Modeling the Rate of Attachment of Listeria monocytogenes, Pantoea agglomerans, and Pseudomonas fluorescens to, and the Probability of Their Detachment from, Potato Tissue at 10{degrees}C

The rate of attachment of bacteria to, and their subsequentdetachment from, the cut surface of raw potato tissue was measuredand modeled by using mathematical approaches that allowed detailedobjective comparisons of adhesion processes under differentconditions. Attachment was rapid and reached equilibrium aftercontact for 60 min. A new method to measure the probabilityof detachment was developed and modeled, revealing that theprobability of detachment for Pseudomonas fluorescens remainedunchanged for contact times between less than 5 s and 60 min.Listeria monocytogenes, however, was more easily removed initially,with the probability of detachment decreasing over the first2 min of contact but remaining constant and equivalent to thatfor Pseudomonas fluorescens thereafter. For all of the bacteriatested, the number of bacteria attached after 2 min of contactwas proportional to the inoculum concentration raised to thepower of 0.79.

INTRODUCTION

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Introduction

There is a growing body of literature reporting the bacterialcontamination of ready-to-eat prepared vegetable tissues. Suchtissues often become contaminated in the field. Despite postharvesttrimming and decontamination processes, this contamination persistsduring commercial preparation, and the products can containin excess of 10⁶ viable bacteria g^–1 at the time of packing(6). Contaminants are predominantly Pseudomonas spp. and Pantoeaspp. (5, 6), but bacteria of public health significance, suchas Escherichia coli, Yersinia spp., and Listeria spp., may alsobe present (1, 4, 5, 12, 14; K. Sizmur and C. W. Walker, Letter,Lancet i:1167, 1988).

Scanning electron microscopy of prepared leaf tissues has shownthat bacteria can be found on the abaxial and adaxial surfaces.However, the bacteria preferentially occupy the cut surfaces,where they multiply throughout storage of the products to forman extensive layer, sometimes many cells thick (6). Growth ofPseudomonas spp. and Pantoea spp. on cut surfaces is followedby production by these bacteria of an extracellular polysaccharide,creating a protective layer which is resistant to chemical andphysical decontamination processes (10). Colonization of thecut surface is a two-stage process; initial adhesion occursrapidly and is followed by the production of an extracellularpolysaccharide by the adhered bacteria.

Most studies of colonized vegetable tissue surfaces emphasizethe mature biofilm stage, and little work has concerned theinitial colonization process. However, commercial decontaminationprocesses are typically applied to prepared vegetable tissueswithin a few minutes of chopping, shredding, or dicing. Theseprocesses usually involve washing in potable water with, insome countries, added biocidal compounds, such as chlorine (11).Such washing occurs considerably before the establishment ofthe protective extracellular polysaccharide, and yet the washingprocess still fails to remove all of the bacteria (2, 6, 9,13).

Here we report studies of the measurement and modeling of therate of adhesion of bacteria to, and the probability of theirdetachment from, vegetable tissue during the early stages ofcolonization.

MATERIALS AND METHODS

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Materials and Methods

Organisms.
Listeria monocytogenes strain F4642 (Scott A) serotype 4b wassupplied by R. Weaver of the Centers for Disease Control andPrevention, Atlanta, Ga. L. monocytogenes strains 10403S andDP-L189 (EGD), both serotype 1/2a, were supplied by D. A. Hodgsonof the University of Warwick, Warwick, United Kingdom. Pantoeaagglomerans VCM was isolated from mung bean sprouts, and Pseudomonasfluorescens A36 was isolated from green peppers; both foodswere obtained from commercial vegetable processing sites.

Culture media.
Stock cultures were maintained on heart infusion agar (Difco)slants incubated at 25°C for 48 h and subsequently storedat 1°C. Fresh slants were subcultured monthly.

Cultures for measurement of the duration of the lag phase ofbacteria, the rate of attachment, and the probability of detachmentwere grown in Pseudomonas broth F (PBF), which contained 10g of tryptone (Difco), 10 g of proteose peptone number 3 (Difco),1.5 g of dipotassium hydrogen phosphate (BDH), 3.1 g of magnesiumsulfate heptahydrate (BDH), and 10 g of glycerol (BDH). Componentswere dissolved in 1 liter of glass-distilled water by boilingthem and then sterilized by autoclaving them at 121 to 124°Cfor 15 min. Inocula were prepared in Trypticase soy broth (TSB;Baltimore Biological Laboratory).

Preparation of inocula.
Bacteria were grown successively in 10 ml of TSB at 25°Cfor 24 h and then at 20°C for 24 h. Inocula for measurementof the rates of attachment and probabilities of detachment wereprepared by dilution of the culture in PBF, previously cooledto 10°C, to give suspensions that contained between 10⁴and 10⁸ viable bacteria ml^–1.

Determination of the lag phase of bacteria.
PBF (100 ml) was transferred to sterile 250-ml Erlenmeyer flasks,which were closed with a rubber bung fitted with a filter toallow gaseous exchange and a device for aseptic sampling ofthe culture. The flask and its contents were then precooledto 10°C overnight. An inoculum (1 ml) grown in TSB was addedto the PBF to give a suspension that contained approximately10³ to 10⁴ viable bacteria ml^–1. Flasks were incubatedat 10°C on an orbital platform shaker (New Brunswick Scientific,Edison, N.J.) rotating at 100 rpm. At intervals throughout incubation,a sample of culture was removed, and the number of viable bacteriawas determined by spreading 50 µl of this suspension,or dilutions of it made in peptone salt dilution fluid (PSDF)(8), onto the surfaces of plates with plate count agar (PCA)(Oxoid CM325), by using a model D spiral plate maker (SpiralSystems, Inc.). The plates used for the enumeration of L. monocytogenescells were incubated at 35°C for 24 h, and those used forthe enumeration of Pseudomonas fluorescens and Pantoea agglomeranscells were incubated at 25°C for 48 h.

Growth curves were fitted using a nonautonomous differentialequation (the Baranyi D model) (3) in order to estimate thelag time and the initiation of the logarithmic phase of growth.The nonautonomous differential equation was fitted to the growthdata by a software program (MicroFit [www.ifr.bbsrc.ac.uk/microfit])written specifically for this purpose.

Preparation of sterile potato disks.
Potatoes were chosen as a model vegetable tissue surface becausesterile disks that were of uniform thickness and compositionand had an entire surface composed of damaged plant cells couldbe prepared. Disks of other tissues (such as carrot, green pepper,celery, and lettuce) were also prepared, but these had differentiatedtissues within them and hence were not uniform, had cut surfacesof inconsistent dimensions (for example, only along the edgesof the disks in the case of lettuce leaf tissues), and weredifficult to prepare aseptically. Avoidance of competing bacteriawas considered important in the work reported here. Preliminarystudies showed that the rates of attachment of L. monocytogenes,Pantoea agglomerans, and Pseudomonas fluorescens to potato diskswere identical to their rates of attachment to green pepperdisks (data not shown). However, preparation of sterile disksof green pepper was difficult and could not be achieved reliably.In contrast, potato disks were simple to prepare aseptically.Accordingly, although potato tissue is unlikely to be eatenraw, it was used as a representative model of tissues that areeaten raw.

Potatoes obtained from a local supermarket were sprayed with70% (vol/vol) ethanol, peeled with a sterile potato peeler,sprayed again with 70% (vol/vol) ethanol, and allowed to dryin a sterile airflow on a sterile surface. A cylindrical coreof potato tuber (27-mm diameter) was cut with a sterile corkborer. The core was extruded from the borer with a sterile stainlesssteel rod, and disks of potato tissue were cut to the desiredthickness with a sterile knife. The sterility of the disks waschecked by immersion of representative disks in TSB, which wasincubated at 20°C and examined for turbidity.

Rate of attachment.
Sterile potato disks with a thickness of approximately 1 mmwere prepared. PBF (20 ml) was cooled to 10°C in sterile,disposable plastic petri dishes (90-mm diameter). An inoculumwas added to the PBF, and up to four potato disks were placedinto the inoculated PBF in each petri dish and incubated withoutshaking at 10°C. Typically, 12 disks were incubated foreach experiment. At intervals, a potato disk was removed withsterile forceps and placed in a second petri dish, where itwas rinsed five times with 20 ml of PSDF in order to removebacteria that were loosely associated with, but had not adheredto, the potato tissue. Rinsing involved tilting the petri dishthree times at an angle of 10^o before decanting the rinsingliquid. Bacteria remaining on the disk were then removed byblending them with 10 ml of PSDF for 60 s by using a StomacherLab-Blender. The number of viable bacteria in the resultantsuspension was determined by spreading 50 µl of this suspension,or dilutions of it made in PSDF, onto the surfaces of platesof PCA by using the spiral plate maker. Plates were incubatedas described above.

Modeling the rate of attachment of bacteria to potato tissue.
The kinetics of adsorption were assumed to be first order, anddesorption as well as adsorption was included in the model (equation1).

(1)
Here N_B was the number of bacteriabound to the surface, N was the number concentration of bacteriafree in suspension (which remained very much larger than N_Bthroughout the experiment and was thus taken to be constant),and t was time. The adsorption and desorption rates were k_aand k_d, respectively. For a more general solution, a term involvingthe surface area of the potato would need to be included, butas the potato disks used in this study were identical, no suchterm was required in equation 1. At equilibrium, where the rateof change of the number bound was 0, the number bound at equilibrium(N_B,eqm) was given by rearranging equation 1 to give equation2.

(2)
It would also be possible todetermine a value for the initial rate of adsorption where theinitial number bound was 0 (equation 3).

(3)
Bothof these quantities depended on the inoculum concentration,N. By the integration of equation 1, the insertion of boundaryconditions, and rearrangement, the equation describing the numberof bound bacteria with time (N_B,t) was determined (equation4).

(4)

Probability of detachment.
Sterile potato disks were prepared as described above, witha thickness of approximately 4 mm, and care was taken to ensurethat the cut surface of each was flat. An inoculum in PBF (50ml) was poured into sterile plastic petri dishes, and potatodisks were prepared and incubated, all as described above. Atintervals, a potato disk was removed and rinsed as describedabove and then sequentially blotted facedown for 60 s onto thesurface of each of a series of PCA plates. These had previouslybeen partially dried in a fan oven at 55°C for 60 min andcooled to room temperature. After removal of the disk from eachplate, the bacteria deposited on the agar were spread over theplate surface with a sterile disposable spreader. Plates wereincubated as described above. The potato disk was incubatedon the surface of the final PCA plate of the series in orderto determine if bacteria remained on the disk after completionof the blotting series. The number of CFU on each PCA platein the blotting series was counted, and its logarithm was plottedagainst the plate number in the blotting series. A straightline was fitted to each data set using the linear regressionfunctions of Excel 2000 (Microsoft), from which a slope, anintercept, standard deviations, and the correlation coefficient,R², were determined. The gradient was a measure of the probabilityof removal of bacteria from the surface of the potato disk.For example, in the case of two disks with the same number ofattached bacteria, a higher probability of removal resultedin a steeper gradient, resulting from a higher proportion ofthe bacteria being removed by the first blotting. From the gradientand the intercept, the number of bacteria initially bound tothe disk before blotting was determined by an extension of themethod of Eginton et al. (7).

Modeling the probability of detachment.
It was assumed that the relationship between the number of viablebacteria per plate and the plate number in the blotting seriesfitted an exponential decrease (equations 5 and 6) as describedby Eginton et al. (7).

(5)

(6)
Here, A is a constant, C is the number of CFUper blot plate, i is the plate succession number in the blottingseries, and k is the reduction exponent.

Eginton et al. (7) determined k by linear regression of thedata from plots of the logarithms of the numbers of viable bacteriaon the plates against the plate numbers. They interpreted thevalue of k as the inverse of the strength of attachment of bacteriato the surface of interest from an argument that identifiedk as the ease of removal of the bacteria from the surface. Toarrive at a more accurate interpretation of the gradient measuredin the work reported here, we have considered the probabilityof removal of bacteria from the surface.

The probability that any particular bacterium was removed duringa blotting event was represented by x, where x took a valuebetween 0 and 1. For an initial number of bacteria adhered tothe face of a potato disk (N_F), the number removed after thefirst blotting event (N_d,1) and therefore the number remainingon the disk (N_r,1) were given by equations 7 and 8.

(7)

(8)
This was extendedto the ith plate in the blotting sequence to give equations9 and 10.

(9)

(10)
Takingthe logarithm of equation 9 resulted in a form that was functionallyidentical to that obtained by Eginton et al. (7), but the parameterswere defined differently (equation 11).

(11)

RESULTS

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Results

Growth in broths.
The lag times estimated by Microfit for each organism grownin PBF at 10°C were 3.1 h for L. monocytogenes Scott A,3.6 h for Pseudomonas fluorescens A36, and 4.9 h for Pantoeaagglomerans VCM. Accordingly, no attachment experiment was continuedfor more than 3 h in order to exclude the possibility of complicationof bacterial multiplication during the experiment.

Rate of attachment of bacteria to potato tissue.
Tests confirmed that potato disks were prepared aseptically,and hence all experiments were conducted in the absence of apotentially competing microflora. The numbers of bacteria attachedto the potato tissue disks increased with time for all organismstested and reached a plateau after approximately 1 h, althoughall bacteria showed significant adsorption at times as shortas 5 min (Fig. 1).

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FIG. 1. Effect of time of contact on the adhesion of bacterial cells to disks of raw potato tissue. Numbers of viable bacteria are the numbers per disk per inoculum concentration (numbers of cells per milliliter). ${blacksquare}$ , L. monocytogenes Scott A; ${diamond}$ , L. monocytogenes 10403S; ${diamondsuit}$ , L. monocytogenes DP-L189; ${blacktriangleup}$ , Pantoea agglomerans VCM; •, Pseudomonas fluorescens A36. Data points are from individual experiments performed either singly, in duplicate (for Pseudomonas fluorescens A36 and Pantoea agglomerans VCM), or in triplicate (for L. monocytogenes Scott A).

Curves with the form of equation 4 were fitted to the data fromFig. 1 by using curve-fitting software (Tablecurve 2D version2.03; Jandel Scientific, Erkrath, Germany), which yielded valuesfor the numbers of bacteria bound at equilibrium, N_B,eqm, therates of adsorption, k_a, the rates of desorption, k_d, and thestandard errors of these values (Table 1).

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TABLE 1. Coefficients determined by fitting equation 4 to the data shown in Fig. 1^a

Probability of detachment of bacteria from potato tissue.
The numbers of bacteria removed from the colonized potato tissuedisks by the successive blotting technique is shown in Fig.2 and 3 (for L. monocytogenes) and in Fig. 4 (for Pseudomonasfluorescens), where they are scaled for inoculum concentration.From these data, the probabilities of removal of a bacteriumfrom the tissue during a blotting event, x, and the numbersattached to the face of the disk before blotting commenced,N_F, were obtained (Table 2), with the equation for N_F (equation12) being equivalent to the method of summation developed byEginton et al. (7).

(12)
Figure 2shows the effect of varying the initial numbers of bacteriafrom 8.0 x 10⁴ to 7.6 x 10⁷/ml on the basis of the probabilityof detachment of L. monocytogenes Scott A, with a contact timeof 2 min. The gradients (k), and therefore the probabilitiesof detachment (x) (Table 2), for the three highest inoculumconcentrations were not significantly different from one another(P > 0.20), with the three replicated determinations at aninoculum of 7.6 x 10⁵ viable bacteria ml^–1 showing excellentagreement (P > 0.75). The apparent difference in the probabilitiesof detachment at the two lowest inoculum concentrations wasnot significant, with a P of 0.05.

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FIG. 2. Effect of inoculum concentration on the subsequent removal of L. monocytogenes Scott A from the surfaces of raw potato disks by blotting. Disks were in contact with the inoculum for 2 min. Numbers of viable bacteria are the numbers of cells per blot plate. Initial inoculum concentrations (per milliliter) were 8.0 x 10⁴ (+), 1.3 x 10⁵ ( ${blacktriangleup}$ ), 7.6 x 10⁵ (x), 7.6 x 10⁵ (•), 7.6 x 10⁵ ( ${circ}$ ), 8.0 x 10⁶ ( ${diamondsuit}$ ), and 7.6 x 10⁷ ( ${blacksquare}$ ). Solid lines represent the best fit to the data obtained by linear regression.

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FIG. 3. Effect of time of contact with the inoculum on the subsequent removal of L. monocytogenes Scott A from the surfaces of raw potato disks by blotting. Numbers of viable bacteria are the numbers of cells per blot plate. The initial inoculum concentration was 1.3 x 10⁵/ml. Disks were in contact with the inoculum for <5 s ( ${blacktriangleup}$ ), 30 s (•), 2 min (x), 10 min ( ${blacklozenge}$ ), and 60 min ( ${blacksquare}$ ). Solid lines represent the best fit to the data obtained by linear regression.

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FIG. 4. Effect of inoculum concentration and time of contact with the inoculum on the subsequent removal of Pseudomonas fluorescens A36 from the surfaces of raw potato disks by blotting. Numbers of viable bacteria are the numbers per disk at each inoculum concentration, measured in cells per blot plate. The times of contact and inoculum concentrations were <5 s, 6.2 x 10⁵/ml (•); <5 s, 5.0 x 10⁴/ml ( ${circ}$ ); 1 min, 5.0 x 10⁴/ml ( ${blacksquare}$ ); 2 min, 5.0 x 10⁴/ml ( ${blacklozenge}$ ); 3 min, 5.0 x 10⁴/ml ( ${blacktriangleup}$ ); 5 min, 5.0 x 10⁴/ml (x); 10 min, 5.0 x 10⁴/ml ( ${square}$ ); 15 min, 6.2 x 10³/ml ( ${diamond}$ ); 30 min, 6.2 x 10³/ml ( ${circ}$ ); and 60 min, 6.2 x 10³/ml (+). Solid lines represent the best fit to the data obtained by linear regression.

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TABLE 2. Coefficients describing the probability of detachment of bacteria from disks of potato tissue

From inspection of Fig. 2, it is clear that the initial numberof bacteria adsorbed to each face of the disk, N_F, increasedwith the inoculum concentration. It was assumed that the relationshipobeyed a simple power law (equation 13), so the logarithm ofN_F was plotted against the logarithm of the initial inoculumconcentration (Fig. 5).

(13)
The coefficientsa and b were determined to be 0.79 (standard error, 0.05) and1.418, respectively, by linear regression, with an R² valueof 0.979, which demonstrated that the assumption of the powerlaw relationship was sound.

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FIG. 5. Effect of the initial inoculum concentrations on the numbers of cells of L. monocytogenes Scott A attached to the face of a raw potato disk. The solid line represents the best fit to the data obtained by linear regression.

From Fig. 3, it can be seen that the gradient, and hence theprobability of detachment, x, for L. monocytogenes Scott A,decreased over the first 2 min but then remained constant upto 60 min. At the 5% probability level, the gradients of thezero time sample and the 30-s sample were not significantlydifferent, mostly because the former was defined from only fourpoints, which resulted in a very large uncertainty in the valueof its gradient. However, the gradients of these lines weresignificantly different (P < 0.05) from those of all of thelater samples, which showed the change in detachment probabilityto be real.

In the case of Pseudomonas fluorescens A36 (Fig. 4), the probabilityof detachment remained constant (P < 0.05) after contacttimes between 0 and 60 min.

The two techniques used here (the blotting of disks of tissuethat had been in contact with bacteria for a given time andthe enumeration of bacteria on disks of tissue during continuouscontact) enabled an interesting comparison to be made: the numberof bacteria calculated to populate a disk compared to the numberactually enumerated by their removal with the Stomacher Lab-Blender.The results of this analysis are shown in Fig. 6. The numbersof L. monocytogenes and Pseudomonas fluorescens organisms attachedto the surfaces of potato disks before blotting were calculated(equation 12), and the results showed the expected increasewith time of contact. The number of L. monocytogenes organismsenumerated by removal with the Stomacher Lab-Blender and thenumber enumerated by blotting were comparable throughout theimmersion history, once we corrected for differences in thesurface areas from which the bacteria were removed (i.e., blottingwas from a single face, but use of the Stomacher Lab-Blenderremoved bacteria from both the faces and the edge of the disk).However, the number of Pseudomonas fluorescens organisms enumeratedby blotting was significantly higher than that determined byremoval with a Stomacher Lab-Blender.

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FIG. 6. Effect of time of contact with raw potato disks on the numbers of cells of L. monocytogenes Scott A ( ${blacksquare}$ , ${square}$ ), and Pseudomonas fluorescens A36 (•, ${circ}$ ) adhered to raw potato disks, enumerated by removal with the Stomacher Lab-Blender (filled symbols) or by the blotting technique (open symbols). Data points are from individual experiments performed either singly, in duplicate, or in triplicate.

DISCUSSION

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Discussion

The mathematical approaches adopted here allowed modeling ofthe rate of attachment of bacteria to, and their detachmentfrom, the cut surface of raw potato tissue.

The numbers of bacteria attached to potato disks were significantlydifferent among organisms, with Pseudomonas fluorescens andPantoea agglomerans adsorbing in significantly higher numbersthan L. monocytogenes (P < 0.001). It is possible that thisdifference was due to the difference in cell wall structuresbetween the gram-positive and gram-negative bacteria, whichis under further investigation. It was also clear that the twoserotypes of L. monocytogenes showed considerable differencesin the numbers of bacteria adsorbing, with serotype 4b (ScottA) adsorbing significantly (P < 0.001) higher numbers thanserotype 1/2a (10403S and DP-L189 EDG). These differences arepossibly attributable to differences in the surfaces or surfacestructures of the two serotypes. It was also clear that, forany particular organism, the values obtained could be very varied(e.g., two of the replicate determinations of the attachmentof L. monocytogenes Scott A gave attachment numbers that weresignificantly higher than those from the other two determinationsat the 1% level, although the values for the other two replicateswere very similar to each other). This variation was possiblya function of the natural variability in the vegetable materialsused, which makes these studies inherently difficult.

The assertion by Eginton et al. (7) that k (determined by linearregression of the data from the plots of the logarithms of thenumbers of viable bacteria on the plates, against the platenumbers) can be interpreted as the inverse of the strength ofattachment of bacteria to the surface of interest appears flawed.If this were indeed the case and the concept of a single strengthof attachment were correct, then any detachment technique thathas exceeded a notional threshold force required to remove onebacterium should result in the removal of all bacteria. We believethat our interpretation of the gradient, as a function of theprobability of removal of bacteria from the surface, is preferable.It is of interest, however, that the k determined by Egintonet al. (7) can be equated to the negative logarithm to the base10 of (1 – x) in the probabilistic analysis presentedhere. Overall, the probability of detachment, x, was not affectedsignificantly by changes in inoculum concentration. Our simpleanalysis does not allow for multiple, or distributions of, detachmentprobabilities. If these had more than a single value, our techniquewould measure an average.

The power (a) of the relationship between the number of bacteriaadsorbed to each face of the potato disk (N_F) and the initialinoculum concentration (i.e., where an increase in inoculumconcentration would result in a directly proportional increasein attachment) was significantly different from the expectedvalue of 1.0 (P < 0.0001). It was possible that the lowervalue of 0.79 occurred because the process of adsorption wassaturated (i.e., the rate of adsorption was limited by diffusionof the bacteria to the surface, and then once the inoculum reacheda certain concentration, the addition of more bacteria wouldnot result in as large an increase in collision rate as at lowerconcentrations). Future studies will determine if this relationshipbetween the number of bacteria attached and the inoculum concentrationis true for different times, organisms, and conditions of attachment.

The number of Pseudomonas fluorescens calculated as populatinga disk can be higher than the number counted on the disk byusing the Stomacher Lab-Blender (Fig. 6). It is possible thatbecause this organism attaches rapidly to vegetable tissue,bacteria removed by blending in the Stomacher Lab-Blender wereable to reattach to the tissue before sampling, resulting inan underestimate of the true number of bacteria on the diskby this traditional enumeration technique. However, bacteriawere being successfully removed in the turbulent liquid shearfields produced in the Stomacher Lab-Blender, in which the potatodisks remained intact. This result demonstrated that, althoughthe bacteria had attached (Fig. 3 and 4), effective removalcould be achieved if sufficient force was applied. This findingsuggests that commercial decontamination is possible if veryvigorous washing procedures are applied to the surfaces of thevegetable tissues in order to overcome the attachment phenomenaquantified here. Additional safeguards are then required toprevent reattachment of the bacteria from the washing liquid.

It is anticipated that the experimental methods and the mathematicalmodels developed here will enable a more detailed investigationof the mechanisms responsible for the attachment of bacteriato vegetable tissues. From this will come improved decontaminationprotocols and methods to prevent bacterial adhesion that canbe applied during commercial preparation of ready-to-eat vegetableproducts.

ACKNOWLEDGMENTS

We acknowledge the financial support of the Biotechnology andBiological Sciences Research Council for this work.

We also thank Sam Forget and Stephanie Cuney for valuable technicalassistance.

FOOTNOTES

* Corresponding author. Mailing address: Food Safety Science Department, Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, United Kingdom. Phone: 44 (0) 1603 255399. Fax: 44 (0) 1603 507723. E-mail: tim.brocklehurst{at}bbsrc.ac.uk.

Present address: IP 21, Norwich Research Park, Colney, NorwichNR4 7UT, United Kingdom.

REFERENCES

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