Intense pulsed light (IPL) and UV-C treatments for inactivating Listeria monocytogenes on solid medium and seafoods

Food Research International 54 (2013) 745–752

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Intense pulsed light (IPL) and UV-C treatments for inactivating Listeria monocytogenes on solid medium and seafoods Chan-Ick Cheigh a, Hee-Jeong Hwang b, Myong-Soo Chung b,⁎ a b

Department of Food and Food Service Industry, Kyungpook National University, Sangju 742-711, South Korea Department of Food Science and Engineering, Ewha Womans University, Seoul 120-750, South Korea

a r t i c l e

i n f o

Article history: Received 31 January 2013 Accepted 20 August 2013 Keywords: Listeria monocytogenes Inactivation Solid medium Seafood Intense pulsed light (IPL) UV-C

a b s t r a c t The inactivation effects of intense pulsed light (IPL) on Listeria monocytogenes surface-inoculated on solid medium and on seafoods such as flatfish, salmon, and shrimp fillets were investigated for various light doses (0.11–1.75 mJ/cm2 per pulse), number of pulses (0–9800 pulses, treatment time of 0–1960 s), and total fluences (0–17.2 J/cm2), and also the inactivation characteristics of UV-C irradiation on L. monocytogenes were evaluated for treatment time of 0–1960 s. Besides, any structural damage to the treated cells after IPL and UV-C treatments was evaluated by transmission electron microscopy (TEM). On the solid medium, approximately 4.0- and 6.0-log reductions of L. monocytogenes cells were achieved with UV-C irradiation for 1000 s and with IPL treatment for 180 s (900 pulses) at a fluence of 1.75 mJ/cm2 per pulse, respectively, with a negligible temperature rise (b2.0 °C) during treatment. On the seafood products, IPL treatment at 1.75 mJ/cm2 per pulse produced approximately 2.2-, 1.9-, and 1.7-log reductions of L. monocytogenes cells inoculated onto shrimp, salmon, and flatfish fillets, respectively, for 3600 pulses (720 s, total fluence of 6.3 J/cm2) and approximately 2.4-, 2.1-, and 1.9-log reductions, respectively, for 6900 pulses (1380 s, total fluence of 12.1 J/cm2), with a slight temperature rise (b5.0 °C) and no observable effect on the food color. Meanwhile, UV-C treatment on the inoculated fish fillets did not show significant effect on irradiation time of 0–1960 s. TEM observations clearly indicated destruction of the cell wall, cytoplasm shrinkage, and leakage of the cellular contents from the cytoplasm in IPL-treated L. monocytogenes cells, unlike UV-C treated cells. © 2013 Elsevier Ltd. All rights reserved.

1. Introduction Foodborne infection is a common and generally mild illness, although in some cases it can result in sickness or even death. In particular, seafood-related infectious disease outbreaks account for a large and growing proportion of all food-borne illness incidents (Butt, Aldridge, & Sanders, 2004). Furthermore, the seafood-related outbreaks appear to be on the rise due to increasing seafood consumption (Butt et al., 2004). It is generally accepted that contamination of seafoods with pathogenic microorganisms such as Vibrio spp., Aeromonas hydrophila, Listeria monocytogenes, Clostridium botulinum, Campylobacter spp., and Escherichia coli O157:H7 is a major source of the serious seafood-related illnesses (Butt et al., 2004). Any method that reduces or eliminates pathogenic microorganisms in foods, extends their shelf life, and improves their quality, may have a significant effect on the incidence of seafood-borne diseases (Dunn, Ott, & Clark, 1995). One particular microorganism, L. monocytogenes, is a long-established important food-borne pathogen. The incidence of food-related listeriosis outbreaks has increased consecutively over the last few years (Goulet, Hedberg, Le Monnier, & de Valk, 2008). L. monocytogenes contamination ⁎ Corresponding author. Tel./fax: +82 2 3277 4508. E-mail address: [email protected] (M.-S. Chung). 0963-9969/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodres.2013.08.025

of seafood varies with product category (Jørgensen & Huss, 1998). In particular, ready-to-eat seafoods, such as slices of ratfish, seafood salad, smoked salmon, and minced tuna, have been identified as having a relatively high incidence of contamination by this microorganism (Ben Embarek, 1994; Gombas, Chen, Clavero, & Scott, 2003; Miya, Takahashi, Ishikawa, Fujii, & Kimura, 2010). The microbial inactivation by the conventional UV-C treatment is well known in food industry. However, the conventional UV-C treatment for food sterilization has several disadvantages, such as poor penetration depth, low emission power, high mercury vapor, and especially long treatment time (Oms-Oliu, Martín-Belloso, & Soliva-Fortuny, 2010). Short treatment time for sterilization efficiency is a very important factor related with productivity and earnings in food industry. Moreover, recently, our research group confirmed that UV-C treatment on seafoods did not show significant effect on irradiation time of 0–600 s (data not shown). Therefore, the development of alternative technology for seafood sterilization is required. Intense pulsed light (IPL) has been highlighted as an innovative nonthermal sterilization technology designed to produce stable and safer food products without inducing damage caused by heating (GómezLópez et al., 2005). The IPL technology decontaminates food surfaces by killing microorganisms using short pulses of intense, broad-spectrum, electromagnetic radiation (Dunn et al., 1995; Gómez-López et al., 2005).

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Various terms have been used to describe this technology, including pulsed ultraviolet (PUV), high-intensity broad-spectrum pulsed light, pulsed light (PL), and pulsed white light (Marquenie et al., 2003; Roberts & Hope, 2003; Rowan et al., 1999; Sharma & Demirci, 2003). The emission spectrum of light irradiation from the xenon flash lamp used for IPL treatment ranges from UV to infrared light. The material to be sterilized is exposed to at least one pulse of light with an energy density typically in the range of 0.01–50 J/cm2 at the surface, and electromagnetic energy is distributed over wavelengths from 170 to 2600 nm (Dunn et al., 1995; Gómez-López et al., 2007). The killing effects of pulsed light are caused by the rich and broad-spectrum UV content, the short duration, and the high peak power of the pulsed light (Dunn et al., 1995). The aim of this study was to evaluate the inactivation effects of IPL on L. monocytogenes inoculated onto a solid medium and onto seafoods such as flatfish, salmon, and shrimp fillets, by varying the fluence per pulse, pulse number, and total fluences, and also to seek the possibilities and the basic standards for the practical application of IPL to seafood products by evaluating the inactivation characteristics of L. monocytogenes following IPL and UV-C treatments. Temperature increase on the surface was measured and color change of the samples was used as quality indicator of the treatment, and any structural damage to the treated cells during and after IPL and UV-C treatments was evaluated by transmission electron microscopy (TEM). 2. Materials and methods 2.1. Microorganism and sample preparation L. monocytogenes KCCM 40307 used in this study was obtained from the Korean Culture Center of Microorganisms (KCCM, Seoul, Korea) and cultured on Tryptone Soya Agar (TSA; Oxoid, Basingstoke, Hampshire, UK). One colony from the agar plate was picked and inoculated into an Erlenmeyer flask containing 25 mL of Tryptone Soya Broth (TSB; Oxoid), and then precultured overnight at 37 °C. A 1 mL aliquot of the precultured fluid was then removed and inoculated to another Erlenmeyer flask containing 100 mL of TSB and cultivated for 9 h at 37 °C. When the cells had grown to the later logarithmic phase, the microorganisms were separated by centrifugation at 8000 ×g for 10 min at 4 °C, washed twice with sterile saline solution (0.85%), and used for analysis. The seafoods used in this study were flatfish (Paralichthys olivaceus), salmon (Salmo salar), and shrimp (Litopenaeus vannamei) fillets. The fillets were purchased from Haejin in Busan, Korea, and stored in a freezer at −18 °C until used. The frozen fillets were thawed at 4 °C for 8 h and cut into 4 cm (length) × 2 cm (width) × 0.4 cm (thickness) pieces before inoculation and IPL treatment.

Controller

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Fig. 1. Schematic diagram of the intense pulsed light (IPL) system.

xenon lamp as measured by a spectroradiometer (ILT-900, International Light Technologies, Peabody, MA, USA).

2.3. IPL and UV-C treatments The inactivation effect of IPL treatment on L. monocytogenes in plates was investigated by spreading 100 μL (1.0 × 106 CFU) of the prepared

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A system to generate pulsed light was designed and manufactured as a lab-scale for laboratory use. The system consisted of a controller, chamber including lamp, and power supply (Fig. 1). The electricity source was able to generate a maximum voltage of 50 kV, with the DC and control circuitry enabling adjustment of the frequency and width of the pulse. The input 220-V AC supply source at 25 A was rectified and transformed to a maximum permissible voltage of 50 kV supplied to a 0.12-μF capacitor via a 6-MΩ series resistor. The energy was stored using resonant charging, and a thyratron was used as the switch for momentary discharge. The quartz lamp used to generate the IPL (XAP Series, Type NL 4006, Heraeus Noblelight, Cambridge, UK), which was filled with xenon at a pressure of 450 Torr, was a 145-mm-long cylindrical lamp with an outer diameter of 7.14 mm that exhibits an emission spectrum (200–1100 nm) ranging from UV-C to infrared light. Fig. 2A shows the instantaneous spectral distribution of the

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sample at the predicted dilution with sterile saline solution (0.85%) onto a TSA plate with a diameter of 90 mm without a cover, sealing the chamber, and then treating the sample with IPL under the following conditions: fluence per pulse, 0.11–1.75 mJ/cm2 per pulse; pulse duration (exponential decaying pulse), 1.5 μs; frequency, 5 Hz (pulses/s); number of pulses, 0–1750 pulses (treatment time: 0–350 s); total fluence (energy received from the lamp by the sample per unit area during the treatment), 0–2.2 J/cm2; and distance between the sample surface and the lamp, 80 mm. Besides, the plates inoculated with L. monocytogenes were irradiated for treatment time of 0–1000 s (total fluences: 0–0.08 J/cm2) with UV-C light (G30T8, Sankyo Denki Co., Kanagawa, Japan) at a distance of 80 mm. The intensity of UV-C light ranged from 200 to 280 nm, with a peak at 254 nm, as measured using a spectroradiometer (Fig. 2B). The sterilization effects of IPL and UV-C treatments on seafoods inoculated with L. monocytogenes were determined by spreading the prepared L. monocytogenes culture (1.0 × 104 CFU/g) onto the surfaces (surface area: 4 × 2 cm, weight: 5.0 ± 0.2 g) of flatfish, salmon, and shrimp fillets. As stated above, one of the goals of our study was to seek the possibilities and the basic standards for the practical and commercial application of IPL sterilization method to seafood products. Thus, in this study, the decision of the inoculation density (1.0 × 104 CFU/g) for seafoods was based on data for microbial contamination levels of seafoods distributed in its markets in Korea. And, in our study, the results on inactivation of L. monocytogenes on a solid medium by IPL were used as (1) comparable data for the decontamination of seafood products by the same system and (2) experimental data for the determination of optimal condition and analysis of operating parameters for the practical application of IPL treatment. However, in our preliminary study, IPL treatment on the same inoculation density (1.0 × 104 CFU/g) in solid medium was difficult to analyze operating parameters and determine optimal condition of IPL because the microorganism in plate was inactivated very quickly. Thus, we determined the initial concentrations (1.0 × 106) of L. monocytogenes in solid medium in consideration of the analyzable microbial density. The inoculated fish fillets were kept in a laminar-flow hood at 16 °C for 30 min to allow the cells to attach. Control samples were inoculated with the microorganisms but not subjected to IPL and UV-C. Fish fillet samples in a sterile Petri dish without a cover (inoculated side facing the top) were placed in the treatment chamber, 80 mm away from the xenon lamp. IPL treatment was conducted for 0–9800 pulses (treatment time: 0–1960 s, total fluences: 0–17.2 J/cm2) at a fluence of 1.75 mJ/cm2 per pulse and frequency of 5 Hz. The temperature of the samples was measured before and during the treatment using a temperature-measuring device (Midi Logger GL200, Graphtec, Yokohama, Japan) with a calibrated thermocouple (type K with response time of 1–2 s) which was located at the surface of the samples. UV-C treatment on the inoculated fish fillets was conducted for 0–1960 s (total fluences: 0–0.16 J/cm2) with UV-C light (G30T8, Sankyo Denki Co.) at a distance of 80 mm. In order to determine the optical penetration of IPL and UV-C into fish fillets, slices of salmon (surface area of 4 × 2 cm, thicknesses of 1.24, 2.05, 3.04, 4.06 and 5.21 mm) and flatfish (surface area of 4 × 2 cm, thicknesses of 1.13, 2.17, 3.17, 4.13 and 5.37 mm) were placed over the detector of spectroradiometer, which was 80 mm away from the lamp face and centered in the IPL or UV-C chamber. IPL doses from 500 pulses (100 s) with a fluence of 1.75 mJ/cm2/pulse (frequency of 5 Hz and total fluence of 0.875 J/cm2) and UV-C doses from treatment time of 1000 s (total fluences of 0.08 J/cm2) were measured for each thickness in triplicate. The results were plotted for observing decay tendency of fluence with thickness of fish fillets (Fig. 7). 2.4. Microbiological analysis The populations of L. monocytogenes on the IPL- and UV-C-treated or untreated fish fillets were counted. Each fillet was placed into sterile

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saline solution and pummeled for 120 s at 230 rpm with a stomacher (Model 400, Seward, Thetford, UK). Washed solution was then serially diluted in sterile saline solution and spiral plated onto Listeria Selective Agar (LSA; Oxoid). After incubating the plates for 48 h at 37 °C, those on which 30–300 colonies had formed were counted, with the results quantified as the number of CFU per gram. Reductions of bacteria were calculated as the number of survivors (N) relative to the initial number of microorganisms (N0) after IPL and UV-C treatments (i.e., log N/N0).

2.5. Color measurements Colorimetric analyses of the IPL-treated and untreated samples were performed with a Hunterlab color difference meter (ColorQuest XE, Hunter Association Laboratory, Reston, VA, USA). Measurements were conducted using reflectance mode of Xenon lamp and the size of view measured area was 6 mm diameter. Samples cut into 6 mm diameter were placed at the reflectance port on the front of the sensor for measurement of reflected color. The color space in this system uses lightness (L) and chromaticity coordinates (a and b). L gives a spectrum ranging from 0 (black) to 100 (white), while −a indicates a green color, +a a red color, −b a blue color, and +b a yellow color. A calibrated white plastic fluorescent standard was used in the UV control and checking process.

2.6. Transmission electron microscopy (TEM) TEM analysis of bacterial cells treated by IPL and UV-C in a solid medium was performed using a previously described method with modifications (Lee et al., 2008). The IPL- and UVC-treated or untreated samples were collected with a sterile glass slide or spreader and 1 mL of 0.1 M phosphate buffer. The cell suspensions were centrifuged immediately at 3000 rpm for 5 min at 4 °C and the supernatant was discarded. The bacterial cells were washed twice with sterile saline solution, and then fixed with 2% paraformaldehyde and 2% glutaraldehyde in 0.05 M sodium cacodylate buffer (pH 7.2). The fixed cells were washed three times with cacodylate buffer, postfixed with 1% osmium tetroxide in cacodylate buffer for 2 h at room temperature, and then washed twice with distilled water and embedded in 0.5% uranyl acetate at 4 °C. Dehydration was performed with an acetone series as follows for 10 min each: 50%, 70%, 80%, 90%, and 100%. The dehydrated samples were treated in a rotor with a graded series of increasing concentrations of resin and acetone until the concentration of the resin was 100%. The polymerization of the resin to form specimen blocks was performed at 70 °C for 16 h. Ultrathin sections were made using a diamond knife in an ultramicrotome (MT-X, RMC, Tucson, AZ, USA). The sections were placed on copper grids and then stained with 2% uranyl acetate for 7 min and 0.4% Reynold's lead citrate for 7 min. They were imaged at different magnifications using an energy-filtering transmission electron microscope (LIBRA 120, Carl Zeiss, Oberkochen, Germany) operating at 120 kV.

2.7. Statistical analysis All experiments were conducted in triplicate and data are expressed as mean ± SD values. Statistical analysis was done by Microsoft Excel 2007 (Microsoft, Redmond, WA, USA) using oneway analysis of variance (ANOVA), with differences considered significant when p b 0.05. The inactivation of L. monocytogenes resulting from IPL treatment was modeled by fitting a modified Weibull model proposed by Albert and Mafart (2005) to the experimental data using GInaFiT tool according to Geeraerd, Valdramidis, and Van Impe (2005).

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3. Results and discussion

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The effect of IPL on L. monocytogenes on a solid medium was evaluated as a function of fluence per pulse (0.11–1.75 mJ/cm2 per pulse) for treatment of 0–1750 pulses (0–350 s) and total fluences (energy received from the lamp by the sample per unit area during the treatment, 0–2.2 J/cm2). As shown in Fig. 3A and B, the viability of the tested microorganisms decreased with increasing pulse number, fluence per pulse, and total fluence. In the early stages of IPL treatment (0–250 pulses), little inactivation was observed at fluence of 0.11 and 0.31 mJ/cm2 per pulse (total fluences of 0.03 and 0.08 J/cm2), while an abrupt inactivation was observed at 1.75 mJ/cm2 (total fluence: 0.44 J/cm2). IPL treatment with 1750 pulses at 0.11 and 0.31 mJ/cm2 per pulse (total fluences of 0.2 and 0.5 J/cm2) produced about 1.7- and 3.8-log reductions in the number of cells, respectively. Cell reductions of 1.5- and 5.8-log were also obtained with 250 and 1750 pulses, respectively, at a fluence of 0.70 mJ/cm2 per pulse. Particularly dramatic reductions in L. monocytogenes on solid medium were observed following IPL treatments at 1.75 mJ/cm2 per pulse, with approximately 4.0- and 6.0-log cell reductions at treatment of 300 and 900 pulses (60 and 180 s, total fluences of 0.5 and 1.6 J/cm2), respectively (Figs. 3A, B, and 4). In this study, the killing effects of IPL are caused by the rich and broad-spectrum UV content, the short duration, and the high peak power of the pulsed light produced by the multiplication of the flash power manifold (Dunn et al., 1995; Gómez-López et al., 2005). And, it has been shown previously that the inactivation of cells by the IPL technology is substantially dependent on the susceptibility of the microorganism (Anderson, Rowan, MacGregor, Fouracre, & Farish, 2000; Rowan et al., 1999). The results presented in Fig. 3A indicate that a representative pathogenic microorganism, L. monocytogenes, is very efficiently inactivated by the IPL process. There have been several reports on the efficiency of IPL treatment for inactivating L. monocytogenes on a solid medium. MacGregor et al. (1998), Paskeviciute, Buchovec, and Luksiene (2011), and Rowan et al. (1999) showed that IPL treatment on L. monocytogenes inoculated onto agar plates resulted in 6.2-, 4.4-, and 6.5-log reductions, respectively. In the present study, the temperature change on the surface of the solid medium including L. monocytogenes was measured during IPL treatments at 0.11–1.75 mJ/cm2 per pulse (Fig. 3C). The highest temperature increase (1.8 °C) was observed at a fluence of 0.70 mJ/cm2 per pulse with 1750 pulses (total fluence: 1.2 J/cm2). As suggested by previous studies, heating of the sample by the lamp is perhaps the most important factor limiting the practical applications of the IPL technology (Elmnasser et al., 2007; Fine & Gervais, 2004; GómezLópez et al., 2007; Hillegas & Demirci, 2003; Jun, Irudayaraj, Demirci, & Geiser, 2003). Therefore, the present results confirm that the treatments affect an overall temperature increase of approximately 1.0–2.0 °C, and that this has no influence on the decontamination effects of IPL. The results presented in Fig. 3 demonstrate that the inactivation of L. monocytogenes significantly increases with the IPL light dose (or fluence per pulse), pulse number, and total fluence. It is well known that the inactivation of microorganisms by IPL is influenced by the light dose, pulse number, wavelength, type of microorganism, type of food, and the distance between the lamp and the sample (Barbosa-Cánovas, Schaffner, Pierson, & Zhang, 2000; Marquenie et al., 2003). However, the most important factor determining the effect of IPL is the light intensity (light dose) to which the sample is exposed as shown in this study, confirming the results of previous studies that have also concluded that the inactivation efficacy of IPL depends on the light intensity and the pulse number (Choi, Cheigh, Jeong, Shin, & Chung, 2010; Farrell, Garvey, Cormican, Faffey, & Rowan, 2010).

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Number of pulses Fig. 3. Inactivation of L. monocytogenes as a function of IPL light intensity (fluence per pulse) and pulse number (A), total fluence (B), and temperature profile on the surface of the solid medium during the treatment (C). □, 0.11 mJ/cm2; ■, 0.31 mJ/cm2; ○, 0.70 mJ/cm2; ●, 1.75 mJ/cm2 per pulse. Experiments were conducted in triplicate. Data are mean and SD values.

3.2. Inactivation of L. monocytogenes on a solid medium using UV-C The inactivation characteristics of L. monocytogenes following UV-C treatment were evaluated for treatment times of 0–1000 s (Fig. 4). In the early stage of UV-C irradiation (0–60 s), little microorganism inactivation was observed, whereas about 4-log reduction of L. monocytogenes

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Treatment time (s) Fig. 4. Inactivation profile of L. monocytogenes on solid medium during IPL treatment at 1.75 mJ/cm2 per pulse (●) and UV-C irradiation at 254 nm (Δ). Experiments were conducted in triplicate. Data are mean and SD values.

was achieved with 1000 s of UV-C treatment. Conventional UV-C treatment has several disadvantages, such as poor penetration depth, low emission power, high mercury vapor, and long exposure time (Oms-Oliu et al., 2010).

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3.3. Transmission electron microscopy (TEM) The IPL- and UVC-induced cell damage incurred by L. monocytogenes was identified using TEM. Fig. 5B (IPL treatment for 150 pulses) and C (900 pulses) shows the distinct structural changes in the L. monocytogenes cells caused by IPL treatment at a fluence of 1.75 mJ/cm2 per pulse. And also, the images indicate that the structural damage of the tested cells increased with increasing pulse number of IPL treatment. Especially, destruction of the cell wall, cytoplasm shrinkage, and leakage of the cellular contents from the cytoplasm were observed at several locations in cells treated with IPL for 900 pulses (180 s) at 1.75 mJ/cm2 per pulse. This cell damage induced by IPL could eventually cause cell death. Meanwhile, the shape of L. monocytogenes cell treated for 1000 s with UV-C (Fig. 5D) was similar to that of untreated control cells (Fig. 5A), except for a blurry and indistinct cell wall. Considerable research has been performed on the mechanism underlying microbial inactivation by IPL. Most researchers consider that the lethal action of IPL can be attributable to a photochemical and/or a photothermal mechanism. They explained their results via a photochemical effect, which involves the formation of pyrimidine dimers in the DNA of the target microorganism (Rowan et al., 1999; Wang, MacGregor, Anderson, & Woolsey, 2005). On the other hand, some authors proposed that a photothermal effect caused by the absorption of UV or pulsed light with excessive energy is responsible for the cell disruption and decontamination by IPL (Hiramoto, 1984; Wekhof, 2000). Furthermore, it is possible that both mechanisms coexist, the relative importance of each depending on the energy density and target microorganism (Takeshita et al., 2003; Wuytack et al., 2003). The results from the present study demonstrate that IPL treatment for 900 pulses (180 s) at 1.75 mJ/cm2 per pulse produces a 6.0-log reduction in L. monocytogenes with a controlled temperature increase during the treatment (Figs. 3 and 4), and the microscopic observations show IPL-induced cell wall damage and leakage of the cellular contents at the same fluence per pulse (Fig. 5C). In order to explain relation between microbial inactivation and cell destruction by IPL, however, further detailed investigation is required.

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Fig. 5. Transmission electron micrographs of L. monocytogenes: (A) untreated, (B) treated for 150 pulses (30 s), (C) treated for 900 pulses (180 s) with IPL at a fluence of 1.75 mJ/cm2 per pulse, and (D) treated for 1000 s with UV-C at 254 nm. Bar corresponds to 200 nm.

3.4. Inactivation of L. monocytogenes on seafoods using IPL The sterilization effects of IPL treatment on seafoods inoculated with L. monocytogenes were investigated at fluences of 0.70 and 1.75 mJ/cm2 per pulse, which were quite effective for inactivation of L. monocytogenes

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on solid medium, and total fluences of 0–17.2 J/cm2. The seafoods used in this study were flatfish, salmon, and shrimp fillets. Microbiological analysis on the prepared fish fillets was performed, and L. monocytogenes was not found in the uninoculated samples (data not shown). As shown in Fig. 6A and C, the overall results demonstrate that the viability of the cells inoculated onto seafoods decreased with pulse number in the following order: shrimp, salmon, and

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flatfish fillets. Approximately 2.0-, 1.8-, and 1.6-log reductions of L. monocytogenes cells inoculated onto shrimp, salmon, and flatfish fillets, respectively, were achieved with IPL treatment for 9800 pulses (1960 s) at 0.70 mJ/cm2 per pulse. And also, IPL treatment at the fluence of 1.75 mJ/cm2 per pulse resulted in 2.2-, 1.9-, and 1.7-log reductions of the cell inoculated onto shrimp, salmon, and flatfish fillets, respectively, for 3600 pulses (720 s, total fluence of 6.3 J/cm2) and 2.4-, 2.1-, and 1.9-log reductions, respectively, for 6900 pulses (1380 s, total fluence of 12.1 J/cm2); the death rate increased exponentially with the pulse number at the early stage of treatment (0–2400 pulses) with similarly shaped inactivation curves. However after 3600 pulses (720 s) at 1.75 mJ/cm2 per pulse (total fluence: 6.3 J/cm2), all curves exhibited pronounced tailing. The inactivation of L. monocytogenes observed in this study was modeled by fitting the experimental data of cell reduction as a function of total fluence (0–17.2 J/cm2) to a log-linear model (based on firstorder kinetics), a Weibull model, and modified Weibull model. Initial analysis indicated that the log-linear model and the Weibull model failed to accurately estimate the inactivation of L. monocytogenes (data not shown), whereas the modified Weibull model showed good fitting performance of the inactivation data (Fig. 6C). The modified Weibull model proposed by Albert and Mafart (2005) is able to describe concave, convex, or linear curves followed by a tailing effect. The model can be written as follows:

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where N0 and Nres are the initial bacterial number and the residual bacterial number, respectively. The δ (J/cm2) parameter represents the fluence for the first decimal reduction, and p is a shape parameter describing convex and concave curves. The root mean sum of squared errors (RMSE) was used as a measure of goodness-of-fit and parameters of the model are shown in Table 1. Consequently, the modified Weibull model adequately describes the inactivation of L. monocytogenes on seafoods by IPL treatment. The temperature change on the surface of the fish fillets inoculated with L. monocytogenes was measured during IPL treatments at 0.70 and 1.75 mJ/cm2 per pulse (Fig. 6B). The initial temperature before IPL treatment was 18.0 ± 0.1 °C on the surface of each fish fillet, and the surface temperature during 6900 pulses of treatment reached 20.6 ± 0.2, 20.4 ± 0.1, and 20.1 ± 0.2 °C on the salmon, shrimp, and flatfish fillets, respectively, at 0.70 mJ/cm2 per pulse (total fluence: 4.8 J/cm2) and 22.9 ± 0.3, 22.6 ± 0.2, and 22.2 ± 0.3 °C, respectively, at 1.75 mJ/cm2 per pulse (total fluence: 12.1 J/cm2). As stated above, because heat from the IPL lamp and light absorption by the food can induce an increase in temperature at the food surface, an important consideration regarding the application of the IPL technology to food is controlling the temperature of the food products. The results indicate that the IPL-induced Table 1 Goodness-of-fit and kinetics parameters of the modified Weibull model for L. monocytogenes inactivation on seafoods by IPL treatment.

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12

16

Fluence (J/cm2) Fig. 6. Inactivation of L. monocytogenes on seafoods as a function of pulse number at a 0.70 (open symbols) and 1.75 mJ/cm2 per pulse (closed symbols) (A), the temperature profile on the surface of the fish fillets during the treatment (B), and fit of a modified Weibull model to IPL inactivation data as a function of total fluence (C). ○, ●, shrimp fillets; △, ▲, salmon fillets; □, ■, flatfish fillets. Experiments were conducted in triplicate. Data are mean and SD values.

RMSE R2b Log10 N0c δd pe Log10 Nresf a b c d e f

Seafoods Salmon fillet

Flatfish fillet

Shrimp fillet

0.0777 0.9924 4.03 ± 1.87 ± 0.73 ± 1.95 ±

0.0733 0.9902 4.01 ± 1.33 ± 0.54 ± 2.13 ±

0.0886 0.9907 4.03 ± 1.58 ± 0.74 ± 1.66 ±

0.08 0.23 0.09 0.05

0.07 0.22 0.08 0.05

RMSE: root mean sum of squared errors. R2: coefficient of determination. Log10 N0: predicted logarithm of initial microbial cell density (CFU/g). δ: fluence for the first decimal reduction (J/cm2). p: shape parameter describing convex and concave curves. Log10 Nres: predicted logarithm of residual microbial cell density (CFU/g).

0.10 0.24 0.10 0.06

C.-I. Cheigh et al. / Food Research International 54 (2013) 745–752

Table 2 Colorimetric analyses of lightness (L) and chromaticity coordinates (a and b) for IPLtreated and untreated salmon, flatfish, and shrimp fillets. Seafood typea

Parameterb L

a

b

Salmon fillet Untreated Treated

59.18 ± 2.73 58.45 ± 3.82

28.97 ± 1.96 29.23 ± 1.81

30.15 ± 1.51 31.71 ± 1.74

Flatfish fillet Untreated Treated

84.52 ± 4.52 83.76 ± 3.27

1.15 ± 0.19 1.02 ± 0.21

3.87 ± 0.21 3.95 ± 0.55

Shrimp fillet Untreated Treated

56.93 ± 3.16 55.35 ± 2.88

26.73 ± 1.55 25.81 ± 1.43

28.39 ± 2.12 27.64 ± 1.79

a

The sterilization effects of UV-C (254 nm) treatment on seafoods inoculated with L. monocytogenes were investigated. However, UV-C treatment on the inoculated fish fillets did not show a significant sterilizing effect on irradiation time of 0–1960 s (total fluences: 0–0.16 J/cm2) (data not shown). This result might be attributable to the poor penetration depth and low emission power of UV-C treatment (Oms-Oliu et al., 2010). 3.6. Optical penetration depth As shown in Fig. 7A, IPL doses inside the fish fillets were decayed exponentially with the thickness and the optical penetration depths (defined as the depth at which the fluence decreases to 1/e or 37% of its initial value) of salmon and flatfish were estimated to be 3.1 and 3.6 mm, respectively (Uesugi & Moraru, 2009). And, the optical penetration of UV-C through salmon and flatfish was estimated to be 0.7 and 0.9 mm, respectively (Fig. 7B). This factor could be considered to determine an appropriate light dose for effective decontamination of opaque solid foods such as seafood and meat products. 4. Conclusion There has been insufficient investigation of the practical application of IPL to seafood sterilization, although IPL treatment for foods was

0.90

(A)

Fluence (J/cm2)

0.75 0.60 0.45 0.30 0.15 0 0

1

2

3

4

5

0.08

6

(B)

0.06

0.04

0.02

0 0

Each replication consisted of three samples with two readings per sample. L represents the lightness, −a indicates a green color, +a indicates a red color, −b indicates a blue color, and +b indicates a yellow color. b

3.5. Inactivation of L. monocytogenes on seafoods using UV-C

Fluence (J/cm2)

increases in temperature on the fish fillets during the treatments are well controlled within the range of 2.1–4.9 °C, and that these small temperature changes have no influence on the IPL-induced decontamination of the seafood samples (Fig. 6B). To determine whether IPL treatment had any negative effects on the color of the seafoods, as a quality indicator, the IPL-treated and untreated samples were analyzed immediately after 6900 pulses at 1.75 mJ/cm2 per pulse (Table 2). As mentioned above, the highest L. monocytogenes inactivation efficiency was observed on the shrimp fillets (Fig. 6A and C) and the temperature increase was highest for the salmon fillets (Fig. 6B). However, as shown in Table 2, the present results indicate that none of these values differ significantly between the treated and untreated fish fillets, and consequently it can be concluded that the IPL treatment causes no observable change to color of the tested seafoods. The findings of the present study demonstrate that the decontamination of seafoods such as shrimp, salmon, and flatfish fillets is quite positive, but is less than that on a solid medium (Figs. 3 and 6). This might be attributable to the opaqueness, surface properties, and composition of the seafoods (Elmnasser et al., 2007; Gómez-López et al., 2007). In general, the efficiency of IPL treatment depends upon microbial exposure, and food products including seafoods have opaque, rough, and irregular surfaces. Since pathogenic microorganisms can penetrate the surfaces upon which they lie via crevices or pores present on the food surface, seafoods with opaque and irregular surface properties can only be decontaminated externally due to the shadowing effect (Elmnasser et al., 2007; Lagunas-Solar, Piña, MacDonald, & Bolkan, 2006; Marquenie et al., 2003; Ozer & Demirci, 2006). Besides, as shown in Fig. 6A and C, a significant reduction of L. monocytogenes counts exhibited in shrimp fillets in particular among the tested seafoods might also be due to their surface properties. The shrimp fillets used in this study had a fairly even and smooth surface compared to the salmon and flatfish fillets, and therefore the shadowing effects that occur during IPL treatment could have been considerably lower for the shrimps. The practical application of IPL clearly merits fullest consideration in the point of view of food stability and shelf-life extension. Oms-Oliu et al. (2010) reported that a 0.7-log reduction of yeast cell in fresh-cut mushrooms by IPL treatment could prolong the shelf-life of the mushrooms by 3–4 days. Nevertheless, for practical applications of IPL, further research should be conducted to overcome shadowing effect that affects the decontamination of food products. All the technology using light is limited by the shadowing effect. Therefore, specialized engineering solutions, such as the use of relatively high peak powers, the optimization of pulse width and frequency, the optimization of operating conditions, and the use of treatment chamber designed differently for type and characteristic of foods, will help to overcome the shadowing effect.

751

1

2

3

4

5

6

Thickness (mm) Fig. 7. Optical penetrations of IPL (A) and UV-C (B) through slices of salmon (○) and flatfish (□) fillets. Fluences of IPL and UV-C at thickness 0 mm (no fish fillets) were 0.875 and 0.08 J/cm2, respectively. Experiments were conducted in triplicate.

752

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approved by the USFDA under code 21CFR 179.41 in 1996. The consideration for the use of the IPL system was originally for finding an alternative method to the UV system, so it was necessary to compare the abilities of microbial inactivation of both systems. The results of the present study show that the IPL treatment inactivated L. monocytogenes more rapidly and effectively than did conventional UV-C treatment. These inactivation effects of IPL might be attributable to the comparatively higher penetration depth and emission power than conventional UV-C. IPL has a high peak power produced by the multiplication of the flash power manifold, resulting in the light intensity of at least 100 times than that of conventional UV light during the same operating time. Short treatment time for sterilization efficiency is a very important factor related with productivity and earnings in food industry. The findings presented herein suggest a lot of potential for the commercial application of IPL for the decontamination of seafood products. However, for the commercial applicability of IPL, further studies are required to overcome limiting factors that affect the decontamination of seafood products, such as the shadow effect, and evaluate its applicability for the sterilization of representative food pathogens in various food products. Acknowledgment This research was supported by a grant (10162KFDA995) from Korea Food & Drug Administration in 2012. References Albert, I., & Mafart, P. (2005). A modified Weibull model for bacterial inactivation. International Journal of Food Microbiology, 100, 197–211. Anderson, J. G., Rowan, N. J., MacGregor, S. J., Fouracre, R. A., & Farish, O. (2000). Inactivation of food-borne enteropathogenic bacteria and spoilage fungi using pulsed-light. IEEE Transactions on Plasma Science, 28, 83–88. Barbosa-Cánovas, G. V., Schaffner, D., Pierson, M.D., & Zhang, H. Q. (2000). Pulsed light technology. Journal of Food Safety, 65, 82–85 (Suppl.). Ben Embarek, P. K. (1994). Presence, detection and growth of Listeria monocytogenes in seafood: A review. International Journal of Food Microbiology, 23, 17–34. Butt, A. A., Aldridge, K. E., & Sanders, C. V. (2004). Infections related to the ingestion of seafood part I: Viral and bacterial infections. The Lancet Infectious Diseases, 4, 201–212. Choi, M. S., Cheigh, C. I., Jeong, E. A., Shin, J. K., & Chung, M. S. (2010). Nonthermal sterilization of Listeria monocytogenes in infant foods by intense pulsed light treatment. Journal of Food Engineering, 97, 504–509. Dunn, J., Ott, T., & Clark, W. (1995). Pulsed-light treatment of food and packaging. Food Technology, 49, 95–98. Elmnasser, N., Guillou, S., Leroi, F., Orange, N., Bakhrouf, A., & Federighi, M. (2007). Pulsed-light system as a novel food decontamination technology: A review. Canadian Journal of Microbiology, 53, 813–821. Farrell, H. P., Garvey, M., Cormican, M., Faffey, J. G., & Rowan, N. J. (2010). Investigation of critical inter-related affecting the efficacy of pulsed light for inactivating clinically relevant bacterial pathogens. Journal of Applied Microbiology, 108, 1494–1508. Fine, F., & Gervais, P. (2004). Efficiency of pulsed UV light for microbial decontamination of food powders. Journal of Food Protection, 67, 787–792. Geeraerd, A. H., Valdramidis, V. P., & Van Impe, J. F. (2005). GInaFiT, a freeware tool to assess non-log-linear microbial survivor curves. International Journal of Food Microbiology, 102, 95–105.

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