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

Pillar 2: Control and intervention strategies which can be implemented along the fork to farm chain to ensure safe beef Laboratory of Microbiology and

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Pillar 2:

Control and intervention strategies which can be implemented along the

fork to farm chain to ensure safe beef

Laboratory of Microbiology and Biotechnology of Foods Agricultural University of Athens

Workpackage 2.2: Processing strategies

- Progress during the last 6 months -

Objectives-AUA 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

Laboratory of Microbiology and Biotechnology of Foods Agricultural University of Athens

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 different traditional marinades to control Evaluation of the effect of different traditional marinades to control S. S. Typhimurium in Typhimurium in beef filletsbeef fillets

Evaluation of the effect of different traditional marinades to control Evaluation of the effect of different traditional marinades to control S. S. Typhimurium in Typhimurium in beef filletsbeef 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

Laboratory of Microbiology and Biotechnology of Foods Agricultural University of Athens

Evaluation of the effect of different traditional marinades to control Evaluation of the effect of different traditional marinades to control S. S. Typhimurium in Typhimurium in beef filletsbeef fillets

Results

2

2.5

3

3.5

4

4.5

0 4 8 12 16 20 24

marination time (hrs)

Log

CFU

/g

olive oil

wine

olive oil & wine

olive oil & 0.3% EO thyme

wine & 0.3% EO thyme

olive oil & wine & 0.3% EO thyme

Survival of S. Typhimurium as affected by the type and the duration of marination

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 4 h

•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

MOST EFFECTIVE DURATION:

12 h

0

1

2

3

4

5

6

7

8

0 10 20 30 40 50 60 70 80 90 100

marination & storage (hrs)

Lo

g C

FU

/g

olive oil wine

olive oil & wine olive oil & 0.3% EO thyme

wine & 0.3% EO thyme olive oil & wine & 0.3% EO

•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

Survival of S. Typhimurium as affected by the type of marinades and the addition of EO during storage

Evaluation of the effect of different traditional marinades to control Evaluation of the effect of different traditional marinades to control S. S. Typhimurium in Typhimurium in beef filletsbeef fillets

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 Foods Agricultural University of Athens

Evaluation of the effect of wine-based marinades to control S. Typhimurium and to extend the shelf life of meat

Laboratory of Microbiology and Biotechnology of Foods Agricultural University of Athens

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: A 3-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), at 5°C

Sampling and sensory evaluation: 12 h, 2, 4, 7, 11, 14 and 19 d

Laboratory of Microbiology and Biotechnology of Foods Agricultural University of Athens

Evaluation of the effect of wine-based marinades to control S. Typhimurium and to extend the shelf life of meat

Control

Wine Wine+0.3% EO

1) Air, non-acidadapted cells

Laboratory of Microbiology and Biotechnology of Foods Agricultural University of Athens

Evaluation of the effect of wine-based marinades to control S. Typhimurium and to extend the shelf life of meat

Control

Wine

4.50

5.00

5.50

6.00

6.50

7.00

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

10.00

0 3 6 9 12 15 18 21TVC-NA-Air Pseudomonas -NA-Air LAB-NA-Air

Salmonella-NA-Air pH- NA-Air

Wine+0.3% EO

2) Air, acidadapted cells

Laboratory of Microbiology and Biotechnology of Foods Agricultural University of Athens

Evaluation of the effect of wine-based marinades to control S. Typhimurium and to extend the shelf life of meat

Control

Wine Wine+0.3% EO

3) MAP, non-acidadapted cells

Laboratory of Microbiology and Biotechnology of Foods Agricultural University of Athens

Evaluation of the effect of wine-based marinades to control S. Typhimurium and to extend the shelf life of meat

Control

Wine Wine+0.3% EO

4) MAP, acidadapted cells

Laboratory of Microbiology and Biotechnology of Foods Agricultural University of Athens

Evaluation of the effect of wine-based marinades to control S. Typhimurium and to extend the shelf life of meat

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

10.00

0 2 4 6 8 10 12 14 16 18 20

LOG

cfu/

g

days

AIR

Control-NA Control-AA Wine-NA

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

10.00

0 2 4 6 8 10 12 14 16 18 20

LOG

cfu/

g

days

MAP

Control-NA Control-AAWine-NA Wine-AAWine+0.3%Thyme-NA Wine+0.3%Thyme-AA

Effect of marination on the behavior of Salmonella Typhimurium inoculated on beef slices, stored under aerobic and MAP atmosphere

Conclusions

•Marination significantly reduced the levels of S. Typhimurium and 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. Typhimurium during the time-course

Evaluation of the effect of wine-based marinades to control S. Typhimurium and to extend the shelf life of meat

Laboratory of Microbiology and Biotechnology of Foods Agricultural University of Athens

Laboratory of Microbiology and Biotechnology of Foods Agricultural University of Athens

Task 2.2.2: Safety of marinades

Monitoring the survival of S. Typhimurium strains

in marinated beef fillets

Monitoring the survival of S. Typhimurium in marinated beef fillets

Laboratory of Microbiology and Biotechnology of Foods Agricultural University of Athens

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), at 5°C

Sampling: 0.5, 7, 14 d

Strain typing: by PFGE

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

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 Foods Agricultural University of Athens

0%

20%

40%

60%

80%

100%

AIR-NA

4|74

DT 193

DSM 554

12h 7d 14d

0

20

40

60

80

100

MAP-NA

4|74

DT 193

DSM 554

12h 7d 14d

0

20

40

60

80

100

AIR-AA

4|74

DT 193

DSM 554

12h 7d 14d

0

20

40

60

80

100

MAP-AA

4|74

DT 193

DSM 554

12h 7d 14d

•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

Monitoring the survival of S. Typhimurium in marinated beef fillets