Seminar SBG

7/30/2019 Seminar SBG

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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


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Principles of operation of CAP

technology

Cold atmospheric plasmas can be produced byselective 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


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How effects of the :-

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

growth phase.

growth temperature.

chemical treatment regime.


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Food-borne human illnesses resulting from contaminated

fresh produce have been reported in several Countries .

Most reporting countries identified leafy greens andberries 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


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Bacterial culture and sample

inoculationThe 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-exponentialphase

late-exponentialphase

(28 h)

(10 h)

(14 h)


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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).


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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)


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lettuce and strawberry surfaces and potatotissue, with no detectable initial levels of S.Typhimurium cells, were used to study the

efficacy of CAP treatment for theinactivation of this microorganism on realfood matrices.

Fresh produce transportedimmediately to the laboratory, where theywere kept in a refrigerator overnight at 4 Cbefore use

Electron micrographshowing Salmonella in thepores of a lettuce leaf


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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 thefood samples and spread.

Membrane filters and food samples were thenallowed to dry for up to 45 min in a laminar

flow cabinet before plasma treatment.


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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 :-

nitrogen plasma jet

1. large bandwidth

2. High impedance voltageprobe


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The plasma system also allows the grid tobe positively and negatively biased using an

external voltage source, therebydetermining the chemical treatment regime

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

reduction process is favoredover oxidationPositive bias

oxidation process is favoredover reduction.Negative bias


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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 perminute, at approximately 1 W output

power.


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Inoculated membrane filters and food

discs were treated with plasma forvarious 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 cellviability remained

constant throughout the nitrogentreatment period


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Following plasma exposure, each membrane

filter and vegetable disc was carefully removedfrom the agar with sterile forceps and placed intoa stomacher bag containing 10 mL of PSDF.

Plasma treated cells were recovered from themembrane filters through agitation using a

stomacher at medium speed for 1 min (LabSystem, England).

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

Viable cells from treated foods were determinedon Xylose Lysine Deoxycholate agar (XLD agar,Oxoid) to select for Salmonella. Preliminaryresults


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showed that XLD yielded the same

rate of Salmonella recovery as thenon-selective media PCA.

The PCA and XLD plates were

incubated at 25 C for 48 h and 37 Cfor 24 h, respectively, before

enumeration.

All experiments were conducted intriplicate using three biologically

independent cultures.


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Scanning electron microscopy

(SEM)

Food samples no more than 2 mm thick were usedand membrane filters were cut and fixed with 3%glutaraldehyde in 0.1 M cacodylate buffer (pH7.2) for 2 h.

The fixative was then replaced with 3 changes of0.1 M cacodylate buffer.

This was followed by dehydration for at least 20min in a series of ethanol solutions (10, 20, 30,40, 50, 60, 70, 80, 90, 3 100%).


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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 thefollowing

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 thebacterial

survival CFU to be reduced by 1 log cycle..


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Experimental parameters can

influence the inactivation efficiency ofSalmonella by CAP treatment these

include the:

Microbial load

Type of substrateon which

bacteria aredeposited

Processparameters such

as gascomposition,

relative humidity,flow rate, input

power and typeof discharge

,Temp., pH etc.


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The physiological state of the bacterial

cells, including growth phase, has alsobeen 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


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In order to study the effect of the growthphase on the subsequent CAP resistance ofS. Typhimurium, cultures were grownaerobically at 20 C in LB and harvested atmid-logarithmic phase, late-logarithmic phase

and stationary phase and deposited ontomembrane filters.

To study the influence of the growthtemperature, cultures were grown to

stationary phase at 20, 25, 37 and 45 C andwere CAP treated following deposition ontomembranefilters.

Survival curves obtained under the aboveconditions


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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 D-

values (min) obtained for the cells grown at 20 C and harvested at

stationary,

late-log and mid-log phase were (1.10 0.08), (1.000.07) and

(1.10 0.08), respectively. In relation to the effect of thegrowth

temperature the D-values (min) obtained were (1.100.07),

(1.00 0.10), (1.10 0.07) and (1.20 0.13) for the cellsgrown at


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20, 25, 37 and 45 C, respectively.Notably, these data show that the

growth phase of S. Typhimurium did

not significantly affect the CAPinactivation rates (p > 0.05). Similarly,

the growth temperature, in the range

from 20 to 45 C, did not significantlyaffect (p > 0.05) the resistance of S.

Typhimurium to CAP inactivation.


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The effect of the chemical treatment on the inactivationof

Salmonella by CAP plasma was investigated byapplying bias

potential to a metal grid placed under the sample. Theexperimental

set-up allowed the grid to be positively biased at 30 V

(reduction) using an external voltage source, therebyfiltering

electrons from the plasma and repelling positive ions,further

minimizing the interaction of these species with thesample.

Similarly, the grid can be negatively biased at 30 V(oxidation),

thereby repelling anions and avoiding their interaction

with the


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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) betweenpositive,

negative and zero bias. This observation indicates thatcharged

particles do not play a major role in the inactivation ofSalmonella

by nitrogen CAP treatment. Although the effect ofvarious plasma

components on the bacterial inactivation has recentlybeen investigated,

the contribution of each type of reactive species to the

inactivation is not well understood


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The inactivation effect of CAPtreatment on lettuce,strawberry, and

potato tissue superficially

contaminated with Salmonella,compared to the inactivation on

membrane filters, is shown in Fig. 6.


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Survival curves showed tailing (decline in inactivationrate) with increasing exposure time. Specifically, asharp decrease in populations was typically observedafter plasma treatment for the initial exposure times(15s, 1 min and 2 min for Salmonella inoculated onstrawberry,lettuce and potato, respectively), followed by

a tailing effect,observed after longer treatments. Themaximum reduction, obtainedafter 15 min of treatment,was 2.72 0.31, 1.76 0.67,0.94 0.30 log CFU forSalmonella inoculated on lettuce, strawberryand potato,respectively. When compared with inactivationcurvesobtained for Salmonella inoculated onto

membrane filters, thoseobtained from real producerequired much longer exposure timesto obtain the samereduction levels. For example, the time requiredtoinactivate 2.7 log cycles of Salmonella population onmembranefilterswas 5 times shorter that the timeneeded to achieve the samelevel of inactivation for

Salmonella inoculated on lettuce disks.


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the influence of the surfacetopography 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 aninoculated membrane filter, where


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Salmonella cells are spread evenly on the smoothsurface. Micrographs

B, C and D show inoculated lettuce, strawberry andpotato

disks, respectively. The micrographs show that theconvoluted

surface features of these tissues result in sequestrationof the

bacterial cells. Different food structures, such as thestomata of

lettuce, the convolutions of strawberry surfaces andthewalls of the

eukaryotic cells of potato tissue, can obscure someSalmonella cells

and/or create physical barriers that are likely to protectbacteria

from CAP inactivation.


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Food surfaces are rougher thanmembrane filter surfaces, which,

together with the possibility of

penetration of microorganisms withinstomata or irregularities, could explain

the longer times needed to

decontaminate foods.


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The intrinsic features of food surfacessuch as waxy cuticles, or othercomponents could also contribute todiffering degrees of CAPdecontamination.

The results obtained from our SEMstudies, also supported by other work

(Noriega et al., 2011), show theimportant influence of substratetopography on the efficacy of CAPinactivation of contaminating bacteria.


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In conclusion,

emerging technology, CAP, has thepotential to decontaminate food

produce and therefore to replace or

augment traditional preservationtechniques in the future. However, the

efficiency of inactivation by CAP s

related to food surface structures,perhaps suggesting combined ..


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decontamination approaches may achieve even greaterlevels of

decontamination.

A further point is that the plasma device used in this studywas a laboratory model and food treatment on a commercialscale would require scale-up devices that shouldsignificantly 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 toconfirm that no harmful by-products are generated by CAPtreatment.

Examination of the sensory properties of cold plasma-treated products and more information about the potentialnutritional and chemical changes occurring as a result oftreatment are also necessary to determine to what extent this

process affects product quality and shelf-life.


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The results reported here indicate that charged particlesdo not play a major role in the inactivation of S.Typhimurium by nitrogen CAP treatment and we alsoshow that bacterial growth phase and growthtemperature play a minor role in the efficiency ofinactivation.

In summary, many questions concerning the species ofantimicrobial compounds generated during plasmadischarge and their specific role in the mechanism ofmicrobial inactivation still remain to be addressed. Inaddition, a more complete understanding of the role ofcell structure, physiology and bacterial stress resistancemechanisms involved in plasma resistance is alsoneeded.


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Conclusion APP is an emerging non-thermal technology for

reducing microbial population on the surface offresh and processed foods.

Various reactive spices of plasma interact to

biological cell to cause changes on cell wall andmorphology of the microorganisms that lead todeath.

Because of the limit information about thenutritional and chemical changes in food

products treated with this technology, specially,sensitive food which has high amount of lipid andvitamins additional issues concerning food qualityand safety must be considered.


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Microbial inactivation mechanism of plasma Several mechanisms are considered to be responsible for

microbial inactivation. During plasma treatment, killingmicroorganisms are result of direct contact to antimicrobialactive 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 leadto microbial death or injury.

In addition to reactive spices, UV photons can modify DNA ofmicroorganisms and as a result disturb cell replication.

Contribution of mentioned mechanisms depends on plasmacharacteristics and to the 276 type of microorganisms.

The former includes voltage, working gas, water content inthe gas, distance of the microorganism from the dischargeglow, etc. where the latter takes account of Gram-positive,Gram-negative, spores and other types.

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