For Heaters:

UV Lights in the heater are used to kill germs, viruses, mold spores, bacteria and fungi as they pass through the heater.

Ultraviolet (UV) light disinfection is getting a lot of attention during the coronavirus pandemic. The main benefit: its ability to kill pathogens like viruses and bacteria.

While UV-C is an extremely effective option for disinfecting, it does come with a safety warning. Many UV-C products use 254 nm, which can penetrate the skin and eyes. Exposure to UV-C can cause burns.

Most products should only be used in empty rooms, which can be challenging for specific industries where there is little downtime.

Right now, there is a threshold limit for the dose of UV in an occupied space over an eight hour period. That may limit the amount of disinfection provided to surfaces. Near UV can help reduce bacterial infection rates in medical facilities and senior care centers.

Killing bacteria and viruses with UV light is particularly effective because it kills germs regardless of drug resistance and without toxic chemicals. It is also effective against all germs, even newly-emerging pathogen strains.

These wavelengths are very close to the visible light spectrum and are believed to be safe for humans.The researchers concluded that the number of bacteria and virus reduction depended on how the device was used.
While it performed well overall (killing between 94.98% and 99.99% of germs), "education on the risks of incorrect application should be included in the travel medical consultation."

The UV light heater will protect you from germs, bacteria, viruses, and other destructive bio-aerosols.

For vegetable washers:

Lettuce is a popular and important leafy vegetable in the human

diet because of its delicious and nutritious characteristics. Accord

ing to a U.S. Dept. of Agriculture report, about 2 million tons

of lettuce were produced in the U.S. in 2014, and the value of

production was over 1 billion dollars (Natl. Agricultural Statistic

Service 2014). Fresh lettuce can be contaminated with foodborne

pathogens at farm by manure and irrigation water. Lettuce, as a

minimally processed and ready-to-eat (RTE) food product, can act

as a vehicle for transmitting foodborne disease. The Centers for

Disease Control and Prevention (CDC) reported a multistate Es

cherichia coli O157:H7 outbreak in 2011 which was directly linked

to romaine lettuce. According to CDC’s investigation, pathogenic

microorganisms such as E. coli O157:H7, Listeria monocytogenes,

Salmonella, and Cyclospora might attach to fresh vegetables during

farming and postharvest storages (CDC 2012). Therefore, an ef

fective washing method during postharvest handling is necessary to

enhance the safety of fresh produce, thereby reducing foodborne


Electrolyzed (EO) water is a strong and versatile antimicrobial

agent with a wide range of applications in medicine, agriculture,

and food industry (Hricova and others 2008). It is generated from

anodic electrolysis of dilute saline solutions (NaCl) in an elec

trolytic cell (Park and others 2004). During electrolysis, NaCl

in the saline solution is disassociated. The negative ions, such as

MS 20160122 Submitted 1/21/2016, Accepted 5/12/2016. Authors are with

the Dept. of Food Science and Technology, The Univ. of Georgia, 1109 Experiment

Street, Griffifin, GA 30223-1797, U.S.A. Direct inquiries to author Hung (E-mail:

chlorine (Cl¯) and hydroxide (OH¯), move to the anode, and

give up electrons. These ions form the components of EO wa

ter including chlorine gas (Cl2), hypochlorite ion (OCl¯), and

hypochlorous acid (HOCl) that contributed to its antimicrobial

activity. EO water is considered an environmental friendly san

itizer, because it reverts to normal water after use. In addition,

many studies have shown that EO water does not lead to negative

effects on the organoleptic properties such as color, aroma, flflavor,

or texture, in various treated food products (Hricova and others

2008; Waters and Hung 2010; Eda and others 2015).

Ozone is a powerful antimicrobial agent which can inactive

bacteria, virus, fungi, and spores at relatively low concentrations

(Alexopoulos and others 2013). The strong oxidation capability of

ozone could rapidly oxidize DNA, lipid, and protein of microor

ganisms, causing their death in a short time (Khadre and others

2001; Patil and others 2014). In 2000, the U.S. Food and Drug

Administration (FDA) approved both gas and aqueous ozone as

a direct food additive for treatment, storage, and processing of

foods. One of ozone’s special properties is that ozone is not sta

ble and can be easily transformed to oxygen in a few minutes,

making it an excellent sanitizer because it leaves no residues in

food (Khadre and others 2001). Several studies on ozone appli

cation have demonstrated that ozone can effectively inhibit the

growth of microorganisms, thus enhancing the safety and quality

of fresh produce (Alexopoulos and others 2013; Patil and others


Ultraviolet-C (UV-254nm) light has been applied with ozone

to enhance the inactivation of E. coli O157:H7. UV-C has strong

microbicidal properties and it does not leave any residues on

food. The FDA has approved UV-C for inactivation of microor

ganisms on food product surfaces (US-FDA 2011; Syamaladevi


2016 Institute of Food Technologists


doi: 10.1111/1750-3841.13364

Vol. 00, Nr. 0, 2016 Journal of Food Science M1

Further reproduction without permission is prohibited

M: Food microbiology

& safetyProduce washing using UV-ozonated water . . .

and others 2013). UV-C exposure causes irreversible damage to

bacterial DNA. It may have a synergistic effects when combined

with ozone for microbial inactivation (Otto and others 2011).

However, the UV-C radiation could be absorbed by ozone and

dissociate ozone molecules to form free oxygen atoms and oxy

gen molecules, which neutralize ozone (Vig 1985). Therefore,

one of the objectives of this study was to investigate if UV-C

can work with aqueous ozone and achieve a synergistic antimi

crobial effect on E. coli O157:H7. This study was also conducted

to evaluate the effificacy of using ozonated and slightly acidic EO

water (SAEW) as spray water to reduce E. coli O157:H7 on ro

maine and iceberg lettuces. The synergistic antimicrobial effect

of combined applications of UV and ozonated water was also


Materials and Methods

Bacterial culture

Three nalidixic acid-resistant strains E. coli O157:H7, includ

ing 932 (human isolate), 1 (beef isolate), and E009 (beef isolate),

were used as inocula. Each strain was propagated separately in

10 mL tryptic soy broth (TSB; Difco/Becton Dickinson, Sparks,

Md., U.S.A.) with 50 mg/L of nalidixic acid (Sigma-Aldrich, St.

Louis, Mo., U.S.A.) at 37 °C for 24 h. The use of nalidixic acid

resistant strains of E. coli O157:H7 was to differentiate from the

microflflora naturally present on lettuce samples in the presence

of nalidixic acid during sampling, thus facilitating the detection

and recovery of the inoculated pathogens. Following incubation,

each strain was individually transferred into a sterile centrifuge

tube and sedimented by centrifugation at 3900 × g at 5 °C for

15 min (Centra-CL3, Intl. Equipment Co., Needham Height,

Mass., U.S.A.). The supernatant was discarded and cell pellets

were resuspended in 10 mL of sterile 0.1 M phosphate buffered

saline (PBS, pH7). The optical density of resuspended bacterial

suspensions was measured using a spectrophotometer to insure all

3 strains had the same initial optical density. Equal volumes (3 mL)

of each strain suspension were combined in a sterile centrifuge

tube to form a 9 mL inoculum.

SAEW produce washer

SAEW at pH 6.0 were freshly generated by electrolyzing a di

lute NaCl solution (ca. 0.03%) using an EAU EO water generator

(Model #P30HST44T, EAU, Ga., U.S.A.) for each experiment.

The pH of SAEW was measured using an ACCUMET pH meter

(AR50, Fisher Scientifific, Pittsburgh, Pa., U.S.A.), and its free chlo

rine concentration was determined using the DPD-FEAS method

(Hach Co., Loveland, Colo., U.S.A.). Appropriate dilutions were

made to achieve 50 mg/L free chlorine concentration in SAEW.

About 100 L SAEW was prepared and stored in a water tank for the

spray experiments in a custom built spray cabinet as described in

Jadeja and Hung (2014). Instead of a handheld sprayer, the same

kind of nozzle (Full cone nozzle #460.526, Lechler Inc., West

Chicago, Ill., U.S.A.) used in the UV-ozonated water treatment

was used. The distance between the nozzle and lettuce samples

was set at 20 cm. SAEW was sprayed at 1.5 L per min.

UV and ozonated water produce washer

A UV-chilled ozonated water washer (UV-ozone washer,

Sanichill Prototype Operating System; Sanist Technologies LLC,

Pittsburgh, Pa., U.S.A.) was used in this study. The refrigeration

system, ozone gas generator, and 2 UV-C (254 nm) lamps (UV Air

Lamp Assembly ASIH1003 17 T3; Light Spectrum Enterprises

Inc., Feasterville-Trevose, Pa., U.S.A.) were 3 major components

of this washer (Figure 1). The refrigeration system maintained

the temperature of wash water at 4 °C. The UV-C intensity at

1.19 mW/cm2 measured by a UV radiometer (Model UVX-25;

UVP LLC, Upland, Calif., U.S.A.) was applied in this study. The

distance between the UV lamps and the lettuce samples was 20

  1. There were 6 nozzles (Full cone nozzle #460.526) in the

spray chamber. To reduce water consumption, 2 water tanks were

set up in this system to recycle water. The ozonation tank (1st

tank) was used to continuously generate ozonated water by inject

ing ozone gas from the ozone generator, and the recycling tank

(2nd tank) was used to collect spray water from the spray chamber.

The recycled water was fifiltered and regenerated in the ozonation

tank. The concentration of O3 in the ozonated water (0.5 mg/L)

was determined using the Indigo method with high-range ozone

Accu Vac Ampuls (Hach Co., Loveland, Colo., U.S.A.) using a

Figure 1–The UV-chilled ozonated water washer


M2 Journal of Food Science Vol. 00, Nr. 0, 2016

M: Food microbiology

& safetyProduce washing using UV-ozonated water . . .

Table 1–Antimicrobial effect of SAEW on romaine and iceberg lettuce with different holding time after inoculation.

Holding timea

2 h 26 h

SAEW treatment Recovery Reduction Recovery Reduction

Lettuce type Treatment time (min) (log CFU/g) (log CFU/g) (log CFU/g) (log CFU/g)

Romaine lettuce Control A6.17


0.08a A6.25



5 A2.70


0.21a 3.5 A3.20


0.69a 3.0

10 A1.37


1.06a 4.8 A2.76


0.72a 3.5

15 A1.59


0.48a 4.6 A2.70


0.41b 3.6

Iceberg lettuce A6.12


0.08a A6.14



5 B4.34


0.13a 1.8 B4.04


0.66a 2.1

10 B3.70


0.59a 2.4 B3.99


0.90a 2.2

15 B3.86


0.51a 2.3 B3.23


0.18a 2.9

a Mean values with the same capital letter in each column with the same treatment time indicates no signifificant difference between romance and iceberg lettuce (P > 0.05). Mean

values with the same lowercase letter in the same row are not signifificantly different (P > 0.05).

Table 2–Antimicrobial effects of UV, O3 water, and their combination on romaine lettuce.a

Outside Inside

Treatment time Recovery Reduction Recovery Reduction

Treatment method (min) (log CFU/g) (log CFU/g) (log CFU/g) (log CFU/g)

UV Control A6.11


0.07a A5.81



5 A4.18


0.23a 1.9 A3.91


0.84a 1.9

10 A4.17


0.43a 2.0 A3.45


0.42a 2.4

15 A3.97


0.31a 2.1 A3.04


1.06b 2.8

Ozonated water A6.16


0.16a A6.27



5 A4.12


0.36a 2.0 AB3.26


0.01a 3.0

10 A3.46


0.32a 2.7 A3.89


0.31a 2.4

15 A3.43


0.28a 2.7 A3.47


0.21a 2.8

UV + ozonated water A5.97


0.16a A5.97



5 A3.77


0.67a 2.2 B2.31


1.17a 3.7

10 B1.45


1.35a 4.5 B2.02


0.69a 4.0

15 B1.14


0.79a 4.8 B1.10


0.60a 4.9

a Mean values in each column at the same treatment time with the same capital letter indicates no signifificant difference between treatment methods (P > 0.05). Mean values for the

same treatment time and method that are followed by the same lowercase letter in the same row indicates no signifificant difference between inoculation locations (P > 0.05).

colorimeter (model DR/890; Hach Co.). The washer was turned

on for 10 min before every treatment to allow the ozone in the

water to reach a steady level.

Preparation and inoculation of romaine and iceberg


Romaine (Lactuca sativa L. var. longifolia) and iceberg (Lactuca

sativa L.) lettuces were purchased from a local grocery store, stored

at 4 °C, and used within 48 h. The outer 2 or 3 damaged lettuce

leaves were discarded, and the next few intact leaves were collected

and trimmed to 20 ± 1 g/leaf. Sample leaves were placed on a

sanitized tray with either outer (abaxial) side or inside (adaxial)

side surface facing up and spot inoculated with 100 µL of the

  1. coli O157:H7 cocktail by placing 15 to 20 inoculum drops on

the surface of each leaf. To study the effect of drying time (2 vs. -

26 h) after inoculation on microbial inactivation, both romaine

and iceberg lettuces were inoculated on the outer surface and

then air-dried in a laminar flflow hood for 2 h at room temperature

(22 ± 2 °C). Half of the lettuce leaf samples was immediately

treated with SAEW, whereas the remaining half of the samples

was covered with plastic wrap and held at 4 °C for additional

24 h before SAEW treatment. The inoculated romaine lettuce

leaves were also used to study the effects of inoculum location

(outer vs. inside surface) on bacterial inactivation after 2 h drying

at room temperature (22 ± 2 °C). Furthermore, the effificacy of

different washing treatments (for example, UV, ozonated water,

SAEW, and a combination of UV and ozonated water) was also


Procedures for treating lettuce

Romaine and iceberg lettuces inoculated with E. coli O157:H7

only on the outer surface and dried for either 2 or 26 h in a lam

inar flflow hood were treated with SAEW for 5, 10, and 15 min.

Treated leaves were placed into a 1.5 L of Whirl-Pak bag contain

ing (Nasco, Fort Atkinson, Wis., U.S.A.) 80 mL of Dey-Engley

(DE) neutralyzing broth (Difco/Becton Dickinson) to stop the

SAEW reaction before subjecting to microbiological analysis.

Romaine lettuce leaves, inoculated with E. coli O157:H7 on

both outside and inside surfaces, were treated with UV, ozonated

water, and their combination. Two lettuce leaves were placed side

by-side directly under the UV light and ozonated water spray

nozzles for each treatment. Each treatment had 3 treatment times,

5, 10, and 15 min. Treated leaves were immediately placed into a

1.5 L Whirl-Pak bag (Nasco, Fort Atkinson, Wis., U.S.A.) with

80 mL of DE broth before subjecting to microbiological analysis.

Both romaine and iceberg lettuce inoculated with E. coli

O157:H7 on the outside surface only were treated with either

SAEW or UV-ozonated water combination for 5, 10, and 15 min.

After treatments, the treated leaves were placed in a 1.5 L of Whirl

Pak bag with 80 mL of DE broth before subjecting to microbio

logical analysis.

Vol. 00, Nr. 0, 2016 Journal of Food Science M3

M: Food microbiology

& safetyProduce washing using UV-ozonated water . . .

Microbiological analysis

After washing treatment, all lettuce samples in DE broth were

pummeled in a stomacher (Stomacher 400 Circulator, Seward,

London, U.K.) for 3 min at the speed of 230 rpm. The samples

were serially diluted with PBS and plated onto Sorbital Mac

Conkey agar (Hardy Diagnostics, Santa Maria, Calif., U.S.A.)

containing 50 µg/mL nalidixic acid (SMACN) to determine the

surviving population of E. coli O157:H7. Undiluted DE/lettuce

homogenates (250 µL) were spread-plated in quadruplicate onto

SMACN, to enumerate the low surviving populations of E. coli

O157:H7 from lettuce samples.

Data analysis

Experiments were replicated at least 3 times and each repli

cate consisted of 2 samples for each treatment. Means of bacterial

counts were calculated as log CFU/g using Microsoft Excel


One way analysis of variance was used for data analysis followed

by Duncan’s multiple range test to compare means using SAS soft

ware 9.4 (SAS Inst. Inc., Cary, N.C., U.S.A.) with the proc anova

procedure. A P 0.05 was considered to be statistically signifificant.

Results and Discussion

Antimicrobial effect of SAEW

The pH, oxidation reduction potential, and free chlorine con

centration of SAEW are 6.08 ± 0.19, 949 ± 21 mV, and 51


0.6 mg/L, respectively. Table 1 shows the surviving popula

tion of

  1. coli O157:H7 on romaine and iceberg lettuce after

SAEW treatments. With a 2 h drying time after inoculation, a

reduction of 3.5 and 1.8 log CFU/g in E. coli O157:H7 was ob

served in romaine and iceberg lettuce, respectively, after a 5 min

treatment with SAEW. Longer treatment (10 and 15 min) led

to an additional 1 and 0.5 log CFU/g reductions in the bacterial

counts in romaine and iceberg lettuce samples, respectively. With a

longer drying time (26 h) after inoculation, a reduction of 3.0 and

2.1 log CFU/g in the bacterial counts were observed in romaine

and iceberg lettuce after a 5 min treatment with SAEW. Longer

treatment times (10 and 15 min) led to an additional 0.5 log

CFU/g in the reduction of bacterial counts in romaine lettuce.

For iceberg lettuce, a 10 min treatment did not cause additional

inactivation of the bacteria, although a 15 min treatment led to

an additional reduction of 0.8 log CFU/g of E. coli O157:H7.

A similar result was reported by Afari and other’s (2015), which

stated that the reduction of E. coli O157:H7 on romaine lettuce

was higher than on iceberg lettuce by 1 log when both samples

were treated with SAEW.

Shown in Table 1, no signifificant difference was observed on the

reduction of E. coli O157:H7 for both romaine and iceberg lettuce

regardless of holding time after inoculation (2 h vs. 26 h) except

romaine lettuce treated by SAEW for 15 min. Therefore, holding

time (2 h vs. 26 h) had little impact on the viability or attachment

of E. coli O157:H7 on lettuce. In other studies, similar observation

was also reported (Farber and others 1998; Lang and others 2004).

Hence, the rest of the experiments in this report were conducted

using only samples with 2 h holding time after inoculation.

Antimicrobial effect of UV, ozonated water, and their


Table 2 shows the surviving population of E. coli O157:H7 on

romaine lettuce after the treatments with UV, ozonated water, and

their combination. For romaine lettuce, similar E. coli O157:H7

reductions were achieved for the same treatment regardless of the

inoculum location (inside or outside surface). For all types of treat

ments (UV, ozonated water, and their combination), there was no

signifificant difference in the reduction of E. coli O157:H7 between

the samples with inside and with outside surface inoculation, ex

cept for the UV-C treatment for 15 min. Therefore, for the rest

of the study only lettuce leaves inoculated on the outside surface

were used.

UV-C treatment for 5 min resulted in about 1.9 log reduc

tions for both inside and outside surface inoculated romaine let

tuce. However, no further signifificant increase on reduction was

observed after the samples were treated for longer time (10 or

15 min). This may be due to the limitation of penetration depth

of UV-C and bacteria hiding in debris, shaded areas due to the

topology of leaves, or internalized in stomata and cut edges.

A 5-min treatment by ozonated water was able to reduce the

population of E. coli O157:H7 by 2 logs for the leaves inoculated

on the outside surface, whereas a 3 log reductions was observed for

the leaves inoculated on the inner surface. Extending the treatment

to 10 min resulted in an additional 0.7 log reductions for leaves

Figure 2–The comparison of SAEW and

UV-ozonated water combination antimicrobial

effects on romaine and iceberg lettuce. Error

bars represent standard deviations. Capital

letters not followed by the same letter in the

same treatment time indicate signifificant

difference (P 0.05).

M4 Journal of Food Science Vol. 00, Nr. 0, 2016

M: Food microbiology

& safetyProduce washing using UV-ozonated water . . .

inoculated on outside; however. no further reduction was observed

beyond 10 min treatment time. This indicates that ozonated water

are more effective than UV-C treatment at longer treatment time

(10 min). This may be due to ozonated water can wash away

bacteria and organic matter residing on the surface of leaves that

offers protection to bacteria from UV-C lights. Ozonated water

may also able to reach to the shaded areas of leaves that could

not be reached by UV-C light due to the fifixed projection angle.

In addition, ozone gas might vaporize from the ozonated water

and penetrate lettuce surfaces. However, the concentration of the

ozonated water (0.5 mg/L) used in this study may be too low and

hence no further signifificant reduction was observed for longer

treatment time.

According to Table 2, the combination of UV and ozonated

water treatment had signifificant higher reduction than either UV

or ozonated water treatment alone. Although no signifificant differ

ence was observed in the surviving population of E. coli O157:H7

on romaine lettuce among 3 treatments at 5 min, the combi

nation of UV and ozonated water achieved an additional 1.8 to

2.5 log CFU/g reductions at 10 min treatment and 2.1 to 2.7

log CFU/g additional reductions at 15 min of treatment when

compared to either UV or ozonated water treatment alone for the

same treatment time. The synergistic effect of UV and ozonated

water combination treatment may be due to ozonated water plus

ozone gas released from ozonated water can get to the folded

leaves where UV-C light was not able to reach. Therefore, the

combination treatment of UV and ozonated water could deliver a

close to 5 log reductions of E. coli O157:H7 on romaine lettuce

compared to 2 to 3 log reductions using UV or ozonated water

treatment alone.

Comparison between UV-ozonated water combination

and SAEW

The antimicrobial effect of UV-ozonated water combination

and SAEW on both romaine and iceberg lettuces are presented in

Figure 2. For romaine lettuce, both treatments were able to deliver

close to 5 log reductions on E. coli O157:H7 at 10 and 15 min

treatments. However, for iceberg lettuce, a signifificant difference in

microbial inactivation was observed between UV-ozonated water

combination treatment and SAEW. UV-ozonated water combina

tion could achieve similar antimicrobial effect on iceberg lettuce

as compared to the romaine lettuce, whereas SAEW can only

achieve up to 2.5 log reductions on iceberg lettuce regardless

of treatment time. This difference may be due to the different

behavior of bacteria on romaine lettuce compared with iceberg

lettuce (Kroupitski and others 2009; Kroupitski and others 2011).

Kroupitski and others (2009) inoculated Salmonella Typhimurium

onto the surface of both romaine and iceberg lettuce leaves and

observed the distribution of bacteria using both scanning electron

microscopy and confocal microscopy. They found that bacteria

tend to aggregate near and into stomata of iceberg lettuce leaves,

whereas no such attraction of bacteria to stomata was found on

romaine lettuce leaves. In this study, lower antimicrobial effificacy

on iceberg lettuce than romaine lettuce for SAEW may be due to

SAEW cannot reach/penetrate into the stomata on iceberg lettuce

where the bacterium was internalized. However, for UV-ozonated

water combination, UV can stimulate the opening of the stom

ata as suggested by Eisinger and others (2000) and hence allowed

ozonated water and/or ozone gas to inactivate bacteria that may

be internalized in the stomata. Hence, a 5 log reductions of E. coli

O157:H7 using UV-ozonated water combination was observed

for both iceberg lettuce and romaine lettuce. Similar results were

reported by Afari and others (2015). They used neutral pH EO

water (NEO, 155 mg/L free chlorine, pH 7.5) in an automated

produce washer at 65 rpm for 30 min and achieved 4.2 and 3.2 log

CFU/g reductions for romaine and iceberg lettuce, respectively.

They demonstrated that when washing romaine lettuce with NEO

achieved additional one log reduction than iceberg lettuce.


This study demonstrated both UV-ozonated water combina

tion and SAEW washing treatments has strong antimicrobial effect

and achieved approximately 5 log reductions of E. coli O157:H7

on romaine lettuce. UV-ozonated water combination treatment

achieved similar reductions on iceberg lettuce whereas less than

2.5 log reductions on E. coli O157:H7 were achieved by SAEW.

This difference may be due to bacteria aggregation near and within

stomata for iceberg lettuce but not romaine lettuce. The UV light

will stimulate the opening of the stomata for the UV-ozonated wa

ter treatment and hence achieve better bacter