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In the site of action, the plasma has the ability to break the covalent bonds in biological molecules and cause different chemical reactions (Moisan et al., 2002). Heat, ultraviolet radiation, charged particles and reactive species have been considered main contributors to the interaction between CAP and bacteria at different levels. However, as reviewed by Laroussi et al. (2008), both thermal effects and UV radiation does not play a significant direct role in the sterilization process by low temperature air plasmas. As most CAPs are designed to operate at ambient temperature, thermal damage is not considered the mechanism of injury. Although UV radiation is one of the powerful disinfectants, it only plays a modest role in plasma sterilization (Laroussi, 2005). Additionally, UV is able to cause DNA damage so that its emission has been limited in most CAP designs (Stolz, 2007). Charged particles have been suggested to play a very significant role in the rupture of the outer membrane of bacterial cells. The electrostatic force caused by charge accumulation on the outer surface of the cell membrane could overcome the tensile strength of the membrane and cause its rupture. Short living free radical, such as reactive oxygen species (ROS), was shown play a very important role in the inactivation of bacteria. They are considered to be the prime plasmaborne disinfectants for air plasma at atmospheric pressure (Lim et al., 2007). In the case of "direct exposure", in addition to the effects of neutral species, the charged species may play a role in rupturing the outer membrane of bacterial cells (Laroussi et al., 2008).
1.4.4 Decontamination effects of Cold Atmospheric Plasma
The main interesting characteristic of the CAP system is that it results from the action of reactive oxygen radicals instead of UV light (Lee et al., 2006). The inactivation capability of non- thermal plasmas has been reported to a wide range of microorganism, including Gram- positive (Wintenberg et al., 1998; Moreau et al., 2000; Philip et al., 2002; Hermann et al., 2002; Birmingham, 2004; Laroussi, 2005), Gram-negative and biofilm- forming bacteria (Vleugels et al., 2005).
The inactivation effects of CAP have been found to be dependent on many factors, such as the sample type and bacterial species. Though, a few general statements with regard to the efficacy of plasma inactivation were suggested by Stoffels (2008). These statements can be used to predict a priori, which draws the superficial nature of the plasma action and bacterial species structure.
Vegetative cells are easier to destroy than spores.
Gram negative species are easier to deactivate than the Gram positive ones. This is because the membrane of Gram positive species is much thicker and harder to penetrate in prime injury mechanism taken by chemical etching or charge- induced damage. Though, there is no significant difference between the killing efficacies between these species.
Aerobic species are more difficult to destroy than anaerobes. The reason is that the aerobes are less sensitive to ROS so that they can stand oxygen- rich conditions.
Direct exposure gains higher efficiency than the post- discharge treatment, and the sterilization is often only on the surface.
Charged species may play a role in decontamination effect. However, this only happen on vegetative bacteria which are close to the sample surface in direct plasma treatment (Stoffels, 2008).
1.4.5 Cold Atmospheric plasma applications
Various designs of cold atmospheric plasma have become hot issues of current non-thermal plasma research due to their wide range of. Inactivation efficacy of these low temperature plasma have been shown in different microorganisms, including fungus, bacteria, bacterial spores, plant cells, cancer cells, proteinaceous matters, and even genetic DNA (Kim et al., 2009).
Cold gas plasmas are of special interest for application with foods due to their chemically reactive plasma species generated are short lived (typical lifetimes from microseconds to milliseconds) so that there is no remaining residues on the surface of the food product after treatment. Moreover, the decontamination effect of CAP has been achieved in a very wide range of microorganisms make it become more interesting technology. The finding that atomic oxygen was one of the most lethal species present in CAP offers the prospect of enhancing the microbiocidal potential of CAP (Perni et al., 2008).
This project was aimed at understanding the effects of CAP on food- borne pathogens on meat. E. coli O157:H7 was used as a model organism in these expects because O157 is a well recognized pathogen found in beef and therefore is a good model for understanding the effects of CAP. Beef steak surface is a very interesting rough surface as it has a very high surface area to weight ratio. Beef steak was chosen for use in this study precisely because it does represent a challenging environment to the antimicrobial properties of any food preservative agent.
CHAPTER 2: MATERIALS AND METHODS
2.1 MEAT SAMPLE PREPARATION
Fresh beef steak bought from the supermarket was aseptically sliced into small slices with a size of 2cmx2cm and 1cm thick. The knife used for cutting was disinfected with ethanol solution 70% (vol/vol). Each slice was placed into a Petri dish and stored at -20oC. Before each experiment, the appropriate numbers of beef slices were defrosted at room temperature for 30 minutes.
2.2 BACTERIAL STRAINS AND GROWTH CONDITIONS
E. coli O157:H7 SAKAI strains, which were Shigella toxin minus strains obtained from Dr Neil Doherty, were used. They were stored in frozen stocks and propagated on LB agar and LB broth. E. coli 0157:H7 oxyR single gene mutant was constructed by Dr Neil Doherty that a kanamycin resistance gene cassette was used to replace the oxyR gene. Wild-type E. coli 0157:H7 cells were stored at 4oC on solid Luria-Bertani plates (LB; 10 g/l NaCl, 10 g/l tryptone and 5 g/l yeast extract) while the E. coli 0157:H7 oxyR mutant was stored on plates containing 1 Âµl/ ml kanamycin to select against loss of the antibiotic resistance gene that had replaced the oxyR genes. For each strain, a small loopfull of cells was used to inoculate 10ml fresh LB broth with antibiotics as required and incubated at 37oC with shaking at 200 rpm for 3 hours. Cultivation under these conditions yielded final concentrations of about 108 colony forming units/ml. Cells were then diluted to 104 colony forming units/ml in sterile Ringer's solution ( 4.5 g/l Sodium Chloride, 0.21g/l Potassium Chloride, 0.24 g/l Calcium Chloride, 0.1 g/l Sodium Bicarbonate) (Oxoid).
2.3 IN VITRO GROWTH CURVES
For each strain of both the wild types and the oxyR single gene mutant of E. coli O157:H7 SAKAI, a loopfull of cells was used to inoculate 10ml fresh LB broth with antibiotics as required and incubated at 37oC with shaking at 200 rpm overnight. 50 Âµl of the overnight growth was suspended into 9,950 Âµl of LB broth and incubated at 37oC with shaking at 200 rpm for 24h. Starting at time point one, 100 Âµl of samples were removed at every 1 h, suspended into 900 Âµl LB broth and determined by OD600. The final OD was corrected for this 10 fold dilution. The media for E. coli 0157:H7 oxyR mutant always contained 1 Âµl/ ml kanamycin in order to select against loss of the antibiotic resistance genes that had replaced the oxyR genes. Aliquots (100Âµl) of cells suspension or appropriate dilution in sterile Ringer's solution were spread over the surface of the LB agar plates and incubated at 30oC.
The meat was dipped into diluted bacterial suspension of 104 CFU/ml so that it is totally immersed. Then meat was transferred into an empty vessel and allowed surface liquid to drain away. Meat were removed and cut into two portions. One half was treated with cold atmospheric plasma while the other half was used as negative control.
2.5 PLASMA TREATMENT
The gas used were helium at a flow rate of 7 SLM (standard liter per minute) mixed with an O2 flow at 35 SCCM (standard cubic centimetre per minute). Applied voltage was 37 V with a peak to peak voltage of 9-10 V, and the distance between the jet nozzle and the meat surface was fixed at 1cm. Plasma lethality was deliberately adjusted to be submaximal in order for differences in the responses of E. coli and its mutant to be detected. The exposure time of the sample to CAP-pen treatment were taken at 0 minute (negative control), 1 minute and 5 minutes.