samar ghazawi >>cold plasma >> my seminar>>2012/2013

By : Samar Al-Ghazawi Supervisor: Prof. Dr. Samih Tamimi

Inactivation of Salmonella typhimurium on Fresh

Produce By Cold Atmospheric

Nitrogen Plasma Technology

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, gas atoms, molecules in the ground or excited state and photons. It is considered to be the fourth state of material in the world

Thermal and nonthermal plasma

Thermal and nonthermal plasmas

If Te ≈ Tion then we have a thermal plasma

If Te » Tion then plasma is nonthermal or nonequilibrium or cold plasma

Cold Atmospheric Nitrogen Plasma Technology

(CAP) Treatment of particular interest for

The decontamination of food surfaces since it doesn't require extreme process conditions compared to classical preservation methods such as heat treatments, which have a negative impact on vegetable tissues

There are environmental and health risks

associated with its by-products.

Increasing number of consumers are demanding

products which are free from harmful chemicals

use of non-thermal cold gas

plasmas.

Why we use the Cold Atmospheric Nitrogen Plasma Technology?

Chlorine products are used to decontaminate fresh fruit and vegetables in the produce industry .

However,

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

Most reporting countries identified leafy greens as the main vector, and either Salmonella, Escherichia coli or norovirus as the target pathogens.

A wide range of fresh fruit and vegetable products have been implicated in Salmonella infections in recent years, such as lettuce, tomatoes, and strawberry .

The objective of the present work was to study the effect of :-

on the inactivation of (S. typhimurium) by Nitrogen CAP.

Growth phase.

Growth temperature.

Chemical treatment regimem.

Influence of the surface topography

MATERIAL AND METHODS

Bacterial culture and sample inoculation

The bacterium used in this study was S. typhimurium

Cultures were grown aerobically in Luria Bertani broth (LB) at 20º C to:_

stationary phase

mid-exponential phase

late-exponential phase

• (28 h)

• (10 h)

• (14 h)

placed on LB agar plates (LBA, Oxoid)

30 mL were deposited onto Whatman polycarbonate membrane

Dilutions of harvested cells were prepared in :

peptone salt dilution fluid (PSDF)

Fresh produce transported immediately to the laboratory

conserved in a refrigerator overnight at 4º C before use.

30 mL of the diluted bacterial culture were carefully pipetted onto the centre of the food samples and spread.

Food discs deposited on LB agar plates.

lettuce

strawberry

potato

Membrane filters and food samples were then allowed to dry for up to 45 mins in a laminar flow cabinet before plasma treatment.

Plasma inactivation procedure Plasma treatments were carried out in a commercially available nitrogen plasma jet.

nitrogen plasma jet

Electrical potential is perturbed by the afterglow of a jet of excited state nitrogen produced by a high voltage discharge typically sampled at a rate of 1 kHz

The electrode is located 25 mm above the gas outlet.

A grid near the electrode, held at the same potential, is used to remove space charge .

The plasma system also allows the grid to be positively and negatively biased using an external voltage source, that way determining the chemical treatment regimem

• reduction processPositive bias

• oxidation processNegative bias

• as a result of the equilibrium state or at “zero” bias redox reactions

The experimental conditions:-

temperature of the samples never exceeded 35º C.

atmospheric pressure and nitrogen throughput of 12 standard litters per minute,

at approximately 1 W output power.

Inoculated membrane filters and food discs

were treated with plasma for

different time periods.

As a control, bacteria

were exposed to nitrogen

(discharge turned “off”)

according to the same time periods.

It was found that in all

cases cell viability remained constant

throughout the nitrogen

treatment period

Plasma treated cells were recovered from the membrane filters through agitation using a

stomacher at medium speed for 1 min.

placed into a stomacher bag containing 10 mL of PSDF.

Following plasma exposure, each membrane filter and vegetable disc was carefully removed from the

agar with sterile forceps

Diluted aliquots were spread on Plate Count Agar (PCA) plates to allow enumeration of surviving bacteria.

25 ºC for 48 h

PCA plate was incubated at:-

Viable cells from treated foods were determined on

Xylose Lysine Deoxycholate agar (XLD agar, Oxoid) to

select for Salmonella.

XLD plate was incubated at:- 37 ºC for 24 h

Preliminary results

showed that XLD yielded the same rate of Salmonella recovery as the non-selective media

PCA.

Scanning electron microscopy (SEM)

we evaluated the physical structure and microbial

distribution of S. typhimurium on the surfaces of the target

foods using Scanning Electron Microscopy (SEM).

The microstructure of the food surfaces and its effect on the

distribution of S. typhimurium suggest a possible reason for

the food-specific variability of CAP inactivation

Data analysis

D-values, expressed as the mean value of three

independent experiments and standard deviation, were

useful for the

comparing microbial CAP resistance.

determine significant differences between D-

values at p < 0.05

Results

The D-values (min) obtained for the cells grown at 20 º C and harvested at stationary, late-log and mid-log phase

Cold plasma inactivation curves and D-values of S. typhimurium cells harvested at different growth phases.Data show the growth phase of S. typhimurium didn’t significantly affect the CAP inactivation rates (p > 0.05).

The effect of the growth temperature the D-values (min) obtained were for the cells grown at 20, 25, 37 and 45 C

Cold plasma inactivation curves and D-values of S. typhimurium cellsgrown at different temperatures. Data show, didn’t significantly affect the resistance of S. typhimurium to CAP inactivation (p > 0.05)

The D-values obtained under the differentchemical treatment regimems

Cold plasma inactivation curves and D-values of S. typhimurium cells treated under different chemical regimems. Data show the charged particles do not play a major role in the inactivation of Salmonella by nitrogen CAP treatment.

This Data, show that we need 2 mins treatment resulted in a 2.71 log-reduction of S. Typhimurium viability on membrane filters whereas a 15 mins treatment was necessary to achieve 2.72 log-reductions of viability on lettuce, 1.76 log-reductions strawberry and 0.94 log-reductions potato

Cold plasma inactivation curve and maximum log reduction-values of S.Typhimurium cells treated on different substrates

The intrinsic features of food surfaces such as waxy cuticles.

Food surfaces are rougher than membrane filter surfaces.

possibility of penetration of microorganisms within stomata.

Could explain the longer times needed to decontaminate foods.

The influence of the surface topography onthe efficiency of CAP treatment was observed using SEM.

Different food structures

• Stomata of lettuce

• Convolutions of strawberry surfaces.

• Walls of the eukaryotic cells of potato tissue.

Can hide some Salmonella cells and/or create physical barriers that are likely to protect bacteria from CAP inactivation.

Magnification membrane filters at 250x membrane filters 10,000x

Micrograph shows the surface of an inoculated membrane filter, where Salmonella cells are spread evenly on the smooth surface

The micrographs show Stomata of lettuce

Magnification lettuce 250 x lettuce 10,000 x

Magnification strawberry at 250 x strawberry 10,000 x

The micrographs show Convolutions of strawberry surfaces.

Magnification potato at 250 x potato 10,000 x

The micrographs show walls of the eukaryotic cells of potato tissue.

Conclusion

emerging technology, CAP, has the potential to decontaminate food produce and therefore to replace

traditional preservation techniques in the future.

The efficiency of inactivation by CAP related to food surface structures.

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.

A further point

Waste water treatment

Sterilization of packaging materials

Surface decontaminati

on of eggs

The application of the Cold atmospheric gas plasma

technology

ThanksThanksfor your attention