1. Lee Young Jun Korea University Lab of Food Microbiology 2007. 5. 16
Influence of acid tolerance response on survival, growth, and thermal cross-protection of Escherichia coli O157:H7 in acidified media and fruit juices
Lee Young Jun
Korea University
Lab of Food Microbiology

2. Table of Contents Introduction Objective Experimental design
Phase Ⅰ
Survival of control, acid-adapted, acid-shocked cells of E. coli O157:H7 inoculated into TSB acidified with acetic or lactic acid
Phase Ⅱ
Growth pattern of E. coli O157:H7 in apple cider and orange juice incubated at 5 and 25℃
Phase Ⅲ
D-values of three type of E. coli O157:H7 cells in TSB, apple cider, and orange juice at 52 and 54℃
Conclusions

3. E. coli O157:H7 Characteristics Diseases Reservoirs Gram negative
Motile rods
Facultative aerobes
Diseases
Diarrhea, Hemorrhagic colitis,
Hemolytic uremic syndrom
Reservoirs
Cattle, Domestic animals,
Humans

4. Acid-adapted? Acid-shocked?
Acid-adapted cells are those which have been exposed to a gradual decrease in environmental pH
Acid-shocked?
Acid-shocked cells are those which have been exposed to an abrupt shift from high pH to low pH

5. D-value Time required to reduce population of microorganism to 1/10
at certain temperature

6. Objective
To determine if survival and growth patterns of acid-adapted and acid-shocked cells of E. coli O157:H7 differ in acidified TSB and in fruit juices
To determine if acid adaptation of acid shock alter heat tolerance of cells in juices

7. Experimental design Phase Ⅰ
Survival of control, acid-adapted, acid-shocked cells of E. coli O157:H7 inoculated into TSB acidified with acetic or lactic acid
Phase Ⅱ
Growth pattern of E. coli O157:H7 in apple cider and orange juice incubated at 5 and 25℃
Phase Ⅲ
D-values of three type of E. coli O157:H7 cells in TSB, apple cider, and orange juice at 52 and 54℃

8. Phase Ⅰ
Survival of control, acid-adapted, acid-shocked cells of E. coli O157:H7 inoculated into TSB acidified with acetic or lactic acid

9. Materials and Methods Strains Cell types Media pH & Incubation time(h)
- E. coli O157:H7
Cell types
- control, acid-adapted, acid-shocked cells
Media
- Tryptic soy broth(TSB)
- Tryptic soy agar(TSA)
- TSA containing 3% sodium chloride(TSAS)
pH & Incubation time(h)
- pH3.4 : 0, 1, 2(acetic)
0, 1, 3, 6, 8(lactic)
- pH3.9 : 0, 5, 9, 12, 18, 24

10. Control cells Acid-adapted cells Acid-shocked cells
- grown in TSB for 18h at 37℃
Acid-adapted cells
- grown in TSB supplement with 1% glucose for 18h at 37℃
Acid-shocked cells
- grown in TSB culture for 16h (pH6.0)
- adjusted to pH4.8 by adding 2M lactic acid
- incubating for 2h at 37℃

11. (adjusted to pH 3.9 or 3.4 with acetic or lactic acid)
E. coli O157:H7
TSB
(adjusted to pH 3.9 or 3.4 with acetic or lactic acid)
TSA and TSAS
Incubated at 37℃ for 24~48 h

12. Table 1. Number of control, acid-adapted, and acid-shocked cells of E
Table 1. Number of control, acid-adapted, and acid-shocked cells of E. coli O157:H7 inoculated into TSB acidified at pH 3.9 or 3.4 with acetic or lactic acids, incubated for up to 24h, and recovered on TSA or TSAS

13. Fig. 1. Differences(log10cfu/ml) in populations of control cells(■), acid-adapted cells(●), and acid-shocked cells(▲) of E. coli O157:H7 recovered from TSB acidified at pH 3.9 with lactic acid incubated at 37℃ for up to 12h when plated on TSA and TSAS

14. Phase Ⅱ
Growth pattern of E. coli O157:H7 in apple cider and orange juice incubated at 5 and 25℃

15. Materials and Methods Strains Cell types Juice types pH Temparature
- E. coli O157:H7
Cell types
- control, acid-adapted, acid-shocked cells
Juice types
- Apple cider (AC1, AC2, AC3)
- Orange juice (OJ1, OJ2, OJ3) pH
- AC1:3.56, AC2:3.98, AC3:3.73
- OJ1:3.82, OJ2:3.86, OJ3:3.86
Temparature
- 5 and 25℃

16. Apple cider and orange juice
E. coli O157:H7
Apple cider and orange juice
TSA
Incubated at 37℃ for 24~48 h

17. Table 2. Populations(log10cfu/ml) of E
Table 2. Populations(log10cfu/ml) of E. coli O157:H7 in apple cider(AC1, AC2, AC3) and orange juice(OJ1, OJ2, OJ3) incubated at 5 and 25℃

18. Phase Ⅲ
D-values of three type of E. coli O157:H7 cells in TSB, apple cider, and orange juice at 52 and 54℃

19. Materials and Methods Strains Cell types Juice types
- E. coli O157:H7
Cell types
- control, acid-adapted, acid-shocked cells
Juice types
- Apple cider (AC1, AC2)
- Orange juice (OJ1, OJ2)
Temparature (D-value)
- 52 and 54℃

20. Apple cider and orange juice
E. coli O157:H7
Apple cider and orange juice
(adjusted to 52 and 54℃)
Heating (10, 20, 30 min)
TSA
Incubated at 37℃ for 48 h
Counting Colony

21. Table 3. D-values of control, acid-adapted, and acid-shocked cells of E. coli O157:H7 cells in TSB(pH7.2), apple cider(AC1, AC2), and orange juice(OJ1, OJ2) at 52 and 54℃

22. Conclusions
Acid-adapted E. coli O157:H7 cells have enhanced tolerance to lactic acid but not acetic acid
Regardless of test parameter, there is not a strong indication that control, acid-adapted, and acid-shocked cells differ in tolerance to the acidic environment in apple cider or orange juice
D-values of acid-adapted cells were considerably high than those of acid-shocked or control cells