Phages for Food Protection Controlling Listeria monocytogenes in Meat, Cheese, Salmon & Vegetables with organic Phage solutions

Transcription

1 Phages for Food Protection Controlling Listeria monocytogenes in Meat, Cheese, Salmon & Vegetables with organic Phage solutions Food Safety Event 6 October 2016 Bert de Vegt

2 Outline Introductions Micreos Phage 101 Listex Phages for Listeria control Meats & seafood Vegetables Dairy Phages for Bio-film removal Regulatory

3 Micreos Dutch company Global leader in applied phage technology Incorporated in employees Targeted anti-bacterial solutions Phages have applications in: - Human Health MR(SA) - Food Safety - Animal Health - Agriculture Amsterdam Wageningen

4 Micreos Wageningen: Food Safety Bilthoven: Human Health

5 Strong Scientific Background and Connections

6 Video

7 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

8 Phages are everywhere Most abundant micro-organisms on earth (>10 30 ) 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 individual phages making it perhaps the most densely populated ecological niche in nature. Leave no footprint- composed of protein and DNA/RNA Breaks down into amino and nucleic acids

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10 Life cycle of lytic phages 1. Adsorption 2. Infection Bacterial cell Lytic cycle 3. Replication 5. Host lysis and Progeny phage release 4. Maturation and assembly Note: Treating food products with PhageGuard Listexand PhageGuard S, we do not rely on the lytic cycle

11 Pathogens of Concern Listeria Salmonella E-coli Campylobacter

12 estimated foodborn deseases per year 9.4 Million Bacteria; 39% Virus; 59% Foodborne pathogens estimated foodborne disease per year Listeria incidence in the USA 255 death per year Annual burden is estimated at $2.8 billion a year Cost per case has been estimated at $1.8 million. estimated foodborn illnesses per year Hospitalizations: 35,798 4% 3% 4% 7% Campylobacter; 24% Salmonella; 55% Scallan et al, 2011

13 foodborn illness due to zoonose Confirmed Cases: 341,455 Salmonella; 26% 1% Campylobacter; 69% Foodborne pathogens EFSA and the European Center for Disease Prevention and Control 2014 incidence data foodborn illness due to zoonose Death: 313 Campylobacter; 8% Campylobacter Salmonella Yersinia E.coli Listeria Other foodborn illness due to zoonose Hospitalizations: 30,581 3% Listeria; 67% Salmonella; 21% Salmonella; 32% Campylobacter; 60% Campylobacter Salmonella Yersinia E.coli Listeria Other Campylobacter Salmonella Yersinia E.coli Listeria Other Food categories most sensitive for Lm: Fish (smoked), RTE meats and cheese

14 3D s for Success Dose or concentration Distribution Dwell Time or contact time

15 Phages provide protection in all zones 1. Food 2. Food Contact Surfaces 3. Environment

16 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

17 Listex Phages on Foods

18 Meat & Poultry

19 Time efficacy of LISTEX in killing L. Monocytogenes on hot dogs L. mono log CFU/cm 2 4,8 4,4 4,0 3,6 3,2 2,8 2,4 2,0 Internal study (Micreos Food Safety, 2015) Time (h) Control LISTEX 0.5% (1x10 9 PFU/mL) Applied at 5 µl/cm 2 LISTEX 1% (2x10 9 PFU/mL) Applied at 5 µl/cm 2 Both concentrations of LISTEX showed an immediate reduction of L. mono

20 Incubation time LISTEX Application on hotdog surface Dr. C.Iancu, Micreos Application Labs h 16 h 6 h 5% LISTEX 1% LISTEX 0,5% LISTEX LISTEX has a fast killing effect at low concentration: >1log reduction for 0,5% solution after 30 min 2 h 30 min 0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 L. Monocytogenes WSLC10403S reduction, logcfu/cm2 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%

21 Hot dogs Spray into the vacuum packages before sealing. Vacuum-sealing of the packages distributes the antimicrobial evenly around the product.

22 Hot dogs Phages can be sprayed directly into a hopper

23 Bagged whole muscle products Phages are sprayed into the package before sealing. Vacuum-sealing distributes the antimicrobial evenly around the product.

24 LISTEX on cooked ham Dose dependent control of Listeria (1x10 7 and 5x10 7 pfu/cm2) during shelf life.

25 Seafood

26 Reduction of Listeria monocytogenes at 4C on raw salmon fillet tissue Kill after 30 minutes 2.5x10 7 = 2 log reduction 2.5x10 8 = 3.5 log reduction Journal of Food Protection 2010, Vol. 73

27 Listeria Kill on raw salmon at different Listex concentrations applied via electrostatic spray 1,00E+05 Listeria concentration, CFU/cm 2 1,00E+04 1,00E+03 1,00E+02 1,00E+01 0,86 log kill 1,26 log kill 1,52 log kill Log4 CFU/cm 2 inoculation level; Treatment with 1% LISTEX solution; Treatment applied with electrospray system at: - 2 µl/cm 2 4 x 10 6 PFU/cm 2 ; - 5 µl/cm 2 1 x 10 7 PFU/cm 2; - 10 µl/cm 2 2 x 10 7 PFU/cm 2; 1,00E+00 Control 4 x 10^6 1 x 10^7 2 x 10^7 Treatment, PFU/cm 2 Micreos Application Center- July 2016 Dr. C. Lancu

28 Dairy & Cheese

29 Efficacy of phage A511 (P100) on Camembert cheese during ripening P100=phage A511 Phage dilution: 15% Phage dosage: 3 x 10 8 PFU/cm 2 (at 10 µl/cm 2 ) Control Phage ph 1 day: 2 log reduction Data generated by ETH (Swiss Federal Institute of Technology Zurich)

30 International Journal of Food Microbiology 155 (2012) Reduction of Listeria monocytogenes in queso fresco cheese by a combination of listericidal and listeriostatic GRAS antimicrobials Kamlesh A. Soni, Monil Desai, Ademola Oladunjoye, Frederick Skrobot, Ramakrishna Nannapaneni Department of Food Science, Nutrition and Health Promotion, POB 9805, Mississippi State University, Mississippi State, MS 39762, United States Fig. 1. Reduction of Listeria monocytogenes in QFC after surface treatment with bacteriophage P100 ( ) or lauric arginate ( ) or their combination ( ) compared to untreated control ( ). The concentration of phage P100 at 108 PFU/g and LAE at 200 ppm/g was used for surface application of QFC. Fig. 3. Reduction of Listeria monocytogenes in QFC after surface treatment with bacteriophage P100 ( ) or PL SD ( ) or their combination ( ) compared to untreated control ( ). The concentration of phage P100 at 108 PFU/g and PL SD (2.8% PL and 0.2% SD) was used for surface application of QFC.

31 Listeria, Log CFU/cheese Effect of LISTEX on Listeria monocytogenes reduction in Belgioioso mozzarella at 24h 2, %: 1 x 10 8 PFU/mL 1,5.5 log.9 log 0.025%: 5 x 10 7 PFU/mL 0.005%: 1 x 10 7 PFU/mL 1 0,5 0 2 logs Start Untreated 0,005% 0,025% 0,050% 0.05% LISTEX solution resulted in a 2 log reduction of L. monocytogenes 2/2

32 Red smear cheese 3 different inoculation levels Use of Listex results in 1-2 log kill in 24h (Guenther et al. 2011)

33 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 2x % 2x10 9 0,05% 1x10 8

34 Listeria Phages for use on Biofilms & Equipment Deep Cleaning & Hot Spot treatment

35 Reduction of L. mono from stainless steel surfaces using PhageGuard ListexP100 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

36 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 : (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 (10 9 PFU/ml) Surface in presence of food residues in 0.2 mm grooves

37 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

38 Phages on Biofilms & Equipment to combat Listeria Use on food contact surfaces and equipment to achieve 3-5 log reductions Use to reduce or eliminate listeria biofilms in the presence or absence 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 are not affected by food debris Phages can be used during regular production are organic and have no effect on the treated food considered innocuous for humans and animals

39 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

40 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 m 2 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.

41 Regulatory Status

42 Processing aid around the world Can No CH USA Isr GCC Ind Arg Aus NZ

43 Thank you for your attention Bert de Vegt