antibacterial and antiviral activities of thymol against salmonella and coliphage ms2 on grape ...

131 Pages·2011·0.53 MB·English

ANTIBACTERIAL AND ANTIVIRAL ACTIVITIES OF THYMOL AGAINST
SALMONELLA AND COLIPHAGE MS2 ON GRAPE TOMATOES, BOTH
ALONE AND IN COMBINATION WITH ORGANIC ACIDS AND
SURFACTANTS

by

Yingjian Lu

A thesis submitted to the Faculty of the University of Delaware in partial
fulfillment of the requirements for the degree of Master of Science in Food Science

Spring 2011

Copyright 2011 Yingjian Lu
All Rights Reserved
ANTIBACTERIAL AND ANTIVIRAL ACTIVITIES OF THYMOL AGAINST
SALMONELLA AND COLIPHAGE MS2 ON GRAPE TOMATOES, BOTH
ALONE AND IN COMBINATION WITH ORGANIC ACIDS AND
SURFACTANTS

by

Yingjian Lu

Approved: _________________________________________________________
Changqing Wu, Ph.D.
Professor in charge of thesis on behalf of the Advisory Committee

Approved: _________________________________________________________
Jack Gelb, Ph.D.
Chair of the Department of Animal and Food Sciences

Approved: _________________________________________________________
Robin Morgan, Ph.D.
Dean of the College of Agriculture and Natural Resources

Approved: _________________________________________________________
Charles G. Riordan, Ph.D.
Vice Provost for Graduate and Professional Education
ACKNOWLEDGMENTS
I am thankful to my advisor Dr. Changqing Wu for all her support and
encouragement during the course of my degree. I am also thankful to Dr. Haiqiang
Chen and Dr. Kali Kniel for their help, advice, and for serving as members of my
committee. A sincere appreciation is extended to Dr. Dallas G. Hoover and Dr. Rolf
Joerger for all their assistance.
I also want to thank some really true friends, such as Xin Dong, Hudaa
Neetoo, Jie Wei and Alex Turley I made over the past two years as well as my parents
who have always been supportive of everything that I have done.
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TABLE OF CONTENTS
LIST OF TABLES ....................................................................................................... viii
LIST OF FIGURES ..................................................................................................... viii
ABSTRACT .................................................................................................................. ix

Chapter

1 INTRODUCTION .............................................................................................. 1
REFERENCES ................................................................................................... 5

2 LITERATURE REVIEW ................................................................................... 8
2.1 U.S. OUTBREAKS ASSOCIATED WITH TOMATOES ........................... 8
2.2 MICROORGANISMS OF CONCERN ..................................................... 10
2.2.1. Salmonella ..................................................................................... 10
2.2.2. Hepatitis A virus (HAV) ................................................................ 11
2.3 WASHING TREATMENT ......................................................................... 12
2.4 CHLORINE ................................................................................................ 13
2.5 PLANT ESSENTIAL OILS AND THEIR COMPONENTS ..................... 15
2.6 ORGANIC ACIDS ..................................................................................... 17
REFERENCES ................................................................................................. 21

3 REDUCTION OF SALMONELLA ENTERICA CONTAMINATION
ON GRAPE TOMATO BY WASHING WITH THYME OIL, THYMOL
AND CARVACROL AS COMPARED TO CHLORINE ................................. 33
ABSTRACT ..................................................................................................... 33
3.1 INTRODUCTION ...................................................................................... 34
3.2 MATERIALS AND METHODS ................................................................ 36
3.2.1 Reagents and media ........................................................................ 36
3.2.2 Bacterial strains and inoculun preparation ..................................... 37
3.2.3Tests of antimicrobial activities ....................................................... 37
3.2.4 Washing tests of surface inoculated tomato using natural
antimicrobial solutions ................................................................ 39
3.2.5 Residual S. enterica in the washing solution .................................. 40
3.2.6 Sensory evaluation ......................................................................... 41
3.2.7 Color and pH measurement ............................................................ 42
iv
3.2.8 Total phenolic and ascobic acid content ......................................... 42
3.2.9 Statistical analysis .......................................................................... 44
3.3 RESULTS ................................................................................................... 44
3.3.1 Antibacterial activity and efficiency of natural antimicrobial
agents to prevent S. enterica cross-contamination of grape
tomato .......................................................................................... 44
3.3.2 Sensory analysis ............................................................................. 48
3.3.3 Color and pH .................................................................................. 49
3.3.4 Content of total phenolic and ascorbic acid ................................... 50
3.4 DISCUSSION ............................................................................................. 50
REFERENCES ................................................................................................. 52

4 REDUCTION OF SALMONELLA ENTERICA ON GRAPE
TOMATOES DURING WASHING BY THYMOL AND ITS
COMBINATIONS ............................................................................................ 57
ABSTRACT ..................................................................................................... 57
4.1 INTRODUCTION ...................................................................................... 58
4.2 MATERIALS AND METHODS ................................................................ 60
4.2.1 Tests of antibacterial activities ....................................................... 60
4.2.1.1 Reagents and media ......................................................... 60
4.2.1.2 Bacterial strains and inoculum preparation ..................... 61
4.2.1.3 MIC and MBC tests ......................................................... 61
4.2.1.4 Washing tests of surface inoculated tomato using
thymol-based antimicrobials ........................................ 63
4.2.1.5 Residual S. enterica in the washing solution for
scale-up ......................................................................... 66
4.2.2 Shelf life of grape tomatoes after washing ..................................... 66
4.2.2.1 Sensory evaluation ........................................................... 67
4.2.2.2 Color and pH measurement ............................................. 67
4.2.2.3 Texture analysis ............................................................... 68
4.2.2.4 Microbiological spoilage ................................................. 68
4.2.3 Statistical analysis .......................................................................... 69
4.3 RESULTS AND DISCUSSION ................................................................. 70
4.3.1 Antibacterial activities of antimicrobial agents .............................. 70
4.3.1.1Single antimicrobial .......................................................... 70
4.3.1.2 Combination of multiple antimicrobials .......................... 72
4.3.1.3 Small-scale washing on grape tomatoes .......................... 74
4.3.1.4 Scale-up washing on grape tomatoes .............................. 77
4.3.2 Shelf-life of tomatoes ..................................................................... 80
4.3.2.1 Sensory analysis .............................................................. 80
v
4.3.2.2 Color and pH ................................................................... 83
4.3.2.3 Texture ............................................................................. 84
4.3.2.4 Microbial spoilage ........................................................... 84
REFERENCES ................................................................................................. 89

5 REDUCTION OF COLIPHAGE MS2 ON GRAPE TOMATOES
DURING WASHING BY THYMOL AND ITS COMBINATIONS ............... 95
ABSTRACT ..................................................................................................... 95
5.1 INTRODUCTION ...................................................................................... 96
5.2 MATERIALS AND METHODS ................................................................ 99
5.2.1 Reagents and media ........................................................................ 99
5.2.2 Tests of antiviral activities against Coliphage MS2 ..................... 100
5.2.2.1 Bacteriophage MS2 preparing ....................................... 100
5.2.2.2 Bacteriophage MS2 enumeration .................................. 100
5.2.2.3 Washing tests of inoculated grape tomatoes .................. 101
5.2.3 Statistical analysis ........................................................................ 102
5.3 RESULTS AND DISCUSSION ............................................................... 103
5.3.1 Anti-MS2 activities of antimicrobial agents ................................. 103
5.3.1.1 Single antimicrobial agent ............................................. 103
5.3.1.2 Combination of multiple antimicrobials ........................ 105
5.3.1.3 Small-scale washing on grape tomatoes ........................ 107
5.3.1.4 Scale-up washing on grape tomatoes ............................ 109
REFERENCES ............................................................................................... 113

6 FUTURE WORKS ......................................................................................... 119
REFERENCES ............................................................................................... 122

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LIST OF TABLES
Table 3.1 Minimum inhibitory concentrations (MICs, mg/ml) and minimum
bactericidal concentrations (MBCs, mg/ml) of natural
antimicrobial agents against four Salmonella enterica serovars.
................................................................................................................. 45
Table 3.2 Sensory test of unwashed grape tomatoes and grape tomatoes
washed with natural antimicrobial solutions . ......................................... 49
Table 4.1 Minimum inhibitory concentrations (MICs, mg/ml) and minimum
bactericidal concentrations (MBCs, mg/ml) of single natural
antimicrobial agents against S. Typhimurium . ....................................... 71
Table 4.2 Inhibition zones of combinations . .......................................................... 74
Table 4.3 Washing test on tomatoes (small-scale) . ................................................. 77
Table 4.4 Washing test on tomatoes (scale-up) . ..................................................... 79
Table 4.5 Residual Salmonella in washing solutions scale-up . .............................. 80
Table 4.6 Sensory test of grape tomatoes after washing (stored in room
temperature) . ........................................................................................... 81
Table 4.7 Sensory test of grape tomatoes after washing (stored in 4oC)) . ............. 82
Table 4.8 pH, texture, color and total bacteria counts of grape tomatoes after
washing (stored in room temperature) . ................................................... 86
Table 4.9 pH, texture, color and total bacteria counts of grape tomatoes after
washing (stored in 4 oC) . ......................................................................... 87
Table 5.1 Anti-MS2 activities of single antimicrobial agent . ............................... 105
Table 5.2 Anti-MS2 activities of multiple antimicrobial agents . ......................... 107
Table 5.3 Washing test on tomatoes (small-scale) . ............................................... 109
Table 5.4 Washing test on tomatoes (scale-up) . ................................................... 112

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LIST OF FIGURES
Figure 3.1 Recovery of 4 Salmonella enterica serovars from surface
inoculated grape tomatoes following 5 min-rinses of natural
antimicrobial solutions ........................................................................... 46
Figure 3.2 Recovery of 4 Salmonella enterica serovars from surface
inoculated grape tomatoes following 10 min-rinses of natural
antimicrobial solutions ........................................................................... 47
Figure 3.3 Residual Salmonella populations in natural antimicrobial
solutions ................................................................................................. 48

vi ii
ABSTRACT
Grape tomato has been classified a high-risk produce for Salmonella
enterica and hepatitis A virus (HAV) contamination in recent years. The objectives of
our research were therefore to develop a formula of thymol (THY) and its combination
with organic acid or surfactant as effective washing solutions to reduce S. enterica and
coliphage MS2 (MS2) as a surrogate for HAV on grape tomatoes. Also, the impact on
the quality of grape tomatoes after washing with those antimicrobial solutions was
studied. Based on our study, THY 0.2 mg/ml + sodium dodecyl sulfate (SDS) 5%+
acetic acid (AA) 2 mg/ml washing treatment not only achieved greater than 7 log
reduction of S. Typhimurium on both small and large numbers of inoculated grape
tomatoes, but also reduced the risk of cross-contamination by residual Salmonella in
the antimicrobial solution. Furthermore, it did not significantly affect the taste, aroma,
visual quality, pH and color of the grape tomato. Also, it could reduce more than 1.8
log population PFU/ml of MS2 on both of small and large scales of inoculated grape
tomato. Thus, THY 0.2 mg/ml +SDS 5%+ AA 2 mg/ml could inactivate both S.
Typhimurium and MS2 on grape tomatoes, and it might be applied in industry as good
natural alternative to more toxic chlorine-based antimicrobial agents in the future.

ix
Chapter 1
INTRODUCTION
Many reported outbreaks and cases of foodborne disease are associated
with pathogenic bacteria such as Salmonella and Escherichia coli O157:H7 as well as
viruses such as hepatitis A virus (HAV) and norovirus. From 1999 to 2006, multistate
outbreaks of S. enterica Typhimurium, Enteritidis and other Salmonella strains were
traced to tomatoes in the US (Bidol et al., 2007; FDA, 2008), and resulted in serious
economic losses for the tomato industry and losses in consumer confidence in safety of
tomato and its products. Acute infectious hepatitis (caused by HAV) and gastroenteritis
(caused by norovirus) have been epidemiologically linked to the consumption of raw and
minimally processed tomatoes (Cliver et al., 2006), and it is estimated that these enteric
viruses are responsible for approximately two-thirds of all foodborne illness with a
known etiology (Mead et al., 1999). Thus, it is interesting to study an effective mitigation
method to reduce Salmonella and enteric viruses on fresh produce such as tomatoes.

Fruits and produce intended for minimal processing are often rinsed or
washed in water. Chlorine is commonly used as sanitizing solution to reduce the number
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