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Ecological approaches for managing microbial diversity to improve safety of traditional food.

Ecological approaches for managing microbial diversity to improve safety of traditional food. Marie-Christine Montel, Unité de Recherches fromagères INRA Aurillac. Example of cheese from experiments performed in WP2A of Truefood project.

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Ecological approaches for managing microbial diversity to improve safety of traditional food.

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  1. Ecological approaches for managing microbial diversity to improve safety of traditional food. Marie-Christine Montel, Unité de Recherches fromagères INRA Aurillac Example of cheese from experiments performed in WP2A of Truefood project

  2. Improvement of microbial safetyWP2A Truefood project Task 2A.1 Improvement microbial safety of milks through reduction of mastitis and the use of antibiotics by feeding regimes and other management practices( ISS, INRA,) Task 2A.2 Management of microbial diversityfor inhibiting pathogenic bacteria (L. monocytogenes, S. aureus) in traditional cheeses ( INRA, UL, TUM, DRI) Task 2A.3 Improvement of environmental conditions governing cheese ripening taking account process efficiency and cheese quality(INRA, DRI) Task 2A.4 Development of a bio-preservation (using lactic acid bacteria) for inhibiting food pathogens on pork muscle tissues (ADIV)

  3. Why an ecological approach? • Because • Cheeses and milk are microbial ecosystems • Cheese is a system in which microbial populations (living or biotic part ) live and have relationships between them and with their surrounding environments ( abiotic part) . • Microbial populations interact each other in synergy or antagonism and their life dpends on nutriments, temperature, humididity, oxygen. • The life of these microbes is determinant for the qualities of cheeses.

  4. Microbial safety Pathogenic bacteria : commercialised raw milk cheeses have to comply with EC regulation Listeria monocytogenes Salmonella Staphylococus aureus ( no enterotoxins production ) Eschercihia coli Opportunist pathogenic bacteria Metabolite productions Enterotoxins Mycotoxins Biogenic amines Transfertof antibioresistance -

  5. Above all, Benefits refer to pleasure and happiness of consumers who require authenticity , traceability, sensorial properties, safety and health values. great taste,

  6. To eliminate pathogenic bacteria EC regulation for 4 species To preserve microbial community: during manufacturing and ripening Increase Benefits MinimiseRisks Microbial Safety Gustative pleasure Health Why to manage microbial diversity ? • A challenge for industrial producing Traditional Fermented products (Cheese, sausage…) for conciliating all these aspects and meet all these requirements

  7. Our behaviour in managment of microbial diversity for microbial safety must have always in mind the benefits of microbial diversity for cheeses-Healthy aspects?-Contribution in hurdle technology -Diversity of their sensorial properties

  8. Management of microbial diversity • Elimination of pathogenic micro-organisms and maintain of microbial diversity having an interest through all the process • control of raw material : microbial quality of milk, meat... • control of microbial dynamics during ripening • Tools for tracking and monitoring microbial diversity

  9. Molecular PCR-SSCP,TGGE, TTGE, DGGE, lH-PCR… Milk , cheeses Classical Approaches get rich each other How to identify and monitor microbial community • Picture depending on culture media, cultavibility of strains • Identification by • phenotypic tests, • genomic tests (speciesPCR, 16s or 23s DNAr sequencing) • Physical spectra ( FTIR) • Monitor microbial dynamics • Picture biased by • Dominant population detected • DNAExtraction • PCR amplification • Coelution in the same peaks

  10. What approaches for biopreservation of traditional products ? • Selection of strains or metabolites (example : nisine) • Understanding microbial ecosystems having antagonist activities • Example of milk and raw milk cheese ecosystems

  11. Selection of strains or inhibiting metabolites Validation at industrial scale Milks , cheeses 1 Strains Collections Challenge tests in experimental cheese Detection of bacteriocin genes In vitro screening of inhibitory activity GAP here!!!! Coculture in milk or other media Test on agar well Agar diffusion Bioluminescence Identification of inhibiting substances bacteriocins, H2O2,

  12. Selection of strain inhibiting • Time consuming and fastidious • Frequent fails due to gap between the results obtained in vitro and those obtained in cheeses • Consortia with high antilisteria activities by in vitro test but not at the surface of cheeses • Low number of strains selected for example in Truefood project able to inhibit and without effect on sensorial properties

  13. Strategies by ecological approach • Rely on a general principle in ecology indicating that: • the whole is more than the sum of each individu as many interactions - synergy, antagonism, competition..- can occur • Hypothesis that preservation of the microbial community with the wholeness of its diversity is important for the different functions -inhibition of pathogens, production of aromatic compounds

  14. Understanding microbial ecosystem having antilisteria activity Advice for milk production or cheese ripening proposal of consortium Screening of milks, or cheese surface on experimental cheeses 1 4 4 Test of simplified microbial consortia in experimental cheese Analysis of the most inhibitory microbial consortia 2 3 Identification of microbial and biochemical part of the ecosystem

  15. Examples of microbial cheese ecosystems with antilisteria activities • Microbial consortia from Surfaces of cheeses • Smear cheeses: Munster • Farm Saint-nectaire cheeses • Microbial consortium from Raw milk from Saint-Nectaire area

  16. First example consortia of microbes from the surface of Saint-Nectaire Study of Inhibition of Listeria monocytogenes at the surface

  17. SN15 SN11 SN26 SN19 SN13 SN9 SN22 SN30 SN24 SN25 SN23 SN12 SN29 SN6 SN8 SN34 SN20 SN28 SN16 SN32 SN2 SN17 SN1 SN35 SN31 SN27 SN3 SN14 SN33 SN18 SN10 SN5 SN4 SN7 Antilisteria activities of surfaces of Saint-Nectaire cheese microbial consortia 0,5 0 -0,5 -1 -1,5 -2 -2,5 • Comparison of inhibitory effect of consortia (34) selected from farm Saint-Nectaire surface cheeses.(Trials on surface of non cooked pressed cheeses ripened at 7°C during 28 days) No inhibition Inhibition DLog • Great diversity in the antilisteria activities observed without relation with pH values • 5.9<pH28d<8.4 • Selection of one consortium SN15 High inhibition

  18. Stability over storage of the inhibiting effect of SN15 consortia against Listeria monocytogenes Time (months) ΔLog Lm (ufc/cm²) ΔLog Lm = Log (Lm SN15) - Log (Lm control) Microbial consortium SN15 still inhibitory after 24 months storage at -20°C

  19. D A D D C C B B Identification of microbial consortium (SN15) inhibiting L. monocytogenes • Identification by phenotypic test, RFLP and 16S DNAr sequencing A=Lactic acid bacteria (6 species) Lb. casei Lb. curvatus Ln. mesenteroides Carnobacterium mobile Marinilactibacillus psychrotolerans E. faecalis D=Yeasts (4 species) Candida sake Yarrowia lipolytica Debaryomyces hanseniiGeotrichum sp. B= Micrococcaceae/ Corynebacteriaceae (7 species)Arthrobacter nicotianae Arthrobacter bergeri Staphylococcus pulvereri Staphylococcus xylosusBrevibacterium linens or casei/ Brevibacterium antiquum Brachybacterium C=Gram negative bacteria (3 species) Proteus vulgarisSerratia proteomaculans Pseudomonas fluorescens or syrinqae

  20. Reconstitution of complex consortium SN15 from the surface • Constitution of consortium with 19 strains (one strain by species) inoculated at 2 Log /cm2 at the surface of cheeses • Comparison of L. monocytogenes growth at the surface of cheeses with the natural complex or reconstituted consortia 3 yeasts 6 Lactic acid bacteria 3 Gram negative D D C 7 Micrococcaceae/ Corynebacteriaceae B reconstituted Complex more inhibitory than the reconstituted Inhibition DLog Complex Study in progress for understanding why?

  21. Second example microbial consortium from raw milk Study Inhibition of L. monocytogenes in the core of cheeses Complex consortium selected in previous study : Millet al, 2006; Saubusse et al, 2007

  22. D A D D C C B B Composition of Microbial consortium from raw milk having antisteria activty ( Saubusse et al, 2007) Great diversity : 29 microbial species A=Lactic acid bacteria (9 species) Lb. casei / Lb. farciminis Lb. curvatus Lb. plantarum Ln. pseudomesenteroides Ln. citreum E. hirae E. Faecalis A. viridans D=Yeasts (5 species) Rhodosporium babjevae Debaryomyces hansenii Candida pseudointermediaC. pararugosa C. deformans B= Micrococcaceae/ Corynebacteriaceae (10 species) Staph. saprophyticus/ Staph. equorum/ Staph haemolyticusCorynebacterium casei/Coryne. flavescensArthrobacter nicotianaeBrevibacterium linensExiguobacterium sp.Brachybacterium rhamnosusMacrococcus caseolyticus C=Gram negative bacteria (5 species) Pseudomonas putidaEnterobacter amnigenus Acinetobacter sp.Chryseobacterium spStenotrophomonas maltophilia

  23. Simplification of reconstituted consortia from raw milk Micrococcaceae/ Corynebacteriaceae Lactic acid bacteria A B Inoculation in pasteurised milk with S. thermophilus (St) Gram negative D C D yeast Manufacturing non coocked pressed cheeses A B A B B A D D C D C C Comparison ofL. monocytogenes to a control with only St thermophilus A B D B D A Microbial and biochemical analysis A B

  24. Inhibition of L. monocytogenes with simplified consortia in core of cheeses 1,5 AB AD A ABC ABD BDC BD B ABCD 1 0,5 A=Lactic acid bacteria 0 B=Micrococcaceae/ Corynebacteriaceae -0,5 ΔLog Lm -1 C=Gram negative -1,5 D=yeast -2 -2,5 ΔLog Lm = Log (Lm assay) - Log (Lm control) • Synergy between lactic acid bacteria and non lactic acid bacteria in the inhibition • Loss of inhibition in simplified consortia without lactic acid bacteria

  25. Link between [L. monocytogenes] and [organic acids]- [volatil compounds]- pH 5 L lactate 4 D lactate Acetic acid ABCD 3 AB Low count of listeria 2 3-methylbutyric acid 1 pH 0 Fact. 2 : 32,90% B Butyric acid A High count of listeria -1 2-heptanol 2-butanol 2-pentanol control ALCOOLS -2 2-hexanone 4-methyl-2-pentanone 2,3-pentanedione 2,3-butanedione 2-butanone -3 KETONES AD ALDEHYDES Hexanoic acid -4 ESTERS 2-methyl-propanal butanal Ethyl formiate Ethyl butanoate -5 -6 -7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6 Active • Hypothesis on the role of acetic acid and D lactate in the inhibition of L. monocytogenes in cheeses • Important variation in volatiles profils according to consortia inoculated Fact. 1 : 51,77%

  26. Inhibition by several hurdles during ripening pH>5.3 <8mg/g <2mg/g <0.3mg/g Alcohols Ethyl esters Ac. acetic Bacteriocins H202 Ac. lactic D Ac. lactic L pH • Besides these microbial factors, environnemental factors can also limit the development of pathogenes • Temperature, relative humidity during ripening

  27. What are the populations involved in the production ? • Microbial dynamics studied by culture methods associated with molecular tools • Example of microbial balance in cheese with consortium AB the most inhibitory • 19 species inoculated still present BUT • Difficulties to quantifye species of this group Lactobacillus Lactobacillus Leuconostoc Leuconostoc 9 9 8 8 Enteroccoccus Enteroccoccus 7 7 Nmax Log UFC/g Nmax Log UFC/g Corynebacteriaceae Micrococcaceae Corynebacteriaceae Micrococcaceae 6 6 5 5

  28. In conclusion, what applications for traditional fermented food? • Arguments for maintaining microbial diversity as a thumb for safety of fermented products ( ex cheese) • High inhibitory potentialities of complex microbial ecosytem from the surface of Cheese but application still limited by difficuties to reconsitute it • Further studies needed to understand why • Scientific data to think about suitable balance between microbial populations in milk • Further studies to adapt milk production practices

  29. In conclusion, what applications for traditional fermented food? • Proposal a simplified microbial consortium still complex associating lactic acid bacteria ( 8 species) and non lactic acid bacteria ( 12 species?) for inhibiting L. monocytogenes in the core of cheese but its industrial use need some improvments • Optimise the preparation of the consortium and insure its stability overtime • Validate it use at industrial scale • Evaluate its effect on sensorial properties • Develop rapid methods to quantifye non lactic acid bacteria in cheese WP6 of Truefood

  30. Thank you for your attention and coming instead of visiting the museum • Partners of WP2A of Truefood project and especially Cécile Callon for her helpful contribution in this presentation Financial support : European Commission under the 6th Framework programme for RTD ( contrat N° Food CT-2006-016264)

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