Kalliopi Rantsiou and Luca Cocolin Faculty of Agriculture University of Turin, Italy

1. Contribution of molecularmethods in understandingmicrobialecology of fermentedsausages and dynamicsduringfermentation Kalliopi Rantsiou and Luca Cocolin Faculty of Agriculture University of Turin, Italy

2. MolecularBiology • Study of the molecularbasis of biologicalactivity • Study of structure and function of cellularmacromolecules (DNA, RNA, proteins)

3. Milestones of MolecularBiology KaryMullis, 1983 1995 2001 Watson and Crick, 1953 Mendel, 1856-63 Griffith, 1928 1972 PCR DNA, hereditary material Inheritance of phenotypic traits Structure of DNA Molecular cloning Sequencing Haemophilusinfluenzae Human Genome Sequencing

4. Central Dogma of MolecularBiology Describes the flow of information in biological systems

5. pyrosequencing

6. Application of molecularbiologymethods in meatfermentations

7. Conventional vs. molecular • Phenotype • variable • interpretation of results • time • Genotype • constant • unequivocal identification • precise characterization

8. Species-specific PCR • Primershavebeendevelopedformain LAB and CNC speciesinvolved in meatfermantations • Targets: 16S rDNA, randomfragments (asidentifiedby RAPD), sodA, hsp-, lipase-encodinggenes • Identification of largenumber of isolates, presumptivetobelongtocertainspecies • Possibilitytodistinguishbetweencloselyrelatedspecies, difficultto separate byphenotype

9. PCR –D(T)GGE • Species-specificmigrationin PCR-DGGE allowsdirectidentification(bycomparisontoprofiles of knowncontrolstrains) or grouping of isolatesbased on profile and subsequentidentificationbyrDNAsequencing • applicationfor LAB, CNC aswellasyeasts, isolatedfromfermentedmeats

10. ISR-PCR • Study of the polymorphism of the 16S-23S IntergenicSpacerRegion (ISR) • applicationfor CNC • Combinedwithotheridentificationmethods

11. Characterization of isolates • Sub-speciescharacterization • todetermineintra-speciesbiodiversity (oftenwith the purpose of developingautochthonousstarters) • Study the performance of inoculated starter cultures

12. Characterization of isolates • RestrictionFragmentLengthPolymorphism (RFLP), plasmidprofile, PulsedField Gel Electrophoresis (PFGE) • PCR-based: RandomlyAmplifiedPolymorphic DNA (RAPD), RepetitiveExtragenicPalindromic (rep)-PCR

13. RFLP Identifiedmicrobial isolate fromfermentedproduct Isolate A isolate B isolate C

14. PFGE

15. Plasmidprofiling or fingerprinting

16. RAPD - PCR

17. rep - PCR

18. Starter culture performance

19. Mainfindings of characterizationmethods • Discrimination of strains with technologically relevant activities (for example, nitrate reduction, amino acid decarboxylase activity, temperature of growth)

20. Mainfindings of characterizationmethods • environmentalparametersselectforspecificbiotypes, whichprevail at the end of the fermentation–selectionpressure of a particularniche • plant-specificpopulations (may originate fromingredients and mayberesponsiblefor the sensorialcharacteristics of the finalproduct)

21. Mainfindings of characterizationmethods • identification of differentbiotypeswithin a starter culture and determination of the oneresponsiblefor the fermentation • Possibility to discriminate the added strains from those naturally present during the ripening of the product

22. Culture-independentmethodstostudymicrobialecology of fermentedmeats

23. Advantages of direct approaches • no cultivation takes place and therefore, the bias, associated with the use of conventional microbiological media for enumeration/isolation, is negated, • compared to the classic approach used so far in microbiology, that is based on isolation of strains from the food matrix and identification, either by physiological/phenotypical tests or by molecular methods, they are less time-consuming and require less effort, • they allow a parallel description of the populations of different microbial groups.

24. The use of RNA DNA may persist in any given environment, some times long after a microorganism is dead. In contrast, RNA is rapidly degraded after cell death and as a consequence, the application of RT-PCR-DGGE gives the fingerprint (or profiles) of alive and metabolically active populations

25. DNA RNA Cocolin et al. 2004, Appliedand EnvironmentalMicrobiology, 67, 5113-5121

26. rDNA: the mostcommonlyemployed target • Universal • Highlyconserved • regionsaswellasvariable • Largesequencedatabasesavailable

27. Application of PCR - DGGE Throughout the fermentationprocess–Populationdynamics Finalproduct–Biodiversity

28. Lactobacilluscurvatus Lactobacillus paracasei Rantsiou et al.2005 , Applied and EnvironmentalMicrobiology, 71, 1977-1986 Lactobacillussakei

29. Rantsiou et al. 2005, Applied and EnvironmentalMicrobiology, 71, 1977-1986

30. Comparison of finalproducts Silvestri et al. 2007, Meat science, 77, 413-423

31. Predominance of LAB in culture independentmethods Cocolin et al. 2009, Meat science, 82, 125-132

32. Yeastdynamics Rantsiou et al. 2005, Applied and EnvironmentalMicrobiology, 71, 1977-1986

33. Bacterialecologybyspecies-specific PCR • 12 sets of primersfor detection of 6 LAB and 6 CNC speciescommonlyencountered in fermentedmeatproducts • Application in the fermentedsausagehomogenate, before and after 24 hourenrichmentperiod (in MRS and Mannitol Salt broth) Aymerich et al. 2003, Applied and EnvironmentalMicrobiology, 69, 4583-4594

34. Quantification of Lactobacillussakeibyspecies-specificqPCR • Quantification of L.sakeiduringfermentation, without the need of cultivation • Applicationto 11 commercial samples of sausages and meat batter Martin et al. 2006, Applied and EnvironmentalMicrobiology, 72, 6040-6048

35. FinalConclusions • Unequivocal species identification • Possibilitytofollow a certainbiotypeduringfermentation • Biases of cultivation are by-passed • Possibilitytodetermine the metabolicallyactivepopulations