pathogen should be determined

by the degree of UV dose eventually.

50

In conclusion, these overall results suggest that conventional UV lamp for

the inactivation of food-borne pathogens can be fully substituted by DUV-

LED technology. Spectral characteristics of DUV-LED such as fast

stabilizing intensity and a property of insensitive to temperature should be

well appreciated for a strong point. DUV-LED leads to effective inactivation

of E. coli O157:H7, S. Typhimurium and L. monocytogenes both on the

surface of medium and in the water system at various conditions.

Furthermore, from our knowledge, this is the first report applying DUV-LED

technology directly to inactivating food-borne pathogens. In addition, DUV-

LED treatment for these large-capacity and high flow speed have never been

reported previously. DUV-LED could be a very promising alternative

technology for UV lamp in the field of controlling food-borne pathogens.

Moreover, applying to the real food sample and comparing disinfection

effectiveness of UV lamp must be studied in the future.

51

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Medical Association 288 (8), 951–953.

58

VI. 문초록

식품 살균 한 UV 사는 2000 미 FDA 에 해 승

았다. 지 지 UV 처리에는 253.7 nm

지닌 압 UV 램프가 사 고 다. 하지만 러한 램프 타

수 누 가능 , 짧 수 과 낮 에 지 효 문 를 갖고

UV 수 램프에 한 안 , DUV-LED 는 살균

야에 큰 심 고 다. 본 연 에 는 DUV-LED

본 특 악하고 고체 지에 뿐만 아니라 물에

UV-C 사 식 독 병원균 감화 효과를 확 하 다.

Escherichia coli O157:H7, Salmonella Typhimurium 과 Listeria

monocytogenes 균 혼합한 cocktail 각각 택 지에

도말하거나 처리 는 수 처리 샘플에 하 다. 연 결과,

도가 가함에 라 LED 는 큰 변화가 없는 , LP

램프는 약 30 °C 지 가 가한 후 다시 감 하는 경향

보 다. UV 사량 1.67 mJ/cm2 지 가할수 , 균

5 log CFU/ml 수 감 를 보 다. 처리 도가 가할

경우 Escherichia coli O157: H7 Salmonella Typhimurium

감화는 가 (P < 0.05) 하 나 Listeria

59

monocytogenes 는 감화 양상 보 지 않았다 (P >

0.05). 90% 상 습도 건에 E. coli O157: H7 만 30%,

60% 처리 환경과 비 하여 (P < 0.05) 감화 효과를

보 다. 3 mJ/cm2 UV 처리량 경우 10ml 수 처리에 는 든

병원균 검 한계 (1.0log CFU/g) 하 감화하 ,

스케 업 스트에 는 2, 3L 각각 처리 량에 균

5.38, 4.37 log CFU/ml 감 었고, 1L 처리량에 만 검

한계 하 감 하 다. 연 식 수 처리 시스 에 는

사량과 감화 효과 형 상 계를 나타내었다. 러한

결과를 통해 현재 개 단계에 는 DUV-LED 가 다양한

지니고 병원 균 감화하는 효과를

지니고 므 LP 램프를 체할 차 안 술

확 었 보여 다.

주 어: DUV-LED, 사, 식 독 균, 표 살균 수 처리,

친환경 술

학 : 2013-21176

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