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Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Novel approach to the microbial decontamination of strawberries: chlorophyllin-based photosensitization


Z. Luksiene and E. Paskeviciute
Institute of Applied Sciences, Vilnius University, Vilnius, Lithuania

Keywords decontamination, nonthermal, photosensitization, preservation, strawberry. Correspondence Zivile Luksiene, Institute of Applied Sciences, Vilnius University, Sauletekio 10, 10223 Vilnius, Lithuania. E-mail: zivile.luksiene@tmi.vu.lt

Abstract Aims: This study is focused on the possibility to control microbial contamination of strawberries by chlorophyllin (Na-Chl)-based photosensitization. Moreover, photosensitization-induced effects on key quality attributes of treated strawberries was evaluated. Methods and Results: Strawberries were inoculated with Listeria monocytogenes ATCL3C 7644, soaked in 1 mmol l)1 Na-Chl for 5 min and illuminated for 30 min with visible light (k = 400 nm, energy density 12 mW cm)2). Results indicated that the decontamination of strawberries using photosensitization was 98% compared to control sample. Naturally occurring yeasts microfungi and mesophiles were inhibited by 86 and 97%, respectively. The shelf life of treated strawberries was extended by 2 days. The total antioxidant activity of treated strawberries increased by 19%. No impact on the amount of phenols, anthocyanins or surface colour was detected. Conclusions: Photosensitization may be an effective, nonthermal and environmentally friendly microbial decontamination technique which expands the shelf life of strawberries without any negative impact on antioxidant activity, and phenols, anthocyanins or colour formation. Signicance and Impact of the Study: Experimental data support the idea that Na-Chl-based photosensitization can be a useful tool for the future development of nonthermal food preservation technology.

2010 2179: received 1 December 2010, revised 10 February 2011 and accepted 11 February 2011
doi:10.1111/j.1365-2672.2011.04986.x

Introduction Fresh produce is becoming more popular all over the world. Strawberries (Fragaria ananassa Duch.) are a popular and nutritious fruit worldwide. They have been reported to contain the highest antioxidant capacity among twelve fruits analysed (Wang et al. 1996; hko nen et al. 2001). The main contributors to antioxiKa dant activity are phenolic compounds which have positive effects for human health, preventing cancer, cardiovascular diseases, and age-induced oxidative stress (Olsson et al. 2004). Anthocyanins are the most abundant avonoids (phenolics) and are present at high level in mature strawberries (Cordenunsi et al. 2005). Short ripening and senescence periods of strawberries, susceptibility to mechanical injury, contamination during storage with fungi and bacteria reduces signicantly their
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shelf life. Strawberries spoilage losses can be as high as 40% (Satin 1996). Furthermore, fresh produce has been increasingly implicated as a vehicle for transmission of foodborne illnesses. Such foodborne illnesses are estimated to result in $ 69 billion of loss in productivity and medical expenses to the US economy (ERS 2005). Several outbreaks associated with consumption of strawberries have been reported (FDA 1999). A variety of pathogenic bacteria such as Listeria monocytogenes, Salmonella spp. as well as pathogenic Escherichia coli strains may be present on fresh fruits (Knudsen et al. 2001; Johannessen et al. 2002). The most widely known postharvest treatments to reduce microbial spoilage are low temperature and modied atmosphere packaging (Nielsen and Leufven 2008). However, it has been reported that these technologies are not effective enough and can have a negative impact on

2011 The Authors Journal of Applied Microbiology 110, 12741283 2011 The Society for Applied Microbiology

Z. Luksiene and E. Paskeviciute

Decontamination of strawberries by photosensitization

the quality of strawberries (Ayala-Zavala et al. 2007). For instance, carbon dioxide treatment reduced anthocyanins content and changed internal fruit colour (Gil et al. 1997). Washing alone or conventional sanitizers have been shown to have limited efcacy at removing spoilage and pathogenic bacteria from the surface (Yuk et al. 2006). There is a need to develop novel processing technologies that are more effective and do not diminish the organoleptic properties and nutritional value of the treated produce. Photosensitization is a novel nonthermal and ecologically friendly treatment that involves the administration of a photoactive compound (photosensitizer) and visible light. After spraying of the photosensitizer on the surface of fruit or vegetable, most pathogens and harmful bacteria distributed on the surface of the fruit are able to bind the photosensitizer. The following illumination of fruits with visible light induces various photocytotoxic reactions and death of surface-attached micro-organisms without any harmful effects on environment (Luksiene 2005; Luksiene and Zukauskas 2009). Na-Chl is a water-soluble food additive (E141) and is used as food colourant, in dietary supplements and in cosmetics (Lopez-Carballo and Ocio 2008). According to our preliminary data, Na-Chl interacts with bacterial wall, outer membrane and after illumination destroys their integrity. This prevents the opportunity to kill micro-organisms using a nonthermal technology. This study is focused on the possibility to decontaminate strawberries by photosensitization from GramPositive food pathogen L. monocytogenes, naturally distributed yeasts, microfungi and mesophiles. In addition, photosensitization-induced effects on key quality attributes of treated strawberries was evaluated. Material and Methods Pure culture of L. monocytogenes Listeria monocytogenes ATCL3C 7644 was kindly provided by the National Veterinary Laboratory (3rd passage of ATCC7644-test organism) (Vilnius, Lithuania). The bacterial strain was cultured on Tryptone Soya Agar supplemented with 06% Yeast Extract (TSYEA) (Liolchem, Roseto degli Abruzzi, Italy) for 24 h at 30C. For bacterial suspension preparation, L. monocytogenes was grown overnight (c. 14 h) at 37C in 20 ml of Tryptone Soya medium supplemented with 06% Yeast Extract (TSYE) (Liolchem), with agitation of 120 rev min)1 (Environmental Shaker-Incubator ES20; Biosan, Riga, Latvia). The overnight bacterial culture was diluted 20 times by the fresh medium (A = 0164) and grown at 37C to mid-log phase (c. 116 109 colony forming units ml)1

(CFU ml)1), A = 09) in a shaker (120 rev min)1). Bacterial optical density was determined in a 1001 mm glass cuvette at k = 540 nm (Hekios Gamma; ThermoSpectronic, Cambridge, UK). Cells were then harvested by centrifugation (20 min, 5000 g) (PC-6, Moscow, Russia) and resuspended to c. 58 109 CFU ml)1 nal concentration in 01 mol l)1 phosphate buffer saline (PBS, pH = 72). This stock suspension was accordingly PBSdiluted to c. 1 107 CFU ml)1 and used for the further photosensitization experiments. Inoculation of L. monocytogenes on the surface of strawberries Strawberries (F. ananassa Dutch.) were purchased in a local supermarket and used within 1 day. Prepared inoculum (described above) of L. monocytogenes was poured over strawberries and left for 30 min at room temperature for cell attachment. Photosensitization treatment Naturally contaminated or pathogen-inoculated strawberries were soaked in 1 mmol l)1 chlorophyllin Na-salt (Na-Chl) (Roth, Karlsruhe, Germany) solution for 5 min. The dried strawberries were placed in the treatment chamber in a sterile Petri dish uncovered and exposed to light intensity 12 mW cm)2 at k = 400 nm for 20 min. The light source necessary for photosensitization was constructed in the Institute of Applied Sciences of Vilnius University. Light dose delivered to the surface of sample was calculated as light intensity multiplied by time (144 J cm)2). Light power density measurements were carried out using a light energy 3 Sigma meter (Coherent, Santa Clara, CA, USA) equipped with piroelectrical detector J25LP04. Control samples were soaked in Na-Chl solution but not illuminated in the chamber. Illumination (400 nm) of unsoaked in Na-Chl samples (other control) did not have any effect on survival of micro-organisms. Total aerobic micro-organisms count Strawberry samples after treatment were separately mixed with an appropriate volume of 01 mol l)1 PBS (1 g of sample 10 ml buffer) and homogenized in sterile BagPage bags using a BagMixer (model MiniMix 100 VP, Interscience, St. Nom, France). Total aerobic microorganism count was determined by serial dilutions (in 09% NaCl) plated on TSYEA and incubation at 30C for 48 h. Total yeasts and microfungi count were determined by serial dilutions (in 09% NaCl) plated on dichloran glycerol agar and incubation at 30C for 72 h The
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2011 The Authors Journal of Applied Microbiology 110, 12741283 2011 The Society for Applied Microbiology

Decontamination of strawberries by photosensitization

Z. Luksiene and E. Paskeviciute

surviving cell populations were enumerated and expressed by log10 (CFU g)1). Evaluation of shelf life of strawberries To evaluate shelf life of treated strawberries, one part of strawberries was soaked in 1 mmol l)1 Na-Chl salt solution, and the control in sterile distillated water. Samples treated with Na-Chl salt were illuminated for 20 min, dried and stored in refrigerator (+6C). The control samples were not illuminated and stored under the identical conditions. Every experimental group consisted of 30 berries (weight 1015 g). Disease-free (shelf life) period of strawberries was evaluated visually by assessment of colour change (rots, spots), induced by growth of spoilage micro-organisms during 9 days after treatment with berries stored in a refrigerator (+6C). The level of infected berries was scored on a 16 scale. Results were expressed as a disease index between 0 and 100 (0, no infection; 100, all fruits are infected) (Kittemann et al. 2008). Temperature measurement on the surface of berry Precision Celsius temperature sensors (Deltha Ohm, Padua, Italy) were used for temperature measurements on the surface of strawberry. Measurements of total antioxidant capacity Total antioxidant capacity of strawberries was measured by ferric reducing ability of plasma (FRAP) method (Benzie and Strain 1996). Extracts for measurement were prepared by homogenization of 1 g of fruit with 50 ml 96% alcohol (Minimix, Interscience). FRAP working solution included 03 mol l)1 acetate buffer (pH 36), 001 mol l)1 2, 4, 6-tripyridyl-s-triazine (TPTZ) in 004 mol l)1 HCl and 002 mol l)1 FeCl36H2O in distilled water. For measurement of antioxidant activity, 15 ml of FRAP reagent and 50 ll of sample solution were mixed. The reading was performed every 30 s up to 5 min at 593 nm (Hekios Gamma; ThermoSpectronic), 1-cm light path. Fe (II) standard solution was tested in parallel. Total soluble phenols assay Samples of strawberries (310 g) were homogenized for 1 min at maximum speed in a Minimix with 30100 ml of mixture containing acetone, distilled water and acetic acid (70 : 295 : 05). Samples were mixed and allowed to stand for 1 h at room temperature. Extracts were centrifuged at 1640 g for 15 min (Micro 200; Hettich, Beverly, MA, USA), and supernatant was used for total phenols (TP) assay.
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TP concentration was measured using the FolinCiocalteu assay (Asami et al. 2003). In brief, 5 ml of distilled water, 05 ml of sample and 1 ml of FolinCiocalteu reagent were mixed and left at room temperature for 5 min. Then, 10 ml of 7% sodium carbonate solution was added and solution was lled to 25 ml nal volume by the addition of distilled water. Solution was mixed well and left at room temperature for 2 h. Then, the mixture was ltered through 8-layer cheesecloth. After that, the TP concentration using a spectrophotometer monitoring 750 nm (Hekios Gamma; ThermoSpectronic) was measured. TP content was standardized against gallic acid and expressed as milligrams per litre of gallic acid equivalents. Total anthocyanins assay Samples weighing 10 g of treated strawberries were blended in a food processor for 1 min with 75 ml of a mixture of methanol, acetic acid and distilled water at a ratio of 25 : 1 : 24. Mixture was centrifuged at 2000 g for 20 min (Micro 200). The supernatant was removed and mixed with 75 ml M : A : W then centrifuged again, and the supernatant was separated. Each sample was extracted 3 times. Optical density was measured using 1 cm path length quartz cuvette at 535 nm (Hekios Gamma; ThermoSpectronic) (Tiwari et al. 2009). Measurement of colour Possible changes of strawberry colour after photosensitization were evaluated from absorption spectrum measuring optical density (OD) of the sample in visible region of spectrum. Samples weighing 10 g of fresh berry were blended in a food processor for 1 min with 75 ml of a mixture of methanol, acetic acid and distilled water (M : A : W) at a ratio of 25 : 1 : 24. The mixture was then centrifuged at 2000 g for 20 min (Micro 200). Optical density (310650 nm) was measured using 1 cm path length quartz cuvette with spectrophotometer (Hekios Gamma; ThermoSpectronic). Each sample was extracted 3 times. Statistics The experiments were carried out in triplicate for each set of exposure, using different batches of strawberries. The data were analysed with Origin 7.5 software (OriginLab Corporation, Northampton, MA, USA). A standard error was estimated for every experimental point and marked in a gure as an error bar. Sometimes the bars were too small to be visible. To estimate the signicance of inactivation of L. monocytogenes on the surface of strawberries

2011 The Authors Journal of Applied Microbiology 110, 12741283 2011 The Society for Applied Microbiology

Z. Luksiene and E. Paskeviciute

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Decontamination of strawberries by photosensitization

Cell number (Log10 CFU g1)

(Fig. 1) and to compare the amounts of soluble phenolics and anthocyanins in strawberries (Fig. 4b,c), analysis of variance (anova) with Bonferroni test was used. To estimate the signicance of inactivation of total mesophiles, yeasts and fungi (Fig. 2a,b) on the surface of strawberries by photosensitization, shelf life of strawberries (Fig. 3) and the effect of photosensitization on the amount of total antioxidants (Fig. 5a) Students t-test was used. Results Photosensitization-based inactivation of L. monocytogenes inoculated on the surface of strawberry The data depicted in Fig. 1 indicated that the population of inoculated Listeria in untreated control strawberry mergulhados group aumentou grew to 68 log. Soaking of berries in Na-Chl solution or just illumination with light was indifferent and did not affect the bacterial survival. As little as 1 mmol l)1 Na-Chl-based photosensitization reduced the population of Listeria by 18 log (98%). Photosensitization-based inactivation of total aerobic mesophils, yeasts and fungi
se Subsequently, it was determined whether naturally surface-distributed mesophiles, microfungi and yeasts were susceptible to photosensitization. Data presented in Fig. 2a indicated that the growth of total aerobic mesophiles in control group increased from 4 log to 8 log during 8 days. In treated strawberries, the amount of mesophiles reduced by 17 log (97%) and increased less

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7 6 Cell Number, Log10 5 4 3 2 1 0


Figure 1 Inactivation of Listeria monocytogenes ATCL3C 7644 on the surface of strawberries by photosensitization with 1 mmol l)1 Na-Chl (incubation time 5 min, illumination time 20 min). ( ), control; ( ), Na-Chl without light and ( ), after photosensitization with Na-Chl.

Figure 2 Inactivation and regrowth of total mesophils (a), yeasts and fungi (b) on the surface of strawberries by photosensitization with 1 mmol l)1 Na-Chl during 8 days after treatment (incubation time 5 min, illumination time 20 min, storage time 8 days at +6C). ( ) control and ( ) after photosensitization with Na-Chl.

(6 log) during 8 days in comparison with control (8 log). No signicant changes of growth rate (l) of mesophiles were observed during 8 days after treatment: in control l = 115 compare it in treated samples l = 110. Data presented in Fig. 2b indicate that 1 mmol l)1 Na-Chl-based photosensitization can reduce the amount of yeasts and fungi in the strawberries by 086 log (86%). Examination of regrowth of micro-organisms during 8 days after treatment indicates that in control group the yeasts and microfungi increase from 21 log to 71 log during 8 days, where as in treated group their amount during the same period reached 58 log. The regrowth rate of yeast and fungal survivors slightly decreased (in control l = 144, in treated samples l = 127).
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2011 The Authors Journal of Applied Microbiology 110, 12741283 2011 The Society for Applied Microbiology

Decontamination of strawberries by photosensitization

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Z. Luksiene and E. Paskeviciute

30
120 100

25
Temperature (C)
0 1 2 3 4 5 Time (days) 6 7 8 9

Survival distribution (%)

80 60 40 20 0

20 15 10 5 0 0 2 4 6 8 10 12 14 Time (min) 16 18 20 22

Figure 3 Shelf life of strawberries after photosensitization with 1 mmol l)1 Na-Chl in comparison with control. () control and (.....) after photosensitization with Na-Chl.

Figure 4 The increase in temperature on the surface of strawberries placed in LED-based light prototype during 20 min of illumination. Thermometer (Delta Ohm, Italy) was used for temperature measurements on the surface of berry.

Evaluation of shelf life of strawberries after photosensitization It is obvious that the most important advantage of any antimicrobial technology is ability to extend the self-life of treated berries. Shelf life of berries was evaluated visually from surface colour changes. Thus, as depicted in Fig. 3, the disease-free period of treated strawberries was prolonged about 2 days in comparison with control. This is a signicant effect as the Na-Chl concentration was not high (1 mmol l)1). Furthermore, some delay of disease induction in the treated strawberries was observed. Measuring the extension of shelf life of treated strawberries by Kittemann et al. (2008) system (16 scale), 5-day storage makes all control berries (100%) infected and disease index (N) was the highest N = 6, whereas just 25% of treated berries were damaged by spoilage microorganisms, thus disease index was signicantly lowered (N = 15). Measurement of strawberry surface temperature The temperature kinetics on the surface of berry during treatment was evaluated using specially attached surface digital attacher inside the treatment chamber. Data presented in Fig. 4 clearly indicate that during 20 min of treatment the temperature on the surface of strawberry increases very slowly and does not exceed 27C. Total antioxidant activity According to the obtained results depicted in Fig. 5a, the total antioxidant activity in control berries was 18 mmol
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Fe2+ kg whereas in berries treated by photosensitization (1 mmol l)1 Na-Chl) it increased to more than 22 mmol Fe2+ kg. This statistically signicant increase (19%) in total antioxidant activity was observed in the strawberries immediately after photosensitization. Evaluation of the amount of total phenolics and anthocyanins To estimate specic changes of nutritional quality of strawberries, the amount of total phenolics and anthocyanins in the treated and control strawberries was evaluated. As depicted in Fig. 5b, the amount of total soluble phenolics immediately after photosensitization (1 mmol l)1 Na-Chl) did not differ from control (1750 mg per 100 g f.f.). Storage without treatment at +6C in the refrigerator reduced their amount to 1400 mg per 100 g f.f. In addition, the level of total anthocyanins in strawberries after photosensitization treatment was the same as in control berries (Fig. 5c). Storage without treatment of strawberries in refrigerator for 24 h reduced the anthocyanin level from 145 mg PGN 100 g f.f. to 122 mg PGN 100 g f.f. in control as well as in treated strawberries. Measurements of strawberry colour The other important characteristic that can be inuenced by photosensitization is the appearance of berry, especially its colour. Thus, strawberries from the most effective treatments were analysed immediately after treatment to determine whether photosensitization had any negative effects on the colour of the strawberry. For this purpose,

2011 The Authors Journal of Applied Microbiology 110, 12741283 2011 The Society for Applied Microbiology

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Decontamination of strawberries by photosensitization

(a)
24 22 20

mmol Fe2+ kg1 fruit

18 16 14 12 10 8 6 4 2 0

absorption spectroscopy was used to analyse the spectrum of berry extract in visible region. It is evident from Fig. 6 that no signicant colour changes are detected over all visible spectrum region, meaning that photosensitization has no impact on strawberry colour. Discussion During the past decade, the emphasis in postharvest fruit substituida protection has shifted from using chemicals to various alternative techniques including biological control (Sharma et al. 2009) and physical methods such as controlled atmosphere (Zheng et al. 2008; Sandhya 2010) or irradiation (Dionisio et al. 2009). To compare, irradiation of strawberries with 23 kGy suprimir irradiation was found to suppress fungi on stored berries and more than double their shelf life, but unfortunately gave rise to changes in strawberry texture, cell wall composition and colour (Yu et al. 1995). Actually, ionizing radiation has been approved for decontamination of food in the USA. However, it is strongly discouraged in EU as consumers mostly prefer nonirradiated products (Neyssen 2000). Marquenie et al. (2003) studied the combined effect of three physical methods high-power pulsed light, heat and UV-C illumination on strawberry decontamination from fungus Botrytis cinerea. Their results revealed that pulsed light alone was ineffective against selected fungus, although combined treatment of all three techniques reduced visually B. cinerea mycelia and did not affect fruit rmness. This technique also prolonged disease-free period increasing the shelf life by 12 days.
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1800 1600 1400 1200 1000 800 600 400 200 0 Control Na-chl without light Na-chl +5 min light

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Na-chl salt without light

Na-chl salt with 5 min light

00 300 350 400 450 500 550 Wavelength (nm) 600 650

Figure 5 The amount of total antioxidants (a), soluble phenolics (b) and anthocyanins (c) in strawberries after 1 mmol l)1 Na-Chl-based photosensitization in comparison with control during 024 h after treatment keeping them at +6C. ( ) control; ( ) after photosensitization with Na-Chl; ( ) 0 h; ( ) 12 h and ( ) 24 h.

Figure 6 Strawberry colour changes after Na-Chl photosensitization treatment: absorption spectrum of strawberry extract samples in visible region. ( ) control; ( ) Na-Chl without light and ( ) after photosensitization with Na-Chl.

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Decontamination of strawberries by photosensitization

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Z. Luksiene and E. Paskeviciute

Allende et al. (2007) determined the effect of UVC light, gaseous O3, super atmospheric O2 and CO2-enriched atmospheres applied individually and in combination on the shelf life of strawberries. Individual treatments did not affect the microbial contamination, whereas a combination reduced the growth of yeast and microfungi by 1 log for up to 5 days and prolonged the storage period, respectively. Data obtained in our previous studies revealed that photosensitization-based treatment inactivated (6 log) food pathogens L. monocytogenes ATCL3C 7644 and Bacillus cereus ATCC 12826 in vitro when aminolevulinic acid (Buchovec et al. 2009; Le Marc et al. 2009; Luksiene et al. 2009; Buchovec et al. 2010) or Na-Chl (Luksiene et al. 2010a,b) was used as photosensitizers. Moreover, such microbial conditions as spores and biolms were susceptible to this treatment (Luksiene et al. 2010a,b). Effective photosensitization-induced inactivation of microfungi and yeasts in vitro has been reported in our previous studies (Luksiene 2005; Luksiene et al. 2005). These results induzem mais prompted us to investigate further the susceptibility of pathogens, yeasts, microfungi and mesophiles to this treatment in real food systems. According to the data obtained, photosensitizationbased treatment can decontaminate strawberry from the inoculated Listeria by 98% (Fig. 1). Mesophiles naturally distributed on the surface of strawberries have been inactivated by 97% (Fig. 2a). It is important to note that spoilage yeasts and microfungi showed slightly lower susceptibility to photosensitization and were inactivated by 86% (Fig. 2b). These data are in line with results of other studies (Anderson et al. 2000). The overall reduction in this microbial contamination prolongs the disease-free period of berries by 2 days (Fig. 3). The irregularity and the different light-reecting properties of the illuminated strawberry surfaces can possibly account for the different antimicrobial efciency in vitro and treating food matrix (Paskeviciute et al. 2009). Taking into account that photosensitization efciency depends on illumination time and light intensity, the obtained antibacterial efciency can be signicantly enhanced by usage of more powerful light sources (light emitting diodes-LED) or increase in illumination time (Luksiene and Buchovec 2009). Distinguishing feature of photosensitization is its nonthermal action. It should be noted that there was no signicant temperature increase on the surface of strawberries which would be dangerous to the quality of fruit after photosensitization treatment (Fig. 4). During 20 min of treatment, the temperature on the surface of strawberries changed from 20 to 25C. It must be emphasized that to nd a physical antimicrobial technique without thermal effects is a complicated tarefa task. For instance,
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high-power pulsed light is an effective disinfection method, but temperature increase will occur. The temperature on the surface of strawberry increased to 80C after 60 s of high-power pulsed light treatment (Bialka and Demirci 2008). The main benecial properties of fruits and vegetables have been partially attributed to the presence of antioxidant compounds (Tulipani et al. 2008). Antioxidants can scavenge free radicals and reactive oxygen species which usually induce toxic processes in the living cell including oxidative damage to proteins and DNA, membrane lipid oxidation, enzyme inactivation and gene mutation that may nally lead to cancer genesis or other oxidative cardiovascular or inammatory diseases (Eberhardt et al. 2000). Therefore, it might be possible that photosensitization being an effective antimicrobial treatment modality can affect and result in some negative impact on the strawberry nutritional properties. Thus, it was necessary se to investigate whether some changes of antioxidant activity took place after photosensitization in strawberries. According to our data presented in Fig. 5a, photosensitization induced signicant increase in total antioxidant activity (19%) in strawberries, which could be associated with enhanced cellular capacity to detoxify reactive oxygen species. However, the mechanisms of these effects have not been thoroughly elucidated so far. In fact, plant cells usually keep the reactive oxygen species (ROS) level under tight control by production or activation of scavenging enzymes (Bailly 2004). Recently, such phenolic compounds as avonoids (anthocyanins), avanols (catechins) and avonols (quercetin) have attracted increasing attention as potent antioxidants. They can stimulate carcinogen-detoxifying enzymes and counteract inammatory processes (Parr and Bolwell 2000). Because phenolic compounds, as effective antimicrobials and UV screens, are accumulated in epidermal tissue, the question elvantada arise, does photosensitization affect their content in the strawberry fruit? According to our results, the photosensitization did not affect the level of soluble phenolics, and storage for only 24 h at +6C diminished their amount from 1750 mg per 100 g f.f. to 1400 mg per 100 g f.f. That is in agreement with the results of (Nunes et al. 1995). As anthocyaninss prevent cardiovascular and other disorders (Zafra-Stone et al. 2007), to save their concentration stable in strawberries is important. Many factors such as pH, light, oxygen, enzymes and high temperature can induce anthocyanin degradation (Wang and Xu 2007). For instance, Tiwari et al. (2009) studied the effect of ozone on strawberry juice anthocyanins and found that ozone induced signicant reductions in their content (982%). Data obtained in this work clearly indicate that the amount of anthocyanins after photosensitization did

2011 The Authors Journal of Applied Microbiology 110, 12741283 2011 The Society for Applied Microbiology

Z. Luksiene and E. Paskeviciute

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Decontamination of strawberries by photosensitization

not change after soaking of berries in Na-Chl or after photosensitization and remained very close to the initial content (140 lg g)1). Slight decrease was observed even after storage 24 h at +6C (121 lg g)1). Photosensitization increased the total antioxidant activity in strawberries, but this effect had no correlation with amount of total phenolics or their constituent anthocyanins. Gil et al. (1997) studied the effect of carbon dioxide treatment on the amount of anthocyanins and other polyphenols in strawberries. Their results revealed that anthocyanins content of CO2-treated fruit was reduced as compared with air-stored fruit. Odriozola-Serrano et al. (2008) also found that high-intensity pulsed electric eld (HIPEF) affected strawberry nutritional qualities. The loss of phenolic content over the storage time in HIPEF and thermally processed strawberry juices was in the range of 215241 mg per 100 ml after 56 days at 4C. Colour is one of the important quality indicators in fresh strawberry appearance and greatly contributes to fruit quality. The bright red colour of strawberry fruit is because of the presence of anthocyanin pigments in the fruit epidermis and cortex (Nunes et al. 1995). In addition, factors such as copigmentation, pH and anthocyanin metabolism may play a signicant role in the expression of colour in strawberries (Gil et al. 1997). The most effective copigments avonols are located in external tissue (Mazza and Miniati 1993). To determine whether photosensitization had any effect on the colour of strawberry, the fruits were analysed immediately after treatment. According to the obtained data (Fig. 6), the colour of strawberry surface was not markedly affected by the photosensitization as no signicant difference was detected between absorption spectrum of treated and control fruits. Other studies performed on decontamination of strawberries using carbon dioxide treatment indicated that fruit surface colour did not change, but remarkable changes were observed in internal esh colour (Gil et al. 1997). However, high-power pulsed light used to decontaminate strawberries did not induce changes in fruit skin colour (Bialka and Demirci 2008) as light-based technologies including photosensitization act supercially. Preliminary data were obtained on the taste of treated berries. Twenty-one volunteers tested the taste of treated strawberries and compared them with control ones. Eighteen of 21 volunteers found no sweetness or rmness changes in control and treated berries. It can be easily explained as visible light alone is not producing any effects, and Na-Chl is a well-known food additive. Conclusions Data obtained in this study clearly indicate that photosensitization might be useful tool to decontaminate strawber-

ries from Gram-positive food pathogen Listeria, yeasts, microfungi and mesophiles distributed on the surface of strawberries. These reductions are comparable to, if not greater than, the reductions obtained by other methods. One of the main advantages of photosensitization is the absence of any harmful effects on strawberry antioxidant activity, total phenolics, avanoids (anthocyanins) or colour with signicant extension of their shelf life (2 days). In addition, no negative impact on the taste of treated strawberries was found. Importantly, treatment is nonthermal and environmentally friendly. Thus, photosensitization has potential as an antimicrobial tool for the treatment of, for example, ready-to-eat fruits, frozen berries, pastry products or similar. More detail studies need to be conducted on the quality and organoleptic characteristics of the treated fruits in the future. Acknowledgements This study was nancially supported by the European Commission (FP6 STREP project HighQ RTE, No 023140). The authors are thankful Dr V. Gudelis and I. Buchovec for their contribution to this study. References
Allende, A., Marin, A., Buendia, B., Tomas-Barberan, F. and Gil, M.I. (2007) Impact of combined postharvest treatments (UV-C light, gaseous O3, superatmospheric O2 and high CO2) on health promoting compounds and shelf-life of strawberries. Postharvest Biol Technol 46, 201211. Anderson, J.G., Rowan, N.J., Mac Gregor, S.J., Fouracre, R.A. and Farish, O. (2000) Inactivation of food-borne enteropathogenic bacteria and spoilage fungi using pulsed-light. IEEE Trans Plasma Sci 28, 8388. Asami, D.K., Yun-Jeong, H., Barret, D.M. and Mitchell, A.E. (2003) Comparison of the total phenolic and ascorbic acid content of freeze-dried and air-dried marionberry, strawberry, and corn grown using conventional, organic, and sustainable agricultural practices. J Agric Food Chem 51, 12371241. Ayala-Zavala, F.J., Shiow, W.Y., Chien, W.Y. and GonzalezAguilar, G.A. (2007) High oxygen treatment increases antioxidant capacity and postharvest life of strawberry fruit. Food Technol Biotechnol 45, 166173. Bailly, C. (2004) Active oxygen species and antioxidants in seed biology. Seed Sci Res 14, 93107. Benzie, I.F.F. and Strain, J.J. (1996) The ferric reducing ability of plasma (FRAPP) as a measure of Antioxidant power: the FRAP assay. Anal Biochem 239, 7076. Bialka, K.L. and Demirci, A. (2008) Efcacy of pulsed UV-light for the decontamination of Escherichia coli O157: H7 and Salmonella spp. on raspberries and strawberries. J Food Sci 73, 201207.

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Z. Luksiene and E. Paskeviciute

Decontamination of strawberries by photosensitization

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2011 The Authors Journal of Applied Microbiology 110, 12741283 2011 The Society for Applied Microbiology

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