42
Potential of atmospheric cold plasma for biofilm control in food processing Paula Bourke, PhD, Dublin Institute of Technology [email protected] IAFP European Symposium, 30 th March 2017
Potential of atmospheric cold plasma for
biofilm control in food processing
Paula Bourke, PhD, Dublin Institute of Technology [email protected] IAFP European Symposium, 30th March 2017
RATIONALE FOR SEEKING NEW FOOD PROCESSING TECHNOLOGIES
https://makanaka.files.wordpress.com/2013/01/fao-food-waste-campaign-201301.jpg?w=700&h=523
Plasma: an ionized gas consisting of
atoms, electrons, ions, molecules,
molecular fragments, and electronically
excited species (informal definition)
www.geo.mtu.edu/weather/aurora/
STATES OF MATTER
SOLID LIQUID GAS PLASMA
Tightly packed, in a regular pattern
Vibrate, but do not move from place to
place
Close together with no regular
arrangement. Vibrate, move
about, and slide past each other
Well separated with no regular
arrangement. Vibrate and move
freely at high speeds
Has no definite volume or shape
and is composed of electrical charged
particles
What is Plasma ?
What Plasma do we use?
•Thermal
•Non-thermal/Cold Temperature
•Low pressure
•Atmospheric pressure
•High pressure Pressure
•Air
•Oxygen
•Helium, Argon
•MAP gas mixes
Gas
•Microwave
•Radio frequency
•Corona
•Dielectric barrier Discharge
Mode of generation
•In Package / Contained
•In –Line or Tunnel array
•Plasma Activated Water
•Plasma Activated Liquids
•Plasma Activated Substances
•Plasma Deposition
Mode of Delivery
What is in Atmospheric Cold Plasma?
Reactive oxygen species
Reactive nitrogen species –
Effects of Non-Thermal Plasma on Mammalian Cells. 2011 Sameer Kalghatgi et al.
UV radiation, energetic ions, charged particals etc.
HOW DOES ATMOSPHERIC COLD PLASMA WORK?
Adapted from Mai-Prochnow et al. 2014. International Journal of Antimicrobial Agents,
43(6),508–517
Dielectric Barrier Discharge
ROS RNS Energy
Gordillo-Vazquez. 2008. Air plasma kinetics under the influence of sprites. Journal of applied physics, 41, 234016
From 2004 – 2012, European Union reported a total of 198 produce-associated outbreaks
Adapted from Callejon et al. 2015. Foodborne pathogens and disease, 12(1), 32 – 38.
MICROBIOLOGICAL CHALLENGES
Microbiological and Food Matrix Challenges relevant to fresh foods – Why do these risks persist?
Pathogens
Spoilage
Biofilms
Spores
Toxins
Internalisation / Structural protection
Investigations at lab-scale to inform proptotype design
In-package treatment
< 2 ºC rise UV photons Reactive species
Example of plasma streaming during treatment of cherry tomatoes
Advantages of IN package treatment – retention of efficacy, Time for longer lived species to effect target, mitigates recontamination or
cross contamination events
• 7 log reduction in MRD after 20 s of Direct and 45 s of Indirect ACP
Interactive effects of mode of exposure, treatment time and post-treatment storage time, media composition, voltage levels and working gas
Ziuzina, D., Patil, S., Cullen, P.J., Keener, K.M. and Bourke, P*. (2013) Atmospheric cold plasma inactivation of Escherichia coli in liquid media inside a sealed package. Journal of applied microbiology. 2013:114:778-787.
EFFECTS ON E. COLI IN LIQUID MODEL
Atmospheric air High voltage level Post treatment storage 24 h
• Microbes exist predominantly as biofilms
• Community of cells surrounded by a matrix
of extracellular polymeric substances (EPS) that hold microbial cells together to a surface
• Enhanced tolerance to high concentrations of antimicrobial agents
Attachment Maturation Dispersion
Adapted from Monroe D. 2007. PlosBiology, 5(11), e307, 2458 - 2461
Biofilm development
Food spoilage Cross-contamination
Spread of foodborne pathogens
NIH. 2009. NIH Guide: Research on bacterial biofilms www.nih.gov/grants/guide/pa-files/PA-03-047.html
• 80% of human infections are associated with biofilms (NIH)
• A major challenge in food, environmental, pharmaceutical industries and in clinical and healthcare scenarios
BACTERIAL BIOFILMS
XTT assay
P. aeruginosa ATCC 27853 48 h biofilm ACP treatment process parameters: Voltage: 80 kV RMS Mode of exposure: Direct/Indirect Post treatment storage time: 24 h
0
20
40
60
80
100
0 60 120 180 240 300
Surv
ivin
g ce
lls,
XTT
%
Treatment time, s
-1
1
3
5
7
0 60 120 180 240 300Lo
g10
CFU
/ml
Treatment time, s
Direct ACP
Indirect ACP
Control 24h
Treatment of biofilms
Plate count
P. aeruginosa 48 h biofilm
ACP: Air, 80 kV, 5 min treatment, 24 h post treatment storage
Control Direct ACP Indirect ACP
Ziuzina, D., Patil, S., Cullen, P.J., Boehm, D., Bourke, P*. (2014). Dielectric barrier discharge atmospheric cold plasma for inactivation of Pseudomonas aeruginosa biofilms. Plasma Medicine, 4, 89 – 104.
SEM
CLSM (SYTO9/PI)
Green - live cells Red – dead cells
Thickness
23 µm 10 µm 10 µm
Thickness
8 µm 10 µm
Thickness
6 µm
Mechanisms of removal?
Treatment of biofilms
ACP against P. aeruginosa QS-controlled virulence factors and biofilm formation capacity
• Bacterial QS is a population density controlled cell to cell communication system • QS is used by bacteria to coordinate the expression of several genes involved in
virulence, biofilm formation and pathogenicity. • QS inhibition - an alternative antimicrobial target??
What did we find? ACP was effective toward reduction of virulence factors: Pyocyanin – 60 s resulted in almost complete reduction Elastase (Las B) - 5 min reduced by ~ 50 % Biofilm formation capacity was not reduced - ACP did not influence the ability of P.
aeruginosa to form biofilms Cytotoxicity (CHO-K1) - ACP treatment significantly reduced cytotoxic effect of P. aeruginosa
supernatant ACP technology may play an important role in attenuation of virulence of pathogenic bacteria
0
20
40
60
80
100
120
TSB P.a 0hcontrol
P.a 24hcontrol
60 s 120 s 300 s
% A
bso
rban
ce, 6
00 n
m
Direct ACP
Indirect ACP
CHO-K1
0
40
80
120
160
200
0 60 120 180 240 300
% A
bso
rban
ce, 5
20 n
m
Treatment time, s
Pyocyanin DirectIndirectControl
0
50
100
150
200
250
300
0 60 120 180 240 300
% A
bso
rban
ce, 4
94 n
m
Treatment time, s
Las B DirectIndirectControl
Control
Ziuzina, D., Boehm, D., Patil, S., Cullen, P.J., Bourke, P*. PLoS ONE 10(9): e0138209. doi:10.1371/journal.pone.0138209
Mechanism of action
SEM Analysis – PTST –sealed container
E. coli & L. monocytogenes E. coli ATCC 25922
Control 1hr storage 24hr storage
L. monocytogenes NCTC 11994
Control 1hr storage 24hr storage
0
2
4
6
8
0 20 40 60 80 100 120
Lo
g1
0 C
FU
ml-1
Treatment time (s)
1.2
1.3
1.4
1.5
1.6
1.7
0 20 40 60 80 100 120
A 2
60
/28
0
Treatment time (s)
E. coli ATCC 25922: ◆Direct; ◆Indirect
E. coli NCTC 12900:▲ Direct; ▲ Indirect
L. monocytogenes NCTC 11994: ■ Direct; ■ Indirect
Voltage: 50kV; Treatment time: 0~120s;
Post treatment storage time: 24hr
Cell Integrity
Han, L; Patil, S; Cullen, P; Keener, K; Bourke, P* (2014) Bacterial inactivation by Atmospheric Cold Plasma: Influence of process parameters and effects on cell leakage and DNA. Journal of Applied Microbiology. 116 (4), 784-794
More DNA damage in Listeria than E.coli
DNA damage effect of plasma.
Genomic DNA damage of (a) E. coli ATCC 25922; (b) E. coli NCTC 12900; (c) L. monocytogenes
NCTC 11994
16s RNA PCR results of (d) E. coli ATCC 25922; (e) E. coli NCTC 12900; (f) L. monocytogenes
NCTC 11994
Lane 1: Non plasma treatment control; 2: 5s directly treated samples; 3: 5s indirectly treated samples;
4: 30s directly treated samples; 5: 30s indirectly treated samples
Han, L; Patil, S; Cullen, P; Keener, K; Bourke, P* (2014) Bacterial inactivation by Atmospheric Cold Plasma: Influence of process parameters and effects on cell leakage and DNA. Journal of Applied Microbiology. 116 (4), 784-794
Intracellular ROS of G-/G+ Intracellular ROS levels in S. aureus were 3 times those in E. coli with
same treatment time E. coli
treatment time (min) 0 1 3 5
E. coli
IF 8.1 7.5 5.9 0
OF 8.1 7.2 5.6 0
S. aureus
treatment time (min) 0 1 3 5
S. aureus
IF 8 6.9 6.4 1.8
OF 8 6.5 6 1.8
0
500
1000
1500
2000
2500
Control 1min 3min 5min
control
IF
OF
0
200
400
600
800
control 1min 3min 5min
Voltage: 80kV Treatment time: 1,3,5min; Post treatment storage time: None
Proposed Mechanism of action (Han et al, 2016, Applied and Environmental Microbiology)
ROS
HVACP
lipopolysaccharide
peptidoglycan
a Gram negative bacteria
d Gram positive bacteria
peptidoglycan
HVACP
ROS
b
e
c cell leakage main effect
f severe damage to intracellular components (eg. DNA)
Han, L., Patil, S., Boehm, D., Milosavljevic, V., Cullen, PJ., Bourke, P*. Mechanism of Inactivation by High Voltage Atmospheric Cold Plasma Differs between Escherichia coli and Staphylococcus aureus. (2016) Applied and Environmental Microbiology.
Han, L., Ziuzina, D., Heslin, C., Boehm, D., Patange, A., Millan-Sango, D., Valdramidis, V. P., Cullen, P. J., & Bourke, P*. (2016). Frontiers in Microbiology
There is a Protection effect of food matrix
Food based BioFilm Studies Produce Grains Meat
Indirect ACP treatment of 70 kV reduced pathogens attached on produce surface and background microflora of produce
Effect of bacterial type: ACP for 10, 60 s and 120 s eliminated Salmonella, E. coli and L. monocytogenes on tomatoes
Effect of produce surface characteristics: extended treatment time was required for reduction of bacteria as well as background microflora on more complex strawberry surface
Cherry tomatoes Strawberries
0
1
2
3
4
5
6
7
8
0 30 60 90 120
Log1
0 C
FU/s
amp
le
Treatment time, s
Ec
St
Lm
0
1
2
3
4
5
6
7
8
0 60 120 180 240 300
D. Ziuzina, S. Patil, P.J. Cullen, K.M. Keener and P. Bourke* (2014). Atmospheric Cold Plasma inactivation of Escherichia coli, Salmonella enterica serovar Typhimurium and Listeria monocytogenes inoculated on fresh produce. Food Microbiology.
EFFECT OF PRODUCE TYPE
SEM: Strawberries SEM: Tomatoes
Ziuzina, D., Han, L., Cullen, P.J., Bourke, P.* (2015). Cold plasma inactivation of internalised bacteria and biofilms for Salmonella enterica serovar Typhimurium, Listeria monocytogenes and Escherichia coli. International Journal of Food Microbiology, 210, 53-61.
ACP was effective against biofilm populations: 5 min of treatment reduced biofilm populations on lettuce by 5 log10 CFU/sample
Effect of storage conditions for biofilm formation: Temperature, light and time had interactive effects on bacterial proliferation, stress response and susceptibility to the ACP treatment
BIOFILMS AND INTERNALIZED BACTERIA
Resulted in lower incidence of bacterial internalization
RoomTinlight/dark 4°Cinlight/dark 4°Cindark
CLSM
E. Coli XL10 GFP
Supported bacterial internalization inside stomata
Salmonella 48 h biofilms formed on lettuce at RT, light/dark ACP: Air, 80 kV, 5 min treatment 24 h post treatment storage at 4°C
• Importance of maintenance of the appropriate storage conditions (low T°C, minimised light exposure) throughout distribution chain for the assurance of microbiological safety of fresh produce
• Importance of effective microbiological control as microorganisms protected by biofilms or complex
structures of different produce commodities may present major risks of cross-contamination of the environment in food production sites
Control
ACPtreated
SEM
Arrows: Green – intact cells Red – cells debris White – uncolonized stomata
E. coli
NCTC 12900
E. coli
ATCC 25922
B. atrophaeus
var. niger,
NAMSA
B. subtilis
ATCC 6633
L. plantarum
ATCC 8014
L. brevis
ATCC 8287
standard medium
(TSB or MRS) strong moderate strong strong strong strong
wheat model medium moderate weak weak strong strong strong
barley model medium moderate strong moderate weak strong strong
strains classification1:
Bacterial biofilm formation in cereal-based media
E. coli Bacillus ssp. Lactobacillus ssp.
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
E. coli NCTC 12900 E. coli ATCC 25922 B. atrophaeus var.niger, NAMSA
B. subtilis ATCC 6633 L. plantarum ATCC 8014 L. brevis ATCC 8287
Ab
sorb
ance
, 59
0 n
m
standard medium (TSB or MRS) wheat model medium barley model medium
1. Stepanovic, S., Vukovic, D., Dakic, I., Savic, B., Svabic-Vlahovic, M., 2000. A modified microtiter-plate test for quantification of staphylococcal biofilm formation. J. Microbiol. Methods 40, 175–179.
a) WHEAT MODEL MEDIUM
b) BARLEY MODEL MEDIUM
0
1
2
3
4
5
6
7
8
E. coli NCTC 12900 E. coli ATCC 25922 B. atrophaeus var.niger, NAMSA
B. subtilis ATCC6633
L. plantarum ATCC8014
L. brevis ATCC 8287
Rem
ain
ing
cells
Lo
g10
C
FU/m
l Control
Indirect ACP
Direct ACP
- - detection limit
0
1
2
3
4
5
6
7
8
E. coli NCTC 12900 E. coli ATCC 25922 B. atrophaeus var.niger, NAMSA
B. subtilis ATCC6633
L. plantarum ATCC8014
L. brevis ATCC 8287
Rem
ain
ing
cells
Lo
g10
C
FU/m
l Control
Indirect ACP
Direct ACP
- - detection limit
Effect of ~80 kV of direct direct/indirect ACP treatment for 5 min on bacterial biofilms – colony count assay
Despite of weak to moderate biofilm formation in wheat and barley model media, ACP treatment efficacy against of B. atrophaeus was low.
Sporulation within Bacillus spp. biofilms formed in cereal-based media
It was determined that the 72 h biofilms of B. atrophaeus constituted on average 90% of spores using either wheat or barley model media for biofilm formation
92.9 89.6 87.7 81.0 0
20
40
60
80
100
B. atrophaeus B. subtilis
Wheat model medium
Barley model medium
a) WHEAT MODEL MEDIUM
Effect of ~80 kV of direct direct/indirect ACP treatment for 5 min on bacterial biofilms – XTT assay
b) BARLEY MODEL MEDIUM
0
20
40
60
80
100
120
140
160
E. coli NCTC 12900 E. coli ATCC 25922 B. atrophaeus var.niger, NAMSA
B. subtilis ATCC 6633 L. plantarum ATCC8014
L. brevis ATCC 8287
%A
, 48
6 n
m
Control
Indirect ACP
Direct ACP
0
20
40
60
80
100
120
140
160
E. coli NCTC 12900 E. coli ATCC 25922 B. atrophaeus var.niger, NAMSA
B. subtilis ATCC 6633 L. plantarum ATCC8014
L. brevis ATCC 8287
%A
, 48
6 n
m
Control
Indirect ACP
Direct ACP
Inactivation of B. atrophaeus spores on abiotic surfaces that mimic grain surfaces
Treatment - 30 min reduced spores on hydrophobic surface by 6 log. Only 4.2 log reductions were achieved with spores attached to hydrophilic surface.
Optical and electron microscopy showed physical changes of spores following ACP
Direct/Indirect mode
Microbiological control
Treatment time 30 min
Atmospheric air
Voltage 80 kV
Storage 2 h at 15°C
0
2
4
6
8
control IF ACP OF ACP
Log1
0 C
FU/m
l
Glass PEO
pti
cal m
icro
sco
py
Gla
ss
SEM
P
E
Control Direct ACP Indirect ACP
Hydrophobic porous (PE) Hydrophilic
non-porous (glass)
Effect of ACP on B. thermosphacta 48h biofilm in 12% beef extract, treated at 80 kV
(24 PTST)and assessed using plate count and XTT assay. (■) ACP treated, ()
untreated biofilm control.
Inactivation of meat spoilage bacterial biofilm
Controlling Brochothrix thermosphacta as a spoilage risk using in-package atmospheric cold plasma. Food Microbiology (2017) Accepted. Patange A.,Boehm, D., BuenoFerrer C., Cullen, PJ., Bourke P*.
Meat decontamination - Shelf-life study
Cold Plasma Control of Background microflora populations on fresh and cooked meat surfaces
Lamb chop Sliced turkey
Pork loin
Plasma-activated Liquids
37
70% H2O
38
Antimicrobial efficacy – H2O2 and pH
Antimicrobial efficacy is dependent on H2O2 and pH
Neutralization of pH removes antimicrobial activity
Re-acidification can not restore biocidal effect
0
2
4
6
8
CT
L
re-a
cid
ifie
d
pH
neutraliz
ed
PAW
log
CF
U/m
l
0 100 200 300 4000
2
4
6
8
PAW
PAW + cat
PAW + 4.5x PBS
contact time [min]
log
CF
U/m
l
0 50 100 150 2000
2
4
6
8
PAW
PAW + pyruvate
PAW + catalase
contact time [min]
log
CF
U/m
l
Selective PAW/PAL for distinct applications?
E. coli S. aureus0
2
4
6
8CTL
PAW
log
CF
U/m
l
http://www.acpfg.com.au www.slideshare.net
HeLa -0
50
100 CTL
PAW
cell
gro
wth
[%
of
co
ntr
ol]
Mechanism of action? Cell wall/membrane?
Modified plasma device 70µM H2O2
Anti-microbial Not cytotoxic
Possible advantages of ACP for food processing
• Non-thermal - heat sensitive ingredients
• Development of “new” products (e.g., shelf-stable PHF)
• Control Spoilage and pathogens - Extend safe shelf-life
• Can be In –package – Mitigates against Recontamination
• Can be a dry process – no chemical residues
• Can be a wet process – longer term effects?
• Can be built into process or equipment
• Low energy requirement and portability
Dr JP Mosnier Dr Tamara Mathews
Prof Kevin Keener Iowa State University BioCentury Research Farm
Thank you for your Attention Acknowledgments
DIT Applied Plasma Research Group Dr Paula Bourke Dr PJ Cullen Dr James Curtin Dr Daniela Boehm Dr Dana Ziuzina* Dr Peng Lu Dr Vladimir Milosavljevic Dr NN Misra* Dr Sonal Patil* Dr Carmen Bueno Ferrer Dr Shashi Pankaj* Dr Lu Han* Dr Diva Almeida* Caitlin Heslin Gill Conway Chaitanya Sarangapani Apurva Patange Agata Los Laurence Scally James Lalor Miroslav Gulan Juan Perez Roseane Cavalcante
