phages for food protection - food valley expo 2016 for food protection . outline ... efsa and the...
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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
Outline
• Micreos
• Phage 101
• Listex Phages for Listeria control
– Meats & seafood
– Vegetables
– Dairy
• Phages for Bio-film removal
• Regulatory
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
Micreos
Wageningen: Food Safety
Bilthoven: Human Health
Strong Scientific Background and Connections
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
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
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
Pathogens of Concern
E-coli
Listeria
Campylobacter
Salmonella
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
3D’s for Success
• Dose or concentration
• Distribution
• Dwell Time or contact time
Phages provide protection in all zones
3. Environment
2. Food Contact Surfaces
1. Food
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
Listex Phages on Foods
Meat & Poultry
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
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%
Hot dogs
Spray into the vacuum packages before sealing. Vacuum-sealing of the
packages distributes the antimicrobial evenly around the product.
Hot dogs
Phages can be sprayed directly into a hopper
Bagged whole muscle products
Phages are sprayed into the package before sealing.
Vacuum-sealing distributes the antimicrobial evenly around the product.
Seafood
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
Dairy & Cheese
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)
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
Red smear cheese
(Guenther et al. 2011)
• 3 different inoculation levels
• Use of Listex results in 1-2 log kill in 24h
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
Listeria Phages for use on Biofilms & Equipment Deep Cleaning & Hot Spot treatment
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
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
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
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
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
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.
Regulatory Status
Processing aid around the world
USA
Can
Aus
NZ
GCC Ind
Isr
Arg
CH
No
