Temperature dependence of shelf-life as affected by microbial proliferation and sensory quality of equilibrium modified atmosphere packaged fresh produce

Postharvest Biology and Technology 26 (2002) 59–73

Temperature dependence of shelf-life as affected bymicrobial proliferation and sensory quality of equilibrium

modified atmosphere packaged fresh produce

L. Jacxsens, F. Devlieghere *, J. DebevereDepartment of Food Technology and Nutrition, Laboratory of Food Microbiology and Food Preser�ation,

Faculty of Agricultural and Applied Biological Sciences, Ghent Uni�ersity, Ghent, Belgium, Coupure links 653, 9000 Gent, Belgium

Received 7 May 2001; accepted 21 December 2001

Abstract

An objective method to define the shelf-life of Equilibrium Modified Atmosphere (EMA) packages of minimallyprocessed vegetables is presented. The changes in sensory quality and proliferation of human pathogenic and spoilagemicro-organisms on three lightly processed and packaged products as a function of storage temperature weremeasured. Sensory quality limited the shelf-life of mixed lettuce and cucumber slices before the limiting effects ofmicrobial proliferation. Shelf-life periods were: for EMA-packaged cucumber slices, stored at 2, 4, 7 and 10 °C, 4, 7,5 and 4 days, respectively; for EMA-packaged mixed lettuce 9, 7, 5 and 3 days, respectively. For bell peppers, on theother hand, the shelf-life was limited by microbial proliferation due to a lower initial microbial quality of the productand extensive availability of water and nutrients. The pH of the product, microbial load and storage temperature hada profound effect on the proliferation of the psychrotrophic pathogens Listeria monocytogenes and Aeromonas ca�iae.Cucumber slices, stored at 7 and 10 °C, had �2 log units growth of L. monocytogenes during the defined shelf-life.A limited though significant growth of both pathogens was noticed on the mixed lettuce, stored at elevatedtemperatures (7 and 10 °C). On bell peppers, a decrease in L. monocytogenes and survival of Aer. ca�iae was detected.Realistic criteria of presence of L. monocytogenes on day 0 (production day) must guarantee microbial safety duringthe shelf-life of the product, especially when storage temperatures are elevated (7 and 10 °C). In order to guaranteethe quality of the fresh-cut produce, it is recommended to keep the product at 4 °C. © 2002 Elsevier Science B.V.All rights reserved.

Keywords: Quality; Food safety; Fresh produce; Packaging; Temperature abuse

www.elsevier.com/locate/postharvbio

1. Introduction

The demand for fresh, minimally processed veg-etables has led to an increase in the quantity andvariety of products available for the consumer.The fresh nature of these products, together with

* Corresponding author. Tel.: +32-9-264-6178; fax: +32-9-225-5510.

E-mail address: [email protected] (F. De-vlieghere).

0925-5214/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved.

PII: S0 925 -5214 (02 )00004 -2

mailto:[email protected]

L. Jacxsens et al. / Posthar�est Biology and Technology 26 (2002) 59–7360

the mild processing techniques and subsequentstorage conditions, have presented indigenous andhuman pathogenic micro-organisms with newecosystems (Francis et al., 1999). Counts ofmesophilic bacteria after processing range from 103

to 109 cfu/g. Product quality is often acceptabledespite such high counts (Nguyen-the and Carlin,1994; Bennik et al., 1998; Brackett, 1999). Micro-bial species potentially causing spoilage vary withthe kind of minimally processed product. Moresugar-rich vegetables undergo microbial fermenta-tion due to growth of lactic acid bacteria or yeastswhereas others develop soft rot symptoms due toproliferation of Gram negative (pectinolytic) mi-cro-organisms. However, in many cases, spoilagecannot be attributed to one particular micro-or-ganism (Nguyen-the and Carlin, 1994; Varoquauxet al., 1996).

Equilibrium modified atmosphere (EMA) pack-aging is increasingly being employed as a mildpreservation technique (Day, 1990). The positiveeffect of typical EMA storage conditions, e.g. 1–5kPa O2 in combination with 3–10 kPa CO2, on theshelf-life of vegetables is thought to result from alowered product respiration and reduction of theoverall rate of metabolic processes resulting in abetter retention of the physiological state of theproduce and consequently, inhibition of the growthof spoilage micro-organisms (Jacques and Morris,1995; Bennik et al., 1998; Jacxsens et al., 1999a).

The microbial safety of fresh-cut produce shouldbe ensured in addition to maintaining acceptablesensory and microbial quality. A number of food-borne diseases have been attributed to the con-sumption of fresh-cut produce (Brackett, 1999).Psychrotrophic pathogens which are capable ofgrowing or maintaining an infectious potentialunder mild preservation regimes are of particularconcern, Listeria monocytogenes, Yersinia entero-colitica, Clostridium botulinum and Aeromonas hy-drophila/ca�iae being amongst the most notable(Nguyen-the and Carlin, 1994; Francis et al., 1999).Y. enterocolitica has been isolated at high fre-quency (up to 76% of samples), but the strains wererecognised as non-pathogenic on the basis of theirserovar (Nguyen-the and Carlin, 1994). Optimal(aerobic) EMA-packages will not support growthand toxin production of C. botulinum, an obligate

anaerobic micro-organism (Blaschek, 2000). Thegrowth/survival of L. monocytogenes depends onthe type of vegetable, storage temperature andcomposition of atmosphere (Berrang et al., 1989b;Bennik et al., 1995; Francis et al., 1999; Jacxsenset al., 1999a). Aeromonas spp. are considered ubi-quitous in refrigerated produce (Neyts et al., 2000a)and Aer. ca�iae (HG4) causes septicemia or gastro-enteritis as an opportunistic pathogen in humans(Abbott et al., 1994). The safety concerns aboutfresh-cut vegetables, stored for longer periods at re-frigeration temperatures under EMA, are justiedsince extension of the time that the vegetables re-main acceptable for consumption can allow psych-rotrophic pathogens such as L. monocytogenes andAer. ca�iae to proliferate (Jacxsens et al., 1999a).

Storage temperature is probably the most impor-tant factor affecting the growth of micro-organisms in EMA-packaged ready-to-use vegeta-bles, since the applied modified atmospheres (1–5kPa O2 to 5–10 kPa CO2) do not contain enoughCO2 to slow down or stop microbial activity(Church and Parsons, 1995; Jacques and Morris,1995; Bennik et al., 1998; Jacxsens et al., 1999a,b).The growth of the mesophilic microflora can besignificantly reduced as storage temperature de-creases and favours psychrotrophic micro-organ-isms (Nguyen-the and Carlin, 1994; Varoquaux etal., 1996). Storage temperature also determinesrespiration rate of the produce and the permeabil-ity for O2, CO2 and water vapour of the packagingfilm and, therefore, changes in atmospheres withinthe EMA-packages (Exama et al., 1993; Hertog etal., 1998; Jacxsens et al., 2000). Temperature has asignificant effect on the sensory quality of this typeof product. Enzymatic browning and shrivelling,being the most limiting sensory disorders of fresh-cut produce, are inhibited by low temperaturestorage (Willocx, 1995; Garcia-Gimeno et al.,1998).

The objective of the present study was to evalu-ate the effect of different refrigerating temperatures(2, 4 and 7 °C) and temperature abuse (10 °C)on the shelf-life (based on the evaluation of themicrobiological proliferation of spoilage andpathogenic micro-organisms and the sensory qual-ity) of EMA-packaged ready-to-eat vegetables(mixed lettuce, a shredded mixture of red,

L. Jacxsens et al. / Posthar�est Biology and Technology 26 (2002) 59–73 61

green and yellow bell peppers, and cucumberslices).

2. Materials and methods

2.1. Preparation of fresh-cut �egetables

The produce was industrially prepared in avegetable processing facility (Allgro, Sint-LievensHoutem, Belgium). Mixed lettuce was a commer-cially available mixture of 20% endive (Cichoriumendi�ia L.), 20% curled endive (Cichorium endi�iaL.), 20% radicchio lettuce (Cichorium intybus var.foliosum L.), and 20% each of lollo rossa and lollobionta lettuces (Lactuca sati�a var. crispa L. (redand green variety, respectively)). Chopped bellpeppers were a mixture of green, red and yellowshredded bell peppers (Capsicum annuum L.)(0.4×1 cm). Cucumber (Cucumis sati�us L.) wassliced (unpeeled) in the laboratory using a slicingmachine (Kitchen Aid, Philips, Eindhoven, theNetherlands), into 0.3 cm slices.

2.2. Design and packaging of EMA-packages forfresh-cut produce

The EMA-packages were designed to obtain anequilibrium atmosphere of 3 kPa O2 at a storagetemperature of 7 °C. Calculations were based onthe equations published by Jacxsens et al. (1999b,

2000). The applied packaging films and their suit-ability to guarantee aerobic atmospheres in thetemperature range of 2–10 °C have been vali-dated previously (Jacxsens et al., 2000). Theproduct weight, film area, required O2 transmis-sion rate (at 7 °C), applied film and thickness, topackage each type of ready-to-eat vegetable, arelisted in Table 1. The packaging films were non-perforated films, especially developed to obtainintermediate or high O2 transmission rates at lowtemperatures and 90% RH (Hyplast, Hoogstraten,Belgium). Their CO2 permeance is high (�1.08×10−10 mol CO2/m2 s Pa at 7 °C and 90% RH).

Modification of the atmosphere (3 kPa O2–5kPa CO2) in the packages was performed byflushing (gas mixer, WITT M618-3MSO, Ga-setechnik, Germany; vacuum compensationchamber, Multivac A300/42 Hagenmuller KG,Wolfertschwenden, Germany). Air products (Vil-voorde, Belgium) supplied the gases O2, CO2 andN2. The packages were stored at 2, 4, 7 and 10 °Cin a ventilated refrigerator (LMS Cooled Incuba-tor, Sevenoaks, Kent, UK). All tests were con-ducted in duplicate.

2.3. Gas analysis

The O2 and CO2 concentrations in theheadspace of the packages were measured bymeans of a CO2/O2 gas analyser, pumping a 40 mlgas sample immediately from the package through

Table 1Package design and packaging films for fresh-cut produce

FilmPackage area Required oxygenFill weight Oxygen permeability ofType of fresh-cutproduce (kg) thickness (�m)applied packaging filmspermeability to obtain 3(m2)

kPa O2 at 7 °Ca (mol (mol O2/m2 s Pa at 7 °C

O2/m2 s Pa) and 90% RH�95%

confidence interval)

1.04×10−11�1.42×10−131.09×10−110.27×0.1950.25Mixed lettuce 400.19×0.15 40Chopped bell 1.57×10−11�2.85×10−130.15 1.52×10−11

peppersCucumber slices 0.15 0.19×0.15 1.33×10−11 1.38×10−11�4.75×10−14 50

a Based on mathematical equations published by Jacxsens et al. (1999b).


a needle and a silicon tube (Servomex Food Pack-age Analyser, Seri 1400, Crowborough, Sussex,UK). The results were compared with the predic-tions made by the integrated approach obtainedin previous work (Jacxsens et al., 2000).

2.4. E�aluation of sensory quality

The typical sensory properties of each type ofproduce were followed during the storage experi-ments at different temperatures. The evaluationwas performed by a trained sensory panel (8–10people) using a descriptive test. The organolepticproperties (taste, smell/flavour and crispness) werejudged under red light in a taste room with indi-vidual boxes. Additionally, visual characteristics(colour of the cut surfaces, dryness/transparency,general appearance) were scored under daylight.The scale is a numerical representation of a sub-jective evaluation that considers the severity of thesymptom (Kader et al., 1973): rating from 1 (ex-cellent fresh), 2 (very fresh), 3 (fresh), 4 (accept-able), 5 (just acceptable), 6 (just unacceptable), 7(unacceptable), 8 (deteriorated), 9 (completely de-teriorated) and 10 (extremely deteriorated). Thecut-off score was fixed at 5 (Willocx, 1995). Thesample was considered as unacceptable when amean score above five was reached.

2.5. Microbiological analysis

The growth of relevant spoilage micro-organ-isms was followed by manual enumeration meth-ods. After preparation of a dilution series (1/10)in Physiologic Peptone Salt solution (PPS, 8.5 g/lNaCl (Vel 8605, Merck Eurolab, Leuven, Bel-gium)+1 g/l peptone (Oxoid L34, Unipath,Basingstoke, Hampshire, UK)), samples wereplated on specific media. Total aerobic psychro-trophic count was pour-plated on Plate CountAgar (PCA, Oxoid CM325) and incubated at22 °C for 5 days. De Man, Rogosa, Sharpe(MRS, Oxoid CM361), pour-plates with a toplayer were used to count micro-aerophilic lacticacid bacteria (3 days incubation at 30 °C). Aspread-plate with Oxytetracycline Glucose Agar(OGA, Sanofi Diagnostics Pasteur 64894, Mar-nes-La-Coquette, France) with an additional sup-

plement (Oxytetracycline supplement, OxoidSR073A) was used to access yeasts (after 3 daysincubation at 30 °C).

2.6. Challenge tests

Inoculation of the vegetables was performedwith a mixture of different L. monocytogenesstrains (Scott A, ATCC 53 and LM LJ1, a strainisolated out of red and green bell peppers at theLaboratory of Food Microbiology and FoodPreservation, Ghent University, Ghent, Belgium)and a mixture of two Aer. ca�iae (HG4) strainsisolated at the Laboratory of Food Microbiologyand Food Preservation, Ghent University, fromfresh spinach (Aer 9) and garden sorrel (Aer 8)(Jacxsens et al., 1999a; Neyts et al., 2000b). Eachstrain was individually cultured from a refriger-ated slant Trypton Soya Agar (TSA, OxoidCM131) for L. monocytogenes and Nutrient Agar(NA, Nutrient broth (Oxoid CM1) with 10 g/lagar (Oxoid L11)) for Aer. ca�iae and consecu-tively subcultured, twice after 24 h, in Brain HeartInfusion (BHI, Oxoid CM225) at 30 °C for Aer.ca�iae and 37 °C for L. monocytogenes. The sec-ond culture was allowed to adapt to the finaltemperature of 2, 4, 7 or 10 °C for 6 h. Thesecond culture was diluted in PPS to an amountwhere a contamination level of approximately103–104 viable cells/g of vegetable was reached.The vegetables were sprayed with the differentpathogen solutions, using a micropipette(Finnpipette (40–200 �l), Labsystem, Helsinki,Finland) and gently mixed to obtain a homoge-neous distribution of the inoculated patho-gens. The inoculated vegetables were gas-pack-aged in the same way as the non-inoculated veg-etables.

To enumerate L. monocytogenes, Listeria-selec-tive agar base (Oxford formulation, OxoidCM856+Listeria selective supplement—Oxfordformulation, Oxoid SR140E) was applied and thespread-plates were incubated at 37 °C for 48 h.Aer. ca�iae was spread-plated on modified bilesalts irgasan brilliant green agar (mBIBG) (pH8.7) and incubated at 30 °C for 24 h (Neyts et al.,2000b).


Fig. 1. Change in O2 concentration (a) and CO2 concentration (b) as a function of storage time (days) inside the EMA packagesfor mixed lettuce, stored at 2 °C (�), 4 °C (�), 7 °C (�) and 10 °C (× ). Error bars indicate SD. The predicted equilibrium O2

level is indicated by full lines (— (2°C), ---- (4°C), — (7°C), ...... (10°C)).

2.7. pH measurement

A sample of 50 g produce was homogenisedusing a mixer (Commercial blender 8010, Waring,New Hartford, CT, USA). The pH was measuredwith an electrode (PH 915600, Orion, Boston,USA) and measurement unit (model 525A,Ankersmit, Boston, USA).

2.8. Statistics

All tests were performed in duplicate. The indi-vidual scores of the sensory properties given bythe trained taste panel were analysed using Excel2000 for Windows 98. The average and standarddeviation were calculated.

The microbial growth of the spoilage andpathogenic micro-organisms was analysed apply-ing SPSS 9.0 (Statistical Package for the SocialSciences) for Windows 98. The data were fitted tothe Baranyi growth model and the parametersinitial cell number (N0), maximum cell number

(Nmax), maximum specific growth rate (�max) andlag phase (�) were calculated (Grijspeerdt andVanrolleghem, 1999).

3. Results

3.1. Internal gas composition of theEMA-packages

Time-dependent changes in the O2 and CO2

levels obtained were reasonably close for all tem-peratures and products. The atmosphere for pack-aged mixed lettuce was a function of storagetemperatures and time (Fig. 1). Similar resultswere obtained for mixed bell peppers and cucum-ber slices (data not shown). The measured andpredicted equilibrium O2 concentrations at 2, 4, 7and 10 °C are illustrated in Fig. 1a. The CO2 levelreached an equilibrium between 2 and 4 kPa forall storage temperatures while the target levelswere 5–10 kPa (Fig. 1b).


3.2. E�aluation of the sensory quality

The average scores of sensory quality are pre-sented in a radial-type figure for mixed lettuce(Fig. 2a) and cucumber slices (Fig. 2b). The gen-eral appearance (freshness) and the colour wereevaluated as most important visual properties of

the mixed lettuce. These properties determined thedisapproval of the trained panel more than theorganoleptic properties (taste/flavour, odour andtexture/crispness (=mouth feel, perception of thestructure of the plant tissue)): at the end of thestorage period of mixed lettuce, scores for colourand general appearance were between 5 (= just

Fig. 2. Change in sensory properties of EMA-packaged mixed lettuce (a) and cucumber slices (b) as a function of the storage timestored at 2, 4, 7 and 10 °C (score 1=excellent fresh, 5= just acceptable (cut off score), 10=extremely deteriorated).


acceptable) and 9 (=completely deteriorated),while the perception of the texture (crispness) wasstill as acceptable (score 4–5) by the panel (Fig. 2a).The odour only seemed to be important at the endof the shelf-life. The end of the shelf-life of themixed lettuce, based on sensory properties, wasreached after 9, 7, 5 and 3 days, respectively storedat 2, 4, 7 and 10 °C.

For cucumber slices, the flesh transparency wasthe most important visual property at low temper-atures (2 °C). This minimally processed vegetablebecomes glassy during storage. The taste, odourand texture/crispness were followed as organolepticproperties for the cucumber slices (Fig. 2b). Theeffect of storage temperature on the sensory char-acteristics was very clear: at a temperature of 2 °C,the limit was day 4 (transparency), at 4 °C day 7,at 7 °C day 5 and at 10 °C day 4. For the cucumberslices, stored at 4, 7 and 10 °C, not only the visualcharacteristics were rejected but the scores of thecrispness, taste and odour also increased during thestorage time to unacceptable values (Fig. 2b).

Similar results were obtained for the mixture ofshredded red, green and yellow bell peppers (datanot shown). The loss of the crispness and texture,accompanied by an unacceptable water loss, werethe dominant problems occurring during storage.The bell peppers became unacceptable after 7, 9, 6and 2 days of storage at respectively 2, 4, 7 and10 °C.

The values of shelf-life period (ts) when a sensorycharacteristic exceeded its limit of acceptability(average score above five) were derived from theradial-type figures. The limiting sensory character-istic differed from temperature to temperature andtype of vegetable. The shelf-life at 2 °C was nottaken into consideration for cucumber slices andbell peppers. The relationship between the naturallogarithm of ts and storage temperature (K) wasrepresented by a linear equation (Garcia-Gimenoet al., 1998):

ln ts=b−a T, (1)

where ts is the mean shelf-life (days) based onexceeding the average score of five of a specificsensory characteristic at a specific storage temper-ature, T is the storage temperature (K), a is theslope (ln days/K) and b the intercept (ln days).

Fig. 3. Linear regression lines of the shelf-life (ln (t)), based onunacceptable total psychrotrophic count (�108 cfu/g) (�)and on unacceptable sensory characteristics (average �5) (�)as a function of storage temperature for EMAP mixed lettuce(a) and cucumber slices (b).

The Eq. (1) for sensory shelf-life of the threetypes of vegetables are:ln ts= −0.0933 T+27.763 (R2=0.9865)

for cucumber slices,

ln ts= −0.1351 T+39.365 (R2=0.9906)

for mixed lettuce,

ln ts= −0.2507 T+71.751 (R2=0.9339)

for mixed bell peppers.

The equations are illustrated in Fig. 3a and b formixed lettuce and cucumber slices, respectively.These equations were set up for a specific initialquality. Consequently, poorer initial quality willresult in shorter shelf-lives and other values for theparameters a and b will be defined.

3.3. Growth of spoilage micro-organisms

Fig. 4a–c illustrates the growth curves of thetotal psychrotrophic count on lettuce, lactic acidbacteria on bell peppers, and yeasts on cucumber


slices, respectively, at different storage tempera-tures, and fitted by the Baranyi model (Grijs-peerdt and Vanrolleghem, 1999). After a certainstorage period, the microbiological analyses wereinterrupted because of spoilage and unacceptablesensory quality. Table 2 summarises the estima-tions for the initial cell number (N0 in log cfu/g),the maximum cell number (Nmax in log cfu/g),maximum specific growth rate (�max in h−1) and

the lag-phase (� in h) of the fitted growth curvesof the total psychrotrophic count (TPC) on thethree vegetables made in SPSS 9.0. R2 were be-tween 0.9299 and 0.9988. For the mixed bellpeppers at 4 °C, however, results were not fittedby the curve for TPC. The initial contaminationwas already very high (6.24�0.01 log cfu/g) andconsequently, the shape of the growth curve couldnot be fitted (Table 2).

Fig. 4. Total psychrotrophic count (log cfu/g�SD) on mixed lettuce (a), lactic acid bacteria on shredded bell peppers (b) and yeastcount on cucumber slices (c) stored under EMA at 2 (�), 4 (�), 7 (�) and 10 °C (× ). Fitting with the Baranyi equation isindicated by the full lines.


Table 2Initial cell number (N0 in log cfu/g�SD), maximum cell number (Nmax in log cfu/g�SD), maximum specific growth rate (�max inh−1�SD), lag-phase (� in h�SD) of total psychrotrophic count

MaximumTemperature Initial cellLag-phase (h)Type of Maximumminimally processed number(°C) specific growth cell number

(log cfu/g)vegetable (log cfu/g)rate (h−1)

121.25�9.61 0.03�0.0012 2.68�0.45Cucumber slices 8.77�0.114 130.59�32.64 0.05�0.04 2.30�0.14 8.87�0.55

45.59�14.89 0.06�0.067 2.82�0.32 8.51�0.5810 39.23�16.59 0.23�0.14 2.81�0.30 7.86�0.38

Mixed bell peppers 2 40.26�4.32 0.041�0.013 5.96�0.18 8.72�0.14–a –a4 –a –a

–a,b 1.12×10−3�7.52×10−5 6.07�0.04 8.76�0.0677–a,b 6.07×10−4�7.39×10−5 5.76�0.1110 9.04�0.37

Mixed lettuce 2 167.36�10.70 0.07�0.03 6.46�0.07 7.38�0.07262.82�5.79 0.01�0.01 5.98�0.05 9.13�1.67436.84�7.31 0.03�0.0037 6.14�0.21 8.11�1.07

–b 0.07�0.0410 6.62�0.13 7.73�0.16

At different storage temperatures on mixed lettuce, mixed bell peppers and cucumber slices packaged under EMA, fitted with theBaranyi equation:

N(time)=N0+�maxA(time)−ln�

1+exp (�maxA(time)−1)

exp (Nmax−N0)

�,

A(time)= time+1

�max

ln�exp (−�maxtime)+(1/exp (��max−1))

1+(1/exp (��max−1))

�.

a No good estimation was obtained due to too high initial contamination and limited outgrowth.b Estimated value for � was negative.

The values of shelf-life (tm), when a specificgroup of spoilage micro-organisms reached theiracceptable limit (total psychrotrophic count(TPC) 108 cfu/g, lactic acid bacteria (LAB) 107–108 cfu/g and/or yeasts 105 cfu/g), were calculatedfrom the actual growth curves (CNERNA-CNRS,1996; Debevere, 1996; Francis et al., 1999). TPCwas the limiting group of micro-organisms formixed lettuce and cucumber slices, while yeastsand LAB limited the shelf-life for bell peppers.The relationship between the natural logarithm oftm and storage temperature (K) was also repre-sented by a linear equation (Garcia-Gimeno et al.,1998):

ln tm=d–c T, (2)

where tm is the mean shelf-life (days) based onreaching the limit for the count of a group ofspoilage micro-organisms at a specific storagetemperature, T is the storage temperature (K), c isthe slope (ln days/K) and d the intercept (ln days).

The Eq. (2) for total psychrotrophic count are:

ln tm= −0.1536 T+44.898 (R2=0.9656)

for cucumber slices,

ln tm= −0.0397 T+13.32 (R2=0.9926)

for mixed lettuce.

For the mixed lettuce, the result at 10 °C wasnot taken into consideration because the experi-ment was interrupted before the limit of 108 cfu/gof TPC was reached because of unacceptable sen-


sory quality. Again, the values of the parameters cand d are dependable on the initial quality of theprocessed vegetables: higher contaminated prod-ucts will reach the limiting values faster. For themixed bell peppers, no good relation could beestablished between the temperature and the shelf-life, based on exceeding the limit of 108 cfu/g LABor 105 cfu/g yeasts because of varying initial con-tamination (Fig. 4b). The calculated microbialshelf-life for mixed lettuce and cucumber slicesfrom Eq. (2) is illustrated in Fig. 3a and b.

3.4. Proliferation of human pathogens

The growth rate of psychrotrophic pathogensdiffered from vegetable to vegetable and was tem-perature-dependent. L. monocytogenes and Aer.ca�iae were unable to grow on mixed bell peppersstored at 2, 4, 7 and 10 °C, although Aer. ca�iaesurvived the storage period on the bell peppers.Aer. ca�iae showed growth on the cucumber slices

at all temperatures but not at 2 and 4 °C on themixed lettuce (Fig. 5a). On the mixed lettuce andcucumber slices, L. monocytogenes survived at2 °C while growth was possible at the other tem-peratures (Fig. 5b). Increasing temperatures re-sulted in increasing maximum growth rates and indecreasing lag-phases, estimated by the Baranyiequation (Fig. 5a and b). Fitting was not possibleat 2 °C for L. monocytogenes as no growth wasobtained.

From the growth curves of L. monocytogenesand Aer. ca�iae, it was possible to derive theirproliferation during the shelf-life period of theminimally processed vegetable, based on its sen-sory and microbiological quality (equations Eqs.(1) and (2), Fig. 3). Results are given in Table 3.With these data, sanitary risks can be definedwithin the shelf-life period of mixed lettuce, cu-cumber slices and mixed bell peppers, packagedunder equilibrium modified atmospheres (Table3).

Fig. 5. Growth curves of Aer. ca�iae (log cfu/g�SD) (a) and L. monocytogenes (b) on EMA packaged cucumber slices stored atdifferent temperatures (2 (�), 4 (�), 7 (�) and 10 °C (× )). Baranyi fitting is indicated by full lines.


Table 3Relation between the shelf-life (days) of EMAP mixed lettuce, mixed bell peppers and cucumber slices and growth of L.monocytogenes and Aer. ca�iae (log cfu/g)

Shelf-life based onTemperature Shelf-life based on Outgrowth of Aer. ca�iaeOutgrowth of L.(°C) microbiological quality monocytogenes during during shelf-life (log cfu/g)sensorial quality (days)

shelf-life (log cfu/g)(days)

Cucumber slices42 12 (TPC) No growth 0.56/4 days

11 (TPC) 1.86/7 days 3.99/7 days4 77 (TPC) 2.54/5 daysa5 4.69/5 days7

10 4 (TPC)4 4.13/4 days 4.50/4 days

Mixed lettuce11 (TPC) No growth No growth2 99 (TPC) 0.30/7 days7 No growth47 (TPC) 0.58/5 days7 0.65/5 days5–b 1.29/3 days3 0.73/3 days10

Mixed bell peppers3 (LAB)2 No growth7 No growth4 (LAB)4 No growth9 No growth1 (TPC) No growth6 No growth7

210 1 (TPC) No growth No growth

a Bold result indicates a sanitation risk of L. monocytogenes.b Experiment interrupted before limit in microbiological quality (PSY, LAB and/or yeasts) was obtained.

4. Discussion

4.1. Measured �ersus predicted oxygen concentra-tion inside EMA-packages

The values obtained inside the packages weresimilar to those obtained in previous studies forother vegetables (Jacxsens et al., 2000). At the endof the storage period, the O2 concentration in-creased further to 10–12 kPa O2 inside the EMA-packages stored at 2 and 4 °C. During storage,the plant tissue is ageing and respiration declines.This is not taken into consideration in the modelproposed by Jacxsens et al. (2000) and can explainthe underestimation of the O2 concentration in-side the packages stored at low temperatures. Theoverestimation at 10 °C can be explained by thefact that spoilage micro-organisms, proliferatingon the produce during storage, consume O2. Thiswas also not taken into consideration in the math-ematical approach (Jacxsens et al., 2000).

Optimally, a CO2 concentration of 5–10 kPa at7 °C was desired. But as the CO2 permeance ofthe packaging films is very high, it was impossible

to accumulate CO2 inside the packages (Fig. 1b)until antimicrobial levels rose (i.e. �20 kPa)(Church and Parsons, 1995). The choice of aproper packaging film is the most difficult step inEMA-packaging (Exama et al., 1993; Lange,2000).

4.2. Influence of storage temperature on sensoryproperties

The cut surfaces of the lettuce were not dis-coloured pink during EMA storage, due to sup-pression of enzymatic discoloration by low O2

levels (Laurila et al., 1998). But at the end of thestorage period, brown discoloration appeared to-gether with a loss of the fresh look of the lettucesbecause of the growth of Gram negative soft rotcausing micro-organisms (Nguyen-the and Carlin,1994; Jacques and Morris, 1995; Fig. 4a). At thattime, the organoleptic properties also became un-acceptable, except at 2 °C, where microbialgrowth rate was slower (Fig. 4a). Gram negativebacteria, such as Pseudomonas spp. or Enterobac-teriaceae, dominating spoilage of leafy vegetables,


are known to have pectinolytic activity (King et al.,1991; Szabo et al., 2000).

For the three fresh-cut vegetables investigated,4 °C is the optimal storage temperature. A storagetemperature of 2 °C was too low for cucumberslices and mixed bell peppers (Fig. 2a, Table 3), aspreviously published by Morris (1982). At 2 °C,the vegetables suffered from chilling injury ratherthan spoilage due to growth of micro-organisms.This chilling injury was manifested as a loss ofwater for the mixed bell peppers while the cucum-ber slices became transparent (Morris, 1982). Bothtypes of minimally processed vegetables lost theirfirmness as well.

4.3. Influence of storage temperature on spoilagemicro-organisms

Spoilage micro-organisms proliferated duringstorage. The effect of temperature on microbialcounts became very clear during the storage exper-iments under EMA conditions. As the temperatureincreased, the lag-phase decreased, although themicro-organisms did not reach the same maximumpopulation density (Nmax) because of earlier inter-ruption of the experiments due to spoilage andrejection by the taste panel (Fig. 4a–c, Table 2).

Spoilage was dominated by total psychrotrophiccount in the case of mixed lettuce and cucumberslices while yeasts and lactic acid bacteria domi-nated the spoilage of mixed bell peppers. Lacticacid bacteria and yeasts were less important duringspoilage of the lettuce. This could possibly beexplained by competition with other populations,such as pectinolytic Gram negative micro-organ-isms, which have a higher growth rate at lowertemperatures and are better adapted to develop onlettuces because of their pectinolytic enzymes (Bar-riga et al., 1991; Nguyen-the and Carlin, 1994). Thegenus Pseudomonas was most frequently isolatedamong psychrotrophic bacteria in samples of let-tuce and other minimally processed leafy vegetables(Nguyen-the and Carlin, 1994; Jacques and Morris,1995; Bennik et al., 1998). Bell peppers containmore sugar than lettuces and cucumber (Nubel,1999), which stimulates growth of yeasts and lacticacid bacteria (Babic et al., 1992; Fleet, 1992).

4.4. Shelf-life determination

The shelf-life based on sensory and microbiolog-ical properties determined the overall shelf-life ofthe EMA-packaged product (Fig. 3a and b). Theagreement between the shelf-life based on thesensory spoilage and growth in total psychro-trophic count was best at low temperatures formixed lettuce indicating that at higher temperaturesother factors are playing a role in the perception ofthe spoilage by the trained taste panel (such as adecreased O2 concentration under the optimal 3kPa (Fig. 1a)), although the limit for O2 for lettuceis defined as �0.5 kPa (Beaudry, 2000). Thisdifference was also found for green asparagus,stored under modified atmosphere conditions byGarcia-Gimeno et al. (1998), where the total psy-chrotrophic count was also more attributed tospoilage, as were lactic acid bacteria. A relationbetween microbial action and lettuce quality oc-curred after a period of storage in which lettucerespiration rendered the plant tissue less resistantto microbial attack (King et al., 1991), as EMAconditions have no direct effect on slowing downmicrobial growth (Bennik et al., 1995, 1998;Jacxsens et al., 1999a). For the cucumber slices onthe other hand, which suffered from chilling injury,the agreement of the shelf-lives was better at higherstorage temperatures and depended on both theperception of spoilage by the trained taste paneland reaching 108 cfu/g of total psychrotrophiccount. It can be concluded that the overall shelf-lifetime was limited by the sensory properties and notby the microbiological growth (Table 3). Thesefindings have also been reported for fresh-cutproducts (King et al., 1991; Jacxsens et al., 1999a)while not for unprocessed vegetables (Garcia-Gimeno et al., 1998). Bell peppers formed anexception (Table 3) probably because of their highinitial contamination, higher nutrient availabilityon cut surfaces and higher microbial competitioncompared to the mixed lettuce and cucumber slices(Bennik et al., 1998). Moreover, spoilage wasdominated by lactic acid bacteria and their type ofspoilage has a less putritive character compared toGram negative micro-organisms (Kakiomenou etal., 1996).


4.5. Proliferation of human pathogens

A decrease of L. monocytogenes and survivalof Aer. ca�iae on mixed bell peppers was alsofound in previously conducted storage experi-ments with EMA-packaged mixed bell peppers,when followed through a simulated distributionchain (Jacxsens et al., in press). The reason forthis can be found in the high load of spoilagemicro-organisms, dominated by lactic acid bac-teria, and the combination of refrigeration andthe limiting pH of bell peppers. The pH of themixture is 5.07�0.16, while the minimum pHfor growth of L. monocytogenes is 4.6–5.0 andof Aer. ca�iae is 4.5–5.5 (ICMSF, 1996).

A storage temperature of 2 °C was apparentlytoo low for growth of L. monocytogenes andAer. ca�iae. But growth of Aer. ca�iae could besustained at this low storage temperature on cu-cumber slices, which represent a nutrient richand pH friendly environment. The pH of thecucumbers was 6.24�0.28. The protective influ-ence of a low natural pH of the ready-to-eatvegetables and the availability of nutrients oncut surfaces, on the growth of both psychro-trophic pathogens was obvious. Mixed lettucecould also be considered as a pH friendly sub-strate (pH 5.88�0.01) but apparently providesless nutrients to sustain growth of Aer. ca�iae.The applied modified atmosphere in the pack-ages (2–5 kPa O2 and 3–5 kPa CO2) probablydid not directly affect the growth/survival/de-cline of the pathogens because the appliedmodified atmospheres did not contain enoughCO2 to inhibit growth (Church and Parsons,1995; Jacques and Morris 1995; Bennik et al.,1998).

Several authors have defined an increase of 2log units of L. monocytogenes in foods as a risk,based on the fact that under normal conditionsthere is a very low contamination level in foods(CNERNA-CNRS, 1996; Debevere, 1996; Fran-cis et al., 1999; Norrung, 2000). According tothe ICMSF (1994), for people who do not havean increased susceptibility, the presence of lessthan 100 cfu/g of L. monocytogenes does notform a risk. But the majority of minimally pro-cessed vegetables are not cooked before con-

sumption and no other listericidal treatment iscarried out. A 2 log growth during the overallshelf-life period of the minimally processed veg-etable (based on its sensory or microbiologicalquality) was (just) not reached on the cucumberslices stored at 4 °C but was clearly reached at7 and 10 °C. On the mixed lettuce, on the otherhand, slower growth was found and no 2 logincrease was reached during the overall shelf-lifeperiod (Table 3).

For Aeromonas spp. no critical limit is pro-posed, as no infectious dose is yet recognised(Jacxsens et al., 1999a). Recent screening for thebacterial quality in minimally processed lettuceshowed the high incidence of this micro-organ-ism with 55% of the vegetables (n=120) posi-tive for Aer. hydrophila/ca�iae (Szabo et al.,2000). Berrang et al. (1989a) found no differencein growth of Aeromonas spp. between air-storedand EMA-packaged produce on cauliflower, as-paragus and broccoli.

Acknowledgements

This work was partly supported by the IWT(Flemish Institute for the Promotion of Scien-tific and Technological Research in Industry)and HYPLAST N.V., Hoogstraten, Belgium.The plastic packaging films were developed,extruded and supplied by HYPLAST,Hoogstraten, Belgium. The measurement of theoxygen permeability was conducted by Hyplastas well.

References

Abbott, S., Serve, H., Janda, J., 1994. Case of Aeromonas�eronii (DNA group 10) bacteremia. J. Clin. Microb. 32,3091–3092.

Babic, I., Hilbert, G., Nguyen-the, C., Guiraud, J., 1992.The yeast flora of stored ready-to-use carrots and theirrole in spoilage. Int. J. Food Sci. Technol. 27, 473–487.

Barriga, M., Trachy, G., Willemot, C., Simard, R., 1991.Microbial changes in shredded iceberg lettuce stored un-der controlled atmospheres. J. Food Sci. 6, 1586–1588.

Beaudry, R., 2000. Responses of horticultural commoditiesto low oxygen: limits to the expanded use of modifiedatmosphere packaging. Hort. Technol. 10, 491–500.


Bennik, M., Smid, E., Rombouts, F., Gorris, L., 1995.Growth of psychrotrophic food-borne pathogens in asolid surface model system under the influence of carbondioxide and oxygen. Food Microb. 12, 509–519.

Bennik, M., Vorstman, W., Smid, E., Gorris, L., 1998. Theinfluence of oxygen and carbon dioxide on the growth ofprevalent Enterobacteriaceae and Pseudomonas speciesisolated from fresh and modified atmosphere stored veg-etables. Food Microb. 15, 459–469.

Berrang, M., Brackett, R., Beuchat, L., 1989a. Growth ofAeromonas hydrophila on fresh vegetables stored under acontrolled atmosphere. Appl. Env. Microb. 55, 2167–2171.

Berrang, M., Brackett, R., Beuchat, L., 1989b. Growth ofListeria monocytogenes on fresh vegetables stored undera controlled atmosphere. J. Food Prot. 52, 702–705.

Blaschek, H., 2000. Clostridium. In: Robinson, R., Batt, C.,Patel, P. (Eds.), Encyclopedia of Food Microbiology.Academic Press, New York, p. 428.

Brackett, R., 1999. Incidence, contributing factors, and con-trol of bacterial pathogens in produce. Postharvest Biol.Technol. 15, 305–311.

Church, I., Parsons, A., 1995. Modified atmosphere packag-ing technology: a review. J. Sci. Food Agric. 67, 143–152.

CNERNA-CNRS, 1996. Produits de la IVe gamme. In:Jouve, J.L. (Ed.), La qualite microbiologique des ali-ments (maitrise et criteres, 2e edition.), Polytechnica,Paris, pp. 73–98.

Day, B., 1990. A perspective of modified atmosphere pack-aging of fresh produce in Western Europe. Food Sci.Technol. Today 4, 215–221.

Debevere, J., 1996. Criteria en praktische methoden voor debepaling van de houdbaarheidsdatum in de etikettering.In: Temmerman, G., Cremer, C., Thyssen, M., Debevere,J. (Eds.), Etikettering, houdbaarheid en bewaring (voe-dingsmiddelen en recht 2), Die Keure, Brugge, pp. 37–64.

Exama, A., Arul, J., Lencki, R.W., Lee, L.Z., Toupin, C.,1993. Suitability of plastic films for modified atmospherepackaging of fruits and vegetables. J. Food Sci. 58,1365–1370.

Fleet, G., 1992. Spoilage yeasts. Crit. Rev. Biotechnol. 12,1–44.

Francis, G., Thomas, C., O’Beirne, D., 1999. The microbio-logical safety of minimally processed vegetables. Int. J.Food Sci. Technol. 34, 1–22.

Garcia-Gimeno, R., Castillejo-Rodriquez, A., Barco-Alcala,E., Zurera-Cosano, G., 1998. Determination of packagedgreen asparagus shelf-life. Food Microb. 15, 191–198.

Grijspeerdt, K., Vanrolleghem, P., 1999. Estimating theparameters of the Baranyi model for bacterial growth.Food Microb. 16, 593–605.

Hertog, M., Peppelenbos, H., Evelo, R., Tijskens, L., 1998.A dynamic and generic model of gas exchange of respir-ing produce: the effects of oxygen, carbon dioxide andtemperature. Postharvest Biol. Technol. 14, 335–349.

ICMSF, 1994. Choice of sampling plan and criteria for Lis-teria monocytogenes. Int. J. Food Microb. 22, 89–96.

ICMSF, 1996. Micro-organisms in Foods 5. Blackie Aca-demic and Professional, London.

Jacques, M.A., Morris, C., 1995. Bacterial population dy-namics and decay on leaves of different ready-to-usebroad-leave endive. Int. J. Food Sci. Technol. 30, 221–236.

Jacxsens, L., Devlieghere, F., Falcato, P., Debevere, J.,1999a. Behavior of Listeria monocytogenes andAeromonas spp. on fresh-cut produce packaged underEquilibrium-modified atmosphere. J. Food Prot. 62,1128–1135.

Jacxsens, L., Devlieghere, F., Debevere, J., 1999b. Validationof a systematic approach to design equilibrium modifiedatmosphere packages for fresh-cut produce. Food Sci.Technol. (Lebensm.-Wiss. u.- Technol.) 32, 425–432.

Jacxsens, L., Devlieghere, F., De Rudder, T., Debevere, J.,2000. Designing equilibrium modified atmosphere pack-ages for fresh-cut vegetables subjected to changes in tem-perature. Food Sci. Technol. (Lebensm.-Wiss.u.-Technol.)33, 178–187.

Jacxsens, L., Devlieghere, F., Debevere, J., 2001. Predictivemodelling for packaging design: equilibrium modified at-mosphere packages of fresh-cut vegetables subjected to asimulated distribution chain. Int. J. Food Microb. inpress.

Kader, A., Lipton, W., Morris, L., 1973. Systems for scor-ing quality of harvested lettuce. HortScience 8, 408–409.

Kakiomenou, K., Tassou, C., Nychas, G., 1996. Microbio-logical, physiochemical and organoleptical changes ofshredded carrots stored under modified storage. Int. J.Food Sci. Technol. 31, 359–366.

King, A., Magnuson, J., Torok, T., Goodman, N., 1991.Microbial flora and storage quality of partially processedlettuce. J. Food Sci. 2, 459–461.

Lange, D.L., 2000. New film technologies for horticulturalproducts. Hort. Technol. 10, 487–506.

Laurila, E., Kervinen, R., Ahvainen, R., 1998. The inhibi-tion of enzymatic browning in minimally processed veg-etables and fruits. Postharvest News Inform. 9, 53–56.

Morris, L.L., 1982. Chilling injury of horticultural crops: anoverview. HortScience 17, 161–168.

Neyts, K., Huys, G., Uyttendaele, M., Debevere, J., 2000a.Incidence and identification of mesoplilic Aeromonas spp.isolated from retail foods. Lett. Appl. Microb. 31, 359–363.

Neyts, K., Notebaert, E., Uyttendaele, M., Debevere, J.,2000b. Modification of the bile salts-Irgasan-brilliantgreen agar for enumeration of Aeromonas species fromfood. Int. J. Food Microb. 57, 211–218.

Nguyen-the, C., Carlin, F., 1994. The microbiology of mini-mally processed fresh fruits and vegetables. Crit. Rev.Food Sci. Nutr. 34, 371–401.

Norrung, B., 2000. Microbiological criteria for L. monocyto-genes in foods under special consideration of risk assess-ment approaches. Int. J. Food Microb. 62, 217–221.


Nubel, 1999. Belgische voedingsmiddelen tabel. Nubel, Brus-sels, Belgium, pp. 39–44.

Szabo, E., Scurrah, K., Burrows, J., 2000. Survey for psy-chrotrophic bacterial pathogens in minimally processedlettuce. Lett. Appl. Microb. 30, 456–460.

Varoquaux, P., Mazollier, J., Albagnac, G., 1996. The influ-ence of raw material characteristics on the storage life of

fresh-cut butterhead lettuce. Postharvest Biol. Technol. 9,127–139.

Willocx, F., 1995. Evolution of microbial and visual qualityof minimally processed foods: a case study on theproduct life cycle of cut endive. Ph.D Thesis, KatholicUniversity of Leuven, Faculty of Agricultural and Ap-plied Biological Sciences, Leuven, Belgium, p. 228.

Documents

Temperature dependence of shelf-life as affected by microbial proliferation and sensory quality of equilibrium modified atmosphere packaged fresh produce