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 JunKorea University

Lab of Food Microbiology2007. 5. 16

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

E. coli O157:H7

• Characteristics Gram negative

Motile rods

Facultative aerobes

• Diseases Diarrhea, Hemorrhagic colitis, Hemolytic uremic syndrom

• Reservoirs Cattle, Domestic animals,

Humans

• Acid-adapted? 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

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

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

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℃

Phase Ⅰ Survival of control, acid-adapted, acid-shocked cells

of E. coli O157:H7 inoculated into TSB acidified with acetic or lactic acid

Materials and Methods

• Strains - 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

• Control 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℃

TSB

(adjusted to pH 3.9 or 3.4 with acetic or lactic acid)

TSA and TSAS

Incubated at 37℃ for 24~48 h

E. coli O157:H7

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

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℃

Phase Ⅱ

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

Materials and Methods

• Strains - 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℃

Apple cider and orange juice

TSA

Incubated at 37℃ for 24~48 h

E. coli O157:H7

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℃

Phase Ⅲ

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

Materials and Methods

• Strains - 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℃

Apple cider and orange juice

(adjusted to 52 and 54℃)

Heating (10, 20, 30 min)

Incubated at 37℃ for 48 h

E. coli O157:H7

TSA

Counting Colony

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℃

• 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

Conclusions