Pillar 2:

1. Pillar 2: Control and intervention strategies which can be implemented along the fork to farm chain to ensure safe beef Workpackage 2.2: Processing strategies - Progress during the last 6 months - Laboratory of Microbiology and Biotechnology of FoodsAgricultural University of Athens

2. Objectives-AUA • Task 2.2.2:Safety of marinades • Evaluation of the effect of different traditional marinades to control Salmonella Typhimurium in beef fillets • Evaluation of the effect of wine-based marinades to control Salmonella Typhimurium and to extend the shelf life of meat • Monitoring the survival of S. Typhimurium strains in marinated beef fillets Laboratory of Microbiology and Biotechnology of Foods Agricultural University of Athens

3. Evaluation of the effect of different traditional marinades to control S. Typhimurium in beef fillets • Task 2.2.2:Safety of marinades • Evaluation of the effect of different traditional marinades to control Salmonella Typhimurium in beef fillets Laboratory of Microbiology and Biotechnology of FoodsAgricultural University of Athens

4. Evaluation of the effect of different traditional marinades to control S. Typhimurium in beef fillets Experimental procedures Product: beef fillets (non sterilized) Inoculum:A 3-strain cocktail of acid or non-acid adapted S. Typhimurium DT 193: Human isolate, epidemic, multi drug resistance 4/74: Calf bowel DSM 554: Unknown origin Marinades:i)Olive oil ii) Wine iii) Olive Oil : wine (1:2) iv) Olive oil supplemented with 0.1% thyme oil v) Olive oil supplemented with 0.3% thyme oil vi) Wine supplemented with 0.1% thyme oil vii) Wine supplemented with 0.3% thyme oil viii) Olive oil : Wine (1:2) supplemented with 0.1% thyme oil ix) Olive oil : Wine (1:2) supplemented with 0.3% thyme oil Marination time: 4, 12, 24 h, at 4 °C Storage: air, at 15 °C, 96 h Laboratory of Microbiology and Biotechnology of Foods Agricultural University of Athens

5. Evaluation of the effect of different traditional marinades to control S. Typhimurium in beef fillets Results Survival of S. Typhimurium as affected by the type and the duration of marination MOST EFFECTIVE DURATION:12 h • Survival of S. Typhimurium was affected by type & duration of marination • immersion of fillets into the marinades for 12 or 24 h resulted to higher decrease of S. Typhimurium compared to 4h • immersion of inoculated fillets into wine resulted into higher decrease of S. Typhimurium compared to the other two marinades (oil, oil & wine) • Adaptation of cells to acid as well as the addition of EO did not affect the survival of S. Typhimurium Laboratory of Microbiology and Biotechnology of Foods Agricultural University of Athens

6. Evaluation of the effect of different traditional marinades to control S. Typhimurium in beef fillets Survival of S. Typhimurium as affected by the type of marinadesand the addition of EO during storage • Survival of S. Typhimurium during storage was significantly affected by the type of marinade as well as the addition of EO of thyme in the marinade • the combination of wine and 0.3% EO of thyme resulted into the higher destruction of S. Typhimurium (bellow detection limit) compared to the other marinades “wine plus 0.3% thyme oil” was the most effective marinade in reducingS. Typhimurium

7. Evaluation of the effect of wine-based marinades to control S. Typhimurium and to extend the shelf life of meat • Task 2.2.2:Safety of marinades • Evaluation of the effect of wine based marinades to control Salmonella Typhimurium and to extend the shelf life of meat Laboratory of Microbiology and Biotechnology of FoodsAgricultural University of Athens

8. Evaluation of the effect of wine-based marinades to control S. Typhimurium and to extend the shelf life of meat Experimental procedures Product: Beef fillets (non sterilized) Inoculum:A3-strain cocktail of acid and non-acid adapted S. Typhimurium cells (DSM 554, DT 193 and 4/74) (ca. 104 cells/g) Marinades:i)Wine ii) Wine supplemented with 0.3% thyme oil Storage comditions: i) air, ii) MAP (25% CO2 and 75% air), at5°C Sampling and sensory evaluation: 12 h, 2, 4, 7, 11, 14 and 19 d Laboratory of Microbiology and Biotechnology of FoodsAgricultural University of Athens

9. Evaluation of the effect of wine-based marinades to control S. Typhimurium and to extend the shelf life of meat Control 1) Air, non-acid adapted cells Wine Wine+0.3% EO Laboratory of Microbiology and Biotechnology of FoodsAgricultural University of Athens

10. Evaluation of the effect of wine-based marinades to control S. Typhimurium and to extend the shelf life of meat Control 2) Air, acid adapted cells Wine Wine+0.3% EO Laboratory of Microbiology and Biotechnology of FoodsAgricultural University of Athens

11. Evaluation of the effect of wine-based marinades to control S. Typhimurium and to extend the shelf life of meat Control 3) MAP, non-acid adapted cells Wine Wine+0.3% EO Laboratory of Microbiology and Biotechnology of FoodsAgricultural University of Athens

12. Evaluation of the effect of wine-based marinades to control S. Typhimurium and to extend the shelf life of meat Control 4) MAP, acid adapted cells Wine Wine+0.3% EO Laboratory of Microbiology and Biotechnology of FoodsAgricultural University of Athens

13. Evaluation of the effect of wine-based marinades to control S. Typhimurium and to extend the shelf life of meat Effect of marination on the behavior of SalmonellaTyphimurium inoculated on beef slices, stored under aerobic and MAP atmosphere Laboratory of Microbiology and Biotechnology of FoodsAgricultural University of Athens

14. Evaluation of the effect of wine-based marinades to control S. Typhimurium and to extend the shelf life of meat • Conclusions • Marination significantly reduced the levels of S. Typhimuriumand the background flora of beef compared to the control (non-marinated) • The survival of S. Typhimurium and microbial association was significantly affected by the addition of EO of thyme in marinade • Not important differences were detected between acid and non-acid adapted S. Typhimuriumduring the time-course Laboratory of Microbiology and Biotechnology of FoodsAgricultural University of Athens

15. Monitoring the survival of S. Typhimurium in marinated beef fillets • Task 2.2.2:Safety of marinades • Monitoring the survival of S. Typhimurium strains in marinated beef fillets Laboratory of Microbiology and Biotechnology of FoodsAgricultural University of Athens

16. Monitoring the survival of S. Typhimurium in marinated beef fillets Experimental procedure Strains:S. Typhimurium DSM 554, DT 193 and 4/74 (acid and non-acid adapted) Marinades:i)Wine, ii) Wine supplemented with 0.3% thyme oil Storage comditions: i) air, ii) MAP (25% CO2 and 75% air), at5°C Sampling: 0.5, 7, 14 d Strain typing: by PFGE Lanes 2, 5, 9-11: DSM 554; Lanes 7, 13: DT 193; Lanes 3, 4, 6, 8, 12, 14: 4/74 Laboratory of Microbiology and Biotechnology of FoodsAgricultural University of Athens

17. Monitoring the survival of S. Typhimurium in marinated beef fillets • Monitoring of S. Typhimurium DSM 554, DT 193 & 4/74 during storage of fillets revealed similar strain distribution in different storage conditions throughout time-course • Importantly, the epidemic multi-drug resistant DT 193 could compete other strains, like 4/74 originating from calf bowel Laboratory of Microbiology and Biotechnology of FoodsAgricultural University of Athens