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Non-Thermal Processing Technologies Research at the USDA’s Eastern Regional Research Center
Brendan A. Niemira, PhDResearch Leader
Food Safety and Intervention Technologies Research Unit
USDA - Agricultural Research Service Eastern Regional Research Ctr.
600 E. Mermaid Ln, Wyndmoor, PA, USAhttp://www.tinyurl.com/FSIT-RU
Food processing technologies at ERRC
• Microwave, infra-red, radio frequency
• Light: UV, pulsed, high-intensity monochromatic
• High pressure processing• Flash steam treatment• Ozonation, ClO2, MAP
B.A. Niemira
• GRAS and novel antimicrobials• Irradiation• Cold plasma• Biocontrols: predators,
competitors, antagonists, bacteriophage
Food Safety and InterventionTechnologies Research Unit
Food processing technologies at ERRC
• Microwave, infra-red, radio frequency
• Light: UV, pulsed, high-intensity monochromatic
• High pressure processing• Flash steam treatment• Ozonation, ClO2, MAP
B.A. Niemira
• GRAS and novel antimicrobials• Irradiation• Cold plasma• Biocontrols: predators,
competitors, antagonists, bacteriophage
Food Safety and InterventionTechnologies Research Unit
Why irradiation?• Investigated for 100+ years. Developed into a safe,
effective food processing technology– Shelf life extension– Sprout suppression– Antimicrobial efficacy – spoilage and safety
• GAP, GMP, preventative controls are effective, but are only part of the answer
• Chemical sanitizers and physical controls– Limitations of use, efficacy
• Alternative and supplemental interventionsB.A. Niemira
Mode of action• Largest target in organisms is water• High energy electrons break water molecules into
OH• and O• radicals, which disrupt membranes, proteins and nucleic acids
• DNA is also broken directly• High energy photons interact with atoms to eject
high energy electrons• Penetration of photons is much greater than for
electrons - implications for how material is processed
B.A. Niemira
Irradiation applications• Prevention of foodborne illness – e.g. Salmonella and
Escherichia coli• Preservation – inactivate spoilage organisms, extend shelf life• Phytosanitation – destroy insects, larvae, eggs in or on tropical
imports, decreasing need for more harmful pest-control practices
• Delay of sprouting and ripening – e.g. potatoes, onions, bulbs• Sterilization – shelf-stable, sterile packaged foods. Used for
hospitals, military, astronauts• Irradiation equally effective against antibiotic resistant P.
aeruginosa, E. coli O157:H7 (Dharmarha et al., IAFP 2018)
Technologies: E-beam
• Electron beam– Varying designs– Linear, cyclotron,
rhodotron– “On demand”
• X-ray– Tungsten, tantalum– “On demand”
• Gamma ray– “Always on”
Technologies: X-ray
• Electron beam– Varying designs– Linear, cyclotron,
rhodotron– “On demand”
• X-ray– Tungsten, tantalum– “On demand”
• Gamma ray– “Always on”
Technologies: gamma
• Electron beam– Varying designs– Linear, cyclotron,
rhodotron– “On demand”
• X-ray– Tungsten, tantalum– “On demand”
• Gamma ray– “Always on”
Implications of the Max/Min Ratio
• Good packaging– effective penetration of
irradiation from above and below
– relatively even dosage, low Max/Min ratio
• Poor packaging– Too bulky, too dense, too thick
(or all three)– incomplete penetration– uneven dosage, high Max/Min
ratio
Irradiation – electron beam
• Dose uniformity is critical. Misalignment of e-beam can reduce absorbed dose from 1.0 kGy to 0.66 kGy
• Product thickness: depth of penetration must be known for each product (Moriera et al, 2012)
High Max/Min Ratio: Consequences
• Dose too high = sensory damage• Dose too low = inadequate kill/control• Irregularly shaped whole produce can have high Max/Min of ~ 3.0
(Brescia et al., 2003)
• Irregular shapes and variations of density common in produce• Product penetration (one sided)
– X-ray and gamma, 30-40 cm– E-beam, 6-8 cm
• Thick, dense products present a challenge for effective, uniform treatment with e-beam irradiation.– Packaging design is critical
Max/Min logistics
• Product orientation with respect to irradiation source• Individual items vs. commercial bulk packaging (bag, tray, pallet, etc.)
Gamma irradiator
• GrayStar – Co60
source• Below floor level,
water shielded• Process pallet loads
in semi-continuous operation
http://www.graystarinc.com/photos.html
Cold plasma
• What is a plasma?– “Fourth state of matter” – Equivalent to highly energetic ionized gas
B.A. Niemira
Terminology: cold, cool and nonthermal• Cold, cool = operates at or near room temperature• Nonthermal = antimicrobial mode of action other than heat
ENERGY
Plasma
ENERGY ENERGY
Cold plasma• Inputs to the system
– energy (electricity, microwaves, etc.)– carrier gas: air, a pure gas (He, Ar, O2, N2, etc.) or a
defined gas mixture• Output
– self-quenching plasma– resolves to UV light and ozone– chemical residues are expected to be minimal to non-
existent• Relatively new technology for food processing
– adaptation from existing applications– regulatory status
B.A. Niemira
ERRC gliding arc cold plasma
B.A. Niemira
Oxygen
Nitrogen Carbon dioxide
Injected volatiles
Ozone
UV light
NOx
ElementalOxygen
Freeradicalse-
Nanoparticles
Plasma jet: rapid treatments
• Almonds– 20s at 6cm reduced E. coli
O157:H7 C9490 by 1.34 log• Sliced tomatoes
– 20s at 2cm reduced S. Stanley by 0.71 log
• Time x Distance: complex & isolate-dependent.
• Nitrogen was less effective than air
• Optimized trade off of amount of kill vs. speed (Niemira 2012 J Food Sci)
B.A. Niemira
Cold plasma: blueberries• Blueberries - model system for small,
fragile fruits• Inoculated with Norovirus surrogates
Murine Norovirus (MNV), Tulane virus (TV)• Native microflora: TAPC, Y&M• Sensory impact: compression firmness,
surface color, total anthocyanins• Measurements of heat buildup, with and
without active cooling measures• Determine non-thermal kill level (Lacombe
et al., Food Micro 2015)
B.A. Niemira
Cold plasma: viral inactivation
B.A. Niemira
Tulane virus Murine Norovirus
Both surrogates effectively inactivated by cold plasma.◦ Significant (P<0.05) effects after 45s (TV) or 15s (MNV).
Antimicrobial effects not related to heat. Engineering controls allowed for minimizing heat buildup on berries.
Cold plasma: native microflora
• Initial reductions in TAPC followed by continued die-off in cold storage
– Suggests sublethal injury, which suggests potential efficacy for combination treatments
• Cold plasma treatments as short as 15s gave lasting suppression of TAPC & spoilage bacteria. Not significantly effective against Y&M for times tested.
• Novel, promising nonthermal technology for small fruit sanitizing
B.A. Niemira
Cold plasma: in-package treatment
• Romaine lettuce, inoculated with E. coli O157:H7• 5’ treatment, with plasma generated inside the commercial clamshell package• 1.0 – 1.5 log reduction, persistent through 7d refrigerated storage• Suggests new applications for post-packaging antimicrobial treatment(Min et al. 2016. JFP; Min et al. 2017 Food Micro)
B.A. Niemira
Commercial
clamshell container
• Cold plasma reactor with clamshell package
• Seven layers of lettuce leaves in a three-column configuration
• Sampling: Top, Middle, Bottom; Left, Center
Position
Microbial reduction (log CFU/g lettuce)
1-layer 3-layer 5-layer7-layer configuration
No shaking Shaking
TopLeft 0.6 ±
0.4 ab0.6 ±0.4 ab
0.9 ±0.2 a
0.7 ±0.2 ab
Center 0.6 ±0.1 ab
0.6 ±0.3 ab
1.1 ±0.1 a
0.7 ±0.2 ab
MiddleLeft 0.6 ±
0.3 ab0.5 ±0.2 ab
0.6 ±0.1 ab
0.8 ±0.2 ab
Center 0.7 ±0.4 ab
0.8 ±0.3 ab
0.6 ±0.2 ab
0.6 ±0.1 ab
BottomLeft 0.6 ±
0.2 ab0.5 ±0.3 ab
0.5 ±0.3 ab
0.4 ±0.1 b
0.7 ±0.1 ab
Center 0.7 ±0.2 ab
0.6 ±0.1 ab
0.5 ±0.3 ab
0.4 ±0.1 b
0.7 ±0.1 ab
Cold plasma: in-package treatment of bulk Romaine lettuce
Cold plasma: in-package treatment
L. mono declined by an additional 0.6 log CFU/g after being stored at 4 °C for 24 h
Cold storage can enhance treatment effect For bulk packaged lettuce, inhibition rates of E. coli O157:H7 were
consistent in all locations for 1-, 3-, 5-, and shaken 7-leaf stacks. Cold plasma worked in both rigid and flexible commercial packages Current produce containers, packaging materials, MAPs, and
processing lines can be with cold plasma Further optimization and scale-up (Min et al., 2018)
Cold plasma Inactivation of Biofilms
• Salmonella and E. coli O157:H7, adherent biofilms grown for 24, 48 or 72h on a test surface
• Cold plasma applied to a moving conveyor belt. Very short treatments reduced the most durable biofilms– E. coli O157:H7: 1.25 log (5s), 2.46 log (10s), 3.03 log (15s)– Salmonella: 0.92 log (5s), 1.59 log (10s), 2.12 log (15s)
• Rapidly and effectively inactivated persistent contamination on food contact surfaces associated with fruits and vegetables processing. (Niemira et al. 2014 J Food Sci)
B.A. Niemira
Cold plasma Inactivation of Biofilms
• Rapid reductions of adherent biofilms: 99.96% reduction in 15s without sanitizers or scrubbing
• Infra-red imaging confirms a nonthermal process (Niemira et al. 2018 submitted)B.A. Niemira
• Salmonella biofilms grown on glass slides 24h, placed inside food containers
• Corona discharge cold plasma (gentler than plasma jet) for 3 minutes• Biofilm EPS structure degraded and dispersed• Image analysis of Live/Dead confocal microscopy to quantify biofilm
dispersal (Hertrich et al, in preparation)
Image analysisBefore After
Cold plasma Inactivation of Biofilms
• Competitive exclusion– Good bug occupies all the niches, so bad
bugs can’t take hold• Inhibition
– Good bugs stop the bad bugs from growing
• Predation– Good bugs actively kill the bad bugs
Biological controls
Efficacy of P. fluorescens on E. coli O157:H7 (Ec) inoculated
on spinach (20 ºC)(Dr. Modesto Olanya)
Treatment 24 hrs
P. fluorescens (Pf) and Ec Reduction of Ec(Log CFU/g)
Ec 43894 + Pf 2-79
Ec 43894 + Pf Q287
Ec 43894 + Pf Q8R-1
0.95+0.45b
2.10+0.04a
1.60+0.00a
Ec 43895 + Pf 2-79
Ec 43895 + Pf Q287
Ec 43895 + Pf Q8R-1
1.05+0.65b
1.50+0.20ab
0.80+0.16b
Means with the same letters are not significantly different (P<0.05) .
Competitive exclusion
Predatory prokaryotes, bacteria predation
Parameters Bdellovibrio /
HalobacteriovoraxOccurrence Seawater, soil, water
Size & morphology
0.35 x 1.2 µm, curved rods
Motility Single polar sheathflagellum
Site in prey Periplasmic space
Reproduction
Prey hosts
Bdelloplasts, segmentationGram negative bacteria
Host specificity Obligate / host independent forms
Predation of gram negative Vibrio by Halobacteriavorax(Dr. Gary Richards)
Predatory prokaryotes, bacteria predation
Predatory prokaryotes, bacteria predation
Swarming, membrane attachment, cell lysis
Effective against Vibriospp.
Research underway to determine antagonism for E. coli O157:H7 and Salmonella
• Nonthermal interventions can effectively reduce pathogen loads
• Optimization for commodity, desired performance metrics
• Scale-up, commercialization
Summary
B.A. Niemira 45
[email protected]/FSIT-RUwww.tinyurl.com/Niemira
SESSION HIGHLIGHTS
Utilize case studies to learn about specific microbial
interventions and audience interaction to discuss
specific interventions of interest
Discuss challenge studies and validation of
interventions
PRESENTERS
• Alvin Lee, PhD, Director, Center for Processing Innovation,
Associate Professor of Food Science and Nutrition, Institute
for Food Safety and Health
• Brendan A. Niemira, PhD, Research Leader, Food Safety and
Intervention Technologies Research Unit, USDA-ARS Eastern
Regional Research Center
Gary Ades, PhD, President, G&L Consulting Group (Facilitator)
• IRRADIATION/IONIZING
RADIATION (E.G., PRODUCE)
• COLD
PLASMA/NONTHERMAL
PLASMA (NTP) (E.G.,
PRODUCE)
• COMPETITIVE INHIBITION
• NOVEL ANTIMICROBIALS
• HIGH PRESSURE
PROCESSING (HPP)
• CHEMICALS (E.G.,
CHLORINE DIOXIDE)
• PULSE LIGHT
• Temperatures (Heat, Cold)
• Microwave
• Radio Frequency
• Ohmic Heating
• Infrared
• Sonic
• Pulsed Electric Field
• Ultraviolet
• Good sanitation
• Handwashing
• Minimizing cross
contamination
• Active packaging
• Protective packaging, e.g.,
produce boxes
• Supplier food safety
programs and specifications
e.g., treatment of spices-The
main methods are ethylene
oxide gas or propylene oxide
gas, irradiation, steam
treatment and microwave
• Formulations
• Probiotics
• Phages
INTERVENTIONS