Decontamination of a rotating cutting tool during operation by means of atmospheric pressure plasmas

Food Control 21 (2010) 1194–1198

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Decontamination of a rotating cutting tool during operation by means of atmospheric pressure plasmas F. Leipold a,*, Y. Kusano a, F. Hansen b, T. Jacobsen b a b

Risø National Laboratory for Sustainable Energy, Technical University of Denmark, DK-4000 Roskilde, Denmark Danish Meat Research Institute, DK-4000 Roskilde, Denmark

a r t i c l e

i n f o

Article history: Received 26 August 2009 Received in revised form 28 January 2010 Accepted 6 February 2010

Keywords: Dielectric barrier discharge (DBD) Listeria Ozone Sterilization

a b s t r a c t The decontamination of a rotating cutting tool used for slicing in the meat industry by means of atmospheric pressure plasmas is investigated. The target is Listeria monocytogenes, a bacterium which causes listeriosis and can be found in plants and food. The non-pathogenic species, Listeria innocua, is used for the experiments. A rotating knife was inoculated with L. innocua. The surface of the rotating knife was partly exposed to an atmospheric pressure dielectric barrier discharge operated in air, where the knife itself served as a ground electrode. The rotation of the knife ensures a treatment of the whole cutting tool. A log 5 reduction of L. innocua is obtained after 340 s of plasma operation. The temperature of the knife after treatment was found to be below 30 °C. The design of the setup allows a decontamination during slicing operation. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Plasma technology has been shown to successfully replace conventional sterilization methods such as autoclaving, ethylene oxide (EtO) treatment, and ionizing radiation (electron beam, gamma radiation). Especially a non-thermal plasma, which operates at temperatures close to room temperature has gained considerable interest, as it has significant advantages compared to conventional sterilization methods: it is non-destructive to heat sensitive material, toxic chemicals like EtO are avoided, it does not degrade bulk properties of the exposed material during treatment. Several mechanisms were initially suspected for destruction of bacteria and spores: reaction with chemically reactive species (like free radicals), electrical interaction and sputtering with charged particles, and exposure to UV radiation. Inactivation of bacteria or alteration of DNA by means of chemically reactive species generated in plasmas have been observed by many groups (Brandenburg et al., 2007; Herrmann, Henins, Park, & Selwyn, 1999; Laroussi, 1996; Laroussi et al., 1999; Laroussi, 2002; Laroussi, Richardson, & Dobbs, 2002; Laroussi, Mendis, & Rosenberg, 2003; Laroussi & Leipold, 2004; Laroussi, Tendero, Lu, Alla, & Hynes, 2006; Laroussi, Hynes, Akan, Lu, & Tendero, 2008; Lu et al., 2008, 2009; Mendis, Rosenberg, & Azam, 2000; Montie, Kelly-Wintenberg, & Roth, 2000; Moreira et al., 2004; Nagatsu et al., 2005; Tang, Lu, Laroussi, & Dobbs, 2008; Yan et al., 2009; Weltmann et al., 2008; Kelly-Wintenberg et al., 1999). Atomic oxygen, ozone, hydroxyl, peroxyl, * Corresponding author. E-mail address: [email protected] (F. Leipold). 0956-7135/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2010.02.006

and NOx radicals are some of the species with demonstrated effects on cells (Laroussi, 2002). Montie et al. (2000) considered membrane lipid alteration caused by fatty acid peroxide formation and protein oxidation as a possible path to affect the cell. In addition, chemical etching can be expected by reactive oxygen radicals (Moreira et al., 2004; Nagatsu et al., 2005). Laroussi and Leipold (2004) and Mendis et al. (2000) suggested that charged particles can accumulate on the outer cell membrane. Electrostatic forces can then lead to rupture of the cell membrane and subsequently cause cell death. Cell rupture was observed for Escherichia coli, but not for Bacillus subtilis. A possible explanation is that the electrostatic forces are not strong enough to cause rupture of spore shells (Laroussi, 2002; Mendis et al., 2000). UV radiation can affect the DNA of a cell so that the cell is not able to replicate, eventually leading to the death of the cell. UV radiation has been found to be partly responsible for the damage to cells when low pressure plasmas are used for sterilization (Gans et al., 2005; Feichtinger, Schulz, Walker, & Schumacher, 2003; Philip et al., 2002). In contrast, UV exposure was found to play a minor role in atmospheric pressure plasma sterilization (Brandenburg et al., 2009; Herrmann et al., 1999; Laroussi, 1996; Laroussi & Leipold, 2004; Montie et al., 2000). Listeria monocytogenes is a bacterium which can be found in plants and food and is known to cause a serious disease (listeriosis) in humans, especially the elderly and immunocompromised. Also pregnant women are especially prone to L. monocytogenes infection, which leads to damage or even death of the foetus. Research has been done to investigate the inactivation of Listeria using plasmas or reactive species which can be generated in a plasma (Critzer, Kelly-Wintenberg, South, & Golden, 2007; Fan, Song, Mcrae,

F. Leipold et al. / Food Control 21 (2010) 1194–1198

Walker, & Sharpe, 2007; Perni, Shama, & Kong, 2008; Rowan et al., 2007; Vaz-Velho, Fonseca, Silva, & Gibbs, 2001; Wade et al., 2003). The methods involve direct treatment, where the contaminated sample is exposed to the plasma (Perni et al., 2008; Rowan et al., 2007) and remote treatment, where the contaminated surface is exposed to the reactive species (O3 , NO) generated in a plasma (Critzer et al., 2007; Fan et al., 2007; Vaz-Velho et al., 2001). Listeria innocua is often used as a non-pathogenic representative for L. monocytogenes. However, it must be ensured that L. innocua can be used as a biological indicator (BI) instead of L. monocytogenes for a certain treatment. Although L. innocua can serve as a BI for thermal treatments (Fairchild & Foegeding, 1993) it is not of relevance for this work, since we operate at temperatures below 30  C. Vaz-Velho et al. (2001) exposed both strains, L. monocytogenes and L. innocua to an ozone atmosphere. They reported a higher resistance of L. innocua to ozone compared to L. monocytogenes. Consequently, the results obtained with L. innocua provide an additional safety margin. Cross contamination of meat with bacteria by using the same knife for several batches of meat is a major concern in meat industry. A decontamination method which is capable of sterilizing the knife continuously during operation can significantly reduce the risk of cross contamination. The objective of this investigation is to demonstrate the inactivation of L. innocua by direct treatment with an atmospheric pressure plasma on the stainless steel surface of a cutting tool used in an industrial meat slicing unit while it is in operation. Since the slicing area on the knife is only a section of the whole knife surface, the DBD can be installed at another section and operate during the slicing process. A knife from industrial meat slicing unit was used for this demonstration.

2. The experimental setup The surface of a rotating circular knife (see Fig. 1) will be exposed to a plasma generated by a dielectric barrier discharge (DBD). The DBD consists of the knife as a ground electrode and an aluminum electrode. The aluminum electrode is water cooled. A ceramic plate ðAl2 O3 Þ in contact with the aluminum electrode serves as dielectric barrier between the electrode and the knife. The knife has a diameter of approximately 450 mm and the powered aluminum electrode has a size of 100 mm  100 mm. The distance between ceramic plate and knife is not constant, because the

Side View Knife

Knife

2nd DBD

Ceramic

Plasma

Ceramic Electrode

Water Cooling

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surface of the knife forms a shallow cone and the ceramic sheet is flat. The closest distance between ceramic plate and knife is 2 mm (at the center of the electrode, point A in Fig. 1), and the longest distance is 4 mm (at the edge of the electrode, point B in Fig. 1). On top of that, the distance between ceramic plate and knife varies slightly during rotation since the surface of the knife is mechanically not a perfect cone. The fluctuation during one revolution of the knife is ±0.17 mm along the path indicated by the inner dashed line in Fig. 1 and ±0.26 mm along the path indicated by the outer dashed line in Fig. 1. The operation gas is ambient air at atmospheric pressure. Applying a high AC voltage between the electrode and the knife generates a plasma between the electrode and the surface of the knife. Frequency and voltage were set to 21.7 kHz and 10.4 kV zero-peak, respectively. This setting corresponds to a DBD power of 360 W. In order to reduce the power, one sinusoidal voltage curve with a period of 46 ls followed by a pause of 4:6 ms ð9:2 msÞ was applied. The duty cycle of 1:100 (1:200) results in an average power of 3.6 W (1.8 W). The rotation speed of the knife during experiments is 3.4 s per revolution. The rotation speed in industrial slicing units is significantly higher. However, the time in which a microorganism is exposed to the plasma, is independent of the rotation speed. The low rotation speed ensures that microorganisms are not lost due to excessive centrifugal forces during experiments. For these experiments, only one side of the knife is treated, but for industrial applications, this technique can easily be applied to both sides. This requires a second electrode and ceramic sheet on the other side of the knife forming a second DBD. The knife serves as ground electrode for both DBDs. The second DBD is depicted with gray lines in the sketch in Fig. 1. The discharge power was set to 1.8 W, 3.6 W, and 0.36 kW. The temperature of the knife was measured by means of a thermo-couple element and was found to be below 30  C in the case of a 0.36 kW power setting. This ensures that heating does not contribute to inactivation of microorganisms. The ozone concentration between the electrodes are measured by means of UV absorption at 254 nm. Fig. 2 shows a schematic of the ozone diagnostic. A sample of air between the electrodes is pumped into the absorption cell. A Hg-lamp serves as a UV source. The UV light is guided through the cell. An interference filter filters out a small spectral range around 254 nm, where ozone has the highest absorption cross section (Orphal, 2003). The filtered UV light is detected by means of a UV sensitive photo diode. The ozone concentration can be calculated by the ratio of the UV intensity between plasma on and plasma off. The measured ozone concentrations for the various power settings are shown in Table 1. The fluctuation in the ozone concentration is due to the slightly changing distance between knife and electrode during knife rotation and consequently a change in the plasma properties. The NO concentration in the same air sample was measured by means of a detector V-RAE (RAE Systems) and was found to be less than 1 ppm for the power settings 1.8 W and 3.6 W and 102 ppm for a power setting of 0.36 kW (see Table 1). The decrease of the ozone concentration with increasing power cannot be explained in a simple way. The formation of ozone in a DBD operated in air is a part of the complex plasma chemistry and is detailed in

B A

Electrode

Fig. 1. Photography and sketch of the experimental setup. The electrode consists of an aluminum plate with a size of 100 mm  100 mm. The ceramic, Al2 O3 , is in contact with electrode and serves as dielectric layer between the electrode and the knife. The gray lines in the sketch depict an electrode for a second DBD in order to treat both sides of the knife. The distance between ceramic sheet and knife is 2 mm at the position A and 4 mm at the position B. The change of distance along the path indicated by the inner (outer) dashed line during one revolution due to mechanical inaccuracies is 0:17 mm ð0:26 mmÞ.

Fig. 2. UV absorption spectroscopy unit for ozone measurement in the DBD. L: quartz lenses, W: quartz windows, F: interference filter with a transmission maximum at 254 nm, and D: UV detector diode.

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Table 1 Measured O3 and NO concentration in the DBD for various power settings. Power (W)

O3 concentration (ppm)

NO concentration (ppm)

1.8 3.6 360

49–88 78–130 <2

<1 <1 102

the results will not change. Cell detachment during operation has not been investigated. However, since no reduction up to 70 s was observed for a treatment power of 1.8 W, a significant loss of cells during operation is not likely. Therefore we assumed no cell detachment during operation.

4. Results

3. Materials and methods Initially the knife was inoculated by evenly spraying approximately 240 ll of a L. innocua inoculum ð5  107 cfu=mlÞ onto the surface of the slicing knife. The inoculum was prepared by culturing the L. innocua (Danish Meat Research Institute (DMRI), strain 0011) in BHI (OXOID CM0225) for 18–20 h at 30 °C. Afterwards the overnight culture ð5  108 cfu=mlÞ was diluted 1:10 in 0.85% NaCl. After the plasma treatment, the knife was sampled using sterile, gauze swabs (100 mm  100 mm Cutisoft Cotton, BSN Medical 72227-09) moistened with approximately 5 ml FKP (0.85% NaCl + 0.1% peptone). The surface of the knife was wiped with the gauze swab by pressing it firmly on to the knife while rotating it one revolution. Afterwards the swab was placed in a stomacher bag and transported from Risø to DMRI in a cooling box at approximately 4 °C. The control has been performed using the same procedure without switching on the plasma. A change in the cell viability due to the delay between treatment and culturing should not affect the results since both control sample and treated samples were exposed to the same procedure. Each plasma treatment and the control were done in four replicates. Between each sampling, the knife was disinfected by wiping the surface in 70% ethanol. In the laboratory (a few hours later), 25 ml 0.85% NaCl was added to each stomacher bag and gauze swab, and treated for 60 s in a stomacher. The initial suspension was afterwards diluted 10-fold in FKP. For enumerating of Listeria, 0.1 ml from the initial suspension and 0.1 ml of relevant dilutions were spread onto Oxford agar (OXOID CM856 + SR206) using a sterile drigalsky spatula. The Oxford agar plates were incubated at 37 °C for 2–3 days and subsequently the number of typical colonies was recorded. In order to increase the sensitivity by a factor of 10 for the power setting of 360 W, 1 ml of relevant dilutions were spread on three agar plates and the number of colonies on each were added. A reduction of the microorganism sticking to the surface was measured. However, when a constant loss of microorganisms for treated samples and control samples is assumed, the ratio of colonies between treated samples and control is not affected. The same is true for the collect and release efficiency for the gauze swap and stomacher step. As long as the efficiency is constant for treated and control samples,

The results show an almost negligible inactivation of L. innocua for a operation time shorter than 34 s at any power setting. For a reduced power setting of 1.8 W and 3.6 W, a log 2 and 3 reduction could be observed after an operation time of 170 s and eventually leading to a log 4 reduction after 340 s. A fairly high inactivation (log 4) can be detected at a power setting of 360 W of the DBD after 68 s operation time, while a further increase of operation time to 340 s leads only to an enhanced inactivation of one more log unit to a log 5 reduction in total. As the detection limit in the spread plate technique for enumerating L. innocua in the first experiments is 10 cfu/ml, the observed reduction in the experiments with a power setting of 1.8 W and 3.6 W cannot exceed 4 log cfu. The detection limit for the experiment with a power setting of 0.36 kW was improved to 1 cfu/ml, shifting the observed reduction to a maximum of approximately 5 log cfu. Therefore, the actual inactivation could be higher than indicated after 340 s operation time. The results are shown in Fig. 3. The solid symbols represent the average value of the four replicates and the open symbols represent the standard deviation. Due to the geometrical design of this experimental setup, the plasma cannot be considered homogeneous over the surface. This is attributed to the different distances between the ceramic sheet and the knife as indicated in Section 2. Furthermore, since the powered electrode has not the shape of a section of a circle, the microorganisms sitting closer to the center of the knife (along the inner dashed line in Fig. 1) are longer exposed to the plasma than these sitting farer away from the center of the knife (along the outer dashed line in Fig. 1). In addition, the plasma varies slightly at different azimuths of the knife due to distance fluctuation during rotation. The cause of these fluctuations is explained in Section 2. This means that the results presented here is an average over the entire surface of the knife which is exposed to the plasma. The spraying technique for inoculating the knife does not guarantee a homogeneous distribution of bacteria over

0

1

Reduction [log10 ]

Samoilovich, Gibalov, and Kozlov (1997). The ozone and NO concentration were measured after adjusting the power setting and before the experiments were done. In addition, random measurements were recorded during the experiments in order to detect a possible change. Principally, the ozone and NO concentration can be measured continuously. Reactive species like ozone, atomic oxygen, and nitrogen oxide are assumed to contribute most to the decontamination effect in this work as suggested in Laroussi (2002). For higher power settings the contribution of electrostatic forces to cell inactivation can be expected to increase. The operation time of the unit was varied between 34 s and 340 s. The ratio between the discharge area and the plasma overlayed area of the knife during one revolution is 12.2%. Therefore, the exposure time of the knife surface to the plasma in average is only 12.2% of the operation time. The inoculation and evaluation procedure is described in Section 3. A photo and a sketch of the experimental setup is shown in Fig. 1.

2

3

4

5

6

0

50

100

150

200

250

300

350

Operation Time [s] Fig. 3. Inactivation versus the operation time of the plasma. The effective exposure time of the bacteria to the plasma is 12.2% of the operation time. Triangles: 1.8 W, diamonds: 3.6 W, and circles: 0.36 kW. The solid symbols represent the average value and the open symbols represent the standard deviation.

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the knife surface. The bacteria will be concentrated in the droplets on the knife which in turn can cause a multi-layer structure of microorganism. This multi-layer structure can protect microorganism laying underneath. This can make the decontamination method less efficient or requires longer treatment times compared to the decontamination of a single layer of microorganism.

5. Discussion A significant increase of inactivation occurs between 68 s and 170 s of operation time. The reduction after 170 s is between log 4 and 5 for a power setting of 360 W. An increase in treatment time does not significantly improve the inactivation. It was found to be log 5 after an operation time of 340 s for a power setting of 360 W. Although the ozone concentration was found to be low (less than 2 ppm) a significant inactivation rate could be achieved. Increasing the power of the DBD above a certain limit reduces the ozone production but increases the NO production (Samoilovich et al., 1997). In a very simplified picture, this can be explained as follows: The mean electron energy in non-equilibrium dry air discharges is typically in the range between 2 and 6 eV. For low reduced electric fields corresponding to a low mean electron energy (approximately 3 eV), oxygen dissociation is the most efficient reaction (Penetrante et al., 1997) and ozone can form by the reaction O þ O2 ! O3 . The dissociation rate of nitrogen at a mean electron energy of 3 eV is two orders of magnitude less than the dissociation rate for oxygen. Increasing the reduced electrical field corresponding to a mean electron energy of 4 eV (or higher) will cause the dissociation efficiency of nitrogen to increase while the dissociation efficiency of oxygen changes only slightly. Oxygen atoms and ozone can additionally form NO with nitrogen atoms which in turn causes a reduction of the ozone concentration. The complex chemistry of air plasmas is detailed in Samoilovich et al. (1997). According to Laroussi (2002), Critzer et al. (2007), NO is an antimicrobial active species and contributes also to the inactivation process. Increasing the power increases also the number of charged particles. Charged particles can cause cell rupture due to electrostatic forces (Laroussi, 2002; Mendis et al., 2000) and can contribute to the inactivation of microorganisms. Further increase in power will again lead to a reduction of NO (Leipold, Fateev, Kusano, Stenum, & Bindslev, 2006; Samoilovich et al., 1997). Vaz-Velho et al. (2001) exposed a pure culture of L. innocua on agar plates to ozone in a chamber (concentration approximately 50 ppm) and found a log 3.5 reduction after an exposure time of 20 min and above. Although the ozone concentration in the chamber increased after 20 min, a further reduction was not observed. Although this ozone concentration corresponds to the ozone concentration in our DBD for a power setting of 1.8 W, where we achieved a reduction of log 3.5 after approximately 300 s of operation time (37 s of exposure time) it is difficult to compare these results due to the different type of treatment. Similar experiments have been carried out by Fan et al. (2007), where L. innocua cultures were exposed to an ozone atmosphere. The ozone concentration was between 50 ppb and 100 ppb. This is significantly less than the ozone concentration in our DBD. This might explain the rather long exposure time of 1.3 h at 20 °C to obtain a log 2 reduction. Critzer et al. (2007) exposed L. monocytogenes on agar plates to the exhaust of a plasma operated in air and reported a reduction of log 4 after an exposure time of 10 s and a further reduction of log 4.5 after an exposure time of 30 s. In spite of the different methods (remote and direct treatment) these results are in good agreement with the reduction obtained at a power setting of 360 W in this work, where a reduction of log 4 is achieved after 8 s of exposure time and a log 4.5 reduction after an exposure time of 20 s. This comparison supports our initial assumption that chemically reactive species

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contribute most the decontamination effect, since UV radiation and cell rupture caused by electrostatic forces are not present in Critzer’s experiments (Critzer et al., 2007). The slightly shorter exposure time for reduction in our experiments may be attributed to the presence of electrostatic forces as an additional mechanism for cell inactivation. Although the concentrations of reactive species are on the same order for the power settings of 3.6 W and 360 W, the moderate increase in cell inactivation (one log unit for an exposure time of 40 s) can be attributed to inactivation by means of electrostatic forces. This suggests that electrostatic forces contribute only for high power settings significantly (in this case an increase by a factor of 100 is required for an improvement of 1 log unit) and inactivation by means of chemically reactive species is the dominant mechanism for cell inactivation at low power settings. Perni et al. (2008) exposed tissues with L. monocytogenes directly to a low temperature discharge operated in an He=O2 mixture. A reduction below the detection limit was reported after 1.5 s. The high standard deviation in our results is most likely due to the challenging design of the experiment. We have chosen a design, which can principally be applied to a knife in an industrial slicing unit and operated during the slicing process. Changes of the shape of the electrodes may be required in the practical application. Industrial setups require usually higher tolerance for the experimental parameters than laboratory setups. One example is the variation of the distance between the electrodes during operation when one electrode is moving. 6. Conclusion Inactivation of L. innocua can efficiently be performed by direct treatment of a material by means of a DBD. A log 5 reduction of L. innocua could be achieved on an industrial rotating cutting tool while the tool is in operation. The required operation time of the cutting tool was 340 s. This corresponds to an effective treatment time of approximately 41 s, because the DBD does not cover the entire cutting tool. This in turn allows applying this decontamination technique during the slicing process and provides the potential to reduce the risk of cross contamination between separate batches of meat. Acknowledgments This work was funded by the Danish Meat Research Institute, Project No. 18557, and the National Laboratory for Sustainable Energy, Technical University of Denmark, Funds for Innovation No. 200800423. References Brandenburg, R., Ehlbeck, J., Stieber, M., von Woedtke1, T., Zeymer, J., Schluter, O., et al. (2007). Antimicrobial treatment of heat sensitive materials by means of atmospheric pressure rf-driven plasma jet. Contributions to Plasma Physics, 47(1–2), 72–79. Brandenburg, R., Lange, H., von Woedtke, T., Stieber, M., Kindel, E., Ehlbeck, J., et al. (2009). Antimicrobial effects of UV and VUV radiation of nonthermal plasma jets. IEEE Transactions on Plasma Science, 37(6), 877–883. Critzer, F. J., Kelly-Wintenberg, K., South, S. L., & Golden, D. A. (2007). Atmospheric plasma inactivation of foodborne pathogens on fresh produce surfaces. Journal of Food Protection, 70(10), 2290–2296. Fairchild, T. M., & Foegeding, P. (1993). A proposed nonpathogenic biological indicator for thermal inactivation of Listeria monocytogenes. Applied and Environmental Microbiology, 59(4), 1247–1250. Fan, L., Song, J., Mcrae, K. B., Walker, B. A., & Sharpe, D. (2007). Gaseous ozone treatment inactivates Listeria innocua in vitro. Journal of Applied Microbiology, 103(6), 2657–2663. Feichtinger, J., Schulz, A., Walker, M., & Schumacher, U. (2003). Sterilisation with low-pressure microwave plasmas. Surface & Coating Technology, 174, 564–569. Gans, T., Osiac, M., O’Connell, D., Kadetov, V. A., Czarnetzki, U., Schwarz-Selinger, T., et al. (2005). Characterization of stationary and pulsed inductively coupled rf discharges for plasma sterilization. Plasma Physics and Controlled Fusion, 47(Sp. Iss. SI Suppl. 5 A), A353–A360.

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