International Journal of Food Microbiology

Volume 70, Issues 1-2, 22 October 2001, Pages 189-195

doi:10.1016/S0168-1605(01)00538-4 | How to Cite or Link Using DOI

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Short communication

Acid adaptation and temperature effect  on the

survival of E. coli O157:H7 in acidicfruit juice and lactic
fermented milk product
, 
Cheng Hsin-Yi and Cheng-Chun Chou
Graduate Institute of Food Science and Technology, National Taiwan University 59, Lane 144, Keelung Rd.,
Section 4, Taipei 106, Taiwan
Received 29 September 2000;  
revised 27 March 2001;  
accepted 7 May 2001.  
Available online 16 October 2001. 

Abstract
In this study, two strains of Escherichia coli O157:H7, (ATCC 43889 and ATCC 43895) were acid adapted at pH
5.0 in tryptic soy broth (TSB) for 4 h. Commercial products of mango juice (pH 3.2), asparagus juice (pH

3.6),  Yakult —a diluted milk fermented drink (pH 3.6), and low-fat yoghurt (pH 3.9) were
inoculated with acid-adapted or nonadapted cells of E. coli O157:H7. Survival of the inoculated E. coli O157:H7
in these commercial food products during storage at 25 or 7 °C was examined. It was found that although
survival of the acid-adapted and nonadapted E. coli O157:H7 ATCC 43895 in asparagus juice during storage at 7
°C did not show marked difference, in general, acid adaptation and low temperature enhanced the survival of E.
coli O157:H7 in both the commercial fruit juices tested. On the contrary, acid adaptation reduced the survival of

both the strains of the test organism in  Yakult  and low-fat yoghurt stored at 7 °C.
Besides, E. coli O157:H7 ATCC 43895 survived longer than ATCC 43889 in all the products examined,
regardless of the storage temperature and acid adaptation.
Author Keywords: Acid adaptation; E. coli O157:H7; Fermented milk product
Article Outline
1. Introduction
2. Materials and methods
2.1. Microorganism and preparation of inoculum
2.2. Survival studies in fruit juice and lactic fermented milk products
2.3. Enumeration of E. coli O157:H7
2.4. Statistical analysis
3. Results and discussion
Acknowledgements
References

1. Introduction
Escherichia coli O157:H7 is a major food-borne pathogen, which causes hemorrhagic colitis and
hemolytic ureamic syndrome (Riley and Doyle). It has been a public health concern for food processors
 and consumers since its first recognition in 1982, with incidents of food-borne disease associated with
this pathogen occurring in the US, Canada and UK (Doyle and Anonymous). Furthermore, the largest
food-borne outbreak in history caused by E. coliO157:H7 that affected more than 900 individuals and
caused seven deaths was reported to occur in Japan (Anonymous, 1996).
Acidification is one of the important measures commonly employed to control growth and survival of
spoilage and pathogenic microorganisms in food(Brown and Booth, 1991). However, various acidic
foods such as apple cider (Besser et al., 1993), mayonnaise (Weagant et al., 1994) and
yoghurt (Morgan et al., 1993) have been implicated in the outbreaks of food-borne disease caused by E.
coli O157:H7.
Acid adaptation response is a phenomenon by which microorganisms show an increased resistance to
environmental stress after the exposure to a moderate acid environment. Acid adaptation and increased
resistance to acid stress have been observed in various organisms
including Listeria (Kroll; Hilland O), E. coli (Goodson and Cheng) and Salmonella (Foster and Hall,
1990). It was also reported that acid adaptation prolonged the survival of some pathogens in various
food systems, and may have important implications in food safety (Leyer and Tsai).

Mango juice, asparagus juice, low-fat yoghurt and  Yakult,  a diluted cultured milk
drink, all have a relatively low pH value and are among the most popular drinks in Taiwan, Japan and
other countries in Southeast Asia. In the present study, the survival pattern of two E. coli O157:H7
strains, as affected by acid adaptation in these products during storage at 7 or 25 °C, were examined.

2. Materials and methods

2.1. Microorganism and preparation of inoculum

Two strains of E. coli O157:H7 (ATCC 43895 and ATCC 43889) were used as the test organisms. They
were obtained from Center Collection and Research Center, Food Research and Development Institute,
Hsinchu, Taiwan.
After two successive transfers of the test organism in tryptic soy broth (TSB, Difco, Detroit, MI, USA) at
37 °C for 24 h, the activated culture was inoculated into TSB and incubated at above temperature for 18
h, when the population was ca. 109 cfu/ml. The culture was the serially diluted with Butterfield's buffer
phosphate diluent (BPD) and served as the inoculum of the control (nonadapted).
To prepare the acid-adapted cells of E. coli O157:H7, essentially, the procedure described by Tsai and
Ingham (1997) was adapted. Ten milliliters of the culture was centrifuged at 8800×g 12 min after
growing at 37 °C for 18 h in TSB. The supernatant was discarded. The cell pellets were washed with
BPD, and were suspended in 10 ml of pH 5.0 TSB (pH adjusted with 6 N HCl) and then incubated at 37
°C for 4 h. The acid-adapted cells were then diluted in the same way as the nonadapted cells and were
used as the inocula.

2.2. Survival studies in fruit juice and lactic fermented milk products

Commercial products of mango juice (pH 3.2), asparagus juice (pH 3.6),  Yakult  
(pH 3.6), a diluted lactic fermented drink of milk, and low-fat yoghurt (pH 3.9) were purchased from a
local supermarket. The fruit juices were subjected to proper treatments in process plants and can be
stored at room temperature. However, a preliminary study was conducted by plating the fruit juice on
tryptic soy agar (TSA, Difco) and incubated for 24–48 h at 37 °C to verify the absence of background
microorganisms. On the other hand, a viable population of ca. 107–108 cfu/ml was detected when the
fermented milk products were plated on TSA, while none was observed on MacConkey sorbitol agar
(MSA, Difco) (Okrend et al., 1990). This verified the absence of E. coli O157:H7 in the fermented milk
products.
In order to perform the survival study, 100 ml of each food product was inoculated with the prepared
acid-adapted or nonadapted inoculum of E. coliO157:H7 to achieve an initial population of ca. 6.0 log
 cfu/ml. Samples were then stored at 7 or 25 °C. During the storage period, viable populations of E.
coliO157:H7 were determined periodically as specified in Results and discussion.

2.3. Enumeration of E. coli O157:H7

For the enumeration of E. coli O157:H7, samples were serially diluted with BPD. The viable population
of E. coli O157:H7 in the fruit juice samples was then determined by plating 0.1 ml of the serially diluted
samples on TSA and incubated at 37 °C for 24–48 h. On the other hand, MSA instead of TSA was used
as the plating medium to enumerate E. coli O157:H7 in the fermented milk products. Although a
reduced recovery of acid-adapted and nonadapted cells was noted on MSA compared with that noted
on TSA, the extents of reduced recovery did not show significant difference (p>0.05).

2.4. Statistical analysis

The mean values and standard deviation were calculated from data obtained from three separate
experiments. These data were then compared by Duncan's multiple range method (SAS, 1989).

3. Results and discussion

E. coli O157:H7, with an optimum growth at pH around 7.0, can grow in the pH range between 4.5 and
9.0 (Glass et al., 1992). It has been reported that this pathogen can survive in some acidic foods such
as apple cider (pH 3.7) (Miller and Kaspar, 1994), mustard (pH 3.1), sweet pickle (pH 2.8) and ketchup
(pH 3.6) (Tsai and Ingham, 1997), and mayonnaise (pH 3.65) (Weagant et al., 1994) for various periods
of storage. As shown in Fig. 1, regardless of the test strain and acid adaptation, the viable population
of E. coli O157:H7 in mango juice declined quickly when stored at 25 °C. After 5 and 6 days of storage,
no viable cells were recovered from mango juice inoculated with nonadapted ATCC 43889 (Fig. 1A) and
ATCC 43895 (Fig. 1B), respectively. Acid-adapted cells were consistently recovered in greater numbers
than nonadapted cells for both the strains during storage at 25 °C (Fig. 1).

Full-size image (6K)
Fig. 1. Survival of acid-adapted (○) and nonadapted (•) E. coli O157:H7 in mango juice (pH 3.2)
during storage at 25 °C. (A) ATCC 43889; (B) ATCC 43895.

Survival of both E. coli O157:H7 strains, regardless of acid adaptation, were all greater at 7 °C (Fig. 2)
than at 25 °C (Fig. 1). For example, the mango juice inoculated with E. coli O157:H7 ATCC 43889 still
contained ca. 4.73 and 3.70 log cfu/ml of the acid-adapted and nonadapted cells, respectively, after 8
days of storage at 7 °C (Fig. 2A). Viable cells of the nonadapted and the acid-adapted E. coli O157:H7
were undetectable after 5 and 6 days of storage at 25 °C (Fig. 1A).

Full-size image (6K)
Fig. 2. Survival of acid-adapted (○) and nonadapted (•) E. coli O157:H7 in mango juice (pH 3.2)
during storage at 7 °C. (A) ATCC 43889; (B) ATCC 43895.
In general, the same pattern of survival was found with both E. coli O157:H7 strains tested in the
asparagus juice during storage (Fig. 3 and Fig. 4). Test organisms also showed enhanced survival in
asparagus juice stored at 7 °C (Fig. 4) compared to that at 25 °C (Fig. 3). Although acid adaptation did
not cause significant increase in the survival of E. coli O157:H7 ATCC 43895 at 7 °C (Fig. 4B), the
surviving population of the acid-adapted cells of ATCC 43889 was found to be significantly (p<0.05)
higher than its nonadapted counterpart in the asparagus juice at the end of storage at 7 °C (Fig. 4A). At
25 °C, the surviving populations of acid-adapted cells of both test organisms were also found to be
higher than the nonadapted cells in asparagus juice at the end of storage (Fig. 3).

Full-size image (6K)
Fig. 3. Survival of acid-adapted (○) and nonadapted (•) E. coli O157:H7 in asparagus juice (pH 3.6)
during storage at 25 °C. (A) ATCC 43889; (B) ATCC 43895.

Full-size image (6K)
Fig. 4. Survival of acid-adapted (○) and nonadapted (•) E. coli O157:H7 in asparagus juice (pH 3.6)
during storage at 7 °C. (A) ATCC 43889; (B) ATCC 43895.

Regardless of the storage temperature and acid adaptation, the survival of the test organisms was
higher in asparagus juice than in mango juice. For example, asparagus juice still contained ca. 3.68 and
1.79 log cfu/ml of the acid-adapted and nonadapted cells, respectively, of E. coli O157:H7 ATCC 43889
after 14 days of storage at 25 °C (Fig. 3A), while the number of the acid-adapted and nonadapted cells
of ATCC 43889 in mango juice reduced to ca. 1.52 log cfu/ml and undetectable level, respectively, after
5 days storage at similar storage temperature (Fig. 1A). The lower survival of the test organism in
mango juice than in asparagus juice may be attributed to the lower pH of mango juice than asparagus
juice.
The enhanced survival of E. coli O157:H7 with acid adaptation and refrigerated storage in both mango
and asparagus juice, as observed in the present study, is in accordance with the reports of various
investigators. Tsai and Ingham (1997) reported that adaptation to acid and low temperature enhanced
the survival of E. coli O157:H7 and Salmonella strains in ketchup. Leyer and Johnson
(1992) and Weagant et al. (1994), who examined the survival of acid-adapted and nonadapted cells
of E. coli O157:H7 in ketchup and mayonnaise (pH 3.05), respectively, also observed a similar
phenomenon. On the other hand, Raghubeer et al. (1994) showed that although viable cells of the
inoculated E. coli O157:H7 were detectable in pH 4.51 salad dressing stored for 17 days at 4 °C, none
was detectable in the pH 3.9 mayonnaise after 96 h storage at 22 °C.
Raw milk (McDonough et al., 1991) and yoghurt (Morgan et al., 1993) have been identified as sources
of the food-borne illness caused by E. coli O157:H7. Therefore, much concern has been extended by
food processors to keep these products free from contamination and proliferation of E. coli O157:H7.

Fig. 5A shows the survival of acid-adapted and nonadapted E. coli O157:H7 ATCC 43889 in 

Yakult,  the diluted fermented milk drink, during storage at 7 °C. Viable populations of the
acid-adapted and nonadapted in the diluted fermented milk drink declined as the storage period
extended. Although during the first 48 h of storage period, viable populations of the acid-adapted and
the nonadapted cells of E. coli O157:H7 ATCC 43889 did not show significant difference, a higher
survival population of nonadapted than the acid-adapted was noted in the milk drink thereafter.

Full-size image (6K)
Fig. 5. Survival of acid-adapted (○) and nonadapted (•) E. coli O157:H7 in diluted fermented milk

drink ( Yakult,  pH 3.6) during storage at 7 °C. (A) ATCC 43889; (B) ATCC
43895.

Viable population of acid-adapted or nonadapted ATCC 43895 also declined rapidly (Fig. 5B) as did
ATCC 43889 (Fig. 5A) in the diluted cultured milk drink during storage at 7 °C. However, strain 43895
was found to be less sensitive than strain 43889 in this diluted fermented milk drink during storage
period. At the end of 144-h storage period, a significant (p<0.05), higher viable population of E.
coli O157:H7 of ca. 3.5 and 4.3 log cfu/ml was noted in the milk drink, originally inoculated with the acid-
adapted and nonadapted ATCC 43895, respectively, compared to those inoculated with ATCC 43889.
The viable population of the inoculated acid-adapted or nonadapted E. coli O157:H7 declined in yoghurt
during storage at 7 °C (Fig. 6). It was also found that acid adaptation reduced the survival of each E.
coli O157:H7 strain tested in yoghurt. Viable populations of both E. coli O157:H7 strains, regardless of
acid adaptation, reduced more quickly in yoghurt (Fig. 6) than in the diluted fermented milk drink (Fig.
5).

Full-size image (6K)
Fig. 6. Survival of acid-adapted (○) and nonadapted (•) E. coli O157:H7 in yoghurt (pH 3.9) during
storage at 7 °C. (A) ATCC 43889; (B) ATCC 43895.

The reduced survival of the acid-adapted E. coli O157:H7 observed in the fermented milk products is
different from those observed in the acidic fruit juice drinks (Fig. 1, Fig. 2, Fig. 3 and Fig. 4), and is also
contradictory to the earlier reports of Leyer and Johnson (1992) and Gahan et al. (1996). The former
observed that the cells of Salmonella grown in a medium acidified to pH 5.8 survived longer in cheese
kept at 5 °C. The latter indicated that acid adaptation enhanced the survival of L. monocytogenes in
yoghurt (pH 3.9) stored at 4 °C. Differences in the test organisms, acid adaptation conditions and
properties of the food system tested may all contribute to the discrepancy of these results. In addition to
acid, some other antimicrobials such as bacteriocins, hydrogen peroxide, ethanol and diacetyl were

reported to be produced by lactic acid  bacteria  and present in the fermented

products (Frank and Marth, 1988). It was also reported that although acid-adapted S. typhimurium was
less sensitive to acid than its nonadapted counterpart, it was more susceptible to hypochlorous
 acid (Leyer and Johnson, 1997). It is also possible that acid adaptation may lead to the increased
susceptibility of the test organism to the above-mentioned antimicrobials produced by lactic

acid  bacteria  in fermented milk products (Gonz and Varadaraj), although it remains

to be further studied. The  effect  on increasing susceptibility to these antimicrobials,

due to acid adaptation, may outweigh the  effect  of enhancing acid tolerance, and
thus, results in reduced survival of acid-adapted E. coli O157:H7 in the lactic fermented milk products as
observed in the present study.
In conclusion, results of the present study further demonstrated that acid adaptation increased the acid
tolerance of E. coli O157:H7 in acid fruit juices. Acid adaptation and low temperature enhanced the
survival E. coli O157:H7 in the acidic fruit juice during storage. On the other hand, survival of acid-
adapted E. coli O157:H7 was less than its nonadapted counterpart in the lactic fermented products
tested. Furthermore, the viable population of E. coli O157:H7 ATCC 43895 declined more slowly than
ATCC 43889 in the fruit juices and lactic fermented products tested during the storage period. Based on
the data obtained from the present study, much attention should be given to prevent the contamination
of this pathogen in these acidic drinks.

Acknowledgements
This research was supported by the Department of Health, Executive Yuan, ROC (DOH-88-TD-1043).
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 Corresponding author. Tel.: +886-2-2363-0231x2717; fax: +886-2-2362-0849;

email: fstcchou@ccms.ntu.edu.tw

International Journal of Food Microbiology

Volume 70, Issues 1-2, 22 October 2001, Pages 189-195