SURVIVAL OF ESCHARICHIA COLI O157:H7 IN SET AND STIRRED YOGURT AS

INFLUENCED BY AN EXOPOLYSACCHARIDE, COLANIC ACID, PRODUCTION

by

SHAIO MEI LEE

(Under the direction of Dr. Jinru Chen)

ABSTRACT

This study was conducted to determine the role of the exopolysaccharide, colanic

acid (CA), produced by Escherichia coli O157:H7 in the survival of the pathogen in set

and stirred yogurt. Pasteurized milk was inoculated with a wild type E. coli O157:H7, its

CA-deficient mutant, and a 1:1 ratio mixture the two strains, respectively, before

fermentation for both set and stirred yogurt. Stirred yogurt was also inoculated after

fermentation. Samples were stored at 4° or 15°C with sampling done twice a week for 3

weeks. All samples were enumerated for total plate counts, population of E. coli

O157:H7, and starter cultures. The results showed that CA plays a protective role in

protecting E. coli O157:H7 from acid stress in set yogurt. However, the same conclusion

could not be drawn for stirred yogurt. It was also found that survival of the pathogen is

influenced by storage temperature and pH of the yogurt.

INDEX WORDS: Colanic acid, Escherichia coli O157:H7, Acid stress, Yogurt

SURVIVAL OF ESCHERICHIA COLI O157:H7 IN SET AND STIRRED YOGURT AS

INFLUENCED BY AN EXOPOLYSACCHARIDE, COLANIC ACID, PRODUCTION

by

SHIAO MEI LEE

B.S., Washington State University, 2000

A Thesis Submitted to the Graduate Faculty of The University of Georgia in Partial

Fulfillment of the Requirements for the Degree

MASTER OF SCIENCE

ATHENS, GEORGIA

2002

2002

Shiao Mei Lee

All Rights Reserved.

SURVIVAL OF ESCHERICHIA COLI O157:H7 IN SET AND STIRRED YOGURT AS

INFLUENCED BY AN EXOPOLYSACCHARIDE, COLANIC ACID, PRODUCTION

by

SHIAO MEI LEE

Approved:

Major Professor: Jinru Chen

Committee: Larry Beuchat Mark Harrison

Electronic Version Approved:

Gardhan L. Patel Dean of the Graduate School The University of Georgia August 2002

DEDICATION

I would like to dedicate this thesis to my parents, Mr. Kok Kheng Lee and Mrs. Lee Lee

Leow, for their unconditional love and support.

Iv

ACKNOWLEDGEMENTS

I would like to express my utmost gratitude to my advisory committee. Thank you

very much to: Dr. Jinru Chen, for her technical, moral, and financial support throughout

the whole study. Dr. L.R. Beuchat for sharing with me his ideas and opinions. Dr. M.A.

Harrison for his guidance.

I would also like to sincerely thank all my co-workers in the lab. Joy Adams, lab

coordinator who was always there for me when I needed help. Dr. Nutan Mytle, Julie

Yeh, and Zack Rabuck for supporting me and cheering me on.

Last but not least, I would like to thank my parents and friends for the moral

support they have given me and also for believing in me.

v

TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS.................................................................................................v

CHAPTER

1 INTRODUCTION .............................................................................................1

2 LITERATURE REVIEW ..................................................................................4

3 SURVIVAL OF ESCHERICHIA COLI O157:H7 IN SET YOGURT AS

INFLUENCED BY AN EXOPOLYSACCHARIDE, COLANIC ACID,

PRODUCTION................................................................................................41

4 INFLUENCE OF AN EXOPOLYSACCHARIDE, COLANIC ACID, ON

FATE OF ESCHERICHIA COLI O157:H7 IN STIRRED YOGURT ............58

5 SUMMARY AND CONCLUSION ................................................................78

vi

CHAPTER 1

INTRODUCTION

1

2

Enterohemorrhagic Escherichia coli O157:H7 is one of the most important

foodborne bacterial pathogens. Symptoms of E. coli O157:H7 infection range from

asymptomatic to severe illnesses such as hemolytic uremic syndrome (HUS),

hemorrhagic colitis (HC), and thrombotic thrombocytopenic purpura (TTP) (Karmali,

1989). The infection dosage of E. coli O157:H7 is low, as few as 1- 10 CFU cells can

cause illness in human. Various foods have served as vehicles for E. coli O157:H7

infection. A yogurt –associated outbreak of E. coli O157:H7 infection occurred in 1991

in England in yogurt manufactured for children. Sixteen children were infected with 11

out of the 16 children under 10 years old. Five of the children under 10 developed HUS.

All children eventually recovered.

Although growth of E. coli O157:H7 occurs at pH 4.5 (Glass et al., 1992), the

pathogen is able to survive at pH much below this level (Miller and Kaspar, 1994,

Hudson et al., 1997, Dineen 1998). The ability of E. coli O157:H7 to tolerate acid stress

(Miller and Kaspar, 1994, Hudson et al., 1997, Dineen 1998) has been attributed to many

factors including having protective structures on cell surfaces. Exopolysaccharide (EPS)

colanic acid (CA) is secreted by cells of E. coli O157:H7 under stress. CA protected cells

against desiccation (Ophir and Gutnick, 1994) and heat (Mao et al., 2001). Recent

studies conducted in our laboratory revealed that CA protected cells of E. coli O157:H7

against acid stress in microbiological medium and synthetic gastric fluid (Mao, et al.,

2001, Unpublished). However, the protective effect of CA in food has not been

demonstrated. The objective of this study was to determine the role of CA in survival of

E. coli O157:H7 in an acidic food: yogurt.

3

A CA- producing E. coli O157:H7 (W), its CA-deficient counterpart (M), and a

mixture of the two strains (1:1, WM) were used in this study. The research was divided

into two phases. In the first phase of the study, E. coli O157:H7 was inoculated into

pasteurized milk prior to fermentation. Contaminated milk was used to process set

yogurt at 43°C. Stirred yogurt was made in phase two. The W, M or WM was

inoculated either before or after yogurt fermentation. Both set and stirred yogurt was

sampled twice a week during a 3-w storage at 4° and 15°C for total plate counts, as well

as populations of E. coli O157:H7 and starter cultures. Data analysis was conducted

using general linear model. The significant difference between the survival of W vs. M,

W vs. WM, and M vs. WM was determined using a 95% confident level.

CHAPTER 2

LITERATURE REVIEW

4

5

ESCHERICHIA COLI O157:H7

Introduction E. coli is a gram negative, rod-shaped, facultative anaerobic bacterium commonly

found as natural colonic flora in warm-blooded animals, including human. E. coli is

serotyped based on the presence of O (somatic), H (flagellar), and K (capsular) surface

antigens. Although most E. coli is non-pathogenic, some causes illnesses (Doyle et al.,

1997, WHO, 1996). Depending on their pathogenic strategies, E. coli can be sub-divided

into four different groups: enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli

(EHEC), enteroaggregative E. coli (EAEC), and enterotoxigenic E. coli (ETEC). E. coli

O157:H7 was first identified in 1982 in an outbreak that occurred at a fast food restaurant

chain (Riley et al., 1983). It expresses the 157th O antigen and the 7th H antigen (Mead

and Griffin, 1998) and is a member of the EHEC group.

The illness caused by E. coli O157:H7 ranges from asymptomatic to severe

illnesses such as hemorrhagic colitis (HC), hemolytic uremic syndrome (HUS), and

thrombotic thrombocytopenic purpura (TTP) (Karmali, 1989). HUS is the most severe

symptom that often leads to renal failure (Moake, 1994). According to Mead and Griffin,

3-5% of the patients who developed HUS die while 50% need dialysis and 75% require

blood transfusion (1998). HUS may also cause some rare complications such as

pancreatitis, diabetes mellitus, stroke, and seizure (Mead and Griffin, 1998). The average

interval between the consumption of E. coli O157:H7 and symptoms of manifestation is 3

d with the usual incubation period ranging from 1 to 8 d (Mead and Griffin, 1998). The

most susceptible groups of people to E. coli O157:H7 infection are young children,

elderly and immuno-comprised individuals.

6

E. coli O157:H7 usually does not ferment D-sorbitol within 24 h (Bouvet et al.,

1999). However, strains that are able to ferment D-sorbitol are isolated occasionally

(Bouvet et al., 1999). Bouvet et al. (1999) examined the influence of growth temperature

on the ability of E. coli O157:H7 to ferment D-sorbitol, and found that 13 of the sorbitol-

negative-strains were able to ferment D-sorbitol after 3 to 4 days of incubation at 30°C

(Bouvet et al., 1999). Incubation at 37° or 40°C did not affect the ability of E. coli

O157:H7 to utilize D-sorbitol (Bouvet et al., 1999). E. coli O157:H7 also lacks β-

glucuronidase which hydrolyzes 4-methyl-umbeliferyl-D-glucuronide and was unable to

grow at temperature higher than 44.5°C (Doyle et al., 1997). E. coli O157:H7 does not

require a high dosage to cause severe illnesses. The infectious dose required to cause

illness was reported to be as low as 1 to 10 CFU (Nataro and Kaper, 1998).

Route of E. coli O157:H7 Contamination

The major source of E. coli O157:H7 is cattle (Griffin and Tauxe, 1991). Food of

cattle origin such as milk, beef, and dairy products have been implicated in outbreaks of

E. coli O157:H7 infection. Other transmission vehicles include water, vegetables, and

fruit juice.

Milk

Raw milk can be contaminated with E. coli O157:H7 through contact with soil,

animal hide and fecal materials. If pasteurization is not done correctly and adequately, E.

coli O157:H7 may survive, eventually grow at abusive, or even chill temperatures. Milk

can also be re-contaminated post-pasteurization through improper handling and

unsanitary environmental hygiene (Upton and Coia, 1994). Raw milk has served as a

carrier for E. coli O157:H7 infection (Reitsma and Henning, 1996). A raw milk-related

7

outbreak of E. coli O157:H7 infection occurred in 1986 in Ontario, Canada.

Kindergarten students visited an Ontario farm and were treated to unpasteurized milk.

Forty two children and 4 adults were infected with E. coli O157:H7 (Thomas and Powell,

2002). Three children developed HUS and 1 fell into a coma (Thomas and Powell,

2002). All the patients recovered eventually. In August of 2001, 5 children were

infected with E. coli O157:H7 after drinking unpasteurized goat milk on a farm in British

Columbia, Canada (Health Canada, 2002). Two of the children developed HUS (Health

Canada, 2002). Upton and Coia (1994) reported an outbreak of E. coli O157:H7

associated with pasteurized milk. In this outbreak, 100 people were infected with 46

children under 15 years old, 32 under 5. Nine children ranging in age from 9 months to

11 years old developed HUS, 6 required kidney dialysis and 1 elderly lady developed

TTP (Upton and Coia, 1994). E. coli O157:H7 was found on milk-handling pipe and the

equipment used to bottle milk (Upton and Coia, 1994). The milk was probably

inadequately pasteurized or contaminated after pasteurization (Upton and Coia, 1994).

Fermented dairy products

Fermented dairy products have also been implicated in outbreaks of E. coli

O157:H7 infection. The first yogurt-related outbreak of E. coli O157 infection was

reported in England in 1991 (Morgan et al., 1993). Sixteen people were infected with 11

of the patients under 10 years old and 5 of the infected children developed HUS (Morgan

et al., 1993). It was suspected that re-contamination of pasteurized milk might have

occurred because a pump used to pump raw milk was also used to pump pasteurized milk

(Morgan et al., 1993). Milk could also be contaminated through the churns that were

often left on the muddy floor of the production area (Morgan et al., 1993). An outbreak

8

of E. coli O157:H7 infection associated with eating fresh cheese curds occurred in

Wisconsin in June 1998 (CDC, 2000). Fifty-five laboratory confirmed cases were

identified (CDC, 2000). E. coli O157:H7 was isolated from some of the environmental

samples, unopened cheese samples, opened and unopened retail packages of curds

collected from the plant soon after the outbreak (CDC, 2000). Unpasteurized raw milk

was mistakenly used as pasteurized milk to process the cheese curds (CDC, 2000).

Meat and meat products

Fermented meat products also served as a vehicle of E. coli O157:H7 infection.

An outbreak associated with dry-cured salami took place in Washington and California in

1994 (CDC, 1995). A total of 20 cases were reported with 3 hospitalized and a 2 year old

developed HUS (CDC, 1995). All patients had purchased and consumed a certain brand

of dry-cured salami for the delicatessen counters of a local grocery chain (CDC, 1995).

Investigators visited 3 of the grocery stores belonging to the grocery chain and collected

samples (CDC, 1995). No errors in food handling were observed but the samples were

tested positive for E. coli O157:H7 (CDC, 1995).

Beef, especially ground beef has been involved in several outbreaks of E. coli

O157:H7 infection (CDC, 1997, Bell et al., 1994). Beef carcasses can easily be

contaminated during slaughtering and processing. Although carcasses are washed, the

washing is often not sufficient to eliminate the pathogen. Berry and Cutter (2000)

studied the effect of 2% acetic acid spray on E. coli O157:H7. It was found that larger

number of cells remained on beef carcasses following the acid washing (Berry and

Cutter, 2000). Ground beef is more vulnerable for E. coli O157:H7 contamination. The

pathogen on the surface can get into the interior of the meat where elimination of the

9

pathogen becomes more difficult during cooking (Mead and Griffin, 1998). The largest

multistate outbreak of E. coli O157:H7 associated with ground beef occurred in late 1992

to early 1993 (CDC, 1993). The states involved in this outbreak were Washington State,

Idaho, California, and Nevada (CDC, 1993). There were a total of 731 cases with 178

hospitalized, 56 patients developed HUS, and 4 children died (Doyle et al., 1997).

Fresh produce

Consumption of fruits and vegetables are on the rise in the past decade.

According to the Economic Research Service, USDA (2000), per capita consumption of

fruits and vegetables has increased from 659.6 lbs. in 1991 to 707.7 lbs. in 2000. This

increase in consumption may be due to healthier lifestyles that consumers adopt and

convenience that fresh cut produce provides.

Fresh produce can be contaminated with E. coli O157:H7 from animal’s manure

and irrigation water. Since fruits and vegetables are usually consumed raw, they have the

potential to be good vehicle for E. coli O157:H7 infections. An outbreak related to eating

alfalfa sprouts happened in Michigan and Virginia from June to July of 1997 (CDC,

1997). A total of 60 persons in Michigan were infected with 2 persons developed HUS

and one had TTP (CDC, 1997). While in Virginia, 48 cases of infection were reported

(CDC, 1997). The source of E. coli O157:H7 contamination in this outbreak was

contaminated alfalfa seeds (CDC, 1997).

Beverage and water

Apple cider is actually fermented apple juice. Typically, apples harvested from

orchards are washed and inspected before processing. Cleaned apples are then crushed

and pressed to extract the apple juice (Anonymous, 2000). Depending on country, some

10

apple juice is then allowed to ferment either by wild or cultured yeast (Anonymous,

2000). After 3 months of fermentation, the apple cider is filtered and then carbonated

(Anonymous, 2000).

Some of the unpastuerized apple ciders in the market were made from unwashed

or dropped apples that might be contaminated with pathogens (Besser et al., 1993).

According to a survey in New England, the producers of apple cider being surveyed all

used dropped apples to make apple cider (Besser et al., 1993). Out of the 36 producers,

only 12 washed and brushed their apples prior to processing (Besser et al., 1993). It was

estimated that 2.5 million gallons of cider were made from apples that were not washed

and brushed (Besser et al., 1993). However, in the outbreak in Connecticut and New

York, picked apples were washed, brushed before pressing. E. coli O157:H7

contamination might come from a well, because water from one of the wells was tested

positive for E. coli O157:H7 (CDC, 1997). Twelve cases were confirmed in the

Connecticut and New York outbreak. Two patients developed HUS, 5 were hospitalized

and 1 developed TTP. Apple cider-related outbreaks of E. coli O157:H7 infection also

occurred in Massachusetts in 1991 (Besser et al., 1993). Among 23 identified patients in

Massachusetts, 4 infected children developed HUS. An outbreak of E. coli O157:H7

infection associated with drinking of unpasteurized commercial apple juice or juice

mixtures containing apple juice occurred in British Columbia, California, Colorado, and

Washington in October of 1996 (CDC, 1996). A total of 45 cases were reported with 12

persons diagnosed with HUS (CDC, 1996). The extent of the outbreak and how the juice

became contaminated were not clear (CDC, 1996)

11

In 1998, an outbreak of E. coli O157:H7 infection in Alpine, Wyoming was

associated with municipal drinking water from a rural water system (Olsen et al, 1998).

The water was not chlorinated and tested positive for fecal organisms (Olsen et al., 1998).

In this outbreak, 157 people became ill with 45% of the patients having E. coli O157:H7

in their stool and 4 patients developed HUS. A lake water-associated outbreak of E. coli

O157:H7 took place in Illinois in 1995. There were a total of 12 cases, mostly children.

Bloody diarrhea occurred in 9 cases and 3 children developed HUS. All patients had

visited the state park with lake swimming beach (CDC, 1996). E. coli was found in the

lake water, but E. coli O157:H7 was not isolated (CDC, 1996).

Survival of E. coli O157:H7 in Food

Dairy products

Survival of E. coli O157:H7 in dairy food is documented by several researchers

(Hudson et al., 1997, Massa, et al., 1997, Wang et al., 1997, Dineen et al., 1998,

McIngvale et al., 2000). Massa et al. (1999) determined the survival rate of E. coli

O157:H7 in unpasteurized milk stored at 8°C. Seven strains of E. coli O157:H7 were

inoculated into raw milk obtained from a dairy farm (Massa et al., 1999). Three of the 7

strains survived while the other 4 actually grew during a 17-d storage (Massa et al.,

1999). This suggests that E. coli O157:H7 is able to survive, and multiply in raw milk at

chill temperatures. In a separate study, growth of E. coli O157:H7 in unpasteurized or

pasteurized milk was observed at 8°, 15°, and 22°C, but not at 5°C (Wang et al., 1997).

Greater growth rate was obtained at relatively higher storage temperatures. E. coli

O157:H7 grew slower in unpasteurized than in pasteurized milk (Wang et al., 1997),

12

which was probably due to the presence of competitive microflora in the unpastuerized

milk.

Fate of E. coli O157:H7 in fermented dairy food is often studied by inoculation of

milk ingredient before laboratory-scale fermentation or inoculation of fermented products

before storage. Commercial products are frequently used in challenge and survival

studies. Hudson et al. (1997) inoculated a strain of E. coli O157:H7 into 3 commercial

yogurt with pH 4.17, 4.39 and 4.47, respectively. E. coli O157:H7 survived for 8 d at

4°C and 5 d at 10°C in yogurt with pH 4.17, 17 and 15 d in yogurt with pH 4.39 at 4° and

10°C, respectively (Hudson et al, 1997). In yogurt with pH 4.47, E. coli O157:H7

survived up to 17 d at both 4° and 10°C (Hudson et al., 1997). Massa et al. (1997)

contaminated milk with 103 and 107 CFU/mL of E. coli O157:H7 to process yogurt with

Streptococcus thermophilus and Lactobacillus bulgaricus. The populations of E. coli

O157:H7 decreased 0.8 and 1.76 log10CFU/mL in yogurt (pH 4.44) stored at 4°C. E. coli

O157:H7 did not survive the pasteurization step during processing of cottage cheese

(Hudson et al., 1997). However, the pathogen persisted for 27, 30, or 27 d in Feta,

Romano, and Colby cheese, respectively. In a study done by Ramsaran et al. (1998), a

bioluminescent strain of E. coli O157:H7 survived, and grew during fermentation of Feta

and Camembert cheese (Ramsaran et al., 1998). At the end of the 75 d storage period at

2°C, approximately 5.5 to 6.5 log10CFU/mL of E. coli O157:H7 were recovered from

inoculated Feta cheese and 5.25 to 5.5 log 10CFU/mL of E. coli O157:H7 from

Camembert cheese. (Ramsaran et al., 1998). The amounts of starter cultures used and the

acid produced by the starter cultures were suggested to be factors inhibiting the growth of

E. coli O157:H7 (Ramsaran et al., 1998). The ability of E. coli O157:H7 to survive

13

during manufacture and curing of cheddar cheese was evaluated by Reitsma and Henning

(1996). Cheese milk was inoculated with 103 and 100 CFU/mL of E. coli O157:H7. It

was found that at both inoculation levels, the pathogen was able to survive during the

cheddar cheese manufacturing process with the pathogen mostly found in the curd instead

of the whey. At the high inoculation level, the pathogen was detectable after 158 d. At

the low inoculation, however, the pathogen reduced to less than 1 CFU/mL in 60 d

(Reitsma and Henning, 1996).

Dineen et al. (1998) inoculated E. coli O157:H7 (103 CFU/mL) into commercial

buttermilk and found that more than 102 CFU/mL survived for 35 d at pH 4.1. E. coli

O157:H7 was recovered after 22 and 32 d, respectively in buttermilk that was inoculated

at pre- or post-fermentation stage (McIngvale et al., 2000).

Listeria monocytogenes, Salmonella Typhimurium DT104, or E. coli O157:H7

was inoculated into milk used to make labneh before and after fermentation (Issa and

Ryser, 2000). Populations of L. monocytogenes remained unchanged while E. coli

O157:H7 grew (0.46 to 1.19 log10CFU/mL) during fermentation (Issa and Ryser, 2000).

Salmonella grew during the first 2 h of fermentation and the cell numbers started to

decline thereafter (Issa and Ryser, 2000). L. monocytogenes survived 15 d in labneh that

was inoculated after fermentation and stored at 6°C. E. coli O157:H7 was not detectable

in the product inoculated post-fermentation and stored for 4 d at 6°C (Issa and Ryser,

2000).

Water and Beverage

Kerr et al. (1999) inoculated a strain of E. coli O157:H7 into non-carbonated

mineral water, sterile natural mineral water, and sterile distilled deionized water at two

14

inoculation levels, 103 and 106 CFU/mL. E. coli O157:H7 survived the longest (63 d) in

non-carbonated mineral water. The pathogen became undetectable after 70, 49, and 21 d

in non-carbonated mineral water, sterile mineral, and sterile distilled deionized water

respectively during storage at 15°C (Kerr et al., 1999). At higher inoculation level, the

pathogen persisted more than 70 days in all types of water (Kerr et al., 1999).

Ingham and Uljas (1998) studied the effect of prior storage of apple cider or apple

juice on tolerance of E. coli O157:H7 to heat at 61°C. Prior storage of inoculated apple

juice at 21°C for 6 h and at 4°C for 24h decreased the thermotolerance of E. coli

O157:H7 at 61°C (Ingham and Uljas, 1998); The survival of a pressure-resistant strain of

E. coli O157:H7 in orange juice was determined by Linton et al. (1999). The orange

juices were adjusted to a pH of 3.4 to 5.0 and inoculated with the E. coli O157:H7

(Linton et al., 1999). The inoculated orange juice was then pressure-treated and stored at

3°C. The survival of E. coli O157:H7 was dependent on the pH of the orange juice.

High-pressure treatment increased the susceptibility of the pathogen to high acid (Linton

et al., 1999).

Mayonnaise, Salad Dressing and Miscellaneous Food

Several factors affect the survival of E. coli O157:H7 in mayonnaise and salad

dressing (Raghubeer et al., 1995), which include pH of the product, presence of

lysozyme, storage temperature, and levels of inoculation (Raghubeers et al., 1995). The

survival rate of E. coli O157:H7 and coliforms was determined at 4° and 22oC in

commercial mayonnaise and refrigerated ranch salad inoculated with two levels (> 106

and 104 CFU/mL) of E. coli O157:H7 and coliform (Raghubeer et al., 1995). E. coli

O157:H7 was recovered from the salad dressing inoculated with 104 CFU/mL of E. coli

15

O157:H7 after 17 d at 4°C. The higher inoculum of E. coli O157:H7 died off after 96 h

storage in mayonnaise at 22°C (Raghubeer et al., 1995). Coliforms were able to survive

longer in salad dressing stored at 4° than at 22°C (Raghubeer et al., 1995).

Mayerhauser (2001) conducted a study on survival of E. coli O157:H7 in retail

mustard. Three strains of E. coli O157:H7 were inoculated individually in to 3 types of

mustard: dijon, yellow, and deli style retail mustard containing acetic acid (Mayerhauser,

2001). The samples were then stored at 25° and 5°C (Mayerhauser, 2001). E. coli

O157:H7 became undetectable after either a few h or d depending on the strain and the

type of mustard used in the study (Mayerhauser, 2001). E. coli O157:H7 was able to

survive longer at 5° than at 25°C.

The effect of acid adaptation and storage temperature on survival of E. coli

O157:H7 and Salmonella spp. in ketchup, mustard, and sweet pickle relish was

determined by Tsai and Ingham (1997). Acid adaptation enhanced the survival of E. coli

O157:H7 and Salmonella in acidic condiments. The extent of the effect depended on

strain and storage temperature. All tested microorganisms survived better at 5°C instead

of at 23°C. In general, E. coli O157:H7 was more acid tolerant than Salmonella spp..

Acid adaptation and low storage temperature increased the survival of E. coli O157:H7

and Salmonella spp. in ketchup. However, this improved survival was not observed in

sweet pickle relish or mustard.

Del Rosario and Beuchat (1995) determined the survival and growth of E. coli

O157:H7 in watermelon and cantaloupe cubes as well as on the rind of both melons. It

was found that the populations of E. coli O157:H7 increased significantly in both melon

cubes between 4 and 6 h with watermelon cubes giving better support to growth than

16

cantaloupe cubes stored at 25°C. The population of E. coli O157:H7 remained constant

at 5°C. Significant increase in population was also observed on the rinds of melons held

at 25°C. However, the pathogen grew better on cantaloupe than watermelon rind. After

4 d of storage at 5°C, populations of E. coli O157:H7 decreased significantly on the

rinds.

Dry Food

E. coli O157:H7 has the ability to survive well, especially at refrigeration

temperature, in dry foods with wide range of water activity and pH. This conclusion was

drawn by Deng et al. (1988) after studying the influence of temperature and pH on

survival of E. coli O157:H7 in reconstituted infant rice cereal. The death of E. coli

O157:H7 in cereal was enhanced by increased temperature and decreased pH during

storage. The rice cereal was reconstituted with milk and apple juice, respectively. It was

found that E. coli O157:H7 was able to survive in rice cereal reconstituted with milk at

all temperature (5, 25, 35, 45°C) while only at 15, 21, and 30°C in rice cereal

reconstituted with apple juice. The fate of E. coli O157:H7 in potato starch was

determined by Park and Beuchat (2000). The survival of the pathogen was enhanced by

optimal water activity and low storage temperature. Product pH did not have much effect

on the survival of E. coli O157:H7.

Meats

The effects of cure mix, pre-cooking, and drying on E. coli O157:H7 during

processing of beef jerky meat were determined by Harrison et al. (1998). Cure mix that

contains salt and sodium nitrite deterred the survival of E. coli O157:H7 (Harrison, et al.,

1998). Pre-cooking of ground beef before drying was also effective in inactivating the

17

pathogen. However, drying itself was not as effective in destroying the pathogen. Faith

et al. (1998) inoculated ground beef (5 or 20% fat) with approximately 108 CFU/mL of E.

coli O157:H7. The contaminated beef samples were then dried at various temperatures

using a home-style dehydrator. It was found that the percent fat, drying time and

temperature had impacts on the survival of E. coli O157:H7 in beef jerky (Faith et al.,

1998). It took 4 h of drying at 68°C and 8 h of drying at 63°C to reach a 5 log reduction

on populations of E. coli O157:H7 (Faith et al., 1998). The ground beef with lower

percentage of fat achieved the reduction faster than the beef with higher fat content (Faith

et al., 1998). The D-value decreased as drying temperature increased and fat content

decreased (Faith et al., 1998). Survival of E. coli O157:H7 during manufacturing and

drying of pepperoni was studied by Riordan et al (1998). The pathogen was inoculated

into the pepperoni batters that had different salt and sodium nitrite concentrations and

different pH (Riordan et al., 1998). With the common commercial formulation, the

populations of E. coli O157:H7 declined only about 1 log10CFU/g (Riordan et al., 1998),

although the USDA recommended a 5 log reduction on the load of E. coli O157:H7.

Reduction in cell counts became significant when a combination of high salt, presence of

sodium nitrite and low pH were used (Riordan et al., 1998). Although it survived, E. coli

O157:H7 did not grow in pepperoni batters (Riordan et al., 1998).

The influence of background flora in ground beef on growth of E. coli O157:H7

was determined by Vold et al. (2000). Background flora commonly found in ground beef

(Lactic acid bacteria such as Lactobacillus sakei isolated from ground beef) were added

onto ground beef that was later inoculated with E. coli O157:H7 and packaged

aerobically and anaerobically (Vold et al., 2000). It was found that the background flora

18

present in high number have the ability to inhibit the growth of E. coli O157:H7,

especially under anaerobic conditions (Vold et al., 2000). Although the interfering

normal microflora controlled the growth of E. coli O157:H7, they failed to reduce the

load of E. coli O157:H7. Five strains of lactic acid bacteria (LAB) were selected as

protective culture in a cooked meat study to determine if LAB could inhibit the growth of

E. coli O157:H7 in cooked, sliced, and vacuum packed ham (Bredholt et al., 1999). The

meat was inoculated with 5 strains of LAB and 102 – 103 CFU/g of E. coli O157:H7 and

stored for 28 d at 10°C (Bredholt et al., 1999). The LAB inhibited the growth of E. coli

O157:H7 in cooked ham stored for 4 w (Bredholt et al., 1999). However, no decline in

populations of E. coli O157:H7 was observed (Bredholt et al., 1999). Kang and Fung

(1999) inoculated E. coli O157:H7, Salmonella Typhimurium and a starter culture into

microbiological media and meat to test the effect of diacetyl. Diacetyl inhibited both

pathogens especially in the presence of acid. However, it did not affect the starter culture

P. acidilactici added to the media and salami.

Ellajosyula et al., (1998) conducted a study to determine the effects of pH, final

heating temperature and time on survival of E. coli O157:H7 and Salmonella

Typhimurium in Lebanon bologna. The pathogens were inoculated into raw batter. The

inoculated batter was then fermented at 26.7°C and then 37.8°C. When the pH reached

5.2 or 4.7, the batter was heated to various temperatures for a maximum of 20 h. The

results suggest that fermentation without heating caused about <1 log10CFU/g reduction.

Heating without fermentation only reduced the cell counts by ca. 3 log10CFU/g

(Ellajosyula et al., 1998). However, combination of fermentation and heating at high

19

temperature (43.3°C or 46.1°C) for 20 h could reduce the cell counts of both pathogens

by 7.5 log10CFU/mL.

Soudjouck batter was inoculated by Cosansu and Ayhan (2000) with E. coli

O157:H7 to determine the survival of the pathogen during fermentation, drying and

storage. The inoculated batter was stuffed into casing and fermented for 3 d at 24°C and

dried for 5 d at 22°C. Half of the samples were vacuum packed while the other half was

aerobically packed for a 3-m storage (Cosansu and Ayhan, 2000). It was noticed that the

pathogen decreased by 3 log10CFU/mL during the 3-d fermentation and 5-d of drying. E.

coli O157:H7 survived longer in the vacuum packed samples.

Response to Stress

Outbreaks of E. coli O157:H7 are mainly foodborne, therefore E. coli O157:H7

must be able to survive the very acidic condition of the gastric fluid to infect a patient. E.

coli O157:H7 was found in several high acid foods such as apple cider (Besser et al.,

1993, CDC, 1996), yogurt (Morgan et al., 1993), and fresh cheese curd (CDC, 2000).

The pathogen usually does not grow at pH less than 5.5, but when the acid-resistance

genes are induced, it can survive at pH as low as 2.0 (Lin et al., 1995).

E. coli produces 30 proteins that are regulated by the alternate sigma factor (rpoS)

(Hengge-Aronis, 1993). These proteins increase acid, heat and salt tolerance of E. coli

O157:H7 (Cheville et al., 1996) and are produced when the cells enter stationary phase.

Cheville et al. (1996) found that without the rpoS, E. coli O157:H7 could not survive the

high acid they were subjected to. Benjamin and Datta. (1995) suggested that the acid

tolerance of E. coli O157:H7 is dependent on the growth phase. When the cells enter

mid-exponential phase, the acid tolerance decreases. They are most tolerant at late

20

stationary phase followed by early stationary phase. In the study of Arnold and Kaspar

(1995), stationary phase and the starved log-phase cells of E. coli O157:H7 had increased

acid tolerance and were able to survive longer in synthetic gastric fluid. Starved cells at

25°C were more tolerant to acid than the same cells at 4°C. It was believed that proteins

induced from the starved cells at 25oC cross-protected cells of E. coli O157:H7 from acid

stress. At 4°C, however, the cells ceased to synthesize protein because the temperature is

below the minimum temperature, 8°C, for protein synthesis in the cells.

E. coli has three acid-resistance systems: acid-induced oxidative system, acid-

induced arginine-dependent system, and glutamate-dependent system (Lin et al., 1996).

The ariginine-dependent and glutamate-dependent systems play key roles in acid

tolerance of E. coli O157:H7 (Lin et al., 1996). Arginine and glutamate both are amino

acids that, when present in even small quantities, are able to help E. coli survive at low

pH (Jordan et al., 1999). The mechanism on how amino acids protect E. coli cells is not

fully comprehended but it is believed that the decarboxylation of these amino acids in the

cells with the proton from the acidic environment may assists in maintaining the pH

inside the cells (Jordan et al., 1999).

Zook et al., (2001) pre-treated E. coli O157:H7 cultures with 0.1% peroxyacetic

acid, hydrogen peroxide, and acetic acid and then challenged the culture in 80 mM

hydrogen peroxide at 54°C. The results indicated that pre-treatment of cultures with

0.1% peroxyacetic acid and hydrogen peroxide increased tolerance of E. coli O157:H7 to

hydrogen peroxide while the cultures pre-exposed to acetic acid were as susceptible to

hydrogen peroxide as the control. Pre-exposure to 0.1% peroxyacetic acid did not

increase the thermotolerance of E. coli O157:H7. However, adapting cells with mild pH

21

in other studies enhanced the thermotolerance of E. coli O157:H7 (Ryu and Beuchat,

1998). Acid-adapted, acid shocked, and control cells of E. coli O157:H7 were inoculated

into TSB with pH ranging from 3.9 to 3.4 acidified by acetic acid and lactic acid. The

cells were also inoculated into commercial apple cider and orange juice and stored at 5°

or 37°C (Ryu and Beuchat, 1998). The results showed that acid-adapted cells were more

tolerant to the low pH than acid-shocked cells in TSB acidified with lactic acid (Ryu and

Beuchat, 1998). E. coli O157:H7 was tolerant to the low pH of apple cider and orange

juice stored at 5° and 25°C for up to 42 days. Acid adapted cells had relatively higher D-

values during heat treatment (Ryu and Beuchat, 1998).

Ingham and Uljas (1998) examined how prior storage in acidic and /or cold

condition affected the survival of E. coli O157:H7 in synthetic gastric fluid. Three strains

of E. coli O157:H7 were kept in acidified TSB or pH 3.5 apple juice at 4° and 21°C for

approximately 7 d before challenging the cells in synthetic gastric juice (pH 2.5) for 4 h

at 37°C (Ingham and Uljas, 1998). It was observed that the cells of E. coli O157:H7

survived better in apple juice than in TSB acidified with organic acid. Survival of E. coli

O157:H7 in pH 2.5 synthetic gastric juice was enhanced by prior-storage of cells at low

temperature and low acid. Cells stored in refrigerated apple juice were more tolerant to

the low pH of synthetic gastric juice compared to those stored at room temperature.

Among the organic acids used, cells stored in TSB acidified with lactic acid were

completely eliminated upon transfer to the synthetic gastric juice (Ingham and Uljas,

1998).

Brudzinski and Harrison (1998) studied influence of temperature and agitation on

acid tolerance response (ATR) of E. coli O157:H7 and non O157:H7 E. coli. The authors

22

found that ATR of E. coli O157:H7 can enhance the survival of the pathogen up to 1,000

folds at 32°C depending on the strains. Non-O157:H7 E. coli were better protected at

25°C. ATR was more protective for E. coli O157:H7 when the cells were grown

statically while the non-O157:H7 had greater ATR when incubated with agitation

(Brudzinski and Harrison, 1998).

Garren et al., (1997) studied the survival of acid-adapted or acid-shocked E. coli

O157:H7. In order to induce acid tolerance, cells were first grown to stationary phase

and exposed to pH 5.5 followed by exposure to pH 3.5 or 4 (Garren et al., 1997). Acid

shock cells were grown to stationary phase and expose to pH 3.5 or 4 in lactic acid. The

authors found that strains survived better at pH 4 at 25°C. Cells incubated at 32°C at pH

3.5 were undetectable after 21 days.

Duffy et al. (2000) studied the effect of growth pH and fermentation on survival

and themotolerance of E. coli O157:H7. Bacterial cells were grown at pH 5.6 (acid

adapted) and 7.4 and later fermented to pH 4.8 or 4.4 (Duffy et al., 2000). The cells were

then subjected to heat treatment at 55°C (Duffy et al., 2000). The authors found that acid

adapted cells had higher D-value than cells grown at pH 7.4. Cells fermented to pH 4.8

had increased D-value while those in pH 4.4 had decreased D-value. This is probably

because that the cells were injured at the low fermentation pH therefore were unable to

survive under subsequent heat stress.

The survival of E. coli O157:H7 is affected by the type of acidulants and storage

temperature. In a study done by Conner and Kotrola, E. coli O157:H7 was inoculated

into a flask containing TSB acidified with acetic acid, citric acid, lactic acid, malic acid,

mandelic acid, and tartaric acid to pH of between 4.0 and 5.2 (Conner and Kotrola, 1995).

23

The samples were then stored at 25°, 10° or 4°C for 56 d. Populations of E. coli

O157:H7 in all organic acid, except mandelic aicd, increased at 25°C, but not at 10°C and

4°C. Higher cell counts were obtained at 4°C compared to 10°C in all acids. The

pathogen survived better in the presence organic acid at 4°C.

In addition to producing stress-responding proteins, strains of E. coli O157:H7

synthesize surface structures that protect the cells from environmental stresses. E. coli

O157:H7 produces exopolysaccharide (EPS) composed of colanic acid (CA) and forms

slimy colonies at room temperature on minimal glucose agar (Chen, Unpublished,

Junkins and Doyle, 1992). While majority strains of E. coli O157:H7 secrete CA (Chen

et al., unpublished), the quantities of production are quite different (Chen et al.,

Unpublished). A CA-deficient E. coli O157:H7 mutant was constructed by Mao et al.

(2001). It was found that the CA-deficient strain was less tolerant to acid and heat (Mao

et al., 2001). This suggests that CA play a role in protecting the cells from heat and acid

stress. The mechanism where E. coli O157:H7 is protected by CA is not known, but it is

likely that CA gives the bacteria cell surface a strong negative charge and when the cells

are in low pH environment, the CA may act as a buffer to the cells thus preventing the

acid from penetrating the cells (Mao et al., 2001).

Ophir and Gutnick (1994) studied the role of EPS in protecting E. coli,

Acinetobacter calcoaceticus, and Erwinia stewartii from desiccation. Mucoid and non-

mucoid cells were subjected simultaneously to desiccation. EPS is able to bind large

amounts of water and maintain the suitable humid environment for the cells. The mucoid

cells were, therefore more resistant to desiccation.

24

The role of CA in biofilm formation was studied by Danese et al (2000). CA-

proficient and CA-deficient cells were allowed to adhere to polyvinyl chloride (PVC) and

observed both macroscopically and microscopically. The results suggests that EPS

production is not required for the initial attachment of the cells to the surface of PVC but

it is important to the formation and the depth of the biofilm (Danese et al., 2000).

YOGURT

Introduction

A 52-w survey conducted by Information Resources, Inc. revealed that unit sales

of yogurt and refrigerated yogurt drinks have gone up 3.3%. This did not include yogurt

sold in food service, and super-center (Anonymous, 2002). Yogurt can be made from

milk of goat, cow, buffalo, reindeer, sheep, yak, zebu, horse, or camel (Tamime and

Robinson, 1999). No one really knows the exact origin of yogurt but it is believed to

originate from the Middle East (Tamime and Robinson, 1999). There are different types

of yogurt, but set and stirred yogurt are among the more commonly consumed yogurts in

the market. Set yogurt are fermented in the cup while stirred yogurts are fermented in a

vat and distributed into cups after the fermentation process.

According to the Code of Federal Regulation (CFR), yogurt is a food produced by

inoculating dairy ingredients such as milk, cream, partially skimmed milk, or skim milk

with lactic acid producing bacteria, Lactobacillus bulgaricus and Streptococcus

thermophilus (FDA, 2000). Regular yogurt contains no less than 3.25% milk fat and no

less than 8.25% milk solids non-fat before adding flavors. The titratable acidity of yogurt

should not be less than 0.9% lactic acid (FDA, 2000)

25

The main ingredient in milk is water, milk fat, and solid non-fat (protein, lactose

and ash). In general, 87.4% of milk is water followed by 4.7% lactose, 3.9% fat, 3.3%

protein, and 0.7% ash (Tamime and Robinson, 1999). Milk fat affects the texture of the

yogurt. The higher the percent fat, the richer and creamier the yogurt is. Lactose is an

energy source for the starter cultures. Lactose is converted into lactic acid through

fermentation by the starter cultures. Protein is related to the formation of coagulum.

Consistency or viscosity of the yogurt is directly proportioned to the level of protein

available in the milk.

Yogurt Fermentation

The starter cultures that are commonly used in the production of yogurt are

Streptococcus thermophilus and Lactobacillus delbruekii subsp. bulgaricus (Frank and

Hassan, 1998). These two microorganisms produce acid during fermentation, however,

the rate of acid production is slow individually. In order to speed up acidification, the

two starter cultures are used together in the production of yogurt. According to

Rajagopal and Sandine (1990), lactobacilli are able to stimulate the growth of S.

thermophilus by releasing amino acids and peptides from casein in milk. In return for the

free amino acids, S. thermophilus would lower the pH, remove oxygen, and produce

formic acid and pyruvate to stimulate the growth of L. bulgaricus (Radke-Mitchell and

Sandine, 1984). S. thermophilus would usually grow first in the inoculated milk to start

the acidification followed by L. bulgaricus in the later stage of fermentation (Frank and

Hassan, 1998).

S. thermophilus and L. delbruekii subsp. bulgaricus are both homofermenters.

Catabolism of lactose happens in the microbial cell (Tamime and Robinson, 1999).

26

Lactose molecules are brought into the cell by the phosphoenolpyruvate (PEP)-dependent

phosphotransferase system (PTS). During the transport of the lactose into the cell, the

lactose is phosphorylated to lactose-6-phosphate (Monnet et al., 1996). Lactose-6-

phosphate is then hydrolyzed by β-phosphogalactosidase into glucose and galactose

(Tamime and Robinson, 1999). The monomers are further converted through the

Tagatose and Emden-Meyerhof-Parnas (EMP) pathway to form lactic acid (Tamime and

Robinson, 1999).

According to Tamime and Robinson (1999), lactic acid is important in the

production of yogurt. Lactic acid helps destabilize the casein micelles. The colloidal

calcium/phosphate complexes in the micelle are converted to soluble calcium phosphate

and diffuse into the milk. This causes the micelle to lose calcium and lead to coagulation

of the casein at pH 4.6-4.7 (Tamime and Robinson, 1999). In addition, lactic acid

contributes to the sharp and acidic taste of yogurt.

Yogurt Production

Production of set and stirred yogurt involves similar processes. Pasteurized milk

is brought in to the plant and kept in a silo until ready to be used. The milk in the silo is

kept cool to prevent growth of spoilage microorganism in the milk. The milk is first

standardized to achieve the required amount of fat, and non-fat solids. For commercial

yogurt, there should be about 1.5g/ 100g of fat and 8.2 to 8.6g/100g of solids-non-fat in

medium fat yogurt (Tamime and Robinson, 1999). Depending on the amount of fat

targeted or available in the milk, the standardization of fat can be done by removal of fat

from milk, addition of whole to skimmed milk, and cream to whole milk or skimmed

milk. Standardization of solids-non-fat can be done by adding milk powder, non-fat dry

27

milk, whey powder or whey concentrate, casein powder and others. Concentration of the

solids-non-fat can also be done to standardized solids-non-fat by vacuum evaporation and

membrane filtration.

After standardization, the milk mixtures are homogenized. Milk is an oil-in-water

emulsion, the oil therefore has the tendency to separate and float to the top.

Homogenization is a process during which the milk is mixed in high speed and forced

under high pressure through a small orifice. Homogenization breaks up the fat globules

and reduces the size of fat globules to less than 2 µm so that the fat would not clump

together and rise to the top (Tamime and Robinson, 1999). It also has effect on other

milk constituents such as milk protein. According to Tamime and Robinson (1999), milk

proteins (casein and whey) can undergo a few changes due to homogenization.

Denaturation of whey protein can occur and with that denaturation, casein and whey

protein interaction might occur. Casein micelles that are broken up could also stabilize

fat globules.

Heating or pasteurization is the next step in the yogurt-making process. The

commonly used pasteurization temperature/time is 85°C for 30 min or 90-95°C for 5 min

(Tamime and Robinson, 1999). There are several reasons for pasteurizing the milk.

Heating destroys most of the microorganisms in milk (Tamime and Robinson, 1999).

However, spore formers and heat-stable enzymes cannot be destroyed during this process

(Tamime and Robinson, 1999). Properly pasteurized milk will provide a good growth

medium for yogurt starter cultures. Heating milk would produce stimulatory effect on the

starter cultures, especially at 90-95°C for 60-180 min. This may be due to reduction in

toxic sulphides in milk or formation of formic acids. Heating also changes the

28

physiochemical properties of milk. Whey protein denatures at temperature above 80°C.

After denaturation, the whey protein reacts with K-casein to form a more stable micelle

(Tamime and Robinson, 1999). The effect of heat on protein is 2-staged. First the

protein denatures due to the alteration of the protein structure, and then the denatured

proteins will aggregate followed by coagulation. The denaturation of protein is

important for the formation of gel during fermentation.

In heated milk, the gel forms as the casein micelles increase in size and arrange

into a chain matrix (Tamime and Robinson, 1999). These changes result in a uniform

distribution of the protein, the aqueous phase is trapped within the network. This would

give a stable coagulum and decreases syneresis. In unheated milk, the casein micelles

form clusters in which protein is not evenly distributed thus unable to trap the aqueous

phase (Tamime and Robinson, 1999). The coagulum formed with unheated milk is 50%

weaker than that form with heated milk.

After heat treatment, milk is cooled down to 40-45°C for optimum growth of

starter cultures: S. thermophilus and L. bulgaricus. (Tamime and Robinson, 1999). The

starter cultures are usually added to the milk slowly while the milk is being pumped into

the fermentation tank. Manual addition or pumping of starter culture into the milk in the

fermentation tank can also be done if a large volume of milk is used. Incubation at 40°-

45°C shortens the time required for fermentation.

The formation of gel happens during fermentation. Gel formation in yogurt is

complicated. Not only lactic acid produced by the starter cultures but also proteinases in

milk may play a role in the coagulation (Tamime and Marshall, 1997). Once formed, the

gel is irreversible.

29

During fermentation, the gel should be left undisturbed. If disturbed, syneresis

can happen easily. The difference between set yogurt and stirred yogurt is that during

incubation, set yogurt is fermented undisturbed in individual containers resulting in gel

that is a continuous semi-solid mass (Tamime and Robinson, 1999). The gel structure of

the stirred yogurt is broken up after the fermentation period during stirring (Tamime and

Robinson, 1999)

The fermentation period ends when pH of the yogurt drops to below 4.7. The

yogurt is cooled down immediately to slow down or stop the fermentation. The starter

cultures become inactive when the temperature drops below 10°C (usually around 5°C)

(Tamime and Robinson, 1999). The rate of cooling can affect the final pH of the yogurt

and also the structure of the milk gel. If the yogurt is cooled too rapidly, it will lead to

syneresis where the whey separates from the gel (Rasic and Kurmann, 1978). There are

two ways to cool yogurt. Yogurt can be cooled either going through one-phase cooling

or two-phase cooling.

In one-phase cooling, yogurt is cooled directly from the incubation temperature to

temperature below 10°C (Tamime and Robinson, 1999). Two-phase cooling lowers

temperature of yogurt from fermentation temperature to about 20°C. Addition of flavor

and fruits, stirring, and filling are done at this stage. After filling into individual yogurt

cups, the yogurt is then cooled down to below 10°C. One-phase cooling is no longer

widely used because at 20°C, the yogurt is less viscous thus suffers less mechanical

damage.

30

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CHAPTER 3

SURVIVAL OF ESCHERICHIA COLI O157:H7 IN SET YOGURT AS INFLUENCED

BY AN EXOPOLYSACCHARIDE, COLANIC ACID, PRODUCTION1

Lee, S.M., and Chen, J. To be submitted to Applied and Environmental Microbiology.

41

42

The role of an exopolysaccharide (EPS), colanic acid (CA), in protecting cells of

Escherichia coli O157:H7 during processing and storage of set yogurt was determined.

Pasteurized milk was inoculated, before fermentation, with a wild type E. coli O157:H7,

its CA-deficient mutant and a mixture of the two strains, respectively. Set yogurt was

processed according to the procedure described by CHR Hansen, Inc. with slight

modifications. The yogurt was stored at 4° or 15°C for 3 weeks. Samples were

enumerated for total plate counts, population of E. coli O157:H7, and starter cultures. It

was noticed that E. coli O157:H7 survived longer in yogurt stored at 15°C than in yogurt

stored at 4°C and acid stressed or injured cells were more readily recovered on TSA than

SMAC. Mutant died off more rapidly than its parental strain suggesting that CA plays a

role in protecting the cells from acid stress.

43

Escherichia coli O157:H7 is a pathogen that causes serious foodborne illnesses

such as hemolytic uremic syndrome (HUS), hemorrhagic colitis (HC), and thrombotic

thrombocytopenic purpura (TTP) (14). Since the first outbreak of E. coli O157:H7

infection in 1982, this pathogen has caused numerous outbreaks (2, 3, 4, 19). Many

foods are implicated in these outbreaks including those acidic food such as apple juice

(3), apple cider (1), fresh cheese curd (2), salami (4), and yogurt (19).

An outbreak of E. coli O157:H7 associated with consumption of yogurt produced

on a farm in northwest of England occurred in 1991 (19). The yogurt was produced

specifically for children. In this outbreak, 11 out of the 16 infected were children under

age 10. Five out of the 10 children developed HUS. Cross-contamination of pasteurized

milk used to process the yogurt might have occurred, because a pump used to pump raw

milk was also utilized to pump pasteurized milk. Another possible route of cross-

contamination was that the workers might have tracked mud and dirt into the processing

plant with their muddy boots. The churns were sometime put on the floor where debris

and mud could easily come in contact with the yogurt or milk. In addition, this plant did

not have proper records of pasteurization temperatures. Thus, the milk may not have

been properly pasteurized before being used to process yogurt.

It was found in laboratory studies that E. coli O157:H7 is tolerant to many

environmental stresses including low pH (6, 16, 22, 24, 25), and heat (10). It was

reported that E. coli O157:H7 is able to survive in acid food such as apple cider (pH 3.7-

4.0) (18), buttermilk (pH 4.1) (8), yogurt (pH 4.17-4.39) (8, 12), and sour cream (pH 4.3)

(8). E. coli O157:H7 does not grow well at temperature higher than 44.5°C (9).

However, heat of yogurt fermentation is not adequate to inactivate the pathogen.

44

The tolerance of E. coli O157:H7 to stress is attributed to many factors including

synthesis of protective cell surface structures. Cells of E. coli O157:H7 secrete, under

stress, a large amount of CA which forms an irregular layer around cell surfaces. This

slime layer plays a role in protecting the cells from desiccation (20), low pH (15), and

heat (15). Tolerance of E. coli O157:H7 to stress, acid and heat stress in particular, is of

importance for it can influence the virulence, prevalence, and involvement of E. coli

O157:H7 in foodborn outbreaks. The objective of this study was to determine the role of

CA in protecting E. coli O157:H7 during production and storage of set yogurt.

MATERIALS AND METHODS

Culture Preparation. Wild type E. coli O157:H7 (W) and its CA-deficient

mutant (M), both from our laboratory collection, were used in the experiments. The M

was constructed by inserting a kanamycin resistance gene cassette into the wca operon of

the W (Mao et al., 2001). To prepare inocula for the set yogurt experiment, the W was

grown on minimal glucose agar (MGA), whereas M on MGA-kanamycin (100 µg/mL) at

25°C for 48 h. Following incubation, cultures of E. coli O157:H7 were collected by

rinsing the plates with 5 mL of 0.1% peptone buffer. Each suspension was appropriately

diluted in 0.1% peptone and plated on TSA and SMAC. The plates were incubated at

37°C for 24 h. Colonies were enumerated and populations of cells in each suspension

were calculated.

Starter culture YC-180 (CHR Hansen, Inc., Milwaukee, WI), which contained

Streptococcus thermophilus, and Lactobacillus delbruekii subsp bulgaricus and subsp.

lactis. YC-180 came in frozen pellets and was kept at –30°C before use.

45

Set yogurt production. Four gallons of whole milk were purchased from a local

grocery store one day before the experiment and stored at a 4°C walk-in refrigerator. On

the set up day, 2.7 L of whole milk were placed into 4 stainless steel containers (VWR,

Atlanta, GA.), respectively. Following the addition of non-fat dried milk (NFDM; 1%),

the milk was stirred with a magnetic stir bar for 4 min at room temperature.

A water bath (91.44cm x 45.6cm x 30.48cm) (Magni Whirl, Blue M Electric

Company, Blue Island, IL) used for pasteurizing the milk was pre-heated to 98°C. The

level of water in the water bath was approximately the same as the level of milk in the

stainless steel containers. The temperature of the water decreased to about 89°C after all

the containers were placed into the water bath. The milk was heated to 95°C and held for

5 min. After heating, the milk was cooled down in an ice bath to 43°C. Starter culture

YC 180 (0.36g, ca.105 CFU/mL of L. bulgaricus and 106 CFU/mL of S. thermophilus)

and E. coli O157:H7 W, M, or a mixture of W and M in 1:1 ratio (ca. 105 CFU/mL) were

subsequently added to the milk. The inoculated milk was mixed with a magnetic stirring

bar at room temperature for 4 min.

Approximately 170.1g (ca. 6 oz.) of milk were measured into sterile

polypropylene fermentation cups (8 oz; Fisher Brand). Milk in each stainless container

was distributed into 16 cups. Fifteen of the cups were used for the survival study

whereas the remainder was used to monitor fermentation conditions. The milk was

fermented in an incubator at 44°C. When the pH of the yogurt dropped to 4.58 ± 0.05

(approximately 4 h), the cups were removed from the incubator and cooled down to 7°C

in an ice bath before being stored at 4° or 15°C.

46

Sampling and Enumeration. The overall sampling scheme of the study is

illustrated in Figure 3.1. Milk was withdrawn before (sample a) and after (sample b) the

addition of NFDM, after pasteurization (sample c), and after cooling (sample d).

Following milk distribution, fermentation or cooling, cups of milk or yogurt labeled ‘BF’

(before fermentation), ‘AF’ (after fermentation), or ‘I’ (initial sampling after fermentation

and cooling) were assayed. Yogurt stored at 4 or 15oC was sampled twice a week for 3

weeks.

Two ml of milk were withdrawn for sample a, b, c, and d, whereas whole cups of

milk or yogurt were used for sampling of BF, AF, I, and yogurt stored at 4 or 15oC. The

pH and temperature of each sample were measured and recorded prior to microbiological

sampling. At each sampling, milk or yogurt was mixed with 170.1 ml of 0.1% peptone

water in a sterile filter stomacher bag (Spiral Biotech, Norwood, MA) and homogenized

using a stomacher (Seward Limited, London) for 30 sec at normal speed. The

homogenates were appropriately diluted in 0.1% peptone water and spiral plated in

duplicate using Autoplate 4000 (Spiral Biotech, Inc., Nethesda, Maryland). All

samples were enumerated on TSA, SMAC, MRS acidified with 0.25% glacial acetic acid,

and M17 supplemented with 10% lactose (Difco Laboratories, Sparks, Maryland). TSA

and SMAC plates monitored the levels of total plate counts and E. coli O157:H7,

respectively. Acidified MRS agar determined the counts of L. bulgaricus whereas M17

with 10% lactose enumerated the populations of S. thermophillus. TSA and SMAC were

47

Whole Milk

Add 1% NFDM 2 ml sample (a)

2 ml sample (b)

Heat to 95°C and hold for 5 min

Cool down to 43°C in ice water bath

2 ml sample (c)

Inoculate with starter culture 2 ml sample (d)

C

Inoculate with E. coli O157:H7

W M WM

Divide into individual cups (6 oz., 170.1g)

Incubate at 44°C for ~ 4h

Sample (BF)

When pH reaches 4.7, cool down rapidly to 7°C in ice water bath Sample (AF)

Sample (I)

Store at 4°C Store at 15°C

Sample twice a week for 3 weeks

Figure 3.1. Flow chart for set yogurt production

48

incubated at 37°C for 24 h. M17 plates were incubated at 37°C for 48 h while acidified

MRS plates at 45°C for 48 h under anaerobic condition.

Data Analysis. Two replications were conducted. Data collected from the

experiments were analyzed by general linear model procedure using SAS Program (SAS

Institute Inc., Cary, N.C.). Significant differences in survival between W and M, W and

WM, M and WM, respectively, on TSA and SMAC at 4o and 15oC were determined.

RESULTS AND DISCUSSION

Microbial quality of the commercial milk used to process set yogurt was

determined. The populations of cells recovered on TSA, SMAC, MRS and M17 were

2.84, 2.51, 0.65 and 2.43 log10 CFU/mL, respectively. The cells recovered on SMAC

were not E. coli O157:H7. Addition of NFDM slightly increased the counts on M17 and,

in some instances, on TSA (Data not shown). Heating of milk at 95oC for 5 min lowered

the levels of microorganisms in milk to an undetectable level.

The pH, temperature and populations of starter cultures in yogurt stored at 4o and

15oC are summarized in Table 3.1. The counts of L. bulgaricus increased 1.39 to 1.71

log10 CFU/mL during fermentation, and remained approximately unchanged throughout

storage at 4 or 15oC. Fermentation also elevated the populations of S. thermophilus by

2.62 to 3.13 log10 CFU/mL. S. thermophilus and L. bulgaricus has a cooperative

relationship in yogurt where they stimulate each other to grow that results in rapid

acidification (11). S. thermophilus is capable of removing oxygen, lowering pH, and

producing formic acid and pyruvate (21) so that L. bulgaricus could grow. L. bulgaricus

49

Table 3.1. The pH, temperature and levels of starter cultures in set yogurt stored at 4° and 15°C. Sample

PH

Temp (°C)

L. bulgaricus Log10 CFU/mL

S. thermophilus Log10 CFU/mL

Sample pH Temp (°C)

L. bulgaricus Log10 CFU/mL

S. thermophilus Log10 CFU/mL

WaBFe 6.81 N/A 5.08 6.03 WBF 6.81 N/A 5.08 6.03 WAFf 4.61 42 6.79 9.16 WAF 4.61 42 6.79 9.16 WIg N/A 7 6.85 9.37 WI N/A 7 6.85 9.37

W1hai 4.54 4 7.09 9.10 W1a 4.33 15 7.10 9.11 W1bj 4.34 4 6.91 8.99 W1b 4.13 15 7.11 8.94 W2a 4.32 4 6.82 9.00 W2a 4.10 15 7.06 8.89 W2b 4.32 4 6.86 8.84 W2b 4.05 15 6.93 8.75 W3a 4.34 4 6.81 8.84 W3a 4.03 15 6.83 8.62 W3b 4.34 4 6.81 8.69 W3b 4.03 15 6.65 8.48

Mb BF 6.84 N/A 5.21 6.36 MBF 6.84 N/A 5.21 6.36 MAF 4.59 39 6.60 9.11 MAF 4.59 39 6.60 9.11 MI N/A 7 6.65 9.26 MI N/A 7 6.65 9.26

M1a 4.60 4 6.75 8.97 M1a 4.41 15 6.86 8.89 M1b 4.49 4 6.93 8.98 M1b 4.18 15 6.87 9.01 M2a 4.46 4 6.86 9.06 M2a 4.07 15 6.90 9.01 M2b 4.41 4 6.92 9.04 M2b 4.04 15 6.71 8.96 M3a 4.33 4 6.89 8.89 M3a 4.00 15 6.82 8.86 M3b 4.41 4 6.88 8.97 M3b 4.02 15 6.63 8.73

WMcBF 6.81 N/A 5.52 6.10 WMBF 6.81 N/A 5.52 6.10 WMAF 4.53 41 7.17 9.13 WMAF 4.53 41 7.17 9.13 WMI N/A 7 7.36 9.14 WMI N/A 7 7.36 9.14

WM1a 4.56 4 7.43 9.05 WM1a 4.32 15 7.46 9.02 WM1b 4.40 4 7.55 9.05 WM1b 4.08 15 7.52 9.06 WM2a 4.33 4 7.59 9.09 WM2a 4.00 15 7.43 9.01 WM2b 4.37 4 7.58 9.02 WM2b 4.04 15 7.41 8.99 WM3a 4.36 4 7.49 8.97 WM3a 4.01 15 7.28 8.96 WM3b 4.36 4 7.54 8.98 WM3b 4.00 15 7.22 8.67 CdBF 6.83 N/A 5.20 6.44 CBF 6.83 N/A 5.20 6.44 CAF 4.60 39 6.72 9.06 CAF 4.60 39 6.72 9.06 CI N/A 7 6.76 9.04 CI N/A 7 6.76 9.04

C1a 4.65 4 6.93 8.92 C1a 4.50 15 7.03 8.77 C1b 4.54 4 6.97 8.80 C1b 4.28 15 6.83 8.95 C2a 4.47 4 7.14 8.90 C2a 4.10 15 6.87 9.03 C2b 4.36 4 6.99 8.99 C2b 4.10 15 6.91 8.75 C3a 4.41 4 7.02 8.90 C3a 3.99 15 6.76 8.48 C3b 4.41 4 6.90 8.88 C3b 4.08 15 6.65 8.83

a W: wild type; b M: CA-deficient mutant; c WM: wild type and mutant mixture; d C: control

e BF: before fermentation; f AF: after fermentation; g I: after cooling. h Sample 1, 2 and 3: yogurt tested after 1, 2 and 3 weeks into storage. i Sample a: the first sampling of the week; j Sample b: the second sampling of the week.

50

frees up amino acid from casein to be utilized by S. thermophilus in return (11). The

counts of S. thermophilus increased more during fermentation than the counts of L.

bulgaricus (Table 3.1). This could be because S. thermophilus grew at the beginning of

the fermentation followed by L. bulgaricus (11).

S. thermophilus and L. bulgaricus are both homofermenters. The decrease in milk

pH during fermentation is the result of lactose catabolism. Lactose is phosphorylated to

lactose-6-phosphate and brought into the cell by the phosphoenolpyruvate (PEP)-

dependent phosphotransferase system (PTS). During the transport of the lactose into the

cell, the phosophate is hydrolyzed by β-phosphogalactosidase into glucose and galactose

(23). The monomers are further converted through the Tagatose and Emden-Meyerhof-

Parnas (EMP) pathway to form lactic acid (23). The pH levels of the yogurt at 15°C

were lower than at 4°C (Table 3.1). This may be because the starter cultures were still

active at 15°C and were able to further produce lactic acid from remaining lactose in

yogurt.

W, M, and WM grew slightly during fermentation (Figure 3.2 and 3.3). Statistical

analysis revealed that at pre-storage stage (sample BF, AF, and I), the survival of W vs.

M, and M vs. WM as reflected by growth on SMAC were significantly different (P <

0.05). However, differences in survival of the same samples enumerated on TSA were

not statistically significant (P > 0.05).

The survival of W vs. M was significantly different during storage at 4° and 15°C

(P < 0.05; Figure 3.2 and 3.3). The M became undetectable on SMAC after 7 d of

storage at 4°C (Figure 3.2B) or 11 d of storage at 15°C (Figure 3.3B). However, the W

remained detectable on SMAC till 18-d storage at 4°C (Figure 3.2B). The levels of W

51

0

1

2

3

4

5

6

7

8

0 2 4 6 8 10 12 14 16 18 20

Time (Days)

Popu

latio

n of

E. c

oli

O15

7:H

7 (L

og10

CFU

/ml)

WMWM

A

0

1

2

3

4

5

6

7

0 2 4 6 8 10 12 14 16 18 20

Time (Days)

Popu

latio

n of

E. c

oli O

157:

H7

(Log

10 C

FU/m

l)

WMWM

B

Figure 3.2. Survival of E. coli O157:H7 in yogurt stored at 4°C. Enumeration was done on TSA (A) and SMAC (B) plates. BF: Before fermentation when cups were placed into the incubator; AF: After the pH of yogurt reached 4.58 ± 0.05; I: After yogurt samples were cooled down to 7°C. 4-21: days into storage.

52

0

1

2

3

4

5

6

7

8

0 2 4 6 8 10 12 14 16 18 20

Time (Days)

Popu

latio

n of

E. c

oli

O15

7:H

7 (L

og10

CFU

/ml)

WMWM

A

0

1

2

3

4

5

6

7

0 2 4 6 8 10 12 14 16 18 20

Time (Days)

Popu

latio

n of

E. c

oli

O15

7:H

7 (L

og10

CFU

/ml)

WMWM

B

Figure 3.3. Survival of E. coli O157:H7 in yogurt stored at 15°C. Enumeration was done on TSA (A) and SMAC (B) plates. BF: Before fermentation when cups were placed into the incubator; AF: After the pH of yogurt reached 4.58 ± 0.05; I: After yogurt samples were cooled down to 7°C. 4-21: days into storage.

53

were between 104 and 105 CFU/mL on SMAC after storage for 21 d at 15°C (Figure

3.3B). Higher populations of cells were recovered on TSA than on SMAC. This is

because TSA is a nonselective medium, stressed and injured cells are readily recovered

on this medium. M and W were both detectable after 21-d storage at 4oC when

enumerated on TSA (Figure 3.2A and 3.3A). However, the counts of M were much

lower compared to W. At day 21, the populations of W and M stored at 4o and 15oC

were ca. 105 and 101 CFU/mL, respectively. These results suggest that without the

protection of CA, E. coli O157:H7 is more susceptible to acidic conditions. Significant

differences in survival between W and M in set yogurt indicate that CA protects E. coli

O157:H7 from acid stress. This agrees with the previous findings reported by our

laboratory (5, 15).

E. coli O157:H7 was able to grow in milk at 8o and 15oC (7, 17, 26). Cell

populations increased 3-5 log10 CFU/mL after a 3-d storage at 15°C (26). In the present

study, E. coli O157:H7 persisted longer at 15oC, although the pH of the yogurt stored at

this temperature was slightly lower than at 4oC (Table 3.1). Apparently, the decline in

the populations of E. coli O157:H7 in yogurt was resulted from a hurdle effect of several

factors including temperature and pH.

The survival of W vs. WM was not significantly different during fermentation and

storage (P>0.05; Figure 3.2 and 3.3). Since M was more susceptible to acid stress than

W, the M cells died off faster, therefore the behavior of WM at the end of the storage was

probably characterized by the W. As to the survival of M vs. WM, differences in survival

at 4o and 15oC was found to be significant when the yogurt was sampled on SMAC and

54

TSA (P <0.05). These results suggest that the fate of M were not significantly altered by

the presence of CA-producing W in the same environment.

Hudson et al. found that E. coli O157:H7 seeded into commercial yogurt (ca.107

CFU/g) survived 8 d at 4°C and 5 d at 10°C in yogurt with a pH of 4.17, 17 and 15 d in

yogurt with a pH of 4.39 at 4° and 10°C, respectively (12). Massa et al. (16) reported

that after 7 d of storage at 4°C, E. coli O157:H7 decreased 0.8 to 1.76 log10 CFU/mL in

yogurt (pH 4.4-4.5) made with S. thermophilus and L. bulgaricus (16), which agreed with

our finding in which W dropped ca. 2 log10 CFU/mL in 7-d storage at 4oC.

It was observed in this study that CA had a vital role in protecting E. coli

O157:H7 from acid stress. CA-producing E. coli O157:H7 persisted in yogurt for a

relatively longer period of time than its CA-deficient counterpart. Storage temperature

affected the survival of E. coli O157:H7 in yogurt. Selective medium SMAC could not

recover as many E. coli O157:H7 cells from yogurt as TSA, a non-selective medium.

REFERENCES

1. Besser R. E., S. M. Lett, J. T. Weber, M. P. Doyle, T. J. Barrett, J. G. Wells, and P.

M. Griffin. 1993. An outbreak of diarrhea and hemolytic uremic syndrome from

Escherichia coli O157:H7 in fresh-pressed apple cider. J. Am. Med. Assoc.

269:2217-2220.

2. Centers for Disease Control and Prevention. 2000. Outbreak of Escherichia coli

O157:H7 infection associated with eating fresh cheese curds – Wisconsin, June 1998.

Morbid. Mortal. Weekly. Rep. 49:911-913.

55

3. Centers for Disease Control and Prevention. 1996. Outbreak of Escherichia coli

O157:H7 infections associated with drinking unpasteurized commercial apple juice –

British Columbia, California, Colorado and Washington. Morbid. Mortal. Weekly

Rep. 45:975.

4. Centers for Disease Control and Prevention. 1995. Escherichia coli O157:H7

outbreak linked to commercially distributed dry-cured salami – Washington and

California, 1994. Morbid. Mortal. Weekly Rep. 44:157-160.

5. Chen, J., and S. M. Lee. Unpublished. Protective effect of Escherichia coli O157:H7

exopolysaccharide colanic acid to osmotic shock and oxidative stress.

6. Conner, D. E., and J. S. Kotrola. 1995. Growth and survival of Escherichia coli

O157:H7 under acidic conditions. Appl. Environ. Microbiol. 61:382-385.

7. Deng Y., J. Ryu, and L.R. Beuchat. 1998. Influence of temperature and pH on

survival of Escherichia coli O157:H7 in dry foods and growth in reconstituted infant

rice cereal. Int. J. Food Microbiol. 45:173-184.

8. Dineen, S. S., K. Takeuchi, J. E. Soudah, and K. J. Boor. 1998. Persistence of

Escherichia coli O157:H7 in dairy fermentation systems. J. Food Prot. 61:1602-1608.

9. Doyle, M. P., T. Zhao, J. Meng, and S. Zhao. 1997. Escherichia coli O157:H7. In

M.P. Doyle, L.R. Beuchat, and T.J. Montville (eds.) Food Microbioloy Fundamentals

and Frontiers. p. 171-191. ASM Press, Washington DC.

10. Faith N. G., R. K. Wierzba, A. M. Ihnot, A. M. Roering, T. D. Lorang, C. W. Kaspar,

and J. B. Luchansky. 1998. Survival of Escherichia coli O157:H7 in full-and

reduced-fat pepperoni after manufacture of sticks, storage of slices at 4°C or 21°C

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under air and vacuum, and baking of slices on frozen pizza at 135, 191, and 246°C. J.

Food Prot. 61:383-389.

11. Frank, J. F., and A. N. Hassan. 1998. Starter cultures and their use. In E.H. Marth and

J.L. Steele (ed.). Appl. Dairy Microbiol. p. 131-172. Marcel Dekker, Inc. New York,

NY.

12. Hudson, L. M., J. Chen, A. R. Hill, and M. W. Griffiths. 1997. Bioluminescence: A

rapid indicator of Escherichia coli O157:H7 in selected yogurt and cheese varieties. J.

Food Prot. 60:891-897.

13. Karmali, M. A., B. T. Steele, M. Petric, and C. Lim. 1983. Sporadic hemolytic uremic

syndrome associated with fecal cytotoxin and cytotoxin-producing Escherichia coli.

Lancet. 1:619-620.

14. Mao, Y., M. P. Doyle, and J. Chen. 2001. Insertion mutagenesis of wca reduces acid

and heat tolerance of enterohemorrhagic Escherichia coli O157:H7. J. Bacteriol.

183:3811-3815.

15. Massa, S., C. Altieri, V. Quaranta, and R. De Pace. 1997. Survival of Escherichia coli

O157:H7 in yoghurt during preparation and storage at 4°C. Lett. Appl. Micobiol

24:347-350.

16. Massa, S., E. Goffredo, C. Altieri, and K. Natola. 1999. Fate of Escherichia coli

O157:H7 in unpasteurized milk stored at 8°C. Lett. Appl. Microbiol. 28:89-92.

17. Miller, L.G., and C.W. Kaspar. 1994. Escherichia coli O157:H7 acid tolerance and

survival in apple cider. J. Food Prot. 57: 460-464.

57

18. Morgan, D., C. P. Newman, D. N. Hutchinson, A. W. Walker, B. Rowe, and F.

Majid. 1993. Verotoxin producing Escherichia coli O157 infections associated with

the consumption of yogurt. Epidemiol. Infect. 111:181-187.

19. Ophir, T., and D. L. Gutnick. 1994. A role for exopolysaccharide in the protection of

microorganism from desiccation. Appl. Environ. Microbiol. 60:740-745.

20. Radke-Mitchell, L., and W. Sandine. 1984. Associative growth and differential

enumeration of Streptococcus thermophilus and Lactobacillus bulgaricus: a review. J.

Food Prot. 47:245-248.

21. Ramsaran, H., J. Chen, B. Brunke, A. Hill, and M. W. Griffiths. 1998. Survival of

bioluminescent Listeria monocytogenes and Escherichia coli O157:H7 in soft

cheeses. J. Dairy Sci. 81:1810-1817.

22. Tamime, A. Y., and R. K. Robinson. 1999. Biochemistry of fermentation. In Yogurt

Science and Technology. p. 432-475. CRC Press. Boca Raton, FL.

23. Tsai, Y., and S. C. Ingham. 1997. Survival of Escherichia coli O157:H7 and

Salmonella spp. in acidic condiments. J. Food Prot. 60:751-755.

24. Uljas, H. E., and S. C. Ingham. 1998. Survival of Escherichia coli O157:H7 in

synthetic gastric fluid after cold and acid habituation in apple juice or trypticase soy

broth acidified with hydrochloric acid or organic acids. J. Food Prot. 61:939-947.

25. Wang, G., T. Zhao, and M. P. Doyle. 1997. Survival and growth of Escherichia coli

O157:H7 in unpasteurized and pasteurized milk. J. Food Prot. 60:610-613.

CHAPTER 4

INFLUENCE OF AN EXOPOLYSACCHARIDE, COLANIC ACID ON, FATE OF

ESCHERICHIA COLI O157:H7 IN STIRRED YOGURT 1

1Lee, S.M. and Chen, J. To be submitted to Applied and Environmental Microbiology.

58

59

This study was undertaken to determine the role of colanic acid (CA), produced

by E. coli O157:H7 under stress, in protecting the cells from acid stress during processing

and storage of stirred yogurt. A wild-type E. coli O157:H7, its CA-deficient mutant and

a mixture of the two strains were either inoculated before fermentation into pasteurized

milk or after fermentation into yogurt. Processed yogurt stored at 4° or 15°C was assayed

twice each week for 3 weeks for total plate counts, populations of E. coli O157:H7 and

starter cultures. No significant differences were observed between survivals of the 3

inocula in stirred yogurt inoculated pre-fermentation and stored at 4° or 15°C as

monitored by SMAC (P>0.05). However, statistical analysis of population recovered on

TSA gave different results. As to post-fermentation inoculation, data recovered did not

suggest that CA significantly enhanced the survival of E. coli O157:H7 in stirred yogurt.

60

Escherichia coli serotype O157:H7 belongs to a group known as

enterohemorrhagic E. coli (EHEC) that causes severe foodborne illnesses. The pathogen

has been a major public health concern since its first outbreak in the State of Oregon and

Washington in 1982 (11). E. coli O157:H7 is unique among foodborne bacterial

pathogens because it does not require a high infection dosage to cause manifestations

such as hemolytic uremic syndrome (HUS) (18). Young children and immuno-

compromised individuals are particularly susceptible to the illness. Griffin and Tauxe

(11) reported that HUS is a leading cause of acute kidney failure in children and some of

the cases are fatal.

Low pH is traditionally believed to be able not only to inhibit but also to kill

pathogens in food. However, several recent outbreaks linked E. coli O157:H7 with acid

food (3, 4, 6, 7, 16). This suggests that the pathogen has the strategy to combat acid

stress (8, 14, 19, 20, 22), subsequently causes illness in human (16). Outbreaks of E. coli

O157:H7 associated with consumption of an acidic food, yogurt, occurred in England in

1991. Cross-contamination between raw and pasteurized milk and poor environmental

hygiene might be the contributing factors (16).

E. coli O157:H7 produces protective exopolysaccharide (EPS), colanic acid

(CA), under adverse environmental conditions (13). Studies have found that CA assists

the pathogen to combat stress in acidified microbiological media and simulated gastric

fluid (15). However, the influence of CA on fate of E. coli O157:H7 in acidic food has

not yet been determined. The objective of this study was to determine whether

production of CA protects cells of E. coli O157:H7 during fermentation and storage of

stirred yogurt.

61

Yogurt is usually made by inoculating dairy ingredients such as milk, cream,

skimmed milk or partially skimmed milk with lactic acid producing bacteria such as

Streptococcus thermophilus and Lactobacillus bulgaricus (FDA, 2000). While set yogurt

is fermented in yogurt cups, stirred yogurt is fermented in bulk. After fermentation, the

milk curd is stirred, and then distributed into yogurt cups. In this study, stirred yogurt

was contaminated with E. coli O157:H7 W, M, or WM at either pre- or post-fermentation

stage. The survival of the pathogen was monitored during processing and a subsequent

3-w storage at 4 or 15oC.

MATERIALS AND METHODS

Culture preparation. E. coli O157:H7 (W) and its CA-deficient mutant (M)

(Mao et al., 2001) from our laboratory collection were used in this study. W and M were

grown on minimal glucose agar (MGA) and MGA with kanamycin (100 µg/mL),

respectively at 25°C for 72 h. The resulting cultures were harvested with 5 mL of 0.1%

peptone water. The cell suspensions were appropriately diluted and plated on tryptic soy

(TSA) and sorbitol MacConkey agar (SMAC) (Difco, Spraks, MD). The plates were

incubated at 37oC for 24 h. Colonies appeared on each plate were enumerated.

Populations of cells in the W and M suspensions were calculated to ascertain that

approximately an equal level of W and M would be used in the stirred yogurt study.

Starter culture, YC-180, used in this study was provided by CHR Hansen, Inc.

(Milaukee, WI). YC-180 is a mixture of Streptococcus thermophilus and Lactobacillus

delbruekii subsps. bulgaricus and subsp lactis. These strains were blended together and

62

made into concentrated frozen pellet to enable easy inoculation without thawing. The

starter cultures were kept at –30°C until use.

Pre-fermentation contamination. Flow chart of stirred yogurt processing is

illustrated in Figure 4.1. Four gallons of whole milk and a pack (725 g) of non-fat dried

milk (NFDM) were purchased from a local grocery store. The milk (2.7 L) was

distributed into each of the 4 stainless steel containers (VWR, Atlanta, GA) and

subsequently supplemented with NFDM (2%). The milk was stirred for 4 min at room

temperature before being stored overnight at 4°C. In the following morning, the milk

was placed into a water bath (91.44cm x 45.6cm x 30.48cm ) (Magni Whirl, Blue M

Electric Company, Blue Island, IL) that was preheated to 98oC. The milk was heated and

pasteurized at 95°C for 5 min. Temperature of the water and milk was constantly

monitored with thermometers.

After pasteurization, the containers were removed from the water bath to plastic

buckets that were filled with ice slurries. Cooled milk (43°C) was inoculated with YC-

180 (0.36g, ca. 105 CFU/mL of L. bulgaricus and 106 CFU/mL of S. thermophilus) and E.

coli O157:H7 W, M, or WM (ca.105 CFU/mL), and stirred for 4 min with magnetic bars

to distribute the cell cultures. The milk in the stainless-steel containers was then

fermented in an incubator at 43°C. The milk containers were removed from the incubator

when pH of milk curds reached approximately 4.5 (in about 4 h). Yogurt from each

container was poured into a mixing bowl (4L, Walmart, Griffin, GA) and stirred with a

hand mixer (Sunbeam Mixmaster Hand Mixer Model 2486, Sunbeam Products Inc.,

Delray Beach, FL.) at the lowest speed. The yogurt was stirred for 2 min, and let sit for 1

63

Whole milk

Add 2% NFDM

2 ml sample (b)

Heat to 95°C in water bath, hold for 5 min

Cool down rapidly to 43°C in ice bath

2 ml sample ( c )

Inoculate with starter culture Pre-fermentation inoculation

W WM

In

When pH reaches 4.5, pou

Stir using

Let sit for 1 min and stir for

Divide into in

Store at 4°C

Sample tw Figure 4.1. Flow chart for processing

M

cubate at 43°C

r in to mixing bowl im

hand mixer for 2 m

another 1 min until c

dividual cups (6oz., 1

Store at 1

ice a week for 3 wee

of artificially contam

C

2 ml sample (a)

2 ml sample (d)

Sample (BF)

mersed in ice bath

in Post-fermentation inoculation

ool down to 18-20°C

70.1g)

Sample (AF/I) 5°C

ks

inated stirred yogurt.

64

min. This process continued until temperature of the yogurt reached 18-20°C

(approximately 10 min). The stirred yogurt (170.1 g) was then divided into sterile

polypropylene yogurt cups (8 oz.; Fisher Brand). Among the 15 cups of yogurt from

each stainless steel container, 1 was used as sample AF (after fermentation sampling), 7

were stored at 4° and 15°C, respectively. Stored yogurt was sampled twice a week

during a 3-w period.

Post-fermentation contamination. The yogurt-making process was basically

the same as described in pre-fermentation contamination except that W, M or WM were

inoculated after fermentation, before stirring (Figure 4.1).

Sampling and enumeration. As indicated in Figure 4.1, milk samples were

taken before (a), and after (b) the addition of NFDM, after pasteurization (c), and after

cooling (d). Milk samples designated as BF were also collected before fermentation, but

after inoculation with starter culture and E. coli O157:H7. Whereas sample AF (after

fermentation) was assayed after distribution of stirred yogurt into individual yogurt cups.

Two ml of milk were taken for sample a, b, c, d, and BF. A whole cup of yogurt

was used for AF and stored samples. The temperature and pH of the yogurt were

measured and recorded prior to microbiological testing. Milk sample a to d were plated

directly while BF was serially diluted in 0.1% peptone water before plating. Yogurt

samples was poured into a filtered stomacher bag and mixed with 170.1 mL of 0.1%

peptone water. The sample was homogenized for 30 seconds at normal speed in a

stomacher (Seward Limited, London). The homogenates were appropriately diluted and

spiral plated using an automated spiral platter (Autoplate 4000, Spiral Biotech, Inc.,

Bethesda, Maryland) for total plate counts, E. coli O157:H7, L. bulgaricus and S.

65

thermophilus, respectively on tryptic soy (TSA), sorbitol MacConkey (SMAC), acidified

MRS, and M17 agar with 10% lactose (Difco, Sparks, MD), respectively. The TSA and

SMAC plates were incubated at 37°C for 24 h, M17 at 37°C for 48 h, and MRS at 45°C

for 48 h under anaerobic condition in GasPak Jars (Fisher Scientific, Atlanta, GA.). All

plates were counted using an automated colony counter (Q-Count, Spiral Biotech Inc.,

Norwood, MA.) after incubation.

Data Analysis. Two replications were conducted. Data were converted to log10

CFU/ mL before they were analyzed by general linear model procedure using SAS

Program (SAS Institute Inc., Cary, N.C.). Significant differences in survival between W

and M, W and WM, M and WM in stirred yogurt stored at 4o and 15oC were determined

using a 5% confidence level.

RESULTS AND DISCUSSION

Microbial quality of commercial milk. The retail milk used to process stirred

yogurt had an average total plate count of 1.42 log10 CFU/mL. The population of cells

recovered on SMAC, MRS and M17 agar were 1.22, <1 and 1.21 log10 CFU/mL

respectively. Colonies on SMAC were not E. coli O157:H7. Pasteurization of milk at

95oC for 5 min lowered the microbial counts to an undetectable level.

Starter culture and yogurt pH. Table 4.1 and 4.2 summarize the pH, temperature and

starter counts in stirred yogurt during the storage at 4° and 15°C, respectively. The

yogurt inoculated at pre- or post-fermentation stage had a pH of 4.64 ± 0.07 and 4.49 ±

0.02, respectively immediately after the fermentation. L. bulgaricus grew 1.61-2.61 log10

66

CFU/mL whereas S. thermophilus increased 2.46-3.58 log10 CFU/mL during

fermentation. The populations of S. thermophilus and L. bulgaricus in stirred yogurt

were relatively stable throughout the storage period (Table 4.2 and 4.3). However, the

pH of the yogurt continuously declined especially at 15oC. Yogurt stored at 15°C,

therefore, had relatively lower pH (3.99-4.10) compared to samples at 4°C (4.37-4.48).

Pre-fermentation contamination. W and WM grew while M remained

approximately steady during fermentation (Figure 4.3 A and B). Since the population of

M was unchanged, the population increase in WM was probably due to the growth of W

cells in the mixture.

Temperature appeared to be an important factor influencing the survival of E. coli

O157:H7 in stirred yogurt. The pathogen persisted longer in yogurt stored at 4oC than at

15oC. At the end of storage at 4oC, 2.7 log10 CFU/mL of W, 1.25 log10 CFU/mL of M,

and 1.43 log10 CFU/mL of WM, respectively were recovered on TSA (Figure 4.3A).

However, the 3 inocula all died off during storage for 21 d at 15oC (Figure 4.4A). In

yogurt stored for 7 d at 4oC, the populations of W, M and WM on SMAC were 2.2, 1.97,

and 1.47 log10 CFU/mL, respectively (Figure 4.3B). However, the M and WM were

undetectable on SMAC at the same sampling day at 15oC (Figure 4.4B). The relatively

faster decline of E. coli O157:H7 populations at 15oC might be associated with the low

pH of the yogurt stored at this temperature (Table 4.1). At 15°C, YC-180 and E. coli

O157:H7 may still be metabolizing and were able to utilize remaining lactose in yogurt

and further decrease its pH. In addition, cell membranes were relatively more

permissible at 15oC, which allowed more efficient transmembrane migration of

undisassociated organic acids produced during fermentation process.

67

Table 4.1. The pH, temperature and starter counts in stirred yogurt that was inoculated with E. coli O157:H7 before fermentation.

Sample pH Temp (°C)

L. bulgaricus Log10 CFU/mL

S. thermophilus Log10 CFU/mL Sample pH

Temp (°C)

L. bulgaricus Log10 CFU/mL

S. thermophilus Log10 CFU/mL

WaBFe N/A N/A 4.23 6.27 WBF N/A N/A 4.23 6.27 WAFf 4.64 14 5.94 9.37 WAF 4.64 14 5.94 9.37 W1gah 4.75 4 5.82 8.81 W 4.36 15 5.91 8.93 W1bi 4.67 4 5.94 9.44 W 4.29 15 5.94 9.50 W2a 4.49 4 5.90 8.93 W 4.17 15 5.83 8.91 W2b 4.46 4 5.85 8.96 W 4.19 15 5.86 8.99 W2a 4.37 4 5.85 8.87 W 4.06 15 5.79 8.88 W3b 4.44 4 5.87 8.95 W 4.00 15 5.74 8.73 MbBF N/A N/A 5.23 6.07 MBF N/A N/A 5.23 6.07 MAF 4.63 14.5 6.84 9.56 MAF 4.63 14.5 6.84 9.56 M1a 4.71 4 7.39 8.95 M 4.28 15 7.56 8.97 M1b 4.62 4 7.50 9.56 M 4.22 15 7.56 9.54 M2a 4.54 4 7.38 8.92 M 4.13 15 7.31 8.98 M2b 4.58 4 7.40 9.02 M 4.14 15 7.32 9.02 M3a 4.36 4 7.42 9.08 M 4.08 15 7.22 8.96 M3b 4.41 4 7.44 9.04 M 4.05 15 7.21 8.78

WMcBF N/A N/A 3.72 6.03 WMBF N/A N/A 3.72 6.03 WMAF 4.57 14.5 6.33 8.49 WMA 4.57 14.5 6.33 8.49 WM1a 4.66 4 5.72 7.50 WM 4.28 15 5.84 9.02 WM1b 4.54 4 5.83 9.55 WM 4.16 15 5.96 9.44 WM2a 4.47 4 5.78 8.97 WM 4.08 15 5.82 8.93 WM2b 4.50 4 5.80 9.02 WM 4.10 15 5.80 8.96 WM3a 4.49 4 5.79 9.05 WM 4.00 15 5.73 8.89 WM3b 4.37 4 5.73 8.89 WM 3.99 15 5.70 8.92 CdBF N/A N/A 4.91 6.57 CBF N/A N/A 4.91 6.57 CAF 4.71 15 6.88 9.44 CAF 4.71 15 6.88 9.44 C1a 4.87 4 6.78 8.83 C 4.36 15 6.95 8.94 C1b 4.69 4 6.96 9.19 C 4.23 15 7.11 9.60 C2a 4.64 4 6.88 8.87 C 4.13 15 6.97 9.02 C2b 4.67 4 6.87 8.89 C 4.13 15 6.85 8.95 C3a 4.51 4 6.90 8.93 C 4.05 15 6.88 8.99 C3b 4.42 4 6.87 8.98 C 4.00 15 6.64 8.87

a W: wild type; bM: CA-deficient mutant; cWM: wild type and mutant mixture; dC: control e BF: before fermentation; f AF: after fermentation g Sample 1, 2, and 3: number of weeks into storage h Sample a: the first sampling in a week; iSample b: the second sampling in a week

68

Table 4.2. The pH, temperature and counts of starter cultures in stirred yogurt that was inoculated with E. coli O157:H7 after fermentation.

Sample pH Temp (°C)

L. bulgaricus Log10 CFU/mL

S. thermophilus Log10 CFU/mL Sample pH

Temp (°C)

L. bulgaricus Log10 CFU/mL

S. thermophilus Log10 CFU/mL

WaBFe N/A N/A 5.36 6.42 WBF N/A N/A 5.35 6.42 WAFf 4.51 43.25 7.63 9.06 WAF 4.51 43.25 7.63 9.06 W1gah 4.68 4 7.42 8.69 W1a 4.39 15 7.47 8.91 W1bi 4.69 4 7.59 9.33 W1b 4.25 15 7.43 8.98 W2a 4.56 4 7.41 9.11 W2a 4.18 15 7.55 9.30 W2b 4.51 4 7.40 8.89 W2b 4.12 15 7.45 8.99 W3a 4.89 4 7.35 8.92 W3a 4.49 15 7.42 9.03 W3b 4.48 4 7.32 8.87 W3b 4.08 15 7.33 8.81

Mb BF N/A N/A 5.15 6.32 MBF N/A N/A 5.15 6.32 MAF 4.50 43.5 7.36 9.22 MAF 4.50 43.5 7.36 9.22 M1a 4.61 4 7.26 8.86 M1a 4.36 15 7.28 8.93 M1b 4.58 4 7.43 7.92 M1b 4.23 15 8.83 8.99 M2a 4.59 4 7.38 9.27 M2a 4.20 15 7.33 9.38 M2b 4.53 4 7.29 8.88 M2b 4.11 15 7.28 8.99 M3a 4.82 4 7.31 8.96 M3a 4.45 15 7.21 8.85 M3b 4.46 4 7.21 8.83 M3b 4.07 15 8.64 8.78

WMc BF N/A N/A 5.19 6.26 WMBF N/A N/A 5.19 6.26 WMAF 4.47 42.75 7.53 9.14 WMAF 4.47 42.75 7.53 9.14 WM1a 4.56 4 7.32 8.98 WM1a 4.29 15 7.42 8.92 WM1b 4.57 4 7.74 9.51 WM1b 4.20 15 7.41 9.03 WM2a 4.47 4 7.40 8.99 WM2a 4.18 15 7.44 9.04 WM2b 4.44 4 7.42 9.02 WM2b 4.11 15 7.36 9.04 WM3a 4.78 4 7.39 8.88 WM3a 4.46 15 7.23 8.78 WM3b 4.39 4 7.36 8.67 WM3b 4.10 15 7.20 8.54 Cd BF N/A N/A 4.24 6.52 CBF N/A N/A 4.37 6.52 CAF 4.50 43 6.53 9.22 CAF 4.50 43 6.53 9.22 C1a 4.66 4 6.28 8.94 C1a 4.37 15 6.38 8.98 C1b 4.71 4 6.33 9.46 C1b 4.22 15 6.45 9.43 C2a 4.62 4 6.30 9.12 C2a 4.12 15 6.45 9.26 C2b 4.50 4 6.32 8.96 C2b 4.03 15 6.37 9.09 C3a 4.86 4 6.33 9.04 C3a 4.37 15 6.30 9.07 C3b 4.47 4 6.34 8.85 C3b 4.00 15 6.36 8.94

a W: wild type; b M: CA-deficient mutant; c WM: wild type and mutant mixture; d C: control e BF: before fermentation; f AF: after fermentation g Sample 1, 2, and 3: number of weeks into storage h Sample a: the first sampling in a week; i Sample b: the second sampling in a week

69

0

1

2

3

4

5

6

7

0 2 4 6 8 10 12 14 16 18 20

Storage time (Days)

Popu

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n of

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7 (L

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WMWM

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0

1

2

3

4

5

6

7

0 2 4 6 8 10 12 14 16 18 20

Storage time (Days)

Popu

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WMWM

B

Figure 4.3. Survival of W, M and WM in stirred yogurt that was inoculated with E. coli O157:H7 at pre-fermentation stage and stored 4°C. Enumeration was done on TSA (A) and SMAC (B). BF: before fermentation; AF: after fermentation; 4-21: number of days into storage.

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0

1

2

3

4

5

6

7

0 2 4 6 8 10 12 14 16 18 20

Storage time (Days)

Popu

latio

n of

E. c

oli

O15

7:H

7 (L

og10

CFU

/ml)

WMWM

A

0

1

2

3

4

5

6

7

0 2 4 6 8 10 12 14 16 18 20

Storage time (Days)

Popu

latio

n of

E. c

oli

O15

7:H

7 (L

og10

CFU

/ml)

WMWM

B

Figure 4.4. Survival of W, M and WM in stirred yogurt that was inoculated with E. coli O157:H7 at pre-fermentation stage and stored at 15°C. Enumeration was done on TSA (A) and SMAC (B). BF: before fermentation; AF: after fermentation; 4-21: number of days into storage.

71

As observed in the set yogurt study (Chapter 3, this study), more cells were

recovered on TSA than on SMAC. At day 7 and 4oC on TSA, the populations of W, M,

and WM were 5.88, 3.49, and 5.79 log10CFU/mL, respectively (Figure 4.3A), but there

were only 2.2, 1.47 and 1.97 log10CFU/mL of W, M and WM respectively on SMAC

(Figure 4.3B). TSA recovered 2.7 log10CFU/mL of W, 1.43 log10CFU/mL of WM, and

1.25 log10CFU/mL of M, respectively (Figure 4.3A) while W and WM became

undetectable on SMAC (Figure 4.3B) after 21 d at 4°C.

The survival of W vs. M, W vs. WM, and M vs. WM in stirred yogurt stored at 4o

and 15oC were not significantly different as monitored by SMAC (P>0.05). However,

statistical analysis of data collected from TSA plates generated different results. While

the W and WM behaved approximately the same (P>0.05), W and M survived differently

in stirred yogurt at either storage temperatures (P<0.05). The P values for the survival of

M vs. WM were 0.04 at 15oC and 0.07 at 4oC.

Post-fermentation. Inoculated after fermentation, cells of E. coli O157:H7 are

less severely injured. Consequently, yogurt inoculated at post-fermentation stage had

higher counts. In yogurt that was stored for 21 d at 4oC, populations of cells on SMAC

decreased only about 1.66 log10 CFU/mL for W, 4.71 log10CFU/mL for M, and 2.97

log10CFU/mL for WM (Figure 4.5B). Whereas counts of W, M, or WM in pre-

fermentation-inoculated yogurt almost all died off at the same sampling time (Figure

4.3B). Yogurt inoculated before fermentation also yielded lower counts on TSA (Figure

4.3A and 4.5A). These results suggest that the cells that did not go through fermentation

survived better in stirred yogurt. This observation agreed with a previous

72

0

1

2

3

4

5

6

7

0 2 4 6 8 10 12 14 16 18 20

Storage time (Days)

Popu

latio

n of

E. c

oli

O15

7:H

7 (L

og10

CFU

/ml)

WMWM

A

0

1

2

3

4

5

6

7

0 2 4 6 8 10 12 14 16 18 20

Storage time (Days)

Popu

latio

n of

E. c

oli

O15

7:H

7 (L

og10

CFU

/ml)

WMWM

B

Figure 4.5. Survival of W, M and WM in stirred yogurt that was inoculated with E. coli O157:H7 post-processing stage and stored at 4°C. Enumeration was done on TSA (A) and SMAC (B). BF: before fermentation; AF: after fermentation; 4-21: number of days into storage.

73

0

1

2

3

4

5

6

7

0 2 4 6 8 10 12 14 16 18 20

Storage time (Days)

Popu

latio

n of

E. c

oli

O15

7:H

7 (L

og10

CFU

/ml)

WMWM

A

0

1

2

3

4

5

6

7

0 2 4 6 8 10 12 14 16 18 20

Storage time (Days)

Popu

latio

n of

E. c

oli

O15

7:H

7 (L

og10

CFU

/ml)

WMWM

B

Figure 4.6. Survival of W, M and WM in stirred yogurt that was inoculated with E. coli O157:H7 at post-fermentation stage and stored at 15°C. Enumeration was done on TSA (A) and SMAC (B). BF: before fermentation; AF: after fermentation; 4-21: number of days into storage.

74

study done by McIngvale et al. (2000) in which E. coli O157:H7 was recovered after 22

and 33 d, respectively in buttermilk contaminated before or after fermentation.

As observed in pre-fermentation inoculation experiment (this study), counts of all

3 inocula in yogurt contaminated at post-fermentation stage declined faster at 15oC

(Figure 4.6) than at 4oC (Figure 4.5). Cells of W, M, and WM were undetectable on

SMAC after 10, 14 or 17 d of storage at 15oC (Figure 4.6B). At 4oC, however, cells of all

3 inocula persisted the entire storage period (Figure 4.5B). On TSA at 15oC (Figure

4.6A), no colonies were recovered from yogurt inoculated with M and WM at day 14 and

with W at day 21. However, W, M, and WM in yogurt stored at 4oC survived the entire

storage period (Figure 4.5A). Hudson et al., (1997) reported that E. coli O157:H7

survived better at 4oC than at 10oC (12). E. coli O157:H7 survived for 8 d at 4°C and 5 d

at 10°C in yogurt with a pH of 4.17, for 17 and 15 d in yogurt with a pH of 4.39 at 4° and

10°C, respectively (12).

Statistical analysis of data recovered from SMAC and TSA did not suggest that

CA significantly enhanced the survival of E. coli O157:H7 in stirred yogurt. Previous

study indicated that CA is loosely attached to the surfaces of its secreting cells. It was

suggested that CA gives the bacterial cell surface a strong negative charge and when the

cells are in low pH environment (great concentration of protons), the CA may act as a

buffer to the cells thus preventing the acid from penetrating the cells (15).

It is possible that the stirring step during production of stirred yogurt physically detached

CA from cells of E. coli O157:H7. The physical and proton-neutralizing barrier provided

75

by CA was no longer available to protect cells of E. coli O15:H7 during subsequent

storage at 4o or 15oC.

REFERENCES

1. Besser R. E., S. M.. Lett, J. T. Weber, M. P. Doyle, T. J. Barrett, J. G. Wells, and

P. M. Griffin. 1993. An outbreak of diarrhea and hemolytic uremic syndrome

from Escherichia coli O157:H7 in fresh-pressed apple cider. J. Am. Med. Assoc.

269:2217-2220.

2. Centers for Disease Control and Prevention. 2000. Outbreak of Escherichia coli

O157:H7 infection associated with eating fresh cheese curds – Wisconsin, June

1998. Morbid. Mortal. Wkly. Rep. 49:911-913.

3. Centers for Disease Control and Prevention. 1996. Outbreak of Escherichia coli

O157:H7 infections associated with drinking unpasteurized commercial apple

juice – British Columbia, California, Colorado and Washington. Morbid. Mortal.

Weekly Rep. 45:975.

4. Centers for Disease Control and Prevention. 1995. Escherichia coli O157:H7

outbreak linked to commercially distributed dry-cured salami – Washington and

California, 1994. Morbid. Mortal. Weekly Rep. 44:157-160.

5. Conner, D. E., and J. S. Kotrola. 1995. Growth and survival of Escherichia coli

O157:H7 under acidic conditions. Appl. Environ. Microbiol. 61:382-385.

6. Food and Drug Administration. 2000. Requirement for specific standardized milk

and cream. Code of Federal Regulations No.21, Part 131, Sec. 200. U.S.

Government Printing Office, Washington D.C.

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7. Griffin, P. M., and R. V. Tauxe. 1991. The epidemiology of infection caused by

Escherichia coli O157:H7, other entero-haemorrhagic E. coli and the associated

haemolytic uraemic syndrome. Epidemiol Rev. 13:60-98.

8. Hudson, L. M., J. Chen, A. R. Hill, and M. W. Griffiths. 1997. Bioluminescence:

A rapid indicator of Escherichia coli O157:H7 in selected yogurt and cheese

varieties. J. Food Prot. 60:891-897.

9. Junkins, A. D., and M. P. Doyle. 1992. Demonstration of exopolysacchride

production by enterohemorrhagic Escherichia coli. Curr. Microbiol. 25:9-17.

10. Massa, S., C. Altieri, V. Quaranta, and R. De Pace. 1997. Survival of Escherichia

coli O157:H7 in yoghurt during preparation and storage at 4°C. Lett. Appl.

Micobiol 24:347-350.

11. Mao, Y. M. P. Doyle, and J. Chen. 2001. Insertion mutagenesis of wca reduces

acid and heat tolerance of enterohemorrhagic Escherichia coli O157:H7. J.

Bacteriol. 183:3811-3815.

12. McIngvale, S. C., X. Q. Chen, J. L.McKillip, and M. A. Drake. 2000. Survival of

Escherichia coli O157:H7 in buttermilk as affected by contamination point and

storage temperature. J. Food Prot. 63:441-444.

13. Morgan, D., C. P. Newman, D. N. Hutchinson, A. W. Walker, B. Rowe, and F.

Majid. 1993. Verotoxin producing Escherichia coli O157 infections associated

with the consumption of yogurt. Epidemiol. Infect. 111:181-187.

14. Padhye, N. V., and M. P. Doyle. 1992. Escherichia coli O157:H7: epidemiology,

pathogenesis, and methods for detection in foods. J. Food Prot. 55:555-565.

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15. Ramsaran, H., J. Chen, B. Brunke, A. Hill, and M. W. Griffiths. 1998. Survival of

bioluminescent Listeria monocytogenes and Escherichia coli O157:H7 in soft

cheeses. J. Dairy Sci. 81:1810-1817.

16. Ryu, J. and L. R. Beuchat. 1998. Influence of acid tolerance responses on

survival, growth, and thermal cross-protection of Escherichia coli O157:H7 in

acidified media and fruit juices. Int. J. Food Microbiol. 45:185-193.

17. Uljas, H. E., and S. E. Ingham. 1998. Survival of Escherichia coli O157:H7 in

synthetic gastric fluid after cold and acid habituation in apple juice or trypticase

soy broth acidified with hydrochloric acid or organic acids. J. Food Prot. 61:939-

947

CHAPTER 5

SUMMARY AND CONCLUSION

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79

The survival of W and M in set yogurt was significantly different on both TSA

and SMAC. Because W and M are isogenic pair with sole difference in CA production,

CA was, therefore, responsible for the difference in survival. Although it survived better,

E. coli O157:H7 did not grow at 15oC. This result is different from a previous study in

which E. coli O157:H7 grew in milk at temperature above 8°C. The difference might be

due to strain variation or the inhibitory pH of the yogurt.

Survival of W and M in stirred yogurt contaminated at post-fermentation stage

was not significantly different. However, yogurt inoculated before fermentation

generated mixed results. The stirring process may have detached the loosely attached CA

from the surfaces of E. coli O157:H7 cells thus making the W as susceptible to acid stress

as the M. In contrary to what was observed in the set yogurt study, the W and M cell

survived better at 4°C than 15°C. This result coincided with studies conducted by other

researchers who found that E. coli O157:H7 survived better at lower temperature under

acidic condition. At 15°C, the cell membrane may be more permissible, making it easier

for undisassociated acids to penetrate the cells.

TSA was able to recover more cells from set and stirred yogurt. This is because

TSA is a non-selective medium, therefore more suitable for recovering stressed or injured

cells.

In conclusion, CA plays a role in protecting cells of E. coli O157:H7 in set yogurt.

However in stirred yogurt, the result was inconclusive. We suggested that the CA might

be detached by mechanical stirring and cells of E. coli O157:H7 are no longer protected

by CA. Although E. coli O157:H7 cells did not grow, it can survive at refrigeration

temperature, especially in yogurt contaminated post-fermentation. Control measures

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should be taken during the processing of yogurt since E. coli O157:H7 can survive the

low pH of yogurt at refrigeration temperature.