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High voltage atmospheric cold plasma treatment of refrigerated chicken eggs for control of Salmonella Enteritidis contamination on egg shell
LWT - Food Science and Technology xxx (2016) 1e7
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High voltage atmospheric cold plasma treatment of refrigerated chicken eggs for control of Salmonella Enteritidis contamination on egg shell Zifan Wan a, Yi Chen b, S.K. Pankaj a, Kevin M. Keener a, * a b
Center for Crop Utilization Research, Iowa State University, Ames, IA, 50011, USA Purdue University, Nelson Hall of Food Science, West Lafayette, IN, 47907, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 August 2016 Received in revised form 13 October 2016 Accepted 25 October 2016 Available online xxx
Salmonella Enteritidis (SE) contamination is a major risk for U.S. consumers from chicken eggs. This study is primarily focused on evaluation of a novel High Voltage Atmospheric Cold Plasma (HVACP) technology for inactivation of Salmonella and its effect on the egg quality. Spot inoculated chicken eggs were treated with high voltage cold plasma (85 kV) under direct and indirect mode of exposure in dry air and modified atmospheric gas environment. A reduction of 5.53 log cfu/egg was observed for egg surfaces directly treated under modified atmospheric gas for 15 min. SE reductions was found dependent on treatment times, gas type, and mode of exposure of eggs to the plasma. No significant difference (p > 0.05) in direct and indirect mode of exposure was observed on the egg quality after plasma treatment. These results demonstrate the potential of HVACP to be used as a suitable non-thermal treatment for reducing Salmonella from packaged chicken eggs. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Atmospheric cold plasma High voltage cold plasma Salmonella Enteritidis Chicken eggs
1. Introduction Americans consume on an average of 251 eggs annually, and in the year 2013 alone, egg industry produced around 82 billion table eggs (American Egg Board, 2013; National Agricultural Statistics Service, 2014). Salmonella Enteritidis (SE), a predominant Salmonella serotype, is a primary concern for the egg industry (Rodrigue, Tauxe, & Rowe, 1990). Among all the SE outbreaks from 1985 to 2003 in a confirmed food vehicle, 75% of the outbreaks had vehicles that were either primarily egg-based or that contained egg ingredients (Braden, 2006). In 2009e2010, Salmonella infection accounted for 30% of the 790 outbreaks with a laboratory-confirmed illness, out of which Salmonella in eggs were responsible for approximately 70% of all the outbreak-related illness (CDC, 2013). Current commercial egg washing process includes four stages: 1) wetting eggs to enable the softening of debris on the shell of the egg; 2) washing eggs with brushes; 3) rinsing with clean hot water; and 4) drying by drainage or assistance of air jet (Hutchison et al., 2003). In US, the most common chemical used by the majority of egg producers for egg washing is chlorine (sodium hypochlorite)
* Corresponding author. E-mail address: [email protected] (K.M. Keener).
(Al-Ajeeli, Taylor, Alvarado, & Coufal, 2016). Chlorine is widely used because it effectively destroys a wide spectrum of pathogenic organisms by oxidizing cellular materials. However, it has been shown that chlorine can react with natural organic matter resulting in the formation of potentially carcinogenic halogenated disinfec€ tion by-products (Olmez & Kretzschmar, 2009). The environmental concerns and consumer perception towards use of chemical decontaminating agents form the impetus to develop novel nonthermal, environment friendly, low-cost, convenient technologies for egg decontamination. Non-thermal plasma is a novel technology which has recently gained significant attention in the food processing research. The term “plasma” refers to a quasi-neutral ionized gas, primarily composed of photons, ions and free electrons as well as atoms in their fundamental or excited states with a net neutral charge (Pankaj, Bueno-Ferrer, Misra, Milosavljevi c, et al., 2014). Cold plasma can be generated through various methods like corona discharge, microwave plasma, radio-frequency plasma. Dielectric barrier discharge is one of the most common methods for cold plasma generation in which plasma is generated between two metal electrodes among which at least one of them is covered with a dielectric layer. Dielectric layer serves the purpose of limiting the discharge current avoiding arc transition and randomly distributing streamers on the electrode surface for a more homogeneous
http://dx.doi.org/10.1016/j.lwt.2016.10.051 0023-6438/© 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Wan, Z., et al., High voltage atmospheric cold plasma treatment of refrigerated chicken eggs for control of Salmonella Enteritidis contamination on egg shell, LWT - Food Science and Technology (2016), http://dx.doi.org/10.1016/j.lwt.2016.10.051
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treatment (Tendero, Tixier, Tristant, Desmaison, & Leprince, 2006). Based on the principle, a novel high voltage atmospheric cold plasma (HVACP) technology has been developed at Purdue University (US20120183437 A1) (Keener & Klockow, 2010). The HVACP ionizes the gas inside a sealed package into reactive gas species (RGS) such as oxygen plasma will have reactive species like ozone (O3), atomic oxygen (O), superoxide (O2), peroxide (O2 2 or H2O2), and hydroxyl radicals (OH) which have bactericidal properties (Gaunt, Beggs, & Georghiou, 2006). After HVACP treatment, the RGS revert to their ground state within 24 h or less leaving no chemical residues. The exposure of food products with the RGS is responsible for the microbial inactivation. Numerous studies have demonstrated the effectiveness of HVACP treatment in controlling the safety of food products such as spinach (Klockow & Keener, 2009), seeds (Trinetta, Vaidya, Linton, & Morgan, 2011), strawberries (Misra, Patil et al., 2014), and cherry tomatoes (Misra, Keener, Bourke, Mosnier, & Cullen, 2014). The goal of this study was to investigate the effect of HVACP treatment of refrigerated chicken eggs for control of Salmonella Enteritidis (SE) on the shell surface. The effect of HVACP treatment on the survival of SE on the eggshell in air and modified atmosphere gas environments were evaluated. Mode of exposure (direct or indirect) and treatment time (5, 10, 15 min) were the used process variables. Egg quality was also evaluated after HVACP treatments which included Haugh unit, yolk color, albumen pH, yolk pH and vitelline membrane strength. 2. Materials and methods 2.1. Bacterial strain and inoculum preparation Salmonella enterica serovar Enteritidis (strain 190:88) was obtained from Department of Food Science, Purdue University. Stock culture was stored with 15% glycerol at 80 C. Fresh working culture was prepared by inoculating frozen culture in 20 ml Tryptic Soy Broth (TSB) (Difco™, MD, USA) and incubated at 37 C for 24 h. The culture was plated on the Xylose Lysine Deoxycholate (XLD) agar (Difco™, MD, USA). After incubation at 37 C for 24 h, one isolated black colony on XLD agar was transferred into the plastic flask with 100 ml TSB. The culture was grown under gentle shaking at 100 rpm on orbital shaker at 37 C for 22 h. 40 ml of culture was centrifuged (Thermo Scientific, Sorvall Legend XTR 750045520) at 7000 rpm for 5 min at 4 C. The resulting pellet was finally suspended in 4 ml of TSB to a final cell concentration of approximately 1011 CFU/ml. 2.2. Spot inoculation of chicken eggs Medium A grade eggs were purchased from a local grocery store. The egg surfaces were first wiped with 70% ethanol sprayed wipes, and then dipped in 70% ethanol for 1 min to decontaminate the
outer shell. Decontaminated eggs were air dried for at least 30 min before inoculation. 0.1 ml SE inoculum was spot inoculated on the sideway of the eggs within a 3 3 cm area (36 spots) (Chen & Zhu, 2011). Then the eggs were allowed to air dry for 1 h in a laminar flow cabinet at room temperature to allow attachment of bacterial cells. After drying, the eggs were placed in a refrigerator at 5 C for overnight to reach treatment temperature prior to the plasma treatment. The final attached population was around 107 CFU/egg. 2.3. HVACP treatment For HVACP treatment, four inoculated eggs were placed inside a polypropylene ArtBin® box (370 355 520 mm, thickness ¼ 3.18 mm) with inoculum on top, then pillow sealed with a high barrier Cryovac® B2630 (Sealed Air, North Carolina, USA) film (470 400 mm after sealing) leaving a 2.54 cm opening for gas flushing. The pillow pack (box in bag) as a whole was flushed with either dry air or MA65 (65% O2, 30% CO2, 5% N2) to reach <5% relative humidity and oxygen content was measured using a Mocon® oxygen gas analyzer (MOCON Inc, Minnepolis, USA), before complete sealing. The HVACP system used consists of a high voltage transformer with a voltage output of 0e130 kV at a frequency of 60 Hz. The polypropylene box inside the sealed film acted as a sample holder as well as a dielectric barrier. It was placed between two aluminum electrodes of 15 cm diameter. Two Cuisinart® (Cuisinart, New Jersey, USA) polypropylene layers (355 272 2.20 mm) were placed above and below the package as additional dielectric barriers (Fig. 1). There were two modes of HVACP exposure assessed for egg treatment (Fig. 2). For direct HVACP treatment, four eggs were placed between two electrodes directly in the plasma field. For indirect treatment, four eggs were placed outside of the plasma field (in the four corners of the box). A plastic cuvette was placed in the middle of the box to serve as a spacer. The inoculated egg samples were treated at 85 kV for 5, 10 and 15 min in duplicates. All packages were stored at 5 C after treatment for 24 h before microbial recovery. 2.4. Measurements of reactive gas species Reactive gas species were measured at 0 and 24 h using Dr€ ager Tubes system as previously described by Klockow and Keener €ger tubes system includes detector tubes and (2009). Briefly, Dra €ger Safety AG & Co. KGaA, Luebeck, Accuro gas detector pump (Dra Germany) which was calibrated for 100 ml of gas. Ozone tubes (part no. CH21001), Nitrous oxides (NOx) tubes (part no. 6724001), and Carbon monoxide (CO) tubes (part no. 6733051) were used for €ger CO measurement tubes have an interference with analysis. Dra ozone, so CO measurements were only taken at 24 h when no ozone €ger tubes system had a precision of ±15% was present. The Dra (Dr€ ager Safety AG & Co. KGaA, Luebeck, Germany).
Fig. 1. Schematic diagram of high voltage atmospheric cold plasma generator set-up for egg treatment.
Please cite this article in press as: Wan, Z., et al., High voltage atmospheric cold plasma treatment of refrigerated chicken eggs for control of Salmonella Enteritidis contamination on egg shell, LWT - Food Science and Technology (2016), http://dx.doi.org/10.1016/j.lwt.2016.10.051
Z. Wan et al. / LWT - Food Science and Technology xxx (2016) 1e7
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Fig. 2. Two mode of exposure to the high voltage atmospheric cold plasma: Direct (left), Indirect (right).
2.5. Optical emission spectroscopy The emission spectra of the HVACP treatment of shell egg was recorded by a computer controlled Ocean Optics spectrometer (Ocean Optics, Inc. Florida, USA). The light from the plasma was delivered by an Ocean Optics optical fiber with a core diameter of 1000 mm and was suitable to measure light from 200 nm to 1100 nm. The collimating lenses with 5 mm diameter, optimized for light from 200 nm to 2000 nm were used to align the light that enters the optical fiber. The length from the collimating lenses to the edge of the box containing sample was 15 cm. The emission spectrum was collected and saved every 30 s during the 15 min HVACP treatment at 85 kV.
8 mm. Three measurements were taken at three different spots on the yolk. Care was taken to ensure measurements were not made near the chalazae as Lyon, Newell, and Morrison (1972) had identified it being the strongest section of the vitelline membrane. 2.8. Statistical analysis Statistical Analysis was done by General Linear Models (GLM) of SAS 9.3 (SAS Institute Inc., North Carolina, USA). For microbial reduction study, three-way ANOVA option has been used, and the main effects and interactions studied were gas, mode and treatment time. For each quality parameter, one-way ANOVA option has been used. Test of significance was done using Tukey's test and statistical significance was indicated at p < 0.05.
2.6. Recovery and enumeration of survivors on the egg surface 3. Results and discussion For recovery of microbial population, positive control (inoculated, untreated), negative control (non-inoculated, untreated) and treated eggs (inoculated) were analyzed. The samples were placed into a sterile stomacher bag containing 20 ml of 0.1 percent peptone water, and were subjected to mild shaking for 2 min. The resulting suspension was serially diluted in 0.1 percent peptone water. Both XLD agar (specific for Salmonella) and Tryptic Soy Agar (TSA, Difco™, MD, USA) (non-selective) were used for spread plate method. The enumeration was carried out after 24e48 h incubation at 37 C. The differences between recovered SE populations on two media were calculated as the number of injured cells (García et al., 2005). 2.7. Egg quality analysis Haugh units (HU) were measured on eggs using an Egg Multi Tester EMT- 5200 (Robotmation Co., Tokyo, Japan). This machine provided egg weight, HU values, and yolk color based on the Yolk Color Fan (DSM Co., Heerlen, Netherlands). The pH of albumen and yolk were measured using a model 220 Denver Instrument pH meter (Denver Instrument, Denver, CO) with a pH probe, IQ150 (Spectrum Technologies, Plainfield, IL). A twopoint calibration was performed on the pH meter using 7.0 and 10.0 buffers prior to each testing. pH measurement was done in triplicate on eggs after vitelline membrane strength measurements. Vitelline Membrane Strength (VMS) was determined using a TAXT2i texture analyzer (Texture Technologies Corp., Scarsdale, NY). A 5-kg load cell was used, along with a 3-mm round-tipped probe. The setting was based on Return to Start mode, modified from the settings used by Biladeau and Keener (2009). Test speed was 0.5 mm/s with a trigger force of 0.1 g. Pre-test and post-test speeds were 1 mm/s and 10 mm/s, respectively and the distance was
3.1. Optical emission spectroscopy of plasma discharge The emission spectra of the HVACP treatment inside the packages for air and MA65 are shown in Fig. 3. For HVACP in air, the majority of the intense peaks are near UV region (300e400 nm) by the emissions from the second positive system (SPS) N2(C-B) and first negative system Nþ 2 (B-X), which is similar to other studies at atmospheric pressures in air (Misra, Pankaj et al., 2014; Misra, Zuizina, Cullen, & Keener, 2013; Pankaj, Bueno-Ferrer, Misra, O'Neill, et al., 2014; Sarangapani et al., 2016). In HVACP in MA65 spectrum, two excited atomic oxygen (O I) species are more dominant which are at 777 and 844 nm and represent electron transition of O atom from 3p5P -> 3s5S and transition of O atom from 3p3P to 3s3S respectively (Chau & Kao, 1996), while the peaks of atomic oxygen in air plasma are not noticeable which is most likely caused by quenching O I (5P) and O I (3P) in air plasma (Walsh, Liu, Iza, Rong, & Kong, 2010). Table 1 presents a summary of reactive gas species generated during HVACP in air and MA65 at both exposure modes. It shows that HVACP is a source for both reactive nitrogen species (RNS) and reactive oxygen species (ROS). The gas €ger tubes are shown in Fig. 4. The ROS conmeasured using Dra centration increased with increase in treatment time and were consistent with relative gas composition of the mixture. 3.2. Salmonella inactivation Recovery results on selective (XLD) and non-selective (TSA) media from HVACP treatment of SE inoculated chicken eggs are shown in Fig. 5 and Fig. 6, respectively. Under direct exposure, both in air and MA65, HVACP resulted in significant (p < 0.05) reduction in SE population which varied linearly with treatment time. Under
Please cite this article in press as: Wan, Z., et al., High voltage atmospheric cold plasma treatment of refrigerated chicken eggs for control of Salmonella Enteritidis contamination on egg shell, LWT - Food Science and Technology (2016), http://dx.doi.org/10.1016/j.lwt.2016.10.051
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Fig. 3. Optical Emission Spectra of HVACP treatment of shell eggs under direct treatment of Air and MA (A e Air Direct Treatment and B e MA Direct Treatment).
Table 1 Summary of intensities (a.u.) of reactive gas species generated during HVACP treatment of eggs. Wavelength (nm)
Gas Species
Air Direct
Air Indirect
MA Direct
MA Indirect
313 316 337 353 357 370 375 380 393 399 405 426 433 609 777 844
N2C-B(2-1) N2C-B(1-0) N2C-B(0-0) N2C-B(1-2) N2C-B(0-1) N2C-B(2-4) N2C-B(1-3) N2C-B(0-2) Nþ 2 B-X(0-0) N2C-B(0-3) N2 C-B(0-3) N2 C-B(0-3) Nþ 2 B-X(0-1) O IV (4P ) O I (5P) O I (3P)
127.2 ± 24.9 472.6 ± 79.9 1299.9 ± 219.6 311.2 ± 59.4 1080.4 ± 217.1 102.4 ± 0.9 307.5 ± 51.3 467.0 ± 92.7 n.d. 163.9 ± 32.7 180.7 ± 33.3 124.2 ± 25.4 112.6 ± 3.9 n.d. n.d. n.d.
201.0 ± 14.5 742.2 ± 43.1 2196.7 ± 92.6 523.2 ± 23.2 1862.5 ± 82.5 129.3 ± 7.6 509.9 ± 16.7 819.1 ± 21.3 117.5 ± 5.8 279.6 ± 6.5 302.8 ± 5.1 157.8 ± 8.6 123.4 ± 5.9 n.d. n.d. n.d.
n.d. n.d. 74.2 n.d. 74.6 ± 3.8 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 77.7 ± 4.8 248.6 ± 156.9 112.2 ± 3.6
n.d. n.d. 85.3 ± 2.4 n.d. 90.7 ± 16.4 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 96.1 ± 8.6 476.7 ± 114.6 104.4 ± 22.6
n.d. e not detected.
indirect treatment, significant (p < 0.05) SE reductions were observed in MA65. However, significant reduction was only observed at higher treatment times for air. One potential pitfall in evaluating antimicrobial intervention strategies is failure to account for the presence of injured cells. On application of any processing technique, a certain population of microbe might be only injured and not completely inactivated. Under favorable environmental conditions as in a non-selective medium like TSA, injured cells usually undergo repair and become functionally normal compared to selective media like XLD
where injured cells can fail to resuscitate (Kang & Fung, 2000). Hence, injured cells were defined as microbes which were able to reproduce and form colonies on a non-selective media (TSA) but not in media with selective agents (XLD). High proportions of injured cells were observed after plasma treatment for 5 and 10 min. However, the population of injured and viable cells significantly (p < 0.05) decreased after 15 min treatment. No significant difference (p > 0.05) in the injured cells was observed for indirect exposure in any gas environment. Considering the low efficiency of indirect treatment on bacterial inactivation, longer
Please cite this article in press as: Wan, Z., et al., High voltage atmospheric cold plasma treatment of refrigerated chicken eggs for control of Salmonella Enteritidis contamination on egg shell, LWT - Food Science and Technology (2016), http://dx.doi.org/10.1016/j.lwt.2016.10.051
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Fig. 4. Reactive gas concentration for MA and Air Direct treatment vs. HVACP treatment time (Ozone and NOx concentration measured immediately after HVACP treatments (Gas -Mode) for 5,10, 15 min; CO concentration measured at 24 h after HVACP treatments (Gas-Mode) for 5, 10, 15 min).
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Fig. 6. Survival of SE population (log10 CFU/egg) exposed to HVACP treatments (Gas -Mode) for 5, 10, 15 min and recovered on TSA. The detection limit of the applied enumeration method was one log (CFU/egg). Same capital letter on top of each bar indicated no significant difference among each bar with the same pattern (or same Gas-Mode combination). Same small letter on top of each bar indicated no significant difference among each bar with the same treatment time. (p < 0.05).
high concentration of CO2 (such as 30%) in itself inhibit the growth of the microorganisms, increasing the effectiveness of HVACP treatment (Chiper, Chen, Mejlholm, Dalgaard, & Stamate, 2011). 3.4. Effect of treatment time and mode of exposure
Fig. 5. Survival of SE population (log10 CFU/egg) exposed to HVACP treatments (Gas -Mode) for 5, 10, 15 min and recovered on XLD. The detection limit of the applied enumeration method was one log (CFU/egg). Same capital letter on top of each bar indicated no significant difference among each bar with the same pattern (or same Gas-Mode combination). Same small letter on top of each bar indicated no significant difference among each bar with the same treatment time. (p < 0.05).
treatment time was needed to reach the peak of injured cells number. Similar results were also reported in previous studies with cold plasma treatments (Critzer, Kelly-Wintenberg, South, & Golden, 2007; Sharma, Pruden, Yu, & Collins, 2005). 3.3. Effect of gas type Gas type was found to be a key variable for SE inactivation on egg shell. Modified gas environment was found more effective for reducing SE population on egg surface. The effects were more pronounced at higher treatment time. This could be attributed to the significantly higher concentrations of reactive species in MA65 compared to atmospheric air at all treatment times. The significant difference between air and MA65 in terms of reactive oxygen species generation rates were also noted by Keener et al. (2012, pp. 445e455). MA65 has higher oxygen than air and so resulted in more reactive oxygen species generation. However, addition of CO2 in the gas mixture has a two-fold benefit. High concentration of CO generated after HVACP treatment has bactericidal effect and also
Generally, the surviving populations of SE decreased with increase in treatment time. The trend was more pronounced for direct HVACP treatments. Direct HVACP treatment in MA65 for 5 or 10 min resulted in reductions of SE populations by 0.7 and 2.3 log cycles, respectively. However, after 15 min treatment, a significant inactivation with 5.5 log CFU/egg reduction was observed. Increased treatment time leads to more reactive gas species (RGS) generation and increasing contact time between microorganisms and RGS, thus increasing the potential for larger bactericidal effect. As for direct-air combination, 15 min treatment achieved more than 5 log reduction of SE which was comparable to previous air plasmatreated egg studies (Davies & Breslin, 2003; Ragni et al., 2010). Direct plasma mode of exposure was found more efficient in SE inactivation compared to the indirect mode, which was in agreement with other studies (Dobrynin, Fridman, Friedman, & Fridman, 2009; Fridman et al., 2007; Ziuzina, Patil, Cullen, Keener, & Bourke, 2013). Direct treatment for 15 min in MA65 reduced the SE population to 1.84 log CFU/egg compared to 5.65 log CFU/egg under indirect mode. Similarly, in case of air, direct mode of exposure achieved enhanced inactivation of 2.44 log compared to indirect mode of exposure. Higher bacterial inactivation under direct plasma treatment can be attributed to the synergistic effect of charged particles and the generated reactive gas species. Misra et al. (2013) also pointed out that the charged particles, UV radiaþ þ þ tion, and short half-live species (such as O 2 , OH , N2 , N2O ) recombine before reaching the sample under indirect treatment. Only reactive species (such as O3, O2, NO2, NO, and CO), which have longer half-lives, can interact with the sample in indirect treatment. 3.5. Effect on egg quality The quality parameters for the untreated and HVACP treated eggs are shown in Table 2. No significant difference (p > 0.05) was observed in any of the analyzed parameters. HU of the untreated eggs were 85.4 and after treatment they ranged from 81.5 to 93.8. No significant difference (p > 0.05) in weight was observed after the
Please cite this article in press as: Wan, Z., et al., High voltage atmospheric cold plasma treatment of refrigerated chicken eggs for control of Salmonella Enteritidis contamination on egg shell, LWT - Food Science and Technology (2016), http://dx.doi.org/10.1016/j.lwt.2016.10.051
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Table 2 Quality parameters of untreated and HVACP treated eggs. Quality parameters
Untreated
HVACP treated
Haugh unit Egg weight Albumen pH Yolk pH Yolk color Vitelline membrane strength
85.4 ± 0.28a 49.8 ± 2.26b 7.21 ± 0.11c 6.22 ± 0.23d 3 ± 2.8e 0.0026 ± 0.0001f
87.65 ± 6.15a 50.4 ± 3.96b 7.21 ± 0.06c 6.23 ± 0.06d 9.5 ± 7.8e 0.0024 ± 0.0001f
aef
p < 0.05.
HVACP treatment suggesting no significant difference in the evaporation rate or CO2 escape from the albumen after the plasma treatment (Caner, 2005). No significant change (p > 0.05) in albumen or yolk pH was observed after the plasma treatment. No change in the vitelline membrane strength was observed after HVACP treatment and the result were comparable with previously published values (Banerjee, 2010). The grading of yolk color was done according to DSM Yolk Color Fan, which is a 15 scales color index to distinguish the yolk color density. Carotenoids are the natural pigments of the egg yolk, giving it a yellow to dark brilliant orange color (Anton, 2007, pp. 1e6). Therefore, the oxidation of carotenoids resulting in color fading could be a great concern for processed shell eggs. However, in this study, no significant difference (p > 0.05) in yolk color was observed after HVACP treatments. It is important to note at this point that, although no undesirable changes in the standard quality attributes were noticed, RGS interaction with the egg proteins, lipids and other components have not been studied. Effect of HVACP generated RGS on the egg chemical composition need further investigation from the consumer safety perspective. 4. Conclusions HVACP treatment in MA65 for 15 min achieved maximum SE inactivation of 5.53 log CFU/egg on TSA and 6.37 log CFU/egg on XLD. HVACP process parameters including gas type, treatment time, and mode of exposure showed strong interactive effects with microbial inactivation. The occurrence of injury was observed for Salmonella cells by plating on selective agar and nonselective agar. No significant (p > 0.05) difference in the egg quality was observed after the HVACP treatment. This study demonstrates the potential of high voltage atmospheric cold plasma to be used as a novel, nonthermal, and environment-friendly decontamination technology for shell eggs. References Al-Ajeeli, M. N., Taylor, T. M., Alvarado, C. Z., & Coufal, C. D. (2016). Comparison of eggshell surface sanitization technologies and impacts on consumer acceptability. Poultry Science, 95(5), 1191e1197. http://dx.doi.org/10.3382/ps/pew014. American Egg Board. (2013). American egg board annual report 2013. Anton, M. (2007). Composition and structure of hen egg yolk Bioactive egg compounds. Springer. Banerjee, P. (2010). The effect of carbon dioxide on lysozyme activity and quality of chicken eggs. Biladeau, A., & Keener, K. (2009). The effects of edible coatings on chicken egg quality under refrigerated storage. Poultry science, 88(6), 1266e1274. Braden, C. R. (2006). Salmonella enterica serotype enteritidis and eggs: A national epidemic in the United States. Clinical Infectious Diseases, 43(4), 512e517. Caner, C. (2005). The effect of edible eggshell coatings on egg quality and consumer perception. Journal of the Science of Food and Agriculture, 85(11), 1897e1902. CDC. (2013). Tracking and reporting foodborne disease outbreaks. Chau, T., & Kao, K. (1996). Optical emission spectra of microwave oxygen plasmas and fabrication of SiO2 films. Journal of Vacuum Science & Technology B, 14(1), 527e532. Chen, Z., & Zhu, C. (2011). Modelling inactivation by aqueous chlorine dioxide of Dothiorella gregaria Sacc. and Fusarium tricinctum (Corda) Sacc. spores inoculated on fresh chestnut kernel. Letters in Applied Microbiology, 52(6), 676e684. http://dx.doi.org/10.1111/j.1472-765X.2011.03061.x.
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V., & Rowe, B. (1990). International increase in Salmonella enteritidis: A new pandemic? Epidemiology and Infection, 105(1), 21e27. http:// dx.doi.org/10.1017/S0950268800047609. Sarangapani, C., Misra, N., Milosavljevic, V., Bourke, P., O'Regan, F., & Cullen, P. (2016). Pesticide degradation in water using atmospheric air cold plasma. Journal of Water Process Engineering, 9, 225e232. Sharma, A., Pruden, A., Yu, Z., & Collins, G. J. (2005). Bacterial inactivation in open air by the afterglow plume emitted from a grounded hollow slot electrode. Environmental science & technology, 39(1), 339e344. Tendero, C., Tixier, C., Tristant, P., Desmaison, J., & Leprince, P. (2006). Atmospheric pressure plasmas: A review. Spectrochimica Acta Part B: Atomic Spectroscopy, 61(1), 2e30. http://dx.doi.org/10.1016/j.sab.2005.10.003. Trinetta, V., Vaidya, N., Linton, R., & Morgan, M. (2011). 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High voltage atmospheric cold plasma treatment of refrigerated chicken eggs for control of Salmonella Enteritidis contamination on egg shell Zifan Wan a, Yi Chen b, S.K. Pankaj a, Kevin M. Keener a, * a b
Center for Crop Utilization Research, Iowa State University, Ames, IA, 50011, USA Purdue University, Nelson Hall of Food Science, West Lafayette, IN, 47907, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 August 2016 Received in revised form 13 October 2016 Accepted 25 October 2016 Available online xxx
Salmonella Enteritidis (SE) contamination is a major risk for U.S. consumers from chicken eggs. This study is primarily focused on evaluation of a novel High Voltage Atmospheric Cold Plasma (HVACP) technology for inactivation of Salmonella and its effect on the egg quality. Spot inoculated chicken eggs were treated with high voltage cold plasma (85 kV) under direct and indirect mode of exposure in dry air and modified atmospheric gas environment. A reduction of 5.53 log cfu/egg was observed for egg surfaces directly treated under modified atmospheric gas for 15 min. SE reductions was found dependent on treatment times, gas type, and mode of exposure of eggs to the plasma. No significant difference (p > 0.05) in direct and indirect mode of exposure was observed on the egg quality after plasma treatment. These results demonstrate the potential of HVACP to be used as a suitable non-thermal treatment for reducing Salmonella from packaged chicken eggs. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Atmospheric cold plasma High voltage cold plasma Salmonella Enteritidis Chicken eggs
1. Introduction Americans consume on an average of 251 eggs annually, and in the year 2013 alone, egg industry produced around 82 billion table eggs (American Egg Board, 2013; National Agricultural Statistics Service, 2014). Salmonella Enteritidis (SE), a predominant Salmonella serotype, is a primary concern for the egg industry (Rodrigue, Tauxe, & Rowe, 1990). Among all the SE outbreaks from 1985 to 2003 in a confirmed food vehicle, 75% of the outbreaks had vehicles that were either primarily egg-based or that contained egg ingredients (Braden, 2006). In 2009e2010, Salmonella infection accounted for 30% of the 790 outbreaks with a laboratory-confirmed illness, out of which Salmonella in eggs were responsible for approximately 70% of all the outbreak-related illness (CDC, 2013). Current commercial egg washing process includes four stages: 1) wetting eggs to enable the softening of debris on the shell of the egg; 2) washing eggs with brushes; 3) rinsing with clean hot water; and 4) drying by drainage or assistance of air jet (Hutchison et al., 2003). In US, the most common chemical used by the majority of egg producers for egg washing is chlorine (sodium hypochlorite)
* Corresponding author. E-mail address: [email protected] (K.M. Keener).
(Al-Ajeeli, Taylor, Alvarado, & Coufal, 2016). Chlorine is widely used because it effectively destroys a wide spectrum of pathogenic organisms by oxidizing cellular materials. However, it has been shown that chlorine can react with natural organic matter resulting in the formation of potentially carcinogenic halogenated disinfec€ tion by-products (Olmez & Kretzschmar, 2009). The environmental concerns and consumer perception towards use of chemical decontaminating agents form the impetus to develop novel nonthermal, environment friendly, low-cost, convenient technologies for egg decontamination. Non-thermal plasma is a novel technology which has recently gained significant attention in the food processing research. The term “plasma” refers to a quasi-neutral ionized gas, primarily composed of photons, ions and free electrons as well as atoms in their fundamental or excited states with a net neutral charge (Pankaj, Bueno-Ferrer, Misra, Milosavljevi c, et al., 2014). Cold plasma can be generated through various methods like corona discharge, microwave plasma, radio-frequency plasma. Dielectric barrier discharge is one of the most common methods for cold plasma generation in which plasma is generated between two metal electrodes among which at least one of them is covered with a dielectric layer. Dielectric layer serves the purpose of limiting the discharge current avoiding arc transition and randomly distributing streamers on the electrode surface for a more homogeneous
http://dx.doi.org/10.1016/j.lwt.2016.10.051 0023-6438/© 2016 Elsevier Ltd. All rights reserved.
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treatment (Tendero, Tixier, Tristant, Desmaison, & Leprince, 2006). Based on the principle, a novel high voltage atmospheric cold plasma (HVACP) technology has been developed at Purdue University (US20120183437 A1) (Keener & Klockow, 2010). The HVACP ionizes the gas inside a sealed package into reactive gas species (RGS) such as oxygen plasma will have reactive species like ozone (O3), atomic oxygen (O), superoxide (O2), peroxide (O2 2 or H2O2), and hydroxyl radicals (OH) which have bactericidal properties (Gaunt, Beggs, & Georghiou, 2006). After HVACP treatment, the RGS revert to their ground state within 24 h or less leaving no chemical residues. The exposure of food products with the RGS is responsible for the microbial inactivation. Numerous studies have demonstrated the effectiveness of HVACP treatment in controlling the safety of food products such as spinach (Klockow & Keener, 2009), seeds (Trinetta, Vaidya, Linton, & Morgan, 2011), strawberries (Misra, Patil et al., 2014), and cherry tomatoes (Misra, Keener, Bourke, Mosnier, & Cullen, 2014). The goal of this study was to investigate the effect of HVACP treatment of refrigerated chicken eggs for control of Salmonella Enteritidis (SE) on the shell surface. The effect of HVACP treatment on the survival of SE on the eggshell in air and modified atmosphere gas environments were evaluated. Mode of exposure (direct or indirect) and treatment time (5, 10, 15 min) were the used process variables. Egg quality was also evaluated after HVACP treatments which included Haugh unit, yolk color, albumen pH, yolk pH and vitelline membrane strength. 2. Materials and methods 2.1. Bacterial strain and inoculum preparation Salmonella enterica serovar Enteritidis (strain 190:88) was obtained from Department of Food Science, Purdue University. Stock culture was stored with 15% glycerol at 80 C. Fresh working culture was prepared by inoculating frozen culture in 20 ml Tryptic Soy Broth (TSB) (Difco™, MD, USA) and incubated at 37 C for 24 h. The culture was plated on the Xylose Lysine Deoxycholate (XLD) agar (Difco™, MD, USA). After incubation at 37 C for 24 h, one isolated black colony on XLD agar was transferred into the plastic flask with 100 ml TSB. The culture was grown under gentle shaking at 100 rpm on orbital shaker at 37 C for 22 h. 40 ml of culture was centrifuged (Thermo Scientific, Sorvall Legend XTR 750045520) at 7000 rpm for 5 min at 4 C. The resulting pellet was finally suspended in 4 ml of TSB to a final cell concentration of approximately 1011 CFU/ml. 2.2. Spot inoculation of chicken eggs Medium A grade eggs were purchased from a local grocery store. The egg surfaces were first wiped with 70% ethanol sprayed wipes, and then dipped in 70% ethanol for 1 min to decontaminate the
outer shell. Decontaminated eggs were air dried for at least 30 min before inoculation. 0.1 ml SE inoculum was spot inoculated on the sideway of the eggs within a 3 3 cm area (36 spots) (Chen & Zhu, 2011). Then the eggs were allowed to air dry for 1 h in a laminar flow cabinet at room temperature to allow attachment of bacterial cells. After drying, the eggs were placed in a refrigerator at 5 C for overnight to reach treatment temperature prior to the plasma treatment. The final attached population was around 107 CFU/egg. 2.3. HVACP treatment For HVACP treatment, four inoculated eggs were placed inside a polypropylene ArtBin® box (370 355 520 mm, thickness ¼ 3.18 mm) with inoculum on top, then pillow sealed with a high barrier Cryovac® B2630 (Sealed Air, North Carolina, USA) film (470 400 mm after sealing) leaving a 2.54 cm opening for gas flushing. The pillow pack (box in bag) as a whole was flushed with either dry air or MA65 (65% O2, 30% CO2, 5% N2) to reach <5% relative humidity and oxygen content was measured using a Mocon® oxygen gas analyzer (MOCON Inc, Minnepolis, USA), before complete sealing. The HVACP system used consists of a high voltage transformer with a voltage output of 0e130 kV at a frequency of 60 Hz. The polypropylene box inside the sealed film acted as a sample holder as well as a dielectric barrier. It was placed between two aluminum electrodes of 15 cm diameter. Two Cuisinart® (Cuisinart, New Jersey, USA) polypropylene layers (355 272 2.20 mm) were placed above and below the package as additional dielectric barriers (Fig. 1). There were two modes of HVACP exposure assessed for egg treatment (Fig. 2). For direct HVACP treatment, four eggs were placed between two electrodes directly in the plasma field. For indirect treatment, four eggs were placed outside of the plasma field (in the four corners of the box). A plastic cuvette was placed in the middle of the box to serve as a spacer. The inoculated egg samples were treated at 85 kV for 5, 10 and 15 min in duplicates. All packages were stored at 5 C after treatment for 24 h before microbial recovery. 2.4. Measurements of reactive gas species Reactive gas species were measured at 0 and 24 h using Dr€ ager Tubes system as previously described by Klockow and Keener €ger tubes system includes detector tubes and (2009). Briefly, Dra €ger Safety AG & Co. KGaA, Luebeck, Accuro gas detector pump (Dra Germany) which was calibrated for 100 ml of gas. Ozone tubes (part no. CH21001), Nitrous oxides (NOx) tubes (part no. 6724001), and Carbon monoxide (CO) tubes (part no. 6733051) were used for €ger CO measurement tubes have an interference with analysis. Dra ozone, so CO measurements were only taken at 24 h when no ozone €ger tubes system had a precision of ±15% was present. The Dra (Dr€ ager Safety AG & Co. KGaA, Luebeck, Germany).
Fig. 1. Schematic diagram of high voltage atmospheric cold plasma generator set-up for egg treatment.
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Fig. 2. Two mode of exposure to the high voltage atmospheric cold plasma: Direct (left), Indirect (right).
2.5. Optical emission spectroscopy The emission spectra of the HVACP treatment of shell egg was recorded by a computer controlled Ocean Optics spectrometer (Ocean Optics, Inc. Florida, USA). The light from the plasma was delivered by an Ocean Optics optical fiber with a core diameter of 1000 mm and was suitable to measure light from 200 nm to 1100 nm. The collimating lenses with 5 mm diameter, optimized for light from 200 nm to 2000 nm were used to align the light that enters the optical fiber. The length from the collimating lenses to the edge of the box containing sample was 15 cm. The emission spectrum was collected and saved every 30 s during the 15 min HVACP treatment at 85 kV.
8 mm. Three measurements were taken at three different spots on the yolk. Care was taken to ensure measurements were not made near the chalazae as Lyon, Newell, and Morrison (1972) had identified it being the strongest section of the vitelline membrane. 2.8. Statistical analysis Statistical Analysis was done by General Linear Models (GLM) of SAS 9.3 (SAS Institute Inc., North Carolina, USA). For microbial reduction study, three-way ANOVA option has been used, and the main effects and interactions studied were gas, mode and treatment time. For each quality parameter, one-way ANOVA option has been used. Test of significance was done using Tukey's test and statistical significance was indicated at p < 0.05.
2.6. Recovery and enumeration of survivors on the egg surface 3. Results and discussion For recovery of microbial population, positive control (inoculated, untreated), negative control (non-inoculated, untreated) and treated eggs (inoculated) were analyzed. The samples were placed into a sterile stomacher bag containing 20 ml of 0.1 percent peptone water, and were subjected to mild shaking for 2 min. The resulting suspension was serially diluted in 0.1 percent peptone water. Both XLD agar (specific for Salmonella) and Tryptic Soy Agar (TSA, Difco™, MD, USA) (non-selective) were used for spread plate method. The enumeration was carried out after 24e48 h incubation at 37 C. The differences between recovered SE populations on two media were calculated as the number of injured cells (García et al., 2005). 2.7. Egg quality analysis Haugh units (HU) were measured on eggs using an Egg Multi Tester EMT- 5200 (Robotmation Co., Tokyo, Japan). This machine provided egg weight, HU values, and yolk color based on the Yolk Color Fan (DSM Co., Heerlen, Netherlands). The pH of albumen and yolk were measured using a model 220 Denver Instrument pH meter (Denver Instrument, Denver, CO) with a pH probe, IQ150 (Spectrum Technologies, Plainfield, IL). A twopoint calibration was performed on the pH meter using 7.0 and 10.0 buffers prior to each testing. pH measurement was done in triplicate on eggs after vitelline membrane strength measurements. Vitelline Membrane Strength (VMS) was determined using a TAXT2i texture analyzer (Texture Technologies Corp., Scarsdale, NY). A 5-kg load cell was used, along with a 3-mm round-tipped probe. The setting was based on Return to Start mode, modified from the settings used by Biladeau and Keener (2009). Test speed was 0.5 mm/s with a trigger force of 0.1 g. Pre-test and post-test speeds were 1 mm/s and 10 mm/s, respectively and the distance was
3.1. Optical emission spectroscopy of plasma discharge The emission spectra of the HVACP treatment inside the packages for air and MA65 are shown in Fig. 3. For HVACP in air, the majority of the intense peaks are near UV region (300e400 nm) by the emissions from the second positive system (SPS) N2(C-B) and first negative system Nþ 2 (B-X), which is similar to other studies at atmospheric pressures in air (Misra, Pankaj et al., 2014; Misra, Zuizina, Cullen, & Keener, 2013; Pankaj, Bueno-Ferrer, Misra, O'Neill, et al., 2014; Sarangapani et al., 2016). In HVACP in MA65 spectrum, two excited atomic oxygen (O I) species are more dominant which are at 777 and 844 nm and represent electron transition of O atom from 3p5P -> 3s5S and transition of O atom from 3p3P to 3s3S respectively (Chau & Kao, 1996), while the peaks of atomic oxygen in air plasma are not noticeable which is most likely caused by quenching O I (5P) and O I (3P) in air plasma (Walsh, Liu, Iza, Rong, & Kong, 2010). Table 1 presents a summary of reactive gas species generated during HVACP in air and MA65 at both exposure modes. It shows that HVACP is a source for both reactive nitrogen species (RNS) and reactive oxygen species (ROS). The gas €ger tubes are shown in Fig. 4. The ROS conmeasured using Dra centration increased with increase in treatment time and were consistent with relative gas composition of the mixture. 3.2. Salmonella inactivation Recovery results on selective (XLD) and non-selective (TSA) media from HVACP treatment of SE inoculated chicken eggs are shown in Fig. 5 and Fig. 6, respectively. Under direct exposure, both in air and MA65, HVACP resulted in significant (p < 0.05) reduction in SE population which varied linearly with treatment time. Under
Please cite this article in press as: Wan, Z., et al., High voltage atmospheric cold plasma treatment of refrigerated chicken eggs for control of Salmonella Enteritidis contamination on egg shell, LWT - Food Science and Technology (2016), http://dx.doi.org/10.1016/j.lwt.2016.10.051
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Fig. 3. Optical Emission Spectra of HVACP treatment of shell eggs under direct treatment of Air and MA (A e Air Direct Treatment and B e MA Direct Treatment).
Table 1 Summary of intensities (a.u.) of reactive gas species generated during HVACP treatment of eggs. Wavelength (nm)
Gas Species
Air Direct
Air Indirect
MA Direct
MA Indirect
313 316 337 353 357 370 375 380 393 399 405 426 433 609 777 844
N2C-B(2-1) N2C-B(1-0) N2C-B(0-0) N2C-B(1-2) N2C-B(0-1) N2C-B(2-4) N2C-B(1-3) N2C-B(0-2) Nþ 2 B-X(0-0) N2C-B(0-3) N2 C-B(0-3) N2 C-B(0-3) Nþ 2 B-X(0-1) O IV (4P ) O I (5P) O I (3P)
127.2 ± 24.9 472.6 ± 79.9 1299.9 ± 219.6 311.2 ± 59.4 1080.4 ± 217.1 102.4 ± 0.9 307.5 ± 51.3 467.0 ± 92.7 n.d. 163.9 ± 32.7 180.7 ± 33.3 124.2 ± 25.4 112.6 ± 3.9 n.d. n.d. n.d.
201.0 ± 14.5 742.2 ± 43.1 2196.7 ± 92.6 523.2 ± 23.2 1862.5 ± 82.5 129.3 ± 7.6 509.9 ± 16.7 819.1 ± 21.3 117.5 ± 5.8 279.6 ± 6.5 302.8 ± 5.1 157.8 ± 8.6 123.4 ± 5.9 n.d. n.d. n.d.
n.d. n.d. 74.2 n.d. 74.6 ± 3.8 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 77.7 ± 4.8 248.6 ± 156.9 112.2 ± 3.6
n.d. n.d. 85.3 ± 2.4 n.d. 90.7 ± 16.4 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 96.1 ± 8.6 476.7 ± 114.6 104.4 ± 22.6
n.d. e not detected.
indirect treatment, significant (p < 0.05) SE reductions were observed in MA65. However, significant reduction was only observed at higher treatment times for air. One potential pitfall in evaluating antimicrobial intervention strategies is failure to account for the presence of injured cells. On application of any processing technique, a certain population of microbe might be only injured and not completely inactivated. Under favorable environmental conditions as in a non-selective medium like TSA, injured cells usually undergo repair and become functionally normal compared to selective media like XLD
where injured cells can fail to resuscitate (Kang & Fung, 2000). Hence, injured cells were defined as microbes which were able to reproduce and form colonies on a non-selective media (TSA) but not in media with selective agents (XLD). High proportions of injured cells were observed after plasma treatment for 5 and 10 min. However, the population of injured and viable cells significantly (p < 0.05) decreased after 15 min treatment. No significant difference (p > 0.05) in the injured cells was observed for indirect exposure in any gas environment. Considering the low efficiency of indirect treatment on bacterial inactivation, longer
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Fig. 4. Reactive gas concentration for MA and Air Direct treatment vs. HVACP treatment time (Ozone and NOx concentration measured immediately after HVACP treatments (Gas -Mode) for 5,10, 15 min; CO concentration measured at 24 h after HVACP treatments (Gas-Mode) for 5, 10, 15 min).
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Fig. 6. Survival of SE population (log10 CFU/egg) exposed to HVACP treatments (Gas -Mode) for 5, 10, 15 min and recovered on TSA. The detection limit of the applied enumeration method was one log (CFU/egg). Same capital letter on top of each bar indicated no significant difference among each bar with the same pattern (or same Gas-Mode combination). Same small letter on top of each bar indicated no significant difference among each bar with the same treatment time. (p < 0.05).
high concentration of CO2 (such as 30%) in itself inhibit the growth of the microorganisms, increasing the effectiveness of HVACP treatment (Chiper, Chen, Mejlholm, Dalgaard, & Stamate, 2011). 3.4. Effect of treatment time and mode of exposure
Fig. 5. Survival of SE population (log10 CFU/egg) exposed to HVACP treatments (Gas -Mode) for 5, 10, 15 min and recovered on XLD. The detection limit of the applied enumeration method was one log (CFU/egg). Same capital letter on top of each bar indicated no significant difference among each bar with the same pattern (or same Gas-Mode combination). Same small letter on top of each bar indicated no significant difference among each bar with the same treatment time. (p < 0.05).
treatment time was needed to reach the peak of injured cells number. Similar results were also reported in previous studies with cold plasma treatments (Critzer, Kelly-Wintenberg, South, & Golden, 2007; Sharma, Pruden, Yu, & Collins, 2005). 3.3. Effect of gas type Gas type was found to be a key variable for SE inactivation on egg shell. Modified gas environment was found more effective for reducing SE population on egg surface. The effects were more pronounced at higher treatment time. This could be attributed to the significantly higher concentrations of reactive species in MA65 compared to atmospheric air at all treatment times. The significant difference between air and MA65 in terms of reactive oxygen species generation rates were also noted by Keener et al. (2012, pp. 445e455). MA65 has higher oxygen than air and so resulted in more reactive oxygen species generation. However, addition of CO2 in the gas mixture has a two-fold benefit. High concentration of CO generated after HVACP treatment has bactericidal effect and also
Generally, the surviving populations of SE decreased with increase in treatment time. The trend was more pronounced for direct HVACP treatments. Direct HVACP treatment in MA65 for 5 or 10 min resulted in reductions of SE populations by 0.7 and 2.3 log cycles, respectively. However, after 15 min treatment, a significant inactivation with 5.5 log CFU/egg reduction was observed. Increased treatment time leads to more reactive gas species (RGS) generation and increasing contact time between microorganisms and RGS, thus increasing the potential for larger bactericidal effect. As for direct-air combination, 15 min treatment achieved more than 5 log reduction of SE which was comparable to previous air plasmatreated egg studies (Davies & Breslin, 2003; Ragni et al., 2010). Direct plasma mode of exposure was found more efficient in SE inactivation compared to the indirect mode, which was in agreement with other studies (Dobrynin, Fridman, Friedman, & Fridman, 2009; Fridman et al., 2007; Ziuzina, Patil, Cullen, Keener, & Bourke, 2013). Direct treatment for 15 min in MA65 reduced the SE population to 1.84 log CFU/egg compared to 5.65 log CFU/egg under indirect mode. Similarly, in case of air, direct mode of exposure achieved enhanced inactivation of 2.44 log compared to indirect mode of exposure. Higher bacterial inactivation under direct plasma treatment can be attributed to the synergistic effect of charged particles and the generated reactive gas species. Misra et al. (2013) also pointed out that the charged particles, UV radiaþ þ þ tion, and short half-live species (such as O 2 , OH , N2 , N2O ) recombine before reaching the sample under indirect treatment. Only reactive species (such as O3, O2, NO2, NO, and CO), which have longer half-lives, can interact with the sample in indirect treatment. 3.5. Effect on egg quality The quality parameters for the untreated and HVACP treated eggs are shown in Table 2. No significant difference (p > 0.05) was observed in any of the analyzed parameters. HU of the untreated eggs were 85.4 and after treatment they ranged from 81.5 to 93.8. No significant difference (p > 0.05) in weight was observed after the
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Table 2 Quality parameters of untreated and HVACP treated eggs. Quality parameters
Untreated
HVACP treated
Haugh unit Egg weight Albumen pH Yolk pH Yolk color Vitelline membrane strength
85.4 ± 0.28a 49.8 ± 2.26b 7.21 ± 0.11c 6.22 ± 0.23d 3 ± 2.8e 0.0026 ± 0.0001f
87.65 ± 6.15a 50.4 ± 3.96b 7.21 ± 0.06c 6.23 ± 0.06d 9.5 ± 7.8e 0.0024 ± 0.0001f
aef
p < 0.05.
HVACP treatment suggesting no significant difference in the evaporation rate or CO2 escape from the albumen after the plasma treatment (Caner, 2005). No significant change (p > 0.05) in albumen or yolk pH was observed after the plasma treatment. No change in the vitelline membrane strength was observed after HVACP treatment and the result were comparable with previously published values (Banerjee, 2010). The grading of yolk color was done according to DSM Yolk Color Fan, which is a 15 scales color index to distinguish the yolk color density. Carotenoids are the natural pigments of the egg yolk, giving it a yellow to dark brilliant orange color (Anton, 2007, pp. 1e6). Therefore, the oxidation of carotenoids resulting in color fading could be a great concern for processed shell eggs. However, in this study, no significant difference (p > 0.05) in yolk color was observed after HVACP treatments. It is important to note at this point that, although no undesirable changes in the standard quality attributes were noticed, RGS interaction with the egg proteins, lipids and other components have not been studied. Effect of HVACP generated RGS on the egg chemical composition need further investigation from the consumer safety perspective. 4. Conclusions HVACP treatment in MA65 for 15 min achieved maximum SE inactivation of 5.53 log CFU/egg on TSA and 6.37 log CFU/egg on XLD. HVACP process parameters including gas type, treatment time, and mode of exposure showed strong interactive effects with microbial inactivation. The occurrence of injury was observed for Salmonella cells by plating on selective agar and nonselective agar. No significant (p > 0.05) difference in the egg quality was observed after the HVACP treatment. This study demonstrates the potential of high voltage atmospheric cold plasma to be used as a novel, nonthermal, and environment-friendly decontamination technology for shell eggs. References Al-Ajeeli, M. N., Taylor, T. M., Alvarado, C. Z., & Coufal, C. D. (2016). Comparison of eggshell surface sanitization technologies and impacts on consumer acceptability. Poultry Science, 95(5), 1191e1197. http://dx.doi.org/10.3382/ps/pew014. American Egg Board. (2013). American egg board annual report 2013. Anton, M. (2007). Composition and structure of hen egg yolk Bioactive egg compounds. Springer. Banerjee, P. (2010). The effect of carbon dioxide on lysozyme activity and quality of chicken eggs. Biladeau, A., & Keener, K. (2009). The effects of edible coatings on chicken egg quality under refrigerated storage. Poultry science, 88(6), 1266e1274. Braden, C. R. (2006). Salmonella enterica serotype enteritidis and eggs: A national epidemic in the United States. Clinical Infectious Diseases, 43(4), 512e517. Caner, C. (2005). The effect of edible eggshell coatings on egg quality and consumer perception. Journal of the Science of Food and Agriculture, 85(11), 1897e1902. CDC. (2013). Tracking and reporting foodborne disease outbreaks. Chau, T., & Kao, K. (1996). Optical emission spectra of microwave oxygen plasmas and fabrication of SiO2 films. Journal of Vacuum Science & Technology B, 14(1), 527e532. Chen, Z., & Zhu, C. (2011). Modelling inactivation by aqueous chlorine dioxide of Dothiorella gregaria Sacc. and Fusarium tricinctum (Corda) Sacc. spores inoculated on fresh chestnut kernel. Letters in Applied Microbiology, 52(6), 676e684. http://dx.doi.org/10.1111/j.1472-765X.2011.03061.x.
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Please cite this article in press as: Wan, Z., et al., High voltage atmospheric cold plasma treatment of refrigerated chicken eggs for control of Salmonella Enteritidis contamination on egg shell, LWT - Food Science and Technology (2016), http://dx.doi.org/10.1016/j.lwt.2016.10.051