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Controlling Listeria monocytogenes in Meat, Cheese, Salmon & Vegetables with organic Phage solutions
Food Valley Expo – Food Quality & Safety
13 October 2016 Bert de Vegt
Phages for Food Protection
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Outline
• Micreos
• Phage 101
• Listex Phages for Listeria control
– Meats & seafood
– Vegetables
– Dairy
• Phages for Bio-film removal
• Regulatory
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Micreos
• Dutch company
• Global leader in applied phage technology
• Incorporated in 2005
• 50 employees
• Targeted anti-bacterial solutions
• Phages have applications in: - Human Health MR(SA) - Food Safety - Animal Health - Agriculture
Amsterdam
Wageningen
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Micreos
Wageningen: Food Safety
Bilthoven: Human Health
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Strong Scientific Background and Connections
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Phages versus Bacteria
• Greek “for bacteria eater”- Natural enemy of bacteria
• Every 48 hours up to 50% of the bacterial population is killed by phages
• Can be 100x smaller then its bacterial host
• Co-evolved with bacterial host
• Outnumber bacteria by up to 10 fold
• Harmless to everything but the specific bacteria
• Very Targeted & Specific
• Naturally present everywhere
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Phages are everywhere
• Most abundant micro-organisms on earth (>1030)
• Ubiquitous in the environment and in the human body
• 1 ml seawater contains up to 1 billion phages and 60,000 types
• 1 gram of human adult feces contains up to 1 billion phages
• The human gut contains as many as 1015 phages
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Life cycle of lytic phages
1. Adsorption 2. Infection
3. Replication
4. Maturation and assembly
5. Host lysis and Progeny phage release
Lytic cycle
Bacterial cell
Note: Treating food products with PhageGuard Listexand PhageGuard S, we do not rely on the lytic cycle
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Pathogens of Concern
E-coli
Listeria
Campylobacter
Salmonella
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Foodborne pathogens
2014 incidence data
Campylobacter;
69%
Salmonella;
26%
1%
foodbornillnessduetozoonose
ConfirmedCases:341,455
Campylobacter Salmonella Yersinia E.coli Listeria Other
Campylobacter;
60%
Salmonella;
32%
3%
foodbornillnessduetozoonose
Hospitalizations:30,581
Campylobacter Salmonella Yersinia E.coli Listeria Other
Campylobacter;
8%
Salmonella;
21%
Listeria;
67%
foodbornillnessduetozoonose
Death:313
Campylobacter Salmonella Yersinia E.coli Listeria Other
Food categories most sensitive for Lm: Fish (smoked), RTE meats and cheese
EFSA and the European Center for Disease Prevention and Control
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3D’s for Success
• Dose or concentration
• Distribution
• Dwell Time or contact time
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Phages provide protection in all zones
3. Environment
2. Food Contact Surfaces
1. Food
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The Benefits of Listeria Phages
• Leads to 1-3 log10 reductions on food
• 3 - 5 log10 reductions on biofilms and process equipment
• PhageGuard Listex is effective against all Listeria species, not just LM • Prevents cross contamination on food contact surfaces • Does not influence desired bacteria • No organoleptic influence • No need to reformulate products • No trade off between quality and safety • Topical spray application • Non corrosive • Suitable for natural & organic products
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Listex Phages on Foods
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Meat & Poultry
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Time efficacy of LISTEX™ in killing L. Monocytogenes on hot dogs
Internal study (Micreos Food Safety, 2015)
2,0
2,4
2,8
3,2
3,6
4,0
4,4
4,8
0 2 4 6 8 10 12 14 16 18 20 22 24
L. mono log CFU/cm2
Time (h)
Control
LISTEX™ 0.5% (1x109 PFU/mL) Applied at 5 µL/cm2
LISTEX™ 1% (2x109 PFU/mL) Applied at 5 µL/cm2
Both concentrations of LISTEX™ showed an immediate reduction of L. mono
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LISTEX™ Application on hotdog surface Dr. C.Iancu, Micreos Application Labs 2015
LISTEX™ has a fast killing effect at
low concentration: >1log reduction
for 0,5% solution after 30 min
0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5
30 min
2 h
6 h
16 h
24 h
L. Monocytogenes WSLC10403S reduction, logCFU/cm2
Incu
bat
ion
tim
e
5% LISTEX
1% LISTEX
0,5% LISTEX
Treatment 30 min 2 h 6 h 16 h 24 h
0,5% LISTEX 95% 96% 98% 98% 97%
1% LISTEX 98% 98% 98% 99% 99%
5% LISTEX 99% 99% 99% 99% 99%
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Hot dogs
Spray into the vacuum packages before sealing. Vacuum-sealing of the
packages distributes the antimicrobial evenly around the product.
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Hot dogs
Phages can be sprayed directly into a hopper
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Bagged whole muscle products
Phages are sprayed into the package before sealing.
Vacuum-sealing distributes the antimicrobial evenly around the product.
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Seafood
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Reduction of Listeria monocytogenes at
4C on raw salmon fillet tissue
Kill after 30 minutes
2.5x107 = 2 log reduction
2.5x108 = 3.5 log reduction
Journal of Food Protection 2010, Vol. 73
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Dairy & Cheese
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1 day: 2 log reduction
Efficacy of phage A511 (P100) on Camembert cheese during ripening
P100=phage A511
Phage dilution: 15% Phage dosage: 3 x 108 PFU/cm2
(at 10 µL/cm2)
Control
Phage
pH
Data generated by ETH (Swiss Federal Institute of Technology Zurich)
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Effect of LISTEX™ on Listeria monocytogenes reduction in Belgioioso mozzarella at 24h
0.05% LISTEX™ solution resulted in a 2 log reduction of L . monocytogenes 2/2
0.05%: 1 x 108 PFU/mL
0.025%: 5 x 107 PFU/mL
0.005%: 1 x 107 PFU/mL
0
0,5
1
1,5
2
2,5
Start Untreated 0,005% 0,025% 0,050%
List
eria
, Lo
g C
FU/c
he
ese
2 logs
.9 log
.5 log
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Red smear cheese
(Guenther et al. 2011)
• 3 different inoculation levels
• Use of Listex results in 1-2 log kill in 24h
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Phage applications in Cheese
• Listeria contamination is typically found on
the outside of cheese products.
• Surface ripened washed rinds and white
mould cheeses are especially at risk since
growing conditions Listeria are ideal and
there is a large risk for cross contamination.
• A sufficient amount of phage needs to be
present to ensure the statistical probability
that the phage encounter the bacteria.
• Preventive use ensures Listeria is accessible
and not vested within the cheese matrix.
Cheese variety Application Dilution
Soft/Semi-hard
On rind at pH 4,5 - 5,5
1%
White Mold
Prior to mold growth
1%
Queso Fresco
In pasteurized milk during
cheese making
0,05%
Washed Rind
In wash liquid
0,05%
Concentration PFU/(plaque forming
units) per ml
100% Listex 2x1011
1% 2x109
0,05% 1x108
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Listeria Phages for use on Biofilms & Equipment Deep Cleaning & Hot Spot treatment
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Top chart (A)
• Nutrient-limiting conditions – grown for 2 days
• 5-strain mixture of L. mono – 2 days attachment
• 5.5 log kill after 24 hrs
Bottom Chart (B)
• Multilayered biofilm- grown for 7 days
• 5-strain mixture of L. mono
• 3.5 log kill after 24 hrs
Removal of Listeria monocytogenes Biofilms with Bacteriophage P1003 KAMLESH A. SONI AND RAMAKRISHNA NANNAPANENI* Department of Food Science, Nutrition and Health Promotion, Mississippi State University, Mississippi State, Mississippi 39762, USA
Reduction of L. mono from stainless steel surfaces
using PhageGuard ListexP100
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Surface in presence of food residues in 0.2 mm grooves
Inactivation of Listeria monocytogenes by disinfectants and Bacteriophages in suspension and stainless steel carrier tests
N. CHAITIEMWONG, W. C. HAZELEGER,* AND R. R. BEUMER Journal of Food Protection 2014
1: (T=0) 2: before and 3: after exposure to sterile water, 4: sodium dichloroisocyanurate (240 mg/liter), 5: quaternary ammonium compounds (740 mg/liter)
6: PhageGuard ListexP100 (109 PFU/ml)
1
2
5
3
4 6 6 6 6
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Removal of Listeria monocytogenes biofilms with bacteriophage PhageGuard ListexP100
KAMLESH A. SONI AND RAMAKRISHNA NANNAPANENI* Department of Food Science, Nutrition and Health Promotion, Mississippi State University, Mississippi State, Mississippi 39762, USA
Untreated
PhageGuard ListexP100
Results reported are after 24 h phage application
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Phages on Biofilms & Equipment to combat Listeria
• 3-5 log reduction on food contact surfaces and equipment
• Reduce or eliminate Listeria biofilms in the presence of food debris.
– Phage treatment is more effective in eliminating Listeria in deep grooves
/scratches compared to chemical disinfectants
– Disinfectants oxidize on the surface, whereas phage can penetrate into
the biofilm
• Phages can be used during regular production
– are organic and have no effect on the treated food
– considered innocuous for humans and animals
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Listex Phages for Environmental Applications
Food-contact surfaces • For food-contact surfaces and environmental surfaces that are
chemically cleaned daily, LISTEX™ can be applied on a
continuous/daily base.
Biofilms/Hot Spots treatments • For environmental application/hot spots where biofilms are
expected LISTEX™ can be used daily at a 5% concentration and
followed by daily testing/swabbing until Listeria will test negative
for three consecutive days. From this point onwards LISTEX™
should be used weekly or bi-weekly. Biofilm should be
eradicated within 2-7 treatments.
Food-processing environments • For the food-processing environments we recommend a
weekly/bi-weekly application of LISTEX™
1% daily
1%-5% daily
Until negative
1% dilution
Bi-weekly
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Listex for environmental use
• Dilute in tap water free chlorine (level of < 1 ppm)
• Spray on the environmental surface/biofilm until it is thoroughly wet
• 10 liters of a 1% LISTEX™ solution can treat approx. 100 m2 of surface
• For the drains either spray or flush with the solution
• Rinse all chemicals from the area before applying phages
• Phages will continue to work as long as the area is wet so should be applied
at the end of the cleaning shift and left for as long as possible
• LISTEX™ solution does not need to be rinsed off
• Clean the sprayer (in and out) and flush it before preparing a new batch.
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Regulatory Status
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Processing aid around the world
USA
Can
Aus
NZ
GCC Ind
Isr
Arg
CH
No