Protective Bacteria: An Option to Control Listeria monocytogenes in Seafood Products

Protective Bacteria: An Option to Control Listeria monocytogenes in Seafood Products Marie-France Pilet UMR SECALIM, INRA, Oniris, 44307, Nantes, France IAFP Athens 11-13th May 2016

Seafood products : positive aspects High nutritional value Unsaturated fatty acids Oligo -elements Vitamins Proteins

Lightly preserved seafood products (LPSP) 2008 166 000 t Consumption 7, 3 % 2014 177 000 t Salted and/or dried fish Smoked fish Cooked shrimp Surimi (FranceAgriMer, 2015)

Microbiology of LPSP Spoilage bacteria Hafnia alvei Phosphobacterium phosphoreum Serratia liquefaciens Brochothrix thermosphacta Lactobacillus sakei Economical losses Pathogenic bacteria Salmonella spp. Histaminogen bacteria Vibrio spp. Clostridium spp. Staphylococcus aureus Listeria monocytogenes Safety issues

Listeria monocytogenes : a problem in seafood? Listeriosis Severe food-borne disease Fatality rate : 15,6 % in EU (2013) Human listeriosis in EU 1763 cases in 2013 Increasing from 2009 to 2013 : 0.37 to 0.44 cases/100 000 population Contamination during the process Contamination and growth in RTE seafood products Refrigerated temperatures Anaerobiosis ph from 4,5 to 9 High tolerance to NaCl Growth abilities (EFSA, 2015)

Listeria monocytogenes : a problem in seafood? Fishery products : highest levels of non compliance In 2013 : 10.8% of fish samples positive for L. monocytogenes 1.6% at level >= 100 CFU/g (EFSA, 2015)

Main hurdles in lightly preserved seafood products Mild process Raw or mild cooking Salting Preservatives Refrigerated storage under MAP or VP Smoking Shelf-life > 5 days Fresh fish under MAP : 8-10 days Cooked shrimp under MAP : 12-14 days Cold smoked fish under VP : 21 to 42 days Biopreservation as complementary or alternative hurdle?

Protective lactic acid bacteria in seafood products? Culture-dependant and cultureindependant methods (TTGE, 16S rdna metasequencing) Lactic Acid Bacteria : dominant microflora at the end of the shelf life (Jaffrès et al., 2009 ; Macé et al., 2011 ; Chaillou et al., 2014) Cold smoked salmon under VP Cooked peeled shrimp under MAP Fresh salmon fillets under MAP Carnobacterium maltaromaticum Carnobacterium divergens Lactococcus piscium Leuconostoc gasocomitatum/gelidum Lactobacillus sakei Vagococcus fluvialis

Specifications for seafood protective cultures selection Growth on seafood matrix Psychrotrophic behavior No impact on sensorial properties of seafood Sensory analysis with trained panels Axe 2 (1 0.9%) 4.5 4 3.5 3 2.5 2 1.5 1 0.5 0-0.5 rice/crustacean -1-1.5-2 -2.5-3 Non spoiled B1 L1 LB1 L3 LB3 L24 B3 LB24 L31 L1 0 LB7 LB17 LB38 LB14 LB31 L17 L7 LB10 L14 L38 milky floorcloth butter/caramel Global intensity sour/fermented amine cheese/foot B7 pyrrolidin/sperm B14 H 2 S/cabbage Spoiled B10 B24 B17 B31 B38-2 -1 0 1 2 3 4 5 Axe 1 (75.1%)

Cold smoked salmon : a model for biopreservation studies Contamination by L. monocytogenes: Raw salmon During the process : slicing, salting 4 C 8 C Growth of L. monocytogenes No bactericidal step Low inhibiting effect by salting (<3%) and smoking (< 1 mg phenol/100 g) No preservatives

Biopreservation studies in cold smoked salmon Protective bacteria Inhibition References mechanism Carnobacterium piscicola A9b carnobacteriocin B2 Nilsson et al. (2004) Carnobacterium divergens V41 divercin V41 Brillet et al. (2004) Carnobacterium piscicola* V1 piscicocin V1 Brillet et al. (2004) Carnobacterium divergens M35 divergicin M35 Tahiri et al. (2009) Carnobacterium maltaromaticum CS526 piscicocin CS526 Yamazaki et al. (2003) Lactobacillus sakei Lb790 sakacin P Katla et al. (2001) Carnobacterium piscicola* A9B Competition for glucose Nilsson et al. (2005) Lactobacillus sakei Lb790/pMLS114 sakacin P Aasen et al.( 2003) *maltaromaticum

Biopreservation of cold smoked salmon with carnobacteria CHALLENGE-TEST WITH 3 BACTERIOCIN-PRODUCING CARNOBACTERIUM : C. maltaromaticum V1 and SF668, C. divergens V41 4 C 8 C 7. 10 4 CFU/g 18 CFU/g (Brillet et al., 2004; 2005)

Biopreservation of cold smoked salmon with carnobacteria C. divergens V41 : additional hurdle to limit L. monocytogenes growth in cold smoked salmon Inhibition effect : 4 log CFU/g No effect of the PC on the sensory properties of the salmon (trained panel) No significative modification of physico-chemical parameters : ph, TVBN Slight effect on endogenous microflora (enterobacteria) Safety aspects : antibiotic resistance, histamine negative, no cytotoxicity Cb divergens included in the QPS list in 2014 (Brillet et al., 2004; 2005 ; EFSA 2014)

Inhibition by Cb. divergens V41 : mechanism Mutant deficient in bacteriocin production L. mono Cb. divergens V41C9 Challenge-test in cold smoked salmon Comparison with Bac+ strain Cb. divv41 bac + L. mono Cb. div V41C9 bac - L. mono Role of bacteriocin production /strain growth No durable effect of semi-purified bacteriocin Nutritional competition or other mechanism? (Richard et al., 2003)

Biopreservation studies in cooked shrimp Raw frozen shrimp Cooking 75 C 2 to 5 min! Contamination by L. monocytogenes Cooling in salted water Packing under MAP CO 2 -N 2 50/50 Main hurdles for L. monocytogenes : Preservatives (organic acids) Protective culture as alternative? Storage 0-4 C 12-14 days

Protective culture selection for shrimp biopreservation Lactococcus piscium CNCM I-4031 Tmax : 29 C Isolated from fresh salmon fillets under MAP Limitation of bacterial spoilage Inhibition of food-borne pathogens Growth inhibition Brochothrix thermosphacta Serratia proteamaculans Metabiosis Photobacterium phosphoreum Carnobacterium divergens Listeria monocytogenes (Matamoros et al., 2009; Fall et al., 2010a et b; Leroi et al., 2015)

Biopreservation of cooked peeled shrimp under MAP CHALLENGE-TEST WITH Lactococcus piscium CNCM I-4031 at 8 C 10 Log (CFU g -1 ) 9 8 7 6 5 4 4 log inhibition No modification of the sensorial properties Safety requirements 3 2 1 4 7 10 13 16 19 22 25 28 31 Time (days) No bacteriocin detected in supernatant L. monocytogenes Lactococcus piscium CNCM I-4031 in co-inoculation L. monocytogenes in co-inoculation (Fall et al., 2010; 2011)

Mode of action of L. piscium CNCM I-4031 Log10(CFU/ml) Log10(CFU/ml) Shrimp Log (UFC g -1 ) 10,0 9,0 8,0 7,0 6,0 5,0 4,0 3,0 1 4 7 10 13 16 19 22 25 28 31 Temps (jours) Lactococcus piscium L.monocytogenes en co-inoculation Times L.monocytogenes Lactococcus piscium en co-inoculation Reproduction of inhibition conditions in a chemically defined medium at 26 C Glucose, 18 aminoacids 8 vitamins, bases, NaCl, Mg, Fe, buffered Synthetic medium Times

Log (CFU/ml) Mode of action of L. piscium CNCM I-4031 Co- culture L. monocytogenes + L. piscium Inhibition is not due to : bacteriocin production in supernatant, acidification, hydrogen peroxyde nutritional competition Chemically defined medium MSMA Culture of L. piscium CNCM I-4031 Culture of L.monocytogenes 10 9 8 7 6 5 No inhibition in separated cultures Membrane (0,45µm) 4 3 0 5 10 15 20 25 30 35 40 45 50 Time(h) L. monocytogenes in pure culture l L. monocytogenes in DC L. piscium in pure culture L. pisicum in DC INHIBITION REQUIRES CELL TO CELL CONTACT (Saraoui et al., 2016)

Mode of action of L. piscium CNCM I-4031 : hypothesis Genome sequencing of L. piscium CNCM I-4031 RNA Seq in monoculture and 25h co-culture conditions (max inhibition) Antimicrobial components 2 CAZymes 1 Multi-Drug resistance and protease 1 DNA repair 1 Surface bacteriocins? Cell walls 3 Bacterial competence 3 RNA 5 43 overexpressed Protein of unkwon genes function 27 for L. piscium : 5 involved in antimicrobial components and bacterial competence

Conclusions Protective lactic acid bacteria efficient to control L. monocytogenes in seafood : Carnobacterium, Lactococcus, Leuconostoc Few commercial applications : LLO, Lyoflora Effect of PC on sensorial properties, metabiosis effects PC characterization Bacterial ecosystem studies and interactions Safety aspects and action mechanisms To be developped with genomic and «omics» approaches Combination with other preservation methods In progress : European project SafeFishDish

French network on protective culture : Research projects Regulation aspects Consumer perception studies http://www.actia-asso.eu/fiche/rmt-28-florepro.html

Special thanks Ifremer team : Françoise Leroi, Jean-Jacques Joffraud, Frédérique Gigout, Mireille Cardinal, Josiane Cornet PhD students : Anne Brillet, Sébastien Matamoros, Papa Abdoulaye Fall, Taous Saraoui Teams of the Microbiology Lab from SECALIM and IFREMER, members of the sensory panel from IFREMER Our partners and financial supports

THANKS FOR YOUR ATTENTION marie-france.pilet@oniris-nantes.fr @InraSecalim http://www6.angers-nantes.inra.fr/secalim