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The detection of food soils and cells on stainless steel using industrial methods: UV illumination and ATP bioluminescenceKathryn A. Whitehead ⁎ , Lindsay A. Smith, Joanna VerranSchool of Biology, Chemistry and Health Sciences, Manchester Metropolitan University, Chester St, Manchester M1 5GD, UK

A B S T R A C TA R T I C L E I N F O

Article history:Received 2 April 2008Received in revised form 23 June 2008Accepted 23 June 2008

Keywords:SoilFoodConditioning lmATP bioluminescenceUV lightSurface

Open food contact surfaces were subjected to organic soiling to provide a source for transfer of microbial

cells. Rapid industrial methods used for the detection of residual cells and soil e.g. ATP (adenosinetriphosphate) bioluminescence and an ultraviolet (UV) light detection method were assessed for their abilityto detect organic soils, or organic soil – cell mix on surfaces. A range of soils (complex [meat extract, shextract, cottage cheese extract]; oils [cholesterol, sh oil, mixed fatty acids]; proteins [bovine serum albumin, sh peptones casein]; carbohydrates [glycogen, starch, lactose]); was used. Under UV, oily soils, mixed fattyacids, cholesterol and casein were detected at low concentrations, with detection levels ranging from 1% to0.001% for different substances. Glycogen was the most dif cult substance to detect at lower concentrations.Using UV wavelength bands ( λ ) of 330– 380 nm, 510 – 560 nm and 590 – 650 nm, wavelength bands of 330 –

380 nm, illuminated most of the soils well, whilst the wavelength band of 510 – 560 nm illuminated the shextract, cholesterol and fatty acids; the 590 – 650 nm wavelength band illuminated the lactose. Soils at allconcentrations were detected by the ATP bioluminescence method; the complex soils gave the highestreadings. When complex soils were combined with Listeria monocytogenes Scott A or a non-pathogenicEscherichia coli O157:H7, ATP measurements increased by 1 – 2 logs. For UV illumination, the L. monocytogenesand cheese combination was the most intensely illuminated, with E. coli and meat the least.UV illumination is a simple well established method for detecting food soil, with little change in ndingswhen microorganisms are included. Performance can be enhanced in certain circumstances by altering the

wavelength. ATP bioluminescence is a proven system for hygienic assessment being especially useful in thepresence of microorganisms rather than organic soil alone.

© 2008 Elsevier B.V. All rights reserved.

1. Introduction

Most equipment used for food preparation in the food industry isconstructed from stainless steel. Regular cleaning of the equipment isrequired to prevent the build up of adsorbed organic material andmicroorganisms. The material present on a given hygienic foodcontact surface may contain organic material (food soil), inorganicmaterial (residue of cleaning agent), and microorganisms. The natureof this mixture (biological and chemical) of viable and inertcomponents will vary depending on the environment ( Verran andWhitehead, 2006 ). In this paper, commercial ‘kits ’ for soil detectionwere compared.

The development of adsorbedlayers, termedconditioning lms,on asurface is considered to be the rst stage in bio lm formation(Chamberlain, 1992 ). Open food contact surfaces do not normallyprovide a solid – liquid interface for microbial attachment, thus a ‘true ’

bio lm is unlikely to develop ( Verran et al., 2002 ). However, organic

materials in the presence or absence of microorganisms will betransferred onto the surfaces; a process known as soiling. Organicsoiling of a surface will affect cell – substratum interactions, and willintroduce additional cell – soil and soil – substratum interactions ( VerranandWhitehead,2006 ).Thein uence of organic material on substratumproperties and cell attachment and retention will have an impact onsurface fouling and on cleaning regimes.

There are a number of methods available ( Verran et al., 2002;Verran and Whitehead, 2006 ) (in situ and in vitro ) that maybe used todetect and/or quantify food soiling on surfaces, but to date the meritsof a number of these methods for their use in the food industry havenot been directly compared ( Davidson et al.,1997; Verran et al., 2002 ).Methods used to detect soils include theuse of iodine for the detectionof starch, Nile blue for fat ( Verran et al., 2002 ), a visual assessment of margarine using beta carotene ( Anderson et al., 1986 ) and milk andcasein soil via the Lowry method ( Bohner et al., 1991 ). Rapid in situindustrial methods include ATP bioluminescence or a UV lightdetection method. Microscopy in vitro methods include ScanningElectron Microscopy (SEM) ( Gounga et al., 2007 ), and epi uorescentmicroscopy ( Whitehead et al., 2005 ). Methods used to detect changesin the surface physicochemistry due to surface soiling include contact

International Journal of Food Microbiology 127 (2008) 121 – 128

⁎ Corresponding author. Tel.: +44 161 247 1157; fax: +44 161 247 6365.E-mail address: [email protected] (K.A. Whitehead).

0168-1605/$ – see front matter © 2008 Elsevier B.V. All rights reserved.

doi: 10.1016/j.ijfoodmicro.2008.06.019

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angle ( van der Mei et al., 2002 ), surface free energy, dispersive andpolar measurements ( Briandet et al., 2001 ), whilst chemical methodsused to determine surface contamination include such methods asEnergy Dispersive X-ray (EDX) (Reid et al., 1994) and FourierTransform Infrared Spectroscopy (FTIR) ( Koca et al., 2007; Pappaset al., 2008 ). Clearly, some of these techniques are less suitable for useon site in the food industry but supporting data generated may helpidentify the most appropriate of the simpler methods. This, andcurrent work in ourlaboratories compares a rangeof these methods todetermine their merits, shortfalls and relationships in terms of soildetection.

ATP bioluminescence has been widely used for the detection of microbial contamination and food residues in the food industry(Grif th et al., 1994; Davidson et al., 1997 ), providing a real timeestimate of total surface cleanliness including the presence of organicdebris and microbial contamination ( Davidson et al., 1999 ).It has beensuccessfully used for determining cell numbers in sh processingfactories ( Miettinenet al., 2001 ) and the dairy industry( Oulahal-Lagsiret al., 2000 ). The results from thesample aredisplayed in relative lightunits (RLU). When compared to contact agar slide method, results forATP bioluminescence were shown to be poorly correlated to the totalnumberof bacteriawhenused to evaluate thesurface contaminationin sh processing factories ( Miettinen et al., 2001 ). Further the ATPmethod does not detect all components of a given soil ( Lappalainenet al., 2000 ). Clearly if ATP is absent one would speculate that thesurface is clean,but some types of residualsoil mayremainundetected.

UV light (353 nm) can also be used for the detection of residualcells and soiling on industrial surfaces ( Pedersen, 2007 ) although (aswith ATP) no distinction is made between the two components. Themolecular con guration of organic material allows some organicresidues to uoresce when illuminated by UV light ( Adhikari and

Tappel,1975 ). Thus, UV light may be used to detect residual soil whenwork surfaces are illuminated at appropriately 350 nm by highlightingareas in an industrial plant that need a more intensive cleaning. Thismethod is advantageous in that it does not require direct contact withthe surface (which ATP bioluminescence and total viable countsrequire). The ability of different wavelengths to detect separatecomponents has not been previously explored.

Although the presence of soil alone provides an indication of theeffectiveness of cleaning and hygienic procedures, it is the micro-organisms present that impact signi cantly on public health. Highlypublicized outbreaks of foodborne diseases caused by pathogens suchas Escherichia coli O157:H7 and Listeria monocytogenes Scott A haveincreased consumer concerns and interest in food safety ( Sofos andSmith, 1993 ). L. monocytogenes is commonly isolated from food

production plants including sh slaughter houses and sh smoke-

houses where some subtypes may be able to persist for months oreven years ( Vogel et al., 2001; Wulff et al., 2006 ). The potential forready-to-eat foods to become cross-contaminated with L. monocytogenes is well recognized either directly or via surfaces and equipmentthat have been in contact with raw materials ( Mena et al., 2004 ).

E. coli O157:H7 can be transmitted to humans through indirect ordirect contamination of foods ( Bouvet et al., 2001 ). Undercookedgroundbeef andraw milk have been implicated in foodborneinfection(Armstrong et al., 1996 ). Many strains of Shiga toxigenic E. coli arehuman pathogens causing illness ranging in severity from milddiarrhoea to severe renal complications that can result in death ( Rivaset al., 2007 ). Cross-contamination during processing and subsequenthandling and preparation of foods leads to the entry of thesepathogens into the food chain ( Hood and Zottola, 1997; Kumar andArand,1998 ). With increasing concerns over biotransfer potential andwith the low minimum infectious doses for pathogens such as E. coliO157:H7, the detection of low levels of contamination is becomingincreasingly important ( Davidson et al., 1999 ).

The aim of this work was to compare two commercially availablemethods, ATP bioluminescence and a UV light method to determinethe nature and limit of detection when used to detect organic material(food soils) and cell – soil fouling on stainless steel substrata.

2. Materials and methods

In this paper, commercial ‘kits ’ used for soil detection werecompared. The nature of the organic material soiling the surface willbe related to the food materials present. Thus a selection of complexsoils (cheese, sh and meat) was used in this study. These complex

Fig. 1. UV illumination imaging of soiled surfaces. Some components uoresced brightly (a) when illuminated with UV light whilst others were more dif cult to detect (b and c).a) Fish oil (oils), b) glycogen (proteins) and c) lactose (carbohydrate). Image size 20 mm×20 mm.

Table 1The level of soil concentration (%) detected on stainless steel using UV illumination(353 nm)

Organic material (soil) Level of soil detection (%)

Meat 0.1Fish 0.1Cheese 0.01Cholesterol 0.001Fish oil 0.1Fatty acids 0.001BSA 0.05Fish peptone 0.05Casein 0.001Glycogen 1Starch 0.1Lactose 0.05

122 K.A. Whitehead et al. / International Journal of Food Microbiology 127 (2008) 121 –128



organic soils are an ill-de ned heterogeneous mix of molecules, thusfor these studies a range of chemically de ned soil components (oils,proteins and carbohydrates) were also included in the study.

Three complex soils were used in this work (meat, sh and cheese)as were a range of chemically de ned soils including proteins (bovineserum albumin (BSA), sh peptones, casein), oils (cholesterol, sh oil,and a mixture of three fatty acids, myristic [21.79%] palmitic [58.29%]stearic [19.93%]), and carbohydrates (glycogen, starch and lactose).Each soil was made up in 10%, 5%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, 0.005%and 0.001% solutions. Meat, cheese and myristic acid were stored at−20 °C and were defrosted to room temperature before use. Fishextract, BSA, sh oil were stored at 4 °C, whilst the cholesterol,palmitic and stearic acids, sh peptone, casein and carbohydrateswere stored at room temperature. Soils were made up from thefollowing components.

2.1. Soiling components

2.1.1. Extraction of meat exudates (method kindly provided by BrigitteCarpentier AFSSA, France)

One kilogram of fresh rolled beef brisket (CO-OP, UK) was cut into10 mm×10 mm pieces. The meat pieces were put into a stainless steeltray and covered in aluminum foil. The meat was covered by anothertray and weighed down with 8.4kg of stainless steel sheets and frozenat −20 °C for 24 h. The meat was defrosted at room temperature andthe meat exudates were poured off and the meat squeezed to recoversurplus exudates. The meat exudates were stored in 20 ml aliquots at−20 °C until needed.

2.1.2. Fish extract (method kindly provided by Lone Gram, DIFRES,Denmark)

Fish llets (cod) were cut into cubes and 500 ml of tap water wasadded kg − 1 sh. The sh was boiled for 5 min. The juice was pressedfrom the sh esh and the resulting solution was drained off. Theliquid was boiled for 5 min without stirring and left for 5 min until

ocs were seen in the liquid. The liquid was ltered through doubledwood pulp (size 4) coffee lters (CO CO-OP, Manchester, UK). Then0.1 M phosphate buffer was added (7.62 g KH 2 (PO)4 and 7.66 g K2 H(PO)4 L − 1 ). The pH was adjusted to 6.6. The optical density (OD)(600 nm) was measured and adjusted to 0.21. The liquid was sterilisedby autoclaving at 100 °C for 30 min. The extract was cooled to 0 °C –

5 °C, which resulted in clearing of the liquid. The liquid was stored at5 °C.

2.1.3. Cheese extract Cheese extract (Langley Farm low fat natural cottage cheese,

Holmforth, W. Yorkshire) was made by draining off the liquid residuefrom the cottage cheese. The remaining cottage cheese was placedinto a stainless steel metal sieve with a 2 mm pore size and gentlypressed to remove all the liquid. The liquid was stored at −20 °C untiluse when it was defrosted to room temperature.

2.1.4. ChemicalsCholesterol, BSA, glycogen, sh oil, starch, myristic, palmitic,

stearic, casein and lactose were all obtained from Sigma Aldrich(Dorset, UK). Fish peptone was obtained from Fluka Biochemika(Switzerland). For the lactose, sh peptone, BSA, starch and glycogen1 ml of 10% solution was made in an Eppendorf tube using steriledistilled water. Samples that were dif cult to get into solution werevortexed for 5min. The sh oil, mixed fattyacids and cholesterol weremade separately into 10% solutions in Eppendorf tubes using chloro-form. The fatty acids myristic (21.79%), palmitic (58.29%) and stearic(19.93%)were mixed into a percentage solution to represent the majoroils found in cheese ( www.foodcomp.dk ).

2.2. Microbiology

The L. monocytogenes Scott A was a kind gift from Professor LoneGram (Danish Institute of Fisheries Research (DIFRES), TechnicalUniversity of Denmark). A non-pathogenic strain of E. coli O157:H7was a kind gift from Dr Brigitte Carpentier (Agence française desécurité sanitaire des aliments (AFSSA), Maisons-Alfort, France).

Stock cultures were stored at −80 °C. To store at −80 °C, the stockcultures were grown overnight in either tryptone soya broth (TSB)(Oxoid, Hampshire, UK) at 30 °C for L. monocytogenes or for E. coli inbrain heat infusion broth (BHIB) (Lab M, Bury, UK) at 37 °C. Equalamounts of culture and freezing mix were added together and wereincubated at the above temperatures for a least 30 min to 1 h. Sampleswere dispensed into 1.5 ml sterile plastic screw capped tubes, andwere frozen. To resuscitate, the culture was defrosted at roomtemperature and a loop of liquid was taken and streaked out ontopreferred media or broth and was incubated at the appropriatetemperature overnight. The remainder of the culture was returned tothe freezer for future use.The freezing mix was made of twosolutions.Solution A contained di-potassium hydrogen phosphate (K 2 HPO4 )12.6 g L − 1 , potassium di-hydrogen phosphate (KH 2 PO4 ) 3.6 g L − 1 tri-

Table 2UV wavelength bands at which individual organic material (soils) on surfaces weremost easily detected

Organic material (soil) Best detection wavelengthMeat 330 – 380 nmFish extract 510 – 560 nmCheese 330 – 380 nmBSA 330– 380 nmFish protein 330 – 380 nm

Casein 330–

380 nmCholesterol 510 – 560 nmFish oil 330– 380 nmFatty acids 510 – 560 nmGlycogen 330 – 380 nmStarch 330 – 380 nmLactose 590 – 650 nm

Fig. 2. Differentwavelengthbands of UV light on sh oil demonstratingthat speci c bandwavelengths of UV lightilluminate the shoil morebrightly. a)330 – 380nm,b) 510 – 560nm

and c) 590–

650 nm ( n =3).

123K.A. Whitehead et al. / International Journal of Food Microbiology 127 (2008) 121 –128

http://www.foodcomp.dk/

http://www.foodcomp.dk/



sodium citrate (Na 3 C6 H5 O7 ×2H2 O) 0.9 g L − 1 , ammonium sulphate(NH4 )2 SO4 ) 1.8 g L − 1 and glycerol 300 g L − 1 . This mixture wasautoclaved at 121 °C for 15 min. Solution B consisted of 1.8 g L − 1

magnesium sulphate (MgSO 4 ×7H2 O). The solution was lter sterilisedusing a 10 ml Luer-Lok ™ syringe (BDH, Poole, UK) and an Acrodisc® lter (32 mm with 0.2 μ m non-pyrogenic supar® membrane, PallCorporation, Cornwall, UK). Then 1 ml of solution B was added to100 ml of solution A to produce the nal freezer mix. All chemicals

used in the freezing mix were obtained from BDH (Poole, UK).In preparation for retention assays, stock cultures of L. mono-cytogenes were inoculated on tryptone soya agar (TSA) (Oxoid,Hampshire, UK), and incubated at 30 °C overnight. Cultures werestoredat 4 °C.Ten milliliters of TSBwas inoculated with a singlecolonyof L. monocytogenes and incubated at 30 °C overnight. One hundredmicroliters of this culture was used to inoculate 100 ml TSB, whichwas incubated at 30 °C for 18 h.

E. coli was inoculated onto brain heat infusion agar (BHIA, Lab M,Bury, UK). TheE. coliwas grown at 37 °C for 24 h. Cultures were storedat 4 °C. Ten milliliters of BHIB was inoculated with a single colonyof E.coli and incubated at 37 °C overnight. One hundred microliters of thisculture was used to inoculate 100 ml of BHIB, which was incubated at37 °C for 18 h.

Following incubation, cells were harvestedat 716 × g for 10 min andwere washed once, by re-suspension in sterile distilled water,vortexing for 1 min, and then centrifugation at 716 × g for 10 min.Cells were re-suspended to an OD of 1.0 at 540 nm in sterile distilledwater. Colony forming units ml − 1 (cfu ml− 1 ) were determined byserialdilution and were 1.62±0.92×10 8 cfu ml− 1 for L. monocytogenes and1.38±0.68×10 8 cfu ml− 1 for E. coli.

2.3. Surface preparation

2.3.1. Soiling of stainless steelOne hundred microliters of organic material was added to a

50 mm×40 mm 304 2B nish 2 mm thick stainless steel plate. Theorganic material was spread across the surface using a sterile plasticspreader. Samples were dried at room temperature in a class 2 owhood. The top 10 mm of the plate was used for labeling and handling.When cells were also added to the sample, 50 μ l of cells and 50 μ l of soil were mixed together in an Eppendorf tube and were applied tothe surface as above. L. monocytogenes was mixed with the cheese orthe sh extract, whilst E. coli was mixed with the meat exudate. Tovisualise cells alone on stainless steel, 100 μ l of the prepared cellsuspensions was added to the surface and allowed to dry at room

Fig. 3. ATP results on conditioning lms at concentrations of 10%, 5%,1%, 0.5%, 0.1%. 0.05%, 0.01%, 0.005% and 0.001%. a) Complex materials (meat, sh, cheese), b) proteins (BSA, shprotein, casein), c) oils (cholesterol, sh oil, fatty acids) and d) carbohydrates (glycogen, starch, lactose). — indicates level above ( b 29 RLU) in which the surface is not considered

hygienic.




temperature for 2 h in a class 2 safety cabinet. All samples were storedat 4 °C.

2.3.2. Preparation of test pieces for SEM The samples with soils±cells were immersed in 4% v/v gluter-

aldehyde (Agar, Essex, UK) for 24 h at 4 °C. Samples were thoroughlyrinsed with 100 cm 3 distilled H 2 O using a distilled water bottle at a 45°angle, with a 3 mm nozzle. Samples were then dried in a class 2 ow

hood. Samples were stored at room temperature in a phosphorouspentoxide (Sigma Aldrich, Dorset, UK) dessicator. The samples were xed to stubs for gold sputter coating, which was carried out using aPolaron E5100 (Milton Keynes, UK) SEM sputter coater. Samples weresputter coated at a vacuum of 0.0921 mbar, for 3 min, at 2500 V, inargon gas at a power of 18 – 20 mA. Images of substrata were obtainedusing a JEOL JSM 5600LV Scanning Electron Microscopy (Jeol Ltd.,Herts, UK).

2.4. Detection methods

2.4.1. UV detection methodA UV lamp (Labino trac-pack) with a lamp range of 353 nm was

kindly provided by Eigil Appel Pedersen founder of the Bactoforce®Company (Silkeborg, Denmark). When the Bactoforce light wasunavailable, a standard UV lamp (15 W bench lamp, 365 nm,1680 μ W cm− 2 at 305 mm, 115 VAC. 60 Hz, Cole-Parmer, London,UK) with an optimum wavelength of 350 nm was used.

2.4.2. UV wavelengthsIn order to determine the optimum wavelengths of the UV light to

illuminate the different soils, soiled samples were visualised using anepi uorescence microscopy (Nikon Eclipse E600, Surrey, UK). Differ-ent lters, 330 – 380 nm, 510 – 560 nm and 590 – 650 nm were used toselect speci c wavelength bands of UV light. The microscope wasmounted with an F-View II black and white digital camera (SoftImaging System Ltd., Helperby, UK, supplied by Olympus, Hertford-

shire,UK). This system useda Cell F Image Analysispackage (Olympus,Hertfordshire, UK). Dried but unstained samples were visualisedunder the differently ltered UV wavelength bands and imaged.

2.4.3. ATP bioluminescenceATP bioluminescent (Hygiena, Herts, UK) measurements were

carried outas permanufacturer'sinstructions( Hygeina,2007 ); however,the manufacturer recommends swabbing a 100 mm× 100 mm area, but

inthisstudy, a 40mm ×40 mm areawas swabbedforcomparability withtheotherexperiments carried out inthiswork. The40 mm×40mm areaof stainless steel was swabbed using the Ultrasnap ATPsampling device(Herts, UK) and placed into the ATP sample device (Herts, UK) accordingto the manufacturer's instructions.

3. Results

Some materials uoresced more brightly than others ( Fig. 1). At aconcentration of 10% w/v or v/v the sh oil (Fig. 1a), and sh protein uoresced brightly and whilst the cheese, fatty acids, casein, glycogen(Fig. 1b), starch and lactose ( Fig. 1c) were detected, they did not uoresce. The meat exudates, cholesterol, BSA and sh extract weredif cult to detect using this method. Detection levels varied with thedifferent components ( Table1 ). Thedetection levels rangedfrom 1% toa low level of 0.001%. Cholesterol, mixed fatty acids and casein wereeasily detected with the lowest amounts of organic soiling (0.001%)although they were not the most uorescent samples. Glycogen wasdif cult to detect at lower concentrations ( b 1%). No one type of material (i.e. complex, carbohydrates, proteins or oils) was more easilydetectable in comparison with other groups, although there wasvariation between components within the groups.

A range of UV wavelengths was used to identify whether differentsoil components could be better illuminated at particularwavelengthsin the UV spectrum. Filter blocks that selected for 330 – 380 nm, 510 –

560 nm and 590 – 650 nm wavelength bands were used. In someinstances, the different wavelengths of light illuminated certain soils

Fig. 4. SEM images demonstrating the distribution of cells when addedto a fouled soil surface. a) E. colionnakedstainlesssteel, b) E. colion stainless steel fouled with meat exudates,

c) L. monocytogenes on naked stainless steel, d) L. monocytogenes on stainless steel fouled with sh extract and e) L. monocytogenes on stainless steel fouled with cheese extract.




selected will be based on the levels of microorganisms detected onfood surfaces in situ .

UV detection provided a simple, non-invasive, non-quanti ablemethod that could not discriminate between cell and soil fouling. Of the different food components screened, oils were detected particu-larly well. The UV wavelength band of 330 – 380 nm was appropriatefor the detection of residual soil in the meat and cheese industry, but aUV wavelength band of 510 – 560nm could be considered for improved

soil detection in the

sh industry. Thus, performance of UV detectioncould be optimised by using light sources with different wavelengthsin the UV spectra to highly illuminate target soils on food processingequipment in different environments. It would be interesting to testthese UV wavelength bands of light on soils retained in vitro and insitu on other surfaces found in the food industry such as rubber andplastics in order to determine if certain UV wavelengths of light candetect soiled surfaces whilst reducing the auto uorescence of thesurfaces onto which they are retained.

The need for rapid industrial detection methods such as these isimportant in determining the ef ciency of cleaning and disinfectionagainst organic soil and cell retention. Consideration might also bepaid to the nature of soil retention on the substratum. Key to theselection of soil for this work was the retention of speci c soils to ade ned substratum. Wildbrett and Sauerer (1989) showed that themain fouling component for stainless steel was proteins, whilst fatwas the main fouling component of plastics. Thus, in terms of surfacehygiene, from our results, further work might be prudent in order todetermine whether it might be more appropriate to use stainless steelrather than polymer materials in a food environment that is fat rich.

As each food processing environment is likely to have a particularmicrobial ecology re ecting the food preservation conditions(Bagge-Ravn et al., 2003 ), the mixture of microorganisms and soilselected when testing cleaning and disinfection agents in vitroshould be appropriate to the intended environment of use. In ad-dition, behavior of microorganisms such as L. monocytogenes isstrongly in uenced by the food matrix ( Gram et al., 2007 ). Forcomplex organic foulants, work by Gram et al. (2007) showed thatsanitizers were extremely ef cient against L. monocytogenes attachedfrom a sh emulsion, whereas only a marginal effect was seen whenthe same sanitizer was used against the organism attached from ameat emulsion.

The detection of organic material is important since food soilsin uence cell retention to a surface, and also inhibit cleaning anddisinfection processes. Much work has been carried out on the effectof proteins on cell attachment, but less consideration has been givento the effect of lipids and carbohydrates on cell retention. Our worksuggests that although a hygienic reading may be given for a surface(for example the proteins), the surface may still retain a conditioninglayer of material that is only detectable by other methods. This willobviously change substratum properties. Although it has beensuggested that generally adsorbed proteins reduce bacterial attach-ment ( Meadows, 1971; Fletcher, 1976; Orstavik, 1977; Fletcher and

Marshall, 1982; Helke et al., 1993; Hood and Zottola, 1997; Barneset al., 1999 ), increased attachment has also been observed ( Meadows,1971; Almakhla et al., 1994; Verran andWhitehead, 2006 ). Inhibitionof cell attachment to surfaces could be due to masking of the surfacechemical groups, which would stopthe adhesive interactions with thebacteria, or due to stearic stabilization ( Rutter and Vincent, 1984 ).Reduced attachment of cells to surfaces may also be due to proteins inthe bulk uid phase competing for binding sites on the stainless steelsurface ( Flint et al., 2007 ). However, it must be remembered that thenature of the effect of the conditioning lm appeared to vary with theorganism, substratum and protein under investigation ( Barnes et al.,1999 ). The increased attachment of cells to rubber and stainless steelwith either whey proteins or lactose has been demonstrated ( Speersand Gilmour, 1985 ). Lipopolysaccharides inhibited attachment of cells

to substrata if in the presence of cells or adsorbed to the substrata

before attachment, whereas dextrans have only demonstrated inhibi-tion when in the presence of the cells ( Pringle and Fletcher, 1986 ).

Both methods implemented in this study areusedfordetection only,and do not indicate the nature of the material (or microorganisms)present. Under UV, oily soils, mixed fatty acids, cholesterol and caseinwere detected at lowconcentrations,with detection levels ranging from1% to 0.001% for different substances. A wavelength band of 330 –

380 nm, illuminated most of the soils. For combined cell – soil surfaces

the L. monocytogenes and cheese combination was the most intenselyilluminated. Soils at all concentrations were detected by the ATPbioluminescence method; the complex soils gave the highest readings.Shortfalls with these methods include detection of the fouled area oncethelight has been removed or the area has been swabbed(ATP), there isno indication as to where the foulingproblemareas occurred. However,these methods provide a veryuseful screen forcleaning procedures andto highlight dif cult to clean areas. The interactions between substrata,soil components and detection methods warrant further work.

Acknowledgements

The authors would like to give their sincere thanks to Eigil AppelPedersen founder of Bactoforce® for his use of their specialistequipment and to the partners of Work Package 11 (HygienicProcessing Systems). This work has been part of and has been fundedby the European PathogenCombat consortium.

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