22nd International Symposium on Plasma Chemistry
July 5-10, 2015; Antwerp, Belgium
Sterilization of cow’s milk using liquid plasma
S.B. Ponraj1, J. Sharp1, J.R. Kanwar2, A.J. Sinclair2, L. Kviz1, K.R. Nicholas3 and X.J. Dai1
1
Institute for Frontier Materials and 2 School of Medicine, Deakin University, Geelong Waurn Ponds, Victoria
3216, Australia
3
Monash University, Melbourne, Victoria 3001, Australia
Abstract: Liquid plasma was studied for its ability to kill bacteria in milk (pasteurized,
inoculated with Escherichia coli) and raw milk. Plasma was generated in the milk by a
nanosecond pulse generator (18 kV, argon) at two different frequencies: i) 2.5 kHz, and
ii) 4 kHz for 2 minutes. No viable cells were detected in milk (inoculated with E.coli)
stored for up to 4 weeks after treatment and no significant pH change was observed in
either milk type.
Keywords: liquid plasma, nanosecond pulsed generator, milk, bacteria
1. Introduction
Bovine milk is a natural food, rich in nutrients, growth
factors, hormones and other bioactive substances, that
provides many health benefits to humans. The nutritional
benefits as well as the hazards of drinking raw milk are
reported elsewhere [1]. Pasteurization is a well-known
technique to eliminate microbial populations in milk.
However, it has been observed that heat treatment alters
the chemical composition (proteins, lipids and
carbohydrates) [2] by several modifications which affects
the physiochemical characteristics of milk [3]. The
removal of bacteria from milk, without altering its
chemical composition, has long been a challenge [4].
Plasma-based sterilization is a promising non-thermal
technology, which can be applied to decontamination of
foods [5], medical devices [6], dentistry [7], dermatology
[8], agriculture [9] and waste water treatment [10].
However, there are very few studies examining plasma
treatment on the microbial quality and physicochemical
characteristics of milk [11, 12].
It has been reported that atmospheric pressure plasma
produces UV light, free radicals, ions, reactive oxygen
species (ROS) and reactive nitrogen species (RNS), which
all act individually or together as strong sterilization
agents [13]. These agents contribute to killing the
microbes by three possible mechanisms: the destruction
of DNA, etching of the microbial cell surface and
volatilization of compounds [14, 15]. The aim of this
study was to investigate the potential of a nanosecond
pulsed atmospheric pressure plasma (NPAPP) system to
kill bacteria, while maintaining the physicochemical
characteristics, and to increase the shelf life of milk.
2. Material and Method
Commercial pasteurized (full cream) and raw milk
samples were purchased from a supermarket in Geelong,
Australia. The milk was inoculated with Escherichia coli
strain ATCC 11229. The bacterial inoculation solution for
milk was prepared according to a standard procedure [11].
The final bacterial population in the milk was calculated
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using serial dilution (6.57 Log CFU/mL in E.coliinoculated milk and 6.06 Log CFU/mL in raw milk).
Nanosecond Pulsed Atmospheric Pressure Plasma
(Liquid Plasma) System
The reaction vessel contains a needle to plate electrode
configuration to generate plasma. Both electrodes were
immersed in milk and spaced at a distance of 1 cm, as
shown in Scheme 1. Argon gas was used to produce the
plasma. The pulse parameters were 10 ns pulse width,
±9 kV operating voltage. Plasma input was changed by
operating at two different frequencies: i) 2.5 kHz, and
ii) 4 kHz, while keeping the other parameters the same for
2 minutes. The reaction vessel was surrounded with ice to
limit any heating.
Scheme 1. Schematic view of liquid plasma generation
between two electrodes immersed in the milk
Milk samples were assessed for microbial quality and
shelf life at 4 °C. Microbial growth was monitored by
plating an aliquot of milk samples onto plate count agar
media and incubated at 37 °C for 48 hours. The bacterial
colonies were counted and the results were expressed in
log CFU per mL. Temperature and pH were measured
1
3. Results and Discussion
The potential of nanosecond pulsed atmospheric
pressure plasma at 18 kV, 4 kHz for two minutes was
evaluated in 1) pasteurized, E.coli-inoculated milk and 2)
raw milk.
1) Pasteurized, E.coli-inoculated milk
All cells were dead in E.coli-inoculated pasteurized
milk (Fig.1c) after plasma treatment at 4 kHz for 2
minutes, whereas little cell death (Fig.1b) was observed at
2.5 kHz. It is notable that, using plasma (4 kHz)
treatment, a ~6.5 log reduction (Fig.2) relative to the
control was achieved.
Fig. 1. Colony reduction in E.coli inoculated milk, after
two minutes of liquid plasma treatment at different
frequencies a) control (untreated), b) 2.5 kHz and c) 4
kHz
Gurol et.al studied the effect of atmospheric corona
discharge using tungsten electrodes in different types of
milk (whole, semi-skimmed and skimmed) and observed
a ~4 log reduction of E.coli after 20 minutes plasma
treatment [11]. Hyun-Joo Kim et.al used dielectric barrier
discharge plasma to inactivate different aerobic bacteria
(E.coli, Listeria monocytogenes and Salmonella
typhimurium) in milk [12]. They achieved an
approximately 2.40 log reduction and also noticed slight
changes in the physicochemical quality of the milk.
Log reduction of E.coli
Log reduction in E.coli-inoculated milk
7
6
5
4
3
2
1
0
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**
**
2.5
4
Plasma frequency (kHz)
Fig. 2. Log reduction of inoculated E.coli in commercial
milk by liquid plasma in two minutes at different
frequencies i) ~2.5 kHz and ii) ~4 kHz. The values are
statistically significant between the two samples
(P<0.001)
2
2) Raw milk
The same methodology was applied to assess the effect
of plasma treatment in raw milk. Raw milk contains
different bacterial types such as Campylobacter jejuni, S.
typhimurium, L. monocytogenes and E.coli [1].
Interestingly, all types of bacteria were killed after plasma
treatment (4 kHz) of 120 seconds (Fig.3 and Fig.4). The
plasma treatment was further varied from 75 to 120
seconds, keeping the other plasma parameters the same, to
more accurately determine the needed treatment time. The
bacterial count was reduced from 6.06 log CFU/mL (0s,
Fig 3a) to 5.16 (75s, Fig 3b), 4.92 (90s, Fig 3c), 2.51
(105s, Fig 3d), and no viable cells were observed after
120 seconds (Fig 3e). The increased death of cells
correlated with increased plasma treatment time.
Fig. 3. Bacterial colony reduction in raw milk (~4 kHz)
at different treatment times a) control (untreated), b) 75 c)
90, d) 105 and e) 120 seconds
Log reduction in Raw Milk
Log reduction of Bacteria
before and after plasma treatment.
Results are expressed as mean ± one standard deviation
of duplicate determinations. A student t-test was
performed to obtain the statistical significance of
differences between the samples.
***
8
**
4
2
**
***
6
**
**
0
75
90
105
120
Plasma treatment time (seconds)
Fig. 4. Log reduction of bacterial population in raw milk
by liquid plasma treatments (~4 kHz) for different
treatment times a) control, b) 75 c) 90, d) 105 and e) 120
seconds. The values are statistically significant between
the samples (P<0.001)
In this work, argon was used to produce a gas bubble
discharge inside the milk as Ar plasma in this setup
produces a much higher density of H 2 O 2 in DI water (as
will be presented elsewhere in the conference). The gas
bubble movement inside the milk helps to expose all the
bacteria to the effects of the bubble discharge. H 2 O 2 is
well known for its unique bactericidal properties in milk
[16]. The concentration and contribution to bacterial
killing can be varied by the power input, sample volume,
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type of gas, electrode gap and geometry. The role of other
ROS and RNS in killing bacteria has been reported
elsewhere [14, 17].
No significant pH change (6.7±0.1) was observed in
E.coli-inoculated milk and in raw milk at different
treatment times (0, 75, 90, 105 and 120 seconds). This
might be due to the buffering nature of milk, which can
absorb either or both H+ and OH- ions produced by the
discharge.
A moderate increase in temperature (final 46 °C) was
observed after the plasma treatment in both milk types.
This increase in temperature may contribute to the
bacterial killing in milk by plasma treatment and needs
further investigation. However, the temperature rise can
still be regarded as not being sufficiently high, to effect
bacterial killing.
The shelf life of plasma (4 kHz) treated milk stored at 4
°C was assessed every 7 days by plating onto PCA. No
signs of E.coli growth over a period of 4 weeks was
observed. This value is greater than the shelf life of
pasteurized milk (72 °C, 15 seconds) which is generally
allowed to be stored for approximately two weeks.
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
J. Heinlin, et al., JDDG: Journal der Deutschen
Dermatologischen Gesellschaft, 8, 12 (2010).
L. Ling, et al., Sci. Rep., 4, (2014).
M. Hijosa-Valsero, et al., Journal of hazardous
materials, 262, (2013).
C. Gurol, et al., International journal of food
microbiology, 157, 1 (2012).
H.-J. Kim, et al., Food Control, 47, (2015).
M. Moisan, et al., Pure and applied chemistry, 74, 3
(2002).
D. Dobrynin, et al., New Journal of Physics, 11, 11
(2009).
M. Korachi, C. Gurol, and N. Aslan, Journal of
Electrostatics, 68, 6 (2010).
H. Lück, Milk hygiene: Hygiene in milk production,
processing and distribution, (1962).
B.G. David, Journal of Physics D: Applied Physics,
45, 26 (2012).
4. Future work
Analysis of the major milk components (protein,
carbohydrate and fat) of plasma treated milk (E.coli
inoculated and raw) is underway and will be presented at
the conference.
5. Conclusion
The results suggest that a nanosecond pulsed
atmospheric pressure plasma treatment can be effective at
sterilizing milk at low temperature and can increase its
storage life at 4 °C to at least four weeks. However, the
sensory, bioactive and nutritional characteristics of
plasma treated milk and possible effects on individual
components need to be studied in more detail.
6. Acknowledgements
We thank David Rubin de Celis Leal, Robert Lovett,
Alex Orokity, Magnolia Beer, Marion Wright and Steve
Atkinson for technical assistance, D. Fabijanic for 316
stainless steel mesh supply, and Peter Lamb, Jane
Allardyce for editorial support.
7. References
[1] W.L. Claeys, et al., Food Control, 31, 1 (2013).
[2] A. Topcu, E. Numanoglu, and I. Saldamli,
International dairy journal, 16, 6 (2006).
[3] H. Burton, Journal of Dairy Research, 51, 02
(1984).
[4] C. Morris, A.L. Brody, and L. Wicker, Packaging
Technology and Science, 20, 4 (2007).
[5] H.-J. Kim, et al., Current Applied Physics, 13, 7
(2013).
[6] U. Schnabel, et al., Plasma Processes and Polymers,
9, 1 (2012).
[7] B. Yang, et al., Journal of Dentistry, 39, 1 (2011).
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