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Intense light pulses decontamination of minimally processed


International Journal of Food Microbiology 103 (2005) 79 – 89 www.elsevier.com/locate/ijfoodmicro

Intense light pulses decontamination of minimally processed vegetables and their shelf-life
V.M. Gomez-Lopeza,b, F. Devliegherea,T, V. Bonduellea, J. Debeverea
b a Laboratory of Food Microbiology and Food Preservation, Ghent University, Coupure Links, 653, 9000 Gent, Belgium Instituto de Ciencias y Tecnologa de Alimentos, Facultad de Ciencias, Universidad Central de Venezuela, Apartado 47097, Caracas 1041-A, Venezuela

Received 2 April 2004; received in revised form 25 August 2004; accepted 22 November 2004

Abstract Intense light pulses (ILP) is a new method intended for decontamination of food surfaces by killing microorganisms using short time high frequency pulses of an intense broad spectrum, rich in UV-C light. This work studied in a first step the effect of food components on the killing efficiency of ILP. In a second step, the decontamination of eight minimally processed (MP) vegetables by ILP was evaluated, and thirdly, the effect of this treatment on the shelf-life of MP cabbage and lettuce stored at 7 8C in equilibrium modified atmosphere packages was assessed by monitoring headspace gas concentrations, microbial populations and sensory attributes. Proteins and oil decreased the decontamination effect of ILP, whilst carbohydrates and water showed variable results depending on the microorganism. For this reason, high protein and fat containing food products have little potential to be efficiently treated by ILP. Vegetables, on the other hand, do not contain high concentrations of both compounds and could therefore be suitable for ILP treatment. For the eight tested MP vegetables, log reductions up to 2.04 were achieved on aerobic mesophilic counts. For the shelf-life studies, respiration rates at 3% O2 and 7 8C were 14.63, 17.89, 9.17 and 16.83 ml O2/h kg produce for control and treated cabbage, and control and treated lettuce respectively; used packaging configurations prevented anoxic conditions during the storage times. Log reductions of 0.54 and 0.46 for aerobic psychrothrophic count (APC) were achieved after flashing MP cabbage and lettuce respectively. APC of treated cabbage became equal than that from control at day 2, and higher at day 7, when the tolerance limit (8 log) was reached and the panel detected the presence of unacceptable levels of off-odours. Control never reached 8 log in APC and were sensory acceptable until the end of the experiment (9 days). In MP lettuce, APC of controls reached rejectable levels at day 2, whilst that of treated samples did after 3 days. Both samples were sensory unacceptable at day 3, controls because of bad overall visual quality (OVQ), off-odour and leaf edge browning and treated samples due to bad OVQ; browning inhibitors might be proposed to preserve OVQ. Yeasts and lactic acid bacteria counts were low in all the samples. It seems that ILP treatment alone under the conditions used in this work does not increase MP vegetables shelf-life in spite of the reduction in the initial microbial load. D 2005 Elsevier B.V. All rights reserved.
Keywords: Intense light pulses; Minimally processing; Fresh-cut vegetables; Decontamination; Shelf-life

T Corresponding author. Tel.: +32 9 264 61 77; fax: +32 9 225 55 10. E-mail address: Frank.Devlieghere@UGent.be (F. Devlieghere). 0168-1605/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2004.11.028

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1. Introduction Fresh vegetables have been traditionally prepared for consumption at home or at restaurants by washing, trimming, cutting, peeling and so on immediately or few hours before serving. When industry started to carry out these processes, a new type of product was created, called minimally processed (MP) vegetables. Although between processing and consumption a span of several days occurs, consumers still want to have fresh or fresh-like vegetables on their dishes or pans, in spite of the fact that MP vegetables have a shorter shelf-life than their intact counterparts. Processing of vegetables promotes a faster physiological (Brecht, 1995) and microbial (Brackett, 1987) degradation of the product in comparison with the raw commodities, and increases the risk of foodborne disease outbreaks (Alzamora et al., 2000). Industry has to overcome these problems with limited tools since preservation methods should avoid impairing the fresh or fresh-like attributes of the product. In order to slow down a fast physiological degradation, the MP industry can apply modified atmosphere packaging and refrigeration (King et al., 1991). The latter is also necessary to slow down microbial proliferation, although the shift to psychrotrophic microorganisms has to be taken into account. In order to prevent microorganisms from reaching undesirable levels in MP vegetables, contamination should be minimised and initial counts before storage can be decreased by using decontamination treatments, which are limited by their effects on product quality. Washing with chlorinated water has been traditionally applied to decontaminate vegetables, but several reports have questioned its efficacy (Parish et al., 2003), while studies show that toxic compounds are generated when chlorine reacts with organic matter (Richardson et al., 1998). As a consequence, several innovative approaches have been explored for the decontamination of MP vegetables. The bulk of them has been devoted to eliminate pathogens (Beuchat, 2000; Parish et al., 2003), and little research has been performed about spoilage microorganisms and the effect of these methods on the sensory attributes of the MP vegetables and their nutritious value and shelf-life (Li et al., 2001; Allende and Artes, 2003a,b). Intense light pulses (ILP) is a novel decontamination method for food surfaces that could be suitable

for disinfecting MP vegetables. This technique appears recurrently in literature reviews (Fine and Gervais, 2003; Senorans et al., 2003; Parish et al., 2003), as bhaving potential for future useQ (Alzamora et al., 2000) or bimminent commercial applicationsQ (Ohlsson, 2002). The literature on this subject is scarce, especially in experiments on food surfaces, and much of the information comes from industry sources, therefore, independently conducted research is needed (FDA, 2000). ILP kills microorganisms using short time high frequency pulses of an intense broad spectrum, rich UV-C light. Explanations for its mechanism of action have been given in terms of structural changes of microbial DNA, comparable to the effect caused by continous ultraviolet sources, but additional mechanisms seem to be involved (Takeshita et al., 2003; Wuytack et al., 2003). Since the ILP decontamination effect seems to depend on the light absorption by microorganisms, certain food components could also absorb the effective wavelengths and decrease the efficiency of this treatment. ILP has been used to successfully inactivate Escherichia coli O157:H7 on alfalfa seeds (Sharma and Demirci, 2003) and Aspergillus niger spores on corn meal (Jun et al., 2003). Regarding shelf-life of MP vegetables, Hoornstra et al. (2002) achieved more than 2 log reductions in aerobic counts on selected vegetables, and calculated, without showing experimental data, that a reduction of 2 log almost increase the shelf-life at 7 8C of cut vegetables by about 4 more days. Consequently, the present study was designed to (1) study the influence of food components on the decontamination efficiency of ILP, (2) the decontamination effect of ILP on several MP vegetables, and (3) its effect on the shelf-life of two MP vegetables stored under equilibrium modified atmosphere packaging (EMAP) (Jacxsens et al., 1999a,b) and refrigeration, evaluating the microbial as well as the sensory quality.

2. Materials and methods 2.1. ILP equipment ILP processing was done using a 100 W stroboscopic Xenon lamp (ST100-IE, Sysmat Industrie, France), pulse duration of 30 As and a pulse intensity

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of 7J. The emitted spectrum ranged from UV-C to infrared, with 50% of the light in the UV region. The lamp was placed in a metal chamber with reflecting inner walls. The equipment can be operated in two ways, manually or automatically. For the manual mode, one single flash can be generated while at the automatic mode the lamp flashes at 15 Hz. The manual mode was used for in vitro experiments, and the automatic mode for in vivo ones. 2.2. Effect of food components on the decontamination efficiency of ILP 2.2.1. Media preparation To examine the influence of several components of foods on the decontamination efficiency of ILP; oil, starch, water and proteins were added to nutrient agar (CM3, Oxoid, Basingstoke, England). In order to test the effect of oil, maize oil (MaRsolie, Derby, Belgium) was autoclaved separately during 15 min at 121 8C. After cooling down, the oil was poured at 45 8C in the liquid agar medium in which 0.5% Tween 20 was also aseptically added. The mixture was shaken carefully by inversion of the bottle. Then Petri dishes were poured. Plates with concentrations of 0% (control), 1% and 10% were prepared. Water soluble starch (217820, Beckton Dickinson, Le Point de Claix, France) was chosen as an example of soluble polysaccharides. This ingredient was autoclaved in a concentrated solution, was added to autoclaved agar, shaken and plates were poured. Plates with concentrations of 0% (control), 1% and 10% were prepared. To test the influence of the presence of proteins, casein (WP I, Dairsco, 02/2001) was autoclaved separately in powder form during 30 min at 121 8C. The autoclaved powder was added to the autoclaved agar medium and after careful stirring a homogeneous mixture was obtained which was poured into plates. To investigate the influence of a humid surface on the decontamination efficiency of ILP, inoculated agar medium was spread with 0.1 ml and 1 ml of water, immediately before flashing. Controls were inoculated nutrient agar plates in which no extra compound was added. Experiments were done in triplicate. 2.2.2. Inoculation, treatment and enumeration The prepared Petri dishes with 15 ml of agar medium were spread inoculated with 0.1 ml of the

101 dilution of a 24 h culture. Studied microorganisms were the Gram positive Listeria monocytogenes (LMG 13305), the Gram negative Photobacterium phosphoreum (LMG 4233) and the yeast Candida lambica (own isolate from mixed lettuce at 7 8C, PR9). L. monocytogenes as well as P. phosphoreum were cultivated at 30 8C in Brain Heart Infusion broth (CM 225, Oxoid) while C. lambica was cultivated in Sabouraud liquid medium (CM 147, Oxoid) at 30 8C. After 1 h drying, the plates were treated with 50 flashes at 8.4 cm from the strobe. The agar was removed from the dishes in a sterile manner in the laminar flow hood and mixed with physiological saline solution making a 10-fold dilution. This blend was mixed thoroughly by means of a Colworth Stomacher 400 (Steward Laboratory, London, UK) and 10-fold dilutions were prepared, plated on Nutrient Agar, incubated during 48h at 30 8C, and the individual colonies were afterwards counted. For the experiments with casein, selective media had to be applied, as autoclaving of the casein did not result in a sterile product: for L. monocytogenes, Listeria selective agar base (Oxoid, CM 856) with the Listeria selective supplement Oxford medium (Oxoid, SR140E) was used. P. phosphoreum was plated on agar to which crystal violet had been added at a concentration of 0.1 mg per 100 ml agar. C. lambica was plated on Yeast Glucose Chloramphenicol agar (64104, Sanofi Diagnostics Pasteur, Marnes-LaCoquette, France). 2.3. Decontamination effect of ILP on several MP vegetables 2.3.1. Processing of the vegetables Eight types of vegetables were bought in a local wholesale company, stored at 7 8C and processed within 1 day: spinach (Spinacia olaracea L.), celeriac (Apium graveolens var. rapaceum), green bell pepper (Capsicum annuum L.), soybean sprouts (Glycine max L.), radicchio (Cichorium intybus var. foliosum L.), carrot (Daucus carota L.), iceberg lettuce (Lactuca sativa var. capitata L.) and white cabbage (Brassica oleracea var. capitata L.). Processing depended on the vegetable: spinach, radicchio and lettuce were shredded in 1 cm pieces and peppers were chopped in 2–4 cm1–2 cm pieces

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with a sharp knife; celeriac was manually peeled and then grated in 0.30.33–5 cm sticks, peeled carrots were grated in 0.30.32 cm sticks and cabbage was shredded in 1 mm thick pieces using a Compacto Kitchen Cutter (Philips, Eindhoven, The Netherlands). Soybean sprouts were used without size reduction. Vegetables were immersed in tap water for 1 min and dried for 1 min by means of a manual kitchen centrifuge (Zyliss, Bern, Switzerland). At least 3 units of each vegetable were used to make a pool of each. 2.3.2. ILP treatment After size reduction, washing and drying, an amount of sample (see Table 1) was evenly and aseptically distributed on a sterile 1421 cm tray and placed at 12.8 cm from the strobe. The tray dimension fitted with the size of the window of the lamp holder; below it the radiation must be maximal. Treatments were done in two cycles; in the first cycle, samples were treated during 45 or 180 s, equivalent to 675 and 2700 pulses. Then the vegetables were immediately turned upside down on another sterile tray and a second cycle with the same duration as the first was started. Finally, the produce was immediately sampled under aseptic conditions and total aerobic counts were performed according to Section 2.4.3. Untreated samples were

used as controls. Treatments were performed in duplicate. 2.4. Effect of ILP on the shelf-life of MP cabbage and lettuce White cabbage or iceberg lettuce were processed, 30 g per treatment, as indicated in 2.3.1, flashed 45 s/ side according to 2.3.2 and immediately packaged using aseptic conditions under EMAPs designed according to results previously obtained as indicated in Section 2.4.1. 2.4.1. Respiration rate measurements The respiration rate of the MP control and flashed cabbage and lettuce was measured by means of the closed method (Mannapperuma and Singh, 1994). Thirty grams of MP vegetables were placed in airtight glass jars (635F11 ml) in triplicate. The jars were flushed with a mixture of 12% O2, 4% CO2 and 84% N2 as initial gas atmosphere by means of a gas packaging unit (gas mixer, WITT M618-3MSO, Gasetechnik, Germany; gas packaging, Multivac A300/42 Hagenmqller KG, Wolfertschwenden, Germany). Air products (Air Liquid, Amsterdam, The Netherlands) supplied the gases. Jars were stored at 7 8C and a gas sample was taken periodically through an airtight septum and analysed by gas chromatography (MicroGC M200, columns: molecular sieve 5A PLOT at 35 8C and Paraplot Q at 45 8C (Agilent, DE, USA)) and helium as gas carrier (Air Liquide, Liege, Belgium). Data were processed according to Jacxsens et al. (1999a,b) to estimate O2 consumption at 7 8C and 3% O2. 2.4.2. Packaging of the MP vegetables MP lettuce and cabbage were packaged under EMAP conditions, in quadruplicate. The applied packaging films (Hyplast N.V., Hoogstraten, Belgium) were experimental films with a high permeability for oxygen at 7 8C and 90% relative humidity, and were selected based on their oxygen permeability. The packaging configurations were designed by using the method validated by Jacxsens et al. (1999a,b) and are shown in Table 2. Gas samples were periodically taken during the shelf-life experiment and analysed by the microGC to assure that anoxic conditions were never reached.

Table 1 Log reductions achieved in mesophilic aerobic counts after treating minimally processed vegetables by intense light pulses at a distance of 12.8 cm Type of fresh-cut produce Spinach Celeriac Green paprika Soybean sprouts Radicchio Carrot Iceberg lettuce White cabbage
a b

Type of cut

Sample size (g)a

Time of processing (s/side)b 45 180 0.90 – 0.56 0.65 0.79 1.64 1.97 2.04 0.84 1.64

Shredded Grated Chopped Whole Shredded Grated Shredded Shredded

15 30 80 40 25 30 30 19 30 11

0.34 0.21 0.37 0.65 0.66 1.67 1.24 1.29 0.64 1.03

Samples very evenly distributed on a 0.140.21 m2 surface. Samples were treated at both sides of a plane.

V.M. Gomez-Lopez et al. / International Journal of Food Microbiology 103 (2005) 79–89 Table 2 Package design and packaging films, for control and intense light pulses treated minimally processed lettuce and cabbage Type of fresh-cut product Respiration rate Fill weight (kg) Package area (m2)b Required permeability for O2c Applied permeability for O2c
a b c a

83

Shredded Iceberg lettuce Control 9.17F0.17 0.90 0.1550.155 2290 2290 Treated 16.83F1.33 0.90 0.1650.165 3538 3529

Shredded white cabbage Control 14.63F2.25 0.90 0.1650.165 3562 3529 Treated 17.89F1.46 0.90 0.150.20 3542 3529

MeanFS.D. (ml O2/(kg h)) at 7 8C and 3% O2. Single-sided. (ml O2/m2 24 h atm) at 7 8C.

2.4.3. Microbiological analysis of spoilage microorganisms The following media and incubation conditions were used to enumerate the proliferation of the spoilage microorganisms: Plate Count Agar (Oxoid, CM325) for mesophilic aerobic plate count, pour plated and incubated at 30 8C for 3 days. Plate Count Agar for aerobic psychrotropic count, incubated at 22 8C for 5 days. de Man-Rogosa-Sharp medium (Oxoid, CM361) with 0.14% sorbic acid (S-1626, SigmaAldrich, Steinheim, Germany) for lactic acid bacteria, pour plated, overlaid with the same medium and incubated aerobically at 30 8C for 3 days. Yeast

Glucose Chloramphenicol agar (64104, Biorad, Marnes-La-Coquette, France) with 50 mg/l (Tournas et al., 1998) chlortetracycline (Difco, 233331) to enumerate yeasts, spread plated and incubated at 30 8C for 3 days. While agar was solidifying plates were kept covered by aluminium foil to avoid photoreactivation (Cleaver, 2003). Microbiological counts were done by taking 30 g of sample from one bag and mixing it with 270 ml peptone saline solution (8.5 g/l NaCl (8605, Vel, Leuven, Belgium) and 1 g/l peptone (Oxoid, L34)) in a sterile Stomacher bag, and homogenisation for 60 s with the Colworth Stomacher. Tenfold dilution series were made in peptone

Table 3 Sensory evaluation of untreated and treated with intense light pulses minimally processed white cabbage stored at 7 8C packaged under equilibrium modified atmosphere Quality attributes Off-odoura Tasteb Overall visual qualityc Sogginessa Browninga Drynessa Control Treated Control Treated Control Treated Control Treated Control Treated Control Treated Time (days) 0 2.5F1.5 3.4F1.6 1.6F0.7 2.1F1.0 1.0F0.0 1.0F0.0 1.0F0.0 1.0F0.0 1.0F0.0 1.0F0.0 1.1F0.2 1.1F0.2 2 1.8F1.0 2.3F1.2 1.4F0.5 1.8F0.6 1.6F1.0 1.6F0.9 1.3F0.5 1.3F0.5 1.0F0.0 1.1F0.3 1.5F0.5 1.5F0.5 5 2.2F1.1 3.0F1.2 1.8F0.9 2.1F1.1 2.4F0.9 3.3F1.4 1.4F0.5 1.3F0.5 1.2F0.4 1.4F0.7 2.1F0.9 2.1F0.9 7 2.3F1.0 3.4F1.5 1.7F0.9 2.7F0.9 2.9F1.4 3.8F1.8 1.3F0.5 1.3F0.5 1.4F0.8 2.2F0.9 1.8F0.7 1.7F0.5 9 2.1F0.9 3.5F1.3 2.5F0.7 2.7F0.8 4.0F1.4 4.9F2.2 1.4F0.5 1.3F0.5 2.2F0.8 2.7F1.5 2.1F0.8 2.1F0.9

MeanFS.D. Numbers in bold are scores above the acceptability limit. a Off-odour, sogginess, browning and dryness scores: 1=none, 5=severe. b Taste score: 1=fresh, 3=acceptable, 5=spoiled. c Overall visual quality score: 1=excellent, 5=fair, 9=extremely poor.

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saline solution for plating. Three bags were sampled for control and treated samples and for each storage day. Specifications published by Jacxsens et al. (1999b) were used to establish the end of the microbial shelf-life. 2.4.4. Evaluation of sensory quality Sensory evaluation was performed the same day of the microbiological analyses by a semi-trained panel of 4–6 people, who evaluated in triplicate specific sensory attributes (see Tables 3 and 4 for details) for cabbage and lettuce. The end of the sensory shelf-life of the sample was reached when at least one of the sensory attributes scored above the middle point of the respective scale. Attributes potentially determining the sensory shelf-life of each produce were selected according to results from preliminary experiments and previous reports (Kader et al., 1973; Lopez-Galvez et al., 1997; Fan and Sokorai, 2002; Allende and Artes, 2003b; Allende et al., 2004).
Table 4 Sensory evaluation of untreated and treated with intense light pulses minimally processed Iceberg lettuce stored at 7 8C packaged under equilibrium modified atmosphere Quality attributes Off-odour Tasteb Overall visual qualityc Sogginessa Leaf edge browninga Leaf surface browninga Translucencya Wiltnessa
a

3. Results and discussion 3.1. Effect of food components on the decontamination efficiency of ILP Fig. 1 reflects the influence of several food components on the decontamination efficiency of ILP. The presence of proteins or oils had a strongly pronounced impact on the decontamination by ILP. Increasing levels of oil and protein reduced the killing efficiency of ILP for the three tested microorganisms. For example, the obtained decontamination effect of about 1.5 log CFU/cm2 for C. lambica was totally reduced in the presence of 10% oil or 10% casein. Since proteins have strong absorption at about 280 nm as well as at higher wavelengths of the UV-B region, and lipids with isolated or conjugated double bonds absorb UV (Hollosy, 2002), it is possible that part of the radiation that could have killed microorganisms in these experiments had been absorbed by proteins and oils, decreasing the effective radiation dose on microorganisms. When water or carbohydrates were added to the medium, no particular trends were observed. In the presence of water, C. lambica was much more sensitive to ILP whereas P. phosphoreum showed exactly the opposite behaviour; L. monocytogenes was not influenced by water. Mimouni (2000) also reports a better inactivation of A. niger on a moist environment than on a drier environment. Starch had no influence on the decontamination of P. phosphoreum, whereas C. lambica and L. monocytogenes became more susceptible for ILP in the presence of starch. It seems that proteinaceous or fatty foods are inappropriate for decontamination by ILP. On the other hand, foods high in carbohydrates but poor in fat and proteins, such as most fruits and vegetables seems to be very suitable for it. Hence, MP vegetables would be good candidates for decontamination by ILP. 3.2. Decontamination effect of ILP on several MP vegetables Table 1 shows the log reductions achieved after treating several MP vegetables at treatment times up to 45 and 180 s/side at 12.8 cm distance from the strobe. Log reductions were between 0.21 and 1.67 at 45 s/side, and between 0.56 and 2.04 at 180 s/side. Slightly higher reductions (from 1.6 for carrots to N2.6

Time (days) 0 Control Treated Control Treated Control Treated Control Treated Control Treated Control Treated Control Treated Control Treated 1.7F1.1 2.3F1.3 1.3F0.6 1.8F1.0 1.5F0.7 1.6F0.8 1.1F0.2 1.2F0.4 1.4F0.6 1.2F0.4 1.1F0.2 1.2F0.4 1.3F0.8 1.4F0.8 1.1F0.3 1.1F0.2 3 3.1F1.3 2.8F1.4 2.4F1.1 2.2F1.3 5.8F1.5 5.5F1.9 2.2F1.4 2.2F1.3 3.7F1.0 2.9F1.2 2.5F0.8 2.8F0.8 2.0F0.9 2.1F0.9 1.4F0.5 1.4F0.6 5 4.3F1.0 3.7F1.1 3.8F1.2 2.6F1.4 6.9F0.9 6.9F1.6 3.5F0.9 3.2F1.0 3.6F0.7 3.3F1.1 2.6F1.1 3.1F1.2 2.3F0.9 2.0F0.9 2.4F1.2 2.6F0.8

MeanFS.D. Numbers in bold are scores above the acceptability limit. a Off-odour, sogginess, leaf edge browning, leaf surface browning, translucency and wiltness scores: 1=none, 5=severe. b Taste score: 1=fresh, 3=acceptable, 5=spoiled. c Overall visual quality score: 1=excellent, 5=fair, 9=extremely poor.

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A6
Reduction (log CFU/cm2) 5 4 3 2 1 0

Control

Low level

High level

B
Reduction (log CFU/cm2)

Water
5 4 3 2 1 0

Starch Low level

Proteins High level

Oil

Control

C
Reduction (log CFU/cm2)

Water 2 Control

Starch Low level

Proteins High level

Oil

1

0 Water Starch Proteins Oil

Fig. 1. Effect of food components on the killing effect of intense light pulses (50 flashes, 8.4 cm from the strobe) over L. monocytogenes (A), P. phosphoreum (B) and C. lambica (C). Water low level: 0.1 ml, high level: 1 ml; starch low level: 1%, high level: 10%(v/v); proteins low level: 1%, high level:10%(w/v); oil low level: 1%, high level: 10%(v/v). Error bars indicate S.D.

for paprika) were obtained in aerobic counts by Hoornstra et al. (2002) using only 2 pulses (0.15 Joule/cm2 per flash). Sharma and Demirci (2003) achieved a population reduction of E. coli O157:H7 of 4.89 log on a 6.25 mm thick layer of alfalfa seeds treated for 90 s (270 pulses) at 8 cm distance, but only

1.42 log reduction at 13 cm; Jun et al. (2003) obtained a log reduction of 2.95 after treating A. niger spores inoculated on corn meal up to 100 s at 13 cm distance from the strobe. Marquenie et al. (2003) found no suppression of fungal development by treating Botrytis cinerea inoculated on strawberries up to 250 s with equipment similar to that used here. Although differences between experimental conditions advise prudence in making comparisons, it can be concluded that the maximum population reductions obtained in this work are in the usual range for this kind of treatments. Overcoming sample heating was a problem in these experiments, limiting the processing time and the proximity of the sample to the strobe. Treatments longer than 45 s/side heated samples excessively, hence results obtained for 180 s/side provided in Table 1 are only indicative of the potential of this technique to decontaminate vegetables. Such long treatments are not useful to treat samples without a serious impairment of quality unless an effective cooling system could be incorporated to the equipment. Even equipment with blowers such as that used by Jun et al. (2003) can cause sample heating as these authors found that some experimental factor settings resulted in corn meal sample temperatures of 100 8C. The differences in log reduction among samples shown in Table 1 are difficult to explain, but might be related to different resistances of the natural microbial populations of each vegetable (as demonstrated by Anderson et al., 2000 for several pathogens and spoilage fungi), the location of microorganisms on and into the samples (shadow effect by different structures) and/or protective substances of the specific vegetable. No pattern is observable related to the kind of processing (i.e. shredded versus grated), shape (foliar versus grated) or sample size. Differences between decontamination results of several vegetables, as reported by Hoornstra et al. (2002), were also difficult to explain. The high log reduction achieved after treating grated carrots up to 45 s/side was remarkable. However, samples showed obvious signs of dehydration probably due to the absorption of light and consecutive heating. After 5 s immersed in water, carrot sticks recovered a fresh-like appearance, but this rehydration step would complicate the industrial application of ILP, and the carrot tissue could have

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been damaged during dehydration. Therefore, they were disregarded in subsequent experiments. A shadow effect was observable when comparing the reduction in contamination between sample sizes of 11 and 19 g versus 30 g (lettuce and cabbage). Eleven and 19 g corresponded to the maximum number of vegetable pieces possible without overlapping. The 30 g corresponded to a sample size considered practical to the later performed shelf-life studies, given the necessity of processing high amounts of sample using a small capacity equipment. In this case, overlapping occurred although it was minimised. 3.3. Effect of ILP on respiration rates of MP vegetables Table 2 shows the respiration rates of control and flashed MP vegetables as well as the package design used for these commodities in shelf-life studies. Measuring respiration rates is fundamental to design the appropriate packaging configuration to reach an EMAP. ILP increased the respiration rate of lettuce more than 80%, but that for cabbage was not significantly affected, revealing interspecific differences in susceptibility. To date, no report is known on the effect of ILP on vegetable tissues. Moreover, from our results no distinction can be made between possible photothermal and photochemical effects. However, since the lamp used in this work is rich in the UV-C part of the spectrum, some similarities may be established between our results and those obtained using continuous UV-C. Allende and Artes (2003a) reported that after a UV-C radiation dose of 8.14 kJ/ m2 bLollo RossoQ lettuce doubled its respiration rate, the same dose increased 75% the respiration rate of bRead Oak LeafQ lettuce (Allende and Artes, 2003b). Similar result was reported by Erkan et al. (2001) for UV radiated zucchini squash tissues. Therefore, it is likely that the increment of lettuce respiration rate after ILP is related to the UV-C part of the used light spectrum. It is known that UV-C affects plant cells, causing damage to DNA, tissue and photosynthetic apparatus among other effects (Stapleton, 1992) that may alter vegetable respiration. By taking into consideration the change in respiration rate after ILP treatment, the packaging configurations used in these experiments allowed that gas concentrations inside

packages were out of anoxic conditions during the shelf-life studies. 3.4. Microbiological spoilage of the EMAP minimally processed vegetables When samples were processed for shelf-life studies, 0.54 log reductions were achieved in MP white cabbage for aerobic psychrotrophic count (APC) and 0.46 in MP Iceberg lettuce, reductions on yeasts counts were not statistically significants (aV0.05). Lactic acid bacteria (LAB) counts were always below the detection limit of the method (10 CFU/g) in both products; Gram positive bacteria are uncommon in lettuce, King et al. (1991) reported that 97.3% of their bacterial isolates from lettuce were Gram negative rods, mainly Pseudomonas. Moreover Barriga et al. (1991) explained the poor growth of LAB in refrigerated lettuce by the competition with other populations with higher growth rates at low temperatures and a better adaptation to lettuce. Jacxsens et al. (2004) stated that LAB are not so important in the spoilage of green leafy vegetables, but during storage anoxic conditions can favour their growth. LAB are part of the natural microflora of cabbage (Carr et al., 2002), but they are present in very low levels, and storage conditions will have not favoured their growth. Psychrotrophic and yeasts counts during the storage of control and flashed MP cabbage are presented in Fig. 2. At day 2, the benefit of the decontamination achieved by ILP is lost, control and treated samples yielded then the same counts, and from day 7 on flashed samples had the highest psychrotrophic count until the end of the experiment. The possibility that the growth rate of microorganisms in decontaminated vegetables is consistently faster than those in non-treated samples deserves careful study because could hamper the application of some decontamination techniques; since most of the related reports do not include storage studies, few data are available to enable comparison. As an example, Li et al. (2001) studied the changes in the natural microflora of Iceberg lettuce treated in warm, chlorinated water, and during storage at refrigeration temperatures. In spite of the obtained reductions of 0.34–1.27 logs in the aerobic psychrothrophic count following treatments, these counts were higher in treated samples after 4 days storage at 5 8C and 2 days

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10 9 8 7 6 5 4 3 2 1 0 1 2 3 5 4 6 Time (days) 7 8 9

Fig. 2. Aerobic psychrotrophic count (APC) and yeasts (log CFU/ gFS.D.) on untreated and intense light pulses treated (45 s/side, 12.8 cm) minimally processed EMA packaged white cabbage. Control, APC (5), flashed, APC (n), control, yeasts (o), flashed, yeasts ( ). Horizontal line indicates the shelf-life limiting number of 108 CFU/g APC.

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storage at 15 8C. It is possible that some decontamination treatments make vegetable tissues become weaker and more susceptible to microbial degradation. Psychrotrophs reached the limit of acceptability (108 CFU/g) at day 7 in treated samples, whilst controls were acceptable during the 9 days of the experiment; yeasts counts never reached the acceptability limit (105 CFU/g). Therefore, ILP did not extend the microbial shelf-life of MP cabbage stored in EMAP at 7 8C. Fig. 3 shows the aerobic psychrotrophic count and yeast counts during the storage of control and flashed MP lettuce. Psychrotrophic count of treated samples kept lower than that for the controls during the 5 days of the experiment, whilst yeasts count of treated lettuce were higher, although still very limited, than that of controls at the end of the experiment. Control samples reached the acceptability limit before or at day 3, while treated ones did after the third day. From the microbiological point of view, gaining one extra day seems to be possible by using ILP, taking also into account that yeasts counts were low. 3.5. Sensory quality The shelf life of perishable food products should always be established by combining the microbial shelf life and the sensory shelf life. Table 3 shows the evolution of six relevant sensory attributes of control

and flashed MP white cabbage stored on EMAP at 7 8C up to 9 days. The presence of off-odours limited the shelf-life of the treated samples to 7 days (score N3.0), the rest of the tested parameters were always below the rejection limit for both kind of samples up to 9 days. A distinctive off-odour described as bplasticQ was obvious immediately after treating samples by ILP, which faded during the next hours according to preliminary studies. For this reason the shelf-life experiment was continued in spite of the early rejection of the treated samples. The detected off-odour is expected to disappear before the product is consumed. From these data, the shelf-life of the treated product based on sensory quality would be 7 days, equal to the microbiological shelf-life. The results obtained for the evolution of eight relevant sensory attributes in control and flashed MP Iceberg lettuce stored on EMAP at 7 8C up to 9 days are provided in Table 4. In this case, the shelf-life given by sensory characteristics was limited by offodours, overall visual quality (OVQ) and leaf edge browning for control samples, and only by OVQ for flashed ones, all these parameters reached unacceptable scores at day 3. How much browning accounts for the bad OVQ score was not determined in this work, but it is known that browning is the major cause of quality loss reported in MP lettuce, and leaf surface browning and cut edge browning defects have been
10 9 8 7 log CFU/g 6 5 4 3 2 1 0
0 1 2 3 Time (days) 4 5

log CFU/g

Fig. 3. Aerobic psychrotrophic count (APC) and yeasts (log CFU/ gFS.D.) on untreated and intense light pulses treated (45 s/side, 12.8 cm) minimally processed EMA packaged iceberg lettuce. Control, APC (5), flashed, APC (n), control, yeasts (o), flashed, yeasts ( ). Horizontal line indicates the shelf-life limiting number of 108 CFU/g APC.

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V.M. Gomez-Lopez et al. / International Journal of Food Microbiology 103 (2005) 79–89 Allende, A., Artes, F., 2003b. Combined ultraviolet-C and modified atmosphere packaging treatments for reducing microbial growth of fresh processed lettuce. Food Sci. Technol. 36, 779 – 786. Allende, A., Aguayo, E., Artes, F., 2004. Microbial and sensory quality of commercial fresh processed red lettuce throughout the production chain and shelf life. Int. J. Food Microbiol. 91, 109 – 117. Alzamora, S.M., Lopez-Malo, A., Tapia, M.S. 2000. Overview. In: Alzamora, S.M., Tapia, M.S., Lopez-Malo, A. (Eds.) Minimally Processed Fruits and Vegetables. Aspen, Maryland, pp. 1,5. Anderson, J.G., Rowan, N.J., MacGregor, S.J., Fouracre, R.A., Farish, R.A., 2000. Inactivation of food-borne enteropathogenic bacteria and spoilage fungi using pulsed-light. IEEE Trans. Plasma Sci. 28, 83 – 88. Barriga, M.I., Trachy, G., Willemot, C., Simard, R.E., 1991. Microbial changes in shredded iceberg lettuce stored under controlled atmospheres. J. Food Sci. 56, 1586–1588, 1599. Beuchat, L., 2000. Use of sanitizers in raw fruit and vegetable processing. In: Alzamora, S.M., Tapia, M.S., Lopez-Malo, A. (Eds.), Minimally Processed Fruits and Vegetables. Aspen, Maryland, pp. 63 – 78. Brackett, R.E., 1987. Microbiological consequences of minimally processed fruits and vegetables. J. Food Qual. 10, 195 – 206. Brecht, J.K., 1995. Physiology of lightly processed fruits and vegetables. HortScience 30, 18 – 24. Cantos, E., Espn, J.C., Tomas-Barberan, F.A., 2001. Effect of wounding on phenolic enzymes in six minimally processed lettuce cultivars upon storage. J. Food Sci. 49, 322 – 330. Carr, F.J., Chill, D., Maida, N., 2002. The lactic acid bacteria. A literature survey. Crit. Rev. Microbiol. 28, 281 – 370. Cleaver, J.E., 2003. Photoreactivation. DNA Repair 2, 629 – 638. Dunn, J.E., Clark, R.W., Asmus, J.F., Pearlman, J.S., Boyerr, K., Painchaud, F., Hoffman, G.A. 1989. US Patent 4,871,559. Erkan, M., Wang, C.Y., Krizek, D.T., 2001. UV-C irradiation reduces microbial populations and deterioration in Cucurbita pepo fruit tissue. Environ. Exp. Bot. 45, 1 – 9. Fan, X., Sokorai, K.J.B., 2002. Sensorial and chemical quality of gamma-irradiated fresh-cut iceberg lettuce in modified atmosphere packages. J. Food Prot. 65, 1760 – 1765. FDA, 2000. Kinetics of microbial inactivation for alternative food processing technologies. Pulsed light technology. http://vm. cfsan.fda.gov/~comm/ift-puls.html. Accessed March 22, 2004. Fine, F., Gervais, P., 2003. Microbial decontamination of food powders: bibliographic review and new prospects. Sci. Aliments 23, 367 – 393. Hollosy, F., 2002. Effects of ultraviolet radiation on plant cells. Micron 33, 179 – 197. Hoornstra, E., de Jong, G., Notermans, S., 2002. Preservation of vegetables by light. In: Society for Applied Microbiology (Ed.), Conference Frontiers in Microbial Fermentation and Preservation; 9–11 January 2002, Wageningen, The Netherlands, pp. 75 – 77. Jacxsens, L., Devlieghere, F., Debevere, J., 1999a. Validation of a systematic approach to design equilibrium modified atmosphere packages for fresh-cut produce. Food Sci. Technol. 32, 425 – 432.

demonstrated to contribute with a decreased OVQ in MP lettuce (Lopez-Galvez et al., 1996). The choice of not using antibrowning agents in this experiment was taken in view of the possibility that ILP could inactivate polyphenol oxidase (PPO) (Dunn et al., 1989), since it is known that this enzyme is responsible for lettuce browning after tissue injury (Cantos et al., 2001). However, according to our results for leaf browning, it seems that no significant PPO inactivation occurred. Therefore, treating MP lettuce with antibrowning agents before ILP flashing can be recommended in order to avoid that this defect limits the shelf-life of the product.

4. Conclusion This study gives evidence that proteinaceous or oily foods are inappropriate for decontamination by ILP. On the other hand, foods high in carbohydrates such as fruits and vegetables seem to be more suitable for it. This study also provides new data about the effect of ILP to decontaminate MP vegetables. An increase of the respiration rate of MP vegetables after ILP treatment as well as shelf-life studies of ILP treated MP vegetables are reported for the first time. ILP did not prolong the shelf-life of MP white cabbage or MP iceberg lettuce. However, from the microbial point of view, one extra storage day at 7 8C was achieved for MP Iceberg lettuce. It is therefore suggested that the application of an antibrowning treatment in combination with ILP would increase the total shelf-life of MP iceberg lettuce.

Acknowledgments The authors want to thank the Consejo de Desarrollo Cientfico y Humanstico of Universidad Central de Venezuela for the PhD scholarship of V. Gomez.

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