whole room disinfection

R&D report no. 299

Members only

Whole room disinfection

2010

Campden BRI 2010

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R&D report no. 299

Whole room disinfection

A Malinowska and J Holah

2010

SUMMARY

To meet retailer, customer and consumer expectations, there are increasing demands within

the food industry for higher standards of control of microorganisms in food production

environments. This requirement for further reduction of pathogens and the identification of

persistent strains has led to a significant interest in the use of whole room disinfection

techniques to supplement routine cleaning and disinfection.

A range of whole room decontamination systems are available commercially, including

ultraviolet light, titanium dioxide and ultraviolet light, ozone, hydrogen peroxide vapour

(HPV) and ionisation, but these techniques have had little microbiological assessment for

their use in the food and drink industry.

This report describes work undertaken to investigate the efficacy of whole room disinfection

on microorganisms attached to surfaces both in the laboratory and in the factory

environment. The microorganisms assessed were Listeria monocytogenes, Pseudomonas

aeruginosa and Staphylococcus aureus, selected because of their known presence, and in

some cases persistence, in the food factory environment. Bacillus subtilis var. globigii was

also assessed with hydrogen peroxide to test its sporicidal activity. This work was also

undertaken to examine the effect of whole room disinfection techniques on surfaces placed

in different spatial orientations.

Chemical fogging using quality disinfectants was effective at reducing airborne microbial

populations by 2 to 3 log orders in 30 to 60 minutes and attached microorganisms on

horizontal surfaces by up to 6 log orders in 60 minutes, with minimal effect on vertical

surfaces and underneath equipment. The amount of chemical released by nebulisers is so

small (relative to chemical fogging), that the level of decontamination achieved on surfaces

is poor. However, the particle size of chemical produced appears to be small enough to

interact with surfaces of all orientations, over a longer time period in which the particles

remain airborne, so what decontamination it does achieve is approximately the same over

all surface geometries.

At low concentrations of (10 g/m3), the effect of HPV was much more pronounced on the

spores of B. subtilis var globigii than on the vegetative bacteria tested, with an approximate

6 log reduction of globigii spores being achieved. At concentrations of 20 g/m3, an

approximately 3 log reduction was achieved with P. aeruginosa, S. aureus and L.

monocytogenes. At this concentration of HPV, the vegetative microorganisms (all of which

are catalase positive) were all able to partially resist the concentration/volume of disinfectant

that they came into contact with. Spores, however, do not express catalase and therefore

have no effective defence mechanism. When the HPV concentration was increased to 40

g/m3, an increase in susceptibility was seen with P. aeruginosa and L. monocytogenes,

resulting in a reduction in excess of 4 logs for P. aeruginosa and 5 logs for L.

monocytogenes. This is in excess of the 4 log reduction required in the European Surface

Test (EN 13697) for surface disinfectants. S. aureus was significantly more resistant than

the other vegetative organisms and its log reduction was not increased at 30 or 40 g/m3.

For ozone, a relationship between ozone concentration and log reduction was established,

though the effect of ozone on each of the three vegetative strains tested was markedly

different with, at 20ppm, S. aureus being most resistant followed by P. aeruginosa and then

L. monocytogenes being most sensitive. A log reduction of >4 logs was achieved for

L. monocytogenes at an ozone concentration of 20ppm, which equates to the requirements

of the European Surface Test (EN 13697) for chemical disinfectants. A log reduction

approaching 4 logs was also achieved for P. aeruginosa. This would suggest that under the

conditions of the practical trials, ozone is more effective than HPV for vegetative

microorganisms.

For both HPV and ozone, there is little practical difference between the log reductions

achieved on horizontal, vertical and underneath surfaces and as such, they can effectively

penetrate every part of a room, including sites that might prove difficult to gain access to

with conventional liquids and manual disinfection procedures. The major disadvantage of

using gases, such as ozone and HPV, is the potential toxicity at high concentrations, which

precludes using them in areas where people are working. The techniques can therefore

only be used in areas that can be isolated and sealed off during the decontamination

process.

Field trials in which ozone was used as a periodic room decontaminant showed little effect

over 1-3 days using an ozone concentration of 8ppm, and the overall reduction in counts

after disinfection was probably less than 1 log order. After 3 days, however, the results for a

pizza manufacturer potentially showed a downward trend in the numbers of microorganisms

present, both before cleaning and after cleaning and disinfection, throughout the three day

period. This may be because for all areas in which ozone is used, oxidisable material within

the room may create an ozone demand which must be satisfied by oxidation before any

significant decontamination of microorganisms can occur. A dosage equivalent to at least

90 ppm for an hour (e.g. 30 ppm for 3 hours), as postulated from the laboratory studies

undertaken, may be required to produce a 5-6 log reduction in vegetative microorganisms

(e.g. L. monocytogenes) as a periodic room decontaminant.

In a 4 week field trial, there was no evidence that ozone was unable to maintain control of

the microflora of the food contact surfaces after disinfection. TVC counts after disinfection

for ozone treated food contact surfaces compared favourably to the post disinfection counts

of combined disinfectant approval trials conducted at other chilled food plants. No adverse

effects have been reported for ozone on the structure and fabric of the building since the

ozonation equipment has been installed. A daily ozone treatment of 8ppm for 30 minutes

may therefore be appropriate as an adjunct to or replacement of chemical disinfection,

though the use of ozone as a replacement for chemical disinfection must be appropriately

validated for each factory situation.

CONTENTS

Page No.

1. BACKGROUND 1

2. INTRODUCTION 1

3. MATERIALS & METHODS 6

3.1 Laboratory trials 6

3.1.1 Chemical fogging 9

3.1.2 Hydrogen peroxide vapour 10

3.1.3 Ozone 11

3.2 Field trials 12

3.3 Statistical analyses 18

4. RESULTS AND DISCUSSION 18

4.1 Pre-trial 23

4.2 Laboratory trials 25

4.2.1 Chemical fogging 25

4.2.2 Hydrogen peroxide vapour 28

4.2.3 Ozone 31

4.3 Field trials 36

5. CONCLUSIONS 43

6. REFERENCES 47

APPENDICES

Appendix 1 Media recipes

Appendix 2 Laboratory and field trial data

Appendix 3 Equipment manufacturer and contact details

1

1. BACKGROUND

During manufacture, food can be exposed to microbiological cross-contamination from

surfaces and the air, which may give rise to food spoilage and safety issues. The traditional

approach to controlling such contamination has been to target specific sites within the

manufacturing environment with cleaning and disinfection regimes. The primary focus is

typically on food production equipment and much of the rest of the processing area, whilst

cleaned, is not routinely decontaminated. This targeted approach may have been sufficient

to maintain day-to-day control of contamination, but has not eliminated all of the organisms

responsible and, in some instances, microbial strains have become persistent in food

factories, surviving for several years. Previous research at Campden BRI assessed the

microbial flora of the high risk areas of five chilled food factories, all operating good practice

cleaning and disinfection regimes, and identified persistent strains of Listeria spp. and

Escherichia coli that had remained in the processing environment in excess of three years

(Holah et al., 2002; Holah et al., 2004). Clearly, if there was any loss of hygiene control in

these factories, these organisms would always be present and therefore could present a

constant risk to product safety.

In high risk food processing areas, thorough disinfection of surfaces is required in order to

reduce the numbers of microorganisms and to prevent transmission of these contaminants.

The belief is that regular use of novel disinfection techniques that can decontaminate the

whole of the area will reduce the number of environmental microorganisms in the production

areas; this may well improve the quality and safety of the food being produced, thereby

reducing wastage and increasing profitability.

2. INTRODUCTION

There is now a desire to supplement traditional, targeted chemical disinfection with

alternative approaches which will control the processing area in its entirety, a technique that

can be termed as whole room disinfection. Novel disinfection techniques that are able to

disinfect whole areas have been implemented in the pharmaceutical and clinical sectors, but

there is limited information on the ability of these techniques to be applied to the food

processing environment.

The range of techniques designed for whole room disinfection is increasing, but those that

are commercially available include:

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- chemical fogging

- hydrogen peroxide vapour

- ozone

- chlorine dioxide

- ultraviolet light

- titanium dioxide coating and ultraviolet light

- ionisation

The critical factors to address before using these techniques include: identifying areas

where the decontamination processes can be applied, health and safety issues related to

using the technique, any effect on the fabric of the equipment and the environmental

building materials, and the practical considerations related to their use in the food

processing environment.

There is also a need to understand how often a whole room disinfection method will be used

in the production area. The techniques can be used on a daily basis, after the routine

cleaning and disinfection procedure has been implemented or, as seen in some factory

environments, they can be used daily to replace the terminal disinfection step. There is also

the option to use a whole room disinfection technique as part of the periodic cleaning and

disinfection procedures which may occur monthly, quarterly or annually, or they may only be

used for decontaminating an area after a pathogen contamination incident.

The level of disinfection that the whole room disinfection systems can achieve also needs to

be determined, as some may achieve decontamination of all exposed room surfaces, such

as ceilings, walls, floors and food processing equipment, but others may include some

penetration into equipment to contact indirectly exposed surfaces. They may also provide

disinfection of the air in the area being treated. The advantages of using these techniques

are that for some, the decontamination process can be certified, providing documented

evidence that the procedure has taken place.

Chemical fogging, nebulisers, hydrogen peroxide vapour (HPV) and ozone (O3) were

studied in this work.

Chemical fogging

Applying chemical disinfectants to production areas as fogs or mists is a method that has

been used routinely in the food industry to control cross contamination. The purpose of

fogging a production area is to create and disperse a disinfectant aerosol to reduce the

numbers of airborne microorganisms and also to apply disinfectant to surfaces that may be

difficult to reach, such as overhead surfaces. Fogging is done by using either a static,

3

purpose built system in an area of a factory with strategically placed nozzles, or more

commonly by using a mobile unit. The equipment works by supersaturating the atmosphere

with a fog of disinfectant chemical.

The disinfectant fog is generated into the local atmosphere by charging the unit with

compressed air and forcing the disinfectant solution through dedicated nozzles, producing

particles in the range of 10 to 20 m. Research carried out by Burfoot et al. (1999)

demonstrated that fogging was most effective when using compressed air-driven fogging

nozzles producing fog droplets with a median diameter of between 10 and 20 m, as the

droplets in this size range dispersed well and settled within 45 minutes. An air velocity of

100 m s-1 was required at the nozzle for effective dispersal of the chemical. Larger particles

can be used if the air velocity is increased or fans are used to assist with the distribution of

the droplets. Under typical conditions, fogging is carried out for a minimum of 15 to 30

minutes to enable the fog to disperse and the chemical action to occur and after fogging, a

period of 45 to 60 minutes is required to allow the droplets to settle out of the air and onto

the surfaces, reducing the risk of operators inhaling the chemical droplets.

Disinfectants that can be fogged include quaternary ammonium compounds (QACs),

amphoterics and peracetic acid (PAA). Typically, a formulated QAC based disinfectant will

be used for fogging at concentrations between 1 and 3%. However, if the site has a

problem with spores, a QAC disinfectant will not be as effective and an option would be to

use a PAA based disinfectant at 1% concentration. PAA is ideal for fogging, due to its

natural breakdown into water and a low concentration of acetic acid and therefore many

applications do not require rinsing.

Areas of application

Chemical fogging is primarily carried out in high risk processing environments, particularly in

factories manufacturing cheese, salad, sandwiches, ready meals and cooked meats, and in

dairies. It is estimated that more than 50% of chilled food manufacturers conduct this

method of disinfection periodically, in conjunction with the routine cleaning and disinfection

routines. Within the production environment, applications include freezers, chillers, ripening

rooms, storage areas and process lines.

Hydrogen peroxide vapour

Due to its rapid degradation into innocuous by-products, decontamination with hydrogen

peroxide vapour (HPV) is a technique that has been widely used for disinfection of the

pharmaceutical environment, including clean rooms and production filling lines, and

therefore it may be an alternative to chemical fogging for the food industry. HPV is said to

have excellent material compatibility and is safe for use on a wide range of metals, including

4

stainless steel and aluminium, plastics such as polypropylene and polycarbonate, and other

materials, such as electronics. However, the efficacy of HPV is reduced by the presence of

organic matter; therefore, cleaning the area prior to use is essential. The disadvantage of

using HPV is the potential toxicity at high concentrations, which prevents the technique from

being used in areas where people are working. Therefore the decontamination process can

only be used in rooms that can be sealed off or quarantined for the duration of the treatment.

Mobile systems can be used throughout the factory environment, or areas can be equipped

with ports to which the equipment can be docked while the decontamination procedure is

carried out. The most commonly used aqueous solution of H2O2 is 30 or 35% w/w, which is

frequently evaporated to produce H2O2 concentrations ranging from 0.1 to 10 mg L-1

(0.00001 to 0.001%) depending on the exposure temperature, which ranges from 4 to 80oC.

The HPV is applied to the room in a heated carrier gas, initially air, and vapours from the

room are returned to the gas generator where further quantities of the H2O2 are evaporated.

As more vapour is introduced into a room, the pressure and concentration of the

peroxide/water vapour will increase. The gaseous state is therefore advantageous because

it ensures uniform contact with all surfaces, including horizontal and vertical surfaces and

cracks. The term 'dry fog' is sometimes used to describe the vapour with droplet sizes

below 10m. Hydrogen peroxide gas diffuses passively when introduced into a given area

and therefore constant movement of the gas is required to ensure that all surfaces are

contacted. This can be aided at atmospheric pressure by using fans or air-handling

systems, or by introducing a slight positive or negative pressure in the area being fumigated.

Hydrogen peroxide demonstrates a broad spectrum of efficacy against viruses, bacteria,

mycobacteria, fungi and bacterial spores. Its activity as a powerful oxidising agent is known

to damage cellular proteins, lipids and nucleic acids, and the increased reactivity of the

gaseous peroxide may be due to the greater presence of the short lived radicals and ions

that form. It is more effective against Gram positive than Gram negative bacteria; however,

the presence of catalase or other peroxidases, particularly in Gram positive bacteria, such

as Staphylococcus spp., allows increased tolerance, due to enzymatic degradation. Elkins

et al. (1999) carried out a study to assess the significance of catalase expression in the

protection of Pseudomonas aeruginosa against H2O2 and demonstrated that KatA catalase

was important for resistance of planktonic and biofilms of P. aeruginosa to H2O2, particularly

at high concentrations. The sporicidal concentration for HPV has been identified in the

range of 0.1 to 3.0 mg L-1 at room temperature (Meszaros et al., 2005).

Ozone

This chemical has been used for decades for water treatment, as it inactivates a wide range

of micro-organisms through oxidation, but the benefit of using ozone in the food industry is

that it is environmentally friendly, with any residual ozone spontaneously decomposing to

5

oxygen.

Ozone is a triatomic form of oxygen, which is unstable and breaks down into molecular

oxygen. The half-life for this reaction is approximately 20 minutes and this rapid process of

natural degradation is both an asset and a liability. On the plus side, it means that the

ozone disappears literally without a trace, leaving no chemical by-products, but on the minus

side, it means that ozone has a very short effective life. High reactivity, penetrability and

spontaneous decomposition into a non-toxic product make ozone a viable disinfectant for

use in food production areas.

Due to its reactive, unstable nature, ozone is produced at the point of use. Ozone

generators effectively pass air through a high-energy source within the equipment and the

resulting physicochemical reaction leads to the formation of ozone that can be used for area

or surface decontamination. Widely used high-energy sources include UV light,

electrochemical cells or corona discharge. A corona is formed by an electrical discharge

around a gas, which causes ionisation and consequently the formation of ozone. The

production of ozone is most effective in a temperature-controlled environment, since the

stability of ozone decreases as the temperature increases.

Microorganisms inherently vary in their sensitivity to ozone with factors such as temperature,

humidity and presence of chemicals; the amount of organic matter surrounding the cell also

greatly affects the degree of inactivation. At the concentrations typically used, ozone is an

effective bactericide and virucide, with greater resistance observed with mycobacteria and

bacterial spores. Effective sporicidal activity is only seen at high relative humidity (75 to

95%). Yeasts and moulds have been reported to have a wide range of resistance profiles

but are generally less resistant than bacterial spores.

Work by Fan et al. (2007) showed that the average time for a 2 log reduction of Listeria

innocua on solid media was 1.3 hours at 20oC, and 2.5 hours at 5oC at 100 nl L-1 ozone

concentration. Work by Taylor and Chana (2000) indicated a 2 log reduction in both

airborne and surface adhered Pseudomonas aeruginosa in 2 h when exposed to 2 ppm

ozone.

6

3. MATERIALS & METHODS

3.1 Laboratory trials

The purpose of the laboratory trials was to develop methods to examine whether the

systems under assessment were able to decontaminate all surfaces, irrespective of

orientation, throughout the whole room. The laboratory trials protocol was based on the

European Norm surface disinfectant test method BS EN 13697: 2001 - Chemical

disinfectants and antiseptics - Quantitative non-porous surface test for evaluation of the

bactericidal activity and/or fungicidal activity of chemical disinfectants used in food,

industrial, domestic and institutional areas.

There are various types of chemical fogging, hydrogen peroxide vapour and ozone

techniques that are commercially available; representative techniques were used in this

study. Each whole room technique available, however, will differ slightly in its application,

such that there may be small differences in their overall performance. The results from the

techniques used in this study are thus specific to these techniques, but are generally

reflective of the performance of that technology.

Preparation of stock and working cultures

Cultures of Staphylococcus aureus (NCIMB 9518), Pseudomonas aeruginosa (NCIMB

10421) and Listeria monocytogenes (NCTC, NCI03 57-09) were maintained on Tryptone

Soya Agar slopes (TSA; Oxoid CM 131; Lab 305), stored at 4C, and were re-cultured from

beads every month to maintain viability. When a working culture was required, the culture

was sub-cultured onto a TSA slope and incubated for 24 hours at 37C. A first subculture

was used as the working culture and was recovered from the slope by adding 5g of sterile

glass beads (VWR International Ltd., BDH, 2.5-3.5 mm) and 9ml of diluent (BS - see

Appendix 1) to each slope. The slopes were then shaken gently to remove the culture from

the agar surface. The resulting suspension was then filtered through a funnel containing

sterile glass wool and eluted further with diluent to maximise recovery. The optical density

of the bacterial suspension was measured at 420nm (Spectrophotometer Libra S4,

Biochrom Ltd.) and calibration graphs of absorbance against viable count were used to

prepare a concentration of 108cfu ml-1. A 1ml volume of this bacterial suspension was

transferred into 9ml of diluent (10-1 dilution) for further dilution, as required.

Inoculation of surfaces

For each test, 25 stainless steel discs (2 cm diameter, Grade 2 B 1.4301 (EN 10088-1),

EN 10 088-2), previously sterilised, were inoculated with 0.05ml of 108cfu ml-1 test

suspension. The suspension was dried onto the discs at a temperature of 37C for

7

approximately 1 hour. The discs were allowed to equilibrate to room temperature before the

test was commenced. A total of 20 discs were treated and 5 discs were left untreated

(positive controls).

Microbial coverage

Epifluorescence microscopy was used to show the attachment and distribution of S. aureus,

P. aeruginosa and L. monocytogenes dried onto stainless steel discs. Three coupons were

inoculated with 0.05ml of 108cfu ml-1 test suspension of each test microorganism and dried

at a temperature of 37C for approximately 1 hour. The discs were allowed to equilibrate to

room temperature and then stained with Acridine orange (HD Supplies, UK) for 3 minutes.

The stain was then rinsed of using sterile distilled water (SDW). The discs were then left to

dry in the dark and analysed using a x100 oil immersion lens connected to a microscope

(Leica DM 2000) with UV light system (ebq 100) and Leica's Image Processing and Analysis

toolkit (Leica QWin) running under the industry standard Microsoft Windows environment.

Experimental set-up

The experiments were conducted in an aerobiology laboratory, 350 cm wide, 405 cm long

and 300 cm high (volume 43 m3). The 20 discs were positioned on metal stands throughout

the air laboratory in three orientations: horizontally, vertically and underneath the shelf, as

shown in Figure 1. Five discs were left untreated and processed at the same time as test

discs.

8

Figure 1 - The arrangement of test surfaces in the aerobiology laboratory

During experiments, all ventilation systems in the laboratory were switched off and the room

was effectively sealed to outside air movements. Any internal air currents during the trials

were created by the operation of the technology under test.

Disc analysis

After conducting the whole room disinfection process, the discs were aseptically transferred

into sterile plastic universal containers (Sterilin, diameter 4-5cm) containing 5g sterile glass

beads (diameter 2.5-3.5 mm) and 9ml diluent and 1ml sodium thiosulphate inactivator (see

Appendix 1). The containers were agitated on a horizontal surface for 1 minute to recover

the remaining bacteria into suspension. Each sample was serially diluted in diluent to 10-4

and plated out in duplicate using TSA. To validate the bacterial recovery process, each disc

was recovered from its container and rinsed with 10 ml sterile distilled water (SDW). Each

disc was then placed test side up on a pre-poured TSA agar plate and 0.1 ml SDW was

pipetted onto the disc and rubbed over the surface with a pipette tip for 1 minute. The discs

were then over poured with TSA agar. All plates were incubated at 37C for 48 hours.

9

The plates were then enumerated and the colony forming units (cfu) per test surface

calculated. From the test results and those recorded for the positive controls, the log

reduction in bacteria after each treatment was calculated.

3.1.1 Chemical Fogging

Two systems were assessed: a traditional fogging system (H&M Disinfection System Ltd.,

Total Hygiene Solution) and a dry mist system (Nebulair).

The chemical fog was applied using a drum top fogger (H&M Disinfection System Ltd ) using

2% disinfectant solution (B1878 Byosan concentrate) supplied by Byotrol. The fogging

system was connected to compressed air and adjusted to 4 bar working pressure. The

contact time was 40 minutes with an additional 1 hour to ventilate the room.

The Nebulair system from Mercatos used 'dry' mist technology to produce micro-particles

(size 1.22 m) of disinfectant that move in the air currents of the room, enabling the

disinfectant to contact all surfaces. Byosan A1616 (Byotrol) was used in different

concentrations: 10%, 20% and 50%. The Nebulair system was switched on for an hour

treatment and then the room was left for an hour to defog before re-entry.

The Cleanaer device is an aerosol technology that delivers very small quantities of liquid in

the form of charged droplets to the environments treated. The Cleanaer device uses an

electrospray technology that delivers small amounts of liquid into the air without tusing heat

or propellants. At the spray face of the device, an electric field creates fine droplets from a

spray capillary. The electrical field is produced by a high voltage, but there is very low

current drawn, so the power at the spray face is tiny (less than 10mW). The technology is

battery-powered. Droplets produced by the electrospray are partially discharged by the

device and are carried away from the spray face by mutual repulsion and air currents.

Four Cleanaer air aerosol technology battery-operated units were installed in the

aerobiology test area, at a height of ~190 cm, against the side wall. The units were

activated by leading electrical connections outside the room to an external on/off switch.

During the first trial, P. aeruginosa discs were exposed to the treatment for 3 hours. During

the second trial, S. aureus discs were exposed to 6 devices positioned against the walls

(~190 cm and ~60 cm height) for 5 hours. Electrostatically charged particles were released

from the liquid fed devices during approximately 83% of the actual time the devices were

working. One device used approx. 2 ml of the 2% biocide solution supplied.

10

3.1.2 Hydrogen peroxide vapour

All the trials were conducted using the proprietary Bioquell process. The Bioquell Clarus

R/R2 produced HPV which was subsequently micro-condensed onto a surface to give a high

concentration of H2O2. HPV was generated by dropping a measured volume of 30% w/w

liquid onto a vapouriser heated to 130C. The resulting HPV was injected until the air was

saturated, at which point hydrogen peroxide began to condense on a surface at a high

concentration (up to 70%).

The HPV decontamination cycle consisted of three phases, in a one step process:

(i) Conditioning: the vapouriser is heated to operational temperature.

(ii) Injection: HPV is injected for a defined period. HPV is delivered via a dual axis

distribution system to ensure a high velocity and even distribution throughout the room.

When the required amount of H2O2 has been injected, the enclosure is allowed to dwell with

no further injection to ensure adequate H2O2 exposure.

(iv) Aeration: The aeration equipment is activated, which catalyses the decomposition

process by passing the air/vapour mixture through an activated carbon filter and breaking

down the HPV to water vapour and oxygen.

The HPV concentration, temperature and relative humidity within the room were measured

by an instrumentation module and monitored by a control computer situated outside the

room, which provided real time feedback of the cycle progress.

Bioquell's hydrogen peroxide vapour equipment was placed in the aerobiology laboratory.

The room was then sealed with a sally tape supplied by Bioquell. The cycle usually lasted

for several hours. Two hydrogen peroxide concentrations were tested. 10g/m3 was used

for the first set of trials; the concentration was increased to 20 g/m3 for the second set of

trials. Additional one-off experiments were conducted using higher concentrations of 30 and

40 g/m3. All steps were monitored by a control computer situated outside the room.

11

3.1.3 Ozone

All the trials were conducted using the proprietary Steritrox patented process including,

where appropriate, their quench technology. The Radical Small Mobile Unit (UMB) from

Steritrox was a portable machine, which operated independently of mains electricity and was

positioned in the centre of the room. The operator selected the relevant setting from the

touch-screen menu and left the area. The machine auto cycled through all of the phases of

the decontamination cycle with constant visual alarms to communicate that it was in

operation. The ozone decontamination cycle consisted of three phases:

(i) Humidification: for maximum antimicrobial activity of ozone, the area was humidified to

70 to 90%.

(ii) Decontamination: the ozone vapour initially reacts with any volatile organic compounds

present in the atmosphere of the area being decontaminated, a process known as ozone

debt absorption. Having overcome this, the vapour concentration builds rapidly, to a test

concentration (which was 8 to 25 ppm).

(iii) Aeration: a biocidal quenching agent was used during the final phase of the cycle to

mop up the remaining ozone, leaving the room safe for immediate re-occupation.

Steritrox's ozone equipment was placed in the aerobiology laboratory. The room was then

sealed with a sally tape supplied by Steritrox. The cycle lasted from 40 minutes up to

several hours, depending on concentration and contact time. These trials were undertaken

with Steritrox's engineer in assistance.

Steritrox laboratory trials

Ozone trials were also undertaken at Steritrox's laboratory in Pershore, Worcestershire.

For the first trial, 25 stainless steel discs, previously sterilised, were inoculated with 0.05ml

108cfu ml-1 S. aureus test suspension. The suspension was dried onto the discs at a

temperature of 37C for approximately 1 hour. Then the discs were allowed to equilibrate to

room temperature before the test commenced. A total of 20 discs were treated and 5 discs

were left untreated (positive controls).

For the second trial, 10 stainless steel discs for each microorganism, previously sterilised,

were inoculated with 0.05 ml 108cfu ml-1 S. aureus, P. aeruginosa or L. monocytogenes test

suspension. The suspension was dried onto the discs at a temperature of 37C for

approximately 1 hour. Then the discs were allowed to equilibrate to room temperature

12

before the test was commenced. A total of 7 discs were treated and 3 discs were left

untreated (positive controls) for each microorganism.

All test and control surfaces were inoculated on the day of the trial in the Food Hygiene

Laboratory. After inoculation and drying, all samples were packed and transported to the

Steritrox facilities. After treatment, the samples were again packed and transported back to

Campden BRI for processing.

The experiments were conducted in a laboratory with a volume of 75 m3 and the discs were

positioned horizontally on a stainless steel table.

The ozone trials undertaken in both establishments can be summarised as follows:

(i) Laboratory trials at Steritrox

exposure to 25 ppm ozone for 90 minutes, testing against S. aureus only.

exposure to 8 ppm ozone for 30 minutes, testing against S. aureus, P. aeruginosa and L.

monocytogenes.

(ii) Campden BRI laboratory trials

exposure to 20 ppm ozone for 30 minutes, then manual ozone removal using extract

fans, against S. aureus and L. monocytogenes.

exposure to 20 ppm ozone for 30 minutes with natural decay to 15 ppm and manual

removal using extract fans, testing against S. aureus, P. aeruginosa and L.

monocytogenes.

Ozone dosage calculations

The dosage of ozone was calculated by multiplying the ozone concentration used in ppm

and the contact time in hours.

3.2 Field trials

The field trials were conducted in conjunction with representatives from Steritrox. Three

single-use ozone decontamination trials in factories were assessed. Those assessments

included representative factories of the RTE, poultry and dry food industry. In addition, one

4 week, continuous factory trial was completed in a sandwich factory.

The field trial methodology was based on a procedure for the approval of terminal

disinfectants for use in high-care areas of a major retailers food suppliers.

13

Swabs

A total of 10 swabs and 10 contact plates were taken at different sites within the

environment. The sites were chosen to represent flat, open, easily cleanable surfaces, and

those areas not expected to be cleaned easily; this included food contact surfaces and non-

food contact surfaces of a variety of materials of construction.

The swab samples were taken with sterile plastic applicators (Sterilin), using the method

described in the Campden BRI Manual of Hygiene Methods for the Food and Drink Industry

Guideline No. 45 (2003). The swabs were then placed in 9ml of Maximum Recovery

Diluent (MRD; LABM 103) and 1ml of sodium thiosulphate inactivator (See Appendix 1).

On receipt in the laboratory, the samples were vortexed using a Rotamixer (Hook and

Tucker Instruments Ltd.) for 30 seconds to resuspend the micro-organisms.

Total Viable Count (TVC)

A 1 ml sample of the swab resuspension fluid was taken and used to prepare a serial

decimal dilution series in MRD. Duplicate 1 ml aliquots were pour plated with Nutrient Agar

(NA; Oxoid CM3) and incubated at 30oC for 48 hours. The colonies were counted and the

results expressed as colony forming units (cfu) per swab.

Contact plates

Contact plates were taken using the method described in the Campden BRI Manual of

Hygiene Methods for the Food and Drink Industry Guideline No. 45 (2003), using 55 mm

contact plates (Sterilin) filled with NA to give a convex meniscus. Colonies appearing after

incubation at 30oC for 48 hours were counted and the results expressed as cfu per plate.

Controls

On each sampling day, portions of microbiological media were incubated at 30oC for 48

hours (NA) and enumerated as positive and negative controls.

(i) Factory 1: Poultry Factory

Test site: Ventstream chiller, room size 2300m3.

The trial was conducted at the end of production. The environment was cleaned with

detergent and rinsed and then terminal disinfectant was replaced by ozone treatment: a

single, periodic ozone treatment.

14

Machine used: 2 x Radical Small Mobile Unit (UMB)

Ozone treatment: 5ppm 1hour + Quench

A total of 15 swabs and 15 contact plates (Table 1) were taken as detailed below. The sites

were sampled by Campden BRI staff, who took samples before cleaning, after cleaning and

after disinfection.

Table 1 - Swab and contact plate sites

Site Code Sample site - product contact surfaces

1 Plastic shackle (outside)

2 Plastic shackle (inside)

3 White board

4 Back plate of shackle (bird contact)

5 Back of shackle

6 Stainless steel surface (external)

7 Stainless steel surface (internal)

8 Spray head (metal)

9 Plastic crate (internal)

10 Stop plastic button

Site Code Sample site - Environmental samples

1E Stainless steel panel (entrance)

2E Plastic cladding

3E Floor (concrete)

4E Back of door

5E Evaporator fin

(ii) Factory 2: Pastry manufacturer

Test site: Ice slurry room, room size 53m3.

The trial was conducted at the end of production on two occasions. The environment was

cleaned with detergent and rinsed and the terminal disinfectant was replaced by ozone

treatment.


Ozone treatment: 8ppm for 40 minutes + quench

15

A total of 15 swabs and 15 contact plates (Table 2) were taken as detailed below.

The sites were sampled by Campden BRI staff, who took samples before cleaning, after

cleaning and after disinfection.



1 Stainless steel vat (inside)

2 Green painted motor

3 Rod power motor

4 Stainless steel balance (top)

5 Underneath stainless steel table

6 Meat pump nozzle (stainless steel)

7 Inside stainless steel meat pipe

8 V-mag stainless steel exit pipe

9 V-mag stainless steel inside hopper

10 Plastic ice slurry pipes (outside)


1E Drain trap (side)

2E Wall cladding

3E Floor

4E Top of gulley pot (drain)

5E Floor to wall edging (concrete)

(iii) Factory 3: Pizza manufacturer

Test site: High care cook-house, room size 75m3.

The trial was conducted at the end of production on three occasions. The environment was

cleaned with detergent and rinsed and the terminal disinfectant was replaced by ozone

treatment.


Ozone treatment: 8ppm for 40 minutes + quench

A total of 15 swabs and 15 contact plates (Table 3) were taken as detailed below. The sites

were sampled by Campden BRI staff, who took samples before cleaning, after cleaning and

after disinfection.

16



1 Tumbler (inside)

2 Shelf underneath tumbler

3 Red switch on/off (tumbler)

4 Glass mixer - blade

5 Glass mixer - bottom (inside)

6 Glass mixer - outlet

7 Stainless steel table-top

8 Glass mixer - panel

9 Stainless steel table - top

10 Blast Chill Room 18B door handle


1E Floor (stainless steel)

2E Wall (behind glass mixer)

3E Floor (red)

4E Around drain seal to the floor

5E Drain inside (drain's basket)

(iv) Factory 4: Sandwich manufacturer

Test site: High care area, room size 1080m3.

Ozone treatment: Routine end of production cleaning and disinfection. Gross solids

removal, rinse with 3ppm ozonated water, chemical application, rinse with 3ppm ozonated

water, seal room, disinfect with 8ppm for 30 minutes + quench.

Machine used (for gaseous treatment): Installed Radical Whole Room System (generation 1,

now superseded by generation 2).

The trial was 4 weeks in duration with sample sites sampled on 3 occasions each week.

These were on a Wednesday afternoon by Campden BRI staff, who took samples before

cleaning, after cleaning and the next morning after disinfection, and on Monday and Friday

mornings by the factory trained representative, who took samples after ozone disinfection

only (Table 4). Samples taken by Campden BRI staff were stored in the fridge and

processed as soon as possible. Samples taken by the factory representative were collected

by a courier in cool boxes and delivered to Campden BRI on the day, where they were

processed. Due to low microbial level, 'before cleaning' sites had to be changed over the

17

4 week trial (Table 5). In addition to food contact surfaces a number of environmental

surfaces were also sampled (Table 6).

Table 4 - Swab and contact plate sites - first set of samples

Site Code Sample site

1 Floor, away from the ozone unit

2 Floor, away from the ozone unit

3 Flowa line (food contact surface)

4 Surface above the conveyor belt on Line 1 conveyor belt

5 Sandwich slicing table

6 Conveyor belt (away from the ozone unit) on Line 1 conveyor belt

7 Conveyor 2 table

8 Slicer - Grothe (long plate)

9 Mixer

10 Conveyor belt on butter machine

Table 5 - Swab and contact plate sites - second set of samples


1 Waste hatch handle (contact plate taken from door plate or the

widest part of the handle ) 2 Floor by Conveyor 1

3 Goods in hatch handle (contact plate taken from door plate or the

widest part of the handle ) 4 Grothe knob

5 Microwave door handle - can you find different location (I think it is

covered with plastic bag during ozone treatment) 6 Glove dispenser (by Conveyor 1)

7 Scraper blade holder (underneath the conveyor belt)

8 Flow rap wheel (Flowa line)

9 Drain 2 (middle one)

10

Can opener (contact plate taken from the widest part of the can

opener) 1e Conveyor 2 table

2e Conveyor belt on butter machine

18

Table 6 - Swab and contact plate sites - environmental samples


1 Floor by conveyor belt 1

2 Drain 1

3 Cheese table

4 Boot (staff)

5 Floor by Grothe machine

6 Sink (Low Risk area)

7 Floor (Low Risk area)

8 Hatch frame (Low Risk area)

9 Wall (Low Risk area)

10 Drain (Low Risk area)

3.3 Statistical Analyses

The performance of the whole room disinfection techniques was assessed by statistical

analysis in a Minitab programme (version 15) using one-way analysis of variance (ANOVA).

A value of P>0.05 indicates that there is no significant difference between groups whilst

P

19

4 field trials, with Steritrox support, were conducted:

o 4 week factory trial in sandwich manufacturer

o I day trial in poultry factory

o 2 day trial in pastry manufacturer

o 3 day trial in pizza manufacturer

The overall results of the work, expressed as the mean log reduction of microorganisms

achieved, are shown in Table 7.

Each laboratory experiment at Campden BRI's laboratory involved testing of 20 stainless

steel discs (2 cm diameter) located around the testing room in different orientations

(horizontal, vertical and underneath) over different expose methods and times.

Each factory trial involved taking the swab samples from the designated locations before

cleaning, after cleaning and after room decontamination.

Table 7 - Descriptive statistic of log reduction of different microorganisms resulting

from the system used, its concentration and discs orientation

Chemical fogging - Byosan 2%

Microorganism Orientation N Mean StDev Minimum Median Maximum

P.aeruginosa

horizontal 8 5.13 0.53 4.18 5.26 5.96

underneath 6 1.94 2.16 0.43 0.70 5.26

vertical 6 1.69 1.35 0.84 1.21 4.42

S.aureus

horizontal 8 5.08 2.21 0.21 5.78 6.76

underneath 6 0.50 0.57 -0.08 0.43 1.30

vertical 6 0.76 0.86 0.17 0.38 2.44

20

Nebulair system - Byosan 10 %


P.aeruginosa

horizontal 16 0.80 0.38 0.32 0.73 1.59

underneath 12 0.92 0.66 0.16 0.69 2.48

vertical 12 0.74 0.54 0.15 0.69 2.10

S.aureus

horizontal 16 0.08 0.29 -0.84 0.09 0.53

underneath 12 0.58 1.02 -2.04 0.50 1.94

vertical 12 0.39 0.39 -0.05 0.28 1.16



P.aeruginosa

horizontal 24 0.52 0.36 -0.14 0.59 1.32

underneath 18 1.17 0.75 -0.15 1.21 2.44

vertical 18 0.78 0.67 -0.39 0.82 2.18

S.aureus

horizontal 8 0.20 0.47 -0.05 0.06 1.34

underneath 6 1.47 0.77 0.08 1.63 2.38

vertical 6 0.85 0.42 0.13 1.05 1.26



S.aureus

horizontal 16 0.42 0.46 0.01 0.27 1.95

underneath 12 1.25 0.81 0.05 0.99 2.70

vertical 12 0.73 0.44 0.13 0.61 1.34

Atrium Innovations aerosol technology - 2 % biocide


P.aeruginosa

horizontal 8 0.56 0.77 -1.05 0.79 1.48

underneath 6 0.68 0.31 0.38 0.54 1.21

vertical 6 0.54 0.31 0.32 0.43 1.12

S.aureus

horizontal 8 0.24 0.14 -0.02 0.25 0.40

underneath 6 0.11 0.30 -0.40 0.21 0.37

vertical 6 0.01 0.44 -0.59 0.27 0.34

21

Hydrogen peroxide vapour - 10 g/m3


B.subtilis var.globigii vertical 5 5.83 - 5.83 5.83 5.83

P.aeruginosa

horizontal 12 1.75 0.89 0.76 1.33 2.77

underneath 9 1.38 1.07 -0.86 1.29 2.73

vertical 9 1.59 0.68 0.85 1.32 2.73

S.aureus

horizontal 28 1.34 0.50 0.59 1.33 2.52

underneath 21 1.61 0.90 0.76 1.62 5.11

vertical 21 1.41 0.59 0.78 1.27 2.79



P.aeruginosa

horizontal 32 2.85 1.30 1.01 2.30 4.51

underneath 24 3.02 1.23 0.95 2.75 4.51

vertical 24 2.96 1.32 0.93 2.92 4.51

S.aureus

horizontal 32 2.44 1.26 1.06 2.10 6.48

underneath 24 2.26 0.74 1.14 2.12 3.55

vertical 24 2.48 1.36 1.23 2.07 6.48

L. monocytogenes

horizontal 32 2.96 1.40 0.80 2.74 5.96

underneath 24 3.16 1.20 1.31 3.00 5.96

vertical 24 2.96 1.27 1.40 2.67 5.96



P.aeruginosa

horizontal 2 3.44 0.213 3.29 3.44 3.59

underneath 2 3.20 0.55 2.81 3.20 3.59

vertical 2 3.59 0.00 3.59 3.59 3.59

S.aureus

horizontal 2 2.02 0.44 1.71 2.02 2.34

underneath 2 2.15 0.29 1.95 2.15 2.35

vertical 2 2.06 0.35 1.81 2.06 2.30

L. monocytogenes

horizontal 2 4.86 0.21 4.71 4.86 5.01

underneath 2 5.49 0.00 5.49 5.49 5.49

vertical 2 4.43 0.08 4.38 4.43 4.49

22



P.aeruginosa

horizontal 2 4.65 0.00 4.65 4.65 4.65

underneath 2 3.50 0.65 3.04 3.50 3.95

vertical 2 4.13 0.74 3.61 4.13 4.65

S.aureus

horizontal 2 2.37 0.03 2.36 2.37 2.39

underneath 2 2.65 0.30 2.44 2.65 2.86

vertical 2 2.59 0.12 2.50 2.59 2.68

L. monocytogenes

horizontal 2 5.39 0.00 5.39 5.39 5.39

underneath 2 5.39 0.00 5.39 5.39 5.39

vertical 2 4.89 0.71 4.39 4.89 5.39

Ozone - 8 ppm

Microorganism Orientation N Mean StDev Minimum Media

n

Maximum

P.aeruginosa horizontal 7 0.86 0.77 0.41 0.61 2.57

S.aureus horizontal 7 0.65 0.09 0.55 0.66 0.81

L. monocytogenes horizontal 7 0.49 0.22 0.15 0.47 0.75

Ozone - 20 ppm (~ 50 ozone/hours)


n

Maximum

P.aeruginosa

horizontal 31 4.07 1.11 1.61 4.55 5.30

underneath 23 3.56 1.29 1.46 3.85 5.30

vertical 23 2.42 0.98 1.15 2.35 4.89

S.aureus

horizontal 32 1.24 0.50 0.71 1.12 3.48

underneath 24 1.76 0.25 1.36 1.72 2.29

vertical 24 1.32 0.29 1.01 1.19 2.12

L. monocytogenes

horizontal 24 4.39 1.15 1.44 5.04 5.24

underneath 18 4.22 1.16 1.87 4.99 5.24

vertical 18 4.35 1.31 1.81 5.04 5.24

Ozone - 20 ppm (~ 20 ozone/hours)


n

Maximum

S.aureus

horizontal 24 0.97 0.60 0.35 0.84 3.06

underneath 17 1.73 0.98 0.90 1.40 3.89

vertical 18 1.05 0.25 0.77 1.03 1.67

L. monocytogenes

horizontal 16 1.62 0.47 0.65 1.59 2.69

underneath 11 1.94 0.42 1.16 2.07 2.41

vertical 12 1.25 0.30 0.84 1.25 1.66

23

Ozone - 25 ppm


n

Maximum

S.aureus horizontal 60 2.07 1.78 -0.08 1.14 6.15

4.1 Pre-trial

All epifluorescence images of P. aeruginosa, S. aureus and L. monocytogenes indicated that

the cultures were attached as a monolayer to the stainless steel discs. Example images of

P. aeruginosa, S. aureus and L. monocytogenes are shown in Figures 2a-c respectively. No

evidence was seen of clumping of cells in a vertical direction that would have limited

penetration of chemical or gaseous disinfectants.

Figure 2a Distribution of P. aeruginosa dried onto stainless steel discs

24

Figure 2b Distribution of S. aureus dried onto stainless steel discs

Figure 2c Distribution of L. monocytogenes dried onto stainless steel discs

25

4.2 Laboratory trials

4.2.1 Chemical fogging

Exposure of P. aeruginosa and S. aureus to chemical fogging at a concentration of

2% B1878 Byosan concentrate (courtesy of Byotrol).

Each experiment involved the testing of 20 stainless steel discs (2 cm diameter) located

around the testing room in different orientations (horizontal, vertical and underneath); log

reductions are shown in Figure 3.

Organism S.aureusP.aeruginosa

7

6

5

4

3

2

1

0

Lo

g r

ed

ucti

on

horizontal

underneath

vertical

Orientation

95% CI for the Mean

Figure 3 - Effect of chemical fogging on microbiological log reduction

depending on orientation

The results achieved are typical for a chemical fog and indicate that decontamination is

strongest on horizontal surfaces (particularly at lower levels), on which the majority of the

disinfectant aerosols settle.

A good level of disinfection is achieved on horizontal surfaces (5 logs), which is above that

required for surface disinfectants as defined by the European Standard surface disinfectant

test EN 13697, in which a 4 log reduction is required to pass the standard.

26

Exposure of P. aeruginosa and S. aureus to Nebulair System at concentration of 10%,

20% and 50% using Byosan disinfectant




Concentration (%) 502010

1.2

1.0

0.8

0.6

0.4

0.2

0.0

Lo

g r

ed

ucti

on

P.aeruginosa

S.aureus

Organism

95% CI for the Mean

Figure 4 - Effect of Byosan on P. aeruginosa and S. aureus when nebulised

at 3 concentrations in the Nebulair System

The degree of microbial reduction by the Nebulair system, using the same biocide as for

chemical fogging, was very poor in comparison, with less than one log order of

decontamination being achieved. In general, the concentration of Byosan used also

appeared to have little effect on log reduction, though there may be some increase in log

reduction between 10 and 20% Byosan for S. aureus. This may relate to the actual quantity

of disinfectant available for disinfection. For chemical fogging, approximately 10 litres of

Byosan was released into the room during the fogging cycle, whereas only ~120ml of

Byosan was released into the room during the Nebulair System cycle (1L lasted for

approximately 8 hours).

27

Concentration (%)

P.aeruginosaS.aureus

502010502010

2.5

2.0

1.5

1.0

0.5

0.0

Lo

g r

ed

ucti

on

horizontal

underneath

vertical

Orientation

95% CI for the Mean

Figure 5 - Effect of Byosan on P. aeruginosa and S. aureus with respect to their

orientation when nebulised in the Nebulair System

Exposure of P. aeruginosa and S. aureus to aerosol technology at a concentration of

2% biocide (courtesy of Atrium Innovation)




The Atrium Innovation technology achieved a small, 0.5 log reduction in P. aeruginoasa and

a marginal, approximately 0.1 log reduction in S. aureus. As for the Nebulair System, only a

small quantity of chemical, 125 ml, was aerosolised during the trial . Orientation did not

have an effect on decontamination, although the low log reductions achieved would probably

mask any differences.

The effect of orientation of the test samples on decontamination by the Nebulair System

Atrium Innovation technology (Figures 5 and 6) suggests that, unlike chemical fogging,

decontamination by nebulisation is not affected by surface orientation. This may be

because the smaller particles generated by the nebulisers (10-20m for fogged particles as

against 1.22 m for nebulised particles [Nebulair System]) are more able to adhere to, and

28

thus have an effect upon, vertical and underneath surfaces. Smaller particles also remain

airborne longer (their settlement rate is less) and, therefore, have more chance to interact

with non horizontal surfaces.

Organism S.aureusP.aeruginosa

1.25

1.00

0.75

0.50

0.25

0.00

-0.25

-0.50

Lo

g r

ed

ucti

on

horizontal

underneath

vertical

Orientation

Figure 6 - Effect of aerosol technology on microbiological log reduction

depending on orientation

4.2.2 Hydrogen peroxide vapour

Exposure to Hydrogen Peroxide Vapour at concentration of 10 g/m3, 20 g/m3 , 30 g/m3

and 40 g/m3 courtesy of Bioquell

Each experiment at 10 g/m3 and 20 g/m3 involved the testing of 20 stainless steel discs (2

cm diameter) located around the testing room in different orientations (horizontal, vertical

and underneath), whilst each experiment at 30 g/m3 and 40 g/m3 involved the testing of 6

stainless steel discs. Log reductions are shown in Figure 7.

29

Concentration 40 g/m330 g/m320 g/m310 g/m3

6

5

4

3

2

1

0

Lo

g r

ed

ucti

on

B.subtilis var.globigii

L.monocytogenes

P.aeruginosa

S.aureus

Organism

95% CI for the Mean

Figure 7 - Effect of H2O2 vapour on microbiological log reduction

at different concentrations

The results in Figure 7 show that microorganisms respond differently to HPV and that there

was a significant difference between the logarithmic microbial reductions of spores and

vegetative bacteria (p=0.003) at a concentration of 20g/m3 and below. An approximate 6 log

reduction of globigii spores was achieved at 10g/m3, which is equivalent to that required for

spores in the decontamination of aseptic equipment.

A relatively poor log reduction of approximately 1.5 logs was achieved for P. aeruginosa and

S. aureus at 10g/m3, the default concentration for the HPV equipment, though this was

substantially increased to an approximately 3 log reduction when this concentration was

doubled to 20g/m3. There was little difference, however, in the log reductions achieved

between the vegetative microorganisms tested, with S. aureus being slightly more resistant.

When the concentration was increased to 30 and 40g/m3, different survival rates between

the vegetative microorganisms was seen. L. monocytogenes was seen as the least

resistant microorganism and its survival was reduced with these increasing concentrations,

though there was no significant difference (P=0.9091) in survival at 30 or 40 g/m3. The

survival of P aeruginosa also was reduced with concentration, though the log reduction at 40

g/m3 was only marginally significantly different to that at 20 g/m3 (P=0.0804). There is no

significant difference in the susceptibility of S. aureus with increasing HPV concentration

30

above 20g/m3 (p=0.8499 at 30g/m3 and P=0.9837 at 40 g/m3). The results for 30 and 40

g/m3 HPV relate, however, to a mean of only 6 discs per microorganism, whilst those at 10

and 20 g/m3 were the mean of 30-80 discs per microorganism.

Figure 8 shows the effect of surface orientation on the decontamination of P. aeruginosa,

S. aureus and L. monocytogenes by HPV at 20g/m3. No statistical differences were seen

between orientations for all organisms (P=0.828, P=0.884 and P=0.780 respectively).

Organism S.aureusP.aeruginosaL.monocytogenes

4

3

2

1

0

Lo

g r

ed

ucti

on

horizontal

underneath

vertical

Orientation

20 g/m3, 43 min cycle, 60 min dwell time

Figure 8 - Effect of H2O2 vapour on microbiological log reduction

depending on orientation at 20 g/m3

31

4.2.3 Ozone

Exposure to ozone at concentrations 8 ppm, 20 ppm and 25 ppm - courtesy of

Steritrox

The tests conducted at Campden BRI used a Radical Small Mobile Unit (UMB) and involved

the testing of 20 stainless steel discs (2 cm diameter) per experiment which were located

around the testing room in different orientations (horizontal, vertical and underneath) for:-

>20 ppm for 30 min - total time: ~4 hrs (no quench)

>20 ppm for 30 min - total time: ~3.5 hrs (no quench)

The test conducted at the Steritrox laboratory, Birlingham, Worcestershire, used a Meditrox

MDX50 machine and involved the testing for:-

7 stainless steel discs per microorganism located in horizontal orientation. 8ppm for

30 min. - total time: ~70 min from door close to door open + quench

20 stainless steel discs per trial located in horizontal orientation. 25ppm for 90 min -

total time: ~ 2 - 2.5h from door close to door open + quench

The log reductions achieved in all ozone trials are summarised in Figure 9.

Concentration 25 ppm20 ppm8 ppm

5

4

3

2

1

0

Lo

g r

ed

ucti

on

S.aureus

L.monocytogenes

P.aeruginosa

Organism

Figure 9 - Effect of gaseous O3 on microbiological log reduction

at different concentrations

32

The results in Figure 9 show two trends. Firstly, there is a clear relationship between ozone

concentration and log reduction, with the log reduction for S. aureus ranging from 0.7 logs at

8ppm, to 1.5 logs at 20ppm and 2.1 logs at 25ppm. Secondly, the effect of ozone on the

three vegetative strains tested is markedly different with, at 20ppm, S. aureus being

statistically (P=0.000) and practically most resistant, followed by P. aeruginosa, and

L. monocytogenes being most sensitive.

The effect of sample orientation on log reduction at an ozone concentration of 20ppm is

shown in Figure 10.

Organism S.aureusP.aeruginosaL.monocytogenes

5

4

3

2

1

0

Lo

g r

ed

ucti

on

horizontal

underneath

vertical

Orientation

Figure 10 - Effect of gaseous O3 on microbiological log reduction

depending on orientation at 20 ppm long cycle

There is no statistical difference in orientation for L. monocytogenes (P=0.896), though the

orientation log reductions are statistically different for both P. aeruginosa (P=0.000) and

S. aureus (P=0.000). The results for S. aureus, in which a greater log reduction is shown for

the underneath surface, are probably due to test variance. The results for P. aeruginosa are

more varied, with a lower reduction on vertical than underneath surfaces. The reason for

this is not clear, but may be due to air circulation patterns in the laboratory. In any case,

there is little evidence to suggest that ozone is not able to penetrate to all surfaces,

irrespective of their orientation.

33

4:003:303:002:302:001:301:000:300:00

25

20

15

10

5

0

100%

95%

90%

85%

80%

75%

70%

65%

60%

Time

Ozo

ne

Co

nce

ntr

ati

on

(p

pm

)

Hu

mid

ity

(R

H)

Ozone

Humidity

Key

23.09.09

Figure 11 - Ozone concentration and humidity levels at 20 ppm treatment (short cycle)

During the trials it became clear that the effect of ozone on log reductions was a

combination of concentration and contact time. When ozone concentration is plotted against

time, the effect of forced ozone removal from the atmosphere following ozonation to 20ppm

(Figure 11) and natural decay following ozonation to 20ppm (Figure 12) can easily be seen.

The amount of ozone in the air that the microorganisms are exposed to following the applied

20ppm ozone concentration is much greater when the ozone is left to decay naturally than

when the quench cycle is initiated. The quench cycle can be applied when the ozone

concentration drops to 8ppm and a typical profile of the reduction on ozone concentration in

the air following quenching can be seen in Figure 13.

The area under the line in the concentration versus time plots can be described as the

ozone dosage. The ozone dosage can be calculated for all of the ozone experiments and is

shown, along with the log reduction achieved, for L. monocytogenes in Table 8.

34

4:304:003:303:002:302:001:301:000:300:00

25

20

15

10

5

0

95%

90%

85%

80%

75%

70%

65%

60%

Time

Ozo

ne

Co

nce

ntr

ati

on

(p

pm

)

Hu

mid

ity

Ozone Conc.

Humidity

11.11.09

Figure 12 - Ozone concentration and humidity levels at 20 ppm treatment (long cycle)

Figure 13 - Ozone concentration and humidity levels at 8 ppm treatment

(courtesy of Steritrox)

35

Table 8 - Relationship between dose and log reduction for L. monocytogenes

Trial Ozone dosage

(ppmhour)

Mean log

reduction

1 65.35 3.51

2 42.26 4.92

3 25.64 1.84

4 24.72 1.38

5 22.07 4.55

6 5.01 0.49

A graph of ozone dosage versus log reduction can then be plotted and the regression line

calculated for each microorganism. The data for L. monocytogenes are shown in Figure 14.

Whilst it can be seen that the accuracy of the regression line is not good, the concept of

using the line to predict the ozone dose required to produce a given log reduction of a target

microorganism can be seen. Further work would be required to build a sufficiently robust

model to validate the approach.

9080706050403020100

6

5

4

3

2

1

0

Ozone dosage (ppm hour)

Me

an

lo

g r

ed

ucti

on

5.01

22.07

24.72

25.64

42.26

65.35

Figure 14 - Relationship between ozone dose and log reduction for L. monocytogenes

36

4.3 Field Trials

The TVC results from the swab points before cleaning, after cleaning and after disinfection

for the 3 field trials using ozone as a periodic decontaminant are shown in Figure 15a and b

(poultry factory food contact and environmental samples), Figure 16a and b (pastry

manufacture food contact and environmental samples) and Figure 17a and b (pizza

manufacture food contact and environmental samples). Raw data for these three trials are

shown in Appendix 2.

After disinfection After cleaning Before cleaning

8

7

6

5

4

3

Lo

g

10

1

2

3

4

5

6

7

8

9

site

Sample

Figure 15a - Effect of gaseous O3 treatment on microbiological log reduction of food

contact samples within a poultry factory

37

After disinfectionAfter cleaningBefore cleaning

9

8

7

6

5

4

3

2

Lo

g

1S. steel upright (entrance)

2 Plastic cladding

3 Floor (concrete)

4 Back of door

5 Evaporator fin

Sample site

Figure 15b - Effect of gaseous O3 treatment on microbiological log reduction of

environmental contact samples within a poultry factory

The results from the single trial in the poultry factory (Figures 15a and b) are varied with

perhaps 5 of the 10 food contact sampling sites showing a reduction after cleaning with the

other sites showing no change or even an increase in counts. Reductions after disinfection

are clearer for the environmental samples. If an overall reduction in counts is observed for

all samples, it is probably less than 1 log order.

38

After disin

fection 2

After cle

aning 2

Before clean

ing 2

After disin

fection 1

After cle

aning 1

Before clean

ing 1

3.5

3.0

2.5

2.0

1.5

1.0

Lo

g

10

1

2

3

4

5

6

7

8

9

site

Sample


contact samples within a pastry manufacture facility

After d

isinfec

tion 2

After c

lean

ing 2

Before clean

ing 2

After d

is infec

tion 1

After c

lean

ing 1

Before

clean

ing 1

8

7

6

5

4

3

2

1

0

Lo

g

Drain trap (side)

Wall cladding

Floor

Top of gulley pot (drain)

Floor to wall edging

Sample site


environmental samples within a pastry manufacture facility

39

After disin

fection 3

After cle

aning 3

Before

clean

ing 3

After dis in

fection 2

After cle

aning 2

Before clean

ing 2

After disin

fection 1

After cle

aning 1

Before

clean

ing 1

9

8

7

6

5

4

3

2

1

0

Lo

g

10

1

2

3

4

5

6

7

8

9

site

Sample


contact samples within a pizza manufacture facility

After d

isinfection 3

After c

lean

ing 3

Before

clean

ing 3

After d

isinfection 2

After c

lean

ing 2

Before clean

ing 2

After d

isinfection 1

After c

lean

ing 1

Before clean

ing 1

8

7

6

5

4

3

2

1

0

Lo

g

Floor (S.steel)

Wall

Floor

Around drain seal to the floor

Drain inside (drain's basket)

Sample site


environmental samples within a pizza manufacture facility

40

The results for the pastry factory over the two day trial (Figure 16a) are difficult to interpret

as the food contact surface counts appear to increase after cleaning and then decrease after

disinfection with ozone. The counts on the food contact surfaces are, however, relatively

low and small differences in recovery efficiency by the swabbing technique on such low

numbers may mask real trends. The counts on environmental surfaces (Figure 16b) are

higher than on food contact surfaces and may show a reduction after disinfection,

particularly on the second day.

The results for the food contact surfaces of the pizza factory over the three day trial (Figure

17a) show that, on each individual day, average counts are lower after cleaning and then

lower still after disinfection. The results could also show a downward trend for the numbers

of microorganisms present both before cleaning and after cleaning and disinfection

throughout the three day period. This downward trend is perhaps more clearly shown with

the environmental samples (Figure 17b).

The TVC swab results from the 4 week trial in the sandwich manufacture facility in which

ozone was used in lieu of a chemical disinfectant are shown for food contact surfaces in

Table 9 and for environmental surfaces in Table 10.

Table 9 TVC counts for food contact surfaces before cleaning, after cleaning and

after ozone disinfection within a sandwich manufacture facility

SWAB RESULTS (Mean log)

Food Contact surfaces N Before

cleaning N

After

cleaning N

After

disinfection

Flowa line 2 2.84 2 0.70 8 1.17

Surface above the conveyor belt on

Line 1

1 2.88 2 0.70 8 1.29

Sandwich slicing table 2 3.45 2 1.57 7 1.22

Conveyor belt on Line 1 2 1.44 2 2.22 7 1.18

Conveyor 2 table 3 2.52 3 1.49 8 1.15

Slicer - Grothe (long plate) 2 3.07 2 0.70 8 1.27

Inside mixer 2 0.88 2 2.14 8 1.52

Conveyor belt on butter machine 3 0.94 2 0.70 8 1.56

Can opener 1 1.18 1 1.18 1 0.70

Grothe blade wheel - - - - 1 0.70

Cheese table 1 3.76 1 4.70 3 1.70

Sink (LR) 1 2.53 1 5.70 3 2.07

Mean log 2.32 1.98 1.29

41

Table 10 - TVC counts for environmental surfaces before cleaning, after cleaning and

after ozone disinfection within a sandwich manufacture facility

SWAB RESULTS (Mean log)

Environmental samples N Before

cleaning N After cleaning N After disinfection

Floor 2 4.94 2 3.19 8 2.82

Floor 2 5.82 2 4.70 8 2.28

Waste hatch handle 1 1.00 1 cont 1 0.70

Floor by conveyor 1 1 4.69 1 3.25 1 0.70

Goods in hatch handle 1 1.18 1 0.70 1 0.70

Grothe knob 1 3.39 1 1.00 1 0.70

Microwave door handle 1 1.65 1 cont 1 0.70

Glove dispenser 1 3.27 1 0.70 1 0.70

Scraper blade holder 1 2.94 1 2.94 1 0.70

Flow rap wheel (Flowa

line)

1 1.81 1 1.81 1 3.55

Drain 2 1 4.81 1 4.81 2 2.85

Floor by conveyor 1 1 5.81 1 4.3 4 1.59

wip door handle - - - - 1 1.48

Drain 1 1 4.86 1 4.71 3 4.57

Boot (staff) 1 4.78 1 4.48 3 5.87

Floor by Grothe machine 1 4.62 1 5.05 3 4.12

Floor (LR) 1 3.52 1 1.18 3 2.18

Hatch frame (LR) 1 4.35 1 0.70 3 5.88

Wall (LR) 1 0.70 1 0.70 3 1.30

Drain (LR) 1 5.13 1 3.94 3 1.92

Mean log 3.65 2.83 2.27

The mean counts of all samples before cleaning, after cleaning and after disinfection for

both food contact and environmental surfaces for the 4 week field trial are shown in Table

11. As a comparison, the average data from 10 field trials of 8 weeks duration undertaken in

chilled food plants and in which chemical disinfectants were tested for approval by a major

retailer are also shown.

42

Table 11 A schematic comparison of chemical disinfection and O3

Pre

cleaning

Post

cleaning

After

disinfection

Average of counts per swab from

10 separate, 8 week duration

disinfectant trials:

4.73 2.80 1.30

Average of counts per swab from 4

week duration trials using ozone

as a disinfectant (food contact

surfaces):

2.32 1.98 1.29

Average of counts per swab from 4

week duration trials using ozone

as a disinfectant (Environmental

samples):

3.65 2.83 2.27

The results in Table 9 show that the TVC count decreased after cleaning and again after

disinfection. After disinfection, for all food contact surfaces, TVC counts were very low,

though this is correlated to the pre-cleaning results. TVC counts on environmental surfaces

(Table 10) were reduced after cleaning and again after disinfection, though were higher than

on food contact surfaces and were more variable. Variability of TVC counts was dependent

on the count prior to cleaning.

The results in Table 11 show that for food contact surfaces, the counts after disinfection

compare favourably to the post disinfection counts of the combined disinfectant approval

trials. Ozone was thus seen to maintain control of the microflora of the food contact

surfaces.

During the 4 week trial no adverse effects were observed on the structure and fabric of the

building. Indeed, the management of the factory also report no adverse effects since the

ozonation equipment was installed.

43

5. CONCLUSIONS

The results achieved for Byosan during chemical fogging are typical for quality disinfectants

in that good decontamination is achieved on horizontal surfaces but little is achieved on

other surface orientations. These findings are in agreement with those of Campden BRI and

the Silsoe Research Institute which undertook a MAFF funded project in 1988 (Burfoot et al.,

1999). This work suggested that fogging is effective at reducing airborne microbial

populations by 2 to 3 log orders in 30 to 60 minutes and horizontal surfaces up to 6 log

orders in 60 minutes, with minimal effect on vertical surfaces and underneath equipment.

Chemical fogging is therefore useful for the periodic decontamination of the air and as an

automated method to apply disinfectants to accessible, food processing equipment surfaces.

It will have little or no effect, however, on ceilings and overhead structures.

As the amount of chemical released by the Nebulair system and Cleanaer technology is so

small, the level of decontamination on surfaces it achieves is relatively poor. However, the

particle size of chemical they produce appears to be small enough to interact with surfaces

of all orientations, so what decontamination it does achieve is approximately the same over

all surface geometries. Smaller particles will also have longer contact times in the vicinity of

the coupon surfaces, which could lead to particle/surface interactions, because their

settlement rate will be slower. There may be a role for nebulisers in low level, continuous

decontamination of food production areas, if the quantity of disinfectant it produces satisfies

Health and Safety requirements with respect to operatives exposure to the chemical via

breathing the droplets.

At low concentrations of HPV (10 g/m3), the effect of HPV was much more pronounced on

spores of B. subtilis var. globigii than on the vegetative bacteria tested, with an approximate

6 log reduction of globigii spores being achieved. With the vegetative bacteria, a

relationship was established between log reduction and concentration, with an

approximately 3 log reduction being achieved for P. aeruginosa, S. aureus and

L. monocytogenes at 20g/m3. Hydrogen peroxide at high concentrations is considered a

good disinfectant and in parallel trials (results not shown), a 5 log reduction in vegetative test

microorganisms according to the methodology of the disinfectant suspension test BS EN

1276, was achieved at a hydrogen peroxide concentration of 10-15% (30% hydrogen

peroxide is vapourised in the Bioquell units tested). It may be proposed, therefore, that at

the lower concentrations of HPV used in the trials (10-20 g/m3), P. aeruginosa, S. aureus

and L. monocytogenes (all of which are catalase positive) were all able to partially resist the

concentration/volume of disinfectant that they came into contact with.

When the HPV concentration was increased to 30 and 40 g/m3, an increase in susceptibility

was seen for P. aeruginosa and L. monocytogenes, resulting in a log reduction in excess of

4 logs for P. aeruginosa and 5 logs for L. monocytogenes at 40 g/m3. This is in excess of

44

the 4 log reduction required in the European Surface Test (EN 13697) for surface

disinfectants.

S. aureus was significantly more resistant than the other vegetative organisms P.

aeruginosa and L. monocytogenes, and B. subtilis var. globigii spores and its log reduction

was not increased at 30 or 40 g/m3. The reason for this resistance is unclear, but may be

due to the tendency of S. aureus cells to clump together, which may resist chemical

vapours. No statistical differences in log reductions were seen between orientations for all

microorganisms tested.

HPV systems may be able to control pathogens such as Listeria in the environment at high

concentrations (e.g. 40 g/m3), though further trials are needed to support this. They do,

however, offer an excellent option for the control of spore forming bacteria.

With ozone, as with HPV, there is a relationship between concentration and log reduction,

though the effect of ozone on each of the three vegetative strains tested was markedly

different with, at 20ppm, S. aureus being most resistant followed by P. aeruginosa, and L.

monocytogenes being most sensitive. Whilst there appeared to be statistical differences in

the log reductions achieved at different sample orientations, these are not thought to be

practically different.

A reduction of >4 logs was achieved with L. monocytogenes at an ozone concentration of

20ppm, which equates to the requirements of the European Surface Test (EN 13697) for

chemical disinfectants. A reduction approaching 4 logs was also achieved with P.

aeruginosa.

The results suggest that, for each microorganism tested, it could be possible to describe a

relationship between ozone concentration and exposure time that can be described as an

ozone dosage. A graph of ozone dosage versus log reduction can then be plotted and the

regression line calculated for each microorganism. From the tentative data in Figure 13 for

L. monocytogenes, extrapolation suggests that a 6 log reduction could be achieved for L.

monocytogenes with a dosage of approximately 90 ppm hours. This equates, for example,

to an exposure of 10 ppm ozone for 9 hours or 30ppm ozone for 3 hours. Whilst this is

theoretical, if it were to be possible to achieve a 6 log reduction of L. monocytogenes on all

surfaces regardless of orientation in a food processing area (i.e. a treatment to the

environment equivalent to the cooking process of the food product), this would provide a

very powerful technique for contamination control. Again, further trials would be required to

substantiate this dosage in practice.

As an overall conclusion from the laboratory trials, vapours and gases (as typified by HPV

and ozone) have several advantages as they can effectively penetrate every part of a room,

45

including sites that might prove difficult to gain access to with conventional liquids and

manual disinfection procedures. The major disadvantage of using gases, such as ozone, is

the potential toxicity at high concentrations, which precludes using them in areas where

people are working. The technique can therefore only be used in areas that can be isolated

and sealed off during the decontamination process.

The food contact and environmental surface results for the three field trials in which ozone

was used as a periodic room decontaminant showed little effect over 1-3 days and an

overall reduction in counts after disinfection was probably less than 1 log order. This is

perhaps not surprising as when 8ppm ozone was tested under laboratory conditions (Figure

9), a reduction of less than1 log order was observed for the three strains tested under the

same ozone application conditions (8ppm, 30min).

After 3 days, however, the results for the pizza factory could show a downward trend for the

numbers of microorganisms present on food contact and environmental surfaces, both

before cleaning and after cleaning and disinfection, throughout the three day period. Ozone

generation equipment manufacturers have postulated that when ozone is first applied to a

cleaned room, there is a mass of organic material that creates an ozone demand which

must be satisfied by oxidation before any significant oxidation of microorganisms can occur.

In essence, this is no different from the effect of organic matter on traditional chemical

disinfectants, e.g. a chlorine organic break point in water treatment. The ozone demand will

be affected by ozone concentration and if the applied ozone concentration is low, this ozone

demand may require an extended time period before it is fully oxidised.

What is clear is that 8ppm ozone for 30 minutes is insufficient for periodic, whole room

decontamination and a dosage of approximately 90ppm/hours as postulated from the

laboratory studies may be required. Given that the ozone demand of a factory is likely to

exceed that of the relatively clean aerobiology laboratories in which the practical trials were

undertaken, however, the necessary dosage may well exceed this figure.

In the 4 week field trial, after disinfection, for all food contact surfaces, TVC counts were

very low, though this is correlated to the pre-cleaning results. TVC counts on environmental

surfaces were also reduced after cleaning and again after disinfection, though were higher

than on food contact surfaces and were more variable.

The counts after disinfection for ozone treated food contact surfaces compare favourably to

the post disinfection counts of the combined disinfectant approval trials. In the disinfectant

approval trials, only sites with TVC counts of >105/swab were chosen, which is in excess of

whole room disinfection

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