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Low Temperature Plasma for decontamination of E. coli in milk
International Journal of Food Microbiology 157 (2012) 1–5
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International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro
Low Temperature Plasma for decontamination of E. coli in milk C. Gurol a, F.Y. Ekinci b, N. Aslan c, M. Korachi a,⁎ a b c
Department of Genetics and Bioengineering, Yeditepe University, Istanbul Turkey Department of Food Engineering, Yeditepe University, Istanbul Turkey Department of Physics, Yeditepe University, Istanbul Turkey
a r t i c l e
i n f o
Article history: Received 29 September 2011 Received in revised form 15 February 2012 Accepted 20 February 2012 Available online 24 February 2012 Keywords: Plasma Corona discharge Decontamination Milk Processing technology
a b s t r a c t Raw milk is a natural, highly nutritious product and a quick and easy supplement for human dietary requirements. Elimination of bacteria in milk has been a problem for decades and new methods with regards to nonthermal applications which do not harm the chemical composition of milk, are currently under investigation. The objective of the study was to determine the potential use of a novel, Low Temperature Plasma (LTP) system for its capability of killing Escherichia coli in milk with different fat contents. The time dependent effect of atmospheric corona discharge generated with 9 kV of AC power supply on E. coli ATCC 25922 dispersed in whole, semi skimmed and skimmed milk was examined. Plasma was applied at time intervals of 0, 3, 6, 9, 12, 15 and 20 min. A significant 54% reduction in the population of E. coli cells after only 3 min was observed regardless of the fat content of the milk. The initial pre-plasma bacterial count of 7.78 Log CFU/ml in whole milk was decreased to 3.63 Log CFU/ml after 20 min of plasma application. LTP did not cause any significant change to the pH and color values of raw milk samples. No viable cells were detected after one week examination in whole milk samples and remained so over the 6 week storage period. The findings of this study show that the novel LTP system tested was able to significantly reduce E. coli in milk by more than a 3 fold log reduction without significantly affecting pH or color properties. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Infectious diseases caused by the ingestion of pathogenic bacteria in contaminated milk are still a major health concern, especially for children (Tiozzo et al., 2011; Fuguay et al., 2011; Todd and Notermans, 2011). While the nutritional benefits of milk consumption are documented, the hazards of drinking untreated milk are also well known. The most predominant infectious diseases caused by contaminated milk include campylobacteriosis (Fuguay et al., 2011), salmonellosis (Poppe, 2011; Tiozzo et al., 2011), yersiniosis (Greenwood and Hooper, 1990), listeriosis (Todd and Notermans, 2011; Yilmaz et al., 2009), tuberculosis (Doran et al., 2009), brucellosis (Ramos et al., 2008), staphylococcal enterotoxin poisoning (Ostyn et al., 2010; Soejima et al., 2007), streptococcal infections (Wyder et al., 2010) and Escherichia coli 0157:H7 infection (Anand and Griffiths, 2011; Martin et al., 1986). Although, the etiological agents in milkborne diseases are numerous, more than 90% of all reported cases of dairy related illnesses are of bacterial origin (Bean et al., 1996). It is therefore standard procedure to properly treat raw milk, to inactivate these pathogens before consumption, either by pasteurization or Ultra High Temperature (UHT). ⁎ Corresponding author at: Yeditepe University, Faculty of Engineering and Architecture, Genetics and Bioengineering Department, Kayisdagi, Istanbul, Turkey. Tel.: + 90 216 578 2653; fax: + 90 216 578 0829. E-mail address: [email protected] (M. Korachi). 0168-1605/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2012.02.016
However, the current thermal decontamination methods are known to induce changes to the chemical and physiological composition of milk and milk products (Topcu et al., 2006; Datta and Deeth, 2003). These changes include: browning, flavor changes (McKellar, 1981), serum protein denaturation resulting in a whiter appearance fat agglomeration and solid destabilization, freezing point depression, possibility of adulteration with condensate and boiler compounds and partial loss of some vitamins (Topcu et al., 2006). Furthermore, UHT treatment renders the milk unsuitable for further processing such as cheese and yogurt production. Due to its complex structure milk has so far shown to be highly sensitive to many current novel technologies (Grahl and Markl, 1996). Therefore, the lack of a technology which enhances microbial food safety and quality, while simultaneously protecting the nutritional, functional and sensory characteristics of foods, has created a huge interest in low-temperature innovative processing techniques. These innovative technologies mostly rely on physical processes, including high hydrostatic pressures, pulsed electric fields and low-temperature plasmas that inactivate microorganisms at ambient or moderately elevated temperatures and short treatment times (Barbosa-Canovas et al., 1997; Korachi et al., 2009). To date the sterilization efficacy of plasma has been studied on medical instruments (Laroussi, 2002), food sanitation (Basaran et al., 2008), textiles (Morent et al., 2008), and water (Korachi et al., 2009). Plasma corona discharge (PCD) causes the formation of active species such as: UV light, free radicals and ions, ozone and atomic oxygen, which contain sufficient energy to break covalent bonds and
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initiate various chemical reactions (Moisan et al., 2001). The antimicrobial activity of plasma treatment is a result of direct contact with these active species (Moisan et al., 2002). Moreover, these active species have been said to disappear in milliseconds after the plasma system is switched off (Korachi et al., 2009). The three basic mechanisms that have been attributed to the inactivation of microorganisms by plasma are the destruction of DNA, volatilization of compounds, and etching of the cell surface by adsorption of reactive species (Korachi et al., 2010; Korachi and Aslan, 2011). These effects may vary due to the operational conditions and the design of the plasma generator. The ability of plasma to work at low temperatures without increasing the operating temperature has opened up the possibility of using LTP for the treatment of heat-sensitive materials (Song et al., 2009; Morent et al., 2008). The elimination of pathogenic bacteria in milk has been a problem for decades and new methods with regards to non-thermal applications which do not harm the chemical composition of milk, are highly required. In this study, the potential use of a novel, simple, LTP system was investigated for its capability of killing E. coli in milk samples with different fat contents. Although there has been increasing interest into the use of plasmas as a sterilizing technique, this is the first study that tests the use of plasma technology as a decontaminating technique in milk. 2. Materials and methods 2.1. Milk samples and cultures Commercial UHT and raw milk samples were obtained from a local market and farm respectively in Istanbul, Turkey. The UHT samples included whole (3% fat), semi-skimmed (1.5% fat), and skimmed milk (0.1% fat). E. coli ATCC 25922 was provided from Yeditepe University, Genetic and Bioengineering Department Culture Collection (Turkey). E. coli ATCC 25922 was grown on Tryptic Soy Agar (TSA) (Merck, Germany) and in Nutrient Broth (NB) (Merck, Germany) at 37 °C for 48 h. The identity of the strain was confirmed by Gram staining and commercially available identification kits based on biochemical assays. Tests were carried out according to the manufacturer's instructions (Biomerieux, France). Culture was stored in the appropriate growth medium containing 20% glycerol at − 70 °C. Viable counts were performed according to standard methods on TSA and expressed in Log CFU/ml. 2.2. Microbial preparation UHT milk was checked for any bacterial growth at the start of the experiment by plating onto TSA. A bacterial stock solution was prepared by inoculation of the test strain in NB and incubating at 37 °C for 48 h. Following the incubation, 0.1 ml of the bacterial solution was suspended in 4.5 ml of NB and the cultures were grown at
37 °C in a shaking water bath (200 rpm) for 3 h. The growth curve was monitored photometrically by the optical density (OD) at 600 nm (UV/VIS Spectrophotometer, Lambda 3B, Perkin Elmer). When the OD approximately reached 0.2 (the middle of the loggrowth phase), 100 μl of the cell suspension was pipetted into 15 ml of sterile milk in a sterile petri dish. The final concentration of the milk suspension was adjusted to 7.78 Log CFU/ml by performing serial dilutions. 100 μl of bacterial suspension was also plated onto the agar media at zero time and incubated at 37 °C for 48 h as a control. 2.3. Low temperature atmospheric pressure plasma system The atmospheric plasma discharge system previously described (Korachi et al., 2009, 2010; Korachi and Aslan, 2011) was used with some modifications (Fig. 1). Results from previous studies with a similar LTP system (Korachi et al., 2009, 2010; Korachi and Aslan, 2011) showed the DC power supply to be more destructive on test materials. Therefore, a 9 kV AC power supply, a simple ballast circuit and two tungsten electrodes (0.8 mm radius) were used for milk applications. Low Temperature Plasma was applied to the contaminated milk solution by applying high voltage between the upper electrode tip and the liquid surface (in which the other electrode was immersed). The upper electrode, which was sterilized after each application, was rotated by an adjustable DC motor and a motorized stirrer was attached to maintain application homogeneity. The temperature was monitored throughout discharge application by the use of a thermometer and the system shut down whenever the temperature reached the allowed maximum temperature (35 °C). The current of nearly 90 mA in the plasma corona resulted in nearly 10 A/cm 2. In order to cool the system, it was lowered to contact with the surface of an icepack. 2.4. Light emission spectroscopy of plasma discharge system The light emission intensities of the AC plasma discharge were determined by a UV–visible emission spectrometer (Baki, Turkey) manufactured by the Laser Technologies Laboratory of Kocaeli University, Turkey. The light from the discharge was transferred through a fiber cable to the spectrometer and the emissions were recorded between wavelengths of 200 and 900 nm. The peaks appearing in the spectrum indicate the ionization levels of the molecules making up the plasma discharge identified using the NIST Atomic Spectra Database (Yu et al., 2008). 2.5. Plasma application The apparatus described above was placed in a Laminar Flow cabinet (Heal Force, China) to prevent contamination. Following bacterial inoculation, the milk samples were immediately exposed to LTP and
Ballast circuit DC motor
Thermometer
HV + POWER _ SUPPLY
Tungsten electrode
Stirrer
Petri dish Lower electrode Ice pack
Fig. 1. Schematic view of atmospheric plasma corona discharge experimental setup.
C. Gurol et al. / International Journal of Food Microbiology 157 (2012) 1–5
the temperature was kept below 35 °C throughout the experiment. One hundred microliters of samples was taken at each time interval of 3, 6, 9, 12, 15 and 20 min, and plated onto both TSA and violet red bile agar (VRBA). The plates were incubated at 37 °C for 48 h and results were expressed as Log CFU/ml. 2.6. pH analysis The experiment above was repeated for pH analysis using raw milk. The pH was measured with a pH meter (Mettler Toledo, USA) throughout the treatment time. The pH of post-plasma treated and control raw milk was compared to detect any changes in the hydrogen concentration due to plasma discharge. 2.7. Color measurement tests A portable spectrophotometer, CM-600 d (Konica Minolta, USA) was used for the color analysis in raw milk. In this system, an illuminator with 8° diffused illumination and 8° visioning angle was used. The light source used was a pulsed xenon lamb with UV cut filter and the wavelength ranged from 400 nm to 700 nm pitching every 10 nm. The light was detected with a silicon photodiode array. Since temperature affects the color values all samples were warmed to room temperature before color measurement were taken. Results were shown according to the CIE-LAB system which includes L*: luminance or lightness component (ranging from 0 to 100), a*: green to red (ranging from −60 to +60) and the b* component: blue to yellow (ranging from −60 to +60). The overall color difference was expressed by means of Delta-E (dE) which indicates the distance between two colors. Using target L*1, a*1 and b*1 measurements and the sample L*2, a*2, and b*2, dE was calculated using the following equation: dE ¼
h
L2 –L1
2
2 2 i1=2 þ a2 –a1 þ b2 –b1
ð2Þ
Depending on the dE values, the color difference was evaluated as not noticeable (0–0.5), slightly noticeable (0.5–1.5), noticeable (1.5–3) and well visible (3–6). 2.8. Storage test Control, 9 min and 20 min plasma-treated samples were stored under cold conditions (4–7 °C) for a total period of 6 weeks. The microbial load of milk samples were monitored by taking 100 μL samples and cultivating on TSA every 7 days. Viable counts were performed and expressed in Log CFU/ml. 2.9. Statistical analysis All experiments were repeated three times. Minitab software version 16 (Minitab, Inc., State College, PA) was used for data analysis. Kruskal–Wallis was performed to compare the mean values of the response variable and treatment conditions. Evaluations were based on a significance level of P b 0.05. 3. Results and discussion 3.1. Light emission spectroscopy results The light emission intensity spectrum of the discharge used in this study showed peaks indicating the ionization levels of the ions in the discharge (Fig. 2). The data were compared and identified according to NIST Atomic Spectra Database available online (Yu et al., 2008). The emission intensity spectrum of atmospheric pressure AC corona discharges with a frequency of 50 Hz was obtained from the plasma
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surrounding the tip of the electrode in air. Several main peaks were identified belonging to oxygen (O), nitrogen (N) and argon (Ar) molecules as is expected because the plasma system operates in air. Since the emission spectra were taken at the tip of the electrode (~1 cm above the milk surface), no major peaks from organic materials were observed as would be the case if the optical fiber of the spectroscope was placed closer to the milk surface. The oxygen peaks in this system corresponded to the positively charged oxygen ions created by electron impact. It is believed that these ions accelerate towards the liquid surface creating ozone and other active species (with very short lifetimes). It was further observed that since the melting temperature of tungsten is 3422 °C, the tungsten tip did not melt and create deposits on the milk surface as has previously been observed with the use of chrome and nickel electrodes (Korachi et al., 2009). 3.2. Microbiological results The time dependent effect of atmospheric corona discharge generated with 9 kV of AC power supply on E. coli ATCC 25922 dispersed in milk was examined. Table 1 represents the effects of the plasma corona discharge on E. coli cell viability (Log CFU/ml) in whole, semiskimmed, and skimmed milk. A significant 54% reduction in the population of E. coli cells following 3 min LTP treatment was observed for all 3 types of milk-fat samples. The initial pre-plasma bacterial count of 7.78 Log CFU/ml in whole milk (Table 1) was decreased to 3.63 Log CFU/ml at time intervals up to 20 min. Similar significant reductions were also observed in semiskimmed milk (P b 0.007), and skimmed milk (P b 0.009). These results indicate that the LTP system used in this study can reduce the number of E. coli in milk regardless of its fat content. This is a positive factor since several studies have shown that decontamination of milk is more difficult than that of model buffer solutions because of the complex structure of milk (Goff and Hill, 1993; Martin et al., 1997). Tests using PEF technology have shown that the fat content of the milk inversely affected microbial inactivation via protection against electric pulses (Grahl and Markl, 1996). Although a study by Reina et al. (1998) and El-Hag et al. (2008) using PEF to treat whole and skimmed milk inoculated with Staphylococcus aureus and Listeria monocytogenes, found similar results to our findings, these controversial findings are probably due to the different designs and working parameters of different systems. Published studies have shown that the initial bacterial concentrations, treatment duration and the structure of the supporting medium all play a role in the antimicrobial efficacy of LTP applications (Laroussi and Leipold, 2004; Ohkawa et al., 2006). To date, there are no studies on the inactivation of food-borne pathogens in milk by LTP for comparison. However, our findings have revealed that the fat content of milk does not affect the susceptibility of E. coli ATCC 25922 to plasma treatment, thus revealing a potential advantage of this decontamination system for possible use in fatty substrates. Current thermal treatments use processing parameters of 63 °C for 30 min (batch method) or 71 °C for 15 s (flash method) for pasteurization and 135 °C for 1 or 2 s for UHT. Although these parameters are for treatment of milk from all pathogens, a detailed study on the effect of UHT treated milk using direct and indirect methods has shown that significant changes occur to milk's color, nutritional and organoleptic properties (Elliot et al., 2005). Since this novel technology operates at both a lower temperature and induces its effect on E. coli in a short time of 3 min, thereby minimizing the damaging effects on the color and pH properties of milk that has been previously described with traditional thermal treatments (McKellar, 1981; Topcu et al., 2006; Datta and Deeth, 2003). 3.3. pH analysis The hydrogen concentration in a solution is typically important while determining a system's decontamination capabilities. Atmospheric
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20 18
Intensity (au)
16 14 12 10 8 6 4 2 0 3500
4000
4500
5000
5500
6000
6500
7000
7500
8000
8500
Wavelength (A) Fig. 2. The emission intensity spectra of atmospheric pressure AC corona discharge.
Milk samples did not show any important changes in color after 0, 3, 6, 9, 12, and 15 min of 9 kV plasma treatments and only a slight change in comparison with the untreated milk occurred after 20 min (Table 2). The color values of raw milk samples before and after plasma application at 9 and 20 min are shown in Table 2. Analytical measurements confirmed that the same color parameters L*, a*, and b* and total color difference (ΔE) existed between the control samples and respective plasma treated samples after processing. The total color difference for milk after 9 min of plasma treatment with 9 kV was 0.25 while longer exposure to plasma (20 min) caused slightly higher color differences with a ΔE value of 0.52 suggesting that plasma application resulted in no or slightly noticeable color differences in milk samples.
number of E. coli decreased from 4.18 to 3.08 Log CFU/ml after 4 days of storage for 9 min plasma applied samples and reached undetectable levels after day 6 (p b 0.001). On the other hand, the levels of E. coli for 20 min plasma applied samples were undetectable after one day storage and remained thereafter at the end of 6 weeks. No viable cells were detected after one week examination and remained so over the 6 week storage period. The immediate decrease in bacterial growth following plasma application is expected and can be attributed to the effect of the reactive species formed by plasma, that directly target the bacterial cell (Korachi et al., 2009). Of more interest was the total reduction of all microorganisms during the first week of storage. These findings suggest that the surviving bacterial cells, post plasma application were inactivated during storage. The findings correlate with those of Song et al. (2009), where the number of L. monocytogenes decreased during storage following Low Temperature Plasma treatment. The decrease in milk's nutrient content (required for bacterial growth) and the accumulation of bacterial by-products over storage time of 42 days could be a possible contributing factor in cell death. However it is probable that the main factor could be the persistent presence of the active species produced by the LTP system. Further investigations are required in order to gain a better understanding of the predominant reason for cell death during storage.
3.5. Preliminary storage analysis
4. Conclusion
The decontamination technologies for milk aim to reduce the number of harmful bacteria and ultimately achieve microbial safety during shelf life. Preliminary storage tests were therefore carried out for 6 weeks on plasma treated whole milk samples following 9 min and 20 min application. Colony numbers (Log CFU/ml) of E. coli in whole milk during storage at 4 °C for a period of 0–6 weeks. The control of untreated milk showed a decline in the CFU's at the 6th week. This can be explained by the depletion of nutritional content in the milk due to and required for bacterial growth. In comparison, samples analyzed immediately following plasma treatment (at zero time) revealed a significant decrease in the number of CFU's (4.18 after 9 min; 3.63 after 20 min) from an initial concentration of 7.78 Log CFU/ml. The
Food processing technologies are turning to the use of low processing temperatures in order to maintain food's safe, natural and “freshlike” characteristics. However to date no technique has been able to simultaneously decontaminate and extend the product's shelf life while maintaining its beneficial properties. Using the advantages of cold plasma, this system was tested for its ability to decontaminate E. coli in milk. The time and storage parameters of LTP were tested on E. coli ATCC 25922 in different types of milk and during storage. It was found that this plasma system significantly reduced the number of colonies in different types of milk (whole, semi-skimmed, skimmed) while having relatively no negative effect on pH and color measurements. Further experiments need to be carried out in
pressure plasma application with 9 kV, AC power supply did not cause any significant change to the pH values of raw milk samples inoculated with E. coli compared to controls (Data not shown). The pH values of the samples were approximately 6.7 ±0.05 at all intervals of 0, 3, 6, 9, 12, 15 and 20 min of plasma corona discharge application suggesting that cell death was not caused by acidity or alkalinity of the milk following LTP treatment. 3.4. Color measurements
Table 1 Effects of the plasma corona discharge on E. coli cell viability (Log CFU/ml) in whole, semi-skimmed, and skimmed milk. Time (min) Type of milk
Log CFU/ml 0
Whole Semi-skimmed Skimmed
3 a
7.78 ± 0.03 7.78 ± 0.03a 7.78 ± 0.01a
6 b
4.23 ± 0.01 4.14 ± 0.01b 4.23 ± 0.01b
9 b
4.31 ± 0.01 4.06 ± 0.21b 4.15 ± 0.02b
12 b
4.18 ± 0.01 3.96 ± 0.01b 3.98 ± 0.01b
The results were expressed as mean ± S.D. followed by the same letter are not significantly different (P b 0.05). ab means in the same column followed by different superscript letters are significantly different (P b 0.05).
15 b
3.90 ± 0.08 3.82 ± 0.01b 3.70 ± 0.01b
20 b
3.84 ± 0.02 3.80 ± 0.03b 3.64 ± 0.01b
3.63 ± 0.01b 3.40 ± 0.08b 3.34 ± 0.01b
C. Gurol et al. / International Journal of Food Microbiology 157 (2012) 1–5 Table 2 Colorimetric parameters of raw milk after 9 and 20 min of 9 kV plasma treatment.
Raw milk 9 min 20 min
L⁎
a⁎
b⁎
ΔE
81.90a ± 0.03 82.08a ± 0.01 81.39a ± 0.13
− 1.47b ± 0.02 − 1.45b ± 0.03 − 1.46b ± 0.02
6.28c ± 0.12 6.37c ± 0.01 6.36c ± 0.06
0.25 0.52
L*: lightness (ranging from 0 to 100), a*: green to red (ranging from − 60 to + 60) and the b*: blue to yellow (ranging from − 60 to + 60), ΔE: calculated color differences. ab means in the same column followed by different superscript letters are significantly different (P b 0.05).
order to demonstrate the full effects of this system on milk, including studies on other microorganisms and the effects of LTP on the chemical, physical, and compositional properties of milk. Acknowledgments This research was supported by Yeditepe University and the Genetics and Bioengineering, Food Engineering and Physics Departments The authors declare that they have no conflicts of interest. References Anand, S.K., Griffiths, M.W., 2011. In: Fuguay, J.W. (Ed.), Pathogens in Milk, Enterobacteriaceae, Encyclopedia of Dairy Sciences, 2nd Edition. , pp. 67–71. Barbosa-Canovas, G.V., Pothakamury, K.R., Palou, E., Swanson, B.G., 1997. Nonthermal preservation of foods. In: Doyle, M.P. (Ed.), Nonthermal preservation of foods. Marcel Dekker, New York, pp. 1–276. Basaran, P., Basaran-Akgul, N., Oksuz, L., 2008. Elimination of Aspergillus parasiticus from nut surface with low pressure cold plasma (LPCP) treatment. Food Microbiology 25, 626–632. Bean, N.H., Goulding, J.S., Lao, C., Angulo, F.J., 1996. Surveillance for foodborne-disease outbreaks—United States, 1988–1992. Morbidity and Mortality Weekly Report 45 (SS-5), 1–73. Datta, N., Deeth, H.C., 2003. Diagnosing the cause of proteolysis in milk. LebensmittelWissenschaft und Technologie 36, 173–182. Doran, P., Carson, J., Costello, E., More, S.J., 2009. An outbreak of tuberculosis affecting cattle and people on an Irish dairy farm in 2005, following the consumption of raw milk from a cow with Tuberculous mastitis. Irish Veterinary Journal 62, 390–397. El-Hag, A., Otunola, A., Jayaram, S.H., Anderson, W.A., 2008. Reduction of microbial growth in milk by pulsed electric fields. IEEE International Conference on Dielectric Liquids 144, 1–4. Elliot, A.J., Datta, N., Amenu, B., Deeth, H.C., 2005. Heat-induced and other chemical changes in commercial UHT milks. The Journal of Dairy Research 72, 442–446. Fuquay, J.W., Fox, P.F., McSweeney, P.L.H., 2011. In: Fuguay, J.W. (Ed.), Pathogens in Milk, Campylobacter spp., Encyclopedia of Dairy Sciences, 2nd Edition. , pp. 40–46. Goff, H.D., Hill, A.R., 1993. Chemistry and physics. In: Hui, Y.H. (Ed.), Dairy Science and Technology Handbook. VCH Publisher, New York, pp. 1–82. Grahl, T., Markl, H., 1996. Killing of microorganisms by pulsed electric fields. Applied Microbiology and Biotechnology 45, 148–157. Greenwood, M.H., Hooper, W.L., 1990. Excretion of Yersinia spp. associated with consumption of pasteurized milk. Epidemiology and Infection 104, 345–350. Korachi, M., Aslan, N., 2011. The effect of atmospheric pressure plasma corona discharge on pH, lipid content and DNA of bacterial cells. Plasma Science and Technology 13, 99–105.
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Korachi, M., Turan, Z., Senturk, K., Sahin, F., Aslan, N., 2009. An investigation into the biocidal effect of high voltage AC/DC atmospheric corona discharges on bacteria, yeasts, fungi and algae. Journal of Electrostatics 67, 678–685. Korachi, M., Gurol, C., Aslan, N., 2010. Atmospheric plasma discharge sterilization effects on whole cell fatty acid profiles of Escherichia coli and Staphylococcus aureus. Journal of Electrostatics 68, 508–512. Laroussi, M., 2002. Non-thermal decontamination of biological media by atmospheric pressure plasmas: review, analysis, and prospects. IEEE Transactions on Plasma Science 30, 1409–1419. Laroussi, M., Leipold, F., 2004. Evaluation of the roles of reactive species, heat and UV radiation in the inactivation of bacterial cells by air plasmas at atmospheric pressure. International Journal of Mass Spectrometry 223, 81–86. Martin, M.L., Shipman, L.D., Wells, J.G., 1986. Isolation of Escherichia coli O157:H7 from dairy cattle associated with two cases of haemolytic uremic syndrome. Lancet 2, 1043. Martin, O., Qin, B.L., Chang, F.J., Barbosa-Canovas, G.V., Swanson, B.G., 1997. Inactivation of Escherichia coli in skim milk by high intensity pulsed electric fields. Journal of Food Process Engineering 20, 317–336. McKellar, R.C., 1981. Development of off-flavors in ultra-high temperature and pasteurized milk as a function of proteolysis. Journal of Dairy Science 64, 2138–2145. Moisan, M., Barbeau, J., Moreau, S., Pelletier, J., Tabrizian, M., Yahia, L.H., 2001. Low temperature sterilization using gas plasmas: a review of the experiments and an analysis of the inactivation mechanisms. International Journal of Pharmaceutics 226, 1–21. Moisan, M., Barbeau, J., Crevier, M.C., Pelletier, J., Philip, N., Saoudi, B., 2002. Plasma sterilization. Methods and mechanisms. Pure and Applied Chemistry. Chimie Pure et Appliquée 74, 349–358. Morent, R., De Geyter, N., Verschuren, J., De Clerck, K., Kiekens, P., Leys, C., 2008. Non-thermal plasma treatment of textiles. Surface and Coatings Technology 202, 3427–3449. Ohkawa, H., Akitsu, T., Tsuji, M., Kimura, H., Kogoma, M., Fukushima, K., 2006. Pulse modulated high-frequency plasma sterilization at atmospheric-pressure. Journal of Surface Coating 200, 5829–5835. Ostyn, A., De Buyser, M.L., Guillier, F., Groult, J., Félix, B., Salah, S., Delmas, G., Hennekinne, J.A., 2010. First evidence of a food poisoning outbreak due to staphylococcal enterotoxintype E, France. Euro Surveillance 15 Available online: http://www.eurosurveillance.org/ ViewArticle.aspx?ArticleId=19528. Poppe, C., 2011. In: Fuguay, J.W. (Ed.), Pathogens in Milk, Salmonella spp., Encyclopedia of Dairy Sciences, 2nd Edition. , pp. 93–98. Ramos, J.M., Bernal, E., Esguevillas, T., Lopez-Garcia, P., Gaztambide, M.S., Gutierrez, F., 2008. Non-imported brucellosis outbreak from unpasteurized raw milk in Moroccan immigrants in Spain. Epidemiology and Infection 136, 1552–1555. Reina, L.D., Jin, Z.T., Zhang, Q.H., Yousef, A.E., 1998. Inactivation of Listeria monocytogenes in milk by pulsed electric field. Journal of Food Protection 61, 1203–1206. Soejima, T., Nagao, E., Yano, Y., Yamagata, H., Kagi, H., Shinagawa, K., 2007. Risk evaluation for staphylococcal food poisoning in processed milk produced with skim milk powder. International Journal of Food Microbiology 115, 29–34. Song, H.P., Kim, B., Choe, J.H., Jung, S., Moon, S.Y., Choe, W., Jo, C., 2009. Evaluation of atmospheric pressure plasma to improve the safety of sliced cheese and ham inoculated by 3-strain cocktail Listeria monocytogenes. Food Microbiology 26, 432–436. Tiozzo, B., Mari, S., Magaudda, P., Arzenton, V., Capozza, D., Neresini, F., Ravarotto, L., 2011. Development and evaluation of a risk-communication campaign on salmonellosis. Food Control 22, 109–117. Todd, E.C.D., Notermans, S., 2011. Surveillance of listeriosis and its causative pathogen, Listeria monocytogenes. Food Control 22, 1484–1490. Topcu, A., Numanoglu, E., Saldamli, I., 2006. Proteolysis and storage stability of UHT milk produced in Turkey. International Dairy Journal 16, 633–638. Wyder, A.B., Boss, R., Naskova, J., Kaufmann, T., Steiner, A., Graber, H.U., 2010. Streptococcus spp. and related bacteria: Their identification and their pathogenic potential for chronicmastitis- A molecular approach. Research in Veterinary Science 91 (3), 349–357. Yilmaz, T., Moyer, B., MacDonell, R.E., Cordero-Coma, M., Gallagher, M.J., 2009. Outbreaks associated with unpasteurized milk and soft cheese: an overview of consumer safety. Food Protection Trends 29 (4), 211–222. Yu, R., Kramida, A.E., Reader, J., 2008. NIST ASD Team. 2008. NIST Atomic Spectra Database (version 3.1.5). http://physics.nist.gov/asd3.
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International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro
Low Temperature Plasma for decontamination of E. coli in milk C. Gurol a, F.Y. Ekinci b, N. Aslan c, M. Korachi a,⁎ a b c
Department of Genetics and Bioengineering, Yeditepe University, Istanbul Turkey Department of Food Engineering, Yeditepe University, Istanbul Turkey Department of Physics, Yeditepe University, Istanbul Turkey
a r t i c l e
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Article history: Received 29 September 2011 Received in revised form 15 February 2012 Accepted 20 February 2012 Available online 24 February 2012 Keywords: Plasma Corona discharge Decontamination Milk Processing technology
a b s t r a c t Raw milk is a natural, highly nutritious product and a quick and easy supplement for human dietary requirements. Elimination of bacteria in milk has been a problem for decades and new methods with regards to nonthermal applications which do not harm the chemical composition of milk, are currently under investigation. The objective of the study was to determine the potential use of a novel, Low Temperature Plasma (LTP) system for its capability of killing Escherichia coli in milk with different fat contents. The time dependent effect of atmospheric corona discharge generated with 9 kV of AC power supply on E. coli ATCC 25922 dispersed in whole, semi skimmed and skimmed milk was examined. Plasma was applied at time intervals of 0, 3, 6, 9, 12, 15 and 20 min. A significant 54% reduction in the population of E. coli cells after only 3 min was observed regardless of the fat content of the milk. The initial pre-plasma bacterial count of 7.78 Log CFU/ml in whole milk was decreased to 3.63 Log CFU/ml after 20 min of plasma application. LTP did not cause any significant change to the pH and color values of raw milk samples. No viable cells were detected after one week examination in whole milk samples and remained so over the 6 week storage period. The findings of this study show that the novel LTP system tested was able to significantly reduce E. coli in milk by more than a 3 fold log reduction without significantly affecting pH or color properties. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Infectious diseases caused by the ingestion of pathogenic bacteria in contaminated milk are still a major health concern, especially for children (Tiozzo et al., 2011; Fuguay et al., 2011; Todd and Notermans, 2011). While the nutritional benefits of milk consumption are documented, the hazards of drinking untreated milk are also well known. The most predominant infectious diseases caused by contaminated milk include campylobacteriosis (Fuguay et al., 2011), salmonellosis (Poppe, 2011; Tiozzo et al., 2011), yersiniosis (Greenwood and Hooper, 1990), listeriosis (Todd and Notermans, 2011; Yilmaz et al., 2009), tuberculosis (Doran et al., 2009), brucellosis (Ramos et al., 2008), staphylococcal enterotoxin poisoning (Ostyn et al., 2010; Soejima et al., 2007), streptococcal infections (Wyder et al., 2010) and Escherichia coli 0157:H7 infection (Anand and Griffiths, 2011; Martin et al., 1986). Although, the etiological agents in milkborne diseases are numerous, more than 90% of all reported cases of dairy related illnesses are of bacterial origin (Bean et al., 1996). It is therefore standard procedure to properly treat raw milk, to inactivate these pathogens before consumption, either by pasteurization or Ultra High Temperature (UHT). ⁎ Corresponding author at: Yeditepe University, Faculty of Engineering and Architecture, Genetics and Bioengineering Department, Kayisdagi, Istanbul, Turkey. Tel.: + 90 216 578 2653; fax: + 90 216 578 0829. E-mail address: [email protected] (M. Korachi). 0168-1605/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2012.02.016
However, the current thermal decontamination methods are known to induce changes to the chemical and physiological composition of milk and milk products (Topcu et al., 2006; Datta and Deeth, 2003). These changes include: browning, flavor changes (McKellar, 1981), serum protein denaturation resulting in a whiter appearance fat agglomeration and solid destabilization, freezing point depression, possibility of adulteration with condensate and boiler compounds and partial loss of some vitamins (Topcu et al., 2006). Furthermore, UHT treatment renders the milk unsuitable for further processing such as cheese and yogurt production. Due to its complex structure milk has so far shown to be highly sensitive to many current novel technologies (Grahl and Markl, 1996). Therefore, the lack of a technology which enhances microbial food safety and quality, while simultaneously protecting the nutritional, functional and sensory characteristics of foods, has created a huge interest in low-temperature innovative processing techniques. These innovative technologies mostly rely on physical processes, including high hydrostatic pressures, pulsed electric fields and low-temperature plasmas that inactivate microorganisms at ambient or moderately elevated temperatures and short treatment times (Barbosa-Canovas et al., 1997; Korachi et al., 2009). To date the sterilization efficacy of plasma has been studied on medical instruments (Laroussi, 2002), food sanitation (Basaran et al., 2008), textiles (Morent et al., 2008), and water (Korachi et al., 2009). Plasma corona discharge (PCD) causes the formation of active species such as: UV light, free radicals and ions, ozone and atomic oxygen, which contain sufficient energy to break covalent bonds and
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initiate various chemical reactions (Moisan et al., 2001). The antimicrobial activity of plasma treatment is a result of direct contact with these active species (Moisan et al., 2002). Moreover, these active species have been said to disappear in milliseconds after the plasma system is switched off (Korachi et al., 2009). The three basic mechanisms that have been attributed to the inactivation of microorganisms by plasma are the destruction of DNA, volatilization of compounds, and etching of the cell surface by adsorption of reactive species (Korachi et al., 2010; Korachi and Aslan, 2011). These effects may vary due to the operational conditions and the design of the plasma generator. The ability of plasma to work at low temperatures without increasing the operating temperature has opened up the possibility of using LTP for the treatment of heat-sensitive materials (Song et al., 2009; Morent et al., 2008). The elimination of pathogenic bacteria in milk has been a problem for decades and new methods with regards to non-thermal applications which do not harm the chemical composition of milk, are highly required. In this study, the potential use of a novel, simple, LTP system was investigated for its capability of killing E. coli in milk samples with different fat contents. Although there has been increasing interest into the use of plasmas as a sterilizing technique, this is the first study that tests the use of plasma technology as a decontaminating technique in milk. 2. Materials and methods 2.1. Milk samples and cultures Commercial UHT and raw milk samples were obtained from a local market and farm respectively in Istanbul, Turkey. The UHT samples included whole (3% fat), semi-skimmed (1.5% fat), and skimmed milk (0.1% fat). E. coli ATCC 25922 was provided from Yeditepe University, Genetic and Bioengineering Department Culture Collection (Turkey). E. coli ATCC 25922 was grown on Tryptic Soy Agar (TSA) (Merck, Germany) and in Nutrient Broth (NB) (Merck, Germany) at 37 °C for 48 h. The identity of the strain was confirmed by Gram staining and commercially available identification kits based on biochemical assays. Tests were carried out according to the manufacturer's instructions (Biomerieux, France). Culture was stored in the appropriate growth medium containing 20% glycerol at − 70 °C. Viable counts were performed according to standard methods on TSA and expressed in Log CFU/ml. 2.2. Microbial preparation UHT milk was checked for any bacterial growth at the start of the experiment by plating onto TSA. A bacterial stock solution was prepared by inoculation of the test strain in NB and incubating at 37 °C for 48 h. Following the incubation, 0.1 ml of the bacterial solution was suspended in 4.5 ml of NB and the cultures were grown at
37 °C in a shaking water bath (200 rpm) for 3 h. The growth curve was monitored photometrically by the optical density (OD) at 600 nm (UV/VIS Spectrophotometer, Lambda 3B, Perkin Elmer). When the OD approximately reached 0.2 (the middle of the loggrowth phase), 100 μl of the cell suspension was pipetted into 15 ml of sterile milk in a sterile petri dish. The final concentration of the milk suspension was adjusted to 7.78 Log CFU/ml by performing serial dilutions. 100 μl of bacterial suspension was also plated onto the agar media at zero time and incubated at 37 °C for 48 h as a control. 2.3. Low temperature atmospheric pressure plasma system The atmospheric plasma discharge system previously described (Korachi et al., 2009, 2010; Korachi and Aslan, 2011) was used with some modifications (Fig. 1). Results from previous studies with a similar LTP system (Korachi et al., 2009, 2010; Korachi and Aslan, 2011) showed the DC power supply to be more destructive on test materials. Therefore, a 9 kV AC power supply, a simple ballast circuit and two tungsten electrodes (0.8 mm radius) were used for milk applications. Low Temperature Plasma was applied to the contaminated milk solution by applying high voltage between the upper electrode tip and the liquid surface (in which the other electrode was immersed). The upper electrode, which was sterilized after each application, was rotated by an adjustable DC motor and a motorized stirrer was attached to maintain application homogeneity. The temperature was monitored throughout discharge application by the use of a thermometer and the system shut down whenever the temperature reached the allowed maximum temperature (35 °C). The current of nearly 90 mA in the plasma corona resulted in nearly 10 A/cm 2. In order to cool the system, it was lowered to contact with the surface of an icepack. 2.4. Light emission spectroscopy of plasma discharge system The light emission intensities of the AC plasma discharge were determined by a UV–visible emission spectrometer (Baki, Turkey) manufactured by the Laser Technologies Laboratory of Kocaeli University, Turkey. The light from the discharge was transferred through a fiber cable to the spectrometer and the emissions were recorded between wavelengths of 200 and 900 nm. The peaks appearing in the spectrum indicate the ionization levels of the molecules making up the plasma discharge identified using the NIST Atomic Spectra Database (Yu et al., 2008). 2.5. Plasma application The apparatus described above was placed in a Laminar Flow cabinet (Heal Force, China) to prevent contamination. Following bacterial inoculation, the milk samples were immediately exposed to LTP and
Ballast circuit DC motor
Thermometer
HV + POWER _ SUPPLY
Tungsten electrode
Stirrer
Petri dish Lower electrode Ice pack
Fig. 1. Schematic view of atmospheric plasma corona discharge experimental setup.
C. Gurol et al. / International Journal of Food Microbiology 157 (2012) 1–5
the temperature was kept below 35 °C throughout the experiment. One hundred microliters of samples was taken at each time interval of 3, 6, 9, 12, 15 and 20 min, and plated onto both TSA and violet red bile agar (VRBA). The plates were incubated at 37 °C for 48 h and results were expressed as Log CFU/ml. 2.6. pH analysis The experiment above was repeated for pH analysis using raw milk. The pH was measured with a pH meter (Mettler Toledo, USA) throughout the treatment time. The pH of post-plasma treated and control raw milk was compared to detect any changes in the hydrogen concentration due to plasma discharge. 2.7. Color measurement tests A portable spectrophotometer, CM-600 d (Konica Minolta, USA) was used for the color analysis in raw milk. In this system, an illuminator with 8° diffused illumination and 8° visioning angle was used. The light source used was a pulsed xenon lamb with UV cut filter and the wavelength ranged from 400 nm to 700 nm pitching every 10 nm. The light was detected with a silicon photodiode array. Since temperature affects the color values all samples were warmed to room temperature before color measurement were taken. Results were shown according to the CIE-LAB system which includes L*: luminance or lightness component (ranging from 0 to 100), a*: green to red (ranging from −60 to +60) and the b* component: blue to yellow (ranging from −60 to +60). The overall color difference was expressed by means of Delta-E (dE) which indicates the distance between two colors. Using target L*1, a*1 and b*1 measurements and the sample L*2, a*2, and b*2, dE was calculated using the following equation: dE ¼
h
L2 –L1
2
2 2 i1=2 þ a2 –a1 þ b2 –b1
ð2Þ
Depending on the dE values, the color difference was evaluated as not noticeable (0–0.5), slightly noticeable (0.5–1.5), noticeable (1.5–3) and well visible (3–6). 2.8. Storage test Control, 9 min and 20 min plasma-treated samples were stored under cold conditions (4–7 °C) for a total period of 6 weeks. The microbial load of milk samples were monitored by taking 100 μL samples and cultivating on TSA every 7 days. Viable counts were performed and expressed in Log CFU/ml. 2.9. Statistical analysis All experiments were repeated three times. Minitab software version 16 (Minitab, Inc., State College, PA) was used for data analysis. Kruskal–Wallis was performed to compare the mean values of the response variable and treatment conditions. Evaluations were based on a significance level of P b 0.05. 3. Results and discussion 3.1. Light emission spectroscopy results The light emission intensity spectrum of the discharge used in this study showed peaks indicating the ionization levels of the ions in the discharge (Fig. 2). The data were compared and identified according to NIST Atomic Spectra Database available online (Yu et al., 2008). The emission intensity spectrum of atmospheric pressure AC corona discharges with a frequency of 50 Hz was obtained from the plasma
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surrounding the tip of the electrode in air. Several main peaks were identified belonging to oxygen (O), nitrogen (N) and argon (Ar) molecules as is expected because the plasma system operates in air. Since the emission spectra were taken at the tip of the electrode (~1 cm above the milk surface), no major peaks from organic materials were observed as would be the case if the optical fiber of the spectroscope was placed closer to the milk surface. The oxygen peaks in this system corresponded to the positively charged oxygen ions created by electron impact. It is believed that these ions accelerate towards the liquid surface creating ozone and other active species (with very short lifetimes). It was further observed that since the melting temperature of tungsten is 3422 °C, the tungsten tip did not melt and create deposits on the milk surface as has previously been observed with the use of chrome and nickel electrodes (Korachi et al., 2009). 3.2. Microbiological results The time dependent effect of atmospheric corona discharge generated with 9 kV of AC power supply on E. coli ATCC 25922 dispersed in milk was examined. Table 1 represents the effects of the plasma corona discharge on E. coli cell viability (Log CFU/ml) in whole, semiskimmed, and skimmed milk. A significant 54% reduction in the population of E. coli cells following 3 min LTP treatment was observed for all 3 types of milk-fat samples. The initial pre-plasma bacterial count of 7.78 Log CFU/ml in whole milk (Table 1) was decreased to 3.63 Log CFU/ml at time intervals up to 20 min. Similar significant reductions were also observed in semiskimmed milk (P b 0.007), and skimmed milk (P b 0.009). These results indicate that the LTP system used in this study can reduce the number of E. coli in milk regardless of its fat content. This is a positive factor since several studies have shown that decontamination of milk is more difficult than that of model buffer solutions because of the complex structure of milk (Goff and Hill, 1993; Martin et al., 1997). Tests using PEF technology have shown that the fat content of the milk inversely affected microbial inactivation via protection against electric pulses (Grahl and Markl, 1996). Although a study by Reina et al. (1998) and El-Hag et al. (2008) using PEF to treat whole and skimmed milk inoculated with Staphylococcus aureus and Listeria monocytogenes, found similar results to our findings, these controversial findings are probably due to the different designs and working parameters of different systems. Published studies have shown that the initial bacterial concentrations, treatment duration and the structure of the supporting medium all play a role in the antimicrobial efficacy of LTP applications (Laroussi and Leipold, 2004; Ohkawa et al., 2006). To date, there are no studies on the inactivation of food-borne pathogens in milk by LTP for comparison. However, our findings have revealed that the fat content of milk does not affect the susceptibility of E. coli ATCC 25922 to plasma treatment, thus revealing a potential advantage of this decontamination system for possible use in fatty substrates. Current thermal treatments use processing parameters of 63 °C for 30 min (batch method) or 71 °C for 15 s (flash method) for pasteurization and 135 °C for 1 or 2 s for UHT. Although these parameters are for treatment of milk from all pathogens, a detailed study on the effect of UHT treated milk using direct and indirect methods has shown that significant changes occur to milk's color, nutritional and organoleptic properties (Elliot et al., 2005). Since this novel technology operates at both a lower temperature and induces its effect on E. coli in a short time of 3 min, thereby minimizing the damaging effects on the color and pH properties of milk that has been previously described with traditional thermal treatments (McKellar, 1981; Topcu et al., 2006; Datta and Deeth, 2003). 3.3. pH analysis The hydrogen concentration in a solution is typically important while determining a system's decontamination capabilities. Atmospheric
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20 18
Intensity (au)
16 14 12 10 8 6 4 2 0 3500
4000
4500
5000
5500
6000
6500
7000
7500
8000
8500
Wavelength (A) Fig. 2. The emission intensity spectra of atmospheric pressure AC corona discharge.
Milk samples did not show any important changes in color after 0, 3, 6, 9, 12, and 15 min of 9 kV plasma treatments and only a slight change in comparison with the untreated milk occurred after 20 min (Table 2). The color values of raw milk samples before and after plasma application at 9 and 20 min are shown in Table 2. Analytical measurements confirmed that the same color parameters L*, a*, and b* and total color difference (ΔE) existed between the control samples and respective plasma treated samples after processing. The total color difference for milk after 9 min of plasma treatment with 9 kV was 0.25 while longer exposure to plasma (20 min) caused slightly higher color differences with a ΔE value of 0.52 suggesting that plasma application resulted in no or slightly noticeable color differences in milk samples.
number of E. coli decreased from 4.18 to 3.08 Log CFU/ml after 4 days of storage for 9 min plasma applied samples and reached undetectable levels after day 6 (p b 0.001). On the other hand, the levels of E. coli for 20 min plasma applied samples were undetectable after one day storage and remained thereafter at the end of 6 weeks. No viable cells were detected after one week examination and remained so over the 6 week storage period. The immediate decrease in bacterial growth following plasma application is expected and can be attributed to the effect of the reactive species formed by plasma, that directly target the bacterial cell (Korachi et al., 2009). Of more interest was the total reduction of all microorganisms during the first week of storage. These findings suggest that the surviving bacterial cells, post plasma application were inactivated during storage. The findings correlate with those of Song et al. (2009), where the number of L. monocytogenes decreased during storage following Low Temperature Plasma treatment. The decrease in milk's nutrient content (required for bacterial growth) and the accumulation of bacterial by-products over storage time of 42 days could be a possible contributing factor in cell death. However it is probable that the main factor could be the persistent presence of the active species produced by the LTP system. Further investigations are required in order to gain a better understanding of the predominant reason for cell death during storage.
3.5. Preliminary storage analysis
4. Conclusion
The decontamination technologies for milk aim to reduce the number of harmful bacteria and ultimately achieve microbial safety during shelf life. Preliminary storage tests were therefore carried out for 6 weeks on plasma treated whole milk samples following 9 min and 20 min application. Colony numbers (Log CFU/ml) of E. coli in whole milk during storage at 4 °C for a period of 0–6 weeks. The control of untreated milk showed a decline in the CFU's at the 6th week. This can be explained by the depletion of nutritional content in the milk due to and required for bacterial growth. In comparison, samples analyzed immediately following plasma treatment (at zero time) revealed a significant decrease in the number of CFU's (4.18 after 9 min; 3.63 after 20 min) from an initial concentration of 7.78 Log CFU/ml. The
Food processing technologies are turning to the use of low processing temperatures in order to maintain food's safe, natural and “freshlike” characteristics. However to date no technique has been able to simultaneously decontaminate and extend the product's shelf life while maintaining its beneficial properties. Using the advantages of cold plasma, this system was tested for its ability to decontaminate E. coli in milk. The time and storage parameters of LTP were tested on E. coli ATCC 25922 in different types of milk and during storage. It was found that this plasma system significantly reduced the number of colonies in different types of milk (whole, semi-skimmed, skimmed) while having relatively no negative effect on pH and color measurements. Further experiments need to be carried out in
pressure plasma application with 9 kV, AC power supply did not cause any significant change to the pH values of raw milk samples inoculated with E. coli compared to controls (Data not shown). The pH values of the samples were approximately 6.7 ±0.05 at all intervals of 0, 3, 6, 9, 12, 15 and 20 min of plasma corona discharge application suggesting that cell death was not caused by acidity or alkalinity of the milk following LTP treatment. 3.4. Color measurements
Table 1 Effects of the plasma corona discharge on E. coli cell viability (Log CFU/ml) in whole, semi-skimmed, and skimmed milk. Time (min) Type of milk
Log CFU/ml 0
Whole Semi-skimmed Skimmed
3 a
7.78 ± 0.03 7.78 ± 0.03a 7.78 ± 0.01a
6 b
4.23 ± 0.01 4.14 ± 0.01b 4.23 ± 0.01b
9 b
4.31 ± 0.01 4.06 ± 0.21b 4.15 ± 0.02b
12 b
4.18 ± 0.01 3.96 ± 0.01b 3.98 ± 0.01b
The results were expressed as mean ± S.D. followed by the same letter are not significantly different (P b 0.05). ab means in the same column followed by different superscript letters are significantly different (P b 0.05).
15 b
3.90 ± 0.08 3.82 ± 0.01b 3.70 ± 0.01b
20 b
3.84 ± 0.02 3.80 ± 0.03b 3.64 ± 0.01b
3.63 ± 0.01b 3.40 ± 0.08b 3.34 ± 0.01b
C. Gurol et al. / International Journal of Food Microbiology 157 (2012) 1–5 Table 2 Colorimetric parameters of raw milk after 9 and 20 min of 9 kV plasma treatment.
Raw milk 9 min 20 min
L⁎
a⁎
b⁎
ΔE
81.90a ± 0.03 82.08a ± 0.01 81.39a ± 0.13
− 1.47b ± 0.02 − 1.45b ± 0.03 − 1.46b ± 0.02
6.28c ± 0.12 6.37c ± 0.01 6.36c ± 0.06
0.25 0.52
L*: lightness (ranging from 0 to 100), a*: green to red (ranging from − 60 to + 60) and the b*: blue to yellow (ranging from − 60 to + 60), ΔE: calculated color differences. ab means in the same column followed by different superscript letters are significantly different (P b 0.05).
order to demonstrate the full effects of this system on milk, including studies on other microorganisms and the effects of LTP on the chemical, physical, and compositional properties of milk. Acknowledgments This research was supported by Yeditepe University and the Genetics and Bioengineering, Food Engineering and Physics Departments The authors declare that they have no conflicts of interest. References Anand, S.K., Griffiths, M.W., 2011. In: Fuguay, J.W. (Ed.), Pathogens in Milk, Enterobacteriaceae, Encyclopedia of Dairy Sciences, 2nd Edition. , pp. 67–71. Barbosa-Canovas, G.V., Pothakamury, K.R., Palou, E., Swanson, B.G., 1997. Nonthermal preservation of foods. In: Doyle, M.P. (Ed.), Nonthermal preservation of foods. Marcel Dekker, New York, pp. 1–276. Basaran, P., Basaran-Akgul, N., Oksuz, L., 2008. Elimination of Aspergillus parasiticus from nut surface with low pressure cold plasma (LPCP) treatment. Food Microbiology 25, 626–632. Bean, N.H., Goulding, J.S., Lao, C., Angulo, F.J., 1996. Surveillance for foodborne-disease outbreaks—United States, 1988–1992. Morbidity and Mortality Weekly Report 45 (SS-5), 1–73. Datta, N., Deeth, H.C., 2003. Diagnosing the cause of proteolysis in milk. LebensmittelWissenschaft und Technologie 36, 173–182. Doran, P., Carson, J., Costello, E., More, S.J., 2009. An outbreak of tuberculosis affecting cattle and people on an Irish dairy farm in 2005, following the consumption of raw milk from a cow with Tuberculous mastitis. Irish Veterinary Journal 62, 390–397. El-Hag, A., Otunola, A., Jayaram, S.H., Anderson, W.A., 2008. Reduction of microbial growth in milk by pulsed electric fields. IEEE International Conference on Dielectric Liquids 144, 1–4. Elliot, A.J., Datta, N., Amenu, B., Deeth, H.C., 2005. Heat-induced and other chemical changes in commercial UHT milks. The Journal of Dairy Research 72, 442–446. Fuquay, J.W., Fox, P.F., McSweeney, P.L.H., 2011. In: Fuguay, J.W. (Ed.), Pathogens in Milk, Campylobacter spp., Encyclopedia of Dairy Sciences, 2nd Edition. , pp. 40–46. Goff, H.D., Hill, A.R., 1993. Chemistry and physics. In: Hui, Y.H. (Ed.), Dairy Science and Technology Handbook. VCH Publisher, New York, pp. 1–82. Grahl, T., Markl, H., 1996. Killing of microorganisms by pulsed electric fields. Applied Microbiology and Biotechnology 45, 148–157. Greenwood, M.H., Hooper, W.L., 1990. Excretion of Yersinia spp. associated with consumption of pasteurized milk. Epidemiology and Infection 104, 345–350. Korachi, M., Aslan, N., 2011. The effect of atmospheric pressure plasma corona discharge on pH, lipid content and DNA of bacterial cells. Plasma Science and Technology 13, 99–105.
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Korachi, M., Turan, Z., Senturk, K., Sahin, F., Aslan, N., 2009. An investigation into the biocidal effect of high voltage AC/DC atmospheric corona discharges on bacteria, yeasts, fungi and algae. Journal of Electrostatics 67, 678–685. Korachi, M., Gurol, C., Aslan, N., 2010. Atmospheric plasma discharge sterilization effects on whole cell fatty acid profiles of Escherichia coli and Staphylococcus aureus. Journal of Electrostatics 68, 508–512. Laroussi, M., 2002. Non-thermal decontamination of biological media by atmospheric pressure plasmas: review, analysis, and prospects. IEEE Transactions on Plasma Science 30, 1409–1419. Laroussi, M., Leipold, F., 2004. Evaluation of the roles of reactive species, heat and UV radiation in the inactivation of bacterial cells by air plasmas at atmospheric pressure. International Journal of Mass Spectrometry 223, 81–86. Martin, M.L., Shipman, L.D., Wells, J.G., 1986. Isolation of Escherichia coli O157:H7 from dairy cattle associated with two cases of haemolytic uremic syndrome. Lancet 2, 1043. Martin, O., Qin, B.L., Chang, F.J., Barbosa-Canovas, G.V., Swanson, B.G., 1997. Inactivation of Escherichia coli in skim milk by high intensity pulsed electric fields. Journal of Food Process Engineering 20, 317–336. McKellar, R.C., 1981. Development of off-flavors in ultra-high temperature and pasteurized milk as a function of proteolysis. Journal of Dairy Science 64, 2138–2145. Moisan, M., Barbeau, J., Moreau, S., Pelletier, J., Tabrizian, M., Yahia, L.H., 2001. Low temperature sterilization using gas plasmas: a review of the experiments and an analysis of the inactivation mechanisms. International Journal of Pharmaceutics 226, 1–21. Moisan, M., Barbeau, J., Crevier, M.C., Pelletier, J., Philip, N., Saoudi, B., 2002. Plasma sterilization. Methods and mechanisms. Pure and Applied Chemistry. Chimie Pure et Appliquée 74, 349–358. Morent, R., De Geyter, N., Verschuren, J., De Clerck, K., Kiekens, P., Leys, C., 2008. Non-thermal plasma treatment of textiles. Surface and Coatings Technology 202, 3427–3449. Ohkawa, H., Akitsu, T., Tsuji, M., Kimura, H., Kogoma, M., Fukushima, K., 2006. Pulse modulated high-frequency plasma sterilization at atmospheric-pressure. Journal of Surface Coating 200, 5829–5835. Ostyn, A., De Buyser, M.L., Guillier, F., Groult, J., Félix, B., Salah, S., Delmas, G., Hennekinne, J.A., 2010. First evidence of a food poisoning outbreak due to staphylococcal enterotoxintype E, France. Euro Surveillance 15 Available online: http://www.eurosurveillance.org/ ViewArticle.aspx?ArticleId=19528. Poppe, C., 2011. In: Fuguay, J.W. (Ed.), Pathogens in Milk, Salmonella spp., Encyclopedia of Dairy Sciences, 2nd Edition. , pp. 93–98. Ramos, J.M., Bernal, E., Esguevillas, T., Lopez-Garcia, P., Gaztambide, M.S., Gutierrez, F., 2008. Non-imported brucellosis outbreak from unpasteurized raw milk in Moroccan immigrants in Spain. Epidemiology and Infection 136, 1552–1555. Reina, L.D., Jin, Z.T., Zhang, Q.H., Yousef, A.E., 1998. Inactivation of Listeria monocytogenes in milk by pulsed electric field. Journal of Food Protection 61, 1203–1206. Soejima, T., Nagao, E., Yano, Y., Yamagata, H., Kagi, H., Shinagawa, K., 2007. Risk evaluation for staphylococcal food poisoning in processed milk produced with skim milk powder. International Journal of Food Microbiology 115, 29–34. Song, H.P., Kim, B., Choe, J.H., Jung, S., Moon, S.Y., Choe, W., Jo, C., 2009. Evaluation of atmospheric pressure plasma to improve the safety of sliced cheese and ham inoculated by 3-strain cocktail Listeria monocytogenes. Food Microbiology 26, 432–436. Tiozzo, B., Mari, S., Magaudda, P., Arzenton, V., Capozza, D., Neresini, F., Ravarotto, L., 2011. Development and evaluation of a risk-communication campaign on salmonellosis. Food Control 22, 109–117. Todd, E.C.D., Notermans, S., 2011. Surveillance of listeriosis and its causative pathogen, Listeria monocytogenes. Food Control 22, 1484–1490. Topcu, A., Numanoglu, E., Saldamli, I., 2006. Proteolysis and storage stability of UHT milk produced in Turkey. International Dairy Journal 16, 633–638. Wyder, A.B., Boss, R., Naskova, J., Kaufmann, T., Steiner, A., Graber, H.U., 2010. Streptococcus spp. and related bacteria: Their identification and their pathogenic potential for chronicmastitis- A molecular approach. Research in Veterinary Science 91 (3), 349–357. Yilmaz, T., Moyer, B., MacDonell, R.E., Cordero-Coma, M., Gallagher, M.J., 2009. Outbreaks associated with unpasteurized milk and soft cheese: an overview of consumer safety. Food Protection Trends 29 (4), 211–222. Yu, R., Kramida, A.E., Reader, J., 2008. NIST ASD Team. 2008. NIST Atomic Spectra Database (version 3.1.5). http://physics.nist.gov/asd3.