School of Food Science and Technology, Jiangnan University

Food Microbiology

Lecturer: Prof. Tian Fengwei

Cold Atmospheric Plasma Technology for

Food Preservation

Fulfilled: Do Thi Van Thanh

November -2016
Introduction

Food-borne pathogens and spoilage microorganisms are highly problematic to resolve in food industry
because of its significant public health risks and economic impact. There are a lot of sterilization
methods to eliminate these microorganisms(Ragni, Berardinelli et al. 2010). Some of these methods rely
on lethal heat treatment such as steam pasteurization, autoclaving, ohmic heating, etc. Most of the novel
and emerging technologies in food processing inactivate microorganisms by physical treatments other
than heating; these include high hydrostatic pressures, pulsed electric fields, application of UV,
ultrasound and more recently, non-thermal plasmas. This latter treatment is one of the more promising
food preservation technologies. It promotes an efficient inactivation of many different types of
microorganisms including spores and viruses, and some yeasts and fungi (Fernndez and Thompson
2012). And most of the previous work on bacterial inactivation has been conducted with bacteria
deposited on the surface of abiotic materials such as membrane filters and glass slides, data on the
disinfection of foodstuffs is steadily accumulating. However, they exist their own defects, they are
costly and required specialized equipment and trained personnel. Lastly, it would be important to take
into account consumer perception of any novel decontamination technology and before
commercialization of plasma treatment in the food industry could become a reality, this would need to
be addressed.(Noriega, Shama et al. 2011)

Therefore, the objective of this paper is to review a general overview to fundamental aspects of plasma
including definition, generation and classification, decontamination and sterilization mechanism.
Especially mention of atmospheric pressure cold plasma, its application in microbial inactivation in food
preservation and future prospect.

Fundaments of Plasma

Plasma definition, generation

Plasma is a quasi-neutral gas, is considered to be the fourth state of matter, following the more familiar
states of solid, liquid and gas and constitutes more than 99% matter of the universe. It is more or less an
electrified gas with a chemically reactive media that consists of a large number of different species such
as electrons, positive and negative ions, free radicals, gas atoms and molecules in the ground or any
higher state of any form of excited species [fig. 1]. It can exist over an extremely wide range of
temperature and pressure by means of coupling energy to gaseous medium. It can be produced at low-
pressure or atmospheric pressure (Bradley 2014). Plasma can be found in the form of natural
phenomena such stars and lightening or man-made as in the production of fluorescent and neon lights,
plasma television etc. The research areas of plasma technology is fast growing and has been particularly
studied for its use on bio-medical materials and devices , surface modification of textiles , removal of
chemicals on surfaces of devices manufactured from heat sensitive materials, water sterilization and
more recently wound healing and food decontamination(Aslan 2013)
 Fig. 1. Constituents of plasma

Classification
All varieties of plasma-chemical systems are traditionally divided into two major categories: thermal
and non-thermal ones, characterized by their specific advantages and disadvantages. Thermal plasmas
(usually arcs or radio frequency (RF) inductively coupled plasma discharges) are associated with Joule
heating and thermal ionization, and enable the delivery of high power (to over 50 MW per unit) at high
operating pressures. However, low excitation selectivity, very high gas temperature, serious quenching
requirements and electrode problems result in limited energy efficiency and applicability of thermal
plasma sources. Non thermal plasmas (of low pressure glow, RF and microwave discharges) offer high
selectivity and energy efficiency in plasma chemical reactions; they are able to operate effectively at low
temperatures and without any special quenching(Fridman, Chirokov et al. 2005).

Low temperature plasma is further subdivided into thermal plasma, also called quasi-equilibrium plasma,
which is in a local thermal equilibrium (LTE) state, and non thermal plasma (NTP), also called non
equilibrium plasma or cold plasma (Vijay Nehra 2008), shown in Table 1. Cold plasmas refer to the
plasmas where most of the coupled electrical energy is primarily channeled to the electron component of
the plasma, thereby producing energetic electrons instead of heating the entire gas stream.

Non-thermal atmospheric plasmas

Cold or non thermal Plasma (NTP) is a high-energy state of a gas when an electrical current is allowed
to pass due to high voltages, cold atmospheric plasmas (CAP) are produced by the excitation of gas with
electrical discharges at room temperature and atmospheric pressure. Actually, non-thermal plasmas can
be generated by means of microwave, RF (radio frequency), pulsed, AC (alternating current) or DC
(direct current) power sources, and several device geometries, electrode configurations, gas mixtures
and flow rates have been studied (Laroussi 2002). Until relatively recently, plasmas could only be
generated under vacuum at temperatures inappropriate for food treatment. However, the advent of
methods for electrically generating non- thermal gas plasmas at ambient temperature and atmospheric
pressure makes them suitable for the decontamination of foods (Kogelschatz 2002). The application of
non-thermal atmospheric pressure plasma is a discipline with increasing attention in the field of food
processing and an emerging non-thermal technology for reducing microbial load on the surface of fresh
and processed foods. Thus the potential applications of non-thermal atmospheric pressure plasma for the
food industry are manifold and it has specific potential for the treatment of foods (Uta Schnabel 2016).

Table 1 Classification of plasma

An important aspect in the use of NTP for decontamination, is the ability to be effective, without
affecting the material being decontaminated. This is possible due to the weakly ionized nature of the
cold plasma discharge. The antimicrobial efficacy of NTP has been related to the specific type of plasma
technology used including; the power level used to generate the plasma, the gas mixture in the plasma
emitter, the intensity and length of exposure, design of the system, flow rate and pressure. However,
over the last few years technological developments have made it possible to produce cold plasma under
atmospheric pressures providing cost effective systems(Aslan 2013).
To date several types of NTPs that work at atmospheric pressure have been described for bio
decontamination purposes [Fig.2], radio frequency (RF) torches, dielectric barrier discharges (DBD),
corona discharges and gliding arc discharge plasmas.
 Fig. 2. a) dielectric barrier discharge b) gliding arc discharge c) radio frequency torch

All plasma generated agents, including charged particles, come in contact with the sample. Conversely,
indirect or remote exposure requires the sample to be placed at a distance from the plasma discharge or
in an adjacent chamber [Fig 3]. In this configuration, the charged particles do not play a role since they
recombine before reaching the sample, and many of the short-lived neutral reactive species also do not
reach the sample.(Fernndez and Thompson 2012).

Fig. 3. Schematics of a direct (a) and indirect (b) plasma treatment of contaminated agar plates. Grid electrode below the
agar surface traps the charged particles.

Microbial inactivation mechanism of plasma

Atmospheric pressure NTP has been observed to have a killing effect on many kinds of microorganisms
and is therefore becoming the technology of choice for decontamination purposes. Studies have
outlined three basic mechanisms attributed to cell death by plasma(Moisan, Barbeau et al. 2001).

1. Direct destruction by UV irradiation of the genetic material of the microorganism.

2. Erosion of the microorganism, atom by atom, through intrinsic photodesorption. Photon-induced
desorption results from UV photons breaking chemical bonds in the microorganism material and
leading to the formation of volatile compounds from atoms intrinsic to the microorganism. The
volatile by-products of this non-equilibrium chemistry are small molecules (eg CO and CHx should
then be possible).
3. Erosion of the microorganism, atom by atom, through etching. Etching results from the adsorption
of reactive species from the plasma (glow or afterglow) on the microorganism with which they
subsequently undergo chemical re-actions to form volatile compounds (spontaneous etching). The
reactive species can be atomic and molecular radicals, for example O and OH, respectively, and
excited molecules. In certain cases, the etching mechanism is enhanced by UV photons (UV-
induced etching), the photons acting synergistically with the reactive species, thereby accelerating
the elimination rate of microorganisms. This UV-induced chemistry under non-equilibrium
conditions can result in the desorption of radicals and molecules, at both the intermediate and final
stages of oxidation.

These deactivation mechanisms cause several reactions in bacterial cells including lipid peroxidation of
poly-unsaturated fatty acids, oxidation of amino acids and DNA oxidation. The main chemical reactions
leading to the production of reactive species in atmospheric plasma discharges are shown in Table 2.
These reactive species have higher sterilization and chemical potentials than conventional chemical
agents giving plasma its potential antimicrobial effect (Aslan 2013).
Table 2 Possible chemical reactions in the atmospheric plasma discharges.

The ability of non-thermal atmospheric plasmas to inactivate vegetative cells, spores, yeast, fungi and
biofilm formers is clearly evident in the literature with the use of various NTP systems. Perhaps
the most-studied organisms to date have been the bacteria, parasites have received rather less attention
from the gas plasma community (Shama and Kong 2012).
The rates and biocidal effects of NTP have shown great variance based on the system used, material
being treated, the bacterial strains and species. It is known that vegetative cells tend to be more
susceptible to plasma application than spores, as is generally true of most sterilizing systems.
Differences in efficacy of such systems have also been demonstrated regarding gram -positive and gram-
negative bacteria, where gram-negative species have been seen to be more susceptible to plasma
treatment than gram positive ones (Rami Ben Gadria 2000).
However, despite the obvious potential of atmospheric NTP technology the inactivation mechanisms of
these different plasma systems are not yet fully understood. The variance in plasma source, process
parameters and process gases used, results in differences in the produced reactive species, thus making
inactivation mechanisms difficult to compare.

Atmospheric Cold Plasmas in the Food Industry

The most obvious application of gas plasmas in the food industry would be to employ them directly to
decontaminate foods. Evidence has steadily been accumulating to show that gas plasmas can be used to
effect reductions in microbial counts for a wide range of different foods covering virtually all food types
including meat, dairy products and plant foods such as fruits, salad crops and nuts (Shama and Kong
2012). A compilation of such results for a range of foods is shown in table 2.
Salmonella spp. has been reported largely as a potential hazard for egg consumers. Decontamination of
egg surface using barrier discharge plasma was studied by (Ragni, Berardinelli et al. 2010). Samples of
shell eggs experimentally inoculated with Salmonella Enteritidis and Salmonella Typhimurium (5.56.5
Log CFU/eggshell) and placed in the treatment chamber. After 90 min of treatment, reductions up to 2.5
Log CFU/eggshell and 4.5 Log CFU/eggshell were observed for S. enteritidis using air with low and
high moisture contents, respectively. No significant negative effects of the gas plasma were observed on
the egg quality traits. The efficacy of cold atmospheric gas plasmas for decontaminating chicken skin
and muscle inoculated with Listeria innocua was examined. Under optimal conditions, a 10 s treatment
gave > 3 log reductions of L.innocua on membrane filters, an 8 min. treatment gave 1 log reduction on
skin, and a 4 min. treatment gave > 3 log reductions on muscle (Estefana Noriega 2011).
Low Temperature Plasma (LTP) system for its capability of killing Escherichia coli in milk with
different fat contents. 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(Gurol, Ekinci et al.
2012). LTP did not cause any significant change to the pH and color values of raw milk samples.
 Table 3 Foods treated with atmospheric gas plasmas (Shama and Kong 2012)

Fresh fruits and vegetables are indispensable components in our daily diet. Conventional thermal
methods of food sterilization, while effective, are unsuitable for fruits and vegetables, since heating
causes inevitable changes of color, smell, flavor, and a loss of nutritional value. A direct-current
atmospheric-pressure cold plasma microjet (PMJ) was applied to decontaminate slices of cucumbers,
carrots and pears with Salmonella. (Wang, Nian et al. 2012)

Fig. 4. Plasma inactivation of (a) cucumber slice, (b) carrot slice, (c) pear slice.

Effective inactivation was achieved on sliced fruit and vegetables after 1 s plasma treatment. The
physiochemical properties of the slices, such as water content, color parameters, and nutritional content
were monitored before and after plasma treatment. It was found that the physiochemical properties
changes caused by the plasma were within an acceptable range (Wang, Nian et al. 2012).

In one study the effect of an atmospheric cold plasma apparatus (air DBD, 15 kv) on the safety,
antioxidant activity and quality of radicchio (red chicory, Cichorium intybus L) was investigated after 15
and 30 min of treatment (in afterglow at 70 mm from the discharge,at 22c and 60% of RH) and during
storage. Escherichia coli 0157:H7 inoculated on radicchio leaves was signiicantly reduced after 15 min
cold plasma treatment (-1.35 log MPN/cm2). However, a 30 min plasma treatment was necessary to
achieve a signiHcant reduction of Usteria monocytogenes counts ( 2.2 log CFU/cm2). Immediately after
cold plasma treatment, no significant effect emerged in terms of antioxidant activity assessed by the
ABTS and ORAC assay and external appearance of the radicchio leaves (Pasquali, Stratakos et al. 2016).
Table 4. Effect of cold plasma on the antioxidant activity of radicchio leaves assessed by ABTS and ORAC values (M TE/g
dried weight) (n = 6) (Pasquali, Stratakos et al. 2016).

Treatment time (min) Sample ABTS ORAC

C 193 22 a 98 1 a
15 T 219 8a 117 5a
30 T 213 18 a 97 18a

Furthermore, the microbial inactivation efficiency of the plasma jet system at these operating parameters
was successfully tested on Escherichia coli bacteria, inoculated on corn salad surface at 107 cfu cm-2
and 104 cfu cm-2. At 20 W, bacteria with lower initial load could be inactivated by 3.6 (0.6) log-cycles
within 15 s of treatment duration; whereas at the higher initial load of 107 cfu cm-2, bacteria were
reduced by 2.1 (0.2) log-cycles after 30 s (Baier, Foerster et al. 2013). Indirect plasma techniques at
plasma-source to-sample distances at which charged particles cannot reach the sample surface are
potentially better suited for fresh produce than direct treatment.
Naturally contaminated peppercorns and with Bacillus subtilis spores, Bacillus atrophaeus spores and
Salmonella enterica inoculated ones were treated using a plasma jet or a microwave-driven remote
plasma. Surface color and the content of essential oils and piperine was measured. S. enterica, B. subtilis
spores and B. atrophaeus spores were reduced by 4.1, 2.4 and 2.8 log, respectively, after 30 min remote
plasma treatment(Hertwig, Reineke et al. 2015). Direct plasma jet treatment did not result inequivalent
inactivation levels. However, both plasma applications did not considerable affect the quality parameters.

Fig.7. SEM pictures of inoculated Bacillus atrophaeus spores on the surface of black peppercorns, (A and B) untreated, (C)
15 min direct plasma treatment and (D) 30 min remote plasma treatment(Hertwig, Reineke et al. 2015).

A direct plasma treatment for maintaining freshness and safety of fresh-cut lettuce without using any
other chemical preservatives, with a 2 cm distance from the treated sample, which was applied for 1 min,
presented good results in terms of final quality attributes that were assessed immediately after the
treatment but also during the storage period [Fig.8]. Depending on the applied process parameters, the
 quality of the lettuce leaves was less affected by the cold plasma. However, the treatment conditions
have to be adapted to each type of commodity(Irina SMEU and Mona Elena POPA 2012).

Fig.8. Total color differences during storage period for control and 2 cm plasma treated samples(Irina SMEU and Mona
Elena POPA 2012).

The future prospect of Cold Atmospheric Plasmas

The range of foods subject to plasma treatment will doubtless continue to grow. 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. The role of cell structure,
physiology and bacterial stress resistance mechanisms involved in plasma resistance is also poorly
understood at the moment. One of the important aspects of experimental work on CAP is that,
treatment must be confirmed not to have negative impact on the organoleptic and nutritional
properties of food. Therefore, it is a necessary for further studies to specify which CAP affects the
chemical and nutritional properties of foods and its shelf life. Moreover, risk assessment of toxic
residues should be carried out in future works.

However, any large-scale industrial application of cold atmospheric-plasma technology in food

decontamination, after appropriate regulatory approval, would require a suitable 2D plasma source much
larger than the microjet (Wang, Nian et al. 2012).

Conclusion
Atmospheric cold plasma represents a good opportunity for the decontamination of foods as an
alternative method for those products that cannot be sanitized with conventional means. And more
importantly for the application of this technique in 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 plasma treated products and more information about its nutritional and chemical
effects are also necessary to determine to what extent this process affects product quality and shelf-life.
The last but not the least, evaluation of the projected cost of treatment and safety of applied gas for
scaling up this technology in food industry should be addressed to determine the applicability of this
method.

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