Non-thermal atmospheric gas plasma device for surface decontamination of shell eggs

Journal of Food Engineering 100 (2010) 125–132

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Non-thermal atmospheric gas plasma device for surface decontamination of shell eggs Luigi Ragni a, Annachiara Berardinelli a,*, Lucia Vannini b, Chiara Montanari b, Federico Sirri b, Maria Elisabetta Guerzoni b, Adriano Guarnieri a a b

Agricultural Economics and Engineering Department, University of Bologna, Piazza G. Goidanich, 60, 47023 Cesena (FC), Italy Food Science Department, University of Bologna, Viale Fanin, 46, 40127 Bologna, Italy

a r t i c l e

i n f o

Article history: Received 6 October 2009 Received in revised form 19 March 2010 Accepted 24 March 2010 Available online 1 April 2010 Keywords: Gas plasma Non-thermal decontamination Shell eggs Salmonella

a b s t r a c t A resistive barrier discharge (RBD) prototype able to generate gas plasma at atmospheric conditions was set up. The discharge was electrically characterized and the plasma glow was analysed by optical emission spectroscopy. The decontamination power of the device was assessed on samples of shell eggs experimentally inoculated with Salmonella Enteritidis and Salmonella Typhimurium (5.5–6.5 Log CFU/eggshell) and placed in the treatment chamber. Different decontamination times (10, 20, 30, 45, 60 and 90 min) and relative humidity values (RH) of the gas mixture in the chamber (i.e. 35% and 65%, at 25 °C) were considered. All samples were treated in the plasma after-glow chamber where the measured temperature was not much higher than the room temperature, minimizing the risk of egg quality alterations. The discharge was characterized by a potential difference of about 15 kV; the emission spectra showed the presence of very reactive species such as the positive ion Nþ 2 and OH and NO radicals. After 90 min of treatment, reductions up to 2.5 Log CFU/eggshell and 4.5 Log CFU/eggshell were observed for S. enteritidis using air with low and high moisture contents, respectively. No significant negative effects of the gas plasma were observed on the egg quality traits. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Gas plasma is an ionized gas containing free electrons and neutral reactive species such as atoms, molecules and radicals (Moisan et al., 2001). It is usually produced by exposing a gas (or a mixture of gases) to an electric field that accelerates the charged particles (electrons) producing collisions with the heavy species. Ions and reactive species, when they are in an excited state, can lose their internal energy through collisions with other particles or surfaces or by emitting photons in the UV range. These factors, separately or in synergistic combination, are the main ones responsible for the germicidal effect of the gas plasma (Laroussi and Leipold, 2004). Discharges at atmospheric pressure and at low temperature make the decontamination process practical, inexpensive and suitable for applications when product preservation is desired. Well known applications concern the healthcare area, re-usable and heat sensitive medical tools, including surface modification for improving wettability and adhesion of polymers (Baier et al., 1992; Dumitrascu et al., 2005). * Corresponding author. Fax: +39 0547 382348. E-mail address: [email protected] (A. Berardinelli). 0260-8774/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.jfoodeng.2010.03.036

Actually, non-thermal plasmas can be generated by means of microwave, RF (radio frequency), pulsed, AC (alternating current) or DC (direct current) power sources, and several device geometries, electrode configurations, gas mixtures and flow rates have been studied (Laroussi, 2002). Methods that have been researched for their routine use in various industrial plasma processing applications (lighting, surface modification, etching, deposition) are the dielectric barrier discharge (DBD), the resistive barrier discharge (RBD) and the atmospheric pressure plasma jet (APPJ). A DBD device consists of two conductive electrodes covered by a layer of dielectric materials such as alumina (Laroussi and Leipold, 2004), where the latter has a resistivity >1012 X m. The DBD discharge devices usually operate at frequencies between 50 Hz and 500 kHz (Moreau et al., 2008). In the RBD systems one or both electrodes are covered by a high resistive material that prevents arcing, and the apparatus can be driven by DC or AC power supplies (Laroussi et al., 2002). APPJ consists of a capacitive device shaped as a coaxial line where the working gas flows and reacts between the inner and outer conductors. The outer electrode is grounded and the inner one is excited by the RF field (Scutze et al., 1998; Herrmann et al., 1999). In other devices the gas excitation is obtained by the exposure to guided microwaves (Lee et al., 2005). These systems

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can generate relatively large volumes of non-equilibrium, low temperatures plasmas at or near atmospheric pressure (the plasmas produced by these devices typically have electron densities in the 109–1011 cm3 range and plasma power densities in the 10–300 mW/cm3). The electrical and chemical emissions of the plasma devices can be measured by set-upping appropriate measurement chains. The electric field in the time domain is often measured by means of high voltage and current probes connected to an oscilloscope (Laroussi et al., 2003; Lu et al., 2003; Machala et al., 2007). The reactive species can be identified by means of optical emission spectroscopy, mass spectroscopy and gas detection (Laroussi et al., 2003; Lu et al., 2003; Kieft et al., 2004; Dumitrascu et al., 2005; Laroussi and Leipold, 2004; Shimizu and Oda, 2001). Electrical and optical measurements can be used to calculate the electron number density of the plasma (Laroussi et al., 2003). Emission spectra are also useful to determine the rotational and vibrational temperatures of plasma (Machala et al., 2007). Several mechanisms can be regarded as responsible for microbial inactivation. Exposure to UV can cause DNA modifications and consequent improper cell replications. Charged particles play an important role in membrane rupture due to electrostatic forces. Oxidation of membranes (particularly if rich in lipids) and amino acids, and respiration inhibition can be due to reactive oxygen and nitrogen species, such as atomic oxygen (O), ozone (O3), hydroxyl (OH), NO, and NO2. The contribution of each of the above mentioned mechanisms depends on plasma characteristics (voltage, working gas, water content in the gas, distance of the microorganism from the discharge glow, etc.) and to the type of microorganisms (Gram-positive, Gram-negative, spores, etc.) (Mendis et al., 2000; Moisan et al., 2002; Laroussi and Leipold, 2004; Laroussi et al., 2003). Extensive research on the use of cold plasmas to inactivate microorganisms is a relatively recent event and further issues must be investigated such as the effects of plasma on the biochemical support of bacteria. Several researches have also been carried out on the application of cold plasma in the food industry. Scientific contributions concern packaging sterilization (Laroussi, 2002; Deilmann et al., 2008), decontamination of grain and legumes infected by fungi (Selcuk et al., 2008), almond infected by Escherichia coli (Deng et al., 2005), inactivation of pathogens in poultry wash water (Rowan et al., 2007), decontamination of the fruit pericarps from spoilage and pathogenic microorganisms (Perni et al., 2008) and vegetables (Critzer et al., 2007). Gas plasma could represent a good opportunity for the decontamination of foods as an alternative method for those products that cannot be sanitized with conventional means. In particular, shell eggs cannot be washed, or cleaned by any other means, before or after grading in the European Economic Union (EC, 2003). Microorganisms, such as Salmonella enteritidis, represent a potential hazard for egg consumers and alternative methods for decontaminations have to be investigated (Davies and Breslin, 2003). Several alternative approaches for the superficial decontamination of shell eggs have been proposed, including pulsed light technology (Hierro et al., 2009) and heat treatments, i.e. hot air, hot water, infra-red radiation, and atmospheric steam (James et al., 2002; Pasquali et al., 2010), in addition to slightly acidic electrolyzed water (Cao et al., 2009) or ozone and UV radiation (Rodriguez-Romo and Yousef, 2005; Fuhrmann et al., 2010). The present research describes and analyses a prototype based on RBD plasma generation. The surface decontamination by the RBD apparatus of shell eggs deliberately contaminated with S. Enteritidis and Salmonella Typhimurium is also assessed and discussed along with the potential side effects on egg quality traits.

2. Materials and methods 2.1. Plasma source and characterization The prototype set-up for this research works with air at atmospheric pressure and the product to be treated is located in an after-glow position. The glow discharge is generated between three pairs of parallel plate electrodes made of brass. One of the two electrodes is covered by a 5 mm thick glass sheet. The voltage at the electrodes is produced by three high voltage transformers and power switching transistors. The electrodes are confined in a hermetic chamber, with a volume of about 70 dm3, housing the product to be processed. Three fans whose main task is to speedily carry the generated plasma species towards the product are mounted over the electrodes. Each circuit driving a pair of electrodes is supplied by a DC power supply whose voltage can be varied from 2 to 19 V. Fig. 1 shows the electronics and the electrodes of the prototype. Plasma electrical emission was characterized by measuring the voltage at the electrodes in the time domain, using a high voltage probe (Tektronix, P6015A) and a digitizing oscilloscope (GW Instek, GDS820C). The voltage was analysed both in the time and frequency dominium. Optical emission spectra were acquired from 200 to 1100 nm by means of an optic fibre probe (Avantes, FCUV400-2) placed at about 20 mm from the discharge and connected to a spectrometer (Avantes, AvaSpec-2048, resolution of 2.4 nm). The effects of two different levels of the relative humidity (%) in the treatment chamber (RH = 35% and 65%, at 25 °C) on the irradiance of an important reactive species (OH) were analysed. A layout of the instrumental chain is shown in Fig. 2. 2.2. Microbiological procedure 2.2.1. Choice of the microorganism, bacterial culture and inoculum preparation In order to verify the effectiveness of the prototype for the superficial decontamination of table eggs, a target pathogen has been deliberately inoculated onto the surface of the eggs. The choice of the pathogen to be used in this work was based on the fact that Salmonella has long been recognized as an important zoonotic pathogen of economic significance in animals and humans. Although campylobacteriosis continued to be the most commonly reported gastrointestinal bacterial pathogen in humans in the

Fig. 1. RBD prototype cabinet and electronics. In the insets: details of one pair of electrodes during discharge and the egg position under the electrodes during the treatment.

L. Ragni et al. / Journal of Food Engineering 100 (2010) 125–132

Spectrometer

PC

Fibre optic probe 20 mm Digitizing oscilloscope

High voltageprobe

Fig. 2. Layout of the instrumental chain for electric and composition plasma characterization.

European Union in 2008, the number of campylobacteriosis cases in Italy was significantly lower than Salmonella (EFSA, 2010). S. enteritidis MB2509, which is a streptomycin-resistant strain, and S. typhimurium T5 were employed for this experimental work. The strains were cultured in Brain Heart Infusion (Oxoid, Basingstoke, UK) (containing 25 ppm of streptomycin only for S. enteritidis) at 37 °C for 24 h; an aliquot of the grown culture (0.1% inoculum) was subsequently transferred into 150 ml of BHI (with added 25 ppm of streptomycin only for S. enteritidis) and incubated at 37 °C for 24 h with mild agitation. Cells were harvested by centrifugation, washed twice with sterile saline solution (0.9% NaCl, w/v), and re-suspended in saline solution (8.5  108 CFU/ml) before being used for the superficial contamination of shell eggs. 2.2.2. Inoculation of shell eggs Two hundred ninety-four fresh, unfertilized eggs (mass of 68.1 ± 4.2 g) were obtained from hens reared in conventional cages from a local poultry farm (Eurovo, Imola, Italy). The efficacy of the RBD prototype was assessed on eggs experimentally contaminated with S. enteritidis and S. typhimurium. Each egg was washed with sterile distilled deionised water (22–25 °C), and then sanitized by dipping them in ethanol (70%, vol/vol) for 30 min as described by Hammack et al. (1993). Sanitized shell eggs were transferred to metallic grids and aseptically dried at room temperature for approximately 40 min before inoculation. Dried, sanitized shell eggs were dipped for 10 s into the Salmonella saline suspension (20 °C) prepared as previously described, and then air-dried for approximately 1 h before being exposed to the gas plasma. This procedure consistently provided microbial concentrations on the shell at the level ranging between 5.5 and 6.5 Log CFU/eggshell. 2.2.3. Gas-plasma treatments The efficacy of the RBD prototype for superficial decontamination of deliberately contaminated eggs was evaluated by exposing shell eggs to gas plasma for different times: 0, 10, 20, 30, 45, 60 and 90 min. The relative humidity (RH) of the treatment chamber was also modulated by taking into consideration two different RH values, 35% and 65%. For each experimental condition (treatment time and RH value) seven eggs were subjected to the gas-plasma treatments. 2.2.4. Enumeration of viable cells Following each treatment, shell eggs were transferred into a sterile sampling bag (International PBI S.p.A., Milan, Italy) and

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100 ml of sterile saline solution was added. Shell eggs were then hand-rubbed through the bag for 3 min to detach the bacteria according to De Reu et al. (2006) and a 1-ml aliquot was used to prepare decimal serial dilutions. According to three independent preliminary experiments, made on 15 eggs each to evaluate the repeatability of the method used to recover cells from the eggshell, the coefficients of variation were lower than 7.3%. Enumeration of the surviving cells was done by surface plating, in triplicate, 100 ll of the appropriate dilutions onto non-selective Triyptic Soy Agar (TSA) and selective Brilliant Green Agar (BGA) (Oxoid) plates which were incubated at 37 °C for 48 h. Up to five presumptive Salmonella colonies were confirmed by API 20E biochemical testing and by streaking samples onto TSA (containing 25 ppm of streptomycin only for S. enteritidis) and xylose lysine desoxycholate (XLD) agar. After incubation at 37 °C, the growth ability in the presence of the antibiotic was checked and characteristics colony morphology of Salmonella onto XLD was observed. Differences between means at different treatment times and enumeration medium were found by ANOVA (Analysis of Variance) (SPPS, version 15.0 for Windows) at p-level value <0.05. 2.3. Assessment of egg quality traits In order to assess possible negative effects of the gas plasma on egg quality traits, after treatments of 90 min at RH either of 35% or 65% and 25 °C, the cuticle presence and the albumen pH (immediately after treatment), the weight loss (as an indirect method to evaluate the shell membrane integrity) and the yolk index (after 28 days of storage at 25 °C), were measured on batches of treated and untreated eggs (50 eggs/group). Moreover the cuticle presence and shell membranes integrity were deeper investigated by scanning electron microscopy (SEM) technique (Hitachi 501B, magnification 300  500, 15 kV) on treated (90 min at RH = 35%, and 25 °C) and untreated eggs (15 eggs/group). According to Board and Halls (1973) the cuticle presence was measured by dipping the treated and untreated eggs into a green solution containing Tartrazine and Green S (Cuticle Blu; MS Technologies Ltd., Northants, UK). Each egg was immersed in the dye solution for 1 min and then rinsed with tap water and degree of uptake of the dye was then quantified by means of a reflectance colorimeter (Minolta Chroma Meter CR-400, Minolta Italia S.p.A., Milan, Italy); the CIE system color profile of lightness (L*), redness (a*) and yellowness (b*) were considered (1978) (a dark green color indicates the presence of cuticle on eggshell). The albumen pH was measured by a pH meter (CyberScan 510 pH – Eutech Instruments) on thick and liquid portions of albumen. The yolk index, related to the migration of the water through the vitelline membrane as a consequence of ageing, was calculated as the height of the yolk divided by the width of the yolk measured by using a decimal digital calliper (Funk, 1948). This index is related to the quality of the vitelline membrane (Fromm, 1966). For SEM analyses, a 1 cm2 wide strip of eggshell was carefully removed and subdivided into two smaller sections for the assessment of the cuticle and the inner surface of inner shell membrane. All samples were mounted on aluminium stubs using double sided carbon discs or double sided sticky tape, left to dry at room temperature and then coated with gold palladium before being examined. The samples were scored according to four scoring categories for cuticle (1, normal; 2, occasionally patchy; 3, very patchy; 4, little or not cuticle) and both for the inner surface of inner shell membrane (1, normal; 2, membranes fibres occasionally clumped; 3, membranes fibres moderate clumped; 4, membranes fibres extensively clumped). Differences between means at different treatment times were also found by ANOVA at p-level value <0.05.

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process (Kuzmichev et al., 2001). The increase of OH radical irradiance in the emission spectrum of humid (RH = 65%) with respect to the dry (RH = 35%) air plasma is shown in Fig. 7. This increase is attributed to the fact that the transitions of the first electronically

3.1. Plasma characterization

15 14 13 12 11

y = 0.6369x + 2.6905 2

R = 0.9929

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13

14

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N2 C-B (0-0)

50 N2 C-B (1-0)

40

N2 C-B (0-1)

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0 -2 0

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0

200

200

-4

250

300 350 Wavelength (nm)

-10

-6

Time (μs)

-10

19

N2 second positive system

60

10

-8

Fig. 3. Waveform of the voltage at the electrodes of the RBD prototype.

5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0

400

450

Fig. 6. Emission spectra of the plasma glow with a discharge voltage at electrodes of about 15 kV (RH = 35% at 25 °C). N2, 2nd positive system (transition between C3Pu and B3Pg electronic states); Nþ 2 , 1st negative system (transition between 2 þ 2 + B2 Rþ u  X Rg electronic states); OH and NO c systems (transition between A R and 2 X P electronic states). Values in brackets refer to vibrational transition (v0 ? v00 ).

2

RH = 35%

RH = 65%

1.8 2

Irradiance (μW/cm )

Voltage magnitude (kV)

17

Fig. 5. Voltage at the electrodes as function of the voltage at the power supply.

2

Voltage at the electrodes (kV)

10

16

Supply voltage (V)

Irradiance (μW/cm )

The results of the time and frequency domain analyses of the voltage at the electrodes with the device supplied with the maximum voltage (19 V) are shown in Figs. 3 and 4, respectively. From Fig. 3 we can see a peak-to-peak voltage of about 15 kV; the frequency spectrum (Fig. 4) is characterized by the fundamental frequency at 12.7 kHz and by others components at 39.1, 64.6 and 91.0 kHz. The voltage at the electrodes is quasi-linearly correlated to the voltage of the power supply (Fig. 5): with an input voltage of 19 V (near the limit of the prototype), the measured voltage at the electrodes (15 kV) was about 42% higher than the voltage measured with an input of 12 V. As expected with atmospheric air non-equilibrium discharges, N2 peaks were dominant in the emission spectra (Fig. 6) where the bands of neutral nitrogen molecules N2 namely the second positive system (k = 290–440 nm, corresponding to the transitions between C3Pu–B3Pg electronic states), and the emission band of a positive ion Nþ 2 , at k  391.4 nm, were observed. The presence of the positive ion Nþ 2 indicates the formation of N2 long-lived metastable states that are reservoirs of energy promoting plasma chemical reactions. Emission of OH, at k = 305–309 nm and at k = 280–285 nm, and NO c systems, at k = 226–248 nm, were also observed in air plasma corresponding to transitions between the first excited doublet A2R+ and the ground X2P states. As a consequence of their strong reactivity, in atmospheric pressure plasmas OH and NO radicals are greatly involved in the decontamination

Peak-to peak voltage at the electrodes (kV)

3. Results and discussion

1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0

0

50

100

150

200

302

303

304

305

306

307

308

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Wavelength (nm)

Frequency (kHz) Fig. 4. FFT analysis of the voltage at the electrodes of the RBD prototype.

Fig. 7. OH radical emission of the plasma glow with dry (RH = 35%) and humid (RH = 65%) air (at 25 °C; voltage at electrodes of about 15 kV).

L. Ragni et al. / Journal of Food Engineering 100 (2010) 125–132

excited OH radical to its ground state is dependent on the concentration of water within the gas mixture (Laroussi and Leipold, 2004). OH radicals mainly result from the direct dissociation of water molecules by electron impact and the emission at k = 305– 309 nm is a function of the concentration of water vapour (Shin et al., 2000). As expected no differences were observed for NO radicals between spectra acquired at the considered RH conditions. The emission irradiance values increase when the voltage at the electrodes increases: about 7–8-fold passing from 10 to 15 kV; Fig. 8 shows OH emission values measured (at 305.7 nm) for humid (RH = 65%) and dry (RH = 35%) air plasma at different voltage levels at the electrodes. 3.2. Microbiological analysis Data concerning the cell loads of S. Enteritidis immediately after gas-plasma treatments in relation to the exposure time and the relative humidity are reported in Table 1. Results obtained with the two media showed that the mean of counts on the selective (BGA) media in most cases were lower than those obtained with the non-selective one (TSA) regardless the treatment time and RH. However, no significant differences (p > 0.05) were found between means of counts on BGA and TSA except for data on the enumeration after 10 min of exposure at RH = 35% and for the untreated eggs at RH = 65%. The exposure of contaminated eggs to gas plasma for 10–20 min resulted in significant cell load decreases of about 1.0–1.6 Log CFU/ eggshell with respect to the untreated controls. For these treat-

(RH = 35%) OH A-X (R.H.=35%) (RH ==65%) 65%) OH A-X (R.H.

2

Irradiance ( μW/cm )

0.08 0.06 0.04 0.02 0.00 10

11

12 13 14 Voltage at the elecrodes (kV)

15

Fig. 8. Emission irradiance values (OH A-X) as function of the voltage at electrodes (RH = 35% and RH = 65% at 25 °C).

Table 1 Viable cell counts (Log CFU/eggshell) of Salmonella Enteritidis MB2509 following increasing exposure times to gas-plasma afterglow in relation to the relative humidity (RH). Counts were evaluated onto BGA and TSA agar plates. Treatment time (minutes) 0 10 20 30 45 60 90

Viable cell counts (Log CFU/eggshell) RH = 35%

RH = 65%

BGA

TSA

BGA

TSA

6.12a,A (0.24) 4.55b,A (0.26) 4.51b,A (0.36) 5.34c,A (0.39) 4.74b,A (0.41) 3.95d,A (0.45) 3.62d,A (0.64)

6.76a,A (0.60) 5.73b,B (0.86) 5.15b,c,d,A (0.64) 5.10b,c,d,A (0.78) 5.34b,c,A (0.88) 4.53c,d,A (0.74) 4.27d,A (0.67)

5.97a,A (0.19) 4.63b,A (0.40) 4.45b,A (1.00) 3.83b,c,A (1.42) 3.14c,d,A (0.54) 2.52d,e,A (0.38) 2.18e,A (0.15)

6.26a,B (0.21) 4.97b,A (0.18) 4.65b,c,A (0.91) 4.04c,A (1.02) 3.85c,A (0.75) 2.93d,A (0.47) 1.74e,A (0.69)

BGA = brilliant green agar; TSA = tryptone soy agar. Differences between means with the same exponent letter are not significant at p-level value <0.05 (lower case letters indicate differences between means at different treatment times within the same RH and medium; capital letters indicate differences between means evaluated with different media within the same treatment time and RH). Values in parentheses are standard deviations.

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ment times, the decontamination power of the prototype did not seem affected by the RH of the atmosphere. The efficacy of the gas plasma generator increased by increasing the treatment time showing a quasi linear trend. A maximum reduction of 2.2–2.5 Log CFU/eggshell in S. Enteritidis levels were observed following 60–90 min of treatment at 35% RH, regardless of the medium. When the RH was 65%, the effectiveness of the treatments resulted to be enhanced: maximum declines of 3.8 and 4.5 Log CFU/eggshell were achieved after 90 min when BGA and TSA media were used, respectively. Similar to S. Enteritidis, also S. Typhimurium showed a higher sensitivity to gas plasma when the treatments were performed at 65% RH: shell surfaces contaminated by S. Typhimurium were significantly reduced by 3.5 Log CFU/eggshell when treated for 90 min (Fig. 9). On the other hand, different inactivation dynamics were observed for the two microorganisms and S. Enteritidis resulted to be more susceptible to the gas plasma. The comparison of the inactivation dynamics obtained with the two RH values indicates a higher sensitiveness of Salmonella when exposed to the gas plasma in the presence of humid atmosphere. The enhancing effect of increased RH on the efficiency of the treatments can be explained by the presence of oxygen species as detected in the discharge emission spectra. It is well known that OH radicals easily interact with microbial cells giving rise to oxidizing reactions. In particular, active oxygen molecules have been shown to cause damages to DNA, RNA, protein and lipids (Farr and Kogoma, 1991). On the other hand, Moreau et al. (2008) reported that plasma has very similar effects to pulsed electric fields, inducing perforations in the membranes of microorganisms. Sato et al. (1996) identified OH and H2O2 as the chemical species that are created in water under the action of a high voltage electric field, and these species are the same as those formed in humid air plasma. Cho et al. (2004) demonstrated an excellent linear correlation between the OH radicals and the rates of E. coli inactivation in photochemical sterilization, which indicates that the OH radical is the primary oxidant species responsible for microbial inactivation. The humidity level is always a critical factor. Muranyi et al. (2008) observed an improvement of A. niger inactivation with increasing relative humidity up to 70%. On the other hand the opposite effect of humidity observed for B. subtilis spores has been attributed to their high resistance to oxidative agents. An increased amount of water vapour (>80%) reduces the sterilization effect because of the poorer transmissibility of UV radiation in such air and protective water film around the microbial cells. Although microbiological results showed that counts in BGA were lower than the ones obtained with TSA, no significant (p > 0.05) or moderate differences were found between the data obtained with the selective and non-selective media. Several studies have been done to improve techniques for recovering sublethally injured Salmonella, E. coli, Listeria monocytogenes and Klebsiella pneumonia (Hara-Kudo et al., 1999; Kang and Fung, 2000; Restaino et al., 2001). In fact, selective agents such as bile salts or antibiotics, which are added to media in order to detect bacteria in foods, may inhibit repair of injured cells or even kill them thus leading to underestimation of viable cells (Gurtler and Kornacki, 2009; Gurtler, 2009; Chambliss et al., 2006; Kang and Fung, 2000). Ponce et al. (1999) reported important differences between counts made in a selective or non-selective media of S. enteritidis in whole liquid eggs following high hydrostatic pressure– temperature–time combinations. However, such differences tended to be less pronounced when a medium with lower selectivity was used (i.e. BGA instead of SS) and when a specific temperature condition was used, thus suggesting that may be related also to the specific process conditions adopted and the level of injury of the cells. On the contrary, Hierro et al. (2009) did not find any

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7

Cell loads (Log CFU/eggshell)

6

RH = 65% RH = 35%

5 4 3 2 1 0 0

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20

30

45

60

90

Exposure time (min) Fig. 9. Effects of increasing exposure times to gas-plasma afterglow and relative humidity (RH) values during the treatments on cell viability of Salmonella Typhimurium inoculated onto the surface of eggs. Counts were evaluated onto TSA agar plates.

significant difference in decontamination of shell eggs by pulsed light treatment by enumerating S. enteritidis on non-selective medium and both improved TSA and selective media. On the other hand, the results obtained by Kobayashi et al. (2005), who analysed the factors involved in recovery of heat-injured S. enteritidis, highlighted that the understanding of the mechanism in the recovery of injured cells in relation to the specific stress used is necessary for the choice of culture media and conditions allowing the most reliable detection of the surviving cells. Although our data on microbial inactivation are preliminary, findings from the current study indicate that gas plasma may be considered as a promising technique to reduce the population of Salmonella on shell eggs. In fact, the 4.5 and 3.5 Log reductions observed for S. Enteritidis and S. Typhimurium is an important result also taking into consideration that S. enteritidis and S. typhimurium are the most frequent serovars causing human infections in Europe in 2007 and 2008 due to the consumption of contaminated eggs (EFSA, 2009, 2010). As both cleaning or washing with sanitizing agents are not currently allowed within the European Community, several alternative approaches for the superficial decontamination of table eggs have been proposed as previously mentioned including pulsed electric light technology (Hierro et al., 2009) and heat treatments, i.e. hot air, hot water, infra-red radiation, and atmospheric steam (James et al., 2002; Pasquali et al., 2010), in addition to slightly acidic electrolyzed water (Cao et al., 2009). However, some of the reports were mainly focused on the process conditions to be used without causing damages to the shell, protein coagulation sensitive biomolecules, but did not specify the decontamination efficacy (James et al., 2002; Fuhrmann et al., 2010). According to Hierro et al. (2009) when unwashed eggs were pulsed, 24–80% of the sam-

ples showed the maximum decontamination (3.6 Log CFU/egg), depending on the fluence applied. Cao et al. (2009) demonstrated that slightly acidic electrolyzed water (SAEW) with a near-neutral pH value exhibits an equivalent or higher bactericidal activity for shell eggs compared to sodium-hypochlorite solution being able to reduce 6.5 Log CFU/g of S. enteritidis on shell eggs when SAEW containing 15 mg/l available chlorine was used for 3 min. Contaminated eggs treated with ozone at 4–8 °C for 10 min or with UV (1500–2500 lW/cm2) at 22–25 °C for 5 min produced 5.9 or 4.3-log microbial reductions, respectively when compared with contaminated untreated controls (Rodriguez-Romo and Yousef, 2005). However, no evaluation of the effects of such treatments on the quality traits of the treated eggs was made by the authors. Although Fuhrmann et al. (2010) used different equipment and experimental conditions, they reported that the yolk was not significantly affected by ozone treatment. However, the soluble cuticle proteins were completely destroyed even at low ozone doses (10 ml/l for 20 min) and efficient egg decontamination was achieved following a 2 h exposure with 10 ml/l. 3.3. Assessment of eggshell quality After the cuticle dying, a significant difference only emerged between the eggshell color at RH = 35% with higher negative values of a* (more green) for eggshell subjected to the treatment, indicating that in these eggs the cuticle was able to bind more green solution than that in the control eggs. Despite of this unexpected result, the color difference was very low if compared to the maximum value that the a* parameter can assume (±60) and can be attributed to heterogeneity of the substrate of the eggs used for control with respect that used for treatment. No differences emerged in the

Table 2 Influence of the gas-plasma treatments (RH = 35% and RH = 65%, at 21 °C) on shell egg quality traits. Eggshell color after the cuticle presence assessment

RH = 35% Control After 90 min RH = 65% Control After 90 min

Albumen pH

Weight loss (%)

Yolk index

29.2a (1.8) 28.5a (2.3)

9.0a (0.3) 9.0a (0.3)

6.9a (0.9) 7.2a(1.4)

0.16a (0.03) 0.17a (0.02)

29.0a (1.3) 30.0a (1.1)

9.0a (0.2) 9.0a (0.3)

6.0a (0.9) 6.7a (2.3)

0.16a (0.02) 0.17a (0.03)

L*

a*

b*

51.8a (4.0) 52.4a (4.3)

10.0a (10.2) 14.4b (6.4)

53.2a (4.1) 54.6a (3.5)

8.6a (5.0) 6.2a (5.9)

Differences between means with the same exponent letter within a column are not significant at p-level value <0.05. Values in parentheses are standard deviations.

L. Ragni et al. / Journal of Food Engineering 100 (2010) 125–132

131

Score

Fig. 10. SEM scoring categories for cuticle, A (1, normal; 2, occasionally patchy; 3, very patchy; 4, little or not cuticle) and for the inner surface of inner shell membrane, B (1, normal; 2, membranes fibres occasionally clumped; 3, membranes fibres moderate clumped; 4, membranes fibres extensively clumped).

4.5 4 3.5 3 2.5 2 1.5 1 0.5 0

cuticle score

inner membrane score

a

a aaa

a

a

a

control

45 min

90 min

Treatment Fig. 11. Means values of scores for cuticle and the inner surface of the inner shell membrane. Differences between means with the same exponent letter within a column are not significant at p-level value <0.05.

treated samples respect to the control ones for the albumen pH, the weight loss and the yolk index (Table 2). SEM analysis revealed that no significant differences emerged between the treated and the control eggs in terms of scores for both cuticle and inner surface of the inner shell membrane (Figs. 10 and 11). It is well known that the first defense against bacterial infection is the cuticle (Board and Halls, 1973). Several Authors found a relationship between the presence and quality of the cuticle and prevention of bacterial penetration (Alls et al., 1964; Drysdale, 1985; Messens et al., 2005). Therefore we can assume that the immersion in ethanol solution used for the superficial disinfection of eggs before their inoculation, do not promote a deeper penetration of Salmonella into the shell and protect cells from gas-plasma treatment nor the gas plasma compromise the cuticle quality. 4. Conclusions A resistive barrier discharge gas-plasma prototype was shown to generate a low temperature after-glow gas mixture able to reduce the S. Enteritidis and S. Typhimurium population on shell eggs. The discharge at the device electrodes was characterized by a

peak-to-peak voltage of about 15 kV with a fundamental frequency of oscillation at 12.7 kHz. The plasma emission, investigated by optical emission spectroscopy, revealed the presence of OH, NO and other reactive radical species involved in the microbial reduction. The relative humidity of the gaseous atmosphere was shown to have a substantial influence on the decontamination power of the device. Maximum cell load reductions of about 2.5 and 4.5 Log CFU/eggshell were achieved for S. Enteritidis after 90 min of treatment with a relative humidity of 35% and 65%, respectively. Negative effects of the examined treatments on egg quality traits were not observed. A duration of treatment up or more than 90 min should be considered as feasibly for the poultry farmers and industries that usually stock eggs for a much longer period than that necessary for the sanitization and could use off-line gas plasma treating cells. Additional advantages of the prototype set up in this work are the use of atmosphere as working gas and the low temperature (25 °C), which showed make this equipment of interest for the industry.

Acknowledgements This work was funded by the European Community within the FP6 European Project named RESCAPE (Food-CT-2006-036018). The authors would like to thank W. Messens (Institute for Agricultural and Fisheries Science, ILVO, Belgium) for kindly providing the Salmonella Enteritidis strain and M.M. Bain (University of Glasgow, Faculty of Veterinary Medicine, 464 Bearsden Road, Glasgow G61 1QH, Scotland, UK) for her valuable help in SEM analyses.

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