What is plasma ?

Plasma is ionized gas that consists of a large number of different species such as electrons, positive and negative ions, free radicals, and gas atoms, molecules in the ground or excited state and photons. It is considered to be the forth state of matter in the world

Principles of operation of CAP technology

Cold atmospheric plasmas can be produced by selective transfer of the generator power to plasma electrons via very high generation frequency or pulsing of the generation power. The generated plasma contains free radicals and excited or non-excited molecules and atoms, which in combination are able to inactivate microorganisms

How effects of the :growth phase.

growth temperature.

chemical treatment regime.

on the inactivation of Salmonella enterica serovar Typhimurium (S. Typhimurium) by Nitrogen CAP were examined

Food-borne human illnesses resulting from contaminated fresh produce have been reported in several Countries .

Most reporting countries identified leafy greens and berries as the main vectors, and either Salmonella, Escherichia coli O157:H7 or norovirus as the target pathogens. Salmonella was identified as the most frequent cause of food-borne outbreaks .

A wide range of fresh fruit and vegetable products have been implicated in Salmonella infections in recent years, such as lettuce, sprouted seed, melon, tomatoes, pepper and ba

Bacterial culture and sample inoculation

The bacterium used in this study was S. Typhimurium Cultures were grown aerobically in Luria Bertani broth (LB) (Difco) at 20 C to either :_
stationary phase mid-exponential phase late-exponential phase

(28 h)
(10 h) (14 h)

In order to study the effect of the growth temperature, cells were also incubated at 25, 37 and 45 C for 28 h (Fig. 1).

Dilutions of harvested cells were prepared in : 1) peptone salt dilution fluid (PSDF) 2) 30 mL aliquots were deposited onto 0.2 mm pore size 25 mm diameter Whatman polycarbonate membrane placed singly on LB agar plates (LBA, Oxoid)

lettuce and strawberry surfaces and potato tissue, with no detectable initial levels of S. Typhimurium cells, were used to study the efficacy of CAP treatment for the inactivation of this microorganism on real
food matrices.

Electron micrograph showing Salmonella in the pores of a lettuce leaf

Fresh produce transported immediately to the laboratory, where they were kept in a refrigerator overnight at 4 C before use

Food discs were obtained by using a sterile 25 mm - diameter cork borer and also deposited on LBA plates. Aliquots (30 mL) of the diluted bacterial culture were carefully pipetted onto the centre of the food samples and spread. Membrane filters and food samples were then allowed to dry for up to 45 min in a laminar flow cabinet before plasma treatment.

Plasma inactivation procedure

Plasma treatments were carried out in a commercially available nitrogen plasma jet. The CAP system used is based on a copper wire electrode configured as a :1. large bandwidth

2. High impedance voltage probe

nitrogen plasma jet

The plasma system also allows the grid to be positively and negatively biased using an external voltage source, thereby determining the chemical treatment regime
Positive bias
reduction process is favored over oxidation

oxidation process is favored Negative bias over reduction.

redox reactions are favored as a result of the equilibrium state or at zero bias

Under the experimental conditions assayed the temperature of the samples never exceeded 35 C. Experiments were performed at atmospheric pressure and nitrogen throughput of 12 standard litres per minute, at approximately 1 W output power.

Inoculated membrane filters and food discs were treated with plasma for various time periods. As a control, bacteria were exposed to nitrogen (discharge turned off) according to the same time series. It was found that in all cases cell viability remained constant throughout the nitrogen treatment period

Following plasma exposure, each membrane filter and vegetable disc was carefully removed from the agar with sterile forceps and placed into a stomacher bag containing 10 mL of PSDF. Plasma treated cells were recovered from the membrane filters through agitation using a stomacher at medium speed for 1 min (Lab System, England). Diluted aliquots were spread on Plate Count Agar (PCA) plates to allow enumeration of surviving bacteria. Viable cells from treated foods were determined on Xylose Lysine Deoxycholate agar (XLD agar, Oxoid) to select for Salmonella. Preliminary results

showed that XLD yielded the same rate of Salmonella recovery as the non-selective media PCA. The PCA and XLD plates were incubated at 25 C for 48 h and 37 C for 24 h, respectively, before enumeration. All experiments were conducted in triplicate using three biologically independent cultures.

Scanning electron microscopy (SEM)

Food samples no more than 2 mm thick were used and membrane filters were cut and fixed with 3% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) for 2 h.

The fixative was then replaced with 3 changes of 0.1 M cacodylate buffer. This was followed by dehydration for at least 20 min in a series of ethanol solutions (10, 20, 30, 40, 50, 60, 70, 80, 90, 3 100%).

Data analysis

D-values were calculated from the negative inverse slope of the survival curves, obtained from a plot of the log number of survivors vs their corresponding treatment times, using the following equation: logN=N0 t=D where: N bacterial population at any time, t. N0 initial bacterial population. D decimal reduction time, or time in minutes for the bacterial survival CFU to be reduced by 1 log cycle..

Experimental parameters can influence the inactivation efficiency of Salmonella by CAP treatment these include the:
Process parameters such as gas composition, relative humidity, flow rate, input power and type of discharge ,Temp., pH etc.

Microbial load

Type of substrate on which bacteria are deposited

The physiological state of the bacterial cells, including growth phase, has also been reported to play a decisive role in bacterial resistance to adverse environments. For example it is well documented that stationary phase Salmonella bacteria are more resistant to inactivation technologies such as thermal, acidic, high pressure and pulsed electric field treatments compared to exponential phase Salmonella

In order to study the effect of the growth phase on the subsequent CAP resistance of S. Typhimurium, cultures were grown aerobically at 20 C in LB and harvested at mid-logarithmic phase, late-logarithmic phase and stationary phase and deposited onto membrane filters. To study the influence of the growth temperature, cultures were grown to stationary phase at 20, 25, 37 and 45 C and were CAP treated following deposition onto membranefilters. Survival curves obtained under the above conditions

were properly fitted into a first order inactivation kinetic; D-values, expressed as the mean value of three independent experiments and standard deviation, were useful for the purpose of comparing microbial CAP resistance. The Dvalues (min) obtained for the cells grown at 20 C and harvested at stationary, late-log and mid-log phase were (1.10 0.08), (1.00 0.07) and (1.10 0.08), respectively. In relation to the effect of the growth temperature the D-values (min) obtained were (1.10 0.07), (1.00 0.10), (1.10 0.07) and (1.20 0.13) for the cells grown at

20, 25, 37 and 45 C, respectively. Notably, these data show that the growth phase of S. Typhimurium did not significantly affect the CAP inactivation rates (p > 0.05). Similarly, the growth temperature, in the range from 20 to 45 C, did not significantly affect (p > 0.05) the resistance of S. Typhimurium to CAP inactivation.

The effect of the chemical treatment on the inactivation of Salmonella by CAP plasma was investigated by applying bias potential to a metal grid placed under the sample. The experimental set-up allowed the grid to be positively biased at 30 V (reduction) using an external voltage source, thereby filtering electrons from the plasma and repelling positive ions, further minimizing the interaction of these species with the sample. Similarly, the grid can be negatively biased at 30 V (oxidation), thereby repelling anions and avoiding their interaction with the

shows the survival curves and the D-values obtained under the different chemical treatment regimes. According to these data, there was no statistically significant difference (p > 0.05) between positive, negative and zero bias. This observation indicates that charged particles do not play a major role in the inactivation of Salmonella by nitrogen CAP treatment. Although the effect of various plasma components on the bacterial inactivation has recently been investigated, the contribution of each type of reactive species to the inactivation is not well understood

The inactivation effect of CAP treatment on lettuce,strawberry, and potato tissue superficially contaminated with Salmonella, compared to the inactivation on membrane filters, is shown in Fig. 6.

Survival curves showed tailing (decline in inactivation rate) with increasing exposure time. Specifically, a sharp decrease in populations was typically observed after plasma treatment for the initial exposure times(15 s, 1 min and 2 min for Salmonella inoculated on strawberry,lettuce and potato, respectively), followed by a tailing effect,observed after longer treatments. The maximum reduction, obtainedafter 15 min of treatment, was 2.72 0.31, 1.76 0.67,0.94 0.30 log CFU for Salmonella inoculated on lettuce, strawberryand potato, respectively. When compared with inactivation curvesobtained for Salmonella inoculated onto membrane filters, thoseobtained from real produce required much longer exposure timesto obtain the same reduction levels. For example, the time requiredto inactivate 2.7 log cycles of Salmonella population on membranefilterswas 5 times shorter that the time needed to achieve the samelevel of inactivation for Salmonella inoculated on lettuce disks.

the influence of the surface topography on the efficacy of CAP treatment was observed using SEM. Fig. 7 shows images of the microstructure of the inoculated filter, lettuce and strawberry surfaces and potato tissue. Micrograph A shows the appearance of the surface of an inoculated membrane filter, where

Salmonella cells are spread evenly on the smooth surface. Micrographs B, C and D show inoculated lettuce, strawberry and potato disks, respectively. The micrographs show that the convoluted surface features of these tissues result in sequestration of the bacterial cells. Different food structures, such as the stomata of lettuce, the convolutions of strawberry surfaces and thewalls of the eukaryotic cells of potato tissue, can obscure some Salmonella cells and/or create physical barriers that are likely to protect bacteria from CAP inactivation.

Food surfaces are rougher than membrane filter surfaces, which, together with the possibility of penetration of microorganisms within stomata or irregularities, could explain the longer times needed to decontaminate foods.

The intrinsic features of food surfaces such as waxy cuticles, or other components could also contribute to differing degrees of CAP decontamination. The results obtained from our SEM studies, also supported by other work (Noriega et al., 2011), show the important influence of substrate topography on the efficacy of CAP inactivation of contaminating bacteria.

In conclusion,

emerging technology, CAP, has the potential to decontaminate food produce and therefore to replace or augment traditional preservation techniques in the future. However, the efficiency of inactivation by CAP s related to food surface structures, perhaps suggesting combined ..

decontamination approaches may achieve even greater levels of decontamination. A further point is that the plasma device used in this study was a laboratory model and food treatment on a commercial scale would require scale-up devices that should significantly reduce the treatment times and increase inactivation levels It should also be noted that for the application of this technique to the food industry, further studies are needed to confirm that no harmful by-products are generated by CAP treatment. Examination of the sensory properties of cold plasmatreated products and more information about the potential nutritional and chemical changes occurring as a result of treatment are also necessary to determine to what extent this process affects product quality and shelf-life.

The results reported here indicate that charged particles do not play a major role in the inactivation of S. Typhimurium by nitrogen CAP treatment and we also show that bacterial growth phase and growth temperature play a minor role in the efficiency of inactivation.

In summary, many questions concerning the species of antimicrobial compounds generated during plasma discharge and their specific role in the mechanism of microbial inactivation still remain to be addressed. In addition, a more complete understanding of the role of cell structure, physiology and bacterial stress resistance mechanisms involved in plasma resistance is also needed.

Conclusion APP is an emerging non-thermal technology for reducing microbial population on the surface of fresh and processed foods. Various reactive spices of plasma interact to biological cell to cause changes on cell wall and morphology of the microorganisms that lead to death. Because of the limit information about the nutritional and chemical changes in food products treated with this technology, specially, sensitive food which has high amount of lipid and vitamins additional issues concerning food quality and safety must be considered.

Microbial inactivation mechanism of plasma Several mechanisms are considered to be responsible for microbial inactivation. During plasma treatment, killing microorganisms are result of direct contact to antimicrobial active spices. Accumulation of charged particles at the surface of the cell membrane can rupture the cell membrane. Oxidation of the lipids, amino acids and nucleic acids with reactive oxygen and nitrogen spices cause changes that lead to microbial death or injury. In addition to reactive spices, UV photons can modify DNA of microorganisms and as a result disturb cell replication. Contribution of mentioned mechanisms depends on plasma characteristics and to the 276 type of microorganisms. The former includes voltage, working gas, water content in the gas, distance of the microorganism from the discharge glow, etc. where the latter takes account of Gram-positive, Gram-negative, spores and other types.